Synthesis and evaluation of indazole based analog sensitive Akt inhibitors

Abstract

The kinase Akt is a key signaling node in regulating cellular growth and survival. It is implicated in cancer by mutation and its role in the downstream transmission of aberrant PI3K signaling. For these reasons, Akt has become an increasingly important target of drug development efforts and several inhibitors are now reaching clinical trials. Paradoxically it has been observed that active site kinase inhibitors of Akt lead to hyperphosphorylation of Akt itself. To investigate this phenomenon we here describe the application of a chemical genetics strategy that replaces native Akt with a mutant version containing an active site substitution that allows for the binding of an engineered inhibitor. This analog sensitive strategy allows for the selective inhibition of a single kinase. In order to create the inhibitor selective for the analog sensitive kinase, a diversity of synthetic approaches was required, finally resulting in the compound PrINZ, a 7-substituted version of the Abbott Labs Akt inhibitor A-443654.

Fig. 1

(a) Structures of disclosed Akt inhibitors in clinical development. (b) Analog sensitive design strategy. Crystal structure of Akt2 with A443654 shows the native gatekeeper methionine and its proximity to the 7 position of indazole ring. Structures of PP1 derivatives commonly used to inhibit analog sensitive kinases.

Fig. 2

(a) In vitro kinase activity of wt and analog sensitive (as) Akt kinases. (b) in vivo kinase activity of wt and as kinase activity. Myristoylated Akt was transfected into HEK293T cells and blotted for Akt substrates.

Fig. 3

in vivo activity of engineered inhibitors 3-IB-PP1 and PrIDZ in HEK 293 cells transfected with myristoylated analog sensitive Akt2 (a) or Akt3 (b).

Scheme 1

(a) Pd₂(dba)₂, P(o-tol)₃, Et₃N, DMF, 90 °C, over night, (b) TFA, CH₂Cl₂, rt, 1 h, yield: 8–29% for 2 steps.

Scheme 2

(a) Br₂, AcOH, 0 °C, 2.5 h, yield: 88% for Y5b, (b) NaNO₂, HCl, H₂O, −20 °C, 30 min then tBuSH, 0 °C, 1 h, (c) tBuOK, DMSO, rt, over night, yield: 54–71% for 2 steps.

Scheme 3

(a) nBuB(OH)₂, Pd(dba)₂,K₃PO₄, Q-Phos, toluene, 90 °C, over night, (b) TFA, CH₂Cl₂, rt, 1 h, yield: 2% for 2 steps.

Scheme 4

(a) R¹CH=CHB(OH)₂, Pd(PPh₃)₄, Na₂CO₃, DME, 85 °C, over night, (b) H₂, Pd/C, EtOH, rt, over night, yield: 62–76% for 2 steps, (c) Br₂, AcOH, 0 °C, 1 h, yield: 60–68%, (d) NaNO₂, HCl, H₂O, −20 °C, 30 min, then tBuSH, 0 °C, 1 h, (e) tBuOK, DMSO, rt, over night, yield: 71–77% for 2 steps.

Scheme 5

(a) R²B(OH)₂, Pd(dba)₂, K₃PO₄, Q-Phos, toluene, 100 °C, 2.5 h, (b) H₂, Pd/C, EtOH, rt, over night, (c) Br₂, AcOH, 0 °C, 1 h, yield: 24–63% for 3 steps, (d) NaNO₂, HCl, H₂O, −20 °C, 30 min, then tBuSH, 0 °C, 1 h, (e) tBuOK, DMSO, rt, over night, yield: 48–81% for 2 steps.

Scheme 6

(a) NaNO₂, HCl, H₂O, −20 °C, 30 min, then tBuSH, 0 °C, 1 h, (b) tBuOK, DMSO, rt, over night, yield: 86% for 2 steps.

Scheme 7

a) [Ph P-CHMeR⁵]⁺ 3 Br⁻, tBuOK / THF, 2–3 h, (b) H₂, Pd/C, THF, rt, over night (R⁵ = H) or TsNH₂NH₂, THF, 100 °C, 3 h (R⁵ = Me), (c) LDA, THF, −78 °C, 1 h then DMF–THF, 78 °C, 1 h, yield: 47–53% for 3 steps, (d) MeMgBr, Et₂O, 0 °C, 30 min, (e) MnO₂, dioxane, 90 °C, 9 h, yield: 63–88% for 2 steps, (f) NH₂NH₂, DMF, 120 °C, 24 h, yield: 7–11%.