Accepted Manuscript Synthesis and antimalarial evaluation of prodrugs of novel fosmidomycin analogues Ana Maria Faísca Phillips, Fátima Nogueira, Fernanda Murtinheira, Maria Teresa Barros PII: DOI: Reference:

S0960-894X(15)00293-0 http://dx.doi.org/10.1016/j.bmcl.2015.03.077 BMCL 22565

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

4 March 2015 24 March 2015 25 March 2015

Please cite this article as: Phillips, A.M.F., Nogueira, F., Murtinheira, F., Barros, M.T., Synthesis and antimalarial evaluation of prodrugs of novel fosmidomycin analogues, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.03.077

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Graphical Abstract

Synthesis and antimalarial evaluation of novel fosmidomycin analogues

Leave this area blank for abstract info.

Ana Maria Faísca Phillips, Fátima Nogueira, Fernanda Murtinheira and Maria Teresa Barros OH N O O O

O

P O O

O

15 O

IC50 (P. falciparum Dd2) = 27.4 nM LD50 (HepG2) = 25.6 µ M SI = 935

Bioorganic & Medicinal Chemistry Letters j o ur n al h om e p a g e : w w w . e l s e v i er . c o m

Synthesis and antimalarial evaluation of prodrugs of novel fosmidomycin analogues Ana Maria Faísca Phillips a, ∗, Fátima Nogueirab, Fernanda Murtinheirab and Maria Teresa Barros a, ∗ a

LAQV, REQUIMTE, Department of Chemistry, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, Quinta da Torre, 2829-516 Caparica, Portugal b Medical Parasitology Department, GHTM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira nº 100, 1349-008 Lisboa, Portugal

A R T IC LE IN F O

A B S TR A C T

Article history: Received Revised Accepted Available online

The continuous development of drug resistance by Plasmodium falciparum, the agent responsible for the most severe forms of malaria, creates the need for the development of novel drugs to fight this disease. Fosmidomycin is an effective antimalarial and potent antibiotic, known to act by inhibiting the enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), essential for the synthesis of isoprenoids in eubacteria and plasmodia, but not in humans. In this study, novel constrained cyclic prodrug analogues of fosmidomycin were synthesized. One, in which the hydroxamate function is incorporated into a six-membered ring, was found have higher antimalarial activity than fosmidomycin against the chloroquine and mefloquine resistant P. falciparum Dd2 strain. In addition, it showed very low cytotoxicity against cultured human cells.

Keywords: antimalarial fosmidomycin hydroxamates phosphonates phospha-Michael

Malaria is an infectious disease, which the World Health Organization considered a priority to eradicate in its Millennium Development Goals in 2000. As a result of measures adopted since then, in 2013 there were 198 million cases of malaria, which resulted in approximately 584 000 deaths, already a decrease of 30 % relative to 2000.1 Although it is endemic to the tropical and subtropical regions of the World, as a result of travelling, it is estimated that 3.2 billion people are at risk of contracting the disease. Disease control involves both prevention and treatment. Many drugs used in the past, e.g. chloroquine or sulfadoxine-pyrimethamine, can nowadays be used in some parts of the world only, as a result of the development of resistance by Plasmodium falciparum, the agent which causes the severest forms of the disease.2 In the Mekong region of Southeast Asia there have even been reports of resistance to the artemisinins, the latest class of drugs to be developed for malaria treatment,3 even though it is recommended by WHO that they are used as combination therapies only, to avoid the spread of resistance.4 Novel medicines are thus continuously needed, both to replace existing medicines and to provide improved treatments. A substance that is being developed for malaria treatment is fosmidomycin (A), a phosphonohydroxamic acid originally isolated as a natural antibiotic from Streptomyces lavendulae.5 Fosmidomycin has potent antimicrobial activity. It is known to ———

2009 Elsevier Ltd. All rights reserved.

inhibit the enzyme 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, a key enzyme in the non-mevalonate pathway for the synthesis of isoprenoids in Plasmodia, eubacteria and plants.6-7 This pathway is an ideal target for drug development, since it is absent in animals and humans, where isoprenoids are synthesized via the mevalonate pathway.

