Modification of the phenyl ring B of phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates by pyridinyl moiety leads to novel antimitotics targeting the colchicine-binding site.

A series of 8 novel pyridinyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PYRIB-SOs) were designed, prepared and evaluated for their mechanism of action. PYRIB-SOs were found to have antiproliferative activity in the nanomolar to submicromolar range on several breast cancer cell lines. Moreover, subsequent biofunctional assays indicated that the most potent PYRIB-SOs 1-3 act as antimitotics binding to the colchicine-binding site (C-BS) of α, ß-tubulin and that they arrest the cell cycle progression in the G2/M phase. Microtubule immunofluorescence and tubulin polymerisation assay confirm that they disrupt the cytoskeleton through inhibition of tubulin polymerisation as observed with microtubule-destabilising agents. They also show good overall theoretical physicochemical, pharmacokinetic and druglike properties. Overall, these results show that PYRIB-SOs is a new family of promising antimitotics to be further studied in vivo for biopharmaceutical and pharmacodynamic evaluations.

Design, synthesis and biological evaluation of new 2,6-difluorinated phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates as new antimicrotubule agents.

We previously discovered a novel family of antimicrotubule agents designated as phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs). In this study, we evaluated the effect of the difluorination of the aromatic ring bearing the imidazolidin-2-one moiety (ring A) at positions 3, 5 and 2, 6 on their antiproliferative activity on four cancer cell lines, their ability to disrupt the microtubules and their toxicity toward chick embryos. We thus synthesized, characterized and biologically evaluated 24 new difluorinated PIB-SO derivatives designated as phenyl 3,5-difluoro-4-(2-oxoimidazolidin-1-yl)benzenesulfonates (3,5-PFB-SOs, 4-15) and phenyl 2,6-difluoro-4-(2-oxoimidazolidin-1-yl)benzenesulfonates (2,6-PFB-SOs, 16-27). The concentration of the drug required to inhibit cell growth by 50% (IC50) of 3,5-PFB-SOs is over 1000 nM while most of 2,6-PFB-SOs exhibit IC50 in the nanomolar range (23-900 nM). Furthermore, the most potent 2,6-PFB-SOs 19, 26 and 27 arrest the cell cycle progression in G2/M phase, induce cytoskeleton disruption and impair microtubule polymerization. Docking studies also show that the most potent 2,6-PFB-SOs 19, 21, 24, 26 and 27 have binding affinity toward the colchicine-binding site (C-BS). Moreover, their antiproliferative activity is not affected by antimicrotubule- and multidrug-resistant cell lines. Besides, they exhibit improved in vitro hepatic stability in the mouse, rat and human microsomes compared to their non-fluorinated counterparts. They also showed theoretical pharmacokinetic, physicochemical and drug-like properties suited for further in vivo assays. In addition, they exhibit low to no systemic toxicity toward chick embryos. Finally, our study evidences that PIB-SOs must be fluorinated in specific positions on ring A to maintain both their antiproliferative activity and their biological activity toward microtubules.

4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamide salts: Novel hydrosoluble prodrugs of antimitotics selectively bioactivated by the cytochrome P450 1A1 in breast cancer cells.

4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides (PAIB-SAs) are members of a new family of prodrugs bioactivated by cytochrome P450 1A1 (CYP1A1) in breast cancer cells into their potent 4-(2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamide metabolites (PIB-SAs). One of the predominant problems for the galenic formulation and administration of PAIB-SAs in animal studies is their poor hydrosolubility. To circumvent that difficulty, we report the design, the synthesis, the chemical characterization, the evaluation of the aqueous solubility, the antiproliferative activity and the mechanism of action of 18 new Na+, K+ and Li+ salts of PAIB-SAs. Our results evidenced that the latter exhibited highly selective antiproliferative activity toward MCF7 and MDA-MB-468 breast cancer cells expressing endogenously CYP1A1 compared to insensitive MDA-MB-231 and HaCaT cells. Moreover, PAIB-SA salts 1-18 are significantly more hydrosoluble (3.9-9.4 mg/mL) than their neutral counterparts (< 0.0001 mg/mL). In addition, the most potent PAIB-SA salts 1-3 and 10-12 arrested the cell cycle progression in the G2/M phase and disrupted the cytoskeleton's dynamic assembly. Finally, PAIB-SA salts are N-dealkylated by CYP1A1 into their corresponding PIB-SA metabolites, which are potent antimitotics. In summary, our results show that our water-soluble PAIB-SA salts, notably the sodium salts, still exhibit potent antiproliferative efficacy and remain prone to CYP1A1 bioactivation. In addition, these PAIB-SA salts will allow the development of galenic formulations suitable for further biopharmaceutical and pharmacodynamic studies.

