Article Cite This: J. Nat. Prod. 2018, 81, 2647−2653
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Total Synthesis and Biological Evaluation of Sericetin for Protection against Cisplatin-Induced Acute Kidney Injury Eun-Sun Kim,†,§ Hongjun Jang,‡,§ Sun-Young Chang,‡ Seung-Hoon Baek,‡ Ok-Nam Bae,*,† and Hyoungsu Kim*,‡ †
J. Nat. Prod. 2018.81:2647-2653. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/11/19. For personal use only.
College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 15588, Republic of Korea ‡ College of Pharmacy and Research Institute of Pharmaceutical Science and Technology (RIPST), Ajou University, Suwon 16499, Republic of Korea S Supporting Information *
ABSTRACT: A concise synthesis of sericetin (1) was performed in four steps from readily available 3-Obenzylgalangin (4), featuring electrocyclization to produce the tricyclic core and a sequential aromatic Claisen/Cope rearrangement to incorporate the 8-prenyl group of 1. In addition, the therapeutic potential of sericetin (1), isosericetin (2), and three prenylated tetracyclic synthetic intermediates (11, 12, and 14) against cisplatin-induced nephrotoxicity using renal tubular cells were evaluated. Compound 14 showed therapeutic potential against cisplatin-induced kidney damage. renylated flavonoids are a subclass of flavonoids that possess a wide range of biological properties, such as antioxidant, anticancer, antifungal, and estrogenic activities.1 The addition of a lipophilic prenyl group to these flavonoids could increase their lipophilicity, thereby increasing their permeabilities and bioavailabilities in vivo.2 Sericetin (1), a prenylated flavonoid, was first isolated from the root-bark of Mundulea sericea in 1960 (Figure 1). This
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As part of continuing efforts in the synthesis and biological evaluation of flavonoids,6 we have been interested in the synthesis of the tetracyclic flavonoid sericetin (1) and its effect on cisplatin-induced acute kidney injury (AKI). Reported herein are a concise synthesis of 1 exploiting an aromatic [3,3]sigmatropic rearrangement and the evaluation of the effect of synthesized sericetin, isosericetin, and prenylated tetracyclic synthesized intermediates against cisplatin-induced nephrotoxicity.
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RESULTS AND DISCUSSION Chemistry. The retrosynthetic plan for sericetin (1) is outlined in Scheme 1. Sericetin (1) could be synthesized from phenol 3 via a prenylation/aromatic [3,3]-sigmatropic rearrangement. In turn, the tetracyclic phenol 3 could be obtained through electrocyclization of prenal with 5,7dihydroxyflavonol (4), which could be readily prepared from the commercially available phloroglucinol (5) through conventional Friedel−Crafts/pyrone annulation. Initial attempts to synthesize sericetin (1) are shown in Scheme 2. The synthesis commenced with the preparation of 8-prenylflavonol 9. Selective THP (tetrahydropyran) protection of the 7-hydroxy group in the flavonol 4,6h prepared from the commercially available phloroglucinol (5), followed by prenylation of the 5-hydroxy group in the known compound 6,6g afforded the aryl prenyl ether 7 in 77% yield over the two steps. Compound 7 was subjected to sequential aromatic Claisen/Cope rearrangement conditions to afford 8-prenyl-
Figure 1. Structures of sericetin and isosericetin.
