Synthesis, biological profiling and mechanistic studies of 4-aminoquinoline-based heterodimeric compounds with dual trypanocidal–antiplasmodial activity
Graphical abstract
Introduction
Human African trypanosomiasis (HAT or sleeping sickness), one of the 17 so-called neglected tropical diseases, and malaria have an enormous health and socioeconomic impact in the developing world.1, 2, 3 Notwithstanding a wide-scale reduction in the number of infected people over recent years due to public health campaigns, HAT and malaria are still leading causes of morbidity and death and of loss of productivity especially in sub-Saharan Africa.1, 4, 5, 6 Malaria annually kills more than 600,000 people.3 The numbers dying from trypanosomiasis have recently been reduced to around 10,000, but the disease retains the potential for major epidemic outbreaks, and it has a devastating impact on domestic livestock.
HAT and malaria are caused by protozoan parasites of the genera Trypanosoma and Plasmodium, which are transmitted to humans through the bite of blood-feeding infected tsetse flies and female Anopheles mosquitoes, respectively. The most common form of HAT in humans, accounting for nearly 95% of cases, is caused by Trypanosoma brucei gambiense, which results in a chronic infection that can last for years. A less common form of the disease with a more acute clinical presentation is caused by the subspecies Trypanosoma brucei rhodesiense. In the case of malaria, five species of Plasmodium can cause the disease, Plasmodium falciparum being the most common and deadly.
HAT begins with a hemolymphatic stage, where the parasite multiplies within the blood, lymph and subcutaneous tissue, and which is characterized by the appearance of nonspecific symptoms such as fever and headache. Invasion of the central nervous system (CNS) by the parasite, after crossing the blood–brain barrier (BBB), leads to the late-stage meningoencephalitic disease. This gives rise to severe neurological symptoms such as psychiatric, motor and sleep disturbances and loss of consciousness. Without treatment, this results in coma and death.
In malaria, the parasites initially infect the liver, and are then released into the bloodstream. The disease may progress to a severe form, where parasites can become sequestered within brain capillaries, particularly in children, evolving to cerebral malaria and eventually to coma and death.
Vector control and public health interventions remain the main options for prevention, as no licensed vaccine for either infection is available. Worryingly, current chemotherapy against HAT and malaria suffers from serious limitations.1, 4, 7, 8, 9 Although five trypanocidal drugs are currently in use (pentamidine, suramin, melarsoprol, nifurtimox and eflornithine), they are generally effective against only one stage of the disease. In addition, they are relatively expensive, require long-lasting parenteral administration which is often impracticable in poor rural settings, display toxicity and parasite resistance has frequently emerged. In the case of the arsenical melarsoprol,10 the latter two issues have challenged its widespread, safe and efficacious use.11, 12 The emergence of resistance is the reason that chloroquine, after decades of being the mainstay for malaria treatment, is no longer widely used. Resistance is also beginning to threaten the effectiveness of the current first-line treatments, based on artemisinin.1
In this context, it is critically important to ensure that the trypanocidal and antimalarial drug development pipelines are continuously replenished with novel candidates that are devoid of the important flaws of existing drugs. The requirement is for new non toxic and inexpensive chemical entities that are effective against resistant parasite strains and are brain permeable, so that they may be useful against malaria and both disease stages of HAT. Intensive research efforts involving phenotypic whole-cell screening of chemical libraries or newly synthesized compounds,13, 14, 15, 16 identification of novel key biological targets and subsequent target-based screening or rational design campaigns,17, 18, 19, 20, 21 development of multitarget-directed ligands,22, 23, 24 or drug repurposing programs1 are being carried out in the pursuit of novel antiprotozoal compounds.
