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Synthesis and anti-inflammatory activities of 1-O-acetylbritannilactone analogues
Phytochemistry Letters 19 (2017) 248–253
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Synthesis and anti-inflammatory activities of 1-Oacetylbritannilactone analogues Xiao-Peng Weia , Yu-Fen Chena , Hong Zhua,b , Xiao-Ran Wua , Yang Yua , De-Xin Konga,c , Hong-Quan Duana,c,* , Mei-Hua Jina,**, Nan Qinc,** a b c
School of Pharmacy, Tianjin Medical University, Tianjin 300070, People’s Republic of China Department of Pharmacy, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an 223300, People’s Republic of China Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, People’s Republic of China
A R T I C L E I N F O
Article history: Received 16 December 2016 Received in revised form 10 January 2017 Accepted 7 February 2017 Available online xxx Keywords: 1-O-acetylbritannilactone 6-Deoxybrintanilactone Chemistry diversity Nitric oxide production Inducible nitric oxide synthase
A B S T R A C T
Natural product 1-O-acetylbritannilactone (ABL) is a major sesquiterpene in Inula Britannica and Inula Japonica. To investigate the chemistry properties of ABL, 12 analogues were synthesized. Compound 1, a new 6-deoxybrintanilactone with a methylene at C-14 position, was characterized by 1D, 2D NMR and HR-MS spectrum. The studies of anti-inflammatory activities showed that compounds 1 and 4 exhibited significant inhibitory effects on the nitric oxide production and inducible nitric oxide synthase (iNOS) expressions. The preparation of compounds 1 and 2 from ABL was also studied. We also speculated a proposed mechanism for the formation of 1, 2 and 3. © 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
1. Introduction Inula. Britannica is a traditional medicinal herb used for the treatment of bronchitis and inflammation with high sesquiterpenes content. 1-O-acetylbritannilactone (ABL) is a 1,10-secoeudesmanolide sesquiterpene isolated from I. Britannica (Bohlmann et al., 1978) with multiple biological effects including antiinflammatory, antibacterial, antihepatitic, antidiabetes, and antitumour activities (Zhou et al., 1993; Fang et al., 2011; Ho et al., 2003; Je et al., 2004; Liu et al., 2008; Qi et al., 2008). In addition, it has been reported that numerous ABL analogues have wide pharmacological effects (Fig. 1) (Chen et al., 2015; Dong et al., 2014; Han et al., 2016; Tang et al., 2014; Xiang et al., 2016). These analogues of ABL not only enrich the chemistry diversity of natural products, but also present valuable biological activities. To study the chemistry of ABL for sesquiterpenes chemistry diversity, we synthesized 12 ABL analogues through esterification, reduction, oxidation and treated with acids. Accidentally, four novel compounds (1-4) were discovered as side products. Their structures were identified using 1H NMR, 13C NMR, 2D NMR and
* Corresponding author at: School of Pharmacy, Tianjin Medical University, Tianjin, 300070, People’s Republic of China. ** Corresponding author. E-mail addresses: [email protected] (H.-Q. Duan), [email protected] (M.-H. Jin), [email protected] (N. Qin).
HR-MS spectra. Compounds 1 and 8 were new 6-deoxybrintanilactones with a methylene group at C-14 position. In this paper, the methods for the preparation of compounds 1-3 were investigated. The biological activities of compounds 1-12 for the inhibition of NO production were also examined in LPS-induced RAW264.7 cells. 2. Results and discussion Powdered and dried flowers of I. japonica (100.0 kg) were extracted with 75% ethanol under reflux. The extracts were concentrated to give a residue (15 kg), which was suspended in water and extracted with EtOAc. The EtOAc extract (1.1 kg) was chromatographed a silica gel column (1.2 kg, 300–400 mesh, 100 10 cm) and eluted with a gradient solvent system (PE-EtOAc, v/v, from 25% to 100% EtOAc). The fractions which had ABL were separated by high-speed countercurrent chromatography (HSCCC, PE-EtOAc-MeOH-H2O, v/v/v/v, 1/1/1/1, mobile phase: lower phase, stationary phase: upper phase, 500 rpm). The ABL (16 g) were purified by recrystallization from MeOH after the HSCCC isolation. The pure compound (+)-ABL was found to be identical to the natural product by comparison of the spectral and optical rotation data (Dong et al., 2014; Merten et al., 2011). Scheme 1 showed the synthesis route of ABL analogues. Treatment of ABL with hydrochloric acid or Sc(OTf)3 generated compounds 1 and 2 in common organic solvents. However, compounds 2 and 3 were obtained when ABL was treated with
http://dx.doi.org/10.1016/j.phytol.2017.02.003 1874-3900/© 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
X.-P. Wei et al. / Phytochemistry Letters 19 (2017) 248–253
249
Fig. 1. The structures of ABL and ABL analogues.
aqueous hydrochloric acid solution without any organic solvents. The oxidation of ABL could produce compounds 4 or 5. The reduction of ABL could give a C-13 b-methyl analogue 6 of ABL with a stereoselectivity by using NaBH4. The analogue 7 was an
acetyl analogue from 6. Treatment of 6 with Sc(OTf)3 also generated analogues 8 and 9. The esterification of ABL could produce compounds 10-12 with isovaleryl chloride, pivaloyl chloride and 3,3-dimethylbutanoyl chloride.