natural flavonoid has two isoprene units, i.e., an 8-prenyl group and as part of the (2H)-pyran ring.3 The structure of sericetin was proposed to be 1 or 2, based on the results of spectroscopic data analyses. The first synthesis of 1 was achieved by Jain and Zutshi and utilized 6- and 8bisprenylation/DDQ-mediated oxidative cyclization of galangin to afford sericetin (1) as a minor product at less than 1% yield, thereby defining its structure as 1.4 The biological properties of this unique prenylated flavonol natural product had not been reported until Mahidol and Ruchirawat isolated it in 2011 from the whole plant of Dunbaria longeracemosa and showed that it possesses antitumor activities in various cancer cell lines.5 © 2018 American Chemical Society and American Society of Pharmacognosy
Received: May 30, 2018 Published: December 12, 2018 2647
DOI: 10.1021/acs.jnatprod.8b00434 J. Nat. Prod. 2018, 81, 2647−2653
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the natural product (1H and 13C NMR and HRMS),4,5 thereby confirming the structure of 11 (Scheme 3). The structure of isomer 12 was also confirmed through synthesis from known 136f,g via (i) propargylation, (ii) hydrogenation, and (iii) Claisen rearrangement (13 → 12).
Scheme 1. Retrosynthetic Plan
Scheme 3. Determination of the Structures 11 and 12
Scheme 2. Initial Attempts at the Synthesis of Sericetin (1)
The rationale for the observed skeletal rearrangement is shown in Figure 2. The 6,7-pyran 11 was formed from aryl
flavonol 9 in 75% yield over two steps after deprotection of the THP group in 8 under acidic conditions. Having installed the 8-prenyl group, the construction of the D-ring in the natural product was next attempted. Thus, the aryl propargyl ether 10 was prepared through selective propargylation of the 7-hydroxy group in 9 under Claisen rearrangement/cyclization conditions. However, the Claisen rearrangement/cyclization of 10 yielded a roughly 1:1 mixture of 11 and 12 in an 88% combined yield. Analysis of the spectroscopic data revealed that the 1:1 mixture contained the target 8-prenylated 6,7-pyran adduct 11 and the 6-prenylated 7,8-pyran adduct 12. To investigate the mechanism of the unexpected isomerization, 11 and 12 were separately subjected to the reaction conditions of Claisen rearrangement/cyclization. No interconversion was obtained, suggesting that the isomerization must have occurred prior to closing of the D-ring. Compound 11 was debenzylated to afford sericetin (1), whose spectroscopic data were identical to those reported for
Figure 2. Rationale for the observed isomerization.
propargyl ether 10 through a [3,3]-sigmatropic rearrangement, followed by keto−enol tautomerization, a [1,5]-sigmatropic rearrangement, and an oxa-Diels−Alder reaction. In contrast, the route to the formation of 12 appears to be more complicated. Initially, the 7-propargyloxy group migrated to C-8 through Claisen rearrangement to form intermediate A. 2648
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isomer in excellent yield (88%) without any skeletal rearrangement. The optimized synthesis route afforded the penultimate intermediate to sericetin (1) in three steps from 3-Obenzylgalangin (4) with the introduction of two C5-isoprene units to the A ring in of 4. Deprotection of the benzyl group with BCl3 in CH2Cl2 (83%) completed the synthesis of sericetin (1). Biological Assay. Upon completion of the concise total synthesis of sericetin (1), biological activities of sericetin (1), isosericetin (2), and the three prenylated tetracyclic synthetic intermediates (11, 12, and 14) were evaluated. After testing all five compounds via the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) (MTT) assay, their therapeutic potential against cisplatin-induced AKI was assessed. AKI is a representative renal injury induced by drugs or chemicals. It is associated with high mortality and contributes to the development of chronic renal failure.8 There have been intensive efforts to develop therapeutic candidates for treating drug-induced AKI.9,10 Cisplatin is commonly used as an anticancer chemotherapeutic,11 but despite its clinical efficacy, its use is limited due to its adverse effects, including nephrotoxicity.12 The accumulation of high concentrations of cisplatin in the kidneys leads to nephrotoxicity,13 and patients treated with cisplatin may show impaired renal function.14 Cisplatin-induced nephrotoxicity is known to be mediated by oxidative stress, mitochondrial dysfunction, inflammatory tissue damage, and apoptosis.11,13 Cisplatin-induced AKI is a well-established representative model to investigate the therapeutic potential and mechanisms of drug candidates to treat or prevent AKI. Since renal damage is mediated by complicated modes of action, natural products such as flavonoids that have shown multiple biological properties are considered to be good therapeutic candidates against druginduced nephrotoxicity. In fact, several natural products have been suggested as potential therapeutic interventions and/or adjuvants to prevent drug-induced kidney injury.9,15 Based on the cytotoxicity results obtained using the MTT assay, 14 showed the most potent protective activity against cisplatin-induced cytotoxicity in renal tubular cells (Figure 3).
