Article pubs.acs.org/jmc
3‑Hydroxy‑N′‑arylidenepropanehydrazonamides with HaloSubstituted 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*,† †
Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität 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, U.K. ∥ Department of Drug Design and Target Validation, Fraunhofer-Institut für Zelltherapie und Immunologie, Weinbergweg 22, 06120 Halle (Saale), Germany ⊥ 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, California 92093, United States ∇ Centre de Recherches Medicales de Lambaréné, B.P.: 242 Lambaréné, Gabon ○ Swiss Tropical and Public Health Institute, Socinstraße 57, 4002 Basel, Switzerland ◆ University of Basel, CH-4003 Basel, Switzerland S Supporting Information *
ABSTRACT: 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.
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INTRODUCTION
moderate, and thus, at least in the foreseeable future, antimalarial drugs will remain an important tool for treatment and prophylaxis. Currently, artemisinin-based 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 complete treatment of 3 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
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 humanpathogenic 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. High-throughput 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 sub-Saharan 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 © 2017 American Chemical Society
Received: January 26, 2017 Published: June 27, 2017 6036
DOI: 10.1021/acs.jmedchem.7b00140 J. Med. Chem. 2017, 60, 6036−6044
Journal of Medicinal Chemistry
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Figure 1. Optimization strategy for lead compound 19.
Scheme 1. Synthesis of Target Compounds 20−34 Starting from Phenanthrene-9-carbaldehydes 9−11a
Reagents and conditions: (i) ACN, n-BuLi in n-hexane (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, i-PrOH or CH2Cl2, 0 °C to rt; (3) R2CHO, i-PrOH or CH2Cl2, rt or R2COR3, pTSA, CH2Cl2, rt. a
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 10 years ago.5 However, despite huge efforts, relatively few novel clinically validated drug targets (e.g., PfATP4, Pf DHODH) have been discovered in the past decade.6−8 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., arylamino alcohols, to improve their antiplasmodial properties. We recently reported on a series of hydrazonamides structurally related to the class of arylamino 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 4-day Peter’s test along with a mean survival time (MSD) of 10 days (Figure 1).
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 structure−activity relationship (SAR) studies and the stepwise improvement of antiplasmodial properties. Consequently, in the first optimization cycle we modified the 4-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 N-dibutyl moiety of the arylamino alcohol lumefantrine (LF), whereas the basic 3-dialkylaminomethyl-4-hydroxybenzylidene 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 3-((diethylamino)methyl)-4hydroxybenzylidene residue of compound 26 and the 6037
DOI: 10.1021/acs.jmedchem.7b00140 J. Med. Chem. 2017, 60, 6036−6044
Journal of Medicinal Chemistry
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Scheme 2. Synthesis of Building Blocksa
Reagents and conditions: (i) K2CO3, Ac2O, 65 °C, 24 h; (ii) FeSO4, NaOH/H2O, 100 °C, 2 h; (iii) 1. isoamyl nitrite, HCl in ethanol (4 N), −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. a
namic 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 phenathrene-9-carboxylic acids (5, 6). Reduction of 5 and 6 with borane−tetrahydrofuran 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 Mannich-type reaction of 4-hydroxybenzaldehyde with diethylamine and paraformaldehyde (Scheme 2).16
confirmed 4-fluorobenzylidene unit of compound 19 were combined with 1,3-difluoro- and 1,3-dichloro-6-trifluoromethylphenanthrene 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 phenanthrene-9-carbaldehydes, as previously published (Scheme 1).9a,b,10,11 First, nucleophilic addition of lithioacetonitrile to phenanthrene-9-carbaldehydes (9−11) yielded 3-hydroxynitriles (13−15). Subsequent Pinner reactions with dry methanol and hydrogen chloride in diethyl ether 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 nonane-5-one and cycloheptanone required the presence of p-toluenesulfonic acid as catalyst to afford 22 and 23. To elucidate the configurations at both carbon− nitrogen double bonds, an X-ray crystal structure determination of 19 was performed previously. The structure analysis indicates a Z-configuration at the C2−N2 and an Econfiguration at the C1−N1 double bond.9a The difluoro-substituted phenanthrene-9-carbaldehyde (11) was synthesized in an analogous manner as the known dichloro derivative (10) utilizing a published five-step route (Scheme 2).12−15 The initial condensation of 2-nitro-4-(trifluoromethyl)phenyl)acetic acid and 2,4-dihalobenzaldehydes yielded cin-
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BIOLOGICAL EVALUATION Hydrazonamides 20−34 (Tables 1−6) were evaluated for their in vitro activity against the chloroquine-sensitive strain Pf 3D7 and the multidrug-resistant strain Pf Dd2.17 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 P. falciparum dual gamete formation assay and for liver stage activity toward exoerythrocytic 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. 6038
DOI: 10.1021/acs.jmedchem.7b00140 J. Med. Chem. 2017, 60, 6036−6044
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Table 1. In Vitro Evaluation of Compounds 20−30
a
Values 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 95%. vw: very wide. dSelectivity index.
