Design, Synthesis and Phenotypic Profiling of Simplified Gedatolisib Analogues

Targeted antitumour therapy has revolutionized the treatment of several types of tumours. Among the validated targets, phosphatidylinositol-3 kinase (PI3K) deserves to be highlighted. Several PI3K inhibitors have been developed for the treatment of cancer, including gedatolisib (4). This inhibitor was elected as a prototype and molecular modifications were planned to design a new series of simplified gedatolisib analogues (5a-f). The analogues were synthesised, and the comparative cytotoxic activity profile was studied in phenotypic models employing solid and nonadherent tumour cell lines. Compound 5f (LASSBio-2252) stood out as the most promising of the series, showing good aqueous solubility (42.38 μM (pH = 7.4); 39.33 μM (pH = 5.8)), good partition coefficient (cLogP = 2.96), cytotoxic activity on human leukemia cell lines (CCRF-CEM, K562 and MOLT-4) and an excellent metabolic stability profile in rat liver microsomes (t1/2 = 462 min; Clapp = 0.058 mL/min/g). The ability of 5f to exert its cytotoxic effect through modulation of the PI3K pathway was demonstrated by flow cytometry analysis in a comparative manner to gedatolisib.

The design concept of compounds 5a-f ( Figure 2) considered the maintenance of the morpholino-1,3,5-triazine scaffold, the simplification of the functionalized phenyl urea subunit by a phenylpiperazine moiety, and the isosteric replacement of the oxygen atom of the morpholine ring by sulphur (S, 5a), methylene (CH2, 5b) and NH (5c) (Figure 2) [11,12]. Further, homologation strategies [13] were adopted to design compounds 5d, 5e and 5f. In this work, a series of simplified gedatolisib analogues (5a-f) was designed, synthesised and evaluated to establish their comparative cytotoxic activity and their ability to modulate the PI3K pathway in phenotypic models using human tumour cell lines.

Chemistry
The simplified gedatolisib analogues (5a-f) were synthesised through a three-step linear synthesis, exploring cyanuric chloride (6) as starting material. As depicted in Figure  3, the first step was based on an aromatic nucleophilic mono-substitution using morpholine as a nucleophile. A second aromatic nucleophilic substitution (SNAr) was performed using phenylpiperazine to obtain the key intermediate 8. The target compounds 5a-f were obtained in overall yields ranging from 40 to 48% from the SNAr reaction between intermediate 8 and the preselected amines ( Figure 3).

Chemistry
The simplified gedatolisib analogues (5a-f) were synthesised through a three-step linear synthesis, exploring cyanuric chloride (6) as starting material. As depicted in Figure 3, the first step was based on an aromatic nucleophilic mono-substitution using morpholine as a nucleophile. A second aromatic nucleophilic substitution (SNAr) was performed using phenylpiperazine to obtain the key intermediate 8. The target compounds 5a-f were obtained in overall yields ranging from 40 to 48% from the SNAr reaction between intermediate 8 and the preselected amines ( Figure 3).

