Antiproliferative and Chemosensitizing Effects of Diarylheptanoids on

Jan 25, 2019 - Sae Miyagishi† , Yohei Saito† , Masuo Goto*‡ , and Kyoko Nakagawa-Goto*†‡. † School of Pharmaceutical Sciences, College of ...
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Article Cite This: ACS Omega 2019, 4, 2053−2062

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Antiproliferative and Chemosensitizing Effects of Diarylheptanoids on Intractable Tumor Cells Sae Miyagishi,† Yohei Saito,† Masuo Goto,*,‡ and Kyoko Nakagawa-Goto*,†,‡ †

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School of Pharmaceutical Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa 920-1192, Japan ‡ Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7568, United States S Supporting Information *

ABSTRACT: We designed and synthesized 5 symmetric and 16 asymmetric diarylheptanoid derivatives and evaluated their chemosensitizing and antiproliferative activities for intractable cancers such as triple-negative breast and multidrug-resistant (MDR) cancers. Among the synthesized derivatives, compound 3, which potentially induced cell cycle arrest at G1, was the best candidate for both doxorubicin and paclitaxel in MDR tumor cells, arresting the cells dose-dependently at S/ G2/M and G2/M, respectively. Furthermore, the cotreatment of 13, which slightly accumulated at the G2/M phase, with doxorubicin in triple-negative breast cancer cells (MDA-MB231) effectively enhanced the efficacy of antiproliferative activity. On the other hand, compounds 2, 12, and 14 having the 3,5-dialkyl-4-hydroxyphenyl moiety exhibited a potent antiproliferative activity against all tested tumor cell lines, including an MDR subline, with IC50 values of 5.0−8.5 μM.

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INTRODUCTION

Although medical and technological tools to fight cancer have progressed remarkably, we have not yet overcome this problematic disease. Chemotherapy is an essential treatment for cancer; however, its effectiveness is often limited by severe side effects and multidrug resistance. For example, although doxorubicin (DOX) is widely used clinically to treat breast cancers, it is a well-known substrate of the ABC drug transporter P-glycoprotein (P-gp), and resistance to the drug occurs readily. DOX also leads to significant cardiotoxicity.1 One possible solution is to develop chemosensitizers that can enhance the effectiveness of the current cancer drugs. Also, when a chemosensitizer is added to a cancer treatment regimen, the dose of the cancer drug can be minimized, and consequently, the development of serious side effects and drug resistance would be reduced. Diarylheptanoids are characterized by two aromatic rings linked by a chain of seven carbons. These natural phytochemicals are mainly found in the genera Alnus, Alpinia, Curcuma, Myrica, and Zingiber.2 The diarylheptanoids are roughly categorized into linear and cyclic types. A well-known linear diarylheptanoid is curcumin (1, Figure 1), which is isolated from the roots of Curcuma longa and exhibits various biological activities, such as antioxidant,3 antitumor, and chemosensitizing effects.4,5 © 2019 American Chemical Society

Figure 1. Structures of linear diarylheptanoids and curcumin (1).

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RESULTS AND DISCUSSION Symmetric compounds, such as curcumin (1), are easily synthesized by the reaction of a borane complex of acetylacetone with two equivalents of benzaldehyde;6 therefore, research on synthetic diarylheptanoids has focused primarily on symmetric rather than asymmetric analogues. In the few reported biological studies on 3,5-dialkylated derivatives,7−14 all compounds were symmetric. We feel that further research on diarylheptanoids, including asymmetric 3,5dialkyl derivatives, could significantly advance the development of new, more effective chemosensitizing, antitumor, and antioxidant agents. Therefore, we designed new analogues with electron-withdrawing and -donating groups on both aryl groups as well as a functional group on the methylene between the carbonyl moieties. Symmetric diarylheptanoids 2−6 were prepared according to literature methods15,16 using two equivalents of a substituted benzaldehyde and acetylacetone Received: November 20, 2018 Accepted: December 27, 2018 Published: January 25, 2019 2053

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or 3-acetoxyacetylacetone17 as the starting material (Scheme 1). All benzaldehydes, except 3,5-diethyl-4-hydroxybenzalde-

nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) data. All synthesized diarylheptanoids together with 1 and P-gp inhibitors, elacridar and tariquidar, were evaluated for antiproliferative effects against five human tumor cell lines (HTCLs), A549 (lung carcinoma), triple-negative breast cancer (TNBC) MDA-MB-231 (estrogen and progesterone receptors-negative, HER2-negative), MCF-7 [estrogen receptor (ER)-positive and HER2-negative breast cancer], KB [identical to AV-3 (CCL-21)/HeLa-derivative], and multidrug-resistant (MDR) KB subline KB-VIN with overexpression of P-gp (Table 1). Although curcumin (1) did not show antiproliferative effects against the tested HTCLs, compounds 2, 12, and 14 significantly inhibited all tested tumor cell lines, including KB-VIN. Accordingly, among the tested diarylheptanoids, optimal antiproliferative activity was found when symmetric compounds contained a 3,5-dimethyl-4-hydroxyphenyl moiety (e.g., 2) or asymmetric compounds contained 3,5-dialkyl-4hydroxyphenyl and 3,4-dihydroxy-5-methoxy groups (e.g., 12 and 14). The effects of 12 and 14 were comparable; thus, both 3,5-dimethyl- and 3,5-diethyl-4-hydroxy substitution patterns on the phenyl ring were effective. Although a combination of 3-methoxy-4-hydroxy- and 3,5-dimethoxy-4-hydroxyphenyl groups (compound 7) as well as 3,5-dimethyl-4-hydroxyphenyl and 3,4,5-trimethoxyphenyl groups (compound 8) resulted in potent activity against chemosensitive tumor cell lines, the activity was slightly reduced against the MDR cell line, suggesting that these compounds are possibly pumped out from the cells by P-gp. Selected compounds that showed antiproliferative activity were also evaluated for effects on cell cycle progression using flow cytometry (Figure 3). The MDR cell line KB-VIN was treated with the compound for 24 and 48 h at a concentration

Scheme 1. Syntheses of Diarylheptanoids 2−22a

a

Reagents and conditions: (a) NaOAc (2.0 equiv), dimethyl sulfoxide (DMSO), rt, 3 h, 67%; (b) (1) B2O3 (1.5 equiv), EtOAc, 70 °C, 30 min, (2) (BuO)3B (2.0 equiv), Ar1CHO (2.0 equiv), 85 °C, overnight, and (3) BuNH2 (2.0 equiv), 100 °C, 2 h, 18−44%; (c) (1) B2O3 (0.3 equiv), EtOAc, 70 °C, 30 min, (2) (BuO)3B (0.2 equiv), Ar1CHO (0.2 equiv), 85 °C, overnight, and (3) BuNH2 (0.2 equiv), 100 °C, 2 h, 24−68%; (d) (1) B2O3 (1.5 equiv), EtOAc, 70 °C, 30 min, (2) (BuO)3B (1.0 equiv), Ar2CHO (1.0 equiv), 85 °C, overnight, and (3) BuNH2 (1.0 equiv), 100 °C, 2 h, 21−58%.

hyde,18 are commercially available. Asymmetric analogues 7− 22 were obtained by two-step reactions, each using one equivalent of a substituted benzaldehyde. The structures of all synthesized compounds (Figure 2) were confirmed from 1H

Figure 2. Structures of synthesized compounds 2−22 (*BT = benzothiophene). 2054

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Table 1. Antiproliferative Activity cell linea/IC50 (μM) compound

KB-VIN

MDA-MB-231

A549

KB

MCF-7

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 curcumin elacridar eariquidar

