Targeting the erythrocytic and liver stages of malaria parasites with s-triazine-based hybrids

Article abstract of DOI:10.1002/cmdc.201500011

A diversity-oriented library of s-triazine-based hybrids was screened for activity against the chloroquine-resistant Plasmodium falciparum W2 strain. The most striking result was sub-micromolar activity against cultured erythrocytic-stage parasites of hybrid molecules containing one or two 8-aminoquinoline moieties. These compounds were not clearly toxic to human cells. The most effective blood-schizontocidal s-triazine derivatives were then screened for activity against the liver stage of malaria parasites. The s-triazine hybrid containing two 8-aminoquinoline moieties and one chlorine atom emerged as the most potent against P. berghei liver-stage infection, active in the low nanomolar region, combined with good metabolic stability in rat liver microsomes. These results indicate that s-triazine-8-aminoquinoline-based hybrids are excellent starting points for lead optimization as dual-stage antimalarials.

Full text of DOI:10.1002/cmdc.201500011

DOI: 10.1002/cmdc.201500011
Full Papers
Targeting the Erythrocytic and Liver Stages of Malaria
Parasites with s-Triazine-Based Hybrids
Catarina A. B. Rodrigues,[a] Raquel F. M. Frade,[a] InÞs S. Albuquerque,[b] Maria J. Perry,[a]
Jiri Gut,[c] Marta Machado,[b] Philip J. Rosenthal,[c] Miguel PrudÞncio,[b] Carlos A. M. Afonso,*[a]
and Rui Moreira*[a]
A diversity-oriented library of s-triazine-based hybrids was
screened for activity against the chloroquine-resistant Plasmo-
dium falciparum W2 strain. The most striking result was sub-mi-
cromolar activity against cultured erythrocytic-stage parasites
of hybrid molecules containing one or two 8-aminoquinoline
moieties. These compounds were not clearly toxic to human
cells. The most effective blood-schizontocidal s-triazine deriva-
tives were then screened for activity against the liver stage of
malaria parasites. The s-triazine hybrid containing two 8-ami-
noquinoline moieties and one chlorine atom emerged as the
most potent against P. berghei liver-stage infection, active in
the low nanomolar region, combined with good metabolic sta-
bility in rat liver microsomes. These results indicate that s-tria-
zine-8-aminoquinoline-based hybrids are excellent starting
points for lead optimization as dual-stage antimalarials.
Introduction
Malaria is a potentially life-threatening disease caused by infec-
tion with parasites of the genus Plasmodium and transmitted
to humans through the bite of Anopheles mosquitoes.[1,2] There
are five known Plasmodium species that infect humans; the
most common are P. vivax, which is predominant in South
America and Asia, and P. falciparum, which is responsible for
most of the mortality worldwide. A large and increasing prob-
lem is resistance of malaria parasites, in particular P. falciparum,
to almost all available drugs.[3] Thus, the discovery and devel-
opment of new effective and affordable antimalarial drugs re-
mains essential to control and eliminate the disease.[3–5]
tate the ultimate goal of eradicating malaria.[9] The asympto-
matic, obligatory developmental phase of the parasite in the
liver,[10] which precedes the formation of red blood cell infec-
tive forms, is of particular relevance in the context of multi-
stage antimalarial agents. Targeting the liver stage of infection
can provide a causal prophylactic strategy, and this could elim-
inate the parasites before they cause symptomatic blood-
stream infections.[11] Action against liver-stage parasites is also
critical to the control of P. vivax and P. ovale infections, as the
life cycles of these parasites include cryptic hypnozoites that
persist in the liver for long periods of time and that, upon re-
activation, are responsible for relapses of malaria.[12] The 8-ami-
noquinoline primaquine (1, Figure 1) is the only registered
drug for radical cure (elimination of dormant hypnozoites) of
P. vivax and P. ovale malaria, and it is also active against the
transient liver forms of all Plasmodium species.[3,13] Primaquine
is also a potent gametocytocidal agent active against the
blood-circulating sexual forms of the parasite that are transmit-
ted to the mosquito upon a blood meal, and thus it is capable
of interrupting the transmission of infection from the human
host to mosquito vectors.[4,13] Primaquine is also active against
the blood stage of malaria parasites, although its potency is
low;[4,14] this limitation has been addressed by developing mul-
tistage hybrid compounds.[15,16]
The development of hybrid antimalarial agents has been
pursued as a strategy to improve potency and to overcome
the emergence of drug resistance.[6,7] A key feature of hybrid
drugs is the presence of different mechanisms of action
against either a single target or different targets.[8] In addition,
hybrid compounds have the potential to act against different
life-cycle stages of malaria parasites, a required feature to facili-
[a] Dr. C. A. B. Rodrigues, Dr. R. F. M. Frade, Dr. M. J. Perry, Prof. C. A. M. Afonso,
Prof. R. Moreira
Instituto de Investigażo do Medicamento (iMed.ULisboa)
Faculdade de Farmꢀcia, Universidade de Lisboa
Av. Prof. Gama Pinto, 1649-003 Lisboa (Portugal)
E-mail: carlosafonso@ff.ul.pt
s-Triazine (1,3,5-triazine) is a versatile scaffold that has found
a wide range of applications in materials and health scien-
ces.[17] s-Triazine derivatives have been reported to display dif-
ferent pharmacological properties,[18] including antimicrobial,[19]
anticancer,[19,20] antiviral,[21,22] and antimalarial activities.[23–26] Di-
hydrofolate reductase (DHFR), which catalyzes the NADPH-de-
pendent reduction of dihydrofolate to tetrahydrofolate, is con-
sidered the target of s-triazine-based antimalarials,[27] including
cycloguanil (2, Figure 1), the active metabolite of proguanil.[28]
rmoreira@ff.ul.pt
[b] I. S. Albuquerque, M. Machado, Prof. M. PrudÞncio
Unidade de Malꢀria, Instituto de Medicinal Molecular
Faculdade de Medicina, Universidade de Lisboa
Av. Prof. Egas Moniz, 1649-028, Lisboa (Portugal)
[c] Dr. J. Gut, Prof. P. J. Rosenthal
Department of Medicine, San Francisco General Hospital
University of California, San Francisco, Box 0811, CA 94143 (USA)
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500011.
