New Norsesquiterpenoids from Cucubalus baccifer

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

The chemical investigation of Cucubalus baccifer L. afforded three new norsesquiterpenoids, cucubalol, cucubalactone and drummondol-11-O-β-D-glucopyranoside together with two known related compounds, drummondol and 5,7[E]-megastigmadiene-3β,4α,9ξ-triol. Their structures were established based on spectral and chemical evidence. No activity was observed in anti-cancer (CDC25), antibacterial (PEPT) and antifungal (YNG) assays.

Cucubalus baccifer L. is the only species in the genus Cucubalus (Caryophyllaceae) which is known as a source plant for the treatment of pulmonary tuberculosis and scrofula in China folk medicine [1]. Previous chemical studies on this plant have revealed the presence of sucrose galactosides [2], [3], tocopherol and tocotrienol [4]. In our attempts to investigate antituberculosis components, five norsesquiterpenoids (Fig. [1]) were isolated and their structures elucidated.

Cucubalol (1) was deduced as C₁₃H₂₂O₄ from a combination of negative FAB-MS, ¹H-NMR (Table [1]), ¹³C-NMR and DEPT (Table [2]) spectra, which was confirmed by HR-FABMS at m/z = 241.1380 [M - H]^- (calcd. 241.1440). The ¹³C-NMR and DEPT spectra displayed thirteen signals (3 × CH₃, 3 × CH₂, 4 × CH, 3 × C). The signals at δ = 65.9, 87.1, 76.3, 82.4, and 69.0 were characterized as carbons bearing an oxygen atom, respectively. The similarity of the NMR data of 1 and 4 showed that they had an identical carbon skeleton. The main difference in the ¹³C-NMR spectra was the presence of one hydroxy substituent at C-3 in 1 rather than a carbonyl group in 4. The stereochemistry of 1 was determined by comparison of its ¹³C-NMR spectral data with those of 4, a NOESY experiment (Fig. [2]), molecular modeling studies, as well as chemical reaction. The relative rigidity of the molecule maintains the tetrahydrofuran ring on the same face of the six-membered ring no matter what conformation is adapted, and naturally the CH₃-13 and CH₃-14 are of the β-form. The NOESY spectrum interactions of H-9 with H-2β and H-4β implied that the hydroxy group at C-8 must be α-oriented as interpreted by aid of molecular modeling. Additional NOESY spectrum interactions for H-3 with H-2β and H-4β showed that the hydroxy functionality at C-3 was on the α-face. This hypothesis was proved by the coupling constant value of H-3 (d, J = 5.6 Hz) which can be achieved only when the six-membered ring adapted a preferential conformation as a chair-form with 3-OH on the α-face. The chromium trioxide oxidation of 1 and 2 under the same conditions resulted in 1a and 2a, respectively. Hydroxy groups at C-3 and C-11 were oxidized in 1 and only C-11 was oxidized in 2 based on the EI-MS at m/z = 254 [M]⁺ and [M - Ac]⁺ . This result can be explained by stereo hindrance of 2. Thus, the structure of 1 was elucidated as 1β,5β-dimethyl-3α,8α-dihydroxy-8β-(3ξ-hydroxy-1-butenyl)-bicyclo[3.2.1]-6-oxaoctane.

Cucubalactone (2) was deduced as C₁₃H₂₀O₅ from a combination of IR, negative FAB-MS, ¹H-NMR (Table [1]), ¹³C-NMR (Table [2]) and DEPT spectra, which was confirmed by HR-FABMS at m/z = 255.1172 [M - H]^- (calcd. 255.1232). The IR bands at 3382, 3274, and 1744 cm^-1 were characteristic of hydroxy and five-membered ring ester carbonyl functionalities, respectively. The ¹³C-NMR spectra of 2 showed similarity to that of 1. The difference was the presence of a quaternary carbonyl carbon at δ = 181.4 (C-7) in 2 rather than a methylene at δ = 76.3 in 1, which was confirmed by IR characteristics and the HMBC interactions of H-2, H₃-14, with C-7 in 2. The cross peaks between H-9 with H-2β and H-4β in the NOESY spectrum (Fig. [3]) suggested that they were located on the same face of the six-membered ring. NOESY correlations between H-2α, H-4α with H-3 showed that the hydroxy group at C-3 was β-oriented. This was verified by the failure of OH-3 chromium trioxide oxidation due to stereo hindrance. Thus, the structure of 2 was identified to be 1β,5β-dimethyl-3β,8α-dihydroxy-8β-(3ξ-hydroxy-1-butenyl)-7-oxobicyclo[3.2.1]-6-oxaoctane.