Scheme 1. The synthesis of ABL analogues.
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X.-P. Wei et al. / Phytochemistry Letters 19 (2017) 248–253
The 1H NMR spectral data of 1 was similar to those of ABL, except for two terminal olefinic proton signals [5.19, 5.03 (each 1H, s)] and one olefinic proton signal [5.38 (1H, d, J = 4.0 Hz)]. In the HMBC spectrum, the proton signals at dH 5.19 and 5.03 (H-14a,b) were correlated with the carbon signals at dC 36.3 (C-9), 143.6 (C5), while the signal at dH 5.38 (H-6) were correlated with the signals at dC 75.4 (C-8), 138.6 (C-10), and 136.9 (C-11) (Fig. 2). Other proton and carbon signals were assigned by 2D NMR spectral data analysis as described above. Thus, the structure of compound 1 was determined to be 1-O-acetyl-4aH-1,10-secoeudesma-5(6),10 (14),11(13)-trien-12,8b-olide. Compound 3 was obtained as a colorless oil, the HRESIMS showed a molecular ion peak at m/z 313.1417 ([M+Na]+, calcd for C17H22O4Na [M+Na]+, 313.1416). The 1H NMR spectrum of 3 revealed the presence of three aromatic proton signals at dH 7.12, 7.14 (d, J = 8.2 Hz, each 1H), 7.25 (s, 1H), two terminal methylene signals at dH 5.97, 6.46 (s, each 1H), one oxygenated methylene at dH 3.58 (t, J = 6.4 Hz, 2H), and two methyl signals at dH 1.21 (d, J = 6.8 Hz, 3H) and 2.32 (s, 3H). The 13C NMR spectrum showed a terminal double bond (dC 128.2), a carboxylic carbon (dC171.4), one benzene ring (dC 145.0, 140.8, 135.8, 130.1, 125.6, 125.5), and a 1hydroxy-4-disubstituted pentane group obtained by comparing the 13C NMR data with those of ABL. In the HMBC spectrum, the methyl proton at dH 2.32 (H-14) was correlated with the carbon signals at dC 144.96 (C-5), 130.10 (C-9), and 135.79 (C-10), and the signal at dH 1.21 (H3-15) was correlated with the signal at dC 144.96 (C-5), 33.69 (C-3), 34.35 (C-4), which assigned the C-5 position for 1-hydroxy-4-disubstituted pentane group. On the other hand, the H-6 (dH 7.25) was correlated with C10 (135.8), C-8 (125.6), and C-11 (134.1), and the H-13 signal (dH 6.46) was correlated with C-7 (140.8) and C-12 (171.4) (Fig. 3). Thus, the terminal methylene and carboxyl acid were located at C11 and C-12, respectively. Therefore, the structure of 3 was elucidated as shown. The structures of the other analogues including 2, 4, 6-12 were elucidated in the same manner as described above, and their physical and chemical data were listed in the Supporting information. As previously reported, ABL analogues could inhibit NO production in LPS-induced RAW264.7 cells, such as 1,6-O,Odiacetylbritannilactone (OABL) (Chen et al., 2015; Tang et al., 2014). Our investigation suggested that the ABL analogues 1 and 4 had inhibitory effect on NO production in LPS-induced RAW264.7 cells, with IC50 value 1.3 mM and 3.43 mM respectively (Table 1). Compared to compounds 1 and 4, the esterification analogues 10-12 exhibited cytotoxicity at 10 mM in RAW264.7 cells (Supplementary data Table S1). Compounds 2, 3 and 6-9 could not inhibit NO production or show any cytotoxicity. It was speculated that introduction of the lipophilic chains at C-6 position of ABL could increase the cytotoxicity of RAW264.7. This phenomenon was also observed in cancer cells (Dong et al., 2014). The iNOS was a key
Fig. 2. Key HMBC and 1H-1H-COSY corelations of compound 1.
Fig. 3. Key HMBC corelations of 3.
Table 1 Effects of ABL analogues on LPS-induced NO production in RAW264.7 cells. Compound
IC50(mM)
Compound
IC50(mM)
1 2 3 4 5 6 ABL
1.3 NE a NE a 3.43 NE a NE a NE a
7 8 9 10 11 12 OABL
NE a NE a NE a CT b CT b CT b 13.8
a b
NE = No Effect in the test concentrations. CT = Cytotoxicity.
enzyme of NO synthesis. Compounds 1 and 4 downregulated the protein expression of iNOS in RAW264.7 cells (Fig. 4). It was speculated that the active analogues might exhibit anti-inflammation effects by downregulated the protein expression. To investigate the detailed information for the generation of compounds 1-3, a series of experiments were performed by varying conditions including solvents, concentrations and Lewis acids. Compounds 1-3 were obtained accidentally when reaction mixtures treated with aqueous hydrochloric acid. So the concentration of hydrochloric acid was firstly researched. When the concentration was less than 1.5 M, the product was not detected (Table 2, entry 1–3). Nonetheless, the compounds 2 and 3 were obtained if the concentrations were 1.5, 3.0 and 6.0 M (Table 2, entry 4–6). It was concluded that increasing the concentration of hydrochloric acid hardly affect the product selectivity and their yields (Table 2). This results may be due to the poor solubility of ABL in water. Therefore, the organic solvents were introduced to improve the solubility of ABL. As a result, the yields of compound 2
Fig. 4. The inhibition effects of compounds 1 and 4 on iNOS expressions in LPSinduced RAW264.7 cells.
