Synthesis and antimalarial activity of ethyl 3-amino-4-oxo-9- (phenylsubstituted)thieno[2,3-b]quinoline-2-carboxylate derivatives

Article abstract of DOI:10.1002/jhet.5570440320

(Chemical Equation Presented) A series of thieno[2,3-b]quinolone derivatives were synthesized and investigated for their abilities to inhibit β-hematin formation, hemoglobin hydrolysis and in vivo for their efficacy in rodent Plasmodium berghei. Compound 3b was the most promising as inhibitor of hemoglobin hydrolysis, and its effects as inhibitor of β-hematin formation was promising. When the aromatic ring was substituted in 2 (Me), in 3 (CF ₃) or in 2,4 (Cl) the inhibition of hemoglobin proteolysis was maximal (88%), the rest of compounds maintained a low inhibition. The most active compound to emerge in vitro and in murine studies, was 3b suggesting an antimalarial activity via multiple mechanisms.

Full text of DOI:10.1002/jhet.5570440320

May-Jun 2007
639
Synthesis and Antimalarial Activity of Ethyl 3-Amino-4-oxo-9-
(phenylsubstituted)thieno[2,3-b]quinoline-2-carboxylate Derivatives.
Jaime Charris,* Arthur Barazarte, José Domínguez, Gricela Lobo, José Camacho, Rosa
Ferrer, Neira Gamboa, Juan Rodrigues
Laboratorio de Síntesis Orgánica, Unidad de Bioquímica Facultad de Farmacia, Universidad Central de
Venezuela, Aptdo. 47206, Los Chaguaramos 1041-A, Caracas, Venezuela.
Mario V. Capparelli
Escuela de Química, Facultad de Ciencias, Universidad Central de Venezuela, Apartado 47074,
Caracas 1041-A, Venezuela
Received August 3, 2006
O
N
O
N
NH2
CO2Et
CN
HSCH2CO2Et
Et3N, EtOH
S
SCH3
R
R
R: H, CH3, OCH3, Br, Cl, F, CF3
A series of thieno[2,3-b]quinolone derivatives were synthesized and investigated for their abilities to
inhibit ꢀ-hematin formation, hemoglobin hydrolysis and in vivo for their efficacy in rodent Plasmodium
berghei. Compound 3b was the most promising as inhibitor of hemoglobin hydrolysis, and its effects as
inhibitor of ꢀ-hematin formation was promising. When the aromatic ring was substituted in 2 (Me), in 3
(CF3) or in 2,4 (Cl) the inhibition of hemoglobin proteolysis was maximal (88%), the rest of compounds
maintained a low inhibition. The most active compound to emerge in vitro and in murine studies, was 3b
suggesting an antimalarial activity via multiple mechanisms.
J. Heterocyclic Chem., 44, 639 (2007).
Fluoroquinolones, such as ciprofloxacin, gatifloxacin,
moxifloxacin and trovafloxacin have been reported for
their antimalarial activities [8]. We have recently
described the preparation and antimalarial activities of
several tricyclic quinolone analogs [9]. In continuation of
our studies directed toward synthesis of quinolones
annelated with various five and six member heterocycles,
we report here the synthesis of thienoquinolones and their
abilities to inhibit ꢀ-hematin formation and hemoglobin
hydrolysis in vitro and in vivo for their efficacy in rodent
Plasmodium berghei.
INTRODUCTION
Malaria is one of the most important infectious disease
problems of humans, particularly in tropical regions of the
world. As per WHO reports, annually there are more than
1 million deaths and 500 million new cases, resulting in at
least 1-2 million deaths [1,2]. In addition, since resistance
to currently used antimalarials is spreading rapidly, there
is a great need for new effective drugs. Thus, there is a
compelling and urgent need for new antimalarials with
mechanisms of action different from those of existing
ones in order to replace those that are becoming obsolete
and to identify new drug targets [3]. Chloroquine has
recently been shown to inhibit hemozoin formation within
the parasite food vacuole [4]. This process is also thought
to be the molecular target of other quinoline antimalarials
[5]. Hemozoin was originally considered to be formed by
polymerization of heme [6], but it has now been
demonstrated to be a crystalline cyclic dimmer of
ferriprotoporphyrin IX [7]. Thus, hemozoin synthesis, a
process unique to the malaria parasite, offers a logical and
valuable potential target for new antimalarial drug
development. New drugs that attack the same vital target
of chloroquine but that are not subject to the same
resistance mechanism would be highly desirable.
