Synthesis and biological evaluation of new imidazo[1,2-a]pyridine derivatives designed as mefloquine analogues.

Article abstract of DOI:10.1016/S0014-827X(02)01304-6

This paper describes the synthesis and the in vitro antimalarial profile of two new imidazo[1,2-a]pyridine derivatives 4HCl and 13HCl, structurally proposed as mefloquine (1) analogues, by exploring bioisosterism and molecular simplification tools. The synthetic route employed to access the title compounds used, as starting material, the previously described ethyl 2-methylimidazo[1,2-aJpyridine-3-carboxylate derivative (5). These novel heterocyclic derivatives 4HCl and 13HCl presented modest antimalarial activity against the W-2 and D-6 clones of Plasmodium falciparum as well as inhibitors of in vitro heme polymerization compared to mefloquine.

Full text of DOI:10.1016/S0014-827X(02)01304-6

Il Farmaco 57 (2002) 825ꢀ832
/
www.elsevier.com/locate/farmac
Synthesis and biological evaluation of new imidazo[1,2-a]pyridine
derivatives designed as mefloquine analogues
a,b
Patricia C. Lima , Mitchell A. Avery , Babu L. Tekwani , Helio de M. Alves , Eliezer
c
d
a
a,b
J. Barreiro , Carlos A.M. Fraga
a,b,
*
a
Laborat o´ rio de avalia c¸ a˜ o e s ´ı ntese de subst aˆ ncias bioativas (LASSBio), faculdade de farm a´ cia, universidade federal do Rio de Janeiro, PO Box 68006,
Rio de Janeiro, RJ 21944-970, Brazil
b
Instituto de qu ´ı mica, universidade federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
c
Department of medicinal chemistry, school of pharmacy, university of Mississippi, PO Box 1848, Mississippi, MS 38677-1848, USA
d
National center for natural products research, school of pharmacy, university of Mississippi, Mississippi, MS, USA
Received 17 May 2002; accepted 6 July 2002
Abstract
This paper describes the synthesis and the in vitro antimalarial profile of two new imidazo[1,2-a]pyridine derivatives 4×
3×HCl, structurally proposed as mefloquine (1) analogues, by exploring bioisosterism and molecular simplification tools. The
synthetic route employed to access the title compounds used, as starting material, the previously described ethyl 2-
HCl presented
/
HCl and
1
/
methylimidazo[1,2-a]pyridine-3-carboxylate derivative (5). These novel heterocyclic derivatives 4×
/
HCl and 13×
/
modest antimalarial activity against the W-2 and D-6 clones of Plasmodium falciparum as well as inhibitors of in vitro heme
polymerization compared to mefloquine.
´
#
2002 Published by Editions scientifiques et m e´ dicales Elsevier SAS.
Keywords: Antimalarial agents; Mefloquine analogues; Imidazo[1,2-a]pyridine; Bioisosterism; Molecular symplification approach
1
. Introduction
by applying a molecular simplification approach to the
quinuclidine nucleus of the natural antimalarial quinine
(2) (Fig. 1), and was launched in 1985, it has been
employed in multidrug-resistant P. falciparum malaria
treatment [3]. Notwithstanding, in spite of great ex-
pectations, increasing resistance to mefloquine (1) has
been observed since its initial use [4]. Neuropsychiatric
[5,6], cardiovascular [5,7] and hepatic [8] side-effects
associated with the use of 1 have also been reported
since its introduction. Additionally, 1 is photochemically
unstable, promoting light induced toxic reactions in
melanin-rich tissues [9].
Quinoline antimalarials, such as mefloquine (1), have
a mechanism of action related to heme metabolism [10].
According to the most prevalent theory, quinoline
derivatives bind to the toxic ferriprotoporphyrin IX,
interfering with its detoxifying polymerization process
Malaria is a serious infectious disease caused by four
species of protozoan parasites of the genus Plasmodium
1], that affects ca. 300 million people worldwide.
[
Human malaria promoted by Plasmodium falciparum
is the most widespread and dangerous type, being
responsible for annual mortality of between 1.5 and
2
.7 million people, including 1 million young children
1]. Many factors influence the effectiveness of malaria
[
chemotherapy, with regional economics figuring promi-
nently in affordability of medications. In the last years,
the situation of malaria has become even more complex
due to the rising resistance of P. falciparum to conven-
tional antimalarial agents such as chloroquine [2].
Mefloquine
fluoromethyl)-4-quinoline methanol (1)] was designed
[d,l-erythro-a-(2-piperidyl)-2,8-bis(tri-
[10,11]. Recently, Bachhawat et al. [12] suggested that
molecular interaction between quinoline antimalarials
and heme involves the initial electrostatic interaction of
* Correspondence and reprints
E-mail address: cmfraga@pharma.ufrj.br (C.A.M. Fraga).
´
0
014-827X/02/$ - see front matter # 2002 Published by Editions scientifiques et m e´ dicales Elsevier SAS.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 1 3 0 4 - 6

826
P.C. Lima et al. / Il Farmaco 57 (2002) 825ꢀ832
/
Fig. 1. Design concept of new imidazo[1,2-a]pyridine carbinolamine derivative 4.
the quaternary nitrogen atom of antimalarials and an
oxygen atom of the carboxylate group on heme,
followed by a typical hydrophobic interaction of the
quinoline nucleus and the planar porphyrin system. The
protonated nitrogen atom, a consequence of the acidic
internal pH of the food vacuole of the parasite (pH 5.2),
is recognized as the locus of action of quinoline
antimalarials [13].
