Investigations on the antimalarial activity of alkoxylated and hydroxylated chalcones 1

INVESTIGATIONS ON THE ANTIMALARIAL ACTIVITY OF ALKOXYLATED AND HYDROXYLATED CHALCONES LIU MEI (B.Eng., China Pharmaceutical University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2003 To my parents ACKNOWLEDGEMENTS I am greatly indebted to my supervisor, Associate Professor Go Mei Lin, for her constant encouragement, patient guidance, generous help and constructive criticism throughout the whole period of this research. Simply speaking, this work would be mission impossible without her. I am deeply grateful to Professor Prapon Wilairat, Chair of Department of Biochemistry in Mahidol University, Thailand, for his tremendous generosity and hospitality in providing the key screening facilities for this project. Special thanks are extended to Dr. Tan May Chin, Theresa, Assistant Professor in Department of Biochemistry, whose wise advice has been invaluable always. My gratitude also goes to the National University of Singapore for awarding me the Research Scholarship, and, all the lecturers, technical and administrative staff, fellow students and friends in Department of Pharmacy for their great assistance and friendship, in particular, Associate Professor Lim Lee Yong, Dr. Shanti, Miss Ng Sek Eng, Madam Oh Tang Booy, Miss Zhou Qingyu, Mr. Sam Wai Johnn and Mr. Wu Xiang. Sincere appreciation also goes to Dr. Philip J. Rosenthal in University of California, San Francisco, and Dr. Simon Croft in London School of Hygiene and Tropical Medicine for their kind help and sharing their knowledge in biochemical studies in this project. Last but not the least, I would like to express my heart-felt gratitude to my family, especially my beloved Baba and Mama, and one anonymous close friend. Without their immeasurable understanding and sharing the joys and frustration all the way, I would never have been to this cheerful moment. i TABLE OF CONTENTS TITLE ACKNOWLEDGEMENT i TABLE OF CONTENTS ii SUMMARY vii PUBLICATIONS AND PRESENTATION ix TABLE OF CONTENTS SECTION ONE: INTRODUCTION 1-25 1.1 Malaria as a Health Problem 1.2 Targets for Antimalarial Drug Discovery 1.2.1 Targets present in the digestive vacuole of the parasite 1.2.2 Targets involved in the synthesis of macromolecules and metabolites 1.2.3 Targets involved in membrane processes and signaling 1.3 Chalcones 13 15 1.3.1 Antimalarial activity 16 1.3.2 Antileishmanial activity 22 1.3.3 Immunosuppressive activity 22 1.3.4 Anticancer properties 23 SECTION TWO: AIM OF THESIS 26-28 SECTION THREE: DRUG DESIGN AND SYNTHESIS OF TARGET COMPOUNDS 3.1 Introduction 29-42 29 ii 3.2 Rationale of Drug Design 29 3.3 Chemical Considerations 33 3.3.1 Mechanism of reaction 33 3.3.2 Characterization of chalcones 35 3.3.3 Determination of the 13C NMR chemical shift of carbonyl carbon in trimethoxychalcones 3.4 Experimental Methods 36 38 3.4.1 General experimental methods 38 3.4.2 Syntheses of alkoxylated chalcones 39 3.4.3 Syntheses of hydroxylated chalcones 40 3.4.4 Method for determining 13C NMR of carbonyl group 41 SECTION FOUR: EVALUATION OF ANTIMALARIAL ACTIVITY 43-57 4.1 Introduction 43 4.2 Experimental Methods 44 4.2.1 In vitro evaluation of antimalarial activity using P. falciparum (K1) 44 4.2.2 In vivo evaluation of antimalarial activity using mice infected with P. berghei (ANKA) 46 4.2.3 Evaluation of cytotoxicity 46 4.3 Results and Discussion 47 4.3.1 In vitro results 47 4.3.2 In vivo Results 52 4.3.3 Selectivity of antimalarial activity 54 4.4 Conclusion 56 iii SECTION FIVE: STRUCTURE ACTIVITY RELATIONSHIPS 5.1 Introduction 58 5.2 Selection and Determination of Physicochemical Parameters 58 5.2.1 Selection of parameters 58-98 58 5.2.2 Determination of physicochemical parameters of chalcones by experimental methods 62 5.2.2.1 Determination of lipophilicity by reversed phase HPLC 62 5.2.2.2 Determination of the 13C NMR chemical shift of the carbonyl carbon 63 5.2.2.3 Determination of physicochemical parameters of chalcones by molecular modeling methods 63 5.3 Multivariate Analysis of Structure Activity Relationships (SAR) of Antimalarial Chalcones 63 5.3.1 Introduction 63 5.3.2 Statistical methods 65 5.3.3 Results and discussion 65 5.3.3.1 Principal component analysis (PCA) 65 5.3.3.2 Projection to latent structures (PLS) analysis 68 5.3.3.2.1 Trimethoxychalcones 69 5.3.3.2.2 Methoxychalcones 74 5.3.3.2.3 Dimethoxychalcones and ethoxychalcones 5.3.3.2.4 Hydroxy and dihydroxychalcones 75 76 5.3.3.2.5 Active hydroxylated and alkoxylated chalcones 77 5.3.3.3 Summary of findings 80 iv 5.4 Comparison of Structure-Activity Relationships between Antileishmanial and Antimalarial Chalcones 80 5.4.1 Introduction 80 5.4.2 Experimental methods 81 5.4.2.1 Determination of in vitro antimalarial activity 81 5.4.2.2 Determination