Rubiaceae-Type Cyclopeptides from Galianthe thalictroides - Journal

Feb 25, 2016 - Two Rubiaceae-type cyclopeptides, 6-O-methylbouvardin (1) and the new cyclopeptide 5β-hydroxy-RA-III (2), were isolated from the roots...
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Rubiaceae-Type Cyclopeptides from Galianthe thalictroides Patrícia O. Figueiredo,*,† Maria de Fatima C. Matos,‡ Renata T. Perdomo,‡ Wilson H. Kato, Jr.,† Marcos Vinícius G. O. Barros,† Fernanda R. Garcez,† and Walmir S. Garcez† †

Instituto de Química, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS 79074-460, Brazil Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS 79070-900, Brazil

‡

S Supporting Information *

ABSTRACT: Two Rubiaceae-type cyclopeptides, 6-O-methylbouvardin (1) and the new cyclopeptide 5β-hydroxy-RA-III (2), were isolated from the roots of Galianthe thalictroides. Employing the sulforhodamine B assay, compounds 1 and 2 were tested in vitro against three cancer cell lines786-0 (kidney carcinoma), PC-3 (prostate carcinoma), and HT-29 (colon carcinoma)and showed GI50 values ranging from 0.06 to 1.80 μg mL−1. This is the first report on the isolation of Rubiaceae-type cyclopeptides from a genus other than Rubia or Bouvardia.

R

ubiaceae-type cyclopeptides, known potent antitumor agents, were isolated for the first time in 1977 from Bouvardia ternifolia.1 Since then, 54 Rubiaceae-type cyclopeptides have been obtained from only Rubia and Bouvardia species (subfamily Rubioidea, tribes Rubieae and Spermacoceae, respectively) of this large family, which comprises around 620 known genera.2−9 Galianthe Griseb., a genus of the family Rubiaceae, subfamily Rubioidea, tribe Spermacoceae,10 includes 51 subshrub species with restricted world distribution, occurring mainly in South American regions. Twenty-two of these species are exclusive to midwestern and southeastern Brazil.10,11 Galianthe thalictroides (K. Schum.) E. L. Cabral has been popularly used in midwestern Brazil for cancer treatment and prevention.12,13 Previously, two cytotoxic β-carboline alkaloids were isolated as major constituents of the bioactive CHCl3 phase obtained by partitioning the EtOH extract derived from G. thalictroides roots.12,13 This phase, however, proved more cytotoxic than both isolated alkaloids, suggesting that it might contain minor constituents with higher activity. Based on the foregoing information, further investigation of the CHCl3 phase from roots of G. thalictroides, guided by the presence of cyclopeptides,14 has now led to the isolation of 6-O-methylbouvardin (1), a Rubiaceae-type cyclopeptide previously isolated only from Bouvardia ternifolia,15 and of the new cyclopeptide 2 (Figure 1), both of which showed significant cytotoxic activity against cancer cell lines. Compound 2 was obtained as an amorphous solid, and its molecular formula was determined as C41H50N6O11, based on the 13C NMR data and the [M + H]+ ion at m/z 803.3634 (calcd for C41H51N6O11, m/z 803.3610) in the HRESIMS spectrum. © XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. Chemical structures of Rubiaceae-type cyclopeptides obtained from the roots of Galianthe thalictroides.

The 13 C NMR spectrum of 2 showed 39 signals corresponding to 41 carbons, including six amide carbonyl carbons in the δ 167.8−172.5 region, suggesting 2 as a peptide. These data, in association with those obtained from 2D NMR analysis (Table 1), were in accordance with the presence of six amino acid residues, which were identified as two alanines (Ala), one serine (Ser), and three N-methyl-modified tyrosines (Tyr). Accordingly, the 1H and 13C NMR spectra of 2 closely resembled those of 6-O-methylbouvardin (1) (Table S1, Supporting Information), except for the lack of the methyl Received: September 22, 2015

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Table 1. 1D and 2D NMR Data for 5β-Hydroxy-RA-III (2) in CDCl3a C/H D-Ala

