Antimalarial N1, N3-Dialkyldioxonaphthoimidazoliums: Synthesis, Biological Activity, and Structure-activity Relationships

Article abstract of DOI:10.1021/acsmedchemlett.9b00457

Here we report the nanomolar potencies of N¹,N³-dialkyldioxonaphthoimidazoliums against asexual forms of sensitive and resistant Plasmodium falciparum. Activity was dependent on the presence of the fused quinone-imidazolium entity and lipophilicity imparted by the N¹/N³ alkyl residues on the scaffold. Gametocytocidal activity was also detected, with most members active at IC₅₀ < 1 μM. A representative analog with good solubility, limited PAMPA permeability, and microsomal stability demonstrated oral efficacy on a humanized mouse model of P. falciparum.

Full text of DOI:10.1021/acsmedchemlett.9b00457

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Letter
Antimalarial N1,N3-Dialkyldioxonaphthoimidazoliums:
Synthesis, biological activity and structure-activity relationships.
Stephen Ahenkorah, Dina Coertzen, Jie Xin Tong, Kevin Fridianto, Sergio Wittlin, Lyn-
Marie Birkholtz, Kevin S. W. Tan, Yulin Lam, Mei-Lin Go, and Richard K. Haynes
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00457 • Publication Date (Web): 11 Dec 2019
Downloaded from pubs.acs.org on December 13, 2019
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ACS Medicinal Chemistry Letters
Antimalarial N1,N3-dialkyldioxonaphthoimidazoliums: Synthesis, biological activity and
structure-activity relationships.
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Stephen Ahenkorah, ^ ‡ Dina Coertzen, ɸ ‡ Jie Xin Tong, # ‡ Kevin Fridianto, §‡ Sergio
Wittlin,  ¥ Lyn-Marie Birkholtz, ɸ Kevin S.W. Tan, # Yulin Lam, § Mei-Lin Go, ^ * Richard K.
Haynes. ¶ *
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^ Department of Pharmacy, # Department of Microbiology and Immunology and § Department
of Chemistry, National University of Singapore, Singapore 117543;  Institute for Sustainable
Malaria Control, Department of Biochemistry, Genetics and Microbiology, University of
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
Pretoria, South Africa 0028; Swiss Tropical and Public Health Institute, Switzerland 4051; ¥
¶
University of Basel, Switzerland 4003, Centre of Excellence for Pharmaceutical Sciences,
North-West University, South Africa 2531.
KEYWORDS Antimalarials; Dioxonaphthoimidazoliums; Quinones; Drug resistant asexual
parasites; Gametocytocidal activity.
Abstract: Here we report the nanomolar potencies of N1, N3-dialkyldioxonaphthoimidazoliums
against asexual forms of sensitive and resistant Plasmodium falciparum. Activity was dependent
on the presence of the fused quinone-imidazolium entity and lipophilicity imparted by the N1/ N3
alkyl residues on the scaffold. Gametocytocidal activity was also detected, with most members
active at IC50 < 1 µM. A representative analog with good solubility, limited PAMPA permeability
and microsomal stability, demonstrated oral efficacy on a humanized mouse model of P.
falciparum.
Substantial progress has been made in decreasing the global burden of malaria over the past
decade. Worldwide, 20 million fewer cases of malaria were recorded in 2017 as compared to
2010.1 Deaths due to malaria have declined, from 607 000 in 2010 to 435 000 in 2017.1
Furthermore, low attrition rates among recent clinical trial candidates hint at a potentially rich
antimalarial drug pipeline in the offing.2 However, optimism engendered by these encouraging
statistics belie two worrying trends. First, several high burden countries on the African
continent have seen a resurgence in malaria in spite of vigorous efforts to curtail spread.1
Second, the Greater Mekong Region has witnessed intensification of the multidrug-resistant
Plasmodium falciparum co-lineage KEL1/PLA1.3 These events highlight the fragility of gains
made against malaria and the need for constant vigilance if they are to be sustained. Like
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other infectious diseases, the emergence of resistant parasite strains is a question of when
and not if. Regardless of the prevailing optimism invoked by the current status of the drug
pipeline, there is a constant need for new agents to protect vulnerable populations, prevent
relapses of vivax infection and disrupt disease transmission.2 Eradication of malaria which is a
