Novel triterpenoid derivatives from Eucomis bicolor Bak. (Hyacinthaceae: Hyacinthoideae)

Abstract

The bulbs of Eucomis bicolor (Hyacinthoideae) yielded fourteen novel natural compounds, including (17S)-3-oxo-24,25,26,27,28-pentanorlanost-8-en-23,17α-olide, whose structure was determined using the logic for structure determination program, and nine novel lanosterol glycosides. Compounds were screened against the NCI-59 cancer cell panel but showed limited activity.

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Introduction

The genus Eucomis L'Hér. (Hyacinthaceae sensu APGII: Hyacithoideae) has long been one of the most highly regarded sources of ethnomedicines in southern Africa, a notion based largely on its popularity in informal trade.^1–3 Eucomis is a small genus of bulbous plants (ten species and fourteen currently accepted taxa), producing inflorescences that are characteristically topped by a conspicuous coma of leafy sterile bracts.³ All genus members occur in South Africa, with only two extending to south tropical Africa. The Hyacinthoideae are known to exhibit significant infraspecific chemical variation, depending on the geographical location and season of collection.^4–6 This prompted the investigation of naturally-sourced Eucomis bicolor Bak. Previous investigations of E. bicolor, purchased from commercial sources in the Netherlands^7,8 and Japan,⁹ yielded, 5-hydroxy-7-methoxy-3-(4′-methoxybenzyl)-4-chromanone, 3,5-dihydroxy-7-methoxy-3-(4′-methoxybenzyl)-4-chromanone,⁸ (Z)-5,7-dihydroxy-3-(4′-methoxybenzylidene)-4-chromanone,⁷ six eucosterol oligosaccharide derivatives⁹ and scillasponin A.¹⁰

Results and discussion

The current report details the analysis of a collection of Eucomis bicolor Bak. sourced from the foothills of the Drakensberg mountains, KwaZulu-Natal, South Africa. The dichloromethane extract of Eucomis bicolor yielded thirteen compounds, including three known homoisoflavonoids, 3,5,7-trihydroxy-3-(4′-methoxybenzyl)-4-chromanone previously isolated from E. bicolor,⁷ Merwilla dracomontana (Hilliard & B. L. Burtt) Speta (as Scilla dracomontana Hilliard & B. L. Burtt),¹¹ Ledebouria graminifolia (Bak.) Jessop,¹² Eucomis montana Compton and Resnova humifusa (Baker) U. & D. M. -D.,¹³ (R)-5,7-dihydroxy-8-methoxy-3-(4′-methoxybenzyl)-4-chromanone previously isolated from Eucomis autumnalis (Mill.) Chitt. subsp. autumnalis and Eucomis comosa (Houtt.) Wehrh. var. comosa¹⁴ and (R)-5,7-dihydroxy-3-(4′-methoxybenzyl)-4-chromanone previously isolated from E. bicolor,⁸ E. montana, R. humifusa,¹³ and Ledebouria revoluta (L.f.) Jessop,¹⁵ four known nortriterpenoids, eucosterol previously isolated from E. autumnalis,¹⁶ Muscari comosum Mill.,¹⁷ Eucomis pallidiflora Bak. subsp. pole-evansii (N.E.Br.) Reyneke ex J.C. Manning and Eucomis schiiffii Reyneke,³ 3-dehydro-15-deoxoeucosterol and 15-deoxoeucosterol, previously isolated from Scilla scilloides Druce,¹⁸ M. comosum,¹⁷ Merwilla plumbea (Lindl.) Speta (as M. natalensis (Planch.) Speta⁴ and Pseudoprospero firmifolium (Baker) Speta,¹⁹ (23S)-17α,23-epoxy-3β,28,29-trihydrox-27-norlanost-8-en-24-one, previously isolated from M. comosum,²⁰ M. plumbea,⁴ E. schiiffii,³ and Ledebouria zebrina (Bak.) S. Venter (as Scilla zebrina Bak.)⁵ and 2,4-dimethoxy-benzaldehyde, which has not been isolated previously from this family, and five novel triterpenoid derivatives, compounds 1–5 and a range of their glycoside derivatives which were isolated from a complex mixture after acetylation, followed by separation (6Ac–14Ac). The structures of the unacetylated forms of these compounds, as they occur in the plant, are given in Fig. 1 and structures of all compounds isolated are provided in the ESI, S.1.†

Fig. 1 Compounds isolated from Eucomis bicolor.

The HREIMS of compound 1 showed a [M + Na]⁺ peak at m/z 407.2570 indicating a molecular formula of C₂₅H₃₆O₃ and the presence of a pentanortriterpenoid. The IR spectrum showed peaks for lactone and keto group carbonyl stretches respectively at 1767 and 1708 cm⁻¹. The ¹H, ¹³C, ¹H–¹H COSY, ¹H–¹³C HSQC, and ¹H–¹³C HMBC spectral data were used as input for the automatic structure elucidation software LSD.^21,22 The corresponding data file is available as ESI (S.3†). The carbon atoms were numbered in the decreasing order of their ¹³C NMR chemical shift and the hydrogen atoms were numbered so that a carbon and a hydrogen atom that are bound together have their corresponding resonances bearing the same number, according to the HSQC spectrum. The oxygen atom of the keto group was numbered 26, those from the lactone group were given the numbers 27 (sp²) and 28 (sp³). The assignment of the number of attached hydrogen atoms attached to each carbon atom by means of the J-modulated HSQC spectrum led to an overall number of carbonated hydrogens of 36, thus leaving no possibility for the presence of a hydroxy group. Carbons resonances at 213.1, 177.1, 135.8, and 133.1 we produced by sp² hybridized carbon atoms, while the one at 98.4 must be sp³ and bound to O-28. The HSQC and HMBC spectra were recorded with a resolution in the ¹³C domain that was sufficient to avoid any ambiguity on peak positions in this domain. However, peak overlapping in the ¹H domain led to the existence of several HMBC correlations for which the interpretation was ambiguous and considered as such by the LSD algorithm. The COSY spectrum identified the two methyl–methine bonds of the molecule and four other carbon–carbon bonds.

The carbons of the singlet methyl groups were also forced to be bonded to quaternary carbons. These constraints and the correlation data produced a single planar structure solution when processed by the LSD software. The NOESY spectrum showed correlations between the 3H-18 and 3H-19, H-20 and 3H-21 resonances, between the 3H-19/H-4 resonances and between the 3H-28/H-5 and 3H-30/H-5 resonances and between the 3H-21/H-12α resonance. Confirming that the C-21 methyl group was in the alpha orientation. Thus compound 1 was identified as (17S)-24,25,26,27,29-pentanor-3-oxo-lanost-8-en-23,17α-olide. This compound would arise from a Michael-type addition of the oxygen of a C-23 hydroxy group oxygen to C-17 of a ring D α,β-unsaturated system (as seen in 4) followed by the oxidative cleavage of the lanostane C-23/C-24 bond, to yield the pentanortriterpenoid structure.

Compound 2 was isolated in very small amounts, but in larger amounts as its tri-acetyl derivative, 2Ac, after acetylation of a complex mixture. The acetate was identified as (17S)-24,25,26,27-tetranor-3β,28,29-triacetoxy-lanost-8-en-23,17α-olide, 2Ac, hence compound 2 was the novel (17S)-24,25,26,27-tetranor-3β,28,29-trihydroxy-lanost-8-en-23,17α-olide. The HREIMS of compound 2Ac, showed a [M + Na]⁺ peak at m/z 581.3096 for C₃₂H₄₆O₈Na, which indicated a molecular formula of C₂₆H₄₀O₅ for 2.

Compound 2Ac differed from 1 in the structure of ring A. The ¹H NMR spectrum of 2Ac showed that the 3-keto group had been replaced by an acetoxy group with the H-3α resonance occurring at δ 4.89 (dd, J = 11.60, 4.10). The corresponding C-3 resonance (δ 74.0) showed correlations in the HMBC spectrum with two sets of oxymethylene proton resonances (2H-28, δ 4.04, 4.27 ea d, J = 12.00; 2H-29, δ 4.31, 4.40 ea d, J = 12.20). The ¹³C NMR spectrum displayed three acetyl carbonyl carbon resonances at δ_C 170.4, δ_C 170.9 and δ_C 171.2 and HMBC correlations enabled confirmation that acetylation had taken place at C-3, C-28 and C-29.

