Anti-Parasite and Cytotoxic Activities of Chloro and Bromo L-Tyrosine Derivatives

A series of twenty-one L-tyrosine derivatives with modifications in the halogenation pattern of the aromatic ring and different degree of methylations on the amine and phenolic hydroxyl groups were synthesized. The structures of all the intermediates and target compounds were confirmed unambiguous by spectroscopy analysis. Additionally, all compounds were evaluated against Plasmodium falciparum and Leishmania panamensis parasites between 20-702 μg mL. The cytotoxic evaluation was done to determine the selectivity index for each compound. Six compounds had the lower EC50 (effective concentration 50) against L. panamensis. One of these compounds was the most active with an EC50 at 24.13 μg mL (76.07 μM). All derivatives showed no significant activity against P. falciparum and no compound has in vitro antifungal activity at 500 μg mL.


Introduction
Diseases caused by protozoan parasites affect about one billion people, with a notable incidence in tropical countries.Diseases such as malaria and leishmaniasis are more frequent in developing countries of tropical and subtropical regions.These diseases are endemic problems in Colombia and other Latin American countries.
Currently, malaria is still a common cause of death with approximately 445,000 cases reported in 2016 in tropical countries of Africa, Asia and America; and tragically most of the victims are children under the age of five. 1 Despite the decrease in fatal cases recorded by the World Health Organization (WHO) in recent years, an increase in the spread of mosquitoes resistant to common insecticides is observed, and more importantly, the emergence of resistance to current drugs by different strains of Plasmodium. 2 Given that malaria is a disease with global implications and almost half of the world population is at risk of infection, combating malaria becomes one of the priorities of WHO programs.
On the other hand, leishmaniasis disease affect more than 10 million people worldwide, principally tropical countries.Protozoan parasites of the genus Leishmania responsible for the infection are: L. panamensis, L. braziliensis, L. guyanensis, L. mexicana and L. amazonensis. 3Currently, chemotherapies to treat cutaneous leishmaniasis are based on old drugs; unfortunately, all of these drugs have severe toxic effects on patients.Side effects are associated with high therapeutic doses used and long term treatment schemes. 4ompounds isolated from marine organisms are considered as rich sources for drug discovery.6][7][8] We reported isolations and identifications of more than sixteen bromotyrosine isolated from sponges Verongula rigida and Aiolochroia crassa with antiparasitic activity, and we found a close relationship between the degree of bromination in the aromatic ring, the methylation in the amino group and their influence in the antiparasitic activity. 9ue to the emerging resistance against current drugs, it is mandatory to develop new, safer and more effective antiparasitic agents.For that reason, in the present study, the potentials of marine natural products derivatives compounds, based on bioactive L-bromotyrosines, were evaluated as new antiparasitic compounds.The syntheses of novel chloro and bromo L-tyrosine derivatives, with different grade of methylation of the amino group, lead us to realize a structure-activity relationship between the halogenations patterns in the aromatic ring of the L-tyrosine and the substitution of the amino group, looking for the best structural parameters for the development of new antiparasitic compounds.

