Synthesis and Evaluation of DHMEQ Derivatives with Tertiary Hydroxyl Group Instead of Secondary Hydroxyl Group

Abstract

Newly synthesized dehydroxymethyl epoxyquinomycin (DHMEQ) derivatives 6–9, which contain a tertiary hydroxyl group instead of the original secondary hydroxyl group, showed improved solubility in alcohol while maintaining their inhibitory activity against nitric oxide (NO) production, which is used as an indicator of nuclear factor-kappa B (NF-κB) inhibitory activity. We also synthesized a derivative 5 having a cyclopropane ring and a tertiary hydroxyl group and examined its inhibitory activity against NO production. Although it reacted with a nucleophile in a flask, it did not inhibit NO production. The change from a secondary hydroxyl group to a tertiary hydroxyl group contributed to improve the solubility of the compounds while retaining NO inhibitory activity, but had no effect on improving the activity of the cyclopropane form. Compounds in which the secondary hydroxyl group of DHMEQ was converted to a tertiary hydroxyl group would be excellent NF-κB inhibitor candidates because their solubility is improved without decreasing NO inhibitory activity.

Introduction

Dehydroxymethyl epoxyquinomycin (DHMEQ) 1 is a superior nuclear factor-kappa B (NF-κB) inhibitor that was developed by Umezawa and colleagues around the year 2000.^1,2) It is known that NF-κB is constitutively activated in many types of cancer, therefore NF-κB inhibitors are expected to be novel candidates for cancer therapy. Based on this concept, Umezawa et al. have examined the inhibitory activity of DHMEQ (1) against NF-κB not only in vitro but also in animal models and confirmed that 1 would be a great candidate as an antitumor agent.³⁾ Although compound 1 is a potent NF-κB inhibitor, there are two points to be improved. The first point is that 1 is easily reacted with nucleophiles present in a living body and hard to react specifically with the Cys³⁸ of NF-κB. In 2018, Umezawa and colleagues synthesized a new DHMEQ analogue 2 to overcome this problem, considering that high reactivity with many chemicals result from an epoxide moiety.⁴⁾ This newly synthesized compound possesses an α-methylene-γ-lactone moiety and is more stable than DHMEQ 1 and also showed stronger inhibitory activity against NF-κB. Recently Otsuka and Fujita synthesized and evaluated N-(7,8-dioxo-7,8-dihydronaphthalen-1-yl)-2-methoxybenzamide (MBNQ) (3) that contains partial structure of 1 to make a better NF-κB inhibitor utilizing DHMEQ.⁵⁾ The other point is that compound 1 shows low solubility in many solvents. DHMEQ shows anticancer activity in pancreatic cancer models, but is limited to intraperitoneal administration due to its low solubility. To solve this problem, Kitagawa and Umezawa investigated intravenous administration of DHMEQ by polymer aggregation.⁶⁾ We have tried to overcome these problems and synthesized a DHMEQ derivative 4 and assessed its inhibitory activity against NF-κB by estimating cyclooxygenase-2 (COX-2) expression as an indicator.⁷⁾ It was expected that the cyclopropane moiety would react with a cysteine residue of NF-κB, but unfortunately, compound 4 did not inhibit NF-κB activity. Further, the solubility of 4 was similar to that of 1; however, it was found that newly synthesized derivatives 5 and 6–9 improved their solubility and became more soluble in alcohol. We report here a new potential DHMEQ derivatives with better solubility that maintains inhibitory activity against nitric oxide (NO) production (Fig. 1).

Fig. 1. DHMEQ and Its Derivatives

Results and Discussion

During the synthesis of 4, an interesting reaction was observed. Treatment of compound 10 with trimethylsilyl chloride (TMSCl) gave the seven-membered ring compound 11 immediately in good yield⁸⁾ (Chart 1). To examine how this ring expansion reaction occurs, compound 13a with a tertiary hydroxyl group was synthesized. When compound 13a was reacted with TMSCl, the cyclopropane ring showed different reactivity, that is, the addition of chloride ion occurred and a bond outside the six-membered ring was cleaved. This type of reaction was also observed when 13b, a compound with a vinyl group, was used. Further, compound 5, containing a salicylic acid moiety, gave the same type of adduct 16, even though the yield was only 14% (Chart 2). These results suggested that compound 5, containing a cyclopropane ring and a tertiary hydroxyl group, would react with a cysteine residue of NF-κB and show inhibitory activity against NF-κB.

