Discovery of 4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamides as selective, orally active inhibitors of protein kinase B (Akt)

Abstract

Protein kinase B (PKB or Akt) is an important component of intracellular signaling pathways regulating growth and survival. Signaling through PKB is frequently deregulated in cancer, and inhibitors of PKB therefore have potential as antitumor agents. The optimization of lipophilic substitution within a series of 4-benzyl-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amines provided ATP-competitive, nanomolar inhibitors with up to 150-fold selectivity for inhibition of PKB over the closely related kinase PKA. Although active in cellular assays, compounds containing 4-amino-4-benzylpiperidines underwent metabolism in vivo, leading to rapid clearance and low oral bioavailability. Variation of the linker group between the piperidine and the lipophilic substituent identified 4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamides as potent and orally bioavailable inhibitors of PKB. Representative compounds modulated biomarkers of signaling through PKB in vivo and strongly inhibited the growth of human tumor xenografts in nude mice at well-tolerated doses.

Figure 1

Structures of the ATP-competitive inhibitors 1 and 2.

Figure 2

Crystal structures of (A) 10 bound to PKBβ and (B) 21 bound to PKBβ. (C) Overlay of the bound conformations of 2 (gold), 10 (green), and 21 (blue).

Figure 3

Selectivity profiles and Gini coefficients(28) (G) of 2 (A), 10 (B), and 21 (C) against a panel of 22 human kinases, measured at 1 μM concentration of the test compound. Kinase dendrogram(29) reproduced courtesy of Cell SignalingTechnology, Inc. (www.cellsignal.com).

Figure 4

Effect of 21 (A) and 32 (C) (both dosed at 200 mg/kg po 5 days per week) on the growth of U87MG human glioblastoma xenografts in CrTac:Ncr-Fox1(nu) athymic mice. Effect of 21 (B) and 32 (D) on PKB phosphorylation of GSK3β in U87MG human tumor xenografts at 4 h (B) or 6 h (D) following a single dose of 200 mg/kg po.

Scheme 1

Reagents and conditions (yields for R = 4-^tBu): (i) LDA, 4-tert-butylbenzyl bromide, THF, −78 °C, 79%; (ii) AcOH, H₂SO₄, reflux, then Boc₂O, NaOH, dioxane-H₂O, 71%; (iii) PhI(TFA)₂, MeCN-H₂O, 46%; (iv) tert-butylsulfinamide, Ti(OEt)₄ (2 equiv), THF, reflux, then 4-tert-butylbenzylmagnesium bromide (5 equiv), THF, rt, 26%; (v) 4M HCl, dioxane, MeOH, rt, 100%; (vi) 4-chloro-7H-pyrrolo[2,3-d]pyrimidine, Et₃N, n-BuOH, 100 °C, 86%; (vii) 4-fluoro-1-(triisopropylsilyl)-1H-pyrrolo[2,3-b]pyridine, Et₃N, NMP, 160 °C, 37%; (viii) 6-chloro-7H-purin-8(9H)-one, Et₃N, n-BuOH 100 °C, 50%; (ix) ethyl 4-chloro-1H-pyrazolo[3,4-b]pyridine-5-carboxylate, Et₃N, n-BuOH, 100 °C, 66%; (x) KOH, H₂O, 120 °C, 47%.

Scheme 2

Reagents and conditions: (i) LiAlH₄, THF, 0 °C then rt, 47%; (ii) NaH, 4-chlorobenzyl bromide, DMF, 0 °C then rt, 22%; (iii) 4M HCl, dioxane, MeOH, rt; 100%; (iv) 4-chloro-7H-pyrrolo[2,3-d]pyrimidine, Et₃N, n-BuOH, 100 °C, 69−82%; (v) 1-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide, DMF, NH₃ aq, rt, 97%; (vi) BH₃, THF, 0 °C then 60 °C, 6%; (vii) 4-chlorobenzoyl chloride, Et₃N, CH₂Cl₂, rt, 35%; (viii) R¹R²NH, HATU, ⁱPr₂NEt, DMF, rt, (R¹R²NH = 4-chlorobenzylamine, 86%).