3-Hydroxy-N′-arylidenepropanehydrazonamides with Halo-Substituted Phenanthrene Scaffolds Cure P. berghei Infected Mice When Administered Perorally

Article abstract of DOI:10.1021/acs.jmedchem.7b00140

Structural optimization of 3-hydroxy-N′-arylidenepropanehydrazonamides provided new analogs with nanomolar to subnanomolar antiplasmodial activity against asexual blood stages of Plasmodium falciparum, excellent parasite selectivity, and nanomolar activity against the earliest forms of gametocyte development. Particularly, derivatives with a 1,3-dihalo-6-trifluoromethylphenanthrene moiety showed outstanding in vivo properties and demonstrated in part curative activity in the Plasmodium berghei mouse model when administered perorally.

Full text of DOI:10.1021/acs.jmedchem.7b00140

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Article
3-Hydroxy-N'-arylidenepropanehydrazonamides with
halo-substituted phenanthrene scaffolds cure P.
berghei-infected mice when administered perorally
Michael Leven, Tanja C. Knaab, Jana Held, Sandra Duffy, Stephan Meister, Christoph Fischli, Diane
Meitzner, Ursula Lehmann, Beate Lungerich, Krystina Kuna, Petra Stahlke, Michael J. Delves, Mirko
Buchholz, Elizabeth A Winzeler, Vicky M. Avery, Benjamin Mordmüller, Sergio Wittlin, and Thomas Kurz
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00140 • Publication Date (Web): 27 Jun 2017
Downloaded from http://pubs.acs.org on June 28, 2017
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3-Hydroxy-N'-arylidenepropanehydrazonamides with
halo-substituted phenanthrene scaffolds cure P.
berghei-infected mice when administered perorally
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†
Michael Leven,† Tanja C. Knaab, Jana Held,‡ Sandra Duffy,§ Stephan Meister,* Christoph
┴,#
┴,#
Fischli,
Diane Meitzner,¥ Ursula Lehmann,
Beate Lungerich,† Krystina Kuna,† Petra
Stahlke,† Michael J. Delves,$ Mirko Buchholz,¥ Elizabeth A. Winzeler,* Vicky M. Avery,§
Benjamin Mordmüller,‡,║ Sergio Wittlin,┴,# and Thomas Kurz*,†
†Institut für Pharmazeutische und Medizinische Chemie, HeinrichꢀHeineꢀUniversität Düsseldorf,
‡
Universitätsstraße 1, 40225 Düsseldorf, Germany. Institut für Tropenmedizin, Eberhard Karls
$
Universität Tübingen, Wilhelmstraße 27, 72074 Tübingen, Germany. Cell and Molecular
¥
Biology, Department of Life Sciences, Imperial College, London SW7 2AZ, UK. Fraunhoferꢀ
Institut für Zelltherapie und Immunologie, Departement of Drug Design and Target Validation,
§
Weinbergweg 22, 06120 Halle (Saale). Griffith Institute for Drug Discovery, Griffith
University, Brisbane Innovation Park, Don Young Road, Nathan, Queensland 4111, Australia.
*Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla,
║
┴
CA 92093, USA. Centre de Recherches Medicales de Lambaréné, Lambaréné, Gabon. Swiss
#
Tropical and Public Health Institute, Socinstraße 57, 4002 Basel, Switzerland. University of
Basel, CHꢀ4003 Basel, Switzerland
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KEYWORDS. Hydrazonamides, asexual blood stages, gametocytes, in vitro activity, in vivo
efficacy.
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ABSTRACT. Structural optimization of 3ꢀhydroxyꢀN′ꢀarylidenepropanehydrazonamides
provided new analogs with nanomolar to subnanomolar antiplasmodial activity against asexual
blood stages of Plasmodium falciparum, excellent parasite selectivity and nanomolar activity
against the earliest forms of gametocyte development. Particularly, derivatives with a 1,3ꢀdihaloꢀ
6ꢀtrifluoromethylꢀphenanthrene moiety showed outstanding in vivo properties and demonstrated
in part curative activity in the Plasmodium berghei mouse model when administered perorally.
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INTRODUCTION. According to the World Malaria Report 2016, there were estimated 212
million new malaria cases and approximately 429 000 malaria deaths worldwide in 2015.1
Among humanꢀpathogenic Plasmodium species P. falciparum is the strain responsible for most
fatalities. Due to extensive global efforts, both malaria incidence and mortality rate have
decreased significantly between 2000 and 2015. Highꢀthroughput screening campaigns and
vaccine development are two major tools in the worldwide fight against malaria. Recently the
malaria vaccine candidate RTS,S obtained a positive scientific opinion by the European
Medicines Agency (EMA) for the prevention of malaria in young children in subꢀSaharan
Africa.2 Furthermore, the World Health Organization (WHO) recommended pilot
implementation to understand how to best use RTS,S.3 However, its protective efficacy is only
moderate and thus, at least in the foreseeable future, antimalarial drugs will remain an important
tool for treatment and prophylaxis. Currently, artemisininꢀbased combination therapies (ACTs),
which consist of an endoperoxide and a partner drug (e.g. artemether and lumefantrine), are the
WHO recommended treatment for uncomplicated malaria. Parenteral artesunate followed by a
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complete treatment of three days with an ACT is recommended for the treatment of severe
malaria.4 However, P. falciparum resistance to artemisinin has spread across mainland Southeast
Asia from southern Vietnam to central Myanmar and, indeed, resistance now affects all currently
deployed antimalarial drugs. Since introduction of atovaquone in 1992, no new antimalarial drug
has been launched for the treatment of malaria despite intensive efforts. As a result of various
comprehensive phenotypic and to a lesser extent target based screening campaigns, the malaria
drug pipeline is significantly stronger than ten years ago.5 However, despite huge efforts,
relatively few novel clinically validated drug targets (e.g. PfATP4, PfDHODH) have been
discovered in the last decade.6,7,8 Hence, it is also prudent to investigate alternative approaches to
identifying new drugs and drug combinations. One alternative approach is the structural
optimization of established antimalarial drugs, e.g. aryl amino alcohols, to improve their
antiplasmodial properties.
