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New naphthoquinone and monoterpenoid from Plumbago zeylanica
Tetrahedron Letters 55 (2014) 6554–6556
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Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
New naphthoquinone and monoterpenoid from Plumbago zeylanica Susumu Ohira a,⇑, Yoshiaki Yokogawa a, Shinji Tsuji a, Taichi Mitsui a, Tatsuhiko Fukukawa a, Ken-ichiro Hayashi a, Atsuhito Kuboki a, Nobuyasu Matsuura a, Munekazu Iinuma b, Hiroshi Nozaki a,⇑ a b
Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan Department of Pharmacognosy, Gifu Pharmaceutical University, 1-2-54, Daigakunishi, Gifu 501-1196, Japan
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Article history: Received 1 August 2014 Revised 29 September 2014 Accepted 3 October 2014 Available online 23 October 2014 Keywords: Nuclear factor jB Plumbago zeylanica Naphthoquinone Monoterpenoid Chiral synthesis Absolute configuration
a b s t r a c t New naphthoquinone 2 and new monoterpenoid 3 were isolated from the stems of Plumbago zeylanica, and their structures were determined on the basis of 2D NMR spectroscopy. Absolute configuration of 3 was established by synthesis of its enantiomer. The new naphthoquinone 2 showed potent inhibitory activity against nuclear factor jB (NF-jB), equivalent to that of parthenolide, a known potent inhibitor of NF-jB. Ó 2014 Elsevier Ltd. All rights reserved.
Plumbago zeylanica (Plumbaginaceae), a perennial herb, is abundant in the tropical and sub-tropical regions of Asia.1 This plant has traditionally been used in India as a folk medicine to cure indigestion, piles, anascara, diarrhea, and skin diseases.2 Previous studies on the chemical constituents of this plant led to the isolation of several naphthoquinone derivatives.3 One of the major compounds was plumbagin (1), which shows diverse pharmacological effects including anti-inflammatory, anticancer, antibacterial, and antifungal activities.4 Recently, plumbagin was reported to be a potent inhibitor of the nuclear factor jB (NF-jB) activation pathway that leads to the suppression of NF-jB regulated gene products.5 NF-jB is a transcription factor and functions as a key regulatory element in inflammatory and immune responses, and in cancer.6 Hence, agents that can regulate down NF-jB activation are of great interest as lead compounds for the treatment of acute and chronic inflammation. In the course of our continuous search for a new class of inhibitors of NF-jB in plant extracts, we have reported new sesqui- and diterpenoids as significant NF-jB inhibitors from Senecio culcitioides7,8 and Caesalpinia echinata,9 respectively. While looking for other NF-jB inhibitors from Plumbago zeylanica, we succeeded in isolating new naphthoquinone 2, which exhibits NF-jB inhibitory activity, as well as new monoterpene alcohol 3. The structures of these compounds were deduced by spectroscopic
⇑ Corresponding authors. Tel./fax: +81 86 256 9425. E-mail address: [email protected] (S. Ohira). http://dx.doi.org/10.1016/j.tetlet.2014.10.016 0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.
analysis, and the absolute stereochemistry of 3 was determined by the chiral synthesis of its enantiomer. Samples of P. zeylanica were collected in Bangalore, India in 2010. An acetone extract of the stems showed significant NF-jB inhibitory activity. Repeated chromatography yielded new naphthoquinone 2, along with new monoterpene alcohol 3. Stems (3 kg) of P. zeylanica were kept in acetone (18 L) at room temperature and the solvent was removed under reduced pressure. The extract (35 g) was subjected to silica gel column chromatography with a gradient of CHCl3/MeOH (8:1 ? 4:1 ? 2:1 ? 1:1, v/v) to give seven fractions. Active fractions (800 mg) were combined and further purified by silica gel column chromatography eluting with a gradient of benzene/acetone (13:1 ? 8:1 ? 4:1 ? 