Evaluation of the Structure-Activity Relationship of Microtubule-Targeting 1,2,4-Triazolo[1,5- a]pyrimidines Identifies New Candidates for Neurodegenerative Tauopathies

Abstract

Studies in tau and Aβ plaque transgenic mouse models demonstrated that brain-penetrant microtubule (MT)-stabilizing compounds, including the 1,2,4-triazolo[1,5-a]pyrimidines, hold promise as candidate treatments for Alzheimer's disease and related neurodegenerative tauopathies. Triazolopyrimidines have already been investigated as anticancer agents; however, the antimitotic activity of these compounds does not always correlate with stabilization of MTs in cells. Indeed, previous studies from our laboratories identified a critical role for the fragment linked at C6 in determining whether triazolopyrimidines promote MT stabilization or, conversely, disrupt MT integrity in cells. To further elucidate the structure-activity relationship (SAR) and to identify potentially improved MT-stabilizing candidates for neurodegenerative disease, a comprehensive set of 68 triazolopyrimidine congeners bearing structural modifications at C6 and/or C7 was designed, synthesized, and evaluated. These studies expand upon prior understanding of triazolopyrimidine SAR and enabled the identification of novel analogues that, relative to the existing lead, exhibit improved physicochemical properties, MT-stabilizing activity, and pharmacokinetics.

Figure 1.

The structure of MT-stabilizing natural products, epothilone D and dictyostatin, and selected examples of MT-binding triazolopyrimidines and phenylpyrimidines.

Figure 2.

Effect of fluorination of the aryl group at C6 on MT-stabilizing activity of Class I triazolopyrimidines. Black squares indicate the average activity of test compounds at 1 and 10 μM, normalized to the activity of 100 nM of 5 (positive control).

Figure 3.

The effect of both branching of aliphatic acyclic amines and homologation of endo- and exo-cyclic amines on MT-stabilizing activity of Class I triazolopyrimidines. Test compounds are ordered along the X-axis based on the number of carbon atoms in the amine fragment. Black squares indicate the average activity of test compounds at 1 and 10 μM, normalized to the activity of 100 nM of 5 (positive control). Compounds that did not produced a statistically significant elevation in AcTub at either 1 or 10 μM are not plotted and marked as inactive.

Figure 4.

To confirm that the nitrile containing triazolopyrimidine derivatives identified in these studies can stabilize neuronal MTs under conditions of tau loss-of-function, we examined the ability of representative compound, 69, to prevent the MT collapse that occurs from reduced binding of hyperphosphorylated tau to axonal MTs after treatment of neuron cultures with the phosphatase inhibitor, okadaic acid (OA). A. Primary rat cortical neurons treated with 1 μM reference compound 3, or 69 in the absence of OA (−OA) show increased axonal acetyl-tubulin staining relative to those receiving vehicle only. Upon treatment with OA (+OA) in the absence of compound (Vehicle), there is a dramatic reduction in axonal AcTub staining with fragmentation of MTs and neuronal processes (also see). Co-addition of 3 or 69 (1 μM) with OA results in normalization of AcTub staining and axonal processes. B. ELISA determination of AcTub levels in homogenates from primary mouse cortical neurons treated with 1 or 10 μM of 69, or vehicle, in the presence of OA. The higher concentration of 69 resulted in AcTub levels comparable to those in neurons without OA treatment.

Figure 5.

Comparison of selected compounds based on experimental logD_7.4 values (triangles) and MT-stabilizing activity (squares) expressed as the average activity in the AcTub assay at 1 and 10 μM normalized to positive control (i.e., 100 nM 5). LogD_7.4 values were determined via shake flask method (experiments run by Analiza, Inc.)

Figure 6.

Brain and plasma pharmacokinetics of 69 after 5 mg/kg i.p. dosing to CD1 mice (A), and comparison of plasma pharmacokinetics of 69 and 3.

Figure 7.

Different views of the co-crystal structure (PBD: 5NJH) of 54 bound within a tubulin preparation (A); and the docked structures of 30 (B), 3 (C) and 72 (D) within the triazolopyrimidine binding site.

Figure 8.

MMGBSA scores for the Class I compounds vs MT-stabilizing activity in QBI293 cells expressed as the log of the average of the activity at 1 and 10 μM in the AcTub assay, relative to positive control, $\text{log}\left(\frac{Avg\mathit{\text{AcTu}}{b}_{1\&10\mu M}}{\mathit{\text{AcTu}}{b}_{0.1\mu M}\text{of}5}\right)$.