The NOESY spectrum showed similar correlations as in 1, and showed additional correlations between the H-5/H-3α/2H-28 resonances, confirming the configuration of the substituent at C-3 as β.

The HREIMS of 3 showed a [M + Na]⁺ ion at m/z 507.3078 corresponding to the molecular formula C₃₀H₄₄O₅ for the compound. The FTIR spectrum showed absorption bands at 1772 and 1701 cm⁻¹ consistent with a γ-lactone and keto group respectively.²³ The keto group occurred at C-3 (δ_C 221.0) and the HMBC spectrum showed correlations between both the C-3 and C-5 (δ_C 52.1) resonances and two H-29 oxymethylene (δ_H 3.24, d, J = 11.20 Hz; δ_H 4.02, d, J = 11.20 Hz), and the 3H-28 (δ_H 1.28) proton resonances. The eight carbon sidechain was modified into a spiro-γ-lactone as in compounds previously reported from Chionodoxa luciliae Boiss.²³ The C-17 resonance (δ_C 98.7) was seen to correlated with the 3H-18 (δ_H, H-20 (δ_H 2.28), 3H-21 (d, δ_H 1.05) and two H-22 (δ_H 1.78 and δ_H 2.72) resonances in the HMBC spectrum, and the C-23 (δ_C 113.4) resonance showed correlations seen with the H-20, two H-22, two H-24 (δ_H 2.00, δ_H 2.47) and H-25 (δ_H 2.97) resonances. The C-27 lactone carbonyl resonance (δ_C 179.0) showed correlations with the two H-24, H-25 and 3H-26 (δ_H 1.26, d, J = 7.40 Hz) resonances. The molecular formula indicated two rings in the sidechain due to the C-17,23 ether and 27,23-lactone. The NOESY spectrum showed correlations between the 3H-19/3H-18 and 3H-19/H-29 (δ_H 4.02) resonances and H-5/3H-28 resonances as expected. Correlations seen between the 3H-18/H-16β/H-20 resonances confirmed the configuration at C-17 as S and that H-20 was β. Correlations seen between the 3H-26 and two H-22 resonances confirmed the configuration at C-23 as S. Compound 3 was identified as (17S,23S)-17α,23-epoxy-29-hydroxylanosta-8-en-3-on-27,23-olide.

The HREIMS of compound 4 showed a [M + Na]⁺ peak at m/z 497.3225 which corresponded to a molecular formula of C₂₉H₄₆O₅ for the compound. The oxymethine C-3 resonance (δ_C 80.8), showed correlations in the HMBC spectrum with the H-5 (J = 10.9 Hz 1.18), the 3H-28 (δ_H 1.26) and the two H-29 (δ_H 3.35, d, δ_H 4.23, d, both J = 10.9 Hz) resonances, indicating the presence of an oxymethylene group at C-29 as in 3. Correlations seen in the NOESY spectrum between the H-3 (δ_H 3.47, dd, J = 9.90, 4.10 Hz), 3H-28 and H-5 resonances, confirmed that H-3 was α, and a correlation was noted between the two H-29 and 3H-19 resonance (δ_H 0.95). The structure of ring D differed from that of the previous compounds having an α,β-unsaturated ring ketone, showed by resonances at δ_C 186.6 (C-17), δ_C 123.0 (C-16) and δ_C 210.1 (C-15).²⁴ The C-17 resonance showed correlations in the HMBC spectrum with the H-16 (δ_H 5.61), 3H-18 (δ_H 0.94), H-20 (δ_H 2.80), 3H-21 (δ_H 1.13, d, J = 6.9 Hz), and two H-22 (δ_H 1.64 and δ_H 1.73) resonances, and the C-15 resonance showed correlations with the 3H-30 (δ_H 1.20) and H-16 resonances. The COSY spectrum showed coupling between the two H-22, and the oxymethine H-23 resonance (δ_H 3.28), between H-23 and the oxymethine H-24 resonance (δ_H 3.31) and between the H-24/2H-25 (δ_H 1.43, 1.59) and 2H-25/3H-26 (δ_H 0.99, t, J = 7.80) resonances.

The configuration at C-23 of the co-isolated 3 and 10 have been determined unequivocally as S, so it was assumed that if compounds like 4 are their precursors, the configuration at C-23 should be the same (S). An attempt was made to determine the configuration at C-24 of the side chain vic-diol employing acyclic 1,2-diol complexation with [Mo₂(OAc)₄] and subsequent CD measurements.²⁵ However, results were inconclusive. Thus 4 was identified as 3β,23S,24ε,29-tetrahydroxy-27-norlanosta-8-16-diene-15-one.

The HREIMS of compound 5 showed [M + Na]⁺ at m/z 495.3071 which corresponded to the molecular formula C₂₉H₄₄O₅ for the compound. The C-24 hydroxyl group in compound 4 was oxidised to a keto group (δ_C 212.6) in compound 5. Compound 5 was identified as 3β,23S,29-trihydroxy-27-norlanosta-8-16-diene-15,24-dione, which has been prepared previously as a derivative of eucosterol but incomplete ¹H NMR and no¹³ NMR data was provided.²⁴

The HRMS of compound 6Ac gave an [M + Na]⁺ ion at m/z 909.4254 indicating the molecular formula of C₄₇H₆₆O₁₆ for this compound. The FTIR spectrum for this compound showed a broad absorption at 1749 cm⁻¹ for the carbonyl groups, 2937 cm⁻¹ and 2868 cm⁻¹ for the aliphatic CH stretches and 1222 cm⁻¹ for a C–O stretch. The ¹³C NMR spectrum indicated that compound 6Ac was the acetylated form of the C-3β, β-D-glucoside derivative of compound 5. The ¹H NMR spectrum showed acetylation had occurred at the C-29 and the C-23 hydroxy groups by downfield shifts of the H-23 and two H-29 proton resonances compared to those of 5 (Tables 1 and 3). The C-3 (δ_C 90.1) resonance showed a correlation in the HMBC spectrum with an anomeric proton resonance at δ_H 4.54 and the COSY and HSQC spectra enabled assignment of the sugar resonances. The correlations seen in the NOESY spectrum between H-1′/H-3′ and H-2′/H-4′ resonances, and the coupling constants J_1′,2′ (8.0 Hz) and J_3′,4′ (9.6 Hz) confirmed that the sugar present was acetylated β-D-glucose.²⁶ Thus, compound 6Ac is 23S,29-diacetoxy-3β-[2′,3′,4′,6′-tetra-O-acetyl-O-β-D-glucopyranoside]-27-norlanosta-8,16-diene-15,24-dione.

Table 1 ¹H NMR (500 MHz) chemical shifts for compounds 1, 2, 2Ac, 3, 4, 5 (CDCl₃)

No	1	2	2Ac	3	4	5
a Superimposed resonances, J not clear.
1α	1.57	1.21	1.38	1.98	1.22	1.20
1β	2.07	1.74	1.82	1.76	1.75	1.76
2α	2.34	1.78	1.73	2.46	1.78	1.75
2β	2.44	1.66	1.83	2.58	1.82	1.81
3	—	3.76^a	4.89, dd, J = 11.60, 4.10	—	3.47, dd, J = 9.90, 4.10	3.44, dd, J = 12.00, 4.60
4	2.28	—	—	—	—	—
5	1.40	1.22	1.64	1.86	1.18, m	1.18, m
6α	1.46	1.48	1.63	1.50	1.38	1.36
6β	1.78	1.77	1.75	1.69	1.82	1.83
7α	2.04	1.98	2.03	2.05	2.48	2.47
7β	2.11	1.98		2.05	2.56	2.26
11α	2.13	1.99	2.17	2.07	2.13	2.13
11β	2.21	1.99		2.07	2.13	2.13
12α	1.54	1.49	1.52	2.20	1.70	1.72
12β	2.25	2.20	2.26	2.20	2.10	2.11
15α	1.45	1.42	1.45	1.38	—	—
15β	1.75	1.72	1.76	1.68	—	—
16α	2.12	2.07	2.14	1.84	5.48 s	5.61, s
16β	2.72, d, J = 6.40	2.76, d, J = 6.40	2.73	2.50	—	—
18	1.00, 3H, s	0.94, 3H, s	0.96, s	0.92, s	0.94, s	0.94, s
19	1.20, 3H, s	0.96, 3H, s	1.06, s	1.05, s	0.95, s	0.95, s
20	2.49	2.47	2.50	2.28	2.80	2.87
21	1.13, 3H, d, J = 6.80	1.10, d, J = 6.60	1.14, d, J = 6.60	1.05, d, J = 6.80	1.13, d, J = 6.90	1.13, d, J = 6.50
22α	2.75, d, J = 6.40	2.03	2.04	2.72, dd, J = 6.70, 13.50	1.64	1.49
22β	2.03	2.72	2.77, d, J = 6.20	1.78	1.73	2.04
23	—	—	—	—	3.28, bt^a	3.97, d, J = 11.30
24α	—	—	—	2.47	3.31, m^a	—
24β		—	—	2.00	—	—
25α	—	—	—	2.97	1.43^a	2.44^a
25β	—	—	—	—	1.59^a	2.53^a
26	—	—	—	1.26, d, J = 7.40	0.99, t, J = 7.80	1.11, t, J = 7.40
28A	1.02, 3H, d, J = 6.7	3.74^a	4.04, d, J = 12.00	1.26	1.26, s	1.26, s
28B	—	4.37, d, J = 11.60	4.27, d, J = 12.00	—	—	—
29A	—	3.81^a	4.31, d, J = 12.20	3.45, d, J = 11.20	3.38, d, J = 10.90	3.35, d, J = 10.60
29B	—	4.14, d, J = 11.60	4.40, d, J = 12.20	4.02, d, J = 11.20	4.23, d, J = 10.90	4.23, d, J = 10.60
30	1.10, 3H, s	1.09, s	1.11, s	1.05, s	1.20, s	1.21, s
COCH3	—	—	2.2, 2.06 s, 2.08 s	—	—	—

