European Journal of Medicinal Chemistry 85 (2014) 289e292

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Preliminary communication

In vitro antiplasmodial and cytotoxic activities of asymmetrical pyridinium derivatives n Rubio-Ruiz a, 1, Víctor M. Castillo-Acosta b, Guiomar Pe rez-Moreno b, Bele lez-Pacanowska b, Luis M. Ruiz-Pe rez b, Antonio Espinosa a, Dolores Gonza a, * a, * Antonio Entrena , Ana Conejo-García nica, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja s/n, 18071 Granada, Spain Departamento de Química Farmac eutica y Orga pez-Neyra”, Consejo Superior de Investigaciones Científicas, Parque Tecnolo gico de Ciencias de la Salud, Instituto de Parasitología y Biomedicina “Lo Avenida del Conocimiento s/n, 18100 Armilla, Granada, Spain

a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 May 2014 Received in revised form 23 July 2014 Accepted 29 July 2014 Available online 30 July 2014

An in vitro investigation of the antiplasmodial and cytotoxic activities of a series of human choline kinase inhibitors against Plasmodium falciparum is reported. Structureeactivity relationship analyses have allowed us to determine the essential parameters for the antimalarial effect of these asymmetrical pyridinium derivatives. One of the compounds meets the World Health Organization's criteria for hit identification against P. falciparum exhibiting an IC50 of 0.0016 mg/ml and a selectivity index of >3000. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Pyridinium compounds Plasmodium falciparum Antimalarial activity Cytotoxicity

1. Introduction Malaria is an important health problem, responsible for 219 million clinical cases and 660,000 deaths in 2010 [1]. A major obstacle to the eradication of this disease is the emerging resistance of the malaria-causing parasite Plasmodium falciparum to most marketed antimalarial drugs [2,3]. As such, new compounds acting through novel mechanisms of action are urgently needed. Choline kinase (CK) is the first enzyme in the Kennedy pathway for the biosynthesis of phosphatidylcholine, the most essential phospholipid in P. falciparum. The proliferation of this parasite within erythrocytes is concomitant with a massive increase of phosphatidylcholine biosynthesis. It has been demonstrated that the inhibition of PfCK disrupts the Kennedy pathway, which results in parasite death [4,5].

Abbreviations: CK, choline kinase; SAR, structureeactivity relationship; SI, selectivity index. * Corresponding authors. E-mail addresses: [email protected] (A. Entrena), [email protected] (A. ConejoGarcía). 1 Present address: Edinburgh Cancer Research Centre, MRC IGMM, University of Edinburgh, Western General Hospital, Crewe Road, EH4 2XU Edinburgh, United Kingdom. http://dx.doi.org/10.1016/j.ejmech.2014.07.105 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

We have previously reported a series of asymmetrical pyridinium derivatives that target the choline phospholipid metabolism in cancer cells and tumors by inhibiting the human CK [6e8]. Their promising activities against human CK prompted us to evaluate these structures for their in vitro antimalarial effect. In this paper the antiplasmodial activities of these 33 mono- or biscationic compounds and also the cytotoxicity of the most active ones are reported. 2. Results and discussion Chemical structures for compounds 1 to 33 and their corresponding IC50 values against P. falciparum are reported in Tables 1e3. The compounds were classified into three groups according to their structures: series A (1e13), B (14e21) and C (22e33). Series A includes monocationic compounds bearing a 4substituted pyridinium ring linked to the N-9 or N-3 atom of an adenine moiety through an aromatic linker. Among N-9 isomers (1e8), the 4-pyrrolidine derivatives display lower IC50 values than the corresponding dimethylamine analogues, compound 7 being the only exception (Table 1). It is also noteworthy that the antimalarial effect of these compounds is closely related to the linker size. In general, increasing the length of the alkyl chain of the linker

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Table 1 In vitro antimalarial activities of Series A.

Compd

Linker

1 2 3 4 5 6 7 8 9 10 11 12 13

m m m m m m m m m m m m m

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

0, 0, 0, 0, 2, 2, 4, 4, 0, 0, 2, 4, 4,

Pyridinium substituent n n n n n n n n n n n n n

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

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

A A A A A A A A A A A A A

(from 0 to 4 carbon atoms) results in increased inhibition of parasite growth although it slightly varies depending on the substituent at position 4 of the pyridinium ring. If that group is 4-pyrrolidine, the 1,2-diphenylethane linker leads to the highest activity (compound 6, IC50 ¼ 93 nM) whereas if the substituent is 4dimethylamine, the 1,4-diphenylbutane linker is the most suitable one (7, IC50 ¼ 162 nM). Overall, biological results show that the N-3 isomers (9e13) have a similar antiplasmodial effect to their N-9 counterparts. Although a strict structureeactivity relationship (SAR) for 9e13 cannot be established due to the lack of several derivatives of this series, the most active compound (13, IC50 ¼ 84 nM) presents the moieties that stand out in the N-9 isomers: a 1,4-diphenylbutane linker and 4-pyrrolidinopyridinium cationic head. In series B the polar amino group at position 6 of the purine ring is replaced by a benzylthio substituent. The presence of this moiety

Table 2 In vitro antimalarial activities of Series B.

