Chapter 2 Alkaloids with Antiprotozoal Activity

CHAPT ER 2 Alkaloids with Antiprotozoal Activity Edison J. Osorio1,, Sara M. Robledo2 and Jaume Bastida3 Contents I. Introduction II. Tropical Diseases Caused by Protozoan Parasites A. Leishmaniasis B. Chagas’ Disease C. African Trypanosomiasis (Sleeping Sickness) D. Malaria III. Alkaloids with Antiprotozoal Activity A. Quinoline and Isoquinoline Alkaloids B. Indole Alkaloids C. Steroidal and Diterpenoid Alkaloids D. Alkaloid Marine Natural Products E. Other Alkaloids IV. Current Strategies and Recent Developments V. Conclusions and Future Directions Acknowledgments References 113 115 115 118 119 120 122 122 149 160 164 168 171 178 179 179 I. INTRODUCTION Protozoan parasites are among the most common chronic infections that occur primarily in rural and poor urban areas of tropical and subtropical regions 1 Grupo de Investigación en Sustancias Bioactivas, Facultad de Quı́mica-Farmacéutica, Universidad de Antioquia, A. A. 1226, Medellı́n, Colombia 2 Programa de Estudio y Control de Enfermedades Tropicales, Facultad de Medicina, Universidad de Antioquia, Medellı́n, Colombia 3 Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona, 08028 Barcelona, Spain  Corresponding author. E-mail address: josorio48@yahoo.com (E.J. Osorio). The Alkaloids, Volume 66 ISSN: 1099-4831, DOI 10.1016/S1099-4831(08)00202-2 r 2008 Elsevier Inc. All rights reserved 113 114 Osorio et al. around the world. They are responsible for a large number of severe and widespread diseases including malaria, leishmaniasis, Chagas’ disease, and sleeping sickness. These diseases, with the exception of malaria, belong to the group of Neglected Tropical Diseases (NTDs), since they are strongly linked with poverty and there is a lack of commercial markets for potential drugs. Like other NTDs, these diseases affect individuals throughout their lives, causing a high degree of morbidity and physical disability and, in certain cases, gross disfigurement. Patients can face social stigmatization and abuse as a result of contracting these diseases. The disability and the poverty associated with these diseases constitute large burdens on the health and economic development of low-income and middle-income countries in Africa, Asia, and the Americas (1). Strategies to control these diseases are based on surveillance, early diagnosis, vector control, and treatment (2). At present, there are only a few drugs on the market to treat these parasitic diseases, and they are not universally available in the affected areas. In addition, current drug treatments are unsatisfactory due to drug resistance, inefficiency, toxicity, prolonged treatment schedules, high cost, etc. Therefore, there is an urgent need for new treatments, which are safe, effective, cheap, and easy-to-administer, and for new lead compounds with novel mechanisms of action. Living organisms are commonly used as natural sources of novel structures for the discovery and development of new drugs, since they contain countless molecules with a great variety of structures and pharmacological activities (3). Out of 1010 new active substances approved as drugs for medical conditions by regulatory agencies during the past 25 years, 490 (48.5%) were from a natural origin (4). The diversity of natural products with antiprotozoal activities has been illustrated in several reviews covering molecules that are mainly active against malaria, leishmaniasis, Chagas’ disease, or sleeping sickness (5–11). This chapter deals exclusively and thoroughly with the alkaloid natural products that are particularly active against Leishmania sp., Trypanosoma cruzi, Trypanosoma brucei, and Plasmodium falciparum. The alkaloids with antiprotozoal activity are grouped according to their structures or origin in several categories: quinoline and isoquinoline alkaloids, indole alkaloids, steroidal alkaloids, alkaloids from marine organisms, and other alkaloids. A discussion on structure–antiprotozoal activity relationships is included, and the mechanism of action of several of these metabolites is described. Recent developments, as well as new experimental strategies in discovery and development of antiprotozoal compounds, are also discussed. Some of the in vitro antitrypanosomal or cytotoxic activities reported in the literature have been transformed into molar concentrations (mM, mM, or nM) to allow a better comparison, independent of their molecular weight. Nevertheless, direct comparison remains complex due to the different assay procedures used in various laboratories. Alkaloids are one of the most important groups of natural products, already providing many drugs for human use (12). Although they can be seriously toxic for the host, alkaloid-containing plants and their biosynthesized alkaloids have a remarkable potential to provide pharmaceutical and biological agents contributing to the development of future antiparasitic drugs. Alkaloids with Antiprotozoal Activity 115 II. TROPICAL DISEASES CAUSED BY PROTOZOAN PARASITES Leishmaniasis, Chagas’ disease (or American trypanosomiasis), malaria, and African trypanosomiasis (or sleeping sickness) are vector-borne infectious diseases caused by protozoan parasites. Despite considerable control efforts, they are among the most prevalent parasitic diseases worldwide with a heavy social and economic burden (13). Those at greatest risk are populations that are poor or beyond the reach of adequate medical attention. Although these diseases are commonly associated with poverty, they are also a cause of hardship and a major hindrance to economic development (14). As a result of poor market incentives for companies to carry out research to develop new drugs, they are called ‘‘neglected diseases’’ (15). In most endemic countries, official Ministry of Health policy is to provide free treatment to all patients, but this is often unfeasible because the required drugs are in limited supply, especially in rural areas where the diseases mostly occur. Consequently, self-help patient organizations are often used to provide diagnosis and treatment. Thus, people afflicted by neglected diseases are vulnerable to violations of their basic human rights, such as access to health care and essential medicines (16). The health impact of these diseases is measured by severe and permanent disabilities and deformities in affected people. In addition to the physical and psychological suffering they cause, these diseases inflict an enormous economic burden on affected communities owing to lost productivity and the high costs associated with long-term care, which in turn contributes to the entrenched cycle of poverty and ill-health in affected populations (13). Overall global prevalence is approximately 550 million cases, and close to 2.7 billion people living in endemic areas are at risk of contracting any of these diseases. In total, there are about 280 million new cases each year causing important health and socio-economic problems where these diseases are endemic. Chemotherapy remains one of the key measures used to control the intolerable burden of protozoan parasitic and other tropical diseases, but most of the available drugs are no longer effective due to drug resistance. Moreover, some of those that are still effective suffer from problems associated with toxicity, compliance, and high cost, resulting in an urgent need for new drugs. A. Leishmaniasis Leishmaniasis is a group of clinical diseases suffered by millions around the world, and affecting 88 countries in Africa, Asia, Europe, and America, 72 of which are developing countries and 13 are among the least developed. The spectrum of the disease is divided into three major syndromes: cutaneous (CL), mucocutaneous (MCL), and visceral leishmaniasis (VL). Annual incidence is estimated at 1.5 million cases of the cutaneous forms (CL and MCL) and 500,000 cases of the visceral form (VL), resulting in approximately 51,000 deaths per year. Overall prevalence is 12 million people and the population at risk is 350 million. However, this estimated global burden of disease is believed to be inaccurate partly due to the passive case detection data used to 116 Osorio et al. estimate the disease prevalence in many endemic countries. Apparently, for each symptomatic case, there are estimated to be 10 asymptomatic infections. The total burden of Disability Adjusted Life Years (DALY) is 2.09 million, with 840,000 for women and 1.25 million for men (13,17–19). The Special Program for Research and Training in Tropical Diseases (TDR) has classified Leishmaniasis as a group of emerging or uncontrolled diseases (Category I) (13,20). Leishmaniasis is produced by at least 17 species of the protozoan Leishmania (order Kinetoplastida, family Trypanosomatidae). L. donovani and L. infantum are the causative agents of VL, while L. major, L. tropica, L. aethiopica, L. braziliensis, L. panamensis, L. amazonensis, and L. mexicana produce CL (17–19). Leishmania spp. are transmitted by sandflies of the genera Phlebotomus (Old World) and Lutzomyia (New World). The parasite exists in two morphological forms: the amastigote (aflagellated form) that proliferates intracellularly in the mammalian macrophages, and the promastigote (extracellular flagellated form) that proliferates in the gut of its sandfly vector and in the acellular culture medium. Infection occurs when an infected sandfly regurgitates infective promastigotes into the blood while feeding. The promastigotes are phagocytized by macrophages and transformed into amastigotes. The life cycle is continued when a sandfly vector feeding on the blood of an infected individual or an animal reservoir host ingests the macrophages infected with amastigotes (13,17–20). Chemotherapy for leishmaniasis is still deficient. Most of the drugs have one or more limitations such as long-term administration, unaffordable cost, toxicity, or even worse, inefficacy due to development of resistance in the parasite (13,17–24). Pentavalent antimonials (SbV) have become the drug of choice for the treatment of all types of leishmaniasis. Pentostams (sodium stibogluconate) (1) and Glucantimes (meglumine antimoniate) (2), are non-covalent chelates of SbV with improved solubility and uptake properties. However, they require long courses of treatment with parenteral administration, produce toxic side effects, and show variable efficacy. Increasing resistance to antimonials has been documented in several regions, but particularly in northeast India (25). Although these drugs constitute the main antileishmanial chemotherapy and have been used for over 50 years, information about their chemistry or precise mode of action and the identity of the biologically active components is uncertain. The second line drugs are pentamidine isethionate (3), and amphotericin B (4) (21,22). Pentamidine toxicity has mainly been associated with high cumulative doses (26). Toxic side effects of 3 include a sensation of burning, headache, tightness of the chest, dizziness, nausea, vomiting, and hypotension. Other serious side effects of pentamidine are hypoglycemia, hyperglycemia, and acute pancreatitis leading to diabetes (27). Common side effects of amphotericin B (4) include anaphylaxis, thrombocytopenia, flushing, generalized pain, chills, fever, phlebitis, anemia, convulsions, anorexia, decreased renal tubular and glomerular function, and hypokalemia. In order to increase the therapeutic index of amphotericin B, and to reduce toxicity, a lipid formulation of amphotericin B was developed (28,29). Alkaloids with Antiprotozoal Activity 117 Among the new drugs currently under clinical evaluation are miltefosine (5), an antitumor agent being used in India for the oral treatment of VL, and which is under clinical trials in different countries for the treatment of CL (21,30,31). Although miltefosine has proven effective, its long half-life could lead to the rapid emergence of resistance. In addition, miltefosine has demonstrated teratogenicity in animal studies leading to its contraindication in pregnancy, and recommended caution in women of childbearing potential (31). Two other drugs under clinical trials are paromomycin (6), and the 8-aminoquinoline derivative sitamaquine (7). Paromomycin is under clinical trial (phase III) for VL, and is currently used in combination with methyl benzethonium chloride or urea for CL. Sitamaquine is currently in phase II clinical trial for VL (21). COOH HO HO HO OH COOH OH O O O Sb Sb O O O H O H .3Na .9H O O O Sb O O HO OH OH Me 2 HO OH OH NH HO HN pentostam® 1 glucantime® 2 (sodium stibogluconate) (meglumine antimoniate) NH NH H2N NH2 O . HOCH CH SO H O 2 2 3 pentamidine isethionate 3 OH Me HO OH O O Me OH OH OH OH O OH COOH Me amphotericin B 4 O HO O NH2 O O P O Me miltefosine 5 O Me OH Me N Me Me Me 118 Osorio et al. NH2 HO HO O Me O MeO O HO NH2 H2N HO O HO H2N OH O O NH 2 paromomycin 6 OH OH N HN N Et Et sitamaquine 7 B. Chagas’ Disease Chagas’ disease is endemic in Latin America, affecting 18 countries from northern Mexico to southern Argentina. There are about 20 million infected individuals, 120 million are at risk of acquiring the infection, and approximately 8 million are carriers of the disease (32–34). Control programs carried out during the past 15 years have reduced annual incidence of the disease from more than 500,000 new cases every year to around 50,000, resulting in about 14,000 deaths per year (35). Approximately 25 to 30% of those infected will progress to irreversible cardiac, esophageal, and colonic pathology, leading to considerable morbidity and mortality. The total DALY burden is 667,000, affecting men and women equally (32–34). TDR has classified Chagas’ disease in category III, since the control strategy has proven effective and elimination is possible (1). Chagas’ disease is produced by the protozoan T. cruzi (order Kinetoplastida, family Trypanosomatidae), which is transmitted to humans and other mammals mostly by blood-sucking reduviid bugs of the subfamily Triatominae (family Reduviidae). The parasite exists in three morphological forms: amastigotes (aflagellated form) that proliferate intracellularly in the mammalian macrophages, epimastigotes (extracellular flagellated form) that proliferate in the insect vector and the acellular culture medium, and trypomastigotes (the infective flagellate form) found in the blood of the mammalian host and in the terminal part of the digestive and urinary tracts of vectors. Infection occurs when infected metacyclic trypomastigotes enter the body through wound openings or mucous membranes. The trypomastigotes enter various cells, differentiate into amastigotes, and multiply intracellularly. The amastigotes differentiate into trypomastigotes, which are then released back into the bloodstream. The life cycle is continued when a reduviid bug feeds on an infected person and ingests trypomastigotes in the blood meal. Transmission of Chagas’ disease could also occur by blood transfusion or materno-fetal transmission (32–34). Chemotherapy for Chagas’ disease is based on two, empirically discovered, drugs, a nitrofuran derivative nifurtimox (8) and a nitroimidazole derivative benznidazole (9), but it is still insufficient (13,23,32,35). Both of these compounds are usually only effective when given during the acute stage of infection, being Alkaloids with Antiprotozoal Activity 119 able to cure at least 50% of recent infections, as indicated by the disappearance of symptoms, and negativization of parasitemia and serology. Other important drawbacks include selective drug sensitivity in different T. cruzi strains, and a necessity for constant medical supervision due to the gastrointestinal and neurological side effects of nifurtimox, and the rash and gastrointestinal symptoms provoked by benznidazole. In addition, treatment is long (30 to 60 days), drug resistance develops, and these compounds are not effective in the chronic stage of the disease (23,32,34,36). O Me O2 N O N N N SO2 nifurtimox 8 N NH NO2 benznidazole 9 C. African Trypanosomiasis (Sleeping Sickness) African trypanosomiasis, also known as sleeping sickness, affects 36 countries in sub-Saharan Africa, and is fatal if left untreated. Although its precise prevalence is unknown, the population at risk is 60 million, while annual incidence is estimated at 300,000 to 500,000 cases, resulting in approximately 66,000 deaths per year. The total DALY burden is around 1.5 million, with 559,000 for women and 996,000 for men (37–39). TDR has classified African trypanosomiasis in Category I as an emerging or uncontrolled disease (13). In humans, it is caused by two kinetoplastid flagellates, Trypanosoma brucei rhodesiense and T. b. gambiense (order Kinetoplastida, family Trypanosomatidae), which are transmitted by several species of blood-feeding tsetse flies of the genus Glossina. T. b. rhodesiense occurs mainly in east and southern Africa, and T. b. gambiense mainly in west and central Africa. Like T. cruzi, the parasite exists in three morphological forms: amastigote, epimastigote, and trypomastigote. Infection occurs via the bite of the blood-sucking male and female tsetse flies that transfer the parasites from human to human. Following the bite of an infected tsetse, parasites multiply in the skin for 1–3 weeks before invading the hemolymphatic system (37–39). The type of treatment depends on the stage of the disease (37,39). The drugs used in the early hemolymphatic stage are less toxic, easier to administer, and more effective, the current standard treatment being pentamidine (3) for T. b. gambiense or suramin (10) for T. b. rhodesiense (40). Successful treatment in the late stage, when the infection has spread to the central nervous system (CNS), requires a drug that can cross the blood-brain barrier to reach the parasite, but such drugs are quite toxic and complicated to administer. The late stage of the disease is treated by melarsoprol (11) or, alternatively, eflornithine (12) (40,41). All these drugs have to be administered intravenously and produce adverse side 120 Osorio et al. effects. Parasite resistance to melarsoprol has been reported (42). O HO3S O HN NH Me O HN HO3S SO3H Me O NH SO3H SO3H O N H SO3H N H suramin 10 H2N H N N N N NH2 H2N H2N As S S melarsoprol 11 F F HOOC OH eflornithine 12 Unfortunately, no vaccine is currently available for any of these diseases. The existing chemotherapy is unsatisfactory in terms of its lack of effectiveness, and also due to the toxicity associated with long-term treatments with empirically discovered drugs. Drug resistance and varying strain sensitivity to the available drugs is another drawback for clinically accessible chemotherapy. The search for new pharmacological alternatives is therefore a scientific priority in order to improve the health and quality of life of people suffering from these diseases. D. Malaria Malaria is a vector-borne infectious disease caused by protozoan parasites of the genus Plasmodium that are spread from person to person through the bites of several species of infected female mosquitoes of the genus Anopheles. Widespread in tropical and subtropical regions of America, Asia, and Africa, malaria is a public health problem in more than 90 countries inhabited by approximately 2400 million people, representing about 35% of the world population (13,43–46). Annual incidence of malaria is estimated at 300–500 million cases, resulting in approximately 1–2 million deaths per year, mostly in Sub-Saharan Africa, but also in Asia, Latin America, the Middle East, and parts of Europe. The total DALY burden is 46.5 million, spread equally between women and men (13,43–45). The economic burden of malaria is extremely high in countries where the disease is endemic, accounting for an estimated 1.3% reduction of the annual economic growth rate, and a higher than 50% reduction of gross national product (GNP), which has a long-term impact (47). The burden of malaria differs according to age and sex. In Africa, almost all deaths occur in children under 5 years of age, when the