Design, synthesis and structure-activity relationships of (1H-pyridin-4-ylidene)amines as potential antimalarials

Article abstract of DOI:10.1016/j.bmcl.2009.05.017

(1H-Pyridin-4-ylidene)amines containing lipophilic side chains at the imine nitrogen atom were prepared as potential clopidol isosteres in the development of antimalarials. Their antiplasmodial activity was evaluated in vitro against the Plasmodium falciparum W2 (chloroquine-resistant) and FCR3 (atovaquone-resistant) strains. The most active of these derivatives, 4m, had an IC₅₀ of 1 μM against W2 and 3 μM against FCR3. Molecular modeling studies suggest that (1H-pyridin-4-ylidene)amines may bind to the ubiquinol oxidation Q_o site of cytochrome bc₁.

Full text of DOI:10.1016/j.bmcl.2009.05.017

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3476–3480
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and structure–activity relationships
of (1H-pyridin-4-ylidene)amines as potential antimalarials
Tiago Rodrigues a, Rita C. Guedes a, Daniel J. V. A. dos Santos a, Marta Carrasco a, Jiri Gut b,
a
Philip J. Rosenthal b, Rui Moreira a, , Francisca Lopes
*
a iMed.UL, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
b Department of Medicine, San Francisco General Hospital, University of California, San Francisco, Box 0811, CA 94143, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
(1H-Pyridin-4-ylidene)amines containing lipophilic side chains at the imine nitrogen atom were pre-
pared as potential clopidol isosteres in the development of antimalarials. Their antiplasmodial activity
was evaluated in vitro against the Plasmodium falciparum W2 (chloroquine-resistant) and FCR3 (atovaqu-
one-resistant) strains. The most active of these derivatives, 4m, had an IC50 of 1 lM against W2 and 3 lM
against FCR3. Molecular modeling studies suggest that (1H-pyridin-4-ylidene)amines may bind to the
Received 6 April 2009
Revised 5 May 2009
Accepted 5 May 2009
Available online 9 May 2009
ubiquinol oxidation Qo site of cytochrome bc1.
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Antiplasmodial
(1H-Pyridin-4-ylidene)amines
Malaria is a serious global health problem, and the problem is
complicated by the rapid emergence and spread of multidrug-
resistant Plasmodium falciparum.1 The urgent need for new drugs
triggered the search for lead compounds, preferably acting on
underexploited parasite targets.2,3 Targeting the mitochondrial
electron transport chain of the human malaria parasite has proved
to be a valid chemotherapeutic strategy4,5 as shown by atovaquone
(1, Fig. 1), which is used in combination with proguanil to treat
multidrug-resistant P. falciparum infections.6 Atovaquone is a po-
tent inhibitor of the bc1 complex, a mitochondrial multisubunit en-
ergy-transducing membrane protein.7,8 Atovaquone binds
selectively to the Qo site of cytochrome b, close to the site of inter-
action with the Rieske iron-sulfur protein (ISP), displacing ubiqui-
nol and substantially blocking the conformational change of the ISP
that is required for electron transfer to cytochrome c1.8 This inhibi-
tion of electron transfer by atovaquone blocks respiration and pro-
duces a concomitant collapse of mitochondrial transmembrane
potential.8–10
O
O
O
OH
Cl
Cl
Me
N
H
Me
2
Cl
1
R5
R6
O
O
N
Cl
R3
R1
R2
OCF3
Me
N
H
Me
N
R4
4
3
Figure 1. Structures of atovaquone, 1, clopidol, 2, GW844520, 3, and (1H-pyridin-4-
ylidene)amines, 4.
Despite its effectiveness, atovaquone resistant strains swiftly
emerged when it was introduced in clinical therapy.11 Interest in
developing novel inhibitors of parasite respiration as potential
antimalarials led to the re-discovery of the anticoccidial drug clop-
idol, 2, a 4(1H)-pyridone with antiplasmodial activity that inhibits
the mitochondrial respiration.12,13 The structure of clopidol was
modified based on the assumption that it could be acting as ubiqui-
none antagonist.14
Replacing the 5-Cl of clopidol by lipophilic side chains led to the
discovery of GW844520, 3, which displays excellent in vitro activ-
ity against multi-drug resistant P. falciparum strains coupled with
low frequency of spontaneous in vitro resistance.14 We now report
our research on the (1H-pyridin-4-ylidene)amine scaffold, 4, as a
potential isostere of clopidol and as a potential antimalarial.