2. Experimental
2.1. Chemistry
M.p.s were determined with a Quimis 340 apparatus
1
and uncorrected. H NMR spectra were obtained in
CDCl , using Me Si as internal standard with a Brucker
3 4
AC 200 and Brucker/spectrospin 400MHz spectro-
meters. Splitting patterns were as follows: s, singlet; d,
doublet; t, triplet; q, quartet; br, broad; dt, double
triplet; td, triple doublet; ddd, double double doublet;
ddt, double double triplet. C NMR spectra were
obtained in the same spectrometers described above at
In order to obtain new, effective and safe drugs to
combat resistant strains of P. falciparum [14] we
described previously the antimalarial profile of new
1
3
1
H-pyrazolo[3,4-b]pyridine carbinolamine derivatives
(
(
3) [15]. These derivatives were planned as mefloquine
1) analogues and, in fact, did possess in vitro activity
50 and 100 MHz, respectively, using CDCl as internal
3
standard. Infrared spectra were obtained with a Nicolet-
05 Magna spectrophotometer using sodium chloride
cells. High resolution mass spectra (HRMS) were
measured with a Bruker BioApex FTMS system by
direct injection using an electron spray interface
against mefloquine-resistant (D-6 clone) and chloro-
quine-resistant (W-2 clone) strains of P. falciparum (Fig.
5
1
). The similar steric and electronic properties of 1 and 3
were, evidenced by molecular modeling studies which
confirmed the bioisosteric relationships between a
quinoline ring and a pyrazolo[3,4-b]pyridine nucleus
(
ESIꢁ
HPLC system Merck with a L-7100 pump and a L-
450A diode array detector (set at 240 nm), equipped
with a Rexchrom 5 mm RP-18 column (250ꢂ10 mm).
Separation were done in the isocratic mode, using
MeOHꢀwater (v/v) at a flow rate of 1.5 ml/min. The
/
). HPLC analyzes were performed on a Lachrom
[
16]. Following these efforts, we report in this paper, the
7
synthesis and antimalarial profile of the new imi-
dazo[1,2-a]pyridine carbinolamine derivative 4, de-
signed by applying the molecular simplification of the
quinoline ring of (3), through removal of one methine
group (c, Fig. 1). To achieve this goal we explored the
previously related isosteric relationships of quinoline
and imidazo[1,2-a]pyridine rings [17] in order to select a
heterocyclic ring pattern which would avoid formation
of phototoxic ‘quinonoid’ species. The target compound
/
/
HPLC grade solvents were purchased from Tedia,
filtered through a Millipore filter (0.45 mm) and
degassed in an ultrasonic bath prior to use.
The progress of all reactions was monitored by TLC
which was performed on 2.0ꢂ5.0 cm aluminum sheets
/
precoated with silica gel 60 (HF-254, Merck) to a
thickness of 0.25 mm. The developed chromatograms
were visualized under ultraviolet light. Merck silica gel
4 (Fig. 1) was such a molecule as will be described
herein.

P.C. Lima et al. / Il Farmaco 57 (2002) 825ꢀ
/832
827
(
Solvents used in reactions were dried and redistilled
prior to use.
70ꢀ
/
230 mesh) was used for column chromatography.
121.4 (C7), 116.9 (C8), 115.1 (C6), 14.1 (Imꢀ
cm): 1638 n(CꢁO), 1534 and 1495 n(Cꢁ
O), 764 d(NꢁCꢀH); HRMS m/z (ESꢁ) Anal. Calc. for
C H N O [MꢁH] : 161.0637, Found: 161.0636.
/
CH ); IR (/
3
/
/
N), 1253 n(Cꢀ
/
/
/
/
ꢁ
1
/
9
8
2
2.1.1. Ethyl 2-methylimidazo[1,2-a]pyridine-3-
carboxylate (5)
2.1.3. (9)-2-Methylimidazo[1,2-a]pyridin-3-
/
A solution of 2-aminopyridine (1 g, 10.6 mmol) and
ethyl 2-chloroacetoacetate (1.60 g, 1.35 ml, 9.7 mmol) in
absolute EtOH (50 ml) was refluxed for 20 h. The
solvent was removed under reduced pressure and the
yl)(pyridin-2-yl)methanol (11)
To a solution of aldehyde 10 (0.4 g, 2.5 mmol) in dry
THF (5 ml) was added, at ꢄ78 8C, a 0.15 M solution of
/
2-lithiopyridine [18 ml, 2.7 mmol (prepared from reac-
tion of 0.1 M solution of 2-bromopyridine in dry THF
with 1.2 equiv. of a 1.6 M solution of n-BuLi in hexanes]
and then stirred for 2.5 h. Next, a saturated aqueous
brown oil residue was extracted with C H14 (5ꢂ40 ml).
/
6
Combined organic layers were concentrated at half
volume and placed at 0 8C to start the precipitation.
