comercial
8. Conclusiones
8.4 Conclusiones desde la perspectiva de la política
including, sulfanilamide, acetyl acetone and ethyl acetoacetate. The newly synthesized compounds were evaluated for their inhibitory profiles against four carbonic anhydrase isoforms: hCA I, II, IV, and VII. The tested compounds showed selectivity to CA II and one series emerged as the most potent CA II inhibitors with low to sub nanomolar Ki values (0.36-6.9 nM). X-ray crystallographic studies of the compounds against CA II were performed to further understand and rationalize their strong CA II inhibitory profile. X-ray co-crystallographic analysis of the adducts of hCA II with three derivatives was achieved at resolutions of 1.32-1.67 Å. The X-ray crystallographic studies showed defined moieties within the ligand structures specifically interact with the hydrophobic and hydrophilic halves of the CA II active site. As CA II up-regulation is implicated with glaucoma, four of the most active CA II inhibitors (Ki values of 0.36-2.9 nM) were evaluated for their IOP lowering action against DRZ as the standard. Compound (E)-3-oxo-N-(4-sulfamoylphenyl)-2-(thiophen-2-ylmethylene)butanamide showed a comparable IOP lowering effect to DRZ (IOP reduction = 8.5 mmHg), while compounds chlorobenzylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide and (E)-2-(4-methoxybenzylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide were more potent than DRZ with IOP reduction of 12.8 and 12.3 mmHg, respectively. Therefore, this study presents compounds (E)-2-(4-chlorobenzylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide and (E)-2-(4-methoxybenzylidene)-3-oxo-N-(4-sulfamoylphenyl)butanamide as promising candidates for the development of
xxiv therapeutic anti-glaucoma strategies. Chapter 7 has been accepted to J. Med.
Chem. (doi: 10.1021/acs.jmedchem.9b02090).
xxv
List of Abbreviations
1H NMR Proton nuclear magnetic resonance
13C NMR Carbon nuclear magnetic resonance
AAZ Acetazolamide
BST-2 Bone marrow stromal antigen 2
C Capsid
CFTR Cystic fibrosis transmembrane conductance regulator CHIKF Chikungunya fever
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NCDS Australian national communicable diseases surveillance NCI National cancer institute
xxvii
OAG Open angle glaucoma
PAINS Pan-assay interference compounds PDB Protein data bank
PES Potential Energy Surfaces
POPC 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
RNA Ribonucleic acid
ROS Reactive oxygen species RTL Relative to liposomes
SAR Structure activity relationship SCXRD Single crystal X-ray diffraction
SR Stacked rings
TBACl Tetrabutylammonium chloride TCEP Tris(2-carboxyethyl)phosphine TEACl Tetraethylammonium chloride
THF Tetrahydrofuran
TLC Thin layer chromatography TMS Tetramethylsilane
TOFA 5-(Tetradecyloxy)furan-2-carboxylic acid UOW University of Wollongong
UPR Unfolding protein response
Val Valine
WHO World health organization
Vln Valinomycin
xxviii List of Figures
Chapter 1:
Figure 1.1: Schematic representation of arbovirus subclasses taxonomy. 1 Figure 1.2: Schematic diagram of alphavirus life cycle. Reproduced with
permission from reference 4.4
4
Figure 1.3: a) CHIKV nsP2 crystal structure (PDB id: 3TRK) b) The macrodomain (nsP3) of CHIKV (PDB id: 3GPG).
7
Figure 1.4: Structure of CHIKV nsP2 inhibitors identified by in silico studies. 9 Figure 1.5: Some known drugs inhibiting CHIKV replication. 11 Figure 1.6: Selected inhibitors of the CHIKV from the screening. CHIKV EC50
are in parenthesis.
12
Figure 1.7: Purines as of CHIKV inhibitors. 13
Figure 1.8: Selected CHIKV inhibitors with different scaffolds. 14 Figure 1.9: Iterative cycle of drug design and lead identification and optimization.
15
Chapter 2:
Figure 2.1: Examples of heterocyclic classes showing anti-CHIKV activity, including thiazolidineone 2.1 and 2.2, pyrimidine 2.3 and pyrimidine fused rings 2.4 and 2.5-2.7.
25
Figure 2.2: Design of the targeted uracil-rhodanine conjugates. 26 Figure 2.3: 1H NMR spectrum of the MCR product of the two regioisomers of 2.16 in a ratio of 1:5. stabilizing hydrogen bonds of the regioisomer ZA.
30
Figure 2.6: Proposed mechanism for the synthesis of isomers Z and E, the alcohol intermediate of interest (red) and the dehydration step (blue) are highlighted. Note the Z isomer is the product of the reaction in this example.
