One new cyclic hexapeptide named rubipodanin A (1), which is the first identified natural N-desmonomethyl Rubiaceae-type cyclopeptide, together with six known Rubiaceae-type cyclopeptides (2–7) were obtained using the TLC cyclopeptide protosite detection method with ninhydrin from the roots and rhizomes of Rubia podantha. The cyclopeptide structures were elucidated by extensive spectroscopic analysis, including 1D-NMR, 2D-NMR, IR, UV and MS. The solution conformation and biological activities of 1 and RA-V (4) were evaluated, and the results demonstrated that the N9-methyl group plays a vital role in the maintenance of the conformation and bioactivity.
Citation: Wang Z, Zhao S-M, Zhao L-M, Chen X-Q, Zeng G-Z, Tan N-H (2015) Rubipodanin A, the First Natural N-Desmonomethyl Rubiaceae-Type Cyclopeptide from Rubia podantha, Indicating an Important Role of the N9-Methyl Group in the Conformation and Bioactivity. PLoS ONE 10(12): e0144950. https://doi.org/10.1371/journal.pone.0144950
Editor: Surajit Bhattacharjya, Nanyang Technological University, SINGAPORE
Received: August 26, 2015; Accepted: November 25, 2015; Published: December 22, 2015
Copyright: © 2015 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the National Basic Research Program of China (2013CB127505), the Foundation of Chinese Academy of Sciences (Hundred Talents Program, XDA09030301-4), the National Natural Science Foundation of China (U1032602, 91013002, 31470428), the National New Drug Innovation Major Project of China (2011ZX09307-002-02), and the Natural Science Foundation of Yunnan Province (2012GA003).
Competing interests: The authors have declared that no competing interests exist.
Rubiaceae-type cyclopeptides (RAs) are homodicyclohexapeptides mainly composed of one α-D-alanine, one α-L-alanine, three N-methyl-α-L-tyrosines, and one other proteinogenic α-L-amino acid. The most unusual feature is a 14-membered ring formed by a phenolic oxygen linkage between two adjacent tyrosines with a cis peptide bond, and the 14-membered ring is fused to a 18-membered cylic hexapeptide ring [1,2]. In 1977, bouvardin and deoxybouvardin (RA-V), the first two RAs, were isolated from Bouvardin ternifolia (Rubiaceae) with potential antitumor activities . Subsequently, an additional 34 RAs have been isolated from Rubia codifolia, R. yunnanensis and R. akane, and more than 200 analogues have been synthesized [1,4–8]. Studies on antitumor mechanisms of RAs have indicated that RA-VII suppresses protein synthesis through interaction with eukaryotic 80S ribosomes . Furthermore, RA-V and RA-XII inhibit the production of NO and inducible nitric oxide synthase (iNOS) . Moreover, RA-VII has the ability to change the conformational structure of F-actin to induce G2 arrest .
For the unique bicyclic structure and significant antitumor activities in vitro and in vivo [1,2,12], RAs have recently attracted our interest. We have now isolated 28 RAs and synthesized several RA analogues, including 11 novel natural RAs consisting of two new skeleton RAs and one new O-seco-RA from R. yunnanensis and R. schumanniana, respectively [13–17]. We have also evaluated their cytotoxic activities and performed 2D- and 3D-QSAR studies on 54 RAs . Our studies showed that RA-V exhibits anti-inflammatory activity by inhibiting NO production and NF-κB activation induced by TNF-α . Furthermore, RA-V significantly suppresses angiogenesis by down-regulating ERK1/2 phosphorylation in HUVEC and HMEC-1 endothelial cells . In addition, RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis and inhibits cell adhesion and invasion via the PI3K/AKT and NF-κB signaling pathways [20,21]. Importantly, the potential roles of RAs in cancer therapy have been highlighted .