RESULTS AND DISCUSSION
We have prepared (2E) ketene S,N-acetals 1a-k by
reaction of o-chlorobenzoylacetonitrile with phenyl
isothiocyanate respective, methyl iodide and potassium
hydroxide in 1,4-dioxane [10]. The configurations of the
compounds 1a-k were determined by 1H NMR and that of
1a confirmed by X-ray crystallography [11] (Figure 1).
The (2E) ketene S,N-acetals 1a-k were mixed with
potassium carbonate and irradiated without solvent in a
domestic microwave oven to afford compounds 2a-k [12].
The target compounds were easily recovered by adding
water to the final substrate, removing the solid products
by filtration, and recrystallization from ethanol-water 1:1

640
J. Charris, A. Barazarte, J. Domínguez, G.Lobo, J. Camacho, R. Ferrer N. Gamboa,
J. Rodrigues, and M.V. Capparelli
Vol 44
Table 1
to give pure samples. The identities of compounds 2a-k
were established by comparison of their physical and
spectroscopic properties with those reported in the
literature [10,12]. Products 3a-k were obtained when 2a-
k, were reacted with ethyl 2-mercaptoacetate, triethyl-
amine in dry ethanol under an inert atmosphere of
nitrogen (Scheme I).
Inhibition of ß-hematin synthesis (IßHS) and globin proteolysis (IPG) by
quinolone derivatives. The results are expressed by the mean ± standard
error of the mean. †p>0.05 compared to chloroquine (CQ). *p>0.05
compared to leupeptin (LEP). **p<0.05 compared to LEP and pepstatin
(PEP).
No
3a
R
H
% IßHS
%IPG
26.84 ± 1.15
74.42 ± 1.46
26.52 ± 1.16
28.55 ± 1.03
30.43 ± 1.16
30.12 ± 1.67
55.89 ± 1.55
11.81 ± 1.51
22.25 ± 2.96
88.61 ± 1.26 **
84.53 ± 1.1 *
89.06 ± 0.69
92.94 ± 0.66
----
< 5
94.2 ± 0.64 †
< 5
3b
3c
2´-Me
4´-Me
3´-OMe
4´-OMe
3´,4´-OMe
4´-Br
Scheme I
O
O
N
NH2
CO2Et
3d
3e
< 5
CN
SCH3
R
R
< 5
a
O
HN
b
S
3f
N
< 5
3g
SCH3
< 5
R
CN
3h
3i
4´-Cl
< 5
Cl
4´-F
< 5
1a-j
2a-j
3a-j
3j
2´,4´-Cl
3´-CF3
< 5
a.
K2CO3, mw; b. HSCH2CO2Et, Et3N, EtOH, ꢀ
3k
LEP
PEP
CQ
< 5
R: H, CH3, OCH3, Br, Cl, F, CF3
-----
-----
Figure 1. Molecular structure of compound 1a. The intramolecular
hydrogen bond is indicated by dashed line. The displacement
86.6 ± 2.75
a
parameters are drawn at 50% probability.
Figure 2. Effects on globin proteolysis by quinolone derivatives.
MW
A
B
3b 3c 3d 3e 3f 3g 3h 3i 3j 3k LEP PEP
Standard Molecular Weight (MW) is expressed in kilodaltons (14.4
kDa). A: Under graded globin (control hemoglobin without
trophozoites). B: Control hemoglobin with trophozoites of P. berghei.
3b-k: quinolone derivatives. LEP: hemoglobin with trophozoites and
leupeptin. PEP: hemoglobin with trophozoites and pepstatin.
