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In Vitro and in Vivo Antimalarial Activity of Derivatives of 110-Phenanthroline Framework.

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Arch. Pharm. Chem. Life Sci. 2006, 339, 201 – 206
A.-D. Yapi et al.
201
Full Paper
In Vitro and in Vivo Antimalarial Activity of Derivatives of
1,10-Phenanthroline Framework
Ange-Dsir Yapi1, Alexis Valentin2, Jean-Michel Chezal3, Olivier Chavignon3, Bernard Chaillot4,
Roseline Gerhardt2, Jean-Claude Teulade3, Yves Blache4
1
Laboratoire de Chimie Thrapeutique, Facult de Pharmacie, Universit de Cte d’Ivoire, Abidjan, Cte
d’Ivoire
2
UMR 152 IRD-UPS, Pharmacochimie des Substances Naturelles et Pharmacophores redox, Facult de
Pharmacie, Toulouse, France
3
Laboratoire de Chimie Organique Pharmaceutique, Facult de Pharmacie, Clermont-Ferrand, France
4
EA 3660, Unit de Molcules d’IntrÞt Biologique, Facult de Pharmacie, Dijon, France
A series of trisubstituted 1,10-phenanthrolines was prepared. These compounds exhibited mild
to high biological activities in vitro both toward chloroquino-resistant FcB1-Columbia and
FcM29-Cameron strains and Nigerian chloroquino-sensitive strain of Plasmodium falciparum.
Cytotoxicity of the most active compounds was estimated showing that one compound (10) exhibited a selective activity against malaria parasite (selectivity indexes of 52 and 144). Antiplasmodial activity of this derivative was optimized by N-10 alkylation and the phenanthrolinium salt
(15) submitted to an in vivo study using mices infected by P. vinckei petteri showing an ED50 of
7.86 mg/kg/day.
Keywords: 1,10-Phenanthroline / Plasmodium falciparum / 8-Aminoquinoline / In vitro and in vivo antiplasmodial activity /
Received: November 22, 2005; accepted: February 1, 2006
DOI 10.1002/ardp.200500246
Introduction
Of all the human parasitic diseases, the greatest toll has
been exacted by malaria both from the point of view of
mortality and morbidity and from its worldwide occurrence in tropical and subtropical areas. Every year, it
causes clinical illness in 300 to 500 million people from
which 1.5 to 2.7 millions die [1]. In addition, this public
health problem has been exacerbated in the last three
decades by the appearance of multiresistant strains of
the parasite to the currently used drugs [2]. Since this
phenomenon is spreading rapidly, there is a huge need
for the development of new effective antiplasmodial
Correspondence: Prof. Dr. Yves Blache, EA 3660, Unit de Molcules
d’IntrÞt Biologique, Facult de Pharmacie, 7 boulevard Jeanne d’Arc,
BP 89700, F-21079 Dijon, France.
E-mail: yves.blache@u-bourgogne.fr
Fax: + 33 3 80 39-3422
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
pharmacophores. As a part of our program concerning
the synthesis and biological activities of aza-analogs of
alkaloids [3], we were interested in the chemistry of
diaza-analogs of phenanthrene (phenanthrene alkaloids
are obtained from various families of the Angiospermae).
In this context, series of diaza-analogs of phenanthrene
were screened in a previous approach for antimalarial
activity in vitro on a Nigerian chloroquino-sensitive strain
and the chloroquino-resistant FcB1-Columbia and FcM29
strains [4] showing that the 1,10-phenanthroline skeleton (compound 1) exhibited biological activities with
some IC50 in the range of 1.3 to 2.4 lM both on chloroquino-sensitive and on chloroquino-resistant strains
(Fig. 1).
In continuation of these studies, we report in this
paper our results concerning the synthesis and the determination of the biological activities of compounds type
2a possessing an hydrophilic function at the C-3 position,
and compounds type 2b possessing a vinylic group at C-3
position.
202
A.-D. Yapi et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 201 – 206
type cyclization [6] using 8-amino-7-formylquinoline 7 [7]
(Scheme 3).
Treatment of 7 with ethyl acetoacetate gave the acid 8
which was further converted to the ester 3 by treatment
with ethyl iodide in basic media. Similarly, the ketone 9
was obtained by treating 7 with 2,4-pentanedione in the
same conditions.
