Isoxazol-5(2H)-one: a new scaffold for potent human neutrophil elastase (HNE) inhibitors

Figures & data

Figure 1. HNE inhibitors.

Figure 2. X-ray structure of compound 2j.

Scheme 1. Reagents and conditions: (a) R₂-COCl, NaH, anhydrous THF, r.t., 24 h; (b) Lawesson’s reagent, toluene, reflux, 5 h; (c) for 4a,b: 3-methylbenzyl chloride, K₂CO₃, anhydrous CH₃CN, 80 °C, 2 h; for 4c: 3-methylphenyl boronic acid, (CH₃COO)₂Cu, Et₃N, anhydrous CH₂Cl₂, r.t., 24 h.

Table 1. HNE inhibitory activity of isoxazolone derivatives 2a–s, 3a,b, 4a–c.

Compound	R2	R3	R4	IC50 (μM)^a
2a	cC3H5	CH3	CH3	0.12 ± 0.023
2b	m-CH3-Ph	CH3	CH3	10.9 ± 1.2
2c	cC3H5	CH3	C2H5	0.13 ± 0.025
2d	m-CH3-Ph	CH3	C2H5	0.094 ± 0.023
2e	cC3H5	C2H5	CH3	2.2 ± 0.28
2f	m-CH3-Ph	C2H5	CH3	1.8 ± 0.32
2g	cC3H5	COOEt	CH3	0.44 ± 0.14
2h	m-CH3-Ph	COOEt	CH3	39.7 ± 5.1
2i	cC3H5	C2H5	H	0.096 ± 0.022
2j	m-CH3-Ph	C2H5	H	0.043 ± 0.011
2k	C2H5	iC3H7	H	1.1 ± 0.14
2l	cC3H5	iC3H7	H	0.034 ± 0.009
2m	cC5H9	iC3H7	H	0.56 ± 0.15
2n	m-CH3-Ph	iC3H7	H	0.042 ± 0.011
2o	p-CH3-Ph	iC3H7	H	0.020 ± 0.007
2p	m-CN-Ph	iC3H7	H	6.5 ± 1.7
2q	p-CN-Ph	iC3H7	H	18.2 ± 2.5
2r	m-CF3-Ph	iC3H7	H	1.2 ± 0.22
2s	p-CF3-Ph	iC3H7	H	0.034 ± 0.012
3a	cC3H5	–	–	2.1 ± 0.31
3b	m-CH3-Ph	–	–	NA^b
4a	CH2-m-CH3-Ph	CH3	CH3	NA^b
4b	CH2-m-CH3-Ph	iC3H7	H	NA^b
4c	m-CH3-Ph	COOEt	CH3	NA^b
Sivelestat	–	–	–	0.050 ± 0.020

^a IC₅₀ values are presented as the mean ± SD of three independent experiments.

^b NA: no inhibitory activity was found at the highest concentration of compound tested (50 μM).

Table 2. Biological activities, geometric parameters, and docking scores of the enzyme–inhibitor complexes predicted by molecular docking with MVD.

Comp.	IC50 (μM)^a	α	d1	d2	d3	L^b	Docking score ΔE (kcal/mol)
2l^c	0.034	85.2	2.903	2.763, 3.967	2.829	5.592	–58.9
3a	2.1	139.6	4.148	2.379, 3.611	2.560	4.939	–44.0
2n	0.042	81.6	3.861	2.399, 3.627	2.596	4.995	–34.2
3b	NA	148.3	4.659	2.750, 3.962	2.849	5.599	–21.1
2o^c	0.020	104.1	3.134	2.760, 3.975	2.819	5.579	–67.6
2q^c	18.2	89.4	4.500	3.130, 4.319	3.286	6.416	–58.4

^a HNE inhibitory activity.

^b The length of the proton transfer channel was calculated as L=d₃+min(d₂).

^c According to the docking results, a Michaelis complex with Ser195 is formed with participation of the ester carbonyl group.

Figure 3. Docking poses of compounds 2n (lower compound) and 3b (upper compound). The ovals represent the C=S and C=O fragment of the two compounds, respectively. Residues within 6 Å from the co-crystallized ligand are shown.

Figure 4. Docking pose of compound 2o. Residues within 3 Å of the pose are visible. The H-bond is shown by a dashed line and is indicated by the arrow.

Figure 5. Analysis of compound 2i spontaneous hydrolysis. Compound 2i (80 μM) was incubated in 0.05 M phosphate buffer (pH 7.5, 25 °C) supplemented with 0, 50, 200, and 400 μg/ml HNE, as indicated. Spontaneous hydrolysis was monitored by measuring changes in absorbance at 275 nm (absorption maximum of compound 2i) over time. The data are presented as the mean ± SD of triplicate samples from one experiment, which is representative of two independent experiments.

Table 3. Half-life (t_1/2) for the hydrolysis of selected isoxazolone derivatives in aqueous buffer and in the presence of HNE.

		t1/2 (h)
Compd.	Max (nm)^a	Spontaneous hydrolysis	In the presence HNE^b
2d	290	7.7	3.5
2i	275	19.3	4.0
2j	285	5.3	2.1
2l	275	19.3	4.3
2n	285	6.2	2.6
2o	290	8.9	3.4
2s	285	3.1	0.9

^a Absorption maximum of the compounds for monitoring hydrolysis.

^b Hydrolysis was monitored in the presence of 400 mU/ml HNE.

Figure 6. Evaluation of HNE inhibition by selected isoxazolone derivatives. HNE was incubated with the indicated compounds at 5 μM concentrations, and kinetic curves monitoring substrate cleavage catalysed by HNE are shown. Representative curves are from three independent experiments.

Figure 7. Kinetics of HNE inhibition by compound 2o. Representative double-reciprocal Lineweaver–Burk plot from three independent experiments.