Crizotinib induces Par-4 secretion from normal cells and GRP78 expression on the cancer cell surface for selective tumor growth inhibition.

Lung cancer is the leading cause of cancer-related deaths. Lung cancer cells develop resistance to apoptosis by suppressing the secretion of the tumor suppressor Par-4 protein (also known as PAWR) and/or down-modulating the Par-4 receptor GRP78 on the cell surface (csGRP78). We sought to identify FDA-approved drugs that elevate csGRP78 on the surface of lung cancer cells and induce Par-4 secretion from the cancer cells and/or normal cells in order to inhibit cancer growth in an autocrine or paracrine manner. In an unbiased screen, we identified crizotinib (CZT), an inhibitor of activated ALK/MET/ROS1 receptor tyrosine kinase, as an inducer of csGRP78 expression in ALK-negative, KRAS or EGFR mutant lung cancer cells. Elevation of csGRP78 in the lung cancer cells was dependent on activation of the non-receptor tyrosine kinase SRC by CZT. Inhibition of SRC activation in the cancer cells prevented csGRP78 translocation but promoted Par-4 secretion by CZT, implying that activated SRC prevented Par-4 secretion. In normal cells, CZT did not activate SRC and csGRP78 elevation but induced Par-4 secretion. Consequently, CZT induced Par-4 secretion from normal cells and elevated csGRP78 in the ALK-negative tumor cells to cause paracrine apoptosis in cancer cell cultures and growth inhibition of tumor xenografts in mice. Thus, CZT induces differential activation of SRC in normal and cancer cells to trigger the pro-apoptotic Par-4-GRP78 axis. As csGRP78 is a targetable receptor, CZT can be repurposed to elevate csGRP78 for inhibition of ALK-negative lung tumors.

Amino-Substituted 3-Aryl- and 3-Heteroarylquinolines as Potential Antileishmanial Agents.

Leishmaniasis, a disease caused by protozoa of the Leishmania species, afflicts roughly 12 million individuals worldwide. Most existing drugs for leishmaniasis are toxic, expensive, difficult to administer, and subject to drug resistance. We report a new class of antileishmanial leads, the 3-arylquinolines, that potently block proliferation of the intramacrophage amastigote form of Leishmania parasites with good selectivity relative to the host macrophages. Early lead 34 was rapidly acting and possessed good potency against L. mexicana (EC50 = 120 nM), 30-fold selectivity for the parasite relative to the macrophage (EC50 = 3.7 µM), and also blocked proliferation of Leishmania donovani parasites resistant to antimonial drugs. Finally, another early lead, 27, which exhibited reasonable in vivo tolerability, impaired disease progression during the dosing period in a murine model of cutaneous leishmaniasis. These results suggest that the arylquinolines provide a fruitful departure point for the development of new antileishmanial drugs.

Potent Synergistic Effect on C-Myc-Driven Colorectal Cancers Using a Novel Indole-Substituted Quinoline with a Plk1 Inhibitor.

Developing effective treatments for colorectal cancers through combinations of small-molecule approaches and immunotherapies present intriguing possibilities for managing these otherwise intractable cancers. During a broad-based, screening effort against multiple colorectal cancer cell lines, we identified indole-substituted quinolines (ISQ), such as N7,N7 -dimethyl-3-(1-methyl-1H-indol-3-yl)quinoline-2,7-diamine (ISQ-1), as potent in vitro inhibitors of several cancer cell lines. We found that ISQ-1 inhibited Wnt signaling, a main driver in the pathway governing colorectal cancer development, and ISQ-1 also activated adenosine monophosphate kinase (AMPK), a cellular energy-homeostasis master regulator. We explored the effect of ISQs on cell metabolism. Seahorse assays measuring oxygen consumption rate (OCR) indicated that ISQ-1 inhibited complex I (i.e., NADH ubiquinone oxidoreductase) in the mitochondrial, electron transport chain (ETC). In addition, ISQ-1 treatment showed remarkable synergistic depletion of oncogenic c-Myc protein level in vitro and induced strong tumor remission in vivo when administered together with BI2536, a polo-like kinase-1 (Plk1) inhibitor. These studies point toward the potential value of dual drug therapies targeting the ETC and Plk-1 for the treatment of c-Myc-driven cancers.

