Tyrosol and hydroxytyrosol derivatives as antitrypanosomal and antileishmanial agents

Accepted Manuscript Tyrosol and hydroxytyrosol derivatives as antitrypanosomal and antileishmanial agents Efres Belmonte-Reche, Marta Martínez-García, Pablo Peñalver, Verónica Gómez- Pérez, Ricardo Lucas, Francisco Gamarro, José María Pérez-Victoria, Juan Carlos Morales PII: S0223-5234(16)30340-3 DOI: 10.1016/j.ejmech.2016.04.047 Reference: EJMECH 8562 To appear in: European Journal of Medicinal Chemistry Received Date: 3 March 2016 Revised Date: 28 March 2016 Accepted Date: 17 April 2016 Please cite this article as: E. Belmonte-Reche, M. Martínez-García, P. Peñalver, V. Gómez-Pérez, R. Lucas, F. Gamarro, J.M. Pérez-Victoria, J.C. Morales, Tyrosol and hydroxytyrosol derivatives as antitrypanosomal and antileishmanial agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.04.047. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. 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MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Graphical Abstract HO HO OH HO H O H NH N S H O HO O O HO HO O IC50 T.brucei =0.36 ± 0.01 M, SI=106 IC50 T .brucei =0.63 ± 0.07 M, SI=21 IC50 T.brucei =0.74 ± 0.36 M, SI=33 HT 14 29 37 IC50 T .brucei =47.5 ± 16.9 M MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Tyrosol and hydroxytyrosol derivatives as antitrypanosomal and antileishmanial agents Efres Belmonte-Reche a , Marta Martínez-García a , Pablo Peñalver a, b , Verónica Gómez-Pérez a , Ricardo Lucas a, b , Francisco Gamarro a , José María Pérez-Victoria a, * , Juan Carlos Morales a, b, * a Department of Biochemistry and Molecular Pharmacology, Institute of Parasitology and Biomedicine López-Neyra, CSIC, Avda. del Conocimiento, 17, 18016 Armilla-Granada, Spain; b Department of Bioorganic Chemistry, Institute of Chemical Research, CSIC – University of Seville, Avda. Americo Vespucio, 49, 41092 Sevilla, Spain Corresponding author: José María Pérez-Victoria ([email protected].es) Juan Carlos Morales (jcmorale[email protected]) Abstract Trypanosomiasis and leishmaniasis keep being a real challenge for health and development of African countries. Existing treatments have considerable side effects and increase resistance of the parasites. We have measured antitrypanosomal and antileishmanial activity of natural phenols, tyrosol (TYR) and hydroxytyrosol (HT) and several of their esters and metabolites. We found significant IC 50 values against Trypanosoma brucei for HT decanoate ester and HT dodecanoate ester (0.6 and 0.36 µM, respectively). This represents a large increase in activity with respect to HT (79 and 132 fold, respectively). Moreover, both compounds displayed a high selectivity index against MRC-5, a non-tumoral human cell line (118 and 106, respectively). Then, we synthesized a focused library of compounds to explore structure-activity. We found the ether and thiourea analogues of HT decanoate ester and HT dodecanoate ester also showed IC 50 values against T.brucei in the low micromolar range. In conclusion, the di-ortho phenolic ring and medium size alkyl chain are essential for activity whereas the chemical bond among them seems less important. Keywords Antitrypanosomal, antileishmanial, tyrosol, hydroxytyrosol MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1. Introduction Infectious diseases caused by protozoan parasites affect millions of people around the world, especially in tropical and subtropical areas. Among them, leishmaniasis (caused by different species of Leishmania) is a major global health problem with around 12 million people infected and causing 1-2 million new cases every year. Trypanosoma brucei, transmitted by the tsetse fly, is responsible for sleeping sickness in humans and preclude the development of productive livestock and agricultural activity based on domesticated animals where it causes Nagana.