Seeking for Non-Zinc-Binding MMP-2 Inhibitors: Synthesis, Biological Evaluation and Molecular Modelling Studies

Abstract

Matrix metalloproteinases (MMPs) are an important family of zinc-containing enzymes with a central role in many physiological and pathological processes. Although several MMP inhibitors have been synthesized over the years, none reached the market because of off-target effects, due to the presence of a zinc binding group in the inhibitor structure. To overcome this problem non-zinc-binding inhibitors (NZIs) have been recently designed. In a previous article, a virtual screening campaign identified some hydroxynaphtyridine and hydroxyquinoline as MMP-2 non-zinc-binding inhibitors. In the present work, simplified analogues of previously-identified hits have been synthesized and tested in enzyme inhibition assays. Docking and molecular dynamics studies were carried out to rationalize the activity data.

Keywords: MMPs; docking; inhibitors; isoquinoline; molecular dynamics; non-zinc-binding group; quinoline; ureas.

Figure 1

Chemical structures of Hits 1 and 2.

Figure 2

Structural modifications performed on Hit 2.

Figure 3

From isoquinolinyl urea 1h to novel derivatives 2a–e and 5, 7.

Scheme 1

Reagents and conditions: benzyl isocyanate (1 equnivalent (eq)), amine, or aniline (1 eq), MeOH (Method a), or CHCl₃, or dichloromethane (DCM) (Method b), room temperature (r.t.) or refluxing, 1–24 h, 41%–99%.

Scheme 2

Reagents and conditions: 5-aminoisoquinoline (1 eq), phenylalkyl-chloride (1 eq), TEA (1.5 eq), dry DCM, N₂, r.t., 2–24 h, 55%–63%; n = 1, 2a; n = 2, 2b.

Scheme 3

Reagents and conditions: 5-carboxyisoquinoline (1 eq), amine (1 eq), 1-hydroxybenzotriazole (HOBt) (1 eq), N,N-dicyclohexylcarbodiimide (DCC) (1 eq), N-methylmorpholine (NMM) (1 eq), DMF, 0 °C, r.t., 24 h, 58%–70%.

Scheme 4

Reagents and conditions: 5-aminoisoquinoline (1 eq), phenylsulfonyl chloride (1.5 eq), pyridine, 0 °C, 3 h, 61%.

Scheme 5

Reagents and conditions: (a) 4-aminobenzylamine (1 eq), di-tert-butyl-dicarbonate (Boc₂O) (0.8 eq), THF, r.t., 2 h, 66%; (b) phenylsulfonyl chloride (1.2 eq), pyridine, 0 °C, r.t., 24 h, 86%; (c) 4 N HCl, dioxane, r.t., 4 h; (d) (i) TEA (1 eq), triphosgene (0.5 eq), toluene, reflux, 4 h; (ii) 5-aminoisoquinoline (1 eq), DCM, 0 °C, r.t., 18 h, 45%–48%; (e) benzoyl chloride (1.2 eq), pyridine, 0 °C, r.t., 24 h, 72%.

Figure 4

Residual enzyme activity on MMP-2, MMP-8 and MMP-13 of Compounds 1a–α, 2a–e, 5 and 7 at 100 µM.

Figure 5

Docking poses of: (A) 1h in the MMP-2; (B) 1m in the MMP-13; (C) 1h in the MMP-8; and (D) 1h in the MMP-13. MMPs are represented as grey (MMP-2), pale cyan (MMP-8) and pale green (MMP-13) cartoons. The zinc ion is a purple sphere; most relevant residues are represented as lines and ligands as sticks. H-bonds are depicted as yellow dashed lines.

Figure 6

Docking poses of: (A) ligand 5 in the MMP-2; (B) and ligand 7 in the MMP-13. MMPs are represented as grey (MMP-2), and pale green (MMP-13) cartoons. The zinc ion is a purple sphere; most relevant residues are represented as lines and ligands as sticks. H-bonds are depicted as yellow dashed lined.

Figure 7

The molecular surface of the S1’ site of, (A) MMP-2 (pale yellow), (B) MMP-8 (pale cyan) and (C) MMP-13 (pale green) is depicted. MMP-2 has a tunnel-like S1’ site, while the MMP-8 and MMP-13 S1’ loop moves, opening an accessory pocket called the S1’* site. However, the MMP-13 site is much larger, the MMP-8 site being hindered by the Arg222 side chain.

Figure 8

Binding poses of 1h (A) and 1i (B) obtained from the last 90 ns of the MD trajectory. MMP-2 is represented as a grey cartoon and ligands as sticks. H-bonds are depicted as yellow dashed lines.

Figure 9

ROC plots obtained reporting the ranking of active compounds after ensemble docking.