Epigenetic Regulation of Wnt Signaling by Carboxamide-Substituted Benzhydryl Amines that Function as Histone Demethylase Inhibitors.

Aberrant activation of Wnt signaling triggered by mutations in either Adenomatous Polyposis Coli (APC) or CTNNB1 (ß-catenin) is a hallmark of colorectal cancers (CRC). As part of a program to develop epigenetic regulators for cancer therapy, we developed carboxamide-substituted benzhydryl amines (CBAs) bearing either aryl or heteroaryl groups that selectively targeted histone lysine demethylases (KDMs) and functioned as inhibitors of the Wnt pathway. A biotinylated variant of N-((5-chloro-8-hydroxyquinolin-7-yl) (4-(diethylamino)phenyl)-methyl)butyramide (CBA-1) identified KDM3A as a binding partner. KDM3A is a Jumonji (JmjC) domain-containing demethylase that is significantly upregulated in CRC. KDM3A regulates the demethylation of histone H3's lysine 9 (H3K9Me2), a repressive marker for transcription. Inhibiting KDM3 increased H3K9Me2 levels, repressed Wnt target genes, and curtailed in vitro CRC cell proliferation. CBA-1 also exhibited in vivo inhibition of Wnt signaling in a zebrafish model without displaying in vivo toxicity.

Pictet-Spengler condensations using 4-(2-aminoethyl)coumarins.

Androgen-deprivation therapy (ADT) is only a palliative measure, and prostate cancer invariably recurs in a lethal, castration-resistant form (CRPC). Prostate cancer resists ADT by metabolizing weak, adrenal androgens to growth-promoting 5α-dihydrotestosterone (DHT), the preferred ligand for the androgen receptor (AR). Developing small-molecule inhibitors for the final steps in androgen metabolic pathways that utilize 17-oxidoreductases required probes that possess fluorescent groups at C-3 and intact, naturally occurring functionality at C-17. Application of the Pictet-Spengler condensation to substituted 4-(2-aminoethyl)coumarins and 5α-androstane-3-ones furnished spirocyclic, fluorescent androgens at the desired C-3 position. Condensations required the presence of activating C-7 amino or N,N-dialkylamino groups in the 4-(2-aminoethyl)coumarin component of these condensation reactions. Successful Pictet-Spengler condensation, for example, of DHT with 9-(2-aminoethyl)-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one led to a spirocyclic androgen, (3R,5S,10S,13S,17S)-17-hydroxy-10,13-dimethyl-1,2,2',3',4,5,6,7,8,8',9,9',10,11,12,12',13,13',14,15,16,17-docosahydro-7'H,11'H-spiro-[cyclopenta[a]phenanthrene-3,4'-pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin]-5'(1'H)-one. Computational modeling supported the surrogacy of the C-3 fluorescent DHT analog as a tool to study 17-oxidoreductases for intracrine, androgen metabolism.

An Underlying Mechanism of Dual Wnt Inhibition and AMPK Activation: Mitochondrial Uncouplers Masquerading as Wnt Inhibitors.

The importance of upregulated Wnt signaling in colorectal cancers led to efforts to develop inhibitors that target ß-catenin in this pathway. We now report that several "Wnt inhibitors" that allegedly target ß-catenin actually function as mitochondrial proton uncouplers that independently activate AMPK and concomitantly inhibit Wnt signaling. As expected for a process in which mitochondrial uncoupling diminishes ATP production, a mitochondrial proton uncoupler, FCCP, and a glucose metabolic inhibitor, 2-DG, activated AMPK and inhibited Wnt signaling. Also consistent with these findings, a well-known "Wnt inhibitor", FH535, functioned as a proton uncoupler, and in support of this finding, the N-methylated analog, 2,5-dichloro-N-methyl-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535-M), was inactive as an uncoupler and Wnt inhibitor. Apart from suggesting an opportunity to develop dual Wnt inhibitors and AMPK activators, these findings provide a cautionary tale that claims for Wnt inhibition alone require scrutiny as possible mitochondrial proton uncouplers or inhibitors of the electron transport chain.

