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Comparative in vitro and in vivo antimalarial activity of the indole alkaloids ellipticine, olivacine, cryptolepine and a synthetic cryptolepine analog.

ARTICLE INFO

Keywords:

Plasmodium falciparum

Plasmodium berghei

Murine macrophages

Antiplasmodial activity

Indole alkaloids

ABSTRACT

Indole alkaloids ellipticine (1), cryptolepine triflate (2a), rationally designed 11-(4-piperidinamino) cryptolepine hydrogen dichloride (2b) and olivacine (3) (an isomer of I) were evaluated in vitro against Plasmodium falciparum and in vivo in Plasmodium berghei-infected mice. 1-3 inhibited P. falciparum ([IC.sub.50] [less than or equal to] 1.4 [micro]M, order of activity: 2b > 1 > 2a >3). In vitro toxicity to murine macrophages was evaluated and revealed selectivity indices (SI) of 10-12 for 2a and SI > 2.8 x [10.sup.2] for 1, 2b and 3.1 administered orally at 50 mg/kg/day was highly active against P. berghei (in vivo inhibition compared to untreated control (IVI) = 100%, mean survival time (MST) > 40 days, comparable activity to chloroquine control). 1 administered orally and subcutaneously was active at 10 mg/kg/day (IVI = 70-77%; MST= 27-29 days). 3 exhibited high oral activity at [greater than or equal to]50 mg/kg/day (IVI = 90-97%, MST = 23-27 days). Ciyptolepine (2a) administered orally and subcutaneously exhibited moderate activity at 50 mg/kg/day (IVI = 43-63%, MST=24-25 days). At 50 mg/kg/day, 2b administered subcutaneously was lethal to infected mice (MST= 3 days) and moderately active when administered orally (IVI = 45-55%, MST= 25 days). I and 3 are promising compounds for development of antimalarials.

[c] 2012 Elsevier GmbH. All rights reserved.

Introduction

Despite great effort and resources for the eradication of malaria this disease still remains a grave public health problem involving hundreds of thousands of deaths annually (WHO 2010). While research on vaccines is at an advanced stage, drug therapy is still the principle tool for the control and eradication of the disease. The emergence of strains of Plasmodium falciparum and P. vivax which are resistant to first and second line antimalarials (multidrug resistant or MDR) have motivated the search for new drugs representing new and distinct chemical classes and mechanisms of action than those of the antimalarial drugs currently in use. Chemical compounds of novel structure and of natural origin represent a major source for the discovery and development of new drugs for diseases, especially malaria (Kaur et al. 2009; Schmidt et al. 2012a, b).

Historically, plants used in traditional medicine as antimalarials and febrifuges have provided substances which have proved to be useful as antimalarials or have served chemists as structural models for the development of semi-synthetic drugs or purely synthetic analogs. This is true of the most important antimalarial natural products revealed to date: quinine (isolated from the bark of Cinchona spp.) and artemisinin (isolated from Artemisia annua leaves). The therapeutic efficacy and complex molecular structure of quinine lead to the development of purely synthetic analogs chloroquine, primaquine, mefloquine, among others in the last century. More recently, semi-synthetic derivatives (e.g. sodium artesunate, artemether, arteether and dihydroartemisinin) prepared in one or more steps from isolated artemisinin have become key pharmaceutical components in formulations used in what is commonly called artemisinin combination therapy (ACT) for the treatment of resistant and MDR Plasmodium falciparum infections (Plowe 2009: Willcox 2011). The extracts of a large number of plant species including many that are used in traditional medicine have been evaluated for in vitro antiplasmodial activities and some have also been tested in in vivo models, usually in mice infected with Plasmodium berghei, P. yoelii or P. chabaudi. In some cases, the constituent(s) responsible for their activities have been isolated but relatively few have been studied further to assess their potential as lead compounds for the development of new antimalarial drugs (Wright 2005).

In recent years, the monoterpene indole alkaloid ellipticine (1, Fig. 1) has been the subject of a number of pharmacological studies and its derivatives have been studied in clinical trials against different forms of cancer. Ellipticine has been isolated from the alkaline ethanol extract of the bark of the Amazonian tree Aspidosperma vargasii (Apocynaceae) (Andrade-Neto et al. 2007; Henrique et al. 2010) which is used in traditional medicine as an antimalarial (Oliveira et al. 2003). In vitro antiplasmodial activity of I was first reported by Andrade-Neto et al. (2007). Recently, the antimalarial activity of 1 was independently confirmed and the comparable or superior activity of four derivatives of 1 against P. falciparum in vitro was described (Passemar et al. 2011; Pohlit et al. 2012).

The roots of the West African climbing shrub Cryptolepis san-guinolenta (Lindl.) Schltr. (Apocynaceae) are a traditionally used herbal for malaria treatment. Dry aqueous root extracts of C. san-guinolenta have proven efficacy according to clinical trials (Willcox 2011). Cryptolepine (2a, Fig. 1) is the major alkaloid constituent in the roots of this plant and 2a sulfate exhibits in vitro activity ([IC.sub.50] = 0.44 [micro]M) against multidrug-resistant K1 strain of P. falciparum (Wright et al. 2001). However, 2a sulfate failed to cure malaria in mice when given orally and is toxic at a dose of 20 mg/kg to P. berghei-infected mice when administered intraperitoneally (i.p.) (Cimanga et al. 1997; Wright et al. 2001). Also, 2a has in vivo chronic effects causing necrosis of rodent liver cells at a dose of 30 mg/kg and also damages the DNA of lymphocytes in vitro. These and other experimental results do not support the use of the indole alkaloid cryptolepine (2a) as an antimalarial (Gopalan et al. 2011; Willcox 2011).

