4-(3-Phenyl-4-(3,4,5-trimethoxybenzoyl)-1H-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

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 (Ki = 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.


■ INTRODUCTION
Carbonic anhydrases (CAs, EC 4.2.1.1)are ubiquitous enzymes that catalyze the reversible hydration of carbon dioxide to produce monohydrogen carbonate and H + ions. 1 CAs fall into eight different classes, αto ι-CAs, but only the αclass is found in mammals.Several genetically distinct CA isoforms in the α-class family, including the human CA (hCA) isoforms, have been recognized. 2,3They are cytosolic proteins, mitochondrial matrix proteins, transmembrane proteins, or proteins linked by means of glycosylphosphatidylinositol tails to the plasma membrane.The CA isoforms play key roles in many physiological processes, such as pH homeostasis, electrolyte secretion, biosynthesis of several metabolites, signal transduction, cell differentiation and proliferation, and oncogenesis. 4,5ysregulated expression and/or abnormal activity of specific CA isoforms cause onset of several pathological conditions. 6he cytosolic CAs I and II are isoforms ubiquitously spread throughout the human body.The CA II plays a relevant physiological role and is found highly expressed not only in red blood cells, the gastrointestinal tract, the lungs, and the kidneys 7 but also in some types of cancer, such as urothelial carcinoma. 8Both isoforms are involved in glaucoma, 9 epilepsy, 10 and Parkinson's disease. 11The human-membraneassociated CA IX and CA XII enzymes catalyze the hydration of carbon dioxide in the extracellular space.Overexpression of 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 R 1 −R 3 substituents).both the CA IX and CA XII isoforms is triggered by the hypoxia-inducible factor 1 (HIF-1) in many types of cancer. 12A IX is expressed in breast carcinoma, colorectal cancer (CRC), glioblastoma, lung cancer, and cervical squamous cell carcinoma. 13High levels of CA XII have been detected in breast cancer, 14 nonsmall cell lung cancer, 15 cervical cancer 16 and gliomas, hemangioblastomas, and meningiomas. 17A spliced form of CA XII is expressed in brain tumors. 18Cystic fibrosis-like syndrome and hyponatremia have been associated with a mutation in CA XII. 19In CRC, the expression of the CA II and CA XII isoforms has been correlated with patient survival, in that higher expression indicated poorer prognosis, and this suggested their potential role as prognostic biomarkers. 20n our previous studies, we synthesized 1,1′-biphenylsulfonamides as potent inhibitors of the hCA isoforms, with nM K i values. 21,22The biphenylsulfonamide 1 showed strong inhibition of hCA isoforms XII and XIV.In this work, we designed new CA inhibitors by introducing a sulfonamide group at the N1-phenyl of 2−4, 23−25 whose primary activity was the inhibition of tubulin polymerization, to give compounds 10−21 (Figure 1 and Table 1).Compounds 11, 15, and 19 showed strong inhibition of hCA isoforms I, II, IX, and XII with low nM K I values (Table 1).These compounds did not significantly inhibit tubulin polymerization, although sulfonamide 5 was reported by Guo 26 to inhibit tubulin assembly.Compound 11 did not inhibit tubulin assembly at 20 μM, and 15 and 19 showed only partial inhibition at 20 μM (in this assay, combretastatin A-4 inhibited the tubulin assembly with an IC 50 of 0.75 μM) (Table S1, Supporting Information).Overexpression of CA II and β-catenin signaling in urothelial carcinoma was reported by Matsue. 27Although the mechanism was not fully elucidated, the authors hypothesized that CA II may induce epithelial−mesenchymal transition (EMT) upregulating Wnt/β-catenin signaling. 28eference drug acetazolamide 6 (AAZ) was demonstrated to suppress Wnt/β-catenin signaling. 29Sulfonamides 7 and 8 were reported by Fang to inhibit Wnt signaling by interfering with both β-catenin/Tcf4 and β-catenin/LEF1 in protein extracts from HCT116 cells. 30The CA XII isoform is required for optimal activity of the P-glycoprotein (P-gp).Increasing CA XII levels at the plasma membrane of P-gp positive cancer cells were found during the acquisition of chemoresistance. 31-gp-mediated chemoresistance is reversed by CA XII inhibitors. 32The 3,4,5-trimethoxybenzoyl moiety of compound 9 was required for modulating P-gp levels. 33ompound 15, the most potent hCA XII inhibitor within the series, merges key structural features present in compounds 1, 7, 8, and 9, namely, the 4′-benzoyl-(1,1′-biphenyl)-4sulfonamide of 1, the 4-ureidobenzenesulfonamide of 7 and 8, and the 3,4,5-trimethoxybenzoyl moiety of 15.A summary sketch of the elements that have been merged to achieve the multitarget activity of compound 15 is shown in Figure 2. We were intrigued by the ability of compound 15 to interfere with the Wnt/β-catenin pathway and to restore the drug sensitivity of P-gp-overexpressing cancer cells.Indeed, 15 inhibited effectively the Wnt/β-catenin pathway and its target gene MYC, and besides its activity in NCI/ADR-RES cells, restored the sensitivity to doxorubicin (DOX) in HT29/DX and MDCK/P-gp cells.In the HT29/DX model, 15 at 100 nM in combination with DOX decreased the cell viability obtained with DOX alone in these chemosensitive cells.