Some investigations have shown that N-methylated amino acids of cyclopeptides play an important role in the maintenance of their conformation and bioactivity. Our previous NMR studies have indicated that both natural and synthetic RAs have two or four conformers [13–18], which prompted us to obtain the natural N-desmethyl RAs and investigate the functional role of amino acid N-methylation in conformation and bioactivity. In this study, we selected another Rubia plant, Rubia podantha Diels, which has been used as a substitute for the traditional Chinese medicine R. codifolia. The roots and rhizomes have been used to treat tuberculosis, menoxenia, rheumatism, contusion, hematemesis, anemia and lipoma for a long time in China. To the best of our knowledge, however, there is no literature on the chemical and antitumor constitutes of this plant. To expand the distribution of plant resources containing RAs and explore the functional role of N-methylated amino acids in natural RAs in the maintenance of the conformation and bioactivity, we structurally and pharmacologically investigated interesting RAs using the TLC cyclopeptide protosite detection method with ninhydrin  from R. podantha. Fortunately, a new cyclic hexapeptide, rubipodanin A (1), which is the first identified natural N-desmonomethyl RA, together with six known RAs (2–7) were obtained. Here, the isolation and structural elucidation of 1–7 as well as cytotoxic and NF-κB signaling pathway activities of 1 and 4 are described.
Materials and Methods
General experimental procedures
Optical rotations were obtained on a Jasco P-1020 polarimeter. IR spectra were measured by a Tensor 27 spectrometer using KBr pellets. UV spectra were performed using a Shimadzu UV-2401A spectrophotometer. 1D-NMR and 2D-NMR spectra were recorded on a Bruker AVANCE III-600 and Ascend ™ 800 spectrometer at 298K. Chemical shifts (δ) were expressed in parts per million (ppm) with reference to the solvent signals. Mass spectra were obtained on a Waters XEVO-TQD spectrometer or an Agilent G6230 TOF Mass spectrometer. Analytical or semi-preparative HPLC was performed on an Agilent 1100 with a Zorbax Eclipse-C18 (4.6 mm × 150 mm; 9.4 mm × 250 mm; 5 μm).
Column chromatography was performed with silica gel (100–200 mesh and 200–300 mesh, Qingdao Yu-Ming-Yuan Chemical Co. Ltd., Qingdao, China), Sephadex LH-20 (Pharmacia Fine Chemical Co., Uppsala, Sweden) or Lichroprep RP-18 gel (40–63 μm, Merck, Darmstadt, Germany). Fractions were monitored by TLC (GF254, Qingdao Yu-Ming-Yuan Chemical Co. Ltd., Qingdao, China), and orange spots were visualized on the plate by spraying with 2% ninhydrin reagent after hydrolyzed in a drying incubator (110°C) for 30 min by HCl .
The roots and rhizomes of R. podantha were collected in November of 2013 from Mount Chang Chong, Kunming, China. The locations/activities to collect R. podantha did not require specific permission because this studied plant is common in the field in Yunnan, China and is not cultivated in privately owned or protected areas. In addition, the filed studies did not involve endangered and protected species. The material was identified by Professor Ning-Hua Tan of Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (KUN0359279) was deposited in the Herbarium of Kunming Institute of Botany.
Extraction and isolation
The air-dried and powdered roots and rhizomes of R. podantha (13 kg) were extracted three times with methanol (3 × 15 L) under reflux. After removal of the solvent under vacuum, the methanol extract (1.63 kg) was subjected to silica gel column chromatography (CC) eluted with petroleum ether-CHCl3 (1:0, 1:1, 0:1) and CHCl3-MeOH (95:5, 9:1, 8:2, 7:3, 0:1) to afford eight fractions. Fractions containing cyclopeptides (150 g, CHCl3-MeOH, 9:1) were rechromatographed on a silica gel CC eluted with a CHCl3-MeOH gradient system (70:1–8:2) to yield six fractions (Fr.1-Fr.6).
Fr.2 (40 g) was applied to silica gel CC using petroleum ether-acetone (3:1–0:1) to yield five subfractions (Fr.2-1 to Fr.2-5). Fr.2-3 (17 g) was separated using Sephadex LH-20 CC (CHCl3-MeOH, 1:1) and then purified over repeated silica gel CC eluting with petroleum ether-acetone (3:2) to afford RA-V (4) (878 mg).
