Napabucasin and Related Heterocycle-Fused ... - ACS Publications

Article Cite This: J. Nat. Prod. 2018, 81, 1636−1644

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Napabucasin and Related Heterocycle-Fused Naphthoquinones as STAT3 Inhibitors with Antiproliferative Activity against Cancer Cells Hauke Löcken, Cinzia Clamor, and Klaus Müller* Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms University, Corrensstraße 48, D-48149 Münster, Germany

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ABSTRACT: Napabucasin (6) and its angularly anellated isomer (7), for which the synthesis is described, together with related plant-derived naphthoquinones, were evaluated in vitro against human breast cancer (MDA-MB-231) and chronic myelogenous leukemia (K562) cells. As observed for β-lapachone (3), the active naphthoquinones all induced apoptosis in a cell-cycle-independent fashion. In contrast to the pyran-fused β-lapachone (3), however, the most potent furan-fused naphthoquinones were able to redox cycle and generate superoxide in cell-based assays, which was independent of NAD(P)H:quinone oxido-reductase 1. In a homogeneous time-resolved fluorescence (HTRF) assays with MDA-MB-231 cells, both napabucasin (6) and isonapabucasin (7) were identified as targeting STAT3 phosphorylation. In addition, drug affinity responsive target stability assays were performed to validate a direct interaction of the naphthoquinones with STAT3. Isonapabucasin (7) turned out to be twice as potent against STAT3 as napabucasin (6) in the HTRF assay, with an EC50 in the submicromolar range, which was in excellent agreement with the potency of both agents to inhibit the growth of MDA-MB-231 cells. Moreover, molecular docking experiments predicted different binding modes to the STAT3 SH2 domain for the linearly anellated napabucasin (6) and its angularly anellated isomer (7).

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characterized in 1982 and was found to have cytotoxic activity in a KB cell culture.7 Recent research efforts have shown that napabucasin (6) is a potent inhibitor of stem cell activity in cancer cells12 by inhibiting the signal transducer and activator of transcription 3 (STAT3) pathway. 13 As a result, napabucasin (6) has been granted Orphan Drug Designation status for the treatment of pancreatic cancer.14 Besides these lapacho-derived substances, there are unsubstituted furan-fused naphthoquinones found in Avicennia species. Naphtho[2,3-b]furan-4,9-dione (avicequinone-B, 4) was isolated15 from the Indian mangrove species Avicennia alba and has recently been reported as a 5α-reductase inhibitor.16 Furthermore, the gray mangrove (Avicennia marina), which is distributed in Africa, Asia, and Australia, contains the angular analogue of avicequinone-B, naphtho[1,2-b]furan-4,5-dione (NFD, 5),17 for which activity against cancer cell lines has been reported.18 Our group has evaluated previously a comparable set of lapacho-analogous compounds as potential antipsoriatic agents against the hyperproliferation of human keratinocytes (HaCaT).4 Among these structures, β-lapachone (3) was found to be a potent inhibitor of keratinocyte growth and compared well with the antipsoriatic anthralin, while

aphthoquinones are widely distributed as secondary metabolites among bacterial, fungal, and plant species.1 They are associated with a wide range of biological properties, such as antibacterial,2 antitrypanocidal,3 and antipsoriatic4,5 activities, and, in particular, antineoplastic activities. Thus, these molecules continue to be of strong interest for the development of new drugs of natural provenance. The compounds investigated herein are constituents of lapacho, the inner bark of the tree Handroanthus impetiginosus (Mart. ex DC.) Mattos, syn. Tabebuia impetiginosa, which belongs to the family Bignoniaceae.6 Preparations of this inner bark are well known to certain indigenous peoples in South America as “ipê roxo” (red thick bark) or “taheebo” (ant wood) and have been used in folk medicine for the treatment of a variety of diseases.6 Several naphthoquinones were isolated and identified as biologically active compounds in the bark and heartwood of Tabebuia species.7 Most prominent are lapachol (1), the dihydrobenzochromenedione α-lapachone (2), its angular derivative β-lapachone (3), and naphtho[2,3-b]furan-4,9dione napabucasin (6). β-Lapachone (3) has been reported as an effective antitumor agent8 and is currently in clinical trials for the therapy of advanced solid tumors and pancreatic cancer.9,10 Redox activation of β-lapachone (3) by NAD(P)H:quinone oxidoreductase 1 (NQO1) to generate reactive oxygen species (ROS) appears to account for its cancer-cell-killing action.11 The linearly anellated napabucasin (6) was isolated and © 2018 American Chemical Society and American Society of Pharmacognosy

Received: March 22, 2018 Published: July 13, 2018 1636

DOI: 10.1021/acs.jnatprod.8b00247 J. Nat. Prod. 2018, 81, 1636−1644

Journal of Natural Products

Article

sodium dithionite and methyl iodide. Regioselective 2lithiation of the furan ring and subsequent addition of N,Ndimethylacetamide gave the 2-acetyl analogue 5b. Finally, the desired target compound 7 was obtained by oxidative demethylation with diammonium cerium(IV) nitrate. Inhibition of Cancer Cell Growth. The antiproliferative effects of the naphthoquinones against the MDA-MB-231 solid breast cancer cell line and the K562 human chronic myelogenous leukemia cell line were evaluated after 48 h of treatment (Table 1). Cell proliferation was measured by Table 1. Antiproliferative Activity against Human Breast Cancer (MDA-MB-231) and Chronic Myelogenous Leukemia (K562) Cell Lines compound lapachol (1) α-lapachone (2) β-lapachone (3) avicequinone B (4) NFD (5) napabucasin (6) isonapabucasin (7)

napabucasin (6) was even more potent. Based upon the promising results obtained with these naturally occurring structures, investigations have been extended herein on heterocycle-fused naphthoquinones with potential anticancer properties. In the present study, the angular structure of βlapachone (3) and NFD (5) was implemented to the linearly anellated napabucasin (6), and its analogous isomer, 2-acetylnaphtho[1,2-b]furan-4,5-dione (isonapabucasin, 7), was prepared. The antiproliferative activities of the naphthoquinones against the MDA-MB-231 breast cancer cell line and the K562 chronic myelogenous leukemia cell line were determined. With respect to the mechanism of action, the ability of the most potent naphthoquinones to induce apoptosis, influence cell cycle distribution, generate ROS, and inhibit STAT3 activity were examined.