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to target the liver stage. Herein, we report prelimina-
ry structure–activity data from this screen, which led
to the identification of highly potent and metabol-
ically stable dual-stage s-triazine conjugates.
Results and Discussion
Chemistry
The synthesis of substituted s-triazines from cyanuric
chloride is a widely used method owing to the diver-
sity of possible derivatizations.[48] The facile displace-
Figure 1. Structures of primaquine (1), cycloguanil (2), and antimalarial 4-aminoquino-
line-s-triazine hybrids 3 and 4. R1 and R2 are chlorine and/or substituted amines (aryl;
alkyl; and tertiary, secondary, and primary amines).[31–40]
ment of chlorine by various nucleophiles in the pres-
ence of a hydrochloride acceptor is a temperature-
controlled process that allows the stepwise addition
of nucleophiles.[52] The s-triazine library was generat-
ed by stepwise nucleophilic substitution of cyanuric
Resistance of the parasite to cycloguanil and other DHFR inhib-
itors is common and is mainly caused by mutations around
the active site of the enzyme, which leads to dramatic decreas-
es in the activities of the DHFR inhibitors.[29,30] For this reason,
s-triazine-based hybrids have been developed with the aim to
improve antimalarial activity and to prevent the emergence of
resistant P. falciparum strains. Recent studies reported that 4-
aminoquinoline s-triazine conjugates (e.g., compounds 3 and
4, Figure 1) have promising ac-
chloride (5) with amines, alcohols, and one thiol (Figure 2) on
the basis of our previously published procedures (Scheme 1).[48]
Compounds 8c, 8e, and 8 f containing only one 8-aminoqui-
noline moiety were synthesized from intermediates 7a, 7g,
and 7d, respectively, by using a method based on reported
procedures.[48] Compound 7b containing two 8-aminoquino-
line moieties was prepared directly from cyanuric chloride and
was then converted into conjugate 8d by heating at reflux in
tivity against blood-stage P. falci-
parum.[31–40] Triazines have also
been conjugated with endoper-
oxides,[41] a class of antimalarial
agents that are highly active
against the asexual erythrocytic
stage of infection. Endoperox-
ides appear to require activation
by iron(II), which accumulates
inside the parasite food vacuole
after digestion of large quanti-
ties of host hemoglobin, to inter-
act with their target.[42–46] Overall,
the activity of reported s-triazine
hybrids against P. falciparum is
improved relative to that of
chloroquine, but their potential
as dual-stage agents, capable of
acting against the blood and
liver stages of malaria parasites,
remains to be explored.
Our previous experience with
Figure 2. Diversity of s-triazine substituents used in this study.
the synthesis of functionalized s-
triazines[47–49] and our ongoing
research on antimalarial com-
pounds[16,50,51] led us to enlarge
and screen our existing library of
s-triazines against Plasmodium
liver and erythrocyte stages. This
library was enriched with the 8-
aminoquinoline structural motif Scheme 1. Temperature-controlled nucleophilic substitution of cyanuric chloride.
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Figure 3.
