2016 Volume 64 Issue 6 Pages 609-615
Long-term use of nonsteroidal antiinflammatory drugs (NSAIDs) may cause serious side effects such as gastric mucosal damage. Resveratrol, a naturally dietary polyphenol, exhibited anti-inflammatory activity and a protective effect against gastric mucosa damage induced by NSAIDs. In this regard, we synthesized a series of resveratrol-based NSAIDs derivatives and evaluated their anti-inflammatory activity against nitric oxide (NO) overproduction in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. We identified mono-substituted resveratrol–ibuprofen combination 21 as the most potent anti-inflammatory agent, which is more active than a physical mixture of ibuprofen and resveratrol, individual ibuprofen, or individual resveratrol. In addition, compound 21 exerted potent inhibitory effects on the LPS-induced expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Furthermore, compound 21 significantly increased the survival rate in an LPS-induced acute inflammatory model and produced markedly less gastric damage than ibuprofen. It was found that compound 21 may be a potent anti-inflammatory agent for the treatment of inflammation-related diseases.
Nonsteroidal antiinflammatory drugs (NSAIDs, Fig. 1) have been widely used to treat the effects of inflammation over a century. NSAIDs inhibit cyclooxygenase (COX) isoforms, COX-1 and COX-2, which play a key role in the conversion of arachidonic acid to eicosanoids. Long-term use of NSAIDs is limited because of its gastrointestinal toxicity due to the inhibition of the gastroprotective COX-1 present at the gastric mucosa level.1,2) Selective COX-2 inhibitors have been developed to reduce the risk of peptic ulceration, but chronic use of COX-2 inhibitors lead to a significant increase in heart attacks and strokes.3) It is well established that nitric oxide (NO) is able to protect the gastric mucosa by relaxing the blood vessel with lower adhesion of leukocytes.4) NO-releasing NSAIDs (NO-NSAIDs) are being investigated to better control side effects of NSAIDs.5) However, long-term use of NO-NSAIDs can cause nitrate tolerance.6,7)
Resveratrol is a polyphenolic compound present in peanuts, grape skins, plums, and red wine.8) Besides its promising broad-spectrum bioactivities such as cardioprotective effects and antioxidant, anti-inflammatory, anticancer, and neuroprotective activities, resveratrol have a protecting effect against gastric mucosa damage induced by NSAIDs.9,10) Additionally, resveratrol has been shown to be capable of inhibiting lipopolysaccharide (LPS) induced inflammatory response.11,12) A lot of preclinical findings have demonstrated that resveratrol is well tolerated and non-toxic.13) It has been reported that gastric toxicity of NSAIDs are largely dependent on the presence of the carboxylic group,14) therefore, NSAIDs esterified with resveratrol may be a new approach to retaining anti-inflammatory properties while reducing side effects.
In this study, we have synthesized a series of resveratrol-based NSAIDs derivatives and evaluated their anti-inflammatory activity and ulcerogenic properties.
Compounds 8–13 were prepared by direct coupling of resveratrol with corresponding NSAIDs (Fig. 1) using classical 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride (EDCI)/4-dimethylaminopyridine (DMAP) reagents (Chart 1). Initial efforts to obtain mono-substituted conjugate focused on the use of classical EDCI/DMAP reagents. In all cases with various equivalents, the undesired trisubstituted conjugate was found to be the dominant product, because of the increasing reactivity of the three hydroxy groups upon esterification. To obtain mono-substituted resveratrol–NSAIDs conjugates in acceptable yield, targeted mono-substituted conjugates were planned by adapting the existing synthetic strategy for selective syntheses of resveratrol–docosahexaenoic acid (DHA) conjugates.15) Using a procedure described by Acerson and Andrus, resveratrol was regioselectively acylated using NaH and acid anhydrides to give compound 1416) (Chart 2). Treatment compound 14 with trifluoromethanesulfonic acid triisopropylsilyl ester (TIPS-OTf) afforded compound 15 in excellent yield. Deprotection of the acetyl group was carried out using a methanolic solution of NaOMe at room temperature, and gave compound 16 in 95% yields. Compounds 17–20 could be prepared using EDCI coupling from compound 16 in good yield. Cleavage of the TIPS protecting groups afforded the desired resveratrol–NSAIDs conjugates 21–24.
Activated macrophage play a crucial role in the initiation and amplification of inflammation via the sustained overproduction of NO and proinflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β).17) In this study, we examined the effects of these resveratrol-based NSAIDs derivatives on LPS-induced overproduction of NO, TNF-α and IL-1β. Further, active compound was selected to evaluate the protecting effect against LPS-induced death and ulcerogenic properties.18)
Inhibition of NO Production in LPS-Induced MacrophagesResveratrol-based NSAIDs derivatives 8–13 and 21–24 were screened for the inhibitory activity against NO production in LPS-stimulated RAW264.7 macrophages. CCK-8 assays were carried out to determine the non-cytotoxic concentrations of these derivatives. Compounds 8–13 and 21–24 at a concentration of 20 µM had no significant inhibition on cell viability after 24 h incubation (data not shown).
