Mangrove Tirucallane- and Apotirucallane-Type Triterpenoids: Structure Diversity of the C-17 Side-Chain and Natural Agonists of Human Farnesoid/Pregnane–X–Receptor

Abstract

Ten new triterpenoid compounds with structure diversity of the C-17 side-chain, including nine tirucallanes, named xylocarpols A–E (1–5) and agallochols A–D (6–9), and an apotirucallane, named 25-dehydroxy protoxylogranatin B (10), were isolated from the mangrove plants Xylocarpus granatum, Xylocarpus moluccensis, and Excoecaria agallocha. The structures of these compounds were established by HR-ESIMS and extensive one-dimensional (1D) and two-dimensional (2D) NMR investigations. The absolute configurations of 1 and 2 were unequivocally determined by single-crystal X-ray diffraction analyses, conducted with Cu Kα radiation; whereas those of 4, 6–8 were assigned by a modified Mosher’s method and the comparison of experimental electronic circular dichroism (ECD) spectra. Most notably, 5, 6, 7, and 9 displayed potent activation effects on farnesoid–X–receptor (FXR) at the concentration of 10.0 μM; 10 exhibited very significant agonistic effects on pregnane–X–receptor (PXR) at the concentration of 10.0 nM.

1. Introduction

Cholestasis, a clinical syndrome of hepatobiliary diseases, is usually caused by accumulation of bile acids in the liver and systemic circulation [1]. Long-term cholestasis can lead to primary biliary cirrhosis, primary sclerosing cholangitis, and hepatic failure. In clinical practice, abnormal metabolism of bile acids is deemed to be a crucial risk factor that induces cholestasis and cholestatic liver injury [1,2]. Farnesoid–X–receptor (FXR) and pregnane–X–receptor (PXR) are two members of the nuclear receptor family. Due to the regulation capability of a suite of genes involved in the metabolism, transport, and elimination of bile acids, FXR and PXR are considered to be the key target proteins for the treatment of cholestasis and liver injury [1,3,4,5].

Xylocarpus granatum and Xylocarpus moluccensis, true-mangrove species of the family Meliaceae, have been used in folk medicine, particularly in South and Southeast Asian countries, for the treatment of cholera, diarrhea, malaria, and other fever diseases [6]. Mangroves of the genus Xylocarpus are mainly distributed in East Africa, India, Bangladesh, Southeast Asia, Southern China, and Northern Australia [7,8]. Previous chemical investigation of X. granatum and X. moluccensis, collected from different locations, led to the isolation and identification of various limonoids, protolimonoids, alkaloids, and flavanones. To the best of our knowledge, limonoids are the main secondary metabolites of these mangroves [7,8,9].

Excoecaria agallocha, a semi-mangrove species of the family Euphorbiaceae, has been used in Asia as a traditional medicine for the treatment of epilepsy, haematuria, leprosy, and toothache [10,11]. It is mainly distributed along the sea coasts of China, India, Philippines, and Oceania. The poisonous milky latex, exuded from the bark of this plant, can cause blistering of the skin and temporary blindness [12,13,14,15,16]. Previous chemical investigation of this plant resulted in the isolation and identification of diterpenoid, triterpenoid, tanninoid, and flavonoid natural compounds [10,17,18,19,20,21]. To date, only 18 triterpenoid compounds, including types of cycloartane, friedelane, lupane, oleanane, and taraxerane, have been reported from this plant [17].

As part of our ongoing investigation of bioactive natural products from mangrove plants, 10 new triterpenoid compounds, including nine tirucallanes (1–9) and one apotirucallane (10) (Figure 1), were isolated from mangrove plants X. granatum, X. moluccensis, and E. agallocha. We reported the isolation and structure elucidation of these compounds, and their agonistic effects on human FXR and PXR.

2. Results and Discussion

Compound 1 was obtained as colorless crystals. Its molecular formula C₃₀H₅₀O₅ with six indices of hydrogen deficiency was established by the negative HR-ESIMS quasi molecular ion peak at m/z 525.3356 ([M + Cl]⁻, calculated for 525.3352) (Supplementary Materials S6 and S7). According to the ¹H and ¹³C NMR spectroscopic data of 1 (

Table 1. ¹H (400 MHz) NMR spectroscopic data for compounds 1–5 in CDCl₃ (δ in ppm, J in Hz).

No.	1	2	3	4	5
1α	1.46 m	1.46 m	1.47 m	1.13 m	1.46 m
1β	1.99 m	2.00 m	2.00 m	1.67 a	1.99 m
2α	2.25 dt (14.4, 3.2)	2.25 m	2.25 m	1.55 m	2.27 m
2β	2.76 td (14.4, 5.8)	2.77 td (14.8, 5.6)	2.75 td (14.4, 5.4)	1.65 a	2.76 td (14.4, 3.6)
3				3.24 dd (11.2, 4.4)
5	1.73 t (8.4)	1.74 t (8.4)	1.72 t (8.6)	1.30 m	1.72 t (8.4)
6α	2.10 a	2.11 a	2.11 a	2.11 br s	2.10 a
6β	2.10 a	2.11 a	2.11 a	1.94 m	2.10 a
7	5.32 d (2.8)	5.34 br d (3.2)	5.31 br d (2.8)	5.26 br s	5.32 d (3.2)
9	2.30 m	2.29 a	2.29 a	2.19 m	2.28 m
11α	1.63	1.57 a	1.58 a	1.55 m	1.55 m
11β	1.54 a	1.57 a	1.58 a	1.48 a	1.60 m
12α	1.66 m	1.57 a	1.67 m	1.58 m	1.62 m
12β	1.83 m	1.84 m	1.81 m	1.83 m	1.84 m
15α	1.53 a	1.56 a	1.52 a	1.48 a	1.51 a
15β	1.53 a	1.56 a	1.52 a	1.48 a	1.51 a
16α	1.40 m	1.42 m	1.24 m	1.34 m	1.32 m
16β	2.00 m	2.14 m	1.92 m	2.00 m	2.00 m
17	1.81 m	2.01 m	1.56 m	1.84 m	1.92 m
18	0.87 s	0.87 s	0.86 s	0.82 s	0.82 s
19	1.01 s	1.02 s	1.01 s	0.74 s	1.01 s
20	1.97 m	1.87 m	2.05 m	1.64 m	1.60 m
21	0.94 d (6.8)	0.67 d (6.4)	0.91 d (6.4)	0.83 a	0.90 d (6.8)
22a	3.80 br d (8.4)	4.14 s	2.51 br d (14.4)	3.81 d (6.0)	3.74 d (4.0)
22b			2.29 a
23	3.66 dd (8.4, 6.4)			3.96 t (6.0)	4.25 t (5.2)
24a	3.55 br d (6.4)	2.32 a	4.07 s	3.65 d (6.0)	4.35 d (5.6)
24b		2.32 a
25		2.21 m	2.16 m
26	1.35 a	0.93 d (6.4)	0.71 d (6.4)	1.21 a	1.42 s
27	1.35 a	0.96 d (6.4)	1.13 d (6.4)	1.21 a	1.36 s
28	1.12 s	1.13 s	1.12 s	0.85 s	1.12 s
29	1.05 a	1.06 s	1.05 s	0.96 s	1.05 a
30	1.05 a	1.07 s	1.02 s	1.00 s	1.05 a

^a Overlapped signals assigned by ¹H–¹H COSY, HSQC, and HMBC spectra without designating multiplicity.

and

Table 2. ¹³C (100 MHz) NMR spectroscopic data for compounds 1‒5 in CDCl₃ (δ in ppm).