Derivatives of cryptolepine (2a) have been introduced having greater antiplasmodial activity and better toxicity profiles. One of the most promising of these derivatives is 2,7-dibromocryptolepine which has potent in vitro activity ([IC.sub.50] = 50 nM) against K1 strain of P. falciparum and exhibits significant suppression (89-91%) of P. berghei growth in infected mice at doses of 20-25 mg/kg/day over 4 days. While 2,7-dibromocryptolepine does not apparently intercalate DNA bases as does 2a, both these compounds damage lymphocyte DNA based on results of the comet assay (Wright et al. 2001; Gopalan et al. 2011). Recently, cryptolepine triflate (2a triflate) and ten synthetic analogs of 2a containing aminoalkyl side chains at C-11 were synthesized and screened for in vitro antiplasmodial activity and cytotoxicity. One of the most promising of these compounds was 11-(4-piperidinamino)cryptolepine hydrogen dichloride (2b) which exhibited potent inhibitory activity ([IC.sub.50] =44 nM) against P. falciparum W2 strain and was the least toxic of all the compounds tested, including 2a triflate and had the largest cytotoxicity to antiplasmodial inhibition ratio (46.4:1) (Lavrado et al. 2008, 2011).

Olivacine (3) is a rare alkaloid which is isolated from Aspidosperma olivaceum. The antitumor activity of 3 has been the subject of studies for decades. Compound 3 and analogs have also been synthesized (Besselievre & Husson 1981; Chevallier-Multon et al. 1990; Guillonneau et al. 2005).

Mechanistic studies demonstrate that cryptolepine (and it analogs) and ellipticine (1) may have important inhibitory effects on the formation of hemozoin in P. falciparum. Hemozoin formation is a fundamental process related to the survival of this parasite within the red blood cell. Heme is toxic to Plasmodium spp. and is a by-product of digestion of hemoglobin by proteolytic enzymes in the parasite's digestive vacuole. Crystallization of heme to hemozoin is a detoxifying process that occurs naturally within the Plasmodium digestive vacuole and is necessary for the proliferation of these parasites within the red blood cell. Inhibition of hemozoin formation is associated with death of parasites within the red blood cell due to osmotic imbalances and other effects. Early mechanistic studies involving chemical assays showed that ellipticine (1) can inhibit heme crystal growth (that is, hemozoin formation) in the lab (Chong and Sullivan 2003). In other work, it was presumed that cryptolepine (2a) interacts directly with heme molecules in the process of inhibiting hemozoin formation (Kumar et al. 2007; Lavrado et al. 2011).

Previous studies point to the fact that large or small structural differences among analogous indole alkaloids, such as cryptolepine analogs and [beta]-carbolines (harmane analogs), can lead to large differences in in vitro and in vivo antimalarial activity and cytotoxicity of these compounds. Thus, small structural differences probably modulate and ultimately define the primary mechanisms of action of these compounds (DNA intercalation, inhibition of heme polymerization, inhibition of protein synthesis, among other mechanisms yet to be revealed) (Arzel et al. 2001; Ancolio et al. 2002; Van Baelen et al. 2009).

In the present study, the antiplasmodial activity of structurally related indole alkaloids ellipticine (1), cryptolepine derivative 2b and olivacine (3) is investigated for the first time in P. berghei-infected mice and the data are compared to those for cryptolepine triflate (2a triflate). The in vitro antiplasmodial activity against chloroquine-resistant and chloroquine sensitive strains ofP. falciparum and cytotoxicity of these compounds was evaluated in vitro against murine macrophages as a means to comparatively evaluate selectivity of the antimalarial effect. The overall aim of this work was to provide comparative in vitro and especially in vivo antimalarial data for all four compounds which might lead to insights into the relative importance of cryptolepine and ellipticine ring systems/skeletons for the further development of antimalarials.

Materials and methods

Chemicals

Ellipticine (1) used in this work was obtained from two sources. Synthetic 1 was purchased from Sigma--Aldrich (Steinheim, Germany). Also, 1 was isolated from the bark of Aspidosperma vargasii from INPA's Ducke Reserve in Amazonas State, Brazil through an alkaline extraction sequence followed by column chromatography as described previously (Andrade-Neto et al. 2007; Henrique et al. 2010). Cryptolepine triflate (2a triflate) and cryptolepine analog 2b were obtained by synthesis as described previously (Lavrado et al. 2008). Olivacine (3) was isolated from Aspidosperma olivaceum from Minas Gerais State, Brazil, by acid-base extraction. The purity of these compounds was checked by TLC, UPLC-MS and NMR and was > 98%.

