Antimalarial activity of novel 4-cyano-3-methylisoquinoline inhibitors against Plasmodium falciparum: design, synthesis and biological evaluation.

Central to malaria pathogenesis is the invasion of human red blood cells by Plasmodium falciparum parasites. Following each cycle of intracellular development and replication, parasites activate a cellular program to egress from their current host cell and invade a new one. The orchestration of this process critically relies upon numerous organised phospho-signaling cascades, which are mediated by a number of central kinases. Parasite kinases are emerging as novel antimalarial targets as they have diverged sufficiently from their mammalian counterparts to allow selectable therapeutic action. Parasite protein kinase A (PfPKA) is highly expressed late in the cell cycle of the parasite blood stage and has been shown to phosphorylate a critical invasion protein, Apical Membrane Antigen 1. This enzyme could therefore be a valuable drug target so we have repurposed a substituted 4-cyano-3-methylisoquinoline that has been shown to inhibit rat PKA with the goal of targeting PfPKA. We synthesised a novel series of compounds and, although many potently inhibit the growth of chloroquine sensitive and resistant strains of P. falciparum, they were found to have minimal activity against PfPKA, indicating that they likely have another target important to parasite cytokinesis and invasion.

Synthesis and biological evaluation of 2-anilino-4-substituted-7H-pyrrolopyrimidines as PDK1 inhibitors.

PDK1, a biological target that has attracted a large amount of attention recently, is responsible for the positive regulation of the PI3K/Akt pathway that is often activated in a large number of human cancers. A series of second-generation 2-anilino-4-substituted-7H-pyrrolopyrimidines were synthesised by installation of various functions at the 4-position of the 7H-pyrrolopyrimidine scaffold. All compounds were screened against the isolated PDK1 enzyme and dose response analysis was obtained on the best compounds of the series.

Synthesis and biological evaluation of substituted 3-anilino-quinolin-2(1H)-ones as PDK1 inhibitors.

PDK1 is an important regulator of the PI3K/Akt pathway, which has been found frequently activated in a large number of human cancers. Herein we described the preparation of novel substituted 3-anilino-quinolin-2(1H)-ones as PDK1 inhibitors. The synthesis is based around a Buchwald-Hartwig cross-coupling of various 3-bromo-6-substituted-quinolin-2(1H)-ones with three different functionalised anilines. The modular nature of the designed synthesis allowed access to a series of novel inhibitors through derivatisation of a late-stage intermediate. All compounds were screened against isolated PDK1 enzyme, with modest inhibition observed.

Potent inhibitors of phosphatidylinositol 3 (PI3) kinase that have antiproliferative activity only when delivered as prodrug forms.

Prodrugs for PI3K: A series of substituted analogues of the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002 were prepared and found to potently inhibit the isolated enzyme but not MCF7 cell proliferation. Two tetrazolyl-substituted analogues were further derivatized as prodrugs resulting in restoration of cell-based activity. These data provide a conceptual model for development of tumor-targeting prodrug forms of cell-impermeable PI3K inhibitors.

Primaquine synergises the activity of chloroquine against chloroquine-resistant P. falciparum.

In recent years, resistance to the antimalarial drug, chloroquine, has become widespread. It is, therefore, imperative to find compounds that could replace chloroquine or work synergistically with this drug to overcome chloroquine resistance. We have examined the interaction between chloroquine, a 4-aminoquinoline, and a number of 8-aminoquinolines, including primaquine, a drug that is widely used to treat Plasmodium vivax infections. We find that primaquine is a potent synergiser of the activity of chloroquine against chloroquine-resistant Plasmodium falciparum. Analysis of matched transfectants expressing mutant and wild-type alleles of the P. falciparum chloroquine resistance transporter (PfCRT) indicate that primaquine exerts its activity by blocking PfCRT, and thus enhancing chloroquine accumulation. Our data suggest that a novel formulation of two antimalarial drugs already licensed for use in humans could be used to treat chloroquine-resistant parasites.