No.	1	2	3	4	5
1	38.5 CH2	38.5 CH2	38.5 CH2	37.2 CH2	38.5 CH2
2	34.9 CH2	34.9 CH2	34.9 CH2	27.8 CH2	34.9 CH2
3	217.0 qC	217.0 qC	216.9 qC	79.3 CH	217.2 qC
4	47.9 qC	47.9 qC	47.9 qC	39.0 qC	47.9 qC
5	52.3 CH	52.3 CH	52.3 CH	50.6 CH	52.3 CH
6	24.4 CH2	24.4 CH2	24.4 CH2	23.9 CH2	24.4 CH2
7	118.0 CH	118.0 CH	118.1 CH	117.8 CH	117.9 CH
8	145.7 qC	145.7 qC	145.6 qC	145.8 qC	145.8 qC
9	48.5 CH	48.4 CH	48.4 CH	49.0 CH	48.5 CH
10	35.0 qC	35.0 qC	35.0 qC	34.9 qC	35.0 qC
11	18.3 CH2	18.2 CH2	18.2 CH2	18.1 CH2	18.3 CH2
12	33.7 CH2	33.4 CH2	33.5 CH2	33.6 CH2	33.7 CH2
13	43.6 qC	43.3 qC	43.6 qC	43.6 qC	43.3 qC
14	51.3 qC	51.4 qC	51.3 qC	51.2 qC	51.3 qC
15	34.0 CH2	34.0 CH2	34.0 CH2	34.0 CH2	34.0 CH2
16	27.4 CH2	28.0 CH2	28.4 CH2	27.7 CH2	27.7 CH2
17	48.6 CH	48.9 CH	53.0 CH	49.2 CH	48.9 CH
18	22.0 CH3	21.9 CH3	22.0 CH3	21.8 CH3	21.9 CH3
19	12.8 CH3	12.8 CH3	12.8 CH3	13.1 CH3	12.8 CH3
20	36.3 CH	39.3 CH	32.7 CH	37.6 CH	37.4 CH
21	11.5 CH3	12.1 CH3	19.7 CH3	12.4 CH3	12.0 CH3
22	75.8 CH	79.5 CH	45.7 CH2	83.7 CH	74.2 CH
23	70.9 CH	212.3 qC	212.2 qC	72.9 CH	86.2 CH
24	80.1 CH	46.8 CH2	80.1 CH	77.3 CH	73.1 CH
25	74.1 qC	24.5 CH	31.3 CH	80.9 qC	86.0 qC
26	24.6 CH3	22.6 CH3	14.6 CH3	27.7 CH3	28.1 CH3
27	27.7 CH3	22.7 CH3	20.2 CH3	21.3 CH3	21.6 CH3
28	21.6 CH3	21.6 CH3	21.6 CH3	14.7 CH3	21.6 CH3
29	24.8 CH3	24.5 CH3	24.6 CH3	27.5 CH3	24.5 CH3
30	27.6 CH3	27.6 CH3	27.4 CH3	27.6 CH3	27.6 CH3

) (Supplementary Materials S8‒S13), two elements of unsaturation were due to a carbon-carbon double bond and a carbonyl group. Thus, the molecule was tetracyclic. The ¹³C NMR spectroscopic data and the DEPT135 experiment of 1 (Supplementary Materials S14 and S15) revealed the presence of eight methyl groups, seven methylene groups, eight methine groups (including three oxygenated ones at δ_C 75.8, 70.9, and 80.1 ppm, respectively, and an olefinic one at δ_C 118.0 ppm), and seven quaternary carbons (including an oxygenated one at δ_C 74.1, an olefinic one at δ_C 145.7, and a ketone carbon at δ_C 217.0 ppm, respectively).

¹H‒¹H COSY correlations (Supplementary Materials S16‒S18) between H-22/H-23 and H-23/H-24 and HMBC correlations from H-24 to C-25, C-26, and C-27 confirmed the connection of the fragment of C-22(OH)–C-23(OH)–C-24(OH)–C-25(OH)–(Me)₂ (Figure 2a). The NMR spectroscopic data of 1 resembled those of toonaciliatavarin E [22], except for different orientations of three chiral centers on the C-17 side-chain, videlicet C-22, C-23, and C-24.

The relative configuration of the tetracyclic tirucallane core (rings-A, B, C, and D) of 1 was corroborated by diagnostic NOE interactions (Supplementary Materials S28‒S32). Those between H-5/H-9, H-5/H₃-29, H-9/H₃-18, and H₃-18/H-20 revealed the α-oriented H-5, H-9, H₃-18, H-20, and H₃-29. Similarly, NOE interactions between H₃-19/H-2β, H₃-19/H₃-28, H₃-19/H-11β, H₃-30/H-11β, and H₃-30/H-17 assigned the β-oriented H-17, H₃-19, H₃-28, and H₃-30 (Figure 2b).

In order to establish the absolute configuration of the whole molecule of 1, particularly the absolute configuration of three chiral centers on the C-17 side-chain, a single-crystal X-ray diffraction analysis, conducted with Cu Kα radiation (Flack parameter of 0.01(6)) (Figure 3), was employed. Thus, the absolute configuration of 1, named xylocarpol A, was unequivocally assigned as (5R,9R,10R,13S,14S,17S,20R,22S,23S,24S)-22,23,24,25-tetrahydroxytirucalla-7-ene-3-one.

Compound 2 was isolated as colorless crystals. Its molecular formula C₃₀H₄₈O₃ (seven degrees of unsaturation) was established by the positive HR-ESIMS quasi molecular ion peak at m/z 457.3670 ([M + H]⁺, calculated for 457.3676) (Supplementary Material S33). The NMR spectroscopic data of 2 (Table 1 and Table 2) (Supplementary Materials S34‒S39) were closely related to those of aphagranin D [23], the difference being the absence of the 24-OH and 25-OH groups in 2. This deduction were corroborated by the upshifted C-24 (δ_C 82.1 CH in aphagranin D; whereas δ_C 46.8 CH₂ in 2) and C-25 (δ_C 79.0 qC in aphagranin D; whereas δ_C 24.5 CH in 2), and the proton spin-spin system, i.e., H₂-24‒H-25(H₃-26)‒H₃-27, observed in the ¹H‒¹H COSY spectrum of 2 (Supplementary Materials S42‒S44).

NOE interactions (Supplementary Materials S53‒S58) between H-5/H-9, H-5/H₃-29, H-9/H₃-18, and H₃-18/H-20 assigned the α-oriented H-5, H-9, H₃-18, and H₃-29; whereas those between H₃-19/H-2β, H₃-19/H₃-28, H₃-30/H-12β, H₃-30/H-17 concluded the β-oriented H-17, H₃-19, H₃-28, and H₃-30. Finally, single-crystal X-ray diffraction analysis, conducted with Cu Kα radiation (Flack parameter of 0.08(13)) (Figure 4), unambiguously established the absolute configuration of the whole molecule of 2. Therefore, the absolute configuration of 2, named xylocarpol B, was unambiguously determined to be (5R,9R,10R,13S,14S,17S,20R,22S)-22-hydroxytirucalla-7-ene-3,23-dione.