8.5 ± 0.4 20.3 ± 0.4 8.7 ± 0.5 21.7 ± 0.1 >40 22.4 ± 0.8 17.8 ± 0.9 20.4 ± 0.8 21.5 ± 0.8 >40 5.4 ± 0.1 22.6 ± 0.1 6.0 ± 0.2 13.4 ± 0.9 >40 29.2 ± 0.2 >40 >40 >40 >40 >40 >20 >40 23.0 ± 0.3

7.2 ± 0.5 20.9 ± 1.0 9.5 ± 0.0 18.2 ± 1.2 >40 6.3 ± 0.2 6.6 ± 0.2 16.8 ± 2.0 19.3 ± 1.9 >40 7.3 ± 0.0 15.3 ± 3.6 6.4 ± 0.1 7.3 ± 0.7 32.4 ± 0.6 22.0 ± 0.4 23.0 ± 0.1 24.6 ± 0.0 29.4 ± 1.1 19.4 ± 0.3 18.9 ± 0.4 >20 6.4 ± 0.1 4.4 ± 0.1

5.1 ± 0.1 15.0 ± 0.7 9.2 ± 1.6 19.5 ± 0.1 >40 5.6 ± 0.1 4.8 ± 0.1 5.3 ± 0.2 21.4 ± 0.4 >40 5.5 ± 0.2 22.4 ± 0.2 5.0 ± 0.0 16.7 ± 0.2 >40 20.7 ± 0.2 23.8 ± 0.4 >40 >40 39.5 ± 0.1 26.8 ± 0.0 >20 6.4 ± 0.1 5.7 ± 0.0

6.7 ± 0.5 18.4 ± 0.1 8.0 ± 0.2 22.1 ± 0.3 >40 5.1 ± 0.0 5.2 ± 0.1 7.0 ± 1.2 24.0 ± 1.1 >40 5.7 ± 0.2 21.9 ± 0.2 5.0 ± 0.1 10.7 ± 1.1 >40 22.3 ± 0.8 >40 40.7 ± 1.3 >40 40.1 ± 2.2 24.2 ± 0.2 >20 >40 7.3 ± 0.3

7.2 ± 0.1 18.5 ± 0.3 7.4 ± 0.5 22.9 ± 0.3 >40 8.2 ± 0.5 6.3 ± 0.1 11.2 ± 0.8 27.8 ± 1.6 >40 7.7 ± 0.4 24.2 ± 0.6 7.0 ± 0.1 21.6 ± 0.4 32.4 ± 0.3 22.1 ± 0.8 33.1 ± 0.8 34.0 ± 0.4 >40 >40 24.1 ± 0.5 >20 21.8 ± 2.0 8.4 ± 0.1

a Each experiment was independently performed three times. Values are means ±standard deviation. A549: lung adenocarcinoma, MDA-MB-231: TNBC (ER-/PgR-/HER2-), MCF-7: breast cancer (ER+/HER2-), KB: identical to AV-3 (ATCC number, CCL-21) as a HeLa (cervical carcinoma) contaminant, and KB-VIN: MDR KB subline overexpressing P-gp.

Figure 3. Effects of compounds on the cell cycle. KB-VIN cells were treated for 24 (upper panels) and 48 h (lower panels) with the compound at 2- (8) or 3-fold (2, 4, or 14) IC50 concentration. DMSO was used as the control and CA-4 was used at 0.2 μM for 24 h as a mitotic inhibitor arresting cells at G2/M. Fixed and PI-stained cells were analyzed by flow cytometry.

accumulation, whereas Me and OMe groups allowed such accumulation. Next, the less potent compounds 3, 5−7, 9−11, 13, and 16−22, especially those with an IC50 value greater than 20 μM against KB-VIN, in the antiproliferative assay were evaluated for chemosensitizing effects on two clinical anticancer drugs, paclitaxel (PTX) and DOX. PTX is widely used to treat breast, ovarian, lung, bladder, prostate, melanoma, esophageal, and other types of solid cancers and mainly acts as a tubulin depolymerization inhibitor. Meanwhile, DOX is an antitumor antibiotic and acts by intercalating into the DNA double helix. Both the drugs are substrates of P-gp, which leads to lower

of 2- (8) or 3-fold (2, 4, or 14) IC50. Combretastatin A-4 (CA4), a tubulin polymerization inhibitor, was used as a mitotic inhibitory control, arresting cells at G2/M. All compounds, except 14, significantly induced sub-G1 cells in a timedependent manner but did not affect the cell cycle progression. These observations suggest that compounds promote nuclear fragmentation, which result in the induction of apoptosis that generally shows up as sub-G1. Interestingly, the patterns of cell populations found after 48 h of treatment with 2, 4, and 8 were clearly distinguishable, suggesting that each compound induced sub-G1 cells via a different mode of action. These results implied that the presence of a hydroxy group at the meta position (e.g., 14) is not preferred for sub-G1 cell 2055

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Table 2. Chemosensitizing Effects: Cotreatment with PTXa 10 μM cmpd + 1 μM PTX compound 3 5 6 10 11 13 16 17 18 19 20 21 22 curcumin DMSO (PTX only) CTRL PTX IC50 (nM)

KB-VIN −23.6 44.6 90.9 55.2 81.7 63.4 46.4 87.3 91.4 93.0 78.7 73.9 80.3 89.1 97.5 100.0 2007.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 3.9 0.7 0.2 5.4 4.0 0.1 7.3 1.7 1.2 2.1 0.8 1.7 0.4 2.4 0.0 59.6

10 μM cmpd + 1 nM PTX MDA-MB-231 58.7 66.3 87.7 54.1 74.6 45.6 73.8 81.8 91.7 89.4 58.1 61.3 78.2 60.9 97.0 100.0 7.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.5 4.5 4.7 5.5 0.7 5.1 0.2 0.3 1.6 1.6 0.5 3.7 1.3 1.7 3.3 0.0 0.2

A549 36.5 58.4 93.8 69.6 71.3 64.3 76.8 84.8 80.6 83.5 77.2 81.6 78.1 89.3 92.0 100.0 5.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

KB 0.8 2.8 2.2 2.2 0.5 1.1 0.4 1.5 1.6 1.9 6.6 1.3 0.6 3.9 4.4 0.0 0.7

72.8 93.5 102.2 95.6 95.6 101.0 98.6 101.5 100.1 103.9 98.5 100.5 102.4 100.3 107.5 100.0 6.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

MCF-7 0.4 1.8 1.5 1.2 2.1 1.9 0.5 2.7 1.1 4.9 0.7 0.3 0.9 0.3 0.8 0.0 0.1

79.9 90.4 101.7 85.2 95.0 100.2 95.4 93.7 99.6 97.7 94.6 94.1 89.3 97.9 104.5 100.0 14.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 4.2 2.1 0.8 2.0 6.3 0.5 1.8 1.4 1.9 1.6 1.8 0.3 0.2 1.7 0.0 0.4

a Cells were cotreated with 1 μM (KB-VIN) or 1 nM PTX (MDA-MB-231, A549, KB, and MCF-7) in combination with 10 μM test compound. Data are expressed as %growth compared with DMSO alone without PTX as the control (CTRL).