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Table 2 ¹³C NMR (125 MHz) chemical shifts for compounds 1, 2, 2Ac, 3, 4, 5 (CDCl₃)

No	1	2	2a	3	4	5
1	37.1 CH2	35.3 CH2	35.3 CH2	35.6 CH2	35.2 CH2	35.2 CH2
2	38.0 CH2	27.6 CH2	23.9 CH2	29.0 CH2	28.2 CH2	28.2 CH2
3	213.1 C	77.9 CH	74.0 CH	221.0 C	80.9 CH	80.8 CH
4	45.2 CH	46.1 C	44.1 C	51.3 C	43.0 C	42.9 C
5	49.7 CH	47.5 CH	44.5 CH	52.1 CH	51.2 CH	51.2 CH
6	22.1 CH2	18.8 CH2	19.4 CH2	19.1 CH2	18.4 CH2	18.1 CH2
7	25.4 CH2	26.3 CH2	26.8 CH2	26.2 CH2	27.4 CH2	27.7 CH2
8	135.8 C	134.8 C	135.2 C	132.6 C	133.6 C	133.4 C
9	133.1 C	134.6 C	134.5 C	136.3 C	135.8 C	136.1 C
10	36.8 C	36.9 C	37.1 C	37.0 C	37.5 C	37.6 C
11	21.5 CH2	20.8 CH2	20.7 CH2	21.0 CH2	20.6 CH2	20.4 CH2
12	24.7 CH2	24.6 CH2	24.6 CH2	25.0 CH2	23.3 CH2	23.2 CH2
13	50.9 C	50.8 C	50.9 C	48.7 C	51.2 C	51.2 C
14	49.0 C	49.0 C	49.0 C	50.7 C	57.3 C	57.3 C
15	31.5 CH2	31.5 CH2	31.5 CH2	31.9 CH2	210.2 C	210.1 C
16	39.3 CH2	39.4 CH2	39.3 CH2	37.3 CH2	123.0 CH	123.0 CH
17	98.4 C	98.6 C	98.4 C	98.7 C	186.6 C	185.8 C
18	18.2 CH3	17.9 CH3	18.3 CH3	18.9 CH3	28.8 CH3	28.8 CH3
19	17.7 CH3	19.8 CH3	19.3 CH3	19.9 CH3	19.7 CH3	19.8 CH3
20	41.9 CH	41.9 CH	41.9 CH	44.0 CH	30.1 CH	30.2 CH
21	17.9 CH3	17.9 CH3	17.9 CH3	18.8 CH3	21.7 CH3	21.7 CH3
22	39.2 CH2	39.2 CH2	39.2 CH2	32.0 CH2	39.4 CH2	39.6 CH2
23	177.1 C	177.4 C	177.4 C	113.4 C	71.9 CH	74.3 CH
24	—	—	—	45.1 CH2	76.9 CH	212.6 C
25	—	—	—	35.8 CH	26.7 CH2	31.4 CH2
26	—	—	—	15.1 CH3	10.2 CH3	7.8 CH3
27	—	—	—	179.0 C	—	—
28	11.7 CH3	63.9 CH2	62.7 CH2	22.1 CH3	22.5 CH3	22.5 CH3
29	—	71.4 CH2	63.8 CH2	66.1 CH2	64.5 CH2	64.5 CH2
30	25.9 CH3	25.8 CH3	25.7 CH3	26.2 CH3	29.6 CH3	29.6 CH3
1-OAc	—	—	21.4 CH3	—	—	—
2-OAc	—	—	21.3 CH3	—	—	—
3-OAc	—	—	21.2 CH3	—	—	—
1-OAc	—	—	170.4 C	—	—	—
2-OAc	—	—	170.9 C	—	—	—
3-OAc	—	—	171.2 C	—	—	—

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Table 3 ¹H NMR (950 MHz) chemical shifts for compounds 6Ac–14Ac (CDCl₃)