X.-P. Wei et al. / Phytochemistry Letters 19 (2017) 248–253 Table 2 ABL was treated by HCl (aq) with different concentration.a Entry
Acid
2 Yields
1 2 3 4 5 6
0.1 M HCl 0.6 M HCl 1.0 M HCl 1.5 M HCl 3.0 M HCl 6.0 M HCl
NP NP NP 16 16 21
b
(%)
Table 4 ABL was treated by different acids and solvents.a 3 Yields
c
NP NP NP 16 19 27
c c
b
(%)
c c c
a All reactions were carried out with ABL (20 mg, 0.0648 mmol), HCl (aq) (1 mL), T = r.t., t = 3 h. b Isolated yields. c NP = No Product.
increased as the reaction solvents were changed to dichloromethane or toluene (Table 3, entry 4 and 5). Meanwhile, Compound 1, a new 6-deoxybrintanilactone with a methylene at C-14 position, was obtained simultaneously by using dichloromethane, toluene or EtOAc as reaction solvents (Table 3, entry 4– 6). Next, various acids were explored as a catalyst of this reaction. It was found that oxalic acid, CuSO4, CoCl2, TFA, TsOH and Zn(OTf)2 could not perform the reaction under the experimental conditions (Table 4, entry 1–5). It is worth mentioned that compound 2 was produced at a yield of 80% when Sc(OTf)3 was employed as the catalyzer with dichloromethane (Table 4, entry 6). The replacement of dichloromethane with other solvents could slightly affect the selectivity of products. More compound 1 was obtained than compound 2, when toluene was reaction solvent (Table 4, entry 12). Further researches showed that TFA, TsOH and Zn(OTf)2 could all catalyze the reaction and give products 1 and 2 by a long-time reaction with lower yields. After treated with an acid, the ABL could convert to 1, 2 and 3. Further control experiment proved that 1 could not produce 2 under the same condition. Based on the above studies, the proposed mechanism was shown in Fig. 5. Compound 1 and 2 were from same starting material, but underwent two different reaction paths. For path A, which produced the 1 when promoted by an acid, ABL generated the key cation intermediated A with the dehydration progress. After deprotonation, intermediated A could transfer to product 1. For path B, promoted by an acid, the intramolecular SN2 reaction could be performed which produced the other key intermediate B. Under the acidic condition, after the opening of 4member ring, the allylic cation C could be formed which converted to intermediate D with deprotonation progress. After the following two steps, namely protonation and deprotonation, the product 2 could be generated. The product 3 may be generated from the hydrolysis of 2, or after hydrolysis of ABL and then undergo as same process as path B.
1 2 3 4 5 6 a
Solvent
1 Yields
H2O MeOH THF CH2Cl2 Toluene EtOAc
c
NP NP c NP c trace 29 56
b
(%)
2 Yields 16 NP 11 40 60 12
c
b
(%)
Acid
Solvent
1 Yields
1 2 3 4 5 6 7 8
CuSO4 Oxalic acid CoCl2 TFA Zn(OTf)2 Sc(OTf)3 Sc(OTf)3 Sc(OTf)3
CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 EtOAc toluene
NP c NP c NP c NP c NP c trace 28 49
b
(%)
2 Yields NP NP NP NP NP 80 53 36
b
(%)
c c c c c
a All reactions were carried out with ABL (20 mg, 0.0648 mmol) and the acid (0.0648 mmol). T = r.t., t = 0.5 h. b Isolated yields. c NP = No Product.
3. Conclusion ABL analogues were synthesized, compounds 1 and 8 were new 6-deoxybrintanilactones with a methylene at C-14 position. Compounds 1 and 8 enriched the chemical diversity of ABL molecular framework. As a Lewis acid, Sc(OTf)3 could catalyzed the preparation of compounds 1 and 2 from ABL. Compounds 1 and 4 exhibited significant activities for the inhibition of NO productions and iNOS protein expressions, were valuable for anti-inflammation drug discovery associated with NO production. 4. Experimental section 4.1. Materials
c
c
4.2. Methods
3 Yields 16 NP 27 NP NP NP
Entry
All the reagents and solvents were obtained from commercial sources and used without further purification unless mentioned. Reactions were detected by thin-layer chromatography (TLC) using pre-coated silica gel glass plates containing a fluorescence indicator. Isolation and purification of the compounds were performed by flash column chromatography over silica gel (SiO2; 300–400 mesh). The NMR spectra were recorded on a Bruker AVANCE III 400 instrument (1H NMR, 400 MHz; 13C NMR, 100 MHz). Chemical shifts were expressed in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Mass spectra were taken in ESI mode on an Agilent 1200 LC–MS (Agilent, Palo Alto, CA, USA). The plants of I. japonica were collected from Henan Province, China, and identified by Professor Y. Zhou (Department of Pharmacognosy, School of Pharmacy, Tianjin Medical University). A voucher specimen (IJ201105) was deposited at School of Pharmacy, Tianjin Medical University, China. The antibodies specific for iNOS and b-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The enhanced chemiluminescence (ECL) Western blot detection reagent was purchased from Thermo Fisher Scientific (Rockford, IL, USA). The bacterial LPS was purchased from Sigma-Aldrich (Louis, MO, USA). The RAW264.7 macrophage cells were obtained from the Korea Cell Line Bank (Seoul, Korea) and cultured in DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 mg/ mL of streptomycin.
