Ricinodols A–G: new tetracyclic triterpenoids as 11β-HSD1 inhibitors from Ricinodendron heudelotii

Abstract

A search for 11β-HSD1 inhibitors from Ricinodendron heudelotii has afforded seven new tetracyclic triterpenoids ricinodols A–G (1–7), along with three known podocarpane-type diterpenoids (8–10). Ricinodols A (1) and B (2) possess a new carbon skeleton with a novel concurrent rearrangement of Me-19 (10 → 9) and Me-30 (14 → 8). Their structures were elucidated by comprehensive spectroscopic analyses and comparison with reported analogues, while the 24R-configuration of those with a 24-OH group was established by an in situ dimolybdenum CD method. Of these isolates, ricinodol E (5) exhibited the best inhibitory activities against both human and mouse 11β-HSD1 with IC₅₀ values of 0.36 ± 0.26 and 0.84 ± 0.18 μM, respectively.

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Introduction

Ricinodendron heudelotii (Baill.) (family Euphorbiaceae) is a fast-growing tree that originally occurred in the tropical forests of West and Central Africa.¹ In African folk medicine, many parts of R. heudelotii are used for a variety of medicinal purposes. For instance, the bark of the root and the stem are used in decoctions or lotions to treat constipation, coughs, dysentery, etc.¹ Surprisingly, few studies on the chemical constituents and biological activities have been reported for such an important species with both economic and medicinal values. Connolly and co-workers once reported two new dinorditerpenoids from its stem bark and roots.²

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a bi-directional oxidoreductase catalysing the interconversion between active and inactive glucocorticoids. It is well-known that glucocorticoids exert a wide spectrum of actions in various tissues and their abnormal levels would result in a series of metabolic abnormalities such as insulin-resistance, visceral obesity, hypertension and dyslipidaemia.^3,4 We recently carried out a project aiming to identify 11β-HSD1 inhibitors from plant sources, which returned several types of triterpenoids with promising activities.^5–7 A sample extract of the stems and leaves of R. heudelotii collected on Hainan island also came to our attention, as its non-polar fractions showed the presence of triterpenoid constituents in our initial chemical profiling. This inspired us to further explore the chemical entities of this plant for potential 11β-HSD1 inhibitors. Subsequent chemical fractionation of the preparative-scale ethanolic extract led to the isolation and identification of seven new tetracyclic triterpenoids, ricinodols A–G (1–7, Fig. 1), along with three known podocarpane-type diterpenoids, nimbinone (8),⁸ 3β,12-dihydroxy-13-methyl-potocarpane-6,8,11,13-tetraene (9),⁹ and 3β,12-dihydroxy-13-methyl-potocarpane-8,10,13-triene (10).¹⁰ The structures of these terpenoids were assigned by analysis of spectroscopic data and comparison with reported compounds, and the R-configuration of C-24 for 1, 2, 4, and 5 was established by in situ dimolybdenum CD method. Compounds 1 and 2 are the first examples of tetracyclic triterpenoids with concurrent rearrangement of Me-19 (10 → 9) and Me-30 (14 → 8). All the isolates were assessed for their inhibitory activities against human and mouse 11β-HSD1, and most of them showed moderate to significant inhibition against both enzymes. The isolation, structure elucidation, and biological evaluation of these compounds are herein presented.

Fig. 1 Structures of ricinodols A–G (1–7).

Results and discussion

Compound 1 was obtained as a white amorphous powder. The molecular formula of C₃₀H₅₀O₄ was deduced from HRESI(−)MS data with an adduct ion peak at m/z 519.3677 [M + HCO₂]⁻ (calcd 519.3686), and corresponded to six indices of hydrogen deficiency. The IR spectrum revealed the presence of hydroxyl (3502, 3404 cm⁻¹) and olefinic (1630 cm⁻¹) functionalities. The ¹³C and DEPT NMR data revealed 30 resonances including eight methyl, eight methylene, six methine (four oxygenated and one olefinic), and eight quaternary (one oxygenated and three olefinic) carbons. Four carbon resonances at δ_C 127.8, 130.5, 135.9, and 157.3 supported the presence of two carbon–carbon double bonds accounting for two out of six indices of hydrogen deficiency, and the remaining four indicated a tetracyclic framework for 1. Comprehensive analysis of ¹H–¹H COSY and HMBC data (Fig. 2A) enabled the establishment of the planar structure of 1 as shown. In detail, four structural fragments (a–d) as deduced from ¹H–¹H COSY correlations were connected through eight quaternary carbons by HMBC correlations of H₃-28(29)/C-3(δ_C 75.6), C-4, and C-5(δ_C 130.2); H₂-1/C-10(δ_C 134.0); H₃-19/C-8, C-9, C-10, and C-11; H₃-18/C-12, C-13, C-14(δ_C 157.1), and C-17; H-15/C-14; H₃-30/C-7(δ_C 73.8), C-8, C-9, and C-14; H-7/C-5; and H₃-26(27)/C-24(δ_C 78.5) and C-25(δ_C 73.3). In addition, the correlations of H₃-28(29)/C-5 and H₃-19/C-10 located one double bond at Δ⁵⁽¹⁰⁾, while the other one was assigned at Δ¹⁴ by the correlations from H-15 (δ_H 5.67), H₃-30, and H₃-18 to C-14. Furthermore, the molecular composition of 1 required the existence of four hydroxyl groups which were assigned at C-3, C-7, C-24, and C-25 as supported by their respective chemical shifts at δ_C 75.6, 73.8, 78.5, and 73.8. Therefore, the planar structure of 1 was delineated, and compared with a routine triterpenoid scaffold, it possessed a new carbon skeleton featuring a concurrent rearrangement of Me-19 (10 → 9) and Me-30 (14 → 8).