Activity of all tested compounds against P. falciparum (PfW2) and human cancer cell lines from breast (MCF-7), colon (HT-29), and lung (MCI-H460).
dioxane. Novel compounds not previously reported in the liter-
ature were characterized by both NMR spectroscopy and LC–
MS.
phenylmethyl)pyrrolidine groups present the highest potency
among compounds 7, both with an IC50 value of 0.6 mm
(Table 1). Interestingly, compounds 7 containing two 2-amino-
1-phenylethanol (ephedrine) moieties (i.e., compounds 7d and
7e) reveal a strong dependence of activity on the stereochem-
istry at C1 and C2. Whereas derivative 7d with two (1R,2S)-
ephedrine groups displays moderate antiplasmodial activity
(IC50 =7.7 mm), its counterpart with two (1S,2S)-ephedrine moi-
Blood schizontocidal activity and cytotoxicity
The s-triazine-based library was evaluated in vitro for schizo-
ntocidal activity against the P. falciparum W2 strain (Figure 3,
Table 1). The in vitro cytotoxicity
of compounds 7 and 8 was eval-
Table 1. In vitro antimalarial activity and cytotoxicity of s-triazine hybrids.
uated by using mammalian
CHOK1 cells. The selectivity
index (SI) as expressed by the
Compd
LogP[a]
IC50 [mm][c]
PfW2[b]
CC50 [mm][c]
HT-29 NCI-H460
SI[d]
MCF-7
CHOK1
>20
>20
>20
W2
ratio CC50CHOK1/IC50 (CC50 =half-
7b,{b,b}
7c,{c,c}
7d,{d,d}
7v,{o,r}
7w,{o,j}
8b,{a,a,s}
8c,{a,a,b}
8d,{a,b,b}
8e,{c,c,b}
8 f,{d,d,b}
8g,{e,e,t}
8h,{f,f,s}
8k,{v,v,v}
8l,{w,w,w}
ART
6.40
6.21
4.37
6.98
5.66
3.81
3.47
5.97
8.00
6.11
4.23
6.01
6.30
0.59
2.52
5.28
2.76
0.60Æ0.01
0.60Æ0.01
7.7Æ0.2
20.6Æ8.4 >25
>25
>25
>25
>33
>33
>3
>18
>4
>25
>25
>25
>25
maximal cytotoxicity concentra-
tion, IC50 =half-maximal inhibito-
ry concentration) calculated for
these three cell lines ranges
from 1 to >90, and compounds
7b, 7c, 8d, and 8 f display the
highest SI values (>30 to >90;
Table 1). In addition, toxicity was
also assessed against three
cancer cells lines: MCF-7 (breast),
HT-29 (colon), and NCI-H460
(lung). Most of the s-triazines
were poorly active against these
cancer cells lines.
1.1Æ0.3
10.8Æ3.4 >25
<10
20.5Æ1.2
>25 >25
8.95Æ1.7 >20
16.4Æ1.2 11.5Æ1.0
>25
>25
>25
>25
>25
13.6Æ3.7
>25
12.8Æ2.0
ND
>20
>20
>25
2.7Æ0.1
3.5Æ0.1
>20
>20
>20
>20
>20
>20
>20
>6
>4
5.6Æ0.3
11.6Æ1.3 <10.0
0.48Æ0.38
8.3Æ5.2
>25
>25
>25
>25
>25
>25
>42
>2
>91
>12
>3
0.22Æ0.05
1.6Æ0.2
13.8Æ1.0
>25
10.3Æ1.1
ND
12.4Æ1.4
>25
9.47Æ1.62
ND
>20
10.4Æ3.3
>25
3.4Æ0.2
6.4Æ0.3
ND
ND
2.2Æ0.4
>20
>20
8.5Æ2.7
>20
>9
>1000
>283
>6
0.02Æ0.003 >20
0.03Æ0.009 >20
CQ
PQ
3.3[53]
>25
[a] Calculated log P values (source: www.vcclab.org/lab/alogps/start.html). [b] Plasmodium falciparum W2
(PfW2). [c] Data represent the meanÆSD of n=2 independent experiments performed in quadruplicate. [d] Se-
lectivity index (SI): CC50 (CHOK1)/IC50. Abbreviations: artemisinin (ART), chloroquine (CQ), primaquine (PQ), not
determined (ND).
Compounds 7b containing
two 8-aminoquinoline moieties
and 7c with two 2-(hydroxydi-
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eties (i.e., compound 7e) is inactive. Nevertheless, replacement
of the chlorine moiety in 7e with an N-(3-aminopropyl)imida-
zole group leads to a significant increase in potency (8g:
IC50 =1.62 mm). The triphenylmethylamine subseries (i.e., com-
pounds 7t–w) also includes members with good activity, in
particular compound 7v with an IC50 value of 1.1 mm.
tions were 8-aminoquinoline-containing derivatives 7b, 8d,
and 8 f, which, at this dose, led to >50% inhibition of the in-
fection relative to the controls, whereas they almost sup-
pressed infection at 10 mm without significantly affecting Huh7
cell proliferation. These results are in sharp contrast to those
for 8-aminoquinoline-containing derivatives 8c and 8e, which
did not demonstrate liver-stage activity; this indicates that the
presence of the 8-aminoquinoline moiety does not necessarily
convey potency against the liver stage. Compounds 7c, 7v,
and 8g, lacking an 8-aminoquinoline substituent, also dis-
played excellent activity at 10 mm concentrations without af-
fecting Huh7 cell confluence.