Figure 1. Chemical structure of compound 1, 2a and 2b
Results and discussion
Synthesis of ester 3 was firstly attempted in a two-steps
process from 8-aminoquinoline 5 with respect to the retrosynthetic pathways described in Scheme 1 and according the procedure used by Adams et al. for the preparation of ethyl 2-methylquinoline carboxylate [5].
Scheme 1. Synthesis of ester 3 with respect to retrosynthetic
pathway.
Curiously, treatment of 8-aminoquinoline 5 with ethyl
acetoacetate did not lead to the expected enaminoester 4
but to amide 6 (Scheme 2). Change of conditions (808C,
508C, 258C, Na2SO4) did not change the result. The structure of 6 was unambiguously determined by 1H- and 13CNMR spectra. The 1H-NMR spectrum showed two singlets
at d 2.31 (3H) and 3.70 (2H), respectively, assigned to the
methyl and methylene groups, while the 13C-NMR spec-
Preparation of vinylic compounds
For this purpose, our strategy was focused on the use of
basic conditions that supposedly assist the elimination of
chlorhydric acid and to lead the vinylic derivatives, or, in
another way, assist the nucleophilic substitution of the
chlorine atom of the chloroethyl chain and to lead aminoethyl derivatives. In that way, compound 1 was treated
with a 1.5 eq. of several aliphatic amines with a catalytic
amount of potassium carbonate (K2CO3) and potassium
iodide (KI) in N,N-dimethylformamide (DMF). In each
case, these reactions led systematically to the elimination compound 10 without formation of the aminoethyl
derivatives (best yield was obtained using triethylamine,
Scheme 4, Table 1). When using the heterocyclic amines
piperidine and pyrrolidine, a subsequent aromatic
nucleophilic substitution of the 4-Cl atom occured leading to compounds 11 and 12. Same reactivity was
observed by using sodium methoxide which led to the
formation of the 4-methoxy derivative 13 admixed with a
small amount of the hydroxy derivative 14.
Three strains of P. falciparum were used to evaluate the
in vitro antiplasmodial activities of compounds 3, 8, 9, 10,
13, 14. Results are summarized in Table 2. This screening
showed that all compounds were less active on the chloroquino-sensitive strain (Nigerain strain) than chloroquine itself. Compounds 3, 8, 9 were the less active derivatives, while the two vinylic derivatives 10, 13 were the
most active and exhibited IC50 values in the same range
as the parent compound 1.
As 1,10-phenanthrolines are also known for their cytotoxic activities, it was considered that compounds under
investigation may be simply exhibiting a general cyto-
Table 1. Reagents and conditions for synthesis of vinylic compounds.
Scheme 2. Synthesis of amide 6.
trum showed the typical signals at d 30.9 (methyl), 52.2
(methylene), 164.0 (amide) and 203 (carbonyl). Finally,
compound 3 was obtained by means of a Friedlnder-
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Reagents and Conditions (i)
Compound Yield
NH3, KI, K2CO3, DMF
EtNH2, KI, K2CO3, DMF
Et2NH, KI, K2CO3, DMF
Et3N, KI, K2CO3, DMF
Piperidine, KI, K2CO3, DMF
Pyrrolidine, KI, K2CO3, DMF
MeOH, Na
10
10
10
10
11 and 10
12 and 10
13 and 14
46%
51%
60%
65%
28% and 25%
22% and 26%
55% and 26%
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Arch. Pharm. Chem. Life Sci. 2006, 339, 201 – 2060
Antimalarial activity of 1,10-Phenanthrolines
203
Scheme 3. Friedlnder-type cyclization for synthesis of 3.
Table 2. IC50 (lM) of compounds 1 – 14 on three P. falciparum strains.
Compound
1
3
8
9
10
13
14
CQ
IC50 Nigerian
IC50 FcB1
IC50 FcM29
24 h
72 h
24 h
72 h
24 h
72 h
2.4 l 0.4
13.62 l 2.1
12.81 l 1.1
24.63 l 1.3
3.29 l 0.27
NT
6.08 l 0.29
0.076
1.5 l 0.6
4.53 l 0.5
7.94 l 0.8
1.92 l 0.7
2.53 l 0.04
NT
2.97 l 0.99
0.076
2.4 l 0.8
27.6 l 2.1
11.7 l 1.4
0.5 l 0.04
1.91 l 0.40
1.77 l 0.30
5.57 l 2.56
0.145
1.3 l 1.1
68.2 l 3.6
11.48 l 1.3
0.83 l 0.02
1.20 l 1.11
1.77 l 0.30
4.24 l 0.22
0.145
2.3 l 0.4
NT
NT
NT
3.56 l 0.40
NT
8.64 l 4.65
0.210
1.8 l 0.3
NT
NT
NT
0.49 l 0.09
NT
4.40 l 0.33
0.210
NT: not tested, CQ: chloroquine.