Culture and test for in vitro inhibition of P. falciparum parasites

Strains of P. falciparum used in this study were the antimalarial drug-susceptible 3D7 clone of the NF54 isolate and the chloroquine-resistant, pyrimethamine-resistant and cycloguanil-resistant K1 strain. Parasites were cultured according to the method of Trager and Jensen (1976) as modified by Andrade-Neto et al. (2007). The parasite culture was carried out at 37 [degrees]C with a hematocrit of 3-5% and in an atmosphere of 5% [CO.sub.2]. The parasites were maintained in vitro in A+ human red blood cells. The culture medium was RPM! 1640 (Sigma-Aldrich) supplemented with 10% human serum and containing 25 mM HEPES and 2 mM glutamine. The micro-test was performed using the method of Rieckmann et al. (1978) with modifications which were described in Andrade-Neto et al. (2007). Stock solutions of indole alkaloids ellipticine (1), cryptolepine triflate (2a triflate), olivacine (3) and 11-substituted cryptolepine hydrogen dichloride analog 2c were prepared in DMSO at a concentration of 5.0 mg/mi. Seven dilutions were performed in culture medium (RPM1 1640) of each stock sample solution resulting in final test concentrations (well concentrations) of 50-3.2 x [10.sup.-3] [micro]g/ml. Each diluted sample was tested in duplicate in 96-well test plates containing a suspension of parasitized red blood cells ata hematocrit of 3% and initial para-sitemia of 1% of synchronized young trophozoites (ring forms). The final volume of each well was 200 [micro]l. Reference antimalarial compounds chloroquine and quinine were used as positive controls at concentrations recommended by WHO (2001). The test plate was incubated for 48 h at 37 [degrees]C under the same low oxygen gas mixture used for parasite culture. After the incubation period, thin smears were prepared from the contents of each well and evaluated using a microscope. The half maximal inhibitory ([IC.sub.50]) responses compared with the drug-free controls were estimated by interpolation using Microcal Origin [R] software.

Test for in vivo suppression of Plasmodium berghei

In vivo antimalarial activity was evaluated using P. berghei NK65 strain (drug-sensitive). This strain was maintained by successive passages of blood forms from mouse to mouse. The test protocol is based on the 4-day suppressive test as described by Peters (1965). Female Webster Swiss mice weighing 26 [+ or -] 2 g were used in this study. Animals were infected intraperitoneally with 0.2 ml of infected blood suspension containing 1 x [10.sup.5] parasitized erythrocytes and randomly divided into groups of three individuals. Test groups were treated orally and subcutaneously at doses ranging from 100 to 1 mg/kg/day. Positive control groups received a dose of 10 mg chloroquine/kg/day orally or subcutaneously and negative control groups received 0.2 ml of 2% DMSO or saline. The animals were treated for 4 days starting 24 h after inoculation with P. berghei. On days 5 and 7 after inoculation with parasites, blood smears were prepared from all mice, fixed with methanol, stained with Giemsa dye, then microscopically examined (1000x magnification). Parasitemia was determined in coded blood smears by randomly counting 2000-4000 erythrocytes in the case of low par-asitemias ([less than or equal to] 10%); or up to 1000 erythrocytes in the case of higher parasitemias. Overall mortality was monitored daily in all groups during a period of 4 weeks following inoculation. The difference between the average parasitemia of control groups (100%) and test groups was calculated as a percentage of parasite growth suppression (PGS) according to the equation: PGS = 100 x (A--B)/A, where A is the average parasitemia of the negative control group and B corresponds to the parasitemia of the test group.

Cytotoxicity test

For this test, macrophages from Swiss mice were used which were described in Mota et al. (2012), with modifications. The macrophages were obtained at the time of use by collection with cold, sterile phosphate saline solution (PSB) from the exudates of the peritoneal cavity of mice. After centrifuging the peritoneal exudate solutions, the supernatant was discarded and pellet was resuspended in 5 ml of RPMI medium without FBS for counting the macrophages in a Neubauer chamber. 1 x [10.sup.5] cells were added to each well. The plate was incubated in a [CO.sub.2] incubator at 37 [degrees]C for 24 h. The cytotoxicity of the samples was determined using the methylthiazoletetrazolium (MU) colorimetric assay (Mosmann 1983). For the assays, the cells were trypsinized, washed, suspended in DMEM, and distributed into 72 wells per plate (5 x [10.sup.3] cells per well) then incubated for 18 h at 37 [degrees]C. The samples were separately diluted in DMSO and tested in triplicate at the following concentrations: 1.5, 3.1, 6.3, 12.5, 25, 50, and 100 [micro]g/ml. In parallel, we evaluated a control group consisting of RPMI 1640 without RS, a control group consisting of 1% DMSO (vehicle) and a positive control (chloroquine, BS Pharma, Belo Horizonte, MG, Brazil) at the same concentrations used for substances 1-3. After 24 and 48 h of incubation at 37 [degrees]C, 100 [micro]l of MU (5 mg/m1 in RPMI 1640 without FBS and without phenol red) was added to each well. After 3 h in a [CO.sub.2] incubator at 37 [degrees]C, the supernatant was removed and added to 100 [micro]l DMSO in each well. The absorbance of each well was obtained from a spectrophotometric reading at 562 nm. The minimum lethal doses that inhibited 50% of cell growth were obtained from the drug concentration response curves. Results are expressed in mean [+ or -]standard deviation.

Selectivity index

The relative cytotoxicity to antiplasmoclial activity for a given compound was evaluated as a selectivity index (SI), where SI = [IC.sub.50]( murine macrophages)/[IC.sub.50](P.falciparum).

Animals and ethical approval

Adult Webster Swiss albino mice (26 [+ or -] 2g weight) were used for the antimalarial and toxicity tests and received water and food ad libitum. In vivo tests were performed using Guidelines for Ethical Conduct in The Care and Use of Animals of Federal University of Rio Grande do Norte (CEUA 043/2010).

