Antiplasmodial Natural Products

Malaria is a human infectious disease that is caused by four species of Plasmodium. It is responsible for more than 1 million deaths per year. Natural products contain a great variety of chemical structures and have been screened for antiplasmodial activity as potential sources of new antimalarial drugs. This review highlights studies on natural products with antimalarial and antiplasmodial activity reported in the literature from January 2009 to November 2010. A total of 360 antiplasmodial natural products comprised of terpenes, including iridoids, sesquiterpenes, diterpenes, terpenoid benzoquinones, steroids, quassinoids, limonoids, curcubitacins, and lanostanes; flavonoids; alkaloids; peptides; phenylalkanoids; xanthones; naphthopyrones; polyketides, including halenaquinones, peroxides, polyacetylenes, and resorcylic acids; depsidones; benzophenones; macrolides; and miscellaneous compounds, including halogenated compounds and chromenes are listed in this review.


Introduction
Malaria is an infectious disease caused by four protozoan species of the genus Plasmodium (Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax) [1]. Small human infection outbreaks caused by a malaria parasite of monkeys, Plasmodium knowlesi, have also been reported in Southeast Asia [2]. The majority of the cases of malaria and deaths (malaria kills 1-2 million people each year) are caused by P. falciparum. P. vivax is generally considered less OPEN ACCESS dangerous than P. falciparum, although both can cause deadly complications in infected people. Nearly 3 billion people are at risk of infection with the malaria parasite P. vivax. A new map of areas where this parasite has been reported, including risk areas, has been drawn [3]. These grim statistics could become even worse if resistance to the existing antimalarial drugs develops further [4]. According to WHO [5], the elimination of malaria from countries with high transmission rates is a long-term goal that will depend on the success of research and development to deliver a more robust arsenal of tools than those available today -tools of greater potency and effectiveness, especially those with an impact on transmission, and replacements for medicines and insecticides that are being lost to resistance. The growing resistance to existing antimalarial drugs could nullify efforts to eliminate this deadly disease. Unfortunately, there are still very few drugs that are active against malaria (artemisinin, atovaquone, and chloroquine analogues) [4,6] and vaccines against malaria are not yet available [6].
At present, drug resistance of the malaria parasite is widespread, no new chemical class of antimalarials has been introduced into clinical practice since 1996, and there has recently been an increase in parasite strains with reduced sensitivity to the newest drugs [7]. Meanwhile, recent advances in genome-based technologies and in vitro screening of whole parasites and a great number of compounds (natural and synthetic) have broadened the range of therapeutic targets and are accelerating the development of a new generation of treatments for both the control and eradication of malaria [6].
The current review provides an overview of a great number of bioactive natural products that have recently been described in the literature (from January 2009 to November 2010) as showing antiplasmodial activity (in vitro), along with a few compounds that were tested for antimalarial activity in animal models [19] using Plasmodium knowlesi (in simians), Plasmodium yoelii, Plasmodium berghei, Plasmodium chabaudi (in mice), and Plasmodium gallinaceum (in birds). In most of these bioassays, antiplasmodial activities were assessed using different P. falciparum strains, which include chloroquine-sensitive (NF54, NF54/64, 3D7, D6, F32, D10, HB3, FCC1-HN, Ghana, MRC-02, TM4), chloroquine-resistant (BHz26/86, Dd2, EN36, ENT30, FcB1, FCM29, FCR3, FCR-3/A2, FCR3F86, S20, W2,), chloroquine-resistant and pyrimethamine-resistant (K1, TM91C235), pyrimethamine-resistant (HB3), cycloguanil-resistant (CDC1), and chloroquine-and antifolate-resistant (K1CB1). Most of evaluations used the [ 3 H]-hypoxanthine-incorporation assay to assess parasite inhibition of growth in the presence of the test-drugs. Antimalarial activity of new compounds has also been determined by using: i) the fluorometric method based on the intercalation of the fluorochrome PicoGreen (SYBR) in the parasite DNA, [20]; ii) enzyme-linked immunosorbent assays (ELISAs) with monoclonal antibodies, which measure the P. falciparum-specific antigen histidine-rich protein 2 (HRP2) or lactate dehydrogenase protein (pLDH). A chemical reaction using ferriprotoporphyrine biocrystallization (FBTI Inhibition Test) has been used to provide a possible action mechanism for presumed antimalarial compounds [21]. Protein farnesyltransferase (FTase) bioassays have also been used to provide insight into their mode of action against P. falciparum [22]. The effects of natural products on glutathione (GSH), which plays a key role in redox mechanisms, and on cysteine (Cys), which is one of the substrates needed for the de novo synthesis of P. falciparum GSH, as well as their impact on β-hematin formation have been investigated, since GSH participates in heme detoxification [23]. The advantages and disadvantages of the different in vitro screening methods have been discussed by Krettli et al. [24] and Wein et al. [25]. For in vivo bioassays P. berghei and P. chabaudi chabaudi have been used most often. The cytotoxicities of the active compounds compiled in this review have generally been evaluated in HEK293, Vero or HeLa cells. For the details of the methodologies, see the appropriate references cited herein.
Several criteria have been proposed for considering a compound as active. Generally, a compound is considered to be inactive when it shows an IC 50 > 200 μM, whereas those with an IC 50 of 100-200 μM have low activity; IC 50 of 20-100 μM, moderate activity; IC 50 of 1-20 μM good activity; and IC 50 < 1 μM excellent/potent antiplasmodial activity [12]. In this review, regardless of the in vitro or in vivo method adopted for antiplasmodial or antimalarial evaluation, we list the active and moderately active compounds in accordance with the corresponding cited literature (data for inactive compounds are not shown in this review).
Studies on the Carpesium genus (Compositae) suggest that the antiplasmodial activity against P. falciparum is due to the presence of 11(13)-dehydroivaxillin (17) in the EtOAc extracts of C. cernuum. The antimalarial activity of 17 was evaluated against P. berghei in mice. Its LD 50 was determined to be 51.2 mg/kg, while doses of 124 mg/kg and above were found to be lethal to mice. DDV (2, 5, 10 mg/kg/day) exhibited a significant blood schizonticidal activity in 4-day early infection, repository evaluation and in an established infection with a significant mean survival time comparable to that of the standard drug (chloroquine, 5 mg/kg/day) [32].   In tests of 33 sesquiterpene lactones from several genera, including Arnica, Xanthium, and Inula (Asteraceae), Schmidt et al. [33] found that the antiprotozoal activities were significantly correlated with cytotoxicity, and the major determinants of this activity were α,β-unsaturated structural elements. Among the tested sesquiterpene lactones, 29 (compounds 18-46, Figure 3) showed activity against P. falciparum (K1) with IC 50 values of 0.3 to 27.5 µM [33].

