Design, Synthesis, Dynamic Docking, Biochemical Characterization, and in Vivo Pharmacokinetics Studies of Novel Topoisomerase II Poisons with Promising Antiproliferative Activity

We previously reported a first set of hybrid topoisomerase II (topoII) poisons whose chemical core merges key pharmacophoric elements of etoposide and merbarone, which are two well-known topoII blockers. Here, we report on the expansion of this hybrid molecular scaffold and present 16 more hybrid derivatives that have been designed, synthesized, and characterized for their ability to block topoII and for their overall drug-like profile. Some of these compounds act as topoII poison and exhibit good solubility, metabolic (microsomal) stability, and promising cytotoxicity in three cancer cell lines (DU145, HeLa, A549). Compound 3f (ARN24139) is the most promising drug-like candidate, with a good pharmacokinetics profile in vivo. Our results indicate that this hybrid new chemical class of topoII poisons deserves further exploration and that 3f is a favorable lead candidate as a topoII poison, meriting future studies to test its efficacy in in vivo tumor models.


In vitro metabolic stability
In vitro microsomial stability. 10mM DMSO stock solution of test compound was pre-incubated at 37˚C for 15min with mouse liver microsomes added 0.1M Tris-HCl buffer (pH 7.4). The final concentration was 4.6µM. After pre-incubation, the co-factors (NADPH, G6P, G6PDH and MgCl2 pre-dissolved in 0.1M Tris-HCl) were added to the incubation mixture and the incubation was continued at 37˚C for 1h. At each time point (0, 5, 15, 30, 60min), 30µL of incubation mixture was diluted with 200µL cold CH3CN spiked with 200nM of internal standard, followed by centrifugation at 3500g for 15min. The supernatant was further diluted with H2O (1:1) for analysis. The concentration of test compound was quantified by LC/MS-MS on a Waters ACQUITY UPLC/MS TQD system consisting of a TQD (Triple Quadrupole Detector) Mass Spectrometer equipped with an Electrospray Ionization interface. The analyses were run on an ACQUITY UPLC BEH C18 (50x2.1mmID, particle size 1.7µm) with a VanGuard BEH C18 pre-column (5x2.1mmID, particle size 1.7µm) at 40°C, using 0.1% HCOOH in H2O (A) and 0.1% HCOOH in CH3CN (B) as mobile phase. Electrospray ionization (ESI) was applied in positive mode. The percentage of test compound remaining at each time point relative to t=0 was calculated. The half-lives (t½) were determined by an one-phase decay equation using a non-linear regression of compound concentration versus time.
In vitro Plasma Stability. 10mM DMSO stock solution of test compound was diluted 50-fold with DMSO-H2O (1:1) and incubated at 37˚C for 2 h with mouse plasma added 5% DMSO (pre-heated at 37˚C for 10 min). The final concentration was 2µM. At each time point (0, 5, 15, 30, 60, 120min), 50 µL of incubation mixture was diluted with 200µL cold CH3CN spiked with 200nM of internal standard, followed by centrifugation at 3500g for 20min. The supernatant was further diluted with H2O (1:1) for analysis. The concentration of test compound was quantified by LC/MS-MS on a Waters ACQUITY UPLC/MS TQD system consisting of a TQD (Triple Quadrupole Detector) Mass Spectrometer equipped with an Electrospray Ionization interface. The analyses were run on an ACQUITY UPLC BEH C18 (50x2.1mmID, particle size 1.7µm) with a VanGuard BEH C18 precolumn (5x2.1mmID, particle size 1.7µm) at 40°C, using 0.1% HCOOH in H2O (A) and 0.1% HCOOH in CH3CN (B) as mobile phase. Electrospray ionization (ESI) was applied in positive mode. The response factors, calculated on the basis of the internal standard peak area, were plotted over time. When possible, response vs. time profiles were fitted with Prism (GraphPad Software, Inc., USA) to estimate compounds half-life in plasma.