Chemistry
The simplified gedatolisib analogues (5a-f) were synthesised through a three-step linear synthesis, exploring cyanuric chloride (6) as starting material. As depicted in Figure  3, the first step was based on an aromatic nucleophilic mono-substitution using morpholine as a nucleophile. A second aromatic nucleophilic substitution (SNAr) was performed using phenylpiperazine to obtain the key intermediate 8. The target compounds 5a-f were obtained in overall yields ranging from 40 to 48% from the SNAr reaction between intermediate 8 and the preselected amines ( Figure 3).   All compounds were characterized by IR, 1 H-NMR, 13 C-NMR and HR-MS. The relative purity was determined by HPLC.
Once synthesised and characterized, compounds had their aqueous solubility determined at pH 5.8 (duodenum) and 7.4 (plasma). The results are shown in Table 1 in a comparative manner to the cLogP, cLogD 5.8 and cLogD 7.4 values predicted in silico using ACD/Percepta ® software (ACD/LABS, Toronto, Ontario). For comparison purposes, the compound 9 containing two morpholine rings (described in the literature under the code CAS-942789959) was added to the studies. The isosteric exchange of oxygen (9) for sulphur (5a) and methylene (5b) results in increased partition coefficient and decreased aqueous solubility, whereas the exchange by the NH group (5c) results in a significant increase in solubility and similar cLogP. The homologation of compound 5c by the introduction of a methyl group (5d) or hydroxyethyl group (5e) or by a methylene to the piperazine ring (5f) results in a small decrease in aqueous solubility and a respective increase in cLogP values. However, all three compounds (5d-f) showed solubility greater than 30 µM at both duodenum and plasma pH values (Table 1). Table 1. pH-dependent aqueous solubility of compounds 5a-f and their cLogP and cLogD values.
Pharmaceuticals 2023, 16, x FOR PEER REVIEW All compounds were characterized by IR, 1 H-NMR, 13 C-NMR and HR-M relative purity was determined by HPLC.
Once synthesised and characterized, compounds had their aqueous so determined at pH 5.8 (duodenum) and 7.4 (plasma). The results are shown in Tab comparative manner to the cLogP, cLogD5.8 and cLogD7.4 values predicted in silic ACD/Percepta ® software (ACD/LABS, Toronto, Ontario). For comparison purpo compound 9 containing two morpholine rings (described in the literature under t CAS-942789959) was added to the studies. The isosteric exchange of oxygen sulphur (5a) and methylene (5b) results in increased partition coefficient and de aqueous solubility, whereas the exchange by the NH group (5c) results in a sig increase in solubility and similar cLogP. The homologation of compound 5c introduction of a methyl group (5d) or hydroxyethyl group (5e) or by a methylen piperazine ring (5f) results in a small decrease in aqueous solubility and a res increase in cLogP values. However, all three compounds (5d-f) showed solubility than 30 µM at both duodenum and plasma pH values (Table 1). To determine the cytotoxic effect of the new simplified gedatolisib analogue the MTT cell viability assay was performed using the following human cell line MCF7, K562, CCRF-CEM and MOLT-4. In a first approach, the assay was validat two standard drugs: gedatolisib and GSK-1059615.
In the CCRF-CEM (human leukemia cell line), gedatolisib (4) showed a CC50 o similar to the value of 23 nM described previously by Gazi and coworkers [14]. MCF7 (breast cancer cells), compound 4 displayed a CC50 of 70 nM, a value close found by Mallon and coworkers [15], who reported CC50 = 36 nM ( To determine the cytotoxic effect of the new simplified gedatolisib analogues (5a-f), the MTT cell viability assay was performed using the following human cell lines: PC-3, MCF7, K562, CCRF-CEM and MOLT-4. In a first approach, the assay was validated with two standard drugs: gedatolisib and GSK-1059615.