Synthesis and cytotoxic activity of N-[(alkylamino)alkyl]carboxamide derivatives of 7-oxo-7H-benz[de]anthracene, 7-oxo-7H-naphtho[1,2,3-de]quinoline, and 7-oxo-7H-benzo[e]perimidine.

7-Oxo-7H-naphtho[1,2,3-de]quinoline-11-carboxamides and analogues were prepared and evaluated for in vitro and in vivo antitumor activity. Chromophore variations included 'deaza' (7-oxo-7H-benz[de]anthracene) and 'diaza' (7-oxo-7H-benzo[e]perimidine) analogues, and side chain variations included chiral alpha-methyl compounds. The naphthoquinolines were the most cytotoxic, with IC(50) values of 5-20 nM, and showed the strongest DNA binding, with high selectivity for G-C rich DNA. The chiral alpha-methyl analogues were 10-20-fold more cytotoxic than the parent des-methyl compound. Both enantiomers provided substantial growth delays against s.c. colon 38 tumors in mice, with the R-enantiomer more active than the S (tumor growth delays of >35 and 12 days, respectively).

Synthesis and cytotoxic activity of carboxamide derivatives of benzo[b][1,6]naphthyridin-(5H)ones.

A previous reaction leading to 2-substituted 6-methyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthyridine-4-carboxylic acids has been extended to encompass a broad range of 2-substituents. Derived carboxamides, particularly 4-N-[2-(dimethylamino)ethyl], were tested for growth inhibitory properties. Potent cytotoxicity against murine P388 leukemia and Lewis lung carcinoma (LLTC) was retained for compounds bearing a remarkably diverse range of 2-substituents with a number having IC50 values <10 nM. Five of the new compounds were tested in vivo against subcutaneous colon 38 tumors in mice; a single dose (1.8 mg/kg) proved curative for the 2-(4-fluorophenyl) derivative, a further increase in potency over the very effective 2-methyl analogue reported previously.

Synergistic interaction of a chloroquine metabolite with chloroquine against drug-resistant malaria parasites.

We have previously shown that structural modification of chlorpromazine to introduce a basic side chain converts this chloroquine (CQ) resistance-reversing agent into a compound that has activity against Plasmodium falciparum in vitro. In an effort to further dissect the structural features that determine quinoline antimalarial activity and drug resistance-reversing activity, we have studied a series of aminoquinolines that are structurally related to CQ. We have analysed their haematin-binding activities, their antimalarial activities and their abilities to synergise the effect of CQ against drug-resistant P. falciparum. We found that a number of the aminoquinolines were able to interact with haematin but showed no or very weak antiparasitic activity. Interestingly, 4-amino-7-chloroquinoline, which is the CQ nucleus without the basic side chain, was able to act as a resistance-reversing agent. These studies point to structural features that may determine the resistance-modulating potential of weakly basic amphipaths. Interestingly, 4-amino-7-chloroquinoline is a metabolic breakdown product of CQ and may contribute to CQ activity against resistant parasites in vivo.

Synthesis and cytotoxic activity of carboxamide derivatives of benzo[b][1,6]naphthyridines.

The reaction of 4-dimethylaminomethylene-6-methyl-4H-pyrano[4,3-b]quinoline-1,3-dione with a range of primary amines gave rise to a series of 2-substituted 6-methyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthyridine-4-carboxylic acids. The derived 4-N-[2-(dimethylamino)ethyl]carboxamides were tested for growth inhibitory properties against murine P388 leukemia, Lewis lung carcinoma (LLTC), and human Jurkat leukemia cell lines. Most compounds were potent cytotoxins, with some having IC(50) values less than 10 nM. Five were tested in vivo against subcutaneous colon 38 tumors in mice, and a single dose (3.9 mg/kg) proved to be curative for the 2-methyl and 2-(3,4-dimethoxyphenyl) derivatives in this refractory model.

