Metalloantimalarials - ACS Publications - American Chemical Society

Review pubs.acs.org/CR

Metalloantimalarials Paloma F. Salas,† Christoph Herrmann,†,‡ and Chris Orvig*,† †

Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada ‡ Advanced Applied Physics Solutions, TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia V6T 2A3, Canada Malaria is one of the world’s most ancient diseases: the first historical records of symptoms that match those of malaria date back to the ancient Chinese (around 3000 B.C.), ancient Egyptians (around 1550 B.C.), and ancient Greeks (around 413 B.C.).2,3 It is believed that the ancestors of the malaria parasite existed at least one-half a billion years ago.3 Malaria, as a human disease, evolved with mankind through history.3 From its origin in West and Central Africa more than 10 000 years ago, malaria spread to other areas through the journey of man, following CONTENTS human migrations to the Mediterranean, Mesopotamia, the Indian peninsula, South-East Asia, Northern Europe, and the 1. Introduction 3450 Americas.2,3 By the end of the 18th century, malaria had spread 2. Prevention, Diagnosis, and Treatment of Malaria 3451 across North America, and during the 19th century it was present 3. Antimalarial Therapy 3453 almost all over the globe, resulting in millions succumbing to the 3.1. Quinoline-Containing Antimalarial Drugs 3454 disease.2 3.2. Mode of Action of Quinoline-Containing The first record of malaria treatment dates back to the 1600s, Drugs 3455 when the natives of South America used the bark of the Cinchona 3.3. Resistance 3458 tree (originally from the high altitudes of South America) to treat 3.4. Other Traditional Antimalarial Drugs 3458 the symptoms of malaria.4 However, significant progress in 4. Metalloantimalarials 3462 malaria treatment was not achieved until the late 19th century, 4.1. Metal Chelators 3462 when physicians Alphonse Laveran and Ronald Ross identified 4.2. Metal Complexes 3464 the Plasmodium parasite as the agent that causes malaria and the 4.2.1. Metal Complexes of Chloroquine and Anopheles mosquito as the vector of the disease, discoveries that Chloroquine Derivatives 3464 granted each of them the Nobel Prize in Physiology or Medicine 4.2.2. Metal Complexes of Other Antimalarial in 1907 and 1902, respectively.5 Since then, malaria has become Drugs 3466 one of the best-studied diseases in Western medicine until 4.2.3. Metal Complexes of Other Ligands 3466 eclipsed in the mid-20th century. 4.3. Bioorganometallic Compounds 3472 By the 1950s, as a result of the combined action of the now4.3.1. Metallocenes 3473 banned pesticide DDT (dichlorodiphenyltrichloroethane) on 4.3.2. Nonmetallocene Compounds 3480 vector control and the effectiveness of antimalarial treatments, 5. Summary 3485 malaria was almost eradicated from North America and from Author Information 3486 almost all of Europe but persisted in the tropics, especially in Corresponding Author 3486 Africa where the intensity of the transmission was and still is the Notes 3486 highest.2 Following this success, several programs aiming at Biographies 3486 global eradication of malaria were initiated.2 Systematic control Acknowledgments 3487 measures such as widespread spraying with DDT and extensive Abbreviations 3487 use of antimalarial drugs as prophylactics were implemented References 3487 worldwide.4 As a consequence, mortality rates dropped significantly, reducing the global population at risk of malaria to 10% of the world population.2 1. INTRODUCTION Despite these initial successes, the emergence of drug-resistant strains of the parasite, the ban on DDT, the appearance of Malaria is a worldwide neglected infectious disease that, despite mosquito resistance to insecticides and climate changes, in decades of research invested in its prevention and treatment, addition to other social and political factors, led to a collapse of remains one of the main causes of mortality and morbidity in the the eradication campaign.6 From the early 1970s onward, the world. As reported by the WHO (World Health Organization) in situation progressively deteriorated, leading to a 2−3-fold 2011, 3.3 billion people were at risk of malaria, mainly in the 106 increase in malaria cases globally.6 By the 1980s, the situation malaria-endemic countries located in the tropical and subtropical zones of the globe.1 Nearly one-half of the world’s population lives under the constant threat of malaria, with the heaviest toll Received: March 23, 2012 borne by the poorest and most vulnerable. Published: February 21, 2013 © 2013 American Chemical Society

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reached endemicity levels in 108 countries.7,8 Three billion people were at risk of contracting malaria, resulting in an estimated 300−500 million cases annually.7,8A recent study shows that global malaria casualties increased from 995 000 in 1980 to a peak of 1 817 000 in 2004.9 Contrary to previous statistics that emphasized the casualties among children below the age of five, a significantly higher number of casualties in adults was estimated in this new study.9 Malaria also contributed to other health-related issues and to a halt of the economic growth of those countries with the highest burden.10 Only in recent years, after decades of neglect, growing international awareness and funding has led to new efforts toward controlling the disease. In 2008, the WHO and the Roll Back Malaria (RBM) Partnership called for a universal coverage of malaria-control interventions by the end of 2010 in order to bring an end to malaria-related casualties by 2015.11,12 The 2010 and 2011 World Malaria Reports document the prevention efforts in fighting this disease, showing a measurable public health impact.1,13 Approximately 600 million people have received vector protection, and 11 African countries have cut cases and casualties from malaria by one-half. The annual number of malaria cases has decreased to an estimated 216 million, and the number of malaria-endemic countries has been reduced from 108 in 2004 to 106 in 2009.1,13 Even though the WHO reports a total number of 655 000 casualties in 2010,1 a recent study reports 1 238 000 malaria-related casualities in 2010, still representing a decrease of 32% since 2004.9 These successes have been made possible through the combined research coordination and global health care planning efforts by institutions like the WHO’s Partner Roll Back Malaria (RBM), the Global Fund, and programs like “Multilateral Initiative on Malaria” (MIM), “Malaria No More” (MNM), “Malaria Vaccine Initiative” (MVI), “Medicines for Malaria Venture” (MMV), together with the pharmaceutical industry and philanthropic support. This progress remains fragile, however, as the malaria parasite proves to be very adaptable to inclement conditions and as political resolution and financial commitment diminish. Recent findings pointed out that the number of malaria deaths was greatly underestimated by previous studies and that if the current action plan were not to be improved, the best outcome would be a reduction of mortality to less than 100 000 only after 2020.9 This would not meet the WHO’s plan of eradication of malaria by 2015. The current global economic crisis also represents a threat to the malaria program. With the announcement of the cancellation of funding by the Global Fund (one of the major supporters of this program),14,15 the sustainable growth of the malaria health plan falls into question. In addition, resistance of the parasite to drugs is a permanent threat that might lead to a resurgence of malaria in already epidemically controlled countries. Therefore, sustained funding and constant research in the treatment and prevention of this disease is necessary to prevent it from bouncing back. Malaria is caused by protozoan parasites of the genus Plasmodium. From over 300 known species of Plasmodium, only five infect humans and cause the distinct disease patterns of malaria: P. falciparum (malaria tropica), P. vivax (malaria tertiana), P. malariae (malaria tertiana), P. ovale (malaria quartana), and P. knowlesi. P. falciparum is the most lethal of these five and considered responsible for approximately 90% of all reported cases. P. vivax is not nearly as lethal as is P. falciparum but persists for years in the dormant stage in the liver and can cause clinical relapses at regular intervals. Parasites of the species

P. malariae, P. ovale, and P. knowlesi are less common. Generally, the parasites are transmitted by certain species of the female Anopheles mosquito. The complex life cycle of the malaria parasite involves different developmental stages taking place in different tissues of both human and mosquito hosts. A simplified schematic representation of the malaria cycle is depicted in Figure 1.

2. PREVENTION, DIAGNOSIS, AND TREATMENT OF MALARIA Malaria is preventable through methods of malaria vector control.16 The two most important vector control methods recommended by the WHO are long-lasting insecticide-treated

Figure 1. Life cycle of the malaria parasite. When an infected mosquito stings a human host, it injects saliva containing sporozoites of the Plasmodium parasite into the bloodstream (1). Sporozoites first invade the host liver where they mature and multiply (2). In P. vivax and P. ovale, part of the liver-stage parasites stay dormant in the liver as hypnozoites (3). Infected liver cells eventually burst, releasing Plasmodium merozoites back into the bloodstream, where they invade the red blood cells (4). Merozoites reproduce asexually inside the red blood cells (erythrocytes), developing through the stages of rings, trophozoites, and schizonts. Each schizont typically divides into merozoites, which are released by lysis of the erythrocyte immediately invading new erythrocytes (5). Symptoms of uncomplicated malaria such as fever, headaches, diarrhea, vomiting, and anemia develop at this stage. If malaria remains untreated, erythrocytes filled with mature stages of the parasite can adhere to the walls of capillary veins, causing vascular occlusion and cerebral death (cerebral malaria). Some merozoites develop into male and female gametocytes (6). If at this erythrocytic stage an uninfected mosquito bites the host, it will pick up Plasmodium gamete cells. Sexual phase of the Plasmodium life cycle takes place inside the mosquito through fusion of male and female gametes, producing ookinetes that migrate to the midgut of the insect. Oocysts that are formed will reside and mature in the external membrane of the mosquito midgut (7). Division and rupture of the oocysts result in the release of thousands of sporozoites that will travel to the insect salivary glands (8), closing the cycle. 3451


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mosquito nets (LLINs) and indoor residual spraying (IRS).17 Indoor residual spraying (IRS) with WHO-approved chemicals consists of application of residual insecticides to the inner surfaces of dwellings. Currently, 12 insecticides belonging to 4 different chemical classes are recommended by the WHO for IRS.17 Long-lasting insecticide-treated mosquito nets (LLINs or ITNs), also known as “bed nets”, protect individuals against bites at night. ITNs are currently treated with pyrethroids only.17 Malaria can also be prevented by drugs (chemoprophylaxis).16,17 For pregnant women and infants, population at the highest risk, the WHO has established the Intermittent Preventive Treatment (IPT) program, consisting of the periodic administration of an antimalarial drug, regardless of the presence of Plasmodium parasites.18 This program is raising concerns in regard to the emergence of parasitic resistance.18,19 Malaria prevention for individuals traveling from malaria-risk-free zones to malariaendemic zones is strongly suggested.20,21 Prevention measures include the use of insect repellent, long-sleeved clothing, bed nets, and administration of antimalarial drugs.20,21 The Centre for Disease Control (CDC) recommends the use of specific medicines for malaria prevention depending on the visited country.22 Increasing international funding for malaria control has resulted in increased coverage of WHO-recommended malaria control interventions.13 For example, millions of ITNs have been delivered, especially to sub-Saharan Africa, and it is estimated that they protect 75% of the population at risk of contracting malaria.13 This funding still falls short. It is estimated that comprehensive coverage of the resources required for malaria control and eradication would cost $5 billion per year over the next few years; however, the budget in 2011 was only $2 billion.1 Furthermore, it will become increasingly important to sustain the prevention campaign, since the life span of ITNs is only 3−5 years, and failure to replace them could lead to a resurgence of malaria. Certainly, the best long-term prevention of this disease would be a malaria vaccine. Several candidates for a vaccine have been investigated in the past decades, but realization of this has proven elusive. Only recently, some promising malaria vaccine candidates are being developed at the preclinical phase,23 but only one, the GlaxoSmithKline Biologicals’ RTS,S malaria vaccine candidate, is currently in phase III clinical trials at 11 sites in Africa.24 This is the only candidate to have reached this stage.24 Unfortunately, development of many promising vaccine candidates already failed due to difficulties resulting from the complex life cycle of the malaria parasite’s and the parasite potent adaptability to generate resistance against an immune response in the host. Malaria can be treated and cured if diagnosed promptly and accurately. The WHO recommends a parasitological confirmation by microscopy or a rapid diagnostic test (RDT) for all patients with a suspected case of malaria, before commencing treatment, in order to avoid the spread of resistance by administrating medicines to treat nonmalaria-related fever.19 Parasitological confirmation requires a reliable microscope, laborious work, and well-trained and -supported staff, conditions which are not always met in all malaria-afflicted regions.25 On the other hand, RDTs are inexpensive and quality-assured tests that can be used at the community level and do not require any electricity or laboratory.19 Nevertheless, it has proven a challenge to establish these diagnostic policies in the most affected areas such as the majority of the African countries. In the early 2000s, only 5% of suspected malaria cases reported in Africa were

properly confirmed via diagnostic testing, and by 2009, this number increased to only 35%.13 The standard treatment recommended by the WHO as the first-line therapy for uncomplicated P. falciparum malaria worldwide consists of artemisinin-based combined therapy (ACT);19 chloroquine, a well-known antimalarial drug, is only effective to treat P. falciparum malaria in Egypt and in a few countries in Central America and the Middle East.26 The recommended treatment for P. vivax malaria consists of a chloroquine-based therapy where effective or ACT in areas of resistance to chloroquine.19 Artemisinin is a fast-acting and highly effective drug, capable of rapid elimination of malaria parasites. ACT is best described as a combination of artemisinin or an artemisinin derivative and a second drug with a different mechanism of action to enhance efficacy while diminishing the risk of resistance development.27 The five ACTs currently recommended for treatment are Artemether/lumefantrine (Coartem and Riamet), artesunate/amodiaquine (Winthrop and Coarsucam), artesunate/mefloquine (Artequin), artesunate/ sulfadoxine pyrimethamine (Gen-Art SP and Artecospe), and dihydroartemisinin/piperaquine (Artekin and Eurartesim).27 The choice of ACT would depend on the efficacy of the combination in the country or area of intended use.27 In 2007, the WHO officially banned oral artemisinin monotherapies and recommended them to be replaced by ACTs.25 On the basis of previous experience with antimalarial drugs, the WHO concluded that misuse and/or overuse of oral artemisinin-based monotherapies represented a threat to the therapeutic life of ACTs by fostering the spread of resistance to artemisinins.25 By recommending ACTs as the only alternative for malaria therapy, the WHO hopes to reduce the chances of developing resistance to artemisinin and its derivatives by administering the artemisinin derivative that will exterminate most of the parasites with its potent action first and second by administering the partner drug that has a longer retention time in the body, terminating the remaining parasites within the host.25 This would limit exposure of the parasite to larger doses of artemisinin, which would reduce the parasite time to adapt and develop resistance. Even though the manufacture of ACTs has increased since 2005, compliance with these recommendations is still incomplete.19 By November 2011, 25 countries were still allowing the marketing of artemisinin monotherapies and 39 pharmaceutical companies were manufacturing them.1 This might be due to the difficulties associated with the agricultural production of Artemisia annua, from which artemisinin is extracted, and the supply of artemisinin, which is influenced by market instability.28 To date, production of artemisinin has been dependent on the cultivation of Artemisia annua.28 Production of semisynthetic artemisinin, derived from yeast cultures, was expected to become available in 2012 but only to be able to partially cover the global market demand.13 The spread of resistance to antimalarial medicines over the past decades has led to an intensification of efficacy monitoring to allow for an early detection of resistance.19 Yet even in their combination, the highly effective artemisinin derivatives and their partner drugs are susceptible to the development of resistance. In 2009, resistance of P. falciparum parasites to artemisinins was confirmed at the Cambodia−Thailand border.28 Suspected resistance to artemisinins has now been identified in Cambodia, Vietnam, Myanmar, and Thailand.29 It was first reported that the combination of artesunate/ mefloquinethe most used ACT in Southeast Asiawas 3452


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Figure 2. Schematic representation of a P. falciparum infected red blood cell and localization of the different drug targets.

presenting resistance.30,31 Now, the combination of dihydroartemisinin−piperaquine, one of the latest to be introduced, has been reported to show resistance in Papua New Guinea.32 As of today, the drugs still work but take more time to be effective.1,27 Despite the observed changes in parasite sensitivity to artemisinins, the clinical and parasitological efficacy of ACTs has not yet been compromised.27,1 Measures have been taken to limit the spread of the artemisinin-resistant parasites.29 However, if the current generation of ACTs were to fail, there would be no alternative treatment available on the market, because currently developed drugs in the pipeline are mostly artemisinin derivatives that would likely present the same type of resistance.

unique to the parasite or have sufficient differences from the host.35 The biological targets of the many families of antimalarial drugs range from cell functions such as detoxification of toxic metabolites and folate metabolism to the more recently discovered biochemical pathways like fatty acid synthesis or protein farnesylation.36 On the basis of their role in parasite growth and survival, the parasite food vacuole, apicoplast, and mitochondrion have been identified as the major organelles for drug targeting.35 Figure 2 gives an overview of old and new antimalarial targets and their localization during the intraerythrocytic stage of the malaria infection. Most of the existing antimalarial drugs were identified using conventional drug discovery techniques.36 Only after the antimalarial potency had been identified clinically in vitro and/ or in vivo in diverse animal models were these compounds developed into medicinal products and their benefits and risks observed during the process of commercialization.36 Routine screening for antiplasmodial activity can be carried out in vitro in various parasite strains of P. falciparum, P.vivax, and P. ovale isolated all over the world.37 In vivo screening of antimalarial activity is done mainly for the rodent parasite P. berghei in a murine malaria model.37 Animal model studies against Plasmodium species that infect humans can only be carried out in monkeys and are therefore of limited accessibility.37 Only recently, with the complete mapping of the malaria, mosquito, and human genomes, are more rational approaches like the embrace of genomic technologies to target enzymes, involved in important metabolic pathways for the parasite, based on pathogen and host genomic and proteomic information, being employed to identify potential chemotherapeutic targets.38 At present, a number of antimalarial drugs are available, but high cost, toxicity, and, most importantly, slowly originating or already widespread parasitic resistance limit their use. Thus, it is imperative to continue the development of new treatment options and improve the current therapeutics if the progress in

3. ANTIMALARIAL THERAPY Of all factors that have contributed to the recrudescence of malaria in the last 50 years, increasing antimalarial drug resistance is probably the major contributor. This phenomenon, which rapidly depleted the therapies available to fight malaria, has led to intensive research carried out for decades that produced a large pool of antimalarial drugs. Yet, as a consequence of the eradication of malaria from North America and most of Europe and the end of social conflicts in malaria-risk zones, governmental and military research in antimalarial therapy slowed down significantly.33 Additionally, drug development has also suffered a lack of interest from the pharmaceutical industry in investing in the development of drugs for disadvantaged markets and from the limited understanding of the complex biochemistry of the malaria parasite that ultimately hinders rational approaches to new drug targets.34 Nevertheless, over the course of the years, different types of antimalarial drug families were developed addressing the necessity to attack not one but multiple mechanisms of survival of the parasite (preferably in a parallel manner).33,34 A wellestablished method for development of drug targets is identification of biologically relevant pathways that either are 3453