The initial screening demonstrated that NSAIDs 1–6 had no effect on NO production (Table 1). Mono-substituted resveratrol–NSAIDs conjugates 21–24 had a higher inhibitory activity than trisubstituted conjugates 8–13. Compounds 21 and 23 displayed significant inhibitory activities with IC50 of 3.26 and 13.25 µM, respectively. The inhibitory ability of compound 21 was higher than that of resveratrol (IC50=8.31 µM) and compound 21 is more active than an equimolar mixture of 1 and 7.
| Compound | Inhibition of NO (%) at 20 µM | Inhibition of NO IC50 (µM)a) |
|---|---|---|
| 1 | 0.45 | >20 |
| 2 | 0.11 | >20 |
| 3 | 0.32 | >20 |
| 4 | 0.65 | >20 |
| 5 | 1.02 | >20 |
| 6 | 0.03 | >20 |
| 7 | 72.14 | 8.31±0.55 |
| 8 | 5.01 | >20 |
| 9 | 0.11 | >20 |
| 10 | 2.01 | >20 |
| 11 | 0.53 | >20 |
| 12 | 1.22 | >20 |
| 13 | 0.44 | >20 |
| 21 | 86.38 | 3.26±0.15 |
| 22 | 43.17 | >20 |
| 23 | 65.20 | 13.52±0.80 |
| 24 | 25.38 | >20 |
| Dexamethasone | 91.21 | 1.85±0.11 |
a) IC50 values are expressed as the mean±S.E.M. from at least three independent experiments.
Since compound 21 significantly decreased LPS-induced NO production, we determined the effects of compounds 21 and 7 on the production of TNF-α and IL-1β in LPS-stimulated macrophages by enzyme-linked immunosorbent assays (ELISA).
RAW264.7 macrophages were pretreated with compounds 21 and 7 for 6 h and then incubated with LPS for 18 h. It was shown that LPS dramatically increased the production of TNF-α and IL-1β in RAW264.7 macrophages (Fig. 2). The compound 21 dramatically decreased the LPS-induced production of TNF-α and IL-1β at a concentration of 20 µM, and its inhibitory ability was comparable to that of compound 7 at the same concentration.
After incubation with 20 µM of compounds 21 and 7 for 6 h, RAW264.7 macrophages were incubated with LPS for 18 h. The TNF-α and IL-1β levels in the culture medium were measured by commercial ELISA kit. The data are expressed as the mean±S.E.M. from at least three independent experiments. * p<0.05 compared with the LPS group.
Because compound 21 significantly inhibited inflammatory response in LPS-stimulated macrophages, compound 21 was selected to evaluate the protecting effect against LPS-induced death. Mice were treated with a lethal dose of LPS (35 mg/kg, intraperitoneally (i.p.)) in the presence or absence of compounds 7 and 21 and their survival rates were monitored for 6 d. In animals pretreated with 200 mg/kg of compounds 21 and 7, survival rates were significantly increased compared to that of the vehicle-treated group (Fig. 3). In contrast, pretreatment with 50 mg/kg of compounds 21 and 7 failed to increase the survival rate.
Long-term use of NSAIDs may cause serious gastric mucosal damage. Therefore, compund 21 was assessed for its ulcerogenic properties in mice. The severity of gastric damage was expressed as an ulcer index (UI) (Table 2). The parent drug ibuprofen, administrated at 250 mg/kg, produced macroscopically detectable gastric damage (UI=23.5; Fig. 4). The acute gastric toxicity of NSAIDs is largely dependent on the presence of the carboxylic group. It has been reported that ester derivatives of NSAIDs could be presumed to produce markedly less gastric damage than the parent drug. In agreement with our expectations, evidence of gastric damage was not observed for compound 21 (UI=0; Fig. 4).
| Compound | 21 | Ibuprofen | Control |
|---|---|---|---|
| Ulcer indexa) | 0b) | 23.5±2.5b) | 0c) |
a) The average overall length (in mm) of individual ulcers in each stomach±S.E.M., n=6, at 6 h after oral administration of the test compound. b) 250 mg/kg dose. c) 1.0% methylcellulose solution.
We have synthesized a series of resveratrol-based NSAIDs derivatives. All synthesized resveratrol-based NSAIDs derivatives were evaluated for their inhibitory activity against LPS-induced NO production, and compound 21 demonstrated inhibition comparable to that of resveratrol. In addition, compound 21 effectively inhibited LPS-induced TNF-α and IL-1β production. The data acquired in the mouse model demonstrated that compound 21 significantly decreased LPS-induced lethality. Gastric ulcerginicity of compound 21 at the doses of 250 mg/kg was less than the same doses of ibuprofen. It is anticipated that our work will provide a new approach for the rational design of anti-inflammatory drugs.