The filtration of yellow crystals formed furnished ethyl
solution of NH Cl (10 ml) was added to the reaction
4
ester 5 (1.74 g, 87%), m.p. 42ꢀ
/
43 8C (literature [18] 42ꢀ
/
mixture, followed by extraction with EtOAc (3ꢂ
The organic layers were dried over anhydrous Na SO
4
/
15 ml).
1
4
4 8C). H NMR (200 MHz, CDCl ) d: 9.20 (dt, 1H,
3
2
H , Jꢃ
Hz), 7.36 (ddd, 1H, H , Jꢃ
/
7.0 and 1.1 Hz), 7.61 (dt, 1H, H , Jꢃ
/
8.8 and 1.6
9.2, 6.8 and 1.2 Hz), 6.98
7.2 and 1.6 Hz), 4.44 (q, 2H,
CH ), 1.45
and concentrated under reduced pressure. The resulting
dark brown oil was crystallized by addition of C H O (3
ml), yielding alcohol 11 (0.37 g, 60%), as dark yellow
5
8
/
7
3
6
(
td, 1H, H , Jꢃ
/
6
1
ꢀ
/
OCH CH , Jꢃ
/
6.7 Hz), 2.72 (s, 3H, Imꢀ
3
/
cubic crystals, m.p. 173ꢀ
/
175 8C. H NMR (200 MHz,
5.0, 1.7 and 0.9 Hz),
6.8, 1.1 and 1.1 Hz), 7.84 (td, 1H,
7.7, 7.7 and 1.7 Hz), 7.76 (d, 1H, H?; Jꢃ7.8
8.9, 1.1 and 1.1 Hz), 7.25
7.1, 5.0 and 1.5 Hz), 7.13 (ddd, 1H,
8.9, 6.8 and 1.3 Hz), 6.74 (td, 1H, H , Jꢃ6.7,
4.0 Hz), 6.22 (d,
2
3
3
1
3
(
t, 3H, ꢀ
/
OCH CH , Jꢃ
/
6.7 Hz); C NMR (50 MHz,
CDCl ) d: 8.41 (ddd, 1H, H?; Jꢃ
/
2
3
6
CDCl ) d: 161.7 (C ꢁ
/O), 153.0 (C5), 147.1 (C2), 128.2
8.30 (dt, 1H, H , Jꢃ
/
3
8
(
6
C8a), 127.8 (C3), 116.9 (C7), 113.8 (C8), 112.8 (C6),
0.5 (OCH CH ), 17.0 (OCH CH ), 14.8 (ImꢀCH3);
C), 1520 and 1501
CꢀH); HRMS m/z
Anal. Calc. for C H N O [Mꢁ
H?; Jꢃ
/
/
4
3
/
Hz), 7.40 (dt, 1H, H , Jꢃ
/
2
3
2
3
5
IR (/cm): 1683 n(Cꢁ
n(Cꢁ
(
/
O), 1557 n(Cꢁ
/
(ddd, 1H, H?; Jꢃ
/
5
/
N), 1223 n(Cꢀ
/
O), 762 d(Nꢁ
/
/
H , Jꢃ
/
/
6
7
ꢁ
1
ESꢁ
/)
/H]
:
5.6 and 1.3 Hz), 6.29 (d, 1H, ꢀ
/
OH, Jꢃ
/
1
1
12
2
2
1
3
2
05.0899, Found: 205.0785.
1H, CH(OH), Jꢃ
NMR (50 MHz, CDCl ) d: 158.0 (C2?), 147.8 (C6?),
/
3.9 Hz), 2.23 (s, 3H, Imꢀ
/
CH3);
C
3
2
(
.1.2. 2-Methy1imidazo[1,2-a]pyridine-3-carbaldehyde
10)
144.7 (C5), 142.0 (C3), 137.2 (C8a), 124.7 (C7), 124.2
(C4), 122.7 (C2), 120.5 (C6), 119.1 (C8), 116.3 (C3?),
Preparation of reductive solution: a solution of 65%
†
111.3 (C5?), 66.1 (CHꢀ
/
OH), 13.2 (Imꢀ
H), 3079 n(ArꢀCꢀH), 1587, 1571
N), 1050 n(CꢀO), 752 d(NꢁCꢀH);
HRMS m/z (ESꢁ) Anal. Calc. for C H N O [Mꢁ
/
CH ); IR
3
ꢄ1
Red-Al [bis(2-methoxyethoxy)aluminum hydride] in
C H CH (20 ml, 55 mmol) was diluted with dry
(cm ): 3440 n(Oꢀ
/
/
/
and 1502 n(Cꢁ
/
/
/
/
6
5
3
C H CH (20 ml) at 0 8C under nitrogen atmosphere.
/
/
6
5
3
14 13
3
ꢁ
1
After homogenizing, a solution of morpholine (5 g, 60
mmol) in dry C H CH (30 ml) was added dropwise.
H] : 240.1131, Found: 240.1130.
6
5
3
The solution was used immediately.
