Interaction of Chalcones with Ferriprotoporphyrin IX. Lee M.S. Elena

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1 Interaction of Chalcones with Ferriprotoporphyrin IX Lee M.S. Elena Department of Pharmacy, ational University of Singapore 10, Kent Ridge Crescent, Singapore ABSTRACT Chalcones are a group of compounds with various substitution patterns on the two aromatic rings of 1,3-diphenyl-2-propen-1-one. Many chalcones are found to possess antimalarial activity. In this project, the binding affinity of a selected number chalcones to ferriprotoporphyrin IX (FP) is studied. The aims of this investigation are two-fold: firstly, to establish if chalcones bind to FP, and secondly, if the binding affinity is correlated to antimalarial potency. The binding affinity of the chalcones is studied using absorption spectroscopy. The change in FP absorbance upon incubation with chalcones is monitored. A decrease in the FP absorbance is observed in 10 out of the 23 chalcones investigated. This is interpreted to mean that these chalcones bind to FP. Generally, chalcones with naphthalene as Ring A and/or 2-hydroxyphenyl as Ring B demonstrate good affinity for FP. However, it is found that these chalcones do not have good antimalarial activity (low IC 50 ). Thus, no correlation between FP binding and antimalarial potency is observed. The antimalarial activities of chalcones may be mediated by other means. ITRODUCTIO Chalcones are a group of compounds with the following structural formula: O R Ring A Ring B Fig.1: Chalcone The antimalarial activity of chalcones have been widely reported. 2-4 However, the mode of action of these chalcones in malaria remains uncertain. In this project, the possibility of chalcones acting as antimalarial agents by interfering with the breakdown of ferriprotoporphyrin IX (heme) is explored. We would like to find out if the antimalarial activity of chalcones is correlated to ferriprotoporphyrin IX (FP) or heme binding. The antimalarial agent CQ (and possibly other aminoquinolines) is widely thought to act by interfering with the breakdown of FP in the food vacuole of the parasite. 1

2 In this investigation, various chalcones which have been shown to be antimalarial agents of differing potencies (as evaluated by their in vitro inhibition of [ 3 H] hypoxanthine incorporation into P.falciparum) are investigated for their binding to hematin (an OH analogue of FP) by monitoring changes to its Soret band. The aims of this investigation are two-fold: firstly to establish if chalcones bind to FP, and secondly, if binding affinity is correlated to antimalarial potency. Chalcones, which are selected for this study, are synthesized by a graduate student. They are characterized by the presence of a heteroaromatic (pyridine, quinoline) ring or bicyclic (naphthalene) ring as Ring A. Ring B is either alkoxylated or hydroxylated. Table 1 gives the structures of the compounds investigated and their IC 50 for the inhibition of [ 3 H] hypoxanthine uptake which has been reported previously. Ring A Substituent on Ring B, R IC 50 (µm) 2,3,4-trimethoxy quinolinyl 4-quinolinyl 2,4-dimethoxy methoxy ethoxy ,4-dihydroxy hydroxy hydroxy ,3,4-trimethoxy ,4-dimethoxy methoxy ethoxy ,4-dihydroxy hydroxy hydroxy A 2,4-dimethoxy ,4-dihydroxy naphthalenyl 4-hydroxy ,4-dihydroxy hydroxy naphthalenyl 2-hydroxy ,4-dihydroxy hydroxy pyridinyl 2-hydroxy 31.0 Table 1: IC 50 values for inhibition of [ 3 H] hypoxanthine uptake into Plasmodium falciparum (K1) in presence of drug. IC 50 for CQ = µm. 2

3 MATERIALS AD METHOD Chloroquine (CQ) and FP (porcine hematin) were obtained from the Sigma Chemical Co. Buffer solution was prepared from 43% methanol in 10mM sodium acetate, ph 5.5. Stock solutions of 2 mm hematin in 0.1M aoh, 2mM or 0.2mM chalcones in methanol (HPLC grade), and 2mM chloroquine in buffer were prepared. The interaction of the chalcones with FP was examined by monitoring their effect on the absorption profile of FP. Samples were prepared in buffer, and visible absorption spectra were collected using a Hewlett Packard 8453 spectrophotometer. Chalcones were added from stock solutions in methanol whereas CQ was added from stock solution in buffer. Concentrations were corrected for dilution. Buffer is used as a blank and the absorption due to the drugs was recorded for later subtraction. RESULTS AD DISCUSSIOS a) Structural Features of Chalcones Majority of the chalcones do not cause decreases in the absorbance of FP to the same degree as CQ ( = 57% at 128 µm). As an arbitrary guide, chalcones which had percent decreases greater than 15% at 128 µm chalcone were classified as those that bind to FP. When classified in this way (Table 2), only ten chalcones were found to bind to FP. Ring B Ring A 3Q 4Q 1 2 2P TM X X na na na DM X X (53%) na na M X X na na na E X X na na na DH X X (87%) (45%) (32%) 4H X X (48%) (26%) X 2H (32%) (63%) na (59%) (17%) Table 2: Chalcones that show binding with FP. The numbers in parentheses are percent decrease of FP absorbance at 128 µm chalcone. binding is defined when the percent decrease is beyond 15%; na = compound not available. It can be seen from Table 2 that binding is strongly associated with chalcones having a 2-hydroxylphenyl B ring. When ring B is 2-hydroxyphenyl, binding was observed for all the available ring A derivatives (3 and 4-quinolinyl, 2-naphthalenyl, 2- pyridinyl). In the case of Ring A, the quinolinyl derivatives are not good candidates for binding. Except for chalcones that had 2-hydroxyphenyl as ring A, all the other quinolinyl chalcones with other substitutions on Ring A did not show binding affinity to FP. The same cannot be said for the naphthalenyl Ring B derivatives. Of those available for study (Ring B = 4-hydroxyphenyl, 2,4-dihydroxyphenyl, dimethoxyphenyl), all 3