One-Pot Synthesis of B-Ring Ortho-Hydroxylated Sappanin-Type Homoisoflavonoids.

A reliable method for the synthesis of B-ring hydroxylated homoisoflavonoids and 3-hetarylmethyl chromones has been developed. The method involves an initial oxa-Diels-Alder reaction of ortho-quinone methides generated from aryl/hetaryl-substituted ortho-( N, N-dimethylaminomethyl)phenols with (2 E)-3-( N, N-dimethylamino)-1-(2-hydroxyphenyl)prop-2-en-1-ones and the subsequent cascade of reactions. This synthetic strategy avoids conventional multistep protocols and does not require the protection of hydroxyl groups, thus allowing the facile synthesis of a library of various aromatic and heterocyclic analogues of naturally occurring homoisoflavonoids.

Semisynthetic aurones inhibit tubulin polymerization at the colchicine-binding site and repress PC-3 tumor xenografts in nude mice and myc-induced T-ALL in zebrafish.

Structure-activity relationships (SAR) in the aurone pharmacophore identified heterocyclic variants of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, selectivity for the colchicine-binding site on tubulin, and absence of appreciable toxicity. Among the leading, biologically active analogs were (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) that inhibited in vitro PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice using 10 mg/kg of 5a had no effect on mice weight, and aurone 5a did not inhibit, as desired, the human ether-à-go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on molecular docking and confirmed by liquid chromatography-electrospray ionization-tandem mass spectrometry studies, aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5b strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model.

Induction of AMPK activation by N,N'-diarylurea FND-4b decreases growth and increases apoptosis in triple negative and estrogen-receptor positive breast cancers.

PURPOSE: Triple negative breast cancer (TNBC) is the most lethal and aggressive subtype of breast cancer. AMP-activated protein kinase (AMPK) is a major energy regulator that suppresses tumor growth, and 1-(3-chloro-4-((trifluoromethyl)thio)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (FND-4b) is a novel AMPK activator that inhibits growth and induces apoptosis in colon cancer. The purpose of this project was to test the effects of FND-4b on AMPK activation, proliferation, and apoptosis in breast cancer with a particular emphasis on TNBC. MATERIALS AND METHODS: (i) Estrogen-receptor positive breast cancer (ER+BC; MCF-7, and T-47D), TNBC (MDA-MB-231 and HCC-1806), and breast cancer stem cells were treated with FND-4b for 24h. Immunoblot analysis assessed AMPK, acetyl-CoA carboxylase (ACC), ribosomal protein S6, cyclin D1, and cleaved PARP. (ii) Sulforhodamine B growth assays were performed after treating ER+BC and TNBC cells with FND-4b for 72h. Proliferation was also assessed by counting cells after 72h of FND-4b treatment. (iii) Cell death ELISA assays were performed after treating ER+BC and TNBC cells with FND-4b for 72h. RESULTS: (i) FND-4b increased AMPK activation with concomitant decreases in ACC activity, phosphorylated S6, and cyclin D1 in all subtypes. (ii) FND-4b decreased proliferation in all cells, while dose-dependent growth decreases were found in ER+BC and TNBC. (iii) Increases in apoptosis were observed in ER+BC and the MDA-MB-231 cell line with FND-4b treatment. CONCLUSIONS: Our findings indicate that FND-4b decreases proliferation for a variety of breast cancers by activating AMPK and has notable effects on TNBC. The growth reductions were mediated through decreases in fatty acid synthesis (ACC), mTOR signaling (S6), and cell cycle flux (cyclin D1). ER+BC cells were more susceptible to FND-4b-induced apoptosis, but MDA-MB-231 cells still underwent apoptosis with higher dose treatment. Further development of FND compounds could result in a novel therapeutic for TNBC.

A crystal structure of coil 1B of vimentin in the filamentous form provides a model of a high-order assembly of a vimentin filament.

