Synthesis and in vitro and in vivo antimalarial activity of novel 4-anilinoquinoline Mannich base derivatives

Article abstract of DOI:10.1007/s00044-010-9397-z

Development of resistance has severely limited the choice of available antimalarial drugs, which clearly highlights the urgent need of novel chemotherapeutic agents for the treatment of malaria. The purpose of this study was to develop new potential antim

Full text of DOI:10.1007/s00044-010-9397-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:1523–1529
DOI 10.1007/s00044-010-9397-z
ORIGINAL RESEARCH
Synthesis and in vitro and in vivo antimalarial activity
of novel 4-anilinoquinoline Mannich base derivatives
•
Bhupendra Singh Dipak Chetia S. K. Puri
•
•
•
Kumkum Srivastava Anil Prakash
Received: 7 March 2010 / Accepted: 22 July 2010 / Published online: 11 August 2010
Ó Springer Science+Business Media, LLC 2010
Abstract Development of resistance has severely limited
the choice of available antimalarial drugs, which clearly
highlights the urgent need of novel chemotherapeutic agents
for the treatment of malaria. The purpose of this study was to
develop new potential antimalarial agents with 4-anilino-
quinoline ring. A series of novel 4-anilinoquinoline Mannich
base drug molecules have been synthesized. The synthesis
involves the preparation of Mannich base and these bases
subsequently coupled with 4,7-dichloroquinoline to get tar-
geted drug molecules (Burckhalter et al. in J Am Chem Soc
70:1363–1373, 1948). Their structures were confirmed by
IR, NMR, and mass spectral data. The synthesized molecules
were evaluated for in vitro and in vivo antimalarial activity
against the chloroquine sensitive 3D7 (West Africa) and
RKL-2 strain of Plasmodium falciparum and rodent malaria
parasite Plasmodium yoelii (strain N-67) in Swiss mice
model, respectively (Charmot et al. in Prev Med 15:889,
1986). Except one molecule (containing diphenylamine), all
the tested molecules showed activity while one of them
(containing morpholine) showed promising in vitro and
significant in vivo antimalarial activity under given test
conditions.
Keywords 4-Anilinoquinoline ꢀ Mannich base ꢀ
Antimalarial ꢀ In vitro ꢀ In vivo ꢀ P. falciparum
Introduction
Chloroquine (CQ) resistance in Plasmodium falciparum
malaria has become a major health concern of the devel-
oping world. Such resistance has incited a reexamination of
the pharmacology of other antimalarial agents that may be
effective against resistant strains (Watkins et al., 1984;
Olliaro et al., 1996). Amodiaquine (AQ) (2), a Mannich
base derivative, was come into the market in the 1950s, has
been shown to be a superior alternative for CQ (Nevill
et al., 1994; Panali et al., 1994; Muller et al., 1996; White,
1996). In 1980s, the use of AQ declined due to agranulo-
cytosis and hepatitis (Douer et al., 1985; Neftel et al.,
1986). However, further investigations have shown that
AQ is no more toxic than CQ to treat uncomplicated
P. falciparum malaria with therapeutic dose. The data of
clinical trials reflects that adverse drug reactions to AQ are
likely to occur only during prophylaxis (White, 1996).
Despite some clinical evidence of AQ sensitivity, lab-
oratory analysis has revealed cross-resistance between CQ
and AQ which has transformed into clinical resistance in
some settings (Doberstyn, 1985; Smrkovski et al., 1985;
Charmot et al., 1986; LeHesran et al., 1997). The emer-
gence of multidrug-resistant strains of Plasmodia has cre-
ated a near-desperate situation, where the need for new
inexpensive antimalarials to circumvent the parasite’s
resistance mechanism has become vital. Unfortunately, the
design and subsequent synthesis of new antimalarials are
B. Singh ꢀ D. Chetia
Department of Pharmaceutical Sciences,
Dibrugarh University, Dibrugarh, Assam, India
S. K. Puri ꢀ K. Srivastava
Parasitology Division, Central Drug Research Institute,
Lucknow, India
A. Prakash
Regional Medical Research Centre, NE Region (ICMR),
Dibrugarh, Assam, India
B. Singh (&)
Department of Pharmacy, S.N. Medical College,
Agra 282002, Uttar Pradesh, India
e-mail: bhupendra.du@gmail.com
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Med Chem Res (2011) 20:1523–1529
hindered by the fact that the mechanism of resistance is not
fully understood. Therefore, it is imperative to establish the
structural features of the Mannich base antimalarials which
enable a compound to evade the drug-resistance mechan-
sim and yet retain activity. This advantage can then be used
in the redesign of new Mannich base antimalarials. Amo-
pyroquine (3), a structural analog to AQ where the
diethylamino side chain is replaced with a pyrrolidine
group, was shown to be more active than both CQ and AQ
against CQ-resistant strains of P. falciparum isolates
(Thompson et al., 1958; Pussard et al., 1988). Subsequent
research into the synthesis of Mannich base compounds
containing side chains which were less susceptible to
metabolism afforded many diverse Mannich base deriva-
tives, one of which was tebuquine (4, Fig. 1).
