A New 1,2-Naphthoquinone Derivative with Anti-lung Cancer Activity

Abstract

1,2-Naphthoquinone (2-NQ) is a nucleophile acceptor that non-selectively makes covalent bonds with cysteine residues in various cellular proteins, and is also found in diesel exhaust, an air pollutant. This molecule has rarely been considered as a pharmacophore of bioactive compounds, in contrast to 1,4-naphthoquinone. We herein designed and synthesized a compound named N-(7,8-dioxo-7,8-dihydronaphthalen-1-yl)-2-methoxybenzamide (MBNQ), in which 2-NQ was hybridized with the nuclear factor-κB (NF-κB) inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) as a nucleophile acceptor. Although 50 µM MBNQ did not inhibit NF-κB signaling, 10 µM MBNQ induced cell death in the lung cancer cell line A549, which was insensitive to 2-NQ (10 µM). In contrast, MBNQ was less toxic in normal lung cells than 2-NQ. A mechanistic study showed that MBNQ mainly induced apoptosis, presumably via the activation of p38 mitogen-activated protein kinase (MAPK). Collectively, the present results demonstrate that the introduction of an appropriate substituent into 2-NQ constitutes a new biologically active entity, which will lead to the development of 2-NQ-based drugs.

Introduction

1,4-Naphthoquinone (Fig. 1A) is present in various biologically active natural products, and many synthetic derivatives have been reported to date.^1–4) The biological activities of 1,4-naphthoquinone compounds are attributed to their properties, i.e., redox, nucleophile-accepting, and DNA-intercalating properties. However, 1,2-naphthoquinone (2-NQ) (Fig. 1A) has rarely been examined as a bioactive compound despite its structural similarity to 1,4-naphthoquinone.^1–3) 2-NQ found in diesel exhaust particles, which cause air pollution, covalently modifies cysteine residues in a number of cellular proteins and induces a range of toxicities,^5,6) because small 2-NQ non-selectively penetrates the clefts of various proteins and reacts with a cysteine thiolate ion of a protein by reversible Michael reaction and the adduct hydroquinone is further autoxidized to orthoquinone⁵⁾ (Fig. 1B). As the final autoxidation step is virtually irreversible, the protein adduct orthoquinone would not undergo reduction back to hydroquinone nor retro-Michael reaction.

Fig. 1. Structures and Reactivity of Compounds Related to This Study

(A) 1,4-Naphthoquinone and 1,2-naphthoquinone (2-NQ). (B) Mechanism of protein modification by 1,2-naphthoquinone.⁵⁾ (C) DHMEQ/its derivative and their substructures. (D) MBNQ synthesized in this study.

We focused our attention on the inhibitory activity of 2-NQ on nuclear factor-κB (NF-κB) signaling.⁷⁾ We speculated that toxic 2-NQ may be converted to a less toxic and more potent bioactive molecule by the introduction of an appropriate substituent. A well-known NF-κB inhibitor is dehydroxymethylepoxyquinomicin (DHMEQ) (Fig. 1C), which comprises an epoxy-quinone nucleophile acceptor and salicylate moieties.⁸⁾ The epoxy-quinone moiety of DHMEQ covalently binds irreversibly to a cysteine residue within the p65 subunit of the NF-κB heterodimer.^9–11) NF-κB subsequently loses its propensity for nuclear localization, which results in the inhibition of its function. The salicylate moiety may specifically interact with p65. Furthermore, a methoxy derivative of the salicylate moiety, OMe-DHMEQ (Fig. 1C), has been shown to exhibit stronger NF-κB inhibitory activity.¹²⁾ Therefore, we designed a compound named N-(7,8-dioxo-7,8-dihydronaphthalen-1-yl)-2-methoxybenzamide (MBNQ) (Fig. 1D), which has 2-NQ instead of the epoxy-quinone part of OMe-DHMEQ as a nucleophile acceptor. We herein report the synthesis and biological evaluation of MBNQ.

Results

Synthesis and NF-κB Inhibitory Activity of MBNQ

As shown in Chart 1, MBNQ was synthesized starting with 8-amino-2-naphthol 1 and 2-methoxybenzoyl chloride 2. The resulting adduct 3 obtained in 31% was treated with benzeneseleninic acid anhydride according to a previously reported procedure¹³⁾ to give MBNQ in 86% yield.

