Atropisomerism and Conformational Equilibria: Impact on PI3Kδ Inhibition of 2‑((6-Amino‑9<i>H</i>‑purin-9-yl)methyl)-5-methyl-3‑(<i>o</i>‑tolyl)quinazolin-4(3<i>H</i>)‑one (IC87114) and Its Conformationally Restricted Analogs

IC87114 [compound <b>1</b>, (2-((6-amino-9<i>H</i>-purin-9-yl)­methyl)-5-methyl-3-(<i>o</i>-tolyl)­quinazolin-4­(3<i>H</i>)-one)] is a potent PI3K inhibitor selective for the δ isoform. As predicted by molecular modeling calculations, rotation around the bond connecting the quinazolin-4­(3<i>H</i>)-one nucleus to the <i>o</i>-tolyl is sterically hampered, which leads to separable conformers with axial chirality (i.e., atropisomers). After verifying that the a<i>S</i> and a<i>R</i> isomers of compound <b>1</b> do not interconvert in solution, we investigated how biological activity is influenced by axial chirality and conformational equilibrium. The a<i>S</i> and a<i>R</i> atropisomers of <b>1</b> were equally active in the PI3Kδ assay. Conversely, the introduction of a methyl group at the methylene hinge connecting the 6-amino-9<i>H</i>-purin-9-yl pendant to the quinazolin-4­(3<i>H</i>)-one nucleus of both a<i>S</i> and a<i>R</i> isomers of <b>1</b> had a critical effect on the inhibitory activity, indicating that modulation of the conformational space accessible for the two bonds departing from the central methylene considerably affects the binding of compound <b>1</b> analogues to PI3Kδ enzyme

Atropisomerism and Conformational Equilibria: Impact on PI3Kδ Inhibition of 2‑((6-Amino‑9<i>H</i>‑purin-9-yl)methyl)-5-methyl-3‑(<i>o</i>‑tolyl)quinazolin-4(3<i>H</i>)‑one (IC87114) and Its Conformationally Restricted Analogs

IC87114 [compound <b>1</b>, (2-((6-amino-9<i>H</i>-purin-9-yl)­methyl)-5-methyl-3-(<i>o</i>-tolyl)­quinazolin-4­(3<i>H</i>)-one)] is a potent PI3K inhibitor selective for the δ isoform. As predicted by molecular modeling calculations, rotation around the bond connecting the quinazolin-4­(3<i>H</i>)-one nucleus to the <i>o</i>-tolyl is sterically hampered, which leads to separable conformers with axial chirality (i.e., atropisomers). After verifying that the a<i>S</i> and a<i>R</i> isomers of compound <b>1</b> do not interconvert in solution, we investigated how biological activity is influenced by axial chirality and conformational equilibrium. The a<i>S</i> and a<i>R</i> atropisomers of <b>1</b> were equally active in the PI3Kδ assay. Conversely, the introduction of a methyl group at the methylene hinge connecting the 6-amino-9<i>H</i>-purin-9-yl pendant to the quinazolin-4­(3<i>H</i>)-one nucleus of both a<i>S</i> and a<i>R</i> isomers of <b>1</b> had a critical effect on the inhibitory activity, indicating that modulation of the conformational space accessible for the two bonds departing from the central methylene considerably affects the binding of compound <b>1</b> analogues to PI3Kδ enzyme

Basal GDNF levels in cultured mouse striatal neurons and in cultured astrocytes (A).

<p>Expression of phosphoERK1/2 and phospho-Akt in cultured striatal neurons treated with LY379268 (1 µM), LY341495 (1 µM) and LY379268+LY341495 for 15 min (B). Densitometric values are means+S.E.M. of 3–4 determinations. *p<0.05 (One-Way ANOVA+Fisher's PLSD) vs. basal values, #p<0.05 vs. LY379268 values. Treatment of cultured neurons with 1 µM LY379268 enhanced GDNF levels 24 h later (C), and it was abrogated by the co-application of the MEK inhibitor, PD98059, or the PI-3-K inhibitor, LY294002 (C). Application of LY379268 to astrocytes made “reactive” by several passages in culture and by the G5 supplement in the medium did not affect GDNF levels (D).</p

ELISA analysis of GDNF expression in the striatum of mice after treatment with saline, LY379268 (3 mg/kg, i.p.), MPTP (30 mg/kg, i.p.), or MPTP+LY379268.

<p>Animals were killed 1,2,3 or 7 days after treatments. Data of GDNF are the mean+S.E.M. of 8 animals. <i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. the corresponding groups of mice treated with saline (*) or with MPTP or LY379268 alone (#).</p

<i>In situ</i> hybridization of sagittal sections at basal ganglia level showing the expression of mRNA encoding GDNF (A) or NGF (B).