Vimentin is an intermediate filament (IF) protein that is expressed in leukocytes, fibroblasts and endothelial cells of blood vessels. Vimentin filaments contribute to structural stability of the cell membrane, organelle positioning and protein transport. Vimentin self-assembles into a dimer that subsequently forms high-order structures, including tetramers and octamers. The details of IF assembly at crystallographic resolutions are limited to the tetrameric form. We describe a crystal structure of a fragment of a vimentin rod domain (coil 1B) with a dimer of tetramers in the asymmetric unit. Coil 1B in the crystal is in an infinitely high-order filamentous assembly state, in which the tetramers are packed against each other laterally in an antiparallel fashion across the crystal lattice. In one of the directions of lateral packing, the tetramers pack against each other strictly head-to-tail, and in the orthogonal direction the tetramers pack in a staggered manner. This organization of the tetramers of coil 1B in the crystal lattice, together with previously reported biochemical and structural data, yield a model of high-order vimentin filament assembly. DATABASE: Structural data are available in the PDB under the accession number 5WHF.

Phenylethynyl-substituted Heterocycles Inhibit Cyclin D1 and Induce the Expression of Cyclin-dependent Kinase Inhibitor p21Wif1/Cip1 in Colorectal Cancer Cells.

Fluorinated, phenylethynyl-substituted heterocycles that possessed either an N-methylamino or N,N-dimethylamino group attached to heterocycles including pyridines, indoles, 1H-indazoles, quinolines, and isoquinolines inhibited the proliferation of LS174T colon cancer cells in which the inhibition of cyclin D1 and induction of the cyclin-dependent kinase inhibitor-1 (i.e., p21Wif1/Cip1) served as a readout for antineoplastic activity at a cellular level. On a molecular level, these agents, particularly 4-((2,6-difluorophenyl)ethynyl)-N-methylisoquinolin-1-amine and 4-((2,6-difluorophenyl)ethynyl)-N,N-dimethylisoquinolin-1-amine, bound and inhibited the catalytic subunit of methionine S-adenosyltransferase-2 (MAT2A).

Efficient Synthesis of Aurone Mannich Bases and Evaluation of their Antineoplastic Activity in PC-3 Prostate Cancer Cells.

An efficient method for regioselective synthesis of C-7 Mannich bases of 6-hydroxyaurones was accomplished by the N,N-dialkylaminomethylation using aminals prepared from dimethylamine, dipropylamine, bis(2-methoxyethyl)amine, N-methylbutylamine, N-methylbenzylamine, morpholine, piperidine, and 1-methylpiperazine. Further transformation of 7-(N,N-dialkylamino)methyl group in these aurones led to formation of C-7 acetoxymethyl and methoxymethyl derivatives of 6-hydroxyaurones, some of which showed promising inhibition of PC-3 prostate cancer cell proliferation in the high nanomolar to low micromolar range that exceeded that of cisplatin.

Developing antineoplastic agents that target peroxisomal enzymes: cytisine-linked isoflavonoids as inhibitors of hydroxysteroid 17-beta-dehydrogenase-4 (HSD17B4).

Cytisine-linked isoflavonoids (CLIFs) inhibited PC-3 prostate and LS174T colon cancer cell proliferation by inhibiting a peroxisomal bifunctional enzyme. A pull-down assay using a biologically active, biotin-modified CLIF identified the target of these agents as the bifunctional peroxisomal enzyme, hydroxysteroid 17ß-dehydrogenase-4 (HSD17B4). Additional studies with truncated versions of HSD17B4 established that CLIFs specifically bind the C-terminus of HSD17B4 and selectively inhibited the enoyl CoA hydratase but not the d-3-hydroxyacyl CoA dehydrogenase activity. HSD17B4 was overexpressed in prostate and colon cancer tissues, knocking down HSD17B4 inhibited cancer cell proliferation, suggesting that HSD17B4 is a potential biomarker and drug target and that CLIFs are potential probes or therapeutic agents for these cancers.