Drummondol-11-O-β-D-glucopyranoside (3) was deduced as C₁₉H₃₀O₉ from a combination of negative FAB-MS at m/z = 402 [M]^-, ¹H-NMR, ¹³C-NMR and DEPT spectra. The ¹³C-NMR showed nineteen signals (3 × CH₃, 4 × CH₂, 8 × CH, 4 × C). The glucosyl residue was observed in the NMR spectra and verified by acidic hydrolysis. The remaining signals in the ¹H- and ¹³C-NMR spectra (Tabs. [1] and 2) were similar to those of drummondol (4) which was previously isolated from Sesbania drummondii [5]. The low field shift of C-11 (from δ = 68.1 in 4 to 74.5 in 3) was attributed to the glycosidation shift of glucose connected to C-11, this was further confirmed by observation of HMBC correlations of H-1′ to C-11 and H-11 to C-1′. The coupling constant value of H-1′ (J = 7.6 Hz) indicated that the configuration of glycosidic bond was β. Dextral optical activity of glucose via acidic hydrolysis suggested that the absolute configuration was of the D-form. Thus, the structure of 3 was determined as drummondol-11-O-β-D -glucopyranoside.

Two known compounds drummondol (4) and 5,7[E]-megastigmadiene-3β,4α,9ξ-triol (5) [6] were also isolated and their structures identified by spectral analyses.

Pharmacological tests were taken on anti-cancer (CDC25), antibacterial (PEPT) and antifungal (YNG) assays. No bioactivity was observed at concentrations of 25 μg/ml, 96 μg/ml and 4 μg/ml, respectively.

Materials and Methods

M.p.: XRC-1 apparatus and uncorrected; Optical rotations: JASCO-20C digital polarimeter; IR: Bio-Rad FTS-135 spectrometer; FAB-MS: VG Auto Spec-3000 spectrometer; Regular ¹H- and ¹³C-NMR: Bruker AM-400 MHz spectrometer, tetramethylsilane (TMS) as internal standard; 2D NMR: DRX-500 MHz spectrometer; Silica gel: 200 - 300 mesh and 10 - 40 μm, Qingdao Marine Chemical and Industrial Factory, China; Diaion HP 20: Mitsubishi Chemical Industries Limited, Japan; D101 macroporous resin: Tianjin Bone Glue Industrial Factory, China. Whole plants of C. baccifer L. were collected in Chenggong county of Yunnan province, China, in September 1999. They were identified by Dr. Y.M. Shui; a voucher specimen (No. 2) was preserved in the Herbarium of Kunming Institute of Botany.

Air-dried and powdered whole plants (24.0 kg) were extracted with 95 % ethanol under reflux (3 × 90 L) for three times (2 h., 1 h., and 1 h., respectively). After concentration of the combined extracts under vacuum, the residue was suspended in water and partitioned with petroleum ether (60 - 90 °C, 3 × 800 mL), EtOAc (3 × 600 mL) and n-BuOH (3 × 600 mL), respectively. The ethyl acetate extract (400.0 g) was decolored on 500 g Diaion HP 20 with a gradient of aqueous MeOH (0 : 1 - 1 : 0, 1000 mL each eluent). The 70 % MeOH eluate (250.0 g) was subsequently subjected to CC over 2500 g silica gel (200 - 300 mesh) with CHCl₃-MeOH (48 : 1 to 8 : 2, 6500 mL each eluent) to give six fractions (frs. 1 - 6). Fraction 1 (5.0 g) was chromatographed over 400 g silica gel with petroleum ether-EtOAc (10 : 1 - 1 : 1, 1500 mL each eluent) to obtain subfraction (fr.1.1) (500 mg) which was further purified over 25 g silica gel (10 - 40 μm) with CHCl₃-Me₂CO (5 : 1, 300 mL) to yield 4 (15 mg), 5 (30 mg). Fraction 6 (440 mg) was further fractionated over 20 g silica gel (200 - 300 mesh) with CHCl₃-Me₂CO (5 : 1, 350 mL) to furnish frs. 6.1 (80 mg) and 6.2 (50 mg). Frs. 6.1 and 6.2 were both purified by vacuum liquid chromatograph over 10 g silica gel (10 - 40 μm) with CHCl₃-iPrOH (10 : 1 - 5 : 1, 30 mL each eluent) to afford 1 (8 mg) and 2 (15 mg), respectively. The n-BuOH portion (70.0 g) was desugarized on 500 g D 101 macroporous resin with H₂O-MeOH (0 : 1 - 7 : 3, 1500 ml each eluent). The 70 % MeOH eluate (20.0 g) was successively subjected to CC over 500 g silica gel (200 - 300 mesh) with CHCl₃-MeOH (12 : 1, 2800 mL) to provided an about 80 % pure fraction (37 mg,), which was finally purified via vacuum liquid chromatograph over 10 g silica gel (10 - 40 μm) with CHCl₃-MeOH (10 : 1, 200 mL) to yield 3 (30 mg).