Chart 1. Cyclopropane Ring Expansion of Compound 10

Chart 2. Synthesis of Tertiary Alcohols and Their Reaction with TMSCl

The inhibitory activity against NF-κB was examined by measuring NO level in lipopolysaccharide (LPS)-treated RAW 264.7 macrophage cells using Griess reagent.⁹⁾ Cells were treated with LPS, and whether NO production decreases by adding synthesized compound 5 was examined. LPS-induced NO production was decreased by adding DHMEQ without cytotoxicity until 30 µM. However, compound 5 did not decrease LPS-induced NO production (Figs. 2 (A), (B)).

Fig. 2. Effects of DHMEQ Derivatives on NO Production in LPS-Stimulated RAW264.7 Cells

Cells were seeded at 1 × 10⁶ cells mL⁻¹ in 96-well plates and cultured overnight. Then, the indicated concentrations of DHMEQ (A), 5 (B), 6 (C), 7 (D), 8 (E), and 9 (F) were added. After the cells were treated with 1 µg/mL LPS (Santa Cruz Biotech) and incubated for 24 h, NO level was measured using Griess reagent (columns). Their cytotoxicity was measured using MTT assay (circles). Values are the means ±standard deviation (S.D.) of triplicate determinations.

From the results above, although compound 5 did not inhibit NO production, the solubility in methanol was improved. Then, compounds 6–9 having an epoxide and a tertiary hydroxyl group were synthesized aiming for both inhibitory activity and improved solubility (Chart 3). Epoxyquinone 17 was reacted with four kinds of Grignard reagents (MeMgBr, EtMgBr, n-PrMgBr, n-BuMgCl) and compounds 6–9 were obtained. All four newly synthesized compounds were readily soluble in alcohol. For example, compound 6 (5.8 mg) was dissolved in 1 mL of MeOH, though DHMEQ could not be dissolved in MeOH at all. The increased solubility of compounds 5–9 may result from some intramolecular hydrogen bonding being lost due to the addition of alkyl groups, which are bigger than a hydrogen atom.¹⁰⁾ The inhibitory activity of compounds 6–9 against NO production in LPS-stimulated RAW264.7 cells were examined (Figs. 2 (C)–(F)). After treatment with LPS, RAW 264.7 cells were added to various concentrations of one compound (6–9). When cells were treated with compounds (6–9), the NO levels were decreased in a dose-dependent manner. As a result, compounds 6–9 exhibited comparable inhibitory activity towards NO production and improved solubility compared to the original DHMEQ (1).

Chart 3. Synthesis of DHMEQ Derivatives with a Tertiary Hydroxyl Group

Conclusion

We have developed new DHMEQ derivatives 6–9 that inhibit NO production with nearly the same strength as DHMEQ, and the compounds showed improved solubility in alcohol. We believe this provides a clue to improving one of the two problems facing DHMEQ. Conversion of secondary hydroxyl groups to tertiary hydroxyl groups did not inhibit NO production in a compound with cyclopropane. The conformation of the hydroxyl group on the cyclohexenone ring may affect the solubility due to intramolecular hydrogen bonding. We would like to further investigate whether compounds 6–9 can be used for in vivo studies in the future.

Experimental

Synthesis of Compounds

General: ¹H- and ¹³C-NMR spectra were recorded on a JEOL JNM-ECX 400 and JNM-ECZ 400S/L1 spectrometers at 400 and 100 MHz, respectively. Chemical shifts were expressed in δ parts per million with tetramethylsilane as internal standard (δ = 0 ppm) for ¹H-NMR. Chemical shifts of carbon signals were referenced to CDCl₃ (δ_C = 77.0 ppm). The following abbreviations are used: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad. IR spectra were recorded on a SHIMADZU IR Prestige-21. Mass spectra were recorded on a JEOL JMS-GCmate II. Melting points were determined on a Yanagimoto MP-S3 micro melting point apparatus and were uncorrected. All reagents and solvents were purchased from commercial sources and used without further purification.

13a

To a solution of 12 (0.12 g, 0.56 mmol) in tetrahydrofuran (THF) (5 mL) was added methyl magnesium bromide (3.0 M in Et₂O, 0.23 mL, 0.67 mmol) at 0 °C and stirred for 3.5 h. The reaction mixture was quenched with saturated aqueous NH₄Cl and extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with hexane-ethyl acetate (1 : 1) to give 13a (50 mg, 0.21 mmol, 38%).