Molecular Modeling on hCA I, hCA II, hCA IX, and hCA XII Isoforms.Docking experiments were performed to gain insights into the molecular details of the binding modes of the reported compounds.Representative structures for each studied hCA isoform were selected from the protein data bank (PDB) server.The compounds were docked by Vina to the catalytic site of the enzymes.Inspection of the proposed docking binding conformations revealed the crucial interaction between the sulfonamide nitrogen atom and the catalytic zinc atom.The position para, meta, or ortho of the sulfonamide at the 1-phenyl ring markedly affected the binding interaction; the most active compounds all had the sulfonamide at the para position.This resulted in its contact involving the zinc atom of the enzyme and the sulfonamide moiety formed an H-bond between an oxygen atom and the Thr199 residue.The phenyl ring bearing the sulfonamide formed hydrophobic interactions with Leu198, a residue that was conserved in all the studied isoforms, and Val121 which became an Ala residue in hCA I.When the sulfonamide group was at the meta or ortho position, a steric clash resulted in attempting to achieve a proper bond distance from the zinc atom.
Inspection of the binding modes led to the identification of hydrophobic interactions of the central pyrrole or indole ring with Pro202 and Leu198, both of which are conserved in all of the studied isoforms.Other hydrophobic interactions involved residues Leu131 in hCA I, Phe131 in hCA II, Val131, and Ala131 in hCA IX and Ala131 hCA XII.The 3,4,5trimethoxyphenyl formed hydrophobic interactions with Phe131, Val135, and Leu204 in the case of hCA II and with Val131, Leu135, and Ala204 in the case of hCA IX.For hCA XII, along with the hydrophobic stabilization provided by the trimethoxyphenyl, an H-bond interaction between Ser132 and a methoxy group was observed.In the case of hCA I, the presence of the bulkier Tyr204 residue instead of Leu (hCA II), Ala (hCA IX), or Asn (hCA XII) forced the trimethoxyphenyl moiety into a different binding conformation that caused lower-quality binding.The binding modes provided by the docking experiments were consistent with the biological data and led to the identification of crucial structural requirements for inhibition of the enzyme (Figure 4).
Inhibition of Tubulin Polymerization and Docking in the Colchicine Site.At 20 μM, compound 11 did not inhibit tubulin assembly, whereas 15 and 19 resulted in partial inhibition.In the same assay, combretastatin A-4 (CSA4) as a reference compound inhibited the assembly reaction with an IC 50 of 0.75 μM and colchicine binding with 99% inhibition.For comparison, 2, the parent compound of 11, inhibited tubulin assembly with an IC 50 of 1.5 μM and colchicine binding by 81%; 23 3, the parent compound of 15, inhibited tubulin assembly with IC 50 of 1.1 μM and colchicine binding by 66%. 24Docking studies of derivatives 11, 15, and 19 at the colchicine site of the tubulin dimer highlighted that binding modes of 11 and 15 were consistent with those of the previously reported parent compounds 2 23 and 3, 24 respectively; however, the sulfonamide group had a negative effect on binding to the colchicine site.Inspection of the docking poses revealed that while the sulfonamide groups of 11, 15, and 19 could fit into a hydrophobic pocket, there were no stabilizing contacts.This may suggest that the desolvation energy cost was not balanced by the binding energy of the sulfonamide moiety. 39For all derivatives, we hypothesized that the binding fitness got worse upon the introduction of the sulfonamide group mainly due to electronic rather than steric reasons (Figure S1, Supporting Information).