In the CCRF-CEM (human leukemia cell line), gedatolisib (4) showed a CC 50 of 30 nM similar to the value of 23 nM described previously by Gazi and coworkers [14]. For the MCF7 (breast cancer cells), compound 4 displayed a CC 50 of 70 nM, a value close to that found by Mallon and coworkers [15], who reported CC 50 = 36 nM ( Table 2). On the other hand, we demonstrated for the first time the cytotoxic effect of gedatolisib against PC3 (human prostate cancer cell line), MOLT-4 (T lymphoblast cell line) and K562 (myelogenous leukemia cell line). As demonstrated in Table 2, gedatolisib was inactive on K562 (CC 50 > 24.000 nM) and showed reduced cytotoxic potency on PC3 cells (CC 50 = 540 nM). However, it was quite potent on MOLT-4 cells with CC 50 = 20 nM. Data expressed in micromolar (µM) and 95% confidence interval. Emax represents the maximum effect of the compound at its maximum concentration used. Emax values were reported when lower than 100%.
Although gedatolisib and GSK-1059615 have similar potency against inhibition of PI3K and mTOR isoforms [10], in the phenotypic assays GSK-1059615 proved to be less potent than gedatolisib, except on K562 cells. However, as expected, both compounds had the greatest cytotoxic effect on cell lines with mutations in the P3IK pathway (i.e., MCF7, CCRF-CEM and MOLT-4) ( Table 2).
After understanding the response profile of the selected cell lines to the treatment with gedatolisib and GSK-1059615, we investigated the cytotoxic activity of compounds 5a-f at a single concentration (30 µM). Compounds that showed a reduction in cell viability ≥ 50% were selected for CC 50 determination. During the experiments, compounds 5a (LASSBio-2250), 5b (LASSBio-2251) and 9 (CAS-942789959) were insoluble and precipitated on the culture medium. Therefore, they were evaluated also at the screening concentration of 1 µM. All three compounds were inactive at 1 µM. On the other hand, the analogues 5c (LASSBio-2247), 5d (LASSBio-2248) and 5f (LASSBio-2252) had a cytotoxic effect on leukemia cell lines and were selected for determination of their 50% cytotoxic concentration (CC 50 ) ( Table 3). Considering that compounds 5a-f were designed as simplified gedatolisib analogues, the absence of a cytotoxic effect on solid tumour lines, particularly on MCF-7, was not expected. MCF-7 strains are mutated with overexpression of the PI3KCA gene, so they are strains sensitive to PI3Kα inhibitors (p110α). As previously informed, gedatolisib, which inhibits PI3Kα with IC 50 = 0.4 nM [10], showed a CC 50 on MCF-7 cells of 70 nM ( Table 2). This result suggests that compounds 5a-f do not modulate PI3Kα isoform activity or do so with low potency. Considering this hypothesis, cytotoxic response concentration studies were maintained with MCF-7 and PC-3 cells.
In the concentration response study, using the 72 h MTT cell viability assay, 5c and 5d showed the maximum effect (E max ) on PC3 cells of 58% and 72%, respectively, and displayed CC 50 = 92.29 and 91.31 µM, respectively. Similar results were found on MCF-7 (E max = 57% and 77%, respectively, and CC 50 > 100 µM) ( Table 3). These results support the hypothesis that such compounds are weak inhibitors of PI3Kα.
Trying to establish a suitable experimental condition for the flow cytometry study, LASSBio-2252 (5f) and gedatolisib (4) were tested with the 48 h MTT assay on the CCRF-CEM and MOLT-4 cell lines. As depicted in Table 4, there is approximately a 10-fold loss of cytotoxic potency of gedatolisib when assayed at 48 h, whereas the loss of potency of compound 5f was less than 4-fold for MOLT-4 and less than 2-fold for CCRF-CEM (Table 4  versus Table 3). However, there is a clear impact on reducing the maximum cytotoxic effect (E max ) induced by 5f on CCRM-CEM cells. Data expressed in micromolar (µM) and 95% confidence interval. Emax represents the maximum effect of the compound at its maximum concentration used. Emax values were reported when lower than 100%.

