Discovery and Characterization of ACT-451840: an Antimalarial Drug with a Novel Mechanism of Action

Article abstract of DOI:10.1002/cmdc.201600298

More than 40 % of the world's population is at risk of being infected with malaria. Most malaria cases occur in the countries of sub-Saharan Africa, Central and South America, and Asia. Resistance to standard therapy, including artemisinin combinations, is increasing. There is an urgent need for novel antimalarials with new mechanisms of action. In a phenotypic screen, we identified a series of phenylalanine-based compounds that exhibit antimalarial activity via a new and yet unknown mechanism of action. Our optimization efforts culminated in the selection of ACT-451840 [(S,E)-N-(4-(4-acetylpiperazin-1-yl)benzyl)-3-(4-(tert-butyl)phenyl)-N-(1-(4-(4-cyanobenzyl)piperazin-1-yl)-1-oxo-3-phenylpropan-2-yl)acrylamide] for clinical development. Herein we describe our optimization efforts from the screening hit to the potential drug candidate with respect to antiparasitic activity, drug metabolism and pharmacokinetics (DMPK) properties, and in vivo pharmacological efficacy.

Full text of DOI:10.1002/cmdc.201600298

DOI: 10.1002/cmdc.201600298
Full Papers
Discovery and Characterization of ACT-451840: an
Antimalarial Drug with a Novel Mechanism of Action
Christoph Boss,*[a] Hamed Aissaoui,[a] Nathalie Amaral,[a] Aude Bauer,[a] Stephanie Bazire,[a]
Christoph Binkert,[a] Reto Brun,[d, e] Cꢀdric Bꢁrki,[a] Claire-Lise Ciana,[a, c] Olivier Corminboeuf,[a]
Stephane Delahaye,[a] Claire Dollinger,[a] Christoph Fischli,[d, e] Walter Fischli,[a]
Alexandre Flock,[a] Marie-Cꢀline Frantz,[a, b] Malory Girault,[a] Corinna Grisostomi,[a]
Astrid Friedli,[a] Bibia Heidmann,[a] Claire Hinder,[a] Gael Jacob,[a] Amelie Le Bihan,[a]
Sophie Malrieu,[a] Saskia Mamzed,[a] Aurelien Merot,[a] Solange Meyer,[a] Sabrina Peixoto,[a]
Nolwenn Petit,[a] Romain Siegrist,[a] Julien Trollux,[a] Thomas Weller,[a] and Sergio Wittlin[d, e]
More than 40% of the world’s population is at risk of being in-
fected with malaria. Most malaria cases occur in the countries
of sub-Saharan Africa, Central and South America, and Asia. Re-
sistance to standard therapy, including artemisinin combina-
tions, is increasing. There is an urgent need for novel antima-
larials with new mechanisms of action. In a phenotypic screen,
we identified a series of phenylalanine-based compounds that
exhibit antimalarial activity via a new and yet unknown mecha-
nism of action. Our optimization efforts culminated in the se-
lection of ACT-451840 [(S,E)-N-(4-(4-acetylpiperazin-1-yl)benzyl)-
3-(4-(tert-butyl)phenyl)-N-(1-(4-(4-cyanobenzyl)piperazin-1-yl)-1-
oxo-3-phenylpropan-2-yl)acrylamide] for clinical development.
Herein we describe our optimization efforts from the screening
hit to the potential drug candidate with respect to antiparasitic
activity, drug metabolism and pharmacokinetics (DMPK) prop-
erties, and in vivo pharmacological efficacy.
Introduction
Malaria remains a major health problem in large areas of the
world, despite significant efforts toward the identification of
novel treatments over the last decade.[1] Approximately half of
the world’s population live in malaria-endemic areas, and the
World Health Organization (WHO) estimates that 1.2 billion
people are at elevated risk of being infected with a malaria
parasite. In 2015 there were an estimated 214 million malaria
cases and 438000 deaths, which reflects a decrease from 2004,
when death cases peaked at 1.8 million.[2] The recently report-
ed emergence of resistance, or decreased efficacy, of the most
modern endoperoxide-containing drug combinations to treat
malaria[3] emphasize the importance of identifying new antima-
larial drugs with novel mechanisms of action.[4] Novel antima-
larial drugs require high safety and tolerability standards, be-
cause the vast majority of fatal cases are children under the
age of 5 (one child dies from malaria every 40 seconds) and
pregnant women.[5] This and the scarcity of novel drug targets
in the malaria parasites resulted in approaches to optimize the
activity and pharmacokinetic (PK) properties of existing drugs.
This, in turn, resulted in moderate advances to prevent the de-
velopment of resistance against these compounds.[6] The diffi-
culty in developing a safe vaccine[7] leaves pharmacological in-
tervention by drugs as the only current option to treat, cure,
or prevent malaria. Therefore, the need for new, inexpensive,
and safe antimalarial agents with novel mechanisms of action
remains very high.
[a] Dr. C. Boss, Dr. H. Aissaoui, N. Amaral, A. Bauer, S. Bazire, Dr. C. Binkert,
Dr. C. Bꢀrki, Dr. C.-L. Ciana, Dr. O. Corminboeuf, S. Delahaye, C. Dollinger,
Dr. W. Fischli, A. Flock, Dr. M.-C. Frantz, M. Girault, Dr. C. Grisostomi,
A. Friedli, Dr. B. Heidmann, C. Hinder, G. Jacob, A. Le Bihan, S. Malrieu,
Dr. S. Mamzed, A. Merot, Dr. S. Meyer, Dr. S. Peixoto, N. Petit, Dr. R. Siegrist,
J. Trollux, Dr. T. Weller
Actelion Pharmaceuticals Ltd., Drug Discovery & Preclinical Development,
Hegenheimermattweg 91, 4123 Allschwil (Switzerland)
E-mail: christoph.boss@actelion.com
[b] Dr. M.-C. Frantz
Present address: L’Orꢁal Research & Innovation, 1 Avenue Eugꢂne Schueller,
93600 Aulnay-sous-Bois (France)
In humans there are five Plasmodium species with distinct
and differentiated disease patterns.[8] The most significant,
deadly, and virulent is Plasmodium falciparum (malaria tropica).
The others are P. vivax, P. ovale (both resulting in malaria terti-
ana), P. malariae (malaria quartana), and, very recently discov-
ered, P. knowlesi. P. falciparium and P. vivax are responsible for
95% of worldwide malaria infections, and nearly all fatal cases
are associated with P. falciparum infections. Malaria parasites
are transmitted by female Anopheles mosquitos when biting
humans for a blood meal. The mosquito injects sporozoites
[c] Dr. C.-L. Ciana
Present address: Sygnature Discovery Ltd., BioCity, Pennyfoot Street, Not-
tingham NG1 1GF (UK)
[d] Prof. R. Brun, C. Fischli, Dr. S. Wittlin
Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel (Swit-
zerland)
[e] Prof. R. Brun, C. Fischli, Dr. S. Wittlin
University of Basel, Petersplatz 1, 4001 Basel (Switzerland)
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cmdc.201600298.