of in vitro antileishmanial activity 81 5.4.2.3 Determination of physicochemical properties of chalcones by molecular modeling methods 82 5.4.2.4 Statistical and Correlation Analyses 82 5.4.2.5 Comparative molecular field analysis (CoMFA) 83 5.4.3 Results and discussions 83 5.4.3.1 Structural requirements for antileishmanial and antimalarial activities 83 5.4.3.2 Comparative molecular field analysis (CoMFA) of antimalarial and antileishmanial chalcones 5.4.4 Summary of findings 5.5 Conclusion 86 91 92 SECTION SIX: MODE OF ANTIMALARIAL ACTIVITY OF CHALCONES 99-112 6.1 Introduction 99 6.2 Materials and Methods 99 6.2.1 Degradation of [14C] methemoglobin by extracts of P. falciparum (K1) 100 6.2.2 Inhibition of falcipain-2 100 6.2.3 Effect on Soret band of hematin 101 v 6.3 Results 102 6.3.1 Effects on the enzymatic activity of a crude plasmodial extract catalyzing the breakdown of radiolabelled methemoglobin 102 6.3.2 Effects on falcipain-2 and associated changes in the food vacuole on incubation 103 6.3.3 Binding to heme 105 6.4 Discussion 110 6.5 Conclusion 111 SECTION SEVEN: CONCLUSION 113-115 APPENDICES A1-A16 Table A1-A11 Table A12-A15 Table A16 BIBLIOGRAPHY I-VIII vi SUMMARY The objective of this thesis is to establish structure-activity correlations for the antimalarial activity of chalcones and to investigate their possible modes of action against Plasmodium. To this end, 105 chalcones were synthesized by base-catalyzed Claisen-Schmidt condensation and evaluated for their ability to inhibit hypoxanthine uptake into P. falciparum (K1) trophozoites. Physicochemical parameters encompassing steric, lipophilic and electronic properties were experimentally determined or obtained in silico from molecular modeling methods. Structure-activity relationships were established using multivariate tools (principal component analysis, partial least squares projection to latent structures), multiple linear regression and comparative molecular field analysis (CoMFA). The structural requirements for antimalarial activity vary according to the nature of ring B in the chalcone framework. Among the alkoxylated chalcones, differing requirements were found depending on whether ring B is substituted with trimethoxy, dimethoxy or methoxy groups. Greater homogeneity was detected among the hydroxylated chalcones. One surprising observation was that active chalcones (defined as those with IC50 < 10 µM for alkoxylated chalcones and IC50 < 20 µM for hydroxylated chalcones) obtained from different classes share similar physicochemical characteristics, namely a preference for a large-size ring B and ring A substituted with electron withdrawing groups. The chalcones were also found to be antileishmanial in tests against Leishmania donovani amastigotes. Different structural requirements were found for antimalarial and antileishmanial activities. Antileishmanial activity was favored by hydroxylated chalcones carrying large-size ring A in contrast to antimalarial activity which is found predominantly among alkoxylated chalcones with specific requirements vii for both rings A and B. Despite these limitations, two chalcones (8 and 19) were found to combine good antimalarial and antileishmanial activities, with being of particular interest as it was found to increase survivability of P. berghei ANKA infected mice at 100 mg/kg. The chalcones were not cytotoxic against the KB3-1 cell line at 20 and 40 µM, indicating specificity in their antimalarial action. However, there were indications of toxicity against mice macrophages revealed during the course of antileishmanial testing that require further attention. The chalcones were found to inhibit several processes involved in hemoglobin degradation in the Plasmodium digestive vacuole, namely interference with the enzymatic activity of a crude plasmodial extract on the breakdown of methemoglobin, inhibition of recombinant falcipain-2, and binding to hematin. It appeared unlikely that these inhibitory effects contributed significantly to the antimalarial activity of chalcones as there was no discernible trend between the various inhibitory activities and in vitro antimalarial potency. It appears likely that other pathways are affected by the chalcones. viii SECTION FOUR EVALUATION OF ANTIMALARIAL ACTIVITY 4. EVALUATION OF ANTIMALARIAL ACTIVITY 4.1 Introduction This section of the thesis reports on the evaluation of the antimalarial activities of the synthesized chalcones. The synthesized compounds were tested for their ability to inhibit the uptake of radiolabelled hypoxanthine into P. falciparum (K1) trophozoites, a chloroquine resistant strain of Plasmodium. The assay is based on the fact that malarial parasites are not capable of de novo synthesis of purines from nonnucleotide precursors and are dependent on salvage pathways for their supply of preformed purine bases. 