Evaluation of the Cytotoxic Selectivity Index
The cytotoxic selectivity index (IS) represents how selective the compound is for tumour cells in relation to nontumour cells, indicating less possibility of adverse effects [16]. The IS of gedatolisib and compound 5f (LASSBio-2252) was calculated by the ratio between the CC 50 on tumour cells and the CC 50 on nontumour cells. The latter was determined by the 72 h MTT assay using human peripheral blood mononuclear cells (PBMCs). At the highest concentration studied (i.e., 50 µM), gedatolisib was not cytotoxic to PBMCs, reducing the viability of normal mononuclear cells by only 33% (E max = 33%). Concentrations higher than 50 µM could not be studied due to the insolubility of gedatolisib. A similar result was observed for LASSBio-2252 (5f). It was assayed at five concentrations, ranging from 0.03 to 100 µM. Even at 100 µM, LASSBio-2252 (5f) was unable to induce a cytotoxic effect above 26% (E max = 26%) on PBMCs (Figures S19 and S20).

Analysis of Cell Viability by Flow Cytometry at 48 and 72 h
To confirm the cytotoxic effect in a nonmetabolic assay, the comparative cytotoxic potency of 5f (LASSBio-2252) and gedatolisib (4) was restudied, using the Guava Muse Luminex ® benchtop cytometer cell viability kit. The assay was conducted using different concentrations of 4 and 5f (0.003 to 100 µM) and MOLT-4, CCRF-CEM and MCF7 cell lines.
The cytotoxic potency of gedatolisib (4) in the Muse ® Count & Viability Kit at 72 h was lower than in the 72 h MTT (Table 5). In fact, gedatolisib (4) showed 100-fold lower potency than that determined by the MTT method on CCRF-CEM (CC 50 = 6.15 µM) and MOLT-4 (CC 50 = 1.15 µM). For the MCF7 cells, the loss of potency was so great that the CC 50 could not be determined. At 50 µM, gedatolisib displayed a maximum effect (E max ) of only 54%. There was also a loss of potency of LASSBio-2252 (5f), but the difference did not exceed 10-fold for both CCRF-CEM and MOLT-4. Compound 5f was inactive on MCF-7 (Table 5). Data expressed in micromolar (µM) and 95% confidence interval. Emax represents the maximum effect of the compound at its maximum concentration used. Emax values were reported when lower than 100%.
These data confirm the cytotoxic effect of the simplified gedatolisib analogue 5f against PI3K mutated cells lines, using a different methodology than MTT, and whose observed cytotoxic effect depends on the disruption of the cell membrane and no longer on the metabolic activity of mitochondria, on which MTT is based.
The Muse ® Count & Viability Kit was also used to analyse cell viability by flow cytometry at 48 h (Table 5). No significant differences in gedatolisib potency were identified on CCRF-CEM and MOLT-4 when compared to the assay performed at 72 h ( Table 5). The data suggest that the cytotoxicity assessed by the MTT method is incubation time dependent (48 h or 72 h, Tables 2-4), whereas the cytotoxicity measured by the viability and cell counting kit is not incubation time dependent (Table 5). Similar results were found for compound 5f. Another intriguing fact is that the difference in cytotoxic potency between gedatolisib and 5f was about 10-fold by this method, whereas it was 100-fold to 700-fold by 48 h and 72 h via the MTT method, respectively.

Analysis of Phosphatidylserine Externalization and Cell Membrane Integrity
For quantitative analysis of live, early and late apoptosis, and cell death on both the CCRF-CEM and MOLT-4 cell lines, treated with gedatolisib and compound 5f (LASSBio-2252), the Muse ® Annexin V and Dead Cell Kit was used. Compounds were studied using three concentrations. The first was based on the average of the CC 50 values obtained in 48 h and 72 h (Table 5), a second concentration was 10 times lower, and a third was 10 times higher.
As can be seen in Figure 4, at their CC 50 values both gedatolisib and its simplified analogue 5f reduced cell viability by about 50% and caused about 50% total apoptosis of the CCRF-CEM. In contrast, on the MOLT-4 compound 5f reduced the number of viable cells by only 14% when tested at its CC 50 value and by 80% when tested at 10-fold concentration ( Figure 5). On the other hand, gedatolisib reduced cell viability by 90% already at a concentration 10 times lower than its CC 50 . However, both compounds led to late apoptosis of MOLT-4 cells with increasing concentrations. These results suggest that, despite the difference in potency, gedatolisib and its analogue 5f (LASSBio-2252) displayed similarity in the cellular mechanism of action against MOLT-4 and CCRF-CEM.
the CCRF-CEM. In contrast, on the MOLT-4 compound 5f reduced the number of viable cells by only 14% when tested at its CC50 value and by 80% when tested at 10-fold concentration ( Figure 5). On the other hand, gedatolisib reduced cell viability by 90% already at a concentration 10 times lower than its CC50. However, both compounds led to late apoptosis of MOLT-4 cells with increasing concentrations. These results suggest that, despite the difference in potency, gedatolisib and its analogue 5f (LASSBio-2252) displayed similarity in the cellular mechanism of action against MOLT-4 and CCRF-CEM.

Cell Cycle Assessment
To understand in which phase of the cell cycle the compounds can act, the Muse ® Cell Cycle Kit was used to allow the quantitative measurement of the percentage of cells in the G0/G1, S and G2/M phases of the cell cycle. The assay was performed using the  To understand in which phase of the cell cycle the compounds can act, the Muse ® Cell Cycle Kit was used to allow the quantitative measurement of the percentage of cells in the G0/G1, S and G2/M phases of the cell cycle. The assay was performed using the CCRF-CEM cell line treated with doxorubicin, vincristine, gedatolisib and LASSBio-2252 (5f). As demonstrated in Figure 6, doxorubicin at the concentration of 0.5 µM behaves as expected, promoting nonspecific action on the cell cycle. Vincristine at 5 µM promotes increased arrest in the G2/M phase, due to its action as an antimicrotubule agent. The concentration choice was based on previous data in the literature [17][18][19][20]. Gedatolisib led to cell cycle arrest in the G0/G1 phase at both concentrations tested (0.6 µM and 6 µM), in agreement with the behaviour expected for PI3K inhibitors [21]. Similarly, LASSBio-2252 (5f) led to cell cycle arrest in G0/G1 when tested using its CC 50 value (i.e., 50 µM).