Table 3. IC50 (lM) of compounds 10, 13 on Hela cell line and
selectivity index values.
Compound
IC50
24 h
1
10
13
CQ
72 h
17.4 l 3.8
14.0 l 6.2
65.10 l1 8.86 62.43 l 36.98
1.44 l 0.3
1.1 l 0.2
68.61 l 7.85 75.15 l 9.13
Selectivity index (72 h)
IC50 Hela/ IC50 Hela/
IC50 FcB1 IC50 FcM29
11.3
52. 02
0.6
518.27
10.77
144.7
–
357.8
Scheme 4. Preparation of vinylic compounds.
toxic effect. For this reason, their cytotoxicity against
Hela cell line was investigated for 10 and 13. Results are
summarized in Table 3.
As can be seen, compound 10 was less cytotoxic than
the parent compound 1, and exhibited a selective activity
against malaria parasite with selectivity indexes of 52
and 144, while the 4-methoxy derivative 13 showed no
selectivity for the parasite. From these results, compound
10 was selected in view of optimization of its activity. For
this purpose, alkylation on the N-10 nitrogen atom was
investigated, since such quarternarization is well known
to increase the antiplasmodial activities of nitrogen het-
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
erocycles such as 1,10-phenanthrolines [4] and benzo[c]phenanthridines [8]. Quarternarization of 10 was
assumed through a N-10 methylation which was the
most effective group for the parent compound 1. Compound 15 was obtained by treatment of 10 with methyl
iodide in acetonitrile (Scheme 5), and evaluated for its
antiplasmodial and cytotoxic activities. Results are summarized in Table 4.
As expected, the results indicated that the N-10 methylation had an increasing effect on antiplasmodial activity, while only slight effects were observed on cytotoxicity, giving to the new derivative 15 high selectivity
indexes particularly at 72 h. With regard to the values of
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A.-D. Yapi et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 201 – 206
Table 4. IC50 (lM) of compound 15 and selectivity index values.
Nigerian
FcB1
FcM29
Hela
IC50 of compound 15
24 h
72 h
10 alkylation of the heterocycle was shown to be of primordial interest for the enhancement of the antiplasmodial activities and selectivity indexes of such compounds.
0.35 l 0.16
0.40 l 0.04
0.31 l 0.08
20.86 l 2.76
Experimental
0.40 l 0.04
0.12 l 0.08
0.08 l 0.04
26.64 l 10.82
Chemistry
Selectivity index values
Hela/FcB1
Hela/FcM29
52
68
Melting points were determined on a Bchi capillary melting
point apparatus (Bchi Labortechnik, Flawil, Switzerland) and
are not corrected. Elemental analysis was perfomed by Microanalytical Center, ENSCM, Montpellier, France. Spectral measurements were taken using the following instruments: 1H-NMR
spectra were taken on Bruker AC 100 or WM 360 or EM 400WB
(Bruker, Rheinstetten, Germany); 13C-NMR spectra were obtained
at 268C with proton noise decoupling at 25 MHz with a Bruker
AC 100 instrument. Chemical shifts are expressed relative to
residual chloroform. MS spectra were recorded on a LKB 2091
mass spectrometer (LKB) at 15 eV [h (source) = 1808C]. Dichloromethane was dried over activated alumina and distilled from
calcium hydride.
222
341
3-Oxo-N-(quinolin-8-yl)butanamide (6)
Scheme 5. Synthesis of compound 15.
Table 5. ED50 (mg/kg/day) of compound 15.
Compound
Dose
tested
Parasitaemia
(day 4)
Surviving
micea)
15
Estimated ED50: 7.86
0.2
2
20
0.2
2
64 l 14%
67 l 11%
0%
36 l 11%
0%
58 l 17%
0
0
5
1
5
0
CQ
Estimated ED50: 0.45
Controlb)
a)
b)
Numbered at day 12.
RPMI (Section 3 Experimental).
chloroquine, these high values (222 and 341) were considered sufficient to investigate the in vivo antimalarial
activity of this compound. For this purpose, a four-day
suppressive in vivo assay was performed on CD female
mice, using P. vinckei petteri [9] (Table 5).