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from its salivary glands into the human bloodstream. The spor-
ozoites enter the liver and develop into schizonts, which con-
tain large numbers of merozoites. It takes about 10 days until
a schizont ruptures and releases the merozoites into the
bloodstream, where these invade red blood cells (RBCs; eryth-
rocytes). In the erythrocyte, the parasite develops from a ring
stage into a trophozoite stage and finally into a blood schiz-
ont. After 24 (P. knowlesi) or 48 (P. falciparum, P. vivax, P. ovale)
or 72 h (P. malariae), the erythrocyte ruptures and again releas-
es merozoites into the bloodstream, where they invade more
erythrocytes. After a few asexual lifecycles, some merozoites
develop into gametocytes, a sexual form. Upon another mos-
quito bite, gametocytes are transferred back into the vector,
where they undergo sexual reproduction to finally produce
large quantities of sporozoites in the salivary gland, ready to
infect the next subject upon the mosquito’s next blood meal.
When erythrocytes rupture within the infected patient, the par-
asites’ waste products and cell debris are released into the
bloodstream, causing some of the clinical symptoms (fever,
chill, headache, abdominal and back pain, nausea, diarrhea,
vomiting) of malaria.[4d] Additional information about the com-
plex life cycle of the malaria parasite can be found in the litera-
ture or the internet.[9]
heteroaromatic ring systems, and the substitution pattern
needs to be investigated (b). The n-pentylbenzyl unit (a) trig-
gers many ideas and should certainly be replaced by more
drug-like groups. Another important aspect is the amino acid
in the core of the compounds. The influence of chirality (B) on
activity as well as determining whether the phenylalanine can
be replaced by other amino acids (d) needs to be assessed.
The piperazine moiety (C) also inspires ideas for variations
(e.g., ring size), and the benzylamine group (c) invites investi-
gations of phenyl group replacements and phenyl substitution
patterns, as well as clarification as to whether the basic amine
is crucial or if acylation could be an option in this region of
the compounds. Screening hit 1 was considered a very inter-
esting and valuable starting point, as it offers diverse optimiza-
tion options to the medicinal chemist. In addition, chemical ac-
cessibility of 1 and potential derivatives is straightforward
(Scheme 1).[14] This is important in the antimalarial field, as cost
of goods is a crucial parameter.
Scheme 1 summarizes the chemistry used to prepare screen-
ing hit 1 and derivatives thereof. We started by coupling of
tert-butyloxycarbonyl (Boc)-protected phenylalanine 2 with
benzylpiperazine 3 under standard peptide-bond-forming con-
ditions followed by Boc deprotection by trifluoroacetic acid in
dichloromethane to obtain 4. If hydrochloric acid in dioxane
was used to deprotect the amine functionality, we often ob-
served decomposition of the piperazinyl amide. Compound 4
was then treated with aldehyde 5 in a reductive amination re-
action in dichloromethane or acetonitrile with sodium triace-
toxyborohydride as the reducing agent to give the precursor
6, which was readily transformed into the final compound 1 by
a simple acylation reaction with the cinnamic acid derivative 7.
Details about the synthetic procedures are given in the Sup-
porting Information and in the patent literature.[14,15] Following
this strategy, we prepared the compounds depicted in Table 1
by introducing various cinnamic acid moieties in the final step
of the sequence.
Results and Discussion
Our approach toward designing inhibitors of food-vacuolar
plasmepsins was complicated a few years ago,[10] when knock-
out experiments with P. falciparum demonstrated that these
presumed new drug targets were not essential for parasite sur-
vival.[11] We therefore performed an erythrocyte-based pheno-
typic screen[12] of ~5000 compounds at the Swiss Tropical and
Public Health Institute against the chloroquine-resistant K1
strain of Plasmodium falciparum. In this effort several hits were
identified, among them compound 1, a phenylalanine-based
derivative offering several possibilities for structure–activity re-
lationship (SAR) investigations (Figure 1).[13]
For SAR, all compounds were tested for inhibition of parasite
growth in RBCs infected with the P. falciparum parasite strain
K1 (chloroquine resistant) and/or with the chloroquine-sensi-
tive strain NF54. Both assays produced similar results, indicat-
ing that our compounds are active against both chloroquine-
sensitive and -resistant strains. Parasites were cultured in vitro
according to Trager and Jensen.[16] IC50 values were determined
by measuring incorporation of the nucleic acid precursor
[3H]hypoxanthine after 72 h of incubation.[17] IC50 values were
usually determined in two ways: in the presence of 0.5% Albu-
max (a serum substitute corresponding to a final assay concen-
tration of 10% bovine serum albumin), or 50% human serum.
The reason for this parallel approach was to identify com-
pounds that lose in vitro potency in the presence of 50%
human serum, as this could be an indication of high protein
binding.
Scrutinizing the screening hit 1 immediately reveals seven
aspects to be investigated. The cinnamic acid moiety raises the
question about the importance of the C=C double bond (A)
for activity and its potential reactivity as a Michael acceptor. It
also needs to be clarified if the phenyl ring can be replaced by
The small cluster of structurally closely related compounds
depicted in Table 1 was initially prepared in a completely dif-
ferent project, and they were all identified as hits in the phe-
notypic antimalarial screen, adding confidence to the results
and already giving initial insight into the SAR. Variations in the
Figure 1. Exemplary screening hit from the phenotypic RBC assay.
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Scheme 1. Preparation of compound 1 as an illustration of the general synthetic pathway, showing readily accessible and efficient chemistry. (Further details
are given in the Supporting Information).
moved or replaced with saturated ring systems or heteroaryl
Table 1. Activities of the initial hit series.
groups. Representative results are summarized in Table 2. In
the selected examples we also replaced the initially present n-
pentyl substituent with a pyridine moiety as a first step toward
improved molecular properties. Significant activity can be re-
tained by reducing the C=C double bond to a single bond
(12), but adding one more methylene unit to the linker result-
ed in the less active derivative 13. The indane system present
in 14 led to a further decrease in antimalarial activity, and re-
placement of the phenyl ring by cyclohexyl (15), cyclopentyl
(16), or complete removal of the ring system (17) also had
Compd
R
IC50 K1 alb 72 h [nm][a]
negative effects on potency, whereas the introduction of an
indole moiety, as shown in 18, resulted in very potent antima-
larial activity. Small substituents such as cyclopropyl (19) or cy-
clopropylacetyl (20) were not tolerated.
The two compounds 21 and 22 (Figure 2), which differ only
by the chirality of the phenylalanine core, indicate that the S
configuration present in the naturally occurring amino acid
(AA) and in compound 21, result in significantly higher antima-
larial activity than compounds with the unnatural R configura-
tion in the core amino acid such as 22, which is virtually inac-
tive.
1
3.8
8
9
70
375
7.3
19
10
11
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent
experiments; in vitro activity against erythrocytic stages of P. falciparum
was determined with a [3H]hypoxanthine incorporation assay (see the
Supporting Information for assay details).
substitution of the phenyl ring in the cinnamoyl unit showed
that a para substituent as present in 1 and 10 results in more
active compounds than the meta-substituted (8 and 11) or the
ortho-substituted (9) analogues. The next aspect to investigate
was the importance of the C=C double bond, the linker length
between the aryl moiety, and the N terminus of the core
amino acid as well as whether the aryl moiety could be re-
Figure 2. Crucial influence of the chirality of the amino acid core.
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ingful for subsequent planning of in vivo experiments based
on in vitro results.
Table 2. First results with the cinnamic acid double bond removed.