30
Figure 2.7: Structures of three alcohol intermediate conformers (C1 – C3) investigated. ΔrG0 (298 K) energies calculated relative to the starting materials, Compound 2.13 and benzaldehyde, in kcal.mol-1. All methods employed the aug-cc-pVDZ basis set.
31
xxix
Figure 2.8: Energy profile for the formation of compound 2.14(Z) and 2.14(E), calculated with DFT M06-2X/aug-cc-pVDZ. Divergent reaction pathways are colour coded red for E formation and blue for Z formation. SR = stacked rings conformer.
33
Chapter 3:
Figure 3.1: Application of medicinal chemistry concepts to current CHIKV inhibitor MADTP-372 towards new potential anti-CHIKV agents. The assumed core, including the explicit hydrogen, are highlighted green.
50
Figure 3.2: X-ray crystal structure of 3.15 (a, 3.20 (b and 3.24 (c. 52 Figure 3.3: ORTEP depictions of compounds 3.23 and 3.39 at 70% probability. 54 Figure 3.4: Structures and Density-Functional Theory (DFT) minimal pharmacophore models of the [1,2,4]triazolo[4,3-a]pyrimidine (3.53), the azahypoxanthine (3.54), the hypoxanthine (3.55) and the pyrimidone tautomers (3.56a-b) showing the electrostatic potential mapped onto two different electron isodensities to convey both the resolved atoms (middle row) and the diffuse molecular surface as seen by the chemical environment (bottom row). Potential H-bond interactions indicated by coloured arrows. The tautomers shown for 3.54 and 3.55 calculated by DFT to represent 100% of the species in vivo. For the pyrimidones 3.56, the relative concentration of the two tautomers is shown.
59
Figure 3.5: a) Consensus picture of the minimal pharmacophore with inferred important H-bond interactions indicated. For compounds like 3.54 and 3.55 R2 could likely represent an H-bonded H2O molecule. b) The DFT Potential Energy Surfaces of 3.53 – 3.55 for the phenyl torsions as defined by the bold blue bonds in (a.
60
Chapter 4:
Figure 4.1: Structure of the active potential leads. 88 Figure 4.2: Possible modification of NCI_37168 (4.5). 89
Chapter 5:
Figure 5.1: Schematic representation showing the design of switchable anion transporters.
102
Figure 5.2: Reported GSH mediated activation and putative transporters 5.1-5.5. 102 Figure 5.3: X-ray structure of anion transporter 5.2. DMSO complex. 104
Figure 5.4: X-ray structure of complex 5.14. 105
Figure 5.5: X-ray structure of anion complex 5.16. 106
xxx Figure 5.6: X-ray structure of anion transporter 5.17. 107 Figure 5.7: Stack plot of 1H NMR spectroscopic titration of receptor 5.2 (1 mM) with TBACl in CD3CN at 298 K.
108
Figure 5.8: Crystal structure of 5.2 with chloride complex. 109 Figure 5.9: a-c) schematic representation of ISE-based assays used to investigate the mechanism of anion transport of receptors 5.1-5.5. d) Chloride efflux achieved by transporters 5.1-5.5 (1.0 mol%) (rtl) for transporters 5.1-5.4 and 0.8 mol% (rtl) for transporter 5.5. e) Chloride efflux achieved by transporter 5.2 at 1.0 mol%
(rtl) in the absence or presence of cationophores (monensin or valinomycin) monitored over a period of 5 min.
111
Figure 5.10: a-d) Schematic representation of the HPTS-based assays used in the current study e) H+/Cl- symport or OH-/Cl- antiport facilitated by compounds 5.1-5.5 (1.0 mol% (rtl)). f) Different conditions were applied to determine the effect of addition of the protonphore cccp at 0.5 mol% (as a measure of chloride uniport), oleic acid at 1 mol% (as a source of fatty acid) and BSA-treated lipid (to test if the transport is fatty acid dependent) on the rate of chloride transport of receptor 5.2 (1 mol%).
113
Figure 5.11: Observed fluorescence ratio response due to HCl influx in the presence of compounds 5.2 (1 mol%) into vesicles loaded with KCl (100 mM) and suspended in KCl, KBr, KI, KNO3 and KClO4 (100 mM). All external and internal solutions were buffered to pH 7 with HEPES (10 mM).
114
Figure 5.12: Chemical structure of tested compounds and the three reducing agents used in the current study.
115
Figure 5.13: Reduction of complexes 5.13 and 5.16 in organic solvent (DMSO) monitored by a) fluorescence and b) UV-Vis spectroscopies by thiols; namely GSH (reduced glutathione) and DTT (dithiothreitol), and TCEP (tris(2-carboxyethyl)phosphine hydrochloride). Fluorescence readings are averages of three replicates and UV-Vis readings are averages of two replicates, always with standard deviations less than 10%.
