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Figure 1. Chemical
structures of pyrantel, morantel, oxantel, and 1.
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Figure 2. Structures of the target
compounds and their design. 2a–c (cyan
box): repositioning of the m-hydroxyl group of oxantel
within the ring. 2d–g (gold box):
removal or substitution of the m-hydroxyl group of
oxantel; 3a–c (purple
box): exploration of the role of rigidity and N-methylation.
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Scheme 1. Synthesis of Oxantel Analogs 2a–gReagents and conditions:
(i)
1,2-dimethyl-1,4,5,6-tetrahydropyrimidine, ethyl formate, 50 °C
(ii) TFA, CH2Cl2, rt.
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Scheme 2. Synthesis of Oxantel Analogs 3a,bReagents and conditions:
(i)
4,4,5,5-tetramethyl-1,3,2-dioxaborolane, CH2Cl2, Et3N, bis(cyclopentadienyl) zirconium chloride hydride,
60 °C (ii) 1,4,5,6-tetrahydropyrimidine-2-thiol, PdCl2(PPh3)2, 100 °C (iii) Pd/C, H2, MeOH, rt.
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Figure 3. Sample current traces
illustrate PAM behavior of 2e on α4β2. Individual
currents evoked by 5-s application
of increasing concentrations of ACh + 10 μM 2e.
Responses were recorded in the order shown (left to right), all on
the same oocyte, clamped at −60 mV; the breaks between traces
omit the current at baseline, representing wash out with OR2 between
challenges, for space economy. The ACh concentrations ranged 0.03–300
μM in 3.13-fold intervals. The right-most trace (black) was
evoked by 300 μM ACh alone as the internal control. For this
cell, the five highest ACh concentrations in the presence of 10 μM 2e evoked peak responses larger than that for ACh alone. See
also Figure 5E.
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Figure 4. Electrophysiology overview. (A) Values
of EC50 for titrations
of ACh in the presence of oxantel analogs are plotted for the α3β2
and α4β2 subtypes, respectively. Shifts to lower values
relative to ACh alone (dashed line segment) generally indicate positive
allosteric modulation, whereas shifts to higher values indicate negative
modulation. For each subtype, the oxantel (Oxa) behavior (solid line
segment) is shown for reference. Analogs are color-coded by their
design strategy group. (B) Values of Emax for titrations of ACh in the presence of oxantel analogs are plotted
for the α3β2 and α4β2 subtypes, respectively,
using the color coding as in panel A. Shifts to higher values relative
to ACh alone (dashed line segment; greater than 1) generally indicate
positive allosteric modulation, whereas shifts to lower values (less
than 1) indicate negative modulation. For each subtype, the oxantel
(Oxa) behavior (solid line segment) is shown for reference. See also Table 1.
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Figure 5. Full titration curves for α3β2 and α4β2.
Concentration–response data for titrations of ACh in the presence
of 10 μM oxantel analogs for the α3β2 and α4β2
subtypes are shown in panels A–C and D–F, respectively.
Evoked current values, measured as peak current amplitudes, were normalized
to the response for each cell to maximal (3 mM) ACh alone. Symbols
represent means and the error bars indicate the standard error of
the mean. Curves through the data represent best fits to the Hill
equation. The parameters for these fits (EC50 and Emax) are given in Table 1. Panels A and D display experiments for
the analog series 2a–c (hydroxyl
position); B and E show series 2d–g (meta substitutions); and C and F are the series 3a–c (miscellaneous), respectively. In
A and D, open squares (and dashed curve) indicate the titration for
ACh alone and the solid black squares (solid black curve) that for
ACh + 10 μM oxantel. These dashed and solid black curves are
also shown in panels B/E and C/F for reference, with the data points
omitted for clarity. The gold solid curve in C and F represents the
best fit for the 2d compound, with the data points omitted
for clarity.
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Figure 6. Binding mode of oxantel (gray, ball-and-sticks
representation)
at the (A) β2(+)α3(−) and at the (B) β2(+)α4(−)
interfaces. The subunit protein backbones are represented as cartoons
(orange for β2, blue for α3, dark green for α4).
Binding site amino acids surrounding the ligand are depicted in stick
representation (yellow for β2, light blue for α3, green
for α4).
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Figure SI1. Superposition between the proposed binding mode of 1 (light grey, extracted fromthe α4β2 binding site) and the most energetically favored conformation in water of oxantel(green) showing good overlay of the three main pharmacophoric elements (the positivelycharged nitrogen, the aromatic ring and the phenolic group). Distances between the phenolicoxygen and the positively charged nitrogen is represented by a dashed black line and measuredin Å.
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Figure SI2. Comparison between A) an extract of theprimary sequence alignment of theshorter loop C of β2(+) (top) and that of the α4(+) (bottom) subunit, generated with Clustal Ω;B) the 3D structure of the loop C of the β2(+) (green) and of the α4(+) (grey), extracted from5KXI. C) Residues of the allosteric binding sites (α3훽2 and α4훽2) interacting with oxantel fromthe (+) side (훽2) are color-coded as yellow, while residues from the (-) side are highlighted ingreen (α3) or cyan (α4). As a reference, residues of the orthosteric binding sites (α4훽2 and α3훽4)interacting with nicotine (PDB ID 5KXI, 6PV7) or with the reported compound 1 from the(+)side (α4 and α3) and conventionally forming the “aromatic box” are color-coded in red, whileresidues from the (-) side are highlighted in magenta (훽2). Residues in bold and underlined havebeen confirmed to be relevant for oxantel ́s activity by mutational studies
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