key Millennium Development Goal set by the United Nations, is achieved only by breaking
disease transmission.4 Unlike malaria control which treats the symptoms caused by asexual
parasites, eradication focuses on eliminating the sexual forms (gametocytes) which are
directly responsible for infecting the anopheline vector and perpetuating the plasmodial life
cycle. Ideally, antimalarial leads should target both asexual and sexual intraerythrocytic
parasites with comparable potencies.5
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A
B
O
O
O
O
N
NH
N
Br
N
N
N
N
O
N
3
N
O
O
Cyclization
O
Photolysis
O
N
O
1
O
O
O
O
YM155
SJ000030570
EC50 Asexual 32 nM
EC50 Sexual 319 nM
SJ0000561981
EC50 Asexual 1 nM
EC50 Sexual 93 nM
SJ0000541602
EC50 Asexual 1 nM
EC50 Sexual 122 nM
IC50 NF54 332 (7.1) nM
IC50 Dd2 103 (15) nM
C
N
O
N
N
O
Br
N
O
O
N
N
N
N
N
N
Br
N
N
N
N
N
Br
Br
O
O
O
O
4
1
3
2
IC50 NF54 58.9 (1.3) nM
IC50 Dd2 43.5 (9.0) nM
IC50 NF54 6.7 (1.3) nM
IC50 Dd2 13.3 (1.9) nM
IC50 NF54 948 (216) nM
IC50 NF54 > 1000 nM
Figure 1: (A) Stepwise conversion of SJ000030570 to SJ0000541602 and SJ0000561981 and
their EC50s against the asexual and sexual stages of P. falciparum 3D7.6 Structures of (B)
YM155 (C) analogs 1-4 7 and their IC50s (± SD) against asexual stages of drug sensitive NF54
and drug resistant Dd2 P. falciparum. Parasitemia was assessed by the parasite lactate
dehydrogenase assay. Positive controls were chloroquine (IC50 NF54 8.2 ±0.4 nM; IC50 Dd2 118 ±3
nM) and artesunate (IC50 NF54 < 5 nM; IC50 Dd2 18.8 ±1.5 nM).
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In an effort to identify lead compounds with potent activity against both life-cycle stages of P.
falciparum, Tanaka and co-workers reported the demethylation of the diaminonaphthoquinone
SJ000030570 to the monomethylamino analog SJ0000541602 on prolonged exposure to light,
followed by cyclization of the latter to the dioxonaphthoimidazolium SJ0000561981 (Figure
1A).6 Both SJ0000541602 and SJ0000561981 were photostable and exceptionally potent
against asexual ring stages and late stage (Stage III-V) gametocytes (Figure 1A).6
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The dioxonaphthoimidazolium scaffold of SJ0000561981 is present in YM155 (sepantronium
bromide, Figure 1B), an antitumor agent and suppressor of the anti-apoptotic protein survivin.8
This prompted us to ask if YM155 could emulate the antimalarial activity of SJ0000561981 and
more importantly, if the scaffold is an incipient antimalarial pharmacophore. Hence, YM155
and several analogs (1-4) that were previously investigated on human cancer cell lines 7 were
screened against the erythrocytic stages of drug sensitive and drug resistant P. falciparum
(Figure 1B). Disappointingly, YM155 displayed only modest activity but its analogs 1 and 4
proved more potent. We were particularly intrigued by 4 whose outstanding activity is
ostensibly linked to the absence of cyclized entities at the ring nitrogens N1/N3. This led us to
propose modifying the alkyl substituents in 4 by homologation or branching as a means of
optimizing the antimalarial potential of the scaffold. To this end, a series of N1,N3-
dialkyldioxonaphthoimidazoliums (4-35) were synthesized and evaluated against the asexual
and sexual intraerythrocytic stages of P. falciparum.
Briefly, our target compounds were obtained by introducing alkyl groups and their
branched/cyclized variants at one of the imidazolium N atoms while retaining an unchanged
alkyl at the other N atom. For instance, with methyl at N1, we inserted ethyl, propyl, butyl,
isopropyl or cyclopropyl in turn at N3 and this was repeated with each of the aforementioned
N3 substituents at N1. In this way, Series A (4-24) was prepared and evaluated on the asexual
stages of P. falciparum. The results prompted the synthesis of two additional series, namely
the non-quaternized Series B (36-40) in which only N1 was substituted and Series C (25-35)
which has isobutyl at N1, the same groups at N3 as in Series A in addition to longer alkyl side
chains (isobutyl, n-hexyl, n-octyl, n-decyl, n-dodecyl) (Scheme 1). Several members in Series
A-C have been reported in the literature, namely 4,10,19; 9 5,7,9,11,14,16,25,36-40; 10 24,31;
11 12,13, 12 and 15. 13 We followed the method of Ho et al.7 to obtain Series A-C.