OH N

H

O P OH H3C OH

O

A: Fosmidomycin

OH N O

O P OH H OH

OH N

O P OH OH

O

B: FR900098

C

Cl Cl

O OH N

H3C O

H

H

D

O

OH P OH

OH N

H

O P OH OH

O

E

O O O P O O O

OBn N CH3 O

F

Figure 1. Structures of fosmidomycin, FR900098 and some known promising antimalarial analogues. The development of analogues is also a fertile area of research. This involves both modifications of the functional

∗ Corresponding author. Tel.: +351-212949647; fax: +351-212948550; e-mail: [email protected] ∗ Corresponding author. Tel.: +351-212948300; fax: +351-212948550; e-mail: [email protected]

7

groups, e.g. as in FR900098 (B, Fig. 1), or modifications in the carbon chain joining the two functional groups, e.g. as in C.8 FR900098 is about 2-fold more active against P. falciparum in vitro, but all bacteria are more sensitive to fosmidomycin.9 Restriction of rotational freedom is another strategy often used in medicinal chemistry to improve the properties of a drug and conformationally constrained analogues like D10 or E11 have also been developed. The activity of analogues is often tested either against live parasites or the reductoisomerase enzymes PfDXR and E. coliDXR. Due to the high charge which phosphonate groups have at some physiological pHs, conversion into prodrugs which can help improve oral bioavailability or cell penetration and hence provide a higher drug concentration at the target site, are also used.12 Hence, prodrugs involving esterification of the phosphonate hydroxyl groups into esters which are hydrolysed by enzymes, have also been developed. The pivaloyloxymethyl ester group used in conformationally constrained F13 and the aryl ester group14 are examples. An enhancement of in-vitro activity has also been observed in some of these cases.15

O

O

b

Br

Br

1

n

7a n = 0 7b n = 1

n

Phospha-Michael

n H: n = 1, 2

Figure 2. Retrosynthesis of cyclic phosphonohydroxamates G. Since the change from a formyl to an acetyl group leads to an increase in antimalarial activity, as observed for FR900098, and many of the analogues in which the only modification is the presence of alkyl/aryl substituents at the position α to phosphorus still have satisfactory activity,19 we envisaged that enclosing the two ends of the molecule into a ring as in G (Fig. 2), could lead to novel conformationally constrained analogues with good properties. A recent publication describing that a modification of the N-acetyl terminus to N-propionyl gave a more active analogue supports this reasoning.20 The “ideal” 3-carbon spacer between the phosphonyl and the carbamoyl groups would still be present,19 and a tighter structure may “fit” better into the DOXP reductoisomerase binding-pocket. To the best of our knowledge, cyclic phosphonohydroxamates in which the phosphonyl and the carbonyl groups are included in a six-membered-ring and bear a 1,3-relationship have not yet been described.

b

PhS

Br

n

Br

Br

3a n = 0 3b n = 1

O

e

OH N

HO P HO O

OBn N O

d

Ph

O

G: n = 1, 2

2a

O S

O P OH OH

B

Recently we described the synthesis of αhydroxyphosphonates which exhibited moderate antimicrobial activity. 16 In continuation of our program on the development of methodology to synthesize phosphonates with useful biological properties,17 we became interested in phosphonohydroxamates, not only because of the possibility of antimalarial and antimicrobial activity, but also because hydroxamates on their own are important pharmacophores.18 They are metal scavangers, particularly of iron, metallo-enzyme inhibitors and can generate nitric oxide. As a result they can be antimicrobial, antiinflammatory, anti-tumor, metal detoxifying agents and hypotensives, and can even be used to reactivate chymotrypsin and acetylcholinesterase when needed. New structures could lead to the development of new drugs.

a

OH N

2b

c BnONH3Cl (4) BnONHCO2Et (5) O

PhS

N OEt OBn

n

N OEt OBn

6a n = 0 6b n = 1 h, n = 1

f, n = 0

OBn N O

9

g

OBn N O

8

S O

OBn O N Ph

Ph

10

S O

i

OBn O N

11

Scheme 1: Synthesis of key intermediates 9 and 11. Reagents and conditions: (a) HBr, H2SO4, reflux; (b) PhSH, DMF, K2CO3, reflux; (c) ethyl chloroformate, pyridine, r.t.; (d) K2CO3, MeCN, reflux; (e) NaIO4, H2O, MeOH, r.t.; (f) LHMDS, THF, 0 ºC; (g) toluene, reflux; (h) LHMDS, THF, r.t.; (i) toluene, reflux.