¹H-NMR (CDCl₃, 400 MHz) δ: 7.40 (s, 1H), 6.42 (d, J = 1.6 Hz, 1H), 5.92 (m, 1H), 5.36 (dq, J = 17.2, 1.6 Hz, 1H), 5.28 (dd, J = 10.4, 1.2 Hz, 1H), 4.64 (dt, J = 6.0, 1.2 Hz, 2H), 2.88 (s, 1H), 2.04 (m, 1H), 1.90 (dt, J = 8.0, 6.0 Hz, 1H), 1.60 (s, 3H), 1.24 (m, 1H), 1.03 (q, J = 4.4 Hz, 1H); ¹³C-NMR (CDCl₃, 100 MHz) δ: 197.03 (4), 152.46 (4), 152.41 (4), 131.64 (3), 119.04 (2), 103.46 (3), 69.93 (4), 66.49 (2), 31.19 (1), 24.42 (3), 24.04 (3), 13.88 (2); IR (film) 3372, 1744, 1628, 1512, 1204, 1042 cm⁻¹; high resolution (HR)-MS (electron ionization (EI)) Calcd for C₁₂H₁₅NO₄ 237.1001. Found 237.1012.

13b

To a solution of 12 (0.33 g, 1.50 mmol) in THF (15 mL) was added vinyl magnesium bromide (1.0 M in THF, 3.7 mL, 3.7 mmol) at 0 °C and stirred for 10 min. The reaction mixture was quenched with saturated aqueous NH₄Cl and extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with hexane-ethyl acetate (1 : 1) to give 13b (98 mg, 0.39 mmol, 26%).

¹H-NMR (400 MHz, CDCl₃) δ: 6.50 (d, J = 1.2 Hz, 1H), 5.98–5.85 (m, 2H), 5.50 (d, J = 16.8 Hz, 1H), 5.34 (dq, J = 17.6, 1.2 Hz, 1H), 5.27 (dq, J = 11.6, 1.2 Hz, 1H), 5.24 (d, J = 10.0 Hz, 1H), 4.61 (m, 2H), 3.62 (s, 1H), 2.05 (m, 1H), 1.90 (dt, J = 7.6, 6.0 Hz, 1H), 1.27 (m, 1H), 1.12 (q, J = 4.8 Hz, 1H); ¹³C-NMR (100 MHz, CDCl₃) δ: 197.39 (4), 152.37 (4), 150.12 (4), 140.97 (3), 131.63 (3), 118.99 (2), 115.13 (2), 104.44 (3), 71.99 (4), 66.46 (2), 24.03 (3), 23.19 (3), 13.81 (2); IR (film) 3379, 1746, 1622, 1514, 1504, 1204, 1038, 758 cm⁻¹; HR-MS (EI) Calcd for C₁₃H₁₅NO₄ 249.1001. Found 249.1005.

14a

To a solution of 13a (20 mg, 0.084 mmol) in THF (0.84 mL) was added trimethylsilyl chloride (0.053 mL, 0.42 mmol) at 0 °C and stirred for 3 d at room temperature (r.t.). The reaction mixture was quenched with saturated aqueous NaHCO₃ and extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with hexane-ethyl acetate (2 : 3) to give 14a (17 mg, 0.062 mmol, 74%).

Recrystallization from ethyl acetate/hexane; mp 150.0–151.0 °C; ¹H-NMR (400 MHz, CDCl₃/CD₃OD) δ: 6.63 (s, 1H), 5.95 (m, 1H), 5.37 (dq, J = 16.8, 1.6 Hz, 1H), 5.30 (dd, J = 10.4, 1.6 Hz, 1H), 4.66 (d, J = 5.6 Hz, 2H), 4.03 (dq, J = 10.8, 1.6 Hz, 1H), 3.37 (t, J = 10.8 Hz, 1H), 2.91 (d, J = 18.8 Hz, 1H), 2.61 (ddd, J = 18.8, 5.6, 1.6 Hz, 1H), 2.45 (m, 1H), 1.63 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃/CD₃OD) δ: 196.67 (4), 157.56 (4), 152.09 (4), 131.36 (3), 118.95 (2), 107.36 (3), 70.53 (4), 66.52 (2), 48.22 (3), 43.51 (2), 36.94 (2), 28.67 (1); IR (KBr) 3360, 1744, 1628, 1533, 1275, 1227, 1202, 1123, 1101, 1032 cm⁻¹; HR-MS (EI) Calcd for C₁₂H₁₆ClNO₄ 273.0768. Found 273.0795.