Table 3 ABL was treated with 37% HCl (aq) in various solvents.a Entry
251
b
(%)
c c
All reactions were carried out with ABL (20 mg, 0.0648 mmol), 37% HCl (100 mL), various solvents (1 mL), T = r.t. b Isolated yields. c NP = No Product.
4.2.1. Measurement of cell viability Cell viability was assessed by the MTT assay. RAW264.7 cells and BMMCs plated in 96-well plates were treated with several concentrations of isolates for 20 h and 4 h, respectively. Then, MTT
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Fig. 5. Proposed mechanisms for the formation of compounds 1 and 2 from ABL with HCl.
(5 mg/mL) was added and incubated for an additional 4 h. The culture medium was discarded, and the formazan blue that formed in the cells was dissolved in DMSO. The optical densities (OD) at 490 nm were measured using a microplate reader.
The synthesis and characterization of compounds 4-12 and results of cell viability were listed in Supplementary information.
4.2.2. Measurement of NO RAW264.7 cells (2 105 cells/mL) were pre-treated with 1-Oacetylbritannilactone analogues for 1 h prior to stimulation with LPS (200 ng/mL) for 18 h. The culture supernatants were collected to be available for measurement of NO production. NO levels were determined using Griess reagent (1% sulfanilamide, 0.1% N-1naphthylenediamine dihydrochloride, and 2.5% phosphoric acid). The absorbance was measured at 570 nm with a multi-mode microplate reader (Molecular Devices FilterMax F5, Sunnyvale, CA, USA). L-N6-(1-iminoethyl) lysine (L-NIL, a selective inhibitor of iNOS) was used as a positive control.
This work was supported by grants from the National Natural Science Foundation of China (NSFC) (81402802, 81202542) and Tianjin Research Program of Applied Basic and Cutting-edge Technologies (15JCQNJC13700, 13JCYBJC24800). We thank Dr. Chun Zhang, University of Utah, USA and Dr. Chen Ji, Abbott Laboratories, USA, for the helpful discussion of the proposed mechanisms for the formation of compounds 1 and 2.
4.2.3. Western blot analysis The cells were washed with PBS three times and lysed with RIPA lysis buffer containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Equal amounts of protein were separated in 10% SDS-polyacrylamide gel and blotted onto a PVDF membrane. The membrane was then blocked with 5% non-fat dry milk in TTBS (20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween-20) and incubated with various primary antibodies. After overnight incubation with primary antibody, the membrane was hybridized with HRP-conjugated secondary antibody for 1 h and washed three times with TTBS. The immunoreactive bands were visualized using the ECL system.
Acknowledgements
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2017.02.003. References Bohlmann, F., Mahanta, P.K., Jakupovic, J., Rastogi, R.C., Natu, A.A., 1978. New sesquiterpene lactones from Inula species. Phytochemistry 17, 1165–1172. Chen, X., Tang, S.A., Lee, E., Qiu, Y., Wang, R., Duan, H.Q., Dan, S., Jin, M., Kong, D., 2015. IVSE, isolated from Inula japonica, suppresses LPS-induced NO production via NF-kB and MAPK inactivation in RAW264.7 cells. Life Sci. 124, 8–15. Dong, S., Tang, J.J., Zhang, C.C., Tian, J.M., Guo, J.T., Zhang, Q., Li, H., Gao, J.M., 2014. Semisynthesis and in vitro cytotoxic evaluation of new analogues of 1-Oacetylbritannilactone, a sesquiterpene from Inula britannica. Eur. J. Med. Chem. 80, 71–82. Fang, X.M., Liu, B., Liu, Y.B., Wang, J.J., Wen, J.K., Li, B.H., Han, M., 2011. Acetylbritannilactone suppresses growth via upregulation of krüppel-like
X.-P. Wei et al. / Phytochemistry Letters 19 (2017) 248–253 transcription factor 4 expression in HT-29 colorectal cancer cells. Oncol. Rep. 26, 1181–1187. Han, Y.Y., Tang, J.J., Gao, R.F., Guo, X., Lei, M., Gao, J.M., 2016. A new semisynthetic 1O-acetyl-6-O-lauroylbritannilactone induces apoptosis of human laryngocarcinoma cells through p53-dependent pathway. Toxicol. In Vitro 35, 112–120. Ho, C.T., Rafi, M., Dipaola, R.S., Ghai, G., Rosen, R.T., Bai, N., 2003. Induction Apoptosis. Google Patents. Je, K.H., Han, A.R., Lee, H.T., Mar, W., Seo, E.K., 2004. The inhibitory principle of lipopolysaccharide-induced nitric oxide production frominula britannica var. chinensis. Arch. Pharm. Res. 27, 83–85. Liu, B., Han, M., Wen, J.K., 2008. Acetylbritannilactone inhibits neointimal hyperplasia after balloon injury of rat artery by suppressing nuclear factor-kB activation. J. Pharmacol. Exp. Ther. 324, 292–298.