Results

Indole alkaloids ellipticine (1), cryptolepine triflate (2a triflate) and olivacine (3) and synthetic analog 1144-piperidinamino)cryptolepine hydrogen dichloride (2b) were assayed for in vitro activity against Plasmodium falciparum 1<1 e 3D7 strains and cytotoxic activity against murine macrophages. From the [IC.sub.50] values for each substance against murine macrophages and malaria parasite strains it was possible to determine selectivity indices. The in vitro results are presented in Table 1. Olivacine (3) is a previously known indole alkaloid for which antimalarial activity has not been previously described. It was the least active compound in vitro, however, it did significantly inhibit P. falciparum growth ([IC.sub.50] = 1.2 [micro]M against K1 strain). The potent in vitro activity of ellipticine (1) reported previously was confirmed herein ([IC.sub.50] values of 0.81 and 0.35 [micro]M against P. falciparum K1 and 3D7 strains, respectively). Ellipticine (1) and its structural isomer olivacine (3) were the least cytotoxic compounds in this study ([IC.sub.50] >0.41 mM, highest concentration tested). This low cytotoxicity contributed greatly to the high selectivity indices obtained for 1 and 3 against P. falciparum 3D7 (>1.2 x [10.sup.3] and >3.4 x [10.sup.2], respectively). The compound with the most in vitro activity against P. falciparum was rationally designed 11-(4-pipericlinamino)cryptolepine hydrogen dichloride (2b) ([IC.sub.50] =0.10 and 0.087 [micro]M, against P. falciparum 1<1 and 3D7 strains, respectively) which had been selected from among a number of synthetic cryptolepine analogs reported earlier based on its favorable in vitro antimalarial and cytotoxic properties reported in that earlier work (Lavrado et al. 2008). In the present work, cryptolepine analog 2b exhibited an [IC.sub.50] value against murine macrophages of 34[micro]M thus making it the second most cytotoxic compound after 2a triflate. Relatively high cytotoxicity against murine macrophages lead to 2a triflate and 2b exhibiting the lowest SI values.

Table 1 Median inhibition concentrations ([IC.sub.50]) and selectivity
indices (SI) for indole alkaloids 1-3 in vitro against Plasmodium
falciparurn K1 and 3D7 strains and murine macrophages.

Compound           [IC.sub.50]                             SI (a)
                    ([micro]M)

Name          No.           P.               Murine
                    falciparum          macrophages

                            K1     3D7

Ellipticine   1           0.81    0.35       >4.1 x        >5.0 x
                                         [10.sup.2]    [10.sup.2]
                                                         /> 1.2 x
                                                       [10.sup.3]

Cryptolepine  2a          0.80    0.91          9.1         11/10
inflate

Cryptolepine  2b          0.10   0.087           34            33
analog                                               x [10.sup.2]
                                                         /> 3.4 x
                                                       [10.sup.2]

Olivacine     3            1.4     1.2       >4.1 x          >2.9
                                         [10.sup.2]  x [10.sup.2]
                                                          /> 3.4
                                                     x [10.sup.2]

Chloroquine               0.13   0.058        1.4 x         1.1 x
diphosphate                              [10.sup.2]  [10.sup.3] /
                                                            2.4 x
                                                       [10.sup.3]

Quinine                   0.16    0.11         n.t.             -
sulfate

Artemisinin             0.0021  0.0011         n.t.             -

Data values are expressed as two (2) significant figures as per the
precision of the methods used. n.t.: not tested: -: not calculable.

(a.) SI = [IC.sub.50]("macrophages")[IC.sub.50](P.falciparum) and is
reported for K1/3D7 strains.


The indole alkaloids 1-3 were evaluated in vivo in P. berghei-infected mice in the 4-day suppressive test and the result is presented in Table 2. Ellipticine (1) was highly active at an oral dose of 50 mg/kg/day (100% inhibition versus controls on days 5 and 7). At this same dose, the mean survival time (MST) of the animals was >40 days (limit of the observation period and identical to the MST of the control substance chloroquine). Also, 1 had good oral activity on day 5 and good activity via subcutaneous injection on day 7 at 10 mg/kg/clay (77 and 70% inhibition, respectively; MST= 27-29 days) and moderate oral activity at 1 mg/kg/day (61-67% inhibition, MST= 22-23 days). 3 exhibited high oral activity at [greater than or equal to]50 mg/kg/day (90-97% inhibition, MST= 23-27 days) and low to moderate oral and subcutaneous activity at 1 and 10 mg/kg/day (7-64% inhibition, MST= 24-27 days). Cryptolepine triflate (2a triflate) exhibited only moderate oral and subcutaneous activity at 50 mg/kg/day (43-63% inhibition, MST= 24-25 clays). At a dose of 50 mg/kg/day, subcutaneously injected cryptolepine derivative 2b was lethal to infected mice (MST= 3 days) and oral activity at this dose was moderate (45-55% inhibition, MST= 25 days). At 10 mg/kg/day, 2b administered orally and subcutaneously exhibited low to moderate activity (25-60% inhibition, MST= 24 days).

Table 2

In vivo suppression of Plasmodium berghei in infected mice and
average mouse survival time after oral and subcutaneous treatments
with compounds 1-3.

Dose            % Parasite                           Average
(mg/kg/day)     inhibition                           survivi
                                                     time [+
                                                    or -] SD
                                                       (day)

                      Oral       Subcutaneous           Oral

                     Day 5  Day         Day 5  Day
                              7                  7

Ellipticine(1)

50                     100  100            NT   NT       >40

10                      77   42            33   70  27 [+ or
                                                        -] 2

1                       67   61             0   44  22 [+ or
                                                        -] 4

Cryptolepine
(2a)

50                      43   50            63   61  24 [+ or
                                                        -] 5

10                      38   28            21   46   18[+ or
                                                        -] 3

1                        3    5            16   31  17 [+ or
                                                        -] 3

Cryptolepine
analog (2b)

50                      55   44             D    D  25 [+ or
                                                        -] 4

10                      47   25            60   30  24 [+ or
                                                        -] 4

Olivacine (3)

