Natural Products as a Source for Antileishmanial

Send Orders for Reprints to [email protected] Combinatorial Chemistry & High Throughput Screening, 2016, 19, 1-17

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REVIEW ARTICLE

Natural Products as a Source for Antileishmanial and Antitrypanosomal Agents Marcus Tullius Scottia, Luciana Scotti*,a, Hamilton Ishikib, Frederico Fávaro Ribeiroc, Rayssa Marques Duarte da Cruza,d, Michelle Pedrosa de Oliveiraa,d and Francisco Jaime Bezerra Mendonça Juniora,c,d a Federal University of Paraiba, Campus I, João Pessoa, PB, Brazil; bUniversity of Western São Paulo (Unoeste), Presidente Prudente, SP, Brazil; cPos-Graduate Program in Therapeutic Innovation, Center of Biological Sciences, Federal University of Pernambuco, Recife, PE, Brazil; dLaboratory of Synthesis and Drug Delivery, Department of Biological Science, State University of Paraiba, João Pessoa, PB, Brazil

ARTICLE HISTORY Received: January 3, 2016 Revised: April 26, 2016 Accepted: May 10, 2016 DOI: 10.2174/13862073196661605 06123921

Abstract: Natural products are compounds extracted from plants, marine organisms, fungi or bacteria. Many research for new drugs are based on these natural molecules, mainly by beneficial effects on health, health, efficacy, and therapeutic safety. Leishmaniosis, Chagas disease and African sleeping sickness are neglected diseases caused by the Leishmania and Trypanosoma ssp. parasites. These infections mainly affect population of developing countries; they have different symptoms, and may often lead to death. The therapeutic drugs available to treat these diseases are either obsolete, toxic, or have questionable efficacy, possibly thru encountering resistance. Discovery of new, safe, effective, and Luciana Scotti affordable molecules is urgently needed. Natural organisms, as marine metabolites, alkaloids, flavonoids, steroids, terpene, coumarins, provide innumerable molecules with the potential to treat these diseases. This study examines studies of natural bioactive compounds antileishmanial and antitrypanosomal agents.

Keywords: Trypanossomiasis, natural products, marine metabolites, alkaloids, flavonoids, steroids, terpene, coumarins. INTRODUCTION The neglected diseases are a group of 17 endemic diseases in underdeveloped or developing countries. Affecting the poorest of areas, the diseases are particularly related to the poor socioeconomic conditions of the populations involved; leaving more than 500 million people at risk [1-3]. Chagas disease is a serious infection considered neglected diseases caused by a protozoan, Trypanosoma cruzi. The disease is present mainly in 21 countries in Latin America, Canada, United States, Japan, Australia and several European countries [4, 5]. Estimates indicate that around 10 million people are infected by T. cruzi, and another 25 million are at risk of infection [6]. Unfortunately there are no vaccines for prevention of the disease and only two drugs are available for treatment (benznidazole and nifurtimox); both have high toxicity and are inefficient against chronic forms of the disease [2, 5, 7]. Sleeping sickness or Human African trypanosomiasis (HAT) is perhaps the most neglected disease that exists. *Address correspondence to this author at the Health Sciences Center, Federal University of Paraiba, Campus I, 58051-970, João Pessoa, PB, Brazil; Fax: 55-83-3291-1528; E-mail: [email protected] 1386-2073/16 $58.00+.00

HAT is due to infection by the parasite Trypanosoma brucei (T. brucei), which is classified into three subspecies [1, 3]: •

Trypanosoma brucei gambiense; responsible for 95% of cases, more concentrated in Central and West Africa, and can remain asymptomatic for years;

•

Trypanosoma brucei rhodesiense; found in East Africa and acute stages of the disease are very intense;

•

Trypanosoma brucei brucei; usually does not infect humans.

Leishmaniasis is one of neglected diseases caused by infection of protozoan parasites from more than 20 Leishmania species. The symptoms are chronic fever, liver problems, anemia and other blood problems. It is endemic in 88 countries and occur in Africa, Asia, Europe, and the Americas, killing thousands and debilitates millions of people every year [8-10]. There are many studies reporting the importance of these parasitic infections in the natural production of immune cells nevertheless natural defenses are inadequate [11-14]. Drug treatment against tropical parasitic diseases, such as leishmaniasis and trypanossomiasis, is of questionable effectiveness and allows the emergence of resistant strains;

© 2016 Bentham Science Publishers

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moreover they are rarely accessible to infected poor people [15]. Despite this clear need for the development of new drugs for treating trypanossomiasis, a recent study has revealed that during the last 6 years, only one drug has been approved for the treatment of Chagas disease, leishmaniasis, and African sleeping sickness [16]. Historically, natural products are a good strategy when searching for new bioactive compounds, they provide a basis for both design and synthesis of derivative compounds that can optimize biological activity and minimize side effects [1, 17]. Combining the need for new active compounds against trypanossomatids and the known potential of natural products to provide chemical structures which can be used as prototypes for new drugs, this article aims to present a review of studies on natural products published over the last six years, including plants, extracts, marine compounds and natural compound isolates (secondary metabolites), which have been assessed for anti-Trypanosomal activity, and may serve as an inspiration for drug development and design in future Chagas disease and African sleeping sickness research. ALKALOIDS Alkaloids are a class of highly diverse secondary metabolites with many related biological and pharmacological properties. In the last century, ethnobotanical research showed that the use of drug plants by indigenous peoples in tropical America was not only cultural, but due to curative effects. Rodriguez and Cavin [18] in 1982 described pharmacological data related to toxicity and the chemotherapeutic value of alkaloids, supporting the hypothesis that psychoactive indole and isoquinoline alkaloids were effective antagonists of helminth neuromuscular transmitters, inhibiting protozoan parasite disease. The indigenous culture of using the South American plant extract indicates therapeutic efficacy without side effects [18]. Thus, the alkaloids belong to a class of compounds that is known to exhibit useful properties for the treatment of malaria, bacterial infections, and tumors among others [19].

Scotti et al.

•

Menispermaceae, Chondodendron tomentosum;

•

Celastraceae, Maytenus ilicifolia;

•

Malvaceae, Waltheria indica L;

•

Euphorbiaceae, Alchornea glandulosa;

Different plants, solvents, methods were used, providing different results even when the extraction process was the same. In some cases, the activity of the extracts was notable, though usually not as successful as the isolated compounds. Baldé and co-workers [19] described for the first time that alkaloid fractions of Pavetta crassipes have antitrypanosomal activity against T. cruzi. They found that leaf extract fractions exhibit trypanocidal activity with IC50s of 1.75 and 1.90 μg/ml. Despite isolation and identification of the individual constituents present in the alkaloid extracts of Pavetta crassipes not having been performed by the authors, Sanon et al. [20] in a previous study describe the presence of indolomonoterpenic alkaloids elaeocarpidin and hydroxyelaeocarpidin in the leaves of Pavetta crassipes [20]. An investigation of medicinal plants from Saudi Arabia against trypomastigote forms of T. cruzi, revealed good activity and selectivity for the chloroform extract of A. ochroleuca with a IC50 value of 0.3 g/ml and an SI >38.6 [21]. The authors describe that the trypanocidal activity may be attributed to the presence of benzylisoquinoline alkaloids in these extracts. Al-Hayan et al. [22] previously reported the presence of benzylisoquinoline alkaloids. The genus Aspidosperma, used in the northwest of Amazonia to prepare medicines against fever and rheumatism is characterized by the occurrence of indolic alkaloids; its extracts show expressive antiprotozoal activity in vitro, including leishmanicidal and trypanocidal activities [23]. Reina, et al. [24] analyzed the activity of South American species of A. rigidum and A. schultesii which are used in traditional Peruvian medicine, against T. cruzi. The authors isolated nine alkaloids, which were investigated for their antiparasitic and cytotoxic activities. Caboxine B (Figure 1) from A. rigidum, an oxindole alkaloid, was active against T. cruzi with an EC50 value of 10.59 μg/ml, being more active that the reference drug nifurtimox, and futher, showing no toxicity against mammalian CHO cells [24].