Terpenoid benzoquinones and analogues
Terpenoid benzoquinones and analogues 83-89 ( Figure 9) isolated from the root extract of Cordia globifera (Boraginaceae) exhibited antiplasmodial activity against the P. falciparum strain K1 with IC 50 values of 0.8 to 13.9 μM. The antifungal and cytotoxic activities of these compounds were also evaluated [42].

Steroids
The major steroid 90 together with three other steroids 91-93 ( Figure 10) isolated from the marine sponge Callyspongia fibrosa (Callyspongiidae) showed antiplasmodial activity (P. falciparum strains 3D7 and K1 with IC 50 values of 20.5 to 54.8 µM). Parasite growth was assessed as pLDH activity and 90 exhibited better activity against a chloroquine-resistant strain of P. falciparum than on a chloroquine-sensitive strain [43]. Two steroidal peroxides 94 and 95 from another marine sponge Ciocalapata sp. (Halichondriidae) showed antiplasmodial activity against P. falciparum K1 (IC 50 values of 6.28 and 7.13 µM, respectively), and cytotoxicity against human cells in a breast cancer cell line MCF-7 (IC 50 values of 0.025 and 0.003 µM, respectively), with very little toxicity against human fibroblasts [39].
An evaluation of the effects of four steroid derivatives 96-99 and a sapogenin 100 extracted from Solanum nudum (Solanaceae) on the total glutathione (GSH) and cysteine contents in P. falciparum in vitro showed that 96 increased total glutathione and cysteine concentrations while 99 decreased the concentrations of both thiols. Acetylation at C16 was crucial for the effect of 96 while type furostanol and terminal glucosidation were necessary for the inhibitory properties of 99. The combination of steroids and buthionine sulfoximine, a specific inhibitor of a step-limiting enzyme in GSH synthesis, did not modify the glutathione contents. In addition, 96 inhibited more than 80% of β-hematin formation at 5.0 mM, while the other steroids did not show any effect [23].

Limonoids
In vitro antiplasmodial tests using the D10 and W2 strains of P. falciparum showed that gedunin
The in vitro antiplasmodial activities of the main hop chalcone xanthohumol (153) and seven of its derivatives were evaluated against two strains of P. falciparum (poW, Dd2). Xanthohumol had the highest activity, with IC 50 values of 8.2 (poW) and 24.0 M (Dd2) [58].
Glycoalkaloids have been isolated from Solanaceae species, and five of them, chaconine (204)