Fig. 2 Key 2D NMR correlations of ricinodol A (1).

The relative configuration of 1 was established by analysis of proton couplings and ROESY data (Fig. 2B). The β-position of 3-OH was assigned on the basis of the coupling patterns of H-3 with H₂-2 (J_2β/2α,3 = 9.1/3.9 Hz) similar to those (J_2β/2α,3 = 9.2/2.8 Hz) of petatrichol B with the same ring-A.¹¹ Then the ROESY correlations of H-2β/H₃-29, H₃-29/H-6β, H-6β/H-7, H-7/H₃-30, and H₃-19/H₃-30 indicated that they were co-facial and β-oriented. Subsequently, the interactions of H-6α/H₃-18 and H₃-18/H-16α allowed the assignment of α-orientation for H-16α and Me-18, and the strong cross-peak of H-16β/H-17 established H-17 in a β-direction. In particular, the assignment of R-configuration for C-20 was supported by the diagnostic ROESY correlations of H₃-21/H₂-16, H₃-21/H-17, and H₃-18/H-20,^12–16 which were consistent with those of euphol acetate with 20R-configuration,¹⁴ while the 20S-epimer tirucallol acetate showed a typical ROESY correlation of H₃-21/H-12α.¹⁴ Structures of the two aforementioned C-20-epimers were confirmed by X-ray crystallography.¹⁷ As the C-24 stereocenter of the side chain proved unidentifiable due to lack of direct evidences, we employed in situ dimolybdenum CD method developed by Snatzke and Frelek to assign its configuration.^18–21 Theoretically, the vicinal diol in a gauche arrangement easily forms a chiral complex with a transition metal, leading to two possible diastereomorphous structures (Fig. 3A), and the preferred conformation is when the bulkier group is located in a pseudo-equatorial position away from the core of the metal complex. According to an empirical rule, the Cotton effect at around 305 nm (band IV) in the induced CD spectrum is related to the sign of the O–C–C–O torsion angle in the favored conformation. Thus, the negative Cotton effect at 311 nm (Δε −1.7) (Fig. 3B) induced by Mo₂(OAc)₄ allowed the assignment of 24R-configuration for 1. The structure of compound 1 was then elucidated and named ricinodol A.

Fig. 3 (A) Conformations of the Mo₂⁴⁺ complexes of 1 and 4; (B) induced CD spectra of the Mo₂⁴⁺ complexes for 1 and 4.

Compound 2 displayed a sodiated molecular ion peak at m/z 495.2439 [M + Na]⁺ (calcd 495.3450) in HRESI(+)MS analysis, corresponding to a molecular formula of C₃₀H₄₈O₄ indicative of a didehydro derivative of 1. Comparison of ¹H and ¹³C NMR data (Table 1) with those of 1 revealed that they were closely related with the only difference occurring to ring-A, where the 3-OH in 1 was oxidized to a carbonyl in 2. Such assignment was supported by the presence of a strong IR absorption band at 1708 cm⁻¹ and a deshielded carbon signal at δ_C 216.2, as well as the HMBC correlations from H₃-28(29) to C-3(δ_C 216.2). The remaining parts of structure of 2 were assigned to be identical to those of 1, which was further confirmed by analysis of 2D NMR spectra (see ESI Fig. S15–S17†) and the induced CD data (see ESI Fig. S1†). Thus, compound 2 was characterized and named ricinodol B.