The 8-prenyl group in intermediate A sequentially underwent Cope rearrangements to furnish intermediate B, which further participated in a Cope rearrangement to afford C, followed by sequential aromatization, [1,5]-sigmatropic rearrangement, and an oxa-Diels−Alder cyclization to form 12. Consequently, the prenyl group in 10 was transferred from C-8 to C-6 through sequential Claisen/double Cope rearrangements to afford 12 with the prenyl unit at C-6. On the basis of the initial results, the construction of the Dring in sericetin (1) must occur prior to the installation of the prenyl group at C-8 in order to obtain only the target 11 and avoid the previously observed skeletal rearrangement. Thus, the 8-prenyl tetracyclic 11 should be obtainable through the transfer of a prenyl group across the A/D or A/C aromatic ring system in 14 from the C-5 hydroxy group to C-8 through sequential Claisen/Cope rearrangements, as described in Scheme 4. The synthesis of 3, which had been previously Scheme 4. Successful Route to Concise and Efficient Synthesis of Sericetin (1)
Figure 3. Protective effect of compounds (CPDs) 1, 2, 11, 12, and 14 against cisplatin-induced cytotoxicity in rat renal tubular cells. NRK52E cells (rat tubular epithelial cells) were treated with compounds 1, 2, 11, 12, and 14 (20 μM) with or without cisplatin (CP; 30 μM) for 24 h. Cell viability was measured via the MTT assay. DMSO (final concentration 0.2%) was used as a vehicle control. Values are mean ± SEM. **p < 0.01 vs control; ##p < 0.01 vs cisplatin-treated cells. N = 3.
prepared in four steps from 4 in the synthesis of macakurzin C was optimized first.6f,g Recently, electrocyclization was successfully used in the one-step pyran annulation of resorcinol derivatives from α,β-unsaturated aldehydes,7 which prompted application of this method to the synthesis of the D-ring in the natural product. Electrocyclization of the resorcinol derivative 4 with 3,3-dimethylacrylaldehyde smoothly provided the target pyran 3 in 54% yield. Prenylation (95%) of the 5-hydroxy group in 3 and subjecting the resulting arylprenyl ether 14 to the conditions of aromatic Claisen rearrangement afforded the 8-prenyl tetracyclic 11 via intermediates D or E as a single
In order to further evaluate the protective activity of 14, NRK-52E cells were treated with various concentrations of 14. As shown in Figures 4A and B, 14 showed protective effects against cisplatin-induced cytotoxicity in a dose-dependent manner, and the compound was not cytotoxic in the dose range tested (Figure 4C). 2649
DOI: 10.1021/acs.jnatprod.8b00434 J. Nat. Prod. 2018, 81, 2647−2653
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Reactive oxygen species (ROS) generation and mitochondrial dysfunction have been suggested as the main mechanisms for apoptotic cell death in cisplatin-induced nephrotoxicity.14a,16 In this study, cisplatin increased ROS generation, as quantified by the 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) assay, in renal tubular cells, whereas treatment with 14 significantly reduced the ROS generation (Figure 5A). Since impaired redox balance induced by cisplatin could result in mitochondrial damages in tubular cells, mitochondrial membrane potentials using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolo carbocyanine iodide (JC-1) fluorescence was measured. The result showed that green fluorescence significantly increased in tubular cells exposed to cisplatin, which suggests that cisplatin affected the mitochondrial membrane potentials. The increase in green fluorescence was reversed in cells cotreated with 14 and cisplatin, showing that normal membrane potentials were restored (Figure 5B). Cisplatin-induced ROS generation and altered mitochondrial membrane potentials can result in apoptotic cell death in renal tubular cells. As observed with the levels of cleaved caspase-3 (active form), cisplatin activated the apoptotic caspase cascade in tubular cells, whereas 14 significantly reduced the level of cleaved caspase-3 (Figure 5C). The in vivo relevancy of the nephroprotective effects of 14 was demonstrated in a rat model of cisplatin-induced AKI. Consistent with the caspase-3 activation seen in tubular cells
Figure 4. Inhibition of cisplatin-induced cytotoxicity by 14 in renal tubular cells. NRK-52E cells were treated with 14 (0, 1, 10, and 50 μM) for 24 h in the absence or presence of cisplatin (CP; 30 μM). (A) Morphologic changes were observed via microscopy. Scale bar: 100 μm. (B, C) Cell viability was measured via MTT assay. Values are mean ± SEM. ** p < 0.01 vs control. ##p < 0.01 vs cisplatin-treated cells. N = 3.