Fr.3 (37 g) was chromatographed on a silica gel CC using a CHCl3-MeOH system (100:1–8:2) to yield six subfractions (Fr.3-1 to Fr.3-6). Fr.3-3 (21 g) was subjected to RP-18 CC, eluting with MeOH-H2O (20–90%) to afford five subfractions (Fr.3-3-1 to Fr.3-3-5). Fr.3-3-2 (9 g) were separated by Sephadex LH-20 CC (CHCl3-MeOH, 1:1) and purified over repeated silica gel CC eluting with petroleum ether-acetone (2:1) to afford RA-VII (5) (658 mg). Fr.3-3-3 (3 g) was also passed through Sephadex LH-20 CC (CHCl3-MeOH, 1:1) and then silica gel CC (petroleum ether–acetone, 3:2) to yield four subfrations (Fr.3-3-3-1 to Fr.3-3-3-4). Fr.3-3-3-2 (32 mg) was further purified by semi-preparative HPLC (30% CH3CN) to yield rubipodanin A (1) (9 mg). Fr.3-3-3-3 (43 mg) was also further purified by semi-preparative HPLC (35% CH3CN) to yield RA-I (2) (7 mg) and RA-III (3) (8 mg).
Fr.4 (10 g) was subjected to an RP-18 CC, eluting with MeOH-H2O (20–90%) to afford four subfractions. Fr.4-4-2 (4 g) were separated by Sephadex LH-20 CC (CHCl3-MeOH, 1:1) and then further purified by semi-preparative HPLC (30% CH3CN) to yield Allo-RA-V (7) (4 mg).
Fr.5 (32 g) was submitted to silica gel CC eluting with gradient CHCl3-MeOH (50:1–8:2) to yield five subfractions (Fr.5-1 to Fr.5-5). Fr.5-3 (13 g) was further purified by repeated silica gel CC (CHCl3-MeOH, 20:1–8:2) followed by Sephdex LH-20 CC (CHCl3-MeOH, 1:1), and it was further separated by repeated RP-18 CC (MeOH-H2O, 30–70%), which led to the isolation of RA-XII (6) (927 mg).
Rubipodanin A (1): white amorphous powder; [α]18.5 D -215.5 (c 0.06, CHCl3); UV (MeOH) λmax (log ε) 203 (4.84), 222 (4.58), 278 (3.73) nm; IR (KBr) νmax 3428, 2957, 2925, 2871, 1656, 1636, 1514, 1500, 1459, 1411, 1377, 1288, 1246, 1212, 1182, 1160, 1111, 1094, 1033 cm-1; 1H (800 MHz) and 13C (200 MHz) NMR data, see Table 1; positive ESIMS m/z 743.80 [M+H]+; positive HRESIMS m/z 765.3214 [M+Na]+, calcd for C39H46N6NaO9 765.3224.
HeLa, A549, SGC7901 and HEK293T cell lines were purchased from the American Type Culture Collection (ATCC, USA) and were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Life technologies, USA) and 1% (v/v) penicillin-streptomycin (Invitrogen, USA). The cells were maintained at 37°C in a humidified incubator atmosphere of 5% CO2/95% air (v/v).
Cytotoxicity was tested by the Sulforhodamine B (SRB) method (Sigma, USA). The assays were performed as described previously .
Cell transfection and luciferase assay
HEK293T cells were seeded in 24-well plates and transiently transfected with 5×κB-luciferase and pTK-Renilla reporters using Lipofectamine 2000 (Invitrogen, USA) for 18 h. The cells were then incubated with different concentrations of compounds for the indicated time, and subsequently stimulated with 10 ng/ml TNF-α for 2 h. The luciferase activity of the cell lysate was analyzed by the Dual Luciferase Reported Assay System (Promega, USA). Luciferase reporter assays were performed as described previously .