Compound 3 provided the molecular formula C₃₀H₄₈O₃ as deduced from the positive HR-ESIMS quasi molecular ion peak at m/z 457.3654 ([M + H]⁺, calculated for 457.3676) (Supplementary Material S59). The NMR spectroscopic data of 3 (Table 1 and Table 2) (Supplementary Materials S60‒S64) resembled those of 2, except for the different location of the only hydroxy group on the C-17 side-chain. A 24-OH group was observed in 3 instead of the 22-OH function in 2. HMBC correlations (Supplementary Materials S72‒S77) from the oxygenated H-24 (δ_H 4.07, s) to C-23, C-25, C-26, and C-27 confirmed the presence of the 24-OH group. NOE interactions (Supplementary Materials S78‒S82) between H-5/H-9, H-5/H₃-29, H-9/H₃-18, and H₃-18/H-20, and those between H₃-19/H-2β, H₃-19/H₃-28, H₃-30/H-11β, and H₃-30/H-17 established the same tetracyclic tirucallane core (rings-A, B, C, and D) in 3 as that of 2. However, due to the limited amount of 3, the chirality of C-24 could not be determined. Thus, the structure of 3, named xylocarpol C, was assigned as 24-hydroxytirucalla-7-ene-3,23-dione.

Compound 4 gave the molecular formula C₃₀H₅₀O₄ as obtained from the negative HR-ESIMS quasi molecular ion peak at m/z 509.3402 ([M + Cl]⁻, calculated for 509.3403) (Supplementary Materials S83 and S84). The ¹H and ¹³C NMR spectroscopic data of 4 (Table 1 and Table 2) (Supplementary Materials S85‒S90) were closely related to those of odoratone [24], except for the replacement of the C-3 ketone group in odoratone by a hydroxy group in 4. The above deduction was corroborated by the upshifted C-3 (δ_C 216.0 qC in odoratone; whereas δ_C 79.3 CH in 4) and ¹H‒¹H COSY correlations between H₂-2/H-3 (Figure 5a) (Supplementary Materials S95‒S97). The relative configuration of 4 was established by NOE interactions (Supplementary Materials S107‒S110). Those between H-3/H-5, H-5/H-9, H-5/H₃-29, H-9/H₃-18, and H₃-18/H-20 assigned the α-oriented H-3, H-5, H-9, H₃-18, H-20, and H₃-29; NOE interactions between H₃-19/H₃-28, H₃-30/H-11β, H₃-19/H-11β, and H₃-30/H-17 concluded the β-oriented H-17, H₃-19, H₃-28, and H₃-30 (Figure 5b). The above results concluded the same relative configuration for the tetracyclic tirucallane core (rings-A, B, C, and D) in 4 as that of 1–3, except for the additional chiral C-3 in 4. Moreover, NOE interactions between H-23/H₃-21 and H-24/H₃-26 assigned the α-oriented H-23, H-24, and H₃-26; those between H-22/H₃-27 concluded the β-oriented H-22 and H₃-27 (Figure 5b). Thus, the relative configuration of the C-17 side-chain of 4 was determined.

In order to establish the absolute configuration of 4, a modified Mosher’s α-methoxy-α-(trifluoromethyl)phenylacetyl (MTPA) ester method was applied [25]. The (3,23,24)-tri(S)- and (3,23,24)-tri(R)-MTPA esters of 4, videlicet 4s and 4r (Supplementary Materials S280‒S289), were successfully prepared. Based on the MTPA ester rule of Δδ (δ_S − δ_R) values (Figure 6) [25], the absolute configurations of C-3, C-23, and C-24 were assigned as S, R, and S, respectively. Therefore, the absolute configuration of 4, named xylocarpol D, was unequivocally established as (3S,5R,9R,10R,13S,14S,17S,20R,22S,23R,24S)- 3,23,24-trihydroxy-22,25-epoxytirucalla-7-ene. The absolute configuration of the 2,2-dimethyltetrahydrofuran-3,4-diol moiety of odoratone [24] was first clarified as 22S,23R,24S.

The molecular formula of 5 was determined to be C₃₀H₄₈O₄ (seven degrees of unsaturation) by the negative HR-ESIMS quasi molecular ion peak at m/z 507.3245 ([M + Cl]⁻, calculated for 507.3247) (Supplementary Materials S111 and S112). Two elements of unsaturation were due to a carbon-carbon double bond and a ketone group; thus, the molecule was pentacyclic. The NMR spectroscopic data of 5 (Table 1 and Table 2) (Supplementary Materials S113‒S118) were similar to those of 4, the difference being the opposite orientation of the 24-OH group and the replacement of the 3-OH group in 4 by a ketone function in 5. Diagnostic NOE interactions (Supplementary Materials S134‒S137) between H-22/H₃-27 and H-24/H₃-27 assigned the β-oriented H-22 and H-24; whereas those between H-23/H₃-26 concluded the α-oriented H-23 (Figure 7). HMBC correlations (Supplementary Materials S128‒S133) from H₂-1, H₂-2, H₃-28, and H₃-29 to the carbon (δ_C 217.2 qC) of a ketone group confirmed its location at C-3. Therefore, the structure of 5, named xylocarpol E, was assigned as depicted.

The molecular formula of 6 was concluded to be C₃₀H₅₀O₄ by the positive HR-ESIMS quasi molecular ion peak at m/z 475.3790 ([M + H]⁺, calculated for 475.3782) (Supplementary Materials S138 and S139). The ¹H and ¹³C NMR spectroscopic data of 6 (

Table 3. ¹H (400 MHz) NMR spectroscopic data for compounds 6‒10 in CDCl₃ (δ in ppm, J in Hz).