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Scheme 1 a
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7
8
9
R2
O
O
O
O
O
R2
O
Cl
d
Cl
Cl
Br
c
b
NH
a
N
NH
R1
Cl
O
N
R1
O
N
R1
O
)
N
R1
O
10
11
12
13
14
15
16
17
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20
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O
O
41b-51b
41a- 51a
4-35
4a-35a
e
1
1
2
1
2
41a,b
42a,b
R = Me (
), Et (
), n-Pr
20a, 20
R = i-Pr, R = c-Pr (
)
4a, 4
R = Me, R = Me (
1
2
1
2
43a,b
44a,b
45a,b
(
),i-Pr (
), c-Pr (
),
), n-hexyl
49a,b
), n-decyl
21a, 21
R = i-Pr,, R = n-Bu (
)
5a, 5
R = Me, R = Et (
)
O
1
2
1
2
46a,b
47a,b
n-Bu (
), i-Bu (
22a,22
R = c-Pr, R = c-Pr (
)
6a.6
R = Me, R = n-Pr (
)
NH2
1
2
1
2
48a,b
50a,b
(
(
), n-octyl (
23a, 23
R = c-Pr, R = n-Bu (
)
7a, 7
R = Me, R = i-Pr (
)
1
2
1
2
51a,b
)
Cl
O
), n-dodecyl (
24a, 24
R = n-Bu, R = n-Bu (
)
8a, 8
R = Me, R = c-Pr (
)
1
2
1
2
25a, 25
R = i-Bu, R = Me (
)
9a, 9
R = Me, R = n-Bu (
)
52
1
2
1
2
26a, 26
R = i-Bu, R = Et (
)
10a, 10
R = Et, R = Et (
)
1
2
b
1
2
27a, 27
R = i-Bu, R = n-Pr (
)
11a, 11
R = Et, R = n-Pr (
)
O
O
1
2
O
1
2
O
28a, 28
R = i-Bu, R = i-Pr (
)
12a, 12
R = Et, R = i-Pr (
)
O
1
2
f
N
N
1
2
c
NH
29a, 29
R = i-Bu, R = c-Pr (
)
13a, 13
R = Et, R = c-Pr (
)
NH
Cl
1
2
1
2
30a, 30
R = i-Bu, R = n-Bu (
)
14a, 14
R = Et, R = n-Bu (
)
NH
1
2
R1
31a, 31
)
1
2
R = i-Bu, R = i-Bu (
15a, 15
R = n-Pr, R = n-Pr (
)
O
O
R1
O
1
2
1
2
32a, 32
R = i-Bu, R = n-hexyl (
)
16a, 16
R = n-Pr, R = i-Pr (
)
36a-40a
36-40
53
1
2
1
2
33a, 33)
R = i-Bu, R = n-octyl (
17a, 17
R = n-Pr, R = c-Pr (
)
1
1
1
2
1
2
39a, 39
R = i-Pr (
)
36a, 36
R = Me (
)
34a, 34
R = i-Bu, R = n-decyl ( )
18a, 18
R = n-Pr, R = n-Bu (
)
1
1
1
2
1
2
40a, 40
R = n-Bu (
)
37a, 37
R = Et (
)
35a, 35
R = i-Bu, R = n-dodecyl ( )
19a, 19
R = i-Pr, R = i-Pr (
)
1
38a, 38)
R = n-Pr (
a Reagents and conditions (a) R1-amine, EtOH, r.t, 18h; (b) acetic anhydride, conc H2SO4, r.t,
1.5 h; (c) R2-amine, toluene, 45 °C, 4 h; (d) 48% HBr (aq), EtOH + EtOAc, 45 °C , 4h to r.t
overnight; (e) NH3 in MeOH, EtOH, 35 °C, 3 h; (f) 48% HBr (aq), 2M NaOH (2 drops), EtOH,
50 °C, 1h.
The growth inhibitory IC50s of Series A compounds on the asexual forms of Dd2 (drug
resistant) and 3D7 (drug sensitive) strains of P. falciparum were determined by a Hoechst
33342-based method with a fluorescence-activated cell sorter (FACS) to measure DNA
content of infected erythrocytes.14 Encouragingly, all the compounds displayed sub-micromolar
activities on both Dd2 and 3d7 with broadly similar levels of potencies but with some
exceptions (17, 23) (Table 1). In addition, we found excellent differential cytotoxicities in this
series, with most compounds associated with a 100-fold or greater selectivity vis-à-vis
mammalian Chinese hamster ovary (CHO) cells.