MDA-MB-231 IC50a [μmol/L] >10b >10b 4.3 >10b 1.8 2.1 0.74

± 0.44 ± 0.22 ± 0.09 ± 0.01

K562 IC50a [μmol/L] >10b >10b 1.6 5.2 2.8 1.0 1.1

± ± ± ± ±

0.36 0.82 0.26 0.30 0.16

a

IC50, concentration of test compound required for 50% inhibition of cell viability, as determined by the MTT assay after 48 h of treatment. Values are the means (±SD) of three independent experiments. bn = 2.

metabolic transformation of MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) to formazan crystals,21 which reflects the number of viable cells. The antitumor agent β-lapachone (3) was active against both cell lines in the low micromolar range, which is in line with its previously reported activity against cancer cells.22,23 Napabucasin (6) proved to be a more potent inhibitor of cell growth with a similar profile toward both the breast cancer and the leukemia cells. Avicequinone B (4), which is an unsubstituted analogue of 6 lacking the 2-acetyl group, is 5-fold less potent toward K562 cells than 6 and has no appreciable effect on MDA-MB-231 cells, at least at the examined concentrations. The angularly anellated naphtho[1,2-b]furan-4,5-diones, NFD (5) and isonapabucasin (7), show enhanced potency when compared with their corresponding linear analogues 4 and 6, respectively. Furthermore, among the furan-fused naphthoquinones 4−7, i.e., napabucasin (6) and its close congeners, the ortho-quinone scaffold favored activity against the solid breast cancer cells, and the para-quinones were more active against leukemia cells. At the tested concentration range, lapachol (1) and αlapachone (2) were devoid of growth inhibitory activity, despite their reported antiproliferative activity in cancer cells.24,25 However, this finding agrees well with a previous report that extremely high concentrations of these agents were required to induce cytotoxicity in human leukemia cells.26 With respect to structure−activity relationships, there are three pairs of naphthoquinones where the impact of the heterocycle-fusion type can be explored by comparing the corresponding linearly anellated (2, 4, 6) and angularly anellated analogues (3, 5, 7) included in Table 1. In all cases, an angularly anellated ring system seems to be superior to linear anellation, as both 5 and 7 were more potent than their isomeric analogues, 4 and 6, respectively. As compared to the highly potent angular 3, the linear anellation in 2 was

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RESULTS AND DISCUSSION Synthesis of Isonapabucasin. To obtain the angular isonapabucasin (7, Scheme 1), a route was adopted that was used previously to synthesize a large number of linearly anellated lapacho-quinones.5,19 Accordingly, the quinoid precursor 520 was converted to the required electron-rich catechol dimethyl ether 5a by reductive methylation with Scheme 1. Synthesis of Isonapabucasin (7)a

a Reagents: (a) 1. Na2S2O4, EtOH, rt; 2. KOH, DMSO, MeI, 5 °C, N2; (b) n-BuLi, N,N-dimethylacetamide, THF, −15 °C, N2; (c) (NH4)2[Ce(NO3)6], MeCN, H2O, 0 °C.

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napabucasin (6) showed a decrease to just 62% viable cells, but with a higher amount of early apoptotic cells (20%). The most potent compound in the MTT assay (IC50 of 0.74 μmol/L) was also the fastest-acting compound, with a population of 14% late apoptotic cells after 4 h of treatment, increasing to 89% after 24 h. A significant population of early apoptotic cells was not detected. Cell Cycle Distribution. To get insight into the potential mechanism of action, it was investigated whether the naphthoquinones influence the cell cycle distribution of MDA-MB-231 cells, using propidium iodide (PI) staining with flow cytometric analysis.28 PI can penetrate cell membranes and intercalates into DNA, allowing a cell cycle analysis by quantification of cellular DNA contents. Figure 2

accompanied by loss of activity. It is also evident that introduction of a 2-acetyl group onto the naphthofurandione scaffolds of 4 and 5, leading to 6 and 7, improved potency toward both the MDA-MB-231 and K562 cell lines. The 2acetylated 6 exhibited higher potency against leukemia than breast cancer cells, while 7 proved to be the most potent agent against the MDA-MB-231 human breast cancer cells of the naphthoquinones tested. Induction of Apoptosis. MDA-MB-231 cells were exposed to the four most potent naphthoquinones and then analyzed by flow cytometry for time-dependent formation of apoptotic cells. We used a combined annexin V−fluoresceinisothiocyanate (FITC)/7-AAD staining method,27 where annexin V detects externalized phosphatidylserine as a marker for early apoptosis. 7-Aminoactinomycin D (7-AAD) is excluded by early apoptotic cells, while it penetrates disrupted membranes and intercalates into DNA of late apoptotic or necrotic cells. Figure 1 presents a percentage distribution of viable, early and late apoptotic, or necrotic cells after incubation with the test compounds for 0−24 h. The results are in line with those effects obtained in the MTT assay. The angularly anellated naphthoquinones 3, 5, and 7 were most potent, decreasing viable cells to 9−38%, when compared to the control (97%) after 24 h of treatment. However, the linear

Figure 2. Flow cytometric analysis of cell cycle distribution. MDAMB-231 cells were treated with the test compounds (1−30 μmol/L) for 24 h, fixed with ice-cold ethanol (70%), and stained using propidium iodide. Results are presented as a percentage distribution of cells in the sub-G0, G1, S, and G2 phase, respectively. Values are the means (±SD) of three independent experiments.

shows the percentage distribution of sub-G0, G1, S, and G2 phase after 24 h of treatment with the test compounds. No significant effects on cell cycle distribution were observed for naphthoquinones 5−7. β-Lapachone (3) has been reported to effectively kill breast cancer cells by apoptosis, which was independent of the cell cycle status.29 However, Figure 2 indicates a shift from the G1 to the G2 phase, but only at relatively high concentrations of 3 (10 and 30 μmol/L). Therefore, the present data suggest that heterocycle-fused naphthoquinones induce apoptosis in a cell-cycle-independent