Historically, in vivo experiments in antimalarial drug discov-
ery are performed in mice, which are not infected by P. falcipa-
rum, but with the rodent malaria parasite P. berghei. We there-
fore used an in vitro assay to determine the activity of our anti-
malarials toward the rodent malaria parasite P. berghei. This
assay was performed in Albumax containing media and had an
incubation time of 24 h. By that we found that our antimalari-
als were less effective against the murine P. berghei parasites
than they were against the human parasite strains. This finally
forced us to work with a humanized P. falciparum SCID mouse
model to obtain relevant in vivo results with our best com-
pound (see below).
Compd
R
IC50 K1 alb 72 h [nm][a]
12
37
As a general aspect, all compounds listed in Table 3 revealed
significant antimalarial activity under the K1 Albumax assay
conditions, but lower activity was observed in the K1 serum
assay with shifts for the best compounds in the range of 5-
(29, 30) to 70-fold (27). A similar pattern was found when ana-
lyzing the assay data from the NF54 Albumax and NF54 serum
assays, which confirmed the validity of the thinking behind the
approach. Analysis of the data obtained with the rodent para-
site showed that the potency to kill P. berghei was lower for all
investigated compounds than their potency to kill the human
parasite. This finding was surprising to us, as most of the
newer antimalarials work with similar potency against the
human and rodent parasites.
13
14
80
159
15
207
16
17
458
281
The data summarized in Table 3 were analyzed by taking
into account the combined assumptions explained above, in
order to guide us toward compounds that show low shift fac-
tors between the Albumax versus human serum conditions.
This meant that we were looking at the absolute activity
values with second priority only. Therefore, compounds 29, 30,
and 32 were among the most interesting derivatives identified
so far, as their activity shifts were <10-fold for both parasite
strains. In addition to these compounds, derivatives 28, 38,
and 43 also presented attractive datasets with respect to anti-
parasitic activity combined with activity shifts between Albu-
max and human serum conditions. Unfortunately, all three
compounds showed potent inhibition of cytochrome P450
(CYP) 3A4 in an assay using testosterone as marker substrate
(28: 0.31 mm, 38: 0.2 mm, 43: 2.5 mm) and were therefore not
considered further. It seemed that the needle-like shape of the
biphenyl or related moieties, possessing a nitrogen atom in an
exposed position toward the “needle-tip”, confirmed reported
findings of being a perfect binder of the heme unit present in
cytochromes. Therefore, compounds with this structural subu-
nit were often found to suffer from cytochrome inhibition lia-
bilities. For further investigations the 4-N-acetylpiperazine-sub-
stituted benzyl group, as present in 29, resulting in an unpro-
blematic CYP3A4 inhibition of 8.4 mm with testosterone as
marker substrate, was used as substituent of choice whenever
possible. Another strategy to overcome strong cytochrome in-
hibition was applied in 26 and 27, by introducing a substituent
at the ortho position to the ring nitrogen atom, resulting in
values of 5.3 mm for 26 and >50 mm for 27.
18
2.2
19
20
>500
>500
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent
experiments; in vitro activity against erythrocytic stages of P. falciparum
was determined using a [3H]hypoxanthine incorporation assay (see the
Supporting Information for assay details).
Based on the results described above, we decided to investi-
gate n-pentylbenzyl replacements, keeping the compounds
otherwise constant as found in compound 1, with the excep-
tion of working with the S configuration and avoiding race-
mates; results are summarized in Table 3. During these investi-
gations we detected limitations of our screening assay, which
used Albumax as culture medium supplement. Binding of com-
pounds to serum components decreased the available free
compound concentration in the assay. Therefore, the absence
of serum components in our assay often led to overestimation
of the compounds’ potency. We therefore optimized the assay
environment by adding 50% human serum (final assay concen-
tration) to the media. Under these conditions, physicochemical
properties of the compounds became more relevant for anti-
malarial activity. In addition, the results became more mean-
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Table 3. Further replacements of the n-pentylbenzyl substituent.
Compd
R
IC50 [nm][a]
K1 alb 72 h
0.8
K1 ser 72 h
8.7
NF54 alb 72 h
NF54 ser 72 h
P. berghei 24 h
23
24
2.0
3.7
>20
27
1.1
0.9
6.3
18
32
>200
25
26
3.8
2.2
>20
ND
0.4
1.0
10
73
26
11
29
91
27
2.6
28
29
1.3
0.9
24
7.9
5.2
88
28
77
5.2
112
30
8.8
35
41
91
ND
31
32
33
4.8
10
34
66
11
36
14
>100
106
34
ND
ND
104
0.4
<7.8
34
35
36
<7.8
<7.8
<7.8
73
12
17
14
<7.8
3
369
27
ND
107
ND
>20
37
3.3
74
4.7
311
ND
38
39
40
1.5
3.4
14
70
29
<7.8
26
27
243
66
73
ND
<7.8
<7.8
115
41
11
>500
ND
ND
ND
42
43
<7.8
>500
ND
5.4
ND
41
ND
82
1.0
6.5
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent experiments; in vitro activity against erythrocytic stages of P. falciparum was deter-
mined using a [3H]hypoxanthine incorporation assay (see the Supporting Information for assay details). ND: not determined.
Another aspect requiring clarification was the question of
optimal substitution position on the benzyl ring. The results
listed in Table 4 clearly confirm that the best orientation of the
biaryl and related systems is a para arrangement (e.g., 50).
Both the meta (44–47) and ortho arrangements (48, 49) result
in decreased antimalarial potency and were not further pur-
sued.
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group of compounds bearing a para-alkoxy substituent at the
phenyl ring. Generally, the compounds exhibited good antima-
larial potency with low activity differences between the human
and rodent parasites, and a tendency to be slightly less active
against the rodent parasite than the alkyl-substituted deriva-
tives. Fluoroalkoxy substituents, as present in 64 and 65, did
not result in any advantage, as was also the case for the exam-
ples of substitutions given in 66–69.
Table 4. meta-Biaryls and ortho-biaryls.
Compd
44
R
IC50 [nm][a]
K1 alb 72 h
Table 6 summarizes the results with respect to replacement
of the phenylalanine core with unnatural amino acids, mainly
containing heterocycles or heteroaryl groups (5- and 6-mem-
bered) as phenyl replacements.[15a] Analysis of the data re-
vealed that combinations of a morpholinyl moiety as R1 and
a 4-pyridyl unit as R, or a piperidinyl moiety as R1 and a 4-pyr-
idyl unit as R resulted in compounds 73 and 75, which exhibit-
ed potent inhibitory activity. Unfortunately, these were coupled
with very potent CYP3A4 inhibition in an assay using midazo-
lam as marker substrate (73: 0.2 mm, 75: 0.3 mm). Replacement
of the 4-pyridyl by a 2-pyridyl group at the R position, a stan-
dard strategy to avoid strong cytochrome inhibition, resulted
in significant losses in activity, as shown for 72 and 74. Further
substituents, such as N-methylpiperazinyl, pyrazinyl, 2-pyrimi-
dinyl, pyrolyl, pyrazolyl, imidazolyl, or isoxazolyl, tested at the
R1 position, all resulted in less active derivatives. A general
trend was that the 4-pyridyl-containing compounds always re-
sulted in better antimalarial activity than the 2-pyridyl-contain-
ing analogues. Based on these results, it was decided to main-
tain a phenylalanine as the core of the antimalarials.