Between 2010 and 2015, thirteen plant species acting on different T. cruzi life stages (listed below by family, genus, and species), whose trypanocidal activity is attributed to alkaloids, were described in the literature, •

Rubiaceae, Pavetta crassipes K. Schum;

•

Papaveraceae, Argemone ochroleuca;

•

Apocynaceae, Aspidosperma rigidum, Aspidosperma schultesii and Geissospermum reticulatum A. Gentry;

•

Ancistrocladaceae, Ancistrocladus cochinchinensis;

•

Rutaceae, Zanthoxylum chiloperone;

•

Annonaceae, Annona foetida;

•

Amaryllidaceae, Narcissus broussonetii;

O

H O

H

H

N

H HN

H

CH3

H H H

H

COOCH3

H H

H

H

H

H3CO

Fig. (1). Chemical structure of Caboxine B.

Natural Products as a Source for Antileishmanial

Combinatorial Chemistry & High Throughput Screening, 2016, Vol. 19, No. 7 3

The Ancistrocladus plant genus is characterized by the presence of phythylisoquinoline alkaloids. Ancistrocladus cochinchinensis is a tropical liana, endogenous to the rainforests of Vietnam. Bringman et al. [25] isolated, elucidated and evaluated the structure against T. cruzi, with other N-8´-coupled naphythylisoquinoline alkaloids with free phenolic hydroxyl radicals from the bark and leaves of A. ochinchinensis. Shown below are the compounds 4´-Odemethylancistrocladinium A with one free –OH, and 6,4´O-didemethylancistrocladinium A (Figure 2) with two free phenolic groups. MeO

Me

S

in vitro against trypomastigote and amastigote forms of T. cruzi. The authors showed that the percentage of trypomastigote lysis at 250 g/ml for the compounds and extracts were, 78% (for extract), 79% (for canthin-6-one) and 75% (for 5-methoxy-canthin-6-one), values that approach those seen with the reference drug benznidazole (87%). Against the amastigotes forms, the results showed that canthin-6-one and 5-methoxy-canthin-6-one, at a concentration of 15.1 M, showed the highest levels of antiamastigote inhibition activity, respectively 90.0 and 66.4 %. The results were comparable with that of benznidazole, although at a five-fold lower concentration. The in vitro cytotoxicity of the compounds was evaluated using fibroblast cell culture (NTC 929) at a concentration of 15.1 M, and no-significant toxicity was observed [26].

N N

OMe

O

N

Me OMe

Me

N

O

N

O

OH

4_-O-Deme thylancistrocladinium A 5 -Me tho x y-ca n th i n -6 -on e

HO

Me

S

OMe

Fig. (3). Alkaloids isolated from Zanthoxylum chiloperone.

N

Me OMe

Me

Ca n thi n -6 -on e

OH

6,4_-O-Dideme thylancistroc ladinium A

Fig. (2). Phythylisoquinoline alkaloids isolated from Ancistrocladus cochinchinensis.

4´-O-demethylancistrocladinium A exhibited excellent anti-infective properties against amastigote forms of T. cruzi (IC50 = 0.03 M/ml), better than the parent analog the fully O-methylated derivative, and about 70-fold better than the reference drug benznidazole. 6,4´-Odidemethylancistrocladinium A was less active, with an IC50 value of 6.0 M/ml. According to the authors, both the number and spatial arrangement of free OH groups are very important for the anti-T.cruzi properties of these alkaloids. The two compounds were weakly toxic in evaluations made against host cells (rat skeletal myoblast L-6 cells, J774.1 macrophages) with respective IC50 values of 79.1 and >100 M/ml, [25]. The alkaloids canthin-6-one and 5-methoxy-canthin-6one (Fig. 3) have been identified in different parts of Zanthoxylum chiloperone, (native in Paraguay). The compounds and their ethanolic leaf extracts were evaluated

Trypanocidal activity assays with oxoaporphin alkaloids (liriodenine and O-methylmoschatoline) and the pyrimidineB-carboline alkaloid (annomontine) (Figure 4), isolated from dichlorometane extract of Annona foetida (Annonaceae) branches revealed that all tested alkaloids have relevant trypanocidal effects against trypomastigote forms of T. cruzi, even better than the positive control violet crystal. The IC50 values, in μg/mL, observed for liriodenine, Omethylmoschatoline and annomontine were: 4.0 ± 0.2, 3.8 ± 1.8 and 4.2 ± 1.9, respectively. Evaluation of the compounds against epimastigote forms of the parasite revealed that all evaluated compounds were active. O-methylmoschatoline was the most active with an IC50 value of 92.0 ± 18.4 μg/mL [27]. In the Iberian Peninsula and North Africa, Narcissus is the most common genus of the Amaryllidaceae family, comprising 80–100 wild species. The alkaloids found in Amaryllidaceae species possess putative pharmacological properties such as antiprotozoal, antiviral, antitumor, and acetylcholinesterase inhibitory activity [28]. Ethyl acetate extract and certain isolated alkaloids from N. broussonetii (tazettine, lycorine and homolycorine), and those found to a lesser extent, such as 8-Odemethylhomolycorine, omethyllycorenine, papyramine, 6epi-papyramine and obliquine (Figure 5) were evaluated in vitro against trypomastigote forms of T. cruzi. As only the EtOAc extract showed significant anti-T. cruzi activity with an IC50 = 1.77 μg /ml., (reference value of 0.349 μg/ml for benznidazole), the authors believe that the activity could be due to synergic action among the identified alkaloids [28].

4 Combinatorial Chemistry & High Throughput Screening, 2016, Vol. 19, No. 7 H

Scotti et al.

O Me

MeO

OH

MeO H

Me O

HO NMe

N

N

MeO

H

OH

Me O

N

O

O

O

Lycorine

Tazettine

O

H

O

O

O MeN

MeN

H

Liriodenine

O- met hy lm osc hatoline

H

H

MeO H

O

MeO OH

HO

N

H

O

HO

N H

H

MeO

OH

MeO

8-O-Demethylhom ocorine

O-Methlycorenine OMe

Annom ontine H 2N

OMe

N MeO

N

Fig. (4). Alkaloids isolated from Annona foetida.