2:1, v/v) to give an active sub fraction (103 mg), which was further purified by octadecylsilane (ODS) medium-pressure liquid chromatography (MeOH/water 10:1, v/v). Further purification of the active sub fraction (55 mg) was accomplished by silica gel column chromatography (n-hexane/acetone 5:1, v/v) to yield compound 2 (9.5 mg) and compound 3 (10.8 mg). Compound 210 was isolated as a yellow oil, and its molecular formula was found to be C14H14O4 based on HR-EIMS (m/z 246.0891 [M+]). The IR spectrum indicated the presence of a hydroxyl (m 3567 cm 1) and a,b-unsaturated ketone groups (m 1660 cm 1). The 1H NMR spectrum revealed the existence of an olefinic methyl group, two tertiary methyl groups, two orthocoupled aromatic protons on a 1,2,3,4-tetrasubstituted benzene ring, and an olefinic proton attributable to a-substituted-a,
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b-unsaturated ketone. In the 13C NMR spectrum, fourteen carbon signals were present, including two ketone carbonyl carbons and a quaternary carbon bearing a hydroxyl group. These results were similar to those of plumbagin (1) except for the disappearance of the aromatic proton at the 6 position observed in 1. The presence and location of the 2-hydroxy-isopropyl and the olefinic methyl groups were confirmed by HMBC correlations of H-3/C-1, C-4, C4a, H-7/C-10, H-8/C-1, C-4a, H-9/C-1, C-3, H-11/C-6, and H-12/C6. Analysis of HMQC and HMBC spectra enabled the complete assignment of all protons and carbons, as shown in Table 1. These data confirmed that the structure of compound 2 is as presented in Figure 1. Compound 3,11 obtained as a yellow oil ([a]D 17.1°; c 0.5, CHCl3), showed the presence of a pseudomolecular ion [M H2O]+ at m/z 170.1320 in HR-EIMS, corresponding to a molecular formula of C10H20O3. The IR spectrum exhibited an absorption band at 3428 cm 1 and 1636 cm 1 corresponding to the hydroxyl and double bond groups, respectively. The 1H NMR and 1H–1H COSY spectra indicated the presence of two tertiary methyls, a methylene proton connected successively with two carbinyl protons, and two vinyl methyls, one of which was coupled with the vinyl proton at d 5.56 (1H, q, J = 6.4 Hz). These results together with the ten carbon signals including a quaternary carbon adjacent to a hydroxyl group in 13C NMR spectrum, suggested that compound 3 is a linear type monoterpene alcohol with a trisubstituted double bond. Analysis of 2D NMR spectra, including HMQC and HMBC spectra, allowed the assignment of all proton and carbon signals as shown in Table 1. The structure was deduced mainly from the HMBC spectrum. The HMBC correlation (H-1/C-3, H-4/C-2, H-9/C-1, C-2) indicated that one of two secondary and one tertiary alcohol group were located at the C-3 and C-2 positions, respectively. In addition to the correlation of H-5/C-7, C-10, H-8/C-6, C-7, the deshielded carbon, and proton signals (dC 81.7 and dH 4.53) suggested that the other secondary alcohol group was attached to the C-5 position. Thus, the planar structure of 3 was elucidated as shown in Figure 1. The (Z)-geometry of the trisubstituted double bond was deduced from the NOE correlation of H-5/H-8, H-5/H-10, and H7/H-10. The relative configuration at positions C-3 and C-5 could not be determined. This is the first instance where a monoterpenoid was isolated from P. zeylanica (Figs. 2 and 3). The absolute structure of 3 became clear after the synthesis of the enantiomer of 3 and its diastereomer from L-malic acid (4). The hydroxyl group of lactone 5, obtained by a known method from 4,12 was protected as a tert-butyldimethylsilyl (TBS) ether 6.
Table 1 H and 13C NMR data of compounds 2 and 3
1
No.
2 dH
1 2 3 4 4a 5 6 7 8 8a 9 10 11 12
6.81 (1H, s)
7.73 (1H, d, 7.9) 7.62 (1H, d, 7.9) 2.19 (3H, d, 1.5) 1.68 (3H, s) 1.68 (3H, s)
3 dC
dH
dC
184.5 149.9 135.5 191.0
1.24 (3H, s)
21.5 82.6 78.5 38.8
115.1 159.0 143.0 132.2 119.4 130.7 16.4 72.5 29.1 29.1
4.00 (1H, dd, 6.0, 3.6) 1.91 (1H, ddd, 12.8, 6.4, 3.2) 2.09 (1H, ddd, 12.8, 9.2, 6.0) 4.53 (1H, t-like, 7.8) 5.56 (1H, q, 6.4) 1.60 (3H, br d, 6.4)
81.7 135.2 121.3 13.1
1.26 (3H, s) 1.57 (3H, br s)
27.8 11.0
NMR spectra were recorded by 500 MHz NMR in CDCl3 and TMS was used as internal standard.
1
2
3
Figure 1. Structures of compounds 1–3.
Figure 2. HMBC of 2.
Figure 3. HMBC, 1H–1H COSY and NOE of 3.