111 The concentration required to inhibit hypoxanthine uptake by 50% (IC50) was determined for each compound and members that showed promising activity (“actives”) were selected for further testing in mice infected with P. berghei ANKA (a chloroquine-sensitive strain). In general, active compounds have IC50 values < 10 µM (alkoxylated chalcones) and < 20 µM (hydroxylated chalcones). The different criteria are necessary because hydroxylated chalcones are generally less active than their alkoxylated analogues. The actives chalcones are among the top 16% - 36% of each class, with the exception of the 4’-ethoxychalcones and 2’hydroxychalcones. Experimental models of murine Plasmodium are a convenient means of evaluating the in vivo effects of potentially useful antimalarial agents and are widely employed in the screening of antimalarial compounds, despite reservations over their usefulness and validity. 112 It is recognized that the human immune system is different from that of mice, and that drugs may have different pharmacokinetic profiles in man and mice. On the other hand, all mammalian Plasmodium species have similar life cycles and are broadly sensitive to the same drugs, thus justifying the use of murine models for testing. 43 The specificity of the antimalarial action of chalcones is also reported in this section. Since an antimalarial agent is necessarily cytotoxic with respect to the plasmodial life stages in the erythrocyte, it is important to establish that its cytotoxic effects not extend to the cells of the host. Thus the toxicity of the chalcones against the KB3-1 cell line is evaluated using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H-tetrazolium bromide] assay. 113 Cells with functional mitochondria are capable of reducing the yellow tetrazolium dye (MTT) into insoluble purple formazan crystals, which are solubilized by the addition of a detergent. The amount of formazan formed is quantified by spectrophotometric means and is correlated to the viability of the cells. Compounds that are cytotoxic will compromise the ability of the cells to carry out the reduction of the tetrazolium and less formazan will be formed accordingly. 4.2 Experimental Methods 4.2.1 In vitro evaluation of antimalarial activity using P. falciparum (K1) The in vitro antimalarial activities of the chalcones were evaluated by the method of Desjardins, R.E. et al. 114 with modifications. Briefly the assay measures the incorporation of [3H] hypoxanthine by the parasites and the inhibition of the incorporation in the presence of the test compound. A strain of chloroquine resistant (K1) P. falciparum was used in the assay. The parasites were grown in hypoxanthine-free medium prior to the addition of test compounds. The test compounds were dissolved in DMSO and serially diluted 10 fold with complete culture media (RPMI-1640, 5% sodium bicarbonate, and 10% normal type “O” human serum to give a 106-fold concentration range. The diluted drugs (25 µl) were transferred to wells in a 96-well microtitre plate, together with 200 µl of 44 parasitized erythrocytes (1-2% parasitemia and 1.5% hematocrit) and the whole was incubated at 37oC, 24 h in a candle jar. The control wells in each plate contained 25 µl of complete medium and the same amount of DMSO in the drug aliquot instead of test compound. Chloroquine was also tested as a positive control. Another drug that was used as positive control was 2,4-dimethoxy-4’-butoxychalcone (41), which has been reported to possess outstanding antimalarial activities against both human (in vitro) and rodent (in vivo) parasites. 70 After 24 h, 25 µl of [3H] hypoxanthine was added and the plates were incubated for an additional 24 h, after which the cells were filtered onto glass fiber filters (Whatman 934-AH) and counted in a scintillation counter. For each concentration of each test compound, the assay was performed in triplicate before the concentration-response profile of each compound was determined and analyzed by a nonlinear, logistic dose response program to give its IC50 which is the concentration of test compound required to inhibit [3H] hypoxanthine uptake by 50% compared to the control. Figure 4.1 Illustration of in vitro evaluation of antimalarial activity (Reproduction permission from Department of Biochemistry, Mahidol University, Thailand) 45 4.2.2 In vivo evaluation of antimalarial activity using mice infected with P. berghei (ANKA) The in vivo test measures the survival of mice following administration of drug. Swiss albino mice (male, weeks, approximately 25 g) were inoculated intraperitoneally with 107 parasitized erythrocytes (P. berghei ANKA). The test compound was given intraperitoneally (ip), at a daily dose of 100 mg/kg in DMSO, for consecutive days after the day of infection (Day 0). Each compound was tested against mice. Three groups of infected control mice were maintained and they were given chloroquine (52 mg/kg, ip, 0.5% Tween buffer solution, pH 7.4, only on Day 1), DMSO (days 1-3) or 41 (2,4-dimethoxy-4’-butoxychalcone, 100 mg/kg in DMSO, ip, days 1-3). Thin blood smears were made from the tail blood of the mice from Days 114, or until their demise. The blood smears were fixed with 5% Giemsa, examined microscopically and graded according to WHO protocol for evaluating the degree of parasitemia. 