[1-2] For these reasons, this parasite is considered to be one of the major root causes of hunger and poverty in sub-Sahara Africa. Current treatments for both diseases present important drawbacks such as high toxicity, increased resistance and variability on their efficacy depending on the different strain of the parasite. Thus, the development of new antiparasitic therapies continues to be necessary. Natural products have been tested for decades to find new antitrypanosomal and antileishmanial agents. Among them, several phenolics and polyphenols have shown relevant activity. For example, flavonoids such as 7,8-dihydroxyflavone, 3-hydroxyflavone and rhamnetin showed low micromolar IC 50 values for Trypanosoma brucei rhodesiense and others such as luteolin and quercetin also displayed low micromolar IC 50 s for Leishmania donovani[3]. In fact, quercetin glycosides present in the aqueous extract of Kalanchoe pinnata were active by oral administration in experimental cutaneous and visceral leishmaniasis infections produced in vivo[4-5]. Tyrosol (TYR, 1) and hydroxytyrosol (HT, 2) (Figure 1) are natural phenolic antioxidants present in olives and olive oil. They have shown a diverse biological activity such as antibacterial[6-7], antiviral[8], anti-inflammatory[9-12], neuroprotective[13] and anticancer activity,[14] inhibition of human LDL oxidation[15] and prevention of platelet aggregation[16]. Recently, Moradi-Afrapoli et al. reported medium activity of TYR against Trypanosoma brucei rhodesiense (IC 50 >15µM)[17]. To the best of our knowledge, HT has not been examined for activity against T.brucei. Though, moderate antileishmanial activity was reported for HT against both, promastigotes of L. infantum, L. donovani, and L. major, and against L. donovani amastigotes that parasitize J774A.1 macrophages[18]. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Figure 1. Chemical structures of tyrosol (TYR, 1) and hydroxytyrosol (HT, 2). The preparation of chemically-modified natural products to improve their antiparasitic activity is still a very active source of potential new drugs. This is the case of chalcone[19] or caracasine acid derivatives[20], or the preparation of hybrids such as Cinchona alkaloids with bile acids[21] or caffeine-based chalcones[22]. Tyrosol and hydroxytyrosol derivatives have also been synthesized in order to improve the antioxidant and biological properties of the parent compounds. Hydroxytyrosol fatty acid esters increased the protection of proteins and lipids against oxidation caused by peroxyl radicals in a brain homogenate as an ex vivo model[23]. Similarly, hydroxytyrosol acetate was able to reduce the metabolic imbalance induced by a highcholesterol diet in rats to a higher extent than HT[24]. Finally, HT alkyl ether derivatives, more stable under biological conditions than HT, have been reported to exert antiproliferative[25], neuroprotective[26-27], antiplatelet and anti-inflammatory effects[28] that are greater than those of HT. Due to the improved biological activity found for several HT derivatives with respect to HT itself, we decided to investigate the antitrypanosomal and antileishmanial activity of a series of TYR and HT derivatives. We prepared a series of tyrosol and hydroxytyrosol fatty acid esters together with three metabolites of HT and TYR, tyrosol sulfate, tyrosol glucuronate and hydroxytyrosol glucuronate for a first in vitro screening. Later, we synthesized a focused library of compounds to explore structure-activity relevance varying the number of phenolic OH groups, the type of chemical bond between the phenolic ring and the alkyl chain, and the length of the alkyl chain. 2. Results and Discussion 2.1. Chemistry Tyrosol and hydroxytyrosol fatty acid esters (3-7 and 11-16, respectively) were synthesized by enzymatic acylation using Novozym 435 and the corresponding vinyl acyl donor in t-butyl methyl ether as reported previously[29-31] (Scheme 1). Tyrosol and hydroxytyrosol glucuronates 8 and 17, 18 were prepared by glycosylation of the acetyl protected TYR or HT derivatives (3 and 10, respectively). We used the acetyl protected trichloroacetimidate glucuronosyl derivative MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 19 as glycosyl donor and boron trifluoride etherate as the catalyst in the glycosylation step. Final acetyl deprotection yielded glucuronate derivatives 8 and 17,18 as described previously[32]. TYR sulfate 9 was prepared following a