The Grignard reaction of 6 and methyl magnesium bromide, followed by deprotection of the TBS group gave triol 7. After selective protection of vicinal diol, the primary alcohol was oxidized with pyridinium chlorochromate (PCC) to aldehyde 9. Without purification, 9 was treated with the Grignard reagent prepared from (Z)-2bromo-2-butene to form alcohol 10 as a diastereomeric mixture. The isomerization of the acetonide of 1,2-diol (10) to the acetonide of 1,3-diol (11 and 12) was partially achieved with a catalytic amount of PPTS at 50 °C in 3,3-dimethoxypropane (DMP). 11 (32%)13 and 12 (10%)14 were separated by silica-gel column chromatography, and characterized by NMR analysis. Comparison of the chemical shifts of the C-2 carbon of the 1,3-dioxane system (106.6 for 11, 98.8 for 12) indicated that the major isomer is the acetonide of anti-1,3-diol with a twist-boat conformation, and the minor isomer is the acetonide of syn-1,3-diol with a chair conformation.15 Furthermore, observation of the NOE between the C-4 and C-6 protons of the 1,3-dioxane system of 12 (Fig. 4) confirmed their relative stereochemistry. Removal of the acetonide group of
Figure 4. NOE of 12.
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well as plumbagin [10 mM: 100% inhibition], which was simultaneously isolated from this plant during the course of this work. In contrast, inhibitory activity was not observed for compound 3 [20 mM: 0% inhibition]. Thus, the presence of the naphthoquinone skeleton is presumed to play a role in the inhibitory activity, suggesting that naphthoquinone derivatives are promising lead compounds for anti-inflammatory agents (Scheme 1).
References and notes
Scheme 1. Reagent and conditions: (a) TBSCl, imidazole, rt, 2 h (98%); (b) CH3MgBr, Et2O, rt, 4 h (71%); (c) TBAF. THF, rt, 6 h (99%); (d) DMP, PPTS, THF, rt, 2 h (72%); (e) PCC, CH2Cl2, rt, 3 h; (f) (Z)-2-bromo-2-butene, Mg, THF, rt, 6 h (43% in two steps); (g) DMP, PPTS, 50 °C, 24 h (32%, 10%); (h) MeOH, PPTS, 50 °C, 8 h (49%); (i) 3 M HCl, THF, 40 °C 10 h (53%).
11 and 12 gave triol 1316 and 14,17 respectively. The spectral properties of 14 were identical to those of the natural product, and the optical rotation (+19°) was opposite to that of the natural product. At this point, the absolute structure of the natural product 3 was confirmed as shown in Figure 1 The NF-jB inhibitory effects of compounds 2 and 3 on TNF-a regulated gene expression were evaluated using HeLa NF-jB-3 cells that expressed the secreted alkaline phosphatase (SEAP) reporter enzyme under the control of an NF-jB responsive promoter.18 Compounds 2 and 3 were subjected to this assay system at 20 mM and 10 mM, and each value was determined from at least three individual experiments. Compound 2 showed potent activity [20 mM: 100%, 10 mM: 90 % inhibitory] equivalent to that of parthenolide [10 mM: 100%], a known potent inhibitor of NF-jB, as