Hexane and chloroform extracts from Cedrela odorata (Spanish or Cuban cedar), commom cedro of aromatic wood [30]; alkaloid extract from C. tomentosum (velvet horn and spongeweed) green seaweed in the family Codiaceae [31] and chloroform extract from T. serratifolia [32] proved to be the best extracts against epimastigote forms of Trypanosoma cruzi strain Y culture in vitro after 2 days of incubation. Among the analyzed extracts isolated from C. tomentosum were three bisbenzylisoquinoline alkaloids (BBIQs): chondrocurine, (S,S)-12-O-methyl(+)-curine, and cycleanine (Figure 6). The alkaloids were evaluated but none of them exhibited relevant activity when evaluated as isolates [29]. Geissospermum reticulatum (Apocynaceae) is a tree commonly found throughout the South American Amazon rainforest. Alkaloid leaf extract from G. reticulatum, and the isolated indolic alkaloid O-demethylaspidospermine (Figure 7) were tested for their antiparasitic activity against epimastigote forms of the Y strain of T. cruzi. The results indicated that T. cruzi is sensitive to both (extract and isolated alkaloid). The isolated indole alkaloid showed a CI50 value of 41.7 μg/mL. CHO cell cytotoxicity was performed for O-demethylaspidospermine and a higher CI50 value was observed than for the reference drug nifurtimox (16.7 μg/mL versus 13.9 μg/mL) [33].

H N

N

MeO Papyramine

Extracts of 8 plant species from the Peruvian Amazon: Aristolochia pilosa L., Brunfelsia grandiflora L. , Cedrela odorata L., Chondodendron tomentosum Ruiz & Pavón, Paullinia clavigera Schltdl, Tabebuia serratifolia (Bahl)G. Nicholson, Tradescantia zebrine (Rose) D.R. Hunt and Zamia ulei Dammer; currently used in traditional Peruvian medicine, have been tested for their antitrypanosomal activity. The different plant parts were dried, powdered, and extracted by maceration with different solvents. Gonzales and co-workers [29], tested the extracts against epimastigote forms of T. cruzi strain Y.

MeO H MeO OH

6-epi-Papiramine

OH

MeO H NMe O

O N

O

Obliqu ine

OH

Fig. (5). Alkaloids isolated from N. broussonetii.

A new sesquiterpene pyridine alkaloid (ilicifoliunines A) along with the known alkaloid aquifoliunine E-I (Figure 8), were isolated from the root bark of Maytenus ilicifolia (Celastraceae) by Vania et al. [34]. The alkaloids were evaluated for their biological activity against epimastigote forms of T. cruzi (Y strain). Ilicifoliunines A displayed potent antitrypanosomal activity, with an IC50 value of 27.7 μM, while aquifoliunine E-I was moderately active with an IC50 value of 41.9 μM. The data indicate that the antiprotozoal potency was comparable to the positive control benznidazole (IC50 = 42.7 μM). Evaluation of the cytotoxicity of these compounds on mammalian cells, using murine peritoneal macrophages indicated that these two sesquiterpene pyridine alkaloids are non- or low toxic at the evaluated concentrations, displaying a selectivity index of 46.2 and 44.0 for ilicifolius and aquifoliunine, respectively [34].



OH

Dichloromethane extracts of aerial parts and roots of Waltheria indica L. (Malvaceae) were prepared and screened with the aim of discovering new natural products with antitrypanosomal activity. A study by Cretton and colleagues [35] describes the isolation and characterization of 10 quinoline alkaloids from W. indica roots. The isolated alkaloids were tested in vitro for their antitrypanosomal and cytotoxic activities. T. cruzi amastigote growth inhibition (Tulahuen C4 strain) was observed for this series of alkaloids. Indeed, their IC50 values were lower than the reference drug benznidazole (IC50 = 2.9 μM) for all of the compounds except for compound 9 (3.1μM). The results showed that 1,3-dimethoxy-2-methyl-5-(5-phenylpentyl)5,6,7,8-tetrahydroquinolin-4(1H)-one (IC50 = 0.02 μM), waltherione H (IC50 = 0.04 μM), and waltherione K, also (IC50 = 0.04 μM) displayed the most potent anti-Chagas activity (Table 1). However, cytotoxicity against L6 cells was high, and the selectivity indexes (SI) were lower than then required for a compound to be considered a hit [35].

MeN OMe O O NMe OH Chondrocurine

MeO

O Me

MeN OH O O NMe O Me (S,S _)-1 2-O -Met hly( +)c urine

A bioactivity-guided fractioning of Alchornea glandulosa methanolic leaf extract performed by Barrosa et al. [36] afforded a new guanidine alkaloid named alchornedine (Fig. 9). This compound displayed antiprotozoal activity against trypomastigote and intracellular amastigote forms of T. cruzi (Y strain). Alchornedine showed IC50 values of 93 μg/mL (443 μM) (for trypomastigotes) and 27 μg/mL (129 μM) (for amastigotes), being considered equipotent to the standard drug benznidazole for trypomastigotes (IC50 value of 139 μg/mL (440 μM)), and being approximately 3-fold more effective than the standard drug benznidazole. The mammalian cell cytotoxicity of alchornedine was verified against NCTC cells and demonstrated an IC50 of 50 μg/mL (237 μM). Despite the observed toxicity, the alkaloid demonstrated a selective elimination of parasites inside macrophages without affecting the morphology of the host cells.

MeO

O Me

Me N O Me O O MeO Cyc le anine

NMe

MeO

Fig. (6). Alkaloids isolated from Cedrela odorata.

N

Creton et al. [37] isolated the quinolone alkaloid waltherione C (Fig. 9) from the dichloromethane extract of the roots of Waltheria indica L. Evaluation of this alkaloid as an antitrypanosomal agent showed an IC50 value of 1.93 μM, lower than the reference drug benznidazole (IC50 = 2.22 μM) against trypomastigotes forms of T. cruzi. In addition, the cytotoxicity towards the L6 cell line was considered low (IC50 = 101.23 μM) and high SI (> 50), making alkaloid a good hit against T. cruzi, according the WHO/TDR criteria [37, 38].

N

HO O

Fig. (7). Chemical structure of O-demethylaspidospermine. OAc OAc

OAc

OAc

OAc OAc

BzO

O

OH

N H

Ilicifoliunines A

N H Waltherione C

N H

Fig. (9). Guanidine and quinolone alkaloid isolated from A. glandulosa and Waltheria indica L.

O N

O O

Alchornedine

O

O

O

O

N

O O

O

O

OAc

O OH

OAc

BzO

OH O

OAc

N

Marine Alkaloids

Aquifoliun ine E-I

Fig. (8). Sesquiterpene pyridine alkaloid isolated from Maytenus ilicifolia.

for

Natural products are also important source of scaffolds Chagas´ disease..While the literature extensively


Scotti et al.