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Figure 3. Some traditional and novel quinoline-based antimalarial drugs.

recommended drugs to treat the severe form of malaria, one of the most delicate stages of the malaria infection.38 Due to its high solubility, it can be administered as an intravenous formulation when patients are unable to tolerate oral medication.38 Quinidine (2), a stereoisomer of 1, has been used previously for treatment of cardiac arrhythmia and later was shown to be suitable for treatment of malaria.39 Both drugs can be administrated either orally or intravenously and are effective in the treatment of chloroquine-resistant malaria.39 Nevertheless, without a doubt, the most popular representative of this family is chloroquine (3). Once known as the most effective, most frequently used and most important antimalarial in the world, 3 now has been rendered inactive in most parts of the world, remaining 100% effective against P. falciparum in only two countries in the Americas.27 Chloroquine (3) still does remain effective against P. ovale, P. malariae and most cases of P. vivax malaria.38,39 It was first synthesized in 1934 but it was not until 1946 that it was made commercially available. Chloroquine (3) was used widely after World War II and was the drug of choice and first line of treatment for treating malaria worldwide for about five decades.33 This synthetic 4-amino quinoline (3) showed high effectiveness against many parasite strains.38,39 Well-defined pharmaceutical properties of this drug include good tolerance in the patient, better effectiveness, and reduced toxicity compared to quinine (1, its predecessor) and low cost of production.37−39 Chloroquine is usually administered as its diphosphate salt (CQDP). Side effects can include headache, diplopia (double vision), dizziness, and nausea.39 It has a half-life retention time between 7 and 56 days.39 While attempting a global eradication of malaria, in a widespread distribution of the drug, 3 was included even in

malaria control is to be sustained. For that purpose it is important to understand the mechanisms through which the drugs act and resistance is generated. In the following subsections, the bestknown quinoline-based antimalarial drug class will be described along with the current knowledge of mechanism of action and parasite resistance. At the end of this section, other important antimalarial drugs will be briefly described. 3.1. Quinoline-Containing Antimalarial Drugs

This family of antimalarials is formed by organic compounds containing a heterocyclic aromatic quinoline group. This is the oldest class of antimalarial drugs, and it is found in some of the most commonly used antimalarial drugs. Figure 3 shows the main quinoline-based antimalarial drugs that are either commercially available or in clinical development. This class of drugs can be divided into three general subgroups: (i) 4-amino quinolines including chloroquine (3), amodiaquine (4), piperaquine (5), and pyronaridine (6), (ii) quinoline methanols including quinine (1), quinidine (2), and mefloquine (7), and (iii) 8-aminoquinolines that include primaquine (8), pamaquine (9), and tafenoquine (10).39 Chloroquine (3), amodiaquine (4), and pyronaridine (6) are weak bases that are diprotonated and hydrophilic at neutral pH.40 Quinine (1), quinidine (2), and mefloquine (7) are weaker bases and lipid soluble at neutral pH. This family is structurally derived from quinine (1), the active ingredient found in the bark of the Cinchona tree, used for centuries to treat malaria. In 1820, quinine (1) was the first antimalarial drug to be identified and isolated.33 For almost two centuries, it was the only widely available medication for clinical treatment of malaria.33 Despite its relatively low efficacy and tolerability, quinine (1) still plays an important role in the treatment of multidrug-resistant malaria.38,39 It is one of the few 3454


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table salt supplies all over the world, as a prophylactic.37 Possibly as a result of this medicated salt program, the first cases of chloroquine resistance appeared at the end of the 1950s.37,40 The extensive and quick spread of resistance literally destroyed the therapeutic efficacy of this drug. As chloroquine-resistant parasites started appearing, efforts were initiated to develop new antimalarial drugs. Synthetic derivatives included new antimalarial drugs such as the 4-aminoquinoline amodiaquine (4) and the quinoline methanol mefloquine (7) among others. Several of these derivatives, designed in the 1970s and 1980s, have subsequently decreased in efficacy due to developing resistances. Amodiaquine (4) was one of the first chloroquine derivatives synthesized to replace chloroquine (3) but has been used less extensively.39 Unlike chloroquine (3), more serious side effects like hepatotoxicity and skin-related effects are associated with amodiaquine (4) and therefore prevent its use in prophylaxis, and its use has been limited since the mid 1980s.37 Although there is significant cross resistance between amodiaquine (4) and chloroquine (3), many of the chloroquine-resistant strains of P. falciparum remain sensitive to this drug, and it is still used in ACT as one of the drugs in combination with artesunate (19). Piperaquine (5) is a bisquinoline antimalarial drug, introduced in the 1960s, as a replacement for chloroquine in first-line treatment for P. falciparum and P. vivax malaria.35 Unfortunately, its extensive usage led to development of resistance, but due to its good tolerability it is now being used as part of an ACT together with dihydroartemisinin.35 Pyronaridine (6) is a synthetic 1-aza-9-anilino-acridine and one of the newest members of this family. It was developed in China in the 1980s, and it recently received the approval of the European Medicines Agency (EMA) in a fixed-dose combination with artesunate (Pyramax).41 Pyronaridine (6) is to date the most active 4-amino quinoline derivative and can be administrated orally.37 It is generally active against chloroquine-resistant parasite strains42,43 and is to be included as an ACT agent in the future.35,41 Mefloquine (7) is a 4-quinoline methanol that was introduced in the mid-1980s to treat patients suffering from chloroquineresistant malaria. Mefloquine (7) was developed by the U.S. military with the purpose of identifying a suitable synthetic drug to replace quinine (1) and chloroquine (3).37 Mefloquine (7) is administrated orally and used in prophylaxis. In different parts of the world it is sold under the commercial names of Lariam, Mef liam, or Mephaquine. Considered a standard therapeutic agent for chloroquine-resistant malaria, use of 7 is limited by its high cost and the appearance of neuropsychiatric side effects.44 Although it is still effective in most areas, parasites resistant to mefloquine appeared early in its history of use and are now widespread in Southeast Asia and Africa.39 Used for treatment of uncomplicated multidrug-resistant P. falciparum malaria,44 mefloquine (7) is also one of the drugs administrated in combination with artesunate as one of the recommended ACTs. Structurally related to the 4-aminoquinolines and quinoline methanols are the 8-aminoquinolines that include primaquine (8), pamaquine (9), and tafenoquine (10). Since they are structurally related to the 4-amino quinolines, they are suspected to have similar targets and mechanism of action; however, the exact mode of action remains unknown.44,45 Primaquine (8) is extremely effective against the hypnozoite stage of P. vivax and P. ovale (before the erythrocytic infection), a latent form of liverstage parasites commonly seen in P. vivax infections.45 In addition, it also prevents the maturation of fertile gametocytes.45

In the blood stages of the parasite life, this drug is inactive at pharmacologically achievable concentrations.45 Primaquine (8) is administrated orally and has a narrow therapeutic index; it is not suitable for prophylaxis and has toxic side effects such as methemoglobinemia and hemolysis.46 Tafenoquine (10) is a newer drug that offers improved activity, less toxic side effects, and a longer acting time in comparison to primaquine (8).46 In addition, tafenoquine (10) is also active against the blood stages in chloroquine- and multidrug-resistant strains of the parasite.46 Since these two drugs (8, 10) show activity against hepatic-stage parasites, they are ideally suited for treating P. vivax infections either alone or in combination with other antimalarial drugs and also as prophylactics.45,46 The potential toxicity concerns for both primaquine (8) and tafenoquine (10) are methemoglobinemia and hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient people as a potentially life-threatening adverse effect.35 This family of drugs (except for the 8-aminoquinolines 8−10) are also known as schizontocides40 because they kill schizonts, the main form in which the malaria parasite exists in the intraerythrocytic stage of the malaria infection. Members of the artemisinin family are considered schizontocides as well.40 All these quinoline-based drugs offer both advantages and disadvantages either due to reduced activity, as a consequence of development of resistance, or due to detrimental toxic side effects. Nowadays, the disadvantages are usually compensated for by pairing these drugs and others with artemisinin or its derivatives. 3.2. Mode of Action of Quinoline-Containing Drugs

During the intraerythrocytic stage of the malaria infection, the parasite matures asexually from rings to trophozoites to schizonts to merozoites, which are then released by lysis to invade new erythrocytes (Figure 1). Most of the quinoline-containing drugs are active only against the intraerythrocytic stages of the parasite infection and used to rapidly clear parasites from the blood.45 While inhabiting the red blood cells, the parasite is surrounded by the single major cytosolic protein in that media, human hemoglobin. During the trophozoite form and at the early stages of the schizonts, the parasite ingests 60−80% of the hemoglobin present in the red blood cell along with the cytoplasm by a phagocytosis-like mechanism and transports the hemoglobin into its digestive vacuole.47,48 The parasite food vacuole is a unique and sophisticated proteolytic organelle.49 With an acidic pH (5.0−5.4) and equipped with multiple and diverse proteases, this organelle appears to be specialized exclusively in hemoglobin degradation.50 In this parasitic organelle a large variety of processes like acidification, hemoglobin-proteolysis, peptide transport, heme crystallization (hemozoin formation), and detoxification of oxygen radicals take place.51 Inside the vacuole, hemoglobin is digested into small peptides which are subsequently transported to the cytoplasm of the parasite.47,48 Degradation of hemoglobin occurs in a systematic pathway that involves a series of proteases operating in a highly specific pathway for hemoglobin proteolysis. These proteases, present in the parasite food vacuole, can be divided into four groups: aspartic proteases (plasmepsins I, II, and IV), cysteine proteases (falcipain 2 and 3), a histoaspartic protease (HAP), and a zinc metalloprotease (falcilysin).51−53 These proteases act in a semiordered fashion to degrade the hemoglobin tetramer. The first three groups, plasmepsins, HAP, and falcipain, play an important role, initiating catabolism by cleaving hemoglobin, 3455


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Figure 4. Structures of Fe(III)PPIX (A), dimeric unit of β-hematin (B), and β-hematin (synthetic hemozoin, C).

while falcilysin cleaves peptide fragments generated by the upstream proteases, generating the amino acids necessary for parasite survival.54−56 Because the parasite has a limited capacity to synthesize amino acids,51 hemoglobin is thought to be broken down to provide essential amino acids for growth and maturation, making hemoglobin catabolism essential for parasite survival.47,48 For this reason, targeting hemoglobin metabolism appeared as a strategy for antimalarial drugs. Proteolysis of hemoglobin leaves free heme as a waste product, the iron in which is rapidly oxidized from Fe2+ to Fe3+ to give ferriprotoporphyrin IX (PPIX) (presumably present as H2O− Fe(III)PPIX) (Figure 4) as a toxic byproduct of this catabolism. Free heme can cause enzyme inhibition, peroxidation of membranes, production of oxygen free radicals, and impaired leukocyte function.57 Heme oxygenase, used by vertebrates to catabolize heme, is absent or occurs in neglectable concentrations within P. falciparum.57 Plasmodium species have developed different pathways to detoxify heme. The most important and predominant mechanism is the sequestration of heme and its crystallization process into an insoluble and chemically inert crystalline structure called hemozoin or malaria pigment, which accumulates within the parasite food vacuole.52 The pigment is visible under the microscope (black opaque crystals) in parasite stages that are actively degrading hemoglobin, such as trophozoites, schizonts, and gametocytes.52

Hemozoin was formerly considered to be a polymeric chain of heme in the form of β-hematin, a noncovalent coordination complex with the ferric iron of each heme moiety bound to a carboxyl side chain of the adjacent heme.58 A ground-breaking discovery was the report of the crystal structure of β-hematin that confirmed hemozoin as a crystalline arrangement of dimers of five-coordinated Fe(III)PPIXs linked by reciprocal iron− carboxylate bonds to one of the propionate side chains of each porphyrin macrocycle and the dimers forming chains, linked by hydrogen bonds in the crystal (Figure 4).59−61 Different mechanisms of hemozoin formation have been proposed, based on observations from the in vitro synthesis of β-hematin.62 The process is regarded as a nonenzymatic autocatalytic biocrystallization process.63−65 The exact mechanism of hemozoin formation in vivo is unknown, although a large amount of evidence points to an oxidative lipid-catalyzed process66−68 that could start at the transport vesicles that carry hemoglobin into the parasite food vacuole.65,69 Although less strongly supported, a process promoted by malarial histidine-rich proteins has been suggested to take place as well,70,71 but the exact role of these proteins and lipids in the malaria pigment formation has yet to be determined. Despite decades of use of the above-mentioned drugs, the mechanism of action is not completely understood. Many different theories were studied in the past, but nowadays it has been widely accepted that the mechanism of action of these drugs involves the interruption of the formation of hemozoin.45,54 3456


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operate simultaneously yet with different efficiencies.84 Quinoline-containing drugs have been shown to be capable of both inhibiting formation of hemozoin and degradation of heme,84,85 following of course certain differences in their paths of action that could explain the differences on therapeutic responses. Many quinolines, as well as artemisinin derivatives, seem to bind heme.73,85 Quinine (1) and mefloquine (7) bind heme less avidly than does chloroquine (3).85 In addition to chloroquine (3), quinine (1), quinidine (2), mefloquine (7), and tafenoquine (10) have been shown to inhibit the process of formation of hemozoin whereas primaquine (8) does not.86 Thus, the mechanism of action of all quinoline-containing drugs may not be identical to that of chloroquine (3), although they may share some biological effects. The mode of action of these and other antimalarials for inhibition of both pathways, hemozoin formation and heme degradation, has been reviewed extensively in the literature.86−89 Figure 5 shows a schematic representation of the mechanism of formation of hemozoin and the proposed mechanisms of action of quinoline-containing drugs on the hemozoin.

Quinoline-containing drugs such as chloroquine (3) and quinine (1) accumulate in the acid food vacuoles of the intraerythrocytic stage of the malaria parasite and are selectively trapped inside the food vacuole in an ion-trapping mechanism.40,45,54 For example, the weak base chloroquine (pKa1 = 8.1, pKa2 = 10.2) in its nonprotonated form can diffuse across the vacuolar membrane, where it is protonated upon entry.40 As an ionic compound it cannot diffuse out of the vacuole and becomes effectively trapped.40 Chloroquine (3) can accumulate to a large extent inside the vacuole, with a gradient ranging from nanomolar concentrations on the outside to millimolar concentrations inside the digestive vacuole of the intra-erythrocytic trophozoite.72 There is extensive evidence indicating that chloroquine (3) exerts its antiplasmodial activity by interfering with the sequestration of toxic heme, thereby halting the parasite natural detoxification process.40,45 Two distinct pathways have been proposed. The first pathway suggests interference of the drug with formation of hemozoin and can be subdivided into two different modes of action. First, direct interaction and binding between drug and hematin, which ultimately prevents incorporation of hematin into hemozoin, disturbing its crystallization process.73 Second, a mode of action demonstrated for chloroquine in particular suggests the inhibition of hemozoin formation, or heme biomineralization, by chemisorption of the drug onto crystallized hemozoin, capping the fast-growing faces of crystalline hemozoin, through formation of very strong lipophilic and cytotoxic heme−chloroquine coordination complexes.74,75 This results in the build-up of large concentrations of free heme, perturbing the barrier properties of cellular membranes irreversibly, causing electronic and membrane stress, disturbing ion homeostasis, and eventually causing destruction of membranes and parasite death.40 The second pathway antimalarials are thought to disrupt is degradation of heme, another important detoxification process of the parasite. This pathway suggests drug interaction blocking the glutathione- or hydrogen-peroxide-dependent degradation of hematin.76−78 Free heme can be degraded by two nonenzymatic processes: through reaction with hydrogen peroxide, generated from spontaneous oxidation of heme from Fe(II) to Fe(III),76 and through reaction with reduced glutathione, when heme diffuses into the cytoplasm of the parasite.77,78 This mechanism of detoxification was initially proposed to account for removal of about 70−80% of the heme released due to hemoglobin degradation,76 and it was demonstrated that quinolinecontaining drugs block this process under conditions resembling those inside the food vacuole.77 This mechanism was questioned due to a lack of direct and conclusive evidence of the released iron.39 Later studies of total iron measurements in parasitized red blood cells demonstrated that at least 70−95% of the heme released from hemoglobin is sequestered as hemozoin.79,80 There is reference to a disputed third pathway of inhibition of hemozoin formation by quinoline antimalarials. Proposed by Slater and Cerami, this pathway considers the hemozoin formation process via a heme−polymerase enzyme, which could be inhibited by quinoline-based drugs;81 however, subsequently, several quinoline antimalarials have been shown to specifically inhibit β-hematin (synthetic hemozoin) formation in the absence of any protein, demonstrating that no enzyme is likely to be involved in the process.82,83 In addition, the existence of heme−polymerase and its activity has yet to be demonstrated. It has been suggested that both pathways, inhibition of formation of hemozoin and inhibition of degradation of heme,

Figure 5. Schematic representation of the mechanism of formation of hemozoin inside the parasite digestive vacuole. Once chloroquine (CQ) has entered the digestive vacuole, it becomes protonated and is believed to interact with free Fe(III)PPIX and with formed hemozoin, halting the detoxification process of the parasite.