1. Studies in Natural Products Chemistry; Atta-ur-Rhaman, Ed.; Elsevier: Amsterdam, 1988; Vol. 2, pp 211–249. 2. Gupta, A.; Gupta, A.; Singh, J. Pharm. Biol. 1999, 37, 321–323. 3. (a) Kamal, G. M.; Gunaherath, B.; Gunatilaka, A. A. L.; Thomson, R. H. Tetrahedron Lett. 1984, 25, 4801–4804; (b) Gunaherath, G. M. K. B.; Gunatilaka, A. A.; Cox, P. J.; Howie, R. A.; Thomson, R. H. Tetrahedron Lett. 1988, 29, 719–720. 4. Padbye, S.; Dandawate, P.; Yusufi, M.; Admad, A.; Sarkar, F. H. Med. Res. Rev. 2012, 32, 1131–1158. 5. (a) Sandur, S. K.; Ichihara, H.; Sethi, G.; Ahn, K. S.; Aggarwal, B. B. J. Biol. Chem. 2006, 281, 17023–17033; (b) Ahmad, A.; Banerjee, S.; Wang, Z.; Kong, D.; Sarkar, F. H. J. Cell. Biochem. 2008, 105, 1461–1471. 6. Ghosh, S.; May, M. J.; Kepp, E. B. Annu. Rev. Immunol. 1998, 16, 225–260. 7. Nozaki, H.; Hayashi, K.; Kawai, M.; Mitsui, T.; Kido, M.; Tani, H.; Takaoka, D.; Uno, H.; Ohira, S.; Kuboki, A.; Matsuura, N. Nat. Prod. Commun. 2012, 7, 427– 430. 8. Mitsui, T.; Hayashi, K.; Kawai, M.; Kido, M.; Tani, H.; Takaoka, D.; Matsuura, N.; Nozaki, H. Chem. Pharm. Bull. 2013, 61, 816–822. 9. Mitsui, T.; Ishihara, R.; Hayashi, K.; Sunadome, M.; Matsuura, N.; Nozaki, H. Chem. Pharm. Bull. 2014, 62, 267–273. 10. Compound 2, yellow oil; UV(MeOH) kmax nm (log e); 398.0 (3.94), 271.0 (4.14), 248.0 (4.13), 208.0 (4.45); IR (neat) mmax; 3567, 2360, 1734, 1660, 1164, 1044 cm 1; HR-EIMS m/z: 246.0891 [M]+ (calcd for C14H14O4, D0.1 mmu). 11. Compound 3, yellow oil; [a]D 17.1° (c 0.5, CHCl3); IR (neat) mmax; 3428, 2970, 1636, 1456, 1378, 1134 cm 1; HR-EIMS m/z: 170.1320 [M H2O]+ (calcd for C10H18O2, D1.3 mmu). 12. Denmark, S. E.; Yang, S.-M. J. Am. Chem. Soc. 2004, 126, 12432–12440. 13. Compound 11, IR (neat) mmax; 3838, 3728, 3624, 2979, 2271, 1699, 1557, 1370, 1156, 1003 cm 1; HR-EIMS m/z: 228.1738 [M]+ (calcd for C13H24O3, D1.3 mmu); 1H NMR (400 MHz, CDCl3): d 1.10 (3H, s), 1.27 (3H, s), 1.37 (3H, s), 1.43 (3H,s), 1.64 (3H, dd, J = 1.6, 6.8), 1.62 (2H, m), 1.73 (3H, t, J = 1.6), 3.98 (1H, dd, J = 2.4, 10.4), 4.84 (1H, dd, J = 3.2, 9.2), 5.32 (1H, dd, J = 6.8, 14.0); 13C NMR (100 MHz, CDCl3): d 13.0, 18.1, 23.1, 25.9, 26.9, 28.6, 35.0, 66.3, 79.5, 80.0, 106.6, 119.8, 138.2. 14. Compound 12, [a]D 17° (c 0.15, CHCl3); IR (neat) mmax; 3838, 3728, 3624, 2979, 2271, 1699, 1557, 1370, 1156, 1003 cm 1; HR-EIMS m/z: 228.1726 [M]+ (calcd for C13H24O3, D0.1 mmu); 1H NMR (400 MHz, CDCl3): d 1.14 (3H, s), 1.20 (3H, s), 1.42 (3H, s), 1.50 (3H, s), 1.60 (2H, m), 1.65 (3H, dd, J = 1.6, 6.8), 1.71 (3H, t, J = 1.6), 3.69 (1H, dd, J = 2.8, 12.0), 4.80 (1H, dd, J = 2.8, 12.0), 5.36 (1H, m); 13C NMR (100 MHz, CDCl3): d 13.0, 18.3, 19.8, 23.6, 26.0, 28.2, 30.1, 66.9, 71.4, 75.2, 98.8, 122.1, 135.7. 15. (a) Evans, D. A.; Rieger, D. L.; James, R.; Gag, J. R. Tetrahedron Lett. 1990, 31, 7099–7100; (b) Rychnovsky, S. D.; Rogers, B. N.; Richardson, T. I. Acc. Chem. Res. 1998, 31, 9–17. 16. Compound 13, [a]D +14° (c 0.435, CHCl3); IR (neat) mmax; 3851, 3733, 3334, 2970, 2922, 2857, 1636, 1456, 1378, 1134 cm 1; HR-EIMS m/z: 170.1283 [M H2O]+ (calcd for C10H18O3, D2.4 mmu); 1H NMR (400 MHz, CDCl3): d 1.18 (3H, s), 1.22 (3H, s), 1.26 (3H, s), 1.65 (2H, m), 1.72 (3H, m), 3.64 (1H, dd, J = 1.6, 10.4), 4.89 (1H, dd, J = 3.2, 10.0), 5.33 (1H, m); 13C NMR (100 MHz, CDCl3): d 12.9, 17.7, 23.7, 26.3, 35.2, 70.1, 72.6, 78.7, 121.6, 137.2. 17. Compound 14, [a]D +19° (c 0.405, CHCl3); the other spectral properties were essentially identical to those of 3. 18. Matsuura, N.; Murakami, R.; Katayama, T.; Hibino, S.; Choshi, T.; Yamada, M. J. Health Sci. 2009, 55, 311–313.