Table 1 ¹H and ¹³C NMR data (in CDCl₃) for compounds 1–4^a

No.	1		2		3		4
	δH (multi, J in Hz)	δC	δH (multi, J in Hz)	δC	δH (multi, J in Hz)	δC	δH (multi, J in Hz)	δC
a ^1H NMR data were measured at 500 MHz. ^13C NMR data were measured at 125 MHz.
1	2.21 (m)	23.0	2.38 (m)	24.6	α 1.09 (dd, 11.9, 11.9)	45.2	α 1.82 (ddd, 14.9, 13.2, 4.0)	36.8
	2.07 (m)		2.28 (m)		β 1.98 (m)		β 2.19 (m)
2	α 1.82 (m)	26.9	2.55 (ddd, 15.0, 9.6, 5.8)	37.2	3.70 (ddd, 11.8, 9.6, 4.3)	68.8	α 2.34 (m)	34.9
	β 1.73 (m)		2.48 (ddd, 15.0, 14.0, 5.7)				β 2.83 (ddd, 14.7, 14,7, 5.2)
3	3.55 (dd, 9.1, 3.9)	75.6		215.8	3.03 (d, 9.6)	83.7		216.2
4		39.4		47.4		39.3		48.0
5		130.2		130.5	1.41 (dd, 12.1, 5.3)	50.6	1.66 (dd, 11.4, 4.9)	49.9
6	α 1.85 (m)	33.2	α 1.92 (m)	32.6	α 2.14 (brd 17.1)	23.9	2.22 (m)	24.3
	β 2.37 (m)		β 2.38 (m)		β 1.99 (m)		2.16 (m)
7	3.61 (dd, 7.1, 4.8)	73.8	3.67 (dd, 5.8, 3.6)	74.3	5.27 (m)	118.0	5.47 (ddd, 4.5, 4.5, 1.8)	120.8
8		45.2		45.0		145.4		141.1
9		41.1		41.1	2.29 (m)	49.0		145.2
10		134.0		135.9		36.2		36.1
11	1.67 (m)	31.2	1.95 (ddd, 15.7, 10.8, 1.5)	31.3	1.55 (m)	18.2	5.40 (dd, 5.1, 1.8)	117.9
	1.53 (m)				1.54 (m)
			1.54 (m)
12	1.59 (m)	36.2	1.66 (m)	36.0	1.81 (dd, 13.6, 9.8)	33.6	4.14 (d, 5.1)	74.0
	1.51 (m)		1.52 (m)		1.69 (ddd, 13.6, 13.6, 9.0)
13		46.0		46.3		43.5		47.8
14		157.1		157.3		51.3		48.6
15	5.67 (dd, 3.9, 1.6)	127.6	5.69 (dd, 3.8, 1.7)	127.8	1.39 (m)	33.9	1.72 (m)	32.3
					1.38 (m)		1.39 (m)
16	α 1.94 (ddd, 15.5, 10.7, 1.6)	35.0	α 1.96 (m)	34.9	α 1.27 (m)	28.4	α 1.38 (m)	27.1
	β 2.24 (m)		β 2.26 (ddd, 15.5, 7.0, 3.9)		β 1.98 (m)		β 1.96 (m)
17	1.56 (m)	61.4	1.59 (m)	61.4	1.50 (m)	53.2	2.33 (m)	41.5
18	1.05 (3H, s)	20.5	1.08 (3H, s)	20.2	0.84 (3H, s)	22.0	0.61 (3H, s)	17.3
19	0.91 (3H, s)	23.5	0.93 (3H, s)	22.9	0.82 (3H, s)	14.2	1.22 (3H, s)	19.6
20	1.67 (m)	33.6	1.67 (m)	33.6	1.41 (m)	35.5	1.72 (m)	34.2
21	0.88 (3H, d, 6.3)	18.2	0.88 (3H, d, 6.4)	18.3	0.84 (3H, d, 6.3)	18.5	0.90 (3H, d, 6.6)	18.5
22	1.68 (m)	32.2	1.67 (m)	32.2	1.99 (m)	28.9	1.73 (m)	30.7
	1.26 (m)		1.28 (m)		1.23 (m)		1.45 (m)
23	1.41 (m)	28.8	1.41 (m)	28.8	2.49 (ddd, 17.3, 8.9, 6.4)	32.9	1.45 (m)	25.5
	1.41 (m)		1.41 (m)		2.56 (ddd, 17.3, 9.3, 5.3)		1.45 (m)
24	3.37 (dd, 8.5, 4.2)	78.5	3.37 (dd, 8.8, 3.9)	78.5		214.8	3.27 (dd, 7.5, 4.1)	78.8
25		73.3		73.3		76.2		73.3
26	1.22 (3H, s)	26.7	1.22 (3H, s)	26.7	1.38 (3H, s)	26.6	1.19 (3H, s)	26.3
27	1.17 (3H, s)	23.3	1.17 (3H, s)	23.3	1.38 (3H, s)	26.6	1.14 (3H, s)	23.0
28	1.04 (3H, s)	25.1	1.17 (3H, s)	23.8	1.01 (3H, s)	28.1	1.05 (3H, s)	24.4
29	0.99 (3H, s)	21.5	1.15 (3H, s)	23.7	0.89 (3H, s)	15.9	1.14 (3H, s)	22.2
30	1.06 (3H, s)	26.1	1.06 (3H, s)	25.7	0.98 (3H, s)	27.3	1.05 (3H, s)	26.3