No.	6	7	8	9	10
1α	1.41 m	1.41 m	1.41 m	1.14 m	7.14 d (10.4)
1β	1.71 m	1.71 m	1.71 m	1.68 m
2α	1.59 a	1.59 a	1.59 a	1.54 m	5.87 d (10.0)
2β	1.68 m	1.67 m	1.68 m	1.66 m
3	3.22 dd (11.6, 4.0)	3.22 dd (11.6, 4.0)	3.22 dd (11.6, 4.4)	3.25 dd (11.2, 4.0)
5	2.13 s	2.13 s	2.13 s	1.32 m	2.17 m
6α				2.15 m	1.77 m
6β				1.95 m	1.92 a
7	5.70 d (2.4)	5.70 d (2.8)	5.70 d (2.8)	5.26 d (3.6)	5.23 br s
9	2.72 m	2.72 m	2.71 m	2.20 m	2.20 m
11α	1.75 a	1.75 a	1.75 a	0.96 a	1.92 a
11β	1.59 a	1.58 a	1.57 a	1.50 a	1.70 m
12α	1.74 a	1.55 a	1.74 a	1.63 m	1.91 a
12β	1.87 m	1.88 m	1.86 m	1.78 m	1.56 m
15α	1.55 a	1.55 a	1.59 a	1.52 a	5.30 s
15β	1.74 a	1.75 a	1.54 a	1.47 a
16α	1.38 m	1.38 m	1.37 m	1.31 m	2.04 m
16β	2.04 m	2.02 m	2.02 m	1.97 m	2.21 m
17	1.55 a	1.55 a	1.55 a	1.47 a	1.81 m
18	0.84 s	0.84 s	0.84 s	0.81 s	1.08 s
19	0.86 s	0.86 s	0.86 s	0.75 s	1.17 s
20	1.43 m	1.44 m	1.48 m	1.44 m	2.42 a
21	0.91 d (6.0)	0.91 d (6.4)	0.88 d (6.4)	0.89 d (6.0)
22a	1.54 a	1.78 m	2.21 m	1.83 m	2.47 m
22b	1.28 m	1.02 m	1.76 a	1.32 m	2.41 a
23a	1.39 m	1.59 m	5.60 dd (15.6, 4.8)	2.41 m	4.32 d (10.0)
23b	1.39 m	1.18 m		2.28 m	3.95 dd (11.2, 5.6)
24	3.36 br s	3.30 dd (11.6, 1.6)	5.62 d (15.6)
25					2.56 m
26	1.18 s	1.17 s	1.32 a		1.17 s
27	1.23 s	1.23 s	1.32 a		1.19 s
28	1.13 s	1.13 s	1.13 s	0.86 s	1.08 s
29	1.32 s	1.32 s	1 32 a	0.97 a	1.08 s
30	1.06 s	1.05 s	1.04 s	0.97 a	1.19 s
2′					1.95 s

^a Overlapped signals assigned by ¹H–¹H COSY, HSQC, and HMBC spectra without designating multiplicity.

and

Table 4. ¹³C (100 MHz) NMR spectroscopic data for compounds 6‒10 in CDCl₃ (δ in ppm).

No.	6	7	8	9	10
1	37.0 CH2	37.0 CH2	37.0 CH2	37.2 CH2	158.1 CH
2	26.6 CH2	26.6 CH2	26.6 CH2	27.7 CH2	125.6 CH
3	79.1 CH	79.1 CH	79.1 CH	79.3 CH	204.6 qC
4	38.0 qC	38.0 qC	38.0 qC	39.0 qC	44.2 qC
5	65.2 CH	65.2 CH	65.2 CH	50.6 CH	46.1 CH
6	200.0 qC	200.0 qC	200.0 qC	23.9 CH2	23.8 CH2
7	125.0 CH	125.0 CH	125.0 CH	118.0 CH	74.6 CH
8	170.6 qC	170.6 qC	170.5 qC	145.7 qC	42.8 qC
9	50.4 CH	50.4 CH	50.4 CH	48.9 CH	38.4 CH
10	43.8 qC	43.8 qC	43.8 qC	34.9 qC	39.8 qC
11	17.6 CH2	17.6 CH2	17.5 CH2	18.1 CH2	16.7 CH2
12	32.9 CH2	32.8 CH2	32.6 CH2	33.8 CH2	34.1 CH2
13	43.0 qC	43.0 qC	43.0 qC	43.6 qC	46.5 qC
14	52.3 qC	52.3 qC	52.3 qC	51.1 qC	159.1 qC
15	32.8 CH2	32.9 CH2	32.9 CH2	34.0 CH2	118.9 CH
16	27.8 CH2	27.7 CH2	27.6 CH2	28.1 CH2	34.6 CH2
17	52.7 CH	52.6 CH	52.1 CH	52.8 CH	54.6 CH
18	21.9 CH3	21.9 CH3	21.9 CH3	21.9 CH3	19.7 CH3
19	14.3 CH3	14.3 CH3	14.3 CH3	13.1 CH3	19.0 CH3
20	35.8 CH	36.3 CH	36.2 CH	35.7 CH	35.6 CH
21	18.2 CH3	18.5 CH3	18.4 CH3	18.0 CH3	176.3 qC
22	32.8 CH2	33.2 CH2	38.6 CH2	30.9 CH2	35.1 CH2
23	28.4 CH2	28.7 CH2	125.2 CH	31.0 CH2	65.4 CH2
24	78.6 CH	79.5 CH	139.7 CH	178.5 qC	177.1 qC
25	73.2 qC	73.2 qC	70.8 qC		34.1 CH
26	23.3 CH3	23.2 CH3	30.0 CH3		19.0 CH3
27	26.7 CH3	26.6 CH3	29.9 CH3		19.1 CH3
28	14.8 CH3	14.8 CH3	14.8 CH3	14.7 CH3	21.3 CH3
29	28.3 CH3	28.3 CH3	28.3 CH3	27.6 CH3	27.1 CH3
30	24.9 CH3	24.9 CH3	24.9 CH3	27.3 CH3	27.4 CH3
1′					170.1 qC
2′					21.2 CH3

) (Supplementary Materials S140‒S146) resembled those of brumollisol B [26], except for the absence of the 23-OH group, being corroborated by the upshifted C-23 (δ_C 69.5 CH in brumollisol B; whereas δ_C 28.4 CH₂ in 6), ¹H‒¹H COSY correlations (Supplementary Materials S149‒S152) between H₂-23/H-24, and HMBC correlations (Supplementary Materials S157‒S164) from H₃-26 and H₃-27 to C-24 (Figure 8a).

The relative configuration for the tetracyclic tirucallane core (rings-A, B, C, and D) of 6 was established by NOE interactions (Supplementary Materials S165‒S168). Those between H-3/H-5, H-5/H-9, H-5/H₃-29, H-9/H₃-18, and H₃-18/H-20 assigned the α-oriented H-3, H-5, H-9, H₃-18, H-20, and H₃-29; NOE interactions between H₃-19/H₃-28, H₃-30/H-11β, H₃-19/H-11β, and H₃-30/H-17 concluded the β-oriented H-17, H₃-19, H₃-28, and H₃-30 (Figure 8b).

The absolute configuration of 6 was established by the application of the modified Mosher’s MTPA ester method [25]. The (3,24)-di(S)- and (3,24)-di(R)-MTPA esters of 6 were prepared (Supplementary Materials S290‒S297). Based on the MTPA ester rule of Δδ (δ_S − δ_R) values (Figure 9) [25], the absolute configurations of C-3 and C-24 were assigned as S and S, respectively. Hence, the absolute configuration of 6, named agallochol A, was unequivocally assigned as (3S,5R,9R,10R,13S,14S,17S,20S,24S)- 3,24,25-trihydroxytirucalla-7-ene-6-one.

Compound 7 provided the same molecular formula (C₃₀H₅₀O₄) as that of 6 based on the positive HR-ESIMS quasi molecular ion peak at m/z 475.3792 ([M + H]⁺, calculated for 475.3782) (Supplementary Materials S169 and S170). The ¹H and ¹³C NMR spectroscopic data of 7 (Table 3 and Table 4) (Supplementary Materials S171‒S175) resembled those of 6, except for the slight difference of ¹H and ¹³C chemical shifts of CH-24 (δ_H 3.36 (br s), δ_C 78.6 in 6; whereas δ_H 3.30 dd (J = 11.6, 1.6 Hz), δ_C 79.5 in 7), indicating that both compounds are a pair of C-24 epimers. The above deduction was further corroborated by ¹H‒¹H COSY correlations between H-24/H₂-23 (Supplementary Materials S178‒S180) and HMBC interactions between H-24/C-23, H-24/C-25, H₃-26/C-24, and H₃-27/C-24 (Supplementary Materials S184‒S189).