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Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
New naphthoquinone and monoterpenoid from Plumbago zeylanica Susumu Ohira a,⇑, Yoshiaki Yokogawa a, Shinji Tsuji a, Taichi Mitsui a, Tatsuhiko Fukukawa a, Ken-ichiro Hayashi a, Atsuhito Kuboki a, Nobuyasu Matsuura a, Munekazu Iinuma b, Hiroshi Nozaki a,⇑ a b
Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan Department of Pharmacognosy, Gifu Pharmaceutical University, 1-2-54, Daigakunishi, Gifu 501-1196, Japan
a r t i c l e
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Article history: Received 1 August 2014 Revised 29 September 2014 Accepted 3 October 2014 Available online 23 October 2014 Keywords: Nuclear factor jB Plumbago zeylanica Naphthoquinone Monoterpenoid Chiral synthesis Absolute configuration
a b s t r a c t New naphthoquinone 2 and new monoterpenoid 3 were isolated from the stems of Plumbago zeylanica, and their structures were determined on the basis of 2D NMR spectroscopy. Absolute configuration of 3 was established by synthesis of its enantiomer. The new naphthoquinone 2 showed potent inhibitory activity against nuclear factor jB (NF-jB), equivalent to that of parthenolide, a known potent inhibitor of NF-jB. Ó 2014 Elsevier Ltd. All rights reserved.
Plumbago zeylanica (Plumbaginaceae), a perennial herb, is abundant in the tropical and sub-tropical regions of Asia.1 This plant has traditionally been used in India as a folk medicine to cure indigestion, piles, anascara, diarrhea, and skin diseases.2 Previous studies on the chemical constituents of this plant led to the isolation of several naphthoquinone derivatives.3 One of the major compounds was plumbagin (1), which shows diverse pharmacological effects including anti-inflammatory, anticancer, antibacterial, and antifungal activities.4 Recently, plumbagin was reported to be a potent inhibitor of the nuclear factor jB (NF-jB) activation pathway that leads to the suppression of NF-jB regulated gene products.5 NF-jB is a transcription factor and functions as a key regulatory element in inflammatory and immune responses, and in cancer.6 Hence, agents that can regulate down NF-jB activation are of great interest as lead compounds for the treatment of acute and chronic inflammation. In the course of our continuous search for a new class of inhibitors of NF-jB in plant extracts, we have reported new sesqui- and diterpenoids as significant NF-jB inhibitors from Senecio culcitioides7,8 and Caesalpinia echinata,9 respectively. While looking for other NF-jB inhibitors from Plumbago zeylanica, we succeeded in isolating new naphthoquinone 2, which exhibits NF-jB inhibitory activity, as well as new monoterpene alcohol 3. The structures of these compounds were deduced by spectroscopic
⇑ Corresponding authors. Tel./fax: +81 86 256 9425. E-mail address: [email protected] (S. Ohira). http://dx.doi.org/10.1016/j.tetlet.2014.10.016 0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.
analysis, and the absolute stereochemistry of 3 was determined by the chiral synthesis of its enantiomer. Samples of P. zeylanica were collected in Bangalore, India in 2010. An acetone extract of the stems showed significant NF-jB inhibitory activity. Repeated chromatography yielded new naphthoquinone 2, along with new monoterpene alcohol 3. Stems (3 kg) of P. zeylanica were kept in acetone (18 L) at room temperature and the solvent was removed under reduced pressure. The extract (35 g) was subjected to silica gel column chromatography with a gradient of CHCl3/MeOH (8:1 ? 4:1 ? 2:1 ? 1:1, v/v) to give seven fractions. Active fractions (800 mg) were combined and further purified by silica gel column chromatography eluting with a gradient of benzene/acetone (13:1 ? 8:1 ? 4:1 ? 2:1, v/v) to give an active sub fraction (103 mg), which was further purified by octadecylsilane (ODS) medium-pressure liquid chromatography (MeOH/water 10:1, v/v). Further purification of the active sub fraction (55 mg) was accomplished by silica gel column chromatography (n-hexane/acetone 5:1, v/v) to yield compound 2 (9.5 mg) and compound 3 (10.8 mg). Compound 210 was isolated as a yellow oil, and its molecular formula was found to be C14H14O4 based on HR-EIMS (m/z 246.0891 [M+]). The IR spectrum indicated the presence of a hydroxyl (m 3567 cm 1) and a,b-unsaturated ketone groups (m 1660 cm 1). The 1H NMR spectrum revealed the existence of an olefinic methyl group, two tertiary methyl groups, two orthocoupled aromatic protons on a 1,2,3,4-tetrasubstituted benzene ring, and an olefinic proton attributable to a-substituted-a,