Figure 1. Flow cytometric analysis of apoptosis induced by βlapachone (3), NFD (5), napabucasin (6), and isonapabucasin (7) over time using combined annexin V−FITC/7-AAD staining. MDAMB-231 cells were incubated with the test compounds (10 μmol/L) for the indicated period of time. Results are presented as a percentage distribution of viable cells (annexin V−/7-AAD−) and early (+/−) and late apoptotic cells (+/+). Values are the means (±SD) of two independent experiments. 1638



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of NQO1 polymorphisms that result in lack of enzyme expression, there are many cell lines that do not express this enzyme.39 Indeed, MDA-MB-231 cells have an undetectable NQO1 level.40,41 This would also explain why 3 is a less potent inhibitor of MDA-MB-231 cell proliferation (Table 1) than naphthoquinones 5−7. Even though trace levels of NQO1 activity have been reported in K562 leukemia cells,42 the present results indicate that this cell line is also insensitive to NQO1-mediated metabolism of 3. Accordingly, cellular responses to 3 observed in this study are NQO1-independent, which require higher concentrations of 3 and are most likely due to conditions where this compound is metabolized by cytochrome P450 oxidoreductases through one-electron reduction.39 In MDA-MB-231 cells napabucasin (6), its angular isomer 7, and the angular NFD (5) all strongly increased superoxide generation as compared to controls. As expected, fluorescence intensity was not influenced by the NQO1 inhibitor dicoumarol, since these cells do not appreciably express NQO1. Consequently, the furan-fused naphthoquinones are also metabolized in an NQO1-indepent pathway by oneelectron reduction. In K562 cells, however, superoxide generation by 5−7 can be inhibited in the presence of dicoumarol, which reveals that NQO1, at least in part, contributes to redox activation of the furan-fused naphthoquinones in the leukemia cell line. While β-lapachone (3) is selectively activated by NQO1,38 the results of this study reveal that angularly furan-fused naphthoquinones such as 7 can undergo one-electron reduction as well as two-electron reduction, which would produce a semiquinone radical and a catechol form, respectively. Upon autoxidation of the semiquinone and catechol, electron transfer to oxygen would give rise to superoxide as the primary ROS, finally regenerating the parent ortho-quinones (Scheme 2). Further ROS are generated from dismutation of superoxide by superoxide dismutase to yield hydrogen peroxide, which is next reduced through the

manner and interact with their putative cellular target without effecting cell cycle distribution. Intracellular Generation of ROS. Modulation of redox homeostasis in cancer cells can be a useful tool in tumor therapy, since cancer cells exhibit a higher concentration of endogenous ROS than normal cells.30,31 A further rise of ROS levels from the therapeutic treatment is expected to make cancer cells more susceptible to oxidative stress, overwhelming the antioxidant capacity, and, thereby, inducing cell death.32 ROS involvement in cancer is not confined to directly damaging macromolecules, but, more importantly, ROS function as secondary messengers and activate apoptotic signaling.33 Hence, small molecules that enhance intracellular ROS levels can serve to target cancer. 30 As such, naphthoquinones are known to undergo enzyme-mediated redox cycling initiated through one- or two-electron reduction, which occurs in two sequential reactions to form unstable hydroquinones via semiquinone radicals. In such a scenario, the hydroquinone and the semiquinone participate in an apparent nonending futile cycling that would utilize oxygen as an electron acceptor, generating ROS such as superoxide.34 To provide information on the capability of the four most potent naphthoquinones of this study to modulate the redox cycle, the formation of superoxide in cell-based assays was measured. For the assessment of the intracellular levels of superoxide flow cytometry was employed using dihydroethidium (DHE), a specific probe for superoxide.35 To evaluate the involvement of NQO1, which catalyzes formally the two-electron transfer to the quinones, the cells were preincubated with the known NQO1 inhibitor dicoumarol.36 NQO1 is generally considered as a detoxifying enzyme that catalyzes the reduction of quinones to hydroquinones, which are easily conjugated and excreted. Notably, the enzyme is overexpressed in a large number of cancer cells and may therefore be used for specific targeting of tumors.37 Figure 3 reveals that in sharp contrast to the furan-fused naphthoquinones 5−7, β-lapachone (3), which is fused with a

Scheme 2. Redox Cycling of NFD (5) and Isonapabucasin (7)a

Figure 3. Flow cytometric analysis for 2-OH-DHE fluorescence. Cells were incubated for 24 h with the test compounds and stained with DHE for 30 min. (A) MDA-MB-231 cells, treated with the indicated compound (5 μmol/L) or vehicle control (DMSO). (B) K562 cells, treated with the indicated compound (5 μmol/L) or vehicle control (DMSO). Dashed bars show cells preincubated with dicoumarol (5 μmol/L, 30 min). Displayed is the relative fluorescence intensity toward vehicle control of at least three independent measurements. Error bars represent the standard deviation. *p < 0.05.

six-membered dihydropyran, did not generate appreciable amounts of superoxide in MDA-MDB-231 and K562 cells. This is not surprising, since 3 has been demonstrated to induce NQO1-dependent death of cancer cells exclusively through the metabolism of this two-electron reductase.38 However, because

a

CPR = cytochrome P450 oxidoreductase, NQO1 = NAD(P)H:quinone oxidoreductase 1.

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Figure 4. HTRF assay of 6 and 7 on STAT3 phosphorylation. MDA-MB-231 cells were serum-starved for 4 h and then treated with the compounds for an additional 4 h. Cells were lysed, transferred to a 384-well plate, and incubated overnight with donor (Eu-cryptate) and acceptor (d2) antibodies. Results are reported as HTRF ratios and normalization value (phospho/total). Dotted lines represent the negative control (no cells). (A) Dose−response curves of 6 and 7 on phospho-STAT3 level. (B) Dose−response curves of 6 and 7 on total-STAT3 level. (C) Normalized dose−response curves of 6 and 7. The EC50 is the concentration of the compound where 50% of its maximal effect is observed. Values are the means (±SD) of three independent experiments.