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We recently reported on a series of hydrazonamides structurally related to the class of aryl amino
alcohol antimalarials exhibiting potent in vitro activity against asexual blood stages (ABS) and
late stage gametocytes (LSG) of P. falciparum.9a,b The most active compound 19 showed
nanomolar antiplasmodial in vitro activity, good parasite selectivity, and a 99.6% reduction of
parasitemia in the P. berghei mouse model when administered perorally in the 4ꢀday Peter’s test
along with a mean survival time (MSD) of ten days (Figure 1).
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Figure 1. Optimization strategy for lead compound 19.
A structural drawback of the lead compound 19 is its monosubstituted phenanthrene scaffold,
which can form reactive electrophilic metabolites and is responsible for the low water solubility
of 19. However, new analogs of 19 with a monosubstituted phenanthrene scaffold are accessible
according to our previously developed synthetic pathway and thus represent a valuable starting
point for initial structureꢀactivity relationship (SAR) studies and the stepwise improvement of
antiplasmodial properties. Consequently, in the first optimization cycle we modified the 4ꢀ
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fluorobenzylidene moiety of 19 and synthesized analogs with alkylidene and basic arylidene
residues (Table 1, 20-30), respectively. The alkylidene residues of compounds 20−23 were
introduced to mimic the Nꢀdibutyl moiety of the aryl amino alcohol lumefantrine (LF), whereas
the basic 3ꢀdialkylaminomethylꢀ4ꢀhydroxybenzylidene moieties were selected to enhance
hydrophilicity and water solubility at physiological pH (compounds 24−30). After their in vitro
and in vivo evaluation the most promising structural features discovered notably the 3ꢀ
((diethylamino)methyl)ꢀ4ꢀhydroxybenzylidene residue of compound 26 and the confirmed 4ꢀ
fluorobenzylidene unit of compound 19, were combined with 1,3ꢀdifluoroꢀ and 1,3ꢀdichloroꢀ6ꢀ
trifluoromethylꢀphenanthrene moieties that are less susceptible to the formation of reactive
electrophilic metabolites compared to the monosubstituted phenanthrene moiety of 19.
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SYNTHESIS. Target compounds 20−34 were synthesized in three steps starting from
phenanthreneꢀ9ꢀcarbaldehydes, as previously published (scheme 1).9a,b,10,11 First, nucleophilic
addition of lithioacetonitrile to phenanthreneꢀ9ꢀcarbaldehydes (9−11) yielded 3ꢀhydroxynitriles
(13−15). Subsequent Pinner reactions with dry methanol and hydrogen chloride in diethylether
led to the corresponding imidate hydrochlorides (16−18). Finally, imido esters, liberated from
their hydrochlorides, were reacted with hydrazine monohydrate to obtain intermediate
amidrazones, which upon condensation with the appropriate aldehyde provided hydrazonamides
(20, 21, 24−34), whereas condensation with nonaneꢀ5ꢀone and cycloheptanone required the
presence of pꢀtoluene sulfonic acid as catalyst to afford 22 and 23. To elucidate the
configurations at both carbon–nitrogen double bonds, an Xꢀray crystal structure determination of
19 was performed previously. The structure analysis indicates a Zꢀconfiguration at the C2–N2
and an Eꢀconfiguration at the C1–N1 double bond.9a
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Scheme 1. Synthesis of target compounds 20−34 starting from phenanthreneꢀ9ꢀcarbaldehydes
9−11
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Reagents and conditions: (i) ACN, nꢀBuLi in nꢀhexane (1.6 M), THF, −78 °C to rt; (ii) MeOH, HCl in Et2O,
CH2Cl2/THF, −10 °C to rt; (iii) 1. K2CO3, Et2O, 0 °C 2. H2NNH2 × H2O, iꢀPrOH or CH2Cl2, 0 °C to rt 3. R2CHO, iꢀ
PrOH or CH2Cl2, rt or R2COR3, pTSA, CH2Cl2, rt.
The difluoroꢀsubstituted phenanthreneꢀ9ꢀcarbaldehyde (11) was synthesized in an analogous
manner as the known dichloro derivative (10) utilizing a published fiveꢀstep route (scheme
2).12,13,14,15 The initial condensation of 2ꢀnitroꢀ4ꢀ(trifluoromethyl)phenyl)acetic acid and 2,4ꢀ
dihalobenzaldehydes yielded cinnamic acids (1, 2). Subsequent reduction of the nitro group with
iron sulfate yielded anilines (3, 4), which underwent Pschorr cyclization in the presence of
isoamyl nitrite and copper to afford phenathreneꢀ9ꢀcarboxylic acids (5, 6). Reduction of 5 and 6
with boraneꢀtetrahydrofuran complex solution gave the corresponding phenanthrene methanols
(7, 8). Their subsequent oxidation with manganese dioxide provided phenanthrene carbaldehydes
(10, 11). Aldehyde 12 was synthesized via Mannichꢀtype reaction of 4ꢀhydroxybenzaldehyde
with diethylamine and paraformaldehyde (scheme 2).16
Scheme 2. Synthesis of building blocks
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O
H
O
X
O-
Na+
X
OH
HO
X
NO2
O
O
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i
ii
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NO2
NH2
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X
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X
CF3
X = Cl, F
CF3
CF3
2,4-dihalo
benzaldehyde
2-Nitro-4-(trifluoromethyl)
phenyl acetic acid
1: X = Cl
2: X = F
3: X = Cl
4: X = F
X
OH
X
OH
X
O
O
v
iv
iii
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X
X
CF3
CF3
CF3
5: X = Cl
6: X = F
10: X = Cl
11: X = F
7: X = Cl
8: X = F
H
O
H
O
H
O
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vi
N
N
N
OH
OH
OH
4-Hydroxy-
4-Hydroxy-3-
12
benzaldehyde
(diethylaminomethyl)-
benzaldehyde
Reagents and conditions: (i) K2CO3, Ac2O, 65°C, 24h (ii) FeSO4, NaOH/H2O, 100 °C, 2 h; (iii) 1. isoamyl nitrite,
HCl in ethanol (4N), −5 °C, 1 h 2. Cu, NaH2PO4, H2SO4/H2O, 40 °C, 2 h; (iv) BH3, THFabs., N2, 0 °C to rt, 12 h; (v)
MnO2, CHCl3, rt to 40–45 °C, 2 h; (vi) CH2O, NH(CH2CH3)2, EtOH, 80 °C, 24 h.