To a solution of ester 5 (1 g, 6 mmol) in dry C H CH
3
2.1.4. (9
/
)-erythro/threo-2-Methylimidazo[1,2-
a]pyridin-3-yl)(piperidin-2-yl)methanol (4)
6
5
(
60 ml) at ꢄ
solution prepared above. The reaction mixture was
stirred for 30 min between ꢄ40 and ꢄ55 8C, then
/
40 8C was added 46 ml of the reductive
To a hydrogenation vessel containing a solution of
alcohol 11 (0.100g, 0.42 mmol) in absolute EtOH (16 ml)
containing concentrated HCl (0.05 ml) was added
/
/
quenched by addition of 1 ml of H O. The solvent was
evaporated to dryness on a rotary vacuum evaporator,
leading to the formation of a crude residue that was
catalytic amounts of PtO (0.004 g, 0.0176 mmol). The
2
2
vessel was then pressurized to 3 atm. with hydrogen, and
shaken at r.t. for 4 h. The resulting suspension was
filtered through celite, and the filtrate was concentrated
at reduced pressure. The crude yellow oil was neutra-
extracted with EtOAc (4ꢂ/50 ml). The organic extracts
were dried over anhydrous Na SO , evaporated and the
4
2
resulting yellow solid was chromatographed on silica gel
with CH Cl ꢀMeOH (99:1) as eluent to furnish alde-
hyde derivative 10 (0.6 g, 60%), as a yellow solid, m.p.
lized by addition of 10% aq. NaHCO solution (10 ml)
3
/
and extracted with EtOAc (3ꢂ/10 ml). The organic
2
2
extracts were dried over anhydrous Na SO and con-
2
4
1
1
1
1
1
15ꢀ
H, Imꢀ
H, H , Jꢃ
/
116 8C. H NMR (200 MHz, CDCl ) d: 10.00 (s,
centrated to dryness under reduced pressure to give a 3:1
mixture of diastereomers erythro/threo of the alcohol 4
1
(0.072 g, 70% yield), as a light yellow oil. H NMR (200
3
/
CHO), 9.52 (d, 1H, H , Jꢃ
/
6.8 Hz), 7.67 (d,
9.0 Hz); 7.52 (dt, 1H, H , Jꢃ8.9, 7.3 and
6.9, 6.9 and 1.0 Hz), 2.71
CH3); C NMR (50 MHz, CDCl ) d: 177.0
5
/
/
8
7
.3 Hz), 7.06 (dt, 1H, H , Jꢃ
/
MHz, CDCl ) d: 8.50 (d, 1H, H , Jꢃ7.0 Hz), 7.39 (d,
/
6
3
8
1
3
(
(
s, 3H, Imꢀ
/
1H, H , Jꢃ
/
8.9 Hz), 7.09 (t, 1H, H , Jꢃ
/6.8 Hz), 6.70 (t,
3
5
6
C ꢁO), 157.3 (C5), 147.8 (C2), 130.3 (C8a), 128.5 (C3),
/
1H, H , Jꢃ
/
6.8 Hz), 4.90 (d, 1H, ꢀ/CH(OH) erythro,
7

828
P.C. Lima et al. / Il Farmaco 57 (2002) 825ꢀ832
/
Jꢃ
/
7.1 Hz), 4.80 (d, ꢀ
7.0 Hz), 2.97 (m, 4H, ꢀ
H?); 2.50 (m, 2H, H?); 2.21 (s, 3H, Imꢀ
/
CH(OH) threo, Jꢃ
OH, ꢀ
CH erythro),
/
9.6 Hz), 3.71
2.1.7. 2-Methyl-3-(pyridin-2-ylmethyl)imidazo[1,2-
a]pyridine (12)
To a solution of the ketone derivative 8 (0.150 g, 0.6
mmol) in diethyleneglycol (2 ml) containing KOH (0.085
g, 2.2 mmol) was added 80% hydrazine monohydrate
(0.08 ml, 2.6 mmol). The reaction mixture was heated at
(
d, 1H, H?; Jꢃ
/
/
/NH and
2
/
6
3
3
2
2
1
.16 (s, 3H, ImꢀCH threo), 1.92 (m, 2H, H?); 1.54 (m,
/
3
5
1
3
H, H?); C NMR (50 MHz, CDCl ) d: 144.6 (C5),
4
3
40.9 (C3), 126.0 (C8a), 125.8 (C7), 123.9 (C2), 118.9
1
95 8C for 2 h, quenched by addition of water (5 ml)
and extracted with EtOAc (3ꢂ10 ml). The organic
layers were evaporated and the crude oil obtained was
chromatographed on silica gel using CH Cl ꢀMeOH
(
(
(
C6), 116.1 (C8), 69.4 (CHOH), 59.5 (C2? erythro), 58.5
/
C2? threo), 46.6 (C6? erythro), 46.8 (C6? threo), 28.3
ꢄ1
C5?), 25.5 (C3?), 24.0 (C4?), 13.4 (Imꢀ
/
CH ); IR (cm ):
3
/
3
395 n(Oꢀ
/
H), 3263 n(Nꢀ
N), 1195 n(CꢀO), 745 d(Nꢁ
) Anal. Calc. for C H N O [Mꢁ
/
H), 1558 and 1499 n(Cꢁ
/
N),
2
2
(99:1) as eluent, furnishing 12 (0.128 g, 96%) as an
orange oil. H NMR (200 MHz, CDCl ) d: 8.56 (dd,
1
1
136 n(Cꢀ
/
/
/
CꢀH); HRMS
/
1
ꢁ
1
m/z (ESꢁ
2
/
/
H]
:
3
1
4
19
3
H, H?; Jꢃ
/
4.9 and 0.9 Hz), 7.98 (d, 1H, H , Jꢃ6.8
/
5
46.1600, Found: 246.1596.