Quinoline-containing drugs, in particular, chloroquine (3), are known to intercalate into the double helix of DNA through ionic interactions,90,91 decreasing the twist of the double helix, altering the conformation of the DNA,92 as well as the biological and physical properties of the helix.93,94 This initially led to the hypothesis that the effects of this intercalation might have something to do with the mode of action of this antiplasmodial, since intercalation of CQ inhibited both DNA and RNA polymerase reactions as a direct consequence of its interaction with the DNA primer.95 DNA intercalation was ruled out as the primary mode of action of these types of drugs eventually; however, intercalation may still play an important role, especially for metal complexes of CQ, as CQ−Ru compounds have been shown to be efficient cancerostatic drugs, working on the intercalation stage.96 3457


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3.3. Resistance

chromosome 7 codes one of these transporter proteins, termed PfCRT (P. falciparum Chloroquine Resistant Transporter) that could be either a channel or a carrier in the membrane of the food vacuole.106 Sets of point mutations in Pf CRT were found to be associated in vitro with chloroquine resistance in several P. falciparum strains from Africa, South America, and South East Asia.72,100 The principal mutations seem to be lysine to threonine (K76T) and alanine to serine (A220S).107 They were indeed detected in all resistant isolates but not in chloroquine-sensitive isolates when tested in vitro.107 Maybe even more importantly, transfection of this genotype sufficed to confer chloroquine resistance in a parasite strain.107 A mutation from asparagine to tyrosine (N86Y) in the Pf MDR1 gene (Plasmodium falciparum Multi Drug Resistance gene) from the chromosome 5 that codes the transmembrane transporter protein P-glycoprotein homologue 1 Pgh-1 or PfMRD1 is also suspected to be responsible for chloroquine resistance by altering the membrane permeability due to an accelerated drug efflux mechanism.108,109 This mutation was found in 48 out of 56 chloroquine-resistant cases.110 It was observed that Verapamil, a Ca2+ channel blocker and resistance modulator in multidrug-resistant cancer cells, was effective in reversing chloroquine resistance in certain parasite strains.111 By analogy to its mechanism of action in cancer cells, it was proposed that an enhanced expression of an ATP-dependent efflux pump (P-glycoprotein) was leading to more rapid expelling of the drug from the parasite food vacuole.111 Although amplification of Pf MDR1 was observed in a number of CQresistant isolates,109 a correlation across a wide variety of strains could not be substantiated, and therefore, no well-built evidence of amplification of this gene with chloroquine-resistance was found.112,113 Nevertheless, significant amount of evidence indicates that polymorphisms within PfMDR1 could contribute to a certain extent to chloroquine resistance.114,115 Much evidence suggests that the Pf CRT K76T mutation is more strongly associated with chloroquine resistance than is the Pf MDR1 N86Y mutation. It has been considered that the latter mutation plays a secondary role in chloroquine resistance.107 Such pronounced resistance response is ultimately believed to be caused by more than two mutations of the Pf CRT that are able to promote important physiological changes in the digestive vacuole; several resistant strains show closely related Pf CRT alleles that differ from those of sensitive strains in seven or eight point mutations.107 Given the similarities in molecular structure, some of these processes confer resistance to other quinoline-containing drugs such as mefloquine (7) and quinine (1) as well, generating cross resistance to other, structurally different, antimalarials with similar mechanisms of action like the artemisinins.114 In fact, an increase in Pf MDR1 copy numbers is associated with resistance to mefloquine (7) and other quinoline-based drugs.116 Nevertheless, the mechanisms of resistance against this family of antimalarials and others are still an active area of research.

Parasite resistance has rendered many antimalarials ineffective. Chloroquine (3) began to fail only 10 years after being put into use. Chloroquine-resistant (CQ-resistant) P. falciparum started spreading in all directions in Asia as well as to East and eventually to West Africa.27 By 2000, the drug was virtually useless in most parts of the world.27 Chloroquine resistance in a parasite strain has been defined in the literature as those strains presenting IC50 values greater than approximately 110 nM.97 As is the case for the mechanism of action, the mechanism for development of resistance against quinoline-containing drugs is not well characterized. What is known though is that the mechanisms by which the parasite turns chemoresistant to antimalarials generally involve chromosomal mutations.98 A genome analysis revealed that chloroquine-resistant parasites originated at four different sites or events that occurred in different geographic locations and subsequently spread outward, leading to widespread chloroquine resistance.98 A lot of effort has been invested during the last decades into a better understanding of how the malaria parasite developed drug resistance, since this knowledge should facilitate design of new agents that circumvent or reverse known parasitic resistance mechanisms. In numerous chloroquine-resistant parasite strains one characteristic recurs: when compared to chloroquine-sensitive strains, parasites would accumulate much lower (four to ten times) levels of drug inside their digestive vacuoles.99 From this it was concluded that resistance to chloroquine in P. falciparum is not due to a change in heme processing but to an alteration in the chloroquine accumulation in the digestive vacuole.100 This could be caused by lower rates of drug influx, higher rates of drug efflux, or a combination of both.100 To a certain degree, resistance would also be influenced by any reduction in binding affinity of the drug for its ultimate target.72 Changes in the transmembrane proteins of the food vacuole in resistant strains of P. falciparum cause a reduced accumulation of the drug in the parasitic food vacuole and, therefore, reduced accumulation of the toxic heme byproduct.101 How these changes affect the mechanisms of drug uptake are still vague. It was initially proposed that mutations of the digestive vacuole lead to a lowered pH inside the vacuole that consequently affects the magnitude of the H+ gradient across this membrane.102 Lower pH values inside the digestive vacuole promote aggregation of relatively insoluble Fe(III)PPIX dimers that would still be able to crystallize to form hemozoin but would not bind the quinoline as favorably as the free soluble Fe(III)PPIX does.102 Since it was proposed that chloroquine accumulation is determined by its access to free heme,99 it was thought that the chloroquineresistant parasites would accumulate less drug at this lower pH inside the food vacuole; however, this hypothesis has mainly been disregarded.103 Recent studies could not conclusively prove a significant difference between the pH of the digestive vacuole in resistant vs sensitive parasite strains.103 A more accepted theory proposes a direct or an indirect alteration of the structurally specific drug interaction, affecting the flow of drug across the vacuolar membrane.104,105 The observed resistance is probably conferred by mutations in multiple genes encoding transmembrane proteins that are necessary for the transport of drug into the food vacuole of the parasite.106,105 There are at least two documented types of transporter proteins that have been mutated in the case of chloroquineresistant Plasmodium falciparum.105 The Pf CRT gene located on

3.4. Other Traditional Antimalarial Drugs

Several classes of drugs are used to treat malaria. Most of these drugs target the intraerythrocytic stage of the parasitic life cycle, with some exceptions to be mentioned below. The most important classes of antimalarial drugs are the quinolines, antifolates, and artemisinin derivatives. The family of the quinolines have already been discussed in section 3.1. In this section, the remaining principal antimalarial drugs used and developed during the last decades, as well as some 3458


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Figure 6. Antifolate drugs and combinations.

Figure 7. Artemisinin and derivatives.

are the latest class. This family has been extremely important for treatment of malaria in the last 20 years, especially for P. falciparum malaria. Currently, the artemisinin family of drugs is the only recommended treatment for P. falciparum malaria.19 Artemisinin (17) is isolated from the Chinese sweet wormwood (Artemisia annua), a plant used for centuries to treat fever. Artemisinin (17) is a sesquiterpene lactone with an endoperoxide bridge in a seven-membered ring system. It is poorly soluble in water and decomposes in other protic solvents, probably via lactone ring opening.118 It was isolated in 1970 for the first time,118 and the complete chemical synthesis was reported as early as 1983 but deemed too expensive for commercial pharmaceutical production;119 a semisynthetic family of derivatives was developed soon after. The watersoluble and more potent derivatives are artemether (18), artesunate (19), and dihydroartemisinin (20). Artemisinin derivatives are extremely efficient in killing parasites, act faster than any other antimalarial available, have a rapid symptomatic response, and are absorbed and eliminated rapidly form the host bloodstream (t1/2 < 60 min.).120 While the latter pharmacokinetic characteristic is beneficial for development of a drug, since it reduces the exposure time and chances of developing resistance, it also dictates that artemisinin-based drugs be taken frequently in order to be effective. Noncompliance could cause high rates of malaria recrudescence or reappearance. To overcome this major drawback, these compounds are often used in conjunction with other effective agents (for instance, in ACTs) to improve antimalarial efficacy. Also, considerable efforts are now being made to develop new derivatives that are more stable in vivo, resulting in improved bioavailability.35 In contrast to other antimalarials, artemisinin-based drugs show antiplasmodial effects at all stages of the malaria infection: both at the asexual pre-erythrocytic (liver) and erythrocytic (bloodstream) stages inside the host as well as in the sexual stage as gametocytocidal agents (killing Plasmodium gametes inside

of the recently introduced drugs, are outlined emphasizing their main pharmacological properties, therapeutic uses, and some of their limitations. The family of antifolate drugs is formed by drug pairs composed of a sulfa drug (sulfadoxine (11), sulfalene (12) or dapsone (13)) and either pyrimethamine (14), proguanil (15), or chlorproguanil (16) (Figure 6). Antifolates are used in fixedratio combinations. The most common examples are sulfadoxine/pyrimethamine (11/14) (SP, Fansidar), which was the first line drug for uncomplicated P. falciparum malaria right after chloroquine was rendered useless, for about 5 years until resistance against the SP combination developed.37 Chlorproguanil/dapsone (16/13) (CD, Lapdap) is a more recent combination that is projected to eventually replace SP.117 The antimalarial potency of these agents relies on their capacity to inhibit parasite metabolism, in particular by inhibiting folic acid synthesis. Pyrimethamine (14) and the biguanides 15 and 16 inhibit dihydrofolate reductase (DHFR), responsible for reduction of dihydrofolate to tetrahydrofolate, and have a greater affinity with the plasmodial enzyme than with the human enzyme.38 The sulfonamides and sulfones 11, 12, and 13 inhibit dihydropteroate synthetase (DHPS), another enzyme of the folate pathway, absent in mammals.38 These two inhibitors work synergistically depleting the pool of tetrahydrofolate (a cofactor of DNA synthesis) and therefore ultimately interfering with the parasite DNA synthesis.38 One of the advantages of CD (16/13) over SP (11/14) is the short plasma half-life time associated with each component of the combination, which reduces the therapeutic time of administration and the possibility of developing resistance.117 The efficacy of this combination is under clinical study, and SP is at the moment the only combination of this family used in conjunction with artesunate (19, Figure 7) as one of the ACTs. Probably the most important family to be mentioned in this section are artemisinin (17) and its derivatives (Figure 7). Among what can be called the traditional antimalarial drugs, they 3459


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Figure 8. Aryl amino alcohols in clinical use for treatment of malaria.

the mosquito gut), thus lowering transmission rates.120 This unrestricted action of artemisinin and derivatives might be the key to their potency. Although the exact site and mechanism of action of the artemisinins is still being debated, structure−activity relationship (SAR) studies have indicated that the presence of the endoperoxide bridge within the sesquiterpene lactone is essential for antimalarial activity.119,120 Similar to 4-amino-quinoline compounds, artemisinin derivatives bind to hemozoin crystals to unfold their antimalarial activity. Artemisinin does not inhibit formation of hemozoin but instead forms adducts with hemozoin and other biological macromolecules including translationally controlled tumor protein (TCTP) and other higher molecular weight proteins.121 This catalyzes the breakdown of the artemisinin-labile peroxide bridge, generating free radicals that alkylate and oxidize important proteins within the parasite, causing lethal damage to the parasite membrane systems.122 Inhibition of a parasite-specific ATPase essential for its survival is one example.123,124 Unfortunately, artemisinin-based therapy is not readily available and affordable everywhere. As mentioned before, the supply of natural artemisinin varies and it is produced in low yield.28 Semisynthetically produced artemisinin and derivatives are costly and pose the already mentioned challenges associated with agricultural production (section 2).28 In terms of toxicity, even though it appears to be safe in humans, it has been demonstrated to produce long-lived toxic metabolites in animal studies.122 Additionally, based on its radical-activated mechanism, neurotoxicity and cumulative toxicity cannot be ruled out. As with other antimalarials, artemisinin-resistant strains of Plasmodium have already emerged but ACTs offer the possibility to contain these resistant strains. Other antimalarials in clinical use are depicted in Figure 8. Atovaquone (21) is a hydroxynaphthoquinone that selectively inhibits electron transport in the mitochondria of the parasite (affecting respiration) through the cytochrome c reductase complex, without affecting the host mitochondrial functions.125,126 Although highly effective, 21 showed a very high rate of recrudescence and later developed resistance.127 This was subsequently resolved by developing a fixed combination product (Malarone) of 21 with the biguanide proguanil (15), a folate inhibitor (see antifolate and combinations above). The mechanism underlying the synergistic action between 21 and 15 is still unknown, but some data suggests proguanil (15) enhances the atovaquone-induced collapse of the mitochondrial potential.127 Maintenance of this transmembrane potential is key in various metabolic processes. Proguanil (15) by itself does not seem to have any effect on either electron transport or membrane potential in malarial mitochondria.40 Although this combination

is highly effective, even against the pre-erythrocytic (liver) stages of the infection, cases of resistance against this combination have threatened to limit its efficacy.37 Halofantrine (22) and lumefantrine (23) are two structurally related drugs that are very active in the erythrocytic stages of the malaria parasite. Halofantrine (22) has been shown to interact with hematin; therefore, it is suspected that both 22 and 23 act by inhibition of hemozoin formation in the parasite food vacuole.128 Halofantrine (22) is a phenanthrene−methanol derivative, highly effective against multidrug-resistant P. falciparum, but of restricted use due to its cardiotoxicity.35 Lumefantrine (23), previously referred to as benflumetol, is an aryl amino alcohol effective against chloroquine-sensitive and -resistant P. falciparum isolates.35 Although less effective than 22, lumefantrine (23) is commercialized since it does not induce cardiotoxicity.37 Lumefantrine (23) is a long-resident drug (half-life time of 3−6 days)37 that is currently combined with the fast-acting, shortlived artemether (18, Figure 7). Certain antibiotics such as fosmidomycin (24), clindamycin (25), tetracycline (26), and doxycycline (27) (Figure 9) also

Figure 9. Antibiotics in clinical use for treatment of malaria.

exhibit antimalarial activity.35,37 These antibiotics exert their antimalarial effects via inhibition of protein synthesis interfering with the parasite fatty acid biosynthesis, taking advantage of a set of parasitic enzymes that are different from the ones utilized by humans.129 They also inhibit other processes in the apicoplast.129 This plastid-like organelle is more prokaryotic than eukaryotic, so it is exclusively found in the parasite. Because of their slow effect, most antibiotics are used in conjunction with quinine (1, Figure 3460


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Figure 10. Metal chelators used for antimalarial therapy.

3) and other antimalarials to treat uncomplicated P. falciparum malaria.35,37 A thorough discussion of the mechanisms of resistance development of P. falciparum against most of these drugs, with the exception of quinolines, discussed earlier, is outside of the scope of this article, and further information is available elsewhere.40,72,100 In regard to novel approaches of antimalarial chemotherapy, advances in the genome and proteome data acquired from mapping the genetic sequence of Plasmodium have revealed many new potential drug targets specific to the vital functions of the parasite without affecting host systems. Several parasite proteins that are products of nuclear encoded genes have been identified, and due to the critical involvement of these enzymes in parasite development, they are currently being pursued as potential drug targets. Among these targets are inhibitors of proteases, farnesyl transferase, peptide deformylase, fatty acid synthesis, glycolysis, and choline uptake.35,37,54 Proteases such as the plasmepsins are needed to catalyze degradation of host hemoglobin during the intraerythrocytic stage of the malaria infection (section 3.2). Farnesyl transferase catalyzes the transfer of the farnesyl residue in certain proteins that are involved in regulatory processes.130 Peptide deformylase is a protein responsible for removing N-terminal formyl groups during maturation of proteins.130 Enzymes that catalyze fatty acid synthesis of the parasite are located in the parasite-exclusive apicoplast and can therefore can be selectively attacked.131 Due to the absence of a citric acid cycle, the parasites produce the energy they require via anaerobic glycolysis. Choline uptake from

the blood is vital for continuous production of membrane lipid during the intraerythrocytic stage of the infection.131 Some of these processes have already been actively pursued as a target for development of cancer chemotherapeutics and have recently been reinvestigated as targets for malaria.37 At the same time, there have been innovations in traditional antiplasmodials. Research toward new hemozoin formation inhibitors, able to overcome the current resistance situation and new artemisinin derivatives, less prone to hydrolysis, have produced a series of candidates currently in preclinical development.37 In the process of discovering and developing new antimalarial drugs, multiple general approaches were followed.36 A first approach was the optimization of existing agents by improving the dosing regimens or formulations. Furthermore, combination therapy offered advantages: two antimalarials with different sites of action in the parasite led to a simultaneous attack to improve antimalarial activity. In addition, combination therapy reduces the chances of encountering resistance (double-resistant parasites strains are less likely to occur) or developing resistance (a reduced parasitic exposure time to the most effective of the combinations reduces the chances of developing resistance). A second strategy was the development of structural analogues of existing agents. This approach was responsible for the development of many currently existing antimalarials and is still one of the most pursued strategies. Other strategies include the fortuitous identification of antimalarial potency in compounds active against other diseases and the discovery of agents that reverse drug resistance. All these strategies have been and are 3461


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treat hemochromatosis, is the first example of a vast family of iron chelators used for the therapy of malaria. DFO (28) is pharmaceutically well known and widely used in experimental studies and clinical applications.124 It inhibits malaria in vitro and in vivo140 and has been used clinically to treat malaria in humans.141,142 The success of this iron chelator, but also its limitations such as its relatively slow permeation into infected cells and restriction to advanced stages of P. falciparum, prompted development of improved analogues. Early modifications of this compound involved making DFO more lipophilic to improve membrane permeability, leading to some derivatives that were more active, more efficient, and acted against different stages of the infected cells. MA-DFO (29), with an N-methylanthranilic acid attached to the N-terminus of 28, is more lipophilic and has shown considerably higher antimalarial activity compared to 28.143 Another derivatization approach was the synthesis of a zinc−desferrioxamine (Zn−DFO) complex (not pictured), which was considered more permeating than the parent compound, to improve cellular uptake in infected erythrocytes.144 This preformed complex penetrates infected erythrocytes and exchanges Zn(II) for Fe(III) in a transmetalation reaction, forming a more stable complex. Zn−DFO seemed to be superior to metal-free DFO, improving the inhibitory potency against parasite growth to concentrations below 20 mM.144 In addition to DFO and its derivatives, several other classes of Fe(III) chelators have been considered for malaria chemotherapy: catecholate siderophores, aminothiols, 3-hydroxypyridin-4-ones, bis-hydroxyphenyl-triazoles, acylhydrazones, thiosemicarbazones, dihydroxycoumarins, polyanionic amines, aminophenols, bicyclic imides, 2,2′-bipyridyl, and 8hydroxyquinolines.138 Catecholamides and catecholate siderophores have been developed specifically for their antimalarial activity. FR160 (30) is a catechol iron chelator siderophore with remarkable antimalarial activity.145 It is 7−60 times more active than 28 against chloroquine-sensitive and chloroquine-resistant strains of P. falciparum with average inhibitory concentrations (IC50) of 170 nM and 1 mM, respectively.146 A study in 137 isolates of P. falciparum showed IC50 values of 30 ranging from 0.1 to 10 μM and the mean IC50 being 1.48 μM.147 In comparison, DFO (28) had a mean IC50 of 20.7 μM for 73 isolates.147 Within the family of aminothiol multidentate chelators two remarkable examples are described: ethane-1,2-bis(N-l-amino-3ethylbutyl-3-thiol) (BAT, 31) and N′,N′,N′-tris(2-methyl-2mercaptopropyl)-1,4,7-triazacyclononane (TAT, 32) showed IC50 values of 7.6 and 3.3 μM, respectively, against a CQ-sensitive P. falciparum strain.148 These two compounds were 5−10 times more potent than was DFO (28).148 The bidentate 3-hydroxypyridin-4-ones (HPOs) have been extensively studied as orally active Fe(III) chelating agents and investigated for antimalarial activity.149 The best representative of this family is deferiprone (1,2-dimethyl-3-hydroxypyridin-4one, 33) that, when administered in combination with other antimalarial drugs such as quinine (1) or doxycycline (27), works resolving fever and coma faster and better than does DFO alone.150 A large variety of HPOs have been studied, in vivo and in vitro, to demonstrate their capacity as antimalarials.150 The tridentate chelator 4-[(3,5-bis-(2-hydroxyphenyl)-1,24)triazol-1-yl]-benzoic acid (ICL670) (34) is an iron chelator for the long-term oral treatment of iron overload that best combines high oral potency and tolerability in animals and originates from the bis-hydroxyphenyltriazoles, a relatively new class of

being applied to develop new antimalarials. This review will highlight metal-containing compounds that were results of application of the above-mentioned strategies and have been evaluated as potential antimalarial agents.