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Merten, J., Wang, Y., Krause, T., Kataeva, O., Metz, P., 2011. Total synthesis of the cytotoxic 1,10-seco-eudesmanolides britannilactone and 1,6-O,Odiacetylbritannilactone. Chemistry 17, 3332–3334. Qi, J.L., Fu, Y., Shi, X.W., Wu, Y.B., Wang, Y.Z., Zhang, D.Q., Shi, Q.W., 2008. Sesquiterpene lactones and their anti-tumor activity from the flowers of Inula britannica. Lett. Drug Des. Discov. 5, 433–436. Tang, S.A., Zhu, H., Qin, N., Zhou, J.Y., Lee, E., Kong, D.X., Jin, M.H., Duan, H.Q., 2014. Anti-inflammatory terpenes from flowers of Inula japonica. Planta Med. 80, 583–589. Xiang, P., Guo, X., Han, Y.Y., Gao, J.M., Tang, J.J., 2016. Cytotoxic and pro-apoptotic activities of sesquiterpene lactones from inula britannica. Nat. Prod. Commun. 11, 7–10. Zhou, B.N., Bai, N.S., Lin, L.Z., Cordell, G.A., 1993. Sesquiterpene lactones from Inula britannica. Phytochemistry 34, 249–252.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Synthesis and anti-inflammatory activities of 1-Oacetylbritannilactone analogues Xiao-Peng Weia , Yu-Fen Chena , Hong Zhua,b , Xiao-Ran Wua , Yang Yua , De-Xin Konga,c , Hong-Quan Duana,c,* , Mei-Hua Jina,**, Nan Qinc,** a b c
School of Pharmacy, Tianjin Medical University, Tianjin 300070, People’s Republic of China Department of Pharmacy, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an 223300, People’s Republic of China Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, People’s Republic of China
A R T I C L E I N F O
Article history: Received 16 December 2016 Received in revised form 10 January 2017 Accepted 7 February 2017 Available online xxx Keywords: 1-O-acetylbritannilactone 6-Deoxybrintanilactone Chemistry diversity Nitric oxide production Inducible nitric oxide synthase
A B S T R A C T
Natural product 1-O-acetylbritannilactone (ABL) is a major sesquiterpene in Inula Britannica and Inula Japonica. To investigate the chemistry properties of ABL, 12 analogues were synthesized. Compound 1, a new 6-deoxybrintanilactone with a methylene at C-14 position, was characterized by 1D, 2D NMR and HR-MS spectrum. The studies of anti-inflammatory activities showed that compounds 1 and 4 exhibited significant inhibitory effects on the nitric oxide production and inducible nitric oxide synthase (iNOS) expressions. The preparation of compounds 1 and 2 from ABL was also studied. We also speculated a proposed mechanism for the formation of 1, 2 and 3. © 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
1. Introduction Inula. Britannica is a traditional medicinal herb used for the treatment of bronchitis and inflammation with high sesquiterpenes content. 1-O-acetylbritannilactone (ABL) is a 1,10-secoeudesmanolide sesquiterpene isolated from I. Britannica (Bohlmann et al., 1978) with multiple biological effects including antiinflammatory, antibacterial, antihepatitic, antidiabetes, and antitumour activities (Zhou et al., 1993; Fang et al., 2011; Ho et al., 2003; Je et al., 2004; Liu et al., 2008; Qi et al., 2008). In addition, it has been reported that numerous ABL analogues have wide pharmacological effects (Fig. 1) (Chen et al., 2015; Dong et al., 2014; Han et al., 2016; Tang et al., 2014; Xiang et al., 2016). These analogues of ABL not only enrich the chemistry diversity of natural products, but also present valuable biological activities. To study the chemistry of ABL for sesquiterpenes chemistry diversity, we synthesized 12 ABL analogues through esterification, reduction, oxidation and treated with acids. Accidentally, four novel compounds (1-4) were discovered as side products. Their structures were identified using 1H NMR, 13C NMR, 2D NMR and
* Corresponding author at: School of Pharmacy, Tianjin Medical University, Tianjin, 300070, People’s Republic of China. ** Corresponding author. E-mail addresses: [email protected] (H.-Q. Duan), [email protected] (M.-H. Jin), [email protected] (N. Qin).