115 Control infected mice treated with DMSO or chloroquine would normally perish within days and 14-16 days respectively. Mice that received 41 lived on the average for 8-9 days. 4.2.3 Evaluation of cytotoxicity Cell viability was assessed on the KB3-1 cell line using the microculture tetrazolium assay described by Alley et al 113 with some modifications. KB3-1, a drug sensitive cervical carcinoma epithelial cell line, was a gift from Dr. Caroline Lee, Department of Biochemistry, National University of Singapore. The cells were cultured to 60-70% confluence in tissue culture flasks containing Dulbecco’s Modified Eagle’s Medium (supplemented with 10% fetal bovine serum) at 37oC in a humidified 46 atmosphere with 5% CO2. On attaining confluence, the cells were trypsinized and aliquots (100 µl) of cells in growth medium were transferred to 96-well tissue culture plates, at densities of × 10 – x 10 cells /ml. After 24 h incubation, the cells were attached to the wells and could be used for test procedures. The cells remained in the exponential growth phase during this period. Stock solutions of test compounds were prepared in DMSO (2-4 mM) and serial dilutions were made so that the final sample concentration in each well (total volume of 200 µl, maximum of 1% v/v DMSO) was 20 – 80 µM. The test sample was incubated with the cells for 15 h under the usual conditions, with eight replicates for each concentration. Wells without cells and those with only cells in culture media were examined in parallel. At the end of the incubation period, the medium was decanted, the cells were washed carefully with phosphate buffer solution, after which an aliquot of MTT (100 µl, 0.5 mg/ml in pH 7.4 HBSS-HEPES buffer) was added to each well and incubated for a further h. The medium was then removed from each well by careful pipetting. DMSO (150 µl) was quickly added to lyse the cells and dissolve the purple formazan crystals. The absorbance of the formazan product was measured after 10 at 590 nm using a microtitre plate reader. The absorbance values obtained at each concentration were averaged, adjusted by subtraction of blank values and expressed as a percentage of the average absorbance obtained from control incubations (absence of sample). 4.3 Results and Discussion 4.3.1 In vitro results The IC50 values of the chalcones for the inhibition of [3H] hypoxanthine uptake into infected erythrocytes are given in Table 4.1. 47 Table 4.1 Structure and in vitro antimalarial activity of chalcones O R R' Ring B Compound No. 6b 11b 12b 13b 27b, c 28b, c 35b 36b 40b 128b 129b 130b 131b 132b 133b 134 19 22b 23 31b,c 32b,c 38 111 112b 113 114 115 116 117 135 201 202b,c 203 204 205c 206 207c 208b,c 209c 210b,c 211 231 232 233c 234 235 236 237 238 239 241c R’ 2’,3’,4’-trimethoxy 4’-methoxy 2’,4’-dihydroxy 2’-hydroxy R 2,4-dichloro 4-dimethylamino 4-trifluoromethyl 2,4-dimethoxy 4-methyl 4-ethyl 3-quinolinyl 4-quinolinyl 4-methoxy 4-fluoro 4-phenyl 2,4-difluoro 4-nitro 3,4-dichloro 4-chloro 2-chloro 3-chloro H 4-hydroxy 2,4-difluoro 4-methoxy 3-quinolinyl 4-quinolinyl 4-fluoro 2,4-dichloro 4-trifluoromethyl 2,4-dimethoxy 4-methyl 4-nitro 4-dimethylamino 4-cyano H 2,4-dichloro 3-quinolinyl 2,4-difluoro 2,4-dimethoxy 1-naphthalenyl 4-trifluoromethyl 2-pyridinyl 2-naphthalenyl 4-pyridinyl 4-quinolinyl 4-chloro 2,4-dichloro 4-dimethylamino 3-quinolinyl 4-chloro 4-methyl 4-methoxy 2,4-dimethoxy 4-trifluoromethyl 4-fluoro 2-pyridinyl IC50a (µM) 5.4 18.0 3.0 16.5 25.6 16.5 2.0 60.0 25.0 9.5 26.2 18.5 22.5 14.5 14.5 41.5 24.4 15.8 7.0 26.8 21.7 4.8 43.0 14.4 16.0 19.0 6.4 70.0 100.0 70.0 94.5 55.5 68.5 16.1 16.0 56.4 24.8 26.5 19.7 20.0 121.6 92.8 12.3 35.5 188.0 28.0 12.9 62.5 61.5 25.5 35.5 47.0 31.0 Ring A Compound No. 41d 8b 29b,c 30b,c 101 102 103 104 105 106 107b 108b 109 110c 25b 26 33b,c 34b,c 39b 121 122b 123 124b 125 126 127b 136 212b,c 213b,c 214c 215c 216b,c 217 218 219 220 221 222 223b 224 225 226 227 228 229 230 242c 243c 244 R’ 4’-butoxy 2’,4’-dimethoxy 4’-ethoxy 4’-hydroxy 2’-hydroxy R 2,4-dimethoxy 2,4-dichloro 4-trifluoromethyl 2,4-difluoro 2,4-dimethoxy 4-ethyl 3-quinolinyl 4-quinolinyl 4-methyl 4-methoxy 4-dimethylamino 4-fluoro 4-chloro 4-bromo 2-chloro-4-fluoro 3,4-dichloro 4-nitro 1-naphthalenyl 2,4-difluoro 