Asymmetric Dibenzothiophene Sulfones as Fluorescent Nuclear Stains

Oct 10, 2018 - Asymmetric dibenzothiophene S,S-dioxides (DBTOOs) were synthesized and their photophysical properties examined. Through examination ...
0 downloads 0 Views 4MB Size
Download PDF
Note pubs.acs.org/joc

Cite This: J. Org. Chem. XXXX, XXX, XXX−XXX

Asymmetric Dibenzothiophene Sulfones as Fluorescent Nuclear Stains John T. Petroff II, Kristin N. Skubic, Christopher K. Arnatt, and Ryan D. McCulla* Department of Chemistry, Saint Louis University, 3501 Laclede Avenue, St. Louis, Missouri 63108, United States

Downloaded via UNIV OF TEXAS AT EL PASO on October 24, 2018 at 13:03:18 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: Asymmetric dibenzothiophene S,S-dioxides (DBTOOs) were synthesized and their photophysical properties examined. Through examination, the molecules fluoresced at wavelengths between 371 and 492 nm with quantum yields of fluorescence nearing 0.59. Three of the sulfonic acid sodium salt analogues were chosen to be introduced to HeLa cells, resulting in illumination of the nucleus by fluorescent microscopy. These compounds function as nuclear stains while also affording the ability to predict the localization of the corresponding sulfoxide precursor to ground-state atomic oxygen. mall-molecule fluorophores were first identified over 1701 years ago and used in imaging of tissues for the first time 100 years later.2 Some of these small molecules include common backbones including fluorescein, BODIPY, iminocoumarins, and rhodamine.3 These fluorophores can function independently as a stain, be coupled with antibodies, or be attached to proteins for histochemical analysis.4,5 This can afford the user the ability to monitor protein trafficking, nucleic acids, or directly identify organelles among many other applications.6 As noted by Terai and Nagano, predicting the photophysical properties of novel fluorescent molecules is quite difficult, and therefore, subsequent discovery of new dyes often relies on serendipity.5 Thus, there exists a demand for new fluorescent probes with new backbones that possess photophysical properties which lend themselves to improved fluorescence imaging.3 Dibenzothiophene (DBT) (1) is a common sulfur impurity in fossil fuels, which garners significant attention due to its global distribution and environmental implications. When DBT is converted into the corresponding sulfoxide or the sulfone, a diverse array of photochemical reactions is observed. For example, studies into the photochemistry of oxidized DBT derivatives include photodeoxygenation of the dibenzothiophene S-oxide (DBTO), the application of this photodeoxygenation in biological systems, and the use of the DBT scaffold in organic light emitting diodes (OLEDs).7−13 The application of DBT derivatives in OLEDs suggest that a natural extension of applications would be to use similar molecules for cell imaging. Small molecules used in OLEDs are selected based on their luminescent properties, which are also inherently important to small-molecule cell dyes. However, despite this relationship, the translation of DBT OLED small molecules into cell stains is not extensively reported in the literature. In addition to the interest in DBTs with regards to OLEDs and possible application as cell dyes, a strong interest exists to utilize the photochemical activity of dibenzothiophene S-oxide

S

© XXXX American Chemical Society

derivatives in biological environments.10,11,14,15 DBTO is posited to produce ground-state atomic oxygen [O(3P)] upon irradiation with ultraviolet (UV) light.7,16−18 The fluorescent properties of the dibenzothiophene S,S-dioxides (DBTOOs) afford visual verification of the localization of these DBTO analogues in cells. It is likely that the nearly identical structure of the DBTOO and DBTO will afford essentially identical placement in the cell. The combination of the ability to identify the location of the DBTOO in a cell, the DBTO’s ability to generate O(3P), and their nearly identical structure affords the opportunity to monitor and ultimately deliver an O(3P) precursor on an organelle basis in the cell. Water-soluble DBTO analogues, when irradiated in cells, did not afford any measurable oxidized biomolecule products after attempting to target lipids.11 The failure of the cellular DBTO irradiations was posited to result from the DBTO localizing away from the lipids, which were the targets in this particular study. Herein, this study aims to utilize the luminescent properties of dibenzothiophene S,S-dioxides to identify the location of derivatized DBTOOs in cells as a stand-alone cellular stain and an analogue of the O(3P) generating DBTO with the intention of delivering an oxidant to specific organelles. The synthesis of the DBTOO dyes is shown in Scheme 1. The first five steps were previously reported.19 The sequence begins with treating DBT (1) with N-bromosuccinimide (NBS) in dimethylformamide (DMF). This affords 2bromodibenzo[b,d]thiophene (2) in 33% yield following sequential recrystallizations. Then 2 was treated with chlorosulfonic acid at −5 °C in DCM. The precipitating solid was filtered and washed with cold DCM, affording 8bromodibenzo[b,d]thiophene-2-sulfonic acid (3) in 79% yield. Then 3 was refluxed in thionyl chloride in the presence of Received: July 27, 2018 Published: October 10, 2018 A