6
Hz), 7.56 (m, 2H, H and H?); 7.06 (m, 2H, H? and H?);
8
4
3
5
6
.94 (d, 1H, H , Jꢃ
/
7.9 Hz), 6.72 (td, 1H, H , Jꢃ
/6.8
7
6
and 0.8 Hz), 4.42 (s, 2H, CH ), 2.47 (s, 3H, Imꢀ
/
CH3);
2
1
3
2
.1.5. 4×
/
HCl
C NMR (50 MHz, CDCl ) d: 157.4 (C2?), 149.1 (C6?),
3
1
H NMR (400 MHz, CDCl ) d: 8.51 (d, 1H, H
3
5
144.1 (C5), 140.2 (C2), 136.8 (C8a), 123.3 (C4?), 123.7
(C3), 121.7 (C7), 121.9 (C3?), 117.1 (C5?), 116.2 (C8),
erythro, Jꢃ
/
6.9 Hz), 8.46 (d, 1H, H threo, Jꢃ
/
6.9 Hz),
5
7
.35 (d, 1H, H , Jꢃ
/
9.9 Hz), 7.06 (t, 1H, H , Jꢃ
/
8.5 Hz),
8
6
111.6 (C6), 29.5 (CH ), 13.0 (Imꢀ
/
CH ); IR (/cm): 2922
H);
2
3
6
.67 (t, 1H, H , Jꢃ
/
6.5 Hz), 4.91 (d, 1H, CH(OH)
7
n(Cꢀ
HRMS m/z (ESꢁ
224.1109, Found: 224.1177.
/
H), 1593, 1571 and 1503 n(Cꢁ
/
N), 753 d(Nꢁ
/
Cꢀ
/
ꢁ
1
erythro, Jꢃ
/
6.8 Hz), 4.79 (d, 1H, CH(OH) threo, Jꢃ
/
/
) Anal. calc. for C H N [Mꢁ
/
H]
:
1
4
13
3
ꢁ
9
.7 Hz), 3.62 (br, 3H, OH and NH ); 2.97 (m, 2H, H?);
2
2
2
.66 (t, 2H, H? threo, Jꢃ
/
11.8 Hz), 2.50 (t, 2H, H6?
11.6 Hz), 2.17 (s, 3H, ImꢀCH erythro),
.12 (s, 3H, ImꢀCH threo), 1.87 (dd, 2H, H?; Jꢃ
1.9Hz), 1.40 (m, 4H, H? and H?); C NMR (100
6
erythro, Jꢃ
2
/
/
2.1.8. (9
/
)-2-Methyl-3-(piperidin-2-
3
/
/
ylmethyl)imidazo[1,2-a]pyridine (13)
3
3
1
3
1
To a hydrogenation vessel containing a solution of
deoxygenated derivative 12 (0.100 g, 0.45 mmol) in
absolute EtOH (16 ml) containing concentrated HCl
4
5
MHz, CDCl ) d: 144.9 (C8a), 141.4 (C2), 126.0 (C5),
24.3 (C7), 116.7 (C8), 114.3 (C3), 112.0 (C6), 68.2
3
1
(
CHOH), 60.8 (C2?), 46.1 (C6?), 27.8 (C3?), 24.2 (C4?),
(0.05 ml) was added catalytic amounts of PtO (0.004 g,
2
2
3.5 (C5?), 14.3 (Imꢀ
/
CH3).
0.0176 mmol). The vessel was then pressurized to 3 atm.
with hydrogen, and shaken at r.t. for 2 h. The resulting
suspension was filtered through celite, and the filtrate
was concentrated at reduced pressure. The crude yellow
oil was neutralized by addition of 10% aq. NaHCO3
2.1.6. (2-Methylimidazo[1,2-a]pyridin-3-yl)(pyridin-2-
yl)methanone (8)
Active MnO2 (0.545 g, 6.27 mmol) was added in
portions to a solution of previously prepared alcohol 11
solution (10 ml) and extracted with EtOAc (3ꢂ
/10 ml).