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b-unsaturated ketone. In the 13C NMR spectrum, fourteen carbon signals were present, including two ketone carbonyl carbons and a quaternary carbon bearing a hydroxyl group. These results were similar to those of plumbagin (1) except for the disappearance of the aromatic proton at the 6 position observed in 1. The presence and location of the 2-hydroxy-isopropyl and the olefinic methyl groups were confirmed by HMBC correlations of H-3/C-1, C-4, C4a, H-7/C-10, H-8/C-1, C-4a, H-9/C-1, C-3, H-11/C-6, and H-12/C6. Analysis of HMQC and HMBC spectra enabled the complete assignment of all protons and carbons, as shown in Table 1. These data confirmed that the structure of compound 2 is as presented in Figure 1. Compound 3,11 obtained as a yellow oil ([a]D 17.1°; c 0.5, CHCl3), showed the presence of a pseudomolecular ion [M H2O]+ at m/z 170.1320 in HR-EIMS, corresponding to a molecular formula of C10H20O3. The IR spectrum exhibited an absorption band at 3428 cm 1 and 1636 cm 1 corresponding to the hydroxyl and double bond groups, respectively. The 1H NMR and 1H–1H COSY spectra indicated the presence of two tertiary methyls, a methylene proton connected successively with two carbinyl protons, and two vinyl methyls, one of which was coupled with the vinyl proton at d 5.56 (1H, q, J = 6.4 Hz). These results together with the ten carbon signals including a quaternary carbon adjacent to a hydroxyl group in 13C NMR spectrum, suggested that compound 3 is a linear type monoterpene alcohol with a trisubstituted double bond. Analysis of 2D NMR spectra, including HMQC and HMBC spectra, allowed the assignment of all proton and carbon signals as shown in Table 1. The structure was deduced mainly from the HMBC spectrum. The HMBC correlation (H-1/C-3, H-4/C-2, H-9/C-1, C-2) indicated that one of two secondary and one tertiary alcohol group were located at the C-3 and C-2 positions, respectively. In addition to the correlation of H-5/C-7, C-10, H-8/C-6, C-7, the deshielded carbon, and proton signals (dC 81.7 and dH 4.53) suggested that the other secondary alcohol group was attached to the C-5 position. Thus, the planar structure of 3 was elucidated as shown in Figure 1. The (Z)-geometry of the trisubstituted double bond was deduced from the NOE correlation of H-5/H-8, H-5/H-10, and H7/H-10. The relative configuration at positions C-3 and C-5 could not be determined. This is the first instance where a monoterpenoid was isolated from P. zeylanica (Figs. 2 and 3). The absolute structure of 3 became clear after the synthesis of the enantiomer of 3 and its diastereomer from L-malic acid (4). The hydroxyl group of lactone 5, obtained by a known method from 4,12 was protected as a tert-butyldimethylsilyl (TBS) ether 6.
Table 1 H and 13C NMR data of compounds 2 and 3
1
No.
2 dH
1 2 3 4 4a 5 6 7 8 8a 9 10 11 12
6.81 (1H, s)
7.73 (1H, d, 7.9) 7.62 (1H, d, 7.9) 2.19 (3H, d, 1.5) 1.68 (3H, s) 1.68 (3H, s)
3 dC
dH
dC
184.5 149.9 135.5 191.0
1.24 (3H, s)
21.5 82.6 78.5 38.8
115.1 159.0 143.0 132.2 119.4 130.7 16.4 72.5 29.1 29.1
4.00 (1H, dd, 6.0, 3.6) 1.91 (1H, ddd, 12.8, 6.4, 3.2) 2.09 (1H, ddd, 12.8, 9.2, 6.0) 4.53 (1H, t-like, 7.8) 5.56 (1H, q, 6.4) 1.60 (3H, br d, 6.4)
81.7 135.2 121.3 13.1
1.26 (3H, s) 1.57 (3H, br s)
27.8 11.0
NMR spectra were recorded by 500 MHz NMR in CDCl3 and TMS was used as internal standard.
1
2
3
Figure 1. Structures of compounds 1–3.
Figure 2. HMBC of 2.
Figure 3. HMBC, 1H–1H COSY and NOE of 3.