K1 ser 72 h
19
22
451
45
>500
46
47
<7.8
344
18
166
48
49
50
211
375
1.6
>500
>500
ND
In Table 7 (compounds 90–92), the results of a fluorine scan
around the phenyl ring of the phenylalanine core are summar-
ized and indicate no advantage of additional substituents in
this area of the compounds. From the important perspective
of cost of goods, this was considered a very positive point.
Table 8 summarizes the results obtained in investigating the
SAR around the benzyl substituent connected to the pipera-
zine moiety of our antimalarials.[15b] Most of this work was
done in the presence of the initially detected 4-trifluoromethyl-
cinnamoyl group. Analyzing the results of compounds 93–104
(CF3-cinnamoyl) showed that all compounds exhibited good
potencies in both Albumax as well as serum-based assay con-
ditions. In general, a moderate loss in activity was observed
when moving to the human serum conditions, with shifts
below a factor of 10-fold. A few compounds were also investi-
gated with regard to their antimalarial potency toward the
rodent parasite, P. berghei. Compound 97, with a hydroxyethyl-
methylamino substituent showed potent activities. Integrating
the substituent into a benzoxazine moiety, as shown in 98, re-
sulted in a significant loss of potency, which was also the case
upon exchanging it with an ethylene glycol unit, as present in
compound 95. Comparing 96 with 102, in which the cyano
substituent is moved from the meta to the para position,
showed that both regioisomers have similar activities toward
the K1 strain, but the para-substituted compound was found
to be significantly more potent toward the NF54 strain. Finally,
the methylsulfone- and ethylsulfone-substituted compounds
103, 104, 106, and 107 were among the most potent com-
pounds identified so far, especially against the NF54 parasite
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent
experiments; in vitro activity against erythrocytic stages of P. falciparum
was determined using a [3H]hypoxanthine incorporation assay (see the
Supporting Information for assay details). ND: not determined.
Table 5 shows a summary of the investigations performed in
the optimization of the cinnamoyl moiety. Compounds 51–53
contain a 2-substituted pyridine ring as phenyl replacement.
Although the activity shifts between the Albumax and human
serum conditions were low, the absolute antiparasitic activities
were rather disappointing. Compound 54 contains a 4-methyl-
sulfone-substituted phenyl unit, a substituent often used in
medicinal chemistry to improve PK behavior and absorption.
The activity shift for this compound was also low. Unfortunate-
ly, 54 showed only low potency against the rodent malaria par-
asite. The 4-alkyl-substituted-phenyl-containing derivatives
(55–58) are the most promising compounds with respect to
activity shifts in the presence and absence of serum, as well as
absolute activity levels and activity against the rodent parasite.
Derivative 55 can be considered the most promising com-
pound in this subgroup. Results obtained for 59 proved the
beneficial effects of a para substituent at the phenyl moiety of
the cinnamoyl group. Compounds 60–63 summarize the sub-
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Table 5. SAR on the cinnamoyl moiety in combination with the acetylpiperazine unit.
Compd
R
IC50 [nm][a]
K1 alb 72 h
5.8
K1 ser 72 h
16
NF54 alb 72 h
NF54 ser 72 h
46
P. berghei 24 h
51
52
53
54
55
56
57
58
59
47
ND
2.6
6.0
9.0
>7.8
6.1
13
22
52
ND
ND
363
47
45
1.4
9.4
1.9
2.9
2.0
8.0
60
32
0.8
5.3
8.5
1.4
6.8
8.3
115
81
0.7
3.7
9.9
1.2
7.4
52
ND
ND
<7.8
100
>500
60
1.0
5.8
5.4
21
153
61
62
0.7
0.6
3.2
3.7
1.9
1.8
10
11
100
75
63
0.9
5.2
2.4
5.6
88
64
65
0.7
0.7
3.7
5.4
3.3
3.7
21
23
117
90
66
67
68
5.1
6.2
20
13
11
70
66
27
>100
>100
46
ND
ND
ND
<7.8
69
2.2
18
24
>100
ND
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent experiments; in vitro activity against erythrocytic stages of P. falciparum was deter-
mined using a [3H]hypoxanthine incorporation assay (see the Supporting Information for assay details). ND: not determined.
and the rodent P. berghei strain. Activity shifts between the Al-
bumax and serum conditions were small, as with the other de-
rivatives. We therefore cannot really expect a better general
in vivo behavior as we correlate activity shifts with potential PK
properties. Based on the intrinsic activity the compounds
might be interesting tools.
In Table 9 we summarize our investigations with respect to
variations and potential replacements of the benzylpiperazinyl
moiety.[15a,c] Bicyclic systems containing a piperazine ring were
investigated as isosteres, as depicted in 108 or 109. These two
compounds showed less promising antimalarial activity than
the compounds from Table 8. Further bicyclic piperazine-con-
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Table 6. Phenylalanine replacements.
Compd
R
R1
IC50 [nm][a]
K1 alb 72 h
156
K1 ser 72 h
NF54 alb 72 h
70
71
72
73
74
75
76
>500
103
105
18
ND
ND
ND
36
23
<7.8
2.9
<7.8
1.2
76
ND
17
10
236
86
>500
>500
>500
483
ND
ND
ND
ND
77
78
79
156
41
80
81
82
83
84
85
86
24
11
377
90
ND
ND
ND
ND
ND
ND
ND
36
298
16
105
195
167
67
>500
194
>500
87
88
32
270
ND
ND
256
>500
89
88
>500
ND
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent experiments; in vitro activity against erythrocytic stages of P. falciparum was deter-
mined using a [3H]hypoxanthine incorporation assay (see the Supporting Information for assay details). ND: not determined.
taining isosteres are depicted in 116–118, and again resulted
in a significant loss in antimalarial activity. Removing the basic
character of the second piperazine nitrogen atom by changing
to a piperazinone (110), by acylating the piperazine (111), or
by directly attaching the electron-poor pyrimidinyl moiety
(112) also resulted in low activities, especially against the NF54
strain under the more relevant human serum conditions. Re-
placing the benzyl substituent with hydroxyethyl (113), me-
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Table 7. SAR on fluorophenylalanine derivatives.
Compd
R
IC50 [nm][a]
K1 alb 72 h
K1 ser 72 h
84
NF54 alb 72 h
NF54 ser 72 h
90
91
92
<7.8
8.6
>100
<7.8
<7.8
168
69
16
>100
4.9
135
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent experiments; in vitro activity against erythrocytic stages of P. falciparum was deter-
mined using a [3H]hypoxanthine incorporation assay (see the Supporting Information for assay details).
thoxyethyl (114), or phenethyl (115) at the piperazine nitrogen
atom also resulted in a clear loss of activity. Further isosteric re-
placements such as tetrahydroisoquinolinyl (119, 120), mor-
pholinyl (121), or an open-chain analogue of the morpholine
group in 122, were all unsuccessful as well. From these investi-
gations no benzylpiperazine unit replacement could be identi-
fied. Therefore, this building block was kept for further explo-
rations, allowing for additional small substituents at the phenyl
ring only.
4-methylsulfone substituent (in 156) resulted in the least
active derivative.