Compound 3b was tested in mice infected with P.
berghei ANKA, a chloroquine-susceptible strain of
murine malaria. Mice were given the compound
(chloroquine or 3b in 20 mg kg-1, i.p. once daily) for 4
consecutive days (days 0-4 post infection). At day fourth
post infection, the parasitemia was determined; the
survival days were monitored and compared with control
mice receiving saline (untreated mice). Control mice died
between days 10 post-infection, compound 3b increased
the survival time for 24 days, while chloroquine
prolonged the survival time of the infected mice to 30
days. Compound 3b was able to reduce and delay the
progression of malaria 6.2% but did not eradicate the
infection (Table 2). Compounds 3b, j, k were not tested as
specific proteases inhibitors in vitro. The mechanism of
action of these compounds on hemoglobin degradation
could be related to the inhibition of some aspartic,
cysteinic or metalloproteases due to the presence of
globin band. The activity of compound 3b on hemoglobin
degradation and ꢁ-hematin formation could relate the
Eleven analogs of N-phenylthieno[2,3-b]quinolone
derivatives were tested in vitro for their activity against
cultured of ꢁ-hematin formation and hemoglobin
hydrolysis and in vivo for their efficacy in rodent
Plasmodium berghei (Table 1, 2). The in vitro assay was
used to assess the abilities of the N-phenylthieno[2,3-
b]quinolone derivatives to inhibit ꢁ-hematin formation. In
that assay, hemin was allowed to form ꢁ-hematin under
acidic conditions. Among the eleven compounds tested,
ten showed no measurable activity; only compound 3b
inhibited ꢁ-hematin formation to (94%) compared to
chloroquine (87%), and inhibited hemoglobine proteolysis
by (75%).
Consequently compounds 3b-k were tested for
inhibition of globin proteolysis, in an in vitro assay which
uses rich extract of trophozoyte to digest the native
hemoglobin of mice. Electrophoretic analyses indicated
that compounds 3b, j, k were effective as inhibitors of
hemoglobin degradation 74.42, 88.61, 84.53% respec-
tively (band at 14.4 KDa) (Figure 2).

May-Jun 2007 Ethyl 3-Amino-4-oxo-9-(phenylsubstituted)thieno[2,3-b]quinoline-2-carboxylate Derivatives
641
mixture of the appropriate quinolones (1.3 mmol), ethyl
mercaptoacetate (1.3 mmol), and ethylamine (3 mmol) in dry
ethanol 10 ml was refluxed for 5 h. The solvent was evaporated
to dryness under reduced pressure, water was added 10 ml and
the solid thus obtained was collected by filtration. Further
purification was accomplished by recrystallization from ethanol-
water (4/1).
mechanism of the action of 3b with the blockade of
hemoglobinolytic proteases and inhibition of ꢀ-hematin
formation.
Table 2
Effect of quinolone derivatives (20mg/kg) on parasitemias at fourth day
post-infection (%P) and survival days (SD) of P. berghei infected-mice.
The results are expressed by the mean ± standard error of the mean.
*p<0.05 and **p<0.01 compared to untreated mice (saline). n=5
Ethyl 3-amino-4-oxo-9-phenyl-4,9-dihydrothieno[2,3-b]-
quinoline-2-carboxylate (3a). Yield 68%, mp 215-217o; ir:
1
3479 (NH2), 1732 (C=O), 1691 (C=O) cm-1; H nmr: ꢁ 1.27(t,
3H, CH3, J=7.2 Hz), 4.35(c, 2H, CH2, J=7.2 Hz), 6.79(d, 1H, 8-
H, J=8.5 Hz), 7.28-7.63(m, 7H, phenyl protons), 8.46(dd, 1H, 5-
H, J=8.7, 1.5 Hz); 13C nmr: 14.7, 60.1, 89.5, 113.5, 115.8, 123.6,
124.6, 126.9, 128.7, 131.1, 132.7, 132.9, 137.3, 141.6, 153.3,
158.6, 165.2, 175.2. Anal. Calcd. for C20H16N2O3S: C, 65.92; H,
4.43; N, 7.69. Found: C, 66.03; H, 4.48; N, 7.49.