100                     97   90            14   55  26 [+ or
                                                        -] 6

50                      91   90            NT   NT  23 [+ or
                                                        -] 2

10                      48   64            40   44  27 [+ or
                                                        -] 2

1                       25   42             7   45  24 [+ or
                                                        -] 3

Chloroquine

10                      99   98            98   99       >40

Control                  0    0             0    0  20 [+ or
                                                        -] 5

Dose
(mg/kg/day)

                 Subcutaneous

Ellipticine(1)

50                         NT

10              29 [+ or -] 3

1               23 [+ or -] 5

Cryptolepine
(2a)

50              25 [+ or -] 4

10              21 [+ or -] 3

1               22 [+ or -] 2

Cryptolepine
analog (2b)

50               3 [+ or -] 1

10              24 [+ or -] 4

Olivacine (3)

100             27 [+ or -] 2

50                         NT

10              26 [+ or -] 2

1               25 [+ or -] 1

Chloroquine

10                        >40

Control         22 [+ or -] 3

SD: standard deviation: NT: not tested: -: not calculable.


Discussion

Indole alkaloids 1-3 were active against chloroquine-resistant K1 and chloroquine-sensitive 3D7 strains ofP.falciparurn. Thus, 1-3 do not exhibit cross-resistance to chloroquine, though, as discussed below, like chloroquine, they inhibit hemozoin formation.

The in vitro activity of 1 against P. folciparurn was described for the first time by Anclrade-Neto (2007) ([IC.sub.50] = 73 nM, K1 strain) and was recently independently confirmed against the chloroquine-resistant FcM29-Cameroon strain of P. falciparurn ([IC.sub.50] = 1.13 pLM ) (Passemar et al. 2011; see also Pohlit et al. 2012). Presented herein are the first data on the antimalarial activity of olivacine (3) which exhibited important in vitro antimalarial activity and low cytotox-icitv.

During intraerythrocytic infection, P. falciparum parasites crystallize toxic heme released during hemoglobin catabolism resulting in hemozoin formation. In mechanistic studies, 1 (ICG, [IC.sub.50] = 7.9 [micro]M) exhibited greater in vitro inhibition of hemozoin crystal growth (ICG) than quinine (ICG, [IC.sub.50] = 17.1 [micro]M). ICG by 1 may occur by surface binding or a substrate sequestration mechanism (Chong and Sullivan 2003).

Alternatively, 1 may inhibit Plasmodium spp. by interaction with DNA. Highly planar, 1 intercalates DNA bases in vitro with high affinity. Also, 1 strongly inhibits DNA topoisomerase II in vitro (Moody et al. 2007). Recently, formation of covalent DNA adducts mediated by ellipticine oxidation with cytochrome P450 and peroxidases was proposed as a mode of action (Kotrbova et al. 2011).

Cryptolepine triflate (2a triflate) exhibited [IC.sub.50] values of 0.80 and 0.91 [micro]M against the 1(1 and 3D7 strains of P. falciparum, respectively, in agreement with earlier reports for 2a against la (Cimanga et al. 1997; Wright et al. 2001) and 3D7 strains (Lavrado et al. 2011). Herein, 2a triflate was the most toxic compound to murine macrophages ([IC.sub.50] = 9.4 [micro]M) and its selectivity indices (SI) were the lowest of all compounds studied (SI = 10-12).

Lavrado et al. (2008) synthesized analogs of 2a containing diamino-alkane side chains at C-11. The basis for this approach was the observation that a basic amino side chain is a requirement for chloroquine accumulation in the acidic digestive vacuole of the parasite. These analogs of 2a were potent inhibitors ([IC.sub.50] = 20-455 nM) of P. falciparum strains having different drug resistance phenotypes (Lavrado et al. 2008, 2011). Herein, the rationally designed 11-(4-piperidinamino)cryptolepine 2b which has optimal in vitro antimalarial and SI (Lavrado et al. 2008) was the most active compound against P. falciparum in vitro.

Cryptolepine analog 2b was less toxic ([IC.sub.50] = 34 [micro]M) to macrophages than 2a and exhibited good SI. Similarly, 2a tri-flate and 2b exhibited low toxicity to HUVEC cells ([IC.sub.50] = 1.18 and 2.04 [micro]M, respectively) (Lavrado et al. 2008, 2011). In previous work, 2a exhibited high cytotoxic, genotoxic, DNA intercalating and topoisomerase II inhibitory properties (Bonjean et al. 1998). 2a iodide, its derivatives and analogs (e.g. 2,7-dibromocryptolepine) cause DNA damage in lymphocytes, but do not affect human sperm. Due to in vitro DNA damage by 2a, 2,7-dibromocryptolepine and related compounds, these substances may not be suitable for preclinical development as antimalarials (Gopalan et al. 2011).

Ellipticine (1), olivacine (3) and related ellipticine-like compounds have received attention due to their high toxicity to tumor cells and low number of side effects. Thus, derivatives of these compounds are excellent target compounds for clinical studies (Sizum et al. 1988; Jasztold-Howorko et al. 2004). In tumor cells, there is evidence that the mechanism of action involves DNA intercalation and interference with the activity of topoisomerase II with consequent cytotoxic effects which are related to size, shape and flatness of 1 and 3 (Carvalho and Laks 2001; Braga et al. 2004). Importantly, we observed low cytotoxicity for 1 and 3 against mouse macrophages whereas 2a triflate and cryptolepine derivative 2b exhibited relatively high toxicity to macrophages.