disease tends to be atypical and more severe (47). The disease burden associated with pregnancy has an additional impact due to the effect of malaria on 121 Alkaloids with Antiprotozoal Activity the health of the fetus (48). TDR has classified malaria in Category II because, although control strategies are available, the disease persists (13). The main causes of malaria are P. falciparum and P. vivax, but also P. ovale and P. malaria can be involved. P. falciparum is the species of parasite responsible for cerebral malaria. Parasites include mosquito stages (zygote, exflagellated gamete, and sporozoite), and human stages (sporozoites, liver schizonts, trophozoite, blood schizont, merozoite, and gametocyte). Infection in humans begins when infected female anopheline mosquitoes inject sporozoites subcutaneously into the human host during a blood meal (43–45). The sporozoites enter the bloodstream and invade hepatocytes where they differentiate and multiply as merozoites. In hepatocytes, P. vivax and P. ovale sporozoites can also develop into latent hypnozoites, which can lie dormant for months or years before differentiating into merozoites (49). The merozoites invade erythrocytes to establish a bloodstage infection, where they appear initially as a ring stage, followed by a growing trophozoite, which develops into a dividing asexual schizont. Some merozoites may also differentiate into sexual forms, male and female gametocytes, which are picked up by female Anopheles mosquitoes during a blood meal. Within the mosquito midgut, male gametocytes produce flagellated microgametes that fertilize the female macrogamete. The resulting ookinete traverses the mosquito gut wall and encysts on the exterior of the gut wall as an oocyst releasing sporozoites that migrate to the mosquito salivary gland (43–45). Malaria infections are treated by several drugs. Currently available antimalarial drugs for therapy and prophylaxis include: the quinoline-containing drugs, chloroquine (13), quinine (14), amodiaquine (15), primaquine (16), and mefloquine (17); the antifolate drugs, pyrimethamine (18), used in combination with sulfadoxine (19) and proguanil (20); and two newer classes of drugs that have been introduced in the last years, artemisinin (21) and its derivatives artesunate (22), artemether (23), and arteether (24); and the hydroxynaphthoquinone, atovaquone (25) (43–45,50). H2C Me N HN OH Et N H HO Et HN MeO Et N Cl Cl N chloroquine 13 quinine 14 N N amodiaquine 15 H HO MeO N H Cl NH2 N HN N NH2 Me primaquine 16 N CF3 CF3 mefloquine 17 Et N NH2 pyrimethamine 18 Et 122 Osorio et al. NH O S Me H OMe H N HN OMe O N N N H Me O O Me O H HN Me H2 N H O Me O sulfadoxine 19 artemisinin 21 Cl proguanil 20 H Me Me O HO O O O H H O O Me OR Cl artesunate (22) R=CO(CH2)2COOH artemether ( 23) R=Me arteether (24) R=Et atovaquone 25 Resistance to antimalarial drugs has been described for P. falciparum and P. vivax. P. falciparum has developed resistance to almost all antimalarial drugs currently used (51). In those areas where chloroquine is still effective it remains the first choice. Unfortunately, chloroquine-resistance is associated with reduced sensitivity to other drugs, such as quinine and amodiaquine. In some areas, P. vivax infection has become resistant to chloroquine and/or primaquine (44,50,52). Due to the global emergence of drug resistance, there is an urgent need for the development of new antimalarial drugs. III. ALKALOIDS WITH ANTIPROTOZOAL ACTIVITY A. Quinoline and Isoquinoline Alkaloids 1. Quinoline Alkaloids The family Rutaceae is an important source of antiprotozoal quinoline alkaloids. Activity-guided fractionation of the extracts of Galipea longiflora K. Krause (Rutaceae), a Bolivian plant used locally by the Indian Chimaneses for the treatment of cutaneous leishmaniasis, has afforded active compounds identified as 2-substituted quinoline alkaloids, especially chimanine B (26) and chimanine D (27), with an IC90 of around 0.14 mM, and 2-n-propylquinoline (28), with an IC50 of around 0.29 mM, against the promastigote forms of Alkaloids with Antiprotozoal Activity 123 L. braziliensis and the epimastigote forms of T. cruzi (53). Results of in vivo experiments indicate that chimanine D (27) and 2-n-propylquinoline (28), at 100 mg/kg/day for 14 days, resisted infection with each New World cutaneous leishmaniasis-causing strain, including the virulent strains L. amazonensis PH8 and L. venezuelensis H-3 (54). In an experimental treatment of VL in infected BALB/c mice, subcutaneous chimanine D (27) at 0.54 mM/kg/day for 10 days resulted in 86.6% parasite suppression in the liver, while daily oral administration of 0.54 mM/kg of 2-n-propylquinoline (28) for 5 or 10 days to L. donovani-infected mice suppressed parasite burdens in the liver by 87.8 and 99.9%, respectively (55). Further biological and chemical studies of 2-substituted quinoline synthetic alkaloids have shown in vitro and/or in vivo antileishmanial properties in the L. amazonensis cutaneous infection murine model, as well as the L. infantum and L. donovani visceral infection murine models (56–58). Preliminary toxicological evaluations of 2-substituted quinoline alkaloids given to BALB/c mice indicated that the drugs had reasonable therapeutic indices (54), and they are currently being investigated in preclinical studies for the development of an oral treatment of VL (59). These studies suggest that the apparently poor in vitro properties and good in vivo efficiency of 2-n-propylquinoline (28) could be due to the pharmacological activity of its metabolites (59,60). Other Rutaceae quinolines, like the 2-substituted tetrahydroquinoline alkaloids galipinine (29) and galipeine (30), isolated from Galipea officinalis Hancock, a plant native to Venezuela and used in folk medicine against fever, showed an in vitro antimalarial effect (IC50: 0.24–6.12 and 0.33–13.78 mM, respectively) on chloroquine-resistant strains of P. falciparum (61). Bioassayguided separations led to the isolation and structural elucidation of two 4-quinolinone alkaloids, dictyolomide A (31) and B (32), from the stem bark of Dictyoloma peruvianum Planch., a small tree used in folk medicine for the treatment of leishmaniasis. Antileishmanial activity could be attributed to these alkaloids, which induced complete and partial lysis in promastigote forms of L. amazonensis and L. braziliensis, respectively, at 0.35 mM (62). In the same way, seven alkaloids have been isolated from Teclea trichocarpa (Engl.) Engl. (a plant used in Kenyan traditional medicine for malaria treatment), including normelicopicine (33) and arborinine (34), which displayed limited in vitro activities against both chloroquine-sensitive (HB3) and chloroquine-resistant (K1) strains of P. falciparum, with an IC50 of 3.8–14.7 mM (63). Normelicopicine (33) was found to have some activity against P. berghei in mice with 32% suppression of parasitaemia at a dose of 25 mg/kg per day. O N R chimanine B (26) R=CHCHMe 2-n-propylquinoline (28) R=(CH2)2Me N chimanine D 27 Me 124 Osorio et al. N Me OR1 OR2 galipinine (29) R1+R2=CH2 galipeine (30) R1=H, R2=Me O O OH OMe N N R dictyolomide A (31) R=(CH2)2CH=CHCH2Me dictyolomide B (32) R=CHOH(CH2)4Me OMe Me R normelicopicine (33) R=OMe arborinine (34) R=H The decahydroquinolines (DHQs) are unusual quinoline alkaloid derivatives, characterized by the presence of a 2,3,5-trisubstituted cis-fused DHQ ring (64). Simple DHQs were first reported from the skins of dendrobatid frogs (65), and to date approximately 30 cis- and trans-DHQs have been isolated from amphibian sources (65,66). All of these alkaloids contain alkyl substituents attached at C-2 and C-5. The marine natural 2,5-dialkyl-DHQ alkaloids have also been isolated from ascidians (66,67), the flat worm Prosthecergeus villatus and its prey, the tunicate Clavelina lepadiformis (68), and a new tunicate species belonging to the genus Didemnum (69). Among plants, Lycopodium spp. are known to produce DHQs, but as parts of more complex alkaloidal molecules (70). DHQ alkaloids have also been isolated from myrmicine ants (71–73). Some DHQ alkaloids obtained from a tunicate species belonging to the genus Didemnum have shown significant and selective antiplasmodial and antitrypanosomal activity. The most pronounced biological activities were found for the alkaloids lepadin E (35) and F (36). Lepadin F has an IC50 of 0.47 mM against the P. falciparum clone K1, while lepadin E showed an IC50 of 0.94 mM. The low cytotoxicity of these molecules makes them suitable models for the development of potential therapeutic agents (69). Among the quinoline alkaloids are the classic antimalarials such as quinine (14), the first important leading natural compound against malaria. The quinoline-containing drug derived from the bark of the Cinchona tree (Rubiaceae), native to Peru and the Andes, was used to treat fevers as early as the 17th century, although it was not until 1820 that the active ingredient of the bark was isolated and used in its purified form (74). Although quinine was replaced by synthetic compounds, it is again being applied against chloroquineresistant strains, and as a treatment for uncomplicated and severe malaria in many different therapeutic regimens (75). Quinine was used as a template for the antimalarials chloroquine (13) and mefloquine (17), two fully aromatic quinoline Alkaloids with Antiprotozoal Activity 125 type compounds (9). Quinimaxs, which is a combination of quinine (14), quinidine (37) (dextrorotatory diastereomer of quinine), and cinchonine (38), all derived from cinchona bark, is also used (76). Quinine, quinidine, cinchonine, and cinchonidine (39) have significant trypanocidal activity with IC50 values of 4.9, 0.8, 1.2, and 7.1 mM, respectively, in T. b. brucei. For 37 and 38, the selectivity indices are greater than 200, indicating the potential of these alkaloids for further drug development (11). Likewise, quinine (14) completely inhibited the in vitro replication of T. cruzi at 15.4 mM (5). Me Me H2C OH H2C N H HO O H H N H H N H HO R O R1 R2 N N quinidine (37) R=OMe cinchonine (38) R=H cinchonidine 39 lepadin E (35) R1=Me, R2=H lepadin F (36) R1=H, R2=Me Although they are not natural products, the 8-aminoquinoline compounds deserve attention. Primaquine (16), a known 2-substituted 8-aminoquinoline, is the only tissue schizonticide (exoerythrocytic) drug available for radical treatment of P. vivax or P. ovale infections (77). Although primaquine has no clinical utility as a blood schizonticide, substantial efforts have been made to identify an 8-aminoquinoline with a better therapeutic index and activity against blood stages of malaria (77). On the other hand, sitamaquine (7) (originally WR6026), an 8-aminoquinoline in development as an antileishmanial agent (78), has undergone several small Phase 1/2 clinical trials with varying levels of success. For instance, 67% of patients were cured of L. chagasi in Brazil when treated with 2 mg/kg/day for 28 days (79); 92 and 100% of patients were cured of VL when treated with 1.75 mg/kg/day for 28 days in Kenya (80) and with 2 mg/kg/day for 28 days in India (81), respectively. Sitamaquine is rapidly metabolized, forming desethyl and 4-CH2OH derivatives, which might be responsible for its activity (78), but little is known about its mechanism of action (82). The mechanism of action of quinoline derivatives against Leishmania and Trypanosoma species remains unknown. Nevertheless, studies of antimalarial drugs against Plasmodium have revealed that quinoline compounds depend on interactions with heme (ferriprotoporphyrin IX) for their antimalarial action (83–85). The intraerythrocytic malaria parasite digests large quantities of host hemoglobin (86), a globular protein (globin) with an embedded heme group. The hemoglobin digestion process involves degradation of the protein component by proteolytic enzymes (87,88) and release of heme component (89,90). Because 126 Osorio et al. heme is toxic to Plasmodium parasites it is converted into a crystalline compound named malaria pigment or hemozoin, which is harmless to the parasite (91,92). Recent studies on cultured P. falciparum have shown that at least 95% of the hematin is incorporated into hemozoin (93). The antimalarial quinoline-type drugs act by binding to hemozoin crystal faces, which would inhibit their growth and result in a buildup of the toxic heme, thus leading to the death of the parasite (94–96). The mechanism of action of DHQ compounds is unknown and needs to be investigated to determine if it is similar to that of the fully aromatic quinolinetype compounds (69). 2. Aporphine and Oxoaporphine Alkaloids The aporphine and oxoaporphine alkaloids are isoquinoline compounds known to have various pharmacological properties, including antiparasitic activity. Most aporphine derivatives occur in members of the Annonaceae family (97–101), one of the largest of the order Magnoliales, comprising about 120 genera and more than 2000 species (102). Among the active alkaloids, liriodenine (40) and anonaine (41), isolated from the roots and trunk bark of Annona spinescens Mart., have shown significant activity against promastigote forms of L. braziliensis, L. amazonensis, and L. donovani (103). However, while in this report liriodenine showed antileishmania activity with an IC100 of 0.36 mM against the L. donovani strain, in another study, liriodenine isolated from the bark of Rollinia emarginata Schltdl. (104) and Unonopsis buchtienii R.E.Fr. (105) presented IC100 values of 18.16 mM, and 11.33 mM, respectively, against the same promastigote forms of the parasite. Similarly, O-methylmoschatoline (42), isolated from the stem bark of U. buchtienii by activity-guided fractionation, showed an interesting in vitro activity against T. brucei with an IC100 of 19.45 mM, but without selectivity. On the other hand, crude extracts of Uvaria klaineana Engl. & Diels stems showed in vitro activity against P. falciparum. Three alkaloids were identified by bioassay-guided fractionation, the main alkaloid being crotsparine (1-hydroxy-2-methoxynorproaporphine) (43), which showed IC50 values of 7.41, 11.29, and 12 mM against the chloroquine-resistant FcB1 and K1, and the chloroquine-sensitive Thai strains of P. falciparum, respectively (106). Preliminary studies in the genus Guatteria (Annonaceae) have revealed interesting active compounds. Extracts from G. foliosa Benth., commonly known as ‘‘Sayakasi’’ among the Chimane Indians in Bolivia, have demonstrated antiparasitic activity against different strains of Leishmania and against T. cruzi (107). Fractionation of the alkaloid extract resulted in the isolation of several isoquinoline alkaloids, including isoguattouregidine (44), which showed significant activity against the promastigote forms of L. donovani and L. amazonensis, with total lysis of parasites at 0.29 mM. On the other hand, isoguattouregidine, argentinine (an aminoethylphenanthrene) (45), and 3-hydroxynornuciferine (46), evaluated at around 0.8 mM, presented significant trypanocidal activity against the bloodstream form (trypomastigote) of T. cruzi with partial lysis of 92, 81, and 68%, respectively (108). Aporphine alkaloids were also obtained in previous studies of G. amplifolia Triana & Planch. and G. dumetorum R.E.Fr. (109,110), whose extracts showed activity against Leishmania sp. and chloroquine-sensitive Alkaloids with Antiprotozoal Activity 127 and -resistant strains of P. falciparum (111). In the search for compounds to treat leishmaniasis, the alkaloids xylopine (47) and nornuciferine (48) from G. amplifolia and cryptodorine (49) and nornantenine (50) from G. dumetorum, demonstrated significant activity against L. mexicana and L. panamensis. Xylopine (47) and cryptodorine (49) were among the most active compounds (IC50 of 3 mM), and showed a 37- and 21-fold, respectively, higher toxicity towards L. mexicana than macrophages, the regular host cells of Leishmania sp. (110). R3 R3 R2 R2 N R1 NH R1 H O liriodenine (40) R1+R2=OCH2O, R3=H methylmoschatoline (42) R1=R2=R3=OMe R4 anonaine (41) R1+R2=OCH2O, R3=R4=H xylopine (47) R1+R2=OCH2O, R3=H, R4=OMe Me N Me OMe HO HO MeO NH H HO N Me MeO MeO OH O crotsparine 43 OH isoguattouregidine 44 argentinine 45 The aporphine derivatives that occur in members of the Annonaceae are also found in members of the Euphorbiaceae, Hernandiaceae, Lauraceae, Menispermaceae, and Papaveraceae, among others. Bioactivity-guided fractionation of Stephania dinklagei Diels. (Menispermaceae) yielded liriodenine (40) and the zwitterionic oxoaporphine alkaloid N-methylliriodendronine (51). In agreement with previously reported results, N-methylliriodendronine was the most active against L. donovani amastigotes (IC50 ¼ 36.1 mM), while liriodenine showed the highest activity against L. donovani promastigotes and P. falciparum, with IC50 values of 15.0 and 26.2 mM, respectively (105,112). Roemrefidine (52), an aporphine alkaloid isolated from the Bolivian vine Sparattanthelium amazonum Mart. (Hernandiaceae), as well as several members of the Papaveraceae, was also found to be active against both chloroquine-resistant and chloroquine-sensitive P. falciparum strains with IC50 values of 0.58 and 0.71 mM, respectively (113). It has also been shown that roemrefidine acts on the parasite maturation, but has no 128 Osorio et al. effect on the erythrocytic reinvasion and no cumulative influence on the metabolic pathways of the parasite. In addition, the in vitro effect of a crude alkaloid extract of Cassytha filiformis L. (Lauraceae), a sprawling parasitic herb traditionally used to treat African trypanosomiasis, on bloodstream forms of T. b. brucei led to the evaluation of its three major aporphine alkaloids. Actinodaphnine (53), cassythine (54), and dicentrine (55) showed IC50 values of 3–15 mM against T. b. brucei (114). Potent antimalarial activity was also observed for stephanine (56) and two 6a,7-dehydroaporphine alkaloids, dehydrocrebanine (57) and dehydrostephanine (58), isolated from Stephania venosa Spreng. (Menispermaceae) (115). The IC50 values were 0.38, 0.22, and 0.12 mM, respectively, against the T9/94 strain of P. falciparum, thus the 6a,7-dehydro derivatives were about 2–3 times more potent than their parent compounds. R3 R2 NH H R1 R6 R5 R4 3-hydroxynornuciferine (46) R1=R2=OMe, R3=OH, R4=R5=R6=H nornuciferine (48) R1=R2=OMe, R3=R4=R5=R6=H cryptodorine (49) R1+R2=R4+R5=OCH2O, R3=R6=H nornantenine (50) R1=R2=OMe, R3=R6=H, R4+R5=OCH2O actinodaphnine (53) R1+R2=OCH2O, R3=R6=H, R4=OH, R5=OMe cassythine (54) R1+R2=OCH2O, R3=R5=OMe, R4=OH, R6=H norcorydine (62) R1=OH, R2=R5=R6=OMe, R3=R4=H HO O O N Me O Me N Me H O N-methylliriodendronine 51 roemrefidine 52 Among the isoquinoline alkaloids with trypanocidal effect are the aporphine alkaloids predicentrine (59), glaucine (60), and boldine (61), which showed inhibition of the in vitro growth of T. cruzi epimastigotes with IC50 values of 0.08, 0.09, and 0.11 mM, respectively (116). In an attempt to discover new alkaloids with antiplasmodial properties, a number of monomeric isoquinoline alkaloids have also been evaluated. In the aporphine group, norcorydine (62) possessed the highest antiplasmodial activity (IC50 ¼ 3.08 mM), while corydine, its N-methyl derivative, was seven-fold less active. The authors suggest that a secondary Alkaloids with Antiprotozoal Activity 129 amino group and a phenolic substituent enhance the in vitro antiplasmodial activity of aporphines (117). Norcorydine was found to be non-toxic to KB cells (IC50 ¼ 733 mM), clearly showing selective toxicity against the K1 strain of P. falciparum. Antiplasmodial activity was also shown by ushinsunine (63) (IC50 ¼ 5.99 mM) and dehydroocoteine (64) (IC50 ¼ 5.78 mM); the latter alkaloid was six-fold more active than its C6a–C7 saturated derivative ocoteine (117). R2 O NMe H R1 N Me H R1 O R2 R4 R3 dicentrine (55) R1+R2=OCH2O, R3=R4=OMe predicentrine (59) R1=R3=R4=OMe, R2=OH glaucine (60) R1=R2=R3=R4=OMe boldine (61) R1=R4=OMe, R2=R3=OH stephanine (56) R1=H, R2=OMe ushinsunine (63) R1=OH, R2=H R1 OMe MeO O N O Me N Me HO Me R4 R2 R3 dehydrocrebanine (57) R1=R4=H, R2=R3=OMe dehydrostephanine (58) R1=R3=R4=H, R2=OMe dehydroocoteine (64) R1=R3=R4=OMe, R2=H OH melosmine 65 The mechanism of action of aporphine alkaloids seems to be related, at least in part, to the inhibition of topoisomerase II by DNA intercalation or minor groove binding (114,118). The identification of liriodenine (40) as a strong, topoisomerase II catalytic inhibitor and a topoisomerase II poison (119) has led to the search for other topoisomerase II inhibitors among the aporphine alkaloids. Liriodenine, like many strong, topoisomerase II inhibitors, is a very planar molecule, and thus a likely DNA intercalator. However, some derived aporphines lack the structural characteristics normally associated with conventional DNA intercalators, such as the presence of two or three fused aromatic rings. Instead, they only have two aromatic rings separated by saturated rings, making them non-planar molecules. In addition, these two aromatic rings are substituted with methylenedioxy and/or methoxy groups, which may hinder access of the molecule to the intercalation sites (114). On the basis of molecular modeling studies, it has been proposed that such non-planar molecules can be ‘‘adaptative 130 Osorio et al. intercalators,’’ which undergo a conformational change upon binding to DNA to adopt a strained planar conformation (118). It has also been suggested that aporphine alkaloids with phenolic groups could participate as antioxidants and in this way inhibit cellular respiration in Trypanosoma (120). It is conceivable that the sterically hindered phenolic groups of the most active alkaloids may be acting as free radical chain breaking antioxidants and that this property may be related in some way to their antitrypanosomal effects (5). Our results would agree with this statement. As part of our study aimed at the isolation of antiprotozoal compounds from some medicinal plants of Colombia, we directed our attention toward the components of Rollinia pittieri Saff. (Annonaceae), obtaining a series of aporphine alkaloids with potent antiprotozoal activity (data not published). The most active alkaloid, melosmine (65), presented the greatest free radical scavenging activity (121). 