Western blot analysis
Cells were lysed using RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, protease inhibitors and phosphatase inhibitors. After being disrupted on ice for 30 min, the cell lysates were centrifuged at 12,000 rpm for 15 min and boiled with SDS sample buffer at 95°C for 5 min. The samples were equally subjected to SDS-PAGE, electrophoresed, and transferred to PVDF membranes (Millipore, USA). After blocking with 5% nonfat milk in TBST, the membranes were incubated with the indicated antibody for 2 h or overnight at 4°C, and the membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The protein bands were detected with a SuperSingal West Pico Chemiluminescence ECL kit (Pierce, USA).
Results and Discussion
The methanol extract of the air-dried powdered roots and rhizomes of R. podantha was subjected to fractionation using silica gel CC, which yielded the total cyclopeptide fraction guided by the TLC cyclopeptide protosite detection method with ninhydrin . This fraction was repeatedly chromatographed over a series of silica gel, Sephadex LH-20, RP-18 CC, and ODS HPLC to obtain seven RAs (Fig 1), including one new natural N-demonomethyl RA (rubipodanin A, 1) and six known RAs, including RA-I (2), RA-III (3), RA-V (4), RA-VII (5), RA-XII (6) and Allo-RA-V (7). The structure and stereochemistry of 1 was established by 1D-NMR, 2D-NMR, UV, IR and MS, and the structural identification of known compounds 2–7 was performed by comparison with NMR data in the literature [7,13,26–28]. Furthermore, cytotoxic activities of 1 and 4 against three tumor cell lines and inhibitory activities of 1 and 4 on the NF-κB signaling pathway were investigated. The results of the solution conformation and biological activities of 1 and 4 suggested that the N9-methyl group plays a vital role in the maintenance of their conformation and bioactivity.
Characterization of Rubipodanin A (1)
Rubipodanin A (1) was obtained as white amorphous powder, and its molecular formula was established as C39H46N6O9 by its positive HRESIMS (m/z 765.3214, [M+Na]+), indicating 20 degrees of unsaturation. The UV spectrum of rubipodanin A showed absorptions at 203, 222 and 278 nm, which demonstrated the existence of phenyl groups. The IR spectrum exhibited absorption bands at 3428 and 1636 cm-1, which is indicative of OH, NH and CO groups. The 1H and 13C NMR spectra of 1 in C5D5N (Table 1) displayed characteristics of typical RAs without any conformers, but these characteristics are common in natural RAs. Further analysis of 1D-NMR and 2D-NMR spectra data of 1 displayed the signals for the three methyls (δH/δC 1.55/21.8, 1.46/17.0, 1.40/19.5), two amide N-methyls (δH/δC 3.03/29.8, 3.00/30.4), one O-methyl (δH/δC 3.71/55.3), three methylenes (δH/δC 3.85, 3.98/33.9; 2.60, 3.65/36.7; 3.34, 3.54/36.5), six α-amino methines (δH/δC 5.77/54.6, 5.19/46.8, 5.18/48.3, 5.01/58.0, 4.91/49.0, 4.10/57.8), two 1,4-disubstituted benzene rings (δC 158.9, 132.9 and δH/δC 7.33/131.1 × 2, 7.04/114.4 × 2; δC 158.9, 136.2 and δH/δC 7.43/133.5, 7.24/131.0, 6.94/126.5, 6.89/124.6), one 1,2,4-trisubstituted benzene ring (δC 152.7, 145.5, 128.1, and δH/δC 7.22/117.9, 6.76/122.2, 4.60/115.1), six carbonyl signals (δC 173.0, 172.6, 172.0, 171.5, 169.9, 169.9) and four amide protons (δH 10.59, 9.80, 8.90, 7.81). An extensive comparison of 1D-NMR and 2D-NMR spectra data of 1 with those of RA-V (4) in C5D5N indicated that both compounds were similar, except for the third amino acid residue (Tyr3). Further analysis of the HMBC and 1H-1H COSY correlations confirmed the construction of the N-desmethyl Tyr3 residue (Fig 2). In the 1H-1H COSY spectrum, cross-peaks of NH-3/H-3α/H-3β suggested that the amide N9-methyl was absent, which was also confirmed by the HMBC correlation from δH 10.59 (NH-3) to δc 173.0 (C-2-CO). In addition, the sequence of the amino acid residues in 1 was confirmed by the HMBC correlations from δH 9.80 (2-NH) to δc 172.6 (C-1-CO), from δH 10.59 (3-NH) to δc 173.0 (C-2-CO), from δH 7.81 (4-NH) to δc 169.9 (C-3-CO), from δH 3.00 (H-5NCH3) to δc 172.0 (C-4-CO), from δH 3.03 (H-6NCH3) to δc 169.9 (C-5-CO) and from δH 8.90 (1-NH) to δc 171.5 (C-6-CO). In the ROESY spectrum, NOE correlations were observed among 3-NH/H-2α, H-3α, 5-NCH3/H-4α, and H-5α/H-6α, thereby indicating that the peptide bonds between Ala2/Tyr3, Ala4/Tyr5 and Tyr5/Tyr6 were trans, trans, and cis, respectively. Because previous have studies demonstrated that the absolute configurations of Ala1 and Ala4 in RAs were generally D (R) and L (S) , the remaining configurations of Ala2, Tyr3, Tyr5 and Tyr6 were deduced as L (S), L (S), L (S) and L (S), respectively, by observing the following NOE correlations: H-1α/2-NH; 3-NH/H-2α, H-3α; 5-NCH3/H-4α; and H-5α/H-6α. Collectively, the structure of 1 was identified as shown in Fig 1, which is the first identified natural N-desmonomethyl RA.
The N-methylated amino acids of cyclopeptides play an important role for the conformation in solution, and RAs generally possess two or four conformers, including natural RAs, synthetic RAs and O-seco-RAs. In 1995, the DL Boger group synthesized several N-desmethyl derivatives of RA-VII and investigated their conformation property by 1H-NMR spectra, and they reported that both N9- and N15-methyl groups are essential for maintenance of their conformation [29–32], which prompted us to investigate the difference between 1 and 4 in NMR spectra. Consistent with previous studies , the 1H NMR spectra of 4 in C5D5N displayed the presence of two conformers in a ratio of 79:21, and 1 revealed a single solution conformation (Fig 3). The similar phenomenon occurred in 13C NMR spectra (Figure N in S1 File), which demonstrated that the N9-methyl group plays a vital role in the conformational property of RAs.
Some studies have shown that natural bioactive cyclopeptides contain at least one N-methylated amino acid. For example, the N-methyl groups of cyclosporin A (CsA), a cyclic undecapeptide containing seven N-methylated amino acids, play an important role for maintenance of immunosuppressive activity . Sansalvamide A, isolated from a marine fungus of the genus Fusarium, is a cyclic tetrapeptide, and the N-methyl derivatives of sansalvamide A exhibit better antitumor activity .
RAs are a type of potent antitumor agent and often contain three N-methylated tyrosines, which promotes researchers to explore the functional role of N-methyl groups on their biological activity. The DL Boger group evaluated the cytotoxicity of synthetic N-desmethyl RA derivatives and showed that the N15-methyl group is essential for their biological activities . In the present study, we investigated the cytotoxicity against three tumor cell lines and inhibitory activity against the tumor-associated NF-κB signaling pathway of rubipodanin A (1) compared to RA-V (4) by using the SRB assay and the Dual Luciferse Reporter Assay System, respectively. As shown in Fig 4, 1 had weak cytotoxicity against HeLa, A549 and SGC-7901 with IC50 values of 7.22 ± 0.76, 7.14 ± 0.81, 3.80 ± 0.17 μM, respectively, which was 420–650 times less than 4 (Table 2). We also found that 4 exhibited a potent inhibitory activity against the NF-κB pathway activation induced by TNF-α, which was approximately 700 times greater than that of 1. In addition, we examined the effects of 1 and 4 on the expression of NF-κB-associated proteins using western blot analysis. As expected, 1 also had an inhibitory effect on TNF-α-induced IκBα phosphorylation, IκBα degradation and p65 phosphorylation only in the high concentration up to 20 μM, while 4 exhibited similar inhibitory effect at a concentration 200 nM. Taken together, these results suggested that the N9-methyl group is essential for maintenance of biological activities of natural RAs and that down-regulation of the NF-κB pathway might account for the mechanism of the antitumor activities of RAs. Of note, previous reports have shown that the N9-methyl group of synthetic RAs is not critical for biological activities , which was not consistent with our results. So more N-desmethyl derivatives of RAs from plants or by synthesis in future are needed to confirm this difference based on the structure-activity relationship analysis.