Based on the evidence that a lipophilic chain can improve anti-
malarial activity of 4(1H)-pyridones relative to that of clopidol,14
we decided to incorporate an aromatic moiety at the imine nitro-
* Corresponding author. Tel.: +351 217946476; fax: +351 217946470.
E-mail address: rmoreira@ff.ul.pt (R. Moreira).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.017

T. Rodrigues et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3476–3480
3477
R5
R6
OH
OH
NEt2
9
: X = Ac, 95%
OH
(i)
AcHN
XHN
N
HN
N
R3
R1
R2
R3
R1 NEt2
R2
8
(ii)
(i)
(ii)
. HI
10: X = H, 100%
N
Me
6a: R1 = R2 = R3 = H
4a-e
-
+
6b: R1 = R3 = H, R2 = Me
(iii)
PPh3Br
n
Cl
O2N
3
1
2
6c
6d
R3
R1
R2
: R = R = H, R = Me
: R1 = R2 = H, R3 = SO2NH2
X
O2N
12a: 3-NO2, X = H, n = 0, 92%
12b: 3-NO2, X = MeO, n = 0, 98%
11a-f
N
5a-g
R5
12c
: 3-NO2, X = Cl, n = 0, 93%
12d: 3-NO2, X = CF3, n = 0, 95%
12e: 3-NO2, X = OCF3, n = 0, 98%
Cl
N
R3
R1
. CF3SO3H
R2
R3
R1
R2
12f
: 2-NO2, X = H, n = 0, 95%
(iii)
(iv)
12g: 4-NO2, X = H, n = 0, 88%
12h: 3-NO2, X = H, n = 1, 52%
N
N
+
-
R4
R4 CF3SO3
7a: R1 = R2 = H, R3 = Cl
4f-p,r-t
(iv)
7b: R1 = R2 = H, R3 = NH2
: R = R3 = Cl, R3 = H
1
7c
n
Scheme 1. Synthesis of (1H-pyridin-4-ylidene)amines 4a–l. Reagents and condi-
tions: (i) 10, EtOH, reflux, 54–89%; (ii) (a) NaH, DMF, 10 min, rt; (b) MeI, 59–73%;
(iii) MeOTf or EtOTf; (iv) 13a–h, TEA, EtOH, reflux. See Tables 1–3 for structures of
compounds 4.
X
H2N
13a: 3-NH2, X = H, n = 0, 95%
13b: 3-NH2, X = MeO, n = 0, 99%
13c
: 3-NH2, X = Cl, n = 0, 78%
13d: 3-NH2, X = CF3, n = 0, 100%
13e: 3-NH2, X = OCF3, n = 0, 98%
gen atom of the (1H-pyridin-4-ylidene)amine scaffold. A first set of
compounds, 4a–e, was prepared via the appropriate 4-(N-arylami-
no)pyridines 6a–d (Scheme 1). Briefly, 4-chloropyridines 5a–d
were refluxed in EtOH with the Mannich base 10, prepared using
a previously described procedure (Scheme 2),15 to give intermedi-
ates 6a–d in good yield. Reacting compounds 6a–d with methyl io-
dide and sodium hydride in DMF afforded the final products 4a–e,
as iodide salts, in 25–76% yield. For R2 = Me, 6b, methylation gave
the desired 4b as well as the corresponding methylamonium salt,
4c. For 6d (R3 = SO2NH2), methylation occurred at the sulfonamide,
phenol and diethylaminomethyl groups (i.e., giving 4e).