Chart 1. Synthetic Route to MBNQ

a) (C₂H₅)₃N, THF, room temperature (r.t.), 2 h, b) Benzeneseleninic acid anhydride, THF, r.t., 30 min.

The NF-κB inhibitory activity of the compound was then examined using a reporter assay. A plasmid containing κB sequences and the firefly luciferase gene (3κB-tk-luc vector), and another plasmid carrying a β-actin promoter and the Renilla luciferase gene (pRL-Luc vector) were co-transfected into HeLa S3 cells. These cells were treated with MBNQ or control compounds (2-NQ and DHMEQ) (50 µM), followed by a stimulation with interleukin-1α (IL-1α) to activate NF-κB. The dual luciferase activity of the lysed cells was measured. The results obtained are shown in Fig. 2. DHMEQ exhibited the strongest inhibitory activity against the activation of NF-κB. In contrast, the activity of 2-NQ appeared to be weak, although statistical significance is not seen. MBNQ showed weak activity, and 2-NQ and MBNQ did not significantly differ. The weaker NF-κB activity of 2-NQ observed in the present study than that previously reported⁷⁾ may be attributed to differences in the cells used for assays.

Fig. 2. NF-κB Inhibitory Activity of the Compounds

HeLa S3 cells were co-transfected with 3κB-tk-luc and pRL-Luc. At 1 d post-transfection, each compound (50 µM) was added, and the cells were incubated for 1 h. Then IL-1α (10 ng/mL) was added to the medium, and the cells were incubated for an additional 3 h. The cells were lysed, and a dual luciferase assay was conducted (n = 3). Firefly luciferase activity (transcription from 3κB-tk-luc) was normalized to Renilla luciferase activity (transcription from pRL-Luc) to calculate relative luciferase activity. Relative values are shown. ** p < 0.01, *** p < 0.001, n.s.: not significant. These indications on the error bars show the comparison with the sample treated with IL-1α and without compounds.

Anti-lung Cancer Activity of MBNQ

NF-κB inhibitory activity was not enhanced by the introduction of a methoxy benzamide group into 2-NQ. 2-NQ was previously shown to be weakly cytotoxic in the human lung carcinoma cell line A549.^14,15) Therefore, we focused on the cytotoxicity of MBNQ in lung cancer cells.

A549 cells were treated with MBNQ or 2-NQ (1 or 10 µM) followed by an incubation for 5 d. The 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay was then performed. As shown in Fig. 3A, 2-NQ did not exhibit cytotoxicity at these concentrations. On the other hand, 10 µM MBNQ significantly decreased viability in A549 cells. The cellular toxicities of the compounds were evaluated using commercially available normal lung cells. Cell death was observed 1 d after the addition of compounds (10 µM), and, thus, the MTT assay was conducted. In normal cells, the toxicity of MBNQ was weaker than that of 2-NQ (Fig. 3B). To obtain more detailed information on the anti-lung cancer activity of MBNQ, the growth of A549 cells was assessed. Cells were treated with MBNQ (10 µM) and incubated for 3 d. At the initiation of the experiment and every day thereafter, cells attached to a culture dish were detached. Dead cells were stained with trypan blue solution, and unstained cells were counted. As shown in Fig. 3C, the growth of A549 cells without MBNQ plateaued after 2 d. In the presence of MBNQ, slight cell growth was noted after 1 d. Cell numbers were lower after 2 d than at the baseline, demonstrating that MBNQ killed the majority of A549 cells after 1 d.

Fig. 3. Anti-lung Cancer Activity of the Compounds

(A) Evaluation of viability of A549 cells treated with the compounds. After addition of each compound (1 or 10 µM), the cells were incubated for 5 d, and MTT assay was performed (n = 4). (B) Evaluation of viability of normal lung cells treated with the compounds. After addition of each compound (10 µM), the cells were incubated for 1 d, and MTT assay was performed (n = 3). (C) Making growth curve of A549 cells treated with MBNQ. After addition of MBNQ (10 µM), the cells were incubated for 3 d. At the starting point and every 1 d, the cells were detached from a culture dish, and stained with trypan blue solution. The unstained cell numbers were counted, and these were put on the graph. In (A) and (B), relative values are shown. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s.: not significant. These indications on the error bars show the comparison with samples without compounds.