The synthesis of small and medium size monocyclic hydroxamic acids was reviewed recently.21 One publication, describing a new approach to these substances as a means to obtain polydentate chelators called our attention.22 The authors reported that an intermediate six-membered cyclic α-sulfonyl carbamate synthesized, could be converted into an unsaturated Nbenzyloxy lactam of type H via a reverse [3+2] cycloaddition. Such a substance seemed ideal for our purposes, as the 1,4unsaturated system is a potential Michael acceptor, although, so far, to the best of our knowledge, the phospha-Michael reaction of unsaturated hydroxamates does not appear to have been reported yet.23 We set out to try, and report now the results of our findings.

We envisioned that the addition of a phosphorus nucleophile to the unsaturated system could give the desired phosphonohydroxamate. Phospha-Michael reactions that use a dialkyl or trialkyl phosphonate as nucleophiles are known. 23a In the first case the reaction is usually promoted by base, in the second often by heat. For example, tetramethylguanidine (TMG), a commercially available base, has been used to promote 1,4addition to unsaturated esters, nitriles and certain ketones in moderate to good yields and mild conditions (0 ºC or r.t. and short reaction times). 27 Some ketones gave 1,2-addition, e.g. methyl vinyl ketone. In these reactions a large excess (40 fold) of dialkylphosphonate was used and a catalytic amount of base. We were interested to see if this method could be extrapolated to unsaturated hydroxamates and in finding conditions to operate closer to equimolar amounts of phosphite / hydroxamate, which would be more convenient if the method were to be applied to large-scale synthesis. Using a 1.2 M solution of the hydroxamate in acetonitrile, we tried the reaction initially with a 1.5:1 ratio of phosphite to hydroxamate, and five-fold excess of base, at room temperature. After 20 h, the presence of a signal at 27.85 ppm in the 31P NMR spectrum showed that less than 30% of the product had formed. If the reaction was performed at 100 ºC, after 7 h, only 25% of the starting material remained to react, according to proton NMR spectroscopy. However, at the same temperature, if the proportion of base to hydroxamate was lowered to nearly 1:1, even in the presence of a 3-fold excess of phosphite, the reaction rate dropped considerably and only about 30% of the product was obtained after 17 h. Eventually the optimum conditions were found to be a ratio of phosphite : hydroxamate : TMG equal to 2:1:5 in the absence of solvent. After 19 hours the ratio of desired phosphonohydroxamate to unreacted starting material was 85:15, as determined by proton NMR spectroscopy, and it remained basically the same after 24 hours. In the spectrum of the crude product the only signals visible were those of the product and remaining unreacted starting material. Under these conditions, after purification by column chromatography, 12 was obtained in 60% yield (Scheme 2). The remaining starting material could be recovered and reacted again. The structure of 12 was confirmed by 1D and 2D NMR spectroscopy, by IR spectroscopy and elemental analysis.28

The key intermediate for the synthesis of the six-membered ring cyclic fosmidomycin analogue, unsaturated hydroxamate 9, was obtained by a known procedure,22 and all the intermediates and precursors are known compounds (Scheme 1).22, 24-25 For the preparation of key intermediate 11, a similar approach was used (Scheme 1). However, to the best of our knowledge, compounds 3b, 6b-7b and 10-11 have not been previously described in the literature. Sulfide 3b, obtained via base-catalyzed alkylation of thiophenol, was reacted with N-benzyl carbamate 5, to give Nprotected alkylcarbamate 6b in good yield. Sulfide oxidation with sodium metaperiodate proceeded smoothly to give sulfoxide 7b which was pure enough to be used in the next reaction without further purification. Treatment of the sulfoxide dissolved in freshly distilled THF with lithium bis(trimethylsilylamide) (LHMDS) at 0 ºC, produced a stabilized carbanion which, when left to react at room temperature, gave rise to 2-azepanone 10 by intramolecular cyclization. An examination of the 1H NMR spectrum of the crude product showed that it consisted of a nearly 1:1 mixture of diastereoisomers, in which the diastereoisomeric N-benzyloxy methylene protons gave rise to two distinct sets of two doublets each. For identification purposes the diastereoisomers could be separated by chromatography on silica gel. In the linear precursors, this distinct set of doublets collapses into a single singlet. The cyclic β-keto sulfoxide was then heated-up in refluxing toluene to promote a reverse [3+2] cycloaddition22,26 which resulted in the dehydrosulfenylation of 10, to give key intermediate 11 in good yield. OBn O N