1NH 1α 1β 1CO 2 L-Ser 2NH 2α 2βa 2βb 2CO 3 L-Tyr 3N-Me 3α 3β 3γ 3δ 3ε 3ζ 3O-Me 3CO 4 L-Ala 4NH 4α 4β 4CO 5 L-Tyr 5N-Me 5α 5β 5γ 5δa 5δb 5εa 5εb 5ζ 5OH 5CO 6 L-Tyr 6N-Me 6α 6βa 6βb 6γ 6δa 6δb 6εa 6εb 6ζ 6O-Me 6CO a

δ1H (J in Hz)

type, 13C

HMBC (H−C)

COSY (H−H)

NOE

1

6.45, d (7.0) 4.45, quint (7.0) 1.33, d (7.0)

6.90, 4.62, 3.93, 3.81,

m m m m

CH, 47.9 CH3, 20.6 C, 172.5

C-6CO, C-1CO C-1β, C-1CO C-1α, C-1CO

H-1α H-1NH, H-1β H-1α

H-6β′, H-1α H-1NH, H-1β, H-4β H-1α

H-2α H-2βa, H-2βb, H-2NH H-2α, H-2βb H-2α, H-2βa

H-3N-Me H-2βb H-2βa

C-2CO, C-3α C-2CO, C-3NMe, C-3β, C-3CO C-3α, C-3γ, C-2CO

H-3β H-3α

H-2α, H-3α, H-4NH H-3NMe, H-3β, H-3δ, H-4NH H-3α, H-3δ, H-4β

C-3β, C-3γ, C-3ε, C-3ζ C-3δ, C-3γ, C-3ζ

H-3ε H-3δ

H-3ε H-3δ

CH, 48.4 CH2, 61.7 C, 171.9

2.97, s 3.66,dd (10.3, 5.3) 3.36, m 7.08, d (8.3) 6.85, d (8.3) 3.80, s

6.71, d (6.7) 4.88, quint (6.7) 1.10, dd (6.7, 6.7)

3.34, s 5.38, d (2.1) 5.06, dd (6.7, 2.1) 7.50, dd (8.6, 2.3) 7.36 dd (8.3, 2.3) 7.01 dd (8.6, 2.3) 7.25 dd (8.6, 2.3)

CH3, 40.3 CH, 68.4 CH2, 32.7 C, 130.3 CH, 130.2 CH, 114.2 C, 158.4 CH3, 55.3 C, 167.8

CH, 46.4 CH3, 18.6 C, 172.0 CH3, 33.1 CH, 53.9 CH, 78.5 C, 139.3 CH, 128.3 CH, 126.8 CH, 124.3 CH, 125.7 C, 158.9

C-3ζ

H-3ε

C-3CO C-4β, C-4CO C-4α, C-4CO

H-4α H-4β, H-4NH H-4α

H-3α, H-4α, H-4β, H-3N-Me H-4β, H-4NH, H-5N-Me H-1α, H-4NH, H-4α, H-5N-Me

C-4CO, C-5α C-4CO, C-5N-Me, C-5β, C-5CO C-5CO

H-5α H-5β H-5α, H-5OH

H-4α, H-5α H-5N-Me, H-5β, H-5δb, H-6α H-5N-Me, H-5α, H-5δb

C-5β, C-5ζ, C-5δb C-5β, C-5ζ, C-5δa C-5γ, C-5εb, C-5ζ C-5γ, C-5εa, C-5ζ

H-5εa H-5εb H-5δa H-5δa

H-5εa H-5α, H-5β, H-5εb H-5δa, H-6δb H-5δb, H-6δb

6.46, d (6.7)

H-5β C, 170.7

2.75, 4.39, 2.92, 3.15,

s dd (11.9, 3.0) dd (18.3, 11.9) dd (18.3, 3.0)

6.58, dd (8.3, 2.1) 4.34, d (2.1) 6.82, d (8.3)

3.95, s

CH3, 29.2 CH, 57.7 CH2, 35.7 C, 127.6 CH, 121.0 CH, 113.2 CH, 112.5 C, 152.9 C, 146.7 CH3, 56.2 C, 170.0

C-5CO, C-6α C-6γ C-6α, C-6γ, C-6δa, C-6δb C-6β, C-6δb, C-6εa, C-6ζ C-6β, C-6δa, C-6εb, C-6ζ C-6γ, C-εb, C-6ζ