Compd

Linker

14 15 16 17 18 19 20 21

m m m m m m m m

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

0, 0, 0, 0, 2, 2, 4, 4,

Pyridinium substituent n n n n n n n n

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

0 0 1 1 1 1 1 1

A A A A A A A A

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

R1 R2 R1 R2 R1 R2 R1 R2

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

R1 R2 R1 R2 R1 R2 R1 R2 R1 R1 R2 R1 R2

IC50 (mM) 14.307 ± 3.633 6.432 ± 0.428 4.840 ± 0.194 0.191 ± 0.019 0.191 ± 0.009 0.093 ± 0.007 0.162 ± 0.007 0.460 ± 0.016 >22.700 0.949 ± 0.116 0.210 ± 0.035 0.930 ± 0.069 0.084 ± 0.017

in the structure leads to higher antimalarial activity compared with the corresponding adenine derivatives (IC50 14 < 1, 15 < 2, 16 < 3, 17 < 4, 18 < 5 and 21 < 8) with the exception of compounds 19 and 20 (Table 2). The compound that exhibits the best antiplasmodial effect of this series is 21 (IC50 ¼ 54 nM) that also incorporates the 1,4-diphenylbutane linker and the 4-pyrrolidinopyridinium 4pyrrolidine pyridinium cationic head. Series C includes biscationic compounds bearing two different 4-substituted pyridinium rings connected through an aromatic linker. Our results show that the presence of a second cationic head in the structure notably increases the antimalarial potency of the compounds, with IC50 values ranging from 94 nM (22) to 2 nM (25) (Table 3). The antiplasmodial effect of these compounds is dependent on the linker length and also on the substituent at position 4 of the pyridinium rings, being the most active compound (25, IC50 ¼ 2 nM) the one bearing 1,4-diphenylbutane as a linker and 4pyrrolidinopyridinium and 4-dimethylaminopyridinium as cationic heads. Thus, our results denote that the structural requirements of mono- and bis-pyridinium compounds for antimalarial activity are very similar, except that this activity is dramatically increased when introducing the second cationic head which is consistent with previous SAR analyses of other quaternary ammonium salts [10,11]. We also evaluated the cytotoxicity of the most active compounds (23e25, 31 and 32, Table 4). Compound 23 exhibits the best selectivity index (SI) fitting the pattern of the hit-to-lead candidate identification criteria for biological and initial safety data against P. falciparum [12]: IC50 < 0.2 mg/ml and SI > 100 versus IC50 ¼ 0.0016 mg/ml and SI ¼ 3187.5 for 23 (Table 4). Therefore, compound 23 represents a good candidate for further development as an antimalarial agent.

IC50 (mM) 0.122 0.411 3.223 0.119 0.092 0.149 0.500 0.054

± ± ± ± ± ± ± ±

0.014 0.014 0.080 0.001 0.014 0.009 0.029 0.004

3. Conclusions In summary, asymmetrical pyridinium compounds were found to be lethal to P. falciparum in vitro in a dose-dependent manner with an IC50 ranging from >23 mM to as low as 2 nM. Linker size, substituent at the position 4 of the pyridinium ring and the introduction of a second pyridinium moiety are the essential parameters for the

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Table 3 In vitro antimalarial activities of Series C.

