Letter Cite This: Org. Lett. 2018, 20, 656−659
pubs.acs.org/OrgLett
Orychophragines A−C, Three Biologically Active Alkaloids from Orychophragmus violaceus Guang-Jie Zhang,†,§ Bin Li,†,§ Hui-Min Cui,† Li Chen,† Ying Tian,† Shi-Jun Liu,† Bo-Wen Li,†,‡ Min Li,† Zi-Ming Xia,†,⊥ Xing-Xiang Chen,†,⊥ Yong Hou,†,# and Jun-Xing Dong*,† †
Beijing Institute of Radiation Medicine, Beijing 100850, People’s Republic of China Beijing University of Technology, Beijing 100124, People’s Republic of China ⊥ Guangdong Pharmaceutical University, Guangzhou 510006, People’s Republic of China # Anhui Medical University, Hefei 230061, People’s Republic of China ‡
S Supporting Information *
ABSTRACT: Orychophragines A−C (1−3), three new alkaloids with an novel 2-piperazinone-fused 2,4-dioxohexahydro-1,3,5triazine skeleton, were isolated from the seeds of Orychophragmus violaceus. Their structures were established on the basis of spectroscopic analysis and X-ray crystallographic analysis. Orychophragines A (1) exhibited remarkable cytotoxicity against HepG2, A549, Hela, and HCT-116 cells with IC50 values of 7.73, 10.79, 11.91, and 9.93 μM, respectively. Orychophragines C (3) showed moderate 60Co γ radiation protection activity in HUVEC cells. A plausible biosynthetic pathway for 1−3 was proposed.
Orychophragmus violaceus (Brassicaceae), introduced as an annual herb, is mainly distributed in north China. Previous chemical and pharmacological studies on Brassicaceae resulted in the isolation and characterization of a number of structurally diverse alkaloids with several novel skeletons and biological activities. To date, the main natural alkaloids reported from Brassicaceae, including nortropane alkaloids,1 indole alkaloids,2 oxindole alkaloids,3 bisindole alkaloids,4 and hydrodibenzofuran alkaloids,5 exhibited extensive biological activities such as antivirus,4 antioxidant,3 antifungal3 xanthine oxidase (XO)/ tyrosinase inhibiting,3 and nitric oxide (NO) inhibiting6 activities. As a part of our ongoing search on new active alkaloids from O. violaceus, three new alkaloids, orychophragines A−C (1−3), bearing a new structure skeleton, were isolated from the seeds of O. violaceus. Compounds 2 and 3 were the first examples of natural products with a unique 2piperazinone-fused 2,4-dioxohexahydro-1,3,5-triazine skeleton. Herein we report the isolation, characterization and bioactivities of these compounds. A possible biosynthetic pathway for the three compounds is also proposed. The air-dried seeds of O. violaceus (40 kg) extracted with 70% EtOH. The pooled extracts were concentrated to yield a residue (4.0 kg) that was then extracted with petroleum ether (PE), EtOAc, and n- BuOH successively, yielding the n-BuOH extract (800 g). The n-BuOH fraction was further separated by column chromatography (CC) over silica gel, Sephadex LH-20 gel, and preparative HPLC to afford 1 (33.7 mg), 2 (14.4 mg) and 3 (25.2 mg) (Figure 1). © 2018 American Chemical Society
Figure 1. Structures of orychophragines A−C (1−3).