The most effective trisubstituted s-triazines 8b–h, 8k, and 8l
present IC50 values ranging from 0.22 to 8.31 mm. The most
striking result is the high antiplasmodial activity of hybrid
members containing two (i.e., compound 8d) or one (i.e.,
compound 8 f) primaquine moiety, with IC50 values of 0.48 and
0.22 mm, respectively. Interestingly, other trisubstituted s-tria-
zines containing only one 8-aminoquinoline substituent are
significantly less potent than 8 f (i.e., 8c: IC50 =5.63 mm, 8e:
IC50 =8.3 mm), which suggests that the (1R,2S)-ephedrine
groups in 8 f might be beneficial for activity. This result, to-
gether with that obtained for (1R,2S)-ephedrine derivative 7d,
is in line with the recent finding that 2-amino-1-phenylethanol
derivatives are useful scaffolds to develop potent antimalarial
agents.[54] Other trisubstituted s-triazines with good antiplas-
modial activity are those containing dibenzylamine groups,
that is, compounds 8b and 8h, or a N-(3-aminopropyl)imida-
zole group, that is, compound 8g. The remaining compounds
resulting from combinations of the s-triazine core with other
amines, alcohols, and benzylmercaptan were found to be inac-
tive.
With these promising results in hand, we then determined
the IC50 values for 7b and 8 f against the liver stage of P. ber-
ghei infection of Huh7 cells. These compounds were 345 and
8 times more potent than primaquine, respectively (Table 2). Of
Table 2. IC50 values for the inhibition of infection of human hepatoma
cells (Huh7) by P. berghei, and half-lives for the degradation of 7b and 8 f
in rat liver microsomes.
1
Compd
IC50 [nm][a]
t = [h]
kobs [hÀ1
]
2
7b
8 f
PQ
ATV
27.5Æ1.5
1210Æ53.8
9500Æ2300
1.10Æ0.03
2.4
5.9
ND
ND
0.32
0.12
ND
ND
[a] Data represent the meanÆSD of n=3 independent experiments per-
formed in triplicate; ND: not determined.
Liver schizontocidal activity
s-Triazine derivatives active against the erythrocytic stage of in-
fection (Table 1) were further evaluated for their ability to in-
hibit the development of malaria parasites in liver cells. A total
of 14 compounds were assayed at two different concentrations
by using an in vitro infection model that employs a human
hepatoma cell line (Huh7) infected with P. berghei expressing
firefly luciferase. Results were compared with those obtained
with primaquine (Figure 4). Most compounds were poorly
active at the lowest concentration tested (1 or 2 mm). Excep-
note, primaquine is only moderately active in this assay, as the
drug requires metabolic activation to display potent activity.[55]
Also, hepatoma cells are metabolically less competent than
hepatocytes in activating primaquine.[56] Compounds 7b and
8 f were less potent than atovaquone, an inhibitor of parasite
cytochrome bc1 that has potent liver-stage activity and that
does not require metabolic activation.[57] Overall, these results
suggest that the 8-aminoquinoline pharmacophore does not
Figure 4. Luminescence-based measurement of dose-dependent effects of selected s-triazine derivatives on P. berghei-infected Huh7 cells. Bars represent in-
fection loads and dots represent cell confluency. Results are expressed as meanÆSD (triplicate wells). PQ: primaquine, used as positive control. DMSO: sol-
vent-treated control.
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N1,N1’-(6-Chloro-1,3,5-triazine-2,4-diyl)bis[N4-(6-methoxyquinolin-
8-yl)pentane-1,4-diamine] (7b): Primaquine biphosphate was first
treated with aqueous KOH (6 equiv) to remove phosphoric acid.
The solution was then extracted with dichloromethane, dried with
Na2CO3, filtered, and concentrated under vacuum. Dioxane (2 mL)
was added at 08C to a mixture of cyanuric chloride (15.7 mg,
0.085 mmol), primaquine (70 mg, 0.268 mmol, 3.15 equiv), and N,N-
diisopropylethylamine (DIPEA; 51 mL, 0.293 mmol, 3.4 equiv). After
45 min of stirring, the solution was warmed up to room tempera-
ture, stirred for another 1.25 h, and then heated at reflux for 20 h.
After cooling, the solvent was removed under vacuum, and the re-
sulting crude material was purified by preparative TLC (EtOAc/
hexane 1:1); the fraction at Rf =0.50 gave the desired compound
require metabolic activation if associated with the s-triazine
core.
Metabolic stability
The microsomal stability of s-triazine hybrids 7b and 8 f was
determined by using rat liver microsomes (Table 2). Both 7b
and 8 f displayed good metabolic stability, with half-lives of 2.4
and 5.9 h, respectively. Neither primaquine nor its oxidative de-
amination product, carboxyprimaquine,[13] were detected in
the incubation mixtures, which suggests that other metabolic
pathways might be operating for 7b and 8 f. In addition, these
hybrids did not degrade upon incubation in 80% human
plasma for 3 days.