OBn O N

b

OBn N O

e n=1

P O HO OH

P O EtO OEt

17

OBn N O

a

b

n=0

n

P O EtO EtO

9 n=0 11 n = 1

16

OBn N O

P O HO HO

12

13 c

c OBn O N O

OH O N

f

O

O P O O O O O

P O O O O O

18 O

OH N O

d O

O

O

P O

O

O

O

15 O

P O O

O

O

19 O

OBn N O

14 O

Scheme 2: Synthesis of prodrugs 15 and 19. Reagents and conditions: (a) (EtO)2 P(O)H, TMG, neat, r.t.; (b) TMSBr, CH2Cl2, r.t.; (c) MeOH, H2O, r.t.; (d) H2, Pd-C, 3 atm, r.t., 17 h; (e) (EtO)2P(O)H, TMG, MeCN, 100 o C; (f) H2, Pd-C, 2 atm, r.t., 19 h.

We were interested in converting our cyclic analogues into potential prodrugs. Simple alkyl esters of phosphonates are metabolically stable, but the acyloxyalkyl esters, of which F (Fig. 1) is an example, are commonly used as prodrugs. A recent study with FR900098 showed that, after chemical modification of the phosphonic acid group with several acyloxyalkyl ester groups, the pivaloyloxymethyl (POM) group gave the highest antimalarial activity. 29 It is hydrolysed by non-specific esterases to liberate the biologically active phosphonic acid. This functional group is nowadays used in two antiviral phosphonates currently on the market, adefovir dipivoxyl and tenofovir disoproxil.12 We decided to use a similar strategy for our analogues. Hence cyclic phosphonohydroxamate 12 was hydrolysed with TMSBr and the resulting crude phosphonic acid esterified with choloromethyl pivalate in the presence of triethyl amine,30 to give bis(POM) 14 in 75% yield. Palladium catalyzed hydrogenation gave target 15, with a free hydroxamic acid group ready to fulfill its enzyme inhibition role (Scheme 2). 31 Phospha-Michael addition to the seven-membered ring proved to be more problematic. When a 1.5:5:1 phosphite-basehydroxamate mixture was reacted in acetonitrile at 100 ºC, after 24 h there was still more starting material than the desired product, in ca 1.0:0.7 ratio as observed by from a proton NMR spectrum. In addition, another unidentified product was also obtained, resulting presumably from an initial direct 1,2-addition of phosphite to the carbonyl group. In a solventless reaction at room temperature, with 2-fold excess of phosphite and 5-fold excess of base, after 26 h only traces of product were observed. Addition of more reagent in a stepwise manner was also tried, but the yield of cyclic phosphonohydroxamate did not improve. Eventually, to obtain the desired product for preparative purposes, the reaction was performed in acetonitrile for 24 h at 100 ºC, the product isolated, and the crude product reacted again under the same conditions for 17 h more. Under these conditions, pure phosphonohydroxamate 16 was obtained after chromatography on silica gel in 20% yield with respect to the unsaturated hydroxamate used initially (Scheme 2). Unreacted starting material could also be recovered. The structure of the product was confirmed by NMR and IR spectroscopy and by elemental analysis. Hydrolysis of the phosphonate ester groups, esterification with chloromethyl pivalate and debenzylation with Pd-C catalyzed hydrogenation were performed in a similar manner to that used for the six-membered phosphonohydroxamate, to produce prodrug 19 which was afterwards used for biological activity studies. Full experimental details and characterization data are provided in the accompanying supplementary information file. The antimalarial activity of the new phosphonohydroxamates was determined in vitro against a P. falciparum Dd2 strain, resistant to chloroquine and mefloquine. The SYBR Green I assay was used and chloroquine and mefloquine as reference drugs.32-33 The results are reported in Table 1. Literature values for the activity of fosmidomycin and FR900098 against the PfDd2 strain are included for comparison.34,15 The new compounds were also evaluated for their in vitro cytotoxicity against human hepatic (HepG2-A16) cells by the tetrazolium dye (MTT) colorimetric assay for cellular growth and viability,35-36 and the results are also presented in Table 1. Whereas the sixmembered ring cyclic phosphonohydroxamate 12, with ethyl ester substituted phosphonic acid groups, showed no significant inhibition of P. falciparum, POM analogue 15 showed high activity, being even more active than either fosmidomycin or FR900098. The seven-membered ring analogue was much less effective. In compound 15, the chain size between phosphorus and nitrogen has the “ideal” three-carbon length,19 previously