Treatment
%P
SD
11.66 ± 1.66
23.42 ± 2.65 **
11.6 ± 2.69
11 ± 1.67
30
Saline
3b
21.8 ± 2.31
6.2 ± 3.88 *
11.2 ± 3.21 *
16.6 ± 3.75
1.3 ± 0.3
3j
3k
Ethyl 3-amino-4-oxo-9-(2-methylphenyl)-4,9-dihydrothi-
eno[2,3-b]quinoline-2-carboxylate (3b). Yield 55%, mp 178-
CQ
1
180o; ir 3475 (NH2), 1725 (C=O), 1695 (C=O) cm-1; H nmr: ꢁ
In conclusion, we have described the syntheses and
evaluation of a series of N-phenylthieno[2,3-b]quinolone
derivatives, where the intermediates 2a-k were prepared
by using a microwave device. The solvent-free reaction
using microwave proceeds with significant decreases in
reaction times, and comparable high chemical yield [12].
From this evaluation, we could say that ring system
thieno[2,3-b]quinolones 9-substitued seems to be a new
class of potential antimalarial compounds. As shown for
compounds 3d-f, the presence of strong electron-releasing
(OMe) groups results in complete loss of activity at the
maximum concentration used for both experiments.
The halogen substituted compounds 3g-k also have
good activities and selectivities against the blockage of
hemoglobinolytic proteases in vitro. The introduction of a
methyl group in position 2 of the aryl 3b had an
increasing effect on antimalarial activity, which would be
imposed by two different mechanism of action. The active
species may still be working as protease inhibitors in their
antimalarial role, but inhibiting a parasitic enzyme. A
different regimen of drug treatment might be more
effective in order to produce best results in vivo assays.
The relatively short and easy syntheses of these molecules
make them potential candidates for further development.
1.27(t, 3H, CH3, J=7.2 Hz), 1.99(s, 3H, CH3), 4.23(c, 2H, CH2,
J=7.2 Hz), 6.72(d, 1H, 8-H, J=8.2 Hz), 7.28-7.56(m, 6H, phenyl
protons), 8.5(dd, 1H, 5-H, J=8.7, 1.48 Hz); 13C nmr: 14.7, 17.1,
60.1, 89.5, 113.5, 115.8, 123.6, 124.6, 126.9, 128.7, 131.1,
132.7, 132.8, 132.9, 136.8, 137.3, 141.6, 153.3, 158.6, 165.2,
175.2. Anal. Calcd. for C21H18N2O3S: C, 66.65; H, 4.79; N, 7.40.
Found: C, 66.51; H, 4.83; N, 7.36.
Ethyl 3-amino-4-oxo-9-(4-methylphenyl)-4,9-dihydrothi-
eno[2,3-b]quinoline-2-carboxylate (3c). Yield 60%, mp 221-
1
223o; ir 3480 (NH2), 1735 (C=O), 1700 (C=O) cm-1; H nmr: ꢁ
1.26(t, 3H, CH3, J=7.2 Hz), 2.51(s, 3H, CH3), 4.22(c, 2H, CH2,
J=7.2 Hz), 6.85(d, 1H, 8-H, J=7.9 Hz), 7.28(d, 2H, 2'- and 6'-H,
J:8.2 Hz), 7.35(t, 1H, 6-H, J:8.0 Hz), 7.46(d, 2H, 3' and 5'-H,
J:8.2 Hz), 7.51(m, 1H, 7-H), 8.46(d, 1H, 5-H, J:8.1 Hz); 13C
nmr: 14.7, 21.5, 59.9, 89.7, 113.4, 116.4, 123.4, 124.6, 126.6,
128.1, 131.7, 132.5, 136.1, 141.3, 142.3, 153.5, 159.6, 164.6,
175.2. Anal. Calcd. for C21H18N2O3S: C, 66.65; H, 4.79; N, 7.40.
Found: C, 66.73; H, 4.76; N, 7.53.