Figure 5. Protective mechanisms of 14 against cisplatin-induced cellular damage in renal tubular cells. NRK-52E cells were treated with cisplatin (CP; 30 μM) with or without 14 (10 μM) for 24 h. (A) Changes in reactive oxygen species generation were measured via dichlorofluorescein (DCF) staining (left panel). The percentage of ROS generation was calculated with DCF fluorescence signal using an ROI program (right panel). (B) Mitochondria membrane potentials were analyzed using JC-1 dye and fluorescence microscopy (left panel). The fluorescence signals were calculated using an ROI statistics program (right panel). Red fluorescence represents JC-1 aggregates with normal membrane potentials, while green fluorescence indicates JC-1 monomers at disrupted potentials. (C) Expressions of cleaved caspase-3 were determined in Western blot analysis. The densities were calculated by the ImageJ program. Values are mean ± SEM. * p < 0.05, ** p < 0.01 vs control; # p < 0.05, ## p < 0.01 vs cisplatin-treated cells. N = 3. 2650
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cisplatin were evaluated. The data demonstrated that 5-Oprenyl tetracyclic 14 has beneficial effects against cisplatinmediated cytotoxicity through antioxidant, mitochondria preserving, and antiapoptotic properties in renal tubular cells. Notably, the nephro-protective efficacy and the mechanisms of 14 were confirmed in a cisplatin-exposed rat model, with histopathologic and functional improvement. 14 is expected to have therapeutic potential against drug-induced kidney damage.
(Figure 5C), cleaved caspase-3 levels significantly increased in kidney tissues isolated from cisplatin-exposed rats, while the increase of caspase-3 activation was not observed in kidneys of rats treated with 14 and cisplatin (Figure 6A). The level of
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EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were determined with an IA9100 melting point apparatus. 1H and 13C NMR spectra were obtained on a Varian Mercury 400 and JEOL ECZ600R. Chemical shifts are reported in ppm units with Me4Si or CHCl3 as the internal standard. HREIMS data were acquired using a JEOL JMS-700. Unless otherwise noted, materials and all solvents were obtained from commercial suppliers and were used without purification. Toluene, CH2Cl2, and tetrahydrofuran (THF) were dried with 4 Å molecular sieve. Cisplatin (cis-diamminedichloroplatinum II) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cisplatin was freshly prepared as a stock solution dissolved in phosphatebuffered saline (PBS) for the in vitro and in vivo experiments. CMH2-DCFDA and JC-1 were purchased from Invitrogen (Waltham, MA, USA). Primary antibody targeting caspase-3 was obtained from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were purchased from Sigma-Aldrich. Sigmatropic Rearrangement of Aryl Propargyl Ether 10. Aryl propargyl ether 10 (40.1 mg, 0.081 mmol) was dissolved in diethylaniline (10 mL), and the mixture was stirred at 270 °C for 12 h. The mixture was