Cucubalol (1): C₁₃H₂₂O₄; colorless gum; [α]²⁸ _D: -3.12 ° (c 0.40, MeOH); negative FAB-MS: m/z = 241 [M - H]^-; negative HR-FABMS: m/z = 241.1380 [M - H]^-, (calcd. 241.1440); EI-MS: m/z= 224 [M - H₂O]⁺ (20), 206 [M - 2H₂O]⁺ (16), 191 [M - 2H₂O - Me]⁺ (3), 166 (18), 148 (38), 123 (32), 109 (46), 99 (52), 82 (65), 71 (42), 55 (77), 43 (100); ¹H-NMR data (Table [1]); ¹³C-NMR and DEPT data (Table [2]).

Cucubalactone (2): C₁₃H₂₀O₅; colorless solid, m. p. 164 - 166 °C; [α]²⁷ _D: -33.1 ° (c 0.40, MeOH); IR (KBr): ν_max = 3382, 3274, 1744 cm^-1; negative FAB-MS: m/z = 255 [M - H]^-; negative HR-FABMS: m/z = 255.1172, (calcd. 255.1232); EI-MS: m/z = 238 [M - H₂O]⁺ (6), 228 (38), 192 (16), 167 (16), 152 (34), 141 (52), 124 (100), 109 (77), 97 (61), 87 (41), 69 (51), 55 (60); ¹H-NMR data (Table [1]); ¹³C-NMR and DEPT data (Table [2]).

Drummondol-11-O-β- D-glucopyranoside (3): C₁₉H₃₀O₉; colorless gum; [α]²⁸ _D: -39.22° (c 0.15, MeOH); negative FAB-MS: m/z = 805 [2M + H]^- (15), 402 [M]^-; ¹H-NMR data (Table [1]); ¹³C- NMR and DEPT data (Table [2]).

Oxidation of 1: A solution of 1 (2 mg) in Me₂CO (2 mL) was treated with a solution of chromium trioxide (500 mg) in Me₂CO (2 mL) for 2 h at room temperature. The mixture was dumped into ice-water and then extracted with EtOAc. The EtOAc extract was chromatographed over 2 g silica gel with petroleum ether-Me₂CO (4 : 1, 5 mL, 3 : 1, 7 mL) to yield 1a (< 1 mg) as colorless gum; EI-MS: m/z = 238 [M]⁺ (14), 220 [M - H₂O]⁺ (13), 211 (7), 195 [M - Ac]⁺ (3), 180 [M - Ac - Me]⁺ (36), 162 (10), 139 (31), 125 (58), 111 (40), 98 (100), 83 (44), 55 (80).

Oxidation of 2: A solution of 2 (2 mg) in Me₂CO (2 mL) was treated by the same method as described in 1. The treated residue was chromatographed over 2 g silica gel with petroleum ether-Me₂CO (4 : 1, 7 mL, 3 : 1, 7 mL) to yield 2a (< 1 mg) as a colorless solid; EI-MS: m/z = 254 [M]⁺ (2), 236 [M - H₂O]⁺ (11), 224 (6), 211 [M - Ac]⁺ (3), 210 (9), 206 (86), 166 (61), 164 (60), 125 (90), 97 (100).