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The most potent compounds were 11, 14, 16, 19 which had low nanomolar activities (IC50 <
10 nM) against both resistant and sensitive plasmodia. Interestingly, the total carbon count on
R1/R2 of these compounds was either 5 or 6, out of a possible range of 2 to 8. Hence, we
posited that optimal activity resided within a narrow spectrum, tentatively defined by 5-6
carbons, with declining potencies at either end of this range. Indeed, diminished activity was
evident in the dibutyl analog 24 while short hydrophilic fragments like methyl and cyclopropyl
were disproportionally represented at R1/R2 of less potent analogs such as 4, 5, 8 and 13. The
hydrophilicity of the cyclopropyl fragment, anomalous for its carbon count, is attributed to its
unique three-dimensional features.15
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Earlier, we observed significantly diminished antimalarial activity in the non-quinoid analog 3
of YM155 (Figure 1C) which portend a pivotal structure-activity role for the quinone. However,
only modest activity was uncovered in representative quinones like 1,4-naphthoquinone,
menadione and 1,4-benzoquinone against NF54 asexual parasites (20-40% inhibition at 1 µM,
Table S-2). Hence, we posited that it was not the quinone per se but the fusion of the quinone
to the charged imidazolium ring that was required for potent activity. Consequently, we
synthesized Series B (36-40) in which the quinone is fused to an uncharged alkyl substituted
imidazole ring (Scheme 1). Gratifyingly, no member in this series inhibited growth by more
than 50% at 1 µM (Table S-2), thus affirming the essentiality of the fused quinone-imidazolium
for potent activity.
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Table 1: Growth inhibitory activities of Series A (4-24) on asexual ring stages of P. falciparum
Dd2 (drug resistant), 3D7 (drug sensitive) and mammalian Chinese Hamster Ovary (CHO)
cells.
1
2
3
4
5
6
7
8
9
R2
O
Br
N
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
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42
43
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45
46
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57
58
59
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1
R1
N
O
IC50 expressed as Mean ± SD
Dd2 (nM)b 3D7 (nM)b CHO (µM)c
a
No R1 a
R2
4
5
6
7
8
9
CH3
CH3
CH3
CH3
CH3
CH3
CH3
26.5 ±2.6
19.9 ±0.3
14.3 ±0.6
16.1 ±1.1
30.4 ±6.3
14.1 ±0.8
19.1 ±2.8
8.4 ±2.1
22.6 ±1.1
8.4 ±4.7
4.8 ±2.6
4.8 ±2.3
25.7 ±3.3
4.3 ±1.5
6.0 ±0.7
3.7 ±0.5
5.7 ±1.1
3.6 ±2.1
3.9 ±0.6
4.7 ±2.3
3.1 ±0.9
13.1 ±1.1
14.7 ±1.0
8.2 ±0.3
C2H5
n-C3H7
i-C3H7
c-C3H7
n-C4H9
C2H5
d
10 C2H5
11 C2H5
12 C2H5
13 C2H5
14 C2H5
15 n-C3H7
16 n-C3H7
17 n-C3H7
n-C3H7
i-C3H7
c-C3H7
n-C4H9
n-C3H7
i-C3H7
c-C3H7
11.7 ±1.3
35.0 ±5.5
9.1 ±4.2
61.5 ±12.7 15.1 ±1.6
d
66.1 ±19
4.2 ±0.2
26.9 ±20
4.0 ± 0.6
331 ±37
6.6 ±0.5
37.0 ±1.2
16.2 ±0.5
26.8 ±1.9
5.5 ±0.6
11.3 ±3.4
8.0 ±2.0
d
20.7 ±3.5
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ACS Medicinal Chemistry Letters
18 n-C3H7
19 i-C3H7
20 i-C3H7
21 i-C3H7
22 c-C3H7
23 c-C3H7
24 n-C4H9
n-C4H9
i-C3H7
c-C3H7
n-C4H9
c-C3H7
n-C4H9
20.9 ±0.9
18.8 ±3.9
6.5 ±4.0
26.8 ±1.4
56.7 ±1.3
1
2
3
4
5
6
7
8
4.7 ±1.0
d
d
25.0 ±4.3
14.9 ±0.2
15.8 ± 0.8 14.6 ±0.9
11.6 ±7.3
80.3 ±1.9
42.5 ±2.4
10.1 ±0.4
30.4 ±2.1
ND
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
d
d
38.6 ±10.5 41.5 ±2.4
20.8 ±3.6
43.6 ±4.0
45.9 ±1.0
182 ±9
n-C4H9
50.5 ±0.9
38.6 ±6.5
N
O
YM
N
155
Chloroquine
Artesunate
Atovaquone
233 ±4
23.4 ±2.6
22.8 ±2.4
2.8 ±1.2
13.3(±4.5
2.5 ±1.3
30
31
a
b
To note that R1/R2 are interchangeable due to resonance within the imidazolium ring.