Table 3. Chemosensitizing Effects: Cotreatment with DOXa 10 μM cmpd +1 μM DOX compound 3 5 6 10 11 13 16 17 18 19 20 21 22 curcumin DMSO (DOX only) CTRL DOX IC50 (nM)

KB-VIN 34.3 56.1 81.1 63.7 76.4 65.7 80.8 80.4 82.0 85.9 68.0 65.4 74.2 75.6 80.6 100.0 2942.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.4 0.6 2.8 0.5 0.1 1.9 1.5 6.9 1.0 4.2 6.3 0.9 1.0 1.8 0.1 0.0 5.8

10 μM cmpd + 100 nM DOX MDA-MB-231 42.6 43.0 76.6 55.0 62.3 17.3 61.9 64.5 70.7 70.0 41.5 35.6 60.9 41.9 82.6 100.0 451.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.3 1.3 1.3 0.4 4.8 2.0 3.7 1.9 0.1 0.4 0.2 0.0 0.1 0.5 2.2 0.0 22.4

A549 17.5 19.9 36.1 27.7 28.4 21.3 28.9 26.1 25.4 30.2 26.2 23.7 26.2 27.6 44.4 100.0 95.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.8 0.4 0.4 1.4 0.7 0.4 0.1 0.7 0.8 0.2 0.9 1.6 0.2 0.7 5.2 0.0 17.4

KB 45.9 49.9 41.7 51.9 46.8 45.1 43.4 53.2 53.7 53.6 50.8 47.3 53.4 52.8 49.8 100.0 197.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

MCF-7 0.8 0.1 2.6 3.1 1.2 0.2 1.0 2.6 0.7 0.7 0.1 0.3 2.0 0.0 1.0 0.0 34.7

70.8 75.0 94.5 81.1 83.9 69.8 86.4 77.9 82.9 81.9 70.3 73.3 74.5 78.6 96.2 100.0 1070.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.4 0.5 0.3 0.5 1.0 4.2 3.6 2.4 3.7 0.1 0.3 1.3 0.5 0.1 0.3 0.0 83.5

a Cells were cotreated with 1 μM (KB-VIN) or 100 nM DOX (MDA-MB-231, A549, KB, and MCF-7) in combination with 10 μM test compound. Data are expressed as %growth compared with DMSO alone without DOX as the CTRL.

upon cotreatment with DOX at 1 μM (KB-VIN) or 100 nM (remaining four cell lines) (Table 3). By themselves, both compounds inhibited tumor cell growth only weakly (IC50 > 15−20 μM) (Table 1). The preliminary structure−activity relationship concluded that the combination of 3,5-diethyl-4hydroxyphenyl moiety and phenyl ring with the p-hydroxy group might be important for the chemosensitizing activity, whereas the acetoxy group at the center carbon was not suitable. On the basis of the initial results, we felt that diarylheptanoids 3 and 13 merited further investigation. We then conducted dose-response studies with these two compounds as chemosensitizers combined with DOX in two tumor cell lines, MDA-MB-231 and KB-VIN (Table 4). The P-gp inhibitors, elacridar and tariquidar, were used as reference compounds

effectiveness of the drug after the tumor develops multidrug resistance. The 13 selected compounds (10 μM) were coadministered with PTX (1 nM or 1 μM) against chemosensitive tumor cell lines (MDA-MB-231, A549, KB, and MCF-7) or MDR subline KB-VIN (Table 2). PTX was used at a noncytotoxic concentration in these assays. Interestingly, the symmetric 3,5-diethyl-4-hydroxy diarylheptanoid 3 noticeably showed a significant chemosensitizing effect against KB-VIN cells. The asymmetric diarylheptanoid 13, containing 3,5-diethyl-4hydroxy and 3,5-dimethoxy-4-hydroxy substituted phenyl rings, also displayed considerable chemosensitizing effect against MDA-MB-231, a TNBC cell line. Furthermore, the same compounds (3 and 13) exhibited potent chemosensitizing effects against KB-VIN and MDA-MB-231, respectively, 2056

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Table 4. Chemosensitizing Effects of Compounds on DOX against KB-VIN and MDA-MB-231a DOX IC50 (nM)b compound DMSO 3

13

curcumin

elacridar tariquidar

conc.

KB-VIN

MDA-MB-231

intensifying effectd KB-VIN/MDA-MB-231

0.1% 1 μM 5 μM 10 μM 1 μM 5 μM 10 μM 1 μM 5 μM 10 μM 10 μM 10 μM

2638.2 ± 331.4 >1000 628.7 ± 80.6 254.9 ± 2.1 >1000 >1000 >1000 >1000 >1000 >1000 206.6 ± 4.5 135.3 ± 9.9

431.2 ± 56.2 >200 137.3 ± 3.3 77.2 ± 5.8 >200 117.0 ± 8.1 26.3 ± 4.2 >200 >200 110.0 ± 7.9 >200c >200c

NEe/NE 4.2/3.1 10.3/5.6 NE/NE NE/3.7 NE/16.4 NE/NE NE/NE NE/3.9 12.8/NE 19.5/NE

a Cells were cotreated with DOX in combination with either compound 3, 13, curcumin, elacridar, or tariquidar. bIC50 of DOX was determined in the presence of 1, 5, or 10 μM of compounds. cCompound was used at 1 μM because of cytotoxicity at 10 μM against MDA-MB-231. No effect was seen with 1 μM elacridar or tariquidar. dFold of intensifying effect of compound on KB-VIN and MDA-MB-231. eNE, no effect.

Figure 4. Chemosensitizing effect of compound 3 on DOX and PTX against KB-VIN. (A) KB-VIN cells were treated for 24 h with compound 3 (top panels) and DOX (middle panels), at different doses as indicated, or cotreated with 10 μM compound 3 in combination with 0.5, 1, or 4 μM DOX (bottom panels). DMSO or 0.2 μM CA-4 was used as a control or as a mitotic inhibitor, respectively. (B) KB-VIN cells were treated for 24 h with 1 or 4 μM PTX (top panels). Cells were cotreated with 10 μM compound 3 in combination with 0.5 or 1 μM PTX (bottom panels). Fixed and PI-stained cells were analyzed by flow cytometry.

sensitizing the P-gp-overexpressing MDR subline KB-VIN. On the basis of our antiproliferative activity assay (Table 1), both elacridar and tariquidar were cytotoxic against chemosensitive cells and likely functioned as a competitive inhibitor of P-gp. Cotreatment with DOX and 3 at various concentrations (1, 5, and 10 μM) resulted in dose-dependent chemosensitizing activity against both cell lines, whereas curcumin at 10 μM sensitized only MDA-MB-231. Compound 13 also showed dose-dependent enhancement of the antiproliferative activity of DOX against MDA-MB-231 cells. In fact, the combination of DOX and 13 led to the lowest IC50 value (26.3 nM), which was 4-fold better than that found with DOX and 1 and 3-fold better than that found with DOX and 3. However, compound 13 combined with either DOX or PTX (Table S1) was not effective against KB-VIN. These observations in MDA-MB-231 and P-gp-overexpressing MDR subline KB-VIN cells suggested that the chemosensitizing effects of the diarylheptanoids may not result from the inhibition of P-gp. Therefore, we used flow cytometry to investigate how chemosensitization with 3 affected the cell

cycle progression in KB-VIN cells treated with DOX and PTX (Figure 4). DOX alone induced dose-dependent accumulation of cells at the G2/M phase, whereas compound 3 alone did not have a significant effect on cell cycle progression. However, when the cells were treated with DOX in the presence of 3, the impact on the cell cycle progression was dramatic. With DOX (0.5 μM) combined with 3 (10 μM), substantial cell cycle arrest was observed at G2/M. The level of accumulation was comparable to that observed with DOX alone at 4 μM. Interestingly, when the concentration of DOX increased in the combination treatment, dose-dependent accumulation was also seen in S-phase cells (Figure 4A), suggesting that S-phase progression was significantly impaired by the combination treatment. Because DOX alone does not cause S-phase accumulation, these observations imply that the chemosensitizing effects of 3 on DOX against KB-VIN cells do not result simply from intercellular accumulation of DOX. Instead, it is likely that 3 sensitizes specific effector proteins or pathways targeted by DOX. At this point, the specific target is unclear. We speculate that 3 binds to S-phase responsive proteins, 2057