3. Benzylisoquinoline, Protoberberine, and Related Alkaloids Benzylisoquinoline, protoberberine, protopine, benzo[c]phenanthridine, and many other alkaloids all contain the basic building block of the isoquinoline skeleton (122). The benzylisoquinolinic alkaloids are widely distributed in Nature and they have been isolated from different plants commonly used in traditional medicine for the treatment of parasitic diseases (6). Among the active constituents are the phenolic benzylisoquinolines coclaurine (66) and norarmepavine (67), which have been shown to inhibit the growth of T. cruzi epimastigotes in vitro with an IC50 of around 0.30 mM (116). The protoberberines are distributed in plant families such as Papaveraceae, Berberidaceae, Fumariaceae, Menispermaceae, Ranunculaceae, Rutaceae, and Annonaceae, with a few also found in the Magnoliaceae and Convolvulaceae (123–129). Most protoberberine alkaloids exist in plants either as tetrahydroprotoberberines or as quaternary protoberberine salts, although a few examples of dihydroprotoberberines have also been described. The quaternary protoberberine alkaloids (QPA) represent approximately 25% of all the currently known alkaloids with a protoberberine skeleton isolated from natural sources (122). Berberine (68), a QPA initially obtained by Buchner and Herberger in 1830 as a yellow extract from Berberis vulgaris L. (Berberidaceae), is probably the most widely distributed alkaloid of all (122), and is well known for its antiparasitic activity (130). This metabolite is the main constituent in various folk remedies used to treat cutaneous leishmaniasis, malaria, and amebiasis (131). Berberine (68) has been used clinically for the treatment of leishmaniasis for over 50 years, and has been shown to possess significant activity both in vitro and in vivo against several species of Leishmania. At a concentration of 29.7 mM, berberine effectively eliminates L. major parasites in mouse peritoneal macrophages, but shows minimum activity when applied topically on mouse cutaneous lesions caused by L. major. Vennerstrom et al. also tested berberine and several of its derivatives for antileishmanial activity against L. donovani and L. panamensis in vivo in golden hamsters (130). Even though berberine is effective against cutaneous ulcers caused by the New World pathogen L. panamensis in rats, it has been observed that in these cases viable amastigotes persist on the skin, resulting in the Alkaloids with Antiprotozoal Activity 131 reappearance of the lesion (132,133). Similarly, although berberine has an ethnomedicinal history in India for cutaneous leishmaniasis, topical application was ineffective in in vivo tests (134). Canadine (69), a tetrahydroberberine, is controversially less toxic and more potent than berberine against L. donovani, but not as potent as meglumine antimonate (Glucantimes), a therapy standard (10). Pessoine (70) and spinosine (71), members of the small group of catecholic isoquinoline alkaloids with a protoberberine skeleton, were isolated from the trunk bark of Annona spinescens (Annonaceae). Pessoine (70) was evaluated for its trypanocidal activity in vitro and induced a partial lysis (55%) in the trypomastigote forms of T. cruzi at 0.79 mM (103). On the other hand, burasaine (72), an alkaloid isolated from the roots of several species of the Burasaia genus, has shown in vitro antiplasmodial activities against the FcM29 strain of P. falciparum, being two times less potent than quinine (135). In an attempt to discover further alkaloids with antiplasmodial properties, a number of isoquinoline alkaloids were evaluated against P. falciparum (strain K1). The protoberberine group included the alkaloids with the highest antiplasmodial activities. Dehydrodiscretine (73) and berberine were the most active with IC50 values of less than 1 mM (0.64 and 0.97 mM, respectively), while three other QPA, columbamine (74), jatrorrhizine (75), and thalifendine (76) and the protopinetype alkaloid, allocryptopine (77), had values between 1 and 10 mM (117). The low antiplasmodial activity of the non-quaternary alkaloid canadine (69) found in this study, in contrast with the high activity of berberine, may indicate that a quaternary nitrogen is required for antiplasmodial activity in this series of alkaloids. Studies carried out to explain the relationship between structure and antimalarial activity in protoberberine alkaloids (136–138) have pointed out certain features. It appears that a quaternary nitrogen atom, especially in an isoquinolinium, rather than a dihydroisoquinolinium ion, contributes to increased antimalarial activity. This activity is also increased by the aromatization of ring C as a consequence of the quaternization of the ring-B nitrogen, and by the type of oxygen substituents on rings A, C, and D, and the position of the oxygen functions on ring D (136–138). Quaternary benzo[c]phenanthridine alkaloids (QBA) occur mainly in plants of the Papaveraceae and Fumariaceae families, and can also be found in the Caprifoliaceae, Meliaceae, and Rutaceae (139). These elicitor-inducible secondary metabolites are called phytoallexins because of their antimicrobial and antifungal activities (140,141). Their main representatives are the 2,3,7,8-tetrasubstituted alkaloids chelerythrine (78) and sanguinarine (79). This latter alkaloid and berberine (68) have been reported as trypanocidal agents against T. b. brucei with IC50 values of 1.9 and 0.5 mM, respectively. No or little selectivity was observed for either alkaloid (selectivity index, SI ¼ 0.7 and 51.0, respectively) (142). Bioassay-guided fractionation of Toddalia asiatica Lam. (Rutaceae), a plant used by the Pokot tribe of Kenya to treat fevers, resulted in the isolation of the QBA nitidine (80), a well-known cytotoxic agent that shows good activity against some chloroquine-sensitive and chloroquine-resistant strains of P. falciparum (IC50 around 0.12–0.47 mM) (143). 132 Osorio et al. While the mechanism of action and pharmacological activities of berberine (68) have been extensively studied, very little is known about the other protoberberine alkaloids (144). It has been reported that berberine is a potent in vitro inhibitor of both nucleic acid and protein synthesis in P. falciparum (145). Despite its slightly buckled structure due to the partial saturation of the central ring, berberine has been previously characterized as a DNA intercalating agent, and as a cationic ligand, electrostatic forces playing an important role in its interaction with DNA (146–149). In a recent comprehensive study, the mode of binding of berberine to short oligonucleotide duplexes was examined by NMR and molecular modeling techniques. The authors concluded that the drug does not intercalate into DNA, but forms minor groove complexes by partial intercalation due to its buckled structure (150). Recent studies with other protoberberine alkaloids suggest that burasaine (72) forms intercalation complexes, showing a true intercalative mode for the binding with doublestranded DNA (144,151), although targets other than DNA may also be invoked (144). In relation to their mode of action, benzo[c]phenanthridine alkaloids have been shown to be topoisomerase inhibitors (152), so it is possible that their antimalarial action is mediated through the inhibition of the parasite enzyme. They also inhibit microtubule assembly and interact with DNA (153–155). MeO R2 NH H R R3 HO coclaurine (66) R=OH norarmepavine (67) R=OMe R4 R5 berberine (68) R1+R2=OCH2O, R3=R4=OMe, R5=H burasaine (72) R1=R2=R3=R4=OMe, R5=H dehydrodiscretine (73) R1=R4=R5=OMe, R2=OH, R3=H columbamine (74) R1=OH, R2=R3=R4=OMe, R5=H jatrorrhizine (75) R1=R3=R4=OMe, R2=OH, R5=H thalifendine (76) R1+R2=OCH2O, R3=OMe, R4=OH, R5=H R2 R1 N R1 O N N O R6 R3 R4 R5 canadine (69) R1+R2=OCH2O, R3=R4=OMe, R5=H, R6=H pessoine (70) R1=R4=R5=OH, R2=OMe, R3=H, R6=H spinosine (71) R1=R2=OMe, R3=H, R4=R5=OH, R6=H O Me OMe OMe allocryptopine 77 Alkaloids with Antiprotozoal Activity 133 O R3 O N Me R1 chelerythrine (78) R1=R2=OMe, R3=H sanguinarine (79) R1+R2=OCH2O, R3=H nitidine (80) R1=H, R2=R3=OMe R2 4. Bisbenzylisoquinoline Alkaloids The bisbenzylisoquinoline (BBIQ) alkaloids constitute a series of over 430 phenylalanine-derived phytometabolites widespread in nature. They are found in the following botanical families: Annonaceae, Aristolochiaceae, Berberidaceae, Hernandiaceae, Lauraceae, Menispermaceae, Monimiaceae, Nymphaeaceae, and Ranunculaceae (156–159). BBIQ alkaloids present a rich and varied chemistry and pharmacology (160), comprising two isoquinoline moieties (head portions) linked to two benzyl moieties (tail portions). They have been classified into 26 structural types (denoted by roman numerals) according to the nature, number, and attachment point of the bridges. The alkaloids within each group differ from one another by the nature of their oxygenated substituents, the degree of unsaturation of the heterocyclic rings, and the stereochemistry of their two chiral centers, C-1 and C-1u (156–159). A number of biological activities have been reported for BBIQ alkaloids including immunomodulatory effects (161,162), cardiovascular effects (163,164), antithrombosis (165), anti-HIV (166), and antiprotozoal activities, in particular against Leishmania sp. (160,167,168), T. cruzi (142,160,169–175), and Plasmodium sp. (176–192). In an evaluation of 14 isoquinoline alkaloids, especially BBIQs extracted from Annonaceae, Berberidaceae, Hernandiaceae, and Menispermaceae, four are of particular interest. Daphnandrine (81) and limacine (82), isolated from Albertisia papuana Becc. and Caryomene olivascens Barneby & Krukoff, respectively (Menispermaceae), obaberine (83) obtained from Pseudoxandra sclerocarpa Maas (Annonaceae) and gyrocarpine (84), produced by Gyrocarpus americanus Jacq. (Hernandiaceae) showed strong activity against promastigotes of L. braziliensis, L. amazonensis, and L. donovani. Leishmanicidal activity of the first three alkaloids was at an IC100 of nearly 84 mM, and gyrocarpine (84) was active in vitro at 16 mM (167). However, in an in vivo test against L. amazonensis (168), gyrocarpine (84) was less potent than Glucantimes. In the same study, isotetrandrine (85), a metabolite isolated from Limaciopsis loangensis Engl. (Menispermaceae), showed leishmanicidal activity at 16 mM against the promastigote forms of L. braziliensis, L. amazonensis, and L. donovani, and exhibited activity approximately equal to or greater than Glucantimes in BALB/c mice infected with L. amazonensis (168). The structural requirements for the antiprotozoal effects of the BBIQ alkaloids were explored by determining the activity of several BBIQ alkaloids against L. donovani and T. b. brucei (160). The results revealed that 13 BBIQ alkaloids: isotetrandrine (85), fangchinoline (86), funiferine (87), tiliageine (88), oxyacanthine (89), aromoline (90), thalisopidine (91), obamegine (92), dinklacorine (93), 134 Osorio et al. isotrilobine (94), trilobine (95), gilletine (96), and insularine (97), had IC50 values of less than 5 mM against L. donovani promastigotes, with fangchinoline (IC50 ¼ 0.39 mM) being as potent as the standard drug pentamidine (IC50 ¼ 0.4 mM). Of the 15 BBIQ alkaloids tested against L. donovani amastigote forms, phaeanthine (98) showed strong activity (IC50 ¼ 2.41 mM), but at this concentration was toxic to the infected macrophages. Cocsoline (99) was the only alkaloid that showed selective toxicity (IC50 ¼ 12.3 mM) towards L. donovani amastigotes in macrophages, being slightly less potent than the standard drug pentostam (IC50 ¼ 9.75 mg Sb(V)/ mL). Of the 12 BBIQ alkaloids tested for antitrypanosomal activity, eight: isotetrandrine (86), aromoline (90), thalisopidine (91), isotrilobine (94), trilobine (95), phaeanthine (98), daphnoline (100), and berbamine (101), had IC50 values of 1–2 mM against T. b. brucei bloodstream trypomastigote forms. Thalisopidine (91) displayed the strongest trypanocidal activity (IC50 ¼ 1.14 mM), but none were as active as pentamidine (IC50 ¼ 0.3 nM) (160). Similarly, berbamine was active on T. b. brucei in the same concentration range as reported in the previous study, with an IC50 value of 2.6 mM (SI ¼ 7.0) (142). On the other hand, phaeanthine was shown to be inactive in mice infected with the same parasite (169). However, Camacho et al. suggest that the optimal structure for antileishmania and antitrypanosomal activity of the BBIQ alkaloids is not fully understood, and that further examination is necessary in order to define these relationships (160). OMe R1 R4 N N R3 Me O O R2 daphnandrine (81) R1=H, R2=R4=OMe, R3=OH (R,S) obaberine (83) R1=Me, R2=R3=R4=OMe (R,S) gyrocarpine (84) R1=Me, R2=R3=OMe, R4=OH (S,R) oxyacanthine (89) R1=Me, R2=OH, R3=R4=OMe (R,S) aromoline (90) R1=Me, R2=R3=OH, R4=OMe (R,S) daphnoline (100) R1=H, R2=R3=OH, R4=OMe (R,S) cepharanthine (107) R1=Me, R2=OMe, R3+R4=OCH2O (R,S) candicusine (108) R1=Me, R2=R3=OH, R4=OMe (R,R) cycleapeltine (110) R1=Me, R2=R4=OMe, R3=OH (S,S) OMe R1 N R4 N R2 Me O R3 O limacine (82) R1=Me, R2=OH, R3=R4=OMe (R,R) isotetrandrine (85) R1=Me, R2=R3=R4=OMe (R,S) fangchinoline (86) R1=Me, R2=OH, R3=R4=OMe (S,S) obamegine (92) R1=Me, R2=R3=OH, R4=OMe (R,S) phaeanthine (98) R1=Me, R2=R3=R4=OMe (R,R) berbamine (101) R1=Me, R2=R4=OMe, R3=OH (R,S) thalrugosine (113) R1=Me, R2=OH, R3=R4=OMe (R,S) 2-norisotetrandine (114) R1=H, R2=R3=R4=OMe (R,S) penduline (118) R1=Me, R2=R4=OMe, R3=OH (S,S) Alkaloids with Antiprotozoal Activity 135 The chemical and biological investigation of the stem bark alkaloidal extract of Guatteria boliviana H.Winkler (Annonaceae) led to the isolation of five new and four known BBIQ derivatives. The alkaloids were tested on trypomastigote forms of T. cruzi and all were active at 0.40 mM. Three of them, funiferine (87), antioquine (102), and guatteboline (103), with an imine function in the molecule, exhibited an IC90 lower than 0.16 mM (170). The substantial differences among the structures did not allow any structure–activity relationship to be elaborated. OMe Me N H MeO N R1 H O R2 Me R3 funiferine (87) R1=R3=OMe, R2=OH (S,R) tiliageine (88) R1=R2=OH, R3=OMe (S,R) antioquine (102) R1=R3=OH, R2=OMe (S,R) OH OMe Me N H MeO N OMe H Me O O HO thalisopidine (91) (S,S) OMe Me N N O H H O OH Me MeO dinklacorine (93) (R,S) O R1 N H R4 N O H R2 R3 O isotrilobine (94) R1=R3=Me, R2=R4=OMe (S,S) trilobine (95) R1=Me, R2=R4=OMe, R3=H (S,S) cocsoline (99) R1=H, R2=OH, R3=Me, R4=OMe (S,S) 12-O-methyltricordatine (109) R1=R3=Me, R2=OMe, R4=OH (S,S) 136 Osorio et al. OMe Me OH O N Me H O O OMe MeO HN H N H O O H N Me O MeO OMe insularine (97) (R,R) gilletine (96) (S,S) Fournet et al. have described the in vitro trypanocidal activity of some BBIQ alkaloids against the epimastigote forms (171) and bloodstream forms of T. cruzi (172). The alkaloids daphnandrine (81), gyrocarpine (84), phaeanthine (98), cocsoline (99), daphnoline (100), and isochondodendrine (104) completely lysed the trypomastigote forms of T. cruzi at a concentration of ca. 250 mg/mL (0.40–0.45 mM) (172). These authors also reported in vivo studies of some active BBIQ alkaloids (173,174). Five alkaloids, limacine (82), isotetrandrine (85), phaeanthine (98), curine (105), and cycleanine (106), were tested orally for their trypanocidal activity in T. cruzi-infected BALB/c mice in the acute phase; curine and cycleanine showed high efficacy and negative parasitemias 5–7 weeks after inoculation at 10 mg/kg (0.002 mM/kg) daily for 10 days of treatment. The other BBIQ alkaloids showed a relative efficacy against both strains (173). Interestingly, curine and cycleanine have two diphenylether linkages head to tail, while limacine, isotetrandrine and its isomer phaeanthine, which have two diphenylether linkages head to head and tail to tail, did not show the same efficacy. In addition, the activity of daphnoline (100) and cepharanthine (107), as well as benznidazole, was determined in acute and chronic T. cruzi-infected mice (174). Compared to benznidazole, oral treatment with daphnoline was more parasitologically and serologically effective in the infected mice. In the case of chronic infection, the serological cure rate was similar to that of the standard drug. These results were not seen with cepharanthine, an excellent inhibitor of trypanothione reductase, a target for specific chemotherapy against Chagas’ disease (175). OMe MeO HN H MeO N O OH O guatteboline (103) (R) The antimalarial activity reported for a significant number of plants, mainly from the Annonaceae, Lauraceae, Menispermaceae, and Ranunculaceae families, has been attributed to the presence of a variety of BBIQ alkaloids (9). With a few Alkaloids with Antiprotozoal Activity 137 exceptions, these compounds show reasonable antimalarial activity, but this is often coupled with cytotoxicity. The large number of BBIQ derivatives that occur in Nature prompted Angerhofer et al. (176) and Marshall et al. (177) to compare the antiplasmodial and cytotoxic activity of 53 and 24 alkaloids, respectively, isolated from different families. In the first study, the most active alkaloids against the chloroquine-sensitive clone D-6 of P. falciparum were candicusine (108) and 12-O-methyltricordatine (109), with IC50 values of 29 and 30 nM, respectively, whereas cepharanthine (107) and cycleapeltine (110), both with an IC50 of around 67 nM, were the most active against the chloroquine-resistant clone W-2. However, when the SI is considered, the most selective alkaloids were cycleanine (106) and malekulatine (111) (e.g., SI=W460 for cycleanine, as compared with W490 for malekulatine in clone D-6 of P. falciparum) (9,176). In the latter study, eight of the BBIQ alkaloids tested had