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Concentration corresponding to 50% of the net amount of fluorescence of non-treated control
cultures. Mean (± SD) of n = 3 separate determinations. c Concentration required to reduce cell
growth by 50% relative to untreated cultures as determined by a tetrazolium dye assay. Mean
(± SD) of n = 3 separate determinations. d c-C3H7 = cyclopropyl
To further examine the activity moderating effects of the alkyl residues at R1/R2, we prepared
Series C which has isobutyl at R1 and longer alkyl side chains (up to dodecyl) at R2. These
compounds were tested alongside the potent Series A analogs (11,14,16, 19) on drug
sensitive NF4 and drug resistant K1 and W2 strains using the SYBR Green I fluorescence
assay as a proxy of parasite proliferation (Table 2).16 Interestingly, a total carbon count of 5-6
at R1/R2 of Series C was yet again associated with optimal activity. Hence, 25 and 26 which
have methyl/ethyl at R2 have IC50 values that were broadly comparable to 11,14,16,19 of
Series A. Extending the length of R2 caused a gradual and uneven decline in activity.
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Table 2: IC50s of Series C (25-35) and potent Series A analogs (11,14, 16,19) on (i) asexual
stages of P. falciparum NF54 (drug sensitive) and K1,W2 (drug resistant) and (ii) early
gametocytes (EG) and late gametocytes (LG) of transgenic parasite line NF54-Pfs16-GFP-
Luc.
1
2
3
4
5
6
7
8
9
IC50 (nM) b
Gametocytocidal IC50
(nM) c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
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39
40
41
42
43
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46
47
48
49
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58
59
60
No R1 a
R2 a
NF54
n-C3H7 3.45 ±0.31 6.5 ±1.7
n-C4H9 1.64 ±0.2 2.4 ±0.7
K1
W2
EG
LG
11 C2H5
14 C2H5
4.94 ±0.29 390 ±83 709 ±112
2.88 ±0.18 268 ±56 320 ±35
2.20 ±0.12 162 ±52 567 ±87
n-C3H7
16
i-C3H7
2.28 ±0.19 3.1 ±0.8
1.43 ±0.13 2.5 ±1.3
19 i-C3H7 i-C3H7
25 i-C4H9 CH3
26 i-C4H9 C2H5
2.18 ±0.16 72 ±5
653 ±29
1.92 ±0.12 3.40 ±0.40 1.85 ±0.10 41 ±17
1.63 ±0.02 3.10 ±0.50 1.62 ±0.14 33 ±5
1088 ± 26
1019 ±251
27 i-C4H9 n-C3H7 2.97 ±0.10 4.42 ±0.10 2.12 ±0.16 269 ±48 842 ± 251
28 i-C4H9 i-C3H7 2.36 ±0.07 4.22 ±0.21 1.96 ±0.30 229 ±79 689 ±138
29 i-C4H9 c-C3H7 3.94 ±0.23 5.85 ±0.08 2.55 ±0.23 139 ±48 832± 274
30 i-C4H9 n-C4H9 7.20 ±0.40 9.85 ±0.21 5.10 ±0.50 181 ±87 275 ± 66
31 i-C4H9 i-C4H9
9.19 ±0.32 12.5 ±0.7
5.75 ±0.27 337 ±65 812 ± 364
5.28 ±0.21 143 ± 9 551 ± 99
32 i-C4H9 n-C6H13 6.20 ±0.15 10.0 ±0.1
33 i-C4H9 n-C8H17 4.36 ±0.08 8.23 ±1.02 3.60 ±0.80 39 ± 18 164 ±10
34 i-C4H9 n-
C10H21
4.96 ±0.17 13.3 ±2.1
6.10 ±0.40 370 ±73 237 ± 66
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35 i-C4H9 n-
C12H25
4.30 ±0.50 11.2 ±1.0
5.50 ±0.40 361 ±75 174 ± 11
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Chloroquine
Artesunate
10 ±3
154 ±14d
3.26 ±.20
-
233 ±40
2.40 ±0.20 63 ±3
- 95 ±11
-
-
3.0 ±0.29
-
259 ±80
143 ±17
Methylene Blue
a R1 and R2 are as depicted in Table 1. b Concentration corresponding to 50% of the net
amount of SYBR Green I fluorescence of non-treated control cultures. Mean (± SEM) of n = 3
independent biological replicates. c Concentration corresponding to 50% reduction of
luminescence in early and late gametocytes of NF54-Pfs16-GFP-Luc in a luciferase-based
assay. Mean (± SEM) of n = 3 independent biological replicates. d IC50 of CQ on the P.
falciparum strain (K1) was 212 (± 32) on the Hoechst assay, indicating broad equivalence
between the Hoechst and SYBR Green I assay platforms.