Fluorinated N,N'-Diarylureas As Novel Therapeutic Agents Against Cancer Stem Cells.

Colorectal cancer is the second-leading cause of cancer-related mortality in the United States. More than 50% of patients with colorectal cancer will develop local recurrence or distant organ metastasis. Cancer stem cells play a major role in the survival and metastasis of cancer cells. In this study, we examined the effects of novel AMP-activated protein kinase (AMPK) activating compounds on colorectal cancer metastatic and stem cell lines as potential candidates for chemotherapy. We found that activation of AMPK by all fluorinated N,N-diarylureas (FND) compounds at micromolar levels significantly inhibited the cell-cycle progression and subsequent cellular proliferation. In addition, we demonstrated that select FNDs significantly increased apoptosis in colorectal cancer metastatic and cancer stem cells. Therefore, FNDs hold considerable promise in the treatment of metastatic colorectal cancer, through elimination of both regular cancer cells and cancer stem cells. Mol Cancer Ther; 16(5); 831-7. ©2017 AACR.

Bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones: New classes of antibacterial/antifungal agents.

The emergence of multidrug-resistant bacterial and fungal strains poses a threat to human health that requires the design and synthesis of new classes of antimicrobial agents. We evaluated bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones for their antibacterial and antifungal activities against panels of Gram-positive/Gram-negative bacteria as well as fungi. We investigated their potential to develop resistance against both bacteria and fungi by a multi-step resistance-selection method, explored their potential to induce the production of reactive oxygen species, and assessed their toxicity. In summary, we found that these compounds exhibited broad-spectrum antibacterial and antifungal activities against most of the tested strains with minimum inhibitory concentration (MIC) values ranging from <0.5 to >500µM against bacteria and 1.0 to >31.3µg/mL against fungi; and in most cases, they exhibited either superior or similar antimicrobial activity compared to those of the standard drugs used in the clinic. We also observed minimal emergence of drug resistance to these newly synthesized compounds by bacteria and fungi even after 15 passages, and we found weak to moderate inhibition of the human Ether-à-go-go-related gene (hERG) channel with acceptable IC50 values ranging from 1.12 to 3.29µM. Overall, these studies show that bis(N-amidinohydrazones) and N-(amidino)-N'-aryl-bishydrazones are potentially promising scaffolds for the discovery of novel antibacterial and antifungal agents.

Antineoplastic Isoflavonoids Derived from Intermediate ortho-Quinone Methides Generated from Mannich Bases.

The regioselective condensations of various 7-hydroxyisoflavonoids with bis(N,N-dimethylamino)methane in a Mannich reaction provided C-8 N,N-dimethylaminomethyl-substituted isoflavonoids in good yield. Similar condensations of 7-hydroxy-8-methylisoflavonoids led to the C-6-substituted analogs. Thermal eliminations of dimethylamine from these C-6 or C-8 N,N-dimethylaminomethyl-substituted isoflavonoids generated ortho-quinone methide intermediates within isoflavonoid frameworks for the first time. Despite other potential competing outcomes, these ortho-quinone methide intermediates trapped dienophiles including 2,3-dihydrofuran, 3,4-dihydro-2H-pyran, 3-(N,N-dimethylamino)-5,5-dimethyl-2-cyclohexen-1-one, 1-morpholinocyclopentene, and 1-morpholinocyclohexene to give various inverse electron-demand Diels-Alder adducts. Several adducts derived from 8-N,N-dimethylaminomethyl-substituted isoflavonoids displayed good activity in the 1-10 µm concentration range in an in vitro proliferation assay using the PC-3 prostate cancer cell line.

Synthesis of a norcantharidin-tethered guanosine: Protein phosphatase-1 inhibitors that change alternative splicing.