4-methoxy 3-quinolinyl 4-quinolinyl 4-fluoro 2,4-dichloro 4-trifluoromethyl 2,4-dimethoxy 4-methyl 4-nitro 4-dimethylamino 4-cyano H 1-naphthalenyl 3-quinolinyl 2-pyridinyl 4-quinolinyl 2-naphthalenyl 4-chloro 2-chloro 3-chloro 4-methoxy 4-methyl 3-methyl 4-butyl 4-trifluoromethyl 4-nitro 4-fluoro 3,4-dichloro 4-dimethylamino 2,4-dichloro H 1-naphthalenyl 2-naphthalenyl 4-quinolinyl a. IC50 values for inhibition of [3H]hypoxanthine uptake into P. falciparum (K1) in presence of drug. IC50 for chloroquine = 0.265 µM. All readings are the average of or more separate determinations. b. Compounds not listed in Chemical Abstract Databases (1967 to date). c. Ring A = heteroaromatic or polycyclic aromatic ring. The nature of the ring is given in R. d. 2,4-Dimethoxy-4’-butoxychalcone 70 48 IC50a (µM) 108.0 18.8 5.9 6.2 2.1 2.4 2.2 27.0 93.8 128.5 55.3 322.0 342.0 542.5 600.0 297.5 415.0 320.0 28.1 33.0 24.9 100.0 24.1 96.0 24.0 30.0 38.0 39.0 30.0 540.0 43.0 39.9 41.0 16.3 51.0 27.5 38.0 61.7 33.1 32.2 25.4 25.8 20.9 30.4 20.4 21.7 18.4 17.7 24.5 29.6 32.5 29.5 Not Done Some obvious trends are evident from these results and they are discussed in the following paragraphs: First, the hydroxylated chalcones are generally less active than their alkoxylated counterparts. A comparison of 4’-hydroxylchalcones and 4’methoxychalcones shows that there are compounds bearing similar ring A substituents. Of these, only one compound (221) in the 4’-hydroxy series has a lower IC50 values than the corresponding 4’-methoxy derivative (114). A same trend is observed among the 2’,4’-dihydroxy and 2’,4’-dimethoxy chalcones: of the compounds bearing similar rings A, only compounds (205, 211) in the 2’,4’dihydroxy series are better than their 2’,4’-dimethoxy counterparts (110, 105). The most active compound among the hydroxylated chalcones is 4-chloro-2’,4’-dihydroxy chalcone (211) with an IC50 of 12.3 µM. This is in comparison with the most active alkoxylated chalcone, 1-(2’,3’,4’-trimethoxyphenyl)-3-(3-quinolinyl)-2-propen-1-one (27), which has an IC50 of µM. Among the alkoxylated chalcones, there are 12 compounds (2, 3, 5, 6, 7, 8, 19, 27, 29, 31, 36, 113) with IC50 values below 10 µM (12 out of 62 = 19.35%). Seven out of 43 hydroxylated chalcones (16.3%) have IC50 values between 10 µM and 20 µM (202, 203, 207, 211, 214, 227, 228). Another observable trend is the exceptionally poor activity of members in the 4’-ethoxy series (n =13). Unlike the equitable representation of trimethoxy, dimethoxy and methoxy chalcones among the “actives” in the alkoxylated series, no 4’-ethoxy derivative is observed to be present. Thirdly, an interesting difference is observed in the activities of the 3quinolinyl and 4-quinolinyl chalcones. Where comparison permits, better activity is consistently observed for the 3-quinolinyl derivative. The difference in activity ranges from 30-fold for the trimethoxy series to 4-fold for the 4’-ethoxy series (Table 4.2). 49 Table 4.2 Structure and in vitro antimalarial activity of “hybrid” chalcones O Ring A R' Ring B Ring A N 3-quinolinyl N 4-quinolinyl 1-naphthalenyl 2-naphthalenyl N 2-pyridinyl Substituent of Ring B (R’) IC50 (µM) 2,3,4-trimethoxy 2.0 2,4-dimethoxy 2.2 4-methoxy 4.8 4-ethoxy 24.9 2,4-dihydroxy 16.1 4-hydroxy 41.0 2-hydroxy 28.0 2,3,4-trimethoxy 60.0 2,4-dimethoxy 27.0 4-methoxy 43.0 4-ethoxy 100.0 2,4-dihydroxy 92.8 4-hydroxy 51.1 2-hydroxy Not Done 2,4-dimethoxy 320.0 2,4-dihydroxy 24.8 4-hydroxy 39.9 2-hydroxy 32.5 2,4-dihydroxy 20.0 4-hydroxy 27.5 2-hydroxy 29.5 2,4-dihydroxy 19.7 4-hydroxy 16.3 2-hydroxy 31.0 One difference between the isomeric quinolinyl chalcones can be traced to their electronic character. As seen from Figure 4.2, the electron withdrawing effect of the carbonyl oxygen can be transmitted by conjugation to the azomethine nitrogen in 4quinolinyl chalcones. Such an effect is not possible in the 3-quinolinyl chalcones (Figure 4.3). 50 Figure 4.2 The canonical structures of 4-quinolinyl chalcones Ring B R' O O _ R' + N N Ring A O _ R' O _ R' + N + N Figure 4.3 The canonical structures of 3-quinolinyl chalcones O O R' N Ring A R' N Ring B O R' N The chemical shifts of the carbonyl carbon of trimethoxychalcones bearing 3quinolinyl (27) and 4-quinolinyl (28) rings A suggest that these differences exist among the isomeric quinolinyl chalcones. As seen in Table 3.2, Section 3.3.3, the chemical shift differences (∆δ = δR - δH) of the carbonyl carbon in 28 and 27 were found to be –1.329 ppm and 0.040 ppm respectively. Negative sign associated with 28 suggests that the 4-quinolinyl ring has a stronger electron withdrawing effect, which would be the case if electron delocalization takes place as shown in Figure 4.2. On 51 the other hand, such an effect is absent in the 3-quinolinyl derivative 27 and this might explain