14b

To a solution of 13b (50 mg, 0.20 mmol) in THF (2.0 mL) was added trimethylsilyl chloride (0.13 mL, 1.0 mmol) at 0 °C and stirred for 1 d at r.t. The reaction mixture was quenched with saturated aqueous NaHCO₃ and extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with hexane- ethyl acetate (2 : 1) to give 14b (39 mg, 0.13 mmol, 65%).

Recrystallization from ethyl acetate/ hexane; mp 136.0–137.0 °C; ¹H-NMR (400 MHz, CD₃OD) δ: 6.68 (s, 1H), 5.95 (dd, J = 17.6, 10.4 Hz, 1H), 5.86 (m, 1H), 5.33 (d, J = 8.4 Hz, 1H), 5.29 (s, 1H), 5.25 (dq, J = 17.2, 1.2 Hz, 1H), 5.15 (dq, J = 10.8, 1.2 Hz, 1H), 4.54 (d, J = 6.0 Hz, 2H), 3.92 (dd, J = 12.4, 2.4 Hz, 1H), 3.33 (t, J = 11.2 Hz, 1H), 2.63 (dd, J = 17.2, 4.8 Hz, 1H), 2.52 (dd, J = 17.6, 4.8 Hz, 1H), 2.36 (m, 1H); ¹³C-NMR (100 MHz, CD₃OD) δ: 199.30 (4), 156.50 (4), 153.77 (4), 140.36 (3), 133.31 (3), 118.98 (2), 117.69 (2), 110.30 (3), 75.14 (4), 67.54 (2), 47.74 (3), 44.44 (2), 37.26 (2); IR (KBr) 3375, 3194, 1744, 1620, 1526, 1204 cm⁻¹; HR-MS (EI) Calcd for C₁₃H₁₆NO₄Cl 285.0768. Found 285.0771.

5

To a solution of 15 (0.20 g, 0.78 mmol) in THF (6 mL) was added methyl magnesium bromide (3.0 M in Et₂O, 0.90 mL, 2.7 mmol) at −30 °C and stirred for 40 min. The reaction mixture was quenched with saturated aqueous NH₄Cl and extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with CHCl₃-acetone (7 : 1) to give 5 (89 mg, 0.33 mmol, 42%).

Recrystallization from ethyl acetate/hexane; mp 165.1–166.0 °C; ¹H-NMR (400 MHz, CD₃OD/CDCl₃ = 3/1) δ: 7.93 (dd, J = 8.0, 1.6 Hz, 1H), 7.37 (dt, J = 8.0, 1.2 Hz, 1H), 6.97–6.88 (m, 3H), 1.97 (m, 1H), 1.92 (m, 1H), 1.59 (s, 3H), 1.31 (dt, J = 8.4, 5.2 Hz, 1H), 1.10 (q, J = 4.8 Hz, 1H); ¹³C-NMR (100 MHz, CD₃OD/CDCl₃ = 3/1) δ: 201.26 (4), 167.52 (4), 157.59 (4), 156.01 (4), 135.04 (3), 131.76 (3), 120.87 (3), 119.27 (4), 117.56 (3), 106.07 (3), 70.09 (4), 32.06 (1), 24.81 (3), 24.59 (3), 15.43 (2); IR (KBr) 3424, 1740, 1668, 1603, 1528, 1458 cm⁻¹; HR-MS (EI) Calcd for C₁₅H₁₅NO₄ 273.1001. Found 273.0998. The stereochemistry was confirmed by nuclear Overhauser effect (NOE) measurement.

16

To a solution of 5 (68 mg, 0.25 mmol) in THF (2.3 mL) was added trimethylsilyl chloride (0.13 mL, 1.0 mmol) at 0 °C and stirred for 2 h at r.t. The reaction mixture was quenched with saturated aqueous NaHCO₃ and extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with CHCl₃-acetone (10: 1) to give 16 (10 mg, 0.032 mmol, 13%).

recrystallization from ethyl acetate/methanol/hexane; mp 180.0–181.0 °C; ¹H-NMR (400 MHz, dimethyl sulfoxide (DMSO)-d₆) δ: 11.87 (br s, 1H), 11.11 (s, 1H), 7.91 (dd, J = 8.0, 2.0 Hz, 1H), 7.42 (m, 1H), 7.00 (d, J = 7.2 Hz, 1H), 6.95 (t, J = 8.0 Hz, 1H), 6.94 (s, 1H), 6.21 (s, 1H), 4.06 (dd, J = 11.6, 2.0 Hz, 1H), 3.41 (t, J = 11.6 Hz, 1H), 2.72 (dd, J = 18.0, 4.0 Hz, 1H), 2.59 (d, J = 17.6 Hz, 1H), 2.45 (m, 1H), 1.60 (s, 3H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 195.49 (4), 164.92 (4), 156.81 (4), 156.45 (4), 134.25 (3), 131.16 (3), 119.73 (4), 118.43 (3), 117.08 (3), 109.26 (3), 70.56 (4), 47.64 (3), 44.84 (2), 37.00 (2), 28.81 (1); IR (KBr); HR-MS (EI) Calcd for C₁₅H₁₆ClNO₄ 309.0768. Found 309.0767.