All commercial chemicals and solvents are reagent grade and were used without further treatment unless otherwise noted. All reactions were carried out under an atmosphere of dry argon. Melting points were determined on a Tech X-4 digital display micro melting point apparatus and are uncorrected. IR spectra were obtained using Perkin-Elmer Spectrum 2000 FT-IR spectrometer. The high resolution-electrospray ionization (HR-ESI)-MS spectra were recorded on an Agilent 6210 series LC/MSD time-of-flight (TOF) from Agilent Technologies. 1H-NMR spectra were obtained with a Bruker AVANCE HD III-400 spectrometer with tetramethylsilane (TMS) as internal standard. The ESI-MS spectra were recorded on an AB Sciex API 4000 QTRAP LC/MS system. TLC was performed using Merck precoated plates (Si gel 60 F254, Germany) of 0.25 mm thickness. The spots on TLC were detected with 254 and 365 nm UV light.
General Procedure for Synthesis of Trisubstituted Conjugates 8–13To a solution of resveratrol in dry dichloromethane at room temperature, NSAIDs, EDCI and DMAP were added. After 8 h, aqueous NaHCO3 solution was added and the aqueous layer was extracted with dichloromethane. The combined organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to give the target compounds.
(E)-5-(4-((2-(4-Isobutylphenyl)propanoyl)oxy)styryl)-1,3-phenylene Bis(2-(3-isobutylphenyl)propanoate) (8)White solid (69%), mp: 57°C, IR (KBr) cm−1: 2954, 2925, 2863, 1753, 1139. HR-ESI-MS m/z: 793.4467 [M+H]+ (Calcd for C53H60O6: 793.4463). MS (ESI) m/z: 793.4 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 7.41 (d, J=8.6 Hz, 2H), 7.30 (d, J=7.6 Hz, 4H), 7.22 (d, J=8.1 Hz, 2H), 7.15 (d, J=8.1 Hz, 4H), 7.10 (d, J=8.1 Hz, 2H), 6.98 (d, J=7.6 Hz, 2H), 6.95 (d, J=16.4 Hz, 1H), 6.86 (d, J=16.3 Hz, 1H), 6.76 (d, J=1.7 Hz, 1H), 6.71 (d, J=1.6 Hz, 1H), 6.40 (t, J=2.1 Hz, 1H), 3.97–3.88 (m, 2H), 3.72 (q, J=7.1 Hz, 1H), 2.47 (d, J=7.2 Hz, 4H), 2.45 (d, J=7.2 Hz, 2H), 1.82–1.90 (m, 3H), 1.60 (d, J=6.8 Hz, 6H), 1.51 (d, J=7.2 Hz, 3H), 0.91 (d, J=6.8 Hz, 12H), 0.89 (d, J=6.8 Hz, 6H). 13C-NMR (101 MHz, CDCl3) δ: 173.10, 172.72, 172.72, 151.49, 151.49, 150.66, 140.90, 140.90, 140.86, 139.34, 137.19, 136.99, 136.99, 134.38, 129.55, 129.55, 129.55, 129.55, 129.53, 129.53, 127.50, 127.50, 127.50, 127.25, 127.25, 127.25, 127.22, 127.22, 127.14, 121.70, 121.70, 121.70, 116.67, 116.67, 114.07, 45.29, 45.29, 45.29, 45.08, 45.08, 45.08, 30.17, 30.17, 29.70, 22.41, 22.41, 22.41, 22.41, 22.41, 22.41, 18.50, 18.50, 14.11.
(E)-5-(4-((2-(6-Methoxynaphthalen-2-yl)propanoyl)oxy)styryl)-1,3-phenylene Bis(2-(6-methoxynaphthalen-2-yl)propanoate) (9)White solid (81%). mp: 75°C, IR (KBr) cm−1: 2925, 1755, 1650, 1595, 1136. HR-ESI-MS m/z: 865.3375 [M+H]+ (Calcd for C56H48O9: 865.3371). MS (ESI) m/z: 865.3 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 7.76–7.70 (m, 9H), 7.51–7.44 (m, 3H), 7.36 (d, J=8.6 Hz, 2H), 7.18–7.13 (m, 6H), 6.97–6.88 (m, 5H), 6.83 (d, J=16.3 Hz, 1H), 6.64 (t, J=2.0 Hz, 1H), 4.05 (dd, J=13.7, 6.7 Hz, 3H), 3.93 (s, 3H), 3.92 (s, 6H), 1.68 (d, J=6.6 Hz, 6H), 1.66 (d, J=6.6 Hz, 3H). 13C-NMR (101 MHz, CDCl3) δ: 173.08, 172.69, 172.69, 157.83, 157.83, 157.83, 151.46, 151.46, 150.60, 139.40, 135.09, 134.87, 134.87, 134.39, 133.89, 133.89, 129.59, 129.36, 129.36, 129.36, 129.02, 129.02, 127.53, 127.53, 127.45, 127.45, 127.45, 127.44, 127.44, 127.06, 126.21, 126.21, 126.17, 126.12, 126.12, 126.12, 121.68, 121.68, 119.14, 119.14, 119.14, 116.72, 116.72, 114.08, 105.70, 105.70, 105.70, 55.34, 55.34, 55.34, 45.62, 45.59, 45.59, 18.49, 18.49, 18.49.