The remaining compounds containing extended lipophilic side
chains were prepared via the pyridinium triflates 7a–c (Scheme
1). These were reacted with the appropriate anilines 13a–h to give
4f–p and 4r–t, as triflate salts, in 11–97% yield.16 Compounds 13a–
h were synthesised using Wittig chemistry in phase-transfer catal-
ysis conditions (Scheme 2), which afforded alkenes 12 in very good
yields.17 Compounds 12 were then reduced in excellent yields to
the corresponding phenethylanilines 13a–g and 3-(3-phenylpro-
pyl)aniline, 13h, by Pd-C-catalysed hydrogenation with triethylsi-
lane in methanol (Scheme 2).18 The N-hydroxy derivative 4q was
synthesised by oxidation of the 4-(N-arylamino)pyridine 6e with
mCPBA (Scheme 3). The geometry of the imine C@N bond was
established as being E, based on NOESY experiments, a result con-
sistent with the reported X-ray crystal structure for 4e.19
13f
: 2-NH2, X = H, n = 0, 94%
13g: 4-NH2, X = H, n = 0, 87%
13h: 3-NH2, X = H, n = 1, 95%
Scheme 2. Synthesis of anilines required for compounds 4a–t. Reagents and
conditions: (i) DEA, CH2O, EtOH, reflux, 75%; (ii) HCl 6 N, reflux, 100; (iii) (a) NaOH
(1.2 M equiv)/CH2Cl2, rt; (b) ArCHO; (iv) Et3SiH, Pd/C, MeOH, rt.
ethyl group and incorporating a chlorine atom at C-3 led to an
improvement in activity (4f vs 4a; Table 2). We then focused our
SAR study on (1H-pyridin-4-ylidene)amines with a phenylethyl
side-chain. Inspection of the data in Table 2 allows the following
observations:
1. Exchanging the N-methyl for an N-ethyl group (i.e., R4) had rel-
atively little effect on activity against the W2 and FCR3 strains
(3-Cl: 4f vs 4h; 3-NH2: 4n vs 4o).
2. For the 3-Cl series, a 4-OCF3 substituent at the terminal aro-
matic ring increased activity against the W2 and FCR3 strains,
when compared to the unsubstituted counterpart (4l vs 4h).
The substituent effect on the antiplasmodial activity against
both strains was 4-OCF3 (4l) > 4-CF3 (4k) > 4-Cl (4j) > H
(4h) ꢀ 4-MeO (4i), which is similar to that reported for the
SAR of clopidol analogues.14 In contrast, for the 3-NH2 ana-
logues, a 4-OCF3 substituent at the terminal aromatic ring had
All compounds 4a–t were tested in vitro against chloroquine-
resistant W2 and atovaquone-resistant FCR3 P. falciparum
strains.20 For the series based on the Mannich-base moiety (Table
1), derivative 4a, unsubstituted at the 1H-pyridin-4-ylidene moi-
Ph
2
Ph
2
ety, presented IC50 values of 8.6 and 7.9
FCR3 strains, respectively, that is, close to those of clopidol (9.7
and 3.4 M, respectively). Incorporation of a methyl group at C-3
of the 1H-pyridin-4-ylidene moiety (i.e., 4d) led to a significant de-
crease in antiplasmodial activity when compared to the parent 4a.
However, a methyl group at C-2 (i.e., 4b) led to a decrease in activ-
ity against the W2 strain but not against the FCR3 strain. Com-
pounds with cationic side chains (4c and 4e) were inactive.
Increasing lipophilicity of (1H-pyridin-4-ylidene)amines by replac-
ing the Mannich-base side-chain at the imine moiety for a phenyl-
lM against the W2 and
HN
N
N
Cl
l
Cl
Cl
Cl
(i)
(ii)
N
OH
N
5d
6e
4q
Scheme 3. Synthesis of compound 4q. Reagents and conditions: (i) 13a, TEA, EtOH,
reflux; (ii) mCPBA, CHCl, reflux.

