Article pubs.acs.org/jmc Synthesis, Biological Evaluation, and Structure−Activity Relationships of Novel Substituted N-Phenyl Ureidobenzenesulfonate Derivatives Blocking Cell Cycle Progression in S-Phase and Inducing DNA Double-Strand Breaks Vanessa Turcotte,†,‡ Seb́astien Fortin,*,†,‡ Florence Vevey,† Yan Coulombe,§ Jacques Lacroix,† Marie-France Côte,́† Jean-Yves Masson, § and René C.-Gaudreault*,†,∥ †Unité des Biotechnologies et de Bioingeńierie, Centre de Recherche, C.H.U.Q., Hôpital Saint-Franco̧is d’Assise, Queb́ec, QC, G1L 3L5, Canada ‡Faculté de Pharmacie, Université Laval, Pavillon Vandry, Queb́ec, QC, G1V 0A6, Canada §Laboratoire de la Stabilité du Geńome, Centre de Recherche en Canceŕologie de l'Université Laval, Queb́ec, QC, G1R 2J6, Canada ∥Faculté de Med́ecine, Université Laval, Pavillon Vandry, Queb́ec, QC, G1V 0A6, Canada ABSTRACT: Twenty-eight new substituted N-phenyl ureido- benzenesulfonate (PUB-SO) and 18 N-phenylureidobenzene- sulfonamide (PUB-SA) derivatives were prepared. Several PUB- SOs exhibited antiproliferative activity at the micromolar level against the HT-29, M21, and MCF-7 cell lines and blocked cell cycle progression in S-phase similarly to cisplatin. In addition, PUB-SOs induced histone H2AX (γH2AX) phosphorylation, indicating that these molecules induce DNA double-strand breaks. In contrast, PUB-SAs were less active than PUB-SOs and did not block cell cycle progression in S-phase. Finally, PUB-SOs 4 and 46 exhibited potent antitumor activity in HT-1080 ﬁbrosarcoma cells grafted onto chick chorioallantoic membranes, which was similar to cisplatin and combretastatin A-4 and without signiﬁcant toxicity toward chick embryos. These new compounds are members of a promising new class of anticancer agents. ■ INTRODUCTION N-Phenyl-N′-(2-chloroethyl)ureas (1) are members of a class of potent antiproliferative agents acting across a large panel of tumor cell lines and in several animal cancer models (Figure 1). Several subsets of these monoalkylating agents were shown to bind covalently to proteins such as βII-tubulin,1−7 thioredoxin- 1,8−10 prohibitin-1,11 and mitochondrial voltage-dependent anion channel,12 leading to arrest of cell cycle progression in Figure 1. Structure of N-phenyl-N′-(2-chloroethyl)ureas (1), PIB-SOs (2), CA-4 (3), and PUB-SOs (4−10). either G2/M or G0/G1 phase. By use of matrix-assisted laser desorption ionization and electrospray mass spectrometry, N- phenyl-N′-(2-chloroethyl)ureas exhibiting antimicrotubule ac- tivity were shown to bind covalently to microtubules via a unique mechanism of nucleophilic addition involving the esteriﬁcation of a Glu residue at position 198 of human β- tubulin (Gluβ198).13 Of interest, Gluβ198, which is located in a small pocket adjacent to the colchicine-binding site, is involved in microtubule stability and dynamics and is also associated with a mechanism of resistance to Taxotere (docetaxel).14,15 With the objective of developing anticancer agents with optimal biopharmaceutical properties and lower toxicity, we recently modiﬁed the structure of the N-phenyl-N′-(2- chloroethyl)urea scaﬀold by the addition of a benzenesulfonate group and cyclization of the 2-chloroethylurea (CEU) moiety into a 1-phenylimidazolidin-2-one heterocycle. The latter modiﬁcations led to a novel class of potent antimicrotubule agents designated as phenyl 4-(2-oxoimidazolidin-1-yl)- benzenesulfonates (PIB-SOs, 2).16 PIB-SOs, molecules con- taining an imidazolidonyl ring, exhibited antiproliferative activities in the low nanomolar range, blocked cell cycle progression in G2/M phase, and bound to the colchicine- binding site, leading to cytoskeleton disruption and apoptosis. Received: May 9, 2012 Published: June 13, 2012 © 2012 American Chemical Society 6194 dx.doi.org/10.1021/jm3006492 | J. Med. Chem. 2012, 55, 6194−6208