1
as a yellow foam. Yield: 27.3 mg (53%). H NMR (300 MHz, CDCl3):
d=8.51 (s, 1H), 7.89 (d, J=6.7 Hz, 1H), 7.38–7.16 (m, 1H), 6.28 (d,
J=17.2 Hz, 2H), 6.10–5.88 (m, 2H), 3.86 (d, J=6.0 Hz, 3H), 3.61 (s,
1H), 3.37 (s, 2H), 1.68 (s, 4H), 1.26 ppm (d, J=4.9 Hz, 3H). 13C NMR
(75 MHz, CDCl3): d=168.2, 165.9, 159.4, 144.4, 144.9, 135.3, 134.7,
129.9, 121.8, 96.7, 91.6, 55.1, 47.7, 40.8, 33.8, 25.9, 20.5 ppm. IR
(KBr): n˜ =3385, 3263, 2958, 2931, 2856, 1610, 1577, 1544,
1388 cmÀ1. MS (ESI+): m/z: 652.42 [M+Na]+, 630.40 [M+H]+.
HRMS (IEA): m/z: calcd for C33H40ClN9O2: 629.2993; found:
629.3001.
Conclusions
We reported that s-triazines 7 and 8 decorated with 8-amino-
quinoline side chains are a new family of efficient dual-stage
antiplasmodial agents endowed with good metabolic stability.
s-Triazine derivatives inhibited the development of intra-eryth-
rocytic forms of P. falciparum, with IC50 values ranging from the
low micromolar to the nanomolar range, while displaying low
cytotoxicity against mammalian cells. The most effective com-
pounds against the erythrocytic stage of P. falciparum were
then screened for activity against the liver stage. Analysis of
the structure–activity relationships revealed that the presence
of (1R,2S)-ephedrine and 8-aminoquinoline moieties was bene-
ficial for dual antiplasmodial activity. However, the presence of
an 8-aminoquinoline moiety was not an absolute requirement
to deliver dual-acting s-triazines, as some compounds without
this moiety (e.g., 7c, 7v, and 8g) were also active. Compounds
7b and 8 f are the more promising from this library; they pre-
sented potent activity against both blood (IC50 =0.60 and
0.22 mm, respectively) and liver (IC50 =0.028 and 1.21 mm, re-
spectively) stages combined with high selectivity indices.
These compounds are significantly more potent than prima-
quine. Additionally, none of the compounds significantly affect-
ed Huh7 cell proliferation, which indicates that triazine–prima-
quine hybrids are selective and nontoxic antimalarial agents.
N1-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-N4-(6-methoxyquinolin-8-
yl)pentane-1,4-diamine (8c): Dioxane (1 mL) was added at 08C to
a mixture of cyanuric chloride (22.2 mg, 0.119 mmol), primaquine
(30.2 mg, 0.114 mmol, 1 equiv), and DIPEA (75 mL, 0.433 mmol,
3.6 equiv). After stirring for 15 min, the solution was warmed to
room temperature and morpholine (52 mL, 0.602 mmol, 5 equiv)
was added. The mixture was stirred at room temperature for
50 min and then heated at reflux for 19 h. After cooling, the sol-
vent was removed under vacuum, and the resulting crude material
was purified by preparative TLC (EtOAc/hexane 1:1); the fraction at
Rf =0.40 gave the desired compound as a yellow foam. Yield:
38.6 mg (57%). 1H NMR (400 MHz, CDCl3) d=8.54 (dd, J=4.2,
1.6 Hz, 1H), 7.94 (dd, J=8.3, 1.6 Hz, 1H), 7.33 (dd, J=8.2, 4.2 Hz,
1H), 6.35 (d, J=2.5 Hz, 1H), 6.29 (d, J=2.5 Hz, 1H), 6.03 (d, J=
8.3 Hz, 1H), 3.91 (s, 3H), 3.73 (s, 8H), 3.71 (s, 8H), 3.66 (m, 1H),
3.51–3.29 (m, 2H), 1.91–1.62 (m, 4H), 1.32 ppm (d, J=6.3 Hz, 3H).
13C NMR (101 MHz, CDCl3) d=159.4, 145.0, 144.3, 135.3, 134.8,
129.9, 121.8, 96.6, 91.5, 66.8, 55.2, 47.8, 43.6, 40.6, 34.2, 26.6,
20.6 ppm. IR (KBr): n˜ =3368, 2959, 2918, 2895, 2851, 1499, 1431,
1389 cmÀ1. MS (ESI+): m/z: 531.37 [M+Na]+, 509.56 [M+H]+.
HRMS (IEA): m/z: calcd for C26H36N8O3: 508.2910; found: 508.2924.