Aqueous kinetic solubility
The aqueous kinetic solubility was determined from a 10mM DMSO stock solution of test compound in Phosphate Buffered Saline (PBS) at pH 7.4. The study was performed by incubation of an aliquot of 10mM DMSO stock solution in PBS (pH 7.4) at a target concentration of 250µM resulting in a final concentration of 2.5% DMSO. The incubation was carried out under shaking at 25°C for 24h followed by centrifugation at 21.100g for 30min. The supernatant was analyzed by UPLC/MS for the quantification of dissolved compound by UV at a specific wavelength (215nm). The analyses were performed on a Waters ACQUITY UPLC/MS SQD system consisting of a SQD (Single Quadrupole Detector) Mass Spectrometer equipped with Electrospray Ionization interface. The analyses were run on an ACQUITY UPLC BEH C18 column (50x2.1mmID, particle size 1.7µm) with a VanGuard BEH C18 pre-column (5x2.1mmID, particle size 1.7µm), using 10mM NH4OAc in H2O at pH 5 adjusted with AcOH (A) and 10mM NH4OAc in MeCN-H2O (95:5) at pH 5 (B) as mobile phase.

Aqueous thermodynamic solubility
The thermodynamic solubility of 3f was determined by addition of Phosphate Buffered Saline (PBS) at pH 7.4 to an excess of solid compound. The assay was performed by incubation of an aliquot of 2.5mg of test compound in 500µL of PBS at pH 7.4. The suspension was shaken at 300 RPM for 24h at 25°C. At the end of the incubation period, the saturated solution was filtered and analyzed by UPLC/MS for the quantification of dissolved compound (in µM) by UV at a specific wavelength (λmax of the test compound). The analyses were performed on a Waters ACQUITY UPLC/MS SQD system equipped with an ESI ion source. The analyses were run on an ACQUITY UPLC BEH C18 column (50x2.1mmID, particle size 1.7µm) with a VanGuard BEH C18 pre-column (5x2.1mmID, particle size 1.7µm), using H2O + 0.1% HCOOH (A) and CH3CN + 0.1% HCOOH (B) as mobile phase.

Plasma Protein Binding
The assay was conducted in triplicate in human and mouse plasma. An appropriate volume of plasma was spiked with a known amount of 10mM DMSO stock solution of the test compound, thus reaching the incubation concentrations of 0.5, 1 and 5µM. Then 300µL/well of the spiked plasma were added to the corresponding donor well of the RED plate (Rapid Equilibrium Dialysis (RED) Device Single-Use Plate with Inserts, Thermo Scientific), a polypropylene plate preloaded with 48 equilibrium dialysis membrane inserts (8K MWCO). 500µL/well of isotonic buffer (PBS, pH 7.4) were added to the receiver well of the RED plate. The RED plate was sealed with adhesive film and shaken at 500 RPM for 3 hours at 37°C. At the end of the incubation period, the "Donor Spin" plate was prepared by adding 100µL of isotonic buffer, 10µL of blank plasma and 10µL from each donor chamber of the RED device, while the "Receiver Spin" plate was prepared by adding 20µL of blank plasma and 100µL from each receiver chamber of the RED device. The "Standards Spin" plate was prepared by adding 100µL of isotonic buffer, 10µL of spiked plasma and 10µL of blank plasma. 300µL of stop solution (Acetonitrile spiked with 200nM of the appropriate internal standard) were added to each well of the three "spin" plates, that subsequently were centrifuged for 20 minutes at 3750 RPM. At this point, the supernatants were ready for injections in the UPLC-MS/MS system. The analyses were performed on a Waters ACQUITY UPLC/MS TQD system consisting of a TQD (triple quadrupole detector) Mass Spectrometer equipped with an Electrospray Ionization interface and a Photodiode Array eλ Detector. The analyses were run on an ACQUITY UPLC BEH C18 (50x2.1mmID, particle size 1.7µm) with a VanGuard BEH C18 pre-column (5x2.1mmID, particle size 1.7µm) at 40°C, using H2O + 0.1% HCOOH (A) and CH3CN + 0.1% HCOOH (B) as mobile phase. Electrospray ionization (ESI) was applied in positive mode and MRM transitions and collision energies were chosen for the specific test compound. Plasma protein binding was calculated applying appropriate corrections to compensate for dilutions during sampling.