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Next, we assessed the compounds in Table 2 for transmission blocking activity on early
(>95% stage II/III) and late (> 90% stage IV/V) stage gametocytes.17 Most compounds were
gametocytocidal (IC50 < 1000 nM) and generally more potent against early gametocytes (IC50
30-400 nM) than late gametocytes (IC50 160-1100 nM). Indeed, several compounds (19, 25,
26, 33) were as cidal as the positive controls artesunate and methylene blue against early
gametocytes. There was no discernible trend as to how alkyl chain lengths impacted early
gametocytocidal activity in Series C. On the other hand, the potent activity of analogs 33, 35
against late stage gametocytes (comparable to artesunate) alluded to a preference for longer
alkyl side chains (total carbon count ≥ 12) at R1/R2 against this stage of the life cycle.
Noticeably, 33 displayed outstanding potencies against both early and late gametocytes which
may suggest its potential in blocking transmission.
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The preceding discussion on the effect of R1/R2 alkyl chain length on asexual/sexual
potencies pointed to a possible moderating role for lipophilicity. To confirm, the clogP values
and antimalarial IC50s of Series A and C were analyzed for statistical correlations based on
Spearman  coefficients (Tables S-3, S-4). Interestingly, no significant correlation between
clogP and asexual IC50 was detected in Series A but it was observed in Series C (Table S-4).
Very likely, the extended clogP range arising from the inclusion of analogs with longer alkyl
side chains in Series C led to this outcome. The correlation for Series C was positive (smaller
clogP /shorter side chains favored potent activity), recapitulating our preceding anecdotal
inferences. When correlations between lipophilicities and gametocytocidal activities (IC50
EG/IC50 LG) of Series C were examined, none was observed for early gametocytes but a
negative relationship was detected for late gametocytes (longer side chains favored potent
activity) (Table S-4).
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Thus far, most of the compounds in Table 2 qualified as validated antimalarial hits based on
their outstanding potencies against asexual (sensitive and multidrug resistant strains, IC50 <
100 nM) and sexual life stages of the parasite.5 Progression to early lead status would require
evidence of in vivo efficacy (ED90 < 50 mg/kg) against the blood stages of infection in a P.
falciparum infected humanized mouse model.5 To this end, we selected 19 as a representative
member for animal studies. The choice of 19 was primarily motivated by its excellent activity
against asexual parasites, exceeding that of the most favored Series C analog 33 which is
arguably better positioned in terms of gametocytocidal activity.
Table 3: PK parameters for compound 19 administered to male C57BL6 mice (n=3 per dosing
route)
Route
Dose
t 1/2 (h)
CL
Vd
Cmax Tmax
AUC0-last
(min.
% F
(mg/kg)
(mL/min/kg) (L/Kg)
(h)
(µM)
a
µmole/L )b
Intravenous
Oral
2
1.4
70
-
8.3
-
-
-
130
69
-
20
11.3
337
2
5
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a Mice showed no sign of distress 24 h after single dose administration by either route. b AUC
was monitored up to 7 h (intravenous) and 24 h (oral).
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3
4
5
6
7
8
9
Prior to pharmacokinetic (PK) profiling in mice, 19 was assessed for its solubility, PAMPA
permeability and liability to metabolic breakdown by rat liver microsomes. 19 had good water
solubility (> 100 µM, pH 7.4) but no detectable PAMPA permeability. It was remarkably stable
to microsomal degradation (Figure S-1) and additionally, well tolerated in mice when
administered intravenously (2 mg/kg) and orally (20 mg/kg). In line with its hydrophilic nature,
the PK of 19 revealed limited tissue distribution (volume of distribution Vd 8.3 L/kg), rapid
clearance from plasma (CL 70 mL/min/kg) and a relatively short half-life (t1/2 1.4 h) (Table 3).
Oral bioavailability was however limited at 5 %, likely due to its quaternized state which would
limit permeability across the gastrointestinal barrier. Notwithstanding, a plot of plasma
concentration (after oral administration) versus time showed that levels of 19 were well above
its IC50 3D7 for the period of exposure (24 h) (Figure 2).