The phytochemical studies of Rubia podantha were performed using the TLC cyclopeptide protosite detection method with ninhydrin, which led to the identification of one new cyclic hexapeptide (rubipodanin A, 1) and six known ones (2–7). Importantly, 1 is the first identified natural N-desmonomethyl RA. Further studies revealed that 1 had only one solution conformation in NMR but two in RA-V (4), and it also had lower cytotoxicity and inhibitory activity against the NF-κB signaling pathway than 4, thus indicating that the N9-methyl group is essential for the conformation and biological activities of RAs. These findings not only expanded the distribution of plant resources containing RAs but also provided new insights into the role of the N-methyl group in maintaining conformational and biological properties of RAs.
S1 File. Relevant spectra of Rubipodanin A (1) and RA-V (4).
1H NMR Spectrum of Rubipodanin A (1) (Figure A). 13C NMR Spectrum of Rubipodanin A (1) (Figure B). HSQC Spectrum of Rubipodanin A (1) (Figure C). COSY Spectrum of Rubipodanin A (1) (Figure D). HMBC Spectrum of Rubipodanin A (1) (Figure E). ROESY Spectrum of Rubipodanin A (1) (Figure F). ESI Mass Spectrum of Rubipodanin A (1) (Figure G). High Resolution Mass Spectrum of Rubipodanin A (1) (Figure H). UV Spectrum of Rubipodanin A (1) (Figure I). IR Spectrum of Rubipodanin A (1) (Figure J). [α]D Spectrum of Rubipodanin A (1) (Figure K). 1H NMR Spectrum of RA-V (4) (Figure L). 13C NMR Spectrum of RA-V (4) (Figure M). 13C NMR spectra comparison of Rubipodanin A (1, top) and RA-V (4, bottom) (Figure N).
We would like to thank the members of the analytical group of the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany for all of the spectral measurements.
Conceived and designed the experiments: ZW NHT. Performed the experiments: ZW GZZ. Analyzed the data: ZW SMZ GZZ. Contributed reagents/materials/analysis tools: SMZ LMZ XQC. Wrote the paper: ZW NHT.
- 1. Tan NH, Zhou J. Plant cyclopeptides. Chem Rev. 2006; 106: 840–895. pmid:16522011
- 2. Zhao SM, Kuang B, Fan JT, Yan H, Xu WY, Tan NH. Antitumor cyclic hexapeptides from Rubia plants: history, chemistry, and mechanism (2005–2011). Chimia. 2011; 65: 952–956. pmid:22273378
- 3. Jolad SD, Hoffman JJ, Torrance SJ, Wiedhopf RM, Cole JR, Arora SK, et al. Bouvardin and deoxybouvardin, antitumor cyclic hexapeptides from Bouvardia ternifolia (Rubiaceae). J Am Chem Soc. 1977; 99: 8040–8044. pmid:591683
- 4. Lee JE, Hitotsuyanagi Y, Kim IH, Hasuda T, Takeya K. A novel bicyclic hexapeptide, RA-XVIII, from Rubia cordifolia: structure, semi-synthesis, and cytotoxicity. Bioorg Med Chem. 2008; 18: 808–811.