Compd

Linker

22 23 24 25 26 27 28 29 30 31 32 33

m m m m m m m m m m m m

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

0, 0, 2, 4, 0, 0, 2, 4, 0, 0, 2, 4,

Pyridiniums substituents n n n n n n n n n n n n

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

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

A A A A A A A A A A A A

antimalarial effect of the compounds tested. Studies on the biochemical mechanism through which these structures result in the pharmacological response are currently underway. Future work will be focussed on in vivo studies in order to further explore the potential of these compounds as antimalarial agents. 4. Experimental section 4.1. Antimalarial activity Parasites of the P. falciparum strain 3D7 were grown in fresh group 0 positive human erythrocytes, obtained from the Centro n Sanguínea-SAS (Granada, Spain) and susRegional de Transfusio pended at 5% hematocrit in RPMI 1640 containing 2% human serum, 0.2% NaHCO3, 0.5% Albumax II, 150 mM hypoxanthine, and 12.5 mg/ml gentamicin. Flasks were incubated at 37  C, under 5% CO2 and 95% air mixture. The stock culture was synchronized with 5% sorbitol, and then approximately 96 h later, the parasites were determined to be mostly late ring stages and early trophozoites. The stock culture was then diluted with complete medium and non-parasitized erythrocytes to yield a hematocrit of 5% and a parasitemia of 0.5%. Stock solutions of the test drugs were prepared at a concentration of 100 mg/ml in >99.9% DMSO (Merck), serially diluted in complete medium, and dispensed into triplicate test wells to yield final concentrations ranging from 0.0002 to 30 mM. Final well volume was 100 mL for the fluorescence assays with a final concentration of DMSO 0.001%. The compounds in DMSO were evaluated per triplicate in 96well plates. Each plate also contained triplicate wells of negative control (no compounds), and positive controls with 80 nM of

Table 4 Cytotoxic activites of compounds 23e25, 31 and 32. Compd

TC50 L6 (mg/ml)

IC50 Pf (mg/ml)

SI

23 24 25 31 32

5.100 0.123 0.012 0.122 0.100

0.0016 0.0021 0.0014 0.0050 0.0035

3187.5 58.6 8.6 24.4 28.6

SI ¼ TC50 in L6 cells/EC50 in P. falciparum.

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

R1, R1, R1, R1, R2, R2, R2, R2, R1, R1, R1, R1,

B B B B B B B B B B B B

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

R2 R2 R2 R2 R3 R3 R3 R3 R3 R3 R3 R3

IC50 (mM) 0.094 0.003 0.003 0.002 0.092 0.038 0.031 0.037 0.066 0.007 0.005 0.045

± ± ± ± ± ± ± ± ± ± ± ±

0.019 0.0002 0.00008 0.0003 0.006 0.003 0.003 0.004 0.008 0.0006 0.0003 0.003

chloroquine (IC50 ¼ 0.010 mM). Parasite growth inhibition assays and IC50 determinations were carried out following methods using the SYBR Green I nucleic acid staining dye [9]. After 72 h of growth, the plates were stored frozen at 20  C. For the fluorescence assay, 100 ml of SYBR Green I in lysis buffer was added to each well, and the contents were mixed until no visible erythrocyte sediment remained. After 1 h of incubation in the dark at room temperature, fluorescence was measured with a fluorescence multiwell plate reader (SpectraMax Gemini EM) from Molecular Devices, with excitation and emission wavelength bands centered at 485 and 530 nm, respectively by using the accompanying SOFTmaxPro software to yield fluorescence counts for analysis. The counts were plotted against the logarithm of the drug concentration and curve fitting was performed by nonlinear regression using Sigma Plot to yield the drug concentration that produced 50% of the observed decline from the maximum counts in the drug-free control wells (IC50). 4.2. Cytotoxicity assay Cells L6 (a primary cell line derived from rat skeletal myoblasts) were plated at a density of 50,000 cells/well of 96-well plates in 100 ml of RPMI 1640 medium supplemented with 1% L-glutamine (200 mM) and 10% fetal bovine serum, allowed to adhere for 3 h. Serial drug dilutions a range from 10 to 0.01 mg/ml were prepared. After 72 h of incubation, the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. 10 ml of Alamar Blue solution was then added to each well and the plates incubated for another 2 h at 37  C and fluorescence was read using a Spectramax Gemini EMmicroplatefluorometer (excitation: 536 nm, emission: 588 nm). To determine TC50 values, fluorescence was platted against inhibitor concentration. TC50 was determined as the concentration at which cytotoxicity (fluorescence) was half that in the absence of inhibitor. Acknowledgments n, Ciencia y Empresa of We thank The Consejería de Innovacio the Junta de Andalucía (Excellence Research Project no. P07-CTS n of 03210 and BIO-199), The Ministerio de Ciencia e Innovacio

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 n of Spain (FPU Spain (SAF2009-11955), the Ministerio de Educacio grant to B.R-R, AP2007-03115) and the RICET FIS Network (RD12/ 0018/0017) for the financial support.

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In vitro antiplasmodial and cytotoxic activities of asymmetrical pyridinium derivatives.

An in vitro investigation of the antiplasmodial and cytotoxic activities of a series of human choline kinase inhibitors against Plasmodium falciparum ...
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