DOI: 10.1021/acs.joc.8b01931 J. Org. Chem. XXXX, XXX, XXX−XXX

Note

The Journal of Organic Chemistry Scheme 1. Scheme Used for Synthesizing 12−14a

a Reagents and conditions: (a) NBS, DMF, rt; (b) ClSO3H, DCM, −5 °C; (c) SOCl2, DMF (cat), reflux; (d) phenol, DABCO, DCM, rt; (e) R = ArB(OH)2, PdCl2(PPh3)2, NaB4O7, DME/H2O (1:1), reflux; (f) mCPBA, DCM, rt; (g) 2.2 equiv of 2 M NaOH in MeOH, DCM, rt.

Table 1. Photophysical Data of 9−14 compound 9 10 11 12 13 14

λabs nma [log ε(M−1 cm−1)]a 238 286 242 238 245 238

[4.65], [4.16] [4.74] [4.62], [4.75], [4.63],

269 [4.51]

245 [4.61], 265 [4.54] 286 [4.71] 263 [4.51]

λex nma,b [log ε (M−1 cm−1)] (λex nma,b) 274 283 274 268 287 274

[4.45] [4.17] [4.51] [4.40] [4.71] [4.36]

(275) (286) (274) (274) (284) (274)

λem nma (λem nm)b [φd] 426 485 492 371 412 421

(434) (478) (486) (380) (422) (428)

[0.05] [0.59] [0.15] [0.07] [0.29] [0.55]

Stokes shift (nm)a,c 152 202 218 103 125 147

Measured in acetonitrile at 10 μM. bMeasured in EtOH at 10 μM. cCalculated from excitation. dMeasured using the single-point method in EtOH.23

a

8-biphenyldibenzo[b,d]thiophene-2-sulfonate 5,5-dioxide, and phenyl 8-(naphthalen-2-yl)dibenzo[b,d]thiophene-2-sulfonate 5,5-dioxide) (9−11) were afforded in yields from 61 to 92%.20 The formation of the sulfone afforded a dramatic increase in visible luminescence when exposed to UV light. The conversion of the phenyl sulfonate ester moiety to the sulfonic acid salt was achieved by adapting a known hydrolysis.21,22 This was done to increase water solubility because 9−11 had little to none. The intermediate sulfones (9−11) were dissolved in DCM, and 2.2 equiv of sodium hydroxide (NaOH) was added via an aliquot of 2 M NaOH in methanol (MeOH). The resulting slurry was washed with water until the organic layer was clear. The washes were concentrated under reduced pressure and purified over a reverse-phase preparative column, providing the products (sodium 8-phenyldibenzo[b,d]thiophene-2-sulfonate 5,5-dioxide, sodium 8-biphenyldibenzo[b,d]thiophene-2-sulfonate 5,5dioxide, sodium 8-(naphthalen-2-yl)dibenzo[b,d]thiophene-2sulfonate 5,5-dioxide) (12−14) in yields from 34 to 89%.

