b Beijing University of Chemical Technology, Beijing 100029, China;
c Hainan Branch of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China
In our ongoing chemical investigation of new bioactive secondary metabolites from traditional Chinese medicine (TCM) [1-4], ten new ent-kaurane diterpenoids fimbriatols A (1) and B-J with high oxidation were obtained from the extract of Flickingeria fimbriata (B1.) Hawkes . From the structural features of these new diterpenoids and their possibly biogenetic pathway, it suggests that some post-modification enzymes especially some oxidation ones play a vital role to construct these new compounds, and this implies that more other minor analogs might be existed in this medicinal plant. Thus further chemical investigation of this plant has been progressed in our lab, which led to isolation of three new ent-kaurane diterpenoids fimbriatols K-M (3-5, Fig. 1) with different substituted group together with a known analog 1, 2-dehydroabbeokutone (2) . Herein we report the isolation, structure elucidation, and bioactive evaluation of these new entkaurane diterpenoids.
2. Results and discussion
The molecular formula of compound 3 was determined to be C29H38O5 on the basis of HR-ESI MS m/z 489.2607 ([M+Na]+, Δ + 1.0). Analysis of the 1H NMR and 13C NMR data of 3 revealed the presence of structural features similar to those found in 1 (isolated from the same medicinal plant and its absolute configuration was determined by X-ray diffraction) , except that chemical values of H2-19 were in down-field (4.87, and 4.21 ppm) in 3 compared with those in 1 (3.97, and 3.50 ppm), implying that the 19-OH in 1 was replaced by other functionality. In addition, a pair of trans-double bond protons (H-2'=7.64, J=16.2 Hz, H-3'=6.37, J=16.2 Hz) together with a mono-substituted phenyl proton signals were observed in 3. The down-fielded chemical shift values of the transdouble bond protons suggested its connectivity with a carbonyl group (leading to the chemical shift values of H-2' and H-3' to be in down-field). Considering the additionally above-mentioned information, it demonstrated that a cinnamoyl group might be present in compound 1. This hypothesis was further confirmed by the 1H-1H COSY and HMBC correlations of 3 (Fig. 2). The key HMBC correlation from H2-19 to C-1' suggested that the additional cinnamoyl group was connected at C-19 (Fig. 2). The NOESY correlations of H-5 with CH3-18 and H-9, H-15a with H-9 and H-17b, OCH2-19 with Me-20, H-6b with H-19b and H-14a, and H-13 with H-14b put those protons of Me-18, H-5, H-9 and OCH2-17 as β-configurations, whereas OCH2-19, H-6a, H-13 as α-configurations, which were same as that of 1 . Thus the structure of 3 was determined to be as 16, 17-dihydroxy-ent-kaur-3-one-19-ylcinnamate.
|Figure 2. Key 1H–1H COSY, HMBC correlations of 3, 5 and NOESY correlations of 3|
Compound 4 was assigned a molecular formula of C29H36O5 (twelve degrees of unsaturation) on the basis of HRESIMS analysis (m/z 487.2452 [M+Na]+, D+0.8), implying one more degree of unsaturation than that of 3. Analysis of the 1H NMR and 13C NMR data of 4 revealed the presence of essentially the same structural features as those found in 3, except that the one additional double bond was presentin 4. 1H-1H COSYand HMBC NMR data confirmed the -CH2-1-CH2-2-fragment was oxidized to the additional corresponding double bond in 4. Thus the structure of 4 was established as 16, 17-dihydroxy-ent-kaur-1-en-3-one-19-yl-cinnamate.
The molecular formula of 5 was established as C29H40O7 (ten unsaturations) byanalysis of its HR-ESIMS [m/z 523.2680 (M+Na)+; Δ-0.8mmu] and NMR data. Analysis of the NMR data of 5 revealed the same key structure as that of 1 except the chemical shift values of OCH2-19 were down-fielded (4.87, and 4.21ppm), implying that other group was connected with C-19. In the downfield region of 1H NMR, 1', 3', 4'-substituted phenyl group was observed. The HMBC correlations put the two methoxyl moieties on the phenyl ring at C-3' and C-4'. The key HMBC correlation from OCH2-19 to C-1' confirmed that 3, 4-dimethoxybenzoic acetyl was connected the ent-kaurane diterpenoid at C-19 (Fig. 2). Thus the planar structure 5 was determined. The NOESY correlations of 5 revealed its same relative configuration as that 3 and 4. Thus compound 5 was determined to be 16, 17-dihydroxy-ent-kaur-1-en-3-one-19-yl-3, 4dimethoxybenzoate.
The absolute configurations of compounds 3-5 were tried to be determined by CD spectra (see Supporting information), whereas due to the influence of other additional chromophores in 3-5, the CD curve lines in these compounds 1, 3-5 were not completely consistent. Considering structural features of 3-5, they differentiated from compounds 1 and 2 only with different functionalities at C-19, which did not influence the configuration as confirmed by the NOESY correlations. Most importantly, the absolute configuration of compound 1 isolated from the same plant was determined by X-ray diffraction experiment . Thus from the view of biosynthetic pathway, the absolute configurations compounds 3-5 were suggested to be same as those of compounds 1 and 2.
