Design, Synthesis and Phenotypic Profiling of Simplified Gedatolisib Analogues
Abstract
:1. Introduction
2. Results and Discussion
2.1. Chemistry
2.2. Pharmacological Experiments
2.2.1. Cell Viability by MTT and CC50 Determination
2.2.2. Evaluation of the Cytotoxic Selectivity Index
2.2.3. Analysis of Cell Viability by Flow Cytometry at 48 and 72 h
2.2.4. Analysis of Phosphatidylserine Externalization and Cell Membrane Integrity
2.2.5. Cell Cycle Assessment
2.2.6. Analysis of PI3K Activation and Phosphorylation
2.2.7. Microsomal Metabolic Stability
3. Material and Methods
3.1. Synthesis and Characterization
3.1.1. Procedure of the Preparation of 4-(4,6-Dichloro-1,3,5-triazin-2-yl)morpholine (7) (Adapted from Reference [22])
3.1.2. Procedure of the Preparation of 4-(4-Chloro-6-(4-Phenylpiperazin-1-yl)-1,3,5-triazin-2-yl)morpholine (8) (Adapted from Reference [22])
3.1.3. General Procedure for the Preparation of N-Substituted Triazines (5a-f) (Adapted from Reference [22])
4-(4-(4-Phenylpiperazin-1-yl)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)morpholine (LASSBio-2247, 5a)
4-(4-(4-Methylpiperazin-1-yl)-6-(4-phenylpiperazin-1-yl)-1,3,5-triazin-2-yl)morpholine (LASSBio-2248, 5b)
2-(4-(4-Morpholino-6-(4-phenylpiperazin-1-yl)-1,3,5-triazin-2-yl)piperazin-1-yl)ethan-1-ol (LASSBio-2249, 5c)
4-(4-(4-Phenylpiperazin-1-yl)-6-thiomorpholino-1,3,5-triazin-2-yl)morpholine (LASSBio-2250, 5d)
4-(4-(4-Phenylpiperazin-1-yl)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)morpholine (LASSBio-2251, 5e)
4-(4-(1,4-Diazepan-1-yl)-6-(4-phenylpiperazin-1-yl)-1,3,5-triazin-2-yl)morpholine (LASSBio-2252, 5f)
3.2. pH-Dependent Aqueous Solubility
3.3. Cell Culture
3.4. Cancer Cell Viability Assay
3.5. PBMC Viability Assay
3.6. Flow Cytometry Analysis
3.6.1. Count and Viability Test
3.6.2. Analysis of Apoptosis by Annexin V
3.6.3. Cell Cycle Arrest
3.6.4. Evaluation of PI3K Activation and Phosphorylation
3.7. Microsomal Metabolic Stability
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Compounds | R | Solubility pH 7.4 (µM) | Solubility pH 5.8 (µM) | cLogP | cLogD7.4 | cLogD5.8 |
5a | Thiomorpholine | 2.88 | 0.52 | 3.42 | 2.65 | 0.84 |
5b | Piperidine | 0.51 | 2.84 | 4.08 | 3.08 | 1.48 |
5c | Piperazine | 59.13 | 43.35 | 2.58 | 1.08 | −1.61 |
5d | N-methylpiperazine | 50.92 | 30.69 | 2.97 | 1.81 | −0.67 |
5e | Hydroxyethylpiperazine | 49.15 | 31.19 | 2.7 | 1.84 | −0.67 |
5f | Homopiperazine | 42.38 | 39.33 | 2.96 | 0.09 | 1.25 |
9 (CAS−942789959) | Morpholine | 6.03 | 3.92 | 2.54 | −1.77 | −1.25 |
Cell Line | Gedatolisib CC50 72 h (µM) | GSK-1059615 CC50 (µM) |
---|---|---|
PC-3 | 0.54 (0.27–1.08) Emax = 80% | 6.78 (3.46–13.26) Emax: 67% |
MCF7 | 0.07 (0.03–0.20) Emax = 87% | 0.55 (0.27–1.12) Emax: 80% |
K562 | 24.09 (15.84–36.63) Emax = 64% | 1.82 (1.24–2.68) Emax = 84% |
CCRF-CEM | 0.03 (0.01–0.05) Emax = 90% | 0.43 (0.09–1.95) Emax = 93% |
MOLT-4 | 0.02 (0.01–0.03) Emax = 93% | 0.63 (0.13–3.07) Emax = 92% |