observed by others to give the more active compounds, the lipophilicity is higher, and presumably the tighter configuration gives a “better fit” to the enzyme binding pocket, resulting in a more potent analogue. In addition, from the data in Table 1, the selectivity index (SI), defined as the ratio of the LD50 value determined on mammalian cells (cytotoxicity) to the IC50 value determined on P. falciparum (the anti-plasmodium activity), can be calculated. For 15, the calculated SI = 935. Since it is generally considered that pharmacological efficacy is not due to in vitro cytotoxicity when the selectivity index (SI) >10, it can be deduced 37 that 15 has no significant toxicity against cultured human cells. Hence it can be concluded that cyclic phosphonohydroxamate 15 is a promising lead as antimalarial therapeutic agent. Table 1. In vitro growth inhibition (IC50) against the P. falciparum strain Dd2 and cytotoxicity (LD50) against the human hepatic cell line HepG2-A16a

Compound

cLogPb

12

1.26±0.61

NS

NS

25.60

9.36

15

0.50±0.50

27.4

0.8

25.63

6.92

19

0.76±0.66

2240.3

117.3

20.09

3.47

Chloroquine

4.69±0.32

300

80

ND

ND

Mefloquine

2.87±0.83

92

5

ND

ND

Fosmidomycin FR900098 (B, mono Na+ salt) Bis(POM)-B

-2.47±0.63

Pf Dd2 IC50 (nM) mean SD

810

HepG2 LD50 (µM) mean SD

34

89315 1.29±0.64

27215

a

The mean value of three independent determinations is reported; NS = No significant inhibition observed; ND = Not determined; b Log P values were calculated using ACD Labs software.

Acknowledgments Financial support from Fundação para a Ciência e a Tecnologia, Ministério da Educação e Ciência, to Ana Maria Faísca Phillips is gratefully acknowledged. The NMR spectrometers are part of the National NMR Facility, supported by Fundação para a Ciência e a Tecnologia (RECI/BBB-BQB/0230/2012). Supplementary Material Supplementary data associated with this article, namely experimental protocols and full characterization data of all new compounds (3b, 6b-7b, 10-12, 14-16 and 18-19,) and their NMR spectra, can be found, in the online version, at …….. References and Notes 1.

2.

3.

4.

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29.

30. 31.

32.

33.

34.

35.

36.

37.