its small positive ∆δ value. Although not clearly understood at this juncture, it is apparent that differences in antimalarial activities of the isomeric quinolinyl chalcones can be traced to their electronic properties. Another noteworthy observation is the almost similar activities of the isomeric naphthalenyl chalcones, which is in contrast to the marked differences observed with the isomeric quinolinyl chalcones. This can be seen from a comparison of the following 1-napthalenyl and 2-naphthalenyl derivatives: 203 and 208 (2’,4’hydroxylchalcones); 212 and 216 (4’-hydroxychalcones); 242 and 213 (2’hydroxylchalcones). These observations suggest that the azomethine nitrogen in quinoline plays an important part in accounting for the observed differences in the activities of the isomeric quinolinyl chalcones. Lastly, the IC50 of 2, 4-dimethoxy-4’-butoxy-chalcone (41) had been reported to be 8.9 µM and 14.8 µM against a chloroquine susceptible (3D7) and resistant (Dd2) strain of P. falciparum, 70 but in the present in vitro assay, 41 was found to be a poor inhibitor of hypoxanthine uptake, with an IC50 of 108 µM. This could be attributed to differences in the protocols used for the tests, such as the strain of plasmodia. 4.3.2 In vivo results The active chalcones identified from in vitro tests (7 hydroxychalcones, IC50 < 20 µM; 12 methoxychalcones, IC50 < 10 µM) were tested in mice infected with P. berghei ANKA, a chloroquine susceptible strain of murine malaria. The test compound was administered to mice at a dose of 100 mg/kg, ip, for consecutive days (Days 1-3 post-infection) and their survival times were monitored and compared with control mice receiving DMSO (untreated mice), chloroquine (50 mg/kg, dose) or 41. 52 The in vivo test was also carried out on some randomly selected chalcones (38, 130, 201, 208) that were not “actives”. Control infected mice treated with DMSO or chloroquine would normally perish within days and 14-16 days respectively. Mice that received 41 lived on the average for 8-9 days. The results of the tests are given in Table 4.3. Table 4.3 Survivability of P. berghei ANKA infected mice when treated with chalcones (100 mg/kg, ip, days). IC50 Compound No. (µM) a) 27 2.0 2.1 29 2.2 2.4 3.0 31 4.8 5.4 5.9 6.2 113 6.4 19 7.0 36 9.5 211 12.3 38 d) 14.4 130 d) 14.5 203 16.0 202 16.1 214 16.3 228 17.7 227 18.4 207 19.7 208 d) 20.0 201 d) 68.5 Chloroquine 0.27 41 108 T/Cuntreatedb) 1.91 0.98 1.29 2.33c), 1.48c) 1.46 0.98 0.94 1.67 1.04 0.92 1.41 1.37 1.41 2.20c), 0.96c) 1.47 1.73c), 1.44c) 1.08 0.99 0.94 1.28 1.47 1.91c), 1.87c) 1.32 1.81 1.08 T/C41 b) 1.91 0.98 1.29 2.33c), 1.55c) 1.46 1.03 0.94 1.67 1.04 0.97 1.47 1.37 1.48 1.83c), 1.00c) 1.53 1.78c), 1.50c) 1.11 1.03 0.99 0.81 0.93 1.20c), 1.95c) 1.36 1.71 - T/C CQ b) 0.96 0.50 0.65 1.18c), 0.74c) 0.74 0.49 0.48 0.84 0.52 0.46 0.70 0.69 0.71 1.22c), 0.52c) 0.80 1.23c), 0.78c) 0.77 0.49 0.47 0.71 0.81 1.05c), 1.02c) 0.94 0.57 a) IC50 from in vitro tests. b) T/C untreated = Ratio of average life span of drug treated mice to that of mice receiving DMSO; T/C 41 = Ratio of average life span of drug treated mice to that of mice treated with 41 T/C CQ = Ratio of average life span of drug treated mice to that of mice treated with chloroquine c) Results of separate repeat on groups of mice. Tests were repeated if T/C CQ > in the first instance. d) Randomly selected chalcones which were not identified as “actives” from in vitro tests. 53 The survivability of the mice is assessed from the ratio of the average life span of the drug treated animal to that of untreated animals (T/C untreated), chloroquine-dosed animals (T/C CQ) or 41-dosed animals (T/C 41). A T/C ratio of more than indicates that the drug has increased the survivability of the animal relative to the control group. Particular attention is paid to compounds which had T/C CQ ratios >1, as these compounds are as good as chloroquine in prolonging the lifespan of the infected mice under the conditions of the experiment, not withstanding the fact that the drug dosing regimen was different for CQ and the chalcones. Only compounds fall into this category: and 208. Both compounds have T/C untreated and T/C 41 ratios greater than 1.5. There were other compounds, which had large T/C untreated and T/C 41 ratios (> 1.5), but none of them had favorable T/C CQ ratios. The activity of is anticipated in view of its low IC50 (2.4 µM), but the activity of 208 is surprising as it is not particularly active in vitro (IC50 20 µM). Poor in vitro and in vivo correlation is a common problem in the evaluation of antimalarial potency and may be due to several factors, such as pharmacokinetic (bioavailability, distribution, metabolism) factors and possibly, different responses of murine and human plasmodium to the test compound. The data suggest that 208 may have a much higher bioavailability than some of the other compounds tested. Alternatively, it may be converted to a more active metabolite in vivo. The lack of in vitro – in vivo correlation so commonly noted in antimalarial testing emphasizes the importance of concurrently carrying out in vivo testing against murine strains to confirm in vitro findings and thus to facilitate the identification of suitable lead compounds. 