Cell Line | 5c (LASSBio-2247) CC50 (µM) | 5d (LASSBio-2248) CC50 (µM) | 5f (LASSBio-2252) CC50 (µM) |
---|---|---|---|
PC-3 | 92.29 (39.92–213.40) Emax = 58% | 91.31 (60.23–138.40) Emax = 72% | 62.15 (55.72–69.33) Emax = 84% |
MCF7 | >100 Emax: 57% | >100 Emax = 77% | 37.04 (31.82–43.12) Emax = 91% |
K562 | 25.63 (18.83–33.13) Emax = 85% | 50.22 (40.23–62.69) Emax = 80% | 23.41 (16.31–33.59) Emax = 84% |
CCRF-CEM | 37.33 (28.61–48.71) Emax = 94% | 66.50 (61.35–72.09) Emax = 95% | 6.25 (4.10–9.54) Emax = 93% |
MOLT-4 | 31.84 (23.48–43.17) Emax = 93% | 64.07 (57.45–71.45) Emax = 91% | 9.76 (5.44–17.53) Emax = 93% |
Cell Line | Gedatolisib CC50 (µM) | 5f (LASSBio-2252) CC50 (µM) |
---|---|---|
CCRF-CEM | 0.14 (0.07–0.28) Emax = 94% | 11.1 µM (5.53–22.29) Emax = 69% |
MOLT-4 | 0.15 (0.06–0.31) Emax = 92% | 36.48 (27.33–49.76) Emax = 80% |
Cell Line | Gedatolisib CC50 (µM) 72 h | Gedatolisib CC50 (µM) 48 h | 5f CC50 (µM) 72 h | 5f CC50 (µM) 48 h |
---|---|---|---|---|
CCRF-CEM | 6.15 (4.60–8.24) Emax: 94% | 5.88 (4.16–8.32) Emax: 94% | 53.15 (50.30–56.17) Emax: 96% | 48.96 (45.71–52.45) Emax: 95% |
MOLT-4 | 1.15 (0.85–1.56) Emax: 86% | 1.65 (1.24–2.20) Emax: 82% | 60.78 (58.02–63.66) Emax: 92% | 51.01 (47.89–58.67) Emax: 90% |
NADPH-Generating System | Metabolization Rates (%) | Elimination Rate Constant (k) | t1/2 (min) | Clapp (mL/min/g) | Recovery (%) |
---|---|---|---|---|---|
Present | 2.90 | 0.0015 | 462.00 | 0.058 | 101,0903 |
Absent | 9.09 | 0.0017 | 407.65 | 0.066 | 100,4186 |
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Costa, C.M.X.; Aparecida-Silva, C.; Gamba, L.E.R.; de Melo, T.N.; Barbosa, G.; Moraes Junior, M.O.d.; de Oliveira, V.R.T.; de Amorim, C.S.; Moraes, J.A.; Barreiro, E.J.; et al. Design, Synthesis and Phenotypic Profiling of Simplified Gedatolisib Analogues. Pharmaceuticals 2023, 16, 209. https://doi.org/10.3390/ph16020209
Costa CMX, Aparecida-Silva C, Gamba LER, de Melo TN, Barbosa G, Moraes Junior MOd, de Oliveira VRT, de Amorim CS, Moraes JA, Barreiro EJ, et al. Design, Synthesis and Phenotypic Profiling of Simplified Gedatolisib Analogues. Pharmaceuticals. 2023; 16(2):209. https://doi.org/10.3390/ph16020209
Chicago/Turabian StyleCosta, Caroline Marques Xavier, Cristiane Aparecida-Silva, Luis Eduardo Reina Gamba, Thalita Neves de Melo, Gisele Barbosa, Manoel Oliveira de Moraes Junior, Victoria Regina Thomaz de Oliveira, Carolinne Souza de Amorim, João A. Moraes, Eliezer Jesus Barreiro, and et al. 2023. "Design, Synthesis and Phenotypic Profiling of Simplified Gedatolisib Analogues" Pharmaceuticals 16, no. 2: 209. https://doi.org/10.3390/ph16020209
APA StyleCosta, C. M. X., Aparecida-Silva, C., Gamba, L. E. R., de Melo, T. N., Barbosa, G., Moraes Junior, M. O. d., de Oliveira, V. R. T., de Amorim, C. S., Moraes, J. A., Barreiro, E. J., & Lima, L. M. (2023). Design, Synthesis and Phenotypic Profiling of Simplified Gedatolisib Analogues. Pharmaceuticals, 16(2), 209. https://doi.org/10.3390/ph16020209