In order to determine the absolute configurations of C-3 and C-24 of 7, (3,24)-di(S)- and (3,24)-di(R)-MTPA esters of 7 were prepared (Supplementary Materials S298‒S305). Based on the MTPA ester rule of Δδ (δ_S − δ_R) values (Figure 9) [25], the absolute configurations of C-3 and C-24 were assigned as S and R, respectively. Hence, the absolute configuration of 7, named agallochol B, was unambiguously concluded to be (3S,5R,9R,10R,13S,14S,17S,20S,24R)-3,24,25-trihydroxytirucalla-7-ene-6-one.

The molecular formula of 8 was established as C₃₀H₄₈O₃ by the positive HR-ESIMS quasi molecular ion peak at m/z 457.3673 ([M + H]⁺, calculated for 457.3682) (Supplementary Material S194). The NMR spectroscopic data of 8 (Table 3 and Table 4) (Supplementary Materials S195‒S200) were similar to those of 7, except for the presence of a Δ²³⁽²⁴⁾ double bond (δ_H 5.60 (dd, J = 15.6, 4.8 Hz, 1H), 5.62 (d, J = 15.6 Hz, 1H); δ_C 125.2, CH, 139.7, CH) and the absence of the 24-OH group. ¹H‒¹H COSY correlations between H-23/H₂-22 and H-23/H-24 (Supplementary Materials S203‒S206), and HMBC correlations between H-23/C-22, H-23/C-24, H-23/C-25, H-24/C-22, H-24/C-23, and H-24/C-25 in 8 (Supplementary Materials S211‒S217) confirmed the above deduction. In addition, the coupling constant of 15.6 Hz between H-23 and H-24 assigned the E-geometry of the Δ²³⁽²⁴⁾ double bond.

The relative configuration of the tetracyclic tirucallane core (rings-A, B, C, and D) of 8 was established as the same as that of 7 by NOE interactions (Supplementary Materials S218‒S222). Those between H-5/H-3, H-5/H-9, H-5/H₃-29, H-9/H₃-18, H₃-18/H-20, H₃-19/H₃-28, H₃-19/H₃-30, and H₃-30/H-17 assigned the same relative configuration of 8 as that of 7. Moreover, the absolute configuration of 8, particularly that of the tetracyclic tirucallane core (rings-A, B, C, and D), was established to be the same as that of 7, except for the deficiency of the chiral C-24, by the accurate fit of their experimental ECD spectra (Figure 10). Therefore, the structure of 8, named agallochol C, was determined to be (3S,5R,9R,10R,13S,14S,17S,20S)-3,25-dihydroxytirucalla-7,23-diene-6-one.

Compound 9 had the molecular formula C₂₇H₄₄O₃ as determined from its negative HR-ESIMS quasi molecular ion peak at m/z 451.2985 ([M + Cl]⁻, calculated for 451.2984) (Supplementary Materials S223 and S224). The NMR spectroscopic data of 9 (Table 3 and Table 4) (Supplementary Materials S225‒S231) resembled those of a trinortirucalla-7-ene, i.e., sikkimenoid F [27], except for the replacement of the C-24 aldehyde group in sikkimenoid F by a C-24 carboxyl group in 9. The above deduction was corroborated by the upshifted C-24 (δ_H 9.75 (br s), δ_C 203.1 CH in sikkimenoid F; whereas δ_C 178.5 qC in 9) and HMBC correlations from H₂-22 and H₂-23 to the carbonyl carbon (C-24) of this carboxyl group. The relative configuration of the tetracyclic tirucallane core (rings-A, B, C, and D) of 9 was established by NOE interactions (Supplementary Materials S247‒S250). Those between H-5/H-3, H-5/H-9, H-5/H₃-29, H-9/H₃-18, H₃-18/H-20, H₃-19/H₃-28, H₃-19/H₃-30, and H₃-30/H-17 assigned the same relative configuration of the tetracyclic tirucallane core of 9 as that of 8. Thus, the structure of 9, named agallochol D, was assigned as 3β-hydroxy-25,26,27-trinortirucalla-7-ene-24-oic acid.

Compound 10 provided the molecular formula C₃₂H₄₆O₇ based on the positive HR-ESIMS quasi molecular ion peak at m/z 543.3303 ([M + H]⁺, calculated for 543.3316) (Supplementary Material S251), requiring ten degrees of unsaturation. According to the ¹H and ¹³C NMR spectroscopic data of 10 (Table 3 and Table 4) (Supplementary Materials S252‒S258), six of the 10 elements of unsaturation were due to two carbon-carbon double bonds and four carbonyls. Thus, the molecule was tetracyclic. The DEPT135 experiment of 10 (Supplementary Materials S259‒S261) combined with its ¹³C NMR spectroscopic data revealed the presence of eight methyl groups, six methylene groups, nine methine groups (including three olefinic ones), and nine quaternary carbons (including four carbonyl carbons).

The above NMR characteristic features of 10 closely resembled those of an apotirucallane protolimonoid, i.e., protoxylogranatin B [28], except for the absence of the 25-OH group in 10. ¹H–¹H COSY correlations from the proton of a methine moiety (δ_H 2.56 m; δ_C 34.1) to H₃-26 and H₃-27 confirmed the presence of the CH-25 group (Figure 11a). The relative configuration of 10 was established on the basis of NOE interactions (Supplementary Materials S275‒S279). Those between H-5/H-9, H-5/H₃-29, H-9/H₃-18, H-20/H₃-18 assigned the α-oriented H-5, H-9, H₃-18, and H-20; whereas those between H₃-19/H₃-28, H₃-19/H-6β, and H₃-30/H-6β concluded the β-oriented H₃-19 and H₃-30 (Figure 11b). The NOE interaction between H-7/H₃-30, but not between H-7/H-5 and H-7/H₃-18, indicated the β-oriented H-7 and the corresponding α-oriented 7-acetoxy group (Figure 11b). Thus, the structure of 10, named 25-dehydroxy protoxylogranatin B, was established as depicted.

In order to search for natural agonists of human FXR and PXR, most of the above isolated compounds were screened for their agonistic effects on these nuclear receptors. Chenodeoxycholic acid (CDCA) or rifampicin was used as the positive control at the concentration of 80.0 μM or 10.0 μM, respectively (Figure 12 and Figure 13). The results showed that 6 and 7 displayed significant agonistic effects on FXR at the concentration of 1.0 μM; while 5, 6, 7, and 9 exhibited significant agonistic effects on FXR at the concentration of 10.0 μM. Moreover, 1 displayed a moderate significant agonistic effect on FXR at the concentration of 10.0 μM (Figure 12). Compound 10 exhibited a significant agonistic effect on PXR at the concentration of 10.0 nM, and even a higher agonistic effect on PXR as compared to that of the positive control, rifampicin, at the same concentration of 10.0 μM (Figure 13).

