4-Oxo-β-lactams (Azetidine-2,4-diones) Are Potent and Selective Inhibitors of Human Leukocyte Elastase

Abstract

Human leukocyte elastase (HLE) is a serine protease stored in and secreted from neutrophils that plays a determinant role in the pathogenesis of several lung diseases. 4-Oxo-beta-lactams, previously reported as acylating agents of porcine pancreatic elastase, were found to be selective and potent inhibitors of HLE. Structure-activity relationship analysis showed that inhibitory activity is very sensitive to the nature of C-3 substituents, with small alkyl substituents such as a gem-diethyl group improving the inhibitory potency when compared to gem-methyl benzyl or ethyl benzyl counterparts. 4-Oxo-beta-lactams containing a heteroarylthiomethyl group on the para position of an N(1)-aryl moiety afforded highly potent and selective inhibition of HLE, even at a very low inhibitor to enzyme ratio, as shown by the k(on) value of 3.24 x 10(6) M(-1) s(-1) for 6f. The corresponding ortho isomers were 40- to 90-fold less potent.

Synthesis, stability, biochemical and pharmacokinetic properties of a new potent and selective 4-oxo-β-lactam inhibitor of human leukocyte elastase

“…NMR spectra were recorded using a Brucker 400 Ultra-Shield (Brucker ARX 400 spectrometer, 400 MHz, Bruker Instruments, Billerica, MA, USA) spectrometer in CDCl 3 solution; chemical shifts, δ, are expressed in ppm relative to tetramethylsilane as the internal standard, and the coupling constants, J, are expressed in Hz. Low-resolution mass spectra were recorded using an HP5988A spectrometer, by RIAIDT [14]. Thioether 7 was prepared by adding triethylamine (2.2 mmol) to a solution of 6 (2 mmol) and thiophenol (2.2 mmol) in dry tetrahydrofuran (THF) (5 mL).…”

“…Thioether 7 was prepared by adding triethylamine (2.2 mmol) to a solution of 6 (2 mmol) and thiophenol (2.2 mmol) in dry tetrahydrofuran (THF) (5 mL). The reaction was stirred at room temperature, being monitored by TLC, on completion the triethylamine hydrochloride was removed by filtration and the solvent removed under reduced pressure [14]. 3,3-Diethyl-1-[4-(phenylsulphonylmethyl) phenyl]azetidine-2,4-dione, 3, was prepared by slowly adding 3-chloroperbenzoic acid (MCPBA, 0.382 mmol) to a cold solution of the thioether 7 [14] (0.153 mmol, 51.9 mg) in DCM (10 mL).…”

“…Inhibition was observed at inhibitor concentrations > 10[E] and thus, progress curves were fitted to Equation 3. The inhibitory activity of 3, towards papain (1mg/ml) was investigated by the incubation method, ( [3] = 50 μM in the incubation mixture), using the described procedures [14].…”

“…The 4-oxo-β-lactams containing N-aryl or N-heteroarylthiomethyl groups were designed as potential mechanism-based inhibitors featuring a latent electrophilic quinone methide imine function, that upon further reaction with an active site nucleophilic residue (e.g. His-57), may ultimately lead to irreversible inactivation of the enzyme [14]. Structure-activity relationship analysis showed that such 4-oxo-β-lactams afforded highly potent and selective inhibition of HLE, even at a very low inhibitor-to-enzyme ratio, as shown by the second-order rate constant for the inhibitor-enzyme association, k on , of 3.2 × 10 6 M −1 s −1 for derivative 1 (Figure 1).…”

“…Structure-activity relationship analysis showed that such 4-oxo-β-lactams afforded highly potent and selective inhibition of HLE, even at a very low inhibitor-to-enzyme ratio, as shown by the second-order rate constant for the inhibitor-enzyme association, k on , of 3.2 × 10 6 M −1 s −1 for derivative 1 (Figure 1). Despite the excellent inhibitory potency displayed by 1 and related derivatives, stability studies in both phosphate buffer and human plasma suggested that the departure of the thiol leaving group following 4-oxo-β-lactam ringopening does not occur in the HLE inhibition pathway [14]. Moreover, the highly hydrophobic nature of the 4-oxo-β-lactams (e.g.…”