Chemistry
Synthesis of bromo and chloro N-substituted derivatives was made using as starting material L-tyrosine.For the synthesis of 21 products, it was necessary to synthesize initially four intermediates, which have their amino and carboxylic acids protected with a carbamate and methyl ester group, respectively.Methyl esters derivatives were formed by the reaction of L-tyrosine with SOCl 2 /MeOH, 10 later the protection of the amino group was done by the reaction with Boc 2 O/triethylamine (TEA) at room temperature. 11The bromination and chlorination of the aromatic ring, getting mono and di halogenated compounds, were done using N-bromosuccinimide (NBS) and N-chlorosuccinimide (NCS), 12 producing intermediates (Int1-Int4).These intermediates were divided into two sets, one of them were treated with trifluoracetic acid (TFA) in dichloromethane (DCM), 13 to cleavage the amino group (1a-1d).The other intermediates were O-methylated with MeI in basic media, 14 and later treated with TFA in DCM (2a-2d) (Scheme 1).
The 1 H nuclear magnetic resonance (NMR) analysis of Int1-Int4 shows the presence of a signal at 3.80 ppm (s, 3H) that represents the -RCOOCH 3 group of the methyl ester.The signal at 1.40 ppm (s, 9H) represents the three-methyl groups of the carbamate moiety.The mono halogenated compounds show in the aromatic region of the spectra three signals that corresponds to the protons at 2, 5, 6 positions in the aromatic ring, while the di halogenated compounds only show in the aromatic region of the spectra one signal, due to the symmetric ring, for two protons at 2 and 6 position.
The syntheses of primary amines were done by the hydrolysis of compounds 1a-1d, 2a-2d with K 2 CO 3 /MeOH, 15 getting compounds 4-11.The syntheses of methyl ester tertiary amines were done by the reductive amination of compounds 1a-1d, 2a-2d with formaldehyde/ NaCNBH 3 , getting compounds 3a-3h. 16These set of compounds were divided in two, one of them were treated with K 2 CO 3 /MeOH to get the tertiary amines 12-18.The other set was treated with MeI/acetone to get the methyl ester quaternary amines, 17  group in the quaternary amines.O-Methyl derivatives were confirmed by 1 H NMR by the signal at 3.80 ppm (s, 3H).

Biological activity
Compounds were tested against P. falciparum (NF54 chloroquine sensitive strain) and L. panamensis (MHOM/ CO/87/UA140epir GP strain) using chloroquine and amphotericin B as positive control drugs, respectively.A cytotoxic test was done over human promonocytic cells (U-937 ATCC CRL1593.2).For every active compound, effective concentration 50 (EC 50 ) and lethal concentration 50 (LC 50 ), in µM units, were tested and selectivity index (SI) was represented as LC 50 /EC 50 .In Table 1, it is shown the anti-leishmanial and antiplasmodial activity of the 21 derivatives.
Compounds 4, 5, 6, 10, 21 and 24 were the most active against L. panamensis with values lower than 50 µg mL -1 .Derivative 21 were the most active with EC 50 of 76.07 µM.Compounds 5 and 6 have the higher SI with values of 7.43 and 7.86, respectively.The twenty-one derivatives have no activity against P. falciparum.
In this study, it is possible to establish that the type of halogenation, in addition to the degree of methylation on the amino group, have influence in the anti-leishmanial activity of the compounds.Brominated compounds with O-methyl substitution in para and primary amine (such as compounds 8 and 9) were less active than their correlated structure with free hydroxyl and additional amine substitutions, compounds 4 and 5.In general, tertiary and quaternary amines derivatives are less cytotoxic than primary analogs.Mono-chloro compound 6 with O-methyl substitution in para and primary amine is the most promising derivative against L. panamensis.
Compounds were evaluated at a final concentration of 500 µg mL -1 against nineteen fungal strains of medical

General
All the laboratory reagents were analytical grade (Sigma-Aldrich).Column chromatography and HPLC solvents were liquid chromatography grade (Merck).Compound purifications were made by column chromatography using Merck silica gel 60 and mixtures of hexane and ethyl acetate.Purifications of biologically tested compounds were made by HPLC Agilent 1200 with diode array detector (DAD, 254, 280 and 366 nm) and Eclipse XDB-C18 column using a mixture of MeOH and H 2 O 0.1% formic acid as the mobile phase. 1 H and 13 C NMR were recorded on Bruker Ultrashield (300 and 75 MHz) and Ascend III HD (600 and 125 MHz) with 5 mm CryoProbe TCI, using CDCl 3 , D 2 O, and DMSO-d 6 as deuterated solvents.Signals were assigned using two-dimensional heteronuclear correlations (COSY (correlation spectroscopy) and HSQC (heteronuclear single quantum correlation)).High-resolution mass spectra (HRMS) were recorded using electrospray ionization (ESI-MS) in a UPLC-Q-Tof (ultra-performance liquid chromatography quadrupole time of flight) (Xevo-XS-QTof, Waters).The drying and cone gas was nitrogen set to flow rates of 300-30 L h -1 , respectively.Methanol samples solutions (1 × 10 -5 M) were directly introduced into the ESI spectrometer at a flow rate of 10 µL min -1 .A capillary voltage of 3.5 kV was used in the positive scan mode, and the cone voltage (Uc) set to 10 V.