Hydrolysis of 3: A solution of 3 (10 mg) in MeOH (3 mL) was treated with 3 N HCl (3 mL) under reflux for 2 h. The mixture was diluted with 3 mL H₂O and concentrated under vacuum to remove MeOH and HCl. The aqueous solution was extracted with EtOAc to furnish 3a (4 mg) which was identical with 4 by TLC detection, R_f = 0.3 (CHCl₃-Me₂CO, 3 : 1). The aqueous layer was analyzed by TLC and PC, respectively, TLC: R_f = 0.6 (CHCl₃-MeOH-H₂O, 3 : 2 : 0.3), PC: R_f = 0.3 (n-BuOH-HOAc-H₂O, 4 : 1 : 5, upper layer) and R_f = 0.4 (n-BuOH-py,-H₂O, 6 : 4 : 3). The concentrated aqueous layer was purified via CC over 3 g silica gel with CHCl₃-MeOH-H₂O (4 : 1 : 0.2, 10 mL) and detected with 2 % aniline phthalate to afford glucose (ca. < 1 mg), which was dextrorotatory.

Acknowledgements

We are grateful to the analytical group of Kunming Institute of Botany for determining all spectral data, and the cooperative laboratory for screening between Kunming Institute of Botany and Bayer AG (Germany) for bioactivity.

References

• 1 Beijing Institute of Botany of Academia S inica. Iconographia Cormophytorum Sinicorum. 1980: pp.642 Beijing Academic Press Beijing;
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• 2 Courtois J E, Ariyoshi U. Galactosides of sucrose from the roots of Cucubalus baccifer .  Bulletin of Society Chimica Biology. 1960;  42 737-51
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• 4 Ivanov S A, Aitzetmueller K. Tocopherol and tocotrienol composition of the seed lipids of a number of species representing the Bulgarian flora.  Fett/Lipid. 1998;  100 348-52
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• 5 Powell R G, Weisleder D, Smith C R. Drummondones A and B: Unique abscisic acid catabolites incorporating a bicyclo[2.2.2]octane ring system.  The Journal of Organic Chemistry. 1986;  51 1074-6
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• 6 Otsuka H, Kamada K, Yao M, Yuasa K, Takeda Y. Alangionosides C-F, megastigmane glycosides from Alangium premnifolium .  Phytochemistry. 1989;  28 3369
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Prof. Jun Zhou

Laboratory of Phytochemistry

Kunming Institute of Botany

The Chinese Academy of Sciences

Kunming 650204

Yunnan

P. R. China

Email: chyx72930@163.net

Fax: +86-871-5150227

References

• 1 Beijing Institute of Botany of Academia S inica. Iconographia Cormophytorum Sinicorum. 1980: pp.642 Beijing Academic Press Beijing;
  Search in Google Scholar
• 2 Courtois J E, Ariyoshi U. Galactosides of sucrose from the roots of Cucubalus baccifer .  Bulletin of Society Chimica Biology. 1960;  42 737-51
  PubMedSearch in Google Scholar
• 3 Courtois J E, Ariyoshi U. Sucrose galactosides of the roots of pinks.  Bulletin of Society Chimica Biology. 1962;  44 23-50
  PubMedSearch in Google Scholar
• 4 Ivanov S A, Aitzetmueller K. Tocopherol and tocotrienol composition of the seed lipids of a number of species representing the Bulgarian flora.  Fett/Lipid. 1998;  100 348-52
  CrossrefPubMedSearch in Google Scholar
• 5 Powell R G, Weisleder D, Smith C R. Drummondones A and B: Unique abscisic acid catabolites incorporating a bicyclo[2.2.2]octane ring system.  The Journal of Organic Chemistry. 1986;  51 1074-6
  PubMedSearch in Google Scholar
• 6 Otsuka H, Kamada K, Yao M, Yuasa K, Takeda Y. Alangionosides C-F, megastigmane glycosides from Alangium premnifolium .  Phytochemistry. 1989;  28 3369
  CrossrefPubMedSearch in Google Scholar

Prof. Jun Zhou

Laboratory of Phytochemistry

Kunming Institute of Botany

The Chinese Academy of Sciences

Kunming 650204

Yunnan

P. R. China

Email: chyx72930@163.net

Fax: +86-871-5150227