4-(3-Phenyl-4-(3,4,5-trimethoxybenzoyl)-1 H-pyrrol-1-yl)benzenesulfonamide, a Novel Carbonic Anhydrase and Wnt/β-Catenin Signaling Pathway Dual-Targeting Inhibitor with Potent Activity against Multidrug Resistant Cancer Cells

Abstract

We synthesized new pyrrole and indole derivatives as human carbonic anhydrase (hCA) inhibitors with the potential to inhibit the Wnt/β-catenin signaling pathway. The presence of both N1-(4-sulfonamidophenyl) and 3-(3,4,5-trimethoxyphenyl) substituents was essential for strong hCA inhibitors. The most potent hCA XII inhibitor 15 (K_i = 6.8 nM) suppressed the Wnt/β-catenin signaling pathway and its target genes MYC, Fgf20, and Sall4 and exhibited the typical markers of apoptosis, cleaved poly(ADP-ribose)polymerase, and cleaved caspase-3. Compound 15 showed strong inhibition of viability in a panel of cancer cells, including colorectal cancer and triple-negative breast cancer cells, was effective against the NCI/ADR-RES DOX-resistant cell line, and restored the sensitivity to doxorubicin (DOX) in HT29/DX and MDCK/P-gp cells. Compound 15 is a novel dual-targeting compound with activity against hCA and Wnt/β-catenin. It thus has a broad targeting spectrum and is an anticancer agent with specific potential in P-glycoprotein overexpressing cell lines.

Figure 1

Structures 1–21. Compounds: 1, CA inhibitor; 2–5, tubulin polymerization inhibitors; 6, acetazolamide; 7,8: Wnt/β-catenin modulators; 9: P-gp modulator; and 10–21: planned inhibitors (see Table 1 for R₁–R₃ substituents).

Figure 2

Structural elements that have been merged to achieve the multitarget activity of 15.

Scheme 1. Synthesis of Sulfomanides 10–13

(a) NaH, DMF, RT, 50 min, Ar, 90–98%; (b) CuI, Cs₂CO₃, 1,10-phenanthroline, 1,4-dioxane, MW, closed vessel, 210 °C, 200 W, 40 min, 17–61%; (c) TBAF, THF, reflux, 4 h, 70–90%.

Scheme 2. Synthesis of Sulfonamides 14-17 (Top Panel) and 18-21 (Bottom Panel)

(a) CuI, Cs₂CO₃, 1,10-phenanthroline, 1,4-dioxane, MW, closed vessel, 210 °C, 200 W, 40 min, 24–65%; (b) TBAF, THF, reflux, 4 h, 71–98%.

Figure 3

SAR summary of hCA I, hCA II, hCA IX, and hCA XII inhibition by compounds 10–21.

Figure 4

Proposed binding mode of derivatives 11 (orange), 15 (magenta), and 19 (cyan). Enzymes are shown as a colored cartoon: hCA I, light blue; hCA II, green; hCA IX, gray; and hCA XII, sand. The zinc atom is depicted as a green sphere; residues involved in interactions are shown as white sticks; H-bonds are depicted as yellow dot lines. For the sake of clarity, amino acid residue numbers refer to hCA II.

Figure 5

(A) Inhibition of MDA-MB 231 TNBC cell growth by compound 15. (B) Inhibition of MDA-MB 231 TNBC cell growth by reference compound SLC-0111. (C) Inhibition of BT-549 TNBC cell growth by compound 15. (D) Inhibition of BT-549 TNBC cell growth by reference compound SLC-0111.

Figure 6

Human primary T cells were treated with 15 at 30 μM or DMSO for 72 h and analyzed for annexin V by flow cytometry. The frequency of annexin V/propidium iodide double positive cells (top right, late phase apoptotic cells) is shown. Data from two different healthy donors (donor A and donor B) are shown.

Figure 7

Total and cleaved PARP levels and cleaved caspase-3 levels in HCT116 cells after a 72 h treatment with increasing concentrations of compound 15.

Figure 8

HEK-293 cells were transfected with luciferase-based vectors and treated with LiCl (50 mM) together with increasing concentrations of compound 15. Cells were harvested 24 h post-treatment and assayed for luciferase activity. Inhibition levels calculated as the luciferase/renilla ratio of the treated samples vs the luciferase/renilla ratio of the untreated (control) samples. Data are represented as the mean ± SD of three independent experiments, each performed in triplicate. *p < 0.05 and **p < 0.01, as determined by analysis of variance (ANOVA).

Figure 9

Left panel. HCT116 cells were treated with LiCl (50 mM) and compound 15 at the indicated concentrations for 24 h. β-Catenin and c-Myc levels were analyzed by Western blot. Actin was used as a loading control. Right panel. Fgf20 or Sall4 mRNA levels were measured by qPCR and normalized to the expression of β-actin mRNA and expressed as a fold change relative to the control sample. Results represent the mean ± SD of three independent experiments, each performed in triplicate. ***p < 0.001, as determined by the t-test.

Figure 10

P-gp ATPase activity, measured spectrophotometrically on the protein immune-purified from HT29/DX cells (A) or MDCK/P-gp (B) cells, treated 3 h without (“0”) or with compound 15 at 1, 10, or 100 nM. H. Data are means ± SD (n = 3). *p < 0.05 and ***p < 0.001: vs untreated (“0”) cells.

Figure 11

Viability of HT29 and HT29/DX (A), MDCK and MDCK/P-gp (B) cells, incubated with fresh medium (ctrl), 5 μM DOX (dox), alone or coincubated with 1, 10, or 100 nM of 15 for 72 h, measured with a spectrophotometric assay. Data are means ± SD (n = 4). ***p < 0.001: versus untreated HT29 or MDCK cells (“ctrl”); °p < 0.001: versus untreated HT29/DX or MDCK/P-gp cells (“ctrl”); and ^###p < 0.001: versus dox-treated HT29/DX or MDCK/P-gp cells.

Figure 12

Radar plot of drug-like properties of compound 15. The light green colored zone represents the suitable physicochemical space for oral bioavailability. −1 < Log P < 5; 150 < MW < 500; 20 Ų < tPSA < 140 Ų; −10 < Log Sw < 0; 0 < HB D + A < 10; and 0 < RotBonds < 10. The red line represents values for derivative 15.