3. Materials and Methods

3.1. General Methods

Optical rotations were recorded at room temperature on a MCP200 modular circular polarimeter (Anton Paar GmbH, Seelze, Germany). A GENESYS 10S UV–Vis spectrophotometer (Thermo Scientific, Shanghai, China) was used to obtain UV spectra. The NMR spectroscopic data were measured on a Bruker AV-400 NMR spectrometer (Bruker Scientific Technology Co. Ltd., Karlsruhe, Germany) using TMS as the internal standard. Single-crystal X-ray diffraction analyses were carried out on an Agilent Xcalibur Atlas Gemini Ultra-diffractometer with mirror monochromated Cu Kα radiation (λ = 1.54184 Å) at 100 K. An LC-ESI (Bruker Daltonics, Bremen, Germany) and an LC-ESI-QTOF mass spectrometer (SYNAPTTM G2 HDMS, Waters, Manchester, UK) were used to acquire HR-ESIMS data. For electronic circular dichroism (ECD) spectra, a Jasco 810 spectropolarimeter (JASCO Corporation, Tokyo, Japan) was applied with the solvent of acetonitrile. Semi-preparative HPLC was carried out on a Waters 2535 pump equipped with a 2489 UV detector (Waters Corporation, Milford, NY, USA) and an ODS column (YMC, 250 × 10 mm inner diameter, 5 µm). Silica gel (100–200 mesh, Qingdao Mar. Chem. Ind. Co. Ltd., Qingdao, China) and ODS silica gel (A-HG 12 nm, 50 mm, YMC Co. Ltd., Kyoto, Japan) were used for column chromatography.

3.2. Plant Material

The seeds of two mangrove plants, Xylocarpus granatum and Xylocarpus moluccensis, were collected in September 2007 at the mangrove swamps of Krishna estuary, Andhra Pradesh, India. The plant species were identified by Mr. Tirumani Satyanandamurty (Government Degree College at Amadala Valasa, India). The voucher samples (No. IXG-02 for X. granatum) and (No. IXM200701 for X. moluccensis) were kept in the Marine Drugs Research Center, College of Pharmacy, Jinan University.

The stems and twigs of the semi-mangrove plant, Excoecaria agallocha, were collected in December 2003 at the mangrove swamps of Hainan Island, China. The identification of the plant was done by Professor Jun Wu (School of Pharmaceutical Sciences, Southern Medical University). A voucher sample (EA-J1) was kept in Marine Drugs Research Center, College of Pharmacy, Jinan University.

3.3. Extraction and Isolation

The seeds of X. granatum were air-dried (22.0 kg), powdered, and then extracted with 95% EtOH (5 × 75 L) at room temperature. The resulting EtOH extract (4.0 kg) was partitioned between EtOAc/water (3:1, v/v) to afford the EtOAc portion (1100.0 g). The EtOAc portion (250.0 g) was subjected to silica gel column chromatography (120 × 10 cm inner diameter; chloroform/methanol, from 100:0 to 10:1) to give 210 fractions. Fractions 50–82 (20.0 g) were combined and separated by an ODS silica gel column (100 × 7 cm inner diameter; acetone/water, from 40:60 to 100:0) to afford 81 subfractions. The subfraction 52 (308.0 mg) was purified by semi-preparative HPLC (MeCN/H₂O, 40:60, 3.0 mL/min) to give 1 (78.0 mg, t_R = 54.0 min) and 5 (3.0 mg, t_R = 62.0 min); whereas the subfraction 53 (128.0 mg) was separated by semi-preparative HPLC (MeCN/H₂O, 40:60, 3.0 mL/min) to yield 4 (15.5 mg, t_R = 35.8 min).

The seeds of X. moluccensis were air-dried (6.0 kg), powdered, and extracted with 95% EtOH (6 × 15 L) at room temperature. The resulting extract (824.6 g) was partitioned between EtOAc/water (3:1, v/v) to give the EtOAc portion (299.1 g), which was applied to silica gel column chromatography (150 × 8.5 cm inner diameter; chloroform/methanol, from 100:0 to 5:1) to afford 223 fractions. Fractions 9–11 (18.5 g) were combined and further separated by ODS silica gel column chromatography (60 × 3 cm inner diameter; acetone/water, from 50:50 to 100:0) to afford 90 subfractions. The subfraction 61 (800.0 mg) was purified by semi-preparative HPLC (MeOH/H₂O, 82:18, 3.0 mL/min) to yield 2 (6.5 mg, t_R = 45.0 min) and 3 (1.0 mg, t_R = 52.0 min). Fractions 68–71 (22.9 g) were combined and separated by ODS silica gel column chromatography (60 × 3 cm inner diameter.; acetone/water, from 40:60 to 100:0) to give 111 subfractions. The subfraction 75 (210.0 mg) was purified by semi-preparative HPLC (MeCN/H₂O, 58:42, 3.0 mL/min) to afford 10 (0.9 mg, t_R = 46.0 min).

The stems and twigs of E. agallocha were air-dried (10.0 kg), powdered, and extracted with 95% EtOH (5 × 40.0 L) at room temperature. The resulting brown extract (370.0 g)was then suspended in water, and extracted with hexane (3:1, v/v) and EtOAc (3:1, v/v), successively, to give the EtOAc portion (192.0 g), which was separated by silica gel column chromatography (150 × 10.5 cm inner diameter; chloroform/methanol, from 100:0 to 5:1) to yield 285 fractions. Fractions 130 to 170 (17.5 g) were combined and subjected to ODS silica gel column chromatography (110 × 6 cm inner diameter; acetone/water, from 30:70 to 100:0) to give 76 subfractions. The subfraction 35 (1.3 g) was purified by semi-preparative HPLC (MeCN/H₂O, 42:58, 3.0 mL/min) to yield 6 (4.0 mg, t_R = 69.8 min) and 7 (4.0 mg, t_R = 73.1 min).

Fractions 40 to 100 (18.0 g) were combined and further separated by ODS silica gel column chromatography (110 × 6 cm inner diameter; acetone/water, from 55:45 to 100:0) to yield 65 subfractions. The subfraction 33 (76.0 mg) was subjected to semi-preparative HPLC (MeCN/H₂O, 70:30, 3.0 mL/min) to give 9 (3.3 mg, t_R = 36.8 min). Fractions 101 to 129 (45.0 g) were combined and purified by ODS silica gel column chromatography (100 × 10 cm inner diameter; acetone/water, from 40:60 to 100:0) to yield 80 subfractions. The subfraction 44 (190.0 mg) was purified by semi-preparative HPLC (MeOH/H₂O, 82:18, 3.0 mL/min) to afford 8 (4.0 mg, t_R = 30.8 min).

Xylocarpol A (1): (5R,9R,10R,13S,14S,17S,20R,22S,23S,24S)-22,23,24,25-tetrahydroxytirucalla- 7-ene-3-one. Colorless crystals; ${\left[a\right]}_{\mathrm{D}}^{25}$ = −68.0 (c 0.1, acetone); UV (MeCN) λ_max (logε) 193 (4.20) nm; ¹H- and ¹³C-NMR spectroscopic data see Table 1 and Table 2, respectively; HR-ESIMS m/z 525.3356 [M + Cl]⁻ (calculated for C₃₀H₅₀ClO₅, 525.3352).