cooled to room temperature, concentrated in vacuo, and filtered through a pad of silica gel (hexanes/EtOAc, 5:1) to afford the 1:1 mixture of 11 and 12. The mixture was separated by column chromatography (silica gel; hexanes/EtOAc, 50:1 to 30:1) to afford 11 (18.1 mg, 45%) and 12 (17.3 mg, 43%). For 11: light yellow solid (mp 140−141 °C): 1H NMR (600 MHz, CDCl3) δ 12.88 (s, 1H), 8.01−7.97 (m, 2H), 7.48−7.40 (m, 3H), 7.33−7.29 (m, 2H), 7.28−7.23 (m, 2H), 6.75 (d, J = 10.0 Hz, 1H), 5.62 (d, J = 10.0 Hz, 1H), 5.19 (tqq, J = 6.8, 1.6, 1.6 Hz, 1H), 5.06 (s, 2H), 3.45 (d, J = 6.8 Hz, 2H), 1.77 (s, 3H), 1.68 (s, 3H), 1.46 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 179.0, 156.6, 156.0, 154.1, 153.5, 137.6, 136.2, 131.5, 130.7, 130.5, 128.58, 128.51, 128.19, 128.09, 128.00, 127.85, 122.1, 115.7, 107.3, 105.7, 105.0, 77.8, 74.4, 28.3, 25.9, 21.6, 18.1; HREIMS m/z found 494.2095 (calcd for C32H30O5 [M]+ 494.2093). For 12: light yellow solid (mp 114−115 °C): 1H NMR (600 MHz, CDCl3) δ 12.9 (s, 1H), 7.96−7.94 (m, 2H), 7.48−7.41 (m, 3H), 7.30−7.28 (m, 2H), 7.25−7.22 (m, 3H), 6.73 (d, J = 9.9 Hz, 1H), 5.57 (d, J = 10.0 Hz, 1H), 5.26 (tqq, J = 7.3, 1.2, 1.2 Hz, 1H), 5.07 (s, 2H), 3.36 (d, J = 7.4 Hz, 2H), 1.82 (s, 3H), 1.69 (s, 3H), 1.47 (s, 6H); 13C NMR (150 MHz, CDCl3) δ 179.0, 158.7 157.2, 155.9, 149.5, 137.8, 136.3, 131.5, 130.8, 130.6, 128.81, 128.60, 128.33, 128.19, 128.13, 127.0, 122.0, 115.1, 112.5, 105.5, 100.7, 77.9, 74.4, 28.1, 25.8, 21.3, 17.9; HREIMS m/z found 494.2092 (calcd for C32H30O5 [M]+ 494.2093). Synthesis of Sericetin (1). The 8-prenyl-6,7-pyran adduct 11 (120.1 mg, 0.242 mmol) was dissolved in CH2Cl2 (10 mL), and the mixture was cooled to −78 °C. To this solution was added BCl3 (0.484 mL, 1.0 M solution in toluene, 0.484 mmol), and the reaction mixture was stirred for 2 h at the same temperature. The reaction was quenched with H2O (10 mL) and diluted with EtOAc (30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (silica gel; hexanes/EtOAc, 4:1 to 2:1) to afford sericentin (1) (86.1 mg, 88%) as a yellow solid (mp 151−152 °C): 1H NMR (600 MHz, CDCl3) δ 11.9 (s, 1H), 8.22− 8.19 (m, 2H), 7.54−7.51 (m, 2H), 7.48−7.45 (m, 1H), 6.75 (d, J = 10.0 Hz, 1H), 6.67 (s, 1H), 5.65 (d, J = 10.0 Hz, 1H), 5.23 (tqq, J = 7.1, 1.3, 1.3 Hz, 1H), 3.53 (d, J = 7.1 Hz, 2H), 1.83 (s, 3H), 1.69 (s,
Figure 6. In vivo nephroprotection by 14 against cisplatin-induced kidney impairment in rats. Cisplatin (CP; 6 mg/kg) and/or 14 (1 mg/kg) were administered to rats. (A) The level of caspase-3 activation in the kidneys was analyzed via Western blotting. (B) Histological changes in renal tissues were observed via hematoxylin and eosin (H&E) staining. Scale bar: 100 μm. *, Exfoliation and shallowing; #, cell death and tubular damages; arrowhead, cast formation. (C and D) Functional changes in kidney were determined by the levels of blood urea nitrogen (BUN) and serum creatinine. Values are mean ± SEM. * p < 0.05, ** p < 0.01 vs rats in control group; # p < 0.05 vs rats treated with cisplatin. N = 6 rats/group.
