arylidenepropanehydrazonamides - ACS Publications - American

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Blood Schizontocidal and Gametocytocidal Activity of 3‑Hydroxy‑N′‑arylidenepropanehydrazonamides: A New Class of Antiplasmodial Compounds Michael Leven,† Jana Held,‡ Sandra Duffy,§ Serena Tschan,‡ Sibylle Sax,∥,⊥ Jolanda Kamber,∥,⊥ Walter Frank,# Krystina Kuna,† Detlef Geffken,∞ Christoph Siethoff,× Stéphane Barth,× Vicky M. Avery,§ Sergio Wittlin,∥,⊥ Benjamin Mordmüller,‡,○ 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 § Eskitis Institute for Drug Discovery, Griffith University, Brisbane Innovation Park, Don Young Road, Nathan, Queensland 4111, Australia ∥ Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland ⊥ University of Basel, CH-4003 Basel, Switzerland # Institut für Anorganische Chemie und Strukturchemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany ∞ Institut für Pharmazie, Universität Hamburg, Bundesstrasse 45, 20146 Hamburg, Germany × Swiss BioQuant, Kägenstrasse 18, 4153 Reinach, Switzerland ○ Centre de Recherches Médicales de Lambaréné, Lambaréné, Gabon S Supporting Information *

ABSTRACT: 3-Hydroxy-N′-arylidenepropanehydrazonamides represent a new class of antiplasmodial compounds. The two most active phenanthrene-based derivatives showed potent in vitro antiplasmodial activity against the 3D7 (sensitive) and Dd2 (multidrug-resistant) strains of Plasmodium falciparum with nanomolar IC50 values in the range of 8−28 nM. Further studies revealed that the most promising derivative, bearing a 4-fluorobenzylidene moiety, demonstrated in vivo antiplasmodial activity after oral administration in a P. berghei malaria model, although no complete parasite elimination was achieved with a four-dose regimen. The in vivo efficacy correlated well with the plasma concentration levels, and no acute toxicity symptoms (e.g., death or changes in general behavior or physiological activities) were observed, which is in agreement with a >1000-fold lower activity against L6 cells, a primary cell line derived from mammalian (rat) skeletal myoblasts. This indicates that lead compound 29 displays selective activity against P. falciparum. Moreover, both phenanthrene-based derivatives were active against stage IV/V gametocytes of P. falciparum in vitro.

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INTRODUCTION Despite significant reductions in the incidence of malaria in recent years, the global impact of this disease still remains considerable, resulting in the deaths of approximately a million people annually.1 Plasmodium falciparum malaria is the cause of the majority of fatalities and is one of the most frequent reasons for hospital admission of young children in sub-Saharan Africa. Currently, no licensed malaria vaccine is available; thus, antimalarial drugs remain the most important tools for malaria © 2014 American Chemical Society

treatment and prophylaxis. However, the emergence of Plasmodium strains resistant to currently used antimalarials contributes to the sustained presence of malaria and calls for intensive, global research efforts.2 It is particularly alarming that even drugs of the artemisinin family are showing the first signs of reduced antiplasmodial activity. In order to impede the emergence of Received: May 27, 2014 Published: September 8, 2014 7971

dx.doi.org/10.1021/jm500811p | J. Med. Chem. 2014, 57, 7971−7976

Journal of Medicinal Chemistry

Article

Figure 1. Arylamino alcohol antimalarials and 3-hydroxy-N′-arylidenepropanehydrazonamides.

malaria, and thus, the discovery of new leads with transmission blocking properties is of high priority.7 Malaria transmission occurs during a blood meal when mature gametocytes are taken up by a female Anopheles mosquito, where they develop into sporozoites, which are transmitted to a human host through a mosquito blood meal.8 Most available antimalarials target the asexual blood stages, which are responsible for the symptoms and complications of malaria. The only approved drug that is known to clear circulating gametocytes in vivo is primaquine.9 Therefore, the development of new and improved drugs that are active not only against the asexual blood stages but also against liver and/or sexual stages may markedly decrease malaria morbidity, mortality, and the spread of malaria and resistance alleles. Recently, we demonstrated that hydrazonamide 3, synthesized for the first time during a heterocyclic chemistry project aiming at the preparation of 1,3-oxazinan-2-ones, exerts in vitro antiplasmodial activity against the chloroquine-sensitive 3D7 strain of P. falciparum.10 Notably, hydrazonamide 3 resembles some general structural features of arylamino alcohol antimalarials. Here we report the synthesis and biological evaluation of various new derivatives of 3 toward asexual blood stages and late stage (stages IV and V) gametocytes of P. falciparum. Moreover, selected compounds were evaluated for their cytotoxicity, some pharmacokinetic properties, and their in vivo antiplasmodial activity in a P. berghei mouse model.