Because HAT and malaria often affect overlapping populations, development of compounds endowed with dual trypanocidal and antiplasmodial activity can be regarded a feasible economic therapeutic strategy.25 A number of 4-amino-7-chloroquinolines and other aminoquinoline derivatives have been recently synthesized and found to be active against T. brucei and/or P. falciparum.26, 27, 28, 29 This prompted us to assess the antiprotozoal activity of huprines, a novel structural class of compounds featuring a 4-aminoquinoline moiety, which had been initially developed as brain permeable inhibitors of the enzyme acetylcholinesterase (AChE). Huprines in general turned out to be moderately potent and selective trypanocidal agents, with a few also being active against the chloroquine-resistant K1 strain of P. falciparum.30, 31 The so-called huprine Y (1, Fig. 1) exhibited the most potent activity against T. brucei (IC50 = 0.61 μM; IC90 = 2.94 μM) and one of the best selectivity indices over rat myoblast L6 cells (SI = 13), but it was essentially devoid of antiplasmodial activity.30
Next, we turned our attention to the molecular dimerization of huprine Y because this approach had been successfully applied to other 4-aminoquinoline derivatives to overcome drug resistance.32, 33, 34, 35, 36 One of the most interesting huprine dimers was the novel dodecamethylene-linked bis(4-aminoquinoline) compound 2 (Fig. 1), which tripled the potency and selectivity of the parent huprine Y against T. brucei, but remained inactive against P. falciparum.37
The lack of potency of huprine Y and bis-huprines against P. falciparum was rather striking because: (i) the huprine Y unit contains the 4-amino-7-chloroquinoline moiety of chloroquine, which is considered to be the pharmocophoric moiety responsible for the inhibition of heme dimerization by the antimalarial drug,28, 38 and (ii) in other bis(4-aminoquinoline) derivatives dimerization had been reported to increase antiplasmodial potency relative to the 4-aminoquinoline monomeric parent compounds due to the doubling of the number of protonatable nitrogen atoms. This enabled a more efficient trapping of the dimeric compounds in the acidic digestive vacuole of P. falciparum, and hence, a more efficient inhibition of heme dimerization.32, 35 We hypothesized that neither huprine Y nor bis-huprines were able to hit the biological target of chloroquine and other bis(4-aminoquinoline) derivatives, despite their structural similarity.
To explore further the dimerization strategy, and to discover new hits with dual trypanocidal–antiplasmodial activity, we report here: (i) the screening against cultured bloodstream forms of T. brucei and P. falciparum and rat myoblast L6 cells of a small in-house library of brain permeable 4-aminoquinoline-based heterodimeric compounds, belonging to three distinct structural classes (series I–III, Fig. 1); (ii) the synthesis of novel 4-aminoquinoline-based heterodimeric compounds based on the most promising series and evaluation of their T. brucei, P. falciparum, rat myoblast L6 cell and human AChE inhibitory activities and brain permeability; and (iii) the assessment of the putative biological targets in T. brucei and P. falciparum of selected hits of the different series and monomeric huprine Y by the in vitro evaluation of their inhibitory activity against T. brucei trypanothione reductase and β-haematin formation.
Section snippets
Screening of trypanocidal, antiplasmodial and cytotoxic activity of 4-aminoquinoline-based heterodimeric compounds of series I–III
The heterodimers of series I–III (Fig. 1) were recently developed by us as inhibitors of the enzyme AChE, of potential interest for the treatment of Alzheimer’s disease.39, 40, 41 The high AChE inhibitory activity displayed by these heterodimers (IC50 values in the low nanomolar range) is a drawback for antiprotozoal drug development. However, the presence of one or two 4-aminoquinoline moieties in the structures of these heterodimers, and the fact that all of them had been found to be brain
Conclusion
By phenotypic screening of a small library of 27 brain permeable 4-aminoquinoline-based heterodimeric compounds against bloodstream forms of T. brucei and the multidrug-resistant strain K1 of P. falciparum, we have identified a number of dual submicromolar trypanocidal–antiplasmodial compounds. Indeed, all of the tested compounds had submicromolar IC50 values against T. brucei, with the vast majority also displaying submicromolar IC90 values. They were all more potent trypanocidal agents than
Chemistry
Melting points were determined in open capillary tubes with a MFB 595010M Gallenkamp melting point apparatus. 400 MHz 1H/100.6 MHz 13C NMR spectra were recorded on a Varian Mercury 400 spectrometer at the Centres Científics i Tecnològics of the University of Barcelona (CCiTUB). The chemical shifts are reported in ppm (δ scale) and coupling constants are reported in Hertz (Hz). The syn (anti) notation of the protons at position 13 of the huprine moiety of the heterodimers means that the
Acknowledgments
This work was supported by Ministerio de Ciencia e Innovación (MICINN) (CTQ2011-22433) and Generalitat de Catalunya (GC) (2014SGR52). J.M.K. acknowledges funding support from the Wellcome Trust (Grant number WT084175). A fellowship from GC to I.S. is gratefully acknowledged. S.Y.G. is grateful to the Commonwealth Scholarship Secretariat, UK, and to the Government of Ghana for financial support.
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