cleaved caspase-3 was similar between the rats in control group and rats treated with 14 and cisplatin. The protective effects of 14 were revealed through both histopathological analyses of kidney tissues (Figure 6B) and analysis of renal functional parameters. BUN and serum creatinine levels, which are representative clinical markers for renal function, were severely affected in the cisplatin-treated rats, and 14 treatment significantly restored impaired renal function (Figure 6C and 6D). There were no histological or functional changes in rats treated with 14 alone (data not shown). In summary, the concise synthesis of sericetin (1) was accomplished in four steps (39.7% overall yield) from readily available 3-O-benzylgalangin (5), using electrocyclization to obtain the tricyclic core and a sequential aromatic Claisen/ Cope rearrangement to incorporate the prenyl group at C(8). In addition, the therapeutic potential of sericetin (1), isosericetin (2), and three prenylated tetracyclic synthetic intermediates (11, 12, and 14) against in vitro AKI induced by 2651
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3H), 1.48 (s, 6H); 13C NMR (150 MHz, CDCl3) δ 175.7, 157.2, 153.8, 153.0, 144.8, 136.4, 131.8, 131.2, 130.1, 128.6, 128.2, 127.6, 122.1, 115.7, 107.8, 104.9, 103.6, 77.9, 28.3, 25.8, 21.5, 18.1; HREIMS m/z found 404.1625 (calcd for C25H24O5 [M]+ 404.1624). Synthesis of Flavonol 3 by Electrocyclization. To a solution of 4 (248.7 mg, 0.69 mmol) in MeOH (5 mL) were added 3,3dimethylacrylaldehyde (0.67 mL, 6.90 mmol) and Ca(OH)2 (204.4 mg, 2.76 mmol) at room temperature. After being stirred for 58 h at the same temperature, the mixture was concentrated in vacuo, diluted with EtOAc/H2O, and acidified with 5 N HCl. The layers were separated, washed with H2O and brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (silica gel; hexanes/EtOAc, 20:1) to afford known 36f,g (159.2 mg, 54%). Synthesis of Aryl Prenyl Ether 14. Phenol 3 (498.5 mg, 1.169 mmol) was dissolved in DMF (20 mL) at room temperature. To this solution were added sequentially K2CO3 (807.8 mg, 5.845 mmol) and prenyl chloride (0.395 mL, 3.507 mmol), and the resulting mixture was stirred for 14 h at 40 °C. The reaction was quenched with saturated aqueous NH4Cl and diluted with EtOAc. The layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (silica gel; hexanes/EtOAc, 2:1) to afford aryl prenyl ether 14 (547.9 mg, 95%): 1H NMR (400 MHz, CDCl3) δ 7.96−7.92 (m, 2H), 7.43−7.37 (m, 3H), 7.32−7.28 (m, 2H), 7.24−7.19 (m, 3H), 6.76 (d, J = 9.6 Hz, 1H), 6.64 (s, 1H), 5.68 (d, J = 10.0 Hz, 1H), 5.67 (dd, J = 7.6, 7.6 Hz, 1H), 5.07(s, 2H), 3.45 (d, J = 7.2 Hz, 2H), 1.78 (s, 3H), 1.71 (s, 3H), 1.46 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 173.4, 157.66, 157.44, 153.84, 153.66, 139.5, 138.3, 136.5, 130.7, 129.99, 129.97, 128.7, 128.3, 127.96, 127.87, 120.1, 113.4, 112.9, 100.3, 77.5, 74.0, 72.2, 28.3, 25.9, 18.2; HREIMS m/z found 494.2094 (calcd for C32H30O5 [M]+ 494.2093). Synthesis of 11 by Sigmatropic Rearrangement of Aryl Prenyl Ether 14. A solution of aryl prenyl ether 14 (547.9 mg, 1.108 mmol) in diethylaniline (100 mL) was stirred at 270 °C for 4 h. The reaction mixture was