similar strategy to the one used for the glucuronate derivative. Sulfation of tyrosol acetate 3 was carried out with SO 3 •NMe 3 as sulfating reagent, NEt 3 as base, and acetonitrile as solvent at 100 °C under microwave radiation. The reaction afforded the sulfated TYR derivative in good yield. Final acyl deprotection and reverse phase purification gave TYR sulfate 9. Scheme 1 We identified hydroxytyrosol decanoate ester 13 and hydroxytyrosol dodecanoate ester 14 as relevant hit compounds against T.brucei (see below, Table 2) after carrying out the first in vitro screening on T. brucei and L. donovani. We decided then to perform structure-activity studies on 13 and 14 by changing the phenolic hydroxyl groups, the type of chemical bond between the phenolic ring and the alkyl chain, and the length of the alkyl chain (Figure 2) in order to improve its biological activity. Figure 2. Parts of compound 14 to focus structure-activity studies. We synthesized a hydroxyl protected version of compound 14 by formation of the acetal derivative 22 and also a shorter version with a decanoate alkyl chain, compound 21 (Scheme 2). The cyclic acetal intermediate 20 was obtained by reaction of HT with CH 2 Cl 2 in basic conditions and then acylated using the enzymatic conditions used previously for HT yielding the desired compounds. The ether analogue of 14, compound 29, was prepared as reported previously[33] by benzyl protection of the phenolic groups, alkylation with 1-iodododecane under basic conditions and final hydrogenation. The HT ether decanoate derivative 28 was prepared following the same MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT procedure. We also prepared the p-methoxybenzyl (PMB) protected HT derivative 24 trying to improve the yields of the route. However, deprotection with trifluoroacetic acid of the ether intermediate 27 was unsuccessful. The synthesis of the thioether analogue of 14 was attempted next. Tosylation of derivatives 23 and 24 was followed by nucleophilic displacement with 1- dodecanethiol producing the protected thioether derivatives 32 and 33 with moderate yields. Neither hydrogenation nor reaction under strong acidic conditions yielded the final thioether deprotected products. Scheme 2 Dopamine hydrochloride was the starting material for the synthesis of the amide and thiourea analogues of 13 and 14 (compounds 37-40) (Scheme 3). Dopamine was also synthesized to compare its activity with HT. The thiourea derivatives were synthesized by reaction of the isothiocyanate intermediate 36 with the corresponding primary alkylamine to obtain the products in moderate yields. The amide analogues of 13 and 14 were prepared by reaction of dopamine hydrochloride with the corresponding fatty acids using HATU as the coupling reagent. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Scheme 3 2.2. Biological evaluation The in vitro antiparasitic activities of TYR, HT and their derivatives 3-18 were evaluated against T. brucei brucei and against axenic amastigotes of L. donovani. Those compounds exhibiting over 40% inhibition of axenic amastigotes at 20 µM were also evaluated on intracellular amastigotes of L. donovani. The cytotoxicity of these compounds was also evaluated against a human non-tumoral lung cell line (MRC-5). Suramin and amphotericin B were used as positive drug controls for T. brucei and L. donovani, respectively. Selectivity indices (SI) were calculated according to the formula: IC 50 (MRC-5) / IC 50 parasite. The activity gain (AG) of each compound with respect to the reference compound (TYR or HT) was calculated according to the formulas: IC 50 (TYR) / IC 50 compound and IC 50 (HT) / IC 50 compound. Table 1 presents the antiparasitic and cytotoxic data for TYR and its derivatives 3-9. IC 50 values against T. brucei ranged from 10 to 62.7 µM and selectivity indices from 1.5 to 5. None of the TYR derivatives improved the activity of TYR itself as can be observed from the AG values. In the case of L. donovani, compounds 4 and 5 showed inhibition over 40% of axenic amastigotes at 20 µM concentration. These two derivatives were evaluated on intracellular amastigotes and showed IC 50 values >10 µM. Aissa et al.