No	6Ac	7Ac	8Ac	9Ac	10Ac	11Ac	12Ac	13Ac	14Ac
a Overlapped signals.
1α	1.21	1.21	1.21	1.25	1.20	1.24	1.22	1.23	1.22
1β	1.82	1.87	1.79	1.86	1.79	1.85	1.75	1.81	1.82
2α	1.76	1.76	1.93	2.00	1.99	1.99	1.80	1.74	2.27
2β	1.92	1.90			2.06	2.07	1.97	1.90	2.65
3	3.20, dd, J = 4.00, 11.80 Hz	3.22, dd, J = 3.20, 12.30	3.21, dd, J = 4.10, 11.90	3.21, dd, J = 4.00, 12.20	3.19, dd, J = 4.00, 11.60	3.20	3.19, dd, J = 4.00, 11.80	3.21, dd, J = 4.30, 12.00	3.16, dd, J = 4.30, 12.00
4	—	—	—	—	—	—	—	—	—
5	1.17	1.15	1.14	1.17	1.16	1.12	1.14	1.14	1.09
6α	1.56	1.55	1.60	1.60	1.61	1.61	1.85	1.54	1.56
6β	1.88	1.88	1.78	1.83	1.80	1.81	1.98	1.84	1.83
7α	2.42	2.42	1.73	1.75	1.76	1.77	1.78	1.75	1.70
7β	2.58	2.57	1.91	1.92	1.93	1.89	1.92	1.91	1.86
11	2.15	2.16	2.07	2.07	1.98	2.10	2.04	2.11	1.92
12α	1.65	1.65	2.17	2.16	1.43	1.46	1.99	1.60	1.58
12β	2.13	2.11			2.22	2.22	2.08	2.34	2.34
15α	—	—	1.32	1.37	1.35	1.38	—	—	—
15β			1.66	1.69	1.66	1.68	—	—	—
16α	5.60, s	5.60, s	1.79	1.81	1.63	1.70	2.22, d, J = 19.2	2.22, d, J = 19.20	2.20, d, J = 19.20
16β			2.50	2.52	1.99	2.00	2.77, d, J = 19.2	2.77, d, J = 19.20	2.76 d, J = 19.20
18	0.84, s	0.85, s	0.90	0.90, s	0.91	0.88, s	0.94, s	0.94	0.93, s
19	1.02, s	1.02, s	1.00	0.99, s	1.00	0.93, s	1.02, s	1.02, s	1.02, s
20	2.67	2.67	2.24	2.28	2.18	2.21	2.29	2.26	2.24
21	1.15, d, J = 7.20	1.15, d, J = 6.60	1.05, d, J = 6.90	1.07, d, J = 6.70	1.06, d, J = 7.70	1.13, d, J = 6.50	1.11, d, J = 6.70	1.11, d, J = 6.80	1.12, d, J = 6.80
22α	1.84	1.85	1.76	1.79	1.81	1.84	1.89	1.92,	1.86
22β	2.03	2.06	2.71, dd, J = 6.50, 13.90	2.74	1.99	2.00	1.94	1.99	1.99
23	4.86, dd, J = 2.70, 10.50 Hz	4.87, dd, J = 3.00, 10.90	—	—	4.56^a	4.54	4.69, dd, J = 8.70, 10.40	4.68, dd, J = 8.70, 9.90	4.67, dd, J = 9.00, 9.40
24α	—	—	1.99	2.03	—	—	—	—	—
24β			2.47	2.50
25α	2.45	2.41
25β	2.51	2.57	2.94	2.98	2.56, q, J = 7.30	2.57, q, J = 7.40	2.48, q, J = 7.00	2.49, q, J = 7.30	2.48, q, J = 7.30
26	1.07, t, J = 7.30 Hz	1.07, t, J = 6.50	1.25, d, J = 7.10	1.28, d, J = 6.70	1.08, t, J = 7.30	1.11, t, J = 7.40	1.07, t, J = 7.20	1.07, t, J = 7.30	1.07, t, J = 7.30
27	—	—	—	—	—	—	—	—	—
28	1.09, s	1.09, s	1.07	1.10, s	1.08	1.07, s	1.08, s	1.08, s	1.05, s
29α	4.17^a	4.16^a	4.16^a	4.16^a	4.17^a	4.16	4.23^a	4.16, d, J = 11.80	4.15, d, J = 11.90
29β	4.24^a	4.25^a	4.23^a	4.25^a	4.24^a	4.25	4.15^a	4.24, d, J = 11.80	4.21, d, J = 11.90
30	1.19, s	1.19, s	1.00	1.01, s	1.26	1.21, s	1.37, s	1.37, s	1.35, s
Glu 1′	4.54, d, J = 8.00 Hz	4.53, d, J = 8.00	4.54, d, J = 8.00	4.52, d, J = 8.20	4.56, d, J = 8.20	4.52, d, J = 8.10	4.54, d, J = 7.90	4.52, d, J = 8.70	4.36, d, J = 8.10
2′	4.98, dd, J = 9.60, 8.00 Hz	4.95, dd, J = 9.70, 8.00	5.00, dd, J = 8.20, 9.40 Hz	4.95, dd, J = 8.20, 9.70	5.00, dd, J = 8.10, 9.50	4.90	5.00, dd, J = 8.10, 9.80	4.94, dd, J = 8.20, 9.40	4.94, dd, J = 8.10, 9.60
3′	5.18, t, J = 9.60 Hz	5.19, t, J = 9.70	5.19, t, J = 9.50	5.18, t, J = 9.70	5.19, t, J = 9.40	5.18	5.18, t, J = 9.80	5.17, t, J = 9.40	3.78, t, J = 9.30
4′	5.06, t, J = 9.60 Hz	4.93, t, J = 9.70	5.07, t, J = 9.70 Hz	4.93, t, J = 9.70	5.06, t, J = 9.60	4.93	5.05, t, J = 9.80	5.06, t, J = 9.50	4.80, t, J = 9.30
5′	3.68, m	3.68, m	3.66, m	3.69, m	3.66, m	3.68	3.65, m	3.66, m	3.60, m
6′A	4.12, m	3.65, m	4.23^a	3.65, m	4.22^a	3.63	4.13	3.65, m	3.62, m
6′B	4.20, m	3.84, m	4.12^a	3.82, m	4.12^a	3.82	4.22	3.82, m	3.79, m
Arab 1′′		4.50, d, J = 6.60	—	4.50, d, J = 6.70	—	4.49, d, J = 6.10	—	4.49, d, J = 6.80	4.48, d, J = 6.60
2′′		5.16, dd, J = 6.60, 8.80	—	5.14, dd, J = 6.70, 9.00	—	5.15	—	5.14, dd, J = 6.80, 8.90	5.13, dd, J = 6.60, 8.80
3′′		5.03, dd, J = 3.70, 8.80	—	5.03, dd, J = 3.60, 9.07	—	5.03	—	5.01, dd, J = 3.50, 8.90	5.00, dd, J = 3.50, 8.90
4′′		5.25, brs	—	5.25, brs	—	5.26	—	5.28, brs	5.24, brs
5′′α		4.05	—	4.03	—	4.03	—	4.03, m	4.01, dd, J = 3.60, 12.90
5′′β		3.63	—	3.62	—	3.60	—	3.61, dd, J = 3.80, 12.90	3.60, m
Xyl 1′′′			—	—	—	—	—	—	4.53, d, J = 6.20
2′′′									4.75, dd, J = 6.20, 7.80
3′′′			—	—	—	—	—	—	5.03, t, J = 7.80
4′′′			—	—	—	—	—	—	4.85, m
5′′′α			—	—	—	—	—	—	3.34, dd, J = 7.90, 12.00
5′′′β			—	—	—	—	—	—	4.06, dd, J = 4.40, 12.00
OAc	2.05, 2.02, 2.12, 2.10, 2.08, 2.02	1.98, 2.02, 2.02, 2.04, 2.05, 2.08, 2.12, 2.13	2.00, 2.02, 2.02, 2.05, 2.08	1.98, 2.02, 2.02, 2.04, 2.04, 2.07, 2.08	1.99, 2.02, 2.02, 2.04, 2.08	1.98, 2.02, 2.02, 2.04, 2.07, 2.08	2.00, 2.02, 2.02, 2.05, 2.08	1.98, 2.02, 2.02, 2.04, 2.04, 2.07, 2.08	1.98, 2.02, 2.02, 2.04, 2.04, 2.07, 2.08, 2.13, 2.12

---

Compound 7Ac was identified as 23S,29-diacetoxy-3β-[2′,3′,4′-tri-O-acetyl-O-β-D-glucopyranoside-(1′′ → 6′)-2′′,3′′,4′′-tri-O-acetyl-β-D-arabinopyranosyl]-27-norlanosta-8,16-diene-15,24-dione. The HRMS spectrum for compound 7Ac gave a [M + Na]⁺ ion at m/z 1125.4865 indicating a molecular formula of C₅₆H₇₈O₂₂ for this acetylated compound. The aglycone part of compound was found to be the same as for compounds 5Ac and 6Ac. However, in addition to the acetylated β-D-glucose at C-3β, the molecular formula indicated an additional acetylated pentose sugar and this was confirmed by the ¹³C NMR spectrum which displayed two characteristic anomeric carbon resonances at δ_C 102.7 in table (C-1′) and δ_C 101.1 in table (C-1′′) with corresponding anomeric proton resonances at δ_H 4.53 (d, J = 8.0 Hz) and δ_H 4.50 (d, J = 6.6 Hz). The anomeric proton resonance at δ_H 4.50, assigned as H-1′′, was seen to correlate in the HMBC spectrum to C-6′ of β-D-glucose of C-6′ indicating a 1′′ → 6′ linkage. The COSY and HSQC spectra were again used to assign the pentose sugar resonance sand the NOESY spectrum and coupling constants were used to confirm whether the pentose was xylose or arabinose. The coupling constants of J_{1′′,2′′} = 6.60 Hz, J_{,2′′,3′′′} = 8.80 Hz and J_{3′′,4′′} = 3.70 Hz indicated that H-1′′/H-2′′ and H-2′′′/H-3′′′ are trans–diaxial and that H-3′′ and H-4′′ have an axial–equatorial relationship. Hence, the sugar was identified as β-D-arabinose and this was supported by correlations seen in the NOESY spectrum between the H-1′′/H-3′′, H-3′′/H-4′′, H-4′′/H_β-5′′ and H-1′′/H_α-5′′ resonances.

Compound 8Ac was identified as (17S,23S)-29-acetoxy-23,17-epoxy-3β-[2′,3′,4′,6′-tetra-O-acetyl-O-β-D-glucopyranoside]-lanost-8-en-27,23-olide, the 3β-β-D-glucoside derivative of compound 3. The molecular ion could not be observed in the LCMS spectrum for compound 8Ac, but the structure could be determined from NMR studies. The ¹³C NMR chemical shifts for the aglycone part were similar to those of compound 3, except that the C-3 keto group carbon was replaced by an oxymethine resonance (δ_C 90.4) and the C-29 hydroxy group had been acetylated. The HMBC spectrum showed a correlation between the H-3α proton resonances (δ_H 3.21, d, J = 4.1, 11.9 Hz) and an anomeric carbon resonance (δ_C 103.2), which corresponded to the proton resonance at δ_H 4.54 (d, J = 8.0 Hz). The coupling constants and NOESY correlations of the sugar present were the same as those of compound 6Ac, indicating that the sugar present was acetylated β-D-glucose.