Xylocarpol B (2): (5R,9R,10R,13S,14S,17S,20R,22S)-22-hydroxytirucalla-7-ene-3,23-dione. Colorless crystals; ${\left[a\right]}_{\mathrm{D}}^{25}$ = −8.0 (c 0.1, acetone); UV (MeCN) λ_max (logε) 191 (4.16) nm; ¹H- and ¹³C-NMR spectroscopic data see Table 1 and Table 2, respectively; HR-ESIMS m/z 457.3670 [M + H]⁺ (calculated for C₃₀H₄₉O₃, 457.3676).

Xylocarpol C (3): White amorphous power; ${\left[a\right]}_{\mathrm{D}}^{25}$ = −82.0 (c 0.1, acetone); UV (MeCN) λ_max (logε) 193 (3.77) nm; ¹H- and ¹³C-NMR spectroscopic data see Table 1 and Table 2, respectively; HR-ESIMS m/z 457.3654 [M + H]⁺ (calculated for C₃₀H₄₉O₃, 457.3676).

Xylocarpol D (4): (3S,5R,9R,10R,13S,14S,17S,20R,22S,23R,24S)-3,23,24-trihydroxy-22,25- epoxytirucalla-7-ene. White amorphous power; ${\left[a\right]}_{\mathrm{D}}^{25}$ = −70.0 (c 0.1, acetone); UV (MeCN) λ_max (logε) 203 (4.40) nm; ¹H- and ¹³C-NMR spectroscopic data see Table 1 and Table 2, respectively; HR-ESIMS m/z 509.3402 [M + Cl]⁻ (calculated for C₃₀H₅₀ClO₄, 509.3403).

Xylocarpol E (5): White amorphous power; ${\left[a\right]}_{\mathrm{D}}^{25}$ = −8.0 (c 0.1, acetone); UV (MeCN) λ_max (logε) 194 (3.70) nm; ¹H- and ¹³C-NMR spectroscopic data see Table 1 and Table 2, respectively; HR-ESIMS m/z 507.3245 [M + Cl]⁻ (calculated for C₃₀H₄₈ClO₄, 507.3247).

Agallochol A (6): (3S,5R,9R,10R,13S,14S,17S,20S,24S)-3,24,25-trihydroxytirucalla-7-ene-6-one. White amorphous power; ${\left[a\right]}_{\mathrm{D}}^{25}$ = −19.5 (c 0.1, acetone); UV (MeCN) λ_max (logε) 204 (3.61), 246 (3.68) nm; ¹H- and ¹³C-NMR spectroscopic data see Table 3 and Table 4, respectively; HR-ESIMS m/z 475.3790 [M + H]⁺ (calculated for C₃₀H₅₁O₄, 475.3782).

Agallochol B (7): (3S,5R,9R,10R,13S,14S,17S,20S,24R)-3,24,25-trihydroxytirucalla-7-ene-6-one. White amorphous power; ${\left[a\right]}_{\mathrm{D}}^{25}$ = −13.2 (c 0.1, acetone); UV (MeCN) λ_max (logε) 204 (3.55), 246 (3.49) nm; ECD (0.21 mM, MeCN) λ_max (∆ε) 209.0 (−16.1), 242.6 (+ 12.8) nm; ¹H- and ¹³C-NMR spectroscopic data see Table 3 and Table 4, respectively; HR-ESIMS m/z 475.3792 [M + H]⁺ (calculated for C₃₀H₅₁O₄, 475.3782).

Agallochol C (8): (3S,5R,9R,10R,13S,14S,17S,20S)-3,25-dihydroxytirucalla-7,23-diene-6-one. White amorphous power; ${\left[a\right]}_{\mathrm{D}}^{25}$ = −22.4 (c 0.1, acetone); UV (MeCN) λ_max (logε) 202 (3.07), 245 (3.82) nm; ECD (0.37 mM, MeCN) λ_max (∆ε) 209.6 (−10.4), 242.4 (+ 7.5) nm; ¹H- and ¹³C-NMR spectroscopic data see Table 3 and Table 4, respectively; HR-ESIMS m/z 457.3673 [M + H]⁺ (calculated for C₃₀H₄₉O₃, 457.3682).

Agallochol D (9): White amorphous power; ${\left[a\right]}_{\mathrm{D}}^{25}$= − 18.4 (c 0.1, acetone); UV (MeCN) λ_max (logε) 204 (3.19) nm; ¹H- and ¹³C-NMR spectroscopic data see Table 3 and Table 4, respectively; HR-ESIMS m/z 451.2985 [M + Cl]⁺ (calculated for C₂₇H₄₄ClO₃, 451.2984).

25-dehydroxy protoxylogranatin B (10): White amorphous power; ${\left[a\right]}_{\mathrm{D}}^{25}$ = −22.0 (c 0.09, acetone); UV (MeCN) λ_max (logε) 202 (4.20) nm; ¹H- and ¹³C-NMR spectroscopic data see Table 3 and Table 4, respectively; HR-ESIMS m/z 543.3303 [M + H]⁻ (calculated for C₃₂H₄₇O₇, 543.3316).

3.4. Preparation of the (3,23,24)-Tri(S)- and (3,23,24)-Tri(R)-MTPA Esters of 4

Compound 4 (1.0 mg) was treated with (S)-MTPA acid (2.0 mg), 4-dimethylaminopyridine (DMAP) (1.0 mg), and N,N′-dicyclohexylcarbodiimide (DCC) (4.0 mg) in dried dichloromethane (0.5 mL) at room temperature for 72 h. The reaction mixture was concentrated and purified by C₁₈ reversed-phase HPLC (YMC-Pack 250 × 10 mm i.d.) with acetonitrile (MeCN/H₂O, 100:0) to afford the (3,23,24)-tri(S)-MTPA esters of 4, named 4s (1.7 mg). Similarly, the (3,23,24)-tri(R)-MTPA esters of 4, named 4r (1.8 mg), were prepared in the same way.

4s: amorphous powder; ¹H NMR (CDCl₃, 400 MHz): δ_H 1.252 (1H, m, H-1α), 1.705 (1H, m, H-1β), 1.637 (1H, m, H-2α), 1.797 (1H, m, H-2β), 4.730 (1H, dd, J = 11.2, 3.2 Hz, H-3), 1.454 (1H, m, H-5), 0.795 (3H, s, H₃-18), 0.752 (3H, s, H₃-19), 1.647 (1H, m, H-20), 0.910 (3H, overlapped, H₃-21), 3.853 (1H, d, J = 5.2 Hz, H-22), 5.365 (1H, t, J = 5.2 Hz, H-23), 5.000 (1H, d, J = 6.0 Hz, H-24), 1.055 (3H, s, H₃-26), 1.288 (3H, s, H₃-27), 0.891 (3H, overlapped, H₃-28), 0.900 (3H, s, H₃-29), 0.967 (3H, s, H₃-30).

4r: amorphous powder; ¹H NMR (CDCl₃, 400 MHz): δ_H 1.264 (1H, m, H-1α), 1.736 (1H, m, H-1β), 1.751 (1H, m, H-2α), 1.868 (1H, m, H-2β), 4.757 (1H, dd, J = 11.6, 4.4 Hz, H-3), 1.442 (1H, m, H-5), 0.820 (3H, s, H₃-18), 0.778 (3H, s, H₃-19), 1.803 (1H, m, H-20), 0.929 (3H, d, J = 6.0 Hz, H₃-21), 4.056 (1H, d, J = 4.8 Hz, H-22), 5.403 (1H, t, J = 5.2 Hz, H-23), 4.926 (1H, d, J = 6.4 Hz, H-24), 0.989 (3H, s, H₃-26), 1.216 (3H, s, H₃-27), 0.884 (3H, overlapped, H₃-28), 0.806 (3H, s, H₃-29), 0.968 (3H, s, H₃-30).