116
Figure 5.14: Reduction of complexes 5.14 and 5.17 in organic solvent (DMSO) monitored by a) fluorescence and b) UV-Vis spectroscopies by thiols; namely GSH (reduced glutathione) and DTT (dithiothreitol), and TCEP (tris(2-carboxyethyl)phosphine hydrochloride). Fluorescence readings are averages of three replicates and UV-Vis readings are averages of two replicates, always with standard deviations less than 10%.
117
Figure 5.15: Reduction of complexes 5.13 and 5.16 in liposomes monitored by 119
xxxi
a) fluorescence and b) UV-Vis spectroscopies by thiols; namely GSH (reduced glutathione) and DTT (dithiothreitol), and TCEP (tris(2-carboxyethyl)phosphine hydrochloride). Fluorescence readings are averages of three replicates and UV-Vis readings are averages of two replicates, always with standard deviations less than 10%.
Figure 5.16: Reduction of complexes 5.14 and 5.17 in liposomes monitored by UV-Vis spectroscopy by thiols; namely GSH (reduced glutathione) and DTT (dithiothreitol), and TCEP (tris(2-carboxyethyl)phosphine hydrochloride). UV-Vis readings are averages of two replicates, always with standard deviations less than 10%.
120
Figure 5.17: Reduction of complexes 5.13 and 5.16 in GSH encapsulated liposomes Fluorescence readings are averages of three replicates, always with standard deviations less than 10%.
120
Figure 5.18: Reduction of complexes 5.13, 5.14, 5.16 and 5.17 in GSH encapsulated liposomes. UV-Vis readings are averages of two replicates, always with standard deviations less than 10%.
121
Figure 5.19: KCl-KOH liposomal model used to assess the switchable-time
dependent studies. POPC vesicles were loaded with KCl (100 mM), buffered to pH 7.0 with HEPES (10 mM). The test compound was added at 0 s and detergent was added at 200 s.
122
Figure 5.20. Observed fluorescence ratio response due to H+/Cl- symport or Cl -/OH-antiport upon reduction of complexes a) 5.13 and b) 5.14 (1 μM) by GSH (6 μΜ) using KCl-KOH assay from POPC vesicles loaded with KCl (100 mM), buffered to pH 7.0 with HEPES (10 mM) at different time intervals. The test complexes 5.13 and 5.14 (1 mol%) and KOH were added firstly, then GSH was added at 0 s. DMSO, GSH (3 mol%), parent anion transporters 5.2 and 5.3 and complex 5.13 and 5.14 (without addition of DTT) were used as controls.
Detergent was added at 200 s. Ionophore concentrations are shown as ionophore to lipid molar ratios. Error bars represent SD from at least three repeats.
123
Figure 5.21. Observed fluorescence ratio response due to H+/C- symport or Cl -/OH- antiport upon reduction of complexes a) 5.16 and b) 5.17 (1 μM) by GSH (6 μΜ) using KCl-KOH assay from POPC vesicles loaded with KCl (100 mM), buffered to pH 7.0 with HEPES (10 mM) at different time intervals. The test complexes 5.16 and 5.17 (1 mol%) and KOH were added firstly, then GSH was added at 0 s. DMSO, GSH (3 mol%), parent anion transporters 5.2 and 5.3 and complex 5.16 and 5.17 (without addition of DTT) were used as controls.
124
xxxii Detergent was added at 200 s. Ionophore concentrations are shown as ionophore
to lipid molar ratios. Error bars represent SD from at least three repeats.
Chapter 6:
Figure 6.1: Structure of designed fluorescent transporters 6.1-6.4. 150 Figure 6.2: X-ray crystal structure of 6.1 a) ORTEP diagram showing 50%
probability anisotropic displacement ellipsoids at 100 K. b) space-filling models.
153
Figure 6.3: Stack plot of the 1H NMR spectrum of receptor 6.3 (2.0 x 10-3 M) upon titration with chloride (0-17 eq.) added as its tetrabutylammonium salt.
153
Figure 6.4: a-c) schematic representation of ISE-based assays used to investigate the mechanism of anion transport of receptors 1-4 a) Cl-/NO3-antiport, b) and c) cationophore coupled-KCl, valinomycin and monensin to measure the Cl- uniport and M+/Cl+transport, respectively.