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Compound 3 displayed a molecular formula of C₃₀H₅₀O₄ as inferred from the HRESI(−)MS ion peak at m/z 519.3673 [M + HCO₂]⁻ (calcd 519.3686) suggestive of six indices of hydrogen deficiency. The IR spectrum showed the presence of hydroxyl (3431 cm⁻¹), carbonyl (1709 cm⁻¹), and olefinic (1643 cm⁻¹) functionalities. The ¹³C NMR spectrum displayed 30 signals whose multiplicity assignments by analysis of DEPT spectrum revealed eight methyl, eight methylene, seven methine (one olefinic), and seven quaternary (one carbonyl) carbons. A proton signal at δ_H 5.27, together with two carbon resonances at δ_C 118.0 and 145.4, indicated the presence of a trisubstituted double bond. This group and the carbonyl occupied two out of six indices of hydrogen deficiency, and the remaining four required 3 to be tetracyclic. The planar structure of 3 was finally established by examination of ¹H–¹H COSY and HMBC data (Fig. 4A). More specifically, the HMBC correlations of H₃-26(27)/C-24(δ_C 214.8) located the only keto group at C-24, and those of H₃-30/C-8(δ_C 145.4) and H-7(δ_H 5.27)/C-5 and C-9 fixed the only double bond at Δ.⁷ Finally, the chemical shifts for C-2(δ_C 68.8) and C-3(δ_C 83.7), the COSY interaction of H-2/H-3, and the HMBC correlations of H₃-28(29)/C-3 supported the presence of two vicinal hydroxyls as drawn. The flat structure of 3 was thus established to bear one of tirucallane, euphane and lanostane scaffolds which possessed the same planar skeleton.¹²

Fig. 4 Key 2D NMR correlations of ricinodol C (3).

The relative configuration of 3 was established mainly by analysis of its ROESY data (Fig. 4B). The ROESY correlations of H-2 with both H₃-19 and H₃-29 revealed that they adopted mutual 1,3-diaxial relationships in ring-A with a chair-like conformation and were assigned to be β-oriented, while the correlation of H-3/H-5 was also supportive of their diaxial position and thus α-orientation. Subsequently, H-9, Me-18, and H-6 at δ_H 2.14 were determined to be α-positioned via the ROESY networks of H-5/H-9, H-9/H₃-18, H₃-18/H-16α, whereas H-16β, Me-30, and H-17 were then established to be oppositely oriented. As with 1, the diagnostic ROESY interactions of H₂-16/H₃-21, H-17/H₃-21, and H₃-18/H-20 permitted the assignment of R-configuration for C-20.^12–16 The euphane skeleton for 3 was thus assigned due to the configurations of C-13 and C-20.¹² Thus, compound 3 was elucidated and named ricinodol C.

Compound 4 displayed a sodiated molecular ion peak at m/z 495.3440 [M + Na]⁺ (calcd 495.3450) in HRESI(+)MS analysis, supportive of a molecular formula of C₃₀H₄₈O₄. The IR absorption bands at 3477, 1691, and 1658 cm⁻¹ indicated the presence of hydroxyl, carbonyl, and olefinic groups, respectively. The maximum UV absorption at 241 nm suggested the presence of a conjugated diene group.²² Comprehensive analysis of HSQC and HMBC spectra (see ESI Fig. S34 & S35†) finally established the planar structure of 4 as shown. In detail, the HMBC correlations of H₃-19/C-9(δ_C 145.2), H₃-30/C-8(δ_C 141.1), H-7(δ_H 5.47)/C-5, C-9, and C-14, and H-11(δ_H 5.40)/C-8, C-10, and C-13 corroborated the presence of the conjugated diene group that was fixed at Δ⁷ and Δ⁹⁽¹¹⁾. In addition, the strong correlation of H₃-28(29)/C-3(δ_C 216.2) located the only keto group at C-3. Furthermore, the interactions of H₃-26(27)/C-24(δ_C 78.8) and C-25(δ_C 73.3) indicated that the vicinal diol at C-24 and C-25 was also present in 4, while the strong correlation of H₃-18/C-12(δ_C 74.0) allowed the last hydroxyl group to be attached at C-12. The relative configurations of the corresponding chiral centers between 4 and 3 were identified to be the same by examination of ROESY data (see ESI Fig. S36†). The diagnostic correlations of H₂-16/H₃-21, H-17/H₃-21, and H₃-18/H-20 in agreement with those of 1–3 indicated that 4 also featured a euphane skeleton.^12–16 The β-orientation of 12-OH was assigned via the upfield shifted carbon resonances, due to γ-gauche effect, of C-14 (Δδ_C −2.7) and C-17 (Δδ_C −11.7) as compared with those of 3 (Table 1), which was further confirmed by the strong ROESY correlation of H-12/H₃-18. The Mo₂⁴⁺ complex of 4 in DMSO exhibited a negative Cotton effect at 305 nm (Δε −1.5) (Fig. 3B) that allowed the assignment of R-configuration for C-24. The structure of 4 was then established and named ricinodol D.