(E)-5-(4-((2-(3-Benzoylphenyl)propanoyl)oxy)styryl)-1,3-phenylene Bis(2-(3-benzoylphenyl)propanoate) (10)White solid (65%). mp: 48°C, IR (KBr) cm−1: 2925, 1756, 1656, 1594, 1284, 1124. HR-ESI-MS m/z: 937.3374 [M+H]+ (Calcd for C62H48O9: 937.3371). MS (ESI) m/z: 937.3 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 7.86–7.79 (m, 9H), 7.75–7.72 (m, 3H), 7.66–7.56 (m, 6H), 7.53–7.41 (m, 11H), 7.01 (d, J=16.3 Hz, 1H), 7.05–6.97 (m, 4H), 6.91 (d, J=16.3 Hz, 1H), 6.70 (d, J=1.9 Hz, 1H), 4.03 (q, J=7.2 Hz, 3H), 1.66 (d, J=7.2 Hz, 3H), 1.65 (d, J=7.2 Hz, 6H). 13C-NMR (101 MHz, CDCl3) δ: 196.46, 196.42, 196.42, 172.50, 172.13, 172.13, 151.33, 151.33, 150.48, 140.28, 140.06, 140.06, 139.54, 138.19, 138.19, 138.16, 137.45, 137.41, 137.41, 134.46, 132.62, 132.62, 132.62, 131.55, 131.55, 131.53, 130.10, 130.10, 130.10, 130.10, 130.10, 130.10, 129.78, 129.36, 129.36, 129.29, 129.29, 129.27, 129.27, 128.86, 128.86, 128.83, 128.38, 128.38, 128.38, 128.38, 128.38, 128.38, 127.64, 127.64, 127.02, 121.66, 121.66, 116.77, 116.77, 113.94, 45.54, 45.54, 45.54, 18.51, 18.51, 18.51.
(E)-5-(4-((2-((2,3-Dimethylphenyl)amino)benzoyl)oxy)styryl)-1,3-phenylene Bis(2-((2,3-dimethylphenyl)amino)benzoate) (11)White solid (73%). mp: 96°C, IR (KBr) cm−1: 1752, 1695, 1596, 1452, 1240, 1155, 1049. HR-ESI-MS m/z: 898.3854 [M+H]+ (Calcd for C59H51N3O6: 898.3851). MS (ESI) m/z: 898.4 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 9.17 (s, 3H), 8.20 (dt, J=8.0, 1.7 Hz, 3H), 7.59 (d, J=8.6 Hz, 2H), 7.37–7.28 (m, 5H), 7.26–7.20 (m, 2H), 7.17 (d, J=7.1 Hz, 4H), 7.13–7.08 (m, 5H), 7.03–7.05 (m, 3H), 6.81–6.72 (m, 6H), 2.33 (s, 9H), 2.18 (s, 6H), 2.17 (s, 3H). 13C-NMR (101 MHz, CDCl3) δ: 167.36, 167.04, 167.04, 151.65, 151.65, 150.59, 150.51, 150.51, 150.35, 139.81, 138.36, 138.31, 138.31, 138.31, 135.17, 135.17, 135.05, 134.61, 132.82, 132.82, 132.63, 131.94, 131.94, 131.94, 129.82, 127.82, 127.82, 127.33, 127.20, 127.20, 127.08, 126.03, 126.03, 125.99, 123.51, 123.51, 123.51, 123.28, 123.28, 122.40, 122.40, 122.40, 117.51, 117.51, 116.25, 116.25, 116.25, 115.44, 113.85, 113.85, 113.85, 109.56, 109.32, 20.63, 20.63, 20.63, 14.09, 14.09, 14.03.