Figure 9.

(A) QSAR predicted vs experimental MT-stabilizing activity in QBI293 cells for training and test sets. In both cases, MT-stabilizing activity is expressed as the log of the average of the activity at 1 and 10 μM in the AcTub assay, relative to positive control, $\text{log}\left(\frac{Avg\mathit{\text{AcTu}}{b}_{1\&10\mu M}}{\mathit{\text{AcTu}}{b}_{0.1\mu M}\text{of}5}\right)$. (B) The visual representation of the field and steric contributions to predicted activity for compound 72.

Figure 10.

3D summary plot of Class I triazolopyrimidines showing the experimental MT-stabilizing activity in the QBI293 assay, the MMGBSA score value (ΔG bind), and the QSAR predicted activity. The experimental MT-stabilizing activity of test compounds is plotted both via color coding and as the log of the average of the activity at 1 and 10 μM in the AcTub assay, relative to positive control, $\text{log}\left(\frac{Avg\mathit{\text{AcTu}}{b}_{1\&10\mu M}}{\mathit{\text{AcTu}}{b}_{0.1\mu M}\text{of}5}\right)$, see Experimental for further details.

Figure 11.

The effect of the stereo-electronic properties of the substituent in para. Active Class I and II compounds are ranked based on the Hammett σ_p values.

Figure 12.

Activity atlas analysis revealing key features of Class I triazolopyrimidines that are necessary for MT-stabilizing activity: (a) red and cyan colors show positive and negative field regions respectively; (b) brown beige regions show hydrophobic interaction sites required for activity; (c) Activity cliffs analysis revealing a favorable hydrophobic region (green) and a favorable negative electrostatic region (cyan).

Scheme 1.. Reagents and Reaction conditions:

a) For 76–85, 87–89, 91: Ar-X, CuBr, NaH, 1,4-dioxane, 60–100 °C, 12 h, 19–74%; For 86: Ar-CH₂Br, NaH, N,N-dimethylformamide, 0 °C, 1 h, 90%; For 90 and 92: Ar-F, K₂CO₃, N,N-dimethylformamide, 60 °C, 4 h, 84–86%; b) N-tributylamine, 170–180 °C, 2–6 h; c) phosphorus oxychloride, 110–130 °C, 6–16 h, 46–58% over two steps; d) appropriate amine, N,N-dimethylformamide, rt–90 °C, 1–18 h, 12–50%; e) Fe, NH₄Cl, H₂O/MeOH (40/50), 80 °C, 2 h, 69%.

Scheme 2.. Reagents and Reaction conditions:

a) For 24: NaOMe, THF, 0 °C to 60 °C, 14 h, 71%; For 25, 31: ROH or RSH, NaH, DMSO, 60 °C, 3 h, 81–95%; For 28: CH₃SNa, DMSO, 60 °C, 3 h, quant.; b) m-CPBA (1 equiv), CH₂Cl₂, 20 °C, 2 h, 32% of 26, 52% of 27, 70% of 29; c) m-CPBA (3 equiv), CH₂Cl₂, 20 °C, 3 h, 71%; d) TEMPO, BAIB, CH₂Cl₂/H₂O (90/70), 20 °C, 2 h, 59%; e) LiOH, MeOH/H₂O (1/1), 20 °C, 16 h, 39%.

Scheme 3.. Reagents and Reaction conditions:

a) appropriate amine, base, N,N-dimethylformamide, 20–90 °C, 1–18 h, 12–50%.

Scheme 4.. Reagents and Reaction conditions:

a) appropriate ester of 4,4,4-trifluoro-3-methylbutanoic acid, HMDS, n-BuLi, THF, −78 °C, 2.5 h, 70% for 110, 25% for 112; b) TFA, CH₂Cl₂, 20 °C, 16 h, 68%; c) LiCl, DMSO, 130 °C (microwave irradiation), 3 h, 76%.

Scheme 5.. Reagents and Reaction conditions:

a) appropriate amine, N,N-dimethylformamide, r.t., 1 h, 56–86%.

Scheme 5.. Reagents and Reaction conditions:

a) appropriate amine, N,N-dimethylformamide, r.t., 1 h, 56–86%.