Figure 5. Prediction of the binding mode of napabucasin (6) and isonapabucasin (7) to the STAT3 SH2 domain. (A) Cartoon representation of DNA-bound STAT3 monomer (PDB ID: 1bg1). SH2 domain is framed in a red box and pictured with surface. (B) Possible binding mode of linear 6 with STAT3 SH2. (C) Possible binding mode of angular 7 with STAT3 SH2. Docked poses were generated using AutoDock4.2. Visualization was carried out with PyMol 0.99rc6. Red dotted lines represent H bonds between the ligand and receptor.

isonapabucasin (7). MDA-MB-231 cells have been reported to exhibit elevated levels of phosphorylated STAT345,48 and are used widely as a convenient model for the identification of STAT3 inhibitors.46,47,49 Dose−response curves of 6 and 7 on phospho-STAT3 and total-STAT3 levels are presented in Figure 4. Both compounds exhibited a good inhibitory activity against the phosphorylation of STAT3 and thus suppress the activation of STAT3 signaling. Napabucasin (6), as a previously described STAT3 inhibitor,12 showed an effective concentration (EC50) of 2.0 μmol/L (Figure 4A). The angular analogue 7 even demonstrated improved inhibitory potency over napabucasin (6) in the submicromolar range with an EC50 of 0.87 μmol/L. These results are in excellent agreement with the potency of both agents to inhibit the growth of MDA-MB-231 cells. To preclude a decrease of the phospho-STAT3 signal due to reduced expression of overall STAT3, the total-STAT3 level was determined. Figure 4B outlines the effect of 6 and 7 on the content of total STAT3. A slight decrease could be observed with both compounds, yet not a total loss. This effect may be

Fenton-type reaction, yielding the highly reactive hydroxyl radical.30 Inhibition of STAT3 Phosphorylation. Signal transducer and activator of transcription 3 (STAT3) is a cytoplasmic transcription factor. Upon activation by tyrosine kinase mediated phosphorylation, STAT3 forms homodimers that translocate into the nucleus. Bound to DNA, the transcription factor regulates the expression of oncogenes associated with apoptosis, angiogenesis, invasion, and migration.43 Since STAT3 is constitutively activated in a large number of cancer cells and because of its critical role in tumor progression, the development of small-molecule inhibitors of the STAT3 pathway is of great interest for the treatment of cancer.44,45 Several naphthoquinones have recently been reported to inhibit phosphorylation and dimerization of STAT3.13,46,47 To evaluate the inhibitory activity of the furan-fused naphthoquinones on the phosphorylation state and therefore the activity of STAT3, homogeneous time-resolved fluorescence (HTRF) assays were performed with MDA-MB-231 cells incubated with napabucasin (6) and its angular analogue 1640



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due to deceased cell numbers. A normalization value to correct the phosphorylation state was calculated as a ratio from the phospho-STAT3 and total-STAT3 signal. Figure 4C reports the normalization values and underlines that 7 is a highly potent inhibitor of STAT3 phosphorylation with a normalized EC50 value of 0.80 μmol/L and superior to napabucasin (6) with a normalized EC50 value of 1.9 μmol/L. To gain insight into the binding mode and to understand the differences in potency between 6 and 7, molecular modeling studies were performed using the AutoDock4.2 software. As receptor, the SH2 domain of the STAT3 monomer was utilized (Figure 5A), which is a solvent-accessible region that comprises three subpockets.46,50 Blocking this domain is of particular interest, as it acts as a motif of recognition for phosphotyrosine and is essential for the activation by tyrosine kinases as well as for the dimerization of phosphorylated STAT3.44 Figure 5B depicts a predicted binding mode of napabucasin (6) to the STAT3 SH2 domain with an AutoDock Score of −5.27 kcal/mol. The quinoid carbonyl oxygen of the linear 6 forms a weak hydrogen bond (330 pm) with Arg609, the acetyl oxygen with Arg585, addressing the subpocket east of Lys591 that is enclosed by Arg585, Ile634, and Ser636. Isonapabucasin (7), on the other hand, is docked inversely as shown in Figure 5C. The eastward located subpocket is addressed by the phenyl moiety, which seems beneficial because of the hydrophobic residue Ile634. The acetyl group directs toward the subpocket to the west of Lys591 and forms a weak (330 pm) hydrogen bond with Glu612 and a moderate (300 pm) bond with Arg609. In this position, the furan oxygen is capable of forming a moderate (290 pm) hydrogen bond with Arg609. The westward orientation of 7 seems to be favorable, as this pose allows an additional hydrogen bond and slightly closer donor−acceptor distances that result in an enhanced AutoDock Score of −5.82 kcal/mol. This is in accordance with the experimentally determined inhibitory activity on STAT3 phosphorylation. Results from the HTRF assay and docking experiments provide evidence for an inhibitory effect on STAT3 phosphorylation. To validate a direct interaction of the test compounds with STAT3, the drug affinity responsive target stability (DARTS) method was used in combination with Western blot analysis. DARTS provides a simple methodology to identify protein−ligand interactions without further modifications or labeling, based on changes in protease stability upon ligand binding.51,52 Accordingly, a lysate of MDA-MB-231 cells was prepared under nondenaturating conditions and incubated with the test compounds or DMSO as a negative control. The samples were split, and one aliquot was digested using thermolysin, a Zn2+- and Ca2+dependent endoprotease. The second aliquot was left untreated. After separation via SDS page, a Western blot was carried out, using STAT3 antibody. Figure 6 presents the results of this DARTS−Western blot analysis. Lysates treated with napabucasin (6) or isonapabucasin (7) showed bands with a decreased intensity as compared to the DMSO control. Nondigested controls treated with the test compounds without addition of thermolysin indicated no difference toward the DMSO control. These findings indicate that both 6 and 7 interact directly with STAT3 and enhance the thermolysin digestion. Isonapabucasin (7) showed a more drastic reduction in band intensity that emphasizes a more potent effect of 7 than that of 6.