BIOLOGICAL EVALUATION. Hydrazonamides 20−34 (Table 1−6) were evaluated for their in
vitro activity against the chloroquineꢀsensitive strain Pf3D7 and the multidrugꢀresistant strain
PfDd2.17 All compounds were also tested for their in vitro cytotoxicity against HepG2 cells.18
Furthermore, the in vitro gametocytocidal activity against ring stage gametocyte (RSG) and stage
I−III gametocytes of P. falciparum was evaluated.19 Selected compounds were also tested in the
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P. falciparum dual gamete formation assay and for liver stage activity towards exoꢀerythrocytic
forms of P. berghei (PbEEF) (supporting information and Table 7).18,20 The established
antimalarials chloroquine (CQ), mefloquine (MQ), artesunate (AS), and atovaquone (ATQ) were
used as reference compounds for in vitro evaluation. Finally, the in vivo efficacy of selected
compounds (22, 25−34) was evaluated in the P. berghei mouse model using the Peter’s test.
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Table 1. In vitro evaluation of compounds (20−30)
R1
R2
Pf 3D7 a
Pf Dd2a
HepG2b,c
IC50 [µM]
SId
(HepG2/Pf3D7)
IC50 [µM] IC50 [µM]
0.0057±0. 0.2247±0.0 n. d.
0016 948
n. d.
CQ
MQ
AS
19
0.0065±0. 0.0066±0.0 n. d.
0012 014
n. d.
0.0019±0. 0.0020±0.0 n. d.
0004 003
n. d.
H
0.0083±0. 0.00114±0. >10
0033 00001
>1037
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H
0.060±0.0 0.092±0.01 7.6(7.0–
07 8.3)
127
20
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H
0.33±0.01 0.72±0.43
27.4(20.2ꢀ 83
37.3)
21
22
23
24
25
nꢀbutyl
nꢀbutyl
0.0083±0. 0.012±0.00 8.6(7.6ꢀ
0032 96 9.8)
1036
0.18±0.02 0.14±0.02
25.4(17.2ꢀ 139
37.4)
cycloheptylidene
H
H
0.025±0.0 0.036±0.00 16.6(vw)
13 27
664
0.0025±0. 0.00097±0. 16.7(vw)
0002 00005
6680
H
H
0.00097±0 0.0034±0.0 14.3(vw)
14742
2897
26
27
.0003
026
0.0029±0.
0002
8.4(6.5ꢀ
10.7)
0.0054±0.0
028
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H
H
0.029±0.0 0.044±0.01 3.7(3.0ꢀ
128
28
29
005
0
4.5)
0.0052±
0.0009
0.0092±0.0 8.4(7.7ꢀ
028 9.1)
1615
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H
0.0015±0. 0.0033±0.0 14.3(vw)
0005 004
9533
30
aValues are the mean of at least two independent experiments conducted in duplicate, each using 12 point serial
dilutions. bValues are the mean of one experiment conducted on two plates in quadruplicate. cconfidence interval for
d
95%, vw: very wide, selectivity index
In vitro evaluation of 20−30.
Within alkylideneꢀsubstituted compounds 20−23, only the Nꢀdibutylꢀsubstituted analog 22
(Pf3D7 IC50 = 0.0083 µM, PfDd2 IC50 = 0.012 µM) displayed antiplasmodial in vitro activity
comparable to lead compound 19 (Pf3D7 IC50 = 0.0083 µM, PfDd2 IC50 = 0.011 µM) without
showing pronounced cytotoxicity to HepG2 cells (SI (IC50 HepG2 / IC50 Pf3D7) = 1037).
Notably, the 3ꢀdialkylaminomethylꢀ4ꢀhydroxyꢀbenzylidene substituted compounds 25−27, 29,
and 30 (Pf3D7 IC50 = 0.00097−0.0052 µM, PfDd2 IC50 = 0.00097−0.0092 µM) exhibited potent
antiplasmodial activity towards P. falciparum strains 3D7 and Dd2 with IC50ꢀvalues in the low
nanomolar to subnanomolar range. The selectivity indices of compounds 25−27, 29, 30 were
excellent (SI = 14433−1615).
In vivo evaluation of compounds 20-30.
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Based on their nanomolar to subnanomolar antiplasmodial in vitro activity and good to excellent
selectivity indices, compound 22 (alkylideneꢀsubstituted) and compounds 25−30 (arylideneꢀ
substituted) were tested for in vivo activity in P. bergheiꢀinfected mice after intraperitoneal and
peroral administration, respectively. In a standard Peter’s test NMRI mice (n = 3) were dosed 4 ×
(4, 24, 48, and 72 h post infection) with 50 mg/kg of experimental compounds, which were
dissolved or suspended in 70/30 Tween 80 / ethanol and diluted 10 times with water before
administration.21,22
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Table 2. In vivo evaluation of compounds 22 and 25−30 at 4 × 50 mg/kg body weight (for CQ,
AS, and MQ at 4 × 30 mg/kg body weight)
Intraperitoneal administration
Peroral administration
Parasitemia
MSDb
Curesc
Parasitemia
MSDb
Curesc
reduction [%]a
reduction [%]a
CQd n. d
n. d.
n. d.
n. d.
7.0
n. d.
n. d.
n. d.
0/3
99.9
99
24
10
29
0/10
0/10
7/10
ASd, n. d.
MQd, n. d.