resistance a precedent has been set to combine new antimalarial agents with a carefully selected partner drug showing suitable complementary pharmacodynamic and pharmacokinetic properties.3 Despite intensive efforts, no major impacting antimalarials have been introduced since atovaquone in 1992. One potential reason for the lack of new antimalarial drugs may be that the total number of druggable targets among the approximately 5300 gene products of P. falciparum is smaller than initially expected.4 Consequently, the optimization of pharmacokinetic and pharmacodynamic properties of established antimalarials (e.g., arylamino alcohols, 4-aminoquinolines, 8-aminoquinolines, artemisinins) to circumvent their resistance potential remains an important tool in the global campaign against malaria.3 Because of their long and successful history in the prophylaxis and treatment of malaria, arylamino alcohol antimalarials, such as quinine (Q), mefloquine (MQ), halofantrine (HF) and lumefantrine (LF) (Figure 1), are still the subject of intensive drug research. Their mechanism of action is poorly understood but is at least in part associated with the inhibition of heme detoxification in asexual blood stage (ABS) parasites.5 Novel arylamino alcohol analogs, for example, MQ analog 1 and Q analog 2 (Figure 1), have been developed with the intention to reduce adverse effects and to improve antiplasmodial properties.6 Blocking malaria transmission from infected individuals to the Anopheles mosquito is a major goal in the global fight against 7972



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Scheme 1. Synthesis of Hydrazonamidesa

Reagents and conditions: (i) EtOH, HCl(g), Et2O or CH2Cl2, −10 °C; (ii) (1) K2CO3, Et2O, 0 °C; (2) H2NNH2·H2O, EtOH or CH2Cl2, 0 °C to rt; (iii) R2CHO, EtOH or CH2Cl2, rt or R2COR3, pTSA, CH2Cl2, rt.

a

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CHEMISTRY The α- and β-hydroxy imidate hydrochlorides (31a−e) were prepared by Pinner reactions and immediately converted into the corresponding imidate bases by treatment with potassium carbonate as previously reported.11 Their subsequent hydrazinolysis afforded α- and β-hydroxyamidrazones (32a−e), which were directly condensed with (hetero)aromatic aldehydes and aryl alkyl ketones to obtain 2-hydroxy-N′-benzylideneacetohydrazonamides (4, 5) and 3-hydroxy-N′-arylidene(arylalkylidene)propanehydrazonamides (6−29) (Scheme 1). The structures of the novel analogs 4−29 and of 30e were elucidated by IR, 1H, 13C NMR spectroscopy, and mass spectrometry. To elucidate the configurations at both carbon− nitrogen double bonds, an X-ray crystal structure determination of 29 was performed (Supporting Information Figure S1). The structure analysis indicates a Z-configuration at the C2−N2 and an E-configuration at the C1−N1 double bond. A further remarkable feature of the molecular structure is the intramolecular hydrogen bond between the amino group and the hydroxyl group, the latter acting as the hydrogen bond acceptor. The purity was determined by elemental analysis and/or HPLC analysis. The purity of all final compounds determined by HPLC analysis was 95% or higher.