Figure 6. DARTS−Western blot analysis for target validation. Lysate of MDA-MB-231 cells was incubated with napabucasin (6), isonapabucasin (7, 100 μmol/L), or vehicle control (DMSO) for 1 h and digested by thermolysin (1:3000) for 30 min. Nondigested controls were carried out. Samples were subjected to SDS-PAGE. Western blot analysis was performed with a STAT3 antibody.

In summary, of the plant-derived naphthoquinones tested, the 2-acetylated napabucasin (6) and isonapabucasin (7), its synthetic isomer, were the most potent inhibitors of MDAMB-231 and K562 cell growth. Our data strongly suggest that the antiproliferative activity of both naphthoquinones is related to inhibition of STAT3 phosphorylation. In particular, the isomer 7 was identified as being twice as potent of an inhibitor as the orphan anticancer drug 6. In contrast to β-lapachone (3), both furan-fused agents were able to act on the redox cycle and generate ROS in an NQO1-independent manner.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined with a Stuart SMP3 melting point apparatus and are uncorrected. FTIR spectra were recorded on a Shimadzu IR spectrometer equipped with a Miracle 10 ATR unit. Signals are reported as wavenumbers (cm−1). 1H NMR and 13C NMR spectra were recorded on Agilent DD2 spectrometers (400 MHz; 600 MHz). Chemical shifts (δ) are reported in ppm and were calculated using the residual peak of the deuterated solvent. HRMS spectra (APCI) were obtained on a Bruker microTOF-QII mass spectrometer. Thin-layer chromatography (TLC) was conducted on Merck 60 F254 precoated silica gel plates. Chromatography refers to column chromatography, which was performed on Macherey-Nagel silica gel (0.063−0.200 mm). Purity was analyzed by HPLC (Thermo-Fisher UltiMate 3000) on a Syncronis C18 column (150 × 3.00 mm, 17.5 nm mesh, ThermoFisher) and was ≥95% for all tested compounds. Lapachol (1), αlapachone (2), and β-lapachone (3) were kindly provided by Prof. Eufrânio N. da Silva Jr.53 Avicequinone B (4) and NFD (5) were synthesized as reported by Tseng et al.;20 napabucasin (6) was prepared as previously described.5 Synthesis of Isonapabucasin (7). 4,5-Dimethoxynaphtho[1,2b]furan (5a). To a suspension of 5 (1.41 g, 7.12 mmol) in ethanol (50 mL) was added sodium dithionite (7.43 g, 42.7 mmol) in water (25 mL), and the resulting mixture was stirred for 3 h at room temperature. Next, this was extracted with diethyl ether (4 × 100 mL) and dried over Na2SO4, and the solvent was evaporated. The residue and KOH (1.40 g, 25.0 mmol) were dissolved in DMSO (15 mL) under N2. Methyl iodide (2.21 mL, 35.6 mmol) was added under icecooling, and the mixture was stirred for 12 h. Then, it was poured into ice−water (100 mL), extracted with ethyl acetate (4 × 200 mL), washed with a saturated solution of NaCl, and dried over Na2SO4. The solvent was evaporated, and the crude product was purified by chromatography (cyclohexane−ethyl acetate, 9:1) to afford 5a as a pale yellow oil (859 mg, 53%): FTIR νmax 1589, 1504, 1469 1261, 1207, 1099 cm−1; 1H NMR (400 MHz, CDCl3) δ 8.26−8.15 (2H, m, H-6, H-9), 7.72 (1H, d, J = 2.1 Hz, H-2), 7.54−7.49 (2H, m, H-7, H8), 7.02 (1H, d, J = 2.1 Hz, H-3), 4.15 (3H, s, OCH3), 4.02 (3H, s, OCH3); 13C NMR (101 MHz, CDCl3) δ 148.44, 144.37, 143.28, 141.93, 127.54, 125.88, 125.57, 122.82, 120.56, 119.30, 119.05, 106.10, 62.11, 61.23; HRAPCIMS m/z 229.0739 [M + H]+ (calcd for C14H13O3, 229.0786). 1-(4,5-Dimethoxynaphtho[1,2-b]furan-2-yl)ethan-1-one (5b). To a solution of 5a (0.63 g, 2.84 mmol) in anhydrous tetrahydrofuran 1641