3478
T. Rodrigues et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3476–3480
Table 1
Antiplasmodial activity of (1H-pyridin-4-ylidene)amines containing a Mannich-base side chain, 4a–e
R5
N
R6
R3
. HI
N
R2
Me
R2
R3
R5
R6
Yield (%)
IC50 (lM)
a
Compound
W2
FCR3
4a
4b
4c
4d
H
H
H
H
Me
OH
OH
OH
OH
OMe
CH2NEt2
CH2NEt2
59
66
25
73
36
8.61 ( 0.41)
20.7 ( 1.7)
>50
33.0 ( 0.7)
>50
7.90 ( 0.24)
8.09 ( 0.44)
9.86 ( 0.28)
>10
Me
Me
H
CH2NðMeÞEt2þIꢁ
CH2NEt2
4e
H
SO2NMe2
CH2NðMeÞEt2þIꢁ
>10
Clopidol
Atovaquone
Chloroquine
3, GW844520
9.73 ( 0.07)
0.0012
3.37 ( 0.19)
1.89 ( 0.10)
0.051
0.052
0.03 (T9-96 strain)14
Antiplasmodial activities determined as described previously.20
a
4h. Finally, a 3-phenylpropyl side chain increased activity when
compared to the 2-phenylethyl equivalent, particularly against
the FCR3 strain (4t vs 4h).
Table 2
Effect of C-3, C-5, N-1 substitution at the (1H-pyridin-4-ylidene)amine scaffold on the
antiplasmodial activity against P. falciparum W2 and FCR3 strains; compound 4n was
assayed as hydroiodide; compound 4q was obtained in the neutral form
A DFT quantum-chemical study was performed as a first step in
evaluating the stereoelectronic properties of the (1H-pyridin-4-yli-
dene)amine scaffold that might be relevant for the antiplasmodial
activity. The geometries of compounds 4, as well as those of clop-
idol (2) and GW844520 (3) were fully optimised at the B3LYP/6-
31G(d,p) level of theory.21,22 The molecular electrostatic potentials
(MEP)26, for compounds 2–4 were calculated and visually analysed.
3-D MEPs superimposed onto the total electron density provide
useful information for the interpretation of long-range interaction
between molecules, which helps to explain how a ligand binds to
its receptor.27 Figure 2 depicts the MEPs for 2, 3 and 4m, with neg-
ative electrostatic potentials (red) on the pyridone oxygen (i.e., in 2
and 3) and imine nitrogen atoms (i.e., 4m), suggesting that these
atoms may participate in electrostatic or hydrogen bonds. For
example, water-mediated hydrogen-bonding between O-3 and
Glu-282 in the Qo site has been reported for the bc1 inhibitor ato-
vaquone, 1.28 Interestingly, the heterocyclic moieties of clopidol,
GW844520 and 4m present a positive electrostatic potential, par-
N
R3
R1
X
. CF3SO3H
N
R4
Compound
R1
R3
R4
X
Yield (%)
IC50 (lM)
W2
FCR3
4f
H
H
H
H
H
H
H
Cl
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Me
Me
Et
Et
Et
Et
Et
Et
Me
Et
H
MeO
H
MeO
Cl
CF3
OCF3
OCF3
H
59
57
82
87
11
76
76
74
50
97
70
31
4.75 ( 0.38)
5.24 ( 0.68)
3.46 ( 0.28)
3.44 ( 0.08)
3.24 ( 0.24)
1.60 ( 0.15)
1.07 ( 0.07)
0.94 ( 0.12)
1.47 ( 0.10)
1.67 ( 0.15)
3.91 ( 0.03)
>8900
2.89 ( 0.89)
5.12 ( 1.61)
3.53 ( 0.29)
3.95 ( 0.93)
2.17 ( 0.33)
1.87 ( 0.33)
1.70 ( 0.20)
2.80 ( 0.03)
2.21 ( 0.22)
2.08 ( 0.95)
1.83 ( 1.27)
>8900
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
NH2
NH2
NH2
Cl
H
OCF3
H
Et
OH
Table 3
Effect of side chain orientation and length on the antiplasmodial activity of (1H-
pyridin-4-ylidene)amines 4 against P. falciparum W2 strain
a detrimental effect on activity. This result is consistent with
the report that electron-donating substituents at C-3 of 4(1H)-
pyridones led to a significant drop in activity relatively to their
3-Cl analogues.14
N
N
N
N
Ph
Cl
Cl
Ph
. CF3SO3H
. CF3SO3H
3. Incorporating a second chlorine atom at C-5 of the 1H-pyridin-
4-ylidene moiety does not affect significantly the antiplasmo-
dial activity (4l vs 4m).
