Two new taxane-glycosides from the needles of Taxus canadensis

2.1 Isolation and structure elucidation of 1

A methanolic extract of the needles of T. canadensis was purified as described in Section 3. Two pure taxane glycosides (1, 2) were obtained as shown in Fig. 1.

Fig. 1:

Taxane glucosides isolated from the needles of T. canadensis.

Taxane 1 was obtained as colorless amorphous solid in a very small yield from air-dried needles of the Canadian yew. The molecular composition of 1, C₃₇H₅₆O₁₄, was established from the combined analysis of high-resolution fast atom bombardment mass spectrometry (HR-FAB-MS) and 2D NMR spectral data. The ¹H NMR spectrum of 1, shown in Table 1, exhibited three-proton signals due to the four tertiary methyl groups at δ_H = 1.21, 1.69, 2.10 and 0.85 ppm, two of which were correlation spectroscopy (COSY)-correlated peaks as geminal methyls (Me-16 and Me-17), and three acetoxy groups at δ_H = 2.15, 1.99 and 1.96 ppm, which were confirmed by the observation of the signals at δ_C = 20.9, 19.6 and 20.3 ppm, and the corresponding carbonyl carbons at δ_C = 168.6 and 169.5 and 168.8 ppm in the ¹³C NMR spectrum. These findings suggested that 1 had a taxane-type core skeleton [6–8]. Indeed, the heteronuclear multiple-bond correlations (HMBC) from H₃-C(18) to C(11), C(12), C(13); and H₃-C(16) and H₃-C(17) to C(1), C(11), C(15); and H₃-C(19) to C(3), C(7), C(8), C(9) revealed that Me-18 was attached to C(12) whereas Me-16 and Me-17 were attached to C(15) and Me-19 was attached to C(8), implying that 1 has a regular 6/8/6 ring system (Fig. 2). The connectivities of the protons on the skeleton of 1 were determined by the analysis of the ¹H–¹H COSY spectrum. Interpretation of ¹H, ¹³C NMR and HMBC spectra permitted the positional assignment of all the functional groups. The ¹H NMR signals at δ_H = 5.25 (1H, br. s), 4.85 (1H, br. s) and 2.99 ppm (1H, d, J = 6.6 Hz) together with the signals at δ_C = 142.7 and 115.9 ppm in the ¹³C NMR spectrum are characteristic of an exocyclic methylene with a C(3) ring junction proton in a taxa-4(20),11-diene [6, 7]. Using H-C(3) as a reference, the connectivities from C(3) to C(2) to C(1) to C(14) to C(13) were deduced from the ¹H–¹H COSY spectrum. The signal at δ_H = 5.38 ppm (1H, dd, J = 6.6, 2.3 Hz) was attributed to H-C(2); the chemical shift indicated that an acetyl group was positioned at C(2). This was confirmed by the observation of the HMBC from H-C(2) to the carbonyl group at δ_C = 168.8 ppm. The signal at δ_H = 5.01 ppm (1H, dd, J = 9.4, 4.8 Hz) was assigned to H-C(14) and suggested that a side chain was attached to C(14). Indeed, a detailed analysis of the ¹H–¹H COSY spectrum revealed a 3-hydroxy-2-methylbutanoyl group in taxane 1. This moiety has been found in several natural taxane analogs [6–8]. Attachment of the 3-hydroxy-2-methylbutanoyl group at C(14) was deduced from a long-range correlation from H-C(14) to C(1′) (δ_C = 173.3 ppm) in the HMBC experiment. The similarity of the NMR data of the 3-hydroxy-2-methylbutanoyl group with the known taxanes reveals the configuration of C(2′) to be R [6–8, 24, 25]. The signal at δ_H = 5.25 ppm (1H, m) was assigned to H-C(5). The chemical shifts of H-C(5) and C(5) suggested that an acetyl group was attached to C(5). Similarly, using H-C(5) as a starting point, the spin system derived from C(5) to C(7) through C(6) was readily interpreted from the analysis of the ¹H–¹H COSY spectrum. The signal at δ_H = 5.30 ppm (1H, dd, J = 12.3, 5.7 Hz), which showed long-range correlations with C(9), C(12) and C(15) in the HMBC spectrum, was attributed to H-C(10). Taxanes with oxygen substitutions at C(2), C(5), C(10) and C(14) have a downfield chemical shift for C(1) (δ_C = 59.3 ppm), a rather unusual chemical shift value for a non-oxygenated methine in the ¹³C NMR spectrum [6, 7]. Additionally, an anomeric carbon signal at δ_C = 98.5 ppm as well as five oxygenated carbons between δ_C = 63.7 and 77.3 ppm, and six hydrogen signals between δ_H = 3.28 and 4.43 ppm indicated the existence of one sugar moiety in taxane 1. The chemical shift, multiplicity and coupling constant values indicated the presence of a glucopyranosyl unit. The analysis of the 2D-NMR data (COSY, HMBC) as well as the MS fragment of [M+H–180]⁺ confirmed it. The coupling constant J = 7.7 Hz of the anomeric proton H-C(1″) indicated that this moiety was connected to the aglycon via a β-linkage (J = 6–8 Hz) [26, 27]. The anomeric proton H-C(1″) showed a long-range correlation with C(10). In addition, H-C(10) was correlated to C(1″) in the HMBC experiment, establishing that the glucose unit was attached to C(10). The orientations of the substituents on the taxane skeleton were confirmed by the coupling constants in the ¹H NMR and the correlations in the nuclear Overhauser effect spectroscopy (NOESY) experiment. The remaining acetyl group was positioned at C(6″) of the glucose residue as indicated by the chemical shifts of Hα–C(6″) and Hβ–C(6″) of the glucose moiety, which was further confirmed by the long-range H-C correlation of Hα–C(6″) to the carbonyl carbon at δ_C = 169.5 ppm in the HMBC experiment. The NOE correlation and the small coupling constant between H-C(14) and H-C(1) suggested that their dihedral angle was about 90°: the C(14) side chain was therefore β-oriented. The chemical structure of 1 was therefore determined as 2α,5α-diacetoxy-10β-(6′-O-acetyl-β-d-glucopyranosyl)oxy-14β-[(2′R,3′S)-3′-hydroxy-2′-methylbutanoyl]oxytaxa-4(20),11-diene, the 6′-O-acetyl derivative of the taxane isolated from Taxus yunnanensis (Cheng et L. K. Fu) [25].