Inc, USA Mass spectrometer. The m/z values of the more
intense peaks were mentioned.
4-(7-Chloro-quinolin-4-ylamino)-2-morpholin-4-
ylmethyl-phenol (BM-1)
Step I. 4-Hydroxyacetanilide (0.05 mol) was subjected to a
Mannich reaction with a morpholine (0.1 mol) and aqueous
formaldehyde (0.1 mol) in ethanol. After reflux (48 h)
period, the solvent was removed under reduced pressure,
and the residue dissolved in dichloromethane (50 ml). The
organic solution was extracted with dilute hydrochloric
acid (0.1 M, 2 9 75 ml). This solution was basified (pH
9–10) and extracted with dichloromethane (3 9 75 ml).
The combined extracts were washed with water (1 9
100 ml) and dried (anhydrous sodium sulfate) and then
solvent was evaporated under reduced pressure to give the
product and the crude product was re-crystallized with
suitable solvent.
Considering the above said facts, we have designed and
synthesized a novel series 4-anilinoquinoline of Mannich
base derivatives (Table 4) that could modulate the anti-
malarial activity.
Step II. A solution of Step I product (N-(3-substituted-
aminomethyl-4-hydroxy-phenyl)-acetamide) (0.005 mol)
in hydrochloric acid (20%, 15 ml) was heated under reflux
for 1 h. This solution was concentrated under reduced
pressure and then co-evaporated with ethanol. The residue
was dissolved in ethanol (20 ml), 4,7-dichloroquinoline
(0.005 mol) was added, and then the solution heated under
reflux for 2 h. The solution was concentrated under
reduced pressure to give a viscous residue which was
poured into ice-cold ammonium hydroxide (5%, 100 ml).
The sticky solid which separated was dissolved in dichlo-
romethane (50 ml) and separated from the basic solution.
The organic solution was washed with water (75 ml), dried
(anhydrous sodium sulfate), and evaporated to dryness
under reduced pressure to give the crude product (pale
brown solid, 64.86%), and the product was re-crystallized.