4t
4. The N-hydroxy derivative (4q; Table 2) was inactive against W2
and FCR3 strains, in line with the reduced antiplasmodial activ-
ity displayed by N-hydroxy-4(1H)-pyridones when compared to
their NH counterparts.14
5. Regarding the position of the phenylethyl side chain (Table 3),
activity against W2 was reduced both in the 2- and 4-isomers
(4r and 4s, respectively) when compared to the 3-analogue,
Compound
Isomer
Yield (%)
IC50
(
l
M)
W2
FCR3
4h
4r
4s
4t
3-(CH2)2Ph
2-(CH2)2Ph
4-(CH2)2Ph
3-(CH2)3Ph
82
59
53
33
3.46 ( 0.28)
6.77 ( 0.38)
>10
3.53 ( 0.29)
3.19 ( 0.90)
3.74 ( 0.12)
2.25 ( 1.12)
2.43 ( 0.10)

T. Rodrigues et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3476–3480
3479
can accommodate a water-mediated hydrogen bond. These inter-
actions are consistent with the crystal structure of the yeast bc1
complex with another hydroxyquinone bound in the Qo binding
site34 and provide further support to our model.
Modeling the interaction of clopidol with bc1 complex showed
that the carbonyl oxygen atom is at 2.41 Å of Glu-272, which is also
consistent with a water-mediated hydrogen bond (Fig. 3A, purple).
In addition, only weak van der Waals contacts between the methyl
and chlorine groups of clopidol with Tyr-269, Pro-271 and Leu-275
were observed. Docking GW844520 in the bc1 complex revealed
that the top ranking solutions present the 4(1H)-pyridone moiety
almost superimposed with that of clopidol and with the side-chain
at C-5 filling the hydrophobic channel leading to the Qo centre (
Fig. 3A, blue). Enhanced van der Waals contacts between the side
chain with Ile-125, Ile-147, Leu-150, Phe-151, Leu-275, Met-295
and Phe-296 were observed, indicating that the bulk of intermolec-
ular interactions of GW844520 and cytochrome b are essentially
hydrophobic.
Docking (1H-pyridin-4-ylidene)amine 4l revealed that the top
ranking conformations sit in the Qo binding site with the side chain
Figure 2. MEPs for clopidol, GW844520 and 4m.
ticularly at C-2 and C-6. These observations can be extended to all
active compounds 4, thus suggesting that (1H-pyridin-4-yli-
dene)amines are likely to interact in the active site in a similar
way to 4(1H)-pyridones.
The energies of HOMO and LUMO as well as the HOMO–LUMO
gap (HLG) for compounds 4 were also calculated and those of 4m,
clopidol and GW844520 are presented in Table 4. No correlation
could be found between these descriptors and antiplasmodial
activity.
Presuming that cytochrome bc1 inhibition is the likely mecha-
nism of the antimalarial activity displayed by (1H-pyridin-4-yli-
dene)amines 4, we undertook
a docking study of these
compounds as well as of clopidol and GW844520 to gain insight
into their putative binding mode in the bc1 complex using GOLD.29
Presently, only a homology model for the cytochrome bc1 from P.
falciparum is available.30 However, structures of the bc1 complex
from Saccharomyces cerevisiae co-crystallised with various inhibi-
tors have been successfully used to develop a model for the binding
mode of atovaquone to the Qo site and to study cytochrome b
mutations conferring atovaquone resistance in Plasmodium31 and
Pneumocystis.32 In the present study we chose a bc1 structure from
S. cerevisiae co-crystallised with stigmatellin (PDB code: 1KYO33),
which contains the functional homodimer with the Rieske iron-
sulfur protein (ISP) in close contact with cytochrome b, that is, cor-
rectly oriented for electron transport. Each ligand was energy-min-
imised and then subjected to 5000 docking runs. The top 10
solutions (i.e., those with the highest GoldScore) were visually ana-
lysed for the hydrophobic and hydrophilic interactions between
the ligand and enzyme residues.