(E)-5-(4-((2-((3-Chloro-2-methylphenyl)amino)benzoyl)oxy)styryl)-1,3-phenylene Bis(2-((3-chloro-2-methylphenyl)amino)benzoate) (12)White solid (66%). mp: 87°C, IR (KBr) cm−1: 1752, 1697, 1606, 1597, 1506, 1450, 1243, 1218, 1130. HR-ESI-MS m/z: 958.2214 [M+H]+ (Calcd for C56H42Cl3N3O6: 958.2212). MS (ESI) m/z: 958.2 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 9.23 (s, 3H), 8.21 (dd, J=8.0, 1.2 Hz, 3H), 7.59 (d, J=8.6 Hz, 2H), 7.41–7.29 (m, 5H), 7.26–7.22 (m, 9H), 7.17–7.05 (m, 5H), 6.87–6.79 (m, 7H), 2.32 (s, 6H), 2.31 (s, 3H). 13C-NMR (101 MHz, CDCl3) δ: 167.30, 166.97, 166.97, 151.57, 151.57, 150.54, 149.58, 149.58, 149.41, 40.09, 140.02, 139.88, 135.70, 135.70, 135.27, 135.27, 135.13, 134.66, 132.13, 132.13, 132.03, 132.03, 132.03, 131.91, 129.91, 127.83, 127.83, 127.32, 126.96, 126.96, 126.91, 126.19, 126.19, 126.03, 123.50, 123.50, 123.50, 123.23, 123.23, 122.33, 122.33, 117.50, 117.50, 117.15, 117.15, 117.15, 115.31, 114.00, 114.00, 114.00, 110.35, 110.08, 110.08, 15.07, 15.07, 15.07.
(E)-5-(4-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetoxy)styryl)-1,3-phenylene Bis(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetate) (13)White solid (67%). mp: 92°C, IR (KBr) cm−1: 1758, 1684, 1593, 1477, 1321, 1220, 1124. HR-ESI-MS m/z: 1246.2842 [M+H]+ (Calcd for C56H42Cl3N3O6: 1246.2846). MS (ESI) m/z: 1246.3 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 7.68 (d, J=8.5 Hz, 6H), 7.51–7.41 (m, 8H), 7.08–6.96 (m, 8H), 6.94–6.86 (m, 4H), 6.79 (t, J=2.0 Hz, 1H), 6.72–6.67 (m, 3H), 3.91 (s, 2H), 3.89 (s, 4H), 3.84 (s, 3H), 3.82 (s, 6H), 2.46 (s, 3H), 2.45 (s, 6H). 13C-NMR (101 MHz, CDCl3) δ: 169.19, 168.84, 168.84, 168.84, 168.31, 168.31, 156.20, 156.20, 156.20, 151.34, 151.34, 150.53, 139.58, 139.41, 139.41, 136.32, 136.32, 136.26, 134.47, 133.86, 133.82, 131.21, 131.21, 131.21, 131.21, 131.21, 131.21, 130.90, 130.90, 130.50, 130.44, 129.79, 129.18, 129.18, 129.18, 129.18, 129.18, 129.18, 127.64, 127.64, 127.64, 127.05, 121.74, 121.74, 121.74, 116.85, 116.85, 115.06, 115.06, 115.06, 114.08, 111.92, 111.92, 111.92, 111.87, 111.87, 111.87, 111.69, 111.69, 111.69, 101.26, 101.26, 55.77, 55.77, 55.77, 30.66, 30.59, 30.54, 13.69, 13.42, 13.42.
General Procedure for Synthesis of 17–20Compound 16 was prepared according to the procedure previously reported. Compounds 17–20 was prepared as described for compounds 8–13 using compound 16.
(E)-4-(3-((Ethyldiisopropylsilyl)oxy)-5-((triisopropylsilyl)oxy)styryl)phenyl 2-(4-Isobutylphenyl)propanoate (17)White solid (75%). MS (ESI) m/z: 729.4 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 7.45 (d, J=8.5 Hz, 2H), 7.30 (d, J=7.9 Hz, 2H), 7.15 (d, J=7.9 Hz, 2H), 6.98 (d, J=8.4 Hz, 2H), 6.95 (d, J=16.0 Hz, 1H), 6.88 (d, J=16.4 Hz, 1H), 6.62 (d, J=1.8 Hz, 2H), 6.34 (s, 1H), 3.93 (q, J=7.1 Hz, 1H), 2.47 (d, J=7.2 Hz, 2H), 1.87 (dt, J=13.4, 6.7 Hz, 1H), 1.61 (d, J=7.2 Hz, 3H), 1.30–1.21 (m, 6H), 1.11 (d, J=7.2 Hz, 36H), 0.91 (d, J=6.6 Hz, 6H). 13C-NMR (101 MHz, CDCl3) δ: 173.15, 157.09, 157.09, 150.29, 140.83, 138.83, 137.23, 135.00, 129.51, 129.51, 128.99, 127.65, 127.32, 127.32, 127.23, 127.23, 121.57, 121.57, 111.37, 111.37, 111.28, 45.31, 45.07, 30.17, 22.64, 22.39, 22.39, 18.52, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 12.70, 12.70, 12.70, 12.70, 12.70, 12.70.