<p>Autoradiogram showing GDNF expression in the striatum of saline-treated mice or LY379268 (0.25 mg/kg, i.p.)-treated mice (A). The inserts show representative GDNF mRNA labeled cells (black grains) with increased levels of labeling in LY379268-treated mice. Autoradiogram showing NGF expression of saline-treated mice or LY379268 (0.25 mg/kg, i.p.)-treated mice (B). Dose-response curve of GDNF mRNA levels in the striatum of mice treated with saline or LY379268 (0.1, 0.25, 1, 3 or 4 mg/kg, i.p) (C) and time-course of GDNF mRNA levels in the striatum of mice after a single injection of LY379268 (0.25 mg/kg, i.p.) (D); values are means±S.E.M (n = 4–5, animals per group; three independent experiments). Striatal GDNF mRNA levels in mice treated with saline, LY379268 (0.25 mg/kg, i.p), LY341495 (1 mg/kg, i.p) or LY379268+LY341495 (E); value are means±S.E.M (n = 4, animals per group; three independent experiments). *<i>p</i><0.05; **<i>p</i><0.01 (One-way ANOVA+Fisher's PLSD) vs. control mice. Scale bar: A–B = 4 mm. Str, striatum; Ctx, cortex; Hipp, hippocampus.</p

Quantitative real-time PCR analysis of GDNF mRNA in mouse striatum (A) and cortex (B) at 3, 6 or 12 h after systemic treatment with saline, LY379268 (3 mg/kg, i.p.), or LY379268 (3 mg/kg, i.p.)+LY341495 (1 mg/kg, i.p.).

<p>Values were normalized with respect to the amount of β-actin mRNA. Values are mean+S.E.M. of four determinations (each from triplicates). *<i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. saline-treated mice.</p

LY379268 fails to protects against MPTP toxicity in mice unilaterally implanted with anti-GDNF antibodies.

<p>Mice were implanted with a gelfoam (Spongostan) pre-soaked with saline alone (A,B) or a saline solution containing 5 µg of neutralizing anti-GDNF antibodies (A,B) in the left caudate nucleus. Stereological counts of TH-positive neurons in the substantia nigra pars compact in the implantation side (left) or contralateral side (right) in response to i.p. injection of saline, LY379268 (3 mg/kg), MPTP alone (30 mg/kg) or MPTP+LY379268 (injected 30 min prior to MPTP injection). Drugs were administered 24 h after the gelfoam implantation. Mice were killed 7 days after MPTP injection. Values (means+S.E.M.) were calculated from 6 mice per group. <i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. the corresponding values in mice treated with saline (*) or vs. the MPTP values of the right side (#).</p

Double immunolabeling for GDNF and NeuN or GFAP in striatal cells showing the labelling of GDNF within neuronal cells (A).

<p>Arrows in the left panel, NeuN-positive cells containing GDNF mRNA black grains; arrow in the right panel, GDNF mRNA black grains, arrow head in the right panel, GFAP-positive cell. Immunohistochemical analysis of GDNF in the striatum of mice treated with a single injection of LY379268 (3 mg/kg, i.p.) and killed 24 h later (B). In both control mice and mice treated with LY379268, GDNF immunoreactivity is exclusively localized in neurons (note the absence of co-localization between GDNF and GFAP), and the extent of immunostaining increases after drug treatment. GDNF immunostaining in the striatum of mice treated 7 days before with MPTP, 20 mg/kg, i.p., x 3, two h apart (C). This treatment led to reactive gliosis in the striatum, as a result of the degeneration of nigro-striatal dopaminergic neurons. Under these conditions, GDNF immunostaining is localized both in neurons and reactive astrocytes. A single injection of LY379268 (3 mg/kg, i.p.) 7 days following MPTP injection did not enhance GDNF immunoreactivity in reactive astrocytes, but still enhanced immunoreactivity in neurons. Interestingly, the number of GDNF-positive reactive astrocytes is lower 24 h following LY379268 injection. Scale bar = 50 and 10 µm.</p

Immunohistochemical analysis of TH in the pars compacta of substantia nigra of mice injected with a single i.p. dose of 30 mg/kg of MPTP, alone or combined with LY379268 (0.25 or 3 mg/kg in a single i.p. injection, 30 min prior to MPTP injection or 0.25 mg/kg/7 days once a day, i.p.).

<p>Scale bar = 100 µm. Stereological TH-positive cell counts are also shown. Values (means+S.E.M.) were calculated from 7–8 mice per group (10 sections - 10 µm thick, cut every 100 µm, per animal were used for the calculation of the density of TH-positive neurons in the pars compacta of the substantia nigra). *<i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. mice treated with MPTP alone.</p

Western blot analysis of striatal GDNF expression in wild-type, <i>mGlu2^−/−</i> or <i>mGlu3^−/−</i> mice in basal conditions (A) and after treatment with LY379268, 3 mg/kg, i.p. (B).

<p>Animals were killed 24 h later. Densitometric data of GDNF are shown and are the mean+S.E.M. of 3 animals performed two times.*<i>p</i><0.05 (One-way ANOVA+Fisher's PLSD) vs. saline-treated mice.</p