Table 1. Anti-trypanossomal and cytotoxic activity (IC50 in μM) of alkaloids isolated from Waltheria indica L [35]. Name

Chemical Structure (CH 2 )7

Cytotoxicity

SIa

0.49

0.75

1.5

0.91

4.03

1.1

25.7

22.7

0.02

0.64

33.8

0.04

0.26

6.5

2.2

22.1

10.1

0.10

0.19

1.9

0.04

0.07

1.8

O OCH 3

8-deoxoantidesmone N H

(CH 2) 7

T. cruzi activity

O

Waltherione E : 3,8-dimethoxy2-methyl-5-octyl-5,6,7,8tetrahydroquinolin-4(1H)-one

OCH 3

0.23 N H

OCH 3

(CH 2) 7

Waltherione F : 1,3-dimethoxy2-methyl-5-(5-phenylpentyl)5,6,7,8-tetrahydroquinolin4(1H)-one

O OCH 3

N H

OCH 3 Ph (CH 2) 5

O

Waltherione G : 1,3-dimethoxy2-methyl-5-(5-phenylpentyl)5,6,7,8-tetrahydroquinolin4(1H)-one

OCH 3

N

OCH 3

Ph

Waltherione H :1,3,8trimethoxy-2-methyl-5-(5phenylpentyl)-5,6,7,8tetrahydroquinolin-4(1H)-one

(CH 2) 5

O OCH 3

N

OCH 3 (CH 2) 7

Waltherione I : 2(hydroxymethyl)-1,3dimethoxy-5-octyl-5,6,7,8tetrahydroquinolin-4(1H)-one

OCH 3 O OC H 3

N

CH 2 OH

OCH 3

(CH 2) 7

O OCH 3

Waltherione J : 1,3-dimethoxy2-methyl-5-octyl-5,6,7,8tetrahydroquinolin-4(1H)-one N

OC H 3

(CH 2) 7

O OCH 3

Waltherione K : 3,8-trimethoxy2-methyl-5-octyl-5,6,7,8tetrahydroquinolin-4(1H)-one N OCH 3

OC H 3


Combinatorial Chemistry & High Throughput Screening, 2016, Vol. 19, No. 7 7 (Table 1) Contd….

Name

Chemical Structure

T. cruzi activity

Cytotoxicity

SIa

3.1

41.1

13.5

Ph (CH2 ) 5

Waltherione L : 3,4-dimethoxy2-methyl-5-(5-phenylpentyl)5,6,7,8-tetrahydroquinolin-1oxide

OCH 3 OCH 3

N O

Benznidazole a

2.9

SI (selectivity index) = IC50 cytotoxicity/IC50 antitrypanosomal activity.

O

discloses the use of secondary metabolites of plants, the use of algal metabolites is still a largely unexplored universe. A few examples, found in the literature include tunicates, sponges, and marine cyanobacteria [39]. Marine organisms constitute a great source of potentially bioactive compounds. e.g., sponges, have great potential to affording many classes of secondary metabolites with different chemical structure [40, 41].

CHO OH N H

N H

3-h ydroxyac etylin dole

Bromopyrrole alkaloids, are a class of sponge metabolite described in the literature as containing compounds with various promising pharmacological activities such as antihistaminic, anti-serotonergic, antibacterial and antifungal among others [41]. Scala and co-workers [41] isolated bromopyrrole alkaloids from Mediterranean marine sponges (genera Agelas and Axinella). Evaluation of the activity against amastigotes forms of T. cruzi was performed. Most of these alkaloids were inactive, or presented low activity at the highest concentrations assayed (90 μg/mL). Among the evaluated alkaloids, only longamide B (Figure 10) showed promising trypanoissomal activity (IC50 > 33.03 μg/mL). Br

O

Br N NH

COO H

Longam ide B

Fig. (10). Bromopyrole alkaloid isolated from marine sponge.

3-Formylin dole

O

O HN

N-ace tyl--oxotryp tamin e

N H

Fig. (11). Indole alkaloids isolated from Bacillus pumilus .

The synthetic alkaloid monalidine A, and guanidine and pyrimidine alkaloids batzelladines D, batzelladines F, batzelladines L, and nor-batzelladine L (Figure 12) from the marine sponge Monanchora arbuscula were isolated and evaluated as trypanocidal agents against T. cruzi. Monalidine A, batzelladines F, batzelladines L, and nor-batzelladine L were found to be active against trypomastigote forms of T. cruzi with IC50 = 8.0, 5.0, 2.0, and 7.0 μM, respectively. The less active compound was batzelladine D (IC50 = 64 μM), which was as much as 8 times more active than the reference drug benzinidazole (IC50 = 441 μM). In addition, alkaloids batzelladine L and nor-batzelladine L demonstrated SI(s) of 7 and 12, respectively (evaluated in monkey kidney cells (LLC-MK2)) [42]. FLAVONOIDS

A study by Martinez-Luis et al. [39] investigated the potential of heterotrophic bacteria from genus Bacillus (with living in association with corals) as source of antiprotozoal agents. The antitrypanosomal activity of three indolic alkaloids, from Bacillus pumilus were able to inhibit the growth of amastigote forms of T. cruzi. The isolated alkaloids: 3-formylindole, 3-hydroxyacetylindole and Nacetyl--oxotryptamine(Figure 11) showed IC50 values of 20.6, 19.4, and 26.9 μM, respectively, while the reference drug nifurtimox showed IC50 = 1.6 μM. The compounds showed no important cytotoxicity against Vero cells, with IC50 values of 149, 66, and 87 μM, respectively [39].

Flavonoids are secondary plant metabolites which present many biological and pharmalogical activities and properties, as described in the literature. Many plant extracts containing flavonoid fractions, and isolated flavonoids are described as possessing anti-trypanosomal activity as shown below. Baldissera et al. [43] described the in vitro antitrypanosoma activity of various extracts (aqueous, ethanolic and methanolic from Achyrocline satureioides (Marcela) against Trypanosoma evansi at concentrations of 50 and 100 ug/ml, with death of the parasites after 9 hours of exposure.

8 Combinatorial Chemistry & High Throughput Screening, 2016, Vol. 19, No. 7 O H N

H2 N

OCH3

H

H N

O

Batzelladine D

N

(CH2) 7

N H

H

N N

O N H

Batzelladine F: R = (CH2) 5 Batzelladine L: R = (CH2) 7 n or - Batzelladine L: R = (CH2) 6

H

N H

N H

N

O

Fig. (13). Chemical structure of flavanone isolated from Bacharis retusa.

N R

H N

HN

O

OH H

O

H

HO HO

H

H

Scotti et al.

Monalidine A

Fig. (12). Monalidine A and the pyrimidine alkaloids isolated from the marine sponge Monanchora arbuscular.