(E)-4-(3,5-Bis((triisopropylsilyl)oxy)styryl)phenyl 2-(6-Methoxynaphthalen-2-yl)propanoate (18)White solid (62%). MS (ESI) m/z: 753.4 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 7.79–7.71 (m, 3H), 7.50 (dd, J=8.5, 1.8 Hz, 1H), 7.44 (d, J=8.6 Hz, 2H), 7.16 (dt, J=7.3, 2.5 Hz, 2H), 6.96 (d, J=8.1 Hz, 2H), 6.94 (d, J=16.4 Hz, 1H), 6.87 (d, J=16.0 Hz, 1H), 6.62 (d, J=2.4 Hz, 2H), 6.34 (t, J=2.1 Hz, 1H), 4.09 (q, J=7.1 Hz, 1H), 3.93 (s, 3H), 1.70 (d, J=7.1 Hz, 3H), 1.32–1.19 (m, 6H), 1.11 (d, J=7.1 Hz, 36H). 13C-NMR (101 MHz, CDCl3) δ: 173.10, 157.81, 157.09, 157.09, 150.25, 138.82, 135.15, 135.05, 133.86, 129.34, 129.02, 129.02, 127.62, 127.39, 127.34, 127.34, 126.17, 126.14, 121.57, 121.57, 119.11, 111.37, 111.37, 111.29, 105.69, 55.34, 45.64, 18.53, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 17.93, 12.70, 12.70, 12.70, 12.70, 12.70, 12.70, 12.70.
(E)-4-(3,5-Bis((triisopropylsilyl)oxy)styryl)phenyl 2-(3-Benzoylphenyl)propanoate (19)White solid (59%). MS (ESI) m/z: 778.2 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 7.88–7.80 (m, 3H), 7.75–7.72 (m, 1H), 7.65 (d, J=7.8 Hz, 1H), 7.63–7.57 (m, 1H), 7.52–7.46 (m, 5H), 7.00 (d, J=8.8 Hz, 2H), 6.96 (d, J=16.4 Hz, 1H), 6.90 (d, J=16.3 Hz, 1H), 6.63 (d, J=2.1 Hz, 2H), 6.35 (t, J=2.1 Hz, 1H), 4.05 (q, J=7.1 Hz, 1H), 1.66 (d, J=7.2 Hz, 3H), 1.30–1.20 (m, 6H), 1.11 (d, J=7.2 Hz, 36H).
(E)-4-(3,5-Bis((triisopropylsilyl)oxy)styryl)phenyl 2-((2,3-Dimethylphenyl)amino)benzoate (20)White solid (56%). MS (ESI) m/z: 764.4 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 9.18 (s, 1H), 8.19 (dd, J=8.1, 1.6 Hz, 1H), 7.57 (d, J=8.6 Hz, 2H), 7.32 (ddd, J=8.6, 7.2, 1.6 Hz, 1H), 7.21 (d, J=8.6 Hz, 2H), 7.17 (d, J=7.6 Hz, 1H), 7.11 (t, J=7.6 Hz, 1H), 7.02 (d, J=16.4 Hz, 1H), 7.01 (d, J=6.8 Hz, 1H), 6.95 (d, J=16.3 Hz, 1H), 6.79 (d, J=8.6 Hz, 1H), 6.76–6.71 (m, 1H), 6.66 (d, J=2.1 Hz, 2H), 6.36 (t, J=2.1 Hz, 1H), 2.32 (s, 3H), 2.17 (s, 3H), 1.27 (ddd, J=16.6, 8.4, 6.6 Hz, 6H), 1.12 (d, J=7.2 Hz, 36H). 13C-NMR (101 MHz, CDCl3) δ: 167.34, 157.12, 157.12, 150.34, 150.20, 138.87, 138.42, 138.28, 135.19, 134.96, 132.65, 131.92, 129.11, 127.69, 127.53, 127.53, 127.07, 125.97, 123.31, 122.19, 122.19, 116.19, 113.83, 111.40, 111.40, 111.30, 109.70, 20.57, 17.95, 17.95, 17.95, 17.95, 17.95, 17.95, 17.95, 17.95, 17.95, 17.95, 17.95, 17.95, 14.00, 12.72, 12.72, 12.72, 12.72, 12.72, 12.72.
General Procedure for Synthesis of Compounds 21–24To a solution of Compounds 17–20 in dry dichloromethane at room temperature, triethylammonium trihydrofluoride (Et3N·3HF, 3 equiv) was added. After 8 h, EtOAc was added to the mixture, and the organic layer was washed with water and saturated brine. The organic phase was dried were dried over anhydrous MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography to give the target compounds.