Orychophragine A (1) was obtained as colorless amorphous powder. The HR-ESI-MS gave a molecular formula of C14H17O4N3 by m/z 290.1147 [M − H]− (calcd for C14H16O4N3, 290.1141). The 1H NMR data (Table 1) evidenced two methyl signals at δH 2.98 (3H, s, CH3-9) and δH 3.75 (3H, s, CH3-8′) and two nitrogen-bearing protons at δH 8.47 (1H, d, J = 4.8 Hz, NH-4) and 6.81 (1H, t, J = 6.2 Hz, NH-8). An AA′BB′-type aromatic ring δH 7.71 (2H, d, J = 8.8 Received: December 6, 2017 Published: January 17, 2018 656
DOI: 10.1021/acs.orglett.7b03801 Org. Lett. 2018, 20, 656−659
Letter
Organic Letters Table 1. 1H and 13C NMR Data (δ in ppm, J in Hz) for Orychophragines A−C (1−3) in DMSO-d6 1 δH (J, Hz)a
no. 1 2 3 4 5 6 7 8 9 10 11 12 13 1’ 2′,6’ 3′,5′ 4’ 7’ 8’ a
2 δCb
δH (J, Hz)a
3 δCb
δH (J, Hz)a
7.91 s 157.06 s 157.06 s 8.47 3.67 3.64 3.40 6.81 2.98
d (4.8) m; 3.26 dd (11.8, 5.3) m m t (6.2) s
9.79 d (1.3)
3.75 s
152.58 s 9.85 d (1.7)
4.08 dd (13.5,5.7); 3.35 m 3.80 m
33.74 m 5.19 3.40 6.63 2.92
6.66 d (8.8) 7.71 d (8.8)
7.81 d (1.7) 152.58 s 152.20 s
38.55 t 55.16 d 41.63 t
152.10 110.83 130.94 116.17 166.08 51.10
s d d s s m
δCb
d (1.7) m; 3.35 m t (5.8) s
6.70 d (8.8) 7.71 d (8.8)
3.74 s
41.07 t 54.72 d 164.98 s 62.31 d 42.99 t 32.12 151.76 111.30 131.11 116.48 166.37 51.36
m s d d s s m
152.35 s 4.21 d (13.5); 3.21 m 3.60 m
5.13 3.39 6.73 2.97
s m; 3.17 m t (5.8) s
6.66 d (8.8) 7.70 d (8.8)
3.74 s
38.55 t 55.61 d 162.61 s 62.84 d 42.99 t 34.35 151.97 111.16 131.07 116.39 166.31 51.32
m s d d s s m
Data measured at 400 MHz. bData measured at 100 MHz.
Hz, H-3′,5′) and 6.66 (2H, d, J = 8.8 Hz, H-2′,6′) suggested para-disubstituted phenyl rings. The 13C NMR data (Table 1) showed carbon resonances corresponding to the above units and three carbonyl carbons δC 166.08 (C-7′), 157.06 (C-2), and 157.06 (C-4). In the 1H−1H COSY spectrum of 1, correlations of H-4/H-5/H-6/H-7 (Figure 2), combined with HMBC correlations of H-7/C-5,
(13), the absolute configuration of 1 was assigned as 6S (Figure 1).
Figure 3. ORTEP drawing of 1.
Orychophragine B (2) was isolated as white amorphous powder (Figure 4). The molecular formula of 2 was determined as C16H19O5N5 by HR-ESI-MS [M + Na]+ peak at m/z
Figure 2. Key 1 H− 1 H COSY and HMBC correlations of orychophragines A−C (1−3).