[34] measured antileishmanial activity against L. major and L. infantum parasite species for tyrosol fatty acid esters from C2 to C18 chain length. They found the best IC 50 values for medium chain tyrosyl derivatives 5-7 (e.g. tyrosol decanoate 6: 65 and 132 µM, against L. major and L. infantum, respectively). The authors proposed this effect could be due to the surfactant activity reported for these medium chain tyrosyl fatty acid esters.[35] When HT and its derivatives 10-18 were evaluated against T. brucei (Table 2), compounds 13-16 showed IC 50 s in the low micromolar range (0.36-2.43 µM). Remarkably, hydroxytyrosol MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT decanoate 13 and hydroxytyrosol dodecanoate 14 displayed a high selectivity index (118 and 101, respectively) and a large increase in activity with respect to HT (AG values of 79 and 132, respectively). A length of the alkyl chain around 10 to 12 carbons seems to be the optimum on this series. When these compounds were examined against axenic amastigotes of L. donovani, three of them (compounds 11-13) exhibited over 40% inhibition at 20 µM concentration. When they were evaluated on intracellular amastigotes, the best IC 50 value was displayed by hydroxytyrosol decanoate 13 (8.44 µM). Kyriazis et al.[18] had previously reported IC 50 data for hydroxytyrosol of 393 µM against L. donovani and in the same range for L. infantum and L. major. We have observed that the addition of a fatty acid ester group to HT improves its activity with respect to HT against L. donovani although only to IC 50 s in the low micromolar range. Table 1. Cytotoxic, trypanocidal and leishmanicidal activities of tyrosol derivatives. Cytotoxicity MRC-5 T.brucei S-16 L.donovani Axenic amastigotes Intracellular amastigotes Compounds R 1 R 2 IC 50 ( µ M) IC 50 ( µ M) SI AG % inhib. ( 20 µ M ) IC 50 ( µ M) SI AG TYR (1) H H >500 >10 > 5 -- 50 >10 5 -- 3 COCH 3 H 113.6 ± 11.3 79.4 ± 23.1 1.4 0.13 28 n.d. -- -- 4 CO(CH 2 ) 4 CH 3 H 166.4 ± 0.6 59.1 ± 2. 3 2. 8 0.16 44 >10 16.6 1 5 CO(CH 2 ) 6 CH 3 H 133.0 ± 9.8 56.8 ± 8.3 2.3 0.22 46 >10 13.3 1 6 CO(CH 2 ) 8 CH 3 H 171.5 ± 15.7 81.4 ± 19.3 2.1 0.16 34 n.d. -- -- 7 CO(CH 2 ) 10 CH 3 H 67.2 ± 0.2 16.2 ± 3.8 4.1 0.52 32 n.d. -- -- 8 H GlcAc 110.2 ± 14.4 61.5 ± 23.8 1.8 0.17 36 n.d. -- -- 9 H SO 3 - K + 106.4 ± 17.2 41.6 ± 12.6 2.6 0.16 36 n.d. -- -- Suramin -- 0.038 ± 0.003 -- Eflornitin -- 0.026 ± 0.002 -- Amphotericin B -- -- 0.10 ± 0.01 Experiments on L. donovani were carried out in axenic amastigotes and intracellular amastigotes. SI= Selectivity Index (IC 50 MRC-5 / IC 50 parasite); AG= Activity gain (IC 50 tyrosol / IC 50 compound) Table 2. Cytotoxic, trypanocidal and leishmanicidal activities of hydroxytyrosol derivatives. Cytotoxicity MRC-5 T.brucei S-16 L.donovani Axenic amastigotes Intracellular amastigotes Compounds R 1 R 2 , R 3 IC 50 ( µ M) IC 50 ( µ M) SI AG % inhib. (20µM) IC 50 ( µ M) SI AG HT (2) H H, H >50 47.5 ± 16.9 > 1 -- 0 n.d. -- -- 10 COCH 3 H, H >50 43.4 ± 14.0 > 1 1 48 >10 -- -- 11 CO(CH 2 ) 2 CH 3 H, H 71.5 ± 15.5 9.4 ± 1.4 8 5 48 >10 7.1 -- 12 CO(CH 2 ) 6 CH 3 H, H 103.5 ± 15.2 9.6 ± 0.8 11 5 45 >10 10.3 -- 13 CO(CH 2 ) 8 CH 3 H, H 71.1 ± 6.9 0.6 ± 0.24 118 79 54 8.44 ± 1.6 8.4 -- 14 CO(CH 2 ) 10 CH 3 H, H 38.1 ± 2.4 0.36 ± 0.01 106 132 56 >10 -- -- 15 CO(CH 2 ) 12 CH 3 H, H > 50 2.29 ± 0.48 > 22 21 53 >10 -- -- 16 CO(CH 2 ) 14 CH 3 H, H > 50 2.43 ± 0.33 > 21 20 52 >10 -- -- 17-18 H GlcAc, H 118.8 ± 21.5 62.3 ± 16.8 2 0.8 31 n.d. -- -- Experiments on L. donovani were carried out in axenic amastigotes and intracellular amastigotes. SI= Selectivity Index (IC 50 MRC-5 / IC 50 parasite); AG= Activity gain (IC 50 hydroxytyrosol / IC 50 compound) MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 144.5 (C 3arom ), 131.9 (C 1arom ), 121.1 (C 6arom ), 116.9 (C 2arom or C 5arom ), 116.4 (C 2arom or C 5arom ), 35.7 (CH 2 ), 35.7 (CH 2 ), 33.1 (CH 2 ), 30.7 (CH 2 ), 30.4 (CH 2 ), 28.0 (CH 2 ), 23.7 (CH 2 ), 14.4 (CH 3 ); ESIHRMS [M + H] calcd for C 21 H 36 N 2 O 2 S 381.2576, found 381.2563. 4.3. Biological assays. 