The results of this study showed that compound 15
was active in vivo with an ED50 = 7.86 mg/kg/d for 4 days
(0.45 mg/kg/d for 4 days for chloroquine). Furthermore,
this product was well tolerated in mice since no loss was
observed after 4 days of treatment at 20 mg/kg/d (the
highest dose tested).
In conclusion, these studies confirmed the potentiality
of the 1,10-phenanthroline framework for the elaboration of new classes of antiplasmodial compounds. Substituents on the C-3 and C-4 positions were shown to affect
the antiplasmodial and cytotoxic activities, while the N-
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
A stirred solution of 1 g (6.9 mmol) of 8-aminoquinoline, 1.74 g
(8.9 mmol) of ethyl acetoacetate, 0.2 g of p-toluenesulfonic acid
in anhydrous toluene (25 mL) was refluxed for 4 h with a DeanStark apparatus. The resulting mixture was concentrated in
vacuo and the residue was washed with a saturated solution of
sodium bicarbonate. The solution was extracted with dichloromethane, the organic layers dried over sodium sulphate and evaporated. The residue was chromatographed on silica gel, eluted
with dichloromethane-methanol (95%) to give 6 as a yellow powder: yield 47%, m. p.: 129 – 1318C. FT-IR (neat): 1661 cm – 1; 3323
cm – 1. 1H-NMR (CDCl3, 100 MHz) d: 2.31 (3H, s, CH3), 3.70 (2H, s,
CH2), 7.50 (1H, dd, J = 2.0 and 1.0 Hz, H69), 7.58 (2H, m, H59, H79),
8.21 (1H, dd, J = 2.0 and 0.5 Hz, H49), 8.80 (1H, t, J = 1.0 Hz, H39),
8.92 (1H, dd, J = 2.0 and 0,5 Hz, H29), 10.62 (1H, s, NH). 13C-NMR
(CDCl3, 25 MHz) d: 29.8, 52.2, 117.1, 122.19, 127.3, 128.1, 134.3,
136.4, 136.5, 148.15, 148.6, 164.1, 203.3. Anal. Calcd. for
C13H12N202: C, 68.41; H, 5.30; N, 12.27. Found: C, 68.56; H, 5.24; N,
12.39.
3-Carboxy-2-methyl-1,10-phenanthroline (8)
A solution of 8-amino-7-quinolinecarbaldehyde (0.7 g, 4 mmol)
in 20 mL of ethylacetoacetate with a catalytic amount of piperidine was refluxed for 2 h. The resulting mixture was cooled on
ice and filtered off, leading to compound 8 which was used without further purification (87%). An analytic sample was obtained
by cristallization with ether, m. p.: 232 – 2348C. 1H-NMR (CDCl3,
100 MHz) d: 2.75 (1H, s, CH3), 7.55 (3H, m, H5, H6, H8), 8.16 (1H,
dd, J = 8.0 and 1.5 Hz, H7), 8.58 (1H, s, H4), 8.80 (1H, dd, J = 4.3
and 1.5 Hz, H9), 10.91 (1H, s, OH). 13C-NMR (CDCl3, 25 MHz) d:
30.7, 122.2, 124.6, 126.7, 130.1, 130.4, 131.3, 136.6, 138.1, 144.6,
150.1, 161.0, 198.4. Anal. Calcd. for C14H10N202: C, 70.58; H, 4.23;
N, 11.75. Found: C, 70.45; H, 4.44; N, 11.71.
Ethyl 2-methyl-1,10-phenanthroline-3-carboxylate (3)
A solution of 300 mg (1.2 mmol) of 3-carboxy-2-methyl-1,10-phenanthroline, 0.1 mL (1.8 mmol) of ethyl iodide, 20 mg of potaswww.archpharm.com
Arch. Pharm. Chem. Life Sci. 2006, 339, 201 – 2060
sium iodide and 240 mg of potassium bicarbonate in DMF
(20 mL) was stirred at room temperature for 1.5 h. Water (30 mL)
was added and the resulting mixture was extracted with dichloromethane, dried over magnesium sulfate and evaporated. The
resulting residue was purified by chromatography on silica gel
eluted with dichloromethane to give 3: yield 95%, m. p.: 200 –
2028C. 1H-NMR (CDCl3, 100 MHz) d: 1.51 (3H, t, J = 6.0 Hz, CH3),
1.44 (3H, s, CH3), 1.97 (2H, q, J = 6.0 Hz, CH2), 7.46 (3H, m, H8, H5,
H6), 8.12 (1H, d, J = 8.0 Hz, H7), 8.44 (1H, s, H4), 8.94 (1H, d, J =
3.0 Hz, H9). 13C-NMR (CDCl3, 25 MHz) d: 14.7, 31.1, 44.0, 119.1,
122.7, 122.8, 128.2, 128.7, 131.4, 135.4, 136.4, 139.1, 139.7,
142.4, 147.7, 198.4. Anal. Calcd. for C16H14N202: C, 72.16; H, 5.30;
N, 10.52. Found: C, 72.32; H, 5.11; N, 10.46.