4.3.3 Selectivity of antimalarial activity 54 The same set of “active” antimalarial chalcones were evaluated in the MTT assay, namely alkoxylated chalcones (n =12) with IC50 values < 10 µM and hydroxylated chalcones (n = 7) with IC50 values < 20 µM. Some less active members were also included for comparative purposes, namely 28, 32 and 208. The cell line (KB 3-1) used in this study is a human oral epidermoid carcinoma cell line that does not form tight junctions in monolayer culture. It is also characterized by the absence of efflux proteins that are responsible for moving the drug out of the cell. KB cells are used in this assay because they have an intermediate sensitivity to cytotoxic agents which makes them particularly useful for viability studies. 116 The chalcones showed concentration dependent effects on cell viability when evaluated at two concentrations (20, 40 µM or 40, 80 µM). As seen in Table 4.4, for each chalcone, cell viability is maintained at not less than 60% viability at the concentration used nearer its in vitro antimalarial activity. Thus, most of the chalcones are selectively toxic against Plasmodium when compared to their toxicity against KB cells. Other studies have suggested that the α,β-unsaturated linkage in chalcones may be a target of a Michael-type reaction by biological nucleophiles, thus predisposing them towards cell toxicity. As stated in an earlier Section 1.3.1, licochalcone A forms conjugates with thiol containing peptides. 81 Another chalcone, 1-p-chlorophenyl-4, 4dimethyl-5-diethylamino-1-penten-3-one is reported to react irreversibly with high molecular weight protein thiols but reversibly with small molecular weight thiols like glutathione and cysteine. 117 In contrast, a recent report of some ring B halogenated chalcones designed as trypanocidal and leishmanicidal compounds noted that these compounds were non-toxic to mouse peritoneal macrophages in an MTT assay. 118 These chalcones had no cytotoxic effect when tested at concentrations comparable to 55 their antileishmanial /antitrypanocidal IC50 values (10 to 300 µM), with cytotoxicity to macrophages being evident only at concentrations ≥ 1000 µM. It is evident that the cytotoxicity of the chalcone is influenced by the substitution pattern on rings A and B. Table 4.4 Viability of KB cells when treated with chalcones No. Ring B* Ring A* IC50 (µM) (in vitro antimalarial activity) KB3-1 cell viability % (SD) at 20 µM KB3-1 cell viability % (SD) at 40 µM KB3-1 cell viability % (SD) at 80 µM 27 28 36 2,3,4-trimethoxy 2,4-dichloro 4-trifluoromethyl 3-quinolinyl 4-quinolinyl 4-fluoro 5.4 3.0 2.0 60.0 9.5 88.8 (17.5) 80.8 (15.0) 77.8 (23.5) 126.1 (19.7) 75.7 (29.2) 69.3 (11.6) 70.4 (19.7) 72.4 (24.7) 110.4 (25.5) - 29 2,4-dimethoxy 4-trifluoromethyl 2,4-difluoro 2,4-dimethoxy 4-ethyl 3-quinolinyl 5.9 6.2 2.1 2.4 2.2 99.6 (18.3) 107.8(7.5) 84.0 (20.5) 85.4 (8.2) 82.6 (17.1) 106.4 (7.8) 91.2 (17.9) 60.8 (6.8) 72.9 (16.8) 63.8 (21.2) - 19 31 32 113 4-methoxy 4-hydroxy 3-quinolinyl 4-quinolinyl 2,4-dimethoxy 7.0 4.8 43.0 6.4 78.9(16.7) 82.9(15.2) 121.6(13.3) 60.0 (15.1) 73.0 (11.3) 89.4 (14.0) 81.7 (22.7) - 202 203 207 208 211 2,4-dihydroxy 3-quinolinyl 2,4-difluoro 2-pyridinyl 2-naphthalenyl 4-Chloro 16.1 16.0 19.7 20.0 12.3 - 89.3(28.5) 88.9(22.1) 96.4(19.3) 101.9 (35.4) 100.7(32.0) 88.8 (10.7) 83.6 (16.1) 85.7 (17.9) 109.9 (20.0) 90.8 (26.1) 214 227 228 4-hydroxy 2-pyridinyl 3,4-dichloro 4-dimethylamino 16.3 18.4 17.7 - 112.3 (18.2) 90.6 (23.3) 84.8 (32.2) 85.0 (8.2) 80.2 (13.9) 77.2 (20.0) 0.27 - - 104.1 (17.5) Chloroquine * Substituted phenyl ring if not a heterocyclic or polycyclic ring. 4.4 Conclusion Twenty chalcones were found to inhibit hypoxanthine uptake into P. falciparum K1 trophozoites with IC50 values of or less than 20 µM. The most active compound is 1-(2’,3’,4’-trimethoxyphenyl)-3-(3-quinolinyl)-2-propen-1-one (27) which has an IC50 of µM. When tested in mice infected with P. berghei ANKA, 56 only compounds, namely 1-(2’,4’-dimethoxyphenyl)-3-(4-ethylphenyl)-2-propen-1one (8) and 1-(2’,4’-dihydroxyphenyl)-3-(2-naphthalenyl)-2-propen-1-one (208), were found to increase the survivability of the infected mice at a dose of 100 mg/kg (ip, days). Cytotoxicity tests using the MTT assay against the KB3-1 cell line indicate that the chalcones specifically target Plasmodium, as seen from the viability of the KB3-1 cells (60% and greater) when the chalcones were tested at concentrations greater than their in vitro IC50 values. 