99.9
< 40.0
99.8
99.9
99.0
64.8
99.8
99.7
98.7
euthan. 0/3
22
25
26
27
22.0
30.0
16.7
0/3
24.7
28.2
10.3
0/3
0/6
0/3
2/3
0/3
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91.3
97.6
99.8
14.0
18.0
11.0
0/3
0/3
0/3
89.7
98.0
96.7
9.7
0/3
0/3
0/3
28
29
30
16.0
11.0
10
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19
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b
aBlood for parasitemia determination was collected on day 4 (96 h after infection). Mean survival time in days
(MSD). Mice were euthanized on day 4 postinfection in order to prevent death otherwise occurring on day 6.
cNumber of parasiteꢀfree mice on day 30. dFor comparison, clinically used antimalarial drugs administered in this P.
berghei model at four daily oral doses of 30 mg/kg body weight showed a reduction in parasitemia of 99% and
99.9% with a mean survival time of 10 and 29 days.21,22 MSD for control mice infected with P. berghei is 6–7
days.23.
The in vivo efficacy of several compounds bearing a 3ꢀalkylaminomethylꢀ4ꢀhydroxyꢀ
benzylidene moiety (25−30) at the 4 × 50 mg/kg p.o. regimen was high (90.0−99.9% activity
compared to the infected, untreated control group), whereas the alkylideneꢀsubstituted compound
22 was significantly less active irrespective of compound administration method. Compared to
lead compound 19 (MSD of 10.0 d) the MSD after oral administration of compounds 25, 26 and
29 was prolonged two to threeꢀfold. The most active compound 26 exhibited high in vivo activity
(i.p.: 99.7% and p.o.: 99.9%) and a significantly prolonged MSD of 30 and 28.2 days when
administered intraperitoneally and perorally, respectively. Interestingly, intraperitoneal
application of 26 led to a cure rate of 66%; two out of three mice were parasite free at day 30.
Intraperitoneal administration of compound 30 resulted in a temporary reduction of physical
activity and diarrhea. All other compounds showed no signs of acute toxicity.
Compound 26 is characterized by a monosubstituted phenanthrene scaffold and a 3ꢀ
diethylaminomethylꢀ4ꢀhydroxyꢀbenzylidene moiety. Therefore, in the next optimization cycle
the favorable 3ꢀdiethylaminomethylꢀ4ꢀhydroxyꢀbenzylidene moiety and the confirmed 4ꢀ
fluorobenzylidene group were selected and combined with 1,3ꢀdihaloꢀ6ꢀtrifluoromethylꢀ
substituted phenanthrene scaffolds as present in phenanthreneꢀ9ꢀcarbaldehydes 10 and 11. The
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results of the in vitro evaluation of hydrazonamides 31−34 bearing 1,3ꢀdihaloꢀ6ꢀCF3ꢀsubstituted
phenanthrene moieties are shown in Table 3.
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8
9
Table 3. In vitro evaluation of compounds 31−34
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18
19
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22
23
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26
27
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31
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33
34
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R1
X
Pf 3D7a
Pf Dd2a
HepG2b,c
IC50 [µM]
SId
IC50 [µM] IC50 [µM]
(HepG2/Pf3D7)
0.0057±0. 0.2247±0.0 n. d.
0016 948
n. d.
CQ
MQ
AS
31
0.0065±0. 0.0066±0.0 n. d.
0012 014
n. d.
0.0019±0. 0.0020±0.0 n. d.
0004 003
n. d.
Cl
0.0018±0. 0.0025±0.0 43.8(vw)
0011 025
24333
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Cl
0.0069±0. 0.0138±0.0 42.8(vw)
0046 036
6203
12043
3436
32
33
34
F
F
0.0023±0. 0.0029±0.0 27.7(20.9ꢀ
0005 022 36.6)
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11
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22
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31
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0.0039±0. 0.0061±0.0 13.4(vw)
0019 028
aValues are the mean of at least two independent experiments conducted in duplicate, each using 12 serial
dilutions. bValues are the mean of one experiment conducted on two plates in quadruplicate. cconfidence interval for
d
95%, vw: very wide, selectivity index
Compounds with 1,3ꢀdihaloꢀsubstituted phenanthrene scaffolds (31−34) displayed IC50ꢀvalues
in the singleꢀdigit nanomolar range towards ABS of P. falciparum (Pf 3D7 IC50 = 0.0023–0.0069
µM, Pf Dd2 IC50 = 0.0029–0.0138 µM). Again, their selectivity indices were excellent (12174–
1158). Consequently, compounds 31−34 were also evaluated for their in vivo activity utilizing
the Peter’s test at 4 × 50 mg/kg bodyweight. Compounds 31−34 exhibited potent in vivo activity
and reduced parasitemia by at least 99.8% along with mean survival times of at least 22.0 days
when administered perorally (MSD = 22.0−29.7 d). With the exception of chloroꢀsubstituted
derivative (32), all hydrazonamides with haloꢀsubstituted phenanthrene scaffolds (31, 33, 34)
were able to cure 17−66% of the P. bergheiꢀinfected mice, defined as the total absence of
detectable parasites on day 30 postꢀinfection. Interestingly, i.p. application of 34 led to a MSD of
30 d along with a cure rate of 66%. With all compounds in Table 4 no overt signs of toxicity
were observed.
Table 4. In vivo evaluation of compounds 31−34 at 4 × 50mg/kg body weight (for CQ, AS, and
MQ at 30 mg/kg)
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Intraperitoneal administration
Peroral administration
5
6
7
8
9
Parasitemia
[%]a
reduction MSDb Curesc Parasitemia
[%]a
reduction MSDb Curesc
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11
12
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19
20
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22
23
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CQd n. d
n. d.
n. d.
n. d.
25.3
n. d.
n. d.
30.0
n. d.
n. d.
n. d.
2/3
99.9
99
24
0/10
0/10
7/10
2/3
ASd n. d.
MQd n. d.
10
99.9
99.9
99.8
99.9
99.9
29
99.9
n. d.
n. d.