3-Acetyl-2-methyl-1,10-phenanthroline (9)
A solution of 8-amino-7-quinolinecarbaldehyde (0.7 g, 4 mmol)
in 2,4-pentanedione (20 mL) with a catalytic amount of piperidine was refluxed for 6 h. After cooling, 30 mL of a saturated
NaCl solution were added. The resulting mixture was extracted
with dichloromethane, and the organic layers were then dried
and evaporated to give a residual brown oil which was purified
by precipitation in ether and cristallization in ethanol, leading
to 9: yield 63% yield, m. p. 138 – 1408C. 1H-NMR (CDCl3, 100 MHz):
d: 2.69 (3H, s, CH3), 3.01 (3H, s, CH3), 7.62 (1H, dd, J1 = 8.0 and
5.0 Hz, H8), 7.74 (1H, d, J = 8.0 Hz, H5 or H6), 7.77 (1H, d, J =
8.0 Hz, H6 or H5), 8.83 (1H, d, J = 8.0 Hz, H7). 13C-NMR (CDCl3,
25 MHz) d: 25.8, 29.8, 124.1, 126.4, 126.8, 127.0, 133.0, 133.1,
137.0, 138.0, 146.2, 146.1, 150.7, 158.4. Anal. Calcd. for
C15H12N20: C, 76.25; H, 5.12; N, 11.85. Found: C, 76.11; H, 5.34; N,
12.00.
4-Chloro-3-vinyl-2-methyl-1,10-phenanthroline (10)
A solution of 1 (250 mg, 0.86 mmol), triethylamine (262 mg,
2.58 mmol) and a catalytic amount of potassium iodide in DMF
(12 mL) was heated at 608C under stirring for 24 h. The mixture
was cooled and evaporated in vacuo. Water (30 mL) was added
and the resulting solution extracted with dichloromethane. The
organic layers were dried and evaporated. The residual oil was
purified by recrystallization in ether to give 10: yield 65%, m. p.:
142 – 1448C. 1H-NMR (CDCl3, 100 MHz) d: 2.73 (3H, s, CH3); 5.34
(1H, dd, J = 16.0 and 2.0 Hz, H29b), 5.52 (1H, dd, J = 10.0 and
2.0 Hz, H29a), 6.52 (1H, dd, H19), 7.28 (1H, m, H8), 7.40 (1H, d, J =
4.0 Hz, H6), 7.83 (1H, d, J = 4.0 Hz, H5), 7.86 (1H, m, H7), 8.95 (1H,
dd, J = 4.0 and 2.0 Hz, H9). 13C-NMR (CDCl3, 25 MHz) d: 25.5, 122.4,
122.9, 124.9, 126.4, 128.2, 131.5, 131.6, 135.8, 140.3, 145.2,
150.5. Anal. Calcd. for C15H11N2Cl: C, 70.73; H, 4.35; N, 10.10.
Found: C, 70.76; H, 4.21; N, 10.32.
2-Methyl-4-(piperidin-1-yl)-3-vinyl-1,10-phenanthroline
(11)
This compound was obtained in 28% yield admixed with 10
(25%) according the procedure used for the synthesis of 10 and
by using piperidine; brown oil. 1H-NMR (CDCl3, 100 MHz) d: 1.77
(6H, m, H39, H49, H59), 2.80 (3H, s, CH3), 3.12 (2H, m, H29, H69), 5.08
(1H, d, J = 12.0 and 2.0 Hz, H2'b), 5.53 (1H, dd, J = 8.0 and 2.0 Hz,
H29a), 6.81 (1H, dd, H19), 7.47 (1H, dd, J = 8.0 and 4.0 Hz, H8), 7.59
(1H, d, J = 9.0 Hz, H6), 8.11 (1H, d, J = 9.0 Hz, H5), 8.12 (1H, m, H7),
9.10 (1H, dd, J = 4.0 and 2.0 Hz, H9). 13C-NMR (CDCl3, 25 MHz) d:
24.5, 25.4, 26.8, 54.1, 120.4, 122.3, 123.1, 124.2, 128.2, 128.2,
129.1, 134.6, 135.6, 149.9, 154.5, 159.2. Anal. Calcd. for C20H21N3:
C, 79.17; H, 6.98; N, 13.85. Found: C, 79.04; H, 6.97; N, 13.99.