The mechanisms responsible for MBNQ-induced cell death were examined using staining solution including fluorescein isothiocyanate (FITC)-annexin V and ethidium homodimer III. Cells treated with MBNQ (10 µM) were incubated for 12 h or 1 d because the death of a small number of cells was expected at these time points (Fig. 3C), and this timing was considered to be appropriate for investigating the underlying mechanisms. Cells were then treated with the staining solution, followed by fluorescent microscopic observations. The results obtained are shown in Fig. 4A. Representative images at 12 h shown on the left. Apoptotic and necrotic cells should be colored in green and red by FITC-annexin V and ethidium homodimer III, respectively. In this condition, red cells were not seen. Yellow cells were green and red cells merged together, showing the late stage of apoptosis or necrosis. Approximately 100 dead cells cultured in the presence of MBNQ were randomly selected, and the ratios of their colors are summarized in the graphs (Fig. 4A, right). At 12 h, apoptotic cells accounted for approximately 90% of all cells. At 1 d, the number of cells that were not judged to be apoptotic or necrotic (colored in yellow) was elevated. These results demonstrate that MBNQ mainly induced apoptosis at least early time point.

Fig. 4. Mechanism of MBNQ Inducing Death of Lung Cancer Cells

(A) Detection of apoptosis and necrosis in A549 cells treated with MBNQ. After addition of MBNQ (10 µM), the cells were incubated for 12 h or 1 d, then stained using apoptotic/necrotic cells detection kit including FITC-annexin V and ethidium homodimer III. Representative images of fluorescent microscope (12 h incubation) are on the left. A total of approximately 100 cells colored in red (necrosis), yellow (late apoptosis or necrosis) and green (apoptosis) were selected randomly, and their numbers are summarized on the graph (right). (B) Immunoblot analysis of A549 cells treated with the compounds. After addition of each compound (10 µM), the cells were incubated for 6 h followed by their lysis. The lysates were then analyzed by immunoblotting using phospho-p38 MAPK (Thr180/Tyr182) antibody.

We then examined changes in p38 mitogen-activated protein kinase (MAPK) signaling, which responds to extracellular stress, e.g., chemical treatment.^16,17) Cells treated with MBNQ or 2-NQ (10 µM) were incubated for 6 h and then lysed. Immunoblotting was performed on the lysate to detect phospho-p38. As shown in Fig. 4B, the amount of phosphorylated p38 increased in the presence of MBNQ, whereas it did not change with 2-NQ, showing that only MBNQ activated p38 MAPK signaling.

Discussion

We designed MBNQ by the structural combination of 2-NQ and the methoxy benzamide group of DHMEQ (Fig. 1C), with the aim of producing an effective inhibitor of the transcription factor NF-κB, which is a potential target of anti-cancer, anti-inflammatory, and anti-immune drugs.^18–20) The NF-κB inhibitory activity of DHMEQ was attributed to the covalent binding of its epoxy-quinone moiety and the p65 subunit of the NF-κB heterodimer, formed by an epoxide-opening reaction via the nucleophilic attack of a specific cysteine residue in p65. The inhibitory activity of MBNQ against NF-κB was weak, similar to 2-NQ, and was not as potent as that of DHMEQ (Fig. 2). This difference between MBNQ and DHMEQ may be due to the reactivity of the nucleophile acceptor (1,2-NQ or epoxy-quinone) or the distance between the methoxy benzoyl/salicylate part and its binding part in p65 when the nucleophilic reaction occurred.

The present results revealed that MBNQ exhibited stronger cytotoxicity in A549 cells than 2-NQ. Previous studies reported that 2-NQ exhibited cytotoxicity in A549 cells via the production of reactive oxygen species (ROS), such as H₂O₂^14,15); however, clear cytotoxicity was not observed under the experimental conditions employed in the present study (Fig. 3A). In the previous study, the final concentration of dimethyl sulfoxide (DMSO) was 1/1000 volume.¹⁵⁾ Larger amount of DMSO (1/100 volume) might influence the cells oxidative status and less sensitive to oxidant. MBNQ, which has a substituent at position 1 of 2-NQ, appears to exhibit similar oxidizing activity to that of 2-NQ. Therefore, increases in cytotoxicity may not be attributed to the greater production of ROS. This may be related to efficient nucleophilic addition or intercalation into DNA; however, the underlying mechanisms have not yet been elucidated. MBNQ was shown to induce apoptosis in the majority of cells (Fig. 4A). Apoptosis is considered to occur via the activation of p38 (Fig. 4B), which has been shown to play a role in the induction of apoptosis.^16,17) Notably, in our repeated experiment, induction of necrosis was seen depending on the cellular condition.