M.p. = 171–173°C;1H NMR (400 MHz, CDCl3): d 8.44
(d, 1H, J = 4.80 Hz, quinoline-H), d 7.98 (d, 1H,
J = 1.60 Hz, quinoline-H), d 7.91 (d, 1H, J = 8.40 Hz,
quinoline-H), d 7.42 (dd, 1H, J = 8.4, 1.6 Hz, quinoline-
H), d 7.10 (d, 1H, J = 1.20 Hz, quinoline-H), d 6.93 (d,
1H, J = 12.80 Hz, Ar–H), d 6.88 (d, 1H, J = 8.80 Hz,
Ar–H), d 6.64 (s, 1H, Ar–OH/–NH), d 6.66 (dd, 1H,
Materials and methods
Chemistry
All the compounds (BM-1 to BM-7) were synthesized
using synthetic grade chemicals of Rankem and Merck
without further purification and obtained from commercial
suppliers. Analytical thin-layer chromatography (TLC) was
performed on aluminum sheets pre-coated with silica gel
obtained from Merck. Visualization was accomplished by
UV light (254 nm). Infrared (IR) spectra were recorded on
Perkin Elmer—Spectrum RX-I and Shimadzu 8201PC
1
spectrophotometers using KBr pellets. The H NMR and
13C NMR spectra were recorded on Bruker Avance—II
400 NMR spectrometer at 400 and 100 MHz, respectively.
Chemical shift values are given in d (ppm) scale using
1
TMS as an internal standard. Significant H NMR data are
written in order: number of protons, multiplicity (b, broad;
s, singlet; d, doublet; t, triplet; m, multiple), coupling
constants in hertz, assignment. The mass spectra were
recorded on MS 500 IT with 410 Prostar Binary LC, Varian
Fig. 1 Chloroquine (1),
Amodiaquine (2),
Amopyroquine (3), and
Cl
OH
OH
OH
4'
5'
6'
N
N
3'
2'
N
H
N
Tebuquine (4)
H3C
1'
NH
N
NH
NH
N
NH
Cl
Cl
N
Cl
Cl
N
1
2
3
4
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Med Chem Res (2011) 20:1523–1529
1525
J = 5.20, 5.20 Hz, Ar–H), d 3.73 (t, 4H, morpholinyl-H),
d 2.54 (s, 2H, CH2), d 1.58 (t, 4H, morpholinyl-H); 13C
NMR (100 MHz, CDCl3): d 155.63 (C-1), 151.58 (C-20),
149.59 (C-40), 149.42 (C-90), 135.13 (C-4), 129.95 (C-70),
128.46 (C-80), 125.81 (C-60), 125.62 (C-3), 125.40 (C-5),
123.53 (C-2), 121.39 (C-50), 117.48 (C-6), 117.16 (C-100),
101.20 (C-30), 66.64, 59.00, 53.18 (C-7); IR (in KBr disk):
3422, 3201, 1614, 1573, 1454, 1374, 1247, 1116, 1073,
859, 813 cm-1; MS (m/z): 369.9 (m?).
disk): 3405, 3060, 1608, 1560, 1440, 1375, 1310, 1260,
1119, 875, 816 cm-1; MS (m/z): 452.1 (m?).
4-(7-Chloro-quinolin-4-ylamino)-2-
dibutylaminomethyl-phenol (BM-4)
This compound was prepared in a similar manner to BM-1 to
give the product as a light yellow solid residue (65.53%);
m.p. = 126–128°C; 1H NMR (400 MHz, CDCl3): d 8.46 (d,
1H, J = 5.36 Hz, quinoline-H), d 7.99 (d, 1H, J = 2.08 Hz,
quinoline-H), d 7.86 (d, 1H, J = 8.92 Hz, quinoline-H), d
7.40 (dd, 1H, J = 2.08, 2.04 Hz, quinoline-H), d 7.10 (d,
1H, J = 2.52 Hz, quinoline-H), d 6.92 (d, 1H, J = 2.4 Hz,
Ar–H), d 6.86 (d, 1H, J = 8.48 Hz, Ar–H), d 6.75 (s, 1H,
Ar–OH/–NH), d 6.63 (dd, 1H, J = 5.44, 5.36 Hz, Ar–H), d
3.76 (s, 2H, CH2), d 2.53 (t, 2H, di-n-butyl-H), d 1.54 (m,
2H, di-n-butyl-H), d 1.32 (m, 2H, di-n-butyl-H), d 0.919 (t,
3H, di-n-butyl-H); 13C NMR (100 MHz, CDCl3): d 156.65
(C-1), 151.83 (C-20), 149.56 (C-40), 149.44 (C-90), 135.28
(C-4), 129.88 (C-70), 128.74 (C-80), 125.81 (C-60), 125.65
(C-3), 125.39 (C-5), 123.56 (C-2), 121.22 (C-50), 117.47
(C-6), 117.16 (C-100), 101.37 (C-30), 58.03, 53.29 (C-7),
28.43, 20.65, 14.04; IR (in KBr disk): 3420, 3205, 2957,
2872, 1614, 1566, 1454, 1371, 1265, 1122, 876, 818,
749 cm-1; MS (m/z): 412.1 (m?).