Animal models
Male C57B6/J male mice, 8 weeks old, 25-30 g were used (Charles River). All procedures were performed in accordance with the Ethical Guidelines of European Communities Council (Directive 2010/63/EU of 22 September 2010) and accepted by the Italian Ministry of Health. All efforts were made to minimize animal suffering and to use the minimal number of animals required to produce reliable results, according to the "3Rs concept". Animals were group-housed in ventilated cages (n=5 mice per cage) and had free access to food and water. They were maintained under a 12-hour light/dark cycle (lights on at 8:00 am) at controlled temperature (21°C ± 1°C) and relative humidity (55% ± 10%).

Pharmacokinetic studies
Compound 3f was administered orally (P.O.) and intravenously (I.V.) to C57B6/J male mice at 10 and 3 mg/Kg dose. Vehicle was: PEG400/Tween 80/Saline solution at 10/10/80 % in volume respectively. 3f was first dissolved in 100% PEG, then diluted to the final target concentration in this vehicle. The target concentration of 3f in the dosing solution was checked and confirmed by LC-MS. The dosing solutions were diluted differently in the vehicle. The different dilutions were further diluted 4-fold with CH3CN prior to analysis. A reference standard was spiked in the vehicle to prepare a calibration curve over a 0.1-10µM range. The calibrators were also diluted 4-fold with CH3CN as for the dosing solution dilutions. The dosing solutions and calibrators were analysed under the same conditions as for the plasma samples described below. Three animals per dose were treated. Blood samples at 0, 15, 30, 60, 120, 240, and 480 minutes after administration were collected for PO arm. Blood samples at 0, 5, 15, 30, 60, 120 and 240 minutes after administration were collected for IV arm. Plasma was separated from blood by centrifugation for 15 minutes at 3500 rpm a 4°C, collected in a eppendorf tube and frozen (-80°C). Brain samples were homogenized in Phosphate buffered saline and were then split in two aliquots kept at -80 °C until analysis. An aliquot was used for compound brain level evaluations, following the same procedure described below for plasma samples. The second aliquot was kept for protein content evaluation by bicinchoninic acid assay (BCA). Control animals treated with vehicle only were also included in the experimental protocol. Plasma samples were centrifuged at 21.100g for 15min. at 4°C. A 50µl aliquot was transferred into a 96-Deep Well plate and 150µl of extraction solution was added. The extraction solution was consisting of cold CH3CN spiked with 200nM of internal standard. The plate was centrifuged at 3270g for 15min. at 4°C. 80µl of supernatant was then transferred into a 96-Deep Well plate and 80µl of H2O was added. A reference standard of the compound was spiked in naïve mouse plasma to prepare a calibration curve over a 1nM -10µM range. Three quality control samples were prepared by spiking the compound in blank mouse plasma to the final concentrations of 20, 200 and 2000nM. Calibrators and quality controls were extracted with the same extraction solution used for the plasma samples. The samples were analysed on a Waters ACQUITY UPLC/MS TQD system (Waters Inc. Milford, USA) consisting of a TQD (Triple Quadrupole Detector) Mass Spectrometer equipped with an Electrospray Ionization interface and a Photodiode Array eλ Detector. The analyses were run on an ACQUITY UPLC BEH C18 (50x2.1mmID, particle size 1.7µm) with a VanGuard BEH C18 precolumn (5x2.1mmID, particle size 1.7µm) at 40°C. H2O + 0.1% HCOOH (A) and CH3CN + 0.1% HCOOH (B) were used as mobile phase with a linear gradient from 50 to 100%B in 2min. with the flow rate set to 0.5mL/min. Electrospray ionization was applied in positive mode. Plasma levels of the parent compound was quantified by monitoring the MRM peak areas. Figure S2. Binding mode superposition of etoposide (yellow carbons, PDBID: 5GWK) and compound ARN24139 (blue carbons) into topoIIα. Molecular docking calculations reveal a favourable π-cation interaction between the E-ring and Arg487.