Dyary and co-workers [44] also observed antitrypanosomal activity against T. Evansi using aqueous Garcinia hombroniana leaf extracts. In the phytochemical screening it was observed that flavonoids are the most importante constituents with a very high selectivity index of 616.36.

forms of the parasite. Another flavonoid narigenin (5,7,4’trihydroxy-flavanone) (Figure 14) isolated from same plant showed no inhibitory activity against T. cruzi. In order to investigate the influence of radicals on the trypanossomal activity of these flavonoids, sakuranetin-4’methyl ether (with two methoxyl at C-7 and C-4’) (Figure 14) was synthesized from sakuranetin. As significant reduction of antiparasitic activity was observed, the authors concluded that to display anti–T. cruzi activity flavonoids require the presence of a –OH group at C-4’ and a -OMe group at C-7 [49]. OR2 R1O

O

OH

O

Al-Massarani and colleagues [45] also investigated the anti-trypanossomal activity of dichloromethane extract from another Garcinia specie; Garcinia mangostana. The authors found that the flavonoid catechin (3,5,7,3',4'pentahydroxyflavan), among the major constituents of CH2Cl2 extract exhibited IC50 value of 7.6 μg/mL against T. cruzi and of 0.5 μg/mL against T. brucei.

Fig. (14). Structures of flavonoids sakuranetin, naringenin and sakuranetin-4’-methyl ether (Naringenin: R1 = R2 = H; Sakuranetin: R1 = CH3; R2 = H; sakuranetin-4’-methyl ether: R1 = CH3; R2 = CH3).

Another extract described with trypanocidal activity is hexanic extract from the branches of Cipadessa fruticosa (Meliaceae), which contain flavone and 7- methoxyflavone as components. The extract was able to reduce by 100% the trypomastigote forms of Trypanosoma cruzi in a concentration of 4 mg/mL [46]. The flavone and 7methoxyflavone isolated were previously described for trypanocidal activity in the work of Ambrozin et al. [47], which found low activity contra trypomastigote forms of T. cruzi (IC50 values of 2116 and 787 g/mL, respectively).

Flavonoids isolated from Delphinium Staphisagria inhibited epimastigote, amastigote, and trypomastigote forms of T. cruzi with lower IC50 values than the standard drug benznidazole. The flavonoids astragalin hepta-acetate, and 2’’-acetylastragalin demonstrated in vitro activity against epimastigotes with IC50 values of 0.8 and 6.5 μM, respectively. Other compounds presented inhibition of amastigote and trypomastigote infection in percentages of up to 91% and 95% [50].

The flavanone 5,6,7-trihydroxy-4-methoxyflavanone (Figure 13) isolated from Bacharis retusa (Asteraceae)exhibited activity in vitro against trypomastigote forms of Trypanosoma cruzi ( IC50 = 20.39 μg/mL). In this study it was observed that the isolated flavonoid was more potent than the reference drug benznidazole (IC50 of 47.0 μg/mL) [48]. From Baccharis retusa, Grecco et al. [48] also isolated the flavanone sakuranetin (5,4'-dihydroxy-7methoxyflavanone) (Figure 14). Anti-T. cruzi activity was found (IC50 value of 20.17 g/mL) against trypomastigote

Mai and colleagues [51] have described flavonoids with activity against T. brucei. The authors demonstrated that certain flavonoids extracted from Gardenia urville were able to inhibit parasite growth at a concentration of 50 M. At 10 M, only 5,7-dihydroxy-3,3’,4’,6-tetramethoxyflavone (Figure 15) demonstrated potent antiprotozoal activity, with IC50 = 3.7 M. Among the seven flavonoids isolated from Vitex simplicifolia by Nwodo and co-workers [52], only artemetin (Figure 16) showed relevant activity contra T. brucei rhodesiense (IC50 = 4.7 μg/ml). This derivative possesses five methoxyl groups in its structure, which increases the



inhibiton interferes in the NADPH generation and and contributes to increase the trypanosome susceptibility to oxidative stress [56]. The enzymes 24-sterol methyltransferase and sterol 14-demethylase are also other biological targets of sterols [57-59].

OCH3 OCH3 HO

O

CH3O

Gutiérrez et al. [60] studied the methanolic extract from the octocoral Muricea austere. Initially, the crude extract prove to be active contra P. falciparum chloroquine-resistant in in vitro assay.

OCH3 OH

O

Fig. (15). 5,7-dihydroxy-3,3’,4’,6-tetramethoxyflavone.

After fractionation of the extract and purification steps, 3 tyramine derivatives (compounds 1-3), 2 steroidal pregnane glycosides (compounds 4 and 5) and 3 sesquiterpenoids (compounds 6-8) were isolated and identified (Fig. 17).

OCH3 CH3O

O

OCH3

CH3O

OCH3 OH

O

Fig. (16). Artemetin.

lipophilicity and allowing easy entry of the compound into the parasite membrane. STEROIDS Marine Marine sources such as fish, sponges, coral, and microorganisms have many chemicals with interesting biological activities [53, 54]. Marine-derived antiplasmodial therapeutics exhibit their activity through several mechanisms of action on a diverse range of biological targets. The literature describes sterols with antitrypanosomal activity which act by inhibition of the enzyme glucose-6-phosphate dehydrogenase [55, 56]. This

These metabolites were evaluated contraP. falciparum chloroquine-resistant strain[61]. The IC50 values of the active compounds were in the range of 36 M to 80 M; the IC50 value of the control drug chloroquine is 0.07 M. The steroidal glycoside compounds 4 (IC50 = 32 g/mL) and 5 (IC50 = 38 g/mL) showed moderate antiplasmodial activity, and compound 7 showed no activity, and compound 8 showed no measurable activity. It was also noted that peracetylation of compounds 4 and 5 increase the antiplasmodial activity [60]. From the extract, compound 1 (IC50 = 36 M) was the most active. Additionally, all compounds were evaluated against Trypanosoma cruzi; compounds 4, 5 and 6 showed moderated activity respectively at IC50 = 46 g/mL, IC50 = 48 g/mL and IC50 = 45 g/mL. Once more, the peracetylation of glycosides 4 and 5 increased their biological activity. Various active secondary metabolites including: polyphenols, alkaloids, acetogenins, and steroids are produced by Cnidarians. Reimão et al. (2008) [62] evaluated eight MeOH extracts, obtained from cnidarian species , against T. cruzi and L. chagasi . Antiprotozoan activity was observed against both parasites when evaluated the crude

NH O

HO

Compound 1 O

NH O

O

HO

Compound 2 OR3

OR1

OR2

NH O

HO

Compound 3 O

O

O O

O

O O

Compound 6

O

O

Compound 7

Fig. (17). Compounds isolated from the octocoral Muricea austere.

O

Compound 8

Compound 4 R1=R3=H: R2=Ac Compound 5 R1=R2=H: R3=Ac


extracts from Leptogorgia punicea Carijoa riisei,, Macrorhynchia philippina and Heterogorgia uatumani, Two extract (C. riisei, H. uatumani) inhibited 100% of promastigotes form of L. chagasi with IC50 value of 2.8 and 4.4 μg/mL, respectively. The IC50 value The extract from M. philippina showed IC50 = 15.37 μg/mL , and the least active extract was obtained from L. punicea that showed an IC50 of 93.30 μg/mL. For all extracts, the IC50 values against T. cruzi were greater than those observed against Leishmania chagasi. Considering the significant protozoalactivity of the crude extract of C. riisei, Reimão et al. (2008) [52] investigated the activity of the steroid (18-acetoxipregna-1,4,20-trien-3-one( (Fig. 18), [63]. O O

O

Fig. (18). Structural formula of the acethylated steroid 18acetoxipregna-1,4,20-trien-3-one, [63].