203
The pyridinone alkaloid 209 ( Figure 23) has been isolated from an EtOAc extract of a culture medium of the fungus Septoria pistaciarum. It exhibited excellent in vitro antiplasmodial activity against chloroquine-sensitive (D6) and -resistant (W2) strains of P. falciparum (IC 50 values of 0.9 and 0.5 µM, respectively) and cytotoxic activity toward Vero cells [77].
Pyrroloiminoquinone alkaloids, discorhabdins A (210) and C (211), and dihydrodiscorhabdin C (212) (Figure 23), have been isolated from a deep-water Alaskan sponge species of the genus Latrunculia (Latrunculiidae). These alkaloids exhibited anti-HCV activity, antiplasmodial activity against P. falciparum strains D6 and W2 (IC 50 values of 53, 2800, and 170 nM vs. 53, 2000, and 130 nM, respectively), and selective antimicrobial activity. Although compounds 210 and 212 displayed potent and selective in vitro antiprotozoal activity, P. berghei-infected mice did not respond to these metabolites due to their toxicity in vivo [78].    (Figure 24), which were tested against two different strains of the malaria parasite P. falciparum (3D7 and Dd2) and parasite growth inhibition of ~16-41% was achieved at 25 µM. Citotoxicity toward mammalian cell lines (MCF-7 and NFF) was also evaluated, and modest in vitro activity in all assays was observed [80].
The macrocycles 234 and 235 ( Figure 26) are histone deacetylase inhibitors (HDACi) that cause a diverse range of responses in biological systems [84]. The antiparasitic capabilities of these macrocyclic HDACi were determined against malarial and leishmanial pathogens. Antiparasitic activities of macrocyclic HDACi derived from macrolide skeletons are dependent on the length (n) of the spacer group that separates their zinc-binding and surface-recognition moieties. Antimalarial activities peak when n=6 (IC 50 234: 95 nM), whereas antileishmanial activities are optimum when n=8-9 (IC 50 235: ~3.3 µM). This observation could facilitate the identification of other HDACi that are more selective for either parasite [84].

Halenaquinone Derivatives
Compounds 269-271 ( Figure 33) were the most active of a series of halenaquinone derivatives from South Pacific marine sponges of the genus Xestospongia (Petrosiidae). The exhibited antiplasmodial activity did not depend on the chloroquine-sensitivity of the strain tested, since there was no significant difference between the IC 50 values for strains FcB1 (IC 50 1.1, 3. 9, and 9.2 μM, respectively) and 3D7 (IC 50 1. 7, 4.1, and 10.9 μM, respectively). The three active compounds were also active in protein farnesyltransferase bioassays, which may provide insight into their mode of action against P. falciparum [22].  1 µM), the latter of which differs from manadoperoxide B (P. falciparum strain FCR3, IC 50 = 6.8 µM) by only minor structural details. This difference in the antiplasmodial activity has been explained on the basis of a model for the interaction of 1,2-dioxanes with heme and the production of C-centered radicals that are toxic to the parasite. For the manadoperoxides, either the endoperoxide linkage is inaccessible to the heme iron or the O1 radical cannot evolve to produce a C-centered radical [93]. Another polyketide-peroxy, plakortide F (methyl ester 278), has been isolated from a species of Plakortis (Plakinidae) from Jamaica. It exhibited good in vitro antiplasmodial activity (IC 50 values of 3.4 and 2.5 µM against P. falciparum D6 and W2 strains, respectively), whereas a non peroxide-polyketide, plakortone D (279) (Figure 35), exhibited IC 50 values of 7.8 and 8.7 µM, respectively [94]. Five-membered-ring polyketide endoperoxides 280 and 281, and a cyclic peroxide 282 ( Figure 34) have been isolated from the sponge Plakortis halichondrioides (Plakinidae). Biological screening of these cycloperoxides for cytotoxic activity against various human tumor cell lines revealed that compounds 281 and 282 are very active. In assays for antiplasmodial activity, compounds 280-282 also showed good activity against the pathogenic microbe P. falciparum (IC 50 values of 4.0, 0.3 and 3.0 µM, respectively). Compound 280 also showed antitubercular activity against Mycobacterium tuberculosis (IC 50 values of 62 and 71 µM, respectively) [95].

Strobilurins
Strobilurins 348-352, two of which (348 and 349) are monochlorinated (Figure 41), have been obtained from the fungus Favolaschia tonkinensis. In addition to their antifungal and cytotoxic activities, they also exhibited antiplasmodial activity against the P. falciparum strain K1, with IC 50 values of 0.06 to 10.3 µM [106].

Conclusions
A few drugs, alone or in combination-chloroquine, primaquine, mefloquine, halofantrine, artemisinin, atovaquone, among others-have been used in chemotherapy for malaria. However, the evolution of drug-or multidrug-resistance has been a challenge for the effectiveness of such chemotherapy. None of the papers in this review claim to have discovered the next antimalarial drug. Instead, they provide a remarkable diversity of new natural products on which to base the discovery and development of antimalarial drugs. This considerable structural diversity is represented in the 360 relevant structures that we examined (as illustrated in Figures 1-44). Several potent antiplasmodial natural products have been described, and those belonging to alkaloid (manzamine, pyridinone, and pyrroloiminoquinone), polyacetylene, phenylethanoid, anthraquinone, polyketide (endoperoxide), nonpeptide macrocyclic, and β-resorcylic lactone classes have high antiplasmodial activity. Most of the active compounds described here have only been evaluated by in vitro assays, few have been evaluated for cytotoxicity, and still fewer have been assayed in vivo. The compounds listed (Figures 1-44) have been included based on the potency and/or selectivity of their biological properties, and reflect the tremendous effort that is being devoted to recognizing the potential of natural products as lead compounds in the treatment of malaria.