Phosphorylation and dephosphorylation of splicing factors play a key role in pre-mRNA splicing events, and cantharidin and norcantharidin analogs inhibit protein phosphatase-1 (PP1) and change alternative pre-mRNA splicing. Targeted inhibitors capable of selectively inhibiting PP-1 could promote exon 7 inclusion in the survival-of-motorneuron-2 gene (SMN2) and shift the proportion of SMN2 protein from a dysfunctional to a functional form. As a prelude to the development of norcantharidin-tethered oligonucleotide inhibitors, the synthesis a norcantharidin-tethered guanosine was developed in which a suitable tether prevented the undesired cyclization of norcantharidin monoamides to imides and possessed a secondary amine terminus suited to the synthesis of oligonucleotides analogs. Application of this methodology led to the synthesis of a diastereomeric mixture of norcantharidin-tethered guanosines, namely bisammonium (1R,2S,3R,4S)- and (1S,2R,3S,4R)-3-((4-(2-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl)oxy)oxidophosphoryl)oxy)ethyl)-phenethyl)(methyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylate, which showed activity in an assay for SMN2 pre-mRNA splicing.

Par-4 secretion: stoichiometry of 3-arylquinoline binding to vimentin.

Advanced prostate tumors usually metastasize to the lung, bone, and other vital tissues and are resistant to conventional therapy. Prostate apoptosis response-4 protein (Par-4) is a tumor suppressor that causes apoptosis in therapy-resistant prostate cancer cells by binding specifically to a receptor, Glucose-regulated protein-78 (GRP78), found only on the surface of cancer cells. 3-Arylquinolines or "arylquins" induce normal cells to release Par-4 from the intermediate filament protein, vimentin and promote Par-4 secretion that targets cancer cells in a paracrine manner. A structure-activity study identified arylquins that promote Par-4 secretion, and an evaluation of arylquin binding to the hERG potassium ion channel using a [(3)H]-dofetilide binding assay permitted the identification of structural features that separated this undesired activity from the desired Par-4 secretory activity. A binding study that relied on the natural fluorescence of arylquins and that used the purified rod domain of vimentin (residues 99-411) suggested that the mechanism behind Par-4 release involved arylquin binding to multiple sites in the rod domain.

Application of Mannich bases to the synthesis of hydroxymethylated isoflavonoids as potential antineoplastic agents.

The regiospecific Mannich aminomethylation of 7-hydroxyisoflavonoids using bis(N,N-dimethylamino)methane afforded C-8 substituted N,N-dimethylaminomethyl adducts, and the regioselective aminomethylation of 5-hydroxy-7-methoxyisoflavonoids afforded predominantly the C-6 substituted N,N-dimethylaminomethyl adducts. Acetylation of these C-6 or C-8 Mannich bases with potassium acetate in acetic anhydride provided access to the corresponding acetoxymethyl derivatives that were subsequently converted to hydroxymethyl- and methoxymethyl-substituted 5-hydroxy- or 7-hydroxyisoflavonoids related to naturally occurring flavonoids. The C-8 acetoxymethyl, hydroxymethyl or methoxymethyl-substituted isoflavonoids possessed promising inhibitory potency in the low micromolar range in a prostate cancer PC-3 cell proliferation assay.

N-Aryl benzenesulfonamide inhibitors of [3H]-thymidine incorporation and ß-catenin signaling in human hepatocyte-derived Huh-7 carcinoma cells.

Structure-activity relationships (SAR) in 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535) were examined as part of a program to identify agents that inhibit the Wnt/ß-catenin signaling pathway that is frequently upregulated in hepatocellular carcinoma (HCC). FH535 was reported as an inhibitor of both ß-catenin in the Wnt signaling pathway and the peroxisome proliferator-activated receptor (PPAR). A ß-catenin/T-cell factor (TCF)/Lymphoid-enhancer factor (LEF)-dependent assay (i.e., luciferase-based TOPFlash assay) as well as a [(3)H]-thymidine incorporation assay were used to explore SAR modifications of FH535. Although replacing the 2,5-dichlorophenylsulfonyl substituent in FH535 with a 2,6-dihalogenation pattern generally produced more biologically active analogs than FH535, other SAR modifications led only to FH535 analogs with comparable or slightly improved activity in these two assays. The absence of a clear SAR pattern in activity suggested a multiplicity of target effectors for N-aryl benzenesulfonamides.