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(20 mL), cooled to −15 °C under N2, was added dropwise nbutyllithium (2.5 mol/L, 2.34 mL, 5.98 mmol), and the solution was stirred for 15 min. Then dimethylacetamide (0.65 mL, 6.71 mmol) was added, and the mixture was allowed to warm to room temperature within 3 h. The reaction was poured into ice−water (100 mL), extracted with ethyl acetate (3 × 70 mL), washed with a saturated solution of NaCl, and dried over Na2SO4. Then the solvent was evaporated, and the crude product was purified by chromatography (cyclohexane−ethyl acetate, 9:1) to afford 5b as a yellow solid (0.19 g, 32%): mp 91−92 °C; FTIR νmax 1658, 1631, 1504, 1462, 1276, 1246, 1195 cm−1; 1H NMR (600 MHz, CDCl3) δ 8.34−8.37 (1H, m, H-6), 8.17−8.19 (1H, m, H-9), 7.73 (1H, s, H-3), 7.52−7.63 (2H, m, H-7, H-8), 4.16 (3H, s, OCH3), 4.02 (3H, s, OCH3), 2.67 (3H, s, CH3); 13C NMR (101 MHz, CDCl3) δ 187.68, 152.26, 149.38, 142.43, 142.05, 129.50, 127.24, 125.63, 122.54, 120.97, 119.31, 118.75, 112.05, 61.53, 60.91; HRAPCIMS m/z 271.0967 [M + H]+ (calcd for C16H15O4, 271.0965). 2-Acetylnaphtho[1,2-b]furan-4,5-dione (7). A solution of diammonium cerium(IV) nitrate (0.73 g, 1.55 mmol) in acetonitrile− water (1:1, 10 mL) was added dropwise to a suspension of 5b (0.15 g, 0.55 mmol) in acetonitrile−water (17:3, 20 mL) at 0 °C. The reaction was stirred for 1 h, then poured into ice-cold water (50 mL) and extracted with CH2Cl2 (3 × 50 mL). The combined organic layers were washed with a saturated solution of NaCl (2 × 50 mL), dried over Na2SO4, and concentrated. Then, n-hexane was added to precipitate 7 as an orange solid (83 mg, 62%): mp 222−224 °C; FTIR νmax 1674, 1523, 1435, 1369 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 8.04−8.00 (1H, m, H-6), 7.96 (1H, s, H-3), 7.90−7.86 (1H, m, H-9), 7.77−7.83 (1H, m, H-8), 7.64−7.68 (1H, m, H-7), 2.54 (3H, s, CH3); 13C NMR (101 MHz, DMSO-d6) δ 186.05, 178.56, 174.07, 160.79, 152.26, 134.94, 131.40, 130.75, 129.36, 126.76, 122.75, 122.36, 116.22, 26.18; HRAPCIMS m/z 241.0473 [M + H]+ (calcd for C14H9O4, 241.0495). Biological Assay Methods. Cell lines were purchased from DSMZ (Braunschweig, Germany) and were confirmed as mycoplasma negative using a MycoAlert (Lonza, Basel) mycoplasma detection kit. MDA-MB-231 human breast cancer cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM), and human chronic myelogenous K562 leukemia cells were maintained in RPMI 1640 medium. Media were supplemented with 10% fetal bovine serum, penicillin G (100 units/mL), streptomycin (0.1 mg/mL), and Lglutamine (30 mg/L). Cells were stored in a humidified 37 °C incubator with 5% CO2. Cell Proliferation Assay. Growth inhibition of MDA-MB-231 cells and K562 cells was evaluated using the MTT assay as previously described.54 Flow Cytometry. Analysis of the cell cycle status was carried out as previously described in full detail.54 Induction of apoptosis was measured in MDA-MB-231 cells using the annexin V−FITC apoptosis detection kit with 7-AAD (BioLegend) as previously described for keratinocytes.5 Detection of intracellular superoxide generation was carried out with DHE. The assay was performed in MDA-MDB-231 and K562 cells essentially as previously described for keratinocytes.5 HTRF STAT3 Assay. STAT3 phosphorylation was measured using the HTRF-based phospho-STAT3 (Y705) and total-STAT3 signaling kits (Cisbio Bioassays) according to the manufacturer’s instructions. MDA-MB-231 cells (105 cells/well) were plated in 96-well plates and were allowed to adhere overnight. The medium was replaced by serum-free DMEM. Cells were starved for 4 h and then incubated with varying concentrations of the test compounds (0.1−30 μmol/L in DMSO, final concentration of DMSO 0.5%) or vehicle for 4 h at 37 °C. Cells were washed with phosphate-buffered saline (PBS) and lysed with supplemented lysis buffer (25 μL, included in the kit) for 45 min at room temperature with shaking at 400 rpm. After homogenization, 16 μL of lysate was transferred to a 384-well plate. Premixed phospho-STAT3 antibody (4 μL, d2 and cryptate labeled antibody, 1:1, included in the kit) was added. For total-STAT3, the remaining lysate in the 96-well plate was diluted with supplemented lysis buffer (15 μL), and the diluted lysate (16 μL) was transferred to

a 384-well plate. Premixed total-STAT3 antibody (4 μL, d2 and cryptate labeled antibody, 1:1, included in the kit) was added, and the reaction was incubated for 24 h at room temperature in the dark. Fluorescence was measured after excitation (320 nm) at 620 nm for the cryptate donor and 665 nm for the d2-acceptor on an Infinite F200 Pro (Tecan) with the software i-control version 1.10 (Tecan). HTRF ratios were calculated from the signals at 665 and 620 nm. To correct the phosphorylation state, a normalization value is calculated from the quotient of phospho-STAT3 HTRF ratio and total-STAT3 HTRF ratio. DARTS Assay. A DARTS assay was performed for target validation. MDA-MB-231 cells were washed with PBS and collected by scraping. The cell pellet was lysed by M-PER (mammalian protein extraction reagent, Thermo Fisher) with phosphatase inhibitors (PhosSTOP, Roche) and protease inhibitors (Complete Ultra Tablets, Roche) on ice for 10 min. After centrifugation (18000g, 10 min, 4 °C), the supernatant (lysate) was kept on ice. The protein concentration was measured with a Pierce BCA protein assay kit (Thermo Fisher). The lysate was incubated with either vehicle (DMSO) or test compound (100 μmol/L) for 1 h in the dark at room temperature. TNC buffer (50 mmol/L TRIS-HCl pH 8.0, 50 mmol/L NaCl, 10 mmol/L CaCl2) was added, and the samples were digested with thermolysin (Sigma, 1:3000, based on the protein concentration) for 30 min at room temperature. Controls without thermolysin were treated with TNC. Digestion was stopped by addition of protease inhibitor and SDS buffer (62.5 mmol/L Tris-HCl pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol). Samples were heated at 70 °C for 10 min, separated by SDS-PAGE (10 μg protein per lane) on 4−15% precast polyacrylamide gels (BioRad), and transferred onto nitrocellulose membranes (Bio-Rad) using the Trans-Blot semidry transfer cell (Bio-Rad). Membranes were blocked with 5% nonfat dried milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 1 h at room temperature. Membranes were washed with TBST (3 × 5 min) and incubated with rabbit antiSTAT3 primary antibody (Cell Signaling, 1:2000 in 5% bovine serum albumin, TBS, 0.1% Tween 20) for 2 h at room temperature. Membranes were washed again with TBST (3 × 5 min) and were incubated with HRP-conjugated anti-rabbit IgG secondary antibody (Jackson ImmunoResearch) for 1 h. A final washing step with TBST (3 × 10 min) was carried out, and the membranes were incubated with Clarity Western ECL Substrate (Bio-Rad). Signals were detected on a ChemiDoc XRS+ imaging system (Bio-Rad) and analyzed using Image Lab version 5.2.1 (Bio-Rad). Molecular Docking. Ligands were created with Avogadro 1.2.0 and minimized according to the MMFF94 force field. For computational docking, the Software AutoDock 4.2 was used to predict the binding mode and approximate free binding energy.55 As receptor, the SH2 domain of the STAT3 protein (PDB ID: 1bg1) was chosen. For preparation of the macromolecule and ligands, water was excluded and Gasteiger charges were assigned. Torsional root and the rotable bonds of the ligand were identified. An AutoGrid map was precomputed for all atom types in the ligand set. The Grid Box was centered at Ser636 with 64 000 grid points per map. Docking was performed using the Lamarckian Genetic Algorithm with default settings. After 25 million energy evaluations, the top ranked poses were visually inspected. Images were generated using PyMol 0.99rc6. Statistical Analysis. All experiments were performed in triplicate, if not otherwise stated. EC50 and IC50 values were calculated by nonlinear regression against a vehicle-treated control using Prism 3.0 (GraphPad Software) and expressed as means ± SD. Differences were considered significant at p < 0.05.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00247. 1642