Ethyl 3-amino-4-oxo-9-(3-methoxyphenyl)-4,9-dihydro-
thieno[2,3-b]quinoline-2-carboxylate (3d). Yield 38%, mp
1
234-236o; ir 3485 (NH2), 1735 (C=O), 1695 (C=O) cm-1; H
nmr: ꢁ 1.27(t, 3H, CH3, J=7.4 Hz), 3.85(s, 3H, OCH3), 4.23(c,
2H, CH2, J=7.4 Hz), 6.89(d, 1H, 8-H, J=8.3 Hz), 6.93(s, 1H, 2'-
H), 7.00(d, 1H, 4'-H, J=7.2 Hz), 7.16(dd, 1H, 6´-H, J=8.1, 2.0
Hz), 7.35(t, 1H, 6-H, J= 7.2 Hz), 7.48(t, 1H, 7-H, J=7.2 Hz),
7.57(t, 1H, 5'-H, J=7.2 Hz), 8.46(d, 1H, 5-H, J=8.1 Hz); 13C nmr:
14.7, 55.8, 59.9, 90.3, 113.2, 113.7, 116.4, 120.3, 123.5, 124.7,
126.6, 126.7, 131.9, 132.6, 139.6, 142.1, 153.2, 159.8, 161.66,
164.6, 175.2. Anal. Calcd. for C21H18N2O4S: C, 63.94; H, 4.60;
N, 7.10. Found: C, 63.85; H, 4.57; N, 7.29.
EXPERIMENTAL
Melting points were determined on a Thomas micro hot stage
apparatus and are uncorrected. Infrared spectra were determined
as KBr pellets on a Shimadzu model 470 spectrophotometer.
Ethyl 3-amino-4-oxo-9-(4-methoxyphenyl)-4,9-dihydro-
thieno[2,3-b]quinoline-2-carboxylate (3e). Yield 53%, mp
1
238o; ir 3480 (NH2), 1725 (C=O), 1685 (C=O) cm-1; H nmr: ꢁ
1
The H NMR spectra were recorded using a Jeol Eclipse 270
1.26(t, 3H, CH3, J=7.2 Hz), 3.92(s, 3H, OCH3), 4.22(c, 2H, CH2,
J=7.2 Hz), 6.86(d, 1H, 8-H, J=8.2 Hz), 7.14(d, 2H, 2´- and 6´-H,
J=8.8 Hz), 7.33(d, 1H, 4'- and 5'-H, J=8.8 Hz), 7.35(t, 1H, 6-H,
J=7.2 Hz), 7.50(t, 1H, 7-H, J=7.2, 1.5 Hz), 8.46(dd, 1H, 5-H,
J=8.1, 2.4 Hz), 13C nmr: 14.7, 55.8, 59.9, 89.3, 113.4, 116.9,
116.4, 123.4, 124.6, 126.6, 129.6, 131.2, 132.5, 142.5, 153.5,
159.5, 161.0, 164.6, 175.2. Anal. Calcd. for C21H18N2O4S: C,
63.94; H, 4.60; N, 7.10. Found: C, 63.92; H, 4.72; N, 7.17.
Ethyl 3-amino-4-oxo-9-(3,4-dimethoxyphenyl)-4,9-dihydro-
thieno[2,3-b]quinoline-2-carboxylate (3f). Yield 47%, mp 250-
MHz spectrometer. Chemical shifts are expressed relative to
residual chloroform. Elemental analyses were performed by
Central Service of Research, Universidad de Málaga, Málaga,
Spain; results were within ± 0.4% of predicted values for all
compounds. Chemical reagents were obtained from Aldrich
Chemical Co. USA. All solvents were distilled and dried with
the usual desiccant. N-phenylquinolone 2,3-substituted 2a-k
were obtained by following the method previously reported [12].
General Procedure for the Synthesis of Ethyl 3-Amino-9-
Phenylthieno[2,3-b]4-Quinolone-2-Carboxylates (3a-k).
