John N. Alumasa

4-N-, 4-S-, and 4-O-Chloroquine Analogues : Influence of Side Chain Length and Quinolyl Nitrogen pKa on Activity vs Chloroquine Resistant Malaria

Using predictions from heme-quinoline antimalarial complex structures, previous modifications of chloroquine (CQ), and hypotheses for chloroquine resistance (CQR), we synthesize and assay CQ analogues that test structure-function principles. We vary side chain length for both monoethyl and diethyl 4-N CQ derivatives. We alter the pKa of the quinolyl N by introducing alkylthio or alkoxy substituents into the 4 position and vary side chain length for these analogues. We introduce an additional titratable amino group to the side chain of 4-O analogues with promising CQR strain selectivity and increase activity while retaining selectivity. We solve atomic resolution structures for complexes formed between representative 4-N, 4-S, and 4-O derivatives vs mu-oxo dimeric heme, measure binding constants for monomeric vs dimeric heme, and quantify hemozoin (Hz) formation inhibition in vitro. The data provide additional insight for the design of CQ analogues with improved activity vs CQR malaria.

Synthesis and antimalarial activity of new 4-amino-7- chloroquinolyl amides, sulfonamides, ureas and thioureas

We report the synthesis and in vitro antimalarial activities of more than 50 7-chloro-4-aminoquinolyl-derived sulfonamides 3-8 and 11-26, ureas 19-22, thioureas 23-26, and amides 27-54. Many of the CQ analogues prepared for this study showed submicromolar antimalarial activity versus HB3 (chloroquine sensitive) and Dd2 (chloroquine resistant strains of Plasmodium falciparum) and low resistance indices were obtained in most cases. Systematic variation of the side chain length and introduction of fluorinated aliphatic and aromatic termini revealed promising leads that overcome CQ resistance. In particular, sulfonamide 3 exhibiting a short side chain with a terminal dansyl moiety combined high antiplasmodial potency with a low resistance index and showed IC(50)s of 17.5 and 22.7 nM against HB3 and Dd2 parasites.

Overcoming Drug Resistance to Heme-Targeted Antimalarials by Systematic Side Chain Variation of 7-Chloro-4-aminoquinolines

Systematic variation of the branching and basicity of the side chain of chloroquine yielded a series of new 7-chloro-4-aminoquinoline derivatives exhibiting high in vitro activity against four different strains of P. falciparum. Many of the compounds tested showed excellent potency against chloroquine sensitive and resistant strains. In particular 4b, 5a, 5b, 5d, 17a, and 17b were found to be significantly more potent than chloroquine against the resistant strains Dd2 and FCB.

Quinine and chloroquine differentially perturb heme monomer-dimer equilibrium.

Nuclear magnetic resonance (NMR) measurements of magnetic susceptibility have been utilized to study the equilibrium between two forms (high-spin monomer vs the antiferromagnetically coupled mu-oxo dimer) of ferriprotoporphyrin(IX) as a function of pH. The pH dependence of this equilibrium is significantly altered by the addition of either chloroquine or quinine. Chloroquine promotes the mu-oxo dimer whereas quinine promotes the monomer.

Synthesis and antimalarial activity of new chloroquine analogues carrying a multifunctional linear side chain

We report the synthesis and in vitro antimalarial activity of several new 4-amino- and 4-alkoxy-7-chloroquinolines carrying a linear dibasic side chain. Many of these chloroquine analogues have submicromolar antimalarial activity versus HB3 (chloroquine sensitive) and Dd2 (chloroquine resistant strain of Plasmodium falciparum) and low resistance indices were obtained in most cases. Importantly, compounds 11-15 and 24 proved to be more potent against Dd2 than chloroquine. Branching of the side chain structure proved detrimental to the activity against the CQR strain.

Cytostatic versus Cytocidal Activities of Chloroquine Analogues and Inhibition of Hemozoin Crystal Growth

ABSTRACT We report an improved, nonhazardous, high-throughput assay for in vitro quantification of antimalarial drug inhibition of β-hematin (hemozoin) crystallization performed under conditions that are more physiological relative to previous assays. The assay uses the differential detergent solubility of crystalline and noncrystalline forms of heme and is optimized via the use of lipid catalyst. Using this assay, we quantify the effect of pH on the crystal growth-inhibitory activities of current quinoline antimalarials, evaluate the catalytic efficiencies of different lipids, and test for a possible correlation between hemozoin inhibition by drugs versus their antiplasmodial activity. Consistent with several previous reports, we found a good correlation between hemozoin inhibition potency versus cytostatic antiplasmodial potency (50% inhibitory concentration) for a series of chloroquine (CQ) analogues. However, we found no correlation between hemozoin inhibition potency and cytocidal antiplasmodial potency (50% lethal dose) for the same drugs, suggesting that cellular targets for these two layers of 4-aminoquinoline drug activity differ. This important concept is also explored further for QN and its stereoisomers in the accompanying paper (A. P. Gorka, K. S. Sherlach, A. C. de Dios, and P. D. Roepe, Antimicrob. Agents Chemother. 57:365–374, 2013).