Synthesis of intermediates (Int1-Int4)
To MeOH (50 mL) at -5 °C, thionyl chloride (2190 µL, 30 mmol) was dropped slowly to maintain the temperature under 0 °C.Then L-tyrosine (5000 mg, 27.6 mmol) was added.The resulting mixture was stirred at 80 °C for 24 h.As the mixture cooled, the product was precipitated by the addition of diethyl ether (100 mL).The precipitate was collected, washed with ethyl ether (20 mL) and dried to give L-tyrosine methyl ester hydrochloride.The product was tested by thin layer chromatography (TLC) mobile phase n-BuOH:AcOH:H 2 O (4:1:1) showing quantitative conversion.This reaction was done twice.The monobromination of L-tyrosine methyl ester (2000 mg, 8.63 mmol) was done with NBS (2000 mg, 11.21 mmol) in MeOH (20 mL) stirred at room temperature for 24 h.The solvent was removed, and the crude was partitioned with EtOAc:H 2 O.The aqueous phase was recovered and evaporated to get red oil.This crude was tested by TLC mobile phase n-BuOH:AcOH:H 2 O (4:1:1) showing the formation of mono bromine compound.This crude was not purified to the next reaction.Compounds dibrominated, monochlorinated and dichlorinated were obtained in the same way using NBS (3380 mg, 19 mmol), NCS (1500 mg, 11.22 mmol) and NCS (2535 mg, 19 mmol), respectively.The protections of the halogenated compounds were done by the reaction in MeOH (15 mL) with Boc 2 O (2300 µL, 10 mmol) and TEA (2000 µL) for 24 h at room temperature.The solvent was removed, the crude was adjusted to pH = 3 and later extracted with EtOAc.The organic phase was dried with Na 2 SO 4 and purified in column chromatography using as mobile phase hexane:EtOAc (3:1).

Synthesis of compounds 2a-2d
The O-methylations of Int1-Int4 were done by dissolving 1500 mg of each intermediate in acetone with 800 mg K 2 CO 3 and 750 µL MeI.The mixture was stirred for 12 h at room temperature.The solvent was removed and purified in column chromatography using as mobile phase hexane:EtOAc (3:1).The products obtained in the last step were dissolved in DCM (3 mL) at 0 °C.After 5 min, TFA (1000 µL) was added to the mixture and left reacting for 24 h at room temperature.Later the solvents and reagents were evaporated, and the crude of the reaction was adjusted to pH = 7.The product was tested by TLC mobile phase hexane:EtOAc (3:1) showing quantitative conversion for compounds 2a-2d: 2a: 920 mg, two steps yield 80%; 2b: 972 mg, two steps yield 80%; 2c: 885 mg, two steps yield 80%; 2d: 916 mg, two steps yield 80%.

Biological activity assays
The compounds were subjected to in vitro evaluation for their cytotoxicity, anti-leishmanial, antiplasmodial and anti-fungal activity.