(15, 16) and by arylidenealkylidene (17, 18) residues resulted in a significant reduction of in vitro antiplasmodial activity. In all cases, 2-naphthyl-substituted analogs (5, 19−27) were less potent than their 1-naphthyl-substituted counterparts (4, 6−10, 12, 14−16). Within the 1-naphthyl-substituted derivatives, compounds bearing a single substituent at the benzylidene moiety with a negative inductive and a positive mesomeric effect (8, 10, 11) were the most potent growth inhibitors of P. falciparum strain 3D7. In agreement with phenanthrene derivatives 28 and 29, the 4-fluorophenyl-substituted compound 10 was the most potent analog of the 1-naphthyl series. Possible reasons may be the reduced electron density of the phenyl ring as well as dipolar interactions of the 4-fluoro substituent with possible targets. Furthermore, the in vitro evaluation revealed that the chain shortened α-hydroxyhydrazoneamide (4) is 30-fold less potent than 3. Given their potent activity toward asexual blood stages, compounds 10, 28, and 29 were also tested for their gametocytocidal activity against stage IV/V gametocytes of P. falciparum in an image-based assay.13 The 2-naphthyl-substituted derivative 23 was screened to get additional insights into SAR (Supporting Information Figure S2). Compared to their activity against asexual blood stage parasites, the gametocytocidal activity of compounds 10, 28, and 29 toward late stage (stages IV and V) gametocytes was considerably lower but still in the nanomolar range, as demonstrated by IC50 values of 783 nM (10), 180 nM (28), and 370 nM (29), respectively. In contrast compound 23 was almost inactive and displayed only a 25% inhibition at the maximum test concentration of 40 μM. It was noted that compounds 10, 28, and 29 had poor solubility in 4% DMSO at higher screening concentrations and were observed to precipitate out of solution. However, this did not impact the identification of compounds with high levels of activity, as these were still identified at the lower compound screening concentrations where precipitation did not occur. MQ and compounds 10, 28, and 29 all demonstrated a submaximal % inhibition plateau, in comparison with the Emax of the positive control puromycin (Supporting Information Figure S2). Within the images obtained for these compounds, at their maximum inhibition plateau, gametocytes could be seen that appeared to be alive. They remain elongated in structure and had MTR staining at levels comparable to viable control gametocytes. These apparently viable individual gametocytes were surrounded by gametocyte debris and other structures that do not have MTR fluorescence. There are, at least, two possible explanations for this sub Emax % inhibition, namely, that these compounds have gametocyte sex or stage sensitivity. The exact reason for this submaximal inhibition has

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BIOLOGICAL EVALUATION First, the in vitro antiplasmodial activities of compounds 3−29 were evaluated against the chloroquine-sensitive 3D7 strain and the multidrug-resistant Dd2 strain of P. falciparum by measuring growth inhibition based on histidine rich protein 2 (HRP2).12 Chloroquine (CQ), MQ, and artesunate were used as reference compounds. As shown in Table 1, the most potent 3-hydroxy-N′arylidenepropanehydrazonamides 8, 10, 11, 28, and 29 inhibited the growth of P. falciparum strain 3D7 with nanomolar IC50 values between 8.3 and 91 nM. Compounds 28 (P. falciparum 3D7 IC50 = 16 nM, P. falciparum Dd2 IC50 = 28 nM) and 29 (P. falciparum 3D7 IC50 = 8.3 nM, P. falciparum Dd2 IC50 = 11 nM) are particularly interesting. Both compounds are characterized by a phenanthrene system and are 1−2 orders of magnitude more active than the 1-naphthyl-substituted lead compound 3. Notably, 28 and 29 are approximately as active as CQ and the arylamino alcohol MQ against strain 3D7 and retained activity in the multidrug-resistant parasite strain Dd2. Compared to its unsubstituted analog 28, the 4-fluorophenyl-substituted derivative 29 demonstrated stronger in vitro growth inhibition of both P. falciparum strains, while its cytotoxicity was reduced. The structural modification of 3 demonstrated also that the replacement of the benzylidene moiety by heteroarylidene 7973



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Table 1. In Vitro Antiplasmodial Activity and Cytotoxicity

IC50 [μM] n CQ MQ ASf PPTg 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

R1

naphthalen-1-yl naphthalen-1-yl naphthalen-2-yl naphthalen-1-yl naphthalen-1-yl naphthalen-1-yl naphthalen-1-yl naphthalen-1-yl naphthalen-1-yl naphthalen-1-yl naphthalen-1-yl naphthalen-1-yl naphthalen-1-yl naphthalen-1-yl naphthalen-1-yl naphthalen-1-yl naphthalen-2-yl naphthalen-2-yl naphthalen-2-yl naphthalen-2-yl naphthalen-2-yl naphthalen-2-yl naphthalen-2-yl naphthalen-2-yl naphthalen-2-yl phenanthren-9-yl phenanthren-9-yl

R2

Ph Ph Ph 4-Me-Ph 4-CF3-Ph 4-OMe-Ph 3-OEt, 4-OH-Ph 4-F-Ph 3-Br-Ph 2,4-diF-Ph 3,4-diF-Ph 3,4-diCl-Ph pyridin-3-yl furan-2-yl Ph 4-F-Ph 4-Me-Ph 4-CF3-Ph 4-OMe-Ph 3-OEt, 4-OH-Ph 4-F-Ph 2,4-diF-Ph 3,4-diCl-Ph pyridin-3-yl furan-2-yl Ph 4-F-Ph