99.8
25.3
23.0
22.0
29.7
31
32
33
34
n. d.
n. d.
2/3
0/3
1/3
1/6
aBlood for parasitemia determination was collected on day 4 (96 h after infection). Mean survival time in days
b
c
(MSD). Number of parasiteꢀfree mice on day 30. dFor comparison, clinically used antimalarial drugs such as
chloroquine, artesunate, mefloquine, administered in this P. berghei model at four daily oral doses of 30 mg/kg body
weight showed a reduction in parasitemia of 99% and 99.9% with a mean survival time of 10 and 29 days.21,22 MSD
for control mice infected with P. berghei is 6−7 days.23
Finally, the most active compounds with substituted phenanthrene moieties and curative
potential (31, 33, 34) were also evaluated for their in vivo activity at doses of 30, 10 and 3 mg/kg
bodyweight (Table 5). At a dose of 30 mg/kg bodyweight, all compounds retained their
parasitemia reduction potential. The most promising compound 33 exhibited comparable high
activity at doses of 50, 30 and 10 mg/kg bodyweight after peroral administration, including
parasitemia reduction, mean survival time and cure rates. Compound 33 is characterised by a
1,3ꢀdifluoroꢀ6ꢀtrifluoromethylꢀphenanthrene moiety and 4ꢀfluorobenzylidene group.
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Table 5. In vivo evaluation of compounds 31, 33 and 34 at 4 × 30 mg/kg, 4 × 10 mg/kg, 4 × 3
5
6
mg/kg.
7
8
9
Peroral administration
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11
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19
20
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60
Parasitemia reduction [%]a MSD b Curesc
4 × 30 mg/kg 99.4
4 × 10 mg/kg 80.0
4 × 3 mg/kg < 40.0
4 × 30 mg/kg 99.4
4 × 10 mg/kg 99.5
4 × 3 mg/kg 78
13.7
5.0
0/3
0/3
31
33
34
euthan. 0/3
25.7
24.7
7.0
1/3
2/3
0/3
0/3
0/3
4 × 30 mg/kg 99.9
4 × 10 mg/kg 99.4
4 × 3 mg/kg < 40
28.3
10.0
euthan. 0/3
aBlood for parasitemia determination was collected on day 4 (96 h after infection). Mean survival time in days
(MSD). Mice were euthanized on day 4 postinfection in order to prevent death otherwise occurring on day 6.
cNumber of parasiteꢀfree mice on day 30. MSD for control mice infected with P. berghei is 6−7 days.23
b
We observed no doseꢀdependent activity for compound 33, as the highest number of mice were
cured (2/3) at 4 × 10 mg/kg bodyweight but not at the highest dose of 4 × 50 mg/kg. An
explanation for this could be that the rate of compound absorption is not increasing at higher
doses, possibly due to poor solubility.
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In vitro stability and solubility of 33. To study the stability of compound 33 at physiological
pH, 33 was dissolved in Tween 20/ethanol/phosphate buffer pH 7.5 (7/3/70, v/v/v) and
monitored over a period of 72 hours. After 6 hours 33 showed nearly no decomposition (> 95 %
drug content), while after 24 hours 31 % of 33 was decomposed. The solubility of 33 was tested
in phosphate buffer under acidic (pH 2.5) and physiological pH (pH 7.5) (buffer were prepared
according to Ph. Eur. 5.0). 33 was almost insoluble in both media (see supporting information).
hERG potassium channel activity. A well known liability of arylamino antimalarials is their
inhibition of the hERG potassium channel that is associated with cardiotoxic effects. Thus,
analogues 33 and34 were also tested for their activity against the hERG K+channel using in vitro
hERG fluoresence polarisation assay (Table 6). Compound 34 turned out to be a moderate
inhibitor of the hERG K+channel (IC50 = 0.390 µm). The hERG channel inhibition of 34 was
comparable to lumefantrine (LF), which is frequently used in combination with arthemeter for
the treatment of P. falciparum malaria in adults and children, whereas the hERG activity of 33 is
somewhat more pronounced.
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18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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Table 6. hERG potassium channel activity of 33, 34
IC50 [µm]
LFa
HFb
33
0.377
0.156
0.067
0.390
34
a
hERG Fluoresence Polarization Assay, 10ꢀpoint titrations, 3ꢀfold serial dilutions, Lumefantrine (LF), bHalofantrine
(HF)
Gametocytocidal properties and liver stage activity
Several compounds were also tested for their activity towards different gametocyte stages
(PfRSG, PfESG) and against P. berghei liver stages (PbEEF) (Table 7 and supporting
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information). In addition compounds 31, 33 and 34 were also evaluated in the P. falciparum dual
gamete formation assay. The gametocytocidal activity of compounds 31−34 towards PfRSG and
PfESG is depicted in Table 7. As previously reported the late stage gametocyte activity for lead
19 demonstrated a submaximal inhibition plateau in relation to the 100% inhibition by the
positive control puromycin.9a Both live and dead parasites, as represented by the viability marker
MitoTracker® Red CMꢀH2Xros, were present within the compound treated images acquired by
confocal microscopy. It was postulated that this submaximal Emax could be either a result of
differential effects against gametocyte sexes or gametocyte stages. Analogs 31, 33, and 34 were
tested for their activity against mature stage V gametocytes to determine their effect on
conversion to either male or female gametes.20 These compounds demonstrated no activity
against either male or female gametes at a concentration of 1 ꢁM. Thus, the compounds did not
exhibit any effect on mature stage V gametocytes, or their ability to convert to gametes. This
data would indicate that the submaximal Emax obtained for lead compound 19 is related to the
gametocyte stage of development.
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19
20
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22
23
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26
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In light of this data selected compounds were tested against the earliest forms of gametocyte
development, ring stage gametocytes (RSG) and stage I−III (early stage) gametocytes.
Compounds 31−34 exhibited nanomolar activity against the earliest forms of gametocyte
development whereas their P. berghei liver stage (PbEEF) growth inhibition was considerably
weaker.
