Journal of Medicinal Chemistry cell lines, namely, HT-29 colon carcinoma, M21 skin melanoma, and MCF-7 breast carcinoma cells. These cell lines were selected as representatives of tumors originating from the three germ layers (i.e., endoderm (HT-29), mesoderm (M21), and ectoderm (MCF-7)). Antiproliferative activity was evaluated using the sulforhodamine B method according to the NCI/NIH Developmental Therapeutics Program.17 The results are summarized in Table 1 and are expressed as the IC50. The antiproliferative activity of several PUB-SOs (compounds 4−7, 10, 12, 13, 16, 17, 26, 30, 31, 45−47, and 49) was equivalent to or better than with cisplatin (cis-diamminedichloroplatinum- (II), cDDP). In contrast, the antiproliferative activity of PUB- SAs was lower than for PUB-SOs and cDDP. Only two PUB- SAs (compounds 20 and 36) showed antiproliferative activity comparable to that observed with cDDP. Eﬀect on Cell Cycle Progression. Table 1 shows the percentage of Jurkat cells in G0/G1, S, and G2/M phases, respectively, after treatment with PUB-SOs, PUB-SAs, and cDDP for 24 h at optimal concentrations regarding the arrest of cell cycle progression in S-phase. Cell cycle distribution observed for control cells treated with 0.5% DMSO was 43%, 40%, and 17% in G0/G1, S, and G2/M phases, respectively. PUB-SOs 5, 6, 10, 30, 31, 33, 40, 41, 45, and 49 caused an S- phase arrest, thereby increasing the percentage of S-phase cells by 12−27%. PUB-SOs 4, 16, 17, 29, 46, and 47 strongly blocked cell cycle progression and this, to a more eﬃcient extent than cDDP, as measured by an increase in the S-phase fraction of 30−41%. A concentration of 19 μM cDDP blocked 69% of the Jurkat cell population in S-phase. In contrast, PUB- SAs did not induce an S-phase block. Structure−Activity Relationships. As depicted in Table 1 and as previously mentioned, replacing the sulfonyl group bridging phenyl rings A and B with a bioisosteric sulfonamide bridge signiﬁcantly lowered antiproliferative activity and reduced the eﬀect on cell cycle progression. Consequently, the spatial conformations of the two phenyl rings conferred by the bridge between the two phenyl rings are important for the activity. Moreover, our structure−activity relationship study shows that the substitution pattern of the pharmacophoric moiety on the A-ring is an important factor in the antiproliferative activity and cell cycle arrest caused by a given derivative. Transposition of the pharmacophoric CEU, CPU, and EU moieties from C4 to C3 on the A-ring signiﬁcantly decreased antiproliferative activity and abolished the eﬀect on cell cycle progression. Thus, derivatives whose A- rings have steric hindrance at C3 position with CEU, CPU, or EU in general did not entail S-phase arrest. Structure−activity relationship studies revealed that the nature of the pharmacophoric substituting group is also important. Derivatives bearing EU and CEU moieties at C4 position of A-ring exhibited antiproliferative activities in the same range and were more potent than their counterparts bearing a CPU moiety. Consequently, steric hindrance at this speciﬁc position does not seem to aﬀect the biological activity. Interestingly, compounds 40, 41, 45−47, 49, and 53 bearing an EU moiety were potent antiproliferative agents and arrested cell cycle progression in S-phase. These compounds lack an electrophilic chlorine substituent, which is involved in the mechanism of nucleophilic esteriﬁcation of acidic peptide residues such as glutamic and aspartic acids.11,13 Thus, the presence of a chlorine atom and dipole−dipole interactions are not prerequisites for the biological activity of this group of compounds, unlike the G2/M or G0/G1 block that is speciﬁcally Article observed with the N-phenyl-N′-(2-chloroethyl)urea deriva- tives.13 This suggests that the mechanism of action of compounds 40, 41, 45−47, 49, and 53 does not likely proceed via nucleophilic protein alkylation. Another most interesting feature of PUB-SOs lies in the fact that the B-ring can accommodate substitution with either a hydroxyl group at C4 or an alkyl (methyl, ethyl, propyl) group at C2 without signiﬁcant alteration of their cytocidal activity, and therefore, steric hindrances do not aﬀect the C2 position. Thus, we obtained a new class of antiproliferative agents that block the S- phase, with several of its members exhibiting IC50 values that are similar to or better than in the case of cDDP, used here as a positive control. Phosphorylation of H2AX. Since the major event occurring in S-phase is DNA replication, we next assessed whether DNA double-strand breaks are involved in the mechanism of action of PUB-SOs. According to current literature,18−21 phosphorylation of Ser-139 at the C-terminus of histone H2AX (thus yielding γH2AX) occurs upon induction of DNA double-strand breaks. To address the mechanism of action of the novel S-phase inhibitors, we evaluated their ability to induce γH2AX formation. H2AX phosphorylation induced by compounds 4, 16, 17, 29−31, 33, 40, 41, 45−47, and 49, which had displayed the highest antiproliferative activity (IC50 < 55 μM) and the ability to block >60% of the S-phase fraction was assessed by immunoﬂuorescence.22,23 As depicted in Figure 2, the latter group of compounds, when tested at their respective IC50 value, induced H2AX phosphorylation in M21 cells. Indeed, γH2AX was detected as nuclear red spots in nuclei (stained in blue using 4′,6-diamidino-2-phenylindole (DAPI)) of cells treated with 4, 16, 17, 29−31, 33, 40, 41, 45− 47, as well as with cDDP, but was absent from control cells. The latter data support the notion that the active PUB-SOs act via the induction of DNA double-strand breaks, which in turn may account for the S-phase cell arrest induced by these compounds. Research is in progress to determine the molecular mechanism responsible for the induction of DNA double- strand breaks and γH2AX by this category of derivatives. Antitumoral Activity As Measured with CAM Assays. The most potent PUB-SOs in each series of CEU, CPU, and EU that induce an S-phase block (compounds 4, 10, 16, 17, 30, 45, 46, and 47) were tested in ovo using the CAM assay. HT- 1080 human ﬁbrosarcoma cells were selected because they produce solid tumors that are sensitive to antiangiogenic and antitumoral agents.24−29 cDDP and CA-4 were used as positive controls. A mixture of Cremophor EL, 99% ethanol, and PBS (1/1/14 v/v) was used as an excipient to inject cDDP, CA-4, and PUB-SOs. cDDP (10 μg/egg) and CA-4 (1 μg/egg) respectively inhibited tumor growth by 46% and 49% and exhibited toxicity in 6% and 21% of the chick embryos. As shown in Figure 3, compounds 4, 10, 16, 17, 30, and 45−47 administered at 30 μg/egg (except for 4 which was used at 10 μg/egg) signiﬁcantly inhibited tumor growth. Thus, com- pounds 10, 30, 45, and 47 respectively inhibited tumor growth by 69%, 68%, 68%, and 65% and exhibited lethality in 15%, 0%, 9%, and 10% of the chick embryos. Compounds 16 and 17 reduced tumor growth by 49%, i.e., to an extent comparable to that of cDDP and CA-4, but were rather toxic toward chick embryos (causing death in 33% and 36% of embryos, respectively). On the other hand, compounds 4 and 46 inhibited tumor growth by 60% and 45%, respectively, while showing low toxicity in chick embryos (with a 15% and 9% 6198 dx.doi.org/10.1021/jm3006492 | J. Med. Chem. 2012, 55, 6194−6208