The organic extracts were dried over anhydrous Na SO
2
4
and concentrated to dryness under reduced pressure to
afford piperidinyl derivative 13 (0.073 g, 73%), as a light
(
0.100 g, 0.42 mmol) in CH Cl (15 ml). The suspension
2 2
was stirred at r.t. for 2.5 h and then filtered through
celite and washed with CH Cl . Combined organic
1
yellow oil. H NMR (200 MHz, CDCl ) d: 8.90 (d, 1H,
3
2
2
H , Jꢃ
/
6.8 Hz), 7.93 (d, 1H, H , Jꢃ8.9 Hz), 7.54 (t, 1H,
/
5
8
extracts were evaporated under reduced pressure to
furnish 8 (0.086 g, 87%) as a yellow solid, m.p. 72ꢀ
H , Jꢃ
/
6.8 Hz), 4.08 (d, 2H, CH , Jꢃ
/
7.0 Hz), 3.48 (t,
7
2
/
1
H, H?; Jꢃ
/
7.0 Hz), 3.01 (m, 4H, ꢀ
/
NH, H? and H?);
2
2
6
1
7
5 8C. H NMR (200 MHz, CDCl ) d: 9.50 (dt, 1H,
3
2.97 (m, 4H, H? and H?); 2.50 (s, 3H, Imꢀ
/
CH ), 1.98 (m,
5
4
3
1
3
H , Jꢃ
1
/
6.9, 1.2 and 1.2 Hz), 8.72 (ddd, 1H, H?; Jꢃ
/
5.2,
7.7, 7.7 and 1.7
7.8, 1.1 and 1.1 Hz), 7.79 (dt,
8.9, 1.2 and 1.2 Hz), 7.65 (m, 2H, H and
5
6
2H, H?); C NMR (50 MHz, CDCl ) d: 144.4 (C5),
3
3
.6 and 1.0 Hz), 8.08 (td, 1H, H?; Jꢃ
/
4
141.2 (C3), 123.2 (C8a), 123.1 (C7), 117.2 (C2), 116.9
(C6), 111.7 (C8), 55.8 (C2?), 47.1 (C6?), 32.9 (C3?), 31.1
Hz), 7.81 (dt, 1H, H?; Jꢃ
1
/
3
H, H?; Jꢃ
/
5
7
(C5?), 29.8 (CH
3382 n(NꢀH), 2926 n(Cꢀ
1113 n(CꢀN), 756 d(Nꢁ
Anal. Calc. for C H N [MꢁH] : 230.1651, Found:
2
), 24.7 (C4?), 13.7 (Imꢀ
H), 1570 and 1504 n(Cꢁ
CꢀH); HRMS m/z (ESꢁ
/
CH
3
); IR (/cm):
H ), 7.29 (td, 1H, H , Jꢃ
/
6.8 and 1.6 Hz), 1.98 (s, 3H,
CH3); C NMR (50 MHz, CDCl ) d: 184.4 (C ꢁO),
56.9 (C2?), 154.9 (C6?), 148.8 (C5), 147.6 (C2), 137.1
C8a), 129.3 (C3 and C4?), 128.6 (C7), 125.6 (C5?), 122.8
C3?), 116.4 (C8), 114.4 (C6), 17.3 (ImꢀCH ); IR (/cm):
619 n(CꢁO), 1583 n(CꢁC), 1564 and 1493 n(Cꢁ
d(NꢁCꢀH); HRMS m/z (ESꢁ) Anal. Calc. for
C H N O [MꢁH] : 238.0974, Found: 238.0971.
8
6
/
/
/
N),
1
3
Imꢀ
1
/
/
3
/
/
/
/)
ꢁ
1
/
1
4
19
3
(
(
230.1642.
/
3
1
/
/
/
N), 758
2.1.9. 13×
/
HCl
1
/
/
/
H NMR (400 MHz, CDCl ) d: 8.05 (s, 1H, H , Jꢃ
/
3
5
ꢁ
1
/
6.8 Hz), 7.47 (d, 1H, H , Jꢃ8.9 Hz), 7.07 (dd, 1H, H7,
/
1
4
11
3
8

P.C. Lima et al. / Il Farmaco 57 (2002) 825ꢀ
/832
829
Jꢃ
/
8.9 and 6.9 Hz), 6.74 (t, H , 1H, Jꢃ
/
6.4 Hz), 3.00 (d,
11.7 Hz), 2.90 (d, 2H, H?; Jꢃ6.6 Hz),
11.8, 11.8 and 2.6 Hz), 2.44 (s,
1.78 (m, 2H, H?); 1.66ꢀ1.55 (m,
1.25 (m, 2H, H?); C NMR (100 MHz,
ketone derivative 8 was obtained in only 15% yield while
the major product formed was characterized as the
tertiary alcohol 9 (entry 3, Table 1). Despite several
experimental variations in the number of equivalents of
lithium reagent used (Table 1), we were unable to
improve the formation of diarylketone derivative 8.
Indicating that the kinetics of the addition of 2-
lithiopyridine to 8, generating the tertiary alcohol 9, is
faster than its formation from nucleophilic addition on
carbonyl ester group of 5. To circumvent this limitation,
we turned to an alternative pathway exploring the
chemoselective conversion of the ester group of 5 to
the more reactive aldehyde group. Thus, ester 5 was
6
2
2
3
2
H, CH , Jꢃ
.51 (ddt, 1H, H?; Jꢃ
H, Imꢀ
H, H?); 1.30ꢀ
/
/
2
6
/
2
/
CH ), 1.80ꢀ
/
/
3
3
1
3
/
4
5
CDCl ) d: 144.4 (C8a), 141.2 (C2), 123.2 (C5), 123.1
3
(
(
(
C7), 117.2 (C3), 116.9 (C8), 111.7 (C6), 55.8 (C2?), 47.1
C6?), 32.9 (CH ), 31.9 (C3?), 26.0 (C4?), 24.7 (C5?), 13.7
Imꢀ
2
/
CH3).
2
2
.2. Biological assays
.2.1. Antimalarial activity
The in vitro antimalarial assay [28] used for evalua-
†
reduced to the aldehyde 10 in 60% yield using RedAl
tion of the new imidazo[1,2-a]pyridine derivatives 4×
/
partially deactivated by addition of 1 equiv. of morpho-
line [19] (Scheme 1).