2-Butylaminomethyl-4-(7-chloro-quinolin-4-ylamino)-
phenol (BM-2)
This compound was prepared in a similar manner to BM-1 to
give the product as a golden yellow solid residue (73%);
1
m.p. = 59–62°C; H NMR (400 MHz, CDCl3): d 8.69 (d,
1H, J = 4.80 Hz, quinoline-H), d 8.14 (d, 1H, J = 8.80 Hz,
quinoline-H), d 7.99 (d, 1H, J = 3.20 Hz, quinoline-H), d
7.44 (dd, 1H, J = 8.80, 6.00 Hz, quinoline-H), d 7.06 (d,
1H, J = 7.60 Hz, quinoline-H), d 6.93 (d, 1H, J =
15.20 Hz, Ar–H), d 6.85 (d, 1H, J = 8.00 Hz, Ar–H), d 6.75
(s, 1H, Ar–OH/–NH), d 6.63 (dd, 1H, J = 5.20, 4.40 Hz,
Ar–H), d 3.37 (s, 2H, CH2), d 1.25(s, 1H, amine), d 3.91 (m,
2H, n-butyl-H), d 2.70 (m, 2H, n-butyl-H), d 1.56 (t, 2H,
n-butyl-H), d 1.35 (t, 3H, n-butyl-H); 13C NMR (100 MHz,
CDCl3): d 156.48 (C-1), 151.24 (C-20), 149.79 (C-40),
149.58 (C-90), 135.18 (C-4), 129.93 (C-70), 128.28 (C-80),
125.61 (C-60), 125.54 (C-3), 125.30 (C-5), 123.49 (C-2),
121.82 (C-50), 117.44 (C-6), 117.24 (C-100), 101.10 (C-30),
52.13 (C-7), 48.38, 31.44, 20.46, 13.70; IR (in KBr disk):
3423, 3070, 1614, 1575, 1460, 1375, 1256, 1157, 1119, 874,
815, 768 cm-1; MS (m/z): 356.0 (m?).
4-(7-Chloro-quinolin-4-ylamino)-2-
[(diisopropylamino)-methyl]-phenol (BM-5)
This compound was prepared in a similar manner to BM-1
to give the product as a dark brown solid residue (58.33%);
1
m.p. = 131–132°C; H NMR (400 MHz, CDCl3): d 8.37
(d, 1H, J = 5.20 Hz, quinoline-H), d 8.07 (d, 1H,
J = 8.88 Hz, quinoline-H), d 7.89 (d, 1H, J = 1.12 Hz,
quinoline-H), d 7.34 (dd, 1H, J = 1.92, 1.92 Hz, quino-
line-H), d 7.29 (d, 1H, J = 8.16 Hz, quinoline-H), d 6.97
(d, 1H, J = 7.68 Hz, Ar–H), d 6.72 (d, 1H, J = 8.40 Hz,
Ar–H), d 6.85 (s, 1H, Ar–OH/–NH), d 6.58 (dd, 1H,
J = 5.28, 5.20 Hz, Ar–H), d 3.76 (s, 2H, CH2), d 3.10 (m,
2H, diisopropyl-H), d 1.05 (d, 12H, diisopropyl-H);13C
NMR (100 MHz, CDCl3): d 157.00 (C-1), 151.84 (C-20),
149.59 (C-40), 149.46 (C-90), 135.13 (C-4), 129.86 (C-70),
128.68 (C-80), 125.64 (C-60), 125.25 (C-3), 125.09 (C-5),
123.60 (C-2), 121.41 (C-50), 117.51 (C-6), 117.07 (C-100),
101.30 (C-30), 49.56, 48.14 (C-7), 19.77; IR (in KBr disk):
3425, 3061, 2970, 2897, 2876, 1612, 1568, 1454, 1367,
1261, 1123, 874, 812 cm-1; MS (m/z): 384.0 (m?).