N-phenyl ureidobenzenesulfonates, a novel class of promising human dihydroorotate dehydrogenase inhibitors.

N-phenyl ureidobenzenesulfonates (PUB-SOs) is a new class of promising anticancer agents inducing replication stresses and cell cycle arrest in S-phase. However, the pharmacological target of PUB-SOs was still unidentified. Consequently, the objective of the present study was to identify and confirm the pharmacological target of the prototypical PUB-SO named 2-ethylphenyl 4-(3-ethylureido)benzenesulfonate (SFOM-0046) leading to the cell cycle arrest in S-phase. The antiproliferative and the cytotoxic activities of SFOM-0046 were characterized using the NCI-60 screening program and its fingerprint was analyzed by COMPARE algorithm. Then, human dihydroorotate dehydrogenase (hDHODH) colorimetric assay, uridine rescuing cell proliferation and molecular docking in the brequinar-binding site were performed. As a result, SFOM-0046 exhibited a mean antiproliferative activity of 3.5 µM in the NCI-60 screening program and evidenced that leukemia and colon cancer cell panels were more sensitive to SFOM-0046. COMPARE algorithm showed that the SFOM-0046 cytotoxic profile is equivalent to the ones of brequinar and dichloroallyl lawsone, two inhibitors of hDHODH. SFOM-0046 inhibited the hDHODH in the low nanomolar range (IC50 = 72 nM) and uridine rescued the cell proliferation of HT-29, HT-1080, M21 and MCF-7 cancer cell lines in the presence of SFOM-0046. Finally, molecular docking showed a binding pose of SFOM-0046 interacting with Met43 and Phe62 present in the brequinar-binding site. In conclusion, PUB-SOs and notably SFOM-0046 are new small molecules hDHODH inhibitors triggering replication stresses and S-phase arrest.

Stereoselective Synthesis of Fluorinated Galactopyranosides as Potential Molecular Probes for Galactophilic Proteins: Assessment of Monofluorogalactoside-LecA Interactions.

The replacement of hydroxyl groups by fluorine atoms on hexopyranoside scaffolds may allow access to invaluable tools for studying various biochemical processes. As part of ongoing activities toward the preparation of fluorinated carbohydrates, a systematic investigation involving the synthesis and biological evaluation of a series of mono- and polyfluorinated galactopyranosides is described. Various monofluorogalactopyranosides, a trifluorinated, and a tetrafluorinated galactopyranoside have been prepared using a Chiron approach. Given the scarcity of these compounds in the literature, in addition to their synthesis, their biological profiles were evaluated. Firstly, the fluorinated compounds were investigated as antiproliferative agents using normal human and mouse cells in comparison with cancerous cells. Most of the fluorinated compounds showed no antiproliferative activity. Secondly, these carbohydrate probes were used as potential inhibitors of galactophilic lectins. The first transverse relaxation-optimized spectroscopy (TROSY) NMR experiments were performed on these interactions, examining chemical shift perturbations of the backbone resonances of LecA, a virulence factor from Pseudomonas aeruginosa. Moreover, taking advantage of the fluorine atom, the 19 F NMR resonances of the monofluorogalactopyranosides were directly monitored in the presence and absence of LecA to assess ligand binding. Lastly, these results were corroborated with the binding potencies of the monofluorinated galactopyranoside derivatives by isothermal titration calorimetry experiments. Analogues with fluorine atoms at C-3 and C-4 showed weaker affinities with LecA as compared to those with the fluorine atom at C-2 or C-6. This research has focused on the chemical synthesis of "drug-like" low-molecular-weight inhibitors that circumvent drawbacks typically associated with natural oligosaccharides.

Diversity-Oriented Synthesis of Diol-Based Peptidomimetics as Potential HIV Protease Inhibitors and Antitumor Agents.