Compound 5 was assigned a molecular formula of C₃₀H₄₈O₄ as evidenced by HRESI(−)MS analysis at m/z 471.3471 [M − H]⁻ (calcd 471.3474). The UV absorption maximum at 244 nm (log ε 4.02) together with the carbon resonances at δ_C 198.6, 171.0, and 124.9 (Table 2) indicated the presence of an α,β-unsaturated carbonyl group.²³ Comparison of the NMR data (Table 2) with those of 3 and 4 revealed that 5 also possessed the euphane skeleton. This was further supported by analysis of 2D NMR data (see ESI Fig. S43–S45†) with HMBC correlations from H₃-28(29) to C-3(δ_C 214.1) and from H-5(δ_H 2.45) & H-7(δ_H 5.77) to C-6(δ_C 198.6) supportive of two keto groups at C-3 and C-6, respectively. In addition, the HMBC correlations of H₃-30/C-8(δ_C 171.0) and H-7(δ_H 5.77)/C-5, C-9, and C-14 attached the only double bond at Δ.⁷ The configuration of C-24 in 5 was also assigned as R by in situ dimolybdenum CD method which revealed a negative Cotton effect at 308 nm (Δε −2.5) (see ESI Fig. S2†). Compound 5 was then identified as shown.

Table 2 ¹H and ¹³C NMR data (in CDCl₃) for compounds 5–7^a

No.	5		6		7
	δH (multi, J in Hz)	δC	δH (multi, J in Hz)	δC	δH (multi, J in Hz)	δC
a ^1H NMR data were measured at 500 MHz. ^13C NMR data were measured at 125 MHz.
1	α 1.70 (m)	37.6	1.25 (dd, 13.0, 12.7)	48.3	2.23 (d, 12.8)	51.4
	β 2.00 (m)		2.38 (dd, 12.7, 6.3)		2.47 (d, 12.8)
2	α 2.31 (ddd, 14.7, 4.7, 3.2)	34.1	4.59 (dd, 13.0, 6.3)	69.3		211.3
	β 2.76 (m)
3		214.1		216.5	3.96 (s)	82.7
4		47.1		47.3		45.5
5	2.45 (s)	65.3	1.72 (dd, 10.9, 6.5)	53.1	2.00 (m)	49.9
6		198.6	2.10 (m)	24.2	2.28 (m)	24.2
			2.10 (m)		2.04 (m)
7	5.77 (d, 2.7)	124.9	5.30 (m)	117.5	5.31 (m)	118.1
8		171.0		145.9		145.6
9	2.75 (m)	49.6	2.29 (m)	48.6	2.57 (m)	49.4
10		43.2		35.6		42.0
11	1.71 (m)	17.7	1.57 (m)	18.4	1.55 (m)	18.4
	1.61 (m)		1.57 (m)		1.41 (m)
12	1.92 (m)	32.7	1.84 (ddd, 13.7, 13.7, 5.6)	33.7	1.86 (m)	33.6
	1.84 (m)		1.72 (m)		1.72 (m)
13		43.0		43.5		43.6
14		52.5		51.3		51.3
15	1.57 (m)	32.9	1.47 (m)	34.0	1.46 (m)	33.9
	1.57 (m)		1.47 (m)		1.46 (m)
16	α 1.34 (m)	28.0	α 1.30 (m)	28.5	α 1.29 (m)	28.5
	β 2.03 (m)		β 1.95 (m)		β 1.96 (m)
17	1.57 (m)	53.0	1.50 (m)	53.5	1.52 (m)	53.4
18	0.84 (3H, s)	22.0	0.80 (3H, s)	22.2	0.86 (3H, s)	22.2
19	1.10 (3H, s)	13.9	1.08 (3H, s)	13.8	0.76 (3H, s)	13.9
20	1.47 (m)	35.3	1.42 (m)	35.6	1.45 (m)	35.6
21	0.87 (3H, d, 6.4)	18.4	0.84 (3H, d, 6.4)	18.5	0.85 (3H, d, 6.4)	18.5
22	1.68 (m)	31.7	1.67 (m)	31.8	1.67 (m)	31.8
	1.20 (m)		1.15 (m)		1.16 (m)
23	1.41 (m)	28.4	1.37 (m)	28.7	1.39 (m)	28.6
	1.41 (m)		1.37 (m)		1.39 (m)
24	3.36 (dd, 8.9, 3.8)	78.3	3.34 (dd, 9.2, 3.4)	78.4	3.35 (dd, 7.9, 4.7)	78.4
25		73.3		73.2		73.2
26	1.23 (3H, s)	26.7	1.20 (3H, s)	26.6	1.21 (3H, s)	26.7
27	1.17 (3H, s)	23.2	1.15 (3H, s)	23.3	1.16 (3H, s)	23.3
28	1.36 (3H, s)	25.2	1.08 (3H, s)	24.4	1.14 (3H, s)	28.0
29	1.38 (3H, s)	21.7	1.16 (3H, s)	21.5	0.74 (3H, s)	15.6
30	1.10 (3H, s)	25.0	1.01 (3H, s)	27.5	0.98 (3H, s)	27.3