4. METALLOANTIMALARIALS Incorporation of metals into medicines dates back centuries. Metal-containing drugs offer a wide range fine tuning of either the metal or the ligand to achieve or improve an observed efficacy. Many successful examples of bioinorganic compounds currently used in therapy such as the platinum anticancer therapy agent Cisplatin and its successors as well as the technetium- and gadolinium-based diagnostic agents Cardiolite and Magnevist have demonstrated that application of metal-containing compounds in medicine can be beneficial and successful in both therapy and diagnosis.132,133 This approach offers the advantage of possible metal/organic fragment synergisms. The biological activity of the organic drug can be enhanced by masking it upon coordination to a metal ion and therefore extending its time of residence in the organism, allowing it to reach biological targets more efficiently.134 In addition, complexation of the metal can decrease the metalassociated toxicity through targeted transport to the specific site of action.134 Encouraged by the success of many metal compounds as antitumor and antiarthritic agents and the pronounced selectivity that certain metal-containing compounds show for biomolecules exclusively found in parasites,135 diverse metallic compounds, which had already been extensively applied in the treatment of other parasitic diseases in the past, were re-evaluated for antiplasmodial properties. As an example, various inorganic salts of bismuth were previously administered against major tropical diseases such as leishmaniasis as well as malaria itself.131 This trend has led to many metal compounds that have shown promising efficacy as antimalarials, described in this section. To the best of our knowledge, the term “metalloantimalarials” was first coined by Goldberg, Sharma, and co-workers in 2003.136 These compounds have been divided in three categories: metal chelators (section 4.1), intact metal coordination complexes of known antimalarial agents (section 4.2), and bioorganometallic compounds (section 4.3). 4.1. Metal Chelators

These therapeutic agents are administered in the form of free ligands to scavenge and chelate free metabolically active metal ions causing the death of the parasite due to deprivation.137 As for bacteria and other microorganisms, iron is essential for survival and replication of the Plasmodium parasite and must be obtained from sources exogenous to the parasite. For these purposes, these microorganisms secrete siderophores that bind extracellular iron(III) with high affinity and internalize the resulting complexes. The parasite sources of iron include plasma transferrin, host erythrocyte ferritin, and the labile intraerythrocyte iron pool.137,138 It has been suggested that the release of iron from hemoglobin decomposition is also one of the sources of iron for the parasite.139 Thus, iron deprivation caused by iron chelation therapy proved to be an option in malaria treatment. Withholding iron results in inhibition of the growth of P. falciparum by either depletion of iron from its metabolic pathways or in situ formation of toxic iron complexes.137 Some antimalarial metal chelators are represented in Figure 10. Desferrioxamine (DFO, 28), a siderophore useful in management of transfusion-related iron overload and primarily used to 3462


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tridentate iron-selective chelators.151 Studies show that when compared to DFO (28) malarial growth was significantly lower with 34.151 The latter was 4 times more active in growth control (at 48 h, growth relative to control was 53% with 34 and 83% with 28 at concentrations of 30 mM and 20% with 34 compared to 26% with 28 at concentrations of 60 mM) but with IC50 values in the same range.152 The acylhydrazones salicylaldehyde isonicotinoyl hydrazone (SIH, 35), 2-hydroxyl-1-naphthylaldehyde m-fluorobenzoyl hydrazone (HNFBH, 36), and pyridoxal isonicotinoyl hydrazone (PIH, 37) were found to be orally effective and inexpensive antimalarials when used individually and with potentiated action when used in combination with DFO (28), extending their action longer, potentially due to synergistic action.153 SAR studies in a series of derivatives with different functional groups in the aromatic ring and hydrazide functionalities indicated that, regardless of the group attached to the aromatic ring, the hydrazide derivative determines the action, with methylhydrazide and benzylhydrazide being the most active in inhibition of parasite growth.154 The highest antiplasmodial inhibition was obtained from the aromatic derivatives, substituted by tert-butyl, chloro, nitro, methyl, or methoxy groups, highlighting the importance of rather hydrophobic or bulky substituents.154 Structurally related to acylhydrazones, the thiosemicarbazones are another promising class of iron chelators. Aroylhydrazones and thiosemicarbazone iron chelators have shown antimalarial activity in vitro against chloroquine-sensitive and chloroquineresistant strains of P. falciparum, being significantly more active than DFO (28).155 Two of the most successful examples are 2hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311, 38) and 2-hydroxy-1-naphthylaldehyde-4-phenyl-3-thiosemicarbazone (N4pT, 39) with IC50 values of 4.5 and 3.0 μM, respectively, for a CQ-sensitive strain and of 4.75 and 2.5 μM, respectively, for a CQ-resistant strain.156 These values were significantly lower than those of DFO (28) and SIH (35), and these compounds appear to be the most effective of the analogues, having IC50 values four times lower than the parent compound PIH (37).156 Lastly, desferrithiocin (40) and derivatives are as active as 28 in in vitro inhibition of liver schizogony of both rodent (P. yoelii) and human (P. falciparum) malaria.157 One concern is, nonetheless, the toxicity related to the in vivo-formed complex that causes erythrocyte membrane disruption and renal toxicity.158 The mechanisms by which these drugs interfere with the sequestration of iron have been discussed in the literature.124,137 Possible mechanisms involve sequestration of iron(III) from vital sources such as storage proteins, low molecular weight siderophores, iron centers of key parasite enzymes, or the scavenging of iron from degraded hemoglobin.159 These iron chelators seem to extract the intracellular iron after crossing the membranes of red blood cells infected with P. falciparum binding selectively iron(II) and iron(III) ions. Iron has six coordination sites, and several of these ligands are pentadentate or tetradentate chelators that might leave one or two coordination sites open for toxic side reactions that could take place inside the parasite.138 Tridentate and bidentate chelators will form 2:1 or 3:1 complexes with the metal, respectively, and might undergo partial dissociation from iron, exposing coordination sites that could participate in toxic reactions in the inside of the parasite.138 Two mechanism of inhibition of parasite growth have been observed for these iron chelators: depletion of iron essential for

several vital processes or in situ formation of toxic iron complexes.137 Sequestering iron by chelation of free iron or by direct interference with enzymes that hold iron, causing in either case iron deficiency while interfering with parasitic essential metabolic functions, is the mechanism that has been documented for the majority of the known antiplasmodial iron chelators.137 The direct correlation between the lipophilicity and the antiplasmodial potency of these agents supports direct intracellular iron chelation as the mode of action: the higher the affinity to cross lipid membranes, the higher the antimalarial activity.159 Also, incubation of these compounds with other sources of Fe(III) suppressed or decreased the in vitro antimalarial activity.152 The same was observed when preformed complexes of Fe3+ were added. On the other hand, no decrease of activity was detected when incubated with free Cu2+ and Zn2+.124,152 The mode of action of desferrioxamine (DFO, 28) and several other siderophore derivatives has been found to involve fast access to intracellular compartments of parasitized cells, selective and high-affinity chelation of iron(III), followed by a fast exit from the cells while exerting significant and irreversible cell damage.159,160 DFO blocks DNA synthesis by limiting the iron supply to ribonucleotide reductase.161−163 Ribonucleotide reductase is one of the trophozoite enzymes essential for DNA synthesis, an iron-containing enzyme that catalyzes reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, the precursors of DNA. DFO inhibits ribonucleotide by chelating the intracellular iron pool and preventing regeneration of the iron-radical center in the enzyme.164−166 In addition to DNA damage, DFO and other derivatives seem to have a selective inhibitory effect in mitochondrial transcript levels, independent from the DNA effect, which might contribute to their antimalarial properties.167 Unlike mammalian cells, parasite cells are permanently damaged after treatment with these iron chelators, even once drug treatment is removed and iron sources are replenished.138,160 However, as mentioned above, DFO (28) is ineffective on the early stages of the parasite (ring form) due to its poor permeability. Derivatives of DFO of greater lipophilicity are more membrane permeable and show faster penetration and greater capacity for chelation of intracellular iron, causing irreversible damage in rings but not in trophozoites.168,169 When used in combination at any molar ratio, DFO and its more lipophilic derivatives had a synergistic inhibitory effect that was faster and more potent than the sum of their individual potencies.169 Combination of DFO and SIH (35) is also synergistic, with DFO potentiating SIH effects in ring stages by serving as a potential sink for iron extracted from cells by SIH (SIH-bound iron(III) could be transferred to DFO).153 SIH (35) and HNFBH (36) will swiftly shuttle between medium and cells to chelate cell iron and transfer it to extracellular DFO.153 The other mechanism of inhibition of parasite growth that has been observed for these iron chelators involves formation of toxic complexes with iron. This is the mechanism of action of aromatic metal chelators such as 8-hydroxyquinoline, alkylthiocarbamates, 2,2′-bipyridyl, and certain aminophenols that, after forming a complex with iron extracellularly, enter the red cell to produce a lethal free-radical intracellular reaction.170,171 Although it is the mechanism of action investigated for most antimalarials, inhibition of hemozoin growth has been rarely studied for metal chelators, possibly due to reports of DFO promoting in vitro β-hematin formation rather than inhibiting 3463


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Figure 11. Ru and Au complexes of chloroquine.

it.172 Few studies have proposed that iron chelators such as DFO, deferiprone, or ICL670 chelate iron from the hemozoin crystal, in addition to chelate essential iron from parasitic compartments.173,174 Only ICL670 was found to be as effective as CQ inhibiting β-hematin formation in vitro, while neither deferiprone nor DFO showed any inhibition.174 Studies of these iron chelators on other pathways of heme degradation, such as hydrogen peroxide and gluthatione-mediated degradations, showed that only CQ and ICL670 can inhibit peroxidative degradation of hemin while all iron chelators displayed virtually no inhibitory activity on GSH-mediated degradation of hemin.174 Of all described iron(III) chelators, only two have been used in clinical studies of malaria: desferrioxamine (28) and deferiprone (33); however, others have been demonstrated in vitro and in vivo to have far better antiplasmodial properties than either of these. Disadvantages associated with these compounds include the lack of selective targeting properties as well as undesired side effects for the patient, even though many of the described chelators seem to have a direct effect toward depriving the parasite of free iron and show less impact on iron levels of the host.140 Use of free ligands for antimalarial applications may result in formation of complexes with other essential metal cations in the extracellular and intracellular matrices. In addition, the antimalarial activity of these compounds, in the milli- to micromolar range, is not competitive with the nanomolar activities observed for other antimalarial drugs. On the other hand, among the advantages associated with the use of these

compounds are the facts that no resistance or signs of potential cross resistance have been found, either as standalone compounds or in combination with other known antimalarials and, given the large structural differences with other antimalarials, development of cross resistance at a later stage seems unlikely. 4.2. Metal Complexes

4.2.1. Metal Complexes of Chloroquine and Chloroquine Derivatives. One of the most successful approaches in the field of metalloantimalarials so far is the modification of the chloroquine structure with metal-containing fragments. Attachment of a metal to chloroquine (3) can result in a strong modification of the electronic properties of the parent compound while also affecting its bioavailability, transport, and other pharmaceutically relevant properties. A number of transition metal ions have been used to form coordination complexes with chloroquine.134,135,175,176 Complexes of chloroquine and chloroquine derivatives (Figure 11) have been shown to exhibit improved efficacy in both CQ-sensitive and CQ-resistant strains of P. falciparum compared to chloroquine (3). Although the mechanism of action of these metal-based agents is unknown, the presence of the metal ion results in an enhanced activity, notably against CQ-resistant strains. Ruthenium complexes of chloroquine were the first coordination complexes to be documented in this field.159 The dinuclear Ru(II) chloroquine [RuCQCl2]2 complex (41), formed from reaction of ruthenium(III) chloride hydrate and 5 equiv of CQ in the presence of a reducing agent, showed a 3464


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Figure 12. Metal complexes of other antimalarial drugs.

marked inhibition of P. berghei and P. falciparum strains.177 41 is five times more potent than is chloroquine against strains of P. berghei (IC50 = 18 nM).177 In vivo experiments in a rodent malarial model of P. berghei demonstrated 41 to be more potent than the parent drug 3, reducing the parasitemia levels by 94% in comparison to 55% for 3 at an equivalent concentration of 1 mg/ kg relative to CQ.177 No acute toxicity was observed in mice for up to 30 days after treatment.177 Further in vitro studies in chloroquine-resistant FcB1 and FcB2 strains of P. falciparum revealed 41 to be four times more potent than the diphosphate salt of 3 (CQDP) in the FcB1 strain and two times more potent in the FcB2 strain.177 The mechanism of action of [RuCQCl2]2 (41) has been described as the posthydrolytic binding of 41 to hematin in solution, inhibiting its aggregation to β-hematin.178 This inhibitory activity is significantly better than for CQ (when measured at the interface of n-octanol/aqueous acetate buffer mixtures at pH < 5). Partition coefficients indicate that 41 is significantly more lipophilic than is 3 and therefore more likely to accumulate near the water−lipid regions of the parasite digestive vacuole.178 On the basis of findings supporting that aggregation of β-hematin occurs near the water−lipid regions, an environment that is modeled by the interface of n-octanol/aqueous acetate mixture,179 it was proposed that the higher lipophilicity of this compound allows its higher accumulation at this interface leading to higher efficiency both in vitro and in vivo.178 Previous reports on the lower ability of the mutated transmembrane transporter PfCRT to promote efflux of highly lipophilic drugs in CQ-resistant strains of P. falciparum180 led to the suggestion that the lipophilicity of 41 might contribute to its increased activity in these strains.178 Furthermore, complex 41 interacts with DNA by intercalation of the quinoline fragment in a very similar way that CQ does.178 Gold(I) antimalarial chemistry has produced several coordination complexes, most through attachment of a gold phosphine fragment to chloroquine that resulted in improvement over the action of the well-known quinoline drug. These compounds

performed better than CQ (3) and were generally more potent than ruthenium and rhodium chloroquine complexes against both P. berghei and P. falciparum parasite strains. The most representative example is the gold(I) chloroquine complex [Au(PPh3)(CQ)]PF6 (AuCQ) (42). It is formed through reaction of triphenylphosphine gold(I) chloride in the presence of potassium hexafluorophosphate with 2 equiv of CQ under reflux conditions.181 This complex caused marked inhibition in vitro and in vivo in models of P. falciparum and P. berghei.181 In an in vitro model of rodent malaria parasite P. berghei, 42 demonstrated an IC50 value of 3 nM, 22 times more potent than the parent drug CQ (3, IC50 = 67 nM).181 In in vitro cultures of CQ-resistant parasite strains of P. falciparum (FcB1, FcB2) 42 was very potent as well, displaying IC50 values of 5.1 and 23 nM, respectively, making 42 nine or five times more potent than 3.181 In contrast to this earlier report, studies of 42 against FcB1 by the same authors reported only a 1.25 times higher potency compared to chloroquine.182 Complex 42 was screened in vivo against rodent malaria strain P. berghei and found to suppress parasitemia in infected rodents by 84% at a dose of 1 mg/kg body weight relative to CQ, while CQDP (3) in an equivalent treatment dose only afforded a reduction of 44% in the parasitemia.181 No acute toxicity or adverse reaction against 42 was observed.181 The mechanism of action of 42 has been investigated, and it was determined that it interacts, similarly to 41, with heme and inhibits β-hematin formation both in aqueous medium and near water/n-octanol interfaces at pH ∼ 5 to a greater extent than do CQDP or other known metal-based antimalarial agents.183 The higher inhibition activity is probably related to higher lipophilicity of 42 compared to CQ. Interaction with DNA, as does free CQDP, has been reported as well.183 A second generation of gold chloroquine complexes (43−47, Figure 11) was synthesized by variation of the phosphine ligand (43, 44) with the purpose of inducing changes in electronic and steric properties of the complexes, by variation of the counteranion, (NO3−, 45), or by variation of the gold oxidation state to Au(III) (46) and use of other biologically important 3465