The steroid showed against promastigostes form of Leishmania a smaller IC50 value ( ten-fold less) than the IC50 value obtained against Trypanosoma cruzi (IC50 = 50.5 μg/mL). Kossuga et al. (2007) [63] determined the cytotoxic activity of this steroid against SF295, MDA-MB435, HCT8, and HL60 cells and the obtained values of IC50 were consistent with our mammalian cytotoxicity evaluation. The results presented confirm the potential of these steroids as leishmanicidal drug candidates. Similarly to octocoral Carijoa riisei, Regalado et al. [64] studied CH2Cl2/MeOH extract from the marine sponge Pandaros acanthifolium (Poecilosclerida, Microcionidae). From this extract, 12 new compounds were identified: six steroidal glycosides (1-6) and their methyl esters (7-12),

Scotti et al.

formed during the extraction steps. All these compounds were evaluated against some parasitic protozoa include:, Leishmania donovani, Trypanosoma cruzi, Trypanosoma brucei rhodesiense and , Plasmodium falciparum . Compound 9 was the most active against T. cruzi and T. b. rhodesiense, with respective IC50 values of 0.038 and 0.77 M, being more attive than the standard drug benznidazole (IC50 = 2.64 M). Additionally, compound 9 showed high potency (ten-times fold) against L. donovani (IC50 value 0.051 M), than than the reference drug miltefosine (IC50 0.505 M). Compounds 3 and 2 presented also good activity against L. donovani . They IC50 values were respectively 0.8 and 2.4 M contra T. b. rhodesiense, 9.7 and 0.3 M contra T. cruzi, and 1.3 and 4.3 M contra L. donovani. Despite the promising antiprotozoal potential of some compounds, they also possess cytotoxicity. Viegelmann et al. [65] isolated three steroids (Fig. 19) from the sponge Haliclona simulans. The identified steroids were: compound 1 (24-methylenecholesterol) (first isolated from the sponge Chalina arbuscula Verill [66] and precursor of other sterols [67]) showed an MIC of 21.56 ± 11.80 M against T. b. brucei. The second steroid (compound 2) was a saringosterol derivative (first isolated from brown algae [68]) and was was the most active sterol against T. b. brucei , showing a MIC value of 4.58 ± 1.80 μM. Compound 3 (first isolated from the sponge Luffariella cf. variabilis [69]) showed a MIC value of 9.01 ± 0 μM against T. b. brucei [65]. Steroid 2 is a promising compound considering its satisfactory inhibitory activity and its absence of cytotoxicity against Hs27. A certain cytotoxicity was observed for sterols 1 and 3 (58 and 100 μM, respectively). Observations concerning the SAR of sterols with antitrypanosomal activity were made by Viegelmann et al. [65]. An increase in antitrypanosomal activity was observed if hydroxylated side chains were present, as observed for sterol 2 [70]. The peroxide group also contribute to the activity, as observed for sterol 3 [70]. The 3-hydroxy group contributes to the bioactivity, being essential forthe leishmanicidal activity of 24-sterol methyltransferase [71], and sterol methyltransferase [72].

Compound (1) HO OH

Compound (3)

HO O

Compound (2) HO

Fig. (19). Steroids isolated from H. simulans.

O



Animal Many animals are a source for new drugs, their venoms and toxins are extremely potent because they have specific interactions with macromolecular targets. Amphibian cutaneous secretions, for example, are a potential source of compounds active against many diseases and parasites and were studied by Tempone et al. [73] in order to verify antileishmanial and antitrypanosomal activity. Tempone studied steroid compounds that are present in animals and plants and show diverse activities; the bufadienolides [74-77]. Parotid secretions from female specimens of Rhinella jimi were extracted with acetic acid and two antileishmanial compounds were isolated, namely telocinobufagin and hellebrigenin. Hellebrigenin had been previously isolated from other plant sources [78], and telocinobufagin from another Rhinella species [79]. These compounds showed growth inhibition with respective IC50 values of 61.21 g/mL and 126.2 g/mL against Leishmania chagasi promastigotes. Additionally, mammalian cytotoxicity studies demonstrated no hemolytic activity for the steroids at the highest concentration of 200 g/mL. It was observed that telocinobufagin could make ultrastructural damage in the parasites but no cytotoxicity was observed in mammalian cells. The observed antiparasitic effect may be due the miss of a transporter-receptor in the macrophage membrane. [73]. With structural modifications, telocinobufagin and hellebrigenin might contribute for the design of new drugs, and their antileishmanial effect may be increased. Plants Guimarães et al. [80] studied Physalis angulata L. (Solanaceae), an herb abundant in steroid compounds like withanolides and physalins that are broadly distributed in subtropical and tropical regions. Physalins are chemically similar with glucocorticoids that present anti-inammatory and immunosuppressant activity [81, 82]. Considering that many cellular activities were regulated by these compounds [83, 84] they were tested for other diseases [84, 85]. From Physalis angulate L, four physalins, named B, D, F and G, were isolated by Guimarães et al. [80] (Fig 20). It was observed that physalins B and F inhibit Leishmania major and Leishmania amazonensis infection in macrophages and mice. The regulation of Leishmania infection is dependant on nitric oxide production and macrophage activation. Physalins B and F act by inhibiting production of nitric oxide. Additionally, physalins showed anti-inammatory properties that are relevant for the healing process in cutaneous leishmaniasis lesions. It was demonstrated that only physalins B and F may inhibit the NF-kB activation, an substantial transcription factor in inammatory responses. Considering this, observation of the structural formula of these three physalins may suppose that the presence of a double bond, in physalin B, and an epoxy ring, in physalins F, not present in physalin D, are crucial for anti-inammatory activity [80]. The Chromolaena is a kind of about 165 species of shrubs in the aster family, Asteraceae, and can be found in

H

O

H

O

O

O

O

O O

O

O

HO

(B)

O

O

HO

(D)

O O

O O

O OH OH H

O

H

O

O O

HO

(F)

O

O

HO

(G)

O

O

O

O O

O

O

O O

O

O OH

Fig. (20). Structures of physalins B, D, F and G isolated from Physalis angulate L