Analysis of PI3K Activation and Phosphorylation
To assess whether the cytotoxic effect is due to the modulation of the PI3K pathway, the evaluation of the activity on the PI3K pathway was investigated by using the Muse ® PI3K Activation Dual Detection Kit on the CCRF-CEM and MOLT-4 cell lines. As expected, the treatment of CCRF-CEM cells with gedatolisib, at a concentration equivalent to its CC50 (~6 µM) and 10 times lower than its CC50 (0.6 µM) (Table 4), increased the percentage of cells in the inactivated state (i.e., unable to phosphorylate the AKT substrate) by about 75% (Figure 7), whereas LASSBio-2252 (5f) raised the percentage of inactivated cells to 30% and 65% when tested at its CC50 and at a concentration 10 times higher than its CC50, respectively (Figure 7). Interestingly, in MOLT-4 cells, even at the concentration 10 times lower than its CC50, LASSBio-2252 (5f) reduced PI3K-mediated phosphorylation by 79% (Figure 8).

Analysis of PI3K Activation and Phosphorylation
To assess whether the cytotoxic effect is due to the modulation of the PI3K pathway, the evaluation of the activity on the PI3K pathway was investigated by using the Muse ® PI3K Activation Dual Detection Kit on the CCRF-CEM and MOLT-4 cell lines. As expected, the treatment of CCRF-CEM cells with gedatolisib, at a concentration equivalent to its CC 50 (~6 µM) and 10 times lower than its CC 50 (0.6 µM) (Table 4), increased the percentage of cells in the inactivated state (i.e., unable to phosphorylate the AKT substrate) by about 75% (Figure 7), whereas LASSBio-2252 (5f) raised the percentage of inactivated cells to 30% and 65% when tested at its CC 50 and at a concentration 10 times higher than its CC 50 , respectively ( Figure 7). Interestingly, in MOLT-4 cells, even at the concentration 10 times lower than its CC 50 , LASSBio-2252 (5f) reduced PI3K-mediated phosphorylation by 79% (Figure 8).

Synthesis and Characterization
All reagents and solvents were purchased from commercial suppliers. The reactions were monitored by thin-layer chromatography, which was performed on aluminium sheets precoated with silica gel 60 (HF-254, Merck) to a thickness of 0.25 mm. The chromatograms were viewed under ultraviolet light (254-365 nm). For column chromatography, Merck silica gel (230-400 mesh) was used. 1 H-NMR, 13 C-NMR and DEPT-135 spectra were determined in deuterated dimethyl sulfoxide using a Varian NMR at 400 MHz and 500 MHz. Chemical shifts are given in parts per million (d) with tetramethylsilane as the internal standard, and coupling constant values (J) are given in Hertz (Hz). Signal multiplicities are represented by: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet) and br (broad signal).
Infrared (IR) spectra were obtained with a FTIR Thermo Scientific™ Nicolet™ iS10 spectrophotometer in ATR mode using ruby crystal support, and the absorption values were expressed in inverse centimetres (cm −1 ). Melting points were determined by differential scanning calorimetry with a Shimadzu DSC-60 apparatus until 300 • C (heating rate: 20 • C/min). DSC-60 equipment calibration was performed using indium (In) as the standard (m.p. 157.2 • C).
The purity of compounds was determined by HPLC (95%) using the Shimadzu-LC20AD apparatus, a Kromasil 100-5C18 (4,6 mm x 6250 mm) column and the SPD-M20A detector (diode array) at 254 nm for quantification of analyte in a 1 mL/min constant flux. The injector was programmed to inject a volume of 20 µL. Mass spectrometry was obtained by positive ionisation on a BrukerAmaZon SL, and data were analysed in Compass 1.3.SR2 software.
3.1.1. Procedure of the Preparation of 4-(4,6-Dichloro-1,3,5-triazin-2-yl)morpholine (7) (Adapted from Reference [22]) Cyanuric chloride (6) (0.5g, 2.71 mmol) was solubilized in 15 mL of THF and subjected to stirring under an ice bath. A mixture of morpholine (0.2 mL, 2.43 mmol) and TEA (0.4 mL, 2.43 mmol) dissolved in 15 mL of THF was added dropwise with the aid of a standard addition funnel. After the addition, the reaction was kept under constant stirring at 0 • C for 6 h. After the end of the reaction, the reaction mixture was concentrated to half the volume under reduced pressure, and crushed ice was added, leading to the precipitation of a white solid that was filtered and washed with 10 mL of cold distilled water. The product obtained was considered pure and was used in the next step without purification. Compound 7 (300 mg; 1.28 mmol) was solubilized in THF (15 mL) and subjected to stirring under an ice bath. A mixture of phenylpiperazine (1.1 equivalents) and TEA (1.1 equivalents) solubilized in THF (15 mL) was added dropwise with the aid of a standard addition funnel. After the addition, the reaction was kept under constant stirring at 0 • C. After 24 h, the mixture was concentrated, and the residue was treated with 5% Na 2 CO 3 until pH 7-8. The product was extracted with CHCl 3 . The organic phase was dried with Na 2 SO 4 and concentrated to a pale-yellow oil, which was filtered over SiO 2 to obtain 414 mg of the translucent oil as a product. Compound 8 was obtained in 90% yields.