We first validated our model by reproducing the crystallised
pose of stigmatellin and studying the interaction of atovaquone
in the Qo site, which revealed that the 2-hydroxy group binds via
a hydrogen bond to the nitrogen atom of His-181 of the Rieske
ISP.34 On the opposite side of the quinone system, the carbonyl car-
bon atom at C-4 is at 3.97 Å of the oxygen atoms of Glu-272, which
Table 4
HOMO and LUMO energies, and the difference between HOMO and LUMO energy
levels (HLG) for clopidol, GW844520, and 4m
Compound
HOMO (eV)
LUMO (eV)
HLG (eV)
Clopidol
GW844520
4m
ꢁ6.03969
ꢁ5.71534
ꢁ5.26391
ꢁ0.66503
ꢁ0.87673
ꢁ0.93414
ꢁ5.37466
ꢁ4.83861
ꢁ4.32977
Figure 3. (A) binding modes of clopidol (purple), GW844520 (blue); (B) putative
binding modes of 4l (purple) and 4m (blue).

3480
T. Rodrigues et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3476–3480
12. Markley, L.; Van Heertum, J.; Doorenbos, H. J. Med. Chem. 1972, 15, 1188.
13. Fry, M.; Williams, R. B. Biochem. Pharmacol. 1984, 33, 229.
14. Yeates, C. L.; Batchelor, J. F.; Capon, E. C.; Cheesman, N. J.; Fry, M.; Hudson, A. T.;
Pudney, M.; Trimming, H.; Woolven, J.; Bueno, J. M.; Chicharro, J.; Fernández,
E.; Fiandor, J. M.; Gargallo-Viola, D.; Heras, F. G.; Herreros, E.; León, M. L. J. Med.
Chem. 2008, 51, 1845.
15. Raynes, K. J.; Stocks, P. A.; O’Neill, P. M.; Park, K.; Ward, S. A. J. Med. Chem. 1999,
42, 2747.
16. 3-Amino-4-chloro-1-ethylpyridinium triflate, 7b: To a solution of 5b (0.77 mmol,
occupying the hydrophobic channel leading to the Qo centre and
interacting with aliphatic and aromatic side chains of Ile-125, Ile-
147, Leu-150, Phe-151, Leu-275, Met-295 and Phe-296, that is,
similar to GW844520 (Fig. 3B, purple). Interestingly, the iminium
hydrogen atom of 4l is in proximity (2.8 Å) to the nitrogen atom
of His-181, which is compatible with an hydrogen bond. The most
potent compound against the W2 strain, 4m, presented an identi-
cal docking pose to 4l, where the (1H-pyridin-4-ylidene)amine
moiety is occupying the hydrophobic channel (Fig. 3B, blue). The
iminium hydrogen atom of 4m is 4.2 Å away from the oxygen atom
of Glu-272, which is compatible with a water-mediated hydrogen
bond. These results support the hypothesis that (1H-pyridin-4-yli-
dene)amines 4 can bind to the Qo site in cytochrome b, promoting
interactions with the residues that define the hydrophobic channel
leading to the Qo centre in a similar way to 4-pyridone GW844520,
a known bc1 complex inhibitor.30
1 M equiv) in dry toluene (1.5 mL) was added ethyl triflate (0.80 mmol, 103
lL,
1.04 M equiv). The reaction mixture was left reacting at room temperature for
24 h. The precipitate was collected and washed with diethyl ether to afford 7b
as a brown solid: 95%, mp 56–58 °C. 1H NMR (DMSO-d6, 400.13 MHz) d 1.48
(3H, t, J = 7.2, CH3); 4.46 (2H, q, J = 7.2, CH2); 6.92 (2H, br s, NH); 8.04 (1H, d,
J = 6.4, Pyr-H3); 8.20 (1H, d, J = 6.4, Pyr-H2); 8.25 (1H, s, Pyr-H20).
N-[(4E)-3-Amino-1-ethylpyridin-4(1H)-ylidene]-3-{2-[4-(trifluoromethoxy)phenyl]-
ethyl}aniline, 4p: Pyridinium triflate 7b (0.80 mmol, 1 M equiv) and aniline 13e
(0.92 mmol, 1.15 M equiv) were dissolved in ethanol absolute (3.5 mL/mmol).