Inhibition of Growth of HCT-116, SW480, and SW620 Cancer Cells.Compounds 11, 15, and 19 were evaluated as inhibitors of the growth of the HCT116, SW480, and SW620 colorectal carcinoma cells (Table 2 and Figure S2, Supporting Information).All cell lines are characterized by enhanced activity of the Wnt/β-catenin pathway due to the CTNNB1 mutated gene present in the HCT116 cells that encodes βcatenin 40 and mutation in the APC tumor suppressor gene in SW480 and SW620 cells. 41,42Compounds 15 and 19 inhibited the HCT116 cells with IC 50 's of 8.7 and 14.9 μM, respectively, compared with 5-fluorouracil (5-FU) (IC 50 = 8.2 μM).Both SW480 and SW620 cell lines were less sensitive to 15 and 19 than the HCT116 cells, with IC 50 values ranging from 17.7 μM (15, SW480 cells) to 45.4 μM (19, SW620 cells).However, it should be noted that 15 and 19 were remarkably more potent than 5-FU as inhibitors of both the SW480 and SW620 cell lines.As an inhibitor of the SW480 and SW620 cells, 5-FU yielded IC 50 's of 217.5 and 102.6 μM, respectively, and was 27and 13-fold less effective than that in the HCT116 cells.In drug combination studies, each compound was used at the corresponding IC 50 concentration.The combination 15 + 5-FU inhibited the HCT116 cells with an IC 50 of 17.0 μM, twofold higher than 15 as a single agent, while 19 + 5-FU was at the same level as 19 alone.As inhibitors of the SW480 and   -7), the ovarian tumor cell line 8 (OVCAR-8), and its derived NCI/ADR-RES DOXresistant cell line that overexpresses P-gp, resulting in the type 1 multidrug-resistance phenotype (Table 3).Compounds 11, 15, and 19 inhibited the MCF-7, OVCAR-8, and NCI/ADR-RES cancer cells with IC 50 values ranging from 1.9 μM (15, MCF-7 cells; 11 and 15, NCI/ADR-RES and OVCAR-8 cells) to 4.8 (19, OVCAR-8 cells).Compound 15 was almost uniformly active against these three cell lines in the 1.9−2.1 μM range.Noteworthy, compounds 1, 12, and 19 were equipotent as inhibitors of the NCI/ADR-RES DOX-resistant cell line (EC 50 's of 2.1−2.2μM), uniformly more active than the tubulin assembly inhibitor vinorelbine (IC 50 = 5.0 ± 1.0 μM) and slightly less potent than microtubule-stabilizing agent paclitaxel (IC 50 = 1.5 ± 0.7 μM). 43nhibition of TNBC Cancer Cells.Chemotherapy is still the standard of care for triple-negative breast cancer (TNBC).Poor prognosis is often ascribed to the emergence of drug resistance to therapeutic agents.In order to evaluate the biological effect of CA inhibitors in TNBC cells, we used MDA-MB 231 and BT-549 cells, which are representative of highly aggressive TNBC subtypes.As shown in Figure 5, Panel A, treatment with compound 15 exerted a potent antiproliferative effect on MDA-MB 231 cells.The effect of 15 was significantly higher as compared with the reference compound SLC-0111, an ureido-substituted benzenesulfonamide small molecule inhibitor of CA IX in Phase 1 trials in patients with advanced solid tumors 44  Human Primary T Lymphocytes.Potential toxic effects of compound 15 on healthy cells were evaluated by treating human primary T lymphocytes with 15 at 30 μM or with dimethyl sulfoxide (DMSO) as a control vehicle.After 72 h, the frequency of late apoptotic cells was assayed by staining with annexin V and propidium iodide.Flow cytometry analysis showed that compared to the control vehicle, treatment with 30 μM 15 caused very low-level toxicity on healthy/normal cells with only a 6−8% increase in cell death (Figure 6).Inhibition of the Wnt/β-Catenin Signaling Pathway by 15.We wondered whether the observed inhibitory effect of compound 15 on CRC cell viability could be attributed to its ability to affect Wnt signaling.To this end, we first transfected human embryonic kidney (HEK) 293T cells with a reporter vector (M50 Super 8x TOPFlash) containing eight repeats of TCF/LEF-binding sites, or its negative control containing the mutated TCF/LEF binding sites (M51 Super 8x FOPFlash).After transfection, cells were treated with the GSK3 inhibitor LiCl to activate the Wnt pathway and incubated with increasing concentrations of 15.As shown in Figure 8, the compound caused a significant and dose-dependent inhibition of TOP reporter activity but did not significantly prevent the activity of the FOP reporter, indicating the specificity of the effect.
Treatment of HCT116 CRC cells with increasing concentrations of 15 resulted in a marked reduction of β-catenin and of its target gene MYC (Figure 9, left panel).Furthermore, the compound significantly reduced the expression of two additional Wnt target genes, 47 Ffg20 and Sal4 (Figure 9, right panel).Together, these data support the hypothesis that 15 exerts its inhibitory effect on the CRC cell viability and growth by preventing the activation of Wnt/β-catenin signaling.
Inhibition of P-gp by 15.To test the efficacy of compound 15, we chose the DOX-sensitive colon cancer HT29 cells, a cell line expressing low levels of P-gp, and its resistant counterpart, the cell line HT29/DX, selected stepwise in media with increasing concentrations of DOX resulting in expression of a high level of P-gp. 48As a cancer model, the HT29 and HT29/ DX pair has been extensively used and characterized for DOX resistance and pharmacological efficacy by our group, 49,50 and the cell line pair has important translational potential.However, HT29/DX cells also express other ABC transporters   including MRP1, MRP2, MRP3, MRP5, and BCRP. 48Hence, to better elucidate the effect that the compound under study here has on P-gp, we also included the canine kidney MDCK cell line, devoid of any transporter, and the MDCK/P-gp cell line, overexpressing human P-gp only, 51 as an internal control.In a preliminary experiment, we measured the cytotoxicity of compound 15 alone.Compound 15 reduced cell viability in the four cell lines, suggesting that there was not a celldependent effect or a different behavior between DOXsensitive and resistant cell lines.The viability was >75% with 1−100 nM 15 (Figure S5, Supporting Information).We worked at 1, 10, and 100 nM concentrations in the subsequent experiments to avoid any bias related to the intrinsic cytotoxicity induced by the compound.