pH-Dependent Aqueous Solubility
A stock solution of the target compound was prepared by dissolving 1 mg in methanol, and the wavelength of greatest absorption of each compound was obtained by scanning between wavelengths from 200 nm to 500 nm. Six dilutions were prepared from the stock solution and the absorbance reading in the UV spectrophotometer (SpectraMax M5, Molecular Devices, USA). The calibration curve with linear regression was created with the values obtained, and the solubility test was measured by correlating the concentration with the absorbance obtained by ultraviolet. Each compound was added in excess at phosphate buffer solution pH 5.8 and 7.4 to obtain a supersaturated solution that was kept under stirring for 4 h at 37 • C and then was filtered through a 0.45 µm filter for reading at the corresponding wavelengths. The solubility was determined through the equation of the straight line obtained by the linear regression of the calibration curve of the compounds [24,25].
Human cancer cell lines were acquired from the Cell Bank of Rio de Janeiro-BCRJ. PBMCs were isolated from healthy patients and followed the guidelines approved by the Ethics Committee CAAE: 38257914.7.0000.5259.

Cancer Cell Viability Assay
Cell viability was assessed by the MTT assay. The lines selected for this study were plated according to their respective densities, which ranged from 3 × 10 4 to 4 × 10 4 cells per well. Subsequently, they were treated with test compounds at different concentrations (0.00003 µM to 150 µM), gedatolisib and GSK-1059615. The plates were incubated in an oven at 5% CO 2 , at 37 • C for 48 and 72 h.
After incubation, the plates are centrifuged at 440× g for 10 min. Then, 110 µL of the supernatant was withdrawn and each well received 10µL of the MTT solution. The plate was again incubated in an oven at 37 • C and 5% CO 2 for 3 to 4 h. At the end of the incubation time, 100 µL of SDS-HCL detergent was added to solubilize the formazan crystals. The plate was shaken on an automatic overnight shaker and then read at a wavelength of 595 nm on the Molecular Devices Spectramax M5 plate reader. CC 50 values were calculated from means (95% confidence interval (CI)) using GraphPad Prism ® , version 6 [26,27].