The Grignard reaction of 6 and methyl magnesium bromide, followed by deprotection of the TBS group gave triol 7. After selective protection of vicinal diol, the primary alcohol was oxidized with pyridinium chlorochromate (PCC) to aldehyde 9. Without purification, 9 was treated with the Grignard reagent prepared from (Z)-2bromo-2-butene to form alcohol 10 as a diastereomeric mixture. The isomerization of the acetonide of 1,2-diol (10) to the acetonide of 1,3-diol (11 and 12) was partially achieved with a catalytic amount of PPTS at 50 °C in 3,3-dimethoxypropane (DMP). 11 (32%)13 and 12 (10%)14 were separated by silica-gel column chromatography, and characterized by NMR analysis. Comparison of the chemical shifts of the C-2 carbon of the 1,3-dioxane system (106.6 for 11, 98.8 for 12) indicated that the major isomer is the acetonide of anti-1,3-diol with a twist-boat conformation, and the minor isomer is the acetonide of syn-1,3-diol with a chair conformation.15 Furthermore, observation of the NOE between the C-4 and C-6 protons of the 1,3-dioxane system of 12 (Fig. 4) confirmed their relative stereochemistry. Removal of the acetonide group of
Figure 4. NOE of 12.
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well as plumbagin [10 mM: 100% inhibition], which was simultaneously isolated from this plant during the course of this work. In contrast, inhibitory activity was not observed for compound 3 [20 mM: 0% inhibition]. Thus, the presence of the naphthoquinone skeleton is presumed to play a role in the inhibitory activity, suggesting that naphthoquinone derivatives are promising lead compounds for anti-inflammatory agents (Scheme 1).
References and notes
Scheme 1. Reagent and conditions: (a) TBSCl, imidazole, rt, 2 h (98%); (b) CH3MgBr, Et2O, rt, 4 h (71%); (c) TBAF. THF, rt, 6 h (99%); (d) DMP, PPTS, THF, rt, 2 h (72%); (e) PCC, CH2Cl2, rt, 3 h; (f) (Z)-2-bromo-2-butene, Mg, THF, rt, 6 h (43% in two steps); (g) DMP, PPTS, 50 °C, 24 h (32%, 10%); (h) MeOH, PPTS, 50 °C, 8 h (49%); (i) 3 M HCl, THF, 40 °C 10 h (53%).
11 and 12 gave triol 1316 and 14,17 respectively. The spectral properties of 14 were identical to those of the natural product, and the optical rotation (+19°) was opposite to that of the natural product. At this point, the absolute structure of the natural product 3 was confirmed as shown in Figure 1 The NF-jB inhibitory effects of compounds 2 and 3 on TNF-a regulated gene expression were evaluated using HeLa NF-jB-3 cells that expressed the secreted alkaline phosphatase (SEAP) reporter enzyme under the control of an NF-jB responsive promoter.18 Compounds 2 and 3 were subjected to this assay system at 20 mM and 10 mM, and each value was determined from at least three individual experiments. Compound 2 showed potent activity [20 mM: 100%, 10 mM: 90 % inhibitory] equivalent to that of parthenolide [10 mM: 100%], a known potent inhibitor of NF-jB, as