IC50 values of less than 1 mM against the multidrug-resistant K1 strain of P. falciparum in vitro, thalisopidine (91) being the most potent antiplasmodial compound with an IC50 value of 90 nM. Under the same test conditions, the IC50 of chloroquine against the same multidrug-resistant strain of P. falciparum was 200 nM (177). OMe Me N H R1 O O R2 H R3 N MeO isochondodendrine (104) R1=R2=OH, R3=Me (R,R) cycleanine (106) R1=R2=OMe, R3=Me (R,R) N'-demethylcycleanine (112) R1=R2=OMe, R3=H, (R,R) A decoction of the root bark of the shrub Albertisia villosa (Exell) Forman of the Menispermaceae family is used in traditional medicine against malaria and many infectious diseases. In an attempt to explain this use, a root bark alkaloidal extract was analyzed and found to contain three BBIQ alkaloids, cocsoline (99), N-demethylcycleanine (112), and most abundantly, cycleanine (106), which has potent antiplasmodial activity. Two Menispermaceae plants, Cyclea barbata Miers and Stephania rotunda Lour., are used in traditional medicine to treat the fever associated with malaria (179,180). Through bioactivity-guided fractionation of C. barbata, five alkaloids were found to be responsible for the antimalarial activity. The most active compounds, cycleapeltine (110) and thalrugosine (113) were capable of inhibiting the growth of cultured P. falciparum strains D-6 and W-2 with IC50 values of 47.6–66.6 and 106.9–128.1 nM, respectively (179). Similarly, phytochemical studies of S. rotunda have demonstrated the presence of several alkaloids (180–183), cepharanthine (107) being the most active against the W-2 138 Osorio et al. strain with an IC50 of 0.61 mM (180,184). Several BBIQ alkaloids have been isolated from Stephania erecta Craib (185). The antimalarial potential of each of these alkaloids was investigated with cultured P. falciparum strains D-6 and W-2, the most active alkaloid being 2-norisotetrandrine (114) (IC50 ¼ 108.6 and 74.4 nM against D-6 and W-2, respectively) (185). Likewise, investigation of the active methanol fraction of the root of Epinetrum villosum (Exell) Troupin (Menispermaceae), a plant which is taken orally for the treatment of fever and malaria (186), led to the isolation of four BBIQ alkaloids (187). Among them, isochondodendrine (104) was found to have the most potent antiplasmodial activity (IC50 ¼ 0.20 mM with SI 175), while cycleanine and cocsoline also acted against P. falciparum (IC50 ¼ 4.50 and 0.54 mM, respectively). Qualitatively, these results for isochondodendrine correspond with those reported by Mambu et al. (188), who isolated and identified three BBIQ alkaloids from the stem bark of Isolona ghesquierei Cavaco & Keraudr. (Annonaceae). They found that curine (105) and isochondodendrine have strong in vitro antiplasmodial activity against the chloroquine-resistant strain FcM29 of P. falciparum with IC50 values of 0.35 and 0.89 mM, respectively. OMe Me N H OH O OH H O N Me MeO curine (105) (R,R) OMe Me N OH H OMe MeO O HO N Me H MeO OMe malekulatine (111) (S,S) Two species of the Lauraceae, Dehaasia incrassata (Jack) Kosterm. and Nectandra salicifolia Nees, have been investigated for their antimalarial activity (9,189,190). The three alkaloids isolated from the leaves and bark of D. incrassata were tested against the K1 strain of P. falciparum, and one of them, oxyacanthine (89), was active with an IC50 of 0.50 mM (189). On the other hand, 16 alkaloids were isolated from N. salicifolia, but only costaricine (115) showed appreciable Alkaloids with Antiprotozoal Activity 139 activity, with IC50 values of 85.8 nM against the chloroquine-sensitive D-6 clone and 0.50 mM against the chloroquine-resistant W-2 clone of P. falciparum (190). Similarly, two species from Ranunculaceae have been investigated for their antiplasmodial activity. Studies on Thalictrum faberi Ulbr. yielded several aporphine-benzylisoquinoline alkaloids and, among those thalifasine (116) and thalifaberidine (117) showed the strongest antimalarial activity with IC50 values of less than 1 mM against the D-6 and W-2 clones of P. falciparum (191). Likewise, the roots of Isopyrum thalictroides L. (Ranunculaceae) yielded the BBIQ alkaloid penduline (118), which is active against chloroquine-resistant strains of P. falciparum. However, it is also more toxic than chloroquine (192). OMe MeO HN H OH NH H HO OMe O costaricine (115) (R,R) R4 R1 R2 MeO N MeO H Me MeO N H Me O MeO R3 thalifasine (116) R1=R4=OH, R2=R3=OMe (S) thalifaberidine (117) R1=H, R2=R3=OH, R4=OMe (S) Optimal structure–antiplasmodial activity requirements of the BBIQ alkaloids are not fully understood and current results do not reveal any clear structure– activity relationships between alkaloid subgroups. Nevertheless, some features have been pointed out. In general, single-bridged BBIQ alkaloids are less active. Likewise, the status of the nitrogen atoms is fundamental to antiplasmodial activity, as evidenced by the quaternization of one or two nitrogen atoms, which leads to a loss of both toxicity and antimalarial activity. The decrease in lipophilicity of BBIQ alkaloids probably contributes to the lower toxicity (176,177). A study of the conformations assumed by compounds of the same subgroup (i.e., modification of conformation with the change of configuration at C-1 and C-1u) should give more information about the structure–activity relationship (176). Although the mechanism of action of BBIQ alkaloids is not well known, recent studies show the capacity of these compounds to block Ca2+ uptake through the 2+ L-type Ca channels (193–195), an important mechanism for the penetration of the 140 Osorio et al. trypomastigote forms of T. cruzi into the host cell (196,197). Another hypothesis describes BBIQ alkaloids as possible trypanothione reductase inhibitors. Results obtained with daphnoline (100) against this target partially confirm its inhibitor activity against T. cruzi in the bloodstream (172) and in acute or chronically T. cruziinfected mice (198). Another possible explanation of the antiprotozoal properties of BBIQ alkaloids could be the DNA intercalation in combination with the inhibition of protein synthesis (142). Nevertheless, with the exception of the single-bridged BBIQ alkaloids, they all possess a large heterocycle of 18 to 20 atoms that is not totally rigid. They could probably act as adaptive intercalators, adopting a strained planar conformation after binding to the DNA. 5. Naphthylisoquinoline Alkaloids Naphthylisoquinoline (NIQ) alkaloids are axially chiral, natural biaryls isolated from African plants belonging to the Ancistrocladaceae and Dioncophyllaceae families. The Dioncophyllaceae constitute a very small family, with only three species: Triphyophyllum peltatum (Hutch & Dalziel.) Airy Shaw (a ‘‘part-time’’ carnivorous liana), Habropetalum dawei (Hutch & Dalziel.) Airy Shaw, and Dioncophyllum thollonii Baill. Closely related, but not carnivorous, are the Ancistrocladaceae, an even smaller family whose only genus, Ancistrocladus, consists of ca. 25 species found in the palaeotropic rain forests of Africa and Asia (199). These natural biaryl alkaloids have been characterized chemically (unique structural framework, axial chirality, unprecedented origin of isoquinoline alkaloids from acetate), and also pharmacologically due to their wide variety of activities related to, e.g., bilharzia, Chagas’ disease, sleeping sickness, leishmaniasis, river blindness, elephantiasis and, in particular, malaria (11,199,200). Some of the alkaloids are also active against larvae of the vectors Anopheles stephensi and Aedes aegypti (199,201). Among the monomeric NIQ alkaloids tested up to now, dioncophyllines A (119), B (120), and E (121), isolated from the rare West African liana D. thollonii, are the most active, with IC50 values of 2–3 mM against T. b. brucei or T. b. rhodesiense bloodstream forms (202,203). Dioncophyllines A and B were previously known from the related plant species T. peltatum, which is the most phytochemically investigated species in the Dioncophyllaceae family (204–206). Dioncophylline A is also a strong agent against T. cruzi (IC50 ¼ 1.85 mM), T. b. rhodesiense (IC50o0.50 mM), and L. donovani (IC50o32.05 mM) (199). Ancistroealaines A (122) and B (123), two 5,8u-coupled NIQ alkaloids isolated from the stem bark of Ancistrocladus ealaensis (207), were tested in vitro against L. donovani, T. cruzi, and T. b. rhodesiense. Ancistroealaine A displayed good activity against T. cruzi (IC50 ¼ 5.60 mM) and T. b. rhodesiense (IC50 ¼ 8.25 mM). Ancistroealaine B, on the other hand, showed significant trypanocidal activity, with an IC50 of 4.98 mM against T. b. rhodesiense. Both alkaloids exhibited cytotoxic effects in mammalian L6 cells (rat skeletal myoblasts) at much higher concentrations (IC50W215 mM for ancistroealaine A and 220 mM for ancistroealaine B). The first phytochemical investigation of the East African liana A. tanzaniensis Cheek & Frim.-Møll., reported the isolation of NIQ alkaloid derivatives with antiprotozoal activity, including two new alkaloids, ancistrotanzanines A (124) 141 Alkaloids with Antiprotozoal Activity and B (125, atropodiastereomer of ancistroealaine A), and the known alkaloid ancistrotectoriline A (126) (208). The most significant activity was found for ancistrotanzanine A and ancistrotanzanine B against L. donovani, T. cruzi, and T. b. rhodesiense, with IC50 values ranging from 1.73 to 4.44 mM and 1.67 to 3.81 mM, respectively. Ancistrotectoriline A, also obtained from the Southeast Asian species A. tectorius (209), showed weak antitrypanosomal activity against T. b. rhodeniense (IC50 ¼ 4.98 mM). Other NIQs with similar antiprotozoal activity have since been reported from the same species (210). Ancistrocladidine (127) was active against L. donovani (IC50 ¼ 7.15 mM), weaker than the active ancistrotanzanine B (125) by a factor of only 2. Likewise, good antitrypanosomal activity was exhibited by ancistrocladidine and ancistrotanzanine C (128) against the pathogen T. b. rhodesiense (IC50 ¼ 4.93 and 3.19 mM) (210). Unfortunately, all of the natural alkaloids were less active than the standard drugs. Me R1 1′ Me NH 7 OH MeO Me NH 7 6′ OH OH Me R2 Me OMe dioncophylline A (119) R1=Me, R2=OMe (R,R) (P) dioncopeltine A (138) R1=CH2OH, R2=OH (R,R) dioncophylline B (120) (R,R) habropetaline A (139) R1=CH2OH, R2=OMe (R,R) 7-epidioncophylline A (144) R1=Me, R2=OMe (R,R) (M ) 5′-O-demethyldioncophylline A (145): R1=Me, R2=OH (R,R) Me Me P 3′ M NH 7 Me OMe OH OH OH Me NH 7 3′ OH Me Me OMe dioncophylline E (121) (R,R) (P and M rotational isomers) OMe OMe MeO P OH Me 8′ 5 Me N OMe Me ancistroealaine A (122) (S) MeO P OMe Me 8′ 5 Me NH OMe Me ancistroealaine B (123) (S,S) NIQ alkaloids with similar antiprotozoal activity have been isolated from A. congolensis J.Leónard (211), A. heyneanus Wall. (212), A. likoko J.Leónard (213), and 142 Osorio et al. A. griffithii Planch. (214). The three alkaloids obtained from the first species, named as ancistrocongoline A (129), ancistrocongoline B (130), and korupensamine A (131), were tested for their in vitro activity against T. b. rhodesiense (IC50 ¼ 7.55, 6.02, and 4.93 mM, respectively) (211). Three secondary metabolites belonging to the rare 7,3u-coupling type: ancistrocladidine (127), ancistroheynine B (132), and ancistrotanzanine C (128), which has already been previously described from the East African species A. tanzaniensis (210), were isolated from the leaves of the Indian liana A. heyneanus (212). Antitrypanosomal and antileishmanial activities were exhibited by ancistrocladidine against T. b. rhodesiense and L. donovani (IC50 ¼ 4.93 and 7.15 mM). Ancistroheynine B and ancistrotanzanine C, as well as ancistrolikokine D (133), which was isolated from A. likoko, presented relatively high activity against T. b. rhodesiense with IC50 values of 7.41, 3.19, and 6.90 mM, respectively (212,213). Likewise, two monomeric NIQ alkaloids, ancistrogriffine A (134) and ancistrogriffine C (135), and the first dimer of a 7,8u-coupled NIQ, ancistrogriffithine A (136), were isolated from the twigs of A. griffithii and analyzed for their antiprotozoal activity (214). Ancistrogriffine A also showed good activity against L. donovani (IC50 ¼ 7.61 mM), while all three alkaloids were active against T. b. rhodesiense (IC50 ¼ 5.32, 7.55, and 1.15 mM, respectively). On the contrary, other dimers, including the anti-HIV active michellamines, e.g., michellamine B (137), were devoid of any antitrypanosomal activity (11,202). The monomeric dioncopeltine A (138), with its additional alcohol function on the methyl group of the naphthalene ring, has a distinctly lower activity against T. b. brucei bloodstream trypomastigotes (IC50 ¼ 22 mM) (11). Habropetaline A (139), isolated from the roots of T. peltatum and identified previously in the crude extract of the rare and difficult-to-provide, related plant species H. dawei (215), was inactive against T. b. rhodesiense, T. cruzi, and L. donovani (216). These findings, although not as good as the results for other NIQs, are an important contribution to the structure–activity relationship investigations. OMe OMe MeO HO MeO 3′ 5 Me Me Me MeO N 8′ 5 Me Me N OMe Me OMe Me ancistrotanzanine A (124) (S) ancistrotanzanine B (125) (S) OMe OMe Me MeO 8′ 5 Me Me NH OMe Me ancistrotectoriline A (126) (S,S) 143 Alkaloids with Antiprotozoal Activity HO MeO R Me 3′ MeO HO R Me N 7 N 7 3′ ancistrocladidine (127) R=OMe (S) ancistroheynine B (132) R=OH (S) ancistrotanzanine C (128) R=OH (R,S) ancistrotectorine (146) R=OMe (R,S) OMe R3 Me OH Me N OH N OMe Me 7 HO Me MeO Me Me OMe Me MeO ancistrolikokine D (133) (S) Me N Me R1 Me HO 8′ 5 HO ancistrocongoline A (129) R1=Me, R2=R3=OH (R,R) ancistrocongoline B (130) R1=Me, R2=R3=OMe (R,R) korupensamine A (131) R1=H, R2=R3=OH (R,R) 8′ OMe Me 8′ 5 R2 Me OMe Me Me OMe Me Me Me ancistrogriffine A (134) (S,S) MeO 8′ HO NH 7 OH Me ancistrogriffine C (135) (S,S) Very recently, the NIQ alkaloids ancistrocladinium A (140) and ancistrocladinium B (141) and a synthetically prepared isoquinolinium salt 142 were found to present potent activity against intracellular amastigotes of L. major at concentrations in the low submicromolar range (IC50 ¼ 4.90, 1.24, and 2.91 mM, respectively). They are more toxic against J774.1 macrophages and peritoneal macrophages than the reference drug amphotericin B (217). In general, the NIQ alkaloids, with N,C-coupled rings and a hetero-biaryl part, exhibit significantly higher leishmanicidal activities. With regard to structural features, these results demonstrate the importance of the quaternary nitrogen atom, which clearly increases antileishmanial activity (217). Although the results concerning antileishmanial and antitrypanosomal activities of NIQ alkaloids are particularly recent, their antimalarial activities were discovered a few years earlier due to the traditional use of the plants in treating malaria (199). One species of the Dioncophyllaceae, T. peltatum (218–220), and several species of Ancistrocladaceae, viz., A. abbreviatus Airy Shaw (218,219), A. barteri Scott-Elliot (218,219), A. heyneanus (221,222), A. korupensis D.W.Thomas & Gereau (223–225), A. likoko (226), and A. robertsoniorum J.Leónard (227) have been investigated for their antiplasmodial activity. In agreement with the results 144 Osorio et al. obtained from crude extracts, several NIQ alkaloids are known to be highly active against P. falciparum. They were initially described as exhibiting strong growth-inhibiting activities in vitro against P. falciparum and P. berghei (228,229), and in vivo against P. berghei (230). Another remarkable fact is that NIQ alkaloids act against the blood forms of Plasmodium spp., and against the exoerythrocytic forms (199,229,231), a most promising additional perspective for these novel antimalarial agents. For synchronized forms of P. chabaudi chabaudi, stage-specific activities have been found (232). Me Me NH HN Me HO 7 8′ Me Me Me OMe OH OH OH 5 8′ OH OMe OMe OMe OH OMe OH 8′′ 7′′′ MeO Me HO OH Me 8′′ 5′′′ Me NH Me OH HN Me michellamine B 137 Me ancistrogriffithine A 136 MeO Me N MeO Me OH 8′ OMe Me Me OMe OMe ancistrocladinium A (140) (S) OMe N 6′ OMe Me Me ancistrocladinium B (141) (S) Among the most active alkaloids isolated from T. peltatum are dioncophylline B (120) (IC50 ¼ 0.616 mM), dioncopeltine A (138) (IC50 ¼ 0.055 mM), habropetaline A (139) (IC50 ¼ 5.8 nM), dioncophylline C (143) (IC50 ¼ 0.038 mM), 7-epidioncophylline A (144) (IC50 ¼ 0.503 mM), and 5u-O-demethyldioncophylline A (145) (IC50 ¼ 0.935 mM) with excellent antiplasmodial activities against the chloroquine-sensitive strain of P. falciparum (NF54) in vitro, and in some cases, in vivo (9,206,216,219,230,232). The IC50 values obtained for these alkaloids are much lower than those observed for most other plant-derived compounds, and compare Alkaloids with Antiprotozoal Activity 145 well with the IC50 values for antiplasmodial drugs currently in use (chloroquine). The good in vitro activity exhibited by dioncophylline B, dioncophylline C, and dioncopeltine A led to in vivo testing in mice against P. berghei or P. chabaudi chabaudi (228,230,232). A complete cure was achieved by dioncophylline C in malaria-infected mice after a 4-day oral treatment with 50 mg/kg/day, without noticeable toxic effects. Likewise, dioncophylline E (121), an alkaloid isolated from D. thollonii, exhibited good activity against P. falciparum, with nearly identical IC50 values against the chloroquine-sensitive (NF54) and chloroquine-resistant (K1) strains. With values of only 0.06 and 0.058 mM, the activity of the new alkaloid ranges within that of the most active NIQ alkaloids, and is only weaker than the standards artemisinin and chloroquine by a factor of 5–10 (203). OH MeO OMe Me N Me 1′ 5 1′ Me OMe Me NH OH isoquinolinium salt 142 OH Me OMe Me HO N Me NH OH OMe OH OH OMe OMe 8′′ Me Me 8' 5 Me dioncophylline C (143) (R,R) 7′′′ OH HO Me korupensamine B (147) (R,R) Me Me 8′ 5 NH OH Me korundamine A 148 Other NIQs like ancistroealaine B (123) (IC50 ¼ 1.28 mM), ancistrotanzanine A (124) (IC50 ¼ 0.74 mM), ancistrotanzanine B (125) (IC50 ¼ 0.72 mM), ancistrotectoriline A (126) (IC50 ¼ 1.19 mM), ancistrocladidine (127) (IC50 ¼ 0.74 mM), ancistrotanzanine C (128) (IC50 ¼ 0.24 mM), ancistrocongoline A (129) (IC50 ¼ 0.54 mM), ancistrocongoline B (130) (IC50 ¼ 0.37 mM), korupensamine A (131) (IC50 ¼ 0.43 mM), ancistroheynine B (132) (IC50 ¼ 1.27 mM), ancistrogriffine A (134) (IC50 ¼ 0.18 mM), ancistrogriffine C (135) (IC50 ¼ 1.06 mM), ancistrogriffithine A (136) (IC50 ¼ 0.04 mM), ancistrotectorine (146) (IC50 ¼ 1.66 mM), and korupensamine B (147) (IC50 ¼ 0.47 mM) showed significant antimalarial activities with IC50 values lower than 2 mM against the chloroquine- and pyrimethamineresistant K1 strain of P. falciparum (207,208,210–214,223). However, in most cases, 146 Osorio et al. the IC50 values were higher than those of the standards (artemisinin or chloroquine). Only the dimeric NIQ ancistrogriffithine A (136) was found to be more active than the standard chloroquine by a factor of two (214). This result was surprising, since dimeric NIQs rarely show considerable antiplasmodial activities (223). Another exception is korundamine A (148) (IC50 ¼ 1.43 mM), a dimeric NIQ alkaloid isolated from A. korupensis that shows both anti-HIV and antimalarial activity (225). It is noteworthy that some of the studied alkaloids exhibited cytotoxic effects on mammalian cells only at or above 30 mg/mL, which reflects the selectivity of these natural compounds for the protozoan parasite. It seems that the compounds from the Dioncophyllaceae are more active than those isolated from the Ancistrocladaceae. Structure–activity considerations indicate that possible criteria for antiplasmodial activity include an R-configuration at C-3 associated with the absence of an oxygen substituent at C-6 in the isoquinoline moiety and the absence of N-methylation (219,233). Dioncophylline E (121), dioncopeltine A (138), habropetaline A (139), and dioncophylline C (143), maybe the most active NIQs, represent pure Dioncophyllaceae-type alkaloids, all having in common the aforementioned characteristics (234). Nevertheless, not all the alkaloids of the Ancistrocladaceae are of the Ancistrocladaceae-type. In fact, all the West African Ancistrocladus spp. investigated so far contain Dioncophyllaceae-type alkaloids, as well as many hybrid-type forms (234,235). Similarly, structure–activity relationship investigations reveal that in NIQ alkaloids the presence of at least one or two free aromatic hydroxy functions is essential for high antiplasmodial activity (228), and this could partly explain the observed activity. The mechanism of action of NIQ compounds is not very well known. Franc- ois et al. (232) suggest that the antiplasmodial activity of dioncophylline B is due to its inhibition of hemozoin degradation, but this is based only on microscopic observations. Further investigation is required to study the possible mode of action of active alkaloids in the different parasites. Nevertheless, existing results show that NIQ alkaloids are structurally intriguing and pharmacologically promising natural products. 