Journal of Medicinal Chemistry Scheme 1a Article aReagents: (i) relevant phenol, TEA/DCM or relevant aniline, DMAP/CH3CN; (ii) SnCl2·2H2O/EtOH or Fe, HCl/EtOH; (iii) relevant isocyanate and appropriate method; (iv) 1 M TBAF/THF. intermediates in the synthesis of PIB-SOs revealed an unusual arrest of cell cycle progression in S-phase (compounds 4−6 and 10, Table 1) instead of the G2/M phase, as observed with their known antimicrotubule counterparts. That unexpected S phase arrest induced by this new subset of N-phenyl-N′-(2- chloroethyl)ureas prompted us to determine their structure− activity relationships and to investigate their mechanism of action. o-Tolyl 4-[3-(2-chloroethyl)ureido]benzenesulfonate (4) and 4-hydroxyphenyl 4-[3-(2-chloroethyl)ureido]- benzenesulfonate (10) were selected as molecular templates to initiate the structure−activity relationship study. We ﬁrst assessed the role and the substitution pattern of the electrophilic CEU group on ring A (Figure 1) via its substitution with a 3-chloropropylurea (CPU) or an ethylurea (EU) moiety, leading to N-phenyl ureidobenzenesulfonate derivatives (PUB-SOs), molecules containing alkylurea moi- eties. We subsequently replaced the sulfonyl group bridging the phenyl rings A and B of PUB-SOs with a bioisosteric sulfonamide bridge, thereby leading to N-phenylureidobenze- nesulfonamides (PUB-SAs). Moreover, we studied the eﬀects of replacing the methyl substituent with an ethyl or propyl group at the C2 position of the B-ring. We also studied the eﬀect of a hydroxyl moiety at the C4 position of the B-ring. The potential antiproliferative activity and eﬀect on cell cycle progression of these novel compounds were assessed in M21 human skin melanoma, estrogen-dependent MCF-7 breast adenocarcinoma, and HT-29 colon adenocarcinoma cell lines. The most potent inhibitors of S-phase progression among these derivatives were assessed for their potential induction of H2AX phosphorylation, which parallels the induction of DNA double- strand breaks, and for their antitumoral activity in HT-1080 human ﬁbrosarcoma cells grafted onto the CAM assay. ■ RESULTS AND DISCUSSION Chemistry. Scheme 1 depicts the synthetic pathways used for the preparation of substituted PUB-SO and PUB-SA analogues. These compounds were prepared by nucleophilic addition of the appropriate phenols or anilines to nitrobenzene- 1-sulfonyl chloride. Nitrophenyl sulfonates 56−67 and nitro- phenylsulfonamides 68−73 were reduced to the corresponding 6196 dx.doi.org/10.1021/jm3006492 | J. Med. Chem. 2012, 55, 6194−6208