156
Figure 6.5: Chloride efflux achieved by transporters 6.1–6.4 (0.10 mol% for transporter 6.1 and 0.05 mol% for transporters 6.2-6.4) from unilamellar POPC vesicles containing 489 mM KCl buffered to pH 7.2 with 5 mM potassium phosphate salts, suspended in 489 mM KNO3 buffered to pH 7.2 with 5 mM phosphate salts. At the endpoint of each experiment (300 s), the detergent was added to lyse the vesicles and calibrate the electrode to 100% chloride efflux.
Each point represents the average of at least two trials.
156
Figure 6.6: Chloride efflux achieved by transporter 6.3 at 0.05 mol% (rtl) in the absence or presence of cationophores (monensin or valinomycin) monitored over a period of 5 min. POPC vesicles are loaded with 300 mM KCl with 5 mM phosphate salts (pH 7.2) and suspended in a 300 mM potassium gluconate solution with 5 mM phosphate salts (pH 7.2). Electrogenic K+transport by valinomycin can only occur if it is balanced by electrogenic Cl–transport by 6.3.
Electroneutral K+/H+antiport by monensin can only occur if the pH gradient is dissipated by electroneutral H+/Cl– transport by 6.3.
157
Figure 6.7: Schematic representation of the HPTS-based assays used in the current study a) H+/Cl- symport or OH-/Cl-antiport b) the presence of cccp (protonophore) to asses Cl-uniport c) the presence of valinomycin to measure the proton flux d) the effect of fatty acid presence as a fuel on the transport.
158
Figure 6.8: H+/Cl-symport or OH-/Cl- antiport facilitated by compounds 6.1-6.4 (0.02 mol% (rtl) for transporter 6.1 and 0.002 mol% (rtl) for transporters 6.2-6.4 from unilamellar POPC vesicles loaded with 100 mM KCl buffered to pH 7.0 with 10 mM HEPES buffer and 1 mM HPTS internal sensor. The vesicles were suspended in an external solution of 100 mM KCl buffered to pH 7.0 with 10 mM
158
xxxiii
HEPES buffer and a base pulse of KOH (25 μL, 0.5 M) was added to generate a transmembrane pH gradient. At the endpoint of each experiment (200 s), the detergent was added to lyse the vesicles and collapse the pH gradient for calibration of HPTS fluorescence.
Figure 6.9: Using KCl-KOH assay from POPC vesicles loaded with KCl (100 mM), buffered to pH 7.0 with HEPES (10 mM), different conditions were applied including using BSA-treated lipid (to test if the transport is fatty acid dependent) addition of oleic acid at 1 mol% (as a source of fatty acid), addition of the protonphore cccp at 1 mol% (to measure of chloride uniport solely), or addition of valinomycin at 0.05 mol% (as a measure of H+ flux), on the rate of chloride transport of receptor 6.3 (0.001 mol%).
157
Chapter 7:
Figure 7.1: Structures of clinically used CAIs and other inhibitors derived from sulfanilamide (SA).
175
Figure 7.2: Design of dual-tailed sulfonamides targeting hCAs. The molecular surface of hydrophobic and hydrophilic halves of the CA active site are colour ed in red and blue, respectively. The Zn(II) ion is represented as an orange sphere (pdb 1CA2).
177
Figure 7.3: Active site view of the hCA II adduct with A) 7.10 (pdb 6UFB), B) 7.13 (pdb 6UFC) and C) 7.16 (pdb 6UFD). Hydrogen bonds are represented as black dashed lines.
183
Figure 7.4. Drop of intraocular pressure (ΔIOP, mmHg) versus time (min) in hypertonic saline-induced ocular hypertension in rabbits, treated with 50 μL of 1% solution of compounds 7.11-7.13 and 7.16 and DRZ as the standard. Data are analyzed with 2way Anova followed by Bonferroni multiple comparison test. * p<0.05 7.11, 7.16, DRZ vs vehicle at 60' and DRZ vs vehicle at 120'; ** p<0.01 7.13 and vs vehicle at 60' and 7.16 vs vehicle at 120'; **** p<0.001 7.11 and 7.13 vs vehicle at 120'.
184
Chapter 8:
Figure 8.1: Summary of the application to medicinal chemistry concepts. 203 Figure 8.2: Structure-guided drug design workflow in this dissertation. 205 Figure 8.3: Structure of the active potential leads 4.1, 4.5 and 4.11 and optimization of 4.5.
206
Figure 8.4: Future development of the thiazolopyrimidine 2.23 and triazolopyrimidine 3.33.
207
xxxiv Figure 8.5: Future development of the switchable anion transporters 5.13, 5.14,
5.16 and 5.17.
209
Figure 8.6: Drug design of the potent anti-glaucoma derivatives studied in the current thesis.
210
Figure 8.7: possible derivatization and development of the CA inhibitors. 211
xxxv