Ent-kaurane diterpenoids displayed a wide range activity, and some have shown very potent activity against bacteria and tumor cell lines . The structure-activities relationship revealed that α-methylenecyclopentanone moiety was essential for the activity against bacteria attributed to a Michael Addition reaction of this function to a sulfhydryl enzyme in bacteria , and other results confirmed that the α, β-unsaturated keto-group in the ent-kaurane diterpenoids was indispensable in the anti-tumor activities . Compounds 3-5 were evaluated the cytotoxicities against three cancer cell lines of A549, MDA-MB-231, and PANC-1 without any biological activity, which further supported the above-mentioned structure-activity relationship (SAP) conclusions.3. Conclusion
Many ent-kaurane diterpenoids with diverse structure features have been reported from different plants especially from the genus Isodon (=Rabdosia) [7, 9, 10]. In this report, three new ent-kaurane diterpenoids fimbrialtols K-M (3-5) with highly substituted functionalities were isolated from the extract of F. fimbriata (B1.) Hawkes. Though these three compounds do not display cytotoxicities against three cancer cell lines, compounds 3-5 are different all more than 600 known ent-kaurane diterpenoids by containing a cinnamic carboxyl group or benzoic carboxyl group, representing the first report in all known analogues, which implied that the post-modification enzymes indeed play a vital role in diversifying the structural feature of ent-kaurane diterpenoids.4. Experimental 4.1. General experimental procedures
Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were recorded on Beckman Coulter DU 800 spectrometer. IR data were recorded using a Shimadzu FTIR-8400S spectrophotometer. 1H and 13C NMR data were acquired with a Bruker 600 and Varian Inova 600/500 spectrometer using solvent signals (CDCl3; δH 7.26/δC 77.6, DMSO-d6; δH 2.49/δC 39.5) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HR-ESIMS data were acquired using a LTQ Orbitrap XL Mass spectrometer.4.2. Plant material
The plant of F. fimbriata (B1.) Hawkes was collected from Changjiang, Qiongzhong, and Danzhou city, Hainan Province, China, in August 2008. The sample was identified by Dr. Rong-Tao, Li (Hainan Branch of Institute of Medicinal Plant Development), and a voucher specimen (FF2008-1, FF2008-2, FF2008-3) has been deposited in the herbarium of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, China.4.3. Extraction and isolation
The air-dried and smashed stems of F. fimbriata (B1.) Hawkes (3.5 kg) were extracted with 95% EtOH (3 × 14 L) to afford a crude extract (330.0g) after evaporation under vacuum. The extract was suspended in H2O (2.0 L) and then partitioned sequentially with petroleum ether (3 ×3.0 L), CH2Cl2 (3 ×3.0 L), EtOAc (3 ×3.0 L), and n-BuOH (3 ×3.0 L), successively. The CH2Cl2 extract (80.g) was subjected to column chromatography over silica gel (80-100 mesh) eluted with petroleum-acetone (1:3-0:1) as the mobile phase to yield 8 fractions (E1-E8). The E5 (5.4 g) was again subjected to column chromatography over silica gel (200-300 mesh) to obtaincompounds 4 (3.1 mg), 5 (5.0 mg) and 2 (16 mg). E7 fraction wassubjected to column chromatography over silica gel (100-200 mesh) eluted by CH2Cl2-EtOAc to obtain 3 (35.0 mg).
(4R, 16R)-16, 17-dihydroxy-ent-kaur-3-one-19-yl-cinnamate (3): white crystal; mp 219-222 ℃; [α]D25 -25.0 (c 0.05, acetonitrile); UV (MeOH) λmax 216.5 (log ε 0.279), 276 nm (log ε 0.402); IR (KBr) υmax 3557, 2929, 1710, 1600, 1591 cm-1; 1H NMR and 13C NMR see Table 1; HR-ESIMS m/z 489.2607 [M+Na]+ (calcd. for C29H38O5Na, 489.2617).
(4R, 16R)-16, 17-dihydroxy-ent-kaur-1-en-3-one-19-yl-cinnamate (4): white crystal; mp 176-178 ℃; [α]D25 -45.2 (c 0.125, acetonitrile); UV (acetonitrile) λmax 216.5 (log ε 0.805), 276 (log ε 0.805) nm; IR (KBr) υmax 3524, 2936, 1711, 1689, 1600, 1591 cm-1; 1H NMR and 13C NMR see Table 1; HR-ESIMS m/z 487.2452 [M+Na]+ (calcd. for C29H36O5Na, 487.2460).
(4R, 16R)-16, 17-dihydroxy-ent-kaur-3-one-19-yl-3, 4-dimethoxybenzoate acetyl (5): light yellow crystal; mp 251-253 ℃; [α]D25 -9.5 (c 0.1, MeOH); UV (MeOH) λmax 205 (log ε 2.337) nm; IR (KBr) υmax 3524, 2936, 1710, 1600, 1591 cm-1; 1H NMR and 13C NMR see Table 1; HRESI-MS m/z 523.2680 [M+Na]+ (calcd. for C29H40O7Na, 487.2460).Acknowledgments
This work was financially supported by the Chinese National S & T Special Project on Major New Drug Innovation (No. 2013ZX09508104), the Fundamental Research Funds for the Central Scientific Research Institutes for Public Welfare, CAMS Innovation Fund for Medical Sciences (CIFMS, No.2016-I2M-3-015).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.cclet.2016.10.033.
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