To evaluate the impact as chemosensitizer agents, we first measured the intracellular accumulation of DOX, a typical substrate of P-gp. 52As expected, HT29 had a basally higher accumulation of the drug compared to the resistant counterpart HT29/DX cells.Similarly, the intracellular amount of DOX in MDCK cells was significantly higher than that in MDCK/P-gp cells (Figure S6, Supporting Information).Compound 15 had no effects on the amount of DOX retained within HT29 cells, nor in MDCK/P-gp cells, where 15 did not change the intracellular amount of the drug.DOX was basally higher in MDCK/P-gp cells than that in HT29 cells: this difference can be explained because HT29 cells also have other ABC transporters (as MRP1) that, although expressed at low levels, can efflux DOX, while MDCK cells lack other transporters.Differently from what we observed in sensitive cells, 15 induced a strong dose-dependent increase in the DOX accumulation in HT29/DX cells, reaching the same level of intracellular drug detected in sensitive HT29 cells when used at 100 nM.This stronger increase in resistant cells is likely due to their higher levels of the ABC transporters that are the putative targets of compound 15.Indeed, the higher the level of the transporters, the higher the inhibition achieved by the compounds and greater the increase on DOX retention.Part of the effect of 15 is mediated by the inhibition of P-gp, as   indicated by its efficacy in increasing DOX accumulation in MDCK/P-gp cells, where no other transporters than P-gp are present.In this model, also at the highest concentration (100 nM), the compound was not able to fully restore the accumulation of the drug to the intracellular level of MDCK cells, which were devoid of P-gp.This result can be explained at least by following reasons: (1) the higher catalytic efficiency of the protein in MDCK/P-gp and HT29/DX cells (Figure 10) and ( 2) the possible targeting of other ABC transporters, present in HT29/DX cells but not in MDCK/P-gp cells.
In determining DOX resistance, 52 we next investigated if 15 was able to inhibit the catalytic cycle of this transporter.We immunopurified the protein from HT29/DX and MDCK/P-gp cell lines, and we measured the rate of ATP hydrolysis, a step necessary to efflux DOX and considered an index of P-gp activity (Table S2, Supporting Information). 53As shown in Figure 10, P-gp extracted from MDCK/P-gp cells was more active than the protein extracted from the HT29/DX cells.However, in both cell lines, compound 15 reduced P-gp activity, starting at 10 nM concentration.This result suggests that the compound acts as P-gp inhibitors.
Finally, we tested the potential of compound 15 to reverse resistance to DOX in terms of cytotoxicity, by measuring cell viability in cells coincubated with both the compound and 5 μM DOX, a concentration already used to discriminate sensitive and resistant cells. 54Also, in our experimental setting, DOX reduced the viability of sensitive HT29 and MDCK cells below 30%, but it did not affect the viability in resistant HT29/ DX and MDCK/P-gp cells (Figure 11).In HT29 and MDCK cells, 15 did not further reduce cell viability compared with DOX alone, consistent with the absence of a significant increase in intracellular retention of the drug.By contrast, in HT29/DX and MDCK/P-gp cells, compound 15 decreased cell viability in a dose-dependent manner, restoring the sensitivity to DOX.Particularly, in the HT29/DX model, the combination of DOX plus 15 at 100 nM produced the same decrease in cell viability achieved by DOX alone in the respective chemosensitive cells, confirming that this setting fully overcame the resistance to DOX.
Drug-like Properties of Compound 15.Representative drug-like properties of compound 15 were calculated on the SwissADME website.There were no violations of the Lipinski 55 and Veber 56 rules; compound 15 was predicted to have good bioavailability after oral administration (Table 4 and Figure 12).

■ CONCLUSIONS
We synthesized new pyrrole and indole CA inhibitors 10−21 by modulating the scaffold of previously reported tubulin polymerization inhibitors 2−4. 21,22    In summary, we describe the synthesis of new pyrrole and indole derivatives as hCA inhibitors with K i values in the nanomolar range.Compound 15, the most potent hCA XII inhibitor with K i = 6.8 nM, caused significant suppression of the Wnt/β-catenin signaling pathway and induced apoptotic cell death in HCT116 cells.Compound 15 restored the sensitivity to DOX in HT29/DX and MDCK/P-gp cells.As a cell growth inhibitor, 15 was equipotent to 5-FU in HCT116 cells and remarkably more potent in the SW480 and SW620 cell lines and inhibited the NCI/ADR-RES DOX-resistant cell line.Against the MDA-MB 231 and BT-549 TNBC cells, 15 was superior to reference compound SLC-0111.Together, these results highlight compound 15 as a novel hCA/β-catenin pathway dual-targeting broad spectrum anticancer agent with a specific potential to restore sensitivity to P-gp expressing cell lines.These findings highlight 15 as a lead compound of a novel class of dual-targeting broad spectrum anticancer agents and will prompt additional studies to explore its potential for the treatment of cancer.