R3

Pf 3D7a

Pf Dd2a

Pf LSGb

L6

selectivity, L6/Pf 3D7c

H H H H H H H H H H H H H H Pr Me H H H H H H H H H H H

0.0068 0.0065 0.0019 9.2 0.10 3.0 >10 0.19 0.11 0.083 0.25 0.060 0.091 0.19 0.18 0.25 2.7 0.51 0.24 0.40 0.54 >10 0.38 1.1 2.0 4.0 1.9 >10 >10 0.016 0.0083

0.14 0.0066 0.0020 8.3 0.24 3.9 >10 0.062 0.30 0.21 0.45 0.20 0.094 0.19 0.55 0.19 1.1 0.38 0.61 0.74 0.38 >10 0.62 1.3 2.5 >10 0.70 >10 >10 0.028 0.011

44%d 0.10e 0.044 ndh ndh ndh ndh ndh ndh ndh ndh 0.78e ndh ndh ndh ndh ndh ndh ndh ndh ndh ndh ndh ndh 25%i ndh ndh ndh ndh 0.18e 0.37e

78 4.5 24 0.0097 17 300 ndh 6.8 26 6.3 4.2 19 3.9 4.2 17 16 22 145 29 34 103 ndh 69 78 >298 93 100 ndh ndh 6.1 16

11403 686 12391 0.0016 170 100 ndh 36 234 76 17 321 43 22 95 63 8 284 120 86 190 ndh 182 71 >149 23 52 ndh ndh 383 1891

a

Values are the mean of at least two independent experiments conducted in duplicate, each using 12 serial dilutions. bValues are the mean of two independent experiments conducted in duplicate, each using 21 serial dilutions. cSelectivity indices were calculated utilizing unrounded cytotoxicity data. d120 μM. eSubmaximal inhibition plateau in relation to a puromycin 100% inhibition control. fAS, artesunate. gPPT, podophyllotoxin. hnd, not determined. i40 μM.

not yet been conclusively identified, and thus, extensive investigations to elucidate this mechanism are ongoing. On the basis of their chemical structures, promising antiplasmodial properties, and excellent selectivity indices, compounds 10 and 29 were selected for pilot PK studies and tested for oral in vivo activity in the P. berghei-infected mouse model.14 Briefly, compounds were dissolved or suspended in 70/30 Tween 80/ethanol and diluted 10× with water. Experimental groups (n = 3 mice) were treated 4× by the oral (po) route (4, 24, 48, and 72 h postinfection). Blood for parasitemia determination was collected on day 4 (96 h after infection). Parasitemia reduction (activity) and mean survival time in days (MSD) for multidose regimens are reported in Table 2. The in vivo efficacy for compound 29 at the 4 × 50 mg/kg regimen was high (99.6% activity compared to an infected, untreated control group), albeit no complete parasite elimination (parasite-free on day 30) was achieved, indicated by the MSD of 10.0 days. Compound 10 did not show pronounced efficacy in this in vivo model, evidenced by an activity of 40% and a MSD of

Table 2. In Vivo Antimalarial Activities of 10 and 29 in P. berghei Infected Micea control 10 29

parasitized erythrocytes [%]b

activity [%]

survival [days]

69 42 0.30

0 40 99.6

4.0c 6.7 ± 0.6 10.0 ± 0.0

a

A standard Peters test using 50 mg/kg body weight oral dose once per day was conducted for 4 days. Experimental groups (n = 3 mice) were treated 4× by the oral (po) route (4, 24, 48, and 72 h postinfection). Compounds were dissolved or suspended in 70/30 Tween 80/ethanol and diluted 10 times with water. bBlood for parasitemia determination was collected on day 4 (96 h postinfection). cMice were euthanized on day 4 postinfection in order to prevent death otherwise occurring on day 6.