(E)-4-(3,5-Dihydroxystyryl)phenyl 2-(4-Isobutylphenyl)propanoate (21)White solid (63%). mp: 147°C, IR (KBr) cm−1: 2956, 2921, 2870, 1727, 1595, 1161. HR-ESI-MS m/z: 417.2063 [M+H]+ (Calcd for C27H28O4: 417.2060). MS (ESI) m/z: 417.2 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 7.42 (d, J=8.6 Hz, 2H), 7.30 (d, J=8.1 Hz, 2H), 7.15 (d, J=8.1 Hz, 2H), 6.98 (d, J=8.1 Hz, 2H), 6.97 (d, J=16 Hz, 1H), 6.85 (d, J=16 Hz, 1H), 6.52 (d, J=2.2 Hz, 2H), 6.25 (t, J=2.2 Hz, 1H), 3.94 (q, J=7.1 Hz, 1H), 2.47 (d, J=7.2 Hz, 2H), 1.87 (dt, J=13.5, 6.8 Hz, 1H), 1.62 (s, 3H), 0.91 (d, J=6.6 Hz, 6H). 13C-NMR (101 MHz, CDCl3) δ: 173.49, 157.00, 157.00, 150.40, 140.90, 139.80, 137.13, 134.78, 129.55, 129.55, 128.46, 128.18, 127.42, 127.42, 127.23, 127.23, 121.65, 121.65, 106.15, 106.15, 102.38, 45.32, 45.06, 30.17, 22.39, 22.39, 18.48.
(E)-4-(3,5-Dihydroxystyryl)phenyl 2-(6-Methoxynaphthalen-2-yl)propanoate (22)White solid (54%). mp: 154°C, IR (KBr) cm−1: 2925, 2855, 1760, 1655, 1590, 1286, 1146. HR-ESI-MS m/z: 441.1694 [M+H]+ (Calcd for C28H24O5: 441.1697). MS (ESI) m/z: 441.1 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 7.80–7.71 (m, 3H), 7.50 (d, J=8.6 Hz, 1H), 7.40 (d, J=8.5 Hz, 2H), 7.16 (dd, J=11.4, 2.5 Hz, 2H), 6.96 (d, J=8.1 Hz, 2H), 6.94 (d, J=16.0 Hz, 1H), 6.84 (d, J=16.2 Hz, 1H), 6.51 (d, J=1.6 Hz, 2H), 6.25 (s, 1H), 4.05 (dd, J=13.7, 6.7 Hz, 1H), 3.92 (s, 3H), 1.70 (d, J=7.1 Hz, 3H). 13C-NMR (101 MHz, CDCl3) δ: 171.23, 157.82, 157.08, 157.08, 139.77, 135.07, 133.87, 129.34, 129.34, 129.03, 128.41, 128.26, 128.26, 127.41, 127.41, 127.41, 126.17, 126.11, 121.64, 121.64, 119.13, 106.10, 106.10, 105.70, 102.38, 55.34, 45.63, 17.20.
(E)-4-(3,5-Dihydroxystyryl)phenyl 2-(3-Benzoylphenyl)propanoate (23)White solid (58%). mp: 62°C, IR (KBr) cm−1: 2925, 2854, 1753, 1656, 1585, 1448, 1286, 1161. HR-ESI-MS m/z: 465.1694 [M+H]+ (Calcd for C62H48O9: 465.1697). MS (ESI) m/z: 465.2 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 7.87 (s, 1H), 7.84–7.79 (m, 2H), 7.73 (d, J=7.7 Hz, 1H), 7.67–7.58 (m, 2H), 7.53–7.47 (m, 3H), 7.43 (d, J=8.6 Hz, 2H), 6.99 (d, J=8.6 Hz, 2H), 6.96 (d, J=16.3 Hz, 1H), 6.86 (d, J=16.3 Hz, 1H), 6.53 (d, J=2.2 Hz, 2H), 6.26 (t, J=2.2 Hz, 1H), 4.10–4.02 (m, 1H), 1.67 (d, J=7.2 Hz, 3H). 13C-NMR (101 MHz, CDCl3) δ: 196.67, 172.81, 157.05, 157.05, 150.15, 140.33, 139.73, 138.15, 137.40, 135.00, 132.66, 131.60, 130.14, 130.14, 129.33, 129.22, 128.80, 128.38, 128.38, 128.38, 128.32, 127.48, 127.48, 121.53, 121.53, 106.17, 106.17, 102.46, 45.57, 18.48.