HR-MS spectra. Compounds 1 and 8 were new 6-deoxybrintanilactones with a methylene group at C-14 position. In this paper, the methods for the preparation of compounds 1-3 were investigated. The biological activities of compounds 1-12 for the inhibition of NO production were also examined in LPS-induced RAW264.7 cells. 2. Results and discussion Powdered and dried flowers of I. japonica (100.0 kg) were extracted with 75% ethanol under reflux. The extracts were concentrated to give a residue (15 kg), which was suspended in water and extracted with EtOAc. The EtOAc extract (1.1 kg) was chromatographed a silica gel column (1.2 kg, 300–400 mesh, 100 10 cm) and eluted with a gradient solvent system (PE-EtOAc, v/v, from 25% to 100% EtOAc). The fractions which had ABL were separated by high-speed countercurrent chromatography (HSCCC, PE-EtOAc-MeOH-H2O, v/v/v/v, 1/1/1/1, mobile phase: lower phase, stationary phase: upper phase, 500 rpm). The ABL (16 g) were purified by recrystallization from MeOH after the HSCCC isolation. The pure compound (+)-ABL was found to be identical to the natural product by comparison of the spectral and optical rotation data (Dong et al., 2014; Merten et al., 2011). Scheme 1 showed the synthesis route of ABL analogues. Treatment of ABL with hydrochloric acid or Sc(OTf)3 generated compounds 1 and 2 in common organic solvents. However, compounds 2 and 3 were obtained when ABL was treated with
http://dx.doi.org/10.1016/j.phytol.2017.02.003 1874-3900/© 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
X.-P. Wei et al. / Phytochemistry Letters 19 (2017) 248–253
249
Fig. 1. The structures of ABL and ABL analogues.
aqueous hydrochloric acid solution without any organic solvents. The oxidation of ABL could produce compounds 4 or 5. The reduction of ABL could give a C-13 b-methyl analogue 6 of ABL with a stereoselectivity by using NaBH4. The analogue 7 was an
acetyl analogue from 6. Treatment of 6 with Sc(OTf)3 also generated analogues 8 and 9. The esterification of ABL could produce compounds 10-12 with isovaleryl chloride, pivaloyl chloride and 3,3-dimethylbutanoyl chloride.
Scheme 1. The synthesis of ABL analogues.
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The 1H NMR spectral data of 1 was similar to those of ABL, except for two terminal olefinic proton signals [5.19, 5.03 (each 1H, s)] and one olefinic proton signal [5.38 (1H, d, J = 4.0 Hz)]. In the HMBC spectrum, the proton signals at dH 5.19 and 5.03 (H-14a,b) were correlated with the carbon signals at dC 36.3 (C-9), 143.6 (C5), while the signal at dH 5.38 (H-6) were correlated with the signals at dC 75.4 (C-8), 138.6 (C-10), and 136.9 (C-11) (Fig. 2). Other proton and carbon signals were assigned by 2D NMR spectral data analysis as described above. Thus, the structure of compound 1 was determined to be 1-O-acetyl-4aH-1,10-secoeudesma-5(6),10 (14),11(13)-trien-12,8b-olide. Compound 3 was obtained as a colorless oil, the HRESIMS showed a molecular ion peak at m/z 313.1417 ([M+Na]+, calcd for C17H22O4Na [M+Na]+, 313.1416). The 1H NMR spectrum of 3 revealed the presence of three aromatic proton signals at dH 7.12, 7.14 (d, J = 8.2 Hz, each 1H), 7.25 (s, 1H), two terminal methylene signals at dH 5.97, 6.46 (s, each 1H), one oxygenated methylene at dH 3.58 (t, J = 6.4 Hz, 2H), and two methyl signals at dH 1.21 (d, J = 6.8 Hz, 3H) and 2.32 (s, 3H). The 13C NMR spectrum showed a terminal double bond (dC 128.2), a carboxylic carbon (dC171.4), one benzene ring (dC 145.0, 140.8, 135.8, 130.1, 125.6, 125.5), and a 1hydroxy-4-disubstituted pentane group obtained by comparing the 13C NMR data with those of ABL. In the HMBC spectrum, the methyl proton at dH 2.32 (H-14) was correlated with the carbon signals at dC 144.96 (C-5), 130.10 (C-9), and 135.79 (C-10), and the signal at dH 1.21 (H3-15) was correlated with the signal at dC 144.96 (C-5), 33.69 (C-3), 34.35 (C-4), which assigned the C-5 position for 1-hydroxy-4-disubstituted pentane group. On the other hand, the H-6 (dH 7.25) was correlated with C10 (135.8), C-8 (125.6), and C-11 (134.1), and the H-13 signal (dH 6.46) was correlated with C-7 (140.8) and C-12 (171.4) (Fig. 3). Thus, the terminal methylene and carboxyl acid were located at C11 and C-12, respectively. Therefore, the structure of 3 was elucidated as shown. The structures of the other analogues including 2, 4, 6-12 were elucidated in the same manner as described above, and their physical and chemical data were listed in the Supporting information. As previously reported, ABL analogues could inhibit NO production in LPS-induced RAW264.7 cells, such as 1,6-O,Odiacetylbritannilactone (OABL) (Chen et al., 2015; Tang et al., 2014). Our investigation suggested that the ABL analogues 1 and 4 had inhibitory effect on NO production in LPS-induced RAW264.7 cells, with IC50 value 1.3 mM and 3.43 mM respectively (Table 1). Compared to compounds 1 and 4, the esterification analogues 10-12 exhibited cytotoxicity at 10 mM in RAW264.7 cells (Supplementary data Table S1). Compounds 2, 3 and 6-9 could not inhibit NO production or show any cytotoxicity. It was speculated that introduction of the lipophilic chains at C-6 position of ABL could increase the cytotoxicity of RAW264.7. This phenomenon was also observed in cancer cells (Dong et al., 2014). The iNOS was a key
Fig. 2. Key HMBC and 1H-1H-COSY corelations of compound 1.