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Figure 2: Systemic exposure of 19 over 24 h following administration of a single oral dose (20
mg/kg) to healthy mice over 24 h. Dotted lines embedded within plot are the IC50 (1x, 3x) values
of 19 against CQ sensitive 3D7 P. falciparum.
Thus encouraged, we proceeded to evaluate the efficacy of 19 on a humanized mouse
model of P. falciparum (Pf3D7 0087/N9). Briefly, 19 was administered orally at 5 doses ranging
from 1 to 50 mg/kg, with each dose given for 4 days (Days 3-6). Parasitemia was determined
on each treatment day and on Day 7. The effective dose of 19 required to reduce parasitemia
by 90% on Day 7 (ED90) was estimated to be 34 mg/kg (Figure 3). Noting that parasitemia was
not reduced significantly at the highest dose, some caution should be accorded to this value.
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19 could admittedly have greater in vivo efficacy if not for its limited oral bioavailability.
1
2
3
Appropriate formulation strategies may be employed to circumvent this liability.
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Figure 3: In vivo efficacy of 19 on the humanized P. falciparum (Pf3D70067/N9) mouse model.
ED90 is the effective dose (mg/kg) required to reduce parasitemia by 90% on Day 7 compared
to untreated control animals. It was obtained from non-linear fitting of log parasitemia on Day 7
post-infection versus administered oral dose of 19. ED90 was estimated to be 34 mg/kg.
Taken together, we have identified two features in the scaffold that were consequential to
potent antimalarial activity. They are (i) substituents (R1/R2) attached to the ring N atoms and
(ii) the fused quinone-imidazolium ring. For (i), alkyl side chains at R1/R2 are preferred to the
ring based residues found in YM155, 1 and 2. The lipophilicities imparted by the alkyl chains
are important drivers of activity but as noted earlier, in disparate ways that were influenced by
the phase of the plasmodial life cycle. The requirement (ii) for the fused quinone-imidazolium
ring is a puzzle. Quinones are found in several antimalarials and in most instances, they are
assumed to disrupt the redox balance in the parasitized erythrocytes through their propensity
to redox cycle.18 Indeed, the potent activity of the antimalarial lead compound plasmadione
was attributed to redox cycling by its quinone moiety.19 In contrast, the low redox potential of
the antimalarial drug atovaquone abrogated the role of the quinone in sustaining a subversive
substrate-driven redox cycle.20 Rather, atovaquone acts as competitive inhibitor of the
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plasmodial cytochrome b quinol oxidation (Qo) site, 21,22 with the quinone providing additional
sites of contact to the target through hydrogen bonding.23 This prompted us to evaluate the
potent Series A analogs (11,14,16,19) against P. falciparum TM90C2B which carries a
mutation in the cyt b gene, thus rendering atovaquone inactive against it (Table 4).
Interestingly, the compounds were no less active on TM90C2B than on CQ resistant (K1) and
sensitive (NF54) strains, thus precluding the mitochondrial cytochrome bc1 complex as a
putative target. On the other hand, the same compounds were identified as redox cyclers on
the in vitro phenol red horse radish peroxidase assay, 24 albeit less effectively than 1,4-
naphthoquinone and intriguingly, exceeding that of the non-quaternized Series B compounds
which had no detectable redox cycling activity (Table S-5). If these findings are reconfirmed on
other assay platforms, it would signal a novel modulating effect by the charged imidazolium on
the redox status of the quinone and hence a mechanistic rationale for the structural
importance of the quinone-imidazolium entity. Alternatively, noting that YM155 is preferentially
taken up by the solute carrier SLC35F2 into malignant cells,25 it is tempting to accord a
facilitatory role to the charged quinone-imidazolium entity with regard to uptake into parasitized
erythrocytes.
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Table 4: IC50 values of Series A analogs (11,14,16,19) against P. falciparum NF54, K1 and
atovaquone-resistant TM90C2B determined by [3H]-hypoxanthine incorporation.