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Figure 5. Chemosensitizing effect of compounds on DOX against MDA-MB-231. (A) MDA-MB-231 cells were treated for 24 h with compound 3 (middle panels) or 13 (lower panels) at the indicated concentration. DMSO or 0.2 μM CA-4 was used as a control or as a mitotic inhibitor, respectively. (B) Cells were treated for 24 h with 0.1 or 0.5 μM DOX in the absence (top panels) or presence of 10 μM compound 3 (middle panels) or 13 (bottom panels). Fixed and PI-stained cells were analyzed by flow cytometry.

tumor cell lines, including MDR subline KB-VIN, with IC50 values ranging from 5.0 to 8.5 μM. Thus, these three compounds might be good leads for the development of new antitumor agents. Compounds 3 and 13, which also contain a 3,5-diethyl-4-hydroxyphenyl A-ring, showed potential as potent chemosensitizers for DOX and/or PTX against MDR as well as TNBC tumor cell lines. Our results demonstrate that, depending on the pattern of the 4′-hydroxy B-ring, diarylheptanoids with a 3,5-dimethyl or diethyl-4-hydroxy A-ring can act as chemosensitizers for DOX to overcome the MDR phenotype as well as combined agents with DOX to treat breast cancers. On the basis of our findings, compounds 2, 12, and 14 are worth evaluating as antitumor leads as well as compounds 3 and 13 as chemosensitizers for DOX against MDR phenotypes and TNBC tumor in vivo.

related to the metabolic pathway of de novo synthesis of deoxynucleotide, making cells sensitive to DOX and resulting in cell arrest at the S-phase. The effects of PTX + 3 against KBVIN (Figure 4B) also suggest that 3 may sensitize a mitosisrelated protein against PTX. Interestingly, these results also suggest that compound 3 can induce cell cycle arrest at G2/M by inspiring the effects of PTX on microtubule/spindle stabilization. Because 3 and 13 sensitized the MDA-MB-231 TNBC cell line toward DOX (Table 4), we also used flow cytometry to study the effects of these two diarylheptanoids on MDA-MB231 cell cycle progression (Figure 5). Interestingly, compound 3 at 20 μM or 40 μM induced cell cycle arrest at G1 or accumulation at sub-G1, respectively, whereas compound 13 showed a weak dose-dependent effect on G2/M progression (Figure 5A). In contrast, these compounds did not directly affect cell cycle progression in KB-VIN cells; thus, compounds 3 and 13 potentially target a MDA-MB-231-selective protein related to the G1/S and G2/M transitions, respectively. Next, MDA-MB-231 cells were treated with compound 3 or 13 at 10 μM, a concentration without an effect on the cell cycle, in combination with DOX. DOX itself at 0.1 or 0.5 μM induced cell cycle arrest at G2/M or late S/G2/M, respectively. When cells were cotreated with 10 μM 3, accumulation of G1 cells was observed. G1 accumulation was not obvious in cells cotreated with 13, whereas decreasing late S and accumulating G2/M cells were seen (Figure 5B). These effects might result simply from the actions of two different types of bioactive compounds, not by sensitization of a target/pathway of DOX. However, as 10 μM 3 or 13 was a noncytotoxic concentration, DOX might induce the bioactivity of these two compounds in the combination. Thus, a combination of DOX + 3 or 13 showed efficient antiproliferative effects against MDA-MB-231 cells.

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EXPERIMENTAL SECTION General Information. All chemicals and solvents were used as purchased. 1H and 13C NMR spectra were recorded on JEOL JMN-ECA600 and JMN-ECS400 spectrometers with tetramethylsilane as the internal standard, and chemical shifts are stated as δ values in ppm. HRMS data were recorded on a JMS-700 MStation (FAB) or a JMS-T100TD (DART) mass spectrometer. Analytical and preparative thin-layer chromatography was carried out on precoated silica gel 60F254 and RP18F254 plates (0.25 or 0.50 mm thickness; Merck). CombiFlash Rf (Teledyne Isco) system was used for flash chromatography. All target compounds were characterized and determined to be at least >95% pure by 1H NMR, HRMS, and analytical highperformance liquid chromatography. 3-Hydroxy-1,7-bis(4-hydroxy-3,5-dimethylphenyl)-5-oxohepta-1,3,6-trien-4-yl Acetate (4). This compound was obtained as a side product of the reaction of 2,4dioxopentan-3-yl acetate (25) (1.72 mL, 16.6 mmol) and 3,5-dimethyl-4-hydroxybenzaldehyde (71.7 mg, 0.48 mmol) under the same conditions as those described in general procedures for the preparation of asymmetric analogues (8− 22) below. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 10:1) to obtain diketone 26 (R1 = R3 = Me, R2 = OH, X = OAc, 51.2 mg, 37%) and 4 (18.0 mg, 18%) as orange needles. mp 202−204 °C

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CONCLUSIONS In summary, we designed and synthesized 5 symmetric and 16 asymmetric diarylheptanoid derivatives and explored their antiproliferative and chemosensitizing activities. Compounds 2, 12, and 14 containing 3,5-dialkyl-4-hydroxyphenyl groups exhibited significant antiproliferative effects against all tested 2058

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(decomposed); 1H NMR (400 MHz, CDCl3): δ 7.64 (2H, d, J = 15.6 Hz), 7.20 (4H, s), 6.61 (2H, d, J = 15.6 Hz), 4.89 (2H, s), 2.44 (3H, s), 2.28 (12H, s); 13C NMR (150 MHz, CDCl3): δ 176.8, 169.5, 154.6, 142.5, 129.3, 127.3, 123.5, 114.8, 20.7, 15.9; HRMS (m/z): [M + H]+ calcd for C25H27O6, 423.1808; found, 423.1794. 3-Hydroxy-1,7-bis(4-hydroxy-3,5-diethylphenyl)-5-oxohepta-1,3,6-trien-4-yl Acetate (5). 2,4-Dioxopentan-3-yl acetate (40.6 mg, 0.26 mmol) and B2O3 (26.7 mg, 0.38 mmol) were dissolved in EtOAc (4.0 mL). The solution was stirred for 30 min at 70 °C. 3,5-Diethyl-4-hydroxybenzaldehyde (91.3 mg, 0.51 mmol) and (nBuO)3B (0.138 mL, 0.51 mmol) were added, and the mixture was stirred overnight. nBuNH2 (0.051 mL, 0.51 mmol) dissolved in EtOAc (0.5 mL) was added dropwise over 5 min. The stirring was continued for 2 h at 100 °C. The mixture was then hydrolyzed by adding 1 N HCl and stirred for 30 min at room temperature (rt). The reaction mixture was extracted with EtOAc three times, washed with brine, dried over Na2SO4, and then concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 10:1) to give 5 (32.5 mg, 26%) as orange needles. mp 151−152 °C; 1H NMR (400 MHz, CDCl3): δ 7.68 (2H, d, J = 15.6 Hz), 7.22 (4H, s), 6.62 (2H, d, J = 16.0 Hz), 4.96 (2H, s), 2.65 (8H, q, J = 15.2, 7.2 Hz), 2.43 (3H, s), 1.27 (12H, t, J = 7.6 Hz); 13C NMR (150 MHz, CDCl3): δ 176.8, 170.1, 153.8, 142.8, 129.7, 127.6, 127.5, 114.7, 23.0, 20.7, 13.8; HRMS (m/z): [M + H]+ calcd for C29H35O6, 479.2434; found, 479.2419. 1,7-Bis(benzo[b]thiophen-3-yl)-5-hydroxyhepta-1,4,6trien-3-one (6). Acetylacetone (23) (0.052 mL, 0.50 mmol) and B2O3 (53.1 mg, 0.76 mmol) were dissolved in EtOAc (4.0 mL). The solution was stirred for 30 min at 70 °C. Benzo[b]thiophene-3-carboxaldehyde (214.4 mg, 1.32 mmol) and (nBuO)3B (0.27 mL, 1.0 mmol) were added, and the mixture was stirred overnight. nBuNH2 (0.1 mL, 1.0 mmol) dissolved in EtOAc (1.0 mL) was added dropwise over 5 min. The stirring was continued for 2 h at 100 °C. The mixture was then hydrolyzed by adding 1 N HCl and stirred for 30 min at rt. The reaction mixture was extracted with EtOAc three times, washed with brine, dried over Na2SO4, and then concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 20:1) to give 6 (57.9 mg, 30%) as an orange amorphous form. 1H NMR (400 MHz, DMSO-d6): δ 8.45 (2H, s), 8.24 (2H, d, J = 7.6 Hz), 8.09 (2H, d, J = 6.8 Hz), 7.98 (2H, d, J = 16.0 Hz), 7.57−7.47 (4H, m), 7.06 (2H, d, J = 16.0 Hz), 6.45 (1H, s); 13C NMR (150 MHz, CDCl3): δ 183.2, 140.5, 137.3, 132.4, 132.3, 127.7, 125.1, 125.0, 124.6, 123.1, 122.1, 102.1; HRMS (m/z): [M + H]+ calcd for C23H17O2S2, 389.0670; found, 389.0653. General Procedures for the Preparation of Asymmetric Analogues (8−22). Acetylacetone (23) (1.72 mL, 16.6 mmol) and B2O3 (348.0 mg, 5.0 mmol) were dissolved in EtOAc (8.0 mL). The solution was stirred for 30 min at 70 °C. 3,5-Dimethyl-4-hydroxybenzaldehyde (501.3 mg, 3.3 mmol) and (nBuO)3B (0.90 mL, 3.3 mmol) were added, and the mixture was stirred overnight. nBuNH2 (0.33 mL, 3.3 mmol) dissolved in EtOAc (2.0 mL) was added dropwise over 5 min. Stirring was continued for 2 h at 100 °C. The mixture was then hydrolyzed by adding 1 N HCl and stirred for 30 min at rt. The reaction mixture was extracted with EtOAc three times. The combined organic phases were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel

(hexane/EtOAc = 12:1) to obtain diketone 26 (R1 = R3 = Me, R2 = OH, X = H, 434.0 mg, 56%), of which 66.8 mg (0.29 mmol) was dissolved in EtOAc (4.0 mL) together with B2O3 (30.1 mg, 0.43 mmol). The reaction mixture was stirred for 30 min at 70 °C. 3,4,5-Trimethoxybenzaldehyde (57.5 mg, 0.29 mmol) and (nBuO)3B (0.078 mL, 29 mmol) were added, and the mixture was stirred overnight. nBuNH2 (0.028 mL, 0.29 mmol) dissolved in EtOAc (0.5 mL) was added dropwise over 5 min. The stirring was continued for 2 h at 100 °C. The mixture was hydrolyzed with 1 N HCl and stirred for 30 min at rt. The reaction mixture was extracted with EtOAc three times. The combined organic phases were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (hexane/EtOAc = 6:1) to give 8 (68.9 mg, 58%). 5-Hydroxy-1-(4-hydroxy-3,5-dimethylphenyl)-7-(3,4,5trimethoxyphenyl)hepta-1,4,6-trien-3-one (8). Yellow needles. mp 170−174 °C; 1H NMR (400 MHz, CDCl3): δ 7.58 (1H, d, J = 16.0 Hz), 7.57 (1H, d, J = 16.0 Hz), 7.22 (2H, s), 6.79 (2H, s), 6.52 (1H, d, J = 13.0 Hz), 6.48 (1H, d, J = 13.0 Hz), 5.81 (1H, s), 4.90 (1H, s), 3.91 (6H, s), 3.89 (3H, s), 2.28 (6H, s); 13C NMR (100 MHz, CDCl3): δ 184.4, 182.1, 154.4, 153.5, 141.0, 140.1, 139.9, 130.7, 129.0, 127.2, 123.5, 123.4, 121.4, 105.2, 101.4, 61.0, 56.2, 15.9; HRMS (m/z): [M + H]+ calcd for C24H27O6, 411.1808; found, 411.1792. 7-(4-Fluoro-3-methoxyphenyl)-5-hydroxy-1-(4-hydroxy3,5-dimethylphenyl)hepta-1,4,6-trien-3-one (9). 37% yield. Yellow prisms. mp 184−187 °C; 1H NMR (400 MHz, CDCl3): δ 7.58 (1H, d, J = 15.6 Hz), 7.58 (1H, d, J = 15.6 Hz), 7.23 (2H, s), 7.14−7.01 (3H, m), 6.52 (1H, d, J = 15.8 Hz), 6.49 (2H, d, J = 15.8 Hz), 5.80 (1H, s), 4.91 (1H, s), 3.94 (3H, s), 2.28 (6H, s); 13C NMR (150 MHz, CDCl3): δ 184.6, 181.8, 154.5, 148.0, 141.2, 139.0, 131.8, 129.0, 127.2, 123.9, 123.5, 121.4, 116.6, 116.4, 112.3, 101.5, 56.3, 15.9; HRMS (m/ z): [M + H]+ calcd for C22H22FO4, 369.1502; found, 369.1477. 7-(3-Fluoro-4-hydroxyphenyl)-5-hydroxy-1-(4-hydroxy3,5-dimethylphenyl)hepta-1,4,6-trien-3-one (10). 29% yield. An orange amorphous form. 1H NMR (400 MHz, CDCl3): δ 7.57 (1H, d, J = 15.4 Hz), 7.54 (1H, d, J = 15.4 Hz), 7.31 (1H, dd, J = 11.4, 1.7 Hz), 7.24 (1H, d, J = 1.7 Hz), 7.23 (2H, s), 7.04−7.00 (1H, m), 6.49 (1H, d, J = 16.0 Hz), 6.47 (1H, d, J = 16.0 Hz), 5.77 (1H, s), 5.36 (1H, s), 4.89 (1H, s), 2.28 (6H, s); 13C NMR (100 MHz, acetone-d6): δ 156.7, 153.7, 151.3, 147.8, 147.6, 141.9, 139.6, 129.9, 128.8, 127.5, 126.6, 125.4, 123.6, 121.8, 118.9, 116.0, 115.8, 102.0, 16.6; HRMS (m/z): [M + H]+ calcd for C21H20FO4, 355.1346; found, 355.1315. 5-Hydroxy-1-(4-hydroxy-3,5-dimethylphenyl)-7-(4-hydroxy-3-nitrophenyl)hepta-1,4,6-trien-3-one (11). 44% yield. An orange amorphous form. 1H NMR (400 MHz, CDCl3): δ 10.73 (1H, s), 8.29 (1H, d, J = 2.0 Hz), 7.79 (1H, dd, J = 8.8, 2.0 Hz), 7.60 (1H, d, J = 12.8 Hz), 7.53 (1H, d, J = 12.8 Hz), 7.24 (2H, s), 7.21 (1H, d, J = 8.8 Hz), 6.57 (1H, d, J = 15.6 Hz), 6.51 (1H, d, J = 15.6 Hz), 5.81 (1H, s), 4.90 (1H, s), 2.28 (6H, s); 13C NMR (150 MHz, acetone-d6): δ 156.9, 156.1, 142.4, 137.7, 136.7, 135.5, 130.0, 128.9, 127.4, 125.7, 125.6, 125.4, 121.8, 121.5, 102.3, 16.6; HRMS (m/z): [M + H]+ calcd for C21H20NO6, 382.1291; found, 382.1259. 7-(3,4-Dihydroxy-5-methoxyphenyl)-5-hydroxy-1-(4-hydroxy-3,5-dimethylphenyl)hepta-1,4,6-trien-3-one (12). 21% yield. A red amorphous form. 1H NMR (400 MHz, DMSOd6): δ 9.16 (1H, s), 8.98 (1H, s), 8.90 (1H, s), 7.48 (1H, d, J = 15.6 Hz), 7.45 (1H, d, J = 15.6 Hz), 7.32 (2H, s), 6.89 (1H, d, J = 1.8 Hz), 6.77 (1H, d, J = 1.8 Hz), 6.68 (1H, d, J = 15.8 2059