6.7 days, which is similar to the MSD of control mice (6.0 days) (Table 2). For comparison, clinically used antimalarial drugs such as artesunate and mefloquine, administered in this P. berghei model 7974



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experiment. Spots were visualized by irradiation with ultraviolet light (254 nm). Melting points (mp) were taken in open capillaries on a Mettler FP 5 melting-point apparatus and are uncorrected. IR spectra were recorded on a Varian FT-IR Scimitar series. Proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker Avance 500 (500.13 MHz for 1H and 125.76 MHz for 13C) using DMSO-d6 as solvent. Chemical shifts are given in parts per million (ppm), δ relative to residual solvent peak for 1H and 13C and to external tetramethylsilane. Elemental analyses were performed on a PerkinElmer PE 2400 CHN elemental analyzer. X-ray diffraction experiments were performed with an Oxford Diffraction Xcalibur (EOS) diffractometer. High-resolution mass spectrometry (HRMS) analyses were performed on a UHR-TOF 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, 254 nm; column, Phenomenex Luna C-18(2) 5 μm particle (250 mm × 4.6 mm), supported by Phenomenex SecurityGuard cartridge kit C18 (4.0 mm × 3.0 mm); mobile phase, linear gradient of acetonitrile (70−100%) in water at a flow rate of 1 mL/min over 15 min. The purity of all final compounds determined by HPLC was 95% or higher. Experimental Data. Synthesis and experimental data of compounds 30e, 31e, and 29 are listed below. 3-Hydroxy-3-(phenanthren-9-yl)propanenitrile (30e) was prepared according to the procedure of Kaiser.16 3-Hydroxy-3-(phenanthren-9-yl)propanenitrile (30e). Colorless solid; yield = 78%; mp = 147 °C. 1H NMR (500.13 MHz, DMSOd6): δ [ppm] = 8.90 (d, J = 7.8 Hz, 1H), 8.82 (d, J = 8.1 Hz, 1H), 8.21 (d, J = 7.5 Hz, 1H), 8.08 (s, 1H), 8.03 (d, J = 6.7 Hz, 1H), 7.87−7.54 (m, 4H), 6.24 (d, J = 4.5 Hz, 1H), 5.72 (ddd, J = 6.0, 4.7, 4.7 Hz, 1H), 3.17 (dd, J = 16.9, 4.5 Hz, 1H), 3.01 (dd, J = 16.9, 6.5 Hz, 1H). 13C{1H} NMR (125.76 MHz, DMSO-d6): δ [ppm] = 136.56, 130.80, 130.08, 129.60, 128.80, 128.75, 127.04, 126.61, 124.13, 123.81, 123.60, 122.76, 118.64, 65.02, 26.78. IR (KBr): ν̃ [cm−1] = 3468 (O−H), 2251 (CN). Anal. Calcd for C17H13NO: C 82.57, H 5.30, N 5.66. Found: C 82.37, H 5.48, N 5.67. Ethyl 3-hydroxy-3-(phenanthren-9-yl)propanimidate hydrochloride (31e) was prepared according to the procedure of Khankischpur.17 All imidate hydrochlorides were characterized by IR spectroscopy and used for the next step without further purification. Ethyl 3-Hydroxy-3-(phenanthren-9-yl)propanimidate Hydrochloride (31e). Colorless solid; yield = 92%; mp = 113 °C. IR (KBr): ν̃ [cm−1] = 1655 (CN). Procedure for the Synthesis of Target Compound 29. Imidate hydrochloride 31e (1.0 mmol) was suspended in diethyl ether (10 mL) and converted into the free base with saturated potassium carbonate solution (5 mL) and ice. The organic layer was separated, and the aqueous layer was extracted two times with diethyl ether (2 × 10 mL). The combined organic layers were dried over magnesium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was dissolved in anhydrous dichloromethane (7.5 mL). The solution was cooled to 5 °C, and hydrazine hydrate (1.0 equiv) was added. After 6 h of stirring at ambient temperature, the resulting suspension was filtered; the solid was washed once with anhydrous diethyl ether and suspended in anhydrous dichloromethane (5 mL). 4-Fluorobenzaldehyde (1.5 equiv) was added, and the mixture was stirred overnight. The solvent was evaporated under reduced pressure, and the residue was purified by column chromatography on silica gel with ethyl acetate/n-hexane 3/7 as eluent. (1Z)-N′-[(E)-4-Fluorobenzylidene]-3-hydroxy-3-(phenanthren-9-yl)propanehydrazonamide (29). Colorless solid; yield = 45%; mp = 169 °C. 1H NMR (500.13 MHz, DMSO-d6): δ [ppm] = 8.90 (dd, J = 6.0, 3.4 Hz, 1H), 8.82 (d, J = 8.0 Hz, 1H), 8.44−8.26 (m, 2H), 8.06 (s, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.97−7.84 (m, 2H), 7.72 (dd, J = 6.1, 3.1 Hz, 2H), 7.70−7.57 (m, 2H), 7.26 (t, J = 8.7 Hz, 2H), 6.89 (br s, 2H), 5.95 (br s, 1H), 5.82 (d, J = 8.5 Hz, 1H), 2.83 (dd, J = 14.7, 2.6 Hz, 1H), 2.62 (dd, J = 14.7, 9.3 Hz, 1H). 13C{1H} NMR (125.76 MHz, DMSO-d6): δ [ppm] = 163.22 (d, J = 246.8 Hz), 162.21, 150.79, 139.39, 132.74 (d, J = 2.9 Hz), 131.48, 130.46, 129.90 (d, J = 8.2 Hz), 129.75, 129.62, 128.95, 127.29, 127.18, 127.01, 126.77, 124.53, 123.88, 123.80,