catalytic amounts of DMF to produce 8-bromodibenzo[b,d]thiophene-2-sulfonyl chloride (4) in 91% yield. The sulfonyl chloride analogue (4) was then converted to the sulfonate ester through the addition of phenol and DABCO in DCM. Following purification with column chromatography, phenyl 8bromodibenzo[b,d]thiophene-2-sulfonate (5) was isolated in 85% yield. The addition of the aromatic substituent at the 8position was achieved through Suzuki coupling, where yields ranged from 65 to 72%. This process produced phenyl 8phenyldibenzo[b,d]thiophene-2-sulfonate (6), phenyl 8-([1,1′biphenyl]-4-yl)dibenzo[b,d]thiophene-2-sulfonate (7), and phenyl 8-(naphthalen-2-yl)dibenzo[b,d]thiophene-2-sulfonate (8). The oxidizing process involved the addition of an excess of meta-chloroperoxybenzoic acid (mCPBA) (5 equiv) to a solution of the given sulfide (6−8) in methylene chloride (DCM). Upon completion of the reaction, and subsequent purification by flash chromatography, the products (phenyl 8phenyldibenzo[b,d]thiophene-2-sulfonate 5,5-dioxide, phenyl B

DOI: 10.1021/acs.joc.8b01931 J. Org. Chem. XXXX, XXX, XXX−XXX

Note

The Journal of Organic Chemistry

Figure 1. Images of 12−14 in HeLa cells. (A) Bright-field image of cells stained with 13; no UV. (B) Fluorescence image of HeLa cells stained with 13. (C) Merged bright-field and fluorescence images of HeLa cells stained with 13. (D) Bright-field image of cells stained with 12; no UV. (E) Fluorescence image of HeLa cells stained with 12. (F) Merged bright-field and fluorescence images of HeLa cells stained with 12. (G) Bright-field image of cells stained with 14; no UV. (H) Fluorescence image of HeLa cells stained with 14. (I) Merged bright-field and fluorescence images of HeLa cells stained with 14.

Figure 2. Images of 12 and 13 in HeLa cells showing cellular mitosis. (A) Bright-field image of cells stained with 13; no UV. (B) Fluorescence image of HeLa cells stained with 13. (C) Merged bright-field and fluorescence images of HeLa cells stained with 13. (D) Bright-field image of cells stained with 12; no UV. (E) Fluorescence image of HeLa cells stained with 12. (F) Merged bright-field and fluorescence images of HeLa cells stained with 12.

nm, which did not overlap with the excitation spectra well. This indicated that the absorption at wavelengths near λmax excites the molecules to excited states above S1 that have nonradiative deactivation pathways to the ground state. The wavelength of λex for the excitation scan corresponds to excitation to S1, which leads to emission. This λex for most of the molecules was red-shifted from the peak absorption by up to 39 nm.

The target molecules (12−14) and their parent molecules (9−11) all fluoresce in the visible region, and their optical properties were determined (Table 1). The observed luminescence is most likely fluorescence. This came into question due to the large Stokes shift. Small-molecule phosphorescence in solution is rare; however, the emissive state was not conclusively established. All the listed compounds’ λmax absorption ranged between 238 and 250 C