DOI: 10.1021/acsomega.8b03215 ACS Omega 2019, 4, 2053−2062

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(18). 42% yield. An orange amorphous form. 1H NMR (400 MHz, CDCl3): δ 7.68 (2H, d, J = 12.0 Hz), 7.64 (2H, d, J = 12.0 Hz), 7.21 (2H, s), 7.13−7.07 (3H, m), 6.66 (1H, d, J = 16.2 Hz), 6.62 (1H, d, J = 16.2 Hz), 4.95 (1H, s), 3.94 (3H, s), 2.43 (3H, s), 2.28 (6H, s); 13C NMR (150 MHz, CDCl3): δ 178.5, 174.8, 170.0, 154.9, 148.0, 143.3, 140.8, 131.8, 129.4, 127.5, 127.1, 123.6, 121.2, 121.1, 117.5, 116.5, 114.7, 113.4, 56.4, 20.7, 15.9; HRMS (m/z): [M + H]+ calcd for C24H24FO6, 427.1557; found, 427.1535. 1-(3,4-Dihydroxy-5-methoxyphenyl)-3-hydroxy-7-(4-hydroxy-3,5-dimethylphenyl)-5-oxohepta-1,3,6-trien-4-yl Acetate (19). 21% yield. A red amorphous form. 1H NMR (400 MHz, CDCl3): δ 7.69 and 7.65 (1H, d, J = 15.6 and 15.4 Hz), 7.66 and 7.60 (1H, d, J = 15.6 and 15.4 Hz), 7.20 (2H, s), 6.71−6.59 (4H, m), 6.63 (2H, s), 6.59 (1H, s), 5.97 (1H, s), 5.72 and 5.66 (0.2:1, 1H, s), 5.35 and 5.32 (1:0.2, 1H, s), 4.97 and 4.92 (0.2:1, 1H, s), 3.93 (3H, s), 2.43 and 2.31 (1:0.2, 3H, s), 2.28 and 2.26 (1:0.2, 6H, s); 13C NMR (150 MHz, CDCl3): δ 190.9 and 189.5, 177.5 and 175.9, 170.1 and 169.4, 155.0 and 154.7, 147.0 and 146.9, 146.7 and 146.6, 144.1 and 144.0, 142.8 and 142.2, 134.8, 130.0 and 129.3, 127.3, 127.2, 126.3, 123.6 and 123.5, 119.0 and 117.9, 115.9 and 115.9 and 114.8, 110.3, 108.0, 104.8, 103.9, 56.3 and 56.3, 20.8 and 20.7, 15.9 and 15.8; HRMS (m/z): [M + H]+ calcd for C24H25O8, 441.1549; found, 441.1533. 3-Hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-1,3,6-trien-4-yl Acetate (20). 21% yield. A red prism. mp 122−125 °C; 1H NMR (400 MHz, CDCl3): δ 7.74 and 7.69 (1H, d, J = 15.8 and 15.6 Hz), 7.71 and 7.66 (1H, d, J = 15.8 and 15.6 Hz), 7.17 and 7.16 (1H, dd, J = 8.2, 2.0 and 8.2, 1.6 Hz), 7.08 and 7.01 (1H, d, J = 2.0 and 1.6 Hz), 6.94 and 6.93(1H, d, J = 8.2 and 8.2 Hz), 6.90 and 6.62 (1H, d, J = 15.6 and 15.6 Hz), 6.89 and 6.60 (1H, d, J = 16.0 and 15.6 Hz), 6.83 and 6.79 (2H, s), 6.00 (2H, s), 5.95 and 5.86 (1:0.7, 1H, s), 5.89 and 5.80 (1:0.7, 1H, s), 3.95 and 3.95 (3H, s), 3.94 and 3.94 (6H, s), 2.41 and 2.31(1:0.7, 3H, s); 13C NMR (150 MHz, CDCl3): δ 189.6 and 189.4, 176.9 and 176.3, 170.0 and 169.3, 149.1 and 149.1, 148.2 and 148.1, 147.2, 146.9 and 146.8, 146.8 and 146.7, 142.7 and 142.7, 137.5, 127.6 and 127.3, 126.6, 125.5, 124.7, 122.6, 118.6 and 118.2, 115.4, 115.2, 114.9 and 114.8, 110.7 and 110.7, 109.9, 106.0 and 105.5, 56.4 and 56.4, 56.1 and 56.0, 20.8 and 20.6; HRMS (m/z): [M + H]+ calcd for C24H25O9, 457.1499; found, 457.1492. 7-(3,5-Diethyl-4-hydroxyphenyl)-3-hydroxy-1-(4-hydroxy3,5-dimethoxyphenyl)-5-oxohepta-1,3,6-trien-4-yl Acetate (21). 44% yield. An orange amorphous form. 1H NMR (400 MHz, CDCl3): δ 7.70 (1H, d, J = 15.4 Hz), 7.65 (1H, d, J = 15.4 Hz), 7.22 (2H, s), 6.79 (2H, s), 6.63 (1H, d, J = 15.8 Hz), 6.60 (1H, d, J = 15.8 Hz), 5.80 (1H, s), 5.00 (1H, s), 3.94 (6H, s), 2.65 (4H, q, J = 14.8, 7.6 Hz), 2.42 (3H, s), 1.27 (6H, t, J = 7.6 Hz); 13C NMR (150 MHz, CDCl3): δ 178.0, 175.2, 171.4, 153.9, 147.2, 142.5, 141.8, 137.4, 129.7, 127.5, 127.4, 126.8, 115.5, 114.6, 105.5, 98.5, 56.4, 23.0, 20.7, 13.8; HRMS (m/z): [M + H]+ calcd for C27H31O8, 483.2019; found, 483.2011. 7-(3,5-Diethyl-4-hydroxyphenyl)-1-(3,4-dihydroxy-5-methoxyphenyl)-3-hydroxy-5-oxohepta-1,3,6-trien-4-yl Acetate (22). 50% yield. A red amorphous form. 1H NMR (400 MHz, CDCl3): δ 7.73 and 7.69 (1H, d, J = 16.4 and 15.8 Hz), 7.66 and 7.60 (1H, d, J = 16.4 and 15.8 Hz), 7.27 and 7.22 (2H, s), 6.91 and 6.90 (1H, s), 6.71 and 6.63 (1H, d and s, J = 2.0 Hz), 6.90 and 6.62 (1H, d, J = 15.8 and 15.8 Hz), 6.89 and 6.61 (1H, d, J = 15.8 and 15.8 Hz), 5.98 (1H, s), 5.73 and 5.66