Table 12 depicts the results obtained when trying to replace
the cinnamoyl moiety by bicyclic bioisosteres (144–148). These
attempts resulted in significantly less potent derivatives and
were stopped at this level.
A few further isosteric replacements investigated for the cin-
namoyl moiety are given in Table 13. Compounds 149 and
150, containing a 1,4-dimethyl-substituted thiazole moiety to
replace the substituted phenyl ring of the cinnamoyl unit,
showed lower potencies in the NF54 assays than previously
discussed analogues. Compound 151, bearing a methylsulfone
substituent at the para position of the phenyl ring, was the
most interesting example out of a series of antimalarials con-
taining a phenoxyacetic acid group as an isosteric replace-
ment. All other examples from this series, lacking the methyl-
sulfone substituent, exhibited significant activity losses be-
tween the artificial Albumax conditions and the more physio-
logical human serum conditions. Derivative 151 was tested for
its in vitro metabolic stability and CYP3A4 inhibition and un-
fortunately could not be further pursued based on the ob-
tained results (HLM=974 mL(minmg)ꢁ1, CYP3A4T=1.4 mm).
To further cross-check our SAR we prepared the compounds
summarized in Table 14. The purpose of this was to confirm
that the acetylpiperazine is the best option for the R1 position.
The investigation focused on thiomorpholines (152, 153), vari-
ously substituted piperazines (154–157, 168), and nitrogen-
containing heteroaryls such as pyridines (158–166) and pyrimi-
dines (167) combined with either the trifluoromethyl-substitut-
ed cinnamoyl or the isopropoxy-substituted cinnamoyl unit,
and in one case the tert-butyl-substituted cinnamoyl moiety.
None of the compounds listed in Table 14 exhibited an im-
proved activity profile over our most promising compounds
(e.g., 132). Comparison of 156, 157, and 168 showed that the
cinnamoyl moiety has only a moderate effect on activity in
combination with the isopropyl-substituted piperazinyl group
We decided to repeat the optimization on the cinnamoyl
portion of compounds by implementing the results obtained
thus far into the selection of the remaining parts of the mole-
cules. This work is summarized in Table 10. The 4-cyanobenzyl-
piperazine was combined with phenylalanine and the 4-(4-ace-
tylpiperazinyl)benzyl substituent to give the template to be
combined with a set of diversely para-substituted cinnamic
acids. The small series of final compounds were assessed for
their activity against the NF54 parasite strain under both the
Albumax and human serum conditions and against the P. ber-
ghei rodent parasite strain. All compounds exhibited excellent
antiparasitic potency below 4 nm in the most artificial Albumax
assay, except 133, containing the methylsulfone substituent.
This substituent provided highly potent compounds when at-
tached to the benzylpiperazinyl unit (103, 104, 106, and 107),
but was not tolerated here. Most of the compounds also
showed excellent potency in the NF54 human serum contain-
ing assay, whereas the situation with respect to the activity in
the rodent P. berghei assay was more complex. With respect to
absolute potencies and activity shifts between assays, 132 was
the most promising derivative from this series.
Table 11 summarizes further optimization efforts of the cin-
namic acid substituent on a slightly different scaffold. This
series of compounds confirmed the findings from Table 10
wherein the 4-tert-butylphenyl moiety of 162 resulted in the
best overall profile. Compound 162 showed an even more
promising overall profile than compound 127. And again, the
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Table 8. SAR on benzylpiperazine substitutions.
Compd
R1
R
IC50 [nm][a]
K1 alb 72 h
1.8
K1 ser 72 h
6.2
NF54 alb 72 h
NF54 ser 72 h
21
P. berghei 24 h
93
94
4.0
2.6
ND
1.4
3.2
7.5
29
14
34
ND
34
95
11
96
97
4.2
0.5
15
18
47
ND
18
4.7
0.9
5.3
98
99
3.9
1.9
36
15
17
ND
35
ND
ND
5.6
100
101
102
1.8
2.1
1.6
16
15
16
4.3
3.7
1.8
>20
48
ND
ND
54
14
103
ND
ND
0.5
3.5
13
104
105
106
ND
ND
ND
ND
ND
ND
0.8
1.9
0.2
3.9
11
7.0
64
1.8
0.9
107
ND
ND
0.5
1.8
3.7
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent experiments; in vitro activity against erythrocytic stages of P. falciparum was deter-
mined using a [3H]hypoxanthine incorporation assay (see the Supporting Information for assay details). ND: not determined.
as R1, confirming that the correct combination of both sub-
stituents is key for highly potent compounds.
ed in Tables 15 and 16 are given in the Supporting Informa-
tion.
As the in vivo rat hepatic clearance was high for the majority
of the compounds investigated, the in vitro/in vivo extrapola-
tion (IVIVE) of hepatic clearance in rat using the well-stirred
liver model[18] was evaluated in order to establish how the in
vitro intrinsic clearance (CLint) data could predict in vivo clear-
ance (CLH), to allow selection of the most promising com-
pounds to be tested in pharmacology in the murine P. berghei
malaria model. The following Equation (1) was used:
DMPK and pharmacology of a selected set of compounds
In the course of optimizing the in vitro antimalarial potency,
the most interesting compounds were investigated with re-
spect to their rat in vitro and in vivo as well as human in vitro
drug metabolism and pharmacokinetics (DMPK) properties. A
selection of results is summarized in Table 15. Descriptions of
the experimental procedures used to generate the data depict-
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Table 9. Benzylpiperazine replacements/alternatives.
Compd
R
IC50 [nm][a]
NF54 alb 72 h
K1 alb 72 h
K1 ser 72 h
29
NF54 ser 72 h
113
108
109
<7.8
28
30
294
49
ND
ND
110
<7.8
36
236
111
112
ND
ND
59
28
>100
<7.8
176
118
113
114
45
102
41
ND
43
ND
<7.8
220
115
116
39
233
ND
ND
ND
ND
213
>500
117
97
>500
ND
ND
118
119
ND
ND
ND
ND
182
>500
8.3
47
120
ND
ND
38
98
121
122
ND
ND
18
42
20
>100
<7.8
135
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent experiments; in vitro activity against erythrocytic stages of P. falciparum was deter-
mined using a [3H]hypoxanthine incorporation assay (see the Supporting Information for assay details). ND: not determined.
ꢀ
ꢀ
ꢁ
ꢁ
invitroCLintꢂSF
in which QH =liver blood flow; SF=scaling factor (quantity of
microsomes/hepatocytes in the body); fub =free fraction in
blood; fup =free fraction in plasma; fuinc =fraction unbound in
QH ꢂ
QH þ
ꢂ fub
ꢂ fub
fuinc
CLH ¼
ð1Þ
invitroCLintꢂSF
fuinc
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Table 10. Cinnamic acid optimization.