A

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J. Charris, A. Barazarte, J. Domínguez, G.Lobo, J. Camacho, R. Ferrer N. Gamboa,
J. Rodrigues, and M.V. Capparelli
Vol 44
252o; ir 3475 (NH2), 1715 (C=O), 1695 (C=O) cm-1; H nmr: ꢀ
1.29(t, 3H, CH3, J=7.2 Hz), 3.89(s, 3H, OCH3), 4.02(s, 3H,
OCH3), 4.24(c, 2H, CH2, J=7.2 Hz), 6.87(s, 1H, 2'-H), 6.91(d,
1H, 8-H, J=8.2 Hz), 7.01(d, 1H, 5'-H, J=7.9 Hz), 7.10(dd, 1H,
6'-H, J=7.9, 2.3 Hz), 7.37(t, 1H, 6-H, J=7.2 Hz), 7.52(t, 1H, 7-H,
J=7.2 Hz), 8.48(dd, 1H, 5-H, J=8.7, 1.4 Hz); 13C nmr: 14.7, 55.8,
56.3, 56.4, 59.9, 89.5, 112.2, 113.2, 120.3, 116.4, 123.5, 124.7,
126.6, 131.2, 132.6, 138.4, 142.7, 150.7, 150.9, 153.2, 158.9,
164.9, 175.3. Anal. Calcd. for C22H20N2O5S: C, 62.25; H, 4.75;
N, 6.60. Found: C, 62.41; H, 4.77; N, 6.76.
Biological assays
1
Inhibition of ꢁ-hematin formation. The ꢁ-hematin
formation assay was performed according to [13], briefly, a
solution of hemin chloride (50 μL, 4mM), dissolved in DMSO
(5.2 mg/mL), was distributed in 96-well micro plates. Different
concentrations (50-5 mM) of the compounds dissolved in
DMSO, were added in triplicate in test wells (50 μL). Controls
contained either water (50 μL) or DMSO (50 μL). ꢁ-hematin
formation was initiated by the addition of acetate buffer (100 μL
0.2 M, pH 4.4). Plates were incubated at 37 ºC for 48 h to allow
for completion of the reaction and centrifuged (4000 RPM x 15
minutes, IEC-CENTRA, MP4R). After discarding the
supernatant, the pellet was washed twice with DMSO (200 μL)
and finally, dissolved in NaOH (200 μL, 0.2 N). The solubilized
aggregates were further diluted 1:2 with NaOH (0.1 N) and
absorbance recorded at 405 nm (Micro plate Reader, BIORAD-
550). The results were expressed as a percentage of inhibition of
ꢁ-hematin formation.
Ethyl 3-amino-4-oxo-9-(4-bromophenyl)-4,9-dihydrothieno-
[2,3-b]quinoline-2-carboxylate (3g). Yield 39%, mp 278-280o;
1
ir 3480 (NH2), 1725 (C=O), 1685 (C=O) cm-1; H nmr: ꢀ 1.27(t,
3H, CH3, J=6.9 Hz), 4.24(c, 2H, CH2, J=6.9 Hz), 6.82(d, 1H, 8-
H, J=8.3 Hz), 7.34(d, 2H, 2'- and 6'-H, J=8.2 Hz), 7.36(t, 1H, 6-
H, J=7.2 Hz), 7.50(t, 1H, 7-H, J=7.2 Hz), 7.82(d, 2H, 4'- and 5'-
H, J=8.2 Hz), 8.45(dd, 1H, 5-H, J=8.7, 1.5 Hz); 13C nmr: 14.7,
59.9, 89.6, 113.6, 116.0, 123.8, 124.6, 126.8, 128.4, 130.3,
132.8, 134.6, 137.5, 141.9, 154.0, 158.4, 164.5, 175.1. Anal.
Calcd. for C20H15BrN2O3S: C, 54.19; H, 3.41; N, 6.32. Found: C,
53.97; H, 3.67; N, 6.46.
Parasite, experimental host and strain maintenance. Male
Balb-C mice, weighing 18-22
g were maintained on a
commercial pellet diet and housed under conditions approved by
Ethics Committee. Plasmodium berghei (ANKA strain
chloroquine sensible), a rodent malaria parasite, was used for
infection. Mice were infected by i.p. passage of 1 x 106 infected
erythrocytes diluted in phosphate buffered saline solution (PBS,
10 mM, pH 7.4, 0.1 mL). Parasitemia was monitored by
microscopic examination of Giemsa stained smears [14].