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Antimalarial activity of 1,10-Phenanthrolines
205
2-Methyl-4-(pyrrolidin-1-yl)-3-vinyl-1,10-phenanthroline
(12)
This compound was obtained in 22% yield mixed with 10 (26%)
according the procedure used for the synthesis of 10 and by
using pyrrolidine; brown oil; 1H-NMR (CDCl3, 100 MHz) d: 2.01
(4H, m, H39, H49), 2.93 (3H, s, CH3), 3.43 (4H, m, H19, H59), 5.57 (2H,
m, H29a, H29b), 6.76 (1H, m, H19), 7.52 (4H, m, H5, H6, H7, H8),
9.17 (1H, d, J = 2.0 Hz, H9). Anal. Calcd. for C19H19N3: C, 78.86, H,
6.62; N, 14.52. Found: C, 78.99; H, 6.57; N, 14.44.
4-Methoxy-2-methyl-3-vinyl-1,10-phenanthroline (13) and
4-hydroxy-2-methyl-3-vinyl-1,10-phenanthroline (14)
To a freshly prepared solution of sodium methanoate in methanol (15 mL), sodium (40 mg), was added a solution of 1 (250 mg)
in methanol (15 mL). The resulting solution was refluxed for 2 h.
After cooling, the solvent was evaporated and the residual oil
purified by chromatography on neutral alumina eluted with
diethyl ether/petroleum ether (95/5) to give 13 as a brown oil
with 55% yield. 1H-NMR (CDCl3, 100 MHz) d: 2.84 (3H, s, CH3), 3.83
(3H, s, OCH3), 5. (2H, m, H2'a, H2'b), 6.68 (1H, dd, J = 17.0 and
11.0 Hz, H1'), 7.38 (2H, m, H6, H8), 7.92 (2H, m, H5, H7), 9.04 (1H,
d, J=4.0 Hz, H9). 13C-NMR (CDCl3, 25 MHz) d: 25.0, 61.0, 120.4
121.7, 122.2, 122.4, 124.1, 125.1, 128.4, 129,7, 131.3, 135.6,
137.3, 145.4, 145.7, 150.0. Anal. Calcd. for C16H14N20: C, 76.78; H,
5.64; N, 11.19. Found: C, 76.87; H, 5.66; N, 11.00. Further elution
gave 14 (brown oil, 26%); MS (m/z): 236 [M+]. 1H-NMR (CDCl3,
100MHz) d: 2.66 (3H, CH3), 5.47 (1H, dd, J = 11.0 and 2.0 Hz, H2'a),
6.19 (1H, dd, J = 18.0 and 2.0 Hz, H29b), 6.66 (1H, dd, H19), 7.51
(1H, m, H8), 7.54 (1H, d, J = 10.0 Hz, H6), 8.19 (1H, m, H7), 8.34
(1H, d, J = 10.0 Hz, H5), 8.85 (1H, d, J = 5.0 Hz, H9), 10.08 (1H, s,
OH). Anal. Calcd for C15H12N20: C, 76.25; H, 5.12; N, 11.85. Found:
C, 76.01; H, 5.31; N, 11.73.