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ligands (47).182,184 The change in oxidation state of the metal center of 46 and 47 leads to a new coordination through the Nterminus of CQ, rather than through the quinoline nitrogen. In vitro studies on the activity of these chloroquine gold compounds were carried out against several strains of P. falciparum of different CQ sensitivity, with all compounds displaying similar or superior activity than CQ, especially in CQresistant strains.182 From the comparison of the activities observed, it can be concluded that the nature of the anion is irrelevant for the activity of the complexes.182 Varying the substituent of the phosphine ligand did not indicate a clear trend either;182 however, one of the highest activities within this group of derivatives was found for [Au(PEt3)(CQ)]PF6 (44), which was 5-fold more effective than chloroquine and 4-fold more effective than the parent complex [Au(PPh3)(CQ)]PF6 (42) against the chloroquine-resistant strain FcB1.183 It was observed that some of these complexes can protect uninfected erythrocytes from parasite infection, something not observed for chloroquine.184 No clear structure− activity correlations could be established for this series of compounds.184 As discussed above for 42, the mechanism of action of these gold−chloroquine compounds has been considered to involve inhibition of hemozoin formation and DNA intercalation. Other less likely theories involve inhibition of DNA, RNA, and protein synthesis.184 4.2.2. Metal Complexes of Other Antimalarial Drugs. Complexes of two traditional antimalarials like amodiaquine (4) and primaquine (8) with a selection of metal ions that included VO(II), Cr(III), Fe(III), Cu(II), Co(II), Ni(II), Zn(II), Cd(II), Hg(II), Rh(III), Pd(II), Au(III), Ag(I), Mn(II), Sn(II), and Pt(II) of general formula [M2+(amodiaquine)(Cl)2] (48) and [Mn+(primaquine)2(X)n(H2O)y] (49) (Figure 12) were prepared and studied as potential antimalarial agents.185 The compounds were screened in vitro against the FAN-5 strain of P. falciparum. Antimalarial activity was reported as minimum inhibitory concentration (MIC). Expressed as an average for all complexes of each drug, complexation of either primaquine or amodiaquine did not represent any increase in potency since the activity of the complexes was identical to the parent drugs: MIC values obtained were 0.1 μM for amodiaquine (4) and its coordination compounds and 1 μM for primaquine (8) and its coordination compounds.185 The highest activity in this study was attributed to the salts of toxic metal ions HgCl2 and CdCl2. No cytotoxicity or selectivity studies were reported.185 Mixed complexes of chloroquine (CQ, 3) and mefloquine (MQ, 7) with Co(II), Ni(II), and Fe(III) were also synthesized and studied for their anti-infective properties.186 Complexes formed were proposed to have a stoichiometry of 1:1:1 with a general formula of [M(II)(CQ)(MQ)Cl2] for MCo2+ (50) and Ni2+ (51) and [M(III)(CQ)(MQ)Cl3] for MFe3+ (52). The nature of the bonding of the mixed ligands and structures was based on spectroscopic evidence, and no structural formulas were provided in any case.186 Structures 50−52 in Figure 12 were deduced from the information provided. Even though the complexes contain two well-known antimalarial drugs, no growth inhibition studies against P. falciparum were performed.186 Results from screening the complexes in vitro and in vivo against several other microorganisms resulted in [Co(CQ)(MQ)Cl2] (50) and [Fe(CQ)(MQ)Cl3] (52) having the best anti-infective properties and showing greater potency than their parent compounds 3 and 7.186 The in vivo toxicity of these compounds was monitored by the levels of activity of the enzyme alkaline phosphatase (ALP) in rat serum, liver, and kidney.186,187 The

membrane-bound ALP enzyme is often used to assess the integrity of tissues; high concentrations in the serum and small concentrations inside the kidney and liver indicate tissue damage, and it is taken as a sign of toxicity.187 Compounds [Co(CQ)(MQ)Cl2] (50) and [Fe(CQ)(MQ)Cl3] (52) demonstrated less toxicity than the Ni(II) analogue and the corresponding ligands CQ and MQ.186 A similar study was carried out with the coordination complex of Co(II) and mefloquine (MQ, 7) of the proposed formula [Co(MQ)Cl2]. This complex showed an 80% reduction in parasitemia upon treatment of an in vivo model of Plasmodium yoelii nigeriensis. However, toxicity was reported with damage suffered in the kidney, liver, or heart of the treated animals.188 The corresponding complex of Ni(II) with mefloquine hydrochloride of the formula [Ni(MQ)Cl2·6H2O] was also studied for anti-infective properties, but again no Plasmodium growth inhibition studies were performed.189 Yet, studies revealed that both mefloquine and the metal complex show toxicity, particularly on the liver and kidney with the metal complex group being of mild toxicity.189 Finally, the antiplasmodial properties of a series of complexes of Fe(III), Cu(II), and Zn(II) with quinine (1) and mefloquine (7) were studied in vivo in a mice model of Plasmodium berghei.190 A selection of these compounds showed antimalarial activity, increased compared to the activity presented by the parent ligands. The toxic effects of these compounds were studied in terms of the level of alkaline phosphatase (ALP) activity of kidney, liver, and serum. The levels of ALP present no particular change, representing no particular toxicity of the ligands or complexes.190 4.2.3. Metal Complexes of Other Ligands. 4.2.3.1. Gold Complexes. Several gold(I) thiolate and phosphine complexes ([Au(PEt3)Cl] (53), sodium aurothiomalate (54), gold(III) cyclam (AuCyclam) (55), and auranofin (56)) (Figure 13) in vitro inhibit the growth of P. falciparum. Sodium aurothiomalate (54) was previously shown to trigger the premature death of Plasmodium-infected erythrocytes.191 Studies probing its influence on the in vitro intraerythrocytic parasite development of P. falciparum and the in vivo behavior against P. berghei in a murine

Figure 13. Gold(I) and gold(III) phosphine and thiolate antimalarial complexes. 3466


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Figure 14. Thiosemicarbazone−Au(I) and −Au(III) complexes with antimalarial activity.

Figure 15. Schiff-base phenol and amine−phenol complexes of Fe(III), Ga(III), and In(III) with antiplasmodial activity.

growth at very low concentrations (IC50 = 142 nM), outperforming the other tested gold(I) complexes: [Au(PEt3)]Cl (53) (IC50 = 2.1 μM), aurothiomalate (54) (IC50 = 168 μM), and AuCyclam (55) (IC50 = 439 μM)).184,192 It was suggested that the antimalarial effect of these compounds is caused by direct inhibition of P. falciparum thioredoxin reductase (TrxR), which induces severe oxidative stress and cytotoxic effects;192 this

model demonstrated that exposure to 54 significantly decreased the in vitro parasitemia of P. falciparum-infected human erythrocytes and delayed the lethal course of P. berghei malaria leading to survival of more than 50% of the mice 30 days after infection.191 Auranofin (AF, 56), a clinically established antiarthritic drug, caused a strong and nearly complete inhibition of P. falciparum 3467


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complexes (66) was tested against CQ-sensitive HB3 as well as against CQ-resistant FCR-3, Indo-1, and Dd2 parasite strains.195 IC50 values for all strains ranged between 1 and 1.5 μM. The 4,6dimethoxy analogues proved to be the most potent against the resistant strains, with 4,6-dimethoxy-ENBPI Fe(III) (Fe-66b) being the most active of all Fe(III) complexes.195 Another potent compound was the gallium(III) analogue 3-methoxy-ENBPA Ga(III) (Ga-67a) complex, with amine instead of imine side arms, which exhibited potent activity against all chloroquineresistant parasite strains despite poor antimalarial activity in the chloroquine-sensitive clones and the lack of activity observed for the ENBPI Ga(III) analogue 66.195 Susceptibility to this agent, mapped in a genetic cross, had a perfect correlation with the chloroquine-resistance phenotype, suggesting that a locus for 3MeO-ENBPA Ga(III) (Ga-67a) susceptibility would be located in the same segment of chromosome 7 as for the chloroquineresistance determinant. The ability of compounds 4,6dimethoxy-ENBPI Fe(III) (Fe-66b) and 3-methoxy-ENBPA Ga(III) (Ga-67a) to inhibit hemozoin formation was measured in vitro, and it was found to correlate with the antiplasmodial activity as well.195 The complex Ga-67a, which from now on we will list separately as [Ga-3-Madd]+ (68), [{1,12-bis(2-hydroxy-3methoxybenzyl)-1,5,8,12-tetraazadodecane}gallium(III)] + as it was renamed by the authors, targets CQ-resistant organisms and demonstrates a mechanism of action similar to that of chloroquine (3) by inhibiting heme sequestration (hemozoin formation).195,196 This particularly interesting activity motivated further studies to determine the extent of its antimalarial capacity. The IC50 values obtained for 68 were >20 μM for the chloroquine-sensitive HB3 strain but 0.5 and 0.6 μM, respectively, for the chloroquine-resistant FCR-3 and Indo-1 parasite strains.195 In its ability to inhibit β-hematin formation, 68 was demonstrated to be a potent inhibitor (IC50 = 1.2 μM), with 2−3-fold higher activity than chloroquine.195 These findings suggest that 68 follows a similar mechanism as do other quinoline-type drugs but overcomes resistance. From the results of structural studies, both in the solid and in the solution state, the authors were able to confirm that the selective biotransport and localization properties of this compound reside in the spatial orientation of the peripheral regions of the aromatic moieties rather than the central core.196 Further studies demonstrated that the overall charge of the complex is also critical to the drug’s ability to inhibit hemozoin formation and that this process is possible because of formation of a drug-hematin propionate salt.197 From the results of fluorescence and competition studies, it was postulated that the mechanism of hemozoin inhibition relies on formation of this salt, limiting the number of propionate groups available to form the critical axial linkage, necessary in the dimeric unit of hemozoin.197 There was further interest in other derivatives based on this ligand scaffold; synthesis and antiplasmodial activity studies were pursued by the same research group and others. One group of compounds studied against the CQ-sensitive HB3 and CQresistant Dd2 parasite strains were the moderately hydrophobic [{1,12-bis(2-hydroxy-3-ethyl-benzyl)-1,5,8,12tetraazadodecane}metal(III)] complexes of Fe(III) ([Fe-3Eadd]+, 69) and Ga(III) ([Ga-3-Eadd]+, 70).136 The octahedral geometry of 69 and 70 (Figure 15) was confirmed by X-ray crystal structure determination.136 The IC50 values obtained for 69 and 70 against HB3 were 80 and 86 nM respectively, a large improvement compared to the parent [Ga-3-Madd]+ (68) that displayed only weak activity (IC50(HB3) > 20 μM).195 The

hypothesis has yet to be confirmed. Furthermore, there is evidence (isobolographic analysis) that 56 has a synergistic role with another inductor of intracellular oxidative stress, artemisinin (17). Compared to artemisinin (16 nM), auranofin is less potent, but when tested for synergistic action, the combination of artemisinin/50 nM auranofin and artemisinin/100 nM auranofin had IC50 values of 12 and 9 nM, respectively, improving the action of artemisinin alone.184,192 In another study, gold(I) complexes of the well-studied antiparasitic thiosemicarbazones (TSCs) were synthesized (Figure 14) and screened for antiplasmodial activity.193 The gold−TSC complexes were tested against the CQ-sensitive D10 and the CQ-resistant W2 strains of P. falciparum. The TSC ligands alone were relatively inactive in both parasite strains (IC50> 10 μM), but their gold(I) complexes 57−60 had an improvement of up to 16-fold in activity in both parasite strains.193 Inhibitory activity of 57−60 against plasmodial falcipain protease was enhanced when compared to that of the ligands. The most effective gold(I) complex, 58, had an IC50 value of 0.87 μM against falcipain; however, no correlation could be found between antiplasmodial activity and falcipain-2 inhibition for these complexes, thereby suggesting that falcipain-2 might not be the primary target for these compounds.193 Even though the activity was increased compared to the TSC ligands, the gold(I) complexes could not match the efficacy of CQ (3) in both parasite strains (IC50 = 0.0173 and 0.095 μM) or the proteinase inhibitor E64 (0.034 μM) used as positive controls.193 Recently, another series of gold(III) thiosemicarbazone complexes (61−65) derived from a gold dimethylaminoethylphenyl scaffold was synthesized and tested in vitro for antimalarial and antitubercular activity.194 Again, incorporation of the gold center enhanced the antimalarial activity of the TSC derivatives against the CQ-resistant W2 strain of P. falciparum, with most of the TSC ligands virtually inactive at the tested concentration. The most active compounds are shown in Figure 14. These TSC−gold(III) complexes display IC50 values in the range of 3.04−7.22 μM against the W2 strain, 3−7 times more active than the corresponding ligands but much less active than chloroquine (3) and artemisinin (17).194 It was observed that replacing the chloro with a bromo substituent on the aromatic ring led to a 2-fold improvement in antiplasmodial activity and that replacing the methyl for the ethyl moiety at the imine carbon did not alter the activity.194 4.2.3.2. Iron and Gallium Complexes. Schiff-base phenol and amine−phenol ligands have been investigated for their antimalarial properties.175 These compounds present a hexadentate environment with a N4O2 donor core, ideal for metal coordination including Fe(III), Ga(III), In(III), and Al(III). This class of N4O2M(III) complexes has favorable cell membrane permeability properties and is known to interact with membrane transporters such as Pgh1, involved in chloroquine sequestration inside the parasite digestive vacuole.175 The hexadentate Schiff-base phenol metal(III) coordination compounds, ethylenediamine-N,N′-bis[propyl(2-hydroxy-(R)benzylimino)]metal(III) complexes [(R)-ENBPI-M(III)] (66), and the corresponding [(R)-benzylamino)] analogue [(R)ENBPA-M(III)] (67) were synthesized and studied for their intraerythrocytic antimalarial properties.195 Complexes with Al(III), Fe(III), Ga(III), and In(III) of the general structure seen for 66 and 67 (Figure 15) were synthesized from the Schiffbase precursor and the acetylacetonates of hydrated metal(III) salts. The antimalarial activity of the (R)-ENBPI metal(III) 3468


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compounds were found to be 10−30-fold less potent than was 68 against the CQ-resistant Dd2 strain (IC50 = 2.5 (69) and 0.8 μM (70)).136 This is the opposite trend observed for the parent [Ga3-Madd]+ (68): 70 is more active in the CQ-sensitive strain and less active in the CQ-resistant strain. These results would suggest that these compounds, unlike 68, are recognized by the PfCRT. Both Fe(III) (69) and Ga(III) (70) complexes were also found to be inhibitors of hemozoin formation.136 Another example of this inversion of trend is represented by the compound [{1,12-bis(2-hydroxy-5-methoxybenzyl)1,5,8,12-tetra-azadodecane}gallium(III)] + [Ga-5-Madd] + (71).198 Screened for antiplasmodial activity, 71 showed modest IC50 values of ∼2 and >15 μM against the CQ-sensitive HB3 and the CQ-resistant Dd2 parasite strains, respectively.198 The inversion of trend in 70 and 71 whose only structural difference to 68 is the methoxy group and its position indirectly confirms the hypothesis that the high activity observed for 68 is strongly related to the spatial orientation of the peripheral regions of the aromatic parts of the complex. Small variations of presence and position of the methoxy functionality on the aromatic rings are able to significantly alter the complexes’ antimalarial activity, thus leading to the conclusion that variations in shape and the electronic changes caused by this substitution are determinant for the activity.198 Furthermore, the same phenomenon was observed in a derivative where a quinoline ring was incorporated into the organic scaffold in an attempt to further enhance the hemozoin inhibitory action of [Ga-3-Madd]+ (68).199 The Ga(III) aminophenol complex 72 was synthesized and screened for antimalarial activity against CQ-sensitive (HB3, IC50 = 0.6 μM) and CQ-resistant (Dd2, IC50 = 1.4 μM) parasite strains of P. falciparum.199 For HB3, the obtained activity constitutes an improvement by a factor of 33, compared to 68, whereas the activity against Dd2 is not significantly different from that of 68 (IC50 = 1.8 μM).199 4.2.3.3. Copper Complexes. Various Cu(II) coordination compounds, depicted in Figure 16, also exhibited notable antimalarial activity. For example, copper(II) complexes of pyridine-2-carboxamidrazones (73 and 74) possess moderate antiplasmodial activity.200 Carboxamidrazones are structurally related to the acylhydrazones and thiosemicarbazones mentioned above. The antimalarial activity of the complexes observed against the CQ-sensitive P. falciparum 3D7 strain showed an increase of approximately 5−20-fold compared to the activity of the ligands; 73 (IC50 of 0.34 μM) deviated from this trend, being the most potent of the group with a 360-fold increase in activity upon copper complexation.200 This high activity seems to be associated with the four-coordinate planar geometry. The only example of five-coordinate copper(II), 74, was 7−80 times less active than the four-coordinated complexes, whose planarity is suggested to promote their facile internalization.200 A second factor proposed to contribute to the enhanced antimalarial activity of the copper compounds is the positive reduction potential that facilitates intracellular reduction to Cu(I) species, which are detrimental to parasite growth.200 Similar to the acylhydrazones, alkyl hydroxynaphthoquinones have been investigated as antimalarial agents via redox activity, thus potentially targeting the mitochondrial electron transport in the parasite.201 Metal complexes of 3-aryl-azo-4-hydroxy-1,2naphthoquinones with Cu(II) were synthesized and evaluated in vitro for their antimalarial activity.202 X-ray crystal structure determination of the copper(II) complex 75 established the geometry depicted in Figure 16, with the central copper atom

Figure 16. Cu(II) coordination compounds with antimalarial activity.

coordinated by the deprotonated hydroxyl group and the azo nitrogen rather than through the 1,2-carbonyl oxygens, forming a six-membered chelate ring. The metal atom is four-coordinated with two additional axial molecules and an overall pseudooctahedral geometry.202 Complex 75 was found to be the most active of all hydroxynaphthoquinone copper complexes, with an IC50 of 5.4 μM against the chloroquine-sensitive 3D7 P. falciparum strain. Compared with the uncomplexed ligand, 75 showed a 20-fold enhancement in activity.202 The cytotoxicity evaluated in normal KB cells revealed a large degree of toxicity, with a selectivity index for 75 of only 2.5 (3D7 over KB), associated with the methyl group of the azo group, which is required for antimalarial activity.202 It should be noted that 75 has the most positive redox potential, correlating well with other antimalarials for which antiplasmodial activity is observed to relate to electron transfer properties.202 A series of copper(II) nanohybrid solids LCu(CH3COO)2 (76) and LCuCl2 (77) with particle sizes of 5−10 and 60−70 nm, respectively, were synthesized using bis(benzimidazole) diamide as the capping ligand L.203 When evaluated for their in vitro antimalarial activity against a CQ-sensitive MCR2 isolate of P. falciparum, IC50 values of 0.059 and 0.048 μM for 76 and 77 were reported, respectively.203 These are the first literaturedescribed examples of copper complexes with IC50 values comparable to that of chloroquine (3, IC50 = 0.039 μM vs MCR2). These benzimidazole copper derivatives are expected to function as inhibitors of plasmepsins, the aspartic proteases involved in the catabolism of hemoglobin inside the parasite digestive vacuole. A study on the interaction between these nanohybrid solids and the protease Plasmepsin II by UV−vis spectroscopy and inhibition kinetics indicated that both 76 and 77 inhibit Plasmepsin II competitively with regard to substrate. P. falciparum blood stage development and hemoglobin digestion were monitored visually as well, following swelling and staining 3469