(E)-4-(3,5-Dihydroxystyryl)phenyl 2-((2,3-Dimethylphenyl)amino)benzoate (24)White solid (53%). mp: 161°C, IR (KBr) cm−1: 2921, 2854, 1756, 1690, 1590, 1454, 1216, 1150, 1044. HR-ESI-MS m/z: 452.1852 [M+H]+ (Calcd for C29H25NO4: 452.1856). MS (ESI) m/z: 452.2 [M+H]+. 1H-NMR (400 MHz, CDCl3) δ: 9.15 (s, 1H), 8.20 (dd, J=8.0, 1.2 Hz, 2H), 7.52 (d, J=8.5 Hz, 2H), 7.32 (t, J=7.8 Hz, 1H), 7.20 (d, J=8.5 Hz, 2H), 7.16 (d, J=7.7 Hz, 1H), 7.11 (t, J=7.6 Hz, 1H), 7.03 (d, J=7.2 Hz, 1H), 7.00 (d, J=16.4 Hz, 1H), 6.88 (d, J=16.3 Hz, 1H), 6.80 (d, J=8.6 Hz, 1H), 6.74 (t, J=7.6 Hz, 2H), 6.53 (d, J=1.9 Hz, 1H), 6.26 (s, 1H), 2.32 (s, 3H), 2.16 (s, 3H). 13C-NMR (101 MHz, CDCl3) δ: 167.44, 157.03, 157.03, 150.37, 139.89, 138.37, 138.30, 135.04, 135.04, 134.91, 132.64, 131.93, 128.59, 128.26, 127.63, 127.63, 127.10, 125.98, 123.30, 122.29, 122.29, 116.22, 113.85, 109.59, 106.21, 106.21, 102.39, 20.57, 14.19.
Cell CultureThe RAW264.7 cells were obtained from the Shanghai Institute of Cell Biology (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, NY, U.S.A.) supplemented with 10% fetal bovine serum (FBS) (PAA, Australia) and penicillium/streptomycin at 37°C in humidified 5% CO2 incubator.
Cell Viability AssayCell proliferation was determined using CCK-8 dye (Dojindo Laboratories, Japan) according to manufacturer’s instructions. Briefly, RAW264.7 cells were plated in 96-well plate, and allowed to adhere overnight. After incubation with tested compounds for 24 h, 10 µL CCK-8 dyes then was add to each well, and cells were incubated at 37°C for 1 h. The absorbance was finally determined at 450 nm using a microplate reader (TECAN Infinite M200).
AnimalsKunming mice (4–6 weeks old) were obtained from the Experimental Animal Center of Guangdong Province (Guangzhou, China). The use of mice was reviewed and approved by the Ethics Committee for Animal Experimentation of the Guangdong University of Technology (Guangzhou, China) and was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were housed at 25°C with a 12/12 h light–dark cycle and fed with a standard rodent diet and water. The animals were acclimatized to the laboratory for at least 7 d before used in experiments.
Nitrite MeasurementRAW264.7 macrophages were pretreated with or without various concentrations of test compounds for 6 h. After a 6 h treatment, the cells were treated with 100 ng/mL LPS. After 18 h, the NO concentration in medium was determined using the Griess reagent (Promega, U.S.A.) according to the manufacturer’s instructions.
Determination of TNF-α and IL-1βRAW264.7 macrophages were pretreated with or without various concentrations of test compounds for 6 h. After a 6 h treatment, the cells were treated with 100 ng/mL LPS. After 18 h, the TNF-α and IL-1β levels in medium were determined with an ELISA kit (eBioScience, San Diego, CA, U.S.A.) according to the manufacturer’s instructions.
LPS-Induced Endotoxic ShockEach group has ten mice. Compounds 21 and 7 were suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na). The mice were orally administered with compound 21 (50, 200 mg/kg of body weight), compound 7 (50, 200 mg/kg), or the control (0.5% CMC-Na). Mice were injected i.p. with 35 mg/kg LPS at 1 h after administration. Survival rates were monitored for 6 d after LPS injection at an interval of 12 h.
Gastric Mucosal DamageThe ability to produce gastric damage was evaluated according to the slightly modified method of Velázquez et al.5) Ibuprofen and compound 21 were suspended in 1% methylcellulose solution. The fasting mice (n=6) were oral administration of ibuprofen (250 mg/kg) or compound 21 (250 mg/kg). The control mice were administered with the same volume of 1% methylcellulose solution. The mice were sacrified 6 h after administration. The stomachs were removed, cut out along the greater curvature of the stomach, gently rinsed with water, and placed on ice. The number and the length of ulcers were determined using a magnifier lense. Each individual gastric lesion was measured along its greatest length (<1 mm=rating of 1; 1–2 mm=rating of 2; >2 mm=rating according to their length in mm). The overall total was designated as the “ulcer index.” The data were expressed as the mean±standard error of the mean (S.E.M.).
Statistical AnalysisData were presented as the mean±S.E.M. The statistical significance of the differences (p<0.05) from a one-way ANOVA followed by Tukey’s or Dunnett’s test.
This work was supported by the National Natural Science Foundation of China (Nos. 21402030, 21402031 and 21272043).
The authors declare no conflict of interest.