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Compound 6 had a molecular formula of C₃₀H₅₀O₄ as determined by HRESI(−)MS data with an adduct ion peak at m/z 519.3685 [M + HCO₂]⁻ (calcd 519.3686). Comparison of ¹H and ¹³C NMR data (Table 2) with those of 3 revealed that they were structural analogues with the differences occurring to ring-A and C-17 side chain, where the C-3 carbonyl (δ_C 216.5) and the CH-24 oxymethine (δ_H 3.34 & δ_C 78.4) in 6 were in place of the CH-3 oxymethine (δ_H 3.03 & δ_C 83.7) and the C-24 carbonyl (δ_C 214.8) in 3. Such assignments were confirmed by the HMBC correlations of H₃-28(29)/C-3(δ_C 216.5) and H₃-26(27)/C-24(δ_C 78.4) (see ESI Fig. S53†). The corresponding stereocenters in 6 were assigned the same as their counterparts in 3 on the basis of proton couplings and ROESY data. The β-axial orientation of H-2 was first determined via the large coupling constant of J_1α,2 (13.0 Hz) and was further confirmed by the ROESY correlations of H-2/H₃-19 and H-2/H₃-29. The α-hydroxy-ketone group at C-2 and C-3 compete with the vicinal diol at C-24 and C-25 in forming metal complex, which prevented us from using in situ dimolybdenum CD method to assign the C-24 configuration. Nevertheless, the high NMR similarity to its cometabolites (1, 2, 4 & 5) and a biogenetic consideration allowed us to tentatively assign a 24R-configuration for 6. Thus, the structure of 6 was characterized and named ricinodol F.

Compound 7 showed a molecular formula of C₃₀H₅₀O₄ same as 6, which was indicated by the HRESI(−)MS ion at m/z 519.3675 [M + HCO₂]⁻¹ (calcd 519.3686). Comparison of ¹H and ¹³C NMR data (Table 2) to those of 6 revealed that the structure of 7 was very similar to that of 6. The main differences occurred to ring-A, where signals of a double doublet oxymethine (δ_H 4.59, δ_C 69.3, CH-2) and a carbonyl (δ_C 216.5, C-3) in 6 were replaced by those of a carbonyl (δ_C 211.3, C-2) and a singlet oxymethine (δ_H 3.96, δ_C 82.7, CH-3) in 7, and meanwhile the multiplicities of both H₂-1 in 7 were simplified to doublets. The structure of 7 was finally confirmed by interpretation of 2D NMR data (see ESI Fig. S61–S63†). In particular, the α-orientation of H-3 was supported by the ROESY correlation of H-3/H-5. Thus, compound 7 was characterized and named ricinodol G.

Preliminary testing of compounds 1–10 at 10.0 μM using scintillation proximity assay (SPA)^24,25 revealed that ricinodols E–G (5–7) showed >50% inhibition against both human and mouse 11β-HSD1, while ricinodols A (1), C (3) and D (4) selectively exhibited >50% inhibition against human 11β-HSD1 (see ESI Tables S1 and S2†). Of these compounds, ricinodol E (5) showed the best inhibitory activities against both enzymes with values of 96.2% and 94.5%. Further detailed bioassays on ricinodol E (5) returned IC₅₀ values of 0.36 ± 0.26 and 0.84 ± 0.18 μM against human and mouse 11β-HSD1, respectively. However, all the three known diterpenoids exhibited weak inhibitions with values <50% at 10.0 μM. The above-discussed activities of the triterpenoid compounds reveals a brief but clear structure–activity relationship that, compared with other analogues, the 6-keto group in ricinodol E (5) significantly improves its inhibitory effect on 11β-HSD1.

Conclusion

In summary, we have described in this article the isolation and elucidation of seven new triterpenoids ricinodols A–G (1–7) and three known diterpenoids (8–10), as well as the evaluation of their inhibitory effects against human and mouse 11β-HSD1. The structure characterization was based on detailed spectroscopic analyses and in situ dimolybdenum CD method. In particular, ricinodols A (1) and B (2) possessed an unprecedented backbone with a rare concurrent rearrangement of Me-19 (10 → 9) and Me-30 (14 → 8) which, to the best of our knowledge, was only found in a pentacyclic triterpenoid petatrichol B.¹¹ All compounds exhibited mild to significant inhibitory activities against both human and mouse 11β-HSD1, and particularly ricinodol E (5) displayed the strongest inhibitory effect at submicromolar level. The discovery of these triterpenoids adds new members to the family of 11β-HSD1 inhibitors, which might find potential applications in future development of new drug leads for metabolic disorders.