Fig. 3. Key HMBC corelations of 3.
Table 1 Effects of ABL analogues on LPS-induced NO production in RAW264.7 cells. Compound
IC50(mM)
Compound
IC50(mM)
1 2 3 4 5 6 ABL
1.3 NE a NE a 3.43 NE a NE a NE a
7 8 9 10 11 12 OABL
NE a NE a NE a CT b CT b CT b 13.8
a b
NE = No Effect in the test concentrations. CT = Cytotoxicity.
enzyme of NO synthesis. Compounds 1 and 4 downregulated the protein expression of iNOS in RAW264.7 cells (Fig. 4). It was speculated that the active analogues might exhibit anti-inflammation effects by downregulated the protein expression. To investigate the detailed information for the generation of compounds 1-3, a series of experiments were performed by varying conditions including solvents, concentrations and Lewis acids. Compounds 1-3 were obtained accidentally when reaction mixtures treated with aqueous hydrochloric acid. So the concentration of hydrochloric acid was firstly researched. When the concentration was less than 1.5 M, the product was not detected (Table 2, entry 1–3). Nonetheless, the compounds 2 and 3 were obtained if the concentrations were 1.5, 3.0 and 6.0 M (Table 2, entry 4–6). It was concluded that increasing the concentration of hydrochloric acid hardly affect the product selectivity and their yields (Table 2). This results may be due to the poor solubility of ABL in water. Therefore, the organic solvents were introduced to improve the solubility of ABL. As a result, the yields of compound 2
Fig. 4. The inhibition effects of compounds 1 and 4 on iNOS expressions in LPSinduced RAW264.7 cells.
X.-P. Wei et al. / Phytochemistry Letters 19 (2017) 248–253 Table 2 ABL was treated by HCl (aq) with different concentration.a Entry
Acid
2 Yields
1 2 3 4 5 6
0.1 M HCl 0.6 M HCl 1.0 M HCl 1.5 M HCl 3.0 M HCl 6.0 M HCl
NP NP NP 16 16 21
b
(%)
Table 4 ABL was treated by different acids and solvents.a 3 Yields
c
NP NP NP 16 19 27
c c
b
(%)
c c c
a All reactions were carried out with ABL (20 mg, 0.0648 mmol), HCl (aq) (1 mL), T = r.t., t = 3 h. b Isolated yields. c NP = No Product.
increased as the reaction solvents were changed to dichloromethane or toluene (Table 3, entry 4 and 5). Meanwhile, Compound 1, a new 6-deoxybrintanilactone with a methylene at C-14 position, was obtained simultaneously by using dichloromethane, toluene or EtOAc as reaction solvents (Table 3, entry 4– 6). Next, various acids were explored as a catalyst of this reaction. It was found that oxalic acid, CuSO4, CoCl2, TFA, TsOH and Zn(OTf)2 could not perform the reaction under the experimental conditions (Table 4, entry 1–5). It is worth mentioned that compound 2 was produced at a yield of 80% when Sc(OTf)3 was employed as the catalyzer with dichloromethane (Table 4, entry 6). The replacement of dichloromethane with other solvents could slightly affect the selectivity of products. More compound 1 was obtained than compound 2, when toluene was reaction solvent (Table 4, entry 12). Further researches showed that TFA, TsOH and Zn(OTf)2 could all catalyze the reaction and give products 1 and 2 by a long-time reaction with lower yields. After treated with an acid, the ABL could convert to 1, 2 and 3. Further control experiment proved that 1 could not produce 2 under the same condition. Based on the above studies, the proposed mechanism was shown in Fig. 5. Compound 1 and 2 were from same starting material, but underwent two different reaction paths. For path A, which produced the 1 when promoted by an acid, ABL generated the key cation intermediated A with the dehydration progress. After deprotonation, intermediated A could transfer to product 1. For path B, promoted by an acid, the intramolecular SN2 reaction could be performed which produced the other key intermediate B. Under the acidic condition, after the opening of 4member ring, the allylic cation C could be formed which converted to intermediate D with deprotonation progress. After the following two steps, namely protonation and deprotonation, the product 2 could be generated. The product 3 may be generated from the hydrolysis of 2, or after hydrolysis of ABL and then undergo as same process as path B.