Examples of 2-NQ derivatives as bioactive compounds have been very limited, in contrast to the 1,4 type. One exception is the anti-cancer compound β-lapachone, which has already entered clinical trials.²¹⁾ 2-NQ is present in diesel exhaust particles, which cause air pollution.^5,6) This negative association appears to have prevented the development of 2-NQ derivatives. In the present study, 2-NQ exhibited cytotoxicity in normal lung cells (Fig. 3B). However, we introduced a methoxy salicylate group, which successfully attenuated toxicity against normal cells and enhanced anti-lung cancer activity. Furthermore, the anti-cancer activity of MBNQ against a panel of sixty human cancer cell lines has already been examined by the Developmental Therapeutics Program in National Cancer Institute (NCI, U.S.A.). The findings obtained showed that MBNQ was highly active against some leukemic cells as well as ovarian/renal cancer cells. These findings will be presented elsewhere.

Conclusion

A newly synthesized 10 µM of 2-NQ derivative was herein shown to exhibit cytotoxicity by induction of apoptosis against lung cancer cells. As a future prospect, improvements in its structure may convert MBNQ into a more potent anti-cancer drugs without toxicity against normal cells. While 2-NQ has not been recognized as a pharmacophore of bioactive compounds, the present study provides novel insights into 2-NQ-based compounds as drug leads.

Experimental

General Procedure Pertaining to Synthesis

TLC was performed on precoated plate [Merck (Darmstadt, Germany) TLC sheets silica 60 F₂₅₄]. Chromatography was carried out on Silica Gel 60N (40–100 mesh) (Kanto Chemical, Tokyo, Japan). NMR spectra were recorded on a Bruker (Billerica, MA, U.S.A.) Avance 600 (600 MHz). Chemical shifts were referenced to tetramethylsilane (TMS). Melting points were determined on a Yanaco (Kyoto, Japan) Melting Point Apparatus. High-resolution mass spectra (HRMS) were recorded by a JEOL (Tokyo, Japan) JMS-DX303HF using positive fast atom bombardment (FAB) with 3-nitrobenzyl alcohol (NBA) as the matrix.

Synthesis of N-(7-Hydroxynaphthalen-1-yl)-2-methoxybenzamide (3)

A solution of 8-amino-2-naphthol 1 (1.00 g, 6.28 mmol), 2-methoxybenzoyl chloride 2 (1.07 g, 6.28 mmol) and triethylamine (1.31 mL, 9.42 mmol) in tetrahydrofuran (THF) (25 mL) were stirred at ambient temperature for 2 h.²²⁾ Upon completion of the reaction, the solvent was evaporated till dryness. The mixture was dissolved in 40 mL of CH₂Cl₂ and washed with water (10 mL×2), dried over anhydrous MgSO₄, and concentrated in vacuo. The residue was purified by silica gel column chromatography (CH₂Cl₂ : EtOAc = 9 : 1) to afford 3 (570 mg, 31%) as a yellowish powder. ¹H-NMR (600 MHz, MeOD) δ: 7.99 (dd, J = 7.4, 1.7 Hz, 1H), 7.74 (dd, J = 7.4, 1.1 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.48 (ddd, J = 8.3, 7.4, 1.7 Hz, 1H), 7.21–7.16 (m 3H), 7.05–7.01 (m, 2H), 4.05 (s, 3H). ¹³C-NMR (150 MHz, MeOD) δ: 166.89, 159.18, 157.29, 134.67, 132.73, 132.63, 131.35, 131.05, 130.65, 127.13, 123.49, 123.28, 123.26, 122.32, 119.51, 113.18, 104.51, 57.04. Mp. 185 °C. HRMS (FAB). Calcd for: C₁₈H₁₆NO₃. 294.1130; Found: 294.1127.