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(20) Tseng, C.-H.; Lin, C.-S.; Shih, P.-K.; Tsao, L.-T.; Wang, J.-P.; Cheng, C.-M.; Tzeng, C.-C.; Chen, Y.-L. Bioorg. Med. Chem. 2009, 17, 6773−6779. (21) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63. (22) Li, C. J.; Wang, C.; Pardee, A. B. Cancer Res. 1995, 55, 3712− 3715. (23) Silvers, M. A.; Deja, S.; Singh, N.; Egnatchik, R. A.; Sudderth, J.; Luo, X. Q.; Beg, M. S.; Burgess, S. C.; DeBerardinis, R. J.; Boothman, D. A.; Merritt, M. E. J. Biol. Chem. 2017, 292, 18203− 18216. (24) Epifano, F.; Genovese, S.; Fiorito, S.; Mathieu, V.; Kiss, R. Phytochem. Rev. 2014, 13, 37−49. (25) Krishnan, P.; Bastow, K. F. Cancer Chemother. Pharmacol. 2001, 47, 187−198. (26) Salustiano, E. J. S.; Netto, C. D.; Fernandes, R. F.; da Silva, A. J. M.; Bacelar, T. S.; Castro, C. P.; Buarque, C. D.; Maia, R. C.; Rumjanek, V. M.; Costa, P. R. R. Invest. New Drugs 2010, 28, 139− 144. (27) Zembruski, N. C. L.; Stache, V.; Haefeli, W. E.; Weiss, J. Anal. Biochem. 2012, 429, 79−81. (28) Riccardi, C.; Nicoletti, I. Nat. Protoc. 2006, 1, 1458−1461. (29) Pink, J. J.; Wuerzberger-Davis, S.; Tagliarino, C.; Planchon, S. M.; Yang, X.; Froelich, C. J.; Boothman, D. A. Exp. Cell Res. 2000, 255, 144−155. (30) Dharmaraja, A. T. J. Med. Chem. 2017, 60, 3221−3240. (31) Wellington, K. W. RSC Adv. 2015, 5, 20309−20338. (32) Sabharwal, S. S.; Schumacker, P. T. Nat. Rev. Cancer 2014, 14, 709−721. (33) Chio, I. I. C.; Tuveson, D. A. Trends Mol. Med. 2017, 23, 411− 429. (34) Song, Y.; Buettner, G. R. Free Radical Biol. Med. 2010, 49, 919− 962. (35) Zielonka, J.; Vasquez-Vivar, J.; Kalyanaraman, B. Nat. Protoc. 2008, 3, 8−21. (36) Tan, A. S.; Berridge, M. V. Free Radical Biol. Med. 2010, 48, 421−429. (37) Pink, J. J.; Planchon, S. M.; Tagliarino, C.; Varnes, M. E.; Siegel, D.; Boothman, D. A. J. Biol. Chem. 2000, 275, 5416−5424. (38) Bey, E. A.; Bentle, M. S.; Reinicke, K. E.; Dong, Y.; Yang, C.-R.; Girard, L.; Minna, J. D.; Bornmann, W. G.; Gao, J.; Boothman, D. A. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 11832. (39) Bey, E. A.; Wuerzberger-Davis, S. M.; Pink, J. J.; Yang, C.-R.; Araki, S.; Reinicke, K. E.; Bentle, M. S.; Dong, Y.; Cataldo, E.; Criswell, T. L.; Wagner, M. W.; Li, L.; Gao, J.; Boothman, D. A. J. Cell. Physiol. 2006, 209, 604−610. (40) Hsieh, T. C. Anticancer Res. 2010, 30, 4169−4176. (41) Parkinson, E. I.; Hergenrother, P. J. Acc. Chem. Res. 2015, 48, 2715−2723. (42) Yan, C.; Dufour, M.; Siegel, D.; Reigan, P.; Gomez, J.; Shieh, B.; Moody, C. J.; Ross, D. Biochemistry 2011, 50, 6678−6688. (43) Furtek, S. L.; Backos, D. S.; Matheson, C. J.; Reigan, P. ACS Chem. Biol. 2016, 11, 308−318. (44) Yue, P.; Turkson, J. Expert Opin. Invest. Drugs 2009, 18, 45−56. (45) Banerjee, K.; Resat, H. Int. J. Cancer 2016, 138, 2570−2578. (46) Debnath, B.; Xu, S.; Neamati, N. J. Med. Chem. 2012, 55, 6645−6668. (47) Yu, W.; Li, C.; Zhang, W.; Xia, Y.; Li, S.; Lin, J.-y.; Yu, K.; Liu, M.; Yang, L.; Luo, J.; Chen, Y.; Sun, H.; Kong, L. J. Med. Chem. 2017, 60, 2718−2731. (48) Berishaj, M.; Gao, S. P.; Ahmed, S.; Leslie, K.; Al-Ahmadie, H.; Gerald, W. L.; Bornmann, W.; Bromberg, J. F. Breast Cancer Res. 2007, 9, R32. (49) Noman, M. A. A.; Hossain, T.; Ahsan, M.; Jamshidi, S.; Hasan, C. M.; Rahman, K. M. J. Nat. Prod. 2018, 81, 236−242. (50) Chun, J.; Li, R. J.; Cheng, M. S.; Kim, Y. S. Cancer Lett. 2015, 357, 393−403. (51) Lomenick, B.; Hao, R.; Jonai, N.; Chin, R. M.; Aghajan, M.; Warburton, S.; Wang, J.; Wu, R. P.; Gomez, F.; Loo, J. A.; Wohlschlegel, J. A.; Vondriska, T. M.; Pelletier, J.; Herschman, H.

