Design, synthesis, and structure-activity relationship studies of 6 H-benzo[ b]indeno[1,2- d]thiophen-6-one derivatives as DYRK1A/CLK1/CLK4/haspin inhibitors

Abstract

A series of sulfur-containing tetracycles was designed and evaluated for their ability to inhibit protein kinase DYRK1A, a target known to have several potential therapeutic applications including cancers, Down syndrome or Alzheimer's disease. Our medicinal chemistry strategy relied on the design of new compounds using ring contraction/isosteric replacement and constrained analogy of known DYRK1A inhibitors, thus resulting in their DYRK1A inhibitory activity enhancement. Whereas a good inhibitory effect of targeted DYRK1A protein was observed for 5-hydroxy compounds 4i-k (IC₅₀ = 35-116 nM) and the 5-methoxy derivative 4e (IC₅₀ = 52 nM), a fairly good selectivity towards its known DYRK1B off-target was observed for 4k. In addition, the most active compound 4k, having an ATP-competitive mechanism of action, proved to be also a potent inhibitor of CLK1/CLK4 (IC₅₀ = 20 and 26 nM) and, to a lesser extent, of haspin (IC₅₀ = 76 nM) kinases. In silico docking studies within the DYRK1A, CLK1/CLK4 and haspin ATP binding sites were carried out to understand the interactions of our tetracyclic derivatives 4 with these targets. Antiproliferative activities on U87/U373 glioblastoma cell lines of the most potent compound 4k showed a moderate effect (IC₅₀ values between 33 and 46 μM). Microsomal stabilities of the designed compounds 4a-m were also investigated, showing great disparities, depending on benzo[b]thiophene ring 5-substitution.

Scheme 1. Access to benzo[b]thiophenes 9. Reagents and conditions: (a) bromoacetaldehyde diethyl acetal, K₂CO₃, acetone, rt, 18 h, 96% for 10a (R⁵ = OMe), 69% for 10b (R⁵ = F). (b) Polyphosphoric acid, chlorobenzene, 130 °C, 18 h, 85% for 9a (R⁵ = OMe), 60% for 9b (R⁵ = F).

Scheme 2. Access to ortho-iodobenzaldehydes 8b,d. Reagents and conditions: (a) I₂/H₅IO₆, acetic acid/H₂SO₄, 70 °C, 16 h, 46%. (b) n-BuLi, I₂, Et₂O, 0 °C then rt, 2 h, 63%. (c) PDC, silica, CH₂Cl₂, rt, 6 h, 76%.

Scheme 3. Access to ortho-iodobenzaldehydes 8a,c,e. Reagents and conditions: (a) NaNO₂/HCl_aq, KI, 0 °C then 90 °C, 1.5 h, quant. for 16a, quant. for 16b. (b) BH₃·Me₂S, B(OMe)₃, THF, rt, 16 h, 65% for 13c, 47% for 13e. (c) PDC, silica, CH₂Cl₂, rt, 6 h, 98% for 8a, 89% for 8c, 61% for 8e.

Scheme 4. Synthesis of bis(het)arylcarbinols 7a–m.

Scheme 5. Preparation of keto derivatives 6 and tetracyclic compounds 4. Reagents and conditions: (a) MnO₂ 6 eq., acetonitrile, rt, 6 h, 67–96%. (b) Pd(OAc)₂ 5 mol%, Cy₃P·HBF₄ 10 mol%, K₂CO₃, DMF, 130 °C, 16 h, 51–87%.

Fig. 1. Structures of some known DYRK1A inhibitors (IC₅₀ for DYRK1A is indicated).

Fig. 2. Drug design strategy for the preparation of tetracycles 4 (IC₅₀ for DYRK1A are indicated).

Fig. 3. Synthetic strategy for the preparation of tetracyclic targets 4.

Fig. 4. Top docking poses obtained for the 5 most potent hits 4e, i, j, k, l, (A, B, C, D and E respectively), obtained from the study with the X-ray structure of DYRK1A (PDB: 5AIK). Stabilizing interactions are highlighted in orange. Compounds 4a–m representations are available in ESI (Fig. S3).

Fig. 5. Superposition of reference ligands described in the literature with compound 4k. These images are generated from the docking of the molecules with the X-ray structure of DYRK1A (PDB: 5AIK), whose surface is represented in dark blue. Compound 4k is depicted with thick sticks in turquoise, harmine in orange, INDY in magenta and compound 3a (R′ = OH) in yellow (A: side view of the ATP cleft. B: Top view).

Fig. 6. Top docking poses of compound 4k within the X-ray structures of DYRK1A (PDB: 5AIK) in dark blue (A), CLK1 (PDB: 6RAA) in cyan-grey (B), CLK4 (PDB: 6FYV) in magenta (C) and haspin (PDB: 3IQ7) in wheat (D). The kinases are shown as ribbon with their surfaces in transparency. The results showed marked homology in the stabilization of compound 4k within the 4 proteins. Residues engaged in stabilizing interactions are highlighted in orange. Haspin kinase exhibits a slightly different binding system.