Compd
R
IC50 [nm][a]
NF54 alb 72 h
NF54 ser 72 h
P. berghei 24 h
123
124
3.8
1.7
14
54
36
1.9
125
1.9
19
126
126
127
128
1.1
0.8
0.8
3.3
3.2
5.4
29
33
32
129
1.7
4.4
72
130
131
132
0.5
3.3
0.4
2.8
8.9
6.7
27
78
14
133
18
33
ND
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent experiments; in vitro activity against erythrocytic stages of P. falciparum was deter-
mined using a [3H]hypoxanthine incorporation assay (see the Supporting Information for assay details). ND: not determined.
incubation. The blood-to-plasma ratio was not measured, but
predicted by the GastroplusTM software package.
the analytical LC–MS/MS systems. The volumes of distribution
(Vss) in rats were all above the volume of total body water, indi-
cating a vast tissue distribution, consistent with the high lipo-
philicity of the compounds. These facts limit the concentration
of compound in the systemic compartment. Plasma protein
binding values for the compounds were high and also in ac-
cordance with the elevated cLogP values. The exposures of the
compounds measured after i.v. administration were all in the
same range. This indicated that the structural modifications of
the discussed set of compounds did not have a pronounced
influence on the clearance and the distribution of the drug.
The bioavailability for the selected derivatives ranged from
1 to 44%, even though exposures after i.v. administration were
very similar. The oral bioavailability is usually limited by high
clearance. The high bioavailability observed for some com-
pounds (e.g., 130 and 132) was not completely understood. A
potential explanation could be that bioavailability (F) was cal-
culated with lower AUCi.v. values than the AUCp.o. values, which
potentially neglects saturation of some hepatic clearance pro-
cesses. We have the impression that working with compounds
The data used for the prediction and the calculated CLint
values are summarized in Table 16. More accurate predictions
were obtained for the highly protein-bound compounds rela-
tive to those showing a higher free fraction, which is rather un-
common, as a low fu (fraction unbound) value should propa-
gate in calculation errors. The factor of the underprediction of
the rat plasma clearance ranged from <1.4 to 7.8, showing
that the investigation of new compounds within this structural
class on CLint in rat liver microsomes (RLM) would not help to
identify differences in the in vivo clearance data. In the human
liver microsomal (HLM) assay, the CLint value determined for
the investigated compounds was in the same range as for the
values obtained in the RLM assay. IVIVE would therefore pre-
dict higher in vivo clearance in humans than in rats. The half
lives (t1/2) in rat after intravenous (i.v.) administration were all in
the same range of 0.76–2.5 h, in accordance with high clear-
ance of the compounds. The terminal half lives might not have
been captured properly due to the limit of quantification of
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Table 11. Optimization of the cinnamic acid in combination with the 4-(N-methyl-N-2-hydroxyethyl)benzylpiperazine moiety.
Compd
R
IC50 [nm][a]
NF54 alb 72 h
10
NF54 ser 72 h
P. berghei 24 h
134
33
58
135
136
4
12
69
24
1.5
5.7
137
138
139
5.8
1.9
1.3
11
102
14
6.6
4.0
11
140
141
1.6
3
3.3
8.2
14
17
142
143
1.2
0.8
6.2
4.5
12
5.6
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent experiments; in vitro activity against erythrocytic stages of P. falciparum was deter-
mined using a [3H]hypoxanthine incorporation assay (see the Supporting Information for assay details).
at the edge of the rule of five, for example, exhibiting values
for three or more parameters above the defined upper limits,
increases the difficulties in predicting in vivo PK behavior
based on in vitro assessments of the compounds.[19]
regimen) to prevent death typically occurring on day 6. Ani-
mals were considered cured if there were no detectable para-
sites on day 30 post-infection. Compound 23 was inactive at
the lower dose of 30 mgkgꢁ1, independent of the route of ad-
ministration, whereas 97 showed signs of antimalarial activity
at 30 mgkgꢁ1 when administered subcutaneously (s.c.) (77%)
as well as per os (p.o.) (66%). At a dose of 100 mgkgꢁ1, 23
showed activity after p.o. administration (82%), but, to our sur-
prise, was inactive after s.c. administration (21%). This behavior
was confirmed in a second independent experiment. We
assume that this is due to the solid-state properties of com-
pound 23 which are different from those of the other com-
pounds of the series. Compound 97 showed remarkable activi-
ty at 100 mgkgꢁ1 of 98% (p.o.) and 99.2% (s.c.), which resulted
in a minutely prolonged survival of the animals up to 7.7 days
in the p.o. experiment. At 100 mgkgꢁ1, compound 100
showed lower activity (75% p.o.; 85% s.c.), but still prolongs
survival of the mice to nearly the same extent as 97.
Based on the pharmacology results obtained with several
examples, especially 63, 97, and 132, we have shown that
large molecules may still have the potential to become drugs,
but rational approaches are rather difficult under these circum-
stances.
With a number of compounds we assessed antimalarial ef-
fects in vivo using a mouse malaria model infected with P. ber-
ghei. Using this test system, both antimalarial activity and sur-
vival time can be analyzed (Table 17). Compounds were admin-
istered 24 h post-infection (single-dose regimen) or 24, 48, and
72 h post-infection (triple-dose regimen). With the single-dose
regimen, blood was collected on day 3 (72 h post-infection).
Samples for the triple-dose regimens were collected on day 4
(96 h post-infection). Antimalarial activity was calculated as the
difference between the mean percent parasitemia for the con-
trol (n=5 mice) and treated groups (n=3 mice) expressed as
a percent relative to the control group. Control mice were eu-
thanized on day 3 (single-dose regimen) or day 4 (triple-dose
The antimalarial activities of 26, 29, and 43 were compared
in two different p.o. dosing regimens of either one dose of
100 mgkgꢁ1, or three dosings of 100 mgkgꢁ1 on three consec-
utive days. Compounds 26 and 43 were inactive in both
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were tested under standardized conditions (formulated in
Tween-EtOH/water) except example 63, which was formulated
in corn oil due to physicochemical limitations.
Table 12. Cinnamic acid isosteres.
Compounds 63 (activity 98%), 126 (activity 99.3%), 127 (ac-
tivity 99.2%), 130 (activity 99.0%), 132 (activity 99.2%), and
143 (activity 99.4%) all showed significant effects on mouse
survival of at least nine days. For compound 126 and 127, 10.3
and 13.3 days were reached, respectively. The combination of
the mouse in vivo results with the in vitro activity data against
P. berghei and P. falciparum and the results obtained in the PK
experiments (Table 15) led us to decide to further profile and
investigate 63, 97, and 132 in vivo under various dosing regi-
mens summarized below. The standard formulation was
changed to corn oil, based on the results obtained for 63.
In a single-administration dose-escalating experiment with
63, the survival time improved in a dose-dependent manner,
reaching 13.0 days after a single administration of 300 mgkgꢁ1
(Table 18). In the experimental setting with repeated adminis-
tration on three consecutive days, average survival times of 22
(3ꢃ100 mgkgꢁ1) and 25 days (3ꢃ300 mgkgꢁ1) were obtained
with some of the animals reaching cure. For the first time we
had confirmation that it was possible to cure P. berghei infect-
ed mice with compounds of the class described in this report.
Compound 97 was our frontrunner for a certain period of
the project and was therefore broadly investigated. The phar-
macological characterization of 97 is summarized in Table 19.