Ethyl 3-amino-4-oxo-9-(4-chlorophenyl)-4,9-dihydrothieno-
[2,3-b]quinoline-2-carboxylate (3h). Yield 43%, mp 268-270o;
1
ir 3475 (NH2), 1725 (C=O), 1700 (C=O) cm-1; H nmr: ꢀ 1.27(t,
3H, CH3, J=7.1 Hz), 4.21(c, 2H, CH2, J=7.1 Hz), 6.82(d, 1H, 8-
H, J=8.2 Hz), 7.35(t, 1H, 6-H, J=7.3 Hz), 7.40(d, 2H, 2'- and 6'-
H, J=8.4 Hz), 7.51(t, 1H, 7-H, J=7.2 Hz), 7.66(d, 2H, 4'- and 5'-
H, J=8.4 Hz), 8.44(dd, 1H, 5-H, J=8.7, 1.5 Hz); 13C nmr: 14.7,
59.9, 89.1, 113.6, 116.0, 123.7, 124.6, 126.8, 130.0, 131.6,
132.8, 137.0, 139.7, 142.0, 153.6, 158.6, 164.5, 175.1. Anal.
Calcd. for C20H15ClN2O3S: C, 60.22; H, 3.79; N, 7.02. Found: C,
60.17; H, 3.76; N, 7.17.
Parasite extracts. Blood of infected animals, at a high level
of parasitaemia (30-70%), was collected by cardiac puncture
with a heparinized syringe and the blood pool was centrifuged
(500g x 10 minutes, 4 ºC). Plasma and buffy coat were removed
and the red blood cells (RBC) pellet was washed twice with
chilled PBS-Glucose (5.4 %). The washed RBC pellet was
centrifuged on a discontinuous percoll gradient (80-70% percoll
in PBS-Glucose, 20000g x 30 min x 4 ºC) [15]. The upper band
(mature forms) was removed by aspiration, collected in
eppendorf tubes and washed twice with chilled PBS-Glucose
and the infected erythrocytes were lysed with the nonionic
detergent saponin (0.1% in PBS x 10 min). Cold PBS (1 mL)
was added and the samples were centrifuged (13000g x 5
minutes, 4 ºC) to remove erythrocyte cytoplasm content
(including erythrocyte hemoglobin). The free parasites were
mixed PBS-Glucose (5.4 %), and subjected to three freeze-thaw
cycles (-70ºC + 37ºC). The final homogenate was used in the
hemoglobin hydrolysis inhibition assay [16].
Ethyl 3-amino-4-oxo-9-(4-fluorophenyl)-4,9-dihydrothieno-
[2,3-b]quinoline-2-carboxylate (3i). Yield 37%, mp 210-212o;
1
ir 3475 (NH2), 1715 (C=O), 1695 (C=O) cm-1; H nmr: ꢀ 1.27(t,
3H, CH3, J=6.9 Hz), 4.23(c, 2H, CH2, J=6.9 Hz), 6.81(d, 1H, 8-
H, J=8.4 Hz), 7.34-7.45(m, 5H, phenyl protons), 7.51(t, 1H, 7-H,
J=7.8 Hz), 8.45(dd, 1H, 5-H, J=7.8, 1.5 Hz); 13C nmr: 14.7, 59.9,
89.3, 113.6, 116.0, 118.4, 123.8, 124.6, 126.8, 130.7, 132.7,
134.5, 142.2, 153.5, 158.9, 163.6, 164.5, 175.1. Anal. Calcd. for
C20H15FN2O3S: C, 62.82; H, 3.95; N, 7.33. Found: C, 63.01; H,
3.83; N, 7.43.
Ethyl 3-amino-4-oxo-9-(2,4-dichlorophenyl)-4,9-dihydro-
thieno[2,3-b]quinoline-2-carboxylate (3j). Yield 34%, mp 196-
1
198o; ir 3485 (NH2), 1725 (C=O), 1695 (C=O) cm-1; H nmr: ꢀ
1.28(t, 3H, CH3, J=7.1 Hz), 4.23(c, 2H, CH2, J=7.1 Hz), 6.69(d,
1H, 8-H, J=8.4 Hz), 7.39(m, 1H, 6-H), 7.46-7.59(m, 3H, phenyl
protons), 7.75(d, 1H, 3'-H, J=2.2 Hz), 8.48(dd, 1H, 5-H, J=8.7,
1.5 Hz). 13C nmr: 14.7, 60.3, 89.9, 113.8, 115.3, 123.9, 124.6,
127.0, 129.9, 131.6, 132.1, 133.0, 134.3, 134.8, 137.9, 141.2,
153.2, 157.9, 164.4, 175.2. Anal. Calcd. for C20H14Cl2N2O3S: C,
55.44; H, 3.26; N, 6.47. Found: C, 55.48; H, 3.29; N, 6.51.