C-6ζ

H-6βa, H-6βb H-6βb H-6α, H-6β′ H-6εa H-6δa

H-6βb H-5α, H-6βa, H-1NH H-6βb H-6N-Me, H-6βa, H-6δa H-6βb, H-6εa H-5α, H-5εb, H-6N-Me, H-6βa H-6O-Me

H-6εa

Recorded at 500/125 MHz. Chemical shifts referenced to residual CHCl3.

and methine signals ascribed to Ala2 in 1, showing instead signals attributable to a serine residue (Figure 1). The COSY 1H−1H correlations between the Hα of the alanine and serine moieties and their respective NH protons (Table 1), in addition to the HMBC correlations between the N-Me Tyr protons and their respective α-carbons, allowed all NH and N-Me hydrogens of each amino acid residue to be

assigned. The HMBC correlations between the NH and N-Me amino acid hydrogens and the carbonyl carbon of their preceding amino acid residues, as well as the NOESY correlations observed for these protons (Table 1), revealed the amino acid sequence of 2 as (cyclo)−Ala1−Ser2−Tyr3− Ala4−Tyr5−Tyr6−. The presence of an unusual shielded aromatic hydrogen resonance for Tyr6−Hδb (δH 4.31) could B

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Figure 2. (A) General MS fragmentation pathways of cyclopeptides.17,18 (B) Proposed fragments for 6-O-methylbouvardin (1) and 5β-hydroxy-RAIII (2), based on HRESIMS/MS analysis, positive mode.

bonds in Ser2−Tyr3, Ala4−Tyr5, and Tyr5−Tyr6 were trans, trans, and cis, respectively, in the major conformer of 2. The absolute configurations of Ala1, Ser2, and Ala4 were characterized as D(R), L(S), and L(S), respectively, by applying Marfey’s method.16 The remaining configurations of Tyr3, Tyr5, and Tyr6 were determined as L(S) for all three residues, based on NOE data. The foregoing data allowed the structure of 2 to be established as shown in Figure 1. The structure of 2, as well as of 6-O-methylbouvardin (1), was also confirmed by the evidence of peptide fragments in their HRESIMS/MS data, as shown in Figure 2 (Figure S 2, Supporting Information).17−20 Compound 2, which was named 5β-hydroxy-RA-III, is being reported as a new Rubiaceae-type cyclopeptide. In addition, Galianthe is only the second non-Rubia genus and the seventh species that produces this class of compounds. Although most of the Rubiaceae-type cyclopeptides have been isolated from Rubia species, none of these Rubia peptides bear a hydroxy substituent at Cβ of Tyr5. This substitution pattern is however observed in known cyclopeptides from B. ternifolia, which,1,15 like G. thalictroides, belongs to the tribe Spermacoceae.9,10 Compounds 1 and 2, as well as fraction F45, from which both were isolated, and the CHCl3 phase, were tested against 786-0 (kidney carcinoma), PC-3 (prostate carcinoma), and

be justified as an anisotropic shielding effect of the aromatic ring of the Tyr5 residue over this proton,15 suggesting that these residues were linked to each other, forming an additional ring (Figure 1). This is in accordance with the chemical skeleton of the Rubiaceae-type cyclopeptides;2 thus, an ether linkage was established between the C-5ζ of Tyr5 and C-6εb of Tyr6 of 2, based on the HMBC and COSY correlations, which inferred for the above-mentioned carbons the deshielded resonances at δC 158.9 and 152.9, respectively. The NOESY correlations observed between Tyr3-N-Me (δH 2.97) and Ser2-Hα (δH 4.62); Tyr3-N-Me and Tyr3-Hα (δH 3.66); Tyr3-Hβ (δH 3.36) and Ala4-Hβ (δH 1.10); Tyr5-N-Me (δH 3.34) and Ala4-Hα (δH 4.88); Tyr5-N-Me and Tyr5-Hα (δH 5.38); Tyr5-Hα (δH 5.38) and Tyr6-Hα (δH 4.39); and Ala4-Hβ (δH 1.10) and Ala1-Hα (δH 4.39) suggested that Ala1 has an inverse configuration in relation to the other amino acid residues, which is in accordance with the configuration of most Rubiaceae-type cyclopeptides.2 Some of these connectivities, namely, from Tyr3-N-Me (δH 2.97) to Ser2-Hα (δH 4.62) and Tyr3-Hα (δH 3.66), from Tyr5N-Me (δH 3.34) to Ala4-Hα (δH 4.88), and from Tyr6-Hα (δH 4.39) to Tyr5-Hα (δH 5.38), also indicated that the peptide C