DOI: 10.1021/acs.joc.8b01931 J. Org. Chem. XXXX, XXX, XXX−XXX

Note

The Journal of Organic Chemistry The commonalities of this library of compounds exist only in their absorbance spectra. Their emission spectra are more varied with regard to the peak wavelength of emission. The larger the chromophore, the more the emission spectra shift further to the red. The phenyl sulfonate compounds (9− 11) have a λmax of emission ranging from 426 to 494 nm, whereas the sulfonic acid salt analogues (12−14) have λmax values between 371 and 428 nm. The transformation of the sulfonate ester to acid shifts the fluorescent emission toward the UV and concomitantly reduces the Stokes shift. This transformation does not consistently alter the quantum yield of fluorescence (φ), which was determined using the single point method.23 The phenyl analogues (9 and 12) maintain a low quantum yield, 0.05 and 0.07, respectively, independent of the phenyl sulfonate ester. In the case of the biphenyl analogues, the phenyl sulfonate ester analogue (10) has a quantum yield of 0.59, and the sulfonic acid analogue (13) has a quantum yield of 0.29. The reduction of quantum yield seen with the transformation of the phenyl sulfonate ester to the sulfonic acid salt with regard to 9 and 12 is not seen with 11 and 14 as the phenomenon is inverted. The naphthyl analogues (11 and 14) display a higher quantum yield 0.55 when it is a sulfonic acid and a lower quantum yield of 0.15 when it is a phenyl sulfonate ester. The inconsistencies of quantum yield related to structure make it difficult to note any relation except that the larger the aromatic substituent the greater the quantum yield. This uncertain relationship between quantum yield and size of aromatic substituent is consistent with previous reports.24 To determine the viability of 12−14 as dyes for biological imaging, HeLa cells were treated with fresh culture medium containing the respective compound at 1.0 μM (0.10% DMSO final concentration) in a volume of 2.0 mL. The cells were left exposed to the compound for 2 h, washed with PBS, and then fixed in 4% paraformaldehyde. Prepared slides were then imaged using a Leica DM 400 B microscope with a DFC3000G camera. The samples were excited with UV light centered at 350 nm. The subsequent images, shown in Figure 1, show fluorescence with varying intensities among the three compounds. Although the relative fluorescence intensities for compounds 12−14 vary significantly, all three stain within the nuclear envelope of the cells (Figure 1). When overlaid with the bright-field images of the cells (Figure 1C,F,I), the observed fluorescence stays largely within the nucleus of the cells. Interestingly, even though 14 possesses the highest quantum yield of fluorescence of the three stains, it has a weaker fluorescence intensity in cells. However, both 12 and 13 appear to be good nuclear stains, which may have specific affinity for DNA. Figure 2 shows HeLa cells stained with 12 or 13 (Figure 2E,B, respectively), which indicate the compounds follow the DNA in the stages of mitosis. For 12, the fluorescence forms a pillar shape indicative of the chromosomes lining up at the metaphase plate (Figure 2E). In Figure 2B, the fluorescence of 13 indicates that the cell is in anaphase or telophase of mitosis. The toxicity of compounds 12−14 was then determined in HeLa cells. The HeLa cells were treated with varying concentrations of the respective dye for 72 h. The IC50 for all of the compounds is in excess of 100 μM, as seen in Figure 3. The biphenyl analogue (13) begins to show some degree of toxicity at 100 μM; however, 12 and 14 do not show any decrease in cell viability at any concentration tested. Overall, 12 and 14 are promising nuclear stains with low cytotoxicity.

Figure 3. Antiproliferation assay showing the cytotoxicity of HeLa cells after being treated for 72 h with dibenzothiophene S,S-dioxide compound derivatives along with controls. (*) Denotes a P < 0.05 in comparison to the vehicle control and other treated cells (two-way ANOVA by Tukey’s mean comparison).

In summary, it has been shown that dibenzothiophene S,Sdioxides are a viable backbone for fluorescent dyes. DBTOO derivatives are inexpensive to make and are easy to modify synthetically. Additionally, these molecules afford modest to excellent photophysical properties, which can be exploited in histochemical studies. In this study, DBTOO derivatives can function as nuclear stains with a nearly exclusive affinity toward DNA. This opens the door to more specific staining protocols with these dyes and future modifications to target other cellular organelles. Extending beyond the obvious application the DBTOO dyes possess in research, this study visually confirms the ability of a DBT to specifically target an organelle and interact with a cell in a biomolecular basis. This result should lead to subsequent investigations into the generation of ground-state atomic oxygen [O(3P)] with respect to cellular DNA by the sulfoxide analogue of these dyes.

■

EXPERIMENTAL SECTION

Synthesis of 8-Bromodibenzo[b,d]thiophene-2-sulfonyl Chloride (4).19 8-Bromodibenzo[b,d]thiophene-2-sulfonic acid (7.69 g, 0.022 mol) and 150 mL of thionyl chloride were combined in a 250 mL round-bottom flask and stirred. To the mixture was added 0.5 mL of DMF. The reaction was refluxed for 24 h. The reaction solution was then poured over 1 L of crushed ice and stirred until the bubbling ceased and the yellowish solid stopped precipitating. The aqueous mixture was washed with dichloromethane (3 × 500 mL) and dried with MgSO4. The solvent was evaporated under reduced pressure, yielding a yellow tinted solid. The crude solid was purified on a normal-phase preparative column using dichloromethane as the eluent, producing a white powder (5.5 g, 68%): 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 8.66 (d, J = 1.7 Hz, 1H), 8.57−8.59 (m, 1H), 7.97−8.03 (dd, 2H), 7.79 (dd, J = 8.3, 1.7 Hz, 1H), 7.67 (dd, J = 8.6, 2.0 Hz, 1H); 13C{1H} NMR (DMSO-d6, 101 MHz) δ (ppm) 145.6, 139.4, 138.0, 137.0, 133.2, 129.7, 125.5, 125.0, 124.8, 122.4, 119.3, 118.2; HRMS (FAB-Magnetic Sector) m/z [M]+ calcd for C12H6BrClO2S2 359.8681; found 359.8681. Typical Procedure for Oxidizing Sulfides to Sulfones. The sulfide 6 (25.2 μmol) was dissolved in enough methylene chloride to maintain a homogeneous solution at −30 °C, which was 3 mL. The solution was then cooled to approximately −30 °C using a 3:2 H2O/ MeOH dry ice bath. To the cooled solution was added mCPBA (5 equiv). The stirring solution was maintained at −30 °C for 4 h and then allowed to warm to room temperature overnight. After being warmed, the solution was washed with saturated sodium bicarbonate water (3 × 5 mL). The washed organic layer was combined with Celite and evaporated under reduced pressure to afford a homogeneous mixture of crude product and Celite. The crude mixture was then purified using normal-phase flash chromatography D