6

To a solution of 17 (0.11 g, 0.41 mmol) in THF (6 mL) was added methyl magnesium bromide (3.0 M in Et₂O, 0.45 mL, 1.35 mmol) at −30 °C and stirred for 40 min. The reaction mixture was quenched with saturated aqueous NH₄Cl and extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with CHCl₃-acetone (20 : 1) to give 6 (43 mg, 0.16 mmol, 39%).

Recrystallization from ethyl acetate/methanol/hexane; mp 196.2–196.6 °C; ¹H-NMR (400 MHz, CD₃OD) δ: 7.98 (dd, J = 7.6, 2.0 Hz, 1H), 7.41 (m, 1H), 7.04 (d, J = 2.4 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 6.95 (d, J = 5.6 Hz, 1H), 3.71 (d, J = 4.4 Hz, 1H), 3.44 (dd, J = 4.0, 2.0 Hz, 1H), 1.50 (s, 3H); ¹³C-NMR (100 MHz, CD₃OD) δ: 196.82 (4), 167.49 (4), 157.83 (4), 157.59 (4), 135.49 (3), 132.39 (3), 121.30 (3), 119.73 (4), 117.83 (3), 106.22 (3), 70.59 (4), 58.77 (3), 54.75 (3), 27.69 (1); IR (KBr) 3277, 3238, 3150, 1655, 1636, 1609, 1520, 1497, 1231, 1206 cm⁻¹; HR-MS Calcd for C₁₄H₁₃NO₅ 275.0794. Found 275.0797. The stereochemistry was confirmed by NOE measurement.

7

To a solution of 17 (53.4 mg, 0.21 mmol) in THF (2 mL) was added ethyl magnesium bromide (1.0 M in THF, 0.63 mL, 0.63 mmol) at −30 °C and stirred for 4 h. The reaction mixture was quenched with saturated aqueous NH₄Cl and extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with benzene-acetone (5 : 1) to give 7 (10.0 mg, 0. 035 mmol, 17%).

Recrystallization from ethyl acetate/hexane; mp 81.6–81.9 °C; ¹H-NMR (400 MHz, CD₃OD) δ: 7.98 (dd, J = 7.6, 1.6 Hz, 1H), 7.40 (m, 1H), 7.14 (d, J = 2.4 Hz, 1H), 6.97 (t, J = 7.2 Hz, 1H), 6.94 (d, J = 7.2 Hz, 1H), 3.68 (d, J = 4.4 Hz, 1H), 3.45 (dd, J = 4.0, 2.0 Hz, 1H), 1.88 (dq, J = 7.2, 2.8, 2H), 0.91 (t, J = 7.6 Hz, 3H); ¹³C-NMR (100 MHz, CD₃OD) δ: 197.32 (4), 167.37 (4), 158.05 (4), 155.42 (4), 135.49 (3), 132.41 (3), 121.20 (3), 119.76 (4), 117.91 (3) 107.77 (3), 73.57 (4), 58.02 (3), 54.88 (3), 34.38 (2), 8.40 (1); IR (KBr) 3370, 3260, 2972, 2938, 1643, 1605, 1518, 1308, 1287, 1234, 1190 cm⁻¹; HR-MS Calcd for C₁₅H₁₅NO₅ 289.0940. Found 289.0950. The stereochemistry was confirmed by NOE measurement.

8

To a solution of 17 (0.10 g, 0.40 mmol) in THF (4 mL) was added propyl magnesium bromide (1.0 M in THF, 1.93 mL, 1.93 mmol) at −30 °C and stirred for 40 min. The reaction mixture was quenched with saturated aqueous NH₄Cl and extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with hexane-ethyl acetate (3 : 2) to give 8 (9.4 mg, 0.031 mmol, 8%).