As seen above, compounds 1-3 inhibit P. falciparum in vitro. Only cryptolepine has been studied previously using in vivo antimalarial models. So, the effects of 1-3 in P. berghei-infected mice were explored. Ellipticine (1) was the most active compound in vivo suppressing parasitemia by 100% and providing MST of >40 days at an oral dose of 50 mg/kg/day. Remarkably, this was the same result obtained for control compound chloroquine at 10 mg/kg/day for all animals. Also, up to 77% inhibition of parasitemia was observed for 1 at doses of 10 mg/kg/day. Few compounds (e.g. chloroquine) significantly reduce parasitemia in mice infected with P. berghei. At the highest dose of 1 (50 mg/kg/day), no mortality or other signs of intoxication were observed. The in vivo antimalarial activity of 3 was lower than that of its structural isomer 1. No toxic effects were observed in mice which were administered 3 at up to 100 mg/kg/day.

The in vivo antimalarial activity of 2a triflate was low to moderate. Thus, 2a triflate administered subcutaneously (50 mg/kg/day) inhibited P. berghei by 63% (day 5). Similarly, Kirby et al. (1995) observed that parasitemia in P. berghei-infected mice was not reduced by subcutaneously administered cryptolepine (113 mg/kg/day).

Cryptolepine iodide (2a iodide) was toxic to P. berghei-infected mice by intraperitoneal injection at 12.5 mg/kg/day (Wright et al. 2001). Toxicity is related to the intraperitoneal route of administration. However, no deaths were reported when 2a iodide was administered subcutaneously (at 113 mg/kg/day) or orally (50 mg/kg/day) (Wright et al. 2001). Herein, 2a triflate exhibited no toxic effects.

Cryptolepine analog 2b exhibited moderate activity in vivo and acute toxicity. Orally at 50 mg/kg/day, 2b inhibited the growth of P. berghei by 55%. However, subcutaneously at 50 mg/kg/day, 2b killed all mice after the second dose. Subcutaneously, at 10 mg/kg/day, 2b caused ulceration at the place of injection and inhibited P. berghei only moderately (60%).

Cryptolepine triflate (2a triflate) and derivatives (e.g. 2,7-dibromocryptolepine and 2b) exhibit limited efficacy in P. berghei-infected mice. Also, cytotoxicity, acute toxicity and genotoxicity are potential drawbacks to their use as drugs. Importantly, structural isomers 1 and 3 exhibited good in vitro SI and 1 provided P. berghei-infected mice with mean survival times greater than 40 days. These data reveal the potential of ellipticine (1) and olivacine (3) as antimalarial leads.

Acknowledgements

This work was supported by grants from the Brazilian Malaria Network (CNIki 555665/2009-7 and CNN 555669/2009-2), Bionorte CNPci 554317/2010-9, MCT/INPA/INPA, PRONEX/FINEP 023/2009 and The Science and Technology Foundation of Portugal (PTDC/SAU-FAR/114864/2009 and PEst-OE/SAU/UI4013/2011). LFRS, AMP, AM, RCNA, GD and DCS thank FAPEAM and/or CNN for individual fellowships received.

References

Ancolio, C., Azas, N., Mahiou, V., 011ivier, E., Di Giorgio, C., Keita, A., Timon-David, P., Balansard, G.. 2002. Antimalarial activity of extracts and alkaloids isolated from six plants used in traditional medicine in Mali and Sao Tome. Phytotherapy Research 16, 646-649.

Andrade-Neto, V.F., lit, A.M., Pinto, A.C., Silva, E.C., Nogueira. K.L., Melo, M.R., Hen- rique, M.C., Amorim, R.C., Silva, LF., Costa, M.R., Nunomura, R.C., Nunomura, S.M., Alecrim, W.D., Alecrim, M.G., Chaves, F.C., Vieira, P.P., 2007. In vitro inhibition of Plasmodium falciparum by substances isolated from Amazonian antimalarial plants. Memorias do Instituto Oswaldo Cruz 102.359-365.

Arzel, E., Rocca, P., Grellier, P., Labaeid, M., Frappier, F., Gueritte, F., Gaspard, C., Mar-sais, F., Godard, A., Queguiner, G., 2001. New synthesis of benzo-8-carbolines, cryptolepines, and their salts: in vitro cytotoxic, antiplasmodial, and antitry-panosomal activities of 8-carboli nes, benzo-8-carbolines, and cryptolepines. Journal of Medicinal Chemistry 44, 949-960.

Besselievre, R., Husson, H.-P., 1981. Syntheses in the ellipticine-olivacine series: a possible biogenetic model. Tetrahedron 37 (Suppl. 1), 241-246.

Bonjean, K., De Pauw-Gillet, M.C., Defresne, M.P., Colson, P., Houssier, C., Das-sonneville, L, Bailly, C., Greimers, R., Wright, C., Quetin-Leclercq, J., Tits, M., Angenot, L. 1998. The DNA intercalating alkaloid cryptolepine interferes with topoisomerase ll and inhibits primarily DNA synthesis in B16 melanoma cells. Biochemistry 37, 5136-5146.

Braga, S.F.. Melo, L.C., Baroneb, P.M.V.B., 2004. Semiempirical study on the electronic structure of antitumor drugs ellipticines, olivacines and isoellipticines. Journal of Molecular Structures 710, 51-59.

Carvalho, A.C.M., Laks, B., 2001. Investigation of geometry and some electronic properties of AZA analogues of the ellipticine and olivacine derivatives. Journal of Molecular Structures 539, 273-278.