- 5. Lee JE, Hitotsuyanagi Y, Fukaya H, Kondo K, Takeya K. New cytotoxic bicyclic hexapeptides, RA-XXIII and RA-XXIV, from Rubia cordifolia L. Chem Pharm Bull. 2008; 56: 730–733. pmid:18451569
- 6. Lee JE, Hitotsuyanagi Y, Takeya K. Structures of cytotoxic bicyclic hexapeptides, RA-XIX, -XX, -XXI, and -XXII, from Rubia cordifolia L. Tetrahedron 2008; 64: 4117–4125.
- 7. Hitotsuyanagi Y, Odagiri M, Kato S, Kusano JI, Hasuda T, Fukaya H, et al. Isolation, structure determination, and synthesis of Allo-RA-V and Neo-RA-V, RA-series bicyclic peptides from Rubia cordifolia L. Chem A Eur J. 2012; 18: 2839–2846.
- 8. Hitotsuyanagi Y, Kusano JI, Kim IH, Hasuda T, Fukaya H, Takeya K. O-Seco-RA-XXIV, a possible precursor of an antitumor peptide RA-XXIV, from Rubia cordifolia L. Phytochem Lett. 2012; 5: 335–339.
- 9. Morita H, Yamamiya T, Takeya K, Itokawa H, Sakuma C, Yamada J, et al. Conformational recognition of RA-XII by 80S ribosomes: a differential line broadening study in 1H NMR spectroscopy. Chem Pharm Bull. 1993; 41: 781–783. pmid:8508481
- 10. Tao J, Morikawa T, Ando S, Matsuda H, Yoshikawa M. Bioactive constituents from Chinese natural medicines. XI. Inhibitors on NO production and degranulation in RBL-2H3 from Rubia yunnanensis: structures of rubianosides II, III, and IV, rubianol, and rubianthraquinone. Chem Pharm Bull. 2003; 51: 654–662. pmid:12808242
- 11. Fujiwara H, Saito SY, Hitotsuyanagi Y, Takeya K, Ohizumi Y. RA-VII, a cyclic depsipeptide, changes the conformational structure of actin to cause G2 arrest by the inhibition of cytokinesis. Cancer Lett. 2004; 209: 223–229. pmid:15159025
- 12. Morita H, Takeya K. Bioactive cyclic peptides from higher plants. Heterocycles 2010; 80: 739–764.
- 13. Fan JT, Su J, Peng YM, Li Y, Li J, Zhou YB, et al. Rubiyunnanins C–H, cytotoxic cyclic hexapeptides from Rubia yunnanensis inhibiting nitric oxide production and NF-κB activation. Bioorg Med Chem. 2010; 18: 8226–8234. pmid:21044847
- 14. Fan JT, Chen YS, Xu WY, Du LC, Zeng GZ, Zhang YM, et al. Rubiyunnanins A and B, two novel cyclic hexapeptides from Rubia yunnanensis. Tetra Lett. 2010; 51: 6810–6813.
- 15. Kuang B, Fan JT, Zhao SM, Tan NH. Cyclopeptides from Rubia schumanniana. Chin J Chin Mater Med. 2012; 37: 2563–2570.
- 16. Huang MB, Zhao SM, Zeng GZ, Kuang B, Chen XQ, Tan NH. Rubischumanins A-C, new cytotoxic cyclopeptides from Rubia schumanniana. Tetrahedron 2014; 70: 7627–7631.
- 17. Liu NN, Zhao SM, Zhao JF, Zeng GZ, Tan NH, Liu JP. Synthesis and conformation studies of rubiyunnanin B analogs. Tetrahedron 2014; 70: 6630–6640.
- 18. Yan H, Pan XL, Tan NH, Fan JT, Zeng GZ, Han HJ. 2D- and 3D-QSAR studies on 54 anti-tumor Rubiaceae-type cyclopeptides. Eur J Med Chem. 2009; 44: 3425–3432. pmid:19303172
- 19. Yue GG, Fan JT, Lee JK, Zeng GZ, Ho TW, Fung KP, et al. Cyclopeptide RA-V inhibits angiogenesis by down-regulating ERK1/2 phosphorylation in HUVEC and HMEC-1 endothelial cells. British J Pharm. 2011; 164: 1883–1898.