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H and 13C NMR spectra of compounds 5a, 5b, and 7; representative dot-plots of annexin V/7-AAD costained cells (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +49 251-833-3324. E-mail: [email protected]. ORCID

Klaus Müller: 0000-0003-3694-4137 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The kind gifts of lapachol, α-lapachone, and β-lapachone by Prof. Eufrânio N. da Silva Jr., Department of Chemistry, Federal University of Minas Gerais, Brazil, are gratefully acknowledged.

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REFERENCES

(1) Petr, B.; Vojtech, A.; Ladislav, H.; Rene, K. Curr. Pharm. Anal. 2009, 5, 47−68. (2) Sánchez-Calvo, J. M.; Barbero, G. R.; Guerrero-Vásquez, G.; Durán, A. G.; Macías, M.; Rodriguez-Iglesias, M. A.; Molinillo, J. M. G.; Macías, F. A. Med. Chem. Res. 2016, 25, 1274−1285. (3) Diogo, E. B. T.; Dias, G. G.; Rodrigues, B. L.; Guimaraes, T. T.; Valenca, W. O.; Camara, C. A.; de Oliveira, R. N.; da Silva, M. G.; Ferreira, V. F.; de Paiva, Y. G.; Goulart, M. O. F.; Menna-Barreto, R. F. S.; de Castro, S. L.; da Silva, E. N., Jr. Bioorg. Med. Chem. 2013, 21, 6337−6348. (4) Müller, K.; Sellmer, A.; Wiegrebe, W. J. Nat. Prod. 1999, 62, 1134−1136. (5) Reichstein, A.; Vortherms, S.; Bannwitz, S.; Tentrop, J.; Prinz, H.; Müller, K. J. Med. Chem. 2012, 55, 7273−7284. (6) Gómez Castellanos, J. R.; Prieto, J. M.; Heinrich, M. J. Ethnopharmacol. 2009, 121, 1−13. (7) Rao, M. M.; Kingston, D. G. I. J. Nat. Prod. 1982, 45, 600−604. (8) Hussain, H.; Green, I. R. Expert Opin. Ther. Pat. 2017, 27, 1111−1121. (9) Beg, M. S.; Huang, X.; Silvers, M. A.; Gerber, D. E.; Bolluyt, J.; Sarode, V.; Fattah, F.; Deberardinis, R. J.; Merritt, M. E.; Xie, X.-J.; Leff, R.; Laheru, D.; Boothman, D. A. J. Surg. Oncol. 2017, 116, 83− 88. (10) https://clinicaltrials.gov/ct2/show/NCT01502800. (11) Li, J. Z.; Ke, Y.; Misra, H. P.; Trush, M. A.; Li, Y. R.; Zhu, H.; Jia, Z. Toxicol. Appl. Pharmacol. 2014, 281, 285−293. (12) Li, Y.; Rogoff, H. A.; Keates, S.; Gao, Y.; Murikipudi, S.; Mikule, K.; Leggett, D.; Li, W.; Pardee, A. B.; Li, C. J. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 1839−1844. (13) Cafferkey, C.; Chau, I. Expert Opin. Invest. Drugs 2016, 25, 1023−1031. (14) Oncology Times 2016, 38, 25, DOI: 10.1097/ 01.COT.0000511606.84427.7b. (15) Ito, C.; Katsuno, S.; Kondo, Y.; Tan, H. T.; Furukawa, H. Chem. Pharm. Bull. 2000, 48, 339−343. (16) Karnsomwan, W.; Netcharoensirisuk, P.; Rungrotmongkol, T.; De-Eknamkul, W.; Chamni, S. Chem. Pharm. Bull. 2017, 65, 253−260. (17) Sutton, D. C.; Gillan, F. T.; Susic, M. Phytochemistry 1985, 24, 2877−2879. (18) Urbanek, R. A.; Suchard, S. J.; Steelman, G. B.; Knappenberger, K. S.; Sygowski, L. A.; Veale, C. A.; Chapdelaine, M. J. J. Med. Chem. 2001, 44, 1777−1793. (19) Bannwitz, S.; Krane, D.; Vortherms, S.; Kalin, T.; Lindenschmidt, C.; Zahedi Golpayegani, N.; Tentrop, J.; Prinz, H.; Müller, K. J. Med. Chem. 2014, 57, 6226−6239. 1643



Article

R.; Clardy, J.; Clarke, C. F.; Huang, J. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 21984−21989. (52) Pai, M. Y.; Lomenick, B.; Hwang, H.; Schiestl, R.; McBride, W.; Loo, J. A.; Huang, J. Methods Mol. Biol. 2015, 1263, 287−298. (53) de Castro, S. L.; Emery, F. S.; da Silva, E. N., Jr. Eur. J. Med. Chem. 2013, 69, 678−700. (54) Prinz, H.; Chamasmani, B.; Vogel, K.; Böhm, K. J.; Aicher, B.; Gerlach, M.; Günther, E. G.; Amon, P.; Ivanov, I.; Müller, K. J. Med. Chem. 2011, 54, 4247−4263. (55) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. J. Comput. Chem. 2009, 30, 2785− 2791.

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