The Hydroxyl Functionality and a Rigid Proximal N are Required for Forming a Novel Non-Covalent Quinine-Heme Complex †

Quinoline antimalarial drugs bind both monomeric and dimeric forms of free heme, with distinct preferences depending on the chemical environment. Under biological conditions, chloroquine (CQ) appears to prefer to bind to μ-oxo dimeric heme, while quinine (QN) preferentially binds monomer. To further explore this important distinction, we study three newly synthesized and several commercially available QN analogues lacking various functional groups. We find that removal of the QN hydroxyl lowers heme affinity, hemozoin (Hz) inhibition efficiency, and antiplasmodial activity. Elimination of the rigid quinuclidyl ring has similar effects, but elimination of either the vinyl or methoxy group does not. Replacing the quinuclidyl N with a less rigid tertiary aliphatic N only partially restores activity. To further study these trends, we probe drug-heme interactions via NMR studies with both Fe and Zn protoporphyrin IX (FPIX, ZnPIX) for QN, dehydroxyQN (DHQN), dequinuclidylQN (DQQN), and deamino-dequinuclidylQN (DADQQN). Magnetic susceptibility measurements in the presence of FPIX demonstrate that these compounds differentially perturb FPIX monomer-dimer equilibrium. We also isolate the QN-FPIX complex formed under mild aqueous conditions and analyze it by mass spectrometry, as well as fluorescence, vibrational, and solid-state NMR spectroscopies. The data elucidate key features of QN pharmacology and allow us to propose a refined model for the preferred binding of QN to monomeric FPIX under biologically relevant conditions. With this model in hand, we also propose how QN, CQ, and amodiaquine (AQ) differ in their ability to inhibit Hz formation.

Antiproliferative and antimalarial anthraquinones of Scutia myrtina from the Madagascar forest.

Bioassay-guided fractionation of an ethanol extract of a Madagascar collection of the bark of Scutia myrtina led to the isolation of three new anthrone-anthraquinones, scutianthraquinones A, B and C (1-3), one new bisanthrone-anthraquinone, scutianthraquinone D (4), and the known anthraquinone, aloesaponarin I (5). The structures of all compounds were determined using a combination of 1D and 2D NMR experiments, including COSY, TOCSY, HSQC, HMBC, and ROESY sequences, and mass spectrometry. All the isolated compounds were tested against the A2780 human ovarian cancer cell line for antiproliferative activities, and against the chloroquine-resistant Plasmodium falciparum strains Dd2 and FCM29 for antiplasmodial activities. Compounds 1, 2 and 4 showed weak antiproliferative activities against the A2780 ovarian cancer cell line, while compounds 1-4 exhibited moderate antiplasmodial activities against P. falciparum Dd2 and compounds 1, 2, and 4 exhibited moderate antiplasmodial activities against P. falciparum FCM29.

Antimalarial drugs and heme in detergent micelles: An NMR study.

Proton nuclear magnetic resonance relaxation times were measured for the protons of micelles formed by the detergents sodium dodecyl sulfate, dodecyltrimethyl ammonium bromide, and polyethylene glycol sorbitan monolaureate in the presence of ferriprotoporphyrin IX and the antimalarial drugs chloroquine, 7-chloro-4-quinolyl 4-N,N-diethylaminobutyl sulfide, and primaquine. Diffusion coefficients were extracted from pulsed gradient NMR experiments to evaluate the degree of association of these drugs with the detergent micelles. Results indicate that at low or neutral pH when the quinolyl N is protonated, chloroquine does not associate with neutral or cationic detergent micelles. For this reason, chloroquines interaction with heme perturbs the partitioning of heme between the aqueous medium and detergent micelles.

Antiplasmodial and Antiproliferative Pseudoguaianolides of Athroisma proteiforme from the Madagascar Dry Forest

Investigation of extracts from the plant Athroisma proteiforme (Humbert) Mattf. (Asteraceae) for antimalarial activity led to the isolation of the five new sesquiterpene lactones 1-5 together with centaureidin (6). The structures of the new compounds were deduced from analyses of physical and spectroscopic data, and the absolute configuration of compound 1 was confirmed by an X-ray crystallographic study. Athrolides C (3) and D (4) both showed antiplasmodial activities with IC50 values of 6.6 (3) and 7.2 μM (4) against the HB3 strain and 5.5 (3) and 4.2 μM (4) against the Dd2 strain of the malarial parasite Plasmodium falciparum. The isolates 1-6 also showed antiproliferative activity against A2780 human ovarian cancer cells, with IC50 values ranging from 0.4 to 2.5 μM.