Ethyl 3-amino-4-oxo-9-(3-trifluoromethylphenyl)-4,9-
dihydrothieno[2,3-b]quinoline-2-carboxylate (3k). Yield
48%, mp 274-276o; ir 3475 (NH2), 1725 (C=O), 1685 (C=O)
Mice native hemoglobin. Native hemoglobin from non-
infected mice was obtained by treating one volume of pellet
erythrocytes with two volumes of water. The resulting solution
was used as the substrate in the inhibition of the hemoglobin
hydrolysis assay.
Inhibition of hemoglobin hydrolysis. The proteolytic effect
of the parasite extract on the native mice hemoglobin was
assayed using 96-wells tissue culture plate (Greiner Bio-One).
The assay mixture contained: mice native hemoglobin (10 μL),
parasite extract (50 μL), GSH (10 μL, 10 μM), and acetate
buffer (0.2 M, pH 5.4) to a final volume of 200 μL. The
compounds, chloroquine, leupeptin and pepstatin (2.5 mM) were
incorporated in the incubation mixture dissolved in DMSO. The
incubations were carried out at 37 ºC for 18 hours and the
reactions were stopped by addition of reduced sample buffer.
The degree of digestion was evaluated electrophoretically by
1
cm-1; H nmr: ꢀ 1.28(t, 3H, CH3, J=7.4 Hz), 4.23(c, 2H, CH2,
J=7.4 Hz), 6.31(d, 1H, 8-H, J=8.4 Hz), 7.01-7.39(m, 6H, phenyl
protons), 7.95(dd, 1H, 5-H, J=7.7, 1.5 Hz); 13C nmr: 14.7, 59.9,
89.8, 115.2, 115.3, 116.8, 117.0, 120.8, 122.8, 125.6, 125.7,
131.2, 132.5, 132.7, 135.6, 135.7, 142.2, 153.8, 160.3, 164.3,
176.4. Anal. Calcd. for C21H15F3N2O3S: C, 58.33; H, 3.50; N,
6.48. Found: C, 58.47; H, 3.53; N, 6.23.

May-Jun 2007 Ethyl 3-Amino-4-oxo-9-(phenylsubstituted)thieno[2,3-b]quinoline-2-carboxylate Derivatives
643
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SDS-PAGE by visual comparison of the globin bands (14.4
kDa). A DMSO control was electrophoreses at the same time.
Once we get the bands, the densitometer registers the band
density values and it reports as intensity/mm2 ± SD, so we
proceed to check de densities of them in order to have a % of
inhibition of hemoglobin hydrolysis.
Four-day suppressive test. NIH mice (18-22 g) were
infected i.p with 1 x 107 Plasmodium berghei parasites. Two
hours after infection, treatment began with the best compounds
tested in the globin hydrolysis assay. These were dissolved in
DMSO (0.1 M), diluted with Saline-Tween 20 solution (2 %).
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19.712(5) Å. Relevant torsion angles: N(2)-C(2)-C(3)-C(6), 178.3(13)°;
S(1)-C(2)-C(3)-C(4), -178.6(10)°; N(2)-C(2)-C(3)-C(4), -0.3(19)°;S(1)-
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Data analysis. Data were statistically analyzed using t-tests
for specific group comparisons, assuming 95 % of confidence
according GraphPad Prism 3.02
Acknowledgements. We thank the Instituto de
Investigaciones Farmacéuticas (IIF-FF), Consejo de Desarrollo
Científico y Humanístico (CDCH-UCV) and FONACIT PCP
programe. Grants IIF: 03.2006, CDCH-UCV. PI 06-30-5100-
2006 and PG 06-30-5125-2003) for financial support.
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