3.5. Preparation of the (3,24)-Di(S)- and (3,24)-Di(R)-MTPA Esters of 6 and 7

Compound 6 (1.0 mg) was treated with (S)-MTPA acid (2.0 mg), DMAP (1.0 mg), and DCC (4.0 mg) in dried dichloromethane (0.5 mL) at room temperature for 72 h. The reaction mixture was concentrated and purified by C₁₈ reversed-phase HPLC (YMC-Pack 250 × 10 mm i.d.) with aqueous acetonitrile (MeCN/H₂O, 84:16) to afford the (3,24)-di(S)-MTPA esters of 6, named 6s (0.5 mg). The (3,24)-di(R)-MTPA esters of 6, named 6r (0.7 mg), was prepared in the same way.

6s: amorphous powder; ¹H NMR (CDCl₃, 400 MHz): δ_H 1.497 (1H, m, H-1α), 1.717 (1H, m, H-1β), 1.619 (1H, m, H-2α), 1.780 (1H, m, H-2β), 4.672 (1H, dd, J = 11.2, 4.0 Hz, H-3), 2.220 (1H, s, H-5), 5.699 (1H, d, J = 2.8 Hz, H-7), 0.806 (3H, s, H₃-18), 0.867 (3H, s, H₃-19), 1.358 (1H, m, H-20), 0.784 (3H, d, J = 6.0 Hz, H₃-21), 1.353 (1H, m, H-22a), 0.876 (1H, m, H-22b), 1.613 (1H, m, H-23a), 1.547 (1H, m, H-23b), 4.964 (1H, dd, J = 10.0, 2.4 Hz, H-24), 1.163 (3H, s, H₃-26), 1.228 (3H, s, H₃-27), 1.220 (3H, s, H₃-28), 1.139 (3H, s, H₃-29), 1.032 (3H, s, H₃-30).

6r: amorphous powder; ¹H NMR (CDCl₃, 400 MHz): δ_H 1.520 (1H, m, H-1α), 1.762 (1H, m, H-1β), 1.749 (1H, m, H-2α), 1.850 (1H, m, H-2β), 4.703 (1H, dd, J = 11.6, 4.4 Hz, H-3), 2.222 (1H, s, H-5), 5.697 (1H, d, J = 2.4 Hz, H-7), 0.825 (3H, s, H₃-18), 0.895 (3H, s, H₃-19), 1.416 (1H, m, H-20), 0.854 (3H, d, J = 6.0 Hz, H₃-21), 1.445 (1H, m, H-22a), 1.009 (1H, m, H-22b), 1.710 (1H, m, H-23a), 1.645 (1H, m, H-23b), 4.981 (1H, dd, J = 10.0, 2.4 Hz, H-24), 1.148 (3H, overlapped, H₃-26), 1.180 (3H, s, H₃-27), 1.148 (3H, overlapped, H₃-28), 1.148 (3H, overlapped, H₃-29), 1.035 (3H, s, H₃-30).

Compound 7 (1.0 mg) was treated with (S)-MTPA acid (2.0 mg), DMAP (1.0 mg), and DCC (4.0 mg) in dried dichloromethane (0.5 mL) at room temperature for 72 h. The reaction mixture was concentrated and purified by C₁₈ reversed-phase HPLC (YMC-Pack 250 × 10 mm i.d.) with aqueous acetonitrile (MeCN/H₂O, 90:10) to afford (3,24)-di(S)-MTPA esters of 7, named 7s (1.5 mg). The (3,24)-di(R)-MTPA esters of 7, named 7r (1.4 mg), was prepared in the same way.

7s: amorphous powder; ¹H NMR (CDCl₃, 400 MHz): δ_H 1.505 (1H, m, H-1α), 1.722 (1H, m, H-1β), 1.625 (1H, m, H-2α), 1.787 (1H, m, H-2β), 4.673 (1H, dd, J = 11.6, 4.0 Hz, H-3), 2.220 (1H, s, H-5), 5.694 (1H, d, J = 2.4 Hz, H-7), 0.815 (3H, s, H₃-18), 0.864 (3H, s, H₃-19), 1.333 (1H, m, H-20), 0.890 (3H, d, J = 6.4 Hz, H₃-21), 1.853 (1H, m, H-22a), 1.052 (1H, m, H-22b), 1.861 (1H, m, H-23a), 1.444 (1H, m, H-23b), 4.951 (1H, dd, J = 9.6, 2.4 Hz, H-24), 1.151 (3H, s, H₃-26), 1.183 (3H, s, H₃-27), 1.228 (3H, s, H₃-28), 1.137 (3H, s, H₃-29), 1.029 (3H, s, H₃-30).

7r: amorphous powder; ¹H NMR (CDCl₃, 400 MHz): δ_H 1.523 (1H, m, H-1α), 1.747 (1H, m, H-1β), 1.747 (1H, m, H-2α), 1.859 (1H, m, H-2β), 4.699 (1H, dd, J = 11.2, 4.0 Hz, H-3), 2.214 (1H, s, H-5), 5.692 (1H, d, J = 2.8 Hz, H-7), 0.791 (3H, s, H₃-18), 0.893 (3H, s, H₃-19), 1.272 (1H, m, H-20), 0.845 (3H, d, J = 6.4 Hz, H₃-21), 1.743 (1H, m, H-22a), 0.945 (1H, m, H-22b), 1.716 (1H, m, H-23a), 1.397 (1H, m, H-23b), 4.948 (1H, dd, J = 10.0, 2.4 Hz, H-24), 1.162 (3H, s, H₃-26), 1.224 (3H, s, H₃-27), 1.148 (3H, overlapped, H₃-28), 1.145 (3H, overlapped, H₃-29), 1.028 (3H, s, H₃-30).

3.6. X-Ray Crystal Data for Xylocarpols A–B (1–2)

Xylocarpol A (1): orthorhombic, C₃₁H₅₄O₆ (C₃₀H₅₀O₅·CH₃OH), space group P2₁, a = 35.3234(5) Å, b = 11.41820(10) Å, c = 7.38890(10) Å, α = 90°, β = 90°, γ = 90°, V = 2980.16(6) ų, Z = 4, D_calcd = 1.165 g cm⁻³, μ = 0.624 mm⁻¹. Crystal size: 0.14 × 0.13 × 0.12 mm³, 33,526 measured reflections, 5916 (R_int = 0.0475) independent reflections, 351 parameters, 0 restraints, F (000) = 1152.0, R₁ = 0.0429, wR₂ = 0.1120 (all data), R₁ = 0.0426, wR₂ = 0.1118 (I > 2σ(I)), and goodness-of-fit (F²) = 1.030. The absolute structural parameter was 0.01 (6), the Flack x parameter was 0.01(6), and the Hooft y was 0.01(5).

Xylocarpol B (2): orthorhombic, C₃₀H₄₈O₃, space group P2₁, a = 6.61860