Antimalarial effects, with respect to survival, were similar at 1ꢃ
100 mgkgꢁ1 and 3ꢃ33 mgkgꢁ1 at t=0, 8, and 16 h. Adminis-
tration of 3ꢃ100 mgkgꢁ1 at the same schedule enhanced sur-
vival time to 11.3 days. A further prolongation of survival to 14,
19, and 27 days respectively, was observed when 97 was ad-
ministered at 100, 350, and 500 mgkgꢁ1 at three consecutive
days. The same dosing regimen with 97 at 700 mgkgꢁ1 result-
ed in a survival of 30 days, with no detectable parasites, which,
Compd
R
IC50 NF54 alb 72 h [nm][a]
144
92
145
178
146
147
148
50
69
253
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent
experiments; in vitro activity against erythrocytic stages of P. falciparum
was determined using a [3H]hypoxanthine incorporation assay (see the
Supporting Information for assay details).
dosing regimens tested. Compound 29 exhibited weak activity
(66%) when dosed once at 100 mgkgꢁ1 and 85% when dosed
three times 100 mgkgꢁ1 on three consecutive days, resulting
in a minute prolongation of survival time of the P. berghei in-
fected mice. Compounds 149 and 150 were found to be inac-
tive in vivo at single and triple dosings of 100 mgkgꢁ1, consis-
tent with the finding that they were poorly active in vitro
against P. berghei. The other examples summarized in Table 17
Table 13. Further cinnamic acid isosteres.
Compd
149
R
R1
IC50 [nm][a]
NF54 alb 72 h
K1 alb 72 h
23
K1 ser 72 h
43
NF54 ser 72 h
418
P. berghei 24 h
>500
253
150
8
44
95
>100
ND
151
<7.8
9.8
5.6
6.7
1281
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent experiments; in vitro activity against erythrocytic stages of P. falciparum was deter-
mined using a [3H]hypoxanthine incorporation assay (see the Supporting Information for assay details). ND: not determined.
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Table 14. Importance of the acetylpiperazine moiety.
Compd
R
R1
IC50 [nm][a]
NF54 alb 72 h
12
NF54 ser 72 h
P. berghei 24 h
152
153
18
268
10
20
ND
51
154
155
156
2.9
3.9
3.2
56
4.4
81
47
ND
157
158
159
86
17
8.8
149
49
ND
ND
ND
69
160
161
9.4
9.0
72
76
ND
ND
162
163
<7.8
114
27
ND
ND
3.8
164
2.3
>20
ND
165
166
167
5.0
4.7
3.3
70
73
80
ND
ND
ND
168
39
54
ND
[a] Data are the geometric mean (ꢀ2-fold) of at least three independent experiments; in vitro activity against erythrocytic stages of P. falciparum was deter-
mined using a [3H]hypoxanthine incorporation assay (see the Supporting Information for assay details). ND: not determined.
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Table 15. Selected DMPK parameters and properties of pharmacologically investigated compounds.[a]
Compd
CL
Vss
t1/2
AUCp.o.
AUCi.v.
Cmax
tmax
F
HLM
RLM
hPPB
rPPB
124
127
130
132
136
137
139
143
51
79
53
57
87
75
79
57
3.7
7.7
6.1
7.6
4.6
3.5
5.3
7.5
1.8
1.6
2.5
2.3
0.76
0.87
1.9
2
411
580
996
1270
228
23.6
274
327
212
317
291
192
223
211
291
151
223
326
288
93
10.6
85.2
157
0.5
1
1
0.5
0.63
0.5
2
13
27
31
44
12
1
275
306
276
267
392
314
304
211
336
289
181
111
276
118
204
129
99.8
99.6
99.9
99.9
99.7
91.1
99.8
99.9
99.8
99.7
99.9
99.9
99.8
96.4
99.9
99.9
13
23
681
2
[a] In vivo data generated in rats; dosing was as follows: 10 mgkgꢁ1 p.o. and 1 mgkgꢁ1 i.v.
Table 16. Data used for prediction and calculated intrinsic clearance (CLint).
Compd
cLogP[a]
B/P
ratio
fu [%]
rat plasma
CLint
RLM
Rat pl. CL
Fold
underprediction
fu [%]
hum. plasma
CLint
HLM
Hum. pl. CL
pred.
RLM
pred.
obsd.
124
127
130
132
136
137
139
143
5.21
5.66
5.86
6.34
4.92
3.84
5.35
6.09
0.65
0.65
0.65
0.65
0.70
0.72
0.70
0.70
0.2
0.3
<0.1
<0.1
0.2
3.6
0.1
<0.1
5.6
2.3
0.3
<0.1
3.7
34
336
289
181
111
276
116
204
129
14
27
<32
NC[b]
17
15
13
51
79
53
57
87
75
79
57
3.6
2.9
<1.7
NC[b]
5.1
5.0
6.6
<1.4
0.2
0.4
<0.1
<0.1
0.3
8.9
0.2
<0.1
275
306
276
267
392
314
304
211
14
27
<32
NC[b]
17
15
13
<41
2.1
0.1
<41
[a] Calculated by GastroplusTM ver. 0.0 (Simulations Plus Inc.). [b] Plasma clearance could not be calculated, as the fu values in microsomes and in plasma
were measured below the 0.1% limit of quantification.
Table 17. Summary of in vivo experiments and results.[a]
Compd
Dose [mgkgꢁ1
]
Administration
Parasitized RBC/100
% of Ctrl
Activity [%]
Survival days
Repeats
Route
100
100
30
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
3ꢃ
1ꢃ
3ꢃ
1ꢃ
3ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
s.c.
p.o.
s.c.
32.34
7.49
79.06
18.32
98.10
73.57
63.79
47.61
33.73
14.56
80.73
64.70
1.55
<40
82
3.0
6.0
3.0
3.0
3.0
3.0
7.0
7.3
3.0
4.0
9.0
7.7
6.0
6.0
6.0
6.7
6.7
3.0
3.0
6.7
7.7
3.0
10.3
13.3
6.7
7.0
9.0
6.7
9.7
23
40.13
30.09
22.88
33.20
12.10
10.16
28.95
45.12
0.27
0.34
0.93
9.93
13.95
5.98
10.13
35.08
31.92
7.67
0.33
38.81
0.25
0.29
0.86
4.10
0.54
<40
<40
<40
<40
66
30
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.[b]
s.c.
p.o.
s.c.
p.o.
s.c.
100
100
100
100
100
100
100
100
100
30
26
29
85
<40
<40
98
99.2
98
77
66
85
75
<40
<40
79
99.1
<40
99.3
99.2
98
89
99
97
99.2
43
63
0.84
2.28
97
22.96
34.10
14.63
24.76
85.75
78.03
20.77
0.89
105.14
0.73
0.78
2.33
11.12
1.46
30
100
100
30
p.o.
s.c.
100
30
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
123
124
125
126
127
128
129
130
131
132
100
100
100
100
100
100
100
100
100
100
0.94
0.29
2.56
0.79
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Table 17. (Continued)
Compd
Dose [mgkgꢁ1
]
Administration
Parasitized RBC/100
% of Ctrl
Activity [%]
Survival days
Repeats
Route
134
135
136
137
138
139
140
141
142
143
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
3ꢃ
1ꢃ
3ꢃ
1ꢃ
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
32.67
16.56
2.20
32.28
0.61
0.63
1.23
7.53
0.42
94.15
47.72
6.33
93.02
1.77
1.81
3.55
21.70
1.22
0.61
83.81
63.82
92.01
86.20
92.08
<40
52
94
<40
98
98
6.0
6.0
6.0
5.0
6.0
7.0
7.7
6.0
6.0
9.7
3.0
4.0
3.0
4.0
3.0
96
78
99.0
99.4
<40
<40
<40
<40
<40
0.21
26.62
33.77
27.58
45.61
33.99
149
150
151
[a] Mice were euthanized at the end of the experiment, i.e., after the number of survival days indicated. Standard experiments were performed with three
mice per treatment or control group. Compounds were formulated in [Tween-EtOH]/water (10:90). [b] Formulation in corn oil.