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Figure 17. Other antimalarial coordination compounds.

characteristics in infected erythrocytes.203 Polyacrylamide gel electrophoresis showed that parasites treated with 76 and 77 contained much more undegraded hemoglobin than did the control group.203 Finally, 76 and 77 were found to be both nontoxic against human hepatocellular carcinoma cells with a remarkable selectivity index of 8906 and 12 640, respectively, and highly selective for Plasmepsin II over Human Cathepsin D with a protease index of 12 and 6.1, respectively.203 It has been proposed that these nanohybrid solids with copper(II) capped by a benzimidazole could resemble the histidine group of copper proteins and thus target and inhibit enzymes via binding, leading to triggering of an antimalarial mechanism.203 4.2.3.4. Other Complexes. As mentioned in section 4.2.3.1, thiosemicarbazones (TSC) are compounds that have shown moderate antimalarial activity in conjunction with gold. Derivatives of TSC have been recently employed as ligands for

mononuclear complexes of various other metal(II) ions. Metal complexes of Pt(II), Pd(II), Cu(II), and Co(II) of general formula [MLCl2] (78) and [ML2Cl2] (79) (Figure 17) bearing four functionalized furyl−thiosemicarbazones (a and b) were synthesized and studied for their antiparasitic properties.204 These compounds were tested against the CQ-resistant W2 parasite strain of P. falciparum. Co(II) and Pt(II) complexes were less active than were the uncoordinated furyl−thiosemicarbazones, while the Pd(II) derivatives were up to 3 times more active than the metal-free furyl−thiosemicarbazones. The most active set of compounds was the Cu(II) complexes of general formula [CuL2Cl2] (Cu-79). These compounds had a 2.5−7.6-fold increase in activity compared to the metal-free furyl− thiosemicarbazones with IC50 values in the range of 4.6−7.8 μM.204 The compounds were also studied for their cytotoxicity in BALB/c mouse splenocyte mammalian cells, showing that 3470


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Zn(II), and Sn(IV) (83) (Figure 17) were also investigated as antimalarial agents and as inhibitors of β-hematin formation in vitro.210 The central metal ion played an important role in the efficacy of the metalloporphyrins. The metalloporphyrins MPPIX (83) of Mg(II), Zn(II), and Sn(IV) were found to be as effective as Fe(II)PPIX and up to 6- and 4-fold more effective than the metal-free porphyrin and chloroquine (3), respectively, in the inhibition of β-hematin formation.210 Less active were the MPPIX complexes 83 of Cu(II) and Mn(II), followed by the MPPIX complexes 83 of Cr(III) and Co(III), which did not present an advantage over metal-free PPIX.210 The efficacy of these compounds was found to correlate well with the exchange rate of water at the central metal ion of the octahedral porphyrin aqua complexes.210 Aggregation studies were carried out to probe the interaction between heme and the MPPIX complexes 83 using UV−vis and fluorescence spectroscopy, which supported the occurrence of acid-triggered π-stacking of heme−MPPIX assemblies, responsible for hemozoin inhibition, by disrupting interactions critical to the stability of the hemozoin.210 No direct information from the in vitro study of antiplasmodial activity was available for 83. Despite this observed inhibition of β-hematin formation, in a separate in vitro study of the synergistic effect of artemisinins and metalloporphyrins in antiplasmodial activity, the Mn(III) and Fe(II) complexes of the sulfonated derivatives of tetraphenylporphyrin (TPPS, 84) and tetramesitylporphyrin TMPS (85) (Figure 17) were found to display only moderate antiplasmodial activity against the two chloroquine-resistant P. falciparum strains FcB1 and FcM29.211 The iron complexes exhibited higher activities than did the manganese complexes with IC50 values that range from 6 to 412 μM. Nevertheless, the activity of artemisinin was increased 11fold by the Mn−TPPS complex, showing synergistic effects for this drug and the manganese porphyrin.212 It is believed that Mn−TPPS “activates” the antimalarial trioxane feature of artemisinin close to or at the drug site of action, thus producing this synergistic effect.212 In a study to determine which metals had the larger contribution to antimalarial activity, direct comparison of the antiplasmodial activity of a diverse group of “soft” metal compounds against the CQ-sensitive 3D7 P. falciparum strain was carried out.213 The spectrum of compounds studied comprised mononuclear and dinuclear Au(III), Ru(III), and Ru(II) complexes as well as Bi(III), Sb(III), and As(III) compounds.213 This group represented a variety of metal centers with diverse structural motifs and metal coordination environments with most of these complexes having been evaluated as anticancer agents before. All tested compounds caused nearly complete inhibition of P. falciparum growth when used in concentrations of 100 μM. At a lower concentration of 1 μM, selected Sb(III), Ru(III), and dinuclear Au(III) complexes showed the best capacity to inhibit parasite growth. IC50 values indicated that Aubipy (86), the three dinuclear gold(III) compounds 87, 88, and 89, and bismuth citrate (90) (Figure 17) manifested a significant potency with IC50 values in the 2−12 μM range.213 NAMI A (91) and SbCl3 (92) (Figure 17) were the most effective agents, with IC50 values of 680 and 220 nM, respectively. These significantly good values were bested by Auranofin (56, IC50 = 142 nM) and attributed to the less ‘‘soft” character of the gold(III) center.213 Modes of action of these compounds could be related to direct interactions of the metal center with specific biomolecular targets within the parasite, like thioredoxin reductase and falcipain, or by inducing severe oxidative stress.

copper(II) complexes were considerably more toxic than the ligands alone.204 Structure−activity relationship studies were carried on the most potent agents, based on measurement of properties such as the saline/n-octanol partition coefficient (log P), DNA interaction, and inhibitory effects on the β-hematin formation.204 It had been discussed that the antiplasmodial activity seemed to be governed by lipophilicity. For this set of compounds though, a correlation between the potency and the lipophilicity was not observed.204 Further studies of the Pd(II) compounds demonstrated that complexation is required to achieve full antimalarial potency, since a mixture of furyl− thiosemicarbazone ligand and metal precursor only had one-half of the antiplasmodial effect compared to the preformed complex.204 DNA interaction was observed, and none of the complexes inhibited β-hematin formation to a significant extent, suggesting a different mechanism of action than for quinoline drugs, potentially involving disruption of the DNA structure in the parasite.204 The coordination compounds of 4-amino-N-(2-pyrimidinyl) benzene sulfonamide (sulfadiazine, SD) and metal ions Cu(II), Zn(II), Mn(II), Ni(II), Pd(II), Pt(II), and VO(II) (80)205 and Fe(III), Ru(III), Rh(III), and Cr(III) (81)206 were prepared and studied in vitro. Sulfadiazine is clinically used to prevent bacterial infection, and its silver(I) coordination compound (Silvadene) is used commercially for treatment of topical burns. The coordination compounds, chelated by bidentate SD ligands, have a square planar (80) or octahedral (81) geometry, depending on the metal ion (Figure 17).205 The metal complexes were screened for their antiprotozoal activity against the chloroquine-resistant P. falciparum strain K1 among other protozoa.205,206 The metal complex [Mn(SD)2(H2O)2] (Mn80) was the most active with an IC50 value of 2.127 μM, an activity only moderately higher than the 2.4 times less potent sulfadiazine. [Fe(SD)3(H2O)3] (Fe-81) was only slightly more active (IC50 = 4.60 μM) than the rest of the metal complexes with sulfadiazine, which displayed IC50 values of >5 μM.205,206 Coordination complexes of buparvaquone (a member of the family of hydroxynaphthoquinones and a potent antitheilerial agent) or 3-trans-(4-tert-butylcyclohexyl)methyl-2-hydroxy-1,4naphthoquione with diverse metal ions such as Cu(II) (82), Ni(II), Co(II), Fe(II), and Mn(II) were synthesized and studied for their in vitro antimalarial activity against P. falciparum 3D7 (CQ-sensitive) and K1 (CQ-resistant) strains.207 All synthesized metal complexes except for Ni(II) showed enhanced activities.207 [Cu(buparvaquone)2(C2H5OH)2] (82) was found to be the most active compound, showing a 1000-fold enhancement of activity compared to the parent quinone against 3D7 and a 3-fold improvement against the CQ-resistant K1 strain.207 Compound 82 was also significantly less toxic than was the parent quinone toward KB cells. Further evaluation of 82 for its in vivo antimalarial activity in a mouse model of P. berghei showed 90% clearance of parasitemia at a dose of 15 mg/kg, while the standard drug chloroquine required a 10 mg/kg dose for complete clearance.207 Complex 82 also exhibited significantly higher growth inhibition than did the parent ligand, both in vitro and in vivo.207 Previous in vitro studies showed that metal-free porphyrins are able to inhibit conversion of heme to β-hematin through a π−π interaction of free hematin in solution and through coordination of the iron atom of hematin by the hydroxyl groups of porphyrin.208 Ferrous−protoporphyrin IX [Fe(II)PPIX] also inhibited hemozoin formation.209 Metalloporphyrins MPPIX of metals ions such as Cr(III), Co(III), Mn(II), Cu(II), Mg(II), 3471


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Figure 18. Ferrocene chloroquine derivatives showing antimalarial action.

complexes conceived with application to medicine in mind, salvarsan (arsphenamine) and neo-salvarsan, were developed by Paul Ehrlich. These compounds, which feature a direct As−C bond, were used to treat syphilis and trypanosomiasis and later became the first bioorganometallic chemotherapeutic agents. In contrast to coordination compounds of transition metals as metallodrugs, organometallic complexes are thought to be unstable or insoluble or both in aqueous media and not well adapted for biological systems. While certainly accurate for many organometallic compounds, many others are suitable for biological applications. Among those a selection of metallocenes and organometallic complexes has proven their potential as pharmaceutical agents experimentally and clinically. One wellknown example is ferrocifen, a ferrocene derivative of tamoxifen, a chemotherapeutic agent for treatment of breast cancer that demonstrated not only greater potency than the parent drug but was also the first chemotherapeutic agent to be active in both hormone-dependent and hormone-independent breast cancer tissue.216,217 Thus, the biological activity of certain organometallic compounds attracted the attention that fostered development in this field. During the last 15 years, significant progress on the

Metal coordination compounds of antimalarial drugs represent in most cases an improvement compared to the activity of the parent drugs against chloroquine-sensitive and -resistant parasite strains. Other advantages of these compounds are the possibility of metal−drug synergism and the toxic effects of the metal ion against the parasite, possibly by inducing electronic stress and oxidative damage in the parasite. Given their resemblance to their parent antimalarial drugs, there is the potential disadvantage of developing cross resistance. Speaking against these compounds is the possibility of them being not very stable under in vivo conditions, their selectivity and, since the complexes are charged in many cases, permeability through membranes might be hindered. Coordination compounds in general improved the activity observed for metal chelators, but their activities remained for the most part in the micromolar range, with a few exceptions in the nanomolar range. 4.3. Bioorganometallic Compounds

Organometallic compounds, with a direct metal−carbon (σ or π) bond, have played an important role in biology and medicine in the last decades, even though they are better known for their use in catalysis and other applications.214,215 The first organometallic 3472


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to the “new” drug if there is resistance for the “old” drug) observed between the responses of 93 and drugs such as chloroquine, amodiaquine, quinine, mefloquine, halofantrine, artesunate, atovaquone, cycloguanil, and pyrimethamine were too low to suggest cross resistance among multiple field isolates.225,228,229 Also, susceptibility of P. falciparum field isolates to ferroquine is not associated with a specific polymorphism or a modulation of expression of the Pf CRT gene.230 A more detailed discussion on the pharmacological properties that made 93 a candidate for human clinical trials can be found elsewhere.231,232 In summary, the antimalarial activity of ferrochloroquine (93) was a remarkable discovery. This compound presents: (i) activity at nanomolar concentrations,225 greater potency than any quinoline-containing antimalarial drug, and potency comparable to artemisinins against chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro; (ii) activity in vivo against strains of P. berghei, P. yoeli, and P. vinckei;134 (iii) inhibition of the in vivo development of both chloroquinesensitive and -resistant P. vinckei strains and protection from lethal infection in mice, with a curative effect that was 5−20 times more potent than chloroquine;225,227 (iv) high selectivity and therapeutic indexes;231,232 and (v) unlikely development of resistance, so it can be used as monotherapy or in combination with existent therapies.233 In terms of its cytotoxicity, the acute toxicity and lethality observed in studies performed with 93 seemed to be dependent on gastric surfeit.155 Ferroquine (93 SSR97193) is now in phase IIb clinical trials under the aegis of Sanofi-Aventis.234 To the best of our knowledge, clinical trials were to be carried out examining the efficacy of an ACT based on artesunate (19) and ferroquine (93) in malaria patients.235 More data about the metabolization and activity of metabolites of 93 as well as detailed studies on clinical isolates and human clinical trials have been published.235 Following the success of ferroquine (93), other structural analogues (94−105, Figure 18) were synthesized in order to delineate structure−activity relationships.221−223,236−239 These studies have shown that incorporation of a ferrocenyl moiety as an integral part of the side chain of chloroquine between the two exocyclic nitrogens of a chloroquine derivative has superior efficacy to other analogues in which the moiety is attached terminally on the side chain239 (97) or bound to the quinoline nitrogen (95).222,223 In order to have antiplasmodial activity, ferrocene requires covalent linkage to the 4-aminoquinoline and the alkyl amine moieties.221 Thus, while ferrocene alone does not show any efficacy, ferrocene incorporation enhanced the potency of chloroquine when enclosed within the molecule.223 The pattern of substitution of the ferrocene, as either 1,2- (93) or 1,1′-disubstituted (102) ferrocene, seems to have little effect on the activity. The 1,1′-disubtituted analogues presented only a slightly lower activity in the examined chloroquine-resistant strains.237 Also, each pure enantiomer of the 1,2-ferrocene derivative (93) that possesses planar chirality is equally active yet slightly less potent than the racemic mixture.240 A conclusive explanation for the increased activity of the racemic mixture of 93 has not yet been drawn, but it has been related to an additive or synergistic effect between the two enantiomers of ferroquine.215,240 Among the many derivatives of ferroquine (94− 105), hydroxyferroquine (HFQ, 105) is probably the most successful and promising candidate, almost as ideal as ferroquine but not improving the toxicity related to it.238 Recent publications have reviewed in great detail the diverse list of ferrochloroquine analogues.231,235,241 Most of these derivatives show better activities than does the parent drug chloroquine 3

treatment of malaria with bioorganometallic compounds has been achieved. Among all the organometallic compounds synthesized and tested, ferrocene antimalarial drug conjugates demonstrated the most promising activity. In this section the bioorganometallic compounds that exhibit antimalarial activity are described in two main sections: the metallocenes, with special focus on ferrocene derivatives, and the nonmetallocene organometallic compounds. 4.3.1. Metallocenes. In a metallocene, a transition metal ion is associated with two cyclopentadienyl anions, “framing” the metal ion in a sandwich-like structure. Several examples of metallocene pharmaceuticals include titanocenes (Ti(IV)) and ferrocenes (Fe(II)), such as the already mentioned ferrocifen. Ferrocene has particular properties such as stability under both aqueous and aerobic conditions, nontoxicity, high lipophilicity, small size, accessible redox potential, and ease of derivatization, which make it highly attractive for biological applications.218,219 The activity of ferrocene as part of a drug design to treat malaria has been investigated. Several ferrocenyl analogues of traditional antimalarials or experimental antimalarials have been synthesized during the last 15 years. Results showed in several cases that the presence of ferrocene improves antiplasmodial activity in the parent structure and in some cases can even overcome drug resistance in parasite strains. 4.3.1.1. Ferrocene Chloroquine Derivatives. Ferroquine or ferrochloroquine (FQ, 93) (Figure 18) was the first derivative of chloroquine containing a ferrocene molecule to be reported, and it is undoubtedly the most successful of all metallo-antimalarials synthesized and tested to date. Ferroquine emerged from a collaborative drug discovery project in which more than 50 ferrocene compounds were synthesized. 155 It was first synthesized in 1997 and is the result of incorporation of ferrocene to the lateral side chain of chloroquine, between the two exocyclic nitrogens substituting two alkyl carbons by two Cp carbons of ferrocene.220 FQ (93) shares structural and biological features with chloroquine (3), but their individual responses against several P. falciparum strains tend to be very different. FQ (93) is highly active in chloroquine-sensitive and chloroquine-resistant P. falciparum parasite strains in vitro.220,221 Against chloroquinesensitive parasite strains, 93 was as effective as the parent 3 with IC50 values that ranged from 3.5 to 218 nM. Against chloroquineresistant parasite strains, 93 was up to 20 times more effective than was chloroquine, with IC50 values that ranged from 5 to 241 nM, maintaining the high observed activity from the CQsensitive strains.222−224 The antimalarial activity of ferroquine (93) has been demonstrated extensively against several strains of P. falciparum from both laboratory clones and clinically isolated clones, obtaining an IC50 mean of 10.8 nM from 103 isolates of CQ-sensitive and CQ-resistant P. falciparum.225 Ferroquine (93) was found to be considerably more active than were the commercially available chloroquine diphosphate (35 times higher against CQ-resistant isolates) and many other commonly used antimalarial drugs.134,214 It also exhibited remarkably high antimalarial activity in vivo in mice infected with P. berghei, P. yoeli, and P. vinckei vinckei.226,227 The survival rate of mice after treatment with 93 was far higher than that with current standard drugs and also displayed long-term stable antimalarial activity in the biosystems. In addition, 93 was active via subcutaneous and oral administration.226,227 Furthermore, the positive significant correlations (as a measure of linear dependence, a positive correlation (r2) between the IC50 of two drugs indicates the possibility of cross resistance or the capacity to develop resistance 3473


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Figure 19. Masked drugs and dual drugs based on ferroquine.