4 showed binding to FP. This may be related to the fact that the naphthalene ring is not a polar ring system. The quinoline rings are of the same size as naphthalene but are π deficient. The pyridine ring is smaller and π deficient. In the case of pyridinyl derivatives, binding was observed only for the 2-hydroxy and 2,4-dihydroxy Ring B analogues, but not 4-hydroxy Ring B analogue. b) FP Binding versus Antimalarial Potency, IC 50 Secondly, the correlation between binding to FP and antimalarial potency was considered. It is seen that the chalcones that exhibited binding to FP had moderate antimalarial activity. DHI which caused the greatest decrease in absorbance (87%) at 128 µm has been reported to have an IC 50 (for inhibition of [ 3 H] hypoxanthine uptake into Plasmodium falciparum in presence of drug) of 24.8 µm. DH2P with an IC 50 of 19.7 µm caused only a 32% drop in absorbance. Moreover the compounds identified to have good in vitro activity (TM3Q, IC 50 2 µm) did not show any change in absorbance of FP. Thus it is concluded that the antimalarial activity of chalcones is not related to their interference with the conversion of FP to hemozoin. evertheless, this investigation has identified several 2-hydroxychalcones that bind strongly to FP but are poor antimalarial agents. The 2-hydroxyphenyl ring B present in these compounds may enhance the planarity of the molecule by intramolecular hydrogen bonding with the carbonyl group. Moreover, most of these chalcones have heteroaromatic (quinoline, pyridine) /bicyclic (naphthalene) rings A which are intrinsically planar. Most molecules that bind to FP do so via π-π donor acceptor complexes involving rings which are planar and electron rich. Literature indicates that π- stacking normally involves an aromatic phenyl ring and the aromatic porphyrin ring of FP. This is thought to disrupt the formation of a bond between the iron of one porphyrin and a carboxylate group of an adjacent porphyrin. This bond is required for the formation of hemozoin crystal. It is not clear why these chalcones bind to FP but are still poor antimalarials. It could be that the resulting complex is not stable and dissociates readily. This can be confirmed by determining the equilibrium dissociation constant of the complex and by using molecular modeling to investigate the association of these chalcones with FP. COCLUSIO This investigation has shown that chalcones with 2-hydroxyphenyl ring B and quinoline, naphthalene and pyridine as Ring A cause a decrease in the absorbance of the Soret band of FP at ph 5.5. This is interpreted to mean that these chalcones bind to FP. However, there is no correlation between the binding phenomenon and the antimalarial activity of these compounds. It is also seen that chalcones with good antimalarial activity do not show any change in the absorbance of the Soret band of FP. The antimalarial acitivities of these compounds are likely to be mediated by other means. 4

5 ACKOWLEDGEMETS I wish to express my deepest gratitude and appreciation to Associate Professor Go Mei Lin, my supervisor, for spending countless hours clearing my doubts and molding my trains of thought into coherent arguments. Besides, I want to give special thanks to Liu Mei, a postgraduate, who had guided me patiently and motivated me to reach greater heights in research. REFERECE 1. Chou et al, Ferriprotoporphyrin IX fulfills the Criteria for Identification as the Chloroquine Receptor of Malaria Parasites. Biochem. 19: , Rongshi Li, George L.Kenyon, Fred E.Cohen, In Vitro Antimalarial Activity of Chalcones and Their Derivatives, J. Med. Chem. 38, , Jose. Dominguez, Jaime E. Charris, Gricela Lobo, Synthesis of Quinolinyl Chalcones and Evaluation of Their Antimalarial Activity, Eur. J. Med. Chem., 36, , Mei Liu, Prapon Wilairat, and Mei Lin Go, Antimalarial Alkoxylated and Hydroxylated Chalcones: Structure-Activity Relationship Analysis, J. Med. Chem., 44, , Andrew M. W. Stead, Patrick G. Bray, I. Geoffrey Edwards, Diamidine Compounds: Selective Uptake and Targeting in Plasmodium falciparum, Mol Pharmacol , Martha Kalkanidis, ectarios Klonis, Leann Tilley, Leslie W. Deady, ovel phenothiazine antimalarials: synthesis, antimalarial activity, and inhibition of the formation of β-haematin, Biochemical Pharmacology 63, , Anthony D. Wright, Huiqin Wang, Marion Gurrath, Inhibition of Heme Detoxification Processes Underlies the Antimalarial Activity of Terpene Isonitrile Compounds from Marine Sponges, J. Med. Chem, 44, ,