PBMC Viability Assay
Donor blood was collected and EDTA (ethylenediamine tetraacetic acid) was added to the sterile laminar flow in a 1:20 ratio. In a 15 mL falcon tube, 4.5 mL of Ficoll gradient was added, and then 9 mL of blood was added over the gradient in a 1:2 ratio. The tubes were centrifuged for 40 min at a speed of 750× g with an acceleration of 2 and a brake of 0. The ring of mononuclear cells formed was collected and added to a new tube.
Tubes with PBMCs were centrifuged in an incomplete RPMI medium for 10 min at 580× g with an acceleration of 8 and a brake of 9. The supernatant was discarded, and the cells were resuspended in 1 mL of RPMI medium supplemented with 20% PBS. PBMCs were added to 96-well plates at a concentration of 1 × 10 5 cells per well. Test compounds were added at concentrations ranging from 0.0003 to 100 µM and incubated for 72 h in a CO 2 incubator at 37 • C [28].
After incubation, each well received 20 µL of MTT (5 mg/mL), followed by incubation for 4 h in the CO 2 oven and centrifugation for 12 min at 1200× g. The supernatant from each well was taken, and 100 µL of isopropyl alcohol was added to solubilize the formazan crystals. The absorbance of the plates was measured at 570 nm in the Flex Station equipment. CC 50 values were calculated from means (95% confidence interval (CI)) using GraphPad Prism ® , version 6 [16,29].

Flow Cytometry Analysis
The benchtop cytometer Guava Muse Luminex ® Cell Analyzer (Merck) was used to elucidate the mechanism of action of LASSBio-2252 in the MCF-7, CCRF-CEM and MOLT-4 cells. Cell viability, cell death pattern, cell cycle and detection of PI3K activation were evaluated, and each analysis was performed using specific protocols of the Guava Muse Cell Analyzer.
Cell lines were added in 24-well plates, and each well received 500 µL of cells in suspension with density ranging from 1 × 10 5 to 3 × 10 5 cells per mL, in duplicate wells. After 24 h, each well received concentrations of LASSBio-2252 or the controls gedatolisib, doxorubicin and vincristine (0.003 to 100 µM) and were reincubated in an oven with 5% CO 2 at 37 • C for 48 h and 72 h.

Count and Viability Test
The Count & Viability Kit (catalog n o . MCH100102, LUMINEX) differentiates between viable and nonviable cells based on the permeability of the dyes present in the reagent to the cell.
In total, 20 µL of the cell solution was added to 380 µL of the reagent in 1 mL Eppendorf tubes and incubated for 5 min at room temperature in the dark. Samples from three independent experiments carried out in duplicate were read on the cytometer, followed by statistical analysis of the histograms on Prism6 to determine the CC 50 .

Analysis of Apoptosis by Annexin V
The Annexin V and Dead Cell Kit (catalog n o . MCH100105, Luminex) contains Annexin V, a phospholipid marker phosphatidylserine, and the molecule 7-AAD, a DNA marker for cells with a ruptured membrane. An aliquot of 100 µL of the cell solution was added to microtubes with a density of up to 5 × 10 5 cells/mL and homogenised with 100 µL of Annexin V reagent. After incubation for 20 min at room temperature, the samples were read on the cytometer. The data with the histograms generated contain information on the percentage of viable cells, in early apoptosis, late apoptosis and dead cells. Samples from three independent experiments were performed in duplicate, and then statistical analysis was performed using the Prism6 statistical program.

Cell Cycle Arrest
The Guava Muse Luminex ® Cell Cycle Kit (catalog n o MCH100106, Luminex) contains the DNA marker propidium iodide (PI) and RNAse A to avoid nonspecific labelling.
After 48 h of incubation in an incubator at 37 • C with 5% CO 2 , 1 mL of the MOLT-4 cell solution and 500 µL of the CCRF-CEM cell solution, contained in the wells, were transferred to Eppendorf tubes and centrifuged at 300× g for 5 min. The volumes used ensured that the cell density remained within the recommended range (1 × 10 6 to 4 × 10 6 cells/mL) at the end of the 48 h incubation period.
The supernatant was discarded, and the pellet formed was resuspended in 1 mL of saline PBS, homogenised, and centrifuged at 300× g for 5 min. The formed pellet was resuspended in residual PBS by homogenization. To fix cellular events, 1 mL of ice-cold 70% alcohol was added dropwise into the tubes and vortexed at medium speed. The samples were incubated, before staining, for at least 3 h at −20 • C.
At the end of the incubation time at −20 • C, the samples were centrifuged at 300× g for 5 min, and then the supernatant was discarded and resuspended in 250 µL of PBS. The samples were again centrifuged at 300× g for 5 min and the supernatant discarded, which constituted the washing step. The pellet formed was resuspended in 200 µL of the Guava Muse Luminex ® Cell Cycle, and the tubes were incubated for 30 min at room temperature, away from light.
After incubation, the samples were read in Guava Muse Luminex ® , and the data with the histograms generated, which contain information on the percentage of the different phases of the cell cycle, were analysed in the statistical program Prism6. This methodology was performed in duplicate of three independent experiments.