DOI: 10.1021/acs.joc.8b01931 J. Org. Chem. XXXX, XXX, XXX−XXX

Note

The Journal of Organic Chemistry utilizing a 99:1 methylene chloride/ethyl acetate as the eluent on a Biotage Horizon to produce 9 (91%), 10 (92%), and 11 (61%). Phenyl 8-phenyldibenzo[b,d]thiophene-2-sulfonate 5,5-dioxide (9): 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 9.13 (d, J = 1.5 Hz, 1 H), 8.91 (d, J = 1.2 Hz, 1 H), 8.27 (d, J = 8.1 Hz, 1 H), 8.16 (d, J = 8.1 Hz, 1 H), 8.05 (dt, J = 1.6, 8.1 Hz, 2 H), 7.97−7.91 (m, 2 H), 7.62− 7.47 (m, 3 H), 7.46−7.38 (m, 2 H), 7.37−7.31 (m, 1 H), 7.22−7.13 (m, 2 H); 13C{1H} NMR (101 MHz,DMSO-d6) δ (ppm) 148.9, 146.7, 142.2, 140.2, 137.8, 135.4, 132.9, 130.9, 130.4, 130.2, 130.0, 129.1, 127.9, 127.4, 123.4, 123.2, 122.8, 122.4, 122.1; HRMS (ESIFTIRC) m/z [M + Na]+ calcd for C24H16O5S2Na+ 471.0331; found 471.0332. Phenyl 8-([1,1′-biphenyl]-4-yl)dibenzo[b,d]thiophene-2sulfonate 5,5-dioxide (10): 1H NMR (400 MHz, DMSO-d6) δ (ppm) 9.16 (d, J = 1.3 Hz, 1 H), 8.99 (d, J = 1.1 Hz, 1 H), 8.29 (d, J = 8.1 Hz, 1 H), 8.21−8.03 (m, 5 H), 7.88 (d, J = 8.6 Hz, 2 H), 7.83− 7.75 (m, 2 H), 7.57−7.48 (m, 2 H), 7.47−7.39 (m, 3 H), 7.38−7.32 (m, 1 H), 7.24−7.14 (m, 2 H); 13C{1H} NMR (101 MHz, DMSOd6) δ (ppm) 148.9, 146.1, 142.2, 140.8, 140.2, 139.3, 136.7, 135.4, 132.9, 130.9, 130.3, 129.9, 129.1, 127.9, 127.3, 126.8, 123.4, 123.2, 122.9, 122.2, 122.1; HRMS (ESI-FTIRC) m/z [M + Na]+ calcd for C30H20O5S2Na+ 547.0644; found 547.0644. Phenyl 8-(naphthalen-2yl)dibenzo[b,d]thiophene-2-sulfonate 5,5-dioxide (11): 1H NMR (400 MHz, DMSO-d6) δ (ppm) 9.16 (d, J = 1.2 Hz, 1 H), 9.05 (s, 1 H), 8.53 (s, 1 H), 8.29 (d, J = 8.1 Hz, 1 H), 8.25−8.18 (m, 2 H), 8.14−7.98 (m, 5 H), 7.66−7.56 (m, 2 H), 7.47−7.39 (m, 2 H), 7.38− 7.31 (m, 1 H), 7.23−7.14 (m, 2 H); 13C{1H} NMR (101 MHz, DMSO-d6) δ (ppm) 149.4, 147.0, 142.7, 140.7, 135.9, 135.6, 133.5, 133.4, 131.4, 130.8, 129.2, 129.0, 128.4, 128.1, 127.5, 127.2, 127.1, 125.5, 123.9, 123.7, 123.4, 123.0, 122.6; HRMS (ESI-FTIRC) m/z [M + Na]+ calcd for C28H18O5S2Na+ 521.0488; found 521.0491. Typical Procedure for Converting the Phenyl Sulfonate Ester to the Sulfonic Acid Sodium Salt. The sulfonic acid salts were synthesized by directly adapting a previously published method by Miller.21 This involved dissolving 202.3 mg (386 μmol) of the phenyl ester (10) in 4 mL of DCM. To the solution was added 