Chevallier-Multon, M.C., Jacquemin-Sablon, A., Besselievre, R., Husson, H.P., Le Pecq, J.B., 1990. Comparative cytotoxicities of a series of ellipticine and olivacine derivatives on multidrug resistant cells of human and murine origins. Anti-Cancer Drug Design 5,319-335.

Chong, C.R., Sullivan Jr., D.J.. 2003. Inhibition of heme crystal growth by antimalarials and other compounds: implications for drug discovery. Biochemical Pharmacology 66, 2201-2212.

Cimanga, K., De Bruyne. T., Pieters, L, Vlietinck, A.J., Turger, C.A., 1997. In vitro and in vivo antiplasmodial activity of cryptolepine and related alkaloids from Clyptolepis sanguinolenta. Journal of Natural Products 60, 688691.

Gopalan, R.C., Emerce, E., Wright, C.W., Karahalil, B., Karakaya, A.E., Anderson, D., 2011. Effects of the anti-malarial compound cryptolepine and its analogues in human lymphocytes and sperm in the comet assay. Toxicology Letters 207, 322-325.

Guillonneau, C.. Nault, A., Raimbaud, E., Leonce, S., Kraus-Berthier, L, Pierre, A., Goldstein, S., 2005. Cytotoxic and antitumoral properties in a series of new, ring D modified, olivacine analogues. Bioorganic & Medicinal Chemistry 13, 175-184.

Henrique, M.C., Nunomura, S.M., Pohlit, A.M., 2010. Alcaltiides inderlicos das cas-cas de Aspidosperma vargasii e A. desmanthum. Quimica Nova 33, 22842287.

Jasztold-Howorko, R., Croisy, A., Carrez, D., Jaroszewicz, I., Nasulewicz, A., Pelczyn-ska, M., Opolski, A.. 2004. Synthesis, structure, and cytostatic properties of new olivacine derivatives. Archiv der Pharmazie (Weinheim) 337, 599-604.

Kaur, K., Jain, M., Kaur, T., Jain, R., 2009. Antimalarials from nature. Bioorganic & Medicinal Chemistry 17, 3229-3256.

Kirby, G.C., Paine, A., Warhurst, D.C., Noainese. B., Phillipson, D., 1995. In vitro and in vivo antimalarial activity of cryptolepine, a plant derived indoloquinoline. Phytotherapy Research 9, 359-363.

Kotrbova, V., Mrazova, B., Moserova, M., Martinek, V., Hodek, P., Hudecek, J., Frei, E., Stiborova, M., 2011. Cytochrome b(5) shifts oxidation of the anticancer drug ellipticine by cytochromes P450 1A1 and IA2 from its detoxication to activation, thereby modulating its pharmacological efficacy. Biochemical Pharmacology 82, 669-680.

Kumar, S., Culla, M., Choubey, V., Maity, P., Bandyopadhyay, U., 2007. Antimalarial drugs inhibiting hemozoin (13-hematin) formation: a mechanistic update. Life Sciences 80, 813-828.

Lavrado, J., Cabal, G.G., Prudencio, M., Mota, M.M., Gut, J., Rosenthal, P.J., Diaz, C., Guedes, R.C., Dos Santos. DJ., Bichenkova, E., Douglas, K.T., Moreira, R., Paulo, A.. 2011. Incorporation of basic side chains into cryptolepine scaffold: structure-antimalarial activity relationships and mechanistic studies. Journal of Medicinal Chemistry 54,734-750.

Lavrado. J., Paulo, A., Gut, J., Rosenthal, P.J., Moreira, R., 2008. Cryptolepine analogues containing basic aminoalkyl side-chains at C-11: synthesis, antiplas-modial activity, and cytotoxicity. Bioorganic & Medicinal Chemistry Letters 18, 1378-1381.

Moody, D.L., Dyba, M., Kosakowska-Cholody, T., Tarasova, N.I., Michejda, C.J., 2007. Synthesis and biological activity of 5-aza-ellipticine derivatives. Bioorganic & Medicinal Chemistry Letters 17, 2380-2384.

Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55-63.

Mota, M.L, Lobo, L.T.. Gal berto da Costa, J.M., Costa, LS., Rocha, H.A., Rocha, E., Silva, LF., Pohlit, A.M., Andrade-Neto, V.F., 2012.1n vitro and in vivo antimalarial activity of essential oils and chemical components from three medicinal plants found in northeastern Brazil. Planta Medica 78, 658-664.

Oliveira, F.Q. Junqueira, R.G., Stehmann, J.R., Branclao, M.G.L., 2003. Potencial das plantas medicinais como fonte de novos antimalaricos: especies indicadas na bibliografia etnomedica brasileira. Revista Brasileira de Plantas Medicinais 5, 23-31.

Passemar, C., Salery, M., Soh, P.N., Linas, M.D., Ahond, A., Poupat, C., Benoit-Vical, F., 2011. Indole and aminoimidazole moieties appear as key structural units in antiplasmodial molecules. Phytomedicine 18,1118-1125.

Peters, W., 1965. Drug resistance in Plasmodium berghei Vincke and Lips, 1948. I. Chloroquine resistance. Experimental Parasitology 17, 80-89.

Plowe, C.V., 2009. The evolution of drug-resistant malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene 103 (Suppl. 1), S11-S14.

Pohlit, A.M., Rocha e Silva, LF., Henrique, M.C., Montoia, A., Amorim, R.C.N., Nunomura, S.M., Andrade-Neto, V.F., 2012. Antimalarial activity of ellipticine. Phytomedicine 19, 1049.

Rieckmann, K.H., Campbell, G.H., Sax, LJ., Mrema, j.E., 1978. Drug sensitivity of Plasmodium falciparum. An in vitro microtechnique. Lancet 1,22-23.