IC50 (nM)
K1
IC50 (nM)
Compound
Compound
NF54
(n=2)
1.2
TM90C2B
(n=3)
2.2
NF54
(n=2)
8.6
K1
(n=2)
144
1.4
TM90C2B
(n=2)
2.2
(n=3)
140
11
14
16
19
Chloroquine
Artesunate
Atovaquone
1.0
1.8
1.4
3.2
2.7
1.0
2.1
1.8
0.4
0.5
11500
1.0
1.9
1.6
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In conclusion, we have identified the dioxonaphthoimidazolium scaffold as a promising
antimalarial pharmacophore, with several members displaying exceptional potencies against
sensitive and multidrug resistant asexual parasites (< 100 nM) as well as early and late stage
gametocytes (< 1000 nM). The attractiveness of the scaffold from a medicinal chemistry
perspective is further enhanced by its synthetic accessibility, the excellent comparative
cytotoxicities of its members, and based on a representative potent analog 19, good aqueous
solubility, metabolic stability and in vivo tolerability in mice. The PK profile of 19 revealed
limited tissue distribution, rapid plasma clearance and a relatively short half-life. However, 19
had limited oral bioavailability (5%) which arguably contributed to its modest activity in the
humanized mouse model of P. falciparum. Greater efficacy would conceivably be achieved if
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not for the poor permeability of 19 arising from its quaternized state. Notwithstanding these PK
issues, it is still possible to leverage on the inherent merits of the scaffold by engaging
formulation strategies or structural modifications to optimize lipophilicity. Lastly, we have
provided clarity on the fundamental structure activity requirements of this class of compounds.
Importantly, the quinone is recognized as a necessary but insufficient feature for potent
activity. Rather it is the fused quinone-imidazolium entity that is pivotal, plausibly by facilitating
cellular entry or fulfilling a mechanistic role in enhancing the reactivity of the quinone.
Additionally, lower lipophilicities appear to augment activity against asexual forms whereas
higher lipophilicities favoured activity against late stage gametocytes. Future work will focus on
addressing the PK limitations of the scaffold through structure modification, establishing a
better understanding of the mode of action and exploring interactions of potent members with
other antimalarial agents.
ASSOCIATED CONTENT
SUPPORTING INFORMATION
The Supporting Information is available free of charge on the ACS publications website at DOI:
xxxx. Description of methods used to synthesize Series A-C compounds; characterization of
intermediates and final compounds; purity determination of final compounds; synthesis of
target compound 19, NMR and HPLC Spectra of 19; activity of Series B on asexual stages of
NF54 P. falciparum; cLogP values of Series A and C; Spearman  correlation coefficients of
Series A and C; solubility, PAMPA permeability and microsomal stability of 19; redox cycling
EC50 values of Series A and B as determined by the phenol red horse radish peroxidase
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assay; cytotoxicities of Series A compounds on Chinese Hamster Ovary (CHO) cells; in vitro
assessment of antimalarial activity against asexual forms and gametocytes of P. falciparum ;
pharmacokinetic analysis of 19 in mice; in vivo efficacy of 19 in a humanized mouse model of
P. falciparum;
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AUTHOR INFORMATION
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Corresponding Authors
* Mei-Lin Go; Email phagoml@nus.edu.sg; Tel: +65-65162654.
Richard K. Haynes; Email richard.haynes@nwu.ac.za; Tel: +27(0)182994466.
AUTHOR CONTRIBUTIONS
The manuscript was written through contributions of all authors. ‡ These authors (SA, DC, JXT,
KTF) contributed equally.
NOTES
The authors declare no conflicting financial interests.
ACKNOWLEDGMENT
This work was funded by the Ministry of Education Academic Research Fund R148000234114,
R148000286114 to ML Go and the South African Medical Research Council (MRC) Flagship
Project MALTB-Redox with funds from the National Treasury under its Economic
Competitiveness and Support Package to R.K. Haynes. SA, TJX and KTF gratefully
acknowledged graduate scholarships from the Ministry of Education Singapore. The authors
thank Dr Radhakrishnan Sridhar, Alex CH Gan, Jocelyn HL Tan of NUS for advice and technical
assistance, Drug Development Unit of National University of Singapore for solubility and
PAMPA permeability determinations, Department of Pharmacology, University of Cape Town
for screening the antimalarial activities of YM155, 1-4 on P. falciparum (NF54, Dd2) and Sibylle
Sax for assistance in performing the in vitro antimalarial assays against atovaquone resistant P.
falciparum.
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(trifluoromethyl)benzyl]-menadione are not due to inhibition of the mitochondrial electron
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Table of Contents Graphic
Pf Asexual IC50 nM
NF54 1.4 K1 2.4
W2 2.2 TM90C2B 1.6
Pf Gametocytes IC50 nM
NF54-Pfs 16-GFP-Luc
Early stage 70
O
O
N+
N
Late stage 650
19
l
Pf efficacy in vivo humanized mouse mode 34 mg/kg
18
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