N1,N1’-(6-Morpholino-1,3,5-triazine-2,4-diyl)bis[N4-(6-methoxyqui-
nolin-8-yl)pentane-1,4-diamine] (8d): Dioxane (2 mL) was added
at 08C to a mixture of cyanuric chloride (27.1 mg, 0.147 mmol), pri-
maquine (81.9 mg, 0.315 mmol, 2.1 equiv), and DIPEA (99 mL,
0.566 mmol, 3.8 equiv). After stirring for 20 min, the solution was
warmed to room temperature. The mixture was stirred at room
temperature for 50 min. Morpholine (23 mL, 0.275 mmol, 5 equiv)
was added, and the mixture was heated at reflux for 18 h. After
cooling, the solvent was removed under vacuum, and the resulting
crude material was purified by preparative TLC (EtOAc/hexane 1:1);
the fraction at Rf =0.20 gave the desired compound as a yellow
Experimental Section
Chemistry
General: All reagents used were purchased from Aldrich or Merck
and were used without further purification. Evolution of the reac-
tions was followed by TLC by using silica Merck Kieselgel 60 F254
plates with inspection under ultraviolet light at l=254 and
325 nm or stain solution of phosphomolybdenum acid in ethanol.
Purifications were performed by preparative TLC plates by using
Merck Kieselgel 60 F254 silica for thin-layer chromatography. The
compounds were characterized by NMR spectroscopy with
a Bruker Avance II400 and 300 Ultrashield Plus, by elemental analy-
sis performed at the Faculty of Pharmacy of University of Lisbon
services, by infrared spectroscopy with a Shimadzu IRAffinity-1 FTIR
spectrometer by using KBr platelets, and by mass spectrometry
with a Micromass Quattro Micro triple quadrupole (Waters, Ireland)
with an electrospray (ESI) ion source.
1
foam. Yield: 71.1 mg (63%). H NMR (400 MHz, CDCl3): d=8.52 (dd,
J=4.2, 1.6 Hz, 2H), 7.91 (dd, J=8.2, 1.5 Hz, 2H), 7.29 (dd, J=8.2,
4.2 Hz, 2H), 6.32 (d, J=2.5 Hz, 2H), 6.27 (d, J=2.0 Hz, 2H), 6.01 (d,
J=8.1 Hz, 2H), 3.88 (s, 6H), 3.76–3.54 (m, 10H), 3.36 (d, J=5.7 Hz,
4H), 1.70 (qd, J=14.5, 8.2 Hz, 8H, 4CH2), 1.28 ppm (d, J=6.3 Hz,
6H). 13C NMR (101 MHz, CDCl3): d=159.4, 145.0, 144.3, 135.4, 134.8,
129.91, 121.8, 96.7, 91.6, 66.8, 55.2, 47.8, 43.6, 40.6, 34.1, 26.6,
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20.6 ppm. IR (KBr): n˜ =3377, 3329, 3314, 3267, 3246, 2959, 2926,
2853, 1545, 1501, 1443, 1422, 1387 cmÀ1. MS (ESI+): m/z: 681.63
[M+H]+. HRMS (IEA): m/z: calcd for C37H48N10O3: 680.3911; found:
680.3911.
the cells were fixed in 2% formaldehyde in phosphate-buffered
saline (PBS), transferred into PBS with 100 mm NH4Cl, 0.1% Triton
X-100, 1 nm YOYO-1, and infected erythrocytes were counted in
a flow cytometer (FACSort, Beckton Dickinson; EX 488 nm, EM
520 nm). Values of IC50 based on comparisons with untreated con-
trol cultures were calculated by using GraphPad PRISM software.
Two independent experiments were performed, each with four
replicates for each of the experimental conditions.
{1,1’-[6-({4-[(6-Methoxyquinolin-8-yl)amino]pentyl}amino)-1,3,5-
triazine-2,4-diyl]bis (pyrrolidine-2,1-diyl)}bis(diphenylmethanol)
(8e): A solution of compound 7c (25.3 mg, 0.041 mmol), prima-
quine (11.5 mg, 0.045 mmol, 1.1 equiv), and DIPEA (10 mL,
0.057 mmol, 1.4 equiv) in dioxane (1 mL) was heated at reflux for
20 h. After cooling, the solvent was removed under vacuum, and
the resulting crude material was purified by preparative TLC
(EtOAc/hexane 2:8); the fraction at Rf =0.14 yielded the desired
Antiproliferative assays: Human cancer cell lines from breast (MCF-
7), colon (HT-29), and lung (NCI-H460) were purchased from ATCC
and cultivated in RPMI-1640 with l-glutamine and 10% fetal
bovine serum (FBS) in a humidified atmosphere with 5% CO2 at
378C. Cells were plated in 96-well plates with a density of 5ꢁ104
(NCI-H460), 1ꢁ105 (HT-29), and 1.5ꢁ105 cells per well (MCF-7) and
cultured for approximately 24 h. Stock solutions of the compounds
to be tested were prepared in ethanol and then diluted with the
cell culture medium with 0.5% FBS. Cells were incubated with the
compounds at 0–20 mm concentration for 48 h. Cells were then
washed with PBS and incubated with 0.5% FBS cell culture
medium containing 50 mgmLÀ1 neutral red. Three hours later, cells
were washed again with PBS, and the amount of neutral red re-
tained by the cells was extracted with an organic solution (20 mL
distilled water, 20 mL ethanol, and 400 mL glacial acetic acid). Ab-
sorbance of the samples was measured at l=540 nm in a plate
reader after gentle shaking. Viability was determined by the ratio
of absorbance of treated and control cells. Two independent ex-
periments were performed, each with four replicates for each of
the experimental conditions. IC50 values were determined by using
GraphPad Prism 5.