TEA (0.92 mmol, 1.15 M equiv) was added to the solution that was kept under
reflux temperature for 24 h. The solvent was evaporated under reduced
pressure and the crude product purified by flash chromatography
CH2Cl2:MeOH (9.5:0.5) to afford 4p as a yellow oil: 70%. 1H NMR (CD3OD,
400.13 MHz) d 1.53 (3H, t, J = 7.2, CH3); 2.99 (4H, br s, CH2CH2); 4.23 (2H, q,
J = 7.2, NCH2); 6.85 (1H, d, J = 6.8, Pyr-H3); 7.10 (1H, s, Ar-H); 7.15-7.19 (4H, m,
Ar-H); 7.27 (2H, d, J = 7.6, Ar-H); 7.41 (1H, t, J = 7.6, Ar-H); 7.78 (1H, d, J = 7.6,
Pyr-H2); 7.82 (1H, s, Pyr-H20). 13C NMR (CD3OD, 100.61 MHz) d 15.13, 36.47,
37.00, 53.85, 105.39, 120.55, 121.57, 122.01, 124.14, 124.67, 126.68, 129.62,
129.86, 134.00, 134.20, 137.54, 140.58, 143.47, 145.05, 147.36. ESI/MS (m/z):
402.53 (M+H+). Anal. Calcd for C22H22F3N3OꢂCF3SO3H: C, 50.09; H, 4.20; N,
7.62; S, 5.81. Found: C, 50.34; H, 4.26; N, 7.66; S, 5.86.
In conclusion, a series of (1H-pyridin-4-ylidene)amines, 4,
containing an lipophilic side-chain at the imine nitrogen atom have
been synthesised as potential isosteres of 4(1H)-pyridone antimal-
arials. Some of these (1H-pyridin-4-ylidene)amines display sig-
nificant antiplasmodial activity against the P. falciparum W2
CQ-resistant and FCR3 atovaquone-resistant strains, with IC50
ranging from 0.9 to 7 lM. Docking studies suggest that (1H-pyri-
17. Hwang, J.-J.; Lin, R.-L.; Shieh, R.-L.; Jwo, J.-J. J. Mol. Catal. A: Chem. 1999, 142,
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din-4-ylidene)amines may bind to the Qo site of bc1 complex, and
thus are suitable scaffolds that might find further applicability in
the design of antimalarials.
Acknowledgments
21. Becke, A. D. J. Chem. Phys. 1993, 98, 5648.
22. Geometries of compounds 2–4m were energy-minimised using density
functional theory. These calculations were performed with the B3LYP hybrid
functional and the 6-31G(d,p) basis set implemented in GAUSSIAN03 software
package.23 After geometry optimisations, partial charges were included using
Amber’s Antechamber module24 (included with Chimera software).25
23. Frisch, M. J. et al., GAUSSIAN 03, Revision C.02; Gaussian: Wallingford, CT, 2004.
24. Wang, J.; Wang, W.; Kollman, P. A.; Case, D. A. J. Mol. Graph. Model. 2006, 25,
247.
This work was supported by Fundação para a Ciência e Tecnolo-
gia (FCT, Portugal); T.R. acknowledges FCT for the Ph.D. grant SFRH/
BD/30689/2006. P.J.R. is a Doris Duke Charitable Foundation Dis-
tinguished Clinical Scientist.
25. UCSF Chimera package from the Resource for Biocomputing, Visualisation, and
Informatics at the University of California, San Francisco (supported by NIH
P41 RR-01081); Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S.,
Greenblatt, D. M., Meng, E. C., Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605.
26. Portmann, S.; Lüthi, H. T. Chimia 2000, 54, 766.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.05.017.
27. Jones, G.; Willet, P.; Glen, R. C. J. Mol. Biol. 1995, 245, 43.
28. Kessl, J. J.; Lange, B. B.; Merbitz-Zahradnik, T.; Zwicker, K.; Hill, P.; Meunier, B.;
Palsdottir, H.; Hunte, C.; Meshnick, S.; Trumpower, B. L. J. Biol. Chem. 2003, 278,
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