Fig. 2:

2D NMR analyses of 1. The dashed arrows show the key HMBC (H→C), and the bold bonds indicate the ¹H–¹H COSY correlations. Most protons are omitted for clarity.

2.2 Structure elucidation of 2

Compound 2 was obtained as colorless amorphous solid. Its molecular composition, C₃₀H₄₆O₁₁, was obtained from HR-FAB-MS at m/z = 621.2677 [M+K]⁺. The ¹H and ¹³C NMR spectra of 2 in CD₃OD (Table 2) showed the characteristic signals of C(14) substituted taxanes, H-C(2) and H-C(10) resonating as doublets of doublet; C(1) had an unusual downfield shift at δ_C = 60.5 ppm, and C(14) appeared in the range of oxygenated carbon at δ_C = 71.9 ppm. The NMR data for the glucose unit are similar to those of 1. The main difference is the presence of a C(14) acetyl group instead of the 3-hydroxy-2-methylbutanoyl group in 1, and the chemical shift of Hα–C(6′) and Hβ–C(6′) in the glucose residue shifted to the upfield indicated that the acetyl group on the OH-C(6) of the glucose was removed. The sugar residue on C(10) in 2 is confirmed by the HMBC of H-C(10) to C(1′) and H-C(1′) to C(10). The β-configuration of the glucose unit was determined by the coupling constant (J = 7.7 Hz) of the anomeric proton H-C(1′) [26, 27]. The NOESY correlations showed the same relative orientations as in taxanes 1. Therefore, the structure of 2 is 2α,14β-diacetoxy-10β-(β-d-glucopyranosyl)oxytaxa-4(20),11-dien-5β-ol, the 5-de-O-acetyl derivative of the taxane isolated from T. canadensis [5]. The NMR spectral data in CDCl₃ are also shown in Table 3.

3.1 Plant material

The needles of T. canadensis Marsh were collected in September 1997 at St-Jean, Quebec, Canada. Several specimens (under accession voucher number lz97-03) have been deposited in the herbarium of the Montreal Botanical Garden, Montreal, Canada.