■ EXPERIMENTAL SECTION Chemistry.All reagents and solvents were handled according to the material safety data sheet of the supplier and used as purchased without further purification.Organic solutions were dried over anhydrous sodium sulfate.Evaporation of solvents was carried out on a Buchi Rotavapor R-210 equipped with a Buchi V-850 vacuum controller and a Buchi V-700 vacuum pump.Column chromatography was performed on columns packed with silica gel from the Macherey−Nagel (70−230 mesh).Silica gel thin-layer chromatography (TLC) cards from Macherey-Nagel (silica gel-precoated aluminum cards with a fluorescent indicator visualizable at 254 nm) were used for TLC.Developed plates were visualized with a Spectroline ENF 260C/FE UV apparatus.Melting points (mp) were determined on a Stuart Scientific SMP1 apparatus and are uncorrected.Infrared (IR) spectra were recorded on a PerkinElmer Spectrum 100 FT-IR spectrophotometer equipped with a universal attenuated total reflectance accessory, and IR data were acquired and processed by PerkinElmer Spectrum 10.03.00.0069 software.Band position and absorption ranges are given in cm −1 .Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded with a Bruker Avance (400 MHz) spectrometer in the indicated solvent, and the corresponding fid files were processed with MestreLab Research SL MestreReNova 6.2.1−769 software.Carbon-13 nuclear magnetic resonance ( 13 C NMR) spectra were recorded with a Bruker AVANCE (100 MHz) spectrometer in the indicated solvent, and the corresponding FID files were processed by MestreLab Research SL MestreReNova 6.2.1−769 software.Chemical shifts of 1 H and 13 C NMR are expressed in δ units (ppm) from tetramethylsilane.
Compound purity was checked by high-pressure liquid chromatography (HPLC).Purity of tested compounds was found to be >95%.The HPLC system used (Thermo Fisher Scientific Inc. Dionex UltiMate 3000) consisted of an SR-3000 solvent rack, an LPG-3400SD quaternary analytical pump, a TCC-3000SD column compartment, a DAD-3000 diode array detector, and an analytical manual injection valve with a 20 μL loop.Samples were dissolved in acetonitrile (1 mg/mL).HPLC analysis was performed by using a Thermo Fisher Scientific Inc. Acclaim 120 C18 column (5 μm, 4.6 mm × 250 mm), at 25 ± 1 °C with an appropriate solvent gradient (acetonitrile/water), flow rate of 1.0 mL/min, and signal detector at 206, 230, 254, and 365 nm were used.Chromatographic data were acquired and processed by Thermo Fisher Scientific Inc. Chromeleon 6.80 SR15 Build 4656 software.UHPLC analysis was carried out on an Accela System Thermo Fisher Scientific (San Jose, CA) which consisted of an Accela 1250 Pump, an Accela autosampler, and an Accela PDA photodiode array detector.Chromatographic data were collected and processed using Thermo Xcalibur Chromatography Manager software, version 1.0.A guard cartridge system (Secur-ityGuard Ultra UHPLC) has been connected to an analytical column Kinetex 2.6 μm EVO C18 100 Å 100 × 3.0 mm (L.x I.D.), both from Phenomenex, Torrance, CA, USA.All analyses were performed at 30 °C, and the mobile phase was filtered through 0.2 μm Omnipore filters (Merck Millipore, Darmstadt, Germany).The mobile phase was delivered at a total flow rate of 0.6 mL/min.The analyses were carried out in the elution gradient.Specific mobile phase and gradient are reported for each compound in captions.Each analysis was performed in triplicate (Figures S7−S9 and Table S3, Supporting Information).Materials: Acetonitrile (HPLC gradient grade), methanol (HPLC gradient grade), water (HPLC gradient grade), and trifluoroacetic acid (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO).
General Procedure for the Preparation of Compounds 10−21.

4-(3-Phenyl-4-(3,4,5-trimethoxybenzoyl)-1H-pyrrol-1-yl)-N,N-bis-((2-(trimethylsilyl)ethoxy
)methyl)benzenesulfonamide. (34).It was synthesized as 27 starting from 32 and 24.Yield 65%, oil. 1   (35).It was synthesized as 27 starting from 32 and 25.Yield 61% as an oil. 1   (39).It was synthesized as 27 starting from 37 and 24.Yield 17% as an oil. 1   (40).It was synthesized as 27 starting from 38 and 24.Yield 40% as an oil. 1   (41).It was synthesized as 27 starting from 38 and 24.Yield 39% as an oil. 1 H NMR (DMSO-d 6 , 400 MHz): δ 0.14 (s, 18H), 0.65 (s, 4H), 3.76 (s, 3H), 3.87 (s, 6H), 4.78 (s, 4H), 7.22 (s, 2H), 7.39 (s, 2H), 7.55 (s, 1H), 7.83−7.85(m, 1H), 7.96−8.05(m, 2H), 8.15 (s, 1H), 8.35 (s, 2H) ppm.IR: 1452 and 2892 cm −1 .(42).It was synthesized as 27 starting from 38 and 26.Yield 61% as an oil. 1  General Procedure for the Preparation of Compounds 24−26.Example: 4-Bromo-N,N-bis((2-(trimethylsilyl)ethoxy)methyl)benzenesulfonamide (24).4-Bromobenzenesulfonamide (1.0 g, 4.24 mmol) in DMF (12 mL) was added dropwise to a suspension of sodium hydride (60% in mineral oil, 408 mg, 9.32 mmol) at 0 °C in DMF (12 mL) under an argon stream.The mixture was allowed to stir at room temperature for 20 min.