southeastern US, northern Mexico and in Brazil. Some studies show that crude extracts from Asteraceae have antiprotozoal activity [86]. Therefore, the dichloromethane and ethanolic crude extracts from Chromolaena hirsuta that produced ve steroids, fteen avonoids and two triterpenes were tested for antiprotozoal activity against L. amazonensis promastigote and T. cruzi trypomastigote forms [86]. The ethanolic and dichloromethane extracts of owers and leaves of C. hirsuta demonstrated substantial activity against T. cruzi trypomastigotes and L. amazonensis promastigotes. The steroids stigmasterol, campesterol, 7stigmastenol, –sitosterol, and spinasterol were conrmed by GC and NMR analysis. However, only avonoids isolated from ethanolic and dichloromethane leaf extracts were assayed against L. amazonensis promastigotes and T. cruzi trypomastigotes forms. The Adansonia digitata tree is found in areas of South Africa, Botswana, Namibia, Mozambique and other African tropical countries where suitable habitats occur. The methanol seed extracts of Adansonia digitata were investigated by Ibrahima et al. [87] in order to verify its invivo anti-trypanosomal activity. Phytochemical screening of the crude extract indicated the presence of steroids, carbohydrates, triterpenes, glycosides, cardiac glycosides, saponins, alkaloids and flavonoids. The tests were realized in albino mice infected with T. b. brucei. The extracts obtained exhibited mild to moderate antitrypanosomal activity. Since the active compound(s) were not isolated, the mechanism by which the extracts of this plant exert their trypanocidal activity is unknown. Three triterpenes namely: cycloeucalenone, 31-norcyclolaudenone and 24-methylenecicloartanol, and a mixture of two sterols: stigmasterol and -sitosterol were isolated and identified from Musa paradisiaca (banana) fruit peel ethanolic extract [88] The sterols and triterpenes isolated from M. paradisiaca were tested against LLC-MK2 and RAW 264.7 cells. The activities determined were similar in pentamidine (EC50 = 23.71 g/ml). The most active compound was the mixture stigmasterol+-sitosterol (EC50 = 14.35 g/ml) followed by 24-methylene-cycloartanol (EC50 = 16.55 g/ml) and 31norcyclolaudenone (EC50 = 39.29 g/ml). Cycloeucalenone


showed no activity. All assays were performed against the promastigote form of L. infantum chagasi. Considering the EC50 values for mammalian tested cells these cycloartanetype triterpenes and sterols from M. paradisiaca are safe compounds. Leishmanicidal activity of five aqueous plant extracts: Rutagraveolens L., Aloe vera L., Chenopodium ambrosioides L., Pfaffia glomerata (Spreng.) Pedersen, and Hyptispectinata (L.) Poit against Leishmania amazonensis were verified by De Queiroz et al. (2014) [89]. The cytotoxic effects of the aqueous extracts of H. pectinata, A. vera, R. graveolens, P. glomerata, and C. ambrosioides against promastigotes were determined, and they exhibited no activity that was deleterious to the host cell. The activities of these aqueous plant extracts against the extracellular replication of L. amazonensis were evaluated using in vitro assays. The observed growth inhibition percentages of H. pectinata, A. vera, R. graveolens, P. glomerata, and C. ambrosioides extracts were 74.2%, 75.6%, 70.8%, 83.8%, and 82.1%, respectively, at 100 g/mL, but they were less potent than pentamidine (with the maximum effect of 96.5% at 100 M). These five species of plants were active against promastigotes, but only H. pectinata, A. vera, and R. graveolens were active against the intracellular amastigotes of L. amazonensis. Synthetics Three cationic steroid antibiotics (CSAs), namely CSA-8, CSA-13, and CSA-54 (Fig 21) were synthesized [90, 91] and assayed against L. major promastigotes and T. cruzi trypomastigotes forms [92]. The ceragenins or CSAs are amphiphilic compounds with a sterol backbone linked to multiple cationic amine groups and other groups attached to them [93] and designed to mimic the amphiphilic characteristics of anti-microbial peptides [90, 94]. Considering that some anti-microbial peptides show trypanocidal and leishmanicidal activity [95-98] these CSAs where assayed in vitro to verify potential trypanocidal and H2N

O

H H 2N

O

Scotti et al.

leishmanicidal activity [92]. The cytotoxicity of all ceragenins was tested on host cells; higher levels of cytotoxicity were demonstrated with CSA-8- and CSA-13treated LLC-MK2 cells. It was observed that all 3 ceragenins were more effective against promastigote forms of L. major (CSA-8 - LD50 19.4 μM; CSA-13 - LD50 4.9 μM and CSA54 - LD50 12.4 μM) than against trypomastigote forms of T. cruzi (CSA-8 - LD50 61 μM; CSA-13 - LD50 9 μM and CSA-54 - LD50 99 μM). The mechanism of action of the ceragenins is similar to anti-microbial peptides (AMPs) that act by disrupting cell membrane integrity [93]. Some AMPs have shown trypanocidal and leishmanicidal activity [96-98]. Lara et al. [92] observed that the Leishmania spp. promastigote plasma membrane is more negatively charged than that of the T. cruzi trypomastigote; this may be the cause of the different toxicities observed. TERPENE From methylene chloride extract of Reneilmia cincinnata Tchuendem et al. (1999) [99] isolated a new isodaucane sesquiterpenoid, 6,7,10-trihydoxyisodaucane, the known sesquiterpenoids oplodiol, oplopanone, 5E,10(14)germacradien-1,4-diol, 1(10)E,5E-germacradien-4-ol, and eudesman-1,4,7-triol. Two P. falciparum malaria parasite clones, designated Sierra Leone (D-6) and Indochina (W-2) were employed to perform the experiments. The W-2 clone is resistant to sulfadoxine, pyrimethamine, chloroquine, and quinine while the D-6 clone is resistant to mefloquine. The methylene chloride extract gave an IC50 of 25.517 mg/mL for the W-2 clone and 4.136 mg/mL for the D-6 clone. The isolated sesquiterpenoid 6,7,10trihydoxyisodaucane was not tested on these parasite clones. The best results were obtained with the 1(10)E,5Egermacradien-4-ol that showed an IC50 of 1.54 g/mL and 1.90 g/mL, respectively for D-6 and W-2 clones. Mba’ninga et al. (2013) [100] discovered four new sequiterpenoids (salaterpenes) from an extensive H2N

OH

H

H

O

NH2

H2N

O

H2N

O

H O

NH

H O

NH

H O

NH

NH2

CSA-13

CSA-8

H2N

O

NH2

CSA-54 Fig. (21). Structural formula of cationic steroid antibiotics CSA-8, CSA-13 and CSA-54.

NH2



chromatographic purication of CH2Cl2–MeOH (1:1) extract of the seeds of Salacia longipes var. camerunensis. After characterization, these isolated compounds were tested in vitro against the P. falciparum W2 strain. These sequiterpenoids exhibit a moderate in vitro antiplasmodial activity with IC50 values below 2.7 M. From aerial parts of Pseudelephantopus spiralis, seven hirsutinolide-type sesquiterpenoids were extracted and isolated by Girardi et al. (2015): 8-acetyl-13ethoxypiptocarphol (1), diacetylpiptocarphol (2), piptocarphins A (3), F (4), and D (5), (1S*,4R*,8S*,10R*)1,4-epoxy-13-ethoxy-1,8,10trihydroxygermacra-5E,7(11)dien-6,12-olide (6), and piptocarphol (7). Highest antiplasmodial activities against P. falciparum, with an IC50 value of 3.0 mg/mL, were observed from aqueous extract. Weak activity, with an IC50 of 21.1 mg/mL, were observed from ethanolic extract. Farther, high cytotoxicity with median cytotoxicity concentrations of 1.7 mg/mL (aqueous extract) and 2.5 mg/mL (ethanolic extracts) were observed in the cytotoxicity tests evaluated in mammalian VERO cell lines. Against the L. infantum promastigote the aqueous extract was active, IC50 = 13.4 mg/mL, while the ethanolic extract was inactive (IC50 > 50 mg/mL). The isolated compounds 2, 3, 5, 6 and 7 were tested against the L. infantum promastigote and axenic amastigote stages. Compound 3, displayed the strongest activity against the promastigotes and axenic amastigotes of L. infantum with IC50 values of 9.5 and 2.0 mM respectively. Nevertheless, compound, 3, showed to be much more cytotoxic than miltefosine with respective IC50 values of 0.9 and 155.3 mM. COUMARINS The Rutaceae family enclose around 160 genera and approximately 1900 species [101, 102]. Two new compounds, 1 and 2 (Fig 22), and the known coumarins 8(3isopropenyloxiran-2-yl)-7-methoxy-2H-chromen-2one(3,phebalosin), compound 3 [103, 104], 2-(7-methoxy-2oxo-2H-chromen-8-yl)-3-methylbut-2-enal(4,murralongin), compound 4 [105, 106], and 8-(5-isopropenyl-2,2-dimethyl1,3-dioxolan-4-yl)-7methoxy-2H-chromen-2-one, compound 5, (5, threo-murrangatin acetonide) [106, 107] were isolated from leaves extracts of G. panamensis T. S. Elias (Rutaceae).