Experimental

General experimental details

Optical rotations were detected on a Autopol VI polarimeter at room temperature. UV spectra were measured on a Shimadzu UV-2550 UV-visible spectrophotometer. IR spectra were recorded on a Perkin-Elmer 577 or a Thermo IS5 spectrometer using KBr disks. NMR spectra were measured on a Bruker AM-500 spectrometer with TMS as internal standard. ESI(±)MS and HR-ESI(±)MS analyses were carried out on a Bruker Daltonics Esquire 3000 plus LC-MS instrument and a waters Q-TOF Ultima mass spectrometer, respectively. Semipreparative HPLC was carried out on a Waters 1525 binary pump system with a Waters 2489 detector (210 nm) using a YMC-Pack ODS-A column (250 × 10 mm, S-5 μm). Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd), C₁₈ reversed-phase silica gel (150–200 mesh, Merck), MCI gel (CHP20P, a kind of refined polystyrene-based separation stuffing, 75–150 μM, Mitsubishi Chemical Industries, Ltd.), and D101-macroporous adsorption resin (Shanghai Huangling Resin Co. Ltd.) were used for column chromatography (CC). Pre-coated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.) were used for TLC detection. All solvents used for CC were of analytical grade (Shanghai Chemical Reagents Co. Ltd.) and solvents used for HPLC were of HPLC grade (J & K Scientific Ltd.).

Plant material

The stems and leaves of Ricinodendron heudelotii were collected in September 2010 from Hainan Province, People's Republic of China, and were authenticated by Prof. Shiman Huang from Department of Biology, Hainan University. A voucher specimen has been deposited in the Herbarium of Shanghai Institute of Materia Medica, Chinese Academy of Sciences (accession number: RH-2010HN-1Y).

Extraction and isolation

The air-dried powdered stems and leaves (10 kg) of Ricinodendron heudelotii were extracted three times with 95% ethanol at room temperature. After evaporation of solvent under reduced pressure, the residue (260 g) was suspended in 1.0 L water and then partitioned with EtOAc (3 × 1.0 L). The combined EtOAc layer was evaporated under reduced pressure to give a dark material (150 g) which was then subjected to column chromatography (CC) over macroporous resin D-101 (EtOH–H₂O, 30%, 50%, 80% and 95%) to return three fractions. The 80% ethanol elution was separated on a column of MCI gel (MeOH–H₂O, 50–100%) to get four fractions, and the second fraction was then chromatographed over silica gel eluted with petroleum ether–acetone (15 : 1 to 1 : 3) to yield four major subfractions A1 to A4. Fraction A2 was submitted to a silica gel column eluted with CH₂Cl₂–MeOH (200 : 1 to 20 : 1) to afford three fractions A2-1 to A2-3, and the second fraction was first separated on a silica gel CC eluted with CH₂Cl₂–MeOH (200 : 1 to 50 : 1) and then purified with semipreparative HPLC to afford compounds 8 (1.0 mg) and 10 (8.0 mg). Fraction A2-3 was fractionated on a reversed-phase C18 silica gel CC using MeOH–H₂O (60–100%) to get two major fractions A2-31 (70%) and A2-32 (75%), and the first fraction A2-31 was then subjected to silica gel CC eluted with petroleum ether–ethyl acetate (3 : 1 to 1 : 1) to afford four fractions A2-311 to A2-314. Faction A2-312 was purified with semipreparative HPLC with MeCN–H₂O (80–100%, 3.0 mL min⁻¹) to yield compounds 2 (1.8 mg), 6 (13.8 mg), and 7 (4.0 mg). Fraction A2-314 was also purified with semipreparative HPLC with MeCN–H₂O (70–90%, 3.0 mL min⁻¹) to yield compound 5 (2.9 mg). Fraction A2-32 was purified with semipreparative HPLC with MeCN–H₂O (50–70%, 3.0 mL min⁻¹) to yield compound 9 (3.4 mg). Fraction A3 was chromatographed on a reversed-phase C18 silica gel column using MeOH–H₂O system (50–90%) to afford 7 fractions A3-1 to A3-7. Fraction A3-5 was then subjected to a silica gel column eluted with CHCl₃–MeOH (80 : 1 to 20 : 1) to get two fractions A3-51 and A3-52, the latter of which was then purified with semipreparative HPLC with 70% MeCN–H₂O to yield compound 1 (3.0 mg). Upon a silica gel column eluted with CHCl₃–MeOH (80 : 1 to 20 : 1) and then semipreparative HPLC with 90% MeCN–H₂O, fraction A3-6 finally afforded compounds 3 (8.3 mg) and 4 (4.0 mg).

