6,7-Dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline amides and corresponding ester isosteres as multidrug resistance reversers

Abstract Aiming to deepen the structure–activity relationships of the two P-glycoprotein (P-gp) modulators elacridar and tariquidar, a new series of amide and ester derivatives carrying a 6,7-dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline scaffold linked to different methoxy-substituted aryl moieties were synthesised. The obtained compounds were evaluated for their P-gp interaction profile and selectivity towards the two other ABC transporters, multidrug-resistance-associated protein-1 and breast cancer resistance protein, showing to be very active and selective versus P-gp. Two amide derivatives, displaying the best P-gp activity, were tested in co-administration with the antineoplastic drug doxorubicin in different cancer cell lines, showing a significant sensitising activity towards doxorubicin. The investigation on the chemical stability of the derivatives towards spontaneous or enzymatic hydrolysis, showed that amides are stable in both models while some ester compounds were hydrolysed in human plasma. This study allowed us to identify two chemosensitizers that behave as non-transported substrates and are characterised by different selectivity profiles.


Chemical stability data Instrumental
The LC-MS/MS analysis was carried out using a Varian 1200L triple quadrupole system (Palo Alto, CA, USA) equipped by two Prostar 210 pumps, a Prostar 410 autosampler and an Elettrospray Source (ESI) operating in positive ions mode. Raw-data were collected and processed by Varian Workstation Vers. 6.8 software. G-Therm 015 thermostatic oven was used to keep the samples at 37 °C during the degradation tests. Eppendorf microcentrifuge 5415D was employed to centrifuge plasma samples.

Standard solutions and calibration curves
Stock solutions of analytes and verapamil hydrochloride (ISTD) were prepared in acetonitrile at 1.0 mg mL -1 and stored at 4 °C. Working solutions of each analyte were freshly prepared by diluting stock solutions up to a concentration of 10 μM and 1 μM (working solution 1 and 2 respectively) in mQ water: acetonitrile 80:20 (v/v) solution. The ISTD working solution was prepared in acetonitrile at 60 ng mL -1 (ISTD solution). A six levels calibration curve was prepared by adding proper volumes of working solution of each analyte to 300 μL of ISTD solution. The obtained solutions were dried under a gentle nitrogen stream and dissolved in 1.0 mL of 10 mM of formic acid in mQ water: acetonitrile 70:30 (v/v) solution. Final concentrations of calibration levels were: 0, 0.05, 0.10, 0.20, 0.50, 0.75 and 1.00 M of analyte in the sample. All calibration levels were analysed six times by the appropriate LC-MS/MS method.

LC-MS/MS method
The chromatographic parameters employed to analyse the samples were tuned to minimize the run time and were reported as follows: -column, Pursuit C18 length = 30 mm; internal diameter = 2mm; particle size = 3 μm purchased from Agilent Technologies (Palo Alto, CA, USA) -acidic mobile phase, composed by 5 mM of ammonium formate and 10mM of formic acid in mQ water: acetonitrile 90:10 (v/v) solution (solvent A), 5 mM of ammonium formate and 10mM of formic acid in mQ water: acetonitrile 10:90 (v/v) solution (solvent B).
-flow rate and the injection volume were 0.25 mL min -1 and 5 μL respectively. The elution gradient is shown in Table S1. The analyses were acquired in product ion scan, resonant excitation mode, parameters are reported in Table S2, using Nitrogen as collision gas.  Linearity and LOD Calibration curves of analytes were obtained by plotting the peak area ratios (PAR), between quantitation ions of analyte and ISTD, versus the nominal concentration of the calibration solution.
A linear regression analysis was applied to obtain the best fitting function between the calibration points.
The precision was evaluated through the relative standard deviation (RSD%) of the quantitative data of the replicate analysis of highest level of calibration curves. In order to obtain reliable LOD values, the standard deviation of response and slope approach was employed. The estimated standard deviations of responses were obtained by the standard deviation S30 of y-intercepts (SDY-I) of regression lines. The obtained linear regressions, the linearity coefficients, precision and the estimated LOD values for each analyte are reported in Table S3.

Solution stability profiles
The solution stability profiles in PBS and human plasma were obtained by monitoring the variation of analyte concentration at different incubation times. They are reported in Figures S1-S26. Figure S1: Degradation plots of 1 in PBS (blue square) and human plasma (red triangle).