Synthesis of MBNQ

To a suspension of benzeneseleninic acid anhydride (240 mg, 0.666 mmol) in THF (5 mL), a solution of 3 (176 mg, 0.600 mmol) in THF (5 mL) was added dropwise.²³⁾ A spontaneous orange color was evolved and the reaction maintained stirring for 30 min at ambient temperature. The solvent was evaporated and EtOAc was added to the mixture which was washed with aqueous sodium bicarbonate solution (10%, 10 mL×2) and water (20 mL), dried over anhydrous MgSO₄ and evaporated till dryness in vacuo. A flash column chromatography (CH₂Cl₂ : hexane = 20 : 1) afforded MBNQ as dark orange solid (159 mg, 86%). ¹H-NMR (600 MHz, CDCl₃) δ: 12.95 (s, 1H), 9.12 (dd, J = 8.8, 1.0 Hz, 1H), 8.14 (dd, J = 7.8, 1.8 Hz, 1H), 7.63–7.61 (m, 1H), 7.52 (ddd, J = 8.8, 7.8, 1.8 Hz, 1H), 7.43–7.41 (m, 1H), 7.10–7.07 (m, 1H), 7.05–7.04 (m, 2H), 6.44 (d, J = 9.9 Hz, 1H), 4.18 (s, 1H). ¹³C-NMR (150 MHz, CDCl₃) δ: 180.43, 180.30, 165.69, 157.75, 146.68, 145.23, 137.20, 135.23, 133.96, 132.54, 127.08, 125.74, 124.84, 121.96, 121.00, 117.15, 111.42, 55.82. Mp. 165 °C. HRMS (FAB) m/z calcd for C₁₈H₁₄NO₄ [M + H]⁺ 308.0923; Found: 308.0926.

Cell Culture and Addition of Drugs

HeLa S3 cells,²⁴⁾ a subclone of the HeLa (human cervical epithelioid carcinoma) cell line, were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). The human lung carcinoma cell line A549²⁵⁾ [provided by the RIKEN BRC through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology/Japan Agency for Medical Research and Development (MEXT/AMED), Japan (RCB0098)] was maintained in DMEM/F12 supplemented with 5% heat-inactivated FBS. Human pulmonary alveolar epithelial cells (ScienCell Carlsbad, CA, U.S.A.) were purchased and used as normal lung cells. A compound 1,2-naphthoquinone (2-NQ) was purchased from Tokyo Chemical (Tokyo, Japan). For addition of drugs to the cells, the compound was dissolved inDMSO, and the solution was added to their culture medium in a 1/100 volume. As a negative control, the same volume of DMSO was added.

Reproducibility of Biological Experiments

All experiments were repeated three times or more to prepare graphs or to present the representative data.

Evaluation of Cell Viability

A549 cells were plated in 24-well plate (5 × 10⁴ cells/well), and incubated for 1d. Then compounds were added. Relative cell viability was quantified by MTT assay as previously described.²⁶⁾ To count numbers of cells, cell suspension was treated with an equal amount of trypan blue stain (0.4%) (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Then, the cells dyed was excluded as died cells.

Analysis of NF-κB Activity

NF-κB activity was measured by reporter assay as previously described.^27,28) HeLa S3 cells were co-transfected with 1.7 µg of 3κB-tk-luc and 1.7 µg of pRL-Luc by the calcium-phosphate co-precipitation method. At 1 d post-transfection, compounds (50 µM) were added to the medium, and the cells were incubated for 1 h. Then IL-1α (10 ng/mL) (PeproTech, Cranbury, NJ, U.S.A.) was added to the medium, and the cells were incubated for an additional 3 h. The cells were lysed, and a dual luciferase assay was conducted using the luciferase assay system or Renilla luciferase assay system (Promega, Madison, WI, U.S.A.).

Detection of Apoptotic and/or Necrotic Cells and Immunoblot Analysis

Mechanism of cell death was analyzed using apoptotic/necrotic cells detection kit (PromoCell, Heidelberg, Germany) as previously described.²⁹⁾ Immunoblot analysis was performed using cells lysed with RIPA buffer. As an antibody, phospho-p38 MAPK (Thr180/Tyr182) antibody (Cell Signaling, Danvers, MA, U.S.A.) or anti-β-actin clone AC-15 (Sigma-Aldrich, St. Louis, MO, U.S.A.) was used. Immunoreactivity was detected by chemiluminescence using ImmunoStar LD (FUJIFILM Wako, Osaka, Japan).

Acknowledgments

We thank Dr. Shigeki Miyamoto (The University of Wisconsin, U.S.A.) for donating the 3κB-tk-luc vector. The A549 cell line was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan. The study was supported by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (17H03999) and the Grant for Joint Research Project of the Institute of Medical Science, The University of Tokyo. This work was also supported by a Grant for Joint Research Project with Science Farm Ltd.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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