1. Studies in Natural Products Chemistry; Atta-ur-Rhaman, Ed.; Elsevier: Amsterdam, 1988; Vol. 2, pp 211–249. 2. Gupta, A.; Gupta, A.; Singh, J. Pharm. Biol. 1999, 37, 321–323. 3. (a) Kamal, G. M.; Gunaherath, B.; Gunatilaka, A. A. L.; Thomson, R. H. Tetrahedron Lett. 1984, 25, 4801–4804; (b) Gunaherath, G. M. K. B.; Gunatilaka, A. A.; Cox, P. J.; Howie, R. A.; Thomson, R. H. Tetrahedron Lett. 1988, 29, 719–720. 4. Padbye, S.; Dandawate, P.; Yusufi, M.; Admad, A.; Sarkar, F. H. Med. Res. Rev. 2012, 32, 1131–1158. 5. (a) Sandur, S. K.; Ichihara, H.; Sethi, G.; Ahn, K. S.; Aggarwal, B. B. J. Biol. Chem. 2006, 281, 17023–17033; (b) Ahmad, A.; Banerjee, S.; Wang, Z.; Kong, D.; Sarkar, F. H. J. Cell. Biochem. 2008, 105, 1461–1471. 6. Ghosh, S.; May, M. J.; Kepp, E. B. Annu. Rev. Immunol. 1998, 16, 225–260. 7. Nozaki, H.; Hayashi, K.; Kawai, M.; Mitsui, T.; Kido, M.; Tani, H.; Takaoka, D.; Uno, H.; Ohira, S.; Kuboki, A.; Matsuura, N. Nat. Prod. Commun. 2012, 7, 427– 430. 8. Mitsui, T.; Hayashi, K.; Kawai, M.; Kido, M.; Tani, H.; Takaoka, D.; Matsuura, N.; Nozaki, H. Chem. Pharm. Bull. 2013, 61, 816–822. 9. Mitsui, T.; Ishihara, R.; Hayashi, K.; Sunadome, M.; Matsuura, N.; Nozaki, H. Chem. Pharm. Bull. 2014, 62, 267–273. 10. Compound 2, yellow oil; UV(MeOH) kmax nm (log e); 398.0 (3.94), 271.0 (4.14), 248.0 (4.13), 208.0 (4.45); IR (neat) mmax; 3567, 2360, 1734, 1660, 1164, 1044 cm 1; HR-EIMS m/z: 246.0891 [M]+ (calcd for C14H14O4, D0.1 mmu). 11. Compound 3, yellow oil; [a]D 17.1° (c 0.5, CHCl3); IR (neat) mmax; 3428, 2970, 1636, 1456, 1378, 1134 cm 1; HR-EIMS m/z: 170.1320 [M H2O]+ (calcd for C10H18O2, D1.3 mmu). 12. Denmark, S. E.; Yang, S.-M. J. Am. Chem. Soc. 2004, 126, 12432–12440. 13. Compound 11, IR (neat) mmax; 3838, 3728, 3624, 2979, 2271, 1699, 1557, 1370, 1156, 1003 cm 1; HR-EIMS m/z: 228.1738 [M]+ (calcd for C13H24O3, D1.3 mmu); 1H NMR (400 MHz, CDCl3): d 1.10 (3H, s), 1.27 (3H, s), 1.37 (3H, s), 1.43 (3H,s), 1.64 (3H, dd, J = 1.6, 6.8), 1.62 (2H, m), 1.73 (3H, t, J = 1.6), 3.98 (1H, dd, J = 2.4, 10.4), 4.84 (1H, dd, J = 3.2, 9.2), 5.32 (1H, dd, J = 6.8, 14.0); 13C NMR (100 MHz, CDCl3): d 13.0, 18.1, 23.1, 25.9, 26.9, 28.6, 35.0, 66.3, 79.5, 80.0, 106.6, 119.8, 138.2. 14. Compound 12, [a]D 17° (c 0.15, CHCl3); IR (neat) mmax; 3838, 3728, 3624, 2979, 2271, 1699, 1557, 1370, 1156, 1003 cm 1; HR-EIMS m/z: 228.1726 [M]+ (calcd for C13H24O3, D0.1 mmu); 1H NMR (400 MHz, CDCl3): d 1.14 (3H, s), 1.20 (3H, s), 1.42 (3H, s), 1.50 (3H, s), 1.60 (2H, m), 1.65 (3H, dd, J = 1.6, 6.8), 1.71 (3H, t, J = 1.6), 3.69 (1H, dd, J = 2.8, 12.0), 4.80 (1H, dd, J = 2.8, 12.0), 5.36 (1H, m); 13C NMR (100 MHz, CDCl3): d 13.0, 18.3, 19.8, 23.6, 26.0, 28.2, 30.1, 66.9, 71.4, 75.2, 98.8, 122.1, 135.7. 15. (a) Evans, D. A.; Rieger, D. L.; James, R.; Gag, J. R. Tetrahedron Lett. 1990, 31, 7099–7100; (b) Rychnovsky, S. D.; Rogers, B. N.; Richardson, T. I. Acc. Chem. Res. 1998, 31, 9–17. 16. Compound 13, [a]D +14° (c 0.435, CHCl3); IR (neat) mmax; 3851, 3733, 3334, 2970, 2922, 2857, 1636, 1456, 1378, 1134 cm 1; HR-EIMS m/z: 170.1283 [M H2O]+ (calcd for C10H18O3, D2.4 mmu); 1H NMR (400 MHz, CDCl3): d 1.18 (3H, s), 1.22 (3H, s), 1.26 (3H, s), 1.65 (2H, m), 1.72 (3H, m), 3.64 (1H, dd, J = 1.6, 10.4), 4.89 (1H, dd, J = 3.2, 10.0), 5.33 (1H, m); 13C NMR (100 MHz, CDCl3): d 12.9, 17.7, 23.7, 26.3, 35.2, 70.1, 72.6, 78.7, 121.6, 137.2. 17. Compound 14, [a]D +19° (c 0.405, CHCl3); the other spectral properties were essentially identical to those of 3. 18. Matsuura, N.; Murakami, R.; Katayama, T.; Hibino, S.; Choshi, T.; Yamada, M. J. Health Sci. 2009, 55, 311–313.