Table 7. Gametocytocidal properties and liver stage activity of 31−34
R1
X
Pf RSGa
Pf ESGa
PbEEFb,c
IC50 [µM] IC50 [µM] IC50 [µM]
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0.017±0.0 0.063±0.0 n.d.
17 16
CQ
AS
0.003±0.0 0.003±0.0 n. d.
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02
01
n. d.
n. d.
0.00026(0.00
023ꢀ0.00030
ATQ
31
Cl
Cl
F
0.036±0.0 0.031±0.0 13.0(11.0ꢀ
16 18 15.5)
0.345±0.2 0.453±0.0 19.1(vw)
23 95
32
33
34
0.066±0.0 0.200±.10 6.9(2.5ꢀ19ꢀ2)
48
1
F
0.110±0.0 0.218±0.0 5.2(4.3ꢀ6.2)
36 73
aValues are the mean of three independent experiments; P. falciparum ring stage gametocytes, early stage
gametocytes, late stage gametocytes. bValues are the mean of one experiment conducted on two plates in
quadruplicate; P. berghei liver stages. . cconfidence interval for 95%, vw: very wide
In summary, several structurally optimized hydrazonamides showed potent in vitro
antiplasmodial activity against multidrug resistant (Dd2) and sensitive (3D7) strains of P.
falciparum with IC50 values in the singleꢀdigit nanomolar to subnanomolar range. Various
compounds demonstrated excellent selectivity indices (SI: IC50 HepG2 / IC50 Pf3D7) and
nanomolar activity against early stage but not late stage gametocytes, suggesting that the
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molecular target is either only expressed or biologically relevant within asexual and early
gametocyte stages. Hydrazonamides with a 1,3ꢀdihaloꢀ6ꢀtrifluoromethylꢀphenanthrenyl moiety
(31−34) were highly active in vivo. The front runner compound 33 contains a 1,3ꢀdifluoroꢀ6ꢀ
trifluoromethylꢀphenanthrenyl moiety and a 4ꢀfluorobenzylidene group. 33 demonstrated potent
in vivo activity in the P. berghei mouse model when administered perorally at 4 × 10 mg/kg and
cured 66% of the P. bergheiꢀinfected mice along with a MSD of 24.7 days. The high
lipophilicity, hERG K+channel inhibition and the improvable drugꢀlikeness of 33 and 34 will be
addressed along with further SAR and initial MOA studies in a followꢀup lead optimization
program.
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Experimental Section
Experimental data. For experimental procedures and data of compounds 2, 4, 6, 8, 11, 12, 14,
15, 17, 18, 20−30 see supporting information and Leven.9
General procedures. Chemicals and solvents were used as purchased without further
purification. Reaction progress was monitored by thin layer chromatography (TLC) on Merck
precoated silica gel plates (fluorescence indicator UV254) using ethyl acetate / nꢀhexane or
dichloromethane/methanol as mobile phase. Column chromatography was performed with
Teledyn Isco CombiFlash Rf200 on MachereyꢀNagel silica gel M (230−400 mesh ASTM).
Compounds were visualized by irradiation with ultraviolet light (254 nm). Melting points (mp)
were taken in open capillaries on a Mettler FP 5 meltingꢀpoint apparatus and are uncorrected. IR
spectra were recorded on a Varian FTꢀIR Scimitar series. Proton (1H) and carbon (13C) NMR
spectra were recorded on a Bruker Avance 300 (300.13 MHz for 1H and 75.47 MHz for 13C), 500
1
13
1
(500.13 MHz for H and 125.76 MHz for C), 600 MHz (600.22 MHz for H and 151.93 MHz
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for C) using DMSOꢀd6 or chloroformꢀd as solvents. Chemical shifts are given in parts per
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5
6
1
million (ppm), relative to residual solvent peak for H and 13C and to external standard
7
8
9
tetramethylsilane (TMS). Elemental analyses were performed on a Perkin Elmer PE 2400 CHN
elemental analyzer. Elektron Spray Ionization (ESI) and Highꢀresolution mass spectrometry
(HRMS) analyses were performed on a UHRꢀTOF maXis 4G (Bruker Daltonics, Bremen). If
necessary, the purity was determined by high performance liquid chromatography (HPLC);
instrument: Varian ProStar 210 system in combination with a photodiode array detector 330,
220−254 nm; column: Phenomenex Luna C−18 (2) 5 µm particle (250 mm × 4.6 mm), supported
by Phenomenex Security Guard Cartridge Kit C18 (4.0 mm × 3.0 mm). Depending on the
compound one of the following mobile phases was used: mobile phase 1: linear gradient of
methanol (10 to 100%) in water at a flow rate of 1 ml/min over 10 min, isocratic elution with
methanol 100% over 10 min, mobile phase 2: linear gradient of acetonitrile (10 to 95 %) in water
at a flow rate of 1 mL/min over 15 min, isocratic elution of acetonitrile in water 95% over 5 min.
The purity of all final compounds determined by HPLC analysis was 95% or higher.