4-Chloro-3-vinyl-2,10-dimethyl-1,10-phenanthrolinium
iodide (15)
A solution of 4-methoxy-2-methyl-3-vinyl-1,10-phenanthroline
(13) (1 mmol) and methyl iodide (5 mmol) in acetone (15 mL) was
refluxed for 12 h. The resulting mixture was then cooled, a precipitate formed which was filtered and washed with acetone to
give 0.35 g of (16) (80%), (Recrystallization solvent dichloromethane/ether (50/50)); m. p. 208 – 2108C, MS (m/z): 269 [M+]. 1HNMR (CDCl3, 100 MHz) d: 2.97 (3H, s, CH3), 5.58 (3H, s, N-CH3),
5.84 (1H, dd, J = 15.0 and 4.0 Hz, H29b), 6.02 (1H, d, J = 4.0 and
3.0 Hz, H29a), 6.95 (1H, dd, H1'), 8.28 (1H, d, J = 3.0 Hz, H5), 8.50
(1H, dd, J1 = 2.0, J2 =4.0 Hz, H8), 8.67 (1H, d, J = 2Hz, H6), 9.30 (1H,
d, J = 2.0 Hz, H7), 10.37 (1H, d, 1.0 Hz, H9). 13C-NMR (CDCl3,
25 MHz) d: 25.5, 55.6, 125.1, 125.3, 126.0, 127.1, 128.9, 130.6,
132.1, 134.1, 137.0, 139.5, 141.4, 146.6, 152.7, 158.0.
Biology
Parasites were cultured according to the method described by
Trager and Jensen [10] with modifications [11]. Cultures were
synchronized by 5% D-sorbitol lysis (Merck, Darmstadt, Germany) [12, 13]. Nigerian was considered as a chloroquino-sensitive strain (chloroquine IC50: 76 l 12 nM, a 100 nM [14], FcM29
and FcB1-Columbia were considered as chloroquino-resistant
strains (chloroquine IC50: 145 l 11.2 nM and 167 l 32.2 nM,
respectively, a100 nM) [13]. In vitro antimalarial activity testing
was performed by following [3H]-hypoxanthine (Amersham,
France) incorporation as described elsewhere [15].
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A.-D. Yapi et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 201 – 206
The toxicity of the drugs was estimated on human fibroblasts
(Hela). Cell lines were cultured under the same conditions as P.
falciparum, except for the 5% human serum which was replaced
by 5% fetal calf serum. After the addition of drugs at various concentrations, cell growth was estimated by [3H]-hypoxanthine
(Amersham, France) incorporation after 24 and 72 h incubation
time [15].
In order to test the antimalarial activity of the compounds, a
four day suppressive in vivo assay was performed on CD female
mice, using P. vinckei petteri [9]. Mice (mean body weight:
20 l 2 g) were infected with 106 infected red blood cells on day 0.
Groups of five mice were treated intraperitoneally from day 0 – 3
with increasing doses (0.2 mg/kg to 20 mg/kg) of the drugs. On
day 4, Giemsa-stained smears were made for each mouse (by tail
venipunction) and parasitaemia was estimated by visual counting of about 5000 erythrocytes. Control mice were treated with
RPMI alone or chloroquine at various doses. Inhibition percentage was calculated according to the following formula:
[10] W. Trager, J. Jensen, Science 1976, 193, 673 – 675.
(control parasitaemia – parasitaemia with drugs)/(control
parasitaemia)6100.
[11] F. Benoit, A. Valentin, Y. Plissier, C. Marion, Z. Dakuyo,
et al. Trans. Roy. Soc. Trop. Med. Hyg. 1995, 89, 217 – 218.
[3] P. J. Aragon, A. D. Yapi, F. Pinguet, J. M. Chezal, J. C. Teulade, et al. Chem. Pharm. Bull. 2004, 52, 659 – 663 and references cited therein.
[4] A. D. Yapi, M. Mustofa, A. Valentin, O. Chavignon, J. C.
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3017 – 3022.
[8] J. M. Nyangulu, S. L. Hargreaves, S. L. Sharples, S. P.
Mackay, R. D. Waigh, et al. Bioorg. Med. Chem. Lett. 2005,
15, 2007 – 2010.
[9] W. Peters, Exp. Parasitol. 1965, 17, 80 – 89.
[12] J. Jensen, J. Am. Trop. Med. Hyg. 1978, 27, 1274 – 1276.
References
[1] L. S. Hoffman, M. G. Subramanian, F. H. Collins, V. Craig,
Nature 2002, 415, 702 – 709.
[2] B. Greenwood, T. Mutabingwa, Nature 2002, 415, 670 –
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i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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420.
[14] D. Parzy, B. Pradines, A. Keundjian, T. Fusa, J. C. Doury,
Med. Trop. 1976, 55, 211 – 215.
[15] A. Valentin, F. Benoit-Vical, C. Moulis, E. Stanislas, M.
Malli, et al. Antimicrob. Agents Chemother. 1997, 41,
2305 – 2307.
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