C-6 and C-1′ (Figure 2) and NH-8/C-7 and C-6′ confirmed the existence of a −CH2−NH− unit between C-6 and C-1′. HMBC correlations of CH3-9/C-6 indicated that C-9 was attached to N-1. Thus, the planar structure of 1 was confirmed. In order to determine the absolute configuration, 1 was crystallized in a mixture of methanol/acetone/H2O (v/v/v, 3/ 1/4). A single-crystal X-ray diffraction analysis using Cu Kα radiation at 108.0 K confirmed the absolute structure of 1 (Figure 3). On the basis of a Flack parameter value of 0.09
Figure 4. ORTEP drawing of 2. 657
DOI: 10.1021/acs.orglett.7b03801 Org. Lett. 2018, 20, 656−659
Letter
Organic Letters 384.1279 (calcd for C16H19O5N5Na, 384.1284). The 1H NMR data (Table 1) evidenced two methyl signals at δH 2.92 (3H, s, CH3-13) and δH 3.74 (3H, s, CH3-8′) and diagnostic resonances for three nitrogen-bearing protons at δH 9.79 (1H, d, J = 1.3 Hz, NH-3), 7.91 (1H, s, NH-1) and 6.63 (1H, t, J = 5.8 Hz, NH-12). An AA′BB′-type aromatic ring δH 7.71 (2H, d, J = 8.8 Hz, H-3′,5′) and 6.70 (2H, d, J = 8.8 Hz, H2′,6′) suggested a para-disubstituted phenyl rings (ring C). The 13C NMR data (Table 1) showed carbon resonances corresponding to the above units and four carbonyl carbons δC 166.37 (C-7′), 164.98 (C-9), 152.58 (C-2), and 152.20 (C-4). Considering the phenyl ring and four carbonyl groups occupy 8 of 10 degrees of unsaturation in 2, the remaining two degrees of unsaturation should be ascribed to two rings. In the 1H−1H COSY spectrum of 2, homonuclear vicinal coupling correlations of H-6/H-7/H-11/H-12 (Figure 2), combined with HMBC correlations of H-11/C-6, C-7, and C-1′ (Figure 2) confirmed that ring C was linked with ring B via a −CH2NH− unit. HMBC correlations of CH3-13/C-7 and C-9 indicated that C-13 was attached to N-8. The HMBC correlations of H6/C-4, C-7, and C-10 and H-10/C-4 and C-9 demonstrated that ring B was fused with ring A via C-10 and N-5. Thus, the planar structure of 2 was confirmed. Conformational analysis of 2 showed one lowest energy conformer whose relative energy was within 2 kcal/mol. The energies, oscillator strengths, and rotational strengths of the first 20 electronic excitations were calculated using the TDDFT methodology at the B3LYP/6-311+G (d, p) level. The ECD spectra were simulated by the Gaussian function (σ = 0.28 eV). The final ECD spectrum (Figure 6, blue-shifted by 11.5 nm) of 2 was directly accomplished by simulation, while the corresponding theoretical ECD spectrum of the enantiomer of 2 was depicted by inverting that of 2. In the 200−400 nm region, the theoretically calculated ECD spectrum of 2 was in agreement with the experimental ECD spectrum of 2 (Figure 6). Therefore, qualitative analysis of the result allowed the assignment as described, which was further supported by the calculated specific rotation [[α]D −107.5] having the same negative sign [[α]D −31.8 (c 0.20, MeOH)] as the measured data. Thus, the absolute configuration of 2 was determined as 7S, 10S (Figure 1). This conclusion was also confirmed by a single-crystal X-ray diffraction analysis using Cu Kα radiation on the basis of the flack parameter (Figure 4). Orychophragine C (3) was obtained as colorless needle crystals. The HR-ESI-MS gave a molecular formula of C16H19O5N5 by m/z 384.1275 [M + Na]+ (calcd for C16H19O5N5Na, 384.1284), the same as that of 2. The 1H and 13C NMR spectroscopic data of 3 (Table 1) were highly similar to those of 2, indicating that compound 3 might be a stereoisomer of 2. Comparing with 2, the planar structure of 3 was confirmed by detailed 2D NMR data analysis. In order to determine the absolute configuration, 3 was crystallized in a mixture of acetone/H2O (v/v, 2/1). A singlecrystal X-ray diffraction analysis using Cu Kα radiation at 106.5 K confirmed the absolute structure of 3 (Figure 5). The crystal structure showed that 3 possessed a 2-piperazinone-fused 2,4dioxohexahydro-1,3,5-triazine skeleton. On the basis of a Flack parameter value of −0.07 (12), the absolute configuration of 3 was assigned as 7S,10R (Figure 1). Thus, orychophragines A−C (1−3) were structurally elucidated as (S)-methyl 4-(((1-methyl-5,6-dioxopiperazin-2yl)methyl)amino)benzoate, methyl 4-((((7S,10S)-8-methyl2,4,9-trioxooctahydropiperazino[1,3,5]triazin-7-yl)methyl)-
Figure 5. ORTEP drawing of 3.