5.8 Hz, 2 × CH3), 23.61 (d, JCP = 4.3 Hz, C-5), 30.65 (d, JCP = 148.4 Hz, CHP), 32.73 (d, J CP = 4.1 Hz, C-3), 49.99 (d, JCP = 17.8 Hz, C-6), 62.25 (d, JCP = 5.3 Hz, 2 × POCH2 ), 75.95 (s, NOCH2), 128.5 (s, o-C, Ph), 128.8 (s, p-C, Ph), 129.6 (s, m-C, Ph), 135.2 (s, i-C, Ph), 165.2 (d, JCP = 18.9 Hz, C=O) ppm. 31P NMR (CDCl3 ): δ 27.85 ppm. IR (KBr): v 3058, 3056, 2983, 2940, 2903, 1672, 1560, 1570, 1542, 1498, 1492, 1468, 1455, 1408, 1374, 1320, 1287, 1272, 1248, 1207, 1163, 1103, 1056, 1023, 967, 903, 814, 783, 753, 733, 699, 652, 605 cm-1. C16H24 NO5P (341.344): calcd. C 56.30, H 7.09, N 4.10; found C 56.15, H 6.86, N 3.98. Ortmann, R.; Wiesner, J.; Reichenberg, A.; Henschker, D.; Beck, E.; Jomaa, H; Schlitzer, M. Bioorg. Med. Chem. Lett. 2003, 13, 2163. Kurz, T.; Schlüter, K.; Kaula, U.; Bergmann, B.; Walter, R. D., Geffken D. Bioorg. Med. Chem. 2006, 14, 5121. 2,2-Dimethyl-propionic acid (2,2-dimethyl-propionyloxymethoxy)(1-hydroxy-2-oxo-piperidin-4-yl)-phosphinoyloxymethyl ester (15): 1H NMR (CDCl3): δ 1.170 (s, 9 H, 3 × CH3), 1.173 (s, 9 H, 3 × CH3), x-1.85 (m, 1 H, H-5), 2.02-2.11 (m, 1 H, H-5´), 2.11-2.33 (m, 1 H, PCH), 2.41 (dt, J = 12.8, 17.2 Hz,1 H, H-3), 2.48-2.60 (dt, J = 5.2, 17.4 Hz, 1 H, H-3´), 3.25 (td, J = 4.0, 11.6 Hz, 1 H, H6), 3.32-3.45 (m, 1 H, H-6´), 5.54-5.70 (m, 4 H, 2 × OCH2 O), 6.00 (br s, 1 H, N-OH) ppm. 13 C NMR (CDCl3 ): δ 21.95 (s, C-5), 26.85 (s, 6 × CH3), 29.67 (s, C-3), 31.01 (d, JCP = 144.3 Hz, CHP), 38.75 (s, 2 × Cq, tBu), 41.55 (d, JCP = 18.4 Hz, C-6), 81.51 (appar. t, JCP = 7.4 Hz, 2 × CH2 , POCH2 ), 169.2 (d, JCP = 17.7 Hz, NCO), 176.8 (s, CO) ppm. 31P NMR (CDCl3 ): δ 29.04 ppm. IR (CH2Cl2 ): v 3220 (br), 2975, 2932, 2910, 2866, 1755, 1660, 1644, 1633, 1484, 1278, 1257, 1138, 1056, 1027, 989, 962, 900, 844, 755 cm-1. C17 H30NO9 P (423.399): calcd. C 48.23, H 7.14, N 3.31; found C 47.73, H 7.14, N 2.91. Determination of the in vitro antimalarial activity: The antimalarial activity of the compounds was determined using the SYBR Green I assay, as previously described, against a laboratory-adapted P. falciparum Dd2 chloroquine and a mefloquine-resistant strain.33 Stock solutions of the drugs were prepared in DMSO and serially diluted in complete media. Parasitized erythrocytes at the early ring stage were added to a final 1% parasitaemia and 3% hematocrit to each triplicate well of a 96-well plate, and incubated for 48 h at 37 ºC prior to growth assessment with SYBRGreenI. Each compound was analyzed at a final concentration range of 0-10 µM (0.2% DMSO), whereas chloroquine and mefloquine was assayed at a concentration range of 0-1 µM. SYBRGreenI fluorescence was quantified using a multi-mode microplate reader (Dynex Triad) and analyzed by nonlinear regression using GraphPad Prism 5 demo version. Carrasco M. P.; Newton A. S.; Gonçalves L.; Góis A.; Machado M.; Gut J.; Nogueira F.; Hänscheid T.; Guedes R. C.; dos Santos D. J.; Rosenthal P. J.; Moreira R. Eur. J. Med. Chem. 2014, 80, 523. Brücher, K.; Illarionov, B.; Held, J.; Tschan, S.; Kunfermann, A.; Pein, M. K.; Bacher, A.; Gräwert, T.; Maes, L.; Mordmüller, B.; Fischer, M.; Kurz, T. J. Med. Chem. 2012, 55, 6566. Determination of the in vitro cytotoxicity to human cells: HepG2A16 human hepatic cell line viability was determined based on the MTT assay as previously described. 36 An in vitro culture of HepG2 cells was maintained in standard culture conditions. Briefly, cells were seeded in a flat-bottomed 96-well tissue culture plate at a density of 1×104 cells/well and allowed to adhere overnight. After removing the medium, 200 µL of fresh medium containing 7 ten-fold dilutions of each compound were added, and a negative control was performed by adding 200 µL of drug free medium. The plate was incubated for 24 h under standard culture conditions. At the end of the incubation period (24h), 20 µL of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium (MTT; 5 mg/mL in PBS) was added to each well, wells were incubated for 3 h at standard culture conditions, supernatant was removed and 200 µL of acidified isopropanol was added to each well. Absorbance was read at 570 nm, to produce a log dosedependence curve. The LD50 was estimated for each compound by non-linear interpolation of the dose-dependence curve using GraphPad Prism 5 demo version. Paulo A., Figueiras M., Machado M., Charneira C., Lavrado J., Santos S. A., Lopes D., Gut J., Rosenthal P. J., Nogueira F., Moreira R. J. Med. Chem. 2014, 57, 3295. Weniger B.; Robledo S.; Arango G. J.; Deharo E.; Aragón R.; Muñoz V.; Callapa J.; Lobstein A.; Anton R. J. Ethnopharmacol. 2001, 78, 193.