Ethyl 4 aminobenzoate

The compounds methyl benzoate (1), n-propyl benzoate(3), n-butyl benzoate (4), methyl 2-naphthoate (7), phenylacetate (9), ethyl 2-bromobenzoate (10), ethyl 3-bromobenzoate (11), ethyl 4-bromobenzoate (12), methyl 8-fluoro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate (methyl flumazenil ester, 16), methyl 4-aminobenzoate (methyl benzocaine ester, 14), and methyl [1,1′-biphenyl]-4-carboxylate (17) were synthesised at the Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio®, UFRJ, Brazil) with degree of purity ≥98%. Flumazenil (13) was purchased from Cristalia® – Brazil. Formic Acid (96%), acetonitrile and methanol of HPLC grade were purchased from Tedia®-Brazil. Water used for the HPLC analysis was produced using a water purification system (Master System MS2000, Gehaka, Brazil). Ethylbenzoate (2), phenylbenzoate (5), benzylbenzoate(6), methyl 2-phenylacetate (8), ethyl 4-aminobenzoate (benzocaine, 13), bicinchoninic acid kit for protein determination, ethylenediamine-tetra-acetic-acid (EDTA), monobasic and bibasic potassium phosphate, bis(p-nitrophenyl) phosphate sodium salt, magnesium chloride hexahydrate, β-nicotinamide adenine dinucleotide phosphate (NADP), D-glucose-6-phosphate, glucose-6-phosphate dehydrogenase, tetrahydrofuran (THF) and lithium hydroxide (LiOH) were purchased from Sigma Aldrich (St. Louis, MO, USA).

Live F. hepatica were collected
at a local abattoir from freshly slaughtered sheep livers with naturally
acquired infections. Adult F. hepatica were prepared for in vitro maintenance, and whole
ES products, reflective of live and terminated flukes, were obtained,
as described by Morphew et al.^{27 (link)} Briefly,
size-matched adults (1–3 cm length) were selected, and replicates
of 10 flukes were grouped for in vitro maintenance
directly (live) or after termination (dead) in ethyl 4-aminobenzoate
(Sigma-Aldrich, U.K.; 1% (w/v) in ethanol (Fisher Scientific, U.K.)),
with 3 mL of fresh supplemented culture medium per fluke and incubation
at 37 °C. For the extraction of whole ES products, media supernatants
were clarified by 300g centrifugation and precipitated via the TCA method, as previously described.^{27 (link)}

All chemicals used throughout the work were of analytical grade. Salts used throughout this study were placed in a vacuum oven and then stored in vacuum desiccators over phosphorus pentoxide, P₄O₁₀, for several days to remove water, before being used for experimental purposes. p-tert-Butyl calix[4]arene (C₄₄H₅₆O₄, ≥ 97%), ethyl 4-aminobenzoate (H₂NC₆H₄CO₂C₂H₅, 98%), phenol (C₆H₆O, ≥99%), sodium ethanoate trihydrate (C₂H₃NaO₂·3H₂O, ≥99%), sodium nitrite (NaNO₂, 97+ %), aluminium (III) chloride (AlCl₃, 98.5%), tetra-n-butylammonium chloride ((C₄H₉)₄NCl, ≥97.0%), tetra-n-butylammonium flouride hydrate ((C₄H₉)₄NF·H₂O), 98%), tetra-n-butylammonium dihydrogen phosphate ((C₄H₉)₄NH₂PO₄)), 97%), tetra-n-butylammonium hydrogen sulfate ((C₄H₉)₄NHSO₄, 97%) and potassium carbonate (K₂CO₃, 99+ %) were purchased from Sigma Alrich.
Deuterated solvents used in NMR experiments, dimethyl sulfoxide-d₆ (C₂D₆OS, 99.9%) and chloroform-d (CDCl₃, 99.8% + 0.05% v/v TMS) were purchased from Cambridge Isotope Laboratories.