Compound 9Ac was identified as (17S,23S)-29-acetoxy-23,17-epoxy-3β-[2′,3′,4′-tri-O-acetyl-O-β-D-glucopyranoside-(1′′ → 6′)-2′′,3′′,4′′-tri-O-acetyl-O-β-D-arabinopyranosyl]-lanost-8-en-27,23-olide. The HRMS for compound 9Ac gave a [M + Na]⁺ ion at m/z 1097.4880, which indicated the molecular formula of C₅₅H₇₈O₂₁ for this compound. The ¹³C NMR spectrum displayed fifty-five carbon resonances, and showed that the aglycone structure was the same as for 8Ac, but that an extra β-D-arabinose sugar was present and linked as determined for 7Ac.

Compound 10Ac was identified as (17S,23S)-29-acetoxy-23,17-epoxy-3β-[2′,3′,4′,6′-tetra-O-acetyl-O-β-D-glucopyranoside]-27-norlanost-8-en-24-one. The LCMS spectrum for compound 10Ac gave a [M]⁺ ion at m/z 830.4421, which indicated the molecular formula of C₄₅H₆₆O₁₄ for this compound. The aglycone part of the molecule was found to be the 29-acetyl derivative of the co-isolated 15-deoxyeucosterol. The C-17 (δ_C 97.3) resonance showed correlations in the HMBC spectrum with the 3H-19 (δ_H 1.00), H-20 (δ_H 1.26), 3H-21 (δ_H 1.06, d, J = 7.0 Hz), two H-22 (δ_H 1.81 and δ_H 1.99) and H-23 (δ_H 4.56) resonances. A ketone carbon resonance at δ_C 213.3 was assigned as C-24 as it showed correlations with the two H-22, H-23, two H-25 (δ_H 2.56, q, J = 7.3 Hz) and 3H-26 (δ_H 1.08, t, J = 7.3 Hz) resonances. The H-29 protons occurred as a pair of doublets (δ_H 4.17, δ_H 4.24) and H-3 α at δ_H 3.19 (dd, J = 4.0, 11.6 Hz). The C-3 resonance showed a correlation in the HMBC spectrum with the anomeric proton resonance at δ_H 4.56. The chemical shifts, coupling constants and correlations seen in the NOESY spectrum of the sugar present were the same as those of the acetylated β-D-glucose in compounds 6Ac and 8Ac. Oxidative cleavage of the C-23/C-24 bond of 10Ac would lead to the γ-lactone structure seen in 1 and 2.

Compound 11Ac was identified as (17S,23S)-29-acetoxy-23,17-epoxy-3β-[2′,3′,4′-tri-O-acetyl-O-β-D-glucopyranoside-(1′′ → 6′)-2′′,3′′,4′′-tri-O-acetyl-O-β-D-arabinopyranosyl]-27-norlanost-8-en-24-one. The expected [M + Na]⁺ peak at m/z 1069.4984 for this compound was not observed in the LCMS spectrum, however, a base peak at m/z 587.1579, which was attributed to the acetylated disaccharide fragment [C₂₃H₃₁O₁₆Na + H]⁺ was observed. The extra hydrogen is unusual, but was also found in compound 14Ac. It is suggested that this hydrogen comes from the triterpenoid system, solvent or electrospray buffer system. As with compounds 6Ac/7Ac and 8Ac/9Ac, compound 11Ac differed from 10Ac in having an extra acetylated β-D-arabinose group at C-6′. This was confirmed using 2D NMR correlations and ¹H NMR coupling constants.

The HRMS spectrum for compound 12Ac, (17S,23S)-29-acetoxy-23,17-epoxy-3β-[2′,3′,4′6′-tetra-O-acetyl-O-β-D-glucopyrano-side]-27-norlanost-8-ene-15,24-dione, gave a [M + Na]⁺ ion at m/z 867.4147, which indicated the molecular formula of C₄₅H₆₄O₁₅ for this compound. 12Ac was the 15-keto derivative of 10Ac. The C-15 (δ_C 215.6) resonance showed correlations in the HMBC spectrum with the 3H-30 (δ_H 1.37) and pair of H-16 (2.22, 2.77, ea d, J = 19.2 Hz) proton resonances.

Compound 13Ac was identified as the 15-keto derivative of 11Ac, (17S,23S)-29-acetoxy-23,17-epoxy-3β-[2′,3′,4′-tri-O-acetyl-O-β-D-glucopyranoside-(1′′ → 6′)-2′′,3′′,4′′-tri-O-acetyl-O-β-D-arabinopyranosyl]-27-norlanost-8-ene-15,24-dione). The HRMS spectrum gave a [M + Na]⁺ ion at m/z 1083.4739 which corresponded to the molecular formula of C₅₄H₇₆O₂₁ for this compound. The NMR spectra showed one fewer methylene group but an extra C-15 ketone carbonyl carbon resonance (δ_C 215.6) which showed correlations in the HMBC spectrum with the 3H-30 (δ_H 1.37) and two H-16 (δ_H 2.22 and 2.77, ea d, J = 19.2 Hz) resonances. The identification of the sugars was done as for the previous disaccharides isolated.

Compound 14Ac was identified as the acetylated trisaccharide of eucosterol, (17S,23S)-29-acetoxy-23,17-epoxy-3β-[2′,3′,4′-tri-O-acetyl-O-β-D-glucopyranoside-(1′′ → 6′)-2′′,3′′,4′′-tri-O-acetyl-O-β-D-arabinopyranosyl-(1′′′ → 3′)-2′′′,3′′′,4′′′-tri-O-acetyl-O-β-D-xylopyranosyl]-27-norlanost-8-ene-15,24-dione. The expected [M + Na]⁺ peak at m/z 1276.5513 for this compound was not observed in the LCMS spectrum. However, a base peak at m/z 803.2235, which was attributed to the acetylated trisaccharide fragment [C₃₂H₄₃O₂₂Na + H]⁺ was observed, similar to that seen in the MS of compound 11Ac. The ¹³C NMR spectrum displayed sixty-three carbons resonance and the aglycone part was found to be the same as in compounds 12Ac and 13Ac. The ¹³C NMR spectrum displayed three anomeric carbon resonances at δ_C 100.3, δ_C 101.7 and δ_C 102.7, which corresponded to the proton resonances at δ_H 4.48 (d, J = 6.6 Hz), δ_H 4.53 (d, J = 6.2 Hz) and δ_H 4.36 (d, J = 8.1 Hz), respectively, confirming that compound 14Ac possessed three sugar groups. The C-3 resonance was seen to correlate in the HMBC spectrum with the resonance at δ_H 4.36, which was assigned as H-1′. This was used as a starting point to assign the chemical shifts of the first sugar. The COSY and HSQC spectrum enabled all ¹H and ¹³C NMR resonances for the sugar at C-3β to be assigned and the large coupling constants (J_1′,2′ = 8.1 Hz, J_3′,4′ = 9.30 Hz) confirmed the trans–diaxial relationships between H-1/H-2 and H-3/H-4 indicating the presence of an acetylated β-D-glucose unit. This was supported by correlations seen in the NOESY spectrum between the H-1′ and H-3′, H-1′ and H-5′ and H-2′ and H-4′ resonances.

As in previous compounds, the C-6′ resonance showed a correlation in the HMBC spectrum with the anomeric proton resonance at δ_H 4.48 (H-1′′) of β-D-arabinose. All resonances for this sugar could be assigned and the coupling constants of J_{1′′,2′′} = 6.6 Hz and J_{3′′,4′′} = 3.5 Hz indicated trans–diaxial and axial–equatorial relationships respectively, confirmed by correlations seen in the NOESY spectrum between the H-1′′/H-3′′, H-3′/H-4′′, H-4′′/H-5′′β and between H-1′′/H-5′′α resonances identified the sugar. The C-3′ resonance was seen to correlate with the anomeric proton resonance at δ_H 4.53 of the pentose sugar in the HMBC spectrum, which was assigned as H-1′′′, indicating the third sugar was linked to β-D-glucose at C-3′. The J_{1′′′,2′′′} = 6.20 Hz and J_{3′′′,4′′′} = 7.80 Hz indicated that H-1′′′/H-2′′′ and H-3′′′/H-4′′′ both have trans–diaxial relationships, hence, the sugar was identified as β-D-xylose. This was confirmed by correlations seen in the NOESY spectrum between the H-1′′′/H-3′′′ and H-4′′′/H-2′′′ resonances.