57 [...]... P.; Go, M L 61st international congress of International Pharmaceutical Federation (FIP), 20 01 1 Multivariate Analysis of in vitro antimalarial activity of chalcones, Abstract No MC-P-038 2 Heme-binding study of antimalarial hybrid chalcones, Abstract No MC-P-0 41 x SECTION ONE INTRODUCTION 1 INTRODUCTION 1. 1 Malaria as a Health Problem Malaria is undoubtedly one of the most intractable and persistent... described as the “xanthone hypothesis”, the significant increase in antimalarial activity of the rufigallol-exifone combination has been attributed to the production of free radicals from the redox cycling of rufigallol in the food vacuole These reactive oxygen species attack exifone, converting it to a hydroxyxanthone, which is postulated to be the final antimalarial agent Scheme 1. 1 The “xanthone hypothesis”... increased acidification of the digestive vacuole which leads to reduced levels of hematin available for complex formation with CQ 1. 3 Chalcones The term “chalcone” applies to the chemical entity 1, 3-diphenylpropenone (benzylideneacetophenone) and any derivative obtained by substitution of this framework Figure 1. 1 Basic structure of chalcones O R' B A R The aromatic ring nearer the carbonyl carbon is designated... focus of several investigations Chemically, the linkage provides conjugation between the two aromatic rings on both ends of the molecule, confers greater rigidity to the chalcone (compared to the saturated analogue) but still allows the molecule to be flexible and to adopt a more extended conformation Saturation of the double bond appears to have a mixed effect on antimalarial activity, with some investigations. .. 15 19 9 entries (19 66 to May 2003) on the biological properties of chalcones There is obviously a strong interest in the scientific properties of chalcones This may be due to the following reasons Firstly, chalcones are precursors for a vast range of flavonoid derivatives found throughout the plant kingdom Flavonoids have been widely researched 66 They possess a wide array of biological properties and. .. DHODase and the electron transport chain is the target of the antimalarial agent atovaquone Atovaquone, a naphthoquinone derivative, interferes with electron transfer at the level of the cytochrome C reductase complex, thus causing the collapse of the mitochondrial membrane potential 32, 33 The activity of DHODase is also affected because of its dependence on a functional mitochondrial electron transport... groups of cysteine or glutathione This Michael-type reaction would deplete cells of essential thiol-containing entities In fact, this reaction has been cited as the basis of the antibacterial effects of chalcones 80 Scheme 1. 3 The reaction scheme for thiolation of chalcones "R H S O O R' R R' R H O R' SR" O: R SR" H+ R' R 20 Given the toxic potential of the Michael reaction, considerable attention has... substitution pattern on the chalcone skeleton is important to this end but no details were revealed in the report The effect of chalcones on the production of TNF-α may be mediated via their effects on nitric oxide (NO) production Rojas et al reported that some fluorinated chalcones inhibited NO production, not as a result of a direct inhibitory action on enzyme (nitric oxide synthetase) activity but...PUBLICATIONS AND PRESENTATIONS Publications: • Liu, M.; Wilairat, P.; Go, M L Antimalarial alkoxylated and hydroxylated chalcones: structure activity relationship analysis Journal of Medicinal Chemistry 20 01, 44: 4443-4452 • Liu, M.; Wilairat, P.; Croft, S.; Tan, L.C.; Go, M L Structure -Activity Relationships of Antileishmanial and Antimalarial Chalcones Bioorganic & Medicinal Chemistry 2003, 11 : 2729-2738... and are potential drug targets In this context, the catalytic activity of DHODase has received much attention This enzyme catalyzes the only redox reaction in the pyrimidine synthetic pathway (dihydroorotate orotate) As a major source of electrons for the mitochondrial electron transport chain of the parasite, it bridges pyrimidine synthesis and the mitochondrial electron transport system DHODase and . in the food vacuole on incubation 10 3 6.3.3 Binding to heme 10 5 6.4 Discussion 11 0 6.5 Conclusion 11 1 SECTION SEVEN: CONCLUSION 11 3 -11 5 APPENDICES A1-A16 Table 1 A1-A 11 Table 2 A12-A15. (CoMFA) of antimalarial and antileishmanial chalcones 86 5.4.4 Summary of findings 91 5.5 Conclusion 92 SECTION SIX: MODE OF ANTIMALARIAL ACTIVITY OF CHALCONES 99 -11 2 6 .1 Introduction 99. Materials and Methods 99 6.2 .1 Degradation of [ 14 C] methemoglobin by extracts of P. falciparum (K1) 10 0 6.2.2 Inhibition of falcipain-2 10 0 6.2.3 Effect on Soret band of hematin 10 1 vi