3.2 Extraction and isolation

Air-dried needles of T. canadensis were ground (4.0 kg) and extracted with 24 L of methanol for 1 day at room temperature. The ground plants were filtered and extracted again with fresh solvent for another three times (each time with 8 L solvent, total 24 L) in 3 days. The combined organic extracts were evaporated under reduced pressure. Water (3 L) was added and lipids were removed by stirring the mixture with hexane (3 × 3 L). The hexane fraction was condensed into 1500 mL and extracted with 80% methanol four times (each 500 mL). The 80% methanol extract, after re-extracted with hexane two times (each 300 mL), was evaporated under reduced pressure, and 1000 mL of water was added and extracted with ethyl acetate for three times (each 700 mL). The combined ethyl acetate extracts were dried with anhydrous sodium sulfate, filtered and evaporated yielding a dark brown extract 25 g. The aqueous phase was then salted (NaCl, 200 g) and extracted with CH₂Cl₂ (4 × 3 L). The combined CH₂Cl₂ extracts were dried with anhydrous sodium sulfate, filtered and evaporated yielding a dark green extract 115 g. The ethyl acetate extract (25 g) was dissolved in 55 mL acetone and absorbed onto 40 g silica gel and subjected to normal phase column chromatography (silica gel 230–400 mesh, 850 g, 25 × 9 cm²) with elution with a mixture of CH₂Cl₂ and MeOH (9:1, 6:1, 5:1, 4:1, 7:3 v/v). Twenty-seven fractions were obtained (Fr_E-1 to Fr_E-27). Fr_E-2 (1.9 g) was dissolved in 5 mL acetone and absorbed onto 5 g silica gel and subjected to normal phase column chromatography (silica gel 230–400 mesh, 100 g, 29 × 3 cm²) and eluted with a mixture of hexane–EtOAc (600:400 mL). Twenty fractions were obtained (Fr_E-2-1 to Fr_E-2-20). Fr_E-2-2 (400 mg) was subjected to preparative HPLC, eluted with a linear gradient of acetonitrile in water from 25% to 100% in 50 min at a flow rate of 18 mL/min. The material eluted at t_R = 34.52 min was collected, concentrated and applied to preparative TLC (1 × 20 × 20 cm², thickness 0.25 mm). Development with a mixture of hexane–ethyl acetate (7:5) yielded taxane 7 (3.0 mg, R_f = 0.40). A portion of the CH₂Cl₂ extract (50 g) was absorbed onto 110 g silica gel and subjected to column chromatography (silica gel 230–400 mesh, 1320 g). Successive elution with a CH₂Cl₂–MeOH gradient with increasing amounts of MeOH from 5% to 45% (total 15 L) yielded 45 fractions (Fr_D-1 to Fr_D-45). Fractions Fr_D-42 to Fr_D-45 were pooled (1.2 g), decolored with active carbon and partitioned between water and ethyl acetate and obtained ethyl acetate soluble fraction 200 mg, which was further separated by HPLC and afforded 2 (2.2 mg, t_R = 21.1 min) and 1 (2.5 mg, t_R = 28.5 min).

3.3 2α,5α-Diacetoxy-10β-(6′-O-acetyl-β-d-glucopyranosyl)oxy-14β-[(2′R,3′S)-3′-hydroxy-2′-methylbutanoyl]oxytaxa-4(20),11-diene (1)

Amorphous solid. – [α]D22 + 32° (c = 0.10, CHCl₃). – ¹H and ¹³C NMR spectral data; see Table 1 ([D₆]acetone). – HRMS [(+)-FAB]: m/z = 763.3257 (calcd. 763.3307 for C₃₇H₅₆O₁₄K, [M+K]⁺).

3.4 2α,14β-Diacetoxy-10β-(β-d-glucopyranosyl)oxytaxa-4(20), 11-dien-5β-ol (2)

Amorphous solid. – [α]D22 + 29° (c = 0.1, CHCl₃). – ¹H and ¹³C NMR spectral data; see Tables 2 (CD₃OD) and 3 (CDCl₃). – HRMS [(+)-FAB]: m/z = 621.2678 (calcd. 621.2677 for C₃₀H₄₆O₁₁K, [M+K]⁺).