Anti-leishmanial activity assay
The anti-leishmanial activity of compounds was determined according to the ability of the compound to reduce the infection by L. panamensis parasites.For this, the anti-leishmanial activity was tested on intracellular amastigotes of L. panamensis transfected with the green fluorescent protein gene (MHOM/CO/87/UA140-EpiR-GFP strain).Briefly, U-937 human cells at a density of 3 × 10 5 cells mL -1 in Roswell Park Memorial Institute medium (RPMI)-1640 and 0.1 µg mL -1 of PMA (phorbol 12-myristate 13-acetate) were dispensed on 24-wells microplate and then infected with stationary phase growing L. panamensis promastigotes in a 15:1 parasites per cell ratio.Plates were incubated at 34 °C and 5% CO 2 for 3 h and then the cells were washed twice with phosphate buffer solution (PBS) to eliminate not internalized parasites.Fresh RPMI-1640 was added to each well (1 mL) and plates were incubated again to complete infection.After 24 h of infection, the RPMI-1640 medium was replaced by fresh culture medium containing each compound at four serial dilutions (50, 12.5, 3.125 and 0.78 µg mL -1 ) and plates were then incubated at 37 °C and 5% CO 2 during 72 h, then, cells were removed from the bottom plate with a trypsin/ ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (250 mg) solution.The cells were centrifuged at 1100 rpm during 10 min at 4 °C, the supernatant was discarded, and cells were washed with 1 mL of cold PBS and centrifuged at 1100 rpm for 10 min at 4 °C.Cells were washed two times employing PBS, as previously, and after the last wash, the supernatant was discarded, and cells were suspended in 500 µL of PBS.Cells were analyzed by flow cytometry employing a flow cytometer (Cytomics FC 500MPL) reading at 488 (exciting) and 525 nm (emitting) over an argon laser and counting 10,000 events.Infected cells were determined according to the events for green fluorescence (parasites).Infected cells exposed to control drugs (amphotericin B) were used as a control for antileishmanial activity (positive control), while infected cells incubated in absence of any compound or drug were used as a control for infection (negative control).Nonspecific fluorescence was corrected by subtracting fluorescence of unstained cells.All determinations were performed in triplicate in at least two independent experiments. 18,19tiplasmodial activity assay Antiplasmodial activity was evaluated in vitro on asynchronic cultures of P. falciparum (3D7 strain), maintained in standard culture conditions.The effect of each compound over the growth of the parasites was determined by quantification of parasite death, based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells into the supernatant.Briefly, unsynchronized P. falciparum cultures were adjusted to 0.5% parasitemia and 1% hematocrit in RPMI medium enriched with 3% lipid-rich bovine serum albumin (Albumax II).Then, in each well of a 96-wells plate, 100 µL of parasite suspension were dispensed and subsequently exposed against 100 µL of four serial dilutions of compounds (100, 25, 6.25 and 1.56 µg mL -1 ).Dilutions were prepared from a stock solution of 1000 µg mL -1 .Chloroquine (CQ) was used as positive antiplasmodial drug control.Parasites unexposed to any compound were used as a control of both growth and viability (negative control).Plates were incubated for 48 h at 37 °C in N 2 (90%), CO 2 (5%) and O 2 (5%) atmosphere.After incubation, plates were harvested, and parasites were subjected to three 20-min freeze-thaw cycles.Meanwhile, 100 µL of Malstat reagent (400 µL Triton X-100 in 80 mL deionized water, 4 g L-lactate, 1.32 g Tris buffer and 0.022 g acetylpyridine adenine dinucleotide in 200 mL deionized water; pH 9.0) and 25 µL of NBT/PES solution (0.16 g nitroblue tetrazolium salt and 0.08 g phenazine ethosulfate in 100 mL deionized water) were added to each well of a second flat-bottomed 96-well microtiter plate.After freeze-thaw cycles, the culture in each of the wells of the first plate was resuspended by pipetting and 15 µL of each well was taken and added to the corresponding well of the second plate (containing Malstat and NBT/PES reagents).
After an hour of incubation in the dark, color development of the LDH reaction was monitored colorimetrically in a spectrofluorometer (Varioskan, Thermo) reading at 650 nm.The intensity of color in each experimental condition was registered as optical densities (O.D.).Non-specific absorbance was corrected by subtracting O.D. of the blank.Determinations were done in triplicate in at least two independent experiments. 20 Scheme 1. Intermediate synthesis from L-tyrosine.(a) SOCl 2 , MEOH; (b) NBS or NCS, MeOH; (c) Boc 2 O, TEA, MeOH; (d) MeI, K 2 CO 3 , acetone; (e) TFA, DCM.