(R)-5,7-Dihydroxy-8-methoxy-3-(4′-methoxybenzyl)-4-chromanone, eucosterol, (17S,23S)-23,17-epoxy-3β,28,29-trihydroxy-27-norlanost-8-en-24-one, 1, 2Ac, 3, 4 and 5 were evaluated against the NCI 60 developmental Therapeutics Program 60 cancer cell line screen at a single dose of 10⁻⁵ M. Details of the methodology of NCI-60 Human Tumour Cell Line Screen are described at http://dtp.nci.nih.gov/branches/btb/ivclsp.html. None of the compounds showed sufficient activity to progress to the five-dose assay although 5 showed 72.5% inhibition of the H522 non-small lung cell line. One dose mean graphs for the compounds screened are provided in the ESI (S.5†).

This investigation produced typical Eucomis-type constituents, homoisoflavonoids and lanostane triterpenoid derivatives. The variability of constituents isolated from the Hyacinthaceae has been noted by us previously.^4–6 Only two of the compounds isolated in this work had been reported to occur in this species previously, the common homoisoflavonoids 3,5,7-trihydroxy-3-(4′-methoxybenzyl)-4-chromanone⁷ and 5,7-dihydroxy-3-(4′-methoxybenzyl)-4-chromanone.⁸ Compounds 3, 8 and 9 had the same spirocyclic triterpenoid skeleton as scillasponin A,¹⁰ but instead of being a pentaglycoside, occurred as the free triterpenoid (3) or as the mono- (8) or di-glycosides (9). Compounds 10–14 differed from related compounds isolated previously in that the C-28 methyl group was not oxidised, and the sugars differed. Rhamnopyranosyl sugars found in compounds isolated previously from the Japanese collection⁹ were not found in this collection from its natural habitat.

Experimental section

General experiment procedures

1D and 2D NMR spectra were recorded in CDCl₃ on a 500 MHz Bruker AVANCE NMR instrument at room temperature at the University of Surrey and those of the acetylated triterpenoid glycosides were recorded on a 950 MHz Bruker AVANCE NMR instrument at the David H. Murdock Research Institute (DHMRI), North Carolina Research Campus (NCRC) in Kannapolis, North Carolina. The chemical shifts (δ) are expressed in ppm and were referenced to the solvent resonances at 7.26 and 77.23 ppm for CDCl₃, 4.87 and 49.15 for CD₃OD for ¹H and ¹³C NMR analysis respectively. The ESI mass spectra were recorded on a Bruker MicroToF mass spectrometer using an Agilent 1100 HPLC to introduce samples (University of Oxford). The optical rotations were determined in CHCl₃ on a JASCO P-1020 polarimeter. The FTIR spectra were recorded using a Perkin-Elmer (2000) spectrometer.

Plant material

Bulbs of Eucomis bicolor (1.5 kg) were collected from the Drakensberg foothills, South Africa during January 2008. A voucher specimen has been lodged for verification purposes (N. Crouch 1157, NH).

Extraction and isolation of compounds

The whole bulbs of Eucomis bicolor (1.5 kg) were air-dried, chopped into smaller pieces and extracted with methylene chloride and methanol using a shaker for 48 h at room temperature. The extract was then filtered and the solvent was removed under reduced pressure to yield the methylene chloride extract (26.31 g) and the methanol extract (104.87 g). Column chromatography over silica gel (Merck 9385) using a 5 cm diameter gravity column, collecting fractions (75 ml each) using a methylene chloride/methanol step gradient starting with 100% methylene chloride and gradually increasing the methanol concentration to 100%. Final purification was undertaken using 1 cm diameter gravity column. Full details are provided in the ESI (S.2†).

The methylene chloride extract yielded (R)-5,7-dihydroxy-8-methoxy-3-(4′-methoxybenzyl)-4-chromanone (6.5 mg), 3,5,7-trihydroxy-3-(4′-methoxybenzyl)-4-chromanone (7.2 mg) and (R)-5,7-dihydroxy-3-(4′-methoxybenzyl)-4-chromanone (7.9 mg), eucosterol (10.2 mg), (17S,23S)-17,23-epoxy-3β,28,29-trihydrox-27-norlanost-8-en-24-one (3.2 mg), 15-deoxoeucosterol (8.2 mg) and 3-dehydro-15-deoxoeucosterol (8.2 mg) and five novel compounds, 1 (8.5 mg), compound 2 which was acetylated to give compound 2Ac (2.8 mg), 3 (3.3 mg), 4 (3.6 mg) and 5 (2.5 mg). The methanol extract yielded (R)-5,7-trihydroxy-3-(4′-hydroxybenzyl)-4-chromanone (8.2 mg) and nine novel lanosterol glycosides which were isolated as their acetate derivatives after acetylation of a complex mixture to yield 6Ac (1.9 mg), 7Ac (1.2 mg), 8Ac (1.8 mg), 9Ac (1.1 mg) 10Ac (2.3 mg), 11Ac (0.8 mg), 12Ac (2.1 mg), 13Ac (1.7 mg) and 14Ac (1.4 mg). A detailed isolation scheme is given in the ESI (S.2†). Full NMR data for compounds 1–5 and acetates of 2, and 6–14 are given in Tables 1–5 and spectra are given in the ESI (S.4†).

Table 4 ¹³C NMR (237 MHz) chemical shifts for compounds 6Ac–7Ac, 10Ac–12Ac (CDCl₃)

No	6Ac	7Ac	10Ac	11Ac	12Ac
1	35.8 CH2	35.6 CH2	36.0 CH2	36.0 CH2	35.9 CH2
2	27.3 CH2	27.2 CH2	27.2 CH2	27.1 CH2	27.4 CH2
3	90.1 CH	89.9 CH	90.3 CH	90.3 CH	90.3 CH
4	42.3 C	42.3 C	42.4 C	42.4 C	42.3 C
5	51.4 CH	51.1 CH	52.2 CH	51.9 CH	51.3 CH
6	19.5 CH2	19.4 CH2	19.7 CH2	19.5 CH2	19.6 CH2
7	26.5 CH2	26.3 CH2	26.8 CH2	26.8 CH2	26.5 CH2
8	133.4 C	133.3 C	134.2 C	134.3 C	133.5 C
9	136.0 C	136.0 C	135.7 C	135.7 C	135.9 C
10	37.4 C	37.4 C	37.1 C	37.0 C	37.4 C
11	20.5 CH2	20.5 CH2	21.0 CH2	21.2 CH2	20.5 CH2
12	23.0 CH2	22.9 CH2	25.1 CH2	25.1 CH2	23.1 CH2
13	47.8 C	47.6 C	48.7 C	48.8 C	47.6 C
14	57.9 C	57.9 C	50.9 C	50.8 C	57.9 C
15	215.6 C	215.7 C	31.9 CH2	31.9 CH2	215.6 C
16	51.9 CH2	51.7 CH2	39.9 CH2	39.8 CH2	52.1 CH2
17	91.2 C	91.2 C	97.3 C	97.3 C	91.3 C
18	20.7 CH3	20.4 CH3	19.2 CH3	19.5 CH3	20.7 CH3
19	19.1 CH3	19.6 CH3	18.9 CH3	19.2 CH3	19.0 CH3
20	43.5 CH	43.3 CH	44.0 CH	43.7 CH	43.2 CH
21	17.4 CH3	17.3 CH3	17.4 CH3	17.4 CH3	17.3 CH3
22	37.2 CH2	36.7 CH2	37.1 CH2	37.0 CH2	37.0 CH2
23	81.3 CH	81.8 CH	81.8 CH	81.6 CH	81.6 CH
24	212.2 C	212.2 C	213.6 C	213.6 C	212.2 C
25	32.7 CH2	32.5 CH2	32.2 CH2	32.4 CH2	32.5 CH2
26	7.6 CH3	7.5 CH3	7.8 CH3	7.6 CH3	7.4 CH3
28	22.6 CH3	22.7 CH3	22.3 CH3	22.5 CH3	22.6 CH3
29	65.7 CH2	65.5 CH2	65.5 CH2	65.5 CH2	65.8 CH2
30	23.9 CH3	23.7 CH3	25.9 CH3	25.8 CH3	23.9 CH3
Glu 1′	102.9 CH	102.7 CH	103.1 CH	102.9 CH	103.7 CH
2′	71.9 CH	72.7 CH	71.9 CH	71.7 CH	71.8 CH
3′	73.2 CH	80.4 CH	73.1 CH	73.1 CH	73.2 CH
4′	69.2 CH	69.2 CH	68.8 CH	69.0 CH	68.8 CH
5′	73.6 CH	73.4 CH	71.8 CH	73.3 CH	71.9 CH
6′	67.8 CH2	67.6 CH2	62.4 CH2	67.3 CH2	62.3 CH2
Arab 1′′	100.6 CH	100.3 CH	—	100.4 CH	—
2′′	69.2 CH	69.0 CH	—	69.0 CH	—
3′′	70.3 CH	70.0 CH	—	70.0 CH	—
4′′	67.6 CH	67.4 CH	—	67.4 CH	—
5′′	63.1 CH2	62.8 CH2	—	62.9 CH2	—
Xyl 1′′′	—	101.1 CH	—	—	—
2′′′	—	70.0 CH	—	—	—
3′′′	—	70.7 CH	—	—	—
4′′′	—	68.6 CH	—	—	—
5′′′	—	61.7 CH	—	—	—
OAc	20.6, 21.2, 21.3, 20.9, 20.9, 21.1, 20.9	20.6, 21.2, 21.3, 20.9, 21.1, 20.9, 21.2, 21.1	20.5, 20.9, 20.9, 21.0, 21.3	20.6, 21.2, 21.3, 20.9, 20.9, 21.1, 20.9	20.5, 20.7, 20.8, 20.8, 21.0
OAc	170.5, 169.7, 170.3, 170.6, 169.5, 169.4, 171.3	170.5, 169.7, 170.3, 170.6, 169.5, 169.4, 171.3, 169.1, 170.1	170.6, 169.4, 170.9, 169.6, 170.8	170.5, 169.7, 170.3, 170.6, 169.5, 169.4, 171.3	170.6, 169.4, 171.2, 169.6, 170.8