4-(7-Chloro-quinolin-4-ylamino)-2-[(diphenylamino)-
methyl]-phenol (BM-3)
This compound was prepared in a similar manner to BM-1
to give the product as a light gray solid residue (66.67%);
1
m.p. = 44–46°C; H NMR (400 MHz, CDCl3): d 8.70 (d,
1H, J = 5.20 Hz, quinoline-H), d 8.11 (d, 1H, J = 1.6 Hz,
quinoline-H), d 8.00 (d, 1H, J = 1.6 Hz, quinoline-H), d
7.44 (dd, 1H, J = 4.80, 2.00 Hz, quinoline-H), d 7.06 (d,
1H, J = 8.00 Hz, quinoline-H), d d 6.97 (d, 1H, J =
2.00 Hz, Ar–H), d 6.90 (d, 1H, J = 7.20 Hz, Ar–H), d 6.87
(s, 1H, Ar–OH/–NH), d 6.68 (dd, 1H, J = 10.80, 5.20 Hz,
Ar–H), d 3.85 (s, 2H, CH2); d 7.58 (d, 1H, J = 8.80 Hz,
phenyl-H), 7.01 (d, 1H, J = 2.80 Hz, phenyl-H), d 6.94 (d,
1H, J = 10.00 Hz, phenyl-H), 13C NMR (100 MHz,
CDCl3): d 152.50 (C-1), 151.23 (C-20), 149.66 (C-40),
149.40 (C-90), 142.65, 135.65 (C-4), 129.60 (C-70), 129.25,
128.61 (C-80), 125.56 (C-60), 124.97 (C-3), 124.45 (C-5),
123.52 (C-2), 121.42 (C-50), 120.45, 119.86, 117.81 (C-6),
117.16 (C-100), 101.13 (C-30), 40.45 (C-7); IR (in KBr
4-(7-Chloro-quinolin-4-ylamino)-2-(isopropylamino-
methyl)-phenol (BM-6)
This compound was prepared in a similar manner to BM-1
to give the product as a dark brown solid residue (46.78%);
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Med Chem Res (2011) 20:1523–1529
1
Methodology
m.p. = 56–57°C; H NMR (400 MHz, CDCl3): d 8.43 (d,
1H, J = 1.00 Hz, quinoline-H), d 7.99 (d, 1H, J =
2.00 Hz, quinoline-H), d 7.88 (d, 1H, J = 9.00 Hz, quin-
oline-H), d 7.36 (dd, 1H, J = 2.12, 2.12 Hz, quinoline-H),
d 7.07 (d, 1H, J = 2.60 Hz, quinoline-H), d 6.92 (d, 1H,
J = 2.52 Hz, Ar–H), d 6.87 (d, 1H, J = 4.52 Hz, Ar–H), d
6.85 (s, 1H, Ar–OH/–NH), d 6.57 (dd, 1H, J = 4.44,
4.40 Hz, Ar–H), d 3.97 (s, 2H, CH2), d 1.25 (s, 1H, amine),
d 2.91 (m, 1H, isopropyl-H), d 1.16 (d, 6H, J = 3.52 Hz,
isopropyl-H); 13C NMR (100 MHz, CDCl3): d 156.74
(C-1), 151.79 (C-20), 149.65 (C-40), 149.39 (C-90), 135.16
(C-4), 130.02 (C-70), 128.59 (C-80), 125.66 (C-60), 125.13
(C-3), 124.95 (C-5), 123.61 (C-2), 121.45 (C-50), 119.93
(C-6), 117.49 (C-100), 101.29 (C-30), 49.95, 48.38 (C-7),
22.38; IR (in KBr disk): 3423, 3030, 2968, 2889, 1612,
1574, 1443, 1373, 1254, 1159, 1123, 874, 814 cm-1 MS
(m/z): 342.0 (m?).
Giemsa stained slide method (Rieckmann et al., 1978), the
cultures of P. falciparum strain were maintained in medium
RPMI 1640 supplemented with 25 mM HEPES, 1%
D-glucose, 0.23% sodium bicarbonate, and 10% heat inac-
tivated human serum. The asynchronous parasites of
P. falciparum were synchronized after 5% D-sorbitol treat-
ment to obtain only the ring stage parasitized cells. For
carrying out the assay, an initial ring stage parasitemia of
0.8–1.5% at 3% hematocrit in a total volume of 200 ll of
medium RPMI-1640 was uniformally maintained. A stock
solution of 5 mg/ml of each of the test samples was pre-
pared in DMSO, and subsequent dilutions were prepared
with culture medium. The diluted samples in 20 ll volume
were added to the test wells so as to obtain final concen-
trations (at fivefold dilutions) ranging between 0.4 and
100 lg/ml in duplicate wells containing parasitized cell
preparation. The culture plates were incubated at 37°C in a
candle jar. After 36–40 h incubation, the blood smears from
each well were prepared and stained with Giemsa stain.
For BM-1, BM-2, and BM-3 compounds the slides were
microscopically observed to record maturation of ring
stage parasites into trophozoites and schizonts in the
presence of different concentrations of the test agents. The
test concentration which inhibited the complete maturation
into schizonts was recorded as the minimum inhibitory
concentrations (MICs) (Table 1). While for the BM-4, BM-
5, BM-6, and BM-7 compounds, the slides were micro-
scopically observed to record percentage of dead rings and
schizonts in the presence of different concentrations of the
test agents. The test concentration which showed the
complete dead rings and schizonts was recorded (Table 2).