(2-(Chloromethoxy)ethyl)trimethylsilane (1,47 g, 8.8 mmol) was added dropwise at 0 °C, and the mixture was stirred at room temperature for 30 min.The reaction was quenched on crushed ice and extracted with ethyl acetate.The organic layer was washed with brine, dried on anhydrous sodium sulfate, and filtered.Removal of the solvent gave pure 24 (2.0 g) that was used without further purification.Yield 98% as an oil. 1  3-Bromo-N,N-bis( (2-(Trimethylsilyl)ethoxy)methyl)benzenesulfonamide (25).It was synthesized as 24 starting from 3bromobenzenesulfonamide.Yield 97% as an oil. 1 57 Hydrogen atoms, missing side chains, and missing loops were added; unnecessary side chains, water molecules, and other solvents were removed.The 3D structures of the sulfonamide compounds were protonated at physiological pH and minimized using the OPLS4 force field included in the same software. 58AutoDock Vina 1.2.3 59 was selected as a docking tool owing to its capacity of utilizing the specific zinc-coordination potentials included in the AutoDock4Zn force field. 60All prepared proteins were aligned to a chosen reference structure (hCA II, in our case, was used also for numeration of the amino acid residues) using UCSF Chimera, 61 and the docking center was selected by calculating the geometric center of its cocrystallized ligand.AutoDockTools 1.5.7 was used to visually adjust the grid box sizes to the hCA active site.Docking simulations were run using the AutoDock4 62 scoring function with an exhaustiveness of 32.Prepared proteins and ligands structures were converted to the PDBQT format using OpenBabel 3.0.1 63,64 while the grid maps generation and the docking process itself were performed in a batch fashion using a custom Python 2.7.3 script.The docking at the colchicine site of tubulin was performed using 3HKD 65 for derivative 11 and the 1SA0 66 structure for derivative 15 and 19.Proteins were prepared as described for the hCAs.Docking computations were carried out with Plants 67 using the default setting and a binding site of 12 Å.Reported images were generated by PyMol. 68iology.CA Inhibition Screening Assay.An Applied Photophysics stopped-flow instrument was used for assaying the CAcatalyzed CO 2 hydration activity. 37Phenol red (at a concentration of 0.2 mM) was used as an indicator, working at the maximum absorbance of 557 nm, with 20 mM Hepes (pH 7.5) as buffer and 20 mM sodium sulfate (for maintaining constant ionic strength).The initial rates of the CA-catalyzed CO 2 hydration reaction were followed for a period of 10−100 s.The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants.For each inhibitor, at least six traces of the initial 5−10% of the reaction were used for determining the initial velocity.The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates.Stock solutions of the inhibitor (0.1 mM) were prepared in distilled−deionized water, and dilutions up to 0.01 nM were carried out thereafter with distilled−deionized water.Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay to allow for the formation of the enzyme−inhibitor complex.The inhibition constants were obtained by nonlinear least-squares methods using the Cheng− Prusoff equation and represent the mean from at least three different determinations.Standard deviations were in the range of ±5−10% of the reported K i values.−71 The enzyme concentrations in the assay system were as follows: hCA I, 13.2 nM; hCA II, 8.4 nM; hCA IX, 7.9 nM; and hCA XII, 15.2 nM.

2-(3-(3,4,5-Trimethoxybenzoyl)-1H-indol-1-yl)-N,N-bis((2-(trimethylsilyl)ethoxy)methyl)benzenesulfonamide
Tubulin Assembly.The reaction mixtures contained 0.8 M monosodium glutamate (pH 6.6 with HCl in a 2 M stock solution), 10 μM tubulin, 4% (v/v) DMSO, and varying concentrations of compound.Following a 15 min preincubation at 30 °C, samples were chilled on ice, GTP to 0.4 mM was added, and turbidity development was followed at 350 nm in a temperature-controlled recording spectrophotometer for 20 min at 30 °C.The extent of the reaction was measured.Full experimental details were previously described. 72 3 H]Colchicine-Binding Assay.The reaction mixtures contained 1.0 μM tubulin, 5.0 μM [ 3 H]colchicine, and 5.0 μM inhibitor and were incubated for 10 min at 37 °C.Complete details were described previously. 73ell Cultures and Cell Viability Assay.Cell lines were obtained from the American Tissue Culture Collection (ATCC), unless specified otherwise.Cells were grown in Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO 2 .In all experiments, 300,000 cells were plated in 9 cm 2 dishes and exposed to test compound dissolved in DMSO (0.1% final concentration) at the indicated concentrations.The methodology for the evaluation of the growth of human MCF-7 breast carcinoma, OVCAR-8, and NCI/ADR-RES cells, obtained from the National Cancer Institute drug screening laboratory, was previously described.73 For IC 50 determinations, OVCAR-8 and NCI/ ADR-RES cells were grown in RPMI 1640 medium with 5% FBS, 5% CO 2 atmosphere at 37 °C, for 96 h.HCT116, SW480, and SW620 cells were grown in DMEM supplemented with 10% FBS and Pen/ Strep (15,070−063, GIBCO, Thermo FisherScientific,Waltham, MA, USA).Cell viability was evaluated by sodium (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) 74 (HCT116 cells) or 3′-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6nitro)benzene-sulfonic acid hydrate (XTT) 75 (SW480 and SW620 cells).Briefly, cells (range 10−30 × 10 3 cells/well) were seeded in 96well microculture plates and then exposed to increasing concentrations of different compounds (range 0−300 μM) for 48 or 72 h.At the end of the treatment, media were