6. Amaryllidaceae Alkaloids Plants of the Amaryllidaceae family are known to produce structurally unique alkaloids, which have been isolated from plants of all of the genera of this family. The Amaryllidaceae alkaloids represent a large, and still expanding, group of isoquinoline alkaloids, the majority of which are not known to occur in any other family of plants. Since the isolation of the first alkaloid, lycorine, from Narcissus pseudonarcissus L. in 1877, substantial progress has been made in examining the Amaryllidaceae plants, although they are still a relatively untapped phytochemical source (236). At present, over 300 alkaloids have been isolated from plants of this family and, although their structures vary considerably, these alkaloids are considered to be biogenetically related (236,237). The Amaryllidaceae alkaloids present a wide range of interesting physiological effects, including antitumor, antiviral, cytotoxic, acetylcholinesterase 147 Alkaloids with Antiprotozoal Activity inhibitory, immunostimulatory, antiinflammatory, analgesic, and DNA-binding activities, and some have also been used in the treatment of Alzheimer’s disease (236). Some of these alkaloids are of particular interest because of their potential antiprotozoal activity. Thus, lycorine (149), augustine (150), and crinamine (151) were found to be the principal antimalarial constituents in Crinum amabile Donn bulbs (238). Augustine, a 5,l0b-ethano-phenanthridine, appeared to be the most active alkaloid demonstrating significant antimalarial activity in both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum (IC50 ¼ 0.46 and 0.60 mM, respectively). Similarly, the pyrrolophenanthridine-type alkaloid lycorine was found to be very active against different strains of P. falciparum (IC50 of around 1.04–2.44 mM) (238,239). However, compared with the antimalarial control compounds, its selectivity indices were low (238). In other studies of the antimalarial properties of Amaryllidaceae alkaloids (240), hemanthamine (152) and hemanthidine (153) exhibited activity against the chloroquine-sensitive strain T9/96 of P. falciparum (IC50 ¼ 2.33 and 1.10 mM, respectively). In addition to hemanthamine and hemanthidine, lycorine, 3-epihydroxybulbispermine (154), galanthine (155), and pancracine (156) were also highly active against the chloroquine-resistant strain K1 of P. falciparum, with IC50 values lower than 1 mg/mL. Some of these alkaloids were more active than the standards used (240–242). Likewise, hemanthamine and hemanthidine isolated from the bulbs of Cyrtanthus elatus (Jacq.) Traub, and pancracine isolated from N. angustifolius Curt. subspecies transcarpathicus, presented antimalarial activity against the NF54 strain of P. falciparum (IC50 ¼ 2.22, 2.20, and 2.43 mM, respectively) (242,243). R2 O R1 OMe H R4 O H R3 H N O N lycorine (149) R1=R2=OH, R3+R4=OCH2O augustine 150 galanthine (155) R1=OH, R2=R3=R4=OMe 1,2-O-diacetyllycorine (159) R1=R2=OAc, R3+R4=OCH2O pseudolycorine (161) R1=R2=R4=OH, R3=OMe R1 R2 R6 R5 H R4 R3 N OH O O OH N H pancracine 156 crinamine (151) R1=OMe, R2=R3=H, R4+R5=OCH2O, R6=OH hemanthamine (152) R1=R3=H, R2=OMe, R4+R5=OCH2O, R6=OH hemanthidine (153) R1=H, R2=OMe, R3=R6=OH, R4+R5=OCH2O 3-epihydroxybulbispermine (154) R1=R3=H, R2=R6=OH, R4+R5=OCH2O oxomaritidine (158) R1+R2=O, R3=R6=H, R4=R5=OMe 148 Osorio et al. Hemanthidine, also isolated from Zephyranthes citrina Baker, and 3-Oacetylsanguinine (157) isolated from Crinum kirkii Baker bulbs, showed in vitro biological activity against T. b. rhodesiense (strain STIB-900) with similar IC50 values of 3.47 and 3.48 mM, respectively, and against T. cruzi (Tulahuen C4 strain) with IC50 values of 4.35 and 7.29 mM, respectively. On the other hand, galanthine, pancracine, oxomaritidine (158) and 1,2-O-diacetyllycorine (159) showed activity only against T. b. rhodesiense with IC50 values of 9.77, 2.44, 9.81, and 2.69 mM, respectively (241–244). It is interesting to note that pancracine showed no cytotoxicity for L6 cells (rat skeletal myoblasts), which confirms the selective activity of this alkaloid for T. b. rhodesiense and P. falciparum (242). OAc O H O HO O N Me 3-O-acetylsanguinine 157 O N ungeremine 160 These results prompted us to investigate the alkaloids from the bulbs of Phaedranassa dubia (H.B. & K.) J.F.Macbr., and to assay the in vitro antiprotozoal activity against T. b. rhodesiense, T. cruzi, L. donovani, and P. falciparum. Eight alkaloids were obtained, and among them, ungeremine (160) showed the highest activity against three of the four protozoan parasites tested. It was active against T. b. rhodesiense (IC50 ¼ 3.41 mM), T. cruzi (IC50 ¼ 2.99 mM), and P. falciparum (IC50 ¼ 0.32 mM), but not against L. donovani. In spite of showing a degree of cytoxicity against L6 cells (rat skeletal myoblasts, IC50 ¼ 60.85 mM), the SI of ungeremine (IC50 L6/IC50 K1) for P. falciparum was around 190, which confirms its selective activity for this protozoa. Pseudolycorine (161) showed some in vitro biological activity against P. falciparum (IC50 ¼ 0.83 mM), as did hemanthamine against T. b. rhodesiense (IC50 ¼ 1.62 mM) and P. falciparum (IC50 ¼ 2.29 mM), the latter was in agreement with previous reports (240,243). For the last two alkaloids, no cytotoxicity was found for L6 cells, which confirms their selective antiprotozoal activity (data not published). Structure–antiprotozoal activity relationships have not been studied in Amaryllidaceae alkaloids. Some results suggest that the methylenedioxy group and a tertiary non-methylated nitrogen contribute to a higher activity in these alkaloids (240). Nevertheless, we have observed strong activity in alkaloids with quaternary nitrogen. We have also found that the mechanism of antiplasmodial action of Amaryllidaceae alkaloids does not depend on interactions with heme, at least in the case of alkaloids like ungeremine, pseudolycorine, and hemanthamine (data not published). Additional investigation is required to specify the necessary structural requirements for antiparasitic activity, and, in particular, the possible mode of action. Alkaloids with Antiprotozoal Activity 149 B. Indole Alkaloids 1. b-Carboline and Structurally Related Alkaloids b-Carboline alkaloids, also known as harmala alkaloids since they were first isolated from Peganum harmala L. (Zygophyllaceae), constitute a large group of natural and synthetic indole alkaloids with varying degrees of aromaticity and a tricyclic pyrido[3,4-b]indole basic structure, including two heterocyclic nitrogen atoms. In addition to their natural occurrence in plants, b-carbolines have been found in a number of other sources, including foodstuffs, marine organisms, insects, and mammals, as well as human tissues and body fluids (245). They are compounds of great interest because of the variety of actions they evoke in biological systems, such as intercalating into DNA, inhibiting CDK, topoisomerase, and monoamine oxidase, and interacting with benzodiazepine and 5-hydroxy serotonin receptors. Furthermore, b-carbolines also possess a broad spectrum of pharmacological properties including sedative, spasmolytic, anxiolytic, hypnotic, antiplatelet, vasorelaxant, anticonvulsant, antitumor, antiviral, antimicrobial, as well as antiparasitic activities (245–250). One of the simple b-carboline alkaloids reported to possess antiprotozoal activity is the dihydro-b-carboline harmaline (162), the main constituent of a number of plants used in traditional medicine to cure leishmaniasis, including P. harmala and Passiflora incarnata L. (6,251). Harmaline has shown antiparasitic action against the human pathogen L. mexicana amazonensis both in vitro and in vivo (251). Recently, harmaline and two aromatic b-carbolines, harmane (163) and harmine (164), were investigated for their in vitro antileishmanial activity toward parasites of the species L. infantum and L. donovani (252,253). Harmaline weakly inhibited the growth of L. infantum promastigotes (IC50 ¼ 116.8 mM), but it showed an interesting amastigote-specific activity with an IC50 of 1.16 mM and a SI greater than 170. Harmane and harmine displayed weak antileishmanial activity toward both the promastigote and amastigote forms of L. infantum. Harmine was the most efficient alkaloid against the promastigote stage of the parasite (IC50 ¼ 3.7 mM), but both harmane and harmine strongly inhibited the growth of intracellular amastigotes (IC50 ¼ 0.27 and 0.23 mM, respectively) with moderate selectivity (SI ¼ 81 and 73) (252). Results observed in some studies demonstrated that harmaline, harmane, harmine, and other simple b-carboline alkaloids, like harmalol (165) and harmol (166), could also exert antiproliferative effects on parasites of the genus Trypanosoma. The most potent were harmane, which presented trypanocidal activity against T. b. rhodesiense, strain STIB 900 (IC50 ¼ 12.63 mM), and harmine against epimastigotes of two different strains of T. cruzi: Tulahuen and LQ (IC50 ¼ 19.69 and 17.99 mM, respectively) (254,255). On the other hand, harmaline, harmalol, and harmol inhibited the in vitro growth of T. cruzi epimastigotes by 50–90% at around 0.23 mM (5,256). Bioassay-guided fractionation of the bark extract of Annona foetida Mart. (Annonaceae) afforded two antileishmanial pyrimidine-b-carboline alkaloids, annomontine (167) and N-hydroxyannomontine (168). Annomontine was six times more active against the promastigote forms of L. braziliensis 150 Osorio et al. (IC50 ¼ 34.8 mM) than N-hydroxyannomontine (IC50 ¼ 252.7 mM). Both alkaloids were inactive against L. guyanensis (257). R N N H R Me H2N N Me harmane (163) R=H harmine (164) R=OMe harmol (166) R=OH harmaline (162) R=OMe harmalol (165) R=OH N R N H N N H N N Et N flavopereirine 169 annomontine (167) R=H N-hydroxyannomontine (168) R=OH MeO MeO N H N H N HN Et H H N Et 6,7-dihydroflavopereirine 170 OH tubulosine 171 There are fewer reports of b-carboline alkaloids exhibiting antimalarial activity than antileishmanial and/or trypanocidal effects. A series of indole alkaloids was obtained from the bark of Geissospermum sericeum Miers (Apocynaceae), a plant that belongs to a small genus of Amazonian trees native to northern South America, several of which are recognized locally as having antimalarial properties. Among them, the best antiplasmodial activity was observed for the b-carboline alkaloid flavopereirine (169) against the K1 and T9/96 strains of P. falciparum (IC50 ¼ 11.53 and 1.83 mM, respectively) (258,259). Flavopereirine also showed moderate cytotoxicity (IC50 ¼ 10.7 mM) against the human cell line KB. This cytotoxicity had been noted previously because of the ability of the alkaloid to selectively inhibit the synthesis of cancer cell DNA (260). Wright et al. obtained an IC50 value of 3.02 mM for the related derivative 6,7-dihydroflavopereirine (170) against chloroquine-resistant K1 P. falciparum (261). In addition, Pogonopus tubulosus K.Schum. (Rubiaceae), a Bolivian plant used by traditional healers in the treatment of malaria, was found to contain three Alkaloids with Antiprotozoal Activity 151 active alkaloids, tubulosine (171), psychotrine, and cephaeline. Tubulosine, a b-carboline-benzoquinolizidine alkaloid derivative, was the most active compound with an IC50 value of 0.012 mM against a chloroquine-sensitive strain of P. falciparum, and an IC50 value of 0.023 mM against a resistant strain. Tubulosine was also tested in vivo against P. vinckei petteri and P. berghei in mice, and in each case, good results were obtained at concentrations lower than the lethal dose (262). However, this alkaloid also displayed some toxicity against KB cells. The results showed some support for the traditional use of the bark of this plant in the treatment of malaria (9). The mechanisms of action of b-carboline alkaloids as antiprotozoal agents are not known. However, b-carbolines have shown a variety of actions in biological systems related to their interaction with DNA, so it has been postulated that some are able to intercalate DNA (133,252,263). Regarding structure–activity relationships, the results obtained in some studies suggest that unsaturation of the pyridine ring increases the antiprotozoal activity. 1,2,3,4-Tetrahydro-bcarbolines have quite low activities, while 1,2-dihydro-b-carbolines are better growth inhibitors. A fully unsaturated pyridoindole ring system seems to be a necessary condition for optimal activity in these compounds. This could be associated with planarity of the molecule, its redox behavior or its electron density distribution, to mention three possible structural desiderata (254,256). Nevertheless, other mechanisms could be involved, since compounds such as harmaline (162), a dihydro-b-carboline, present greater antiprotozoal activity than fully aromatic compounds. It has been reported that these alkaloids form intermolecular associations with the prosthetic groups of the flavoenzymes FAD and FRN, and that they are also able to interfere with topoisomerase II, or the metabolism of aromatic amino acids, or inhibit the respiratory chain in the parasite (6,254,264,265). These processes would play an important role in the mechanism of action of b-carbolines. In spite of this, due to their possible mutagenic potential, and their activity as inhibitors of monoamino oxidases A and B, which could produce psychopathic effects, b-carbolines cannot be used as therapeutic agents (266,267). 2. Indolemonoterpene Alkaloids The indolemonoterpene alkaloids have been extensively investigated for a wide variety of pharmacological effects, such as contraceptive, antitumor, antiinflammatory, anti-HIV, bactericide, leishmanicidal, and antimalarial activities, as well as a stimulatory action on the CNS (12,268–273). Until now, more than 2000 different alkaloids of this class have been isolated. Due to their effect on the CNS and their potential use as antiaddiction agents (274), together with the anticancer Vinca alkaloids, the Apocynaceae alkaloids coronaridine (172) and ibogaine (173) have been the most studied. Coronaridine was purified by bioassay-guided fractionation from a stem ethanolic extract of Peschiera australis Miers (syn. Tabernaemontana australis, Apocynaceae), which inhibited the growth of L. amazonensis in axenic cultures and infected murine macrophages. The pure alkaloid exhibited potent antileishmanial activity against L. amazonensis promastigotes (IC97 ¼ 35.25 mM) (275). Coronaridine, with an isoquinuclidine 152 Osorio et al. ring fused to an indole moiety, is an iboga-type indole alkaloid found in species used traditionally in folk medicine for the treatment of leishmaniasis, like Peschiera laeta Miers and P. vanheurckii (Müll.Arg.) L.Allorge (271,276–278). Another monoterpene indole alkaloid from the Apocynaceae, but not widely studied, is the aspidofractinine-type alkaloid, pleiocarpine (174) (IC50o63 mM), one of three leishmanicidal alkaloids from Kopsia griffithii King & Gamble was found to show activity against the promastigote forms of L. donovani (253). N R1 N H H N H R2 Et N MeOOC coronaridine (172) R1=H, R2=COOMe ibogaine (173) R1=OMe, R2=H COOMe H pleiocarpine 174 O N N N N H H O Et R1 R2 OMe H OH O MeOOC camptothecin 175 H dihydrocorynantheine (176) R1=H, R2=Et corynantheine (177) R1=H, R2=CHCH2 corynantheidine (178) R1=Et, R2=H MeO N N H H H O H MeOOC OMe O H H OMe reserpine 179 N N H H OMe OMe MeOOC Me O ajmalicine 180 Camptothecin (175), a modified indolemonoterpene alkaloid produced by Camptotheca acuminata Decne. (Cornaceae), Nothapodytes foetida (Wight) Sleumer, Pyrenacantha klaineana Pierre et Exell & Mendonca, Merrilliodendron megacarpum (Hemsl.) Sleumer (Icacinaceae), Ophiorrhiza pumila Champ. ex Benth. (Rubiaceae), Ervatamia heyneana Cooke (Apocynaceae), and Mostuea brunonis Didr. (Loganiaceae), is a known antitumor agent with an unusual heterocyclic structure (279,280). Its structure was elucidated in the mid-1960s (281), and the first total Alkaloids with Antiprotozoal Activity 153 synthesis of optically active 20(S)-camptothecin was reported in 1975 (282). Camptothecin (175) is lethal to T. brucei, T. cruzi, and L. donovani, with EC50 values of 1.5, 1.6, and 3.2 mM, respectively (279). In erythrocytic P. falciparum, camptothecin was cytotoxic with an EC50 value of 32 mM for NF54 (chloroquine-sensitive strain) and 36 mM for FCR3 (chloroquine-resistant strain) (283). The rather broad spectrum of antiparasitic activity of camptothecin is probably due to its inhibitory activity against topoisomerase I. Camptothecin is an established natural antitumor drug and a well-characterized inhibitor of eukaryotic DNA topoisomerase I (280,284). When trypanosomes or leishmania were treated with this alkaloid, both nuclear and mitochondrial DNA were cleaved and covalently linked to protein. This is consistent with the existence of drug-sensitive topoisomerase I activity in both compartments. Camptothecin is an important lead for much needed new chemotherapy, as well as a valuable tool for studying topoisomerase I activity (279). Five indolemonoterpene alkaloids of the yohimbine- and corynantheine-type, obtained from the bark of Corynanthe pachyceras K. Schum. (Rubiaceae), presented marked activity against promastigotes of L. major. Among these alkaloids are dihydrocorynantheine (176), corynantheine (177), and corynantheidine (178), which present IC50 values of below 3 mM. These metabolites do not show significant cytotoxic activity against the drug-sensitive KB-3-1 and multidrugresistant KB-V1 cell lines, indicating an important selectivity in their antiprotozoal activity (285). Due to the absence of previous reports about the leishmanicidal activity of yohimbine- and corynantheine-type indole alkaloids, several structurally related alkaloids were added to this last study, including reserpine (179), ajmalicine (180), and ajmaline (181). The leishmanicidal activity of ajmalicine corresponded to an IC50 value of 0.57 mM, and interestingly, ajmaline was inactive against L. major promastigotes, suggesting that the active alkaloids contain a relatively planar tetracyclic structure. It is proposed that the mechanism of action of the C. pachyceras alkaloids could be based on the inhibition of the respiratory chain of the parasite (285). OH N N H H N N H Me Et H ajmaline 181 N H OH O H H H O strychnine 182 H HO N H Me Me H OHC akagerine 183 Several research groups have studied the antimalarial activity of indolemonoterpene alkaloids to find new antimalarial compounds of plant origin. In this field, indole alkaloids with different skeletons have been tested (Aspidosperma and Picralima from the Apocynaceae family and Strychnos from Loganiaceae), but no clear relationships between structure and activity have been deduced. Interestingly, indolemonoterpene alkaloids such as strychnine (182) from 154 Osorio et al. Strychnos have been tested against Plasmodium, but are