Dipeptide-derived nitriles containing additional electrophilic sites: Potentially irreversible inhibitors of cysteine proteases

Figures & data

Figure 1.  Reaction of 2-alkylthio-4-oxo-quinazolineacetonitriles and analogous thieno[3,2-d]pyrimidineacetonitriles with aliphatic thiols to imidazo-fused tricyclic systems by trapping of thioimidates; X = CH=CH or S¹³.

Scheme 1.  Synthesis of compound 3. Reagents and conditions: (a) (NH₂)₂CS, NaOEt, EtOH, reflux; (b) 1 N NaOH, CH₃I, acetone, room temperature; (c) BrCH₂CN, (nBu)₄NBr, 1 N NaOH, H₂O/CH₂Cl₂.

Scheme 2.  Synthesis of compound 8. Reagents and conditions: (a) (i) N- methylmorpholine, ClCO₂CH₂CH(CH₃)₂, THF; (ii) 25% NH₃, −25°C to room temperature; (b) 4 N HCl/dioxane, room temperature; (c) (i) Boc-Phe-OH, N-methylmorpholine, ClCO₂CH₂CH(CH₃)₂, THF; (ii) 7 in 1 N NaOH/H₂O, −25°C to room temperature; (d) (ClCN)₃, DMF, room temperature; (e) CH₃I, ethyl acetate, room temperature.

Scheme 3.  Synthesis of compound 10. Reagents and conditions: (a) (i) N-methylmorpholine, ClCO₂CH₂CH(CH₃)₂, THF; (ii) H-Asn-NH₂ × HCl, H₂O, 1 N NaOH, −25°C to room temperature; (b) (ClCN)₃, DMF, room temperature.

Figure 2.  The dihedral angles ϕ and ψ at the peptidic backbone. ϕ is defined by the atoms C_{i – 1}, N_i, C_αi, and C_i and ψ by N_i, C_αi, C_i, and N_{i + 1}.

Figure 3.  (A) Three-dimensional representation of the l-phenylalanine-derived enantiomer of compound 3. The values for the dihedral angles are ϕ = −80° and ψ = +124°. (B) For comparison, the corresponding open-chain N-acetyl-phenylalanyl-glycine nitrile in the extended, bioactive conformation (ϕ = −139°, ψ = +135°) is shown. Images prepared with PyMOL²⁹.

Figure 4.  Formation of 2-alkylthio-5-aminopyrroles from succinic dinitrile and thioles (R = CH₂COOH) according to Elnagdi et al.³².

Table 1.  Inhibition of cathepsin L, cathepsin S, cathepsin K, and papain by dipeptide nitriles with electrophilic side chains in the P1 position.

Compound	R	Ki (μM)
		Bovine cathepsin L	Human cathepsin S	Human cathepsin K	Papain
7	CH2SCH3	0.27 ± 0.02	0.65 ± 0.03	3.6 ± 0.6	0.25 ± 0.01
8	CH2S^+(CH3)2	0.52 ± 0.07	0.73 ± 0.03	3.8 ± 0.3	n.d.
10	C=N	0.90 ± 0.02	0.84 ± 0.04	6.3 ± 0.5	1.1 ± 0.1

Figure 5.  (A) Cathepsin S-catalyzed hydrolysis of Z-Val-Val-Arg-NHMec (40 μM) in the presence of increasing concentrations of compound 8 (from top to bottom: 0, 0, 0.3, 0.5, 1.0, 2.0, 3.0, 5.0, 7.0, 10, 20, 100 μM) in 50 mM potassium phosphate pH 6.5, 50 mM NaCl, 2 mM EDTA, 0.01% Triton X-100, 25 μM DTT, 1% DMSO, 37°C. (B) Cathepsin L-catalyzed hydrolysis of Z-Phe-Arg-NHMec (10 μM) in the presence of increasing concentrations of compound 10 (from top to bottom: 0, 0, 1, 2, 3, 4, 5, 7, 10, 15, 50 μM) in 0.1 M sodium phosphate pH 6.0, 0.1 M NaCl, 5 mM EDTA, 0.01% Brij 35, 25 μM DTT, 1% DMSO, 37°C. The reactions were initiated by addition of the enzyme. Fluorescence emission at 440 nm was measured after excitation at 360 nm. Fluorescence units (FU) were corrected for background fluorescence.

Figure 6.  Crystal structure of the complex between cathepsin S and morpholinocarbonyl-leucyl-O-benzylserine nitrile. The thioimidate nitrogen is in hydrogen bond distance to the side chain nitrogen of Gln 19 and the main chain nitrogen of Cys 25. PDB entry 1MS6³⁷, image prepared with PyMOL²⁹.

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