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General procedure for the synthesis of 3-hydroxy-N'-arylidenepropanehydrazonamides
(19−34). The respective imidoester hydrochloride (1.0 mmol) was suspended in diethyl ether (20
mL) and converted into the free base by treatment with a saturated potassium carbonate solution
(5 mL) and ice. The organic layer was separated and the aqueous layer was extracted with
diethyl ether (2 × 30 mL). The combined organic layers were dried over sodium sulfate, filtered
and the solvent was evaporated under reduced pressure. The residue was dissolved in anhydrous
dichloromethane (7.5 mL) (20−30) or anhydrous isopropanol (12 ml) (31−34), cooled to 5 °C and
hydrazine hydrate (1.0 equiv) was added. The mixture was stirred for 6 h before the resulting
suspension was filtered. The solid was washed with anhydrous diethyl ether and suspended in
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anhydrous dichloromethane (5 mL) (20−30) or anhydrous isopropanol (12mL) (31−34). The
appropriate aldehyde was added (1.5 equiv) and the mixture was stirred overnight. The solvent
was evaporated under reduced pressure and the residue was purified by column chromatography
with ethyl acetate/nꢀhexane (31, 33), dichloromethane/methanol (20−22, 23) or
dichloromethane/methanol/triethylamine (24−30, 32, 34) as eluent and by subsequent
crystallization with diethyl ether. Condensation reactions of intermediate amidrazones with
ketones and pꢀtoluenesulfonic afforded hydrazonamides 22 and 23 and were performed
according to previously published procedures.9a,b
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(Z)-3-(1,3-Dichloro-6-(trifluoromethyl)phenanthrene-9-yl)-N'-((E)-4-fluorobenzylidene)-3-
1
hydroxypropanehydrazonamide (31). White solid; yield = 13%; mp = 154 °C. H NMR
(500.13 MHz, DMSOꢀd6): δ [ppm] = 9.38 (s, 1H), 9.23 (s, 1H), 8.62 (d, J = 8.7 Hz, 1H), 8.54 (s,
1H), 8.31 (s, 1H), 8.09 (d, J = 8.8 Hz, 1H), 8.04 (d, J = 1.8 Hz, 1H), 7.89 (dd, J = 8.7, 5.8 Hz,
2H), 7.25 (t, J = 8.9 Hz, 2H), 6.87 (s, 2H), 6.20 (d, J = 3.3 Hz, 1H), 5.95 – 5.80 (m, 1H), 2.81
(dd, J = 14.7, 3.6 Hz, 1H), 2.62 (dd, J = 14.6, 8.8 Hz, 1H). 13C NMR (151.93 MHz, DMSOꢀd6): δ
[ppm] = 163.33 (d, J = 247.8 Hz), 161.77, 150.97, 141.57, 133.12, 132.78 (d, J = 2.9 Hz),
132.39, 132.12, 132.07, 129.99 (d, J = 8.1 Hz), 129.33, 128.52, 128.01 (q, J = 32.0 Hz), 127.52,
126.40, 124.87 (q, J = 272.8 Hz), 124.29–124.12 (m), 123.09, 122.77–122.63 (m), 121.17,
115.95 (d, J = 21.5 Hz), 68.17, 41.90. Anal. calcd. for C25H17Cl2F4N3O: C 57.49, H 3.28, N 8.04,
found: C 57.65, H 3.49, N 7.80.
(Z)-3-(1,3-Dichloro-6-(trifluoromethyl)phenanthren9-yl)-N'-((E)-3-((diethylamino)methyl)-
4-hydroxybenzylidene)-3-hydroxypropanehydrazonamide (32). Light yellow solid; yield =
33%; mp = 149 °C. 1H NMR (600.22 MHz, DMSOꢀd6): δ [ppm] = 9.36 (s, 1H), 9.21 (d, J = 1.9
Hz, 1H), 8.61 (d, J = 8.7 Hz, 1H), 8.54 (s, 1H), 8.18 (s, 1H), 8.09 (dd, J = 8.7, 1.8 Hz, 1H), 8.02
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(d, J = 1.9 Hz, 1H), 7.60–7.51 (m, 2H), 6.71 (m, 3H), 6.24 (s, 1H), 5.86 (dd, J = 8.7, 3.8 Hz, 1H),
3.78 (s, 2H), 2.81 (dd, J = 14.8, 3.8 Hz, 1H), 2.65–2.61 (m, 1H), 2.58 (q, J = 7.1 Hz, 4H), 1.05 (t,
J = 7.1 Hz, 6H). 13C NMR (151.93 MHz, DMSOꢀd6): δ [ppm] = 160.01, 159.42, 151.79, 141.03,
132.57, 131.84, 131.60, 131.54, 128.79, 127.99, 127.94, 127.79, 127.45 (q, J = 31.9 Hz), 127.01
(d, J = 3.8 Hz), 126.34, 125.90, 124.31 (d, J = 272.4 Hz), 123.66 (d, J = 2.1 Hz), 122.66, 122.57,
122.17 (d, J = 3.1 Hz), 120.66, 115.43, 67.59, 55.25, 45.64, 41.20, 10.89. Anal. calcd. for
C30H29Cl2F3N4O2: C 59.51, H 4.83, N 9.25, found: C 59.28, H 5.13, N 8.98.
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(Z)-3-(1,3-Difluoro-6-(trifluoromethyl)phenanthren-9-yl)-N'-((E)-4-fluorobenzylidene)-3-
hydroxypropanehydrazonamide (33). White solid; yield: 50%; mp = 130 °C. 1H NMR (600.22
MHz, DMSOꢀd6): δ [ppm] = 9.26 (s, 1H), 8.80 (d, J = 10.5 Hz, 1H), 8.62 (d, J = 8.7 Hz, 1H),
8.31 (d, J = 6.3 Hz, 2H), 8.08 (d, J = 8.7 Hz, 1H), 7.89 (dd, J = 8.5, 5.9 Hz, 2H), 7.68 (s, 1H),
7.25 (t, J = 8.8 Hz, 2H), 6.87 (s, 2H), 6.13 (s, 1H), 5.93–5.80 (m, 1H), 2.82 (dd, J = 14.7, 3.7 Hz,
1H), 2.65 (dd, J = 14.7, 8.9 Hz, 1H). 13C NMR (151.93 MHz, DMSOꢀd6): δ [ppm] = 162.77 (d, J
= 247.2 Hz), 161.22, 160.58 (dd, J = 245.2, 13.9 Hz), 158.77 (dd, J = 251.4, 13.7 Hz), 150.39,
139.02 , 132.22 (d, J = 2.3 Hz), 132.01 , 131.56 (d, J = 11.1 Hz), 131.53 (d, J = 11.4 Hz), 129.43
(d, J = 8.4 Hz), 128.83 , 127.17 (q, J = 32.0 Hz), 126.07, 124.32 (q, J = 272.7 Hz), 123.61–
123.51 (m), 122.14–122.02 (m), 117.63 (d, J = 15.2 Hz), 116.84–116.74 (m), 115.39 (d, J = 21.6
Hz), 105.27 (dd, J = 20.8, 3.2 Hz), 103.13 (dd, J = 27.9, 24.1 Hz), 67.53, 41.31. Anal. calcd. for
C25H17F6N3O: C 61.35, H 3.50, N 8.58, found: C 61.39, H 3.67, N 8.34. HPLC analysis:
retention time (mobile phase 2) = 11.14 min, peak area = 99,2%. m/z = 490.1353 [M+H]+ calcd.