lipophilicity, basicity, and electronic profile of the parent molecule, which eventually alters its pharmacodynamic behavior. It is also possible that the ring exerts a unique biological effect, not associated with other structural entities.246 A different approach than the one presented above, in which the basic structure of ferroquine (93) is modified to tune the reactivity and other pharmaceutical properties of this drug, was the development of masked pro-drugs or “dual drugs”. One example is 4-aminoquinoline 106 bearing an amino side chain substituted by an aliphatic Mannich base (Figure 19).247 This drug was designed so that the quinoline part facilitates recognition and accumulation inside the digestive vacuole. Once inside the parasite, oxidative N-dealkylation, catalyzed by cytP450 enzymes or other oxidative conditions found inside the parasite, will release the 4-aminoquinoline, whose function will be to inhibit hemozoin formation, intercalate with DNA, and produce oxidative stress (through the thiol-reactive Mannich bases). Preliminary results of the antiplasmodial activity, evaluated in vitro against several P. falciparum parasite strains with different degrees of drug susceptibility, and experiments probing inhibition of β-hematin formation and DNA-binding capabilities indicated that the ferrocenyl Mannich-base 106 is a potent inhibitor of parasite growth, nearly as potent as ferroquine (93).247 However, 106 is also six times more toxic than 93 to

and were, in the most successful cases, as good as ferroquine. Despite the efforts invested to further improve the activity of FQ, none of them to date has out-performed ferroquine (93) in antimalarial potency and pharmacological properties. The mechanism of action of ferroquine (93) has been studied, although it is only partially understood; its therapeutic effectiveness has been attributed to its preferential accumulation in the parasite food vacuole by a factor higher than 50 in comparison to CQ (3).242 It has been postulated that its weaker basic properties, its higher lipophilicity, and its special conformation, influenced by the presence of an intramolecular hydrogen bond, are key characteristics that play an important role in the elevated vacuolar accumulation.243,244 The activity of FQ (93) can be ascribed to a dual mechanism: first, FQ is a strong inhibitor of β-hematin formation, more potent than CQ, and therefore able to halt more efficiently the detoxification mechanism of the parasite; and second, the redox activation from ferrocene to ferrocenium and consequent generation of reactive oxygen species (ROS) via a Fenton-like reaction, which likely irreversibly damages the parasite.242,244 Thus far, the role of the ferrocene ring in the antimalarial activity of ferroquine remains uncertain. The ring system was hypothesized to have an effect on its interaction with PfCRT involved in CQ resistance.242−245 The metallocene might act through modification of shape, volume, 3474


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Figure 20. Heterobimetallic compounds based on ferroquine.

enzymatically or by hydrolysis. In vitro testing against the CQsensitive NF54 and the CQ-resistant K1 P. falciparum strains showed 111−114 to be more potent than are the parent GR inhibitors/GHS depletors but less active than ferroquine (93) and its derivatives.251 Preliminary cleavage assays were also conducted to support the pro-drug strategy. At cytosolic pH (7.4) the dual molecules are stable and the amide bond does not break, but at vacuolar pH (5.2) the bond is broken and both moieties are released; however, contrary to expectation, no inhibition of the P. falciparum GR was detected. In turn, high inhibition of β-hematin formation was observed.251 Nevertheless, these compounds (except for those derived from 112) were potent antimalarials with IC50 values between 27 and 161 nM in both parasite strains, not as active as chloroquine (3) against the NF54 strain (IC50 = 11.9 nM) but up to 8-fold more potent than chloroquine against the K1 strain (IC50 = 289.2 nM).251 A “double-load” approach was followed in the synthesis of ferrocene quinoline derivatives of triazacyclonane (115 and 116, Figure 19), containing two units of a chloroquine derivative or two units of ferrocene.252 In vitro testing against chloroquinesensitive HB3 and chloroquine-resistant Dd2 P. falciparum strains revealed that the bisquinoline 7-chloro-4-[4-(7-chloro-4quinolyl)-7-ferrocenylmethyl-1,4,7-triazacyclononan-1-yl]quinoline (115) proved to be more potent than 116 (a bisferrocenyl derivative).252 Compound 115 showed IC50 values of 110 and 62 nM against the HB3 and Dd2 strains, respectively, being 5.5 times less active than CQ (3) in HB3 but 1.5 times more active than 3 in the Dd2 strain.233 The authors proposed that for 115 the redox behavior of ferrocene as a one-electron system and its high lipophilicity may partially explain the increased activity, whereas for 116 the even higher lipophilicity, high molecular weight, and steric hindrance may contribute to its lower potency by perturbing its transport to the parasitic food vacuole of the parasite and/or destabilizing the interaction of the quinoline fragment with hematin.252 Lastly, a third strategy was the design of heterobimetallic compounds or ferroquine derivatives bearing a second metal center (117−119, a−c, Figure 20). Derivatives of ferrocenyl-4aminoquinoline acted as coordination ligands (L) to form complexes of general formula [Au(L)(PPh3)]NO3, [Au(C6F5)(L)], and [Rh(Cl)(COD)(L)].253 No information on the geometry of coordinating atoms was provided.253 In terms of their capacity to inhibit parasite growth in CQ-resistant K1 and CQ-sensitive D10 strains, coordination of the second metal ion did not lead to a significant improvement.253 On the contrary, for most compounds (117−119) a decrease in activity was observed.

both the human lung MRC-5 and the glioblastoma carmustineresistant NCH89 cell lines.247 The series of hybrid compounds consisting of a 1,2,4-trioxane fragment and ferroquine, coined “trioxaferroquines” (107−110, Figure 19), can be considered as another class of “dual” compounds. These compounds combine the ferroquine moiety (ferrocene and 4-aminoquinoline) and the main feature of the group of artemisinins, the endoperoxide bridge fragment essential for artemisinin activity. These hybrids have the possibility of a dual or synergistic antimalarial action. Trioxaquines, organic hybrids of a quinoline molecule and a 1,2,4-trioxane, have already been shown to be highly active against CQ-resistant strains of P. falciparum.248 A series of trioxaferroquines were screened against the two CQ-resistant strains FcB1 and FcM29, exhibiting potent antimalarial action with IC50 values in the range 16−71 nM for both strains.249 The most potent trioxaferroquine 107 showed IC50 values of 20 and 17 nM against FcB1 and FcM29, respectively. In addition to the trioxaferroquines, a corresponding series of trioxaferrocenes (not containing the quinoline fragment) was also synthesized and evaluated.249 Trioxaferrocenes were much less active inhibitors with IC50 values in the rage of 145−1697 nM against both strains. The 4−10 times reduced activity compared to the corresponding trioxaferroquines proved the importance of the 4-aminoquinoline moiety for antimalarial action, even against chloroquineresistant parasite strains. Despite their good activity, the organometallic trioxaferroquines do not represent an advantage over their organic trioxaquine counterparts (no ferrocene). The antimalarial activity of 107 was furthermore screened in vivo in a murine model of P. vickei petteri.249 After 4 days of treatment with doses of 10 and 25 mg/kg/day, parasitemia was below detectable levels, indicating rapid parasite clearance, but was accompanied by recurrence and recrudescence of infection shortly after the end of the treatment.249 Another example of a pro-drug strategy, as well as a combination of two different classes of antimalarials, is the series of ferroquine analogues conjugated to a gluthatione reductase (GR) inhibitor (111−114, Figure 19). This strategy seeks to combine the effects of oxidative stress caused by ferroquine with depletion of gluthatione (GSH), an enzyme known to protect cells from oxidative damage and associated with increased resistance to CQ in P. falciparum.250 Since GSH levels depend on an efficient reduction of glutathione disulfide (GSSG) by glutathione disulfide reductase (GR), GR inhibitors have been assayed as antimalarial drugs previously.37 Conjugates 111−114 were synthesized by linking a ferroquine derivative to a GR inhibitor or GSH depletor by an amide linker, cleavable 3475


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The most active compounds were the gold complexes of the general formula [Au(C6F5)(L)] (118). The rhodium complexes (119) were significantly less active than 118 and exhibited a moderate to strong antagonistic effect on the efficacy of the parent ferroquine compounds.253 It was noted that the presence of the second metal, specifically gold, made the ferrocenyl moiety far more difficult to oxidize, and the authors postulated this could explain some of the loss in efficacy. No discernible correlation between redox activity and antiplasmodial efficacy can be made yet from what is known from the mode of action of ferrocenyl chloroquine compounds. 4.3.1.2. Ferrocene Conjugates of Other Antimalarial Drugs. Introduction of ferrocene into the core or as a pendant of the structures of other traditional and novel antimalarials is a recurrent strategy to research enhanced activity and overcome drug resistance to commonly utilized drugs. The benefit of combining a ferrocene moiety and an artemisinin derivative could lie in altering the pharmacological properties as well as altering the electrochemical properties, which could induce a more ideal homolytic cleavage of the peroxide bond and a stronger interaction with ferriprotoporphyrin IX Fe(III)PPIX (see section 3.4, mode of action of artemisinins). Two series of derivatives bearing a ferrocene pendant at the core of artemisinin (through the use of a variety of linkers) were prepared and tested in vitro against strains of P. falciparum. The first series (120 and 121, Figure 21) was functionalized at the C-16 position of artemisinin, and the Fc derivatives were found to be 2.5−5-fold more active than the parent artemisinin (17) and as active as

dihydroartemisinin (20) with IC50 values of 1 (120) and 2.1 nM (121).254 The second series of derivatives, represented by 122 and 123 (Figure 21), was functionalized at the carbonyl functionality of artemisinin via an ether or ester bond.255 These artemisinin ferrocenyl derivatives did not perform better when compared to the parent drugs artemisinin (17) or dihydroartemisinin (20). The most active and only compound with amine functionality at the ferrocene, 123, was the only one that matched artemisinin in activity but was still less active than dihydroartemisinin (20).255 All other ferrocenyl derivatives from the second series presented 1.2 to 5.5-fold decreased growth inhibitory capacity in comparison to the parent drugs when were evaluated against the CQ-sensitive HB3 and SGE2 as well as the CQ-resistant Dd2 strains of P. falciparum.255 For both series, a correlation between antimalarial activity and affinity for binding to Fe(III)PPIX was found.254,255 A series of ferrocene aminohydroxynaphthoquinones (124, Figure 22), analogues of the antimalarial drug atovaquone (21), were synthesized and tested for their in vitro activity against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strains of P. falciparum.256 Unfortunately, these derivatives were 4−10-fold less active than the parent drug atovaquone (21).256 Ferrocenyl derivatives of mefloquine 125 and quinine 126 (Figure 22) were tested against chloroquine-sensitive (HB3) and chloroquine- and mefloquine-resistant (Dd2) P. falciparum strains.257 The quinuclidinyl and piperidinyl side chain of quinine and mefloquine, respectively, were substituted with a ferrocene moiety while preserving a basic amino group. These derivatives exhibited lower antimalarial activity than did the parent drugs mefloquine (7) and quinine (1) in either strain, indicating no activity enhancement attributable to the ferrocene unit.257 All solutions of ferrocenyl analogues seemed to be unstable under the employed biological assay conditions. This may be attributed to formation of presumably inactive carbocations at the acidic pH of the food vacuole of the parasite.155 The ferrocene−pyrrolo[1,2-α]quinoxaline compounds (127− 129, Figure 22) are derivatives of bisquinoline and bisacridine antimalarial drugs, drugs that have shown much lower resistance indices than chloroquine, becoming good candidates for chloroquine-resistant malaria therapy.258 In vitro testing against the chloroquine-sensitive F32 and the chloroquine-resistant FcB1 and PFB strains of P. falciparum showed that the best results were observed for the ferrocene−pyrrolo[1,2-α]quinoxalines linked by a bis(3-aminopropyl)piperazine bearing a methoxy group on the pyrrolo[1,2-α]quinoxaline nucleus and an unsubstituted terminal N-ferrocenylmethylamine (127).258 With IC50 values of 9 nM for F32 and 17 nM and 22 nM for FcB1 and PFB, 127 was twice as efficient as was chloroquine against the CQ-sensitive strain and 6−10 times more active than chloroquine against the CQ-resistant strains. When the most potent analogues of this series were tested for their ability to inhibit β-hematin formation in vitro, only 128 exhibited moderate activity.258 Recently, a second generation of ferrocene−pyrrolo[1,2α]quinoxalines, featuring the ferrocene in the 3-position of the heterocycle and a substituted benzyl group, were screened for antiplasmodial activity in vitro against the CQ-sensitive F32 and the CQ-resistant FcB1 and K1 strains of P. falciparum.259 The most potent derivatives were ferrocenyl pyrrolo[1,2-α]quinoxalines linked by a bis(3-aminopropyl)piperazine linker, substituted by a nitro benzyl moiety (129). Against F32, none of these compounds were found to be equally or more effective than

Figure 21. Ferrocene artemisinin derivatives. 3476


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Figure 22. Ferrocenyl derivatives of mefloquine, quinine, aminohydroxynaphthoquinone, and pyrrolo[1,2-α]quinoxaline.

Figure 23. Ferrocene conjugates of compounds with suspected antiplasmodial action.

chloroquine (IC50 = 19.5 nM) with IC50 values of 38−85 nM.259 The same was observed against the CQ-resistant FcB1, with IC50 values ranging between 0.14 and 0.38 μM (IC50(3) = 0.105 μM); however, a 1.25−2.25-fold improvement in potency compared to chloroquine (IC50 = 0.226 μM) was observed against K1.259 The cytotoxicity of these compounds in the MCR-5 cell line was in the micromolar range (IC50 2.12−9.30 μM), well above the antiplasmodial range, leading to good selectivity indices between 22.5 and 120.259 Inhibition of β-hematin formation was studied as well, and the most potent compounds could also be identified as good inhibitors of hemozoin formation, performing superior to CQ.259 4.3.1.3. Ferrocene Conjugates of Compounds with Suspected Antiplasmodial Action. Structurally similar to the previous group, arylpiperazines have been found to display mefloquine-type antimalarial behavior.260 An unsubstituted ferrocenyl benzylpiperazine (130, Figure 23) was synthesized as part of a larger group of arylpiperazines that were evaluated

against the CQ-sensitive D10 and NF54 and the CQ-resistant W2 and FCR3 strains of P. falciparum.260 Compound 130 exhibited remarkable antimalarial activity in the CQ-resistant strains, about 2−20 times greater than the activity of 130 found for CQ-sensitive strains. Despite this superior activity, the difference in activity between the resistant and sensitive strains is much less pronounced than for other studied arylpiperazines.260 In addition, this compound was 2−3 times less active than was chloroquine (3) in CQ-resistant strains and 43−447 less active than 3 in CQ-sensitive strains with IC50 values in the range from 0.78 to 1.9 μM.260 Conjugates of ferroquine and thiosemicarbazones (TSC, 131, Figure 23) were evaluated in vitro against four strains of P. falciparum with different levels of CQ resistance.261 The ferrocenyl−TSC derivatives of 131 were more active than the TSC alone, displaying activity in the micromolar range with IC50 values of 0.2−16.2 μM; however, activity was comparable with the purely organic derivatives of TSC and 4-aminoquinoline, 3477


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Figure 24. Ferrocene conjugates of compounds with suspected antiplasmodial action.

and second due to incorporation of the ferrocene. The most successful derivative 135 was further tested against a larger panel of P. falciparum strains always exhibiting a higher activity (20− 100-fold) compared to ciprofloxacin, with a remarkably constant activity regardless of the level of resistance of the strains to CQ.263 Ferrocenyl chalcones (of the general structure 138 and 139, Figure 24) were evaluated in vitro for antimalarial activity against the chloroquine-resistant K1 P. falciparum strain with the aim of studying the influence of the ferrocene in the chalcone structure.246 The antimalarial activity of chalcones was previously studied in vitro and in vivo, with the most potent derivative being as equally active as CQ in this resistant parasite strain.265 The most active compounds (140 and 141, Figure 24) exhibited moderate antimalarial activity (IC50 4.5 and 5.1 μM, respectively) but were still less active than the parent chalcones.246 When evaluated for size, electronic, lipophilic, and electrochemical character, it was discovered that the location of the ferrocene influences both the redox potential of the central Fe2+ and the polarity of the carbonyl linkage, and these in turn influence on the antiplasmodial activity.266 Hence, it was noted that compounds that are more resistant to oxidation and bear the ferrocene moiety adjacent to the carbonyl linkage (138) were more selective and potent antiplasmodial agents.266 On the basis of these results, ferrocene was dismissed as having a purely structural role as a hydrophobic spacer. Ferrocenyl chalcones also demonstrated radical-quenching properties and hydroxyl adduct formation in EPR experiments.266 Thus, the authors proposed that ferrocene might contribute in a more direct way via electron transfer involving Fe2+ and that this process may, to some degree, contribute to their antiplasmodial activity.266 Conjugates of ferrocene and azolium salts (142 and 143, Figure 24) were designed in the hope of mimicking the redox characteristics of methylene blue,267 which is an antimalarial enzyme inhibitor, active against the glutathione reductase (GR) enzyme of the malaria parasite, and was a chemotherapeutic agent until it was discarded for toxicity reasons. N-Ferrocenyl-

indicating that ferrocene does not increase antimalarial activity.261 These compounds also exhibited low inhibitory potency against parasitic cysteine protease falcipain-2 (involved in hematin degradation). A separate study on gold(I) complexes of TSC (mentioned in section 4.2.3.1) included a heterobimetallic TSC compound with ferrocene (132) that, when tested for antiplasmodial capacity against P. falciparum, was less active than the parent ferrocenyl-TSC compound in CQsensitive and CQ-resistant parasite strains.193 More recently, a series of ferrocenylthiosemicarbazone metallodendrimers (133 and 134, Figure 23) was synthesized through conjugation of ferrocenylthiosemicarbazones to a branched poly(propyleneimine) dendritic scaffold.262 Expectations were to increase the antimalarial activity associated with ferrocenyl−thiosemicarbazones by increasing bioavailability through the peripheral amine groups of the dendrimer. The antiplasmodial activity of these compounds was investigated in vitro against the CQ-resistant W2 strain.262 The dendritic ferrocenyl thiosemicarbazones 133 and 134 displayed moderate antimalarial activity with IC50 values of 6.59 and 1.79 μM, respectively. These activities are far better than those displayed by the nondendritic ferrocenyl-thiosemicarbazones with 3−11fold increases in activity.262 Derivatives of ciprofloxacin (135−137, Figure 24) bearing ferrocene substituents at different positions along the quinolone ring were tested for antiplasmodial activity against the CQsensitive 3D7 and CQ-resistant W2 P. falciparum strains.263 Ciprofloxacin is part of the family of quinolines and fluoroquinolones, common antibacterials that have proven to be active in vitro against CQ-sensitive and CQ-resistant P. falciparum strains.264 All ferrocenyl derivatives were significantly more active than the parent compound, ciprofloxacin.263 The most dramatic increase in activity compared to ciprofloxacin was observed for 135: a 15-fold improvement in 3D7 and a 30-fold improvement in W2 after 48 h of contact. Primarily, this increase in activity is the consequence of an increase in the drug’s lipophilicity as a result of the ester derivatization of ciprofloxacin 3478