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Table 5 ¹³C NMR (237 MHz) chemical shifts for compounds 8Ac, 9Ac, 13Ac and 14Ac (CDCl₃)

No	8Ac	9Ac	13Ac	14Ac
1	36.0 CH2	36.0 CH2	35.7 CH2	35.7 CH2
2	27.1 CH2	27.1 CH2	26.4 CH2	26.3 CH2
3	90.4 CH	90.2 CH	90.1 CH	90.1 CH
4	42.4 C	42.4 C	42.3 C	42.3 C
5	51.9 CH	51.9 CH	51.7 CH	51.7 CH
6	19.7 CH2	19.7 CH2	19.6 CH2	19.6 CH2
7	26.5 CH2	26.5 CH2	28.3 CH2	28.3 CH2
8	134.1 C	134.2 C	133.4 C	133.3 C
9	135.6 C	135.6 C	135.7 C	135.8 C
10	37.0 C	37.0 C	37.6 C	37.6 C
11	20.7 CH2	20.7 CH2	20.3 CH2	20.3 CH2
12	24.8 CH2	24.8 CH2	23.5 CH2	23.5 CH2
13	48.6 C	48.6 C	51.7 C	57.2 C
14	50.7 C	50.6 C	57.2 C	51.2 C
15	31.8 CH2	31.8 CH2	209.4 C	209.4 C
16	37.4 CH2	37.4 CH2	123.6 CH	123.6 CH
17	98.8 C	98.8 C	184.3 CH	184.4 CH
18	18.9 CH3	18.9 CH3	29.7 CH3	29.0 CH3
19	19.7 CH3	19.7 CH3	18.7 CH3	18.7 CH3
20	44.0 CH	44.0 CH	30.3 CH	30.4 CH
21	18.9 CH3	18.9 CH3	21.3 CH3	21.1 CH3
22	45.1 CH2	45.1 CH2	35.8 CH2	35.8 CH2
23	113.7 C	113.7 C	76.6 CH	76.6 CH
24	45.2 CH2	45.2 CH2	207.6 C	207.6 C
25	35.9 CH	35.9 CH	32.1 CH2	32.1 CH2
26	15.2 CH3	15.2 CH3	7.4 CH3	7.4 CH3
27	179.6 C	179.6 C	—	—
28	22.6 CH3	22.6 CH3	22.7 CH3	22.7 CH3
29	65.73 CH2	65.7 CH2	65.8 CH2	65.8 CH2
30	26.0 CH3	26.0 CH3	29.4 CH3	29.4 CH3
Glu 1′	103.2 CH	102.9 CH	103.9 CH	102.9 CH
2′	71.9 CH	71.9 CH	71.8 CH	71.9 CH
3′	73.3 CH	73.2 CH	73.2 CH	73.1 CH
4′	68.8 CH	69.2 CH	68.8 CH	69.0 CH
5′	71.8 CH	73.6 CH	71.9 CH	73.3 CH
6′	62.3 CH2	67.6 CH2	62.3 CH2	67.7 CH2
Arab 1′′	—	100.4 CH	—	100.5 CH
2′′	—	69.2 CH	—	69.2 CH
3′′	—	70.3 CH	—	70.3 CH
4′′	—	67.5 CH	—	67.5 CH
5′′	—	62.9 CH2	—	63.0 CH2
OAc	20.8, 20.8, 20.8, 21.3, 21.9 CH3	20.6, 21.2, 21.3, 20.9, 20.9, 21.1, 20.9	20.8, 21.0, 20.8, 20.8, 20.8, 21.3	20.8, 21.2, 21.3, 20.9, 20.9, 21.1, 20.9, 20.4
OAc	170.6, 169.4, 171.2, 169.6, 170.9 C	170.5, 169.7, 170.3, 170.6, 169.5, 169.4, 171.3	169.4, 169.6, 170.4, 170.7, 170.8, 171.2	170.5, 169.7, 170.3, 170.6, 169.5, 169.4, 171.3, 170.3

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(17S)-24,25,26,27,29-Pentanor-3-oxo-lanost-8-en-23,17α-olide (1). Pale yellow powder; [α]^23.6_D +29.15 (c 0.0094, CHCl₃); IR ν_max (NaCl) cm⁻¹ 2953, 2923 cm⁻¹ (C–H stretches) and 1767, 1708 cm⁻¹ (C=O stretch); HRESIMS m/z 407.2570 [M + Na]⁺ (calc. for C₂₅H₃₆O₃Na 407.2562). NMR data are given in Tables 1 and 2.

(17S)-24,25,26,27-Tetranor-3β,28,29-triacetoxy-lanost-8-en-23,17α-olide (2Ac). Colourless oil; [α]^23.6_D 40.67 (c 0.0009, CHCl₃); IR ν_max (NaCl) cm⁻¹ 2916, 2849 (C–H stretch) and 1778, 1738 (C=O stretch); HRESIMS m/z 581.3096 [M + Na]⁺ (calc. for C₃₂H₄₆O₈Na 581.3090). NMR data are given in Tables 1 and 2.

(17S,23S)-17α,23-Epoxy-29-hydroxylanost-8-en-3-on-27,23-olide (3). Colourless oil; [α]^23.6_D +11.25 (c 0.0032, CHCl₃); IR ν_max (NaCl) cm⁻¹ 2923, 2853 (C–H stretch) 3467 (O–H stretch) and 1772, 1701 (C=O stretch); HRESIMS m/z 507.3078 [M + Na]⁺ (calc. for C₃₀H₄₄O₅Na 507.3086). NMR data are given in Tables 1 and 2.

3β,23S,24ε,29-Tetrahydroxy-27-norlanosta-8-16-diene-15-one (4). Pale Yellow oil; [α]^23.6_D +25.00 (c 0.0008, CHCl₃); IR ν_max (NaCl) cm⁻¹ 2932, 2875 (C–H stretch), 3399 (O–H stretch) and 1692 (C=O stretch); HRESIMS m/z 497.3225 [M + Na]⁺ (calc. for C₂₉H₄₆O₅Na 497.3243). NMR data are given in Tables 1 and 2.

3β,23S,29-Trihydroxy-27-norlanost-8,16-diene-15,24-dione (5). Colourless oil; [α]^23.6_D = +79.8 (c 0.0020, CHCl₃); IR ν_max (NaCl) cm⁻¹ 3433 (O–H stretch) 3402 (O–H stretch) and 1736 (C=O stretch); HRESIMS m/z 495.3071 [M + Na]⁺ (calc. for C₂₉H₄₄O₅Na 495.3086). NMR data are given in Tables 1 and 2.