concentrated in vacuo and purified by column chromatography (silica gel; hexanes/EtOAc, 20:1 to 10:1) to afford 11 (481.3 mg, 88%) In Vitro Study in Renal Tubular Cells. In vitro protective activity and the underlying mechanisms were studied in NRK-52E, rat renal tubular epithelial cell line, purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). After cells were treated with cisplatin and/or analyte such as 14 for 24 h, biochemical assays were conducted to determine the cisplatin-induced cellular changes. Cell viability was determined using the MTT assay.9 Changes in ROS level or mitochondrial membrane potential were determined by fluorescent dyes of DCF and JC-1, respectively.17,18 Activation of caspase-3 was determined by Western blotting analysis of cleaved active caspase-3.9 Details on each experimental procedures are described in the Supporting Information. In Vivo Acute Kidney Injury Models in Rats. Protocols for animal experiments were approved by the Hanyang University Institutional Animal Care and Use Committee (IACUC 20160155A). To induce acute kidney injury, cisplatin (6 mg/kg) was administered to Sprague−Dawley rats (male, body weight 200 ± 10 g) by intraperitoneal injection. Compound 14 was dissolved in 5% DMSO in 0.9% normal saline and injected once at 24 h before cisplatin administration and once a day for five consecutive days after cisplatin treatment. The rats were divided into four groups and treated as follows: vehicle control group treated with 1% DMSO in saline, cisplatin alone with single administration of cisplatin (6 mg/kg) in saline, 14 + cisplatin group treated with cisplatin (6 mg/kg) and 1 mg/kg 14, and 14 alone treated with 1 mg/kg 14. Rats were sacrificed at day 7 after cisplatin treatment. To analyze renal function and/or tissue damage, blood and kidney tissue samples were collected from each rat. The levels of BUN and serum creatinine in serum samples were determined by clinical chemistry analysis in Neodin VetLab (Seoul, Korea). Histologic observations were performed with isolated kidney samples. To detect caspase-3 activation in vivo, isolated rat
kidney tissues were homogenized and used in Western blot. Details on each experimental procedure were described in the Supporting Information.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00434. Experimental procedures and characterization data of compounds 1, 2, 7, 9−12, and 14; copies of 1H and 13C NMR spectra of compounds 1, 2, 7, 9−12, and 14 (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel: 82-31-400-5805. Fax: 82-31-400-5958. E-mail: onbae@ hanyang.ac.kr (O.-N. Bae). *Tel: 82-31-219-3448. Fax: 82-31-219-3435. E-mail:
[email protected] (H. Kim). ORCID
Hyoungsu Kim: 0000-0002-2774-8727 Author Contributions §
E.-S. Kim and H. Jang contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Professor S. Ruchirawat (Chulabhorn Graduate Institute) for providing the spectra of sericetin. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF2017R1A2B4004155 and NRF-2017R1C1B3002626).
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REFERENCES
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DOI: 10.1021/acs.jnatprod.8b00434 J. Nat. Prod. 2018, 81, 2647−2653
