Aromatic 2-chloroethyl urea derivatives and bioisosteres. Part 2: Cytocidal activity and effects on the nuclear translocation of thioredoxin-1, and the cell cycle progression Jessica S. Fortin a,c, * , Marie-France Côté a , Jacques Lacroix a , Éric Petitclerc a,b , René C.-Gaudreault a,b, * a Unité des Biotechnologies et de Bioingénierie, CHUQ, Hôpital Saint-François d’Assise, Québec, Que., Canada G1L 3L5 b Département de Médecine, Faculté de Médecine, Université Laval, Québec, Que., Canada G1K 7P4 c Faculté de Pharmacie, Université Laval, Québec, Que., Canada G1K 7P4 article info Article history: Received 28 February 2008 Revised 8 May 2008 Accepted 4 June 2008 Available online 10 June 2008 Keywords: N-Aryl 2-amino-oxazolines N-Phenyl-N 0 -(2-chloroacetyl)ureas Thioredoxin Alkylating agents abstract Recently, a subset of N-phenyl-N 0 -(2-chloroethyl)ureas (CEU) was found abrogating the nuclear translo- cation of thioredoxin-1 and arresting the cell cycle in G 0 /G 1 phase. Several derivatives were prepared to assess their effect on cell cycle progression and on the intracellular location of Trx-1. Compounds 1–20, 21–40, and 41–60 exhibited GI 50 between 1 and 80 lM. Immunocytochemistry analysis showed com- pounds 4, 6, 8, 10, 11, 23, 24, 26–31, 34, 37, 41, 44, 46–51, 53, 56, and 57 inhibiting the nuclear translo- cation of Trx-1. Our results suggest that increasing the electrophilic character of these molecules might enhance the antiproliferative activity at the expense of the selectivity toward thioredoxin-1 and the G 0 / G 1 phase arrest. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction N-Phenyl-N 0 -(2-chloroethyl)ureas (CEU) are soft alkylating agents developed in our laboratory as potential anticancer drugs. 1–12 These molecules were designed initially from the aro- matic moiety of nitrogen mustards such as chlorambucil, and the non-nitrosated pharmacophore of aliphatic nitrosoureas such as carmustine. CEUs cytotoxicity has been shown to circumvent a large number of mechanisms of chemoresistance, including in- creased P-glycoprotein expression, increased DNA repair, increased intracellular gluthatione-S-transferase activity, and alteration of topoisomerase II activity. 2 Prototypical CEUs such as 1-(2-chloro- ethyl)-3-(4-tert-butylphenyl)urea (tBCEU) and 1-(2-chloroethyl)- 3-(4-iodophenyl)ureas (ICEU) covalently bind to the colchicine- binding site on b-tubulin isoform 2 via a unique acylation of the glutamic acid residue in position 198 that leads to the arrest of the cell cycle progression in G 2 /M transition and to apoptosis. 3,4,13 Our structure–activity relationships and molecular pharmacol- ogy studies unexpectedly showed that a CEU derivative named 1- (2-chloroethyl)-3-(4-cyclohexylphenyl)ureas (cHCEU) was block- ing the cell cycle progression in G 0 /G 1 phase instead of G 2 /M as all other antimicrotubules do. 13 In addition, studies using [ 14 C]cHCEU confirmed that the cHCEU does not bind to the colchi- cine-binding site but instead to the thioredoxin isoform-1 (Trx-1), the mitochondrial voltage-dependent anion channel isoform-1, and to a lesser extent to a few other unidentified cytosolic proteins. Interestingly, Trx-1 is an important protein in the G 1 to G 2 /M phase transition and its alkylation may lead, at least partly, to the inhibi- tion of its presence in the cell’s nucleus and to the arrest of the cell cycle progression into the G 0 /G 1 phase. 13 Interestingly, the arrest of the cell cycle progression in G 0 /G 1 phase is the hallmark of drugs such as vitamin E, 14 acetylsalicylic acid, 15 sulindac, 16 celecoxib, 17 and troglitazone 18 or in dietary agents such as epigallocatechin- 3-gallate, 19 resveratrol, 20,21 capsaicin, 22 and benzyl isothiocya- nate 23 that are exhibiting chemopreventive activities against vari- ous cancers. That observation prompted us to developed new molecules exhibiting similar properties. 24,25 To that end, a new subset of cyto- static CEU derivatives has been prepared to study the effect of the nature and the position of the substituents on the aromatic ring in regard to the inhibition of the intracellular translocation of Trx-1. 25 In addition, we have prepared also a series of analogues and bio- isosteres of CEU, namely, N-phenyl-N 0 -(2-ethyl)ureas (EU), N-phe- nyl-N 0 -(2-chloroacetyl)ureas (CAU), and N-aryl amino-2-oxazoline (4,5-dihydro-N-phenyloxazol-2-amines: OXA) derivatives to study the effect of different alkylating moieties on the translocation of Trx-1 and their antiproliferative activity on three human tumor cell lines, namely, HT-29, M21, and MCF-7 tumor cells. 0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2008.06.006 * Corresponding authors. Tel.: +1 418 525 4485; fax: +1 418 525 4372 (J.S.F.); tel.: +1 418 525 4444x52363; fax: +1 418 525 4372 (R.C.G.). E-mail addresses: jessica.fortin.1@ulaval.ca (J.S. Fortin), rene.c-gaudreault@ crsfa.ulaval.ca (R. C.-Gaudreault). Bioorganic & Medicinal Chemistry 16 (2008) 7477–7488 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc