N-Acyl substituted 7-amino-4-chloroisocoumarin: A peptide degradation model via an imide mechanism Ce´ drik Garino, Fre´ de´ ric Bihel, Florence Souard, Gilles Que´ le´ ver and Jean-Louis Kraus* INSERM U-382, Institut de Biologie du De ´veloppement de Marseille, CNRS-INSERM-Universite ´delaMe ´diterrane ´e, Laboratoire de Chimie Biomole ´culaire, Faculte ´ des Sciences de Luminy, case 907, 13288 Marseille Cedex 09, France Received 13 October 2003; accepted 14 January 2004 Abstract—During the coupling reaction between 3-alkoxy-7-amino-4-chloroisocoumarin and N-acyl alanine dipeptide, an unex- pected deamidation reaction was observed. The proposed mechanism for this reaction involved the formation of an imide inter- mediate which after cleavage led to the release of amino acid moiety. The described deamidation reaction represents the first chemical model involving a non-peptidic moiety, which mimics biological and chemical deamidation processes occurring in proteins or peptides incorporating an asparagine or a glutamine residue. # 2004 Elsevier Ltd. All rights reserved. We have recently reported the remarkable chemical reactivity of 3-alkoxy-7-amino-4-chloroisocoumarin derivatives 1 towards amines 1 as well as their biological activity to inhibit the secretion of b-amyloid peptide, 2 a major component of the senile plaques involved in the Alzheimer’s disease. These chemical and biological activities of such isocoumarin derivatives are related to their ability to react with various nucleophiles through a quinonimine methide intermediates. 3 Continuing our efforts to develop new 4-chloro- isocoumarin derivatives of interest for Alzheimer’s disease therapy, we investigated the feasibility to couple various specific pseudopeptides at the free 7-amino position of the isocoumarin nucleus using standard DCC/ HOBt peptide coupling methodology. N-(Difluoro- phenylacetyl)-alanyl peptide moiety was selected to be coupled to the 7-amino-4-chloroisocoumarin 1. Indeed, it has been reported that N-(3,5-difluorophenylacetyl)- alanine 2 linked to various aromatic heterocyclic nuclei, led to potent b-amyloid peptide inhibitors. 4,5 Besides their use in the design of b-amyloid peptide inhib- itors, the remarkable chemical reactivity of 4-chloro- isocoumarin derivatives could also be used in protein deamidation model. Indeed, in vivo protein deamida- tion processes have drastic consequences in regulatory signal like apoptosis. 6 In this paper, we report a simple chemical model which mimicks well-known in vivo protein deamidation processes. When 7-amino-4-chloroisocoumarin 1 (R=OCH 3 ) was added to N-(3,5-difluorophenylacetyl)-alanine 2 pre- activated with DCC/HOBt in the presence of diisopro- pylethylamine, after standard work up (5% citric acid followed by 5% NaHCO 3 washing solutions), LC/MS analysis of the obtained crude mixture revealed the presence of several reaction products represented on Scheme 1. Besides the expected di-adduct 3, characterized by LC/ MS (peak at (M+H) + =495, (retention time) t r =16.90 min), two other molecular peak ions, that are (M+H) + =424, t r =24.40 min and (M+H) + =341, t r =10.49 min, corresponding respectively to com- pounds 4 and 5 were identified. These results were repeatedly obtained three times. Compounds 3 and 4 were isolated (19% and 11% yields respectively) upon column chromatography, fully and unambiguously characterized by NMR and LC/MS. 7 Compound 5, only present as traces ( < 1%), was identified by LC/MS but not isolated. Besides, starting material 1 was recov- ered and the overall yield of this uncomplete reaction was 30%. 0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2004.01.030 Bioorganic & Medicinal Chemistry Letters 14 (2004) 1771–1774 Keywords: Isocoumarin derivatives; Deamidation reaction; Imide intermediate. * Corresponding author. Tel.: +33-491-82-91-41; fax: +33-491-26-97- 57; e-mail: kraus@luminy.univ-mrs.fr