Chloroquine was used as the standard reference drug.
4-(7-Chloro-quinolin-4-ylamino)-2-n-propylamino-
methyl-phenol (BM-7)
This compound was prepared in a similar manner to BM-1 to
give the product as a brown solid residue (55.56%);
1
m.p. = 58–59°C; H NMR (400 MHz, CDCl3): d 8.43 (d,
1H, J = 5.36 Hz, quinoline-H), d 7.99 (d, 1H, J = 2.04 Hz,
quinoline-H), d 7.88 (d, 1H, J = 2.80 Hz, quinoline-H), d
7.42 (dd, 1H, J = 2.12, 2.08 Hz, quinoline-H), d 7.08 (d,
1H, J = 2.64 Hz, quinoline-H), d 6.93 (d, 1H, J = 2.60 Hz,
Ar–H), d 6.87 (d, 1H, J = 1.72 Hz, Ar–H), d 6.90 (s, 1H,
Ar–OH/–NH), d 6.65 (dd, 1H, J = 5.40, 5.28 Hz, Ar–H), d
3.98 (s, 2H, CH2), d 1.25 (s, 1H, amine), d 2.66 (t, 2H,
n-propyl-H), d 1.58 (m, 2H, n-propyl-H), d 0.96 (t, 3H,
n-propyl-H); 13C NMR (100 MHz, CDCl3): d 156.70 (C-1),
151.75 (C-20), 149.66 (C-40), 149.36 (C-90), 135.21 (C-4),
130.01 (C-70), 128.56 (C-80), 126.45 (C-60), 125.70(C-3),
125.30 (C-5), 123.62 (C-2), 121.47 (C-50), 119.94 (C-6),
117.49 (C-100), 101.31 (C-30), 52.50, 50.62 (C-7), 22.69,
11.65; IR (in KBr disk): 3424, 3205, 2957, 2874, 1606, 1568,
1445, 1371, 1258, 1121, 878, 816, 740 cm-1; MS (m/z):
342.0 (m?).
In vivo antimalarial activity
The in vivo antimalarial activity of the compound BM-1 was
carried out in the CDRI, Lucknow. The aqueous suspensions
of the test sample were prepared after making a paste with
few drops of Tween 80 and a few drops of water. The volume
was adjusted so as to obtain the required dose in 1.0 ml
volume. The treatment to a group of 5 Plasmodium yoelii
infected mice was administered via oral route, once daily for
four consecutive days from days 0 to 3. One group of five
mice was administered the aqueous vehicle used for pre-
paring suspension and served as untreated control.
Pharmacology
In vitro antimalarial activity
The in vitro antimalarial assay of synthesized compounds
was carried out against two different CQ sensitive strains
of P. falciparum; first three compounds (BM-1 to BM-3)
were evaluated against 3D7 (West Africa) strain in the
CDRI, Lucknow, while rest of the compounds (BM-4 to
BM-7) were evaluated against MRC-RKL-2 strain in
RMRC, Dibrugarh.
The thin blood smears were prepared from each animal
on day 4 i.e., 24 h after the last treatment dose. The degree
of infection was microscopically recorded in terms of
number of P. yoelii infected cells per 100 RBC (i.e., per-
cent parasitemia). The mean value determined for a group
of five mice was used to calculate the percent suppression
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Table 1 Antimalarial activity
Compound
Concentrations
evaluated (lg/ml)
Number of parasites/100 infected RBCs
Percent schizont
maturation inhibition
(in vitro) of synthesized
compounds against chloroquine
sensitive P. falciparum (3D7,
West Africa)
Rings
Trophozoites
Schizonts
BM-1
100
50
100
100
100
100
100
100
100
100
100
68
0
0
0
0
100
100
100
100
100
100
100
100
100
73.33
100
100
100
82.22
71.11
38.88
0.05
100
100
97.1
88.4
–
10
0
0
2.0
0
0
1.0
0
0
0.5
0
0
0.25
0.125
0.063
0.031
100
50
0
0
0
0
0
0
8
24
0
BM-2
BM-3
0
100
100
100
23
30
45
15
0
0
0
10
0
0
2.0
55
16
26
55
85
0
1.0
40
100
50
0
0
Chloroquine 0.50
100
100
95
0.25
0
0
0.125
0.0625
3
2
Bold-italic values indicate
minimum concentration at
which hundred percent schizont
maturation inhibition
70
32
10
8
Control-1
–
0
90
acetanilide as starting materials (Burckhalter et al., 1948). In
this series, the 30-diethylamino function of AQ is replaced by
a 30-primary or secondary amino function. The synthesis
involves the preparation of Mannich base of the 4-hy-
droxyacetanilide followed by hydrolysis of the amide func-
tion of the Mannich base. The hydrolysis product (Mannich
substituted 4-aminophenol) is subsequently coupled
with 4,7-dichloroquinoline to provide target compounds
(Table 4). The new compounds were characterized by the
use of TLC and melting point techniques. Moreover, the
structures of the novel compounds were determined using
IR, 1H NMR, 13C NMR, and mass spectroscopy.