Hz), 6.67 (1H, d, J = 15.8 Hz), 6.04 (1H, s), 3.82 (3H, s), 2.19 (6H, s); 13C NMR (150 MHz, acetone-d6): δ 184.8, 184.2, 156.7, 149.2, 146.5, 141.6, 141.5, 137.5, 129.8, 127.5, 127.3, 125.4, 122.5, 121.9, 110.3, 104.7, 101.7, 56.5, 16.6; HRMS (m/ z): [M + H]+ calcd for C22H23O6, 383.1495; found, 383.1491. 1-(3,5-Diethyl-4-hydroxyphenyl)-5-hydroxy-7-(4-hydroxy3,5-dimethoxyphenyl)hepta-1,4,6-trien-3-one (13). 48% yield. Red needles. mp 162−163 °C; 1H NMR (400 MHz, CDCl3): δ 7.61 (1H, d, J = 15.4 Hz), 7.57 (1H, d, J = 15.4 Hz), 7.24 (2H, s), 6.81 (2H, s), 6.50 (1H, d, J = 15.6 Hz), 6.49 (1H, d, J = 15.6 Hz), 5.81 (1H, s), 5.76 (1H, s), 4.95 (1H, s), 3.95 (6H, s), 2.65 (4H, q, J = 15.0, 7.4 Hz), 1.28 (6H, t, J = 7.6 Hz); 13C NMR (150 MHz, CDCl3): δ 183.9, 182.7, 153.5, 147.2, 141.0, 140.5, 137.0, 129.7, 127.5, 127.1, 126.7, 122.2, 121.4, 105.0, 101.2, 56.4, 23.0, 13.8; HRMS (m/z): [M + H]+ calcd for C25H29O6, 425.1964; found, 425.1923. 1-(3,5-Diethyl-4-hydroxyphenyl)-7-(3,4-dihydroxy-5-methoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (14). 50% yield. A red amorphous form. 1H NMR (400 MHz, CDCl3): δ 7.60 (1H, d, J = 15.6 Hz), 7.53 (1H, d, J = 15.6 Hz), 7.24 (2H, s), 6.87 (1H, d, J = 1.6 Hz), 6.68 (1H, d, J = 2.0 Hz), 6.51 (1H, d, J = 15.8 Hz), 6.47 (1H, d, J = 15.8 Hz), 5.80 (1H, s), 5.61 (1H, s), 5.32 (1H, s), 4.95 (1H, s), 3.94 (3H, s), 2.65 (4H, q, J = 15.2, 7.2 Hz), 1.28 (6H, t, J = 7.6 Hz); 13C NMR (150 MHz, CDCl3): δ 184.0, 182.7, 153.5, 147.0, 144.1, 141.1, 140.3, 134.5, 130.0, 127.5, 127.4, 127.1, 122.5, 121.4, 108.7, 103.5, 101.3, 56.3, 23.0, 13.8; HRMS (m/z): [M + H]+ calcd for C24H27O6, 411.1808; found, 411.1799. 1-(Benzo[b]thiophen-3-yl)-5-hydroxy-7-(4-hydroxy-3,5dimethylphenyl)hepta-1,4,6-trien-3-one (15). 54% yield. Orange needles. mp 213−214 °C; 1H NMR (400 MHz, CDCl3): δ 8.06 (1H, d, J = 7.6 Hz), 7.95 (1H, d, J = 15.8 Hz), 7.90 (1H, d, J = 7.6 Hz), 7.79 (1H, s), 7.59 (1H, d, J = 15.8 Hz), 7.52−7.40 (2H, m), 7.24 (2H, s), 6.72 (1H, d, J = 15.8 Hz), 6.51 (1H, d, J = 15.8 Hz), 5.84 (1H, s), 4.90 (1H, s), 2.28 (6H, s); 13C NMR (150 MHz, CDCl3): δ 184.4, 182.1, 154.5, 141.1, 140.5, 137.3, 132.4, 131.6, 129.0, 127.2, 125.0, 124.9, 124.7, 123.6, 123.0, 122.1, 121.4, 101.6, 15.9; HRMS (m/z): [M + H]+ calcd for C23H21O3S, 377.1211; found, 377.1194. 1-(Benzo[b]thiophen-3-yl)-5-hydroxy-7-(3,4,5trimethoxyphenyl)hepta-1,4,6-trien-3-one (16). 25% yield. A red amorphous form. 1H NMR (400 MHz, CDCl3): δ 8.06 (1H, d, J = 7.6 Hz), 7.97 (1H, d, J = 15.8 Hz), 7.90 (1H, d, J = 7.6 Hz), 7.80 (1H, s), 7.61 (1H, d, J = 15.8 Hz), 7.51−7.41 (2H, m), 6.80 (2H, s), 6.73 (1H, d, J = 15.8 Hz), 6.56 (1H, d, J = 15.8 Hz), 5.89 (1H, s), 3.92 (6H, s), 3.90 (3H, s); 13C NMR (150 MHz, CDCl3): δ 183.2, 183.1, 153.5, 140.7, 140.5, 140.1, 137.3, 132.3, 132.2, 130.5, 127.6, 125.1, 125.0, 124.6, 123.4, 123.1, 122.1, 105.3, 101.8, 61.0, 56.2; HRMS (m/z): [M + H]+ calcd for C24H23O5S, 423.1266; found, 423.1246. 3-Hydroxy-7-(4-hydroxy-3,5-dimethylphenyl)-5-oxo-1(3,4,5-trimethoxyphenyl)hepta-1,3,6-trien-4-yl Acetate (17). 49% yield. Yellow needles. mp 193−196 °C; 1H NMR (400 MHz, CDCl3): δ 7.67 (1H, d, J = 15.6 Hz), 7.64 (1H, d, J = 15.6 Hz), 7.21 (2H, s), 6.77 (2H, s), 6.64 (1H, d, J = 15.4 Hz), 6.62 (1H, d, J = 15.4 Hz), 4.94 (1H, s), 3.91 (6H, s), 3.90 (3H, s), 2.42 (3H, s), 2.28 (6H, s); 13C NMR (150 MHz, CDCl3): δ 178.1, 175.2, 170.0, 154.8, 153.4, 143.2, 142.0, 140.4, 130.6, 129.4, 127.4, 127.2, 123.6, 116.9, 114.7, 105.6, 61.0, 56.2, 20.7, 15.9; HRMS (m/z): [M + H]+ calcd for C26H29O8, 469.1862; found, 469.1844. 1-(4-Fluoro-3-methoxyphenyl)-3-hydroxy-7-(4-hydroxy3,5-dimethylphenyl)-5-oxohepta-1,3,6-trien-4-yl Acetate 2060

DOI: 10.1021/acsomega.8b03215 ACS Omega 2019, 4, 2053−2062

ACS Omega

Article

supported in part by the Eshelman Institute for Innovation, Chapel Hill, North Carolina, awarded to M.G.