Figure 6. Experimental ECD spectrum of 2 (black), the calculated ECD spectra of 2 (red), and the enantiomer of 2 (blue).
amino)benzoate and methyl 4-((((7S,10R)-8-methyl-2,4,9trioxooctahydropiperazino[1,3,5]triazin-7-yl)methyl)amino)benzoate. A plausible biogenetic pathway was proposed that the formation of orychophragines involves the following key steps (Scheme 1): (1) a transamination reaction converted serine to Scheme 1. Proposed Biosynthetic Pathway for Orychophragines A−C (1−3)
M1; (2) the enzymic catalytic reaction of M1 and oxalic acid to M2 was catalyzed by N-acetyltransferase; (3) in the existence of Lewis acids, a CN double bond in M3 could form by the leaving of H2O; (4) the NH in the ring was easy to methylation catalyzed by methyl transferase in M4; (5) orychophragine A (1) was derived from M4 by the reaction with a methylated pABA unit; (6) the key intermidate M5 was derived from M2 catalyzed by methyl transferase and N-acetyltransferase; (7) subsequent oxidation of M5 leads to a proposed intermediate M6; (8) M7 was derived from M6 by the reaction with a 658
DOI: 10.1021/acs.orglett.7b03801 Org. Lett. 2018, 20, 656−659
Letter
Organic Letters Table 2. Cytotoxic Activity of 1−3 against HepG2 and A549 Cell Lines in Vitroa IC50 (μM)
a
no.
HepG2
A549
Hela
HCT-116
1 2 3 cisplatin
7.73 ± 0.55*** 43.47 ± 1.37 >50 9.88 ± 0.43
10.79 ± 0.86 48.30 ± 0.91 >50 10.73 ± 0.41
11.91 ± 0.46 >50 >50 8.33 ± 0.35
9.83 ± 0.71 22.47 ± 1.19 >50 9.48 ± 0.98
The results were mean ± SD. ***p < 0.01. Positive control: cisplatin.
Author Contributions
carbamic acid unit; (9) orychophragine B and C (2 and 3) were derived from M7 by the reaction with a methylated pABA unit. In this work, orychophragines A−C (1−3) were evaluated for their cytotoxic activity against HepG2, A549, Hela, and HCT-116 cell lines in vitro with the MTT assay.7 Compound 1 exhibited cytotoxicity with IC50 values of 7.73, 10.79, 11.91, and 9.93 μM (Table 2), respectively. Compounds 1−3 were also evaluated for their anti-HBV activity through inhibiting the secretion of HBsAg in HepG2.2.15 cell line in vitro,8 but they were found inactive at the concentration of 50 μM. The antioxidant capacity of 1−3 were determined in vitro by scavenging ABTS radical assays.9 None of them showed antioxidation activity at the concentration of 50 μM. Compounds 1−3 were evaluated for radiation protection against 60Co γ irradiation in HUVEC cell line.10 Results showed that 3 (at the concentration of 10 μM) increased the survival rate of γ irradiated HUVEC cells from (56.71 ± 0.92) % to (77.64 ± 1.19) % (P < 0.01, GraphPad Prism 5), compared with the control group. In conclusion, orychophragines A−C (1−3), a new family of natural products, were isolated from O. violaceus and represented the first examples of 2-piperazinone-fused 2,4dioxohexahydro-1,3,5-triazine alkaloids. The unusual skeleton of orychophragines A−C indicated that the alkaloids isolated from O. violaceus might imply an interesting biosynthetic mechanism and should be studied further.
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§
G.-J.Z. and B.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Mrs. Mei-Feng Xu and Mrs. Yan Xue of the National Center of Biomedical Analysis for the NMR and HRESIMS measurement.
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REFERENCES
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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.orglett.7b03801. General experimental procedures, 1H and 13C NMR, HSQC, HMBC, COSY, IR, and HRESIMS spectra of 1− 3 (PDF) Accession Codes
CCDC 1574682−1574683 and 1589180 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/ cif, or by emailing
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Guang-Jie Zhang: 0000-0002-2628-7455 Jun-Xing Dong: 0000-0002-7109-2120 659
DOI: 10.1021/acs.orglett.7b03801 Org. Lett. 2018, 20, 656−659