In Vitro Evaluation of 20−30. Within alkylidenesubstituted compounds 20−23, only the N-dibutyl-substituted analog 22 (Pf 3D7 IC50 = 0.0083 μM, Pf Dd2 IC50 = 0.012 μM) displayed antiplasmodial in vitro activity comparable to lead compound 19 (Pf 3D7 IC50 = 0.0083 μM, Pf Dd2 IC50 = 0.011 μM) without showing pronounced cytotoxicity to HepG2 cells (SI (IC50(HepG2)/IC50(Pf 3D7) = 1037). Notably, the 3dialkylaminomethyl-4-hydroxybenzylidene substituted compounds 25−27, 29, and 30 (Pf 3D7 IC50 = 0.00097−0.0052 μM, Pf Dd2 IC50 = 0.00097−0.0092 μM) exhibited potent antiplasmodial activity toward P. falciparum strains 3D7 and
Dd2 with IC50 values 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. On the basis of their nanomolar to subnanomolar antiplasmodial in vitro activity and good to excellent selectivity indices, compound 22 (alkylidene-substituted) and compounds 25−30 (arylidenesubstituted) were tested for in vivo activity in P. berghei-infected 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 after infection) with 50 mg/kg of 6039
DOI: 10.1021/acs.jmedchem.7b00140 J. Med. Chem. 2017, 60, 6036−6044
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experimental compounds, which were dissolved or suspended in 70/30 Tween 80/ethanol and diluted 10 times with water before administration.21,22
The in vivo efficacy of several compounds bearing a 3alkylaminomethyl-4-hydroxy-benzylidene moiety (25−30) at the 4 × 50 mg/kg po regimen was high (90.0−99.9% activity compared to the infected, untreated control group), whereas the alkylidene-substituted 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 2- to 3-fold. The most active compound 26 exhibited high in vivo activity (ip 99.7% and po 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 3-diethylaminomethyl-4-hydroxybenzylidene moiety. Therefore, in the next optimization cycle the favorable 3-diethylaminomethyl-4-hydroxybenzylidene moiety and the confirmed 4-fluorobenzylidene group were selected and combined with 1,3-dihalo-6-trifluoromethyl-substituted phenanthrene scaffolds as present in phenanthrene-9-carbaldehydes 10 and 11. The results of the in vitro evaluation of hydrazonamides 31−34 bearing 1,3-dihalo-6-CF3-substituted phenanthrene moieties are shown in Table 3. Compounds with 1,3-dihalo-substituted phenanthrene scaffolds (31−34) displayed IC50 values in the single-digit nanomolar range toward ABS of P. falciparum (Pf 3D7 IC50 = 0.0023−0.0069 μM, Pf Dd2 IC50 = 0.0029−0.0138 μM).
Table 2. In Vivo Evaluation of Compounds 22 and 25−30 at 4 × 50 mg/kg Bodyweight (for CQ, AS, and MQ at 4 × 30 mg/kg Bodyweight) intraperitoneal administration
compd
parasitemia reduction [%]a
CQd ASd MQd 22 25 26 27 28 29 30
nd nd nd 64.8 99.8 99.7 98.7 91.3 97.6 99.8
MSDb curesc nd nd nd 7.0 22.0 30.0 16.7 14.0 18.0 11.0
nd nd nd 0/3 0/3 2/3 0/3 0/3 0/3 0/3
peroral administration parasitemia reduction [%]a
MSDb
curesc
99.9 99 99.9 95% drug content), while after 24 h 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 was 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 and 34 were also tested for their activity against the hERG K+channel using in vitro hERG fluoresence polarization assay (Table 6). Compound 34 turned out to be a
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 chlorosubstituted derivative (32), all hydrazonamides with halosubstituted phenanthrene scaffolds (31, 33, 34) were able to cure 17−66% of the P. berghei-infected mice, defined as the total absence of detectable parasites on day 30 postinfection. Interestingly, ip 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 × 50 mg/kg Bodyweight (for CQ, AS, and MQ at 30 mg/kg) intraperitoneal administration
compd
parasitemia reduction [%]a
MSD
cures
n. d nd nd 99.9 nd nd 99.8
nd nd nd 25.3 nd nd 30.0
nd nd nd 2/3 nd nd 2/3
d
CQ ASd MQd 31 32 33 34
b
peroral administration
c
parasitemia reduction [%]a
MSDb
curesc
99.9 99 99.9 99.9 99.8 99.9 99.9
24 10 29 25.3 23.0 22.0 29.7
0/10 0/10 7/10 2/3 0/3 1/3 1/6
Table 6. hERG Potassium Channel Activity of 33 and 34a
a
Blood for parasitemia determination was collected on day 4 (96 h after infection). bMean survival time in days (MSD). cNumber of parasite-free 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 bodyweight, 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
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. Gametocytocidal Properties and Liver Stage Activity. Several compounds were also tested for their activity toward different gametocyte stages (Pf RSG, Pf ESG) and against P. berghei liver stages (PbEEF) (Table 7 and Supporting 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 toward Pf RSG and Pf ESG 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 CM-H2Xros, were present within the compound treated images acquired by confocal microscopy. It was postulated that this submaximal Emax could be 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
peroral administration 31
33
34
4 4 4 4 4 4 4 4 4
× × × × × × × × ×
30 mg/kg 10 mg/kg 3 mg/kg 30 mg/kg 10 mg/kg 3 mg/kg 30 mg/kg 10 mg/kg 3 mg/kg
MSDb
curesc
99.4 80.0