removed and incubated at 37 °C in the dark for 4 h in phosphate-buffered saline (PBS) containing 0.2 mg/mL MTT or XTT and PMS (phenazine methosulfate) at a final concentration of 25 μM.Absorbance at 450 nm along with the reference wavelength at 650 nm was measured using a microplate spectrophotometer (Multiskan FC Microplate Photometer, Thermo Scientific, Waltham, MA, USA).The cell growth inhibition rate was calculated utilizing the following formula: inhibition rate (%) = [control OD − (sample OD/control OD)] × 100, where control OD is the absorbance of a negative control, and sample OD is the absorbance of the test sample.The IC 50 values were determined with GraphPad Prism 5 through constructed dose−response curves.Human MDA-MB-231 TNBC cells (ATCC HTB-26) were grown in DMEM supplemented with 10% FCS, while TNBC cells BT-549 (ATCC HTB-122) were grown in RPMI plus 10% FBS and 1 μg/mL bovine insulin.Cells were kept at low passage, returning to the original frozen stocks every 3−4 months.Hypoxic culture conditions were realized in the presence of 1% O 2 and 5% CO 2 .The different cell lines were seeded 10,000 cells/well in 48-well plates and treated in 1% FBS with increasing concentrations of compound 15 or reference SLC-0111.After 72 h of incubation at 37 °C with 1% O 2 and 5% CO 2 , cells were trypsinized, and cell counting was performed with the MACSQuant analyzer (Miltenyi Biotec).76 Potential toxicity on healthy cells was evaluated by treating human primary T lymphocytes from two healthy donors with 30 μM 15 or with a control vehicle (DMSO).Healthy donors' peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Nycomed) gradient centrifugation. Tlymphocytes were negatively selected from PBMCs using magnetic Dynabeads Untouched Human T Cells Kit (Thermo Fisher Scientific) following the manufacturer's instructions.Apoptotic cell death was evaluated using APC Annexin-V Apoptosis Detection Kit with PI (Thermo Fisher Scientific).Briefly, 1.5 × 10 6 / mL T cells were cultured in 48-well plates, untreated or treated with 15 at 30 μM for 72 h.Cells were then stained using annexin-V/APC and propidium iodide according to the manufacturer's instruction.Cell populations were acquired using a FACS Canto II flow cytometer (BD Biosciences). Flw cytometric analysis was performed using Flow Jo Flow Cytometric Analysis Software.Human chemosensitive HT29 colon cancer cells were purchased from ATCC (Manassas, VA).These cells were cultured in RPMI 1640 medium supplemented with 10% v/v FBS, 1% v/v penicillin−streptomycin, and 1% v/v L- glutamine.Human HT29/DX cells were generated by stepwise selection in medium with an increasing concentration of DOX, as described previously, 53 and maintained in culture medium with a final concentration of 200 nM DOX.MDCK and MDCK/P-gp (a gift of Prof. P. Borst, NKI-AVL Institute, Amsterdam, The Netherlands) were grown in DMEM high glucose supplemented with 10% FBS, 1% penicillin, 1% v/v penicillin−streptomycin, and 1% v/v glutamine.All cell lines were authenticated by microsatellite analysis using the PowerPlex kit (Promega Corporation, Madison, WI; last authentication: January 2022).Cells were seeded in 96-well plates.In the first experimental set, cells were incubated for 72 h with DMSO as a solvent or compound 15 at the following concentrations: 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM.In the second experimental set, cells were incubated for 72 h with 5 μM DOX alone or with 15 at 1, 10, or 100 nM.Cell viability was evaluated using the WST-1 assay (Sigma-Merck), as per the manufacturer's instructions, using a Packard EL340 microplate reader (Bio-Tek Instruments, Winooski, VT).The absorbance units of the untreated cells were considered 100%; the absorbance units of the other experimental conditions were expressed as a percentage versus the absorbance units of the untreated cells.
Wnt Reporter Assay.Wnt reporter assay was performed as previously described. 77HEK293T cells were seeded at 2 × 10 4 cells/ cm 2 in triplicate in a 24-well plate.The following day, cells were transfected using DreamFect Gold (OZ Biosciences #DG80500) according to the manufacturer's instructions.25 ng of the TOP reporter vector (M50 Super 8x TOPFlash Addgene #12456) or 25 ng of the FOP control plasmid (M51 Super 8x FOPFlash Addgene #12457) was transfected in each well in combination with 10 ng of TK Renilla (Promega #E2241) and empty vector (pcDNA3.1) up to 100 ng.After 24 h, cells were incubated with starvation media (Opti-MEM reduced serum medium supplemented with 0.5% FBS, 1% penicillin/streptomycin, 1% sodium pyruvate, and 1% nonessential amino acids) for 8 h.After starvation, Wnt/β-catenin signaling was activated by treating cells with lithium chloride (50 mM) in a starvation medium for 24 h.The following day, cells were treated with increasing concentrations of compound 15 or vehicle (DMSO) for 24 h, and, at the end of the experiment, cells were lysed using Passive Lysis Buffer (Biotium #99912).D-Luciferine (#10101 Biotium) was diluted in the noncommercial luciferase buffer 78 at a final concentration of 40 μg/mL.Coelenterazine (#S053 Synchem) was diluted in PBS with Ca 2+ and Mg 2+ at a final concentration of 0.75 μg/ mL.Luminescence was measured using the GloMax Discover Microplate Reader (Promega).