Table 2 Antimalarial activity (in vitro) of synthesized compounds
against chloroquine sensitive P. falciparum (RKL-2)
Compound
Dosase (lg/ml) % Dead rings ? schizontsa
BM-4
100
100
100
100
5.0
3.0
BM-5
BM-6
6.5
BM-7
6.5
Chloroquine (standard)
0.4
54.0
a
Mean of two replicates. Counted against 100 asexual parasites per
replicate
of parasitemia with respect to the untreated control group.
The animals which did not show any parasites on day 4
were subsequently monitored twice weekly till day 28 post-
infection. The animals which did not develop any infection
till day 28 were recorded as cured (Table 3).
Pharmacology
Initially, all the compounds (BM-1 to BM-7) were tested
for in vitro antimalarial activity against the CQ sensitive
3D7 (West Africa) and RKL-2 strain of P. falciparum. All
the compounds showed activity but only two compounds
(BM-1 and BM-2) showed activity with MIC 0.063 and
10 lg/ml, respectively, as compared to Chloriquine
(MIC = 0.25 lg/ml). The antimalarial screening result
reflects that the compound (BM-1) with six member het-
erocyclic ring (Morpholine) at the side chain showed
Result and discussion
Chemistry
In this study, a series of novel 4-anilinoquinoline Mannich
base derivatives were prepared (Fig. 2) by using 4-hydroxy-
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Med Chem Res (2011) 20:1523–1529
Table 3 In vivo antimalarial profile of BM-1 against P. yoelii N67 in Swiss mice
Dose mg/kg No. of mice Percent suppression No. of mice showing nil parasitemia on day
on day 4
Remarks
4
7
10
14
21
28
2/5
500
5
5
100
–
5/5
0/5
5/5
5/5
4/5
2/5
Curative response in 2/5 mice
Control
Fig. 2 Reaction scheme for
the synthesis of novel
4-anilinoquinoline Mannich
base compounds. R1R2NH is
primary or secondary amines
OH
R1
N
R2
OH
OH
R1
N
R2
Mannich reaction
NH
1. Hydrolysis
2. Coupling with
NHCOCH3
4, 7-Dichloroquinoline
NHCOCH3
Cl
N
4-Hydroxyacetanilide
Mannich base
Targeted Compound
Conclusion
Table 4 List of the designed compounds
OH
New 4-anilinoquinoline Mannich base derivatives were
prepared for evaluation of their antimalarial activity. It was
found that morpholine substituted 4-anilinoquinoline
Mannich base derivative (BM-1) exhibited strong sup-
pressive activity against CQ resistant P. yoelii infection in
swice mice.
7
1
R
1
6
5
N
2
3
R
2
4
NH
5´
8´
4´
10´
9´
N
1´
6´
7´
3´
2´
Cl
Acknowledgments We are thankful to M/s Mangalam Drug &
Organics Ltd, Mumbai for supplying the gift samples of 4,7-dichlo-
roquinoline, and also grateful to All India Council for Technical
Education (AICTE) for the providing financial assistance. We extend
our sincere thanks to the Head, Department of Pharmaceutical Sci-
ences, Dibrugarh University, Assam for providing laboratory
facilities.
Compounds
R1
R2
BM-1
BM-2
BM-3
BM-4
BM-5
BM-6
BM-7
–(CH2)2O(CH2)2–
H
n-Butyl
Phenyl
Phenyl
n-Butyl
Isopropyl
H
n-Butyl
Isopropyl
Isopropyl
n-Propyl
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