Potent inhibition of scrapie prion replication in cultured cells by bis-acridines.

Prion diseases are characterized by an accumulation of PrP(Sc), a misfolded isoform of the normal cellular prion protein, PrP(C). We previously reported the bioactivity of acridine-based compounds against PrP(Sc) replication in scrapie-infected neuroblastoma cells and now report the improved potency of bis-acridine compounds. Bis-acridines are characterized by a dimeric motif, comprising two acridine heterocycles tethered by a linker. A library of bis-(6-chloro-2-methoxy-acridin-9-yl) and bis-(7-chloro-2-methoxy-benzo[b][1,5]naphthyridin-10-yl) analogs was synthesized to explore the effect of structurally diverse linkers on PrP(Sc) replication in scrapie-infected neuroblastoma cells. Structure-activity analysis revealed that linker length and structure are important determinants for inhibition of prion replication in cultured scrapied cells. Three bis-acridine analogs, (6-chloro-2-methoxy-acridin-9-yl)-(3-[4-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-piperazin-1-yl]-propyl)-amine, N,N'-bis-(6-chloro-2-methoxy-acridin-9-yl)-1,8-diamino-3,6-dioxaoctane, and (1-[[4-(6-chloro-2-methoxy-acridin-9-ylamino)-butyl]-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-carbamoyl]-ethyl)-carbamic acid tert-butyl ester, showed half-maximal inhibition of PrP(Sc) formation at 40, 25, and 30 nM, respectively, and were not cytotoxic to uninfected neuroblastoma cells at concentrations of 500 nM. Our data suggest that bis-acridine analogs may provide a potent alternative to the acridine-based compound quinacrine, which is currently under clinical evaluation for the treatment of prion disease.

Synthesis and cytotoxic activity of N-(2-diethylamino)ethylcarboxamide and other derivatives of 10H-quindoline.

A series of mono- and dimeric N-methylquindoline carboxamides were prepared by Friedlander condensation between methyl 2-amino-3-formyl benzoate and 3-acetoxy-1-acetylindoles, followed by exhaustive methylation with methyl iodide to give N-methylquindoline esters. Direct amination of these, or hydrolysis to the acids and amine coupling via intermediate imidazolides gave the desired carboxamides. The compounds were evaluated in a panel of cell lines in culture. The monomeric compounds showed similar structure-activity relationships to the known indeno[1,2-b]quinolines, with a 4-methyl group increasing potency several-fold. Bis analogues linked through the carboxamide were more cytotoxic than the corresponding monomers in the human leukemia lines, but N-N linked dimers were generally less potent, except for a tetracationic derivative. The most potent monomeric analogue showed moderate growth delay (ca. 5 days) against sub-cutaneously implanted colon 38 tumors in mice.

Novel phenothiazine antimalarials: synthesis, antimalarial activity, and inhibition of the formation of beta-haematin.

We report the synthesis of a series of novel phenothiazine compounds that inhibit the growth of both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. We found that the antimalarial activity of these phenothiazines increased with an increase in the number of basic groups in the alkylamino side chain, which may reflect increased uptake into the parasite food vacuole or differences in the toxicities of individual FP-drug complexes. We have examined the ability of the parent phenothiazine, chlorpromazine, and some novel phenothiazines to inhibit the formation of beta-haematin. The degree of antimalarial potency was loosely correlated with the efficacy of inhibition of beta-haematin formation, suggesting that these phenothiazines exert their antimalarial activities in a manner similar to that of chloroquine, i.e. by antagonizing the sequestration of toxic haem (ferriprotoporphyrin IX) moieties within the malaria parasite. Chlorpromazine is an effective modulator of chloroquine resistance; however, the more potent phenothiazine derivatives were more active against chloroquine-sensitive parasites than against chloroquine-resistant parasites and showed little synergy of action when used in combination with chloroquine. These studies point to structural features that may determine the antimalarial activity and resistance modulating potential of weakly basic amphipaths.