Characterization of new compounds

Ricinodol A (1). White amorphous powder; [α]²⁶_D −1.8 (c 0.11 in MeOH); IR (KBr disk) ν_max 3502, 3404, 2970, 2961, 2925, 2874, 1630, 1467, 1379 cm⁻¹; ¹H and ¹³C NMR (CDCl₃) Table 1; ESI(+)MS m/z 497.4 [M + Na]⁺; ESI(−)MS m/z 519.5 [M + HCO₂]⁻; HRESI(−)MS m/z 519.3677 [M + HCO₂]⁻ (519.3686, calcd for C₃₁H₅₁O₆).

Ricinodol B (2). White amorphous powder; [α]²⁶_D 4.0 (c 0.15 in MeOH); IR (KBr disk) ν_max 3439, 2970, 2927, 2871, 1708, 1627, 1466, 1378, 1282, 1064 cm⁻¹; ¹H and ¹³C NMR (CDCl₃) Table 1; ESI(+)MS m/z 495.3 [M + Na]⁺; ESI(−)MS m/z 517.6 [M + HCO₂]⁻; HRESI(+)MS m/z 495.3439 [M + Na]⁺ (495.3450, calcd for C₃₀H₄₈O₄Na).

Ricinodol C (3). White amorphous powder; [α]²⁶_D −15.3 (c 0.75 in MeOH); IR (KBr disk) ν_max 3431, 2968, 1709, 1643, 1466, 1375, 1057, 989 cm⁻¹; ¹H and ¹³C NMR (CDCl₃) Table 1; ESI(+)MS m/z 475.3 [M + H]⁺, 971.8 [2M + Na]⁺; ESI(−)MS m/z 519.6 [M + HCO₂]⁻; HRESI(−)MS m/z 519.3673 [M + HCO₂]⁻ (519.3686, calcd for C₃₁H₅₁O₆).

Ricinodol D (4). White amorphous powder; [α]²⁶_D −128.0 (c 0.35 in MeOH); UV (MeOH) λ_max (log ε) 241 (4.17) nm; IR (KBr) ν_max 3477, 2960, 2935, 2879, 1691, 1658, 1454, 1377, 1024, 916 cm⁻¹; ¹H and ¹³C NMR (CDCl₃) Table 1; ESI(+)MS m/z 495.5 [M + H]⁺, 967.9 [2M + Na]⁺; ESI(−)MS m/z 517.7 [M + HCO₂]⁻; HRESI(+)MS m/z 495.3440 [M + H]⁺ (495.3450, calcd for C₃₀H₄₈O₄Na).

Ricinodol E (5). White amorphous powder; [α]²⁶_D 4.0 (c 0.1 in MeOH); UV (MeOH) λ_max (log ε) 244 (4.02) nm; IR (KBr disk) ν_max 3444, 2968, 2933, 2877, 1708, 1664, 1468, 1379, 1078, 1008 cm⁻¹; ¹H and ¹³C NMR (CDCl₃) Table 2; ESI(+)MS m/z 473.4 [M + H]⁺, 967.8 [2M + Na]⁺; ESI(−)MS m/z 517.6 [M + HCO₂]⁻; HRESI(−)MS m/z 471.3471 [M − H]⁻ (471.3474, calcd for C₃₀H₄₇O₄).

Ricinodol F (6). White amorphous powder; [α]²⁶_D −5.6 (c 0.90 in MeOH); IR (KBr disk) ν_max 3442, 2968, 2881, 1710, 1670, 1468, 1386, 1074, 991 cm⁻¹; ¹H and ¹³C NMR (CDCl₃) Table 2; ESI(+)MS m/z 475.4 [M + H]⁺, 971.9 [2M + Na]⁺; ESI(−)MS m/z 519.5 [M + HCO₂]⁻; HRESI(−)MS m/z 519.3685 [M + HCO₂]⁻ (519.3686, calcd for C₃₁H₅₁O₆).

Ricinodol G (7). White amorphous powder; [α]²⁶_D −2.3 (c 0.35 in MeOH); IR (KBr disk) ν_max 3429, 2968, 2941, 2896, 1695, 1637, 1468, 1388, 1120, 1043 cm⁻¹; ¹H and ¹³C NMR (CDCl₃) Table 2; ESI(+)MS m/z 475.4 [M + H]⁺, 971.9 [2M + Na]⁺; ESI(−)MS m/z 519.5 [M + HCO₂]⁻; HRESI(−)MS m/z 519.3675 [M + HCO₂]⁻ (519.3686, calcd for C₃₁H₅₁O₆).

Bioassays

As previously reported.⁷

Acknowledgements

This work was funded by the National Natural Science Foundation (nos 21272244, 81322045) of the People's Republic of China. We thank Prof. S. M. Huang of Hainan University for the identification of the plant materials.

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Footnote

† Electronic supplementary information (ESI) available: IR, MS, and 1D & 2D NMR spectra of compounds 1–7, the preliminary bioassay results, and the induced CD spectra for compounds 2 and 5. See DOI: 10.1039/c5ra01857a

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