R1O

O

O

R2

1

R2 =

R1 =

O

O

2

R1 = CH3

R2 = O

Fig. (22). Structural formula of compounds 1 and 2 isolated by Arango et al. (2010) [102].

Compounds 1-4 were tested against axenic L. panamensis amastigotes. Due to an insufcient amount, compound 5 was not assayed. Compounds 1 and 2 showed the best activity with EC50 = 10 g/mL each. The cytotoxicity against human pro-monocytic U-937 cells of compounds 1 and 2 were CC50 = 9.7 and CC50 = 33.0 g/mL, respectively. The most selective was compound 2 which was tested against intracellular amastigotes and showed an EC50 higher than its CC50 (>33 g/mL). Arango et al. (2010) [102] suggested that both cytotoxic and leishmanicidal activities in chromenone compounds can be differentially controled introducting substituents at positions C-7 and C-8. Ibrar et al. (2015) [108] designed, synthesized and tested a series of new compounds and evaluated them, in vitro. In this work, a coumarin was linked with heterocyclic and non-heterocyclic skeletons, taking into account that the integration of two or more pharmacophoric groups, in the same molecule, is a rational technique for the development of new bioactive coumpounds. The best inhibition was obtained for compound 5i, showing inhibition of 70.4 + 2.2% at 100 mM, whereas amphotericin B, used as a standard drug, showed 79.8% inhibition. Compound 5i attach two methyl groups on the aryl ring linked directly to the thiazole ring. Compound 5c was the least active, displaying 40.6 + 2.2% inhibition at 100 mM. The antiproliferative activity was measured in vitro by cell growth inhibition against lung carcinoma (H-157) and kidney fibroblast (BHK-21) cell lines. Against BHK-21cells, compound 5m showed the highest inhibition of 66.8 + 1.1% and 64.2 + 2.3% at 100 and 10 mM, respectively. Against H157 cell lines, compound 5l showed the highest inhibition of 65.0 + 1.8% and 57.3 + 0.7% at 100 and 10 mM. Vila-Nova et al. (2013) [109] tested the extracts derived from Platymiscium floribundum and Annona muricata against L. donovani, L. Mexicana and L. major. The compound scoparone (6,7-dimethoxycoumarin) was isolated from P. floribundum. From A. muricata the compounds acetogenins, corossolone and annonacinone were isolated. It was observed that these compounds inhibited promastigote growth in all Leishmania species. The activity was based on a dose-dependent response. Annonacinone revealed high leishmanicidal activity (EC50 = 6.72 – 8.00 g/mL) while corossolone (EC50 = 16.14 – 18.73 g/mL), and scoparone (EC50 = 9.11 – 27.51 g/mL) revealed moderate activities. Kayser et al. (2001) [110] tested against Leishmania parasites the extracts and the isolated constituents of Pelargonium sidoides. From the extract, gallic acid and its methyl ester, 7-hydroxy-5,6-dimethoxycoumarin, (+)catechin, 6-hydrox-7-methoxycoumarin, 5,6,7trimethoxycoumarin and 6,8-dihydroxy-5,7dimethoxycoumarin were obtained. Neither the extracts nor the characteristic compounds of P. sidoides showed signicant activity against L. donovani promastigote. However, pronounced leishmanicidal effects against the L. donovani amastigote were observed for the methanol extract. Adittionally, the isolated coumarins were inactive at concentrations up to 25 g/mL. The antileishmanial activity of (-) mammea A/BB, a coumarin isolated from leaves of Calophyllum brasiliense


Cambess (Clusiaceae), were studied by Brenzan et al. (2007) [111]. Previous studies showed in vitro leishmanicidal activity of this compound purified from a dichloromethane crude extract of C. brasiliense leaves [101]. The concentration necessary to inhibit 50% growth of L. amazonensis promastigote and amastigote were 3.0 and 0.88 g/mL, respectively. No cytotoxicity against macrophages J774G8 at CC50 of 63.5 mM was observed. Tiuman et al (2012) [112] demonstrated that mice treated with (-) mammea A/BB intramuscularly or topically, after 8 weeks, showed controlled Leishmania infection similar to animals treated with glucantime; showing that this coumarin may be used in cutaneous leishmaniasis lesions. Napolitano et al (2004) [113] isolated a new coumarin, 7geranyloxycoumarin (Fig 23), called aurapten, from Rutaceae species Esenbeckia febrifuga hexane extract tested against L. major Friedlin promastigotes in axenic cultures. This new compound showed an LD50 of 30 M.

CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS We would like to thank both CNPq and Capes for financial support. REFERENCES [1]

[2]

[3]

O

O

O

[4]

Fig. (23). Chemical structure of aurapten.

CONCLUSIONS The Neglected Diseases are 17 diseases transmitted by virus, protozoa, helminthes, and bacteria, they are infections caused principally by tropical parasites, affecting people who live in poor countries, and having differing symptoms, may often lead to death. Among the neglected diseases, American trypanosomiasis and Human African Trypanosomiasis are highlighted by the large number of infected and severe symptoms people. Both infections leave the patient with unsatisfactory pharmacological treatment. Despite the diseases causing great social problems, there are only a restricted number of effective drugs available, which also carry serious side effects. At the same time, pharmaceutical companies do not invest in research for new drugs that are effective against tropical infections, thus they are called neglected. Natural products of vegetable or animal origins (including marine species) are a rich source of new bioactive compounds that deserve scientific investigation for biological activity. For example, marine and steroids of animals showed be active against promastigota of Leishmania chagasi. This mini-review gathered studies in alkaloids, flavonoids, steroids, terpenes, alkaloids, and coumarins where it was observed that the compounds had greater potential activity against these parasites. With the large number of infections caused by Trypanosoma and Leishmania spp. and because of unsatisfactory chemotherapy, the main objective of this study is to encourage the introduction of natural product agents to the treatment of these infections.

Scotti et al.

[5]

[6]

[7]

[8]

[9]

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