devoid of any antiplasmodial activity, as previously observed for other Strychnos monomers (286–289) (see in following section). Only akagerine (183), a known corynanthean-type indole alkaloid with an N–C17 linkage (290), isolated from S. usambarensis Gilg ex Engl., displayed antiprotozoal activity in mice (286,291). During a phytochemical investigation of two Aspidosperma spp., A. pyrifolium Mart. and A. megalocarpon Müll.Arg., monoterpene indole alkaloids possessing an aspidospermane skeleton were isolated (292,293). In this series of alkaloids, 11 were chosen for their close structural similarity, and their in vitro antiplasmodial activity and toxicity were examined. The most active alkaloid, aspidospermine (184), 10-methoxyaspidospermidine (185), and vallesine (186), presented IC50 values between 3.2 and 6.6 mM after incubation for 72 h against chloroquinesensitive and chloroquine-resistant strains of P. falciparum (294). The SI of the alkaloids 10-methoxyaspidospermidine and vallesine were interesting, with values of 22.7 and 15.6, respectively. On the other hand, extracts from Picralima nitida Th. & H.Dur., a plant extensively used in many folk remedies, e.g., against malaria, have been studied experimentally against Plasmodium both in vitro and in vivo (295–298), with encouraging results. A number of alkaloid constituents have been isolated and identified (299), but only one active alkaloid, akuammine (187), has been reported from the seed extract (300). COOMe N H R3 HO Et R2 N H R1 aspidospermine (184) R1=Ac, R2=OMe, R3=H 10-methoxyaspidospermidine (185) R1=H, R2=R3=OMe vallesine (186) R1=CHO, R2=OMe, R3=H O N H Me N Me akuammine 187 3. Bisindole Alkaloids Members of the Apocynaceae family have been used for centuries in folk medicine, and several of their alkaloids have been isolated and are now in clinical use as separate drugs, such as vinblastine, vincristine, and reserpine (179) (275). Ethnobotanical sources mention that the most common medicinal uses of this family of plants involve its antimicrobial action against infectious diseases such as syphilis, leprosy, and gonorrhea, as well as its antiparasitic action against worms, dysentery, diarrhea, cutaneous leishmaniasis, and malaria (275,301). These effective uses described in traditional medicine are probably due to the presence of indole alkaloids, which are the main secondary metabolites in Apocynaceae (275). Included in the family is the genus Kopsia with some 30 species found mostly in tropical Asia (302). Preliminary screening of extracts from K. griffithii showed strong antileishmanial activity, which had been Alkaloids with Antiprotozoal Activity 155 previously traced to the basic fraction from the ethanol extract of the leaves (303). The active principles responsible for this activity were subsequently identified (253). Although K. griffithii is notable for furnishing a remarkable array of alkaloidal types, only three alkaloids were found to show activity against the promastigote forms of L. donovani: the simple b-carboline, harmane (163), the aspidofractinine-type alkaloid pleiocarpine (174), which was previously discussed, and the quasidimer buchtienine (188) (IC50 between 0.79–3.16 mM) (253). This result, which identifies buchtienine as the principal alkaloid responsible for the leishmanicidal activity, is in agreement with the isolation of buchtienine along with other indole alkaloids from the Bolivian plant Peschiera buchtienii (H.Winkler) Markgr. (syn. Tabernaemontana buchtienii), which is also used locally for the treatment of leishmaniasis (278). Natives of the tropical Bolivian Chapare region also use P. vanheurckii to treat cutaneous leishmaniasis. Preliminary antileishmania screening against L. amazonensis and L. braziliensis showed that extracts from the leaves and stem bark of P. van heurkii exhibited significant activity against parasites in relation to the presence of conodurine-type bisindole alkaloids (277). The strongest leishmanicidal activity was observed with the bisindole alkaloids conodurine (189), N-demethylconodurine ( ¼ gabunine) (190), and conoduramine (191). Weak toxicity toward macrophage host cells, and strong activity against the intracellular amastigote form of Leishmania, were observed for conodurine and N-demethylconodurine, which confirms that the leishmanicidal activity exhibited by these alkaloids is not due to a general antiproliferative effect. In vivo, conodurine was less active than Glucantimes, while N-demethylconodurine was devoid of activity at l00 mg/kg (277). On the other hand, a related bisindole alkaloid, voacamine (192), was the most active alkaloid isolated from Peschiera fuchsiaefolia (A.DC.) Miers (304). It showed good antiplasmodial activity against both the chloroquine-sensitive D-6 strain (IC50 ¼ 0.34 mM) and the chloroquine-resistant W-2 strain of P. falciparum (IC50 ¼ 0.41 mM). Apparently, the crude alkaloid extract from the root bark of the same plant is more active than voacamine (IC50 ¼ 0.18 mg/mL for D-6 and 0.28 mg/mL for W-2 strain), and is particularly rich in bisindole alkaloids (0.22% of the plant material) (304). COOMe H N H N N H H H MeOOC H HN H N R H Me H H N buchtienine 188 Me N MeO N H MeOOC Et H H H conodurine (189) R=Me N-demethylconodurine (190) R=H 156 Osorio et al. COOMe COOMe H N H H N Me H H H Me N N H H Me Me MeO N N MeO Et N H MeOOC H Et N H MeOOC H H H H H voacamine 192 conoduramine 191 Another genus of Apocynaceae investigated for its antiprotozoal activity is Alstonia, whose 43 species are distributed throughout Africa, Central America, Asia, and the Pacific region. A number of species are known to be used by traditional healers in the treatment of malaria though there are conflicting reports in the literature as to their activity (305). More than 130 alkaloids have been isolated from Alstonia spp., only a few of which have been assessed for antimalarial activity. From A. angustifolia Wall., a plant used in Southeast Asia to treat malaria and dysentery, nine alkaloids were isolated and tested, and the one most active against P. falciparum was villalstonine (193) (306). Likewise, 13 indole alkaloids were isolated from the active extract of A. macrophylla Wall., and again villalstonine (IC50 ¼ 0.27 mM) was the most active. This alkaloid, as well as macrocarpamine (194), with an IC50 of 0.36 mM, presented high antiplasmodial activity against the multidrug-resistant K1 strain of P. falciparum cultured in human erythrocytes (307). H H H H N N Me H O Me N Me H N Me H H O N MeOOC O Me villalstonine 193 H H N H H H H N MeOOC H Me H N H H macrocarpamine 194 Me Alkaloids with Antiprotozoal Activity 157 MeO N N H H HO Me H R HN Me H N ramiflorine A (195) R=H ramiflorine B (196) R=H N H H H N H H CH2 N Me H N H N isostrychnopentamine 197 The crude basic extract from the stem bark of Aspidosperma ramiflorum Müll.Arg. showed good antileishmanial activity, which was attributed to the presence of indole alkaloids (308). The fractionation, purification procedures, and the activity of the isolated alkaloids of this species against promastigote forms of the L. amazonensis parasite were described recently (309). The bisindole alkaloids ramiflorines A (195) and B (196) showed good antipromastigote activity, with calculated IC50 values of 34.69 and 10.51 mM, respectively. In comparison with pentamidine, these alkaloids were very active. Although the mode of action of these alkaloids is not known, they are similar in structure to several other bisindole alkaloids, which indicates that their mode of action may also be similar; that is, they would act as inhibitors of protein synthesis, DNA intercalating agents, or as topoisomerase inhibitors (310). Alkaloids with a dihydrotchibangensine structure, e.g., buchtienine (188), are also common in the genus Strychnos. Belonging to the Loganiaceae family, the pantropical Strychnos genus comprises about 200 species and can be subdivided into three geographically separated groups found in Central and South America (at least 73 species), Africa (75 species), and Asia (about 44 species). Plants in this genus have been used in folk medicine, and in arrow and dart poisons, in many parts of the world, and are very well known for providing one of the most famous of all poisons. Strychnine (182), as it was logically named, is one of the numerous indolemonoterpene alkaloids possessing the strychnan skeleton produced by the genus Strychnos (291,311). However, the results obtained in previous studies largely confirm that the monomeric indole alkaloid strychnine and its derivative monomers are devoid of any antiplasmodial activity (286–289), in comparison to the Strychnos bisindole derivatives. N H H N H H N H H N H Et H HN H N ochrolifuanine A 198 N H N Me dihydrousambarensine 199 158 Osorio et al. N N H N H H H H N CH2OH O H H CH2OH O H N H N H N H H H N H Me O H O H O H H strychnogucine B 201 18-hydroxyisosungucine 200 The in vitro antiplasmodial activities of 69 alkaloids from various Strychnos species were evaluated against chloroquine-resistant and chloroquine-sensitive lines of P. falciparum (9). Twelve bisindole alkaloids showed IC50 values of o2 mM against all of the Plasmodium lines tested. Among these, it was possible to distinguish four principal structural classes: usambarine-type (i.e., comprised of two tryptamine units with a single iridoid unit), matopensine-type, longicaudatinetype, and sungucine-type alkaloids. Two alkaloids, isostrychnopentamine (197), an asymmetric bisindole monoterpene alkaloid obtained from the leaves of S. usambarensis, whose pyrrolidine ring is joined to the widely distributed b-carbolinylindolo[2,3-a]quinolizinyl-methane system (312), and ochrolifuanine A (198) exhibited very potent activity of o500 nM against all lines tested. Dihydrousambarensine (199) was selectively highly active (IC50 ¼ 0.032 mM) against the chloroquine-resistant strain W-2. These three alkaloids are usambarine-type bisindole alkaloids (313), but as they possess highly varied structures, no structure– activity relationships could be deduced from these data. However, some structural characteristics related to antiplasmodial activity can be kept in mind: all of the active alkaloids were tertiary dimers, and a certain degree of basicity seems necessary for the antiplasmodial activity of this family of alkaloids. As has been hypothesized for chloroquine, these observations are consistent with the ability of basic alkaloids to accumulate at higher levels in the acidic food vacuole of the parasite, where digestion of hemoglobin takes place. N H N H H H Me O H N H N Me H H H N H Me H N strychnohexamine 202 Alkaloids with Antiprotozoal Activity 159 The liana Strychnos icaja Baill., besides being used as an ordeal poison, is also occasionally used in African traditional medicine to treat malaria. The roots of this species are particularly rich in antiplasmodial bisindole alkaloids. Some bisindole alkaloid derivatives of sungucine, such as 18-hydroxyisosungucine (200) and strychnogucine B (201), and the trisindolomonoterpene alkaloid strychnohexamine (202), all possessing a strychnine substructure, as well as potent and selective antiplasmodial properties, have been isolated from S. icaja (289,314,315). 18-Hydroxyisosungucine and strychnogucine B were highly active against four P. falciparum strains (IC50 values of 0.14–1.26 and 0.08–0.62 mM, respectively). Strychnogucine B was more active against a chloroquine-resistant strain than against a chloroquine-sensitive one (best IC50 was 85 nM against the W-2 strain). In addition, this alkaloid showed selective antiplasmodial activity with 25–180 times greater toxicity toward P. falciparum, relative to cultured human cancer cells (KB) or human fibroblasts (WI38) (315). Likewise, strychnohexamine presented interesting antiplasmodial activity with an IC50 value of nearly 1 mM against the FCA chloroquine-sensitive strain of P. falciparum (289). These results confirmed the reported antiplasmodial activities of Strychnos bisindole alkaloids. Since they are derivatives of strychnine, it was important, in view of their potential use as antimalarial drugs, to assess their possible strychnine-like convulsant properties (316). The convulsant effects of most monomeric derivatives were described in mice many years ago (317), but some bisindolic and trisindolic alkaloids from S. icaja are devoid of such strychninerelated properties, at least in vitro. Further studies will involve in vivo assays aimed at confirming the absence of pro-convulsant or toxic effects of these alkaloids, together with examining their in vivo antimalarial potency (316). 4. Indoloquinoline Alkaloids Cryptolepine (203) is an indoloquinoline alkaloid first isolated from the roots of Cryptolepis triangularis N.E.Br. (Asclepiadaceae) collected in the Congo. Extracts of the roots of the related climbing liana C. sanguinolenta, in which cryptolepine is the main alkaloid, have been used in traditional medicine in West and Central Africa, and clinically in Ghana, for the treatment of malaria (318,319). Cryptolepine, which is an indolo[3,2-b]quinoline (or a benzo-d-carboline), showed potent in vitro activity (IC50 ¼ 0.13 mM) against the multidrug-resistant K1 strain of P. falciparum (261,320). Cimanga et al. reported an in vitro IC50 of 0.14 mM for cryptolepine against the same P. falciparum strain (K1), and also in vivo antiplasmodial activity (significant reduction of parasitemia) against P. berghei in subcutaneously infected mice when cryptolepine or its hydrochloride were orally administered (dissolved in 2.5% Tween 80) daily for 4 days at a dose of 50 mg/kg body weight (321). Grellier et al. also confirmed the antiplasmodial activity of cryptolepine in vivo in a similar 4-day suppressive test in mice infected with P. vinckei petteri or P. berghei (intraperitoneal treatment with 1.25–10 mg/kg body weight) (322). However, this alkaloid also has cytotoxic properties probably due to its ability to intercalate into DNA, as well as inhibit topoisomerase II and DNA synthesis (310). Although initially neocryptolepine (204), a minor alkaloid from C. sanguinolenta (Lindl.) Schltr., was reported to show activity comparable to cryptolepine (IC50 of 0.22 mM, K1 strain) (321), more recent investigations have shown that it is about seven times less active against the chloroquine-resistant 160 Osorio et al. P. falciparum Ghana-strain (323). Another minor alkaloid from the same plant with an indolo[3,2-c]quinoline (or a benzo-g-carboline) structure, which was named isocryptolepine (205), also showed in vitro antiplasmodial properties against various chloroquine-sensitive or -resistant strains of P. falciparum (IC50 values of about 0.8 mM) (322). Me N N cryptolepine 203 N N N Me N Me neocryptolepine 204 isocryptolepine 205 As in the case of the b-carboline alkaloids, DNA and topoisomerase II are two potential targets for other indole alkaloids, such as indolemonoterpenes and indoloquinolines. These alkaloids behave like typical DNA intercalating agents, but their affinities for double-stranded DNA can vary significantly depending on their structural complexity (324). Nevertheless, the activity of cryptolepine (203) is due, at least in part, to a chloroquine-like action that does not depend on intercalation into DNA (325). It has recently been postulated that the antiplasmodial activity of the indoloquinoline alkaoids is due to a combination of at least two mechanisms of action. Inhibition of the heme detoxification process is a selective mechanism, whereas DNA intercalation, a non-selective mechanism, is responsible for the cytotoxicity, and probably also for the activity against the other parasites tested (319), i.e., T. b. brucei bloodstream forms (11). C. Steroidal and Diterpenoid Alkaloids Several different steroidal glycoalkaloids of the solanidane or spirosolane type from plants belonging to the genus Solanum (Solanaceae) have been tested against epimastigote and trypomastigote forms of T. cruzi. Among them, the spirosolanes a-solasonine (206) and a-solamargine (207), as well as the solanidanes, a-chaconine (208) and a-solanine (209), four glycoalkaloids composed of a lipid core and a trisaccharide carbohydrate moiety, almost completely inhibited the growth of the epimastigote form of T. cruzi at 5.7 mM. They were even more effective than ketoconazole at the same concentration (326). Although without providing supporting data, this study suggests that stagespecific membrane constituents are responsible for susceptibility to these alkaloids. Likewise, it was established that the role of the sugar moiety is very important in antitrypanosomal activity. In fact, recent studies confirm that the carbohydrate moiety clearly plays a significant role in the cytolytic properties of a-solasonine and a-solamargine against T. cruzi, by means of specific carbohydrate interactions that lead to the formation and intercalation of sterol complexes into the parasite plasma membrane (327). Alkaloids with Antiprotozoal Activity Me Me HN 161 Me H Me H H OH O HO HO HO OH O O OH Me HO HO H H O O O α-solasonine 206 OH Sarachine (210), another steroidal alkaloid isolated from leaves of the Bolivian plant Saracha punctata Ruiz & Pav. (Solanaceae), completely inhibits the growth of the promastigote forms of L. braziliensis, L. donovani, and L. amazonensis and also the growth of epismastigote forms of T. cruzi (50% inhibition) at a concentration of 25 mM. However, at the same concentration it shows strong toxicity against mouse peritoneal macrophages (328). Sarachine also showed in vitro antiplasmodial activity with an IC50 of 25 nM against a chloroquine-sensitive strain, and an IC50 of 176 nM against INDO-resistant strains of P. falciparum. Despite its high cellular toxicity toward macrophages, sarachine exhibited more selective toxicity against Plasmodium than against KB carcinoma cells (IC50 ¼ 50 mM), thus giving cytotoxicity-activity ratios of 2000 and 286, respectively, for the two strains. The alkaloid was also active in vivo against P. vinckei, with 83% inhibition of the parasitemia at 100 mg/kg/2 days (328). All the above-mentioned studies showed interesting preliminary biological activities for this alkaloid. Me Me HN Me H Me H H OH O H H O O HO O Me O Me O HO HO HO HO OH OH α-solamargine 207 Me Me H H Me H N H OH O H H O O HO O Me O Me O HO HO HO HO OH OH α-chaconine 208 Me 162 Osorio et al. Me Me H H H Me N Me H OH O HO HO Me Me Me H O OH Me HO HO H H HO OH O N H H O O α-solanine 209 O OH Me H N(Me)2 Me Me H H H2N Me R1 H R2HN sarachine 210 H R3 H H compound 2 (211) R1=OH,R2= compound 14 (212) R1=R3=H,R2= O Me O Me, R3=OAc Me Me Me Seventeen natural pregnane-type steroidal alkaloids isolated from Sarcococca hookeriana Baill. (Buxaceae) were recently evaluated for their antileishmanial activity. Some of them, such as compound 2 (211) and compound 14 (212), displayed very similar activity to that of the standard drug amphotericin B against L. major promastigotes (IC50 ¼ 0.40 and 1.01 mM, respectively) (329). In further exploration to develop antileishmanial drugs, eight steroidal alkaloids obtained from the leaves of Holarrhena curtisii King & Gamble (Apocynaceae) exhibited leishmanicidal activity against promastigotes of L. donovani. All eight steroidal alkaloids of the pregnane-type were effective in the concentration ranges investigated, in particular the aminoglycosteroids holacurtine (213) and N-demethylholacurtine (214), and the aminosteroids holamine (215) and 15Rhydroxyholamine (216) (IC50 values below 20 mM) (330). Additional bioassays, however, revealed that these alkaloids also showed cytotoxic activity against the HL-60 cell line. Another species of the family Apocynaceae, Funtumia elastica Stapf, which is commonly used in traditional medicine in West Africa to treat infectious diseases including malaria, was recently analyzed through bioassayguided fractionation, and four steroidal alkaloids, holarrhetine (217), conessine (218), holarrhesine (219), and isoconessimine (220), were isolated (331). Alkaloids with Antiprotozoal Activity 163 They were identified as the active constituents against the chloroquine-resistant strain FcB1 of P. falciparum (IC50 values of 1.13, 1.04, 0.97, and 3.39 mM, respectively); conessine showed the highest SI. Me Ac H Me H Me O RHN O Me Ac H H Me OH H H H R H2N H holamine (215) R=H 15 -hydroxyholamine (216) R=OH OMe holacurtine (213) R=Me N-demethylholacurtine (214) R=H Me Me H H H R2MeN Me N R1 H O holarrhetine (217) R1= O conessine (218) R1=H,R2=Me O holarrhesine (219) R1= O isoconessimine (220) R1=R2=H Me , R2=Me Me Me , R2=H Me On the other hand, there are few reports of antiprotozoal activity in C19-norditerpene (NDAs) and C20-diterpene alkaloids (DAs). The only study of diterpenoid alkaloids suggests that DAs are a class of alkaloids with potential for further development in antiprotozoal therapy (332). From a total of 43 diterpene alkaloids (both NDAs and DAs from several chemical groups), only three atisine-type DAs markedly inhibited the growth of the L. infantum promastigotes. Isoazitine (221) exhibited the highest toxicity against the extracellular L. infantum parasites (IC50 of 22.58 mM) with an IC50 lower than that obtained by the antileishmanial reference drug, pentamidine isethionate. Azitine (222) and 15,22-O-diacetyl-19-oxodihydro-atisine (223) also showed pronounced effects against promastigotes (IC50 of 30.92 and 27.16 mM, respectively). In general, this leishmanicidal activity was associated with a lack of toxicity to murine macrophages (332). It seems that these alkaloids act fundamentally at the level of the cytoplasmic membrane of the parasites, as 164 Osorio et al. well as in some organelle membranes. Additional studies are required to confirm the mechanism of action. CH2 CH2 CH2 OAc H N H Me isoazitine 221 OH H N H Me azitine 222 OH H N O H Me OAc Me 15, 22-O-diacetyl19-oxodihydroatisine 223 D. Alkaloid Marine Natural Products In addition to a wide variety of bioactivities, the structural diversity of marine natural products also provides a rich template for the design and development of new pharmaceutical agents (333,334). Among the alkaloids of marine origin, the manzamines are a group of b-carboline alkaloids first reported in 1986 from the Okinawan sponge of the genus Haliclona (335). These complex metabolites usually contain several fused nitrogen-polycyclic rings, which are attached to a b-carboline moiety (336,337). Since the first report of manzamine A, an additional 30 manzamine-type alkaloids have been reported from nine different sponge genera (338,339). It has been demonstrated that manzamines exhibit a variety of bioactivities including antitumor (340), insecticidal, antibacterial, antimalarial, and leishmanicidal (341,342). Manzamine A (224) and 8-hydroxymanzamine A (225) exhibited potent in vitro bioactivity against D-6 chloroquine-sensitive (IC50 ¼ 0.0082 and 0.011 mM, respectively) and W-2 chloroquine-resistant (IC50 ¼ 0.0014 mM) strains of P. falciparum. Other manzamine alkaloids such as 6-hydroxymanzamine E (226) and manzamine Y (227) also presented antimalarial activity with IC50 values of 1.34–1.50 and 0.74–1.50 mM, respectively, against the same species of P. falciparum (342). In addition to their antiplasmodial activity, the manzamines have been evaluated for their activity against L. donovani promastigotes. The alkaloids manzamine A, manzamine Y, 6-hydroxymanzamine E, manzamine E (228), and manzamine F (229) exhibited in vitro bioactivity with IC50 values below 9.11 mM (342). Other studies have further demonstrated the potential of manzamine alkaloids as novel antimalarial agents that inhibit malaria parasites in vivo (335,343). Manzamine A and its hydroxy derivative, 8-hydroxymanzamine A (225), significantly prolonged survival in P. berghei-infected mice when administered in a single dose of 50 or 100 mmoles/kg (343). On the other hand, the alkaloids ent-8-hydroxymanzamine A (230), ent-manzamine F (231), along with the unprecedented manzamine dimer, neo-kauluamine (232) were assayed in vivo against P. berghei with a single intraperitoneal (i.p.) dose of 100 mmoles/kg. Parasitemia was efficiently reduced by ent-8-hydroxymanzamine Alkaloids with Antiprotozoal Activity 165 A and neo-kauluamine, prolonging the survival of P. berghei-infected mice (by 9–12 days), as compared with untreated controls (2–3 days), and mice treated with artemisinin (2 days) and chloroquine (6 days). The increase in survival of mice treated with manzamines was partly attributed to an observed immunostimulatory effect (343). R1 R1 N N N H H H N H N H H R2 OH H N H N R2 OH N O manzamine A (224) R1=R2=H 8-hydroxymanzamine A (225) R1=H, R2=OH manzamine Y (227) R1=OH, R2=H 6-hydroxymanzamine E (226) R1=OH, R2=H manzamine E (228) R1=R2=H manzamine F (229) R1=H, R2=OH Considering their greater activity compared to the currently used drugs, artemisinin and chloroquine, their immunostimulatory properties, and the absence of significant in vivo toxicity (335,343), the manzamines are clearly valuable candidates for further investigation and development as promising leads against malaria. Furthermore, oral and intravenous pharmacokinetic studies of manzamine A in rats show the alkaloid has low metabolic clearance, a reasonably long pharmacokinetic half-life, and good absolute oral bioavailability of 20.6% (344). Understanding the structure–activity relationships and molecular mechanism of action of this unique class of alkaloids is certain to lead to more effective and safer manzamine-related antimalarial drugs (345). N H N H H OH OH H N N N ent-8-hydroxymanzamine A 230 N H H H N H OH OH N O ent-manzamine F 231 166 Osorio et al. N H N O O OH N N H OH H N HO N H OH H N H N neo-kauluamine 232 Other marine indole alkaloids have also drawn attention due to the significant activity they have shown in cancer or cytotoxicity assays (346), and this class of secondary metabolites may also have tremendous unexplored potential in the treatment of antiprotozoal diseases. Among the investigated indole alkaloids, homofascaplysin (233) and fascaplysin (234), two quaternary b-carboline alkaloids isolated from the sponge Hyrtios erecta, have proven to be potent in vitro inhibitors of NF54 chloroquine-susceptible (0.07 and 0.12 mM) and K1 chloroquine-resistant (0.04 and 0.18 mM) strains of P. falciparum (347). Further biological activity of fascaplysin was found against T. b. rhodesiense (IC50 ¼ 0.60 mM), but it was cytotoxic to L6 cells, which confirms that the antitrypanosomal activity exhibited by this alkaloid is probably due to a general antiproliferative effect (347). N N H R1 R2 homofascaplysin (233) R1 = CH2COMe,R2 = OH fascaplysin (234) R1 + R2= O NH HN N R1 R2 H Me 1,8a;8b,3a-didehydro-8 -hydroxyptilocaulin (235) R1 = OH,R2 = n-Bu 1,8a;8b,3a-didehydro-8 -hydroxyptilocaulin (236) R1 = n-Bu,R2 = OH mirabilinB (237) R1 = H,R2 = n-Bu Alkaloids with Antiprotozoal Activity 167 Guanidine alkaloids are a unique class of sponge-derived metabolites exhibiting a broad range of biological activities (348). Members of this class have been evaluated against different protozoan parasites, for example, three ptilocaulin-type tricyclic guanidine alkaloids: 1,8a;8b,3a-didehydro-8bhydroxyptilocaulin (235), 1,8a;8b,3a-didehydro-8a-hydroxyptilocaulin (236), and mirabilin B (237), which were identified from the marine sponge Monanchora unguifera (349). The mixture of 235 and 236 was active against the parasite P. falciparum with an IC50 of 3.8 mg/mL, while mirabilin B exhibited antiprotozoal activity against L. donovani with an IC50 of 17 mg/mL (65 mM). Another spongederived metabolite, renieramycin A (238), is a new alkaloid from the Japanese sponge Neopetrosia sp., which dose-dependently inhibited recombinant L. amazonensis proliferation (IC50 ¼ 0.34 mM) while showing cytotoxicity at a 10 times higher concentration (350). Jasplakinolide (239), a cyclic peptide alkaloid isolated from the marine sponge Jaspis sp., markedly decreased parasitemia of P. falciparum in vitro due to merozoite interference with the erythrocyte invasion (351). The decrease became evident at day 2 at concentrations of 0.3 mM and above, and the parasites finally disappeared at day 4. OMe O O Me H Me N N MeO O Me H N O N OH O H N Br HO Me O O Me O O Me Me O O Me NH Me Me Me jasplakinolide 239 renieramycin A (238) OMe O Me n-BuO HN O H Me Me H NH HN Me Me H H Me ascosalipyrrolidinone A 240 Me (CH2)6Me prodigiosin 241 This present review also includes some other nitrogen-containing compounds of marine origin with interesting bioactivity. As a result of studies with fungal strains associated with marine algae, the novel nitrogen-containing compound ascosalipyrrolidinone A (240) was isolated from the obligate marine fungus Ascochyta salicorniae. It is noteworthy that ascosalipyrrolidinone A was 168 Osorio et al. active against T. cruzi with a MIC of 2.57 mM, whereas the control (benznidazole) had a MIC of 115.3 mM. It also displayed antiplasmodial activity toward both the chloroquine-resistant strain K1 and chloroquine-susceptible strain NF54 of P. falciparum (1.72 and 0.88 mM, respectively) (352). Further development of this compound is limited by its cytotoxic effects on rat skeletal muscle myoblast cells. On the other hand, in vitro and in vivo antimalarial studies were conducted with the tripyrrole bacterial pigment heptyl prodigiosin (241), purified from the culture of a marine tunicate proteobacteria in the Philippines (353,354). The investigators reported that the in vitro activity of heptyl prodigiosin against P. falciparum 3D7 was similar to chloroquine (IC50 ¼ 0.07 vs. 0.015 mM, respectively). Interestingly, a single subcutaneous administration of 5–20 mg/kg heptyl prodigiosin significantly extended the survival of P. berghei ANKA strain-infected mice, suggesting that the molecule might be used as a lead compound (353,354). E. Other Alkaloids Quinazolinones and their derivatives are now known to have a wide range of useful biological properties, e.g., protein tyrosine kinase inhibitory, cholecystokinin inhibitory, antimicrobial, anticonvulsant, sedative, hypotensive, antidepressant, antiinflammatory, and antiallergy activities (355–357), but the interest of medicinal chemistry in quinazolinone derivatives was stimulated in the early 1950s with the elucidation of a quinazolinone alkaloid, febrifugine (242), from the Asian plant Dichroa febrifuga Lour., an ingredient of a traditional Chinese herbal remedy effective against malaria (358). This was one of the earliest studies of novel antimalarials from a plant source, and also reported the inactive interconvertible isomeric alkaloid isofebrifugine (243). Clinical trials showed that febrifugine was such a powerful emetic that it could not be used successfully as an antimalarial drug. Recently, quinazolinones fused with a simple b-carboline have also been reported to have antiplasmodial activity (359). Bioassay-guided fractionation of Araliopsis tabouensis Aubrév. & Pellegr. (Rutaceae) resulted in the isolation of the two alkaloids, 2-methoxy-13methylrutaecarpine (244) and 5,8,13,14-tetrahydro-2-methoxy-14-methyl-5-oxo7H-indolo[2u,3u:3,4]pyrido[2,1-b]quinazolin-6-ium chloride (245), which exhibited significant antimalarial activity against the chloroquine-sensitive D-6 strain of P. falciparum (IC50 ¼ 5.43 and 9.92 mM, respectively) (359). In addition, tryptanthrin (246), 4-azatryptanthrin (247), and a series of substituted derivatives were studied for their in vitro activity against bloodstream forms of T. b. brucei (360). The unsubstituted derivatives were weakly active (IC50 ¼ 23 and 40 mM for tryptanthrin and 4-azatryptanthrin, respectively). The antitrypanosomal activity was markedly improved by the presence of an electron-withdrawing group (halogen or nitro) at position 8 of the (aza)tryptanthrin ring system: the most active analog of the series, 4-aza-8bromotryptanthrin (248), was up to 100 times more active than the unsubstituted Alkaloids with Antiprotozoal Activity 169 4-azatryptanthrin (IC50 ¼ 0.4 mM) (360). O O H H N N O N H N N OH febrifugine 242 H HN isofebrifugine 243 O O N N MeO OH O H MeO N N N Me HN Me 2-methoxy-13-methylrutaecarpine 244 5,8,13,14-tetrahydro-2-methoxy-14-methyl5-oxo-7H-indolo[2',3':3,4]pyrido[2,1-b] quinazolin-6-iumchloride 245 The well-known component of Piper sp., the alkaloid piperine (249), is the main secondary metabolite in Piper nigrum L. Various biological activities have been attributed to piperine, including insecticidal (361) and nematocidal activity (362), inhibition of liver metabolism (363), and antiprotozoal properties (364,365). Kapil reported a study of piperine activity against promastigote forms of L. donovani (364). Encouraging data were later obtained when testing L. donovaniinfected hamsters in vivo with piperine intercalated into mannose-coated liposomes (366). More recently, lipid nanosphere formulations of piperine have also been assessed in BALB/c mice infected with L. donovani AG83 for 60 days. It was observed that a single dose of 5 mg/kg of these piperine formulations significantly reduced the liver and splenic parasite burden (367). Finally, the trypanocidal effects of piperine and some synthetic derivatives against epimastigote and amastigote forms of T. cruzi (365) have also been reported. Piperine proved to be a potent T. cruzi inhibitor, being more toxic to intracellular amastigotes (IC50 ¼ 4.91 mM) than epimastigotes (IC50 ¼ 7.36 mM). O X O R N O N O N O tryptanthrin (246) X = CH2,R = H 4-azatryptanthrin (247) X = N,R = H 4-aza-8-bromotryptanthrin (248) X = N,R = Br piperine 249 Emetine (250), a benzoquinolizidine alkaloid from Cephaelis ipecacuanha (Brot.) Rich. (Rubiaceae) used in the treatment of amebiasis, exhibited substantial 170 Osorio et al. trypanocidal activity (IC50 ¼ 0.039 mM and 0.43 mM for T. b. brucei and T. congolense, respectively), which was also accompanied by cytotoxic effects in HL-60 cells (142). The in vitro antiplasmodial and antileishmanial activities of other emetine-like alkaloids such as cephaeline (251), psychotrine (252), klugine (253), and isocephaeline (254), four benzoquinolizidine derivatives isolated from the Bolivian medicinal plants Pogonopus tubulosus K.Schum. and Psychotria klugii Standl. (Rubiaceae), have also been reported. Cephaeline, with an IC50 of 0.058 mM against the chloroquinesensitive strain 2087, and an IC50 of 0.023 mM against the chloroquine-resistant strain INDO of P. falciparum, was as potent as chloroquine (262). Likewise, cephaeline and klugine were equally potent against the chloroquine-resistant P. falciparum strain W-2 (IC50 0.099 and 0.059 mM) and the chloroquine-sensitive strain D-6 (IC50 0.081 mM) (368). However, psychotrine was less active, its IC50 values against the strains 2087 and INDO of P. falciparum being below 0.84 mM (262). The relative in vitro inactivity of psychotrine in comparison with cephaeline can be explained by its double bond in ring C, which enhances the coplanar conformation and electron environment (262). This suggests that minor changes in alkaloid structures may significantly affect their antiparasitic activities. Cephaeline was reported to inhibit in vivo parasitemia against P. berghei at a dose of 6 mg/kg/day (262). When tested for in vitro cytotoxic activity against the human cancer cell lines SK-MEL, KB, BT-549, and SK-OV-3 human cancer cell lines, cephaeline was more potent than doxorubicin, while klugine and isocephaeline were devoid of cytotoxic activity against these cell lines (368). The antileishmanial activity evaluation revealed that the alkaloids cephaeline, klugine, and isocephaeline showed strong activity against L. donovani promastigotes. Among these, cephaeline was the most potent (IC50 0.06 mM), being W20- and W5-fold more active than the antileishmanial agents pentamidine and amphotericin B, respectively. On the other hand, emetine displayed potent activity against L. donovani (IC50 0.062 mM), but was more toxic than cephaeline against VERO cells. In addition, klugine and isocephaeline also demonstrated strong activities against L. donovani, with IC50 values of 0.85 and 0.96 mM, respectively, and were devoid of significant toxicity against VERO cells (368). DNA intercalation, in combination with the inhibition of protein synthesis, could be responsible for the observed antiprotozoal and cytotoxic effects of these different alkaloids (142). R1 MeO MeO N H H Et H H N MeO N H N H Et H R3 OMe OMe R2 emetine (250) R1=R2=OMe,R3=H cephaeline (251) R1=OMe,R2=OH,R3=H klugine (253) R1=R2=OH,R3= H isocephaeline (254) R1=OMe,R2=OH,R3=H OH psychotrine 252 Alkaloids with Antiprotozoal Activity O 171 R O N N N Me N OMe O R hadranthine A (255) R=OMe imbiline 1 (256) R=H sampangine (257) R=H 3-methoxysampangine (258) R=OMe OMe O H H N R H HN H (Me)2N OO N HN H O O Et Me ziziphine N (259) R=i-Bu ziziphine Q (260) R=i-Pr An ethanolic extract of the stem bark of Duguetia hadrantha R.E.Fr. (Annonaceae) showed sufficient antimalarial activity to warrant bioassay-guided fractionation. This led to the isolation of antimalarial 4,5-dioxo-1-aza-aporphine alkaloids, hadranthine A (255) and imbiline 1 (256), and the copyrin alkaloids, sampangine (257) and 3-methoxysampangine (258), which were more active against the chloroquine-resistant P. falciparum clone W-2 (IC50 ¼ 0.37, 0.96, 0.29, and 0.36 mM, respectively) than the chloroquine-sensitive clone D-6 (369). Due to an absence of cytotoxicity toward VERO cells, the alkaloids hadranthine A, sampangine, and 3-methoxysampangine could be considered as potential antimalarial lead compounds. Bioassay-guided fractionation of the EtOAc extract of the roots of Ziziphus oenoplia (L.) Mill. (Rhamnaceae) resulted in the isolation of some new 13-membered cyclopeptide alkaloids. Ziziphine N (259) and ziziphine Q (260) demonstrated significant antiplasmodial activity against the multidrug-resistant strain K1 of P. falciparum with IC50 values of 6.41 and 5.86 mM, respectively. Both the methoxyl and the N,N-dimethylamino groups are seemingly crucial for the activity of these alkaloids (370). IV. CURRENT STRATEGIES AND RECENT DEVELOPMENTS The burden of leishmaniasis, malaria, Chagas’ disease, and African trypanosomiasis is very costly in terms of human suffering, as well as contributing to 172 Osorio et al. poverty and underdevelopment. No vaccines are currently available for any of these diseases and therefore treatment remains a key element of disease control. The majority of available drugs have one or more of the following drawbacks: (a) insufficient efficacy or increasing loss of effectiveness due to resistance, (b) a high level of toxicity, (c) high cost, and (d) they are not readily available. Thus, new therapies for all of these protozoal diseases are urgently needed to reduce the mortality and morbidity associated with them (371). However, there is a lack of a robust pipeline of products in discovery and development to deliver drugs that meet the desired target product profiles for these diseases. It has been established that new antiprotozoal drugs should be dosed orally and curative regimens should be short. They should also be inexpensive to ensure routine availability in povertystricken countries where these diseases are endemic. Thus, considering these facts, new drugs must have the following characteristics (372,373):  Malaria: For uncomplicated P. falciparum malaria, drugs must be orally active; be of low cost (BUS$1 per full treatment course); be effective against drugresistant parasites; have a low propensity to generate rapid resistance; be curative within 3 days; have potential for combination with other agents; have a pediatric formulation and be stable under tropical conditions (shelf life of W2 years).  Leishmaniasis: The course of treatment must be short (r14 days), with a single daily dose, alternate day or weekly dosing being acceptable; an oral drug is desired but can be injectable if treatment time is reduced; safety of available treatment should be improved, particularly for children and pregnant women; cost should be less than current treatments (US$200–400) and stability under standard tropical conditions is required (shelf life W2 years).  