Next, aldehyde 10 was condensed with 1 equiv. of 2-
HCl and 13×
/
HCl was an adaptation of the parasite
lactate dehydrogenase (pLDH) based assay developed
originally by Makler et al. [29]. The assay was set up in
lithiopyridine at ꢄ78 8C to furnish the pyridinyl
/
9
6 well microplates. Antimalarial action of each com-
alcohol 11 in 62% yield [15,20,21] (Scheme 1). The final
step of the synthetic route consisted of the selective
heterogeneous catalytic hydrogenation [15] of the pyri-
dine ring, furnishing the piperidine ring of the target
imidazo[1,2-a]-pyridine derivative 4. Treatment of rac-
alcohol (11) with molecular hydrogen in ethanol con-
pounds was tested at least at six different concentration
on two P. falciparum clones, i.e. a chloroquine sensitive
D6-Sierra Leone clone and a chloroquine resistant W2-
Indochina clone. The quinoline antimalarial drugs
mefloquine (1) and quinine (2) were used as the positive
controls and DMSO was tested as the negative control
with each assay. Citotoxicity was conducted on Vero cell
line, using the method previously described by Skehan et
al. [30].
taining 4 mol% of PtO giving a mixture of erythro-4
2
and threo-4 diastereomers, in 70% yield (Scheme 1). The
diastereomeric erythro/threo ratio was determined to be
75:25, respectively, by reversed-phase HPLC analysis.
The assignment of the relative configuration of the
1
2
.2.2. Heme polymerization assay
major diastereomer of 4 was made by H NMR through
Heme polymerization i.e. conversion of heme to b-
hematin in the presence of oleolglycerol was assayed in
comparative analysis of coupling constants (J) of
carbinolic hydrogen signal (Ha) of erythro and threo
stereoisomers (Fig. 2). Is well known [22] that the threo
isomer of mefloquine (1) and analogues presented a
vicinal coupling constant between the carbinolic hydro-
gen (Ha) and 2-piperidinyl hydrogen (Hb) of greater
magnitude than the corresponding erythro isomer (Fig.
2). The doublet signal of Ha of the major isomer of 4
vitro up according to the methods described earlier
[
31,32]. Briefly, the reaction mixture (200 ml) with 100 ml
acetate buffer (100 mM, pH 4.8), 100 mM heme, 0.5 mg
oleolglycerol and the test compound was incubated over
night at 37 8C. Nonpolymerized heme was removed by
washing with Tris/SDS buffer and sodium bicarbonate.
b-Hematin was solubilized in 0.1 N NaOH and OD was
recorded at 405 nm.
appeared at 4.90 ppm (Jꢃ
to the erythro diastereomer, while the minor doublet
signal at 4.80 ppm (Jꢃ9.7 Hz) was attributed to the
threo isomer (Fig. 2).
/
6.5 Hz), and was attributed
/
3
. Results and discussion
In order to evaluate the antimalarial profile of the
dehydroxylated analogue of 4, we oxidized the pyridinyl
alcohol 11 with MnO2 [23] to the corresponding
diheteroarylketone derivative 8, which was subsequently
3
.1. Chemistry
The synthesis of the new imidazo[1,2-a]pyridine
deoxygenated in 96% yield by applying classical Wolfꢀ
/
carbinolamine derivative 4 is outlined in Scheme 1.
The starting material was the imidazo[1,2-a]pyridine-3-
carboxylic acid ethyl ester (5) obtained in 87% yield by
condensation between 2-aminopyridine (6) and ethyl 2-
chloroacetoacetate (7) [18]. The initial approach used to
achieve the construction of the piperidine side-chain
consisted of exploring the controlled nucleophilic addi-
tion of 1 equiv. of 2-lithiopyridine to the carbonyl group
of ester 5. However, under these conditions the desired
Kishner methodology [24,25]. Next, the desired piperi-
dinyl derivative 13 was obtained in 73% yield by PtO2
catalyzed hydrogenation [15]. Before testing target
compounds 4 and 13, they were quantitatively converted
to the corresponding hydrochlorides 4×
/
HCl and 13×
/
HCl
by treatment with gaseous HCl in dichloromethane to
facilitate solubilization in aqueous bioassay conditions.
Considering that the internalization of antimalarial 4-
aminoquinoline derivatives in the parasite acidic vacuole

830
P.C. Lima et al. / Il Farmaco 57 (2002) 825ꢀ832
/
Scheme 1. Synthesis of new imidazo[1,2-a]pyridine derivative 4.
Table 1
Experimental variations of the reaction of the imidazo[1,2-a]pyridine
ethyl ester 5 with 2-lithiopyridine
a
b,c
Entry Experimental conditions
-Lithiopyridine (equiv.)
Product
2
Time (h)
8 (%) 9 (%)
1
2
3
4
5
0.5
0.5
1.0
1.5
2.0
0.5
1.0
2.5
2.5
2.5
10
14
15
20
23
15
23
38
45
50
a
All procedures were made in THF at ꢄ78 8C.
Physical and spectroscopic data of the compound 8 are described
b
in Section 2.
c
Fig. 2. Characterization of the relative configuration of the diaster-
1
eomers of 4 by H NMR.