Evaluation of PI3K Activation and Phosphorylation
The methodology evaluates the activation and phosphorylation of PI3K and was performed from the labelling with the Guava Muse Luminex ® PI3K Activation Dual Detection Kit (catalog n o . MCH200103, Luminex) containing the anti-AKT/PKB-PECy5 antibody to measure total PI3K and the anti-phospho-AKT (Ser473)-Alexa Fluor ® 555 antibody for measurement of activated PI3K.
Following 48 h of incubation in an incubator at 37 • C with 5% CO 2 , 1 mL of the cell solution was transferred to an Eppendorf tube, washed with PBS and centrifuged at 300× g for 5 min. The supernatant was discarded, and the pellet formed was resuspended in 500 µL of PBS. The samples were again centrifuged at 300× g for 5 min and the supernatant discarded.
Then the cell solutions were resuspended in 200 µL of assay buffer (Part no. CS202124), followed by the addition of 200 µL of fixation buffer (Part n o . CS202122), ending up with a total volume of 400 µL per sample. The samples were incubated for 5 min on ice and centrifuged at 300× g for 5 min and the supernatant discarded.
Cells were permeabilized by adding 200 µL of ice-cold permeabilization buffer (Part n o . CS202125) and then incubated on ice for 5 min and centrifuged at 300× g for 5 min, and the supernatant was discarded. The formed pellets were resuspended in 450 µL of assay buffer and vortexed at medium speed. In total, 90 µL of the solution from each sample was aliquoted into a new tube, and 10 µL of the cocktail solution containing Anti-phospho-Akt (Ser473), Alexa Fluor ® 555 and Anti-Akt/PKB, Pecy5 was added. The samples were homogenised and incubated for 30 min in the dark at room temperature.
After the incubation step, 100 µL of assay buffer was added per sample and centrifuged at 300× g for 5 min, and the supernatant was discarded. The formed pellets were resuspended in 200 µL of assay buffer and vortexed, and the samples were read in Guava Muse Luminex ® . Data from three independent experiments performed in duplicate were analysed in the Prism6 program to determine the percentage of cells expressing activated and inactivated PI3K.

Microsomal Metabolic Stability
The metabolic stability of compound 5f (LASSBio-2252) was studied as previously described [30], and 1 mg/mL of human rat microsomes was incubated with compound 2f (10 µM) in the presence and in the absence of the cofactor (i.e., NADPH generating system). After mixing and incubation at 37 • C in a shaking water bath for 1 h, 500 µL cold acetonitrile containing the internal standard was added to terminate the reaction at different time intervals (0, 15, 30, 45 and 60 min). After mixing and centrifugation at 10,500× g for 15 min at 4 ºC, the supernatant was filtered and then was injected into the HPLC system using a mixture of methanol: water acidified (0.1% formic acid, pH 3.0) (50:50, v/v) as the mobile phase at a flow rate of 1.0 mL/min. After analysing the results, the pharmacokinetic parameters were calculated as microsomal half-life (t 1/2 ) and apparent intrinsic clearance (Clapp) [31].

Conclusions
In summary, we describe the identification of a new cytotoxic agent, designed as a simplified analogue of gedatolisib. LASSBio-2252 (5f) was shown to have cytotoxic potency in the low micromolar range and with enhanced cytotoxic action on leukemic versus solid tumour cell lines. The cytotoxic effect was confirmed by different methodologies and in a comparative manner to gedatolisib. The ability of 5f to exert its cytotoxic action through modulation of the PI3K pathway was demonstrated in a phenotypic model compared to gedatolisib. This new analogue has great aqueous solubility and high metabolic stability in rat liver microsomes. Taken together, LASSBio-2252 (5f) can be considered a new lead candidate, whose subsequent structural optimization steps are required to increase its cytotoxic potency.