compound as
a
yellow foam. Yield: 26 mg (76%). 1H NMR
(400 MHz, DMSO, 808C): d=8.52 (dd, J=9.5, 3.4 Hz, 1H), 8.05 (d,
J=8.3 Hz, 1H), 7.46–7.11 (m, 20H), 6.70 (s, 1H), 6.47 (d, J=2.3 Hz,
1H), 6.28 (d, J=1.9 Hz, 1H), 6.09 (d, J=7.2 Hz, 1H), 3.84 (s, 3H),
3.69 (d, J=7.8 Hz, 2H), 3.61 (d, J=5.4 Hz, 1H), 3.20 (s, 2H), 2.97 (s,
2H), 2.00 (s, 2H), 1.85 (s, 2H), 1.71–1.34 (m, 8H), 1.21 (d, J=7.1 Hz,
3H), 1.01 ppm (s, 2H).13C NMR (101 MHz, [D6]DMSO, 808C): d=
164.7, 159.7, 147.5, 145.4, 145.4, 144.6, 135.3, 135.1, 130.1, 128.3,
128.1, 127.6, 127.5, 127, 126.9, 122.4, 96.7, 92.6, 82.1, 55.5, 47.8,
40.6, 34.3, 29.3, 26.5, 22.8, 20.8 ppm. IR (KBr): n˜ =3275, 3169, 2961,
2930, 2874, 1535, 1448, 1425, 1389 cmÀ1. MS (ESI+): m/z: 841.39
[M+H]+. HRMS (ESI-FIA-TOF): m/z: calcd for [C52H56N8O3 +H]+:
841.4548; found: 841.4550.
(1R,1’R,2S,2’S)-2,2’-{[6-({4-[(6-Methoxyquinolin-8-yl)amino]pen-
tyl}amino)-1,3,5-triazine-2,4-diyl]bis(methylazanediyl)}bis(1-phe-
nylpropan-1-ol) (8 f): A solution of compound 7d (101.2 mg,
0.23 mmol), primaquine (66 mg, 0.25 mmol, 1.1 equiv), and DIPEA
(47 mL, 0.27 mmol, 1.2 equiv) in dioxane (2 mL) was heated at
reflux for 24 h. After 24 h, unreacted 7d remained; more prima-
quine (33.2 mg, 0.125 mmol, 0.55 equiv) and dioxane (2 mL) were
added, and the mixture was heated at reflux for an additional 24 h.
After cooling, the solvent was removed under vacuum, and the re-
sulting crude material was purified by preparative TLC (EtOAc/
hexane 1:1); the fraction at Rf =0.53 yielded the desired compound
as a yellow foam. Yield: 51 mg (33%).1H NMR (400 MHz, [D6]DMSO,
808C): d=8.53 (dd, J=4.1, 0.8 Hz, 1H), 8.05 (dd, J=8.3, 1.5 Hz,
1H), 7.40 (dd, J=8.3, 4.2 Hz, 1H), 7.36 (d, J=7.4 Hz, 4H), 7.22 (t,
J=7.3 Hz, 4H), 7.15 (t, J=7.2 Hz, 2H), 6.47 (d, J=2.4 Hz, 1H), 6.29
(d, J=2.3 Hz, 1H), 6.12 (d, J=8.4 Hz, 1H), 5.21 (s, 1H), 4.93 (s, 2H),
4.75 (d, J=5.3 Hz, 2H), 3.83 (s, 3H), 3.65 (s, 1H), 3.29 (d, J=4.4 Hz,
2H), 2.91 (s, 6H), 1.77–1.54 (m, 4H), 1.25 (d, J=6.3 Hz, 3H), 1.14 (d,
J=6.8 Hz, 6H). 13C NMR (101 MHz, [D6]DMSO, 808C): d=165.9,
165.4, 159.7, 145.3, 144.7, 144.6, 135.2, 135.1, 130.1, 128.0, 127.0,
126.7, 122.4, 96.8, 92.7, 75.7, 55.5, 55.3, 47.9, 40.8, 34.4, 30.1, 26.7,
20.7, 12.62 ppm. IR (KBr): n˜ =3385, 3329, 3292, 3265, 3246, 3215,
3171, 3084, 3058, 2961, 2934, 2868, 2552, 2502, 1560, 1518, 1481,
1450, 1391, 1356, 1219, 1163, 1051, 1030, 822, 750, 702 cmÀ1. MS
(ESI+): m/z: 665.36 [M+H]+. HRMS (IEA): m/z: calcd for
C38H48N8O3: 664.3849; found: 664.3857,
Antiplasmodium liver-stage activity: Huh-7 cells, a human hepatoma
cell line, were cultured in 1640 RPMI medium supplemented with
10% v/v fetal calf serum (FCS), 1% v/v nonessential amino acids,
1% v/v penicillin/streptomycin, 1% v/v glutamine, and 10 mm
HEPES, pH 7, and maintained at 378C with 5% CO2. Inhibition of
P. berghei liver-stage infection was determined by measuring the
luminescence of Huh-7 cell lysates 48 h after infection with a firefly
luciferase-expressing P. berghei line, PbGFP-Luccon, as previously
described[11,58] Briefly, cells (12ꢁ103 cells per well) were seeded in
96-well plates the day before drug treatment and infection. Tested
compounds were prepared in the following way: 10 mm stock sol-
utions were obtained by dissolving accurately weighed com-
pounds in MeOH and dilutions were subsequently made with
medium to the desired concentration. Medium was replaced by
fresh medium containing the appropriate concentration of each
compound 1 h prior to infection. Sporozoites (10000 spz per well),
freshly obtained through disruption of salivary glands of infected