Schmidt, T.J., Khal id, S.A., Romanha, A.J., Alves, T.M.A., Biavatti, M.W., Brun, R., Costa, F.B., Castro, S.L., Ferreira, V.F.. Lacerda, M.V.G., Lago, J.H.G., Leon, LL, Lopes, N.P., Amorim, R.C.N., Niehues, M., Ogungbe, I.V., Pohlit, A.M., Scotti, M.T., Setzer, W.N., Soeiro, M.N.C., Steindel, M., Tenwone, A.G., 2012a. The potential of secondary metabolites from plants as drugs or leads against protozoan neglected diseases. Part I. Current Medicinal Chemistry 19,2128-2175.

Schmidt, T.J., Khalid, S.A., Romanha, A.J., Alves, T.M.A., Biavatti, M.W., Brun, R., Andrade Neto, V.F.. Costa, F.B., Castro, S.L., Ferreira, V.F., Lacerda, M.V.G., Lago, J.H.G., Leon, LL, Lopes, N.P., Amorim, R.C.N., Niehues, M., Ogungbe, I.V., Pohlit, A.M., Scotti, M.T., Setzer. W.N., Soeiro, M.N.C., Steindel, M.. Tempone, A.G., 2012b. The potential of secondary metabolites from plants as drugs or leads against protozoan neglected diseases - Part II. Current Medicinal Chemistry 19,2176-2228.

Sizum, P., Auclair, C., Lescot, E., Paoletti, C., Perly, B., Fermandjian, S., 1988. Stacking and edge-to-edge associations of antitumoral ellipticine derivatives are controlled in solution by interactions involving their nitrogen sites. Biopolymers 27, 1085-1096.

Trager, W.Jensen, J.B.,1976. Human malaria parasites in continuous culture. Science 193,673-675.

Van Baelen, G., Hostyn, S., Dhooghe, L, Tapolcsanyi, P., Matyus, P., Lemiere, G., Dommisse, R., Kaiser, M., Brun, R., Cos, P., Maes, L., Hajos, G., Riedl, Z., Nagy, I., Maes, B.U., Pieters, L, 2009. Structure-activity relationship of antiparasitic and cytotoxic indoloquinoline alkaloids, and their tricyclic and bicyclic analogues. Bioorganic & Medicinal Chemistry 17, 7209-7217.

WHO, World Health Organization, 2001. In vitro micro-test (MARK 111) for the response of Plasmodium falciparum to cloroquine, mefloquine, quinine, amodiaquine, sulfadoxineipyrimethamine and arteminisin. In: CTD/MAL/97.20.

WHO, World Health Organization, 2010. World Malaria Report 2010. World Health Organization, Geneva (Switzerland).

Willcox, M., 2011. Improved traditional phytomedicines in current use for the clinical treatment of malaria. Planta Medica 77, 662-671.

Wright, C.W., 2005. Traditional antimalarials and the development of novel antimalarial drugs. Journal of Ethnopharmacology 100,67-71.

Wright, C.W., Addae-Kyereme, J., Breen, A.G., Brown, I.E., Cox, M.F., Croft, S.L., Gokcek, Y., Kendrick, H., Phillips, R.M., Pollet, P.L., 2001. Synthesis and evaluation of cryp-tolepine analogues for their potential as new antimalarial agents. Journal of Medicinal Chemistry 44, 3187-3194.

L.F. Rocha e Silva (a), (b), (c), A. Montoiaa (a), (b) R.C.N. Amorim (a), M.R. Melo (a), M.C. Henrique (a), (b), S.M. Nunomura (a), M.R.F. Costa (d), V.F. Andrade Neto (e), D.S. Costa (a), (c), G. Dantas (e), J. Lavrado (f), R. Moreira (f), A. Paulo (f), A.C. Pinto (g), W.P. Tadei (a), R.S. Zacardi (h), M.N. Eberlin (h), A.M. Pohlit (a), *

(a) National Institute for Amazonian Research, Av. Andre Aratijo, 2936, Aleixo, 69060-001 Manaus, Amazonas, Brazil

(b) Federal University of Amazonas, Av. General Rodrigo Otavio, 3000, Coroado, 69077-000 Manaus, Amazonas, Brazil

(c) University Center of the North, R. Dez de Julho, 873, Centro, 69010-060 Manaus, Amazonas, Brazil

(d) Heitor Vieira Dourado Tropical Medicine Foundation, Av. Pedro Teixeira, 25, Dom Pedro, 69040-000 Manaus, Amazonas, Brazil

(e) Federal University of Rio Grande do Norte, CP. 1524, Campus Universitario Lagoa Nova, 59078-970 Natal, Rio Grande do Norte, Brazil

(f) University of Lisbon, Alameda da Universidade, 1649-004 Lisbon, Portugal

(g) Federal University of Rio de Janeiro, Av. Athos da Silveira Ramos, 149, Cidade Universitdria, 21941-909 Rio de Janeiro, Brazil

(h) State University of Campinas, Campus Universittirio Zeferino Vaz SN, Cidade Universitaria Campinas, 13083-970 Campinas, Silo Paulo, Brazil

* Corresponding author. Tel.: +55 92 3643 3177; tax: +55 92 3b43 3U/V. E-mail addresses: ampohlit@inpa.gov.br, ampohlit@gmail.com (A.M. Pohlit).

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Author:Silva, L.F. Rocha e; Montoiaa, A.; Amorim, R.C.N.; Melo, M.R.; Henrique, M.C.; Nunomura, S.M.; Costa
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Dec 15, 2012
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