Chagas’ disease: The drugs should be active against blood and tissue forms as well as chronic forms of the disease; parental administration with reduced treatment time would be acceptable; an oral drug is desired as well as improved safety over current products (without cardiac effects), with a formulation that can be used for children and during pregnancy; they should be inexpensive and stable under tropical conditions (shelf life W2 years).  African trypanosomiasis: Activity against both major species T. rhodesiense and T. gambiense is required as well as against known resistant strains, for example, melarsoprol failures; treatment for early-stage diseases is acceptable but efficacy against both early- and late-stage is desired, as well as parental administration in late-stage disease and oral formulation for early-stage disease; a cure should be achieved in 14 days or less; the cost should be less than the current treatment for early-stage disease ($100–140); safety for use during pregnancy and stability under tropical conditions (shelf life W2 years) are also required. Since markets for drugs that treat tropical diseases are primarily in poor countries, marketing opportunities are generally considered to be limited. Thus, it is very important to employ a coordinated approach involving multidisciplinary networks of academic and government institutions, researchers in several disciplines, as well as partnerships between large pharmaceutical Alkaloids with Antiprotozoal Activity 173 companies and the public sector in both developed and developing countries. Fortunately, during the past 8 years there has been a dramatic increase in interest in Research and Development (R&D) directed towards producing new drugs for tropical diseases and various partnerships involving academic consortia, industry, governments, and philanthropic organizations are now dedicated to drug discovery, preclinical and late-stage development candidates and drug development in countries where most patients cannot afford to pay prices for the patented drugs they need (2,374–379). One of the first steps in drug discovery and development is the identification of lead compounds that can be taken forward into lead optimization, or drug candidates that can be tested in human clinical trials. However, discovering lead compounds with the potential to become usable drugs is currently a key bottleneck in the pipeline for needed new and improved treatments for these tropical diseases. To improve discovery of active compounds and let them reach clinical application in the near future, different approaches have been identified and established (380–382). These approaches include: (a) the identification of antiprotozoal active compounds from natural products by evaluation of biological activity of plant extracts and metabolites purified from these extracts to identify products as parent compounds for the semi-synthetic or fully synthetic production of new drugs, (b) the development of analogs of existing agents to improve on existing drugs by chemical modifications of these compounds; this approach does not require knowledge of the mechanism of action or the biological target of the parent compound, (c) the identification of new chemotherapeutic targets by using the available genome sequences of human pathogens and their vectors and subsequent discovery of compounds that act on these targets (383–385), (d) the optimization of therapy with available drugs to improve efficacy, providing additive or synergistic antiparasitic activity, or preventing resistance development. The optimization may include new dosing and/or formulations, combination therapies, including newer agents and new combinations of older agents, (e) the secondary use of compounds originally developed to treat other diseases, an approach which usually involves ‘‘whole organism’’ screening of a known drug or compound against the disease-causing parasite. Finally, (f) the identification of drug resistance by combining previously effective agents with compounds that reverse parasite resistance to these agents. All the approaches are being carried out by a number of initiatives, such as the Compound Evaluation and the Natural Products Initiative (CEN) (386), created in 2004 at the Special Programme for Research and Training in TDR. The CEN program coordinates the evaluation of compounds and natural products in validated whole organism and molecular target assays, promotes the use of natural products as starting points for the discovery of new lead compounds, and supports development of new tools and methods to accelerate drug discovery. In response to this initiative, extensive evaluations of natural products as potential new therapies for these tropical diseases are underway. In the same direction, the Genomics and Discovery Research Committees (GDR) (387) at TDR are supporting several new networks for drug discovery involving developed and developing country researchers, institutions, and 174 Osorio et al. industry. These networks include the following: (a) a Compound Evaluation Network to screen thousands of compounds a year for antiparasitic activity, (b) Medicinal Chemistry and Pharmacokinetics Networks to promote the ‘‘hits’’ or ‘‘leads’’ identified through the compound evaluation network, (c) a Drug Target Portfolio Network to create a globally accessible database containing a prioritized list of drug targets across the range of disease-causing parasites that are the focus of TDR research. In addition, new organizations have been recently created to fund research into protozoal diseases and are focused on drug development rather than drug discovery. The Tropical Disease Initiative (TDI) (388), the Institute for One World Health (IOWH) (389), and the World Bank (390) are some of the various organizations committed to the development of new antiprotozoal drugs, working in one or more stages of the drug development process. The open source TDI is asking sponsors, governments, and charities to subsidize developing country purchases at a guaranteed price, as well as creating nonprofit venture-capital firms (Virtual Pharmas) that look for promising drug candidates before pushing drug development through contracts with corporate partners by charities. The IOWH, a not-for-profit pharmaceutical company, based in San Francisco and established in 2000, is focused on developing safe, effective, and affordable new medicines for people with infectious diseases in the developing world. Similarly, the TDR Programme is also supporting drug discovery and development activities by the creation of some public-private partnerships (PPPs) to address the imbalance in funding between developed and developing world diseases (373). In these PPPs, the private partner can expand its business opportunities in return for assuming the new or expanded responsibilities and risks. Among these PPPs are Drug Neglected Drug Initiative (DNDi) (391) and Medicines for Malaria Venture (MMV) (392). The DNDi was created in 2003 by the Oswaldo Cruz Foundation (Brazil), the Indian Council for Medical Research, the Kenya Medical Research Institute, the Ministry of Health of Malaysia, Pasteur Institute (France), Médecins sans Frontières (MSF) (393), and the UNDP/World Bank/WHO/TDR (371) to improve the quality of life and the health of people suffering from neglected diseases. The MMV was established in 1999 to bring public and private sector partners together to fund and provide managerial and logistical support for the discovery, development, and delivery of new medicines to treat and prevent malaria. Pharmaceutical companies are also increasing their participation in this process. GlaxoSmithKline, for example, is focused on discovering, developing, and making new drugs and vaccines available for treatment or prevention of diseases in the developing world, primarily malaria, but also HIV/AIDS and tuberculosis. The Novartis Institute in Singapore and the Wellcome Trust are focusing on malaria in partnership with MMV. Sanofi-Aventis has established the Impact Malaria Programme and continues collaborating with TDR and DNDi. Pfizer Inc. has also been collaborating with TDR to provide compounds and drug discovery support to identify leads for malaria, African sleeping sickness, Chagas’ disease, leishmaniasis, and other tropical diseases. Alkaloids with Antiprotozoal Activity 175 Ongoing examples of drug discovery and development partnerships exploiting some of these approaches are listed below (373). Most of the drugs under study are in the discovery phase, while others are in the post-regulatory label extension phase. Malaria: (a) Discovery phase: Improved 4-aminoquinoline (MMV, GlaxoSmithKline, University of Liverpool); Farnesyltransferase inhibitors (MMV, Bristol-Myers Squibb, University of Washington); Manzamine derivatives (MMV, University of Mississippi); Cysteine protease inhibitors (MMV, University of California San Francisco, GSK); Fatty acid biosynthesis inhibition (MMV, Texas A&M, Albert Einstein/Howard Hughes, Jacobus); Pyridone (MMV, GlaxoSmithKline); New dicationic molecules (MMV, University of North Carolina, Immtech); Dihydrofolate reductase inhibition (MMV, BIOTEC Thailand). (b) Development phase: Rectal artesunate (TDR); Chlorproguanil-dapsoneartesunate (MMV, TDR, GlaxoSmithKline); Pyronaridine-artesunate (MMV, TDR, Shin Poong); Amodiaquine-artesunate (TDR, European Union, DNDi); Mefloqine-artesunate (TDR, European Union, DNDi); Artemisone (MMV, Bayer, University of Hong Kong); Synthetic peroxide (MMV, Ranbaxy, University of Nebraska); i.v. artesunate (MMV, Walter Reed Army Institute of Research); and (c) Post-regulatory label extensions phase: Coartem in children of 5 kg weight (TDR, Novartis); Coartem pediatric form (MMV, TDR, Novartis); Chlorproguanildapsone (TDR, GlaxoSmithKline). Leishmaniasis: (a) Development phase: Paromomycin (IOWH-TDR) and (b) Postregulatory label extensions phase: Miltefosine (TDR-Zentaris). Chagas’ disease: (a) Development phase: Azole compounds (IOWH-Yale University). African trypanosomiasis: (a) Discovery phase: Novel diamidines supported by Bill and Melinda Gates Foundation at University of North Carolina-Immtech; (b) Development phase: Oral eflornithine by TDR-Aventis; Novel diamidine (DB289) by Bill and Melinda Gates Foundation at University of North Carolina-Immtech. It is noteworthy that although in the last 50 years a hundred alkaloidcontaining plants traditionally used to treat parasitic diseases have proven to be biologically active against protozoan parasites in in vitro and in vivo models of infection, relatively few alkaloids have been studied further to assess their potential as lead compounds for the development of new antiprotozoal drugs. What is worse, only a few of these alkaloids have been tested in humans and none are under investigation in advanced stages of the drug development process. Moreover, despite all the analytical techniques available, a high number of alkaloid-containing plant species have not been investigated chemically or biologically in any detail and their mechanisms of action are yet to be properly determined (394). In the specific case of alkaloids active against Leishmania sp., T. cruzi, T. brucei, and Plasmodium sp. parasites, berberine (68) has been evaluated in human malaria and human leishmaniasis, but the available studies for berberine have not been evaluated in controlled and randomized clinical trials and therefore, more studies are required to confirm these results. Chimanine D (27) and 2-n-propylquinoline (28) have reached the clinical evaluation phase 176 Osorio et al. for the treatment of cutaneous leishmaniasis (6), while 4-aminoquinoline, manzamine derivatives and pyridone have been incorporated in the discovery phase for antimalarial drugs (392). Although no plant-derived alkaloid is currently in the drug development phase, alkaloids continue to be useful in lead discovery because of their physicochemical and biological properties. Alkaloids display all the physicochemical properties of a typical drug (395): they have a moderate molecular weight (250–600 Da), the average number of NH and OH groups being 0.97 and 5.55, respectively (fewer than 5 and 10, according to the Lipinski rules). Alkaloids are amenable to standard techniques of purification and spectral analysis and most of them are relatively simple to synthesize, which permits a structure–activity relationship to be explored. Alkaloids can also be made more bioavailable by modulating their biological lipophobicity by substitution of certain chemical groups. Additionally, they display biological activities in the nM range and are derived from a sustainable resource (12). On the other hand, there is substantial ethnomedical and ecological information about plants that might contain alkaloids as their active metabolites, which remains uninvestigated chemically and biologically. Me N O N H N H H N H Me H Ac CH2OH H H O H icajine 261 O isoretuline 262 Me N O Me N H H N H Ac H N H O Me strychnobrasiline 263 Ac H COOMe H O Me malagashanine 264 Additionally, some plant-derived alkaloids have potential as modulators of the multidrug resistance (MDR) phenotype. Transporters of the ATP-Binding Cassette (ABC) family are known to provide the basis of MDR in mammalian cancer cells and in pathogenic yeasts, fungi, bacteria, and parasitic protozoa (396–404). One strategy to reverse the resistance of cells expressing ABC transporters is a combined and simultaneous use of chemotherapeutic agents and modulators. These modulators, also known as chemosensitizers, represent a wide Alkaloids with Antiprotozoal Activity 177 range of chemical structures and can exert different cellular effects; however, they are supposed to act by the same mechanism for the reversal of MDR (405). Among potential candidates that have been screened for their ability to reverse MDR in protozoa are monoterpene indole, BBIQ, and isoquinoline alkaloids. MeO R MeO Me N O MeO MeO MeO N Me H OMe MeO N H Me MeO OMe laudanosine 267 herveline B (265) R=H herveline C (266) R=Me Three alkaloids, icajine (261), isoretuline (262), and strychnobrasiline (263) are able to reverse chloroquine-resistance at concentrations of between 2.5 and 25 mg/mL. Icajine has also proved to be synergistic with mefloquine against a mefloquine-resistant strain of P. falciparum (406). Likewise, the alkaloid malagashanine (264) displayed a good biological profile, selectively enhancing the in vitro antimalarial activity of quinolines (chloroquine, quinine, and mefloquine), aminoacridines (quinacrine and pyronaridine), and a structurally unrelated drug (halofantrine) against chloroquine-resistant strains of P. falciparum (135,407). It was recently demonstrated that malagashanine prevents chloroquine efflux from, and stimulates chloroquine influx into, drug-resistant P. falciparum (408). Limacine (82) and fangchinoline (86), two isomeric BBIQs, are the most efficient potentiating drugs against the chloroquine-resistant strain FcM29 of P. falciparum, with fangchinoline being significantly more active than limacine (409). Moreover, hervelines B (265) and C (266), two alkaloids with moderate activity against P. falciparum, acted as enhancers of chloroquine activity in a dosedependent manner (410,411). Laudanosine (267) also displayed in vitro chloroquine-potentiating action (9). These results showed that the methylation was vital for the potentiating action and that the bioactivity probably resulted mainly from the benzyltetrahydroisoquinoline moiety. Structure–activity relationship studies on MDR modulators have defined pharmacophoric substructures and physicochemical properties for anti-MDR activity. Among them are aromatic ring structures, a basic nitrogen atom, and high lipophilicity (405,412). One recurring tenet in these studies is the requirement of a nitrogen atom in the molecule, although it has since been established that the nitrogen is not an absolute parameter for activity, but exerts an influence through its contribution to hydrogen bond acceptor strength (413). Nevertheless, the derivation of structure–activity relationships for chemosensitisers should be restricted to chemically related compounds, with careful checking that MDR 178 Osorio et al. reversion is due to the same mechanism, and relinquishing efforts to establish general rules (414). Alkaloids should continue to be an important part of drug development well into the future. The sequencing of the human genome opens new territory in terms of our ability to identify the proteins expressed by genes associated with the onset of diseases. These proteins can be used as molecular targets for testing thousands of compounds, including plant-derived alkaloids, in high throughput assays. V. CONCLUSIONS AND FUTURE DIRECTIONS A large number of antiprotozoal alkaloids with immense structural variety have been isolated from plants and marine organisms. However, despite all the analytical techniques available, considerable work is still needed to determine their mechanisms of action. Furthermore, many of these compounds have only been subjected to in vitro testing and in vivo results are still lacking. Similarly, although substantial progress has been made in identifying novel drug leads from the ocean’s resources, great efforts are still needed to advance to clinical applications. In the future, the world’s oceans will surely be playing an important part in alleviating the global protozoa-disease burden. Considering the current interest in screening plants and/or marine organisms for antiprotozoal activity, and the incomplete knowledge of promising antiprotozoal natural alkaloids, potentially useful species and/or compounds should be tested principally in animals in order to determine their effectiveness in whole-organism systems, with particular emphasis on toxicity studies as well as an examination of effects on normal beneficial microbiota. In addition, methods need to be standardized to enable a more systematic search and to facilitate interpretation of results. Major changes are occurring in R&D for drugs to treat tropical and neglected diseases, with the emergence of many organizations involved in product development. However, although this is encouraging, much more remains to be done. These organizations collaborate and form partnerships, but, in addition, some competition for projects and funding can develop. The challenge is to ensure that the products are delivered to the people who need them and to ensure that scientists in endemic countries are involved in the whole process. To guarantee the long-term sustainability of these programs, greater involvement of disease-endemic countries has to be built into the PPP organization model. More focused and result-oriented technology transfer and capacity building will support a future role of disease-endemic countries in discovering and developing the drugs they need. Plant-derived alkaloids have provided many medicinal drugs in the past. They have contributed almost 50% of plant-derived natural products of pharmaceutical and biological significance and remain a potential source of novel therapeutic agents against infectious and non-infectious disorders. Even though there exist an important number of alkaloids from natural sources that Alkaloids with Antiprotozoal Activity 179 have demonstrated potential as possible antiprotozoal agents, most of them do not meet all the requirements considered to be essential for their commercialization. Notwithstanding these problems, natural alkaloids will continue to play an important role in the development of a new generation of antiprotozoal drugs. ACKNOWLEDGMENTS The authors thank Ms. Lucy Brzoska for language revision, as well as Dr. Strahil H. Berkov for his critical review. Edison J. Osorio is grateful for a Fundación Carolina doctoral grant. 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