Design and Synthesis of Water-Soluble and Potent MMP-13 Inhibitors with Activity in Human Osteosarcoma Cells

Abstract

Osteoarthritis is a degenerative disease, often resulting in chronic joint pain and commonly affecting elderly people. Current treatments with anti-inflammatory drugs are palliative, making the discovery of new treatments necessary. The inhibition of matrix metalloproteinase MMP-13 is a validated strategy to prevent the progression of this common joint disorder. We recently described polybrominated benzotriazole derivatives with nanomolar inhibitory activity and a promising selectivity profile against this collagenase. In this work, we have extended the study in order to explore the influence of bromine atoms and the nature of the S1' heterocyclic interacting moiety on the solubility/selectivity balance of this type of compound. Drug target interactions have been assessed through a combination of molecular modeling studies and NMR experiments. Compound 9a has been identified as a water-soluble and highly potent inhibitor with activity in MG-63 human osteosarcoma cells.

Keywords: MMP-13 inhibitors; RMN; metalloproteinases; molecular modeling; organic synthesis; osteoarthritis.

Figure 1

Structure of three published MMP-13 inhibitors.

Figure 2

Chemical structure and IC₅₀ values for compounds 1 and 2.

Figure 3

Chemical structure of compounds 3–5.

Scheme 1

Synthesis of the benzotriazole-derived alkynes. Reagents and conditions: (a) ACN, K₂CO₃, 4-bromobut-1-yne, 24 h, RT.

Scheme 2

Synthesis of the phthalimide-derived alkynes. Reagents and conditions: (a) (i) KMnO₄, KOH, H₂O, 120 °C, MW (ii) Ac₂O, 80 °C, MW (b) 3-butyn-1-amine, EtOH, 100 °C, MW.

Scheme 3

Synthesis of clicked acids and hydroxamates^. Reagents and conditions: (a) CuSO₄, sodium ascorbate, DMF (and TBTA for the synthesis of (9b–d) (b) HCl in MeOH, 0 °C for (9a–e), (10a–e); AcOH, THF, H₂O, 45 °C MW radiation for (10b–d).

Figure 4

PyMOL stick and cartoon representation of the interactions established with the Ω-loop and S1′ pocket of the energetically most favorable binding pose of compounds 9a,e bound to MMP-13 (left) MMP-2 (right). Hydrogen bonds are shown as dashed lines, and the catalytic zinc ion is shown as a grey sphere. For the sake of clarity, hydrogens are not shown.

Figure 4

PyMOL stick and cartoon representation of the interactions established with the Ω-loop and S1′ pocket of the energetically most favorable binding pose of compounds 9a,e bound to MMP-13 (left) MMP-2 (right). Hydrogen bonds are shown as dashed lines, and the catalytic zinc ion is shown as a grey sphere. For the sake of clarity, hydrogens are not shown.

Figure 5

PyMOL stick and cartoon representation and 2D representation of the interactions established with the Ω-loop and S1′ pocket of the energetically most favorable binding poses of compounds 10a–e bound to MMP-13 (left) and MMP-2 (right). Hydrogen bonds are shown as dashed lines, and the catalytic zinc ion is shown as a grey sphere. For the sake of clarity, hydrogens are not shown.

Figure 5

PyMOL stick and cartoon representation and 2D representation of the interactions established with the Ω-loop and S1′ pocket of the energetically most favorable binding poses of compounds 10a–e bound to MMP-13 (left) and MMP-2 (right). Hydrogen bonds are shown as dashed lines, and the catalytic zinc ion is shown as a grey sphere. For the sake of clarity, hydrogens are not shown.

Figure 5

PyMOL stick and cartoon representation and 2D representation of the interactions established with the Ω-loop and S1′ pocket of the energetically most favorable binding poses of compounds 10a–e bound to MMP-13 (left) and MMP-2 (right). Hydrogen bonds are shown as dashed lines, and the catalytic zinc ion is shown as a grey sphere. For the sake of clarity, hydrogens are not shown.

Figure 5

PyMOL stick and cartoon representation and 2D representation of the interactions established with the Ω-loop and S1′ pocket of the energetically most favorable binding poses of compounds 10a–e bound to MMP-13 (left) and MMP-2 (right). Hydrogen bonds are shown as dashed lines, and the catalytic zinc ion is shown as a grey sphere. For the sake of clarity, hydrogens are not shown.

Figure 6

NMR studies corresponding to the interaction between the MMP-13 catalytic domain and compound 9a. Blue: 1D ¹H spectrum of compound 9a and the MMP-13 catalytic domain; green: WaterLOGSY experiment of 9a; red: WaterLOGSY experiment of 9a in the presence of the MMP-13 catalytic domain.

Figure 7

Effect of 9a on MMP-13 activity in MG-63 cells. MG-63 cells were incubated in the absence (DMSO) or the presence of 9a (0.5, 5, and 50 nM). MMP-13 activity was determined in the cell supernatants using the SensoLyte^® Plus 520 MMP-13 assay kit. Results were normalized to total protein content in the supernatants and calculated relative to the treatment with DMSO, considered as 100% in terms of MMP-13 activity. Data are expressed as the mean ± SEM and are representative of three experiments. * p < 0.05 vs. DMSO.