female Anopheles stephensi mosquitoes, were added to the wells
1 h after compound addition. Sporozoite addition was followed by
centrifugation at 1700 g for 5 min. At 24 h post-infection, medium
was again replaced by fresh medium containing the appropriate
concentration of each compound. Parasite load was determined
48 h after infection by luminescence measurement by using Bioti-
um’s Firefly Luciferase Assay Kit. The effect of the compounds on
the viability of Huh-7 cells was assessed by the Alamar Blue assay
(Invitrogen, UK) by using the manufacturer’s protocol. Nonlinear re-
gression analysis was employed to fit the normalized results of the
dose-response curves, and IC50 values were determined by using
SigmaPlot software. IC50 values presented are the mean IC50 value
averaged from three independent experiments, each performed in
triplicate wells.
Biology
Activity against erythrocyte-stage P. falciparum: Human erythrocytes
infected with 1% ring-stage W2-strain P. falciparum strains
synchronized with 5% sorbitol were incubated with test com-
pounds in 96-well plates at 378C for 48 h in RPMI-1640 medium,
supplemented with 25 mm 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid (HEPES) pH 7.4, 10% heat inactivated human serum
(or 0.5% Albumax, 2% human serum), and 100 mm hypoxanthine
under an atmosphere of 3% O2, 5% CO2, and 91% N2. After 48 h,
Metabolism studies: The time courses of unchanged compounds
7b and 8 f in microsomes were determined. Each compound was
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incubated with a reaction mixture (800 mL) consisting of rat liver
microsomal protein (20 mL of 20 mgprotein mLÀ1
suspended in
)
50 mm PBS (160 mL), water (570 mL), NADPH regenerating system
solution A (40 mL, containing 31 mm NADP+, 66 mm glucose 6-
phosphatase, and 0.67 mm MgCl2), and NADPH regenerating
system solution B (8 mL, containing 40 UmLÀ1 glucose-6-phosphate
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378C for 5 min, enzyme reactions were initiated by adding the
parent solution of the drug (2 mL, 10À2 m) in DMSO. Samples were
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room temperature. The supernatants were immediately analyzed,
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Acknowledgements
This work was supported by Fundażo para a CiÞncia e Tecnolo-
gia, Portugal (grants PEst-OE/SAU/UI4013/2014, REDE/1501/REM/
2005, PTDC/CTM-POL/114367/2009, PTDC/SAU-MIC/117060/2010,
and PTDC/SAU-FAR/118459/2010; fellowships SFRH/BD/48145/
2008 to C.A.B.R. and SFRH/BPD/73822/2010 to R.F.M.F.) and Fun-
dażo Luso-Americana (award to R.M.).
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Received: January 13, 2015
Revised: February 15, 2015
Published online on && &&, 0000
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C. A. B. Rodrigues, R. F. M. Frade,
I. S. Albuquerque, M. J. Perry, J. Gut,
M. Machado, P. J. Rosenthal,
M. PrudÞncio, C. A. M. Afonso,*
R. Moreira*
&& – &&
Double-edged sword to kill malaria
parasites: s-Triazine-based hybrid com-
pounds containing a primaquine moiety
were found to be dual-stage antiplas-
modial agents. The two hybrids are
highly potent against both the liver and
blood stages of the parasite’s life cycle
and show excellent metabolic stability.
Targeting the Erythrocytic and Liver
Stages of Malaria Parasites with s-
Triazine-Based Hybrids
ChemMedChem 0000, 00, 0 – 0
www.chemmedchem.org
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ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