for [C25H17F6N3O]+: 490.1349
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(Z)-N'-((E)-3-((Diethylamino)methyl)-4-hydroxybenzylidene)-3-(1,3-difluoro-6-
(trifluoromethyl)phenanthrene-9-yl)-3-hydroxypropanehydrazonamide (34). Light yellow
solid; yield: 57%; mp = 129 °C. 1H NMR (600.22 MHz, chloroformꢀd): δ [ppm] = 8.76 (s, 1H),
8.34 (d, J = 8.7 Hz, 2H), 8.31 (d, J = 8.6 Hz, 1H), 8.05 (d, J = 10.4 Hz, 1H), 7.85 (d, J = 8.6 Hz,
1H), 7.55 (dd, J = 8.2, 2.1 Hz, 1H), 7.42 (s, 1H), 7.12 (t, J = 8.9 Hz, 1H), 6.82 (d, J = 8.3 Hz,
1H), 5.88 (dd, J = 9.3, 2.6 Hz, 1H), 5.53 (s, 1H), 3.82 (s, 2H), 2.85 (dd, J = 15.9, 9.5 Hz, 1H),
2.78 (dd, J = 15.9, 2.6 Hz, 1H), 2.65 (q, J = 7.2 Hz, 4H), 1.13 (t, J = 7.1 Hz, 6H). 13C NMR
(151.93 MHz, chloroformꢀd): δ [ppm] = 161.19 (dd, J = 247.8, 12.6 Hz), 161.06, 159.83 (dd, J =
254.3, 13.8 Hz), 159.80, 155.83, 136.41, 132.05, 131.98 (dd, J = 9.9 Hz), 129.10, 128.94, 128.40
(q, J = 32.8 Hz), 127.90, 125.82, 125.14, 124.24 (q, J = 272.4 Hz), 123.71 (d, J = 2.9 Hz),
122.34, 121.25 (d, J = 4.3 Hz), 118.34 (d, J = 14.6 Hz), 117.97 (d, J = 6.5Hz), 116.49, 103.78
(dd, J = 22.6, 4.0 Hz), 102.79 (dd, J = 27.4, 24.4 Hz), 67.98, 56.82, 46.32, 40.32, 11.15. HPLC
analysis (mobile phase 2): retention time = 6.86 min, peak area = 95.09%. m/z = 573.2280
[M+H]+ calcd. for [C30H29F5N4O2]+: 573.2283
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Biological evaluation. In vitro activity against asexual blood stages was evaluated based on a
histidine rich protein 2 (HRP2) enzyme linked immunosorbent assay (ELISA).9,17 The activity
against P. berghei liver stages was assayed with a LuciferaseꢀBased HighꢀThroughput
Screening.18 Evaluation of gametocytocidal in vitro activity was performed according to
literature procedures.19 The in vitro toxicity was tested via a bioluminescence quantification of
cytotoxicity against HepG2 cells.18 Mouse experiments were conducted according to known
procedures.9a,22, 23 The animal experiments performed at the Swiss Tropical and Public Health
Institute (Swiss TPH) were approved by the Swiss Cantonal Authorities (permission no. 1731).
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ASSOCIATED CONTENT
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Supporting Information Available: Experimental procedures, analytical data for compounds 2,
4, 6, 8, 11, 12, 14, 15, 17, 18, 20−30, gametocytocidal properties and liver stage activity of
19−30, molecular formula strings for compounds 19−34. This material is available free of charge
via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
Thomas Kurz, Phone: +49 211 81 14984. Fax: +49 211 81 13847. Eꢀmail: Thomas.kurz@uniꢀ
duesseldorf.de
Author Contributions
† Michael Leven and Tanja Knaab contributed equally to this publication.
ACKNOWLEDGMENT
We gratefully acknowledge funding and support by Medicines for Malaria Venture (MMV). The
authors thank and acknowledge the Australian Red Cross Blood Bank for the provision of fresh
red blood cells without which this research could not have been completed. This work was
supported by the Australian Research Council (LP120200557 awarded to VMA). The Deutsche
Forschungsgemeinschaft (DFG) is acknowledged for funds used to purchase the UHRꢀTOF
maXis 4G (Bruker Daltonics, Bremen) HRMS instrument used in this research.
ABBREVIATIONS USED
ABS, asexual blood stage; ACT, artemisininꢀbased combination therapy; AS, artesunate; ATQ,
atovaquone; CQ, chloroquine; chloroformꢀd, deuterated chloroform; DMSOꢀd6, deuterated
dimethyl sulfoxide; d, doublet; dd, doublet of doublets; EMA, European Medicines Agency;
ESG, early stage gametocytes; euthan., euthanized; HF, halofantrine; HepG2, hepatocellular
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carcinoma G2; hERG, human Etherꢀaꢀgoꢀgo Related Gene; HPLC, high pressure liquid
chromatography; HRMS, highꢀresolution mass spectroscopy; LF, Lumefantrine; LSG, late stage
gametocytes; mp, melting point; MSD, mean survival time in days; MQ, mefloquine; NMRI,
naval medical research institute; PbEEF, Plasmodium berghei exoerythrocytic form; PfATP4,
Plasmodium falciparum ATPase 4; PfDHODH, Plasmodium falciparum dihydroorotate
dehydrogenase; q, quartet; RSG, ring stage gametocytes; s, singulet; SAR, structureꢀactivity
relationship; SI, selectivity index; t, triplet; UHRꢀTOF, ultra high resolutionꢀtime of flight;
WHO, World Health Organization
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