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at room temperature. After concentration in vacuo, the EtOH extract was partitioned between MeOH/H2O (9:1; 1000 mL) and hexane (3 × 300 mL); MeOH/H2O (1:1; 1000 mL) and CHCl3 (5 × 300 mL); and MeOH/H2O (1:1; 1000 mL) and EtOAc (3 × 400 mL). The hexane, CHCl3, EtOAc, and MeOH(aq) fractions were tested for the presence of cyclopeptides by TLC, as described by Zhou and Tan.12 The CHCl3 phase (3.85 g), proving positive for the presence of cyclopeptides by TLC (red spots), was chromatographed on an RP-18 silica gel column (40−63 μm, 7.0 × 6.5 cm), yielding fractions F-1 (H2O, 250 mL), F-2 (H2O/MeOH, 8:2, 250 mL), F-3 (H2O/MeOH, 6:4, 250 mL), F-4 (H2O/MeOH, 4:6, 250 mL), F-5 (H2O/MeOH, 2:8, 250 mL), F-6 (H2O/MeOH, 1:9, 250 mL), F-7 (MeOH, 250 mL), and F-8 (MeOH/EtOAc, 250 mL). These fractions were subjected to TLC for detection of cyclopeptides.12 Fractions F-4 and F-5 were subsequently combined to give fraction F-45. Fraction F-45 (783.1 mg) was chromatographed on a Sephadex LH20 column (35.5 × 5.0 cm) eluted with CHCl3/MeOH (9:1), yielding 120 fractions of 20 mL each. Fraction 28 (226.6 mg) from this column, which was shown to contain cyclopeptides, was further chromatographed on a silica gel 60 column (230−400 mesh, 25.0 × 3.5 cm) eluted with CHCl3/MeOH (10:0 → 0:10), yielding 68 fractions of 15 mL each. Fraction 28 (38.0 mg) from this column proved positive for cyclopeptides. Compounds 1 (15.0 mg) and 2 (9.3 mg) were obtained as principal components of this fraction, after isocratic semipreparative HPLC (H2O/CH3CN, 55:45 respectively). 6-O-Methylbouvardin (1): white, amorphous powder; [α]23 D = −174.7 (c 1.3, MeOH); UV (MeOH) λmax (log ε) 246 (3.30), 277 (3.22) nm; IR (KBr) νmax 3392, 2936, 2852, 1663, 1514, 1449, 1412 cm−1; 1H and 13C NMR data, see Supporting Information; positive HRESIMS m/z 787.3684 [M + H]+ (calcd for C41H51N6O10, m/z 787.3661). 5β-Hydroxy-RA-III (2): white, amorphous powder; [α]23 D = −84.5 (c 0.7, MeOH); UV (MeOH) λmax (log ε) 242 (3.54), 277 (3.34) nm; IR (KBr) νmax 3397, 2915, 2847, 1736, 1648, 1511 cm−1; 1H and 13C NMR data, see Table 1; positive HRESIMS 803.3634, [M + H]+ (calcd for C41H51N6O11, m/z 803.3610).

HT-29 (colon carcinoma) cell lines, and their respective GI50 values are depicted in Table 2. Table 2. Cytotoxicity against Cancer Cell Lines (GI50 μg mL−1) Exhibited by Cyclopeptide-Containing Fractions and Compounds 1 (6-O-Methylbouvardin) and 2 (5β-HydroxyRA-III) Obtained from Roots of Galianthe thalictroides sample

HT-29

786-0

PC-3

CHCl3 fraction fraction F45 6-O-methylbouvardin (1) 5β-hydroxy-RA-III (2) doxorubicin