(0.9:1, 1H, s), 5.36 and 5.73 (1:0.9, 1H, s), 5.04 and 4.99 (0.9:1, 1H, s), 3.93 and 3.93 (3H, s), 2.65 and 2.63 (4H, q, J = 15.2, 7.4 Hz), 2.42 and 2.31 (1:0.9, 3H, s), 1.27 and 1.26 (6H, t, J = 7.4 Hz); 13C NMR (150 MHz, CDCl3): δ 189.6 and 189.5, 177.6 and 175.8, 170.1 and 169.4, 154.6 and 153.9, 147.0 and 146.9, 146.6, 144.1 and 144.0, 143.1, 142.1, 134.8, 129.8 and 129.7, 128.2, 127.5, 127.4 and 127.3, 127.0, 119.0, 115.9, 114.7, 110.4 and 110.3, 108.1 and 108.0, 104.8, 103.9, 56.4 and 56.3, 23.0 and 23.0, 20.8 and 20.7, 13.8 and 13.7; HRMS (m/z): [M + H]+ calcd for C26H29O8, 469.1862; found, 469.1849. Antiproliferative Activity Assay. A549, MDA-MB-231, MCF-7, and KB cell lines were provided by the Lineberger Comprehensive Cancer Center (UNC-CH) or from ATCC (Manassas, VA). KB-VIN was a generous gift from Professor Y.-C. Cheng (Yale University). The assay for the antiproliferative activity was performed by the sulforhodamine B (SRB) method, as described previously.19 All stock cell lines were cultured in RPMI-1640 medium containing 2 mM L-glutamine and 25 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (Gibco) supplemented with 10% fetal bovine serum (Specialty Medium), streptomycin (100 μg/mL), and penicillin (100 IU) (Corning) at 37 °C with 5% CO2 in air. MDR stock cells (KB-VIN) were maintained in the presence of 100 nM vincristine. Freshly trypsinized cell suspensions were seeded onto 96-well microtiter plates at the densities of 4000− 12 000 cells per well with the compounds and cultured for 72 h. The absorbance at 515 nm was measured using a microplate reader (ELx800, BioTek) operated by Gen5 software (BioTek) after solubilizing the protein-bound SRB with 10 mM Tris base. IC50 was calculated from at least three independent experiments performed in duplicate. Flow Cytometric Analysis. Cells were seeded in a 12-well plate 24 h prior to treatment with compounds for 24 or 48 h. Harvested and 70% EtOH-fixed cells were stained with propidium iodide (PI) containing RNase (BD Bioscience) subjected to flow cytometry (BD LSRFortessa, BD Biosciences).19

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We appreciate the helpful suggestions and editing of the manuscript by Dr. Susan L. Morris-Natschke (UNC-CH).

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03215. 1 H NMR spectra for compounds 1−22 and chemosensitizing effects of compounds 3 and 13 on PTX against KB-VIN and MDA-MB-231 (PDF)

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REFERENCES

(1) Recent review: Pugazhendhi, A.; Edison, T. N. J. I.; Velmurugan, B. K.; Jacob, J. A.; Karuppusamy, I. Toxicity of Doxorubicin (Dox) to different experimental organ systems. Life Sci. 2018, 200, 26−30. (2) Lv, H.; She, G. Naturally occurring diarylheptanoids - A supplementary version. Rec. Nat. Prod. 2012, 6, 321−333. (3) Elsayed, A. S. I. The curcumin as an antioxidant natural herb, with emphasize on its effects against some diseases. Int. J. Appl. Biol. Pharm. Technol. 2016, 7, 26−40. (4) Rezaee, R.; Momtazi, A. A.; Monemi, A.; Sahebkar, A. Curcumin: A potentially powerful tool to reverse cisplatin-induced toxicity. Pharmacol. Res. 2017, 117, 218−227. (5) Mohajeri, M.; Sahebkar, A. Protective effects of curcumin against doxorubicin-induced toxicity and resistance: A review. Crit. Rev. Oncol. Hematol. 2018, 122, 30−51. (6) Pabon, H. J. J. A synthesis of curcumin and related compounds. Recl. Trav. Chim. Pays-Bas 1964, 83, 379−386. (7) Zheng, B.; Yang, L.; Wen, C.; Huang, X.; Xu, C.; Lee, K.-H.; Xu, J. Curcumin analog L3 alleviates diabetic atherosclerosis by multiple effects. Eur. J. Pharmacol. 2016, 775, 22−34. (8) Worachartcheewan, A.; Nantasenamat, C.; Isarankura-NaAyudhya, C.; Prachayasittikul, S.; Prachayasittikul, V. Predicting the free radical scavenging activity of curcumin derivatives. Chemom. Intell. Lab. Syst. 2011, 109, 207−216. (9) Venkateswarlu, S.; Ramachandra, M. S.; Subbaraju, G. V. Synthesis and biological evaluation of polyhydroxycurcuminoids. Bioorg. Med. Chem. 2005, 13, 6374−6380. (10) Ligeret, H.; Barthelemy, S.; Zini, R.; Tillement, J.-P.; Labidalle, S.; Morin, D. Effects of curcumin and curcumin derivatives on mitochondrial permeability transition pore. Free Radical Biol. Med. 2004, 36, 919−929. (11) Youssef, K. M.; El-Sherbeny, M. A.; El-Shafie, F. S.; Farag, H. A.; Al-Deeb, O. A.; Awadalla, S. A. A. Synthesis of curcumin analogues as potential antioxidant, cancer chemopreventive agents. Arch. Pharm. 2004, 337, 42−54. (12) Gafner, S.; Lee, S.-K.; Cuendet, M.; Barthélémy, S.; Vergnes, L.; Labidalle, S.; Mehta, R. G.; Boone, C. W.; Pezzuto, J. M. Biologic evaluation of curcumin and structural derivatives in cancer chemoprevention model systems. Phytochemistry 2004, 65, 2849−2859. (13) Venkatesan, P.; Rao, M. N. A. Structure-activity relationships for the inhibition of lipid peroxidation and the scavenging of free radicals by synthetic symmetrical curcumin analogues. J. Pharm. Pharmacol. 2000, 52, 1123−1128. (14) Nurfina, A. N.; Reksohadiprodjo, M. S.; Timmerman, H.; Jenie, U. A.; Sugiyanto, D.; van der Goot, H. Synthesis of some symmetrical curcumin derivatives and their antiinflammatory activity. Eur. J. Med. Chem. 1997, 32, 321−328. (15) Lin, L.; Shi, Q.; Nyarko, A. K.; Bastow, K. F.; Wu, C.-C.; Su, C.Y.; Shih, C. C.-Y.; Lee, K.-H. Antitumor Agents. 250.†Design and Synthesis of New Curcumin Analogues as Potential Anti-Prostate Cancer Agents. J. Med. Chem. 2006, 49, 3963−3972. (16) Pedersen, U.; Rasmussen, P. B.; Lawesson, S.-O. Synthesis of Naturally Occurring Curcuminoids and Related Compounds. Liebigs Ann. Chem. 1985, 1985, 1557−1569. (17) Valgimigli, L.; Brigati, G.; Pedulli, G. F.; DiLabio, G. A.; Mastragostino, M.; Arbizzani, C.; Pratt, D. A. The effect of ring nitrogen atoms on the homolytic reactivity of phenolic compounds: understanding the radical-scavenging ability of 5-pyrimidinols. Chem.Eur. J. 2003, 9, 4997−5010.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +1-919-843-6325 (M.G.). *E-mail: [email protected]. Phone: +81-76-2646305 (K.N.G.). ORCID

Masuo Goto: 0000-0002-9659-1460 Kyoko Nakagawa-Goto: 0000-0002-1642-6538 Funding

This study was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT KAKENHI, Japan) awarded to K.N.-G. (grant numbers 25293024 and 25670054). This work was also 2061

DOI: 10.1021/acsomega.8b03215 ACS Omega 2019, 4, 2053−2062

ACS Omega

Article

(18) Ieda, N.; Nakagawa, H.; Horinouchi, T.; Peng, T.; Yang, D.; Tsumoto, H.; Suzuki, T.; Fukuhara, K.; Miyata, N. Peroxynitrite generation from a NO-releasing nitrobenzene derivative in response to photoirradiation. Chem. Commun. 2011, 47, 6449−6451. (19) Nakagawa-Goto, K.; Taniguchi, Y.; Watanabe, Y.; Oda, A.; Ohkoshi, E.; Hamel, E.; Lee, K.-H.; Goto, M. Triethylated chromones with substituted naphthalenes as tubulin inhibitors. Bioorg. Med. Chem. 2016, 24, 6048−6057.

2062

DOI: 10.1021/acsomega.8b03215 ACS Omega 2019, 4, 2053−2062