4.3.1. In vitro antitrypanosomal activity Bloodstream forms (BSF) of T. brucei brucei 'single marker' S427 (S16) were grown at 37 ºC, 5% CO2 in HMI-9 medium supplemented with 10% (heat-inactivated fetal bovine serum, hiFBS). Drug susceptibility assay was performed as described in (Carvalho L et al, 2015). Briefly, parasites (1 × 10 4 BSF per mL) were incubated in 96-well plates with increasing concentration of drugs/compounds for 72 h at 37 ºC, 5% CO 2 in culture medium. Cell proliferation was determined using the alamarBlue® assay[42]. The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro. Fluorescence was recorded with an Infinite® F200 microplate reader (Tecan Austria GmbH, Austria) equipped with 550 and 590 nm filters for excitation and emission wavelengths, respectively. 4.3.2. In vitro antileishmanial activity The experiments of drug susceptibility on L. donovani were carried out in axenic amastigotes and intracellular amastigotes. Axenic L. donovani MHOM/ET/67/HU3 amastigote parasites were grown in Schneider medium supplemented with 20% hiFBS, pH 5.4 at 37 ºC and 5% CO 2 . 10 6 axenic amastigotes/ml in a 96-well plate were incubated with increasing concentrations of compounds for 72 h at 37 °C, followed by a resazurin-based assay, as described.[43] Additionally, we use intracellular amastigotes of a L. donovani (MHOM/ET/67/HU3) line with luciferase gene integrated into the parasite genome.[44] The susceptibility of intracellular L. donovani amastigotes to synthesized compounds was determined using the Luciferase Assay System Kit (Promega, Madison, Wis.) as previously described.[43] Briefly, macrophagedifferentiated-THP-1 cells were plated at a density of 3x10 4 macrophages/well in 96-well white polystyrene microplates and were infected at a macrophage/parasite ratio of 1:10 with late-stage MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT promastigotes. Infected cell cultures were incubated at different compound concentrations in RPMI 1640 medium plus 10% hiFBS at 37 °C with 5% CO 2 for 72 h and then lysed. Luminescence intensity was measured as indicative of the intracellular parasite growth, according to the instructions of the supplier. 4.3.3. Cytotoxicity assay Human myelomonocytic cell line THP-1 were grown at 37 ºC and 5% CO 2 in RPMI-1640 supplemented with 10% hiFBS, 2 mM glutamate, 100 U/mL penicillin and 100 µg/mL streptomycin. 3 x 10 4 cells/well in 96-well plates were differentiated to macrophages with 20 ng/mL of PMA treatment for 48 h followed by 24 h of culture in fresh medium.[43] MRC-5 cells, a SV-40 transformed human fetal lung fibroblast cell line, were maintained at 37 ºC and 5% CO 2 in DMEM supplemented with 10% hiFBS, 100 U/mL penicillin and 100 µg/mL streptomycin. Cells were harvested by treatment with 0.05% (w/v) trypsin plus 0.48 mM EDTA for 5 min, diluted to 4 x 10 4 cell/ml in 96-well plates and incubated at 37 ºC and 5% CO 2 before toxicity assay.[43] Cellular toxicity of all compounds was determined using the colorimetric MTT-based assay after incubation at 37 °C for 72 h in the presence of increasing concentrations of compounds.[43] The results are expressed as EC 50 values, as the concentration of compound that reduce cell growth by 50% versus untreated control cells. Acknowledgements We gratefully acknowledge to Junta de Andalucía (Grant No. FQM-7316 (JCM), BIO-1786 (JMPV), CTS-7282 (FG)) and Spanish Ministry of Economy and Competitiveness (Grant No. SAF2011-28215 (JMPV) and SAF2012-34267 (FG)) for financial support. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT References [1] B. S. Hursey, The programme against African trypanosomiasis: aims, objectives and achievements, Trends Parasitol 17 (2001) 2-3. [2] M. J. Vreysen, M. T. Seck, B. 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MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Highlights • Tyrosol and hydroxytyrosol alkyl derivatives were synthesized in 2-3 steps. • Their antitrypanosomal and antileishmanial activity is reported. • Compounds 13 and 14 showed the best IC 50 against Trypanosoma brucei (0.36-0.6 µM). • Compounds 13 and 14 displayed a high selectivity index (SI=101-118). • Two phenol groups and medium size alkyl chain are essential for activity.