ICD-1578

The novel inhibitor 7‐N‐phenylcarbamoylamino‐4‐chloro‐3‐propyloxyisocoumarin (ICD 1578) was tested for its ability to antagonize the zinc metalloprotease activity of botulinum toxin B (BoNT/B). The efficacy of this compound was tested in a cell‐free system using a 50‐mer synaptobrevin peptide as substrate. The peptide, designated as [Pya88] S 39–88, had a fluorescent amino acid analog, l‐pyrenylalanine (Pya), substituted for the normal Phe88 of synaptobrevin‐2. Cleavage by BoNT light chain yielded fragments of 38 and 11 amino acids, respectively. The smaller fragment, containing the Pya fluorophore, was readily separated and quantified by fluorescence spectroscopy at 377 nm. In the presence of 7–200 μM ICD 1578, cleavage of [Pya88] S 39–88 was progressively reduced (IC50=27.6 μM), and 100 μM ICD 1578 produced >95% inhibition. For comparison, captopril, a well‐known zinc metalloprotease inhibitor, generated less than 10% inhibition at a concentration of 5 mM. ICD 1578 is the most potent antagonist of BoNT/B light chain thus far described.

A series of 3-alkoxy-7-amino-4-chloroisocoumarins with various 3-alkoxy substituents have been prepared and evaluated as inhibitors of human leukocyte elastase (HLE). In addition, a new series of acyl, urea, and carbamate derivatives of 7-amino-4-chloro-3-methoxyisocoumarin (1), 7-amino-4-chloro-3-propoxyisocoumarin (3), and 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarin (6) have been synthesized. Most of the synthesized compounds are very potent inhibitors of HLE with kobs/[I] values between 10(4) and 10(6) M-1 s-1. Hydrophobic substituents on the 7-amino position of the isocoumarin ring afford the best selectivity and inhibitory potency for HLE. In the 2-bromoethoxy series, compound 24 with a PhNHCONH 7-substituent had a kobs/[I] value of 1.2 x 10(6) M-1 s-1, was very selective for HLE, and was the most potent inhibitor of HLE tested. Of the extended chain L-phenylalanyl derivatives, the Bz-L-Phe compound 66 with a kobs/[I] value of 1.8 x 10(5) M-1 s-1 was the most potent inhibitor of HLE in the 3-methoxyisocoumarin series and was also very selective for HLE. Our results indicate that a high degree of selectivity, along with potency, can be introduced into mechanism-based isocoumarin inhibitors.