Table 18. In vivo data for compound 63.[a]
Dose [mgkgꢁ1
]
Administration repeats
Parasitized RBC/100
% of Ctrl
Activity [%]
Survival days
300
100
30
10
3
300
100
30
10
3
1ꢃ
1ꢃ
1ꢃ
1ꢃ
1ꢃ
3ꢃ
3ꢃ
3ꢃ
3ꢃ
3ꢃ
0.19
0.27
11.66
18.49
24.42
0.16
1.12
1.55
67.69
107.32
141.76
0.37
99.0
98
13
9.0
3.0
3.0
<40
<40
<40
99.6
99.7
65
3.0
25.0[b]
22.0[c]
7.0
0.14
0.32
14.59
49.70
45.44
34.54
117.62
107.54
<40
<40
4.0
4.0
[a] Three mice per experiment; compound was formulated in corn oil for the experiments and administered p.o. Repeated administration was on consecu-
tive days (mice cured: survival was 30 days, and at this time point mice were parasite free). [b] 2 out of 3 mice cured under the experimental conditions.
[c] 1 out of 3 mice cured under the experimental conditions.
in the P. berghei model, is considered a cure. Single-dose ad-
ministrations, even at high doses such as 500 or 750 mgkgꢁ1
60 mgkgꢁ1 revealed that 132 started to show significant anti-
malarial effects (parasite reduction) in vivo at 20 mgkgꢁ1
,
.
did not result in cure. These findings were in line with the PK
behavior found with compound 97, with a rather short half-
life, which results in fast excretion and allows the parasites to
recur.
When administered repetitively on three consecutive days,
compound 132 exhibited curative activity at 300 mgkgꢁ1 and
significant antimalarial effects already at 100 mgkgꢁ1. With re-
spect to parasitemia, 132 showed excellent reduction at
30 mgkgꢁ1, resulting in 99.80% activity.
Our most promising compound at the end of our drug dis-
covery efforts was 132 (ACT-451840). The results of the phar-
macological experiments obtained with 132 in the P. berghei
infected mouse model are summarized in Table 20. The dose-
escalating experiments with single doses between 10 and
The in vivo activity of 132 was also assessed in the P. falcipa-
rum humanized immunodeficient mouse model established
and performed at GSK (DDW, Tres Cantos, Spain).[20] After esca-
lating oral dosing on four consecutive days, 132 exhibited
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Table 19. In vivo data for compound 97.[a]
Dose [mgkgꢁ1
]
Administration repeats
Parasitized RBC/100
% of Ctrl
Activity [%]
Survival days
100
33
100
100
350
350
500
500
750
1ꢃ
0.98
0.33
0.12
0.07
0.17
0.0
0.21
0.50
0.30
0.15
26.30
3.47
1.16
0.42
0.14
0.56
0.0
0.60
0.70
0.86
0.20
108.31
97
99.0
99.6
99.9
99.4
99.9
99.4
99.3
99.1
99.8
<40
7.0
7.3
11.3
14.0
9.0
19.3
11.6
27.2
14.4
30.0[d]
3.0
3ꢃ[b]
3ꢃ[b]
3ꢃ[c]
1ꢃ
3ꢃ[c]
1ꢃ
3ꢃ[c]
1ꢃ
750
100
3ꢃ[c]
1ꢃ
(ꢁ24 h)[e]
100
1ꢃ
27.99
116.44
<40
3.0
(ꢁ48 h)[f]
[a] Three mice per experiment; compound was formulated in corn oil for the experiments and administered p.o. [b] Repeated administration was at t=
0 h, t=8 h, t=16 h. [c] Repeated administration was on consecutive days. [d] Parasite-free mice on day 30 post-infection were considered cured. [e] Ad-
ministration of 97 24 h prior to infecting the mice. [f] Administration of 97 48 h prior to infecting the mice. [e,f] These two experiments indicate that 97 is
not useful as preventive treatment under the experimental conditions.
Table 20. In vivo data for compound 132 (ACT-451840).[a]
Dose [mgkgꢁ1
]
Administration repeats
Parasitized RBC/100
% of Ctrl
Activity [%]
Survival days
100
60
30
25
20
15
10
300
100
30
10
3
1ꢃ[b]
1ꢃ[c]
0.29
0.10
0.83
2.60
6.37
16.33
23.80
0.17
0.15
0.10
0.79
0.37
3.11
99.2
99.6
97
90
76
<40
<40
99.6
99.6
99.8
<40
<40
9.7
12.7
8.7
6.7
7.0
3.0
3.0
30.0[e]
26.7
13.0
4.0
1ꢃ[c]
1ꢃ[c]
9.71
1ꢃ[c]
23.77
60.99
88.87
0.39
0.35
0.23
1ꢃ[c]
1ꢃ[c]
3ꢃ[c,d]
3ꢃ[c,d]
3ꢃ[c,d]
3ꢃ[c,d]
3ꢃ[c,d]
28.63
50.98
67.75
120.65
4.0
[a] Three mice per experiment. [b] Compound was formulated in [Tween-EtOH]/water (10:90) and administered p.o. [c] Compound was formulated in corn
oil for the experiments and administered p.o. [d] Repeated administration was on consecutive days. [e] 30 days survival is considered as cure.
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Acknowledgements
The authors thank Dr. Stephan Buchmann and his team for the
preparation of formulations for in vivo experiments, Dr. Martine
Clozel for enthusiastic support of the project, and Joerg Hehn,
Pauline Bertin, Julien Hazemann, Jerome Gabillet, Pascal Re-
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Keywords: antimalarial drugs · malaria · phenotypic drug
discovery · phenylalanine derivatives · SCID mouse model
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Received: June 13, 2016
Revised: July 4, 2016
Published online on && &&, 0000
&
ChemMedChem 2016, 11, 1 – 21
www.chemmedchem.org
20
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

FULL PAPERS
C. Boss,* H. Aissaoui, N. Amaral, A. Bauer,
S. Bazire, C. Binkert, R. Brun, C. Bꢀrki,
C.-L. Ciana, O. Corminboeuf, S. Delahaye,
C. Dollinger, C. Fischli, W. Fischli, A. Flock,
M.-C. Frantz, M. Girault, C. Grisostomi,
A. Friedli, B. Heidmann, C. Hinder,
G. Jacob, A. Le Bihan, S. Malrieu,
S. Mamzed, A. Merot, S. Meyer, S. Peixoto,
N. Petit, R. Siegrist, J. Trollux, T. Weller,
S. Wittlin
Do it the phenotypic way: After having
failed by using a target-based rational
approach, we decided to go after malar-
ia parasites by putting phenotypic
assays at the center of our efforts.
Herein you will learn the story behind
the identification of ACT-451840, which
was selected for clinical development as
an antimalarial drug candidate.
&& – &&
Discovery and Characterization of
ACT-451840: an Antimalarial Drug
with a Novel Mechanism of Action
ChemMedChem 2016, 11, 1 – 21
www.chemmedchem.org
21
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