Recrystallization from ethyl acetate/hexane; mp 140.8–141.7 °C; ¹H-NMR (400 MHz, CD₃OD) δ: 7.98 (dd, J = 7.6, 1.2 Hz, 1H), 7.41 (m, 1H), 7.11 (d, J = 2.4 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 6.94 (d, J = 6.8 Hz, 1H), 3.69 (d, J = 4.0 Hz, 1H), 3.44 (dd, J = 4.0, 2.4 Hz, 1H), 1.80 (m, 2H), 1.32 (m, 2H), 0.92 (t, J = 6.8 Hz, 3H); ¹³C-NMR (100 MHz, CD₃OD) δ: 197.28 (4), 167.33 (4), 157.97 (4), 155.76 (4), 135.49 (3), 132.41 (3), 121.23 (3), 119.76 (4), 117.88 (3), 107.56 (3), 73.17 (4), 58.25 (3), 54.89 (3), 43.75 (2), 18.10 (2), 14.53 (1); IR (KBr) 3379, 3254, 2963, 1661, 1605, 1517, 1492, 1456, 1288, 1232, 1207 cm⁻¹; HR-MS Calcd for C₁₆H₁₇NO₅ 303.1106. Found 303.1107. The stereochemistry was confirmed by NOE measurement.

9

To a solution of 17 (0.10 g, 0.39 mmol) in THF (4 mL) was added butyl magnesium chloride (2.0 M in THF, 0.58 mL, 1.16 mmol) at −78 °C and stirred for 50 min. The reaction mixture was quenched with saturated aqueous NH₄Cl and extracted with ethyl acetate. The organic layer was dried over anhydrous MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel eluted with benzene-acetone (4 : 1) to give 9 (33 mg, 0.10 mmol, 26%).

Recrystallization from ethyl acetate/hexane; mp 162.1–162.8 °C; ¹H-NMR (400 MHz, CD₃OD) δ: 7.99 (dd, J = 8.4, 1.6 Hz, 1H), 7.41 (dt, J = 8.4, 1.6 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 3.69 (d, J = 4.4 Hz, 1H), 3.45 (dd, J = 4.0, 2.0 Hz, 1H), 1.83 (m, 2H), 1.30 (m, 4H), 0.87 (t, J = 6.8 Hz, 3H); ¹³C-NMR (100 MHz, CD₃OD) δ: 197.28 (4), 167.29 (4), 157.78 (4), 155.72 (4), 135.49 (3), 132.43 (3), 121.33 (3), 119.75 (4), 117.80 (3), 107.62 (3), 73.17 (4), 58.26 (3), 54.91 (3), 41.23 (2), 26.84 (2), 23.86 (2), 14.16 (1); IR (KBr) 3385, 3233, 1659, 1636, 1526, 1497, 1229, 1206 cm⁻¹; HR-MS Calcd for C₁₇H₁₉NO₅ 317.1263. Found 317.1264. The stereochemistry was confirmed by NOE measurement.

Biological Activity

Murine macrophage RAW264.7 cells were cultured at 37 °C with 5% CO₂ in Dulbecco’s modified Eagle medium (DMEM) (Nissui, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS; GE Healthcare Hyclone Laboratories, Logan, UT, U.S.A.). Cells were seeded at 1 × 10⁶ cells mL⁻¹ in 96-well plates and cultured overnight. The sample solution in MeOH (2 µL) was added to each well (198 µL) and then cells were stimulated with 1 µg/mL LPS (Santa Cruz Biotek, VT, U.S.A.). After the cells were incubated for 24 h, an aliquot of culture medium (100 µL) was mixed with an equal volume of Griess reagent (0.5% sulfamide, 0.05% N-naphthylethylenediamine hydrochloride in 5% phosphoric acid). Optical density at 550 nm was measured with a microplate reader (SYNERGY H1, Agilent, CA, U.S.A.). The cytotoxicity of samples was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. After the aliquot of culture medium was removed for the measurement of NO production, MTT solution was added to the cells. After 4 h, the culture medium was removed and the formazan product was dissolved in DMSO (150 µL). Optical density at 540 nm was measured similarly as above. All assays were performed in duplicate to confirm reproducibility.

Acknowledgments

This study was supported in part by a Grant from the Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sport, Science and Technology, Japan (MEXT), 2014–2018 (S1411005).

We thank Prof. Shinji Nagumo and Prof. Kenji Matsuno for their assistance. We also thank Ms. Rio Ootsuki and Mr. Kazuki Watanabe for their contribution to synthesize some compounds.

Conflict of Interest

The authors declare no conflict of interest.

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