Peptidomimetic HIV protease inhibitors are an important class of drugs used in the treatment of AIDS. The synthesis of a new type of diol-based peptidomimetics is described. Our route is flexible, uses d-glucal as an inexpensive starting material, and makes minimal use of protection/deprotection cycles. Binding affinities from molecular docking simulations suggest that these compounds are potential inhibitors of HIV protease. Moreover, the antiproliferative activities of compounds 33 a, 35 a, and 35 b on HT-29, M21, and MCF7 cancer cell lines are in the low micromolar range. The results provide a platform that could facilitate the development of medically relevant asymmetrical diol-based peptidomimetics.

Homologation of the Alkyl Side Chain of Antimitotic Phenyl 4-(2-Oxo-3-alkylimidazolidin-1-yl)benzenesulfonate Prodrugs Selectively Targeting CYP1A1-Expressing Breast Cancers Improves Their Stability in Rodent Liver Microsomes.

Phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) are a new family of antimitotic prodrugs bioactivated in breast cancer cells expressing CYP1A1. In this study, we report that the 14C-labeled prototypical PAIB-SO [14C]CEU-818 and its antimitotic counterpart [14C]CEU-602 are distributed in whole mouse body and they show a short half-life in mice. To circumvent this limitation, we evaluated the effect of the homologation of the alkyl side chain of the imidazolidin-2-one moiety of PAIB-SOs. Our studies evidence that PAIB-SOs bearing an n-pentyl side chain exhibit antiproliferative activity in the nanomolar-to-low-micromolar range and a high selectivity toward CYP1A1-positive breast cancer cells. Moreover, the most potent n-pentyl PAIB-SOs were significantly more stable toward rodent liver microsomes. In addition, PAIB-SOs 10 and 14 show significant antitumor activity and low toxicity in chorioallantoic membrane (CAM) assay. Our study confirms that homologation is a suitable approach to improve the rodent hepatic stability of PAIB-SOs.

Preparation and biological evaluation of new antimicrotubule agents: Modification of the imidazolidin-2-one moiety of phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates.

We prepared and biologically evaluated 32 novel molecules named phenyl 4-(dioxoimidazolidin-1-yl)benzenesulfonates (PID-SOs) and ethyl 2-(3-(4-(phenoxysulfonyl)phenyl)ureido)acetates (EPA-SOs). The antiproliferative activity of PID-SOs and EPA-SOs was assessed on four cancer cell lines (HT-1080, HT-29, M21, and MCF7). The most potent PID-SOs bearing an imidazolidin-2,4-dione group show antiproliferative activity in the nanomolar to low micromolar range (0.066 - 6 µM) while EPA-SOs and PID-SOs bearing an imidazolidin-2,5-dione moiety are mostly not active, exhibiting antiproliferative activity over 100 µM. The most potent PID-SOs (16-18) arrest the cell cycle progression in G2/M phase and interact with the colchicine-binding site leading to the microtubule and cytoskeleton disruption. Moreover, their antiproliferative activity is not impaired in vinblastine-, paclitaxel-, and multidrug-resistant cell lines. Finally, our study confirms that PID-SOs bearing the imidazolidin-2,4-dione moiety are a new family of promising antimitotics.

Branched alkyl of phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates as unique cytochrome P450 1A1-activated antimitotic prodrugs: Biological evaluation and mechanism of bioactivation.