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Figure 25. Ferrocenyl carbohydrate conjugates with antimalarial activity.

methyl, N′-methyl-2-aryl (or styryl) benzimidazolium iodide salts (142 and 143) were tested against the NF54 P. falciparum strain, resulting in IC50 values between 0.08 and 0.84 μM.267 Finally, conjugates of a chemosensitizer (overcomes chloroquine resistance) like strychnobrasiline and ferrocenyl derivatives were designed in hopes of overcoming the lack of in vivo activity of this chemosensitizer.268 Na-Deacetyl-ferrocenoylstrychnobrasiline (144) showed in vitro activity 15 times higher than the parent strychnobrasiline and significant synergistic effects (with chloroquine) against the chloroquine-resistant strain FCM29;268 however, the compound lacked any in vivo potentiating effect, and an antagonistic effect was even observed at higher doses.268 4.3.1.4. Ferrocene Conjugates of Other Compounds. Ferrocenyl conjugates with nontraditional antimalarial drugs or compounds that were not in investigation for antimalarial properties also exhibited the ability to inhibit growth of the malaria parasite.269−271 Among these serendipious discoveries are unusual conjugates such as ferrocenyl carbohydrate conjugates (Figure 25) which showed moderate antiplasmodial effects. Ferrocenyl ellagitannin derivatives showed antiplasmodial activity against the chloroquine-sensitive FCR3 parasite strain.269 The largest improvement was achieved by 145 that displayed the highest activity with an IC50 value of 0.6 μM, showing an inhibitory activity similar to quinine (0.11 μM) against the FCR3 chloroquine-sensitive strain, while the parent compound ellagitannin, a trideca-O-methyl-α-pedunculagin, displayed no antimalarial activity whatsoever.269 The cytotoxicity of these compounds was also evaluated in a mouse mammary tumor cell line (FM3A) with selectivity for 145 being greater than 25.269 Ferrocenyl carbohydrate compounds with ferrocene substituted with either one or two monosaccharides via different linkers (amide, ester, thioester, 146−148, Figure 25) were synthesized in our group.270,272 The antiplasmodial activity of these compounds was determined against the chloroquinesensitive D10 and 3D7 strains as well as the chloroquine-resistant K1 strain.270 146 was active in D10 (IC50 = 12.5 μM) but lost activity by a factor of 4 against K1. Compound 147, the disubstituted analogue of 146, showed similar behavior but was 3 times more active with IC50 values of 6.7−16.4 μM against the CQ-sensitive and CQ-resistant strains. Compound 148 was the most active of the group with IC50 values of 4.9 and 6.1 μM for

3D7 and K1, respectively. Notably, for compounds showing activity, bisubstituted conjugates were generally more active than their monosubstituted analogues.270 Our group was able to develop these promising results obtained from the various carbohydrate conjugates into new conjugates, fusing a ferrocene moiety with a carbohydrate and a chloroquine derivative. These new derivatives show promising antiplasmodial activity in the nanomolar region in both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum.273,274 3-O-Substituted glucose ferrocenyl derivatives (149 and 150, Figure 25) were synthesized along with other functionalized glucoses to be tested as P. falciparum hexose transporter (PfHT) inhibitors.271 Previous studies established that some 3-Oderivatives of glucose could selectively inhibit PfHT and rapidly disrupt intraparasitic homeostasis while not disrupting host erythrocytes.271 These compounds (149 and 150) were screened for their ability to inhibit uptake of D-glucose mediated by PfHT expression in Xenopus oocytes and thus for their potential as antimalarials. Unfortunately, the presence of a ferrocenyl group resulted in loss of inhibition compared to the parent 3-O-glucose derivative.271 In the first series of organometallic derivatives of indoles, ferrocenyl 2-phenylindole hybrids (151, Figure 26), the ferrocene was introduced into position 3 of the 2-phenylindole scaffold, well known for its antimitotic properties, and tested for cytotoxic and antiplasmodial properties against a large panel of P. falciparum parasite strains.239 Indoles are synthetically attractive small molecules; because they are inexpensive and easy to synthesize, they have already been investigated in their biological properties especially as antitumor agents.239 Regardless of the susceptibility to chloroquine of the P. falciparum strains, the ferrocenyl derivatives only exhibited weak inhibitory effects and no clear improvement was detected between the indole precursor and the ferrocenyl conjugate.275 The most active conjugate, the unsubstituted ferrocenyl indole 152, showed IC50 values in the range of 18.8−28.2 μM. Compounds of the general structure 151 showed only moderate antimalarial activity, the presence of the ferrocenyl moiety having a marginal effect on activity with no observable correlation between anticancer activities and antimalarial activities for these compounds.275 4.3.1.5. Ruthenocene Conjugates of Antimalarial Drugs. With iron and ruthenium, both group 8 transition metals, ruthenocene analogues of ferroquine were a logical consequence. 3479


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radicals, which in vitro and in vivo would create oxidative stress and contribute to the death of the parasite.244,245 This difference in electrochemical behavior between 93 and 153 is, nevertheless, not reflected in a strong difference in antimalarial potency, which would suggest that any redox contribution from the Fe2+/Fe3+ pair may contribute to the overall antimalarial action but not be the determining factor. 4.3.2. Nonmetallocene Compounds. Even though metallocene compounds of ferrocene make up most of the bioorganometallic antimalarials, due to their stability in vivo and beneficial redox properties, other organometallic classes of compounds have been envisioned and described as antimalarials as well. Arene−metal complexes, half-sandwich complexes, and alkenyl-substituted organometallics are now described. 4.3.2.1. Chloroquine Derivatives. In addition to the previously mentioned binuclear chloroquine Ru(II) coordination compound [RuCl2(CQ)]2 (41), organoruthenium compounds have also been evaluated for their antiplasmodial activity.276 A series of organo-Ru(II)−CQ half-sandwich complexes of general formula [Ru(II)(η 6 -arene)(CQ)]L2 (157−161, Figure 28) were found to display different binding

Figure 26. Ferrocenyl 2-phenylindole hybrids as antimalarials.

Slight modifications to the synthetic methodology yielded ruthenoquine (RQ) (153) and its structural derivatives (154− 156, Figure 27).224,236,237 These compounds exhibited good

Figure 27. Ruthenocene chloroquine derivatives with antimalarial action.

efficacy in both chloroquine-sensitive (D10) and chloroquineresistant (K1) strains, and as observed for ferroquine, addition of the ruthenocene was key for overcoming chloroquine resistance.224,236,237 Ruthenoquine (153) was found to be slightly less active than ferroquine (93) in both parasite strains with IC50 values of 23.8 and 6.3 nM for D10 and K1, respectively.223 Nonetheless, this compound was still as effective as chloroquine in D10 and 53 times more potent than CQ in K1, generally showing the same efficacy as ferroquine (93) against both types of parasite strains.223,236,237 In further studies in several strains of P. falciparum of different drug sensitivity it was shown that the activity of FQ and RQ is correlated with each other but not with CQ, confirming a similar mode of action for these metallocenes and a lack of cross resistance.244 One of the main goals of synthesizing the ruthenium analogues, other than to search for more potent analogues of ferroquine, was to establish the importance of the metal. It was suggested that the redox contribution of the ferrocene/ ferrocenium couple can be crucial in explaining the antimalarial activity of ferroquine.244,245 For the redox-pair Ru2+/Ru3+ a much higher potential is expected and oxidation is therefore less likely to occur compared to ferroquine. It was shown that 153 and derivatives 154−156 are not able to facilitate formation of OH

Figure 28. Organoruthenium complexes containing chloroquine.

modes of chloroquine, including the first example of a η6chloroquine binding mode through the carbocyclic ring.276 Compounds 157−161 were synthesized through a bridgesplitting reaction between the well-known dimer [Ru(η6arene)Cl2]2 and CQ, facilitated in certain cases by Ag(I). Compounds 157−160 have a conventional half-sandwich structure motif and can easily undergo ligand exchange of one chloride ligand for solvent molecules but are relatively stable in solution and in the presence of air. Compound 161 has chloroquine coordinated as the CQDP diphosphate salt (the two most basic nitrogens being protonated) in an unprecedented πcoordination through the aromatic ring of quinoline side chain to Ru(II). This proposed novel structure could not be confirmed by X-ray crystal structure analysis; however, NMR and FTIR data together with density functional theory (DFT) calculations indicate this to be the most likely binding mode, supporting a model with coordination of Ru(II) through the carbocyclic rather than the N-heterocyclic ring.276 3480


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Figure 29. Half-sandwich compounds of Rh, Ir, Re, and Cr containing chloroquine.

Figure 30. Organometallic compounds with antimalarial action, not containing chloroquine.

Complexes 157−159 and 161 were active against CQresistant and CQ-sensitive parasite strains of P. falciparum. All compounds proved to be less active than the parent drug chloroquine in all tested CQ-sensitive strains except for 3D7, for which the compounds were twice as active as chloroquine.276 Against the chloroquine-resistant parasite strains, 157−159 and 161 were more potent than chloroquine, successfully overcoming resistance in these strains, the exception being 161 showing slightly lower activity than CQDP against the highly resistant W2 strain. The most potent of this series was compound 159 being 2.4−5-fold more active than chloroquine.276 The same authors that previously investigated the mechanism of action of [RuCl2(CQ)]2 (41) studied the mechanism of action for these organo-Ru(II) compounds and proposed that a balance of physicochemical properties, particularly the lipophilicity and structural features of the drug, plays a determining role in their increased capacity for heme aggregation inhibition.277 Compounds 157 and 161 were also tested for their capacity to inhibit growth of cancer cells in HCT-116 colon cancer and LS141 liposarcoma cancer cell lines.276 157 showed almost twice the activity of 161, with IC50 values of 20 and 8 μM in each cell line. These values were comparable to observations for other organoRu(II) complexes studied as antitumor agents.276

Treatment of bis(cyclooctadiene)rhodium(I) chloride [Rh(COD)Cl]2 with 3 equiv of CQ under mild conditions provided air-stable [Rh(CQ)(COD)Cl] (162, Figure 29).177 Growth inhibition studies of 162 carried out in vitro against P. berghei indicated the rhodium complex as equipotent to chloroquine diphosphate (CQDP), representing no advantage over the action of chloroquine.177 In contrast to these initially discouraging in vitro results, in vivo experiments in a rodent malarial model of P. berghei demonstrated that 162 was more potent than chloroquine, leading to a reduction of parasitemia levels by 73% compared to 55% with chloroquine, using equivalent concentrations of 1 mg/kg CQ.177 Furthermore, 162 did not show any sign of acute toxicity up to 30 days post-treatment.177 Iridium−CQ (163−165, Figure 29) complexes were synthesized and evaluated for antimalarial activity in in vitro cultures of P. berghei.278 IC50 values obtained were 72 (163), 59 (164), and 126 nM (165) (IC50(CQ) = 72 nM). The only compound seemingly improving the action of the parent drug was 164; however, no clear structure−activity relationship could be established from this trend.278 Cyclopentadienyl tricarbonylrhenium derivatives of chloroquine (166 and 167, Figure 29) were evaluated in vitro against the chloroquine-sensitive 3D7 and the chloroquine-resistant W2 3481


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Table 1. Antiplasmodial Activity Data for Metalloantimalarialsa compound quinoline-based drugs 1225 1269 3220 3177 3193 3203 3207 3221 3222 3224,236 3225 3236 3237 3259 3263 (48/96 h) 3266 3275 3276 4185 4225 7225 8225 8185 17184,192 19225 20254 21207 21225 22225 metal chelators 28147 28156 30146 30147 31148 32148 35156 37156 38156 39156 metal complexes 41177 42181 43182 44182 45182 46182 47182 53192 54192 55192 57192 17 + 57192 57193 59193 59193 60193

CQ-sensitive strains

CQ-resistant strains

341 nM (mean from 65 isolates of P. falciparum) 378 nM (SGE2), 176 nM (FG2), 93 nM (FG4), 971 nM (FG3) 72 nM (“P. berghei”) 0.0173 μM (D10) 0.039 μM (MCR2) 19 nM (3D7) 287 nM (SGE2) 26 nM (HB3) 22.9 nM (D10) 370 nM (mean from 102 isolates of P. falciparum) 22.9 nM (D10), 8.52 nM (3D7) 23 nM (D10) 195 nM (F32) 19/21 nM (3D7) 25 nM (3D7) 45.6 nM (FcB1), 39.5 nM (3D7), 58.3 nM (PFB), 8.2 nM (F32) MIC = 0.1 μM (FAN-5) 18.1 nM (mean from 71 isolates of P. falciparum) 8.3 nM (mean from 88 isolates of P. falciparum) 7600 nM (mean from 71 isolates of P. falciparum) MIC = 1 μM (FAN-5) 16 nM 2.9 nM (mean from 65 isolates of P. falciparum) 5 nM (P. falciparum) 3 nM (3D7) 3.3 nM (mean from 67 isolates of P. falciparum) 0.8 nM (mean from 98 isolates of P. falciparum) 20.7 μM (over 73 strains) 22 μM (3D7) 170 nM (various) 1.48 μM (over 137 strains) 7.6 μM (3D7) 3.3 μM (3D7) 40 μM (3D7) 50 μM (3D7) 4.5 μM (3D7) 3.0 μM (3D7)

0.11 μM (FCR3) 2.4 μM (FCM6), 5.5 μM (FCM17), 5.1 μM (FG1) 47 nM (FcB1), 104 nM (FcB2) 0.095 μM (W2)

4365 nM (FCM6), 4555 nM (FCM17) 137 nM (Dd2), 154 nM (FG3), 168 nM (FG4), 184 nM (FCR3), 288 nM (FG1) 352.3 nM (K1) 290 nM, 352.3 nM (K1) 352 nM (K1) 0.105 μM (FcB1), 0.226 μM (K1) 480/462 nM (W2) 0.26 μM (K1) 502 nM (FCM29), 539 nM (W2) 2155 nM (W2), 1184 nM (Dd2), 1883 nM (K1)

18 μM (7G8) 1 mM (various)

4.75 μM (7G8) 2.5 μM (7G8)

18 nM (“P. berghei”) 3 nM 17 nM (F32) 13 nM (F32) 10 nM (F32) 13 nM (F32) 18 nM (F32) 2.1 μM (3D7) 168 μM (3D7) 439 μM (3D7) 142 nM (3D7) 12 nM, 9 nM (3D7) 2.02 μM (D10) 3.84 μM (D10) 3.84 μM (D10) 4.64 μM (D10)

10.5 nM (FcB1), 46.5 nM (FcB2) 5.1 nM (FcB1), 23 nM (FcB2) 40 nM (FcB1), 50 nM (W2), 93 nM (K1) 10 nM (FcB1), 50 nM (W2), 99 nM (K1) 40 nM (FcB1), 40 nM (W2), 58 nM (K1) 43 nM (K1) 54 nM (K1)

3.55 2.81 2.81 3.06

3482

μM μM μM μM

(W2) (W2) (W2) (W2)


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Table 1. continued compound

CQ-sensitive strains

CQ-resistant strains

metal complexes 61−65194 3.04−7.22 μM (W2) 66, 67195 1−1.5 μM (HB3, FCR-3, Indo-1, Dd2) 68195,196 >20 μM (HB3) 0.5 μM (FCR-3), 0.6 μM (Indo-1) 69136 80 nM (HB3) 2.5 μM (Dd2) 69199 1.8 μM (Dd2) 70136 86 nM (HB3) 0.8 μM (Dd2) 71198 2 μM (HB3) >15 μM (Dd2) 72199 0.6 μM (HB3) 1.4 μM (Dd2) 73200 (Cu) 0.34 μM (3D7) 75202 5.4 μM (3D7) 76203 0.059 μM (MCR2) 77203 0.048 μM (MCR2) Mn-80205 2.127 μM (K1) Fe-81205,206 4.60 μM (K1) 82207 0.0002 μg/mL (3D7) 0.01 μg/mL (K1) Fe-84212 6−412 μM (FcB1, FcM29) 86−89213 2−12 μM (3D7) 90213 680 nM (3D7) 91213 220 nM (3D7) bioorganometallic compounds 93(C4H6O6)2220 218 nM (SGE2), 80 nM (FG2), 78 nM (FG4), 129 nM (FG3) 93223,237 17.7 nM (D10), 20.2 nM (HB3) 18.9 nM (Dd2), 8.1 (W2) 93224 16.3 nM (D10) 5.0 nM (K1) 93225 10.8 nM (mean from 103 isolates of P. falciparum) 93236 17.7 nM (D10) 13.5 nM (K1) 93237 18 nM (D10) 14 nM (K1) 93266 71 μM (K1) 93275 3.5 nM (3D7) 6.8 nM (FCM29), 6.6 nM (W2) 95222 143 nM (HB3) 789 nM (Dd2), 428 nM (FG3), 477 nM (FG4), 294 nM (FCR3), 616 nM (FG1) 222 96 27 nM (HB3) 53 nM (Dd2), 58 nM (FG3), 77 nM (FG4), 294 nM (FCR3), 86 nM (FG1) 98221 (R1 = R2 = C2H5) 554 nM (SGE2) 971 nM (FCM6), 1003 nM (FCM17) 98221 (R = cHex) 807 nM (SGE2) 702 nM (FCM6), 1026 nM (FCM17) 98223 12.4−204.8 nM (HB3) 17.0−169.5 nM (Dd2), 16.8−19.2 nM (W2) 99223 45.7−274 nM (HB3) 29.8−120.1 nM (Dd2), 24.0−134.4 nM (W2) 100236 (n = 2, R = H) 33.2 nM (D10),

Metalloantimalarials - ACS Publications - American Chemical Society

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