- 20. Fang XY, Chen W, Fan JT, Song R, Wang L, Gu YH, et al. Plant cyclopeptide RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis through blocking PDK1–AKT interaction. Toxicol and Appl Pharm. 2013; 267: 95–103.
- 21. Leung HW, Wang Z, Yue GG, Zhao SM, Lee JKM, Fung KP, et al. Cyclopeptide RA-V inhibits cell adhesion and invasion in both estrogen receptor positive and negative breast cancer cells via PI3K/AKT and NF-κB signaling pathways. Biochi et Biophy Acta (BBA)-Mole Cell Res. 2015; 1853: 1827–1840.
- 22. Lau CB, Yue GG, Fung KP, Tan NH, Leung PC. The potential role of Chinese herbal medicines in cancer management. Science 2015; 347: S45–S47.
- 23. Zhou J, Tan NH. Application of a new TLC chemical method for detection of cyclopeptides in plants. Chin Sci Bull. 2000; 45: 1825–1831.
- 24. Zeng GZ, Tan NH, Ji CJ, Fan JT, Huang HQ, Han HJ, et al. Apoptosis inducement of bigelovin from Inula helianthus-aquatica on human Leukemia U937 cells. Phytotherapy Res. 2009; 23: 885–891.
- 25. Zhao SM, Wang Z, Zeng GZ, Song WW, Chen XQ, Li XN, et al. New cytotoxic naphthohydroquinone dimers from Rubia alata. Organic Lett. 2014; 16: 5576–5579.
- 26. Itokawa H, Takeya K, Mihara K, Mori N, Hamanaka T, Sonobe T, et al. Studies on the antitumor cyclic hexapeptides obtained from Rubiae Radix. Chem Pharm Bull. 1983; 31: 1424–1427. pmid:6627519
- 27. Itokawa H, Takeya K, Mori N, Sonobe T, Mihashi S, Hamanaka T. Studies on antitumor cyclic hexapeptides RA obtained from Rubiae Radix Rubiaceae. VI: Minor antitumor constituents. Chem Pharm Bull. 1986; 34: 3762–3768. pmid:3815597
- 28. Morita H, Yamamiya T, Takeya K, Itokawa H. New antitumor bicyclic hexapeptides, RA-XI,-XII,-XIII and -XIV from Rubia cordifolia. Chem Pharm Bull. 1992; 40: 1352–1354. pmid:1394660
- 29. Boger DL, Yohannes D, Myers J. Synthesis of N29-desmethyl RA-VII. Identification of the pharmacophore of RA-I-VII and deoxybouvardin and reassignment of the subunit functional roles. J Org Chem. 1992; 57: 1319–1321.
- 30. Boger DL, Yohannes D, Zhou J, Patane MA. Total synthesis of cycloisodityrosine, RA-VII, deoxybouvardin, and N29-desmethyl-RA-VII: identification of the pharmacophore and reversal of the subunit functional roles. J Am Chem Soc. 1993; 115: 3420–3430.
- 31. Boger DL, Patane MA, Zhou J. Total synthesis of bouvardin, O-methylbouvardin, and O-methyl-N9-desmethylbouvardin. J Am Chem Soc. 1994; 116: 8544–8556.
- 32. Boger DL, Zhou J. N-Desmethyl derivatives of deoxybouvardin and RA-VII: synthesis and evaluation. J Am Chem Soc. 1995; 117: 7364–7378.
- 33. Aebi JD, Deyo DT, Sun CQ, Guillaume D, Dunlap B, Rich DH. Synthesis, conformation, and immunosuppressive activities of three analogs of cyclosporin A modified in the 1-position. J Med Chem. 1990; 33: 999–1009. pmid:2308150
- 34. Otrubova K, Lushington G, Vander VD, McGuire KL, McAlpine SR. Comprehensive study of sansalvamide A derivatives and their structure-activity relationships against drug-resistant colon cancer cell lines. J Med Chem. 2008; 51: 530–544. pmid:18186604