1 2 3 4 5 6 a
Solvent
1 Yields
H2O MeOH THF CH2Cl2 Toluene EtOAc
c
NP NP c NP c trace 29 56
b
(%)
2 Yields 16 NP 11 40 60 12
c
b
(%)
Acid
Solvent
1 Yields
1 2 3 4 5 6 7 8
CuSO4 Oxalic acid CoCl2 TFA Zn(OTf)2 Sc(OTf)3 Sc(OTf)3 Sc(OTf)3
CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 EtOAc toluene
NP c NP c NP c NP c NP c trace 28 49
b
(%)
2 Yields NP NP NP NP NP 80 53 36
b
(%)
c c c c c
a All reactions were carried out with ABL (20 mg, 0.0648 mmol) and the acid (0.0648 mmol). T = r.t., t = 0.5 h. b Isolated yields. c NP = No Product.
3. Conclusion ABL analogues were synthesized, compounds 1 and 8 were new 6-deoxybrintanilactones with a methylene at C-14 position. Compounds 1 and 8 enriched the chemical diversity of ABL molecular framework. As a Lewis acid, Sc(OTf)3 could catalyzed the preparation of compounds 1 and 2 from ABL. Compounds 1 and 4 exhibited significant activities for the inhibition of NO productions and iNOS protein expressions, were valuable for anti-inflammation drug discovery associated with NO production. 4. Experimental section 4.1. Materials
c
c
4.2. Methods
3 Yields 16 NP 27 NP NP NP
Entry
All the reagents and solvents were obtained from commercial sources and used without further purification unless mentioned. Reactions were detected by thin-layer chromatography (TLC) using pre-coated silica gel glass plates containing a fluorescence indicator. Isolation and purification of the compounds were performed by flash column chromatography over silica gel (SiO2; 300–400 mesh). The NMR spectra were recorded on a Bruker AVANCE III 400 instrument (1H NMR, 400 MHz; 13C NMR, 100 MHz). Chemical shifts were expressed in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard. Mass spectra were taken in ESI mode on an Agilent 1200 LC–MS (Agilent, Palo Alto, CA, USA). The plants of I. japonica were collected from Henan Province, China, and identified by Professor Y. Zhou (Department of Pharmacognosy, School of Pharmacy, Tianjin Medical University). A voucher specimen (IJ201105) was deposited at School of Pharmacy, Tianjin Medical University, China. The antibodies specific for iNOS and b-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The enhanced chemiluminescence (ECL) Western blot detection reagent was purchased from Thermo Fisher Scientific (Rockford, IL, USA). The bacterial LPS was purchased from Sigma-Aldrich (Louis, MO, USA). The RAW264.7 macrophage cells were obtained from the Korea Cell Line Bank (Seoul, Korea) and cultured in DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 mg/ mL of streptomycin.
Table 3 ABL was treated with 37% HCl (aq) in various solvents.a Entry
251
b
(%)
c c
All reactions were carried out with ABL (20 mg, 0.0648 mmol), 37% HCl (100 mL), various solvents (1 mL), T = r.t. b Isolated yields. c NP = No Product.
4.2.1. Measurement of cell viability Cell viability was assessed by the MTT assay. RAW264.7 cells and BMMCs plated in 96-well plates were treated with several concentrations of isolates for 20 h and 4 h, respectively. Then, MTT
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Fig. 5. Proposed mechanisms for the formation of compounds 1 and 2 from ABL with HCl.
(5 mg/mL) was added and incubated for an additional 4 h. The culture medium was discarded, and the formazan blue that formed in the cells was dissolved in DMSO. The optical densities (OD) at 490 nm were measured using a microplate reader.
The synthesis and characterization of compounds 4-12 and results of cell viability were listed in Supplementary information.
4.2.2. Measurement of NO RAW264.7 cells (2 105 cells/mL) were pre-treated with 1-Oacetylbritannilactone analogues for 1 h prior to stimulation with LPS (200 ng/mL) for 18 h. The culture supernatants were collected to be available for measurement of NO production. NO levels were determined using Griess reagent (1% sulfanilamide, 0.1% N-1naphthylenediamine dihydrochloride, and 2.5% phosphoric acid). The absorbance was measured at 570 nm with a multi-mode microplate reader (Molecular Devices FilterMax F5, Sunnyvale, CA, USA). L-N6-(1-iminoethyl) lysine (L-NIL, a selective inhibitor of iNOS) was used as a positive control.
This work was supported by grants from the National Natural Science Foundation of China (NSFC) (81402802, 81202542) and Tianjin Research Program of Applied Basic and Cutting-edge Technologies (15JCQNJC13700, 13JCYBJC24800). We thank Dr. Chun Zhang, University of Utah, USA and Dr. Chen Ji, Abbott Laboratories, USA, for the helpful discussion of the proposed mechanisms for the formation of compounds 1 and 2.
4.2.3. Western blot analysis The cells were washed with PBS three times and lysed with RIPA lysis buffer containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Equal amounts of protein were separated in 10% SDS-polyacrylamide gel and blotted onto a PVDF membrane. The membrane was then blocked with 5% non-fat dry milk in TTBS (20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween-20) and incubated with various primary antibodies. After overnight incubation with primary antibody, the membrane was hybridized with HRP-conjugated secondary antibody for 1 h and washed three times with TTBS. The immunoreactive bands were visualized using the ECL system.
Acknowledgements
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