0.64 0.22 0.07 1.80 0.27

0.23 0.20 0.07 1.14 0.02

0.24 0.23 0.06 0.26 0.35

Both compounds exhibited cytotoxic activity against all three cancer cell lines, but 1 proved more cytotoxic than 2, and even more than doxorubicin, the positive control. These potent cytotoxic effects are in accordance with findings by Yan et al.,21 who concluded that the presence of −OMe groups linked to Cζ of tyrosine residues 3 and 6 enhances cytotoxicity in Rubiaceaetype cyclopeptides. Furthermore, the presence of bulky or polar side chains in the amino acid residue 2 seems to decrease antitumor activity, which is consistent with the lower cytotoxicity of 2.21 In a previous investigation, 6-O-methylbouvardin was shown to have antitumor activity against P388 mouse leukemia cells (T/C = 134% at 1.0 mg mL−1).15 Therefore, compounds 1 and 2, despite being minor constituents of the CHCl3 phase, might contribute significantly to its strong cytotoxic activity. Studies have been conducted to elucidate the mechanisms underlying the antitumor activity of Rubiaceae-type cyclopeptides. Bouvardin (RA-V) and RA-VII are known to inhibit protein synthesis, acting at the level of 80S ribosomes,22−24 and to display antiangiogenic activity.25,26 The foregoing results emphasize the potential of Rubiaceaetype cyclopeptides as anticancer drugs and reveal a new source of this class of bioactive compounds in rubiaceous genera other than Rubia or Bouvardia.

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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.jnatprod.5b00849. Copies of 1D and 2D NMR, UV, and IR spectra of compounds 1 and 2, their HRESIMS and HRESIMS/MS spectra and analyses, the protocols for the Marfey’s analysis of absolute configuration of amino acids, and the protocol for the in vitro cytotoxicity assay (PDF)

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation was determined on a PerkinElmer 341 polarimeter (Na filter, λ = 589 nm). UV spectra were obtained on a USB4000 UV/vis spectrophotometer (Ocean Optics). IR spectra were recorded on a Nicolet iS5 FT-IR spectrometer (Thermo Scientific). NMR spectra were obtained in CDCl3 (Cambridge Isotope Laboratories) on a DPX-300 spectrometer (Bruker) operating at 300 MHz (1H)/75 MHz (13C) and on a Varian Inova spectrometer operating at 500 MHz (1H)/125 MHz (13C). HRESIMS and HRESIMS/MS data were acquired in the positive ion mode on a micrOTOF-Q II instrument (Bruker Daltonics) by direct injection. Silica gel 60 (230−400 mesh), RP-18 silica gel (40−63 μm), and Sephadex LH-20 were used for column chromatography. Reversed-phase semipreparative HPLC separation was performed with a Shimadzu LC-6AD pump using RP-18 silica gel (5 μm, 21.6 mm × 250 mm) in a Shim-Pack column at a flow rate of 18 mL min−1, with monitoring at 270 nm. Plant Material. Roots of Galianthe thalictroides were collected from Campo Grande, Mato Grosso do Sul, Brazil, in September 2012. The plant material was identified by Prof. Arnildo Pott (Centro de Ciências Biológicas e da Saúde, Universidade Federal de Mato Grosso do Sul, Brazil). A voucher specimen (no. 29003) has been deposited at the CGMS Herbarium of the Universidade Federal de Mato Grosso do Sul. Extraction and Isolation of Cyclopeptides. Air-dried, powdered roots (2.99 kg) of G. thalictroides were extracted with EtOH (3 × 2 L)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +55-67-3345-3599. Fax: +55-67-3345-3552. E-mail: patricia.fi[email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Fundect-MS PRONEM 0088/12, CNPq, and CPq-PROPP-UFMS for their financial support and to CAPES and CNPq for the grants awarded. Thanks are also extended to Prof. M. G. Carvalho, who kindly provided Marfey’s reagent and the D- and L-amino acid standards; Mr. A. J. S. Garcez, for providing the plant material; Prof. A. Pott, for his assistance in identifying the plant material; Prof. J. E. Carvalho, for providing the cancer cell lines; and Prof. J.-L. D

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Wolfender, Dr. E. F. Queiroz, and L. Marcourt for the NMR spectra of compound 2.

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REFERENCES

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