1
Spectroscopic data of the compound 9: H NMR (200 MHz,
DMSO-d
6
) d: 8.57 (dt, 2H, H?; Jꢃ4.8, 1.2 Hz), 7.77ꢀ
/7.65 (m, 6H, H3?;
2
H? and H?); 7.48 (d, 1H, H
5
), 7.30ꢀ
/
7.23 (m, 3H, H and H?); 7.07
8
4
5
6
very similar to mefloquine (1), i.e. pK ꢃ8.6 (water)
[27], suggesting that the drug accumulation rate in the
parasite vacuole would also be similar.
/
(
ddd, 1H, H
, Jꢃ6.9, 1.0 Hz), 1.50 (s, 3H, ImꢀCH
054 and 3004 n(ArꢀCꢀH), 1637 and 1430 n(CꢁN), 1195 n(CꢀO),
47 d(NꢁCꢀH); HRMS m/z (ESꢁ) Anal. Calc. for C19
6
, Jꢃ9.0, 6.7 and 1.1 Hz), 6.55 (s, 1H, OH), 6.47 (td, 1H,
a2
H
7
3
); IR (/cm): 3425 n(OꢀH),
3
7
H
16
N
4
O
ꢁ
1
[
MꢁH] : 3 17.0761, Found: 317.1405.
3.2. Biological activity
is largely dependent of it’s ionization constants [2], we
determined the pK of 4 using the classical UVꢀVis
spectrophotometric method [26]. The ionization con-
The in vitro antimalarial profile [28] of the diaste-
reomeric mixture of the novel imidazo[1,2-a]pyridine
/
a
derivatives 4×HCl and 13×HCl was evaluated in vitro
/
/
stant values found for the compound 4 in water were
against two P. falciparum clones designated as Indo-
china (W-2, mefloquine-sensitive and chloroquine-resis-
tant) and Sierra Leone (D-6, mefloquine-resistant and
5
.80 (pK ) and 8.85 (pK ). The pK value of 4,
a1 a2 a2
referring to the ionization of the piperidinyl group, is

P.C. Lima et al. / Il Farmaco 57 (2002) 825ꢀ
/832
831
Table 2
Antimalarial activities of the novel imidazo[1,2-a]pyridine derivatives 4×HCl and 13×HCl
a
b
a
b
c
Comp.
IC50 (mg/l) D-6 clone
S.I.
IC50 (mg/l) W-2 clone
S.I.
TC50 (mg/l) Vero cells
4
1
×HCl
3×HCl
34669503
30009500
1093
ꢀ14
ꢀ16
ꢀ4760
ꢀ3660
25 00095000
25 00095000
2696
ꢀ1.88
ꢀ1.88
ꢀ1830
ꢀ200
NC
NC
NC
NC
Mefloquine×HCl
Quinine×HCl
1394
238931
a
Values are mean9SD of three observations.
S.I.ꢃselectivity indexꢃIC50 (Vero Cells)/IC50 (P. falciparum).
NCꢃno cytotoxicity up to the highest dose tested (47 000 ng/ml).
b
c
placebo-controlled trial, Br. J. Clin. Pharmacol. 42 (1996) 415ꢀ
21.
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/
chloroquine-sensitive), using the protocol developed by
Makler et al. [29] (Table 2). Compounds 4×HCl and 13×
HCl were inactive against both clones of P. falciparum
when compared with mefloquine (1) and quinine (2). In
the spite of the removal of the pharmacophoric hydroxyl
4
/
/
[
[
/
7] F.M. Gribble, T.M. Davis, C.E. Higham, A. Clark, F.M.
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/
group of the carbinolamine derivative 4×
/
HCl, it’s
[
dehydroxylated analogue 13×HCl presented a similar
/
Chetrit, Mefloquine-induced acute hepatitis, Pharmacotherapy 20
bioactivity profile, indicating that the mechanism of
action against the parasite seems to be mainly dependent
of the nature of the aromatic heterocyclic ring. Com-
(
2000) 1517ꢀ1519.
/
[
9] S. Navaratnam, I. Hamblett, H.H. Tonnesen, Photoreactivity of
biologically active compounds; XVI. Formation and reactivity of
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/
HCl and 13×/HCl did not show Vero cell
(
10] G. Padmanaban, P.N. Rangarajan, Heme metabolism of plasmo-
2000) 25ꢀ38.
/
toxicicity at the concentrations tested [30].
Additionally, the imidazo[1,2-a]pyridine mefloquine
[
dium is a major antimalarial target, Biochem. Biophys. Res.
analogues 4×
/
HCl and 13×
/
HCl reported in this commu-
Commun. 268 (2000) 665ꢀ
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/
668.
nication did not show a noticeable effect on heme
polymerization activity [31,32] in vitro up to a 200 mM
concentration. Mefloquine (1) inhibited the heme poly-
/
[
12] K. Bachhawat, C.J. Thomas, N. Surolia, A. Surolia, Interaction
of chloroquine and its analogues with heme: an isothermal
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/
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Acknowledgements
EMBO J. 30 (1984) 2695ꢀ
/2700.
The authors are grateful to CNPq, CAPES, FAPERJ,
FUJB and PRONEX-0888 (Brazil) for financial support
and fellowships. We are also thankful to NPPN
Analytical Center for spectroscopic facilities.
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/
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(
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