We recently discovered a new family of prodrugs deriving from phenyl 4-(2-oxo-3-imidazolidin-1-yl)benzenesulfonates (PIB-SOs) bioactivatable by cytochrome P450 1A1 (CYP1A1) into potent antimitotics referred to as phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs). PAIB-SOs display significant selectivity toward human breast cancer cells based on the N-dealkylation of PAIB-SOs into their corresponding PIB-SOs by CYP1A1. In this study, we have evaluated the molecular mechanism of the bioactivation of PAIB-SOs into PIB-SOs by branching the linear alkyl chain on the imidazolidin-2-one (IMZ) moiety of PAIB-SOs by branched alkyl groups such as isopropyl, isobutyl and sec-butyl. Our results show that PAIB-SOs bearing an isobutyl group on the IMZ moiety and either a methoxy, a chloro or a bromo group at positions 3, 3,5 or 3,4,5 on the aromatic ring B exhibit antiproliferative activity ranging from 0.13 to 6.9 µM and selectivity toward MCF7 and MDA-MB-468 mammary cancer cells comparatively to other cell lines tested. Moreover, the most potent and selective PAIB-SOs bearing an isobutyl group and either a 3,5-Cl (44), 3,5-Br (45) or a 3,4,5-OMe (46) on the IMZ moiety exhibit antiproliferative activity in the sub-micromolar range and high selectivity ratios toward mammary cancer cells. They stop the cell cycle of MCF7 cells in the G2/M phase and disrupt their cytoskeleton. Furthermore, our studies evidenced that PAIB-SOs bearing either an isopropyl, a sec-butyl or an isobutyl group are hydroxylated on the carbon atom adjacent to the IMZ (Cα-OH) but only PAIB-SOs bearing an isobutyl group are bioactivated into PIB-SOs. Finally, PAIB-SOs 45 and 46 exhibit low toxicity toward normal cells and chick embryos and are thus promising antimitotic prodrugs highly selective toward CYP1A1-expressing breast cancer cells.

Phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonates and phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonamides as new antimicrotubule agents targeting the colchicine-binding site.

We recently designed and prepared new families of potent antimicrotubule agents designated as N-phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs) and phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonamides (PIB-SAs). Our previous structure-activity relationship studies (SAR) focused on the aromatic ring B of PIB-SOs and PIB-SAs leaving the impact of the phenylimidazolidin-2-one moiety (ring A) on the binding to the colchicine-binding site (C-BS) poorly studied. Therefore, the aim of the present study was to evaluate the effect of replacing the imidazolidin-2-one (IMZ) group by a pyrrolidin-2-one moiety. To that end, 15 new phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonate (PYB-SO) and 15 phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonamide (PYB-SA) derivatives were designed, prepared, chemically characterised and biologically evaluated. PYB-SOs and PYB-SAs exhibit antiproliferative activity in the low nanomolar to low micromolar range (0.0087-8.6 µM and 0.056-21 µM, respectively) on human HT-1080, HT-29, M21 and MCF7 cancer cell lines. Moreover, they block cell cycle progression in G2/M phase. Immunofluorescence, tubulin affinity and tubulin polymerisation assays show that they cause microtubule depolymerisation by docking the C-BS. In addition, docking assays with the most potent derivatives show binding affinity toward the C-BS and they also exhibit weak or no toxicity toward chick embryos. Finally, physicochemical properties calculated using the SwissADME algorithm show that PYB-SOs and PYB-SAs are promising new families of antimicrotubule agents.

Preparation, characterisation and biological evaluation of new N-phenyl amidobenzenesulfonates and N-phenyl ureidobenzenesulfonates inducing DNA double-strand breaks. Part 3. Modulation of ring A.

N-Phenyl ureidobenzenesulfonates (PUB-SOs) are a new class of anticancer agents blocking the cell cycle progression in S-phase, inducing replicative stress and DNA double-strand breaks (DSBs). In this study, we evaluate the effect of modifying the nature and the position of different substituents on ring A of PUB-SOs on the antiproliferative activity, pharmacological activity as well as on calculated physicochemical, pharmacokinetics and drug-likeness properties. Modification of the urea group by an amide group led to new PUB-SO analogs designated as N-phenyl amidobenzenesulfonates (PAB-SOs). The 2-chloroethyl moiety on ring A was also substituted by different alkyl, cycloalkyl and chloroalkyl groups. The new PAB-SOs and PUB-SOs blocking the cell cycle progression in S-phase exhibit antiproliferative activity in the submicromolar to low micromolar range (0.14-27 µM) on four human cancer cell lines, namely HT-1080, HT-29, M21 and MCF7. Moreover, selected PUB-SO and PAB-SO derivatives induced the phosphorylation of H2AX in M21 cells and do not exhibit or only slightly alkylating activity as confirmed by the 4-(4-nitrobenzyl)pyridine (NBP) assay. Finally, our results show that structure modifications weakly affect the calculated physicochemical, pharmacokinetics and drug-likeness properties of PAB-SOs and PUB-SOs. Therefore, PAB-SOs and PUB-SOs are promising anticancer agents inducing replicative stress and DNA damage via a mechanism of action unrelated to DNA alkylation.