Figure 2 .
Figure 2. Structural elements that have been merged to achieve the multitarget activity of 15.Scheme 1. Synthesis of Sulfomanides 10−13 a

Figure 4 .
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 6 .
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 .
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 .
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 posttreatment 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 .
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.Fgf 20 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.
Derivatives bearing the sulfonamide at the para position of the N1-phenyl ring showed strong inhibition of the hCA isoforms I, II, IX, and XII with K i values at nanomolar concentrations.Compounds 11, 15, and 19 characterized by the presence of both sulfonamidophenyl and 3,4,5-trimethoxyphenyl substituents at positions 1 and 3, respectively, of the heterocycle were the strongest hCA inhibitors within the series.In particular, 11 inhibited the hCA II and hCA IX isoforms with K i values of 10.2 and 11.5 nM, respectively; 15 inhibited the hCA XII with K i of 6.8 nM; and 19 inhibited the hCA IX with K i of 7.3 nM.Conversely, 11, 15, and 19 had limited ability to inhibit tubulin polymerization: at 20 μM, 15 and 19 produced only partial inhibition of tubulin assembly, while CSA4 inhibited assembly by over 50% at less than 1 μM.The docking poses of compounds 11, 15, and 19 in the binding sites of the four hCA isoforms were consistent with the observed K i values.On the other hand, the docking poses of 11, 15, and 19 into the colchicine site of tubulin revealed that the sulfonamide group did not form stabilizing contacts, suggesting that the desolvation energy cost was not balanced by the binding energy.As inhibitors of the HCT116 cells, compounds 15 and 19 were worthy of comparison with 5-FU, and they were remarkably more potent than 5-FU as inhibitors of the SW480 and SW620 cell lines.Compounds 11, 15, and 19 inhibited the MCF-7 and OVCAR-8 cell lines with IC 50 values in the single digit micromolar range and, notably, were almost equipotent against the NCI/ADR-RES DOX-resistant cell line.Compound 15 potently inhibited MDA-MB 231 and BT-549 TNBC cells and was superior to the reference compound SLC-0111.

Table 1 .
Inhibition Data of Human CA Isoforms I, II, IX, and XII by Compounds 10−21 and Reference Compounds 1 and AAZ c a Mean from three different assays, by a stopped flow technique.b Standard deviations were in the range of ±5−10% of the reported K i values.c No data.d AAZ, acetazolamide.

Table 2 .
Inhibition of Growth of HCT-116, SW480, and SW620 Cancer Cells by Compounds 15 and 19 a

Table 3 .
Inhibition of Growth of MCF-7, OVCAR-8, and NCI/ADR-RES Cell Lines by Compounds 11, 15, and 19 a a Experiments were performed in duplicate or triplicate.b MCF-7 human nonmetastatic breast cancer epithelial cells.c In this assay, CSA4 as a reference compound yielded IC 50 of 15 ± 4 nM.d OVCAR-8: ovarian tumor cell line 8. e NCI/ADR-RES: DOX-resistant cell line derived from OVCAR-8.

■ ASSOCIATED CONTENT * sı Supporting Information The
Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c01424.Inhibition of tubulin polymerization (IC 50 values) by 11, 15, and 19 and references CSA4, 2, and 3: proposed binding mode for derivatives 11, 15, and 19 into the colchicine site on tubulin; effects of 15 on HCT116 cell viability in MTT assay and IC 50 value determined by nonlinear regression; total and cleaved PARP and Caspase-3 levels in HCT116 cells upon 72 h treatment with 15; dose-dependent viability and intracellular DOX accumulation of 15-treated HT29 and HT29/DX, MDCK, and MDCK/P-gp cells; rate of ATP hydrolysis in HT29/DX and MDCK/P-gp cells upon treatment with increasing concentrations of 15; and UHPLC chromatograms of compounds 11, 15, and 19 and relative area (%) recorded at 254 nm (PDF) List of molecular formula string spread sheet for all tested compounds and associated biological data (CSV) Predicted binding mode of 11, 15, and 19 in hCA I Laboratory Affiliated to Istituto Pasteur Italia�Fondazione Cenci Bolognetti, Department of Molecular Medicine, Sapienza University of Rome, Rome 00161, Italy; Email: gianluca.canettieri@uniroma1.it Romano Silvestri − Laboratory Affiliated with the Institute Pasteur Italy�Cenci Bolognetti Foundation, Department of Drug Chemistry and Technologies, Sapienza University of Rome, Roma 00185, Italy; orcid.org/0000-0003-2489-0178;Email: romano.silvestri@uniroma1.it (PDB ID: 7Q0D) (PDB) hCA II (PDB ID: 5E2R) (PDB)