23S,29-Diacetoxy-3β-[2′,3′,4′,6′-tetra-O-acetyl-O-β-D-glucopyranoside]-27-norlanosta-8,16-diene-15,24-dione (6Ac). Clear gum; [α]^23.6_D = +12.2 (c = 0.0007 g ml⁻¹, CHCl₃); IR ν_max (NaCl) cm⁻¹ 2925, 2878 (C–H stretch), 1240 cm⁻¹ (C–O stretch) and 1753 cm⁻¹ (C=O stretch); HRESIMS m/z 909.4254 [M + Na]⁺ (calc. for C₄₇H₆₆O₁₆Na 909.4243, 909.4248). NMR data are given in Tables 3 and 4.

(17S,23S)-29-Acetoxy-23,17-epoxy-3β-[2′,3′,4′-tri-O-acetyl-O-β-D-glucopyranoside-(1′′ → 6′)-2′′,3′′,4′′-tri-O-acetyl-O-β-D-arabinopyranosyl]-lanost-8-en-23,27-olide (7Ac). Clear gum; [α]^23.6_D = +11.3 (c = 0.0007 g ml⁻¹, CHCl₃); IR ν_max (NaCl) cm⁻¹ 2925, 2878 (C– H stretch), 1221 and 1248 cm⁻¹ (C–O stretch) and 1753 cm⁻¹ (C=O stretch). HRESIMS m/z 1125.4865 [M + Na]⁺ (calc. for C₅₆H₇₈O₂₂Na 1125.4882). NMR data are given in Tables 3 and 4.

(17S,23S)-29-Acetoxy-23,17-epoxy-3β-[2′,3′,4′6′-tetra-O-acetyl-O-β-D-glucopyranoside]-lanost-8-en-23,27-olide (8Ac). Clear gum; [α]^23.6_D = +10.5 (c = 0.0012 g ml⁻¹, CHCl₃), IR ν_max (NaCl) cm⁻¹ 2922, 2847 (C–H stretch), 1225 cm⁻¹ (C–O stretch) and 1755 cm⁻¹ (C=O stretch). HRESIMS: [M]⁺ not observed. NMR data are given in Tables 3 and 5.

(17S,23S)-29-Acetoxy-23,17-epoxy-3β-[2′,3′,4′-tri-O-acetyl-O-β-D-glucopyranoside-(1′′ → 6′)-2′′,3′′,4′′-tri-O-acetyl-O-β-L-arabinopyranosyl]-lanost-8-en-23,27-olide (9Ac). Clear gum; [α]^23.6_D = +11.3 (c = 0.0007 g ml⁻¹, CHCl₃); IR ν_max (NaCl) cm⁻¹ 2925, 2878 (C–H stretch), 1221 and 1248 cm⁻¹ (C–O stretch) and 1753 cm⁻¹ (C=O stretch). HRESIMS m/z 1097.4880 [M + Na]⁺ (calc. for C₅₅H₇₈O₂₁Na 1097.4933). NMR data are given in Tables 3 and 5.

(17S,23S)-29-Acetoxy-23,17-epoxy-3β-[2′,3′,4′6′-tetra-O-acetyl-O-β-D-glucopyranoside]-27-norlanost-8-en-24-one (10Ac). Clear gum; [α]^23.6_D = +12.4 (c = 0.0011 g ml⁻¹, CHCl₃); IR ν_max (NaCl) cm⁻¹ 2922, 2857 cm⁻¹ (C–H stretches), 1229 cm⁻¹ (C–O stretch) and 1744 cm⁻¹ (C=O stretch); HRESIMS m/z 830.4421 [M]⁺ (calc. for C₄₅H₆₆O₁₄ 830.4453). NMR data are given in Tables 3 and 4.

(17S,23S)-29-Acetoxy-23,17-epoxy-3β-[2′,3′,4′-tri-O-acetyl-O-β-D-glucopyranoside-(1′′ → 6′)-2′′,3′′,4′′-tri-O-acetyl-O-β-D-arabinopyranosyl]-27-norlanost-8-en-24-one (11Ac). Clear gum; [α]^23.6_D = +19.2 (c = 0.0005 g ml⁻¹, CHCl₃); IR ν_max (NaCl) cm⁻¹ 2937, 2868 cm⁻¹ (C– H stretches), 1223 cm⁻¹ (C–O stretch) and 1749 cm⁻¹ (C=O stretch); HRESIMS [M]⁺ not observed. Base peak at m/z 587.1579, [C₂₃H₃₁O₁₆Na + H]⁺ was observed. NMR data are provided in Tables 3 and 4.

(17S,23S)-29-Acetoxy-23,17-epoxy-3β-[2′,3′,4′6′-tetra-O-acetyl-O-β-D-glucopyranoside]-27-norlanost-8-ene-15,24-dione (12Ac). Clear gum; [α]^23.6_D = +22.9 (c = 0.0016 g ml⁻¹, CHCl₃); IR ν_max (NaCl) cm⁻¹ 2921, 2847 cm⁻¹ (C– H stretches), 1222 cm⁻¹ (C–O stretch) and 1748 cm⁻¹ (C=O stretch); HRESIMS m/z 867.4147 [M + Na]⁺ (calc. for C₄₅H₆₄O₁₅Na 867.4143). NMR data are given in Tables 3 and 4.

(17S,23S)-29-Acetoxy-23,17-epoxy-3β-[2′,3′,4′-tri-O-acetyl-O-β-D-glucopyranoside-(1′′ → 6′)-2′′,3′′,4′′-tri-O-acetyl-O-β-L-arabinopyranosyl]-27-norlanost-8-en-15,24-dione) (13Ac). Clear gum; [α]^23.6_D = +25.4 (c = 0.0011 g ml⁻¹, CHCl₃); IR ν_max (NaCl) cm⁻¹ 2922, 2848 cm⁻¹ (C– H stretches), 1225 cm⁻¹ (C–O stretch) and 1755 cm⁻¹ (C=O stretch); HRESIMS m/z 1083.4739 [M + Na]⁺ (calc. for C₅₄H₇₆O₂₁Na 1083.4776). NMR data are provided in Tables 3 and 5.

(17S,23S)-29-Acetoxy-23,17-epoxy-3β-[2′,3′,4′-tri-O-acetyl-O-β-D-glucopyranoside-(1′′ → 6′)-2′′,3′′,4′′-tri-O-acetyl-O-L-D-arabinopyranosyl-(1′′′ → 3′)-2′′′,3′′′,4′′′-tri-O-acetyl-O-β-D-xylopyranosyl]-27-norlanost-8-ene-15,24-dione (14Ac). Clear gum; [α]^23.6_D = +24.5 (c = 0.0007 g ml⁻¹, CHCl₃), IR ν_max (NaCl) cm⁻¹ 2937, 2868 (C–H stretch) and 1223 cm⁻¹ (C–O stretch) and 1749 cm⁻¹ (C=O stretch). HRESIMS [M]⁺ not observed. Base peak at m/z 803.2235 [C₃₂H₄₃O₂₂Na + H requires 803.2221] was observed. NMR data are provided in Tables 3 and 5.

Acknowledgements

This work was supported by the EPSRC Research Council [grant number EP/P50404X] and the PMI2 Project funded by the UK Department for Business, Innovation and Skills (BIS) for the benefit of the United States Higher Education Sector and the UK Higher Education Sector (grant NPF204, awarded to Malcolm von Schantz). The views expressed are not necessarily those of BIS, nor British Council. This grant funded a visit by ML to the David H. Murdock Research Institute, Kannapolis, USA to record NMR spectra on the 950 MHz NMR instrument and we thank Dr Kevin Knagge for his assistance. Financial support by CNRS, Conseil Regional Champagne Ardenne, Conseil General de la Marne, Ministry of Higher Education and Research (MESR) and EU-programme FEDER to the PlAneT CPER project is gratefully acknowledged. We thank Mr Colin Sparrow of the Mass Spectrometry Research Facility, Department of Chemistry, University of Oxford for MS analysis. The authors wish to acknowledge The Developmental Therapeutics Program (DTP) of the National Cancer Institute of the United States (U.S.A) for performing the screening of compounds.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra28584h

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