at four daily oral doses of 30 mg/kg, showed a reduction in parasitemia of 99.0% and 99.9% with a mean survival time of 10 and 29 days, respectively.15 The plasma concentration levels (experimental procedure in Supporting Information) of compounds 10 and 29 (Table 3) Table 3. Plasma Concentration Levels of 10 and 29 in P. berghei Infected Mice at 1, 4, and 24 h after per Oral Treatmenta concentration [ng/mL] at compd

1h

4h

24 h

10 29

316 4690

145 3100

1000-fold lower activity against L6 cells, a primary cell line derived from mammalian (rat) skeletal myoblasts. This indicates that compound 29 displays selective activity against P. falciparum in vitro and in vivo (Table 1 and Table 3). Moreover, 29 shows druglike properties; its molecular weight of 385.43 g/mol, the calculated log P (4.63, Molinspiration software, version 2011.04), and the number of hydrogen bond donors (3) and acceptors (4) are within the limits of Lipinski’s rule of five. However, to avoid metabolism induced carcinogenicity, the structural modification of the unsubstituted phenanthrene system will be a matter of future research.

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CONCLUSION In summary, the phenanthrene-based 3-hydroxy-N′-arylidenepropanehydrazonamides 28 and 29 represent a new type of antiplasmodial compounds demonstrating activity not only toward asexual blood stages but also against late stage gametocytes of P. falciparum. Because of their gametocytocidal (transmission blocking) properties, novel leads like 29 are of high priority since they contribute to the malaria eradication agenda. Moreover, the most active compound 29 is characterized by an excellent selectivity index and showed in vivo efficacy in a P. berghei malaria model after oral administration. However, no cure was observed as evidenced by a MSD of 10.0 days. Taking all data into account, 29 can be considered as an early lead compound and a starting point for further optimization. To avoid metabolism induced carcinogenicity, the modification of the unsubstituted phenanthrene system will be addressed in future research.

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EXPERIMENTAL SECTION

General Procedures. Chemicals and solvents were used as purchased without further purification. Reaction progress was monitored on precoated Merck silica gel plates (fluorescence indicator UV254) using ethyl acetate/n-hexane as solvent system. Column chromatography was performed with Macherey-Nagel silica gel M (230−400 mesh ASTM) with the solvent mixtures specified in the corresponding 7975



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123.09, 115.86 (d, J = 21.7 Hz), 67.97, 41.93. IR (KBr): ν̃ [cm−1] = 3494, 3380 (N−H, O−H), 1627, 1606 (CN). Anal. Calcd for C24H20FN3O: C 74.79, H 5.23, N 10.90. Found: C 74.68, H 5.39, N 10.80. HPLC analysis: retention time = 8.51 min, peak area = 99.5%.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures, analytical data, and biological evaluation. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 211 81 14985. Fax: +49 211 81 13847. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully thank Xavier Ding and Medicines for Malaria Venture (MMV) for their support. The Deutsche Forschungsgemeinschaft (DFG) is acknowledged for funds used to purchase the UHR-TOF maXis 4G (Bruker Daltonics, Bremen) HRMS instrument used in this research.

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ABBREVIATIONS USED ABS, asexual blood stage; CQ, chloroquine; DMSO, dimethyl sulfoxide; HF, halofantrine; LC−MS, liquid chromatography− mass spectrometry; LF, lumefantrine; LSG, late stage gametocyte; MSD, mean survival time in days; MTR, Mitotracker Red; MQ, mefloquine; pTSA, p-toluenesulfonic acid; Q, quinine

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REFERENCES

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