400 μL of a 2 M solution of NaOH in MeOH. The solution was stirred overnight, forming a paste. The paste was diluted with 2 mL of DCM and then washed with 3 × 5 mL of DI water and centrifuged. The aqueous washes were discarded. The organic layer was then washed with 2 × 5 mL of basic NaOH(aq) water. The remaining organic mixture was dried under a flow of N2(g) to afford the products 12 (34%), 13 (34%), and 14 (89%) as a white solid. Sodium 8phenyldibenzo[b,d]thiophene-2-sulfonate 5,5-dioxide (12): 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 8.67 (d, J = 1.2 Hz, 1H), 8.57 (d, J = 0.9 Hz, 1H), 8.01−8.06 (m, 1H), 7.91−7.99 (m, 4H), 7.84−7.89 (m, 1H), 7.51−7.57 (m, 2H), 7.43−7.51 (m, 1H); 13C{1H} NMR (DMSO-d6, 101 MHz) δ (ppm) 154.4, 146.3, 138.1, 137.2, 135.9, 131.7, 130.9, 129.0, 128.8, 128.1, 126.4, 122.4, 121.6, 121.3, 120.3; HRMS (ESI-FTIRC) m/z [M + Na]+ calcd for C18H11NaO5S2Na+ 416.983781; found 416.9837. Sodium 8-([1,1′-biphenyl]-4-yl)dibenzo[b,d]thiophene-2-sulfonate 5,5-dioxide (13): 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 8.75 (s, 1H), 8.60 (s, 1H), 7.99− 8.12 (m, 5H), 7.92−7.98 (m, 1H), 7.81−7.91 (m, 4H), 7.78 (d, J = 7.2 Hz, 2H), 7.51 (t, J = 7.6 Hz, 3H), 7.41 (s, 1H); 13C{1H} NMR (DMSO-d6, 101 MHz) δ (ppm) 140.5, 139.3, 137.2, 137.0, 135.9, 131.7, 130.9, 129.0, 128.9, 128.2, 128.0, 127.8, 127.2, 126.7, 122.4, 121.6, 121.1, 120.3; HRMS (ESI-FTIRC) m/z [M + Na]+ calcd for C24H15NaO5S2Na+ 493.015081; found 493.0149. Sodium 8-(naphthalen-1-yl)dibenzo[b,d]thiophene-2-sulfonate 5,5-dioxide (14): 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 8.83 (s, 1H), 8.62 (s, 1H), 8.57 (s, 1H), 8.04−8.15 (m, 6H), 7.94−7.98 (m, 1H), 7.87−7.92 (m, 1H), 7.56−7.64 (m, 2H); 13C{1H} NMR (DMSO-d6, 101 MHz) δ (ppm) 154.4, 146.1, 137.3, 136.0, 135.3, 133.2, 132.9, 131.7, 131.0, 129.3, 128.6, 128.5, 128.2, 127.5, 126.8, 126.7, 126.6, 125.1, 122.5, 121.7, 121.4, 120.3; HRMS (ESI-FTIRC) m/z [M + Na]+ calcd for C22H13NaO5S2Na+ 466.9994; found 466.9994. Antiproliferation Studies. HeLa cells were cultured in DMEM (ThermoFisher) supplemented with 10% FBS (ThermoFisher) and 1% penicillin−streptomycin (ThermoFisher 10000 U/mL). Cells (1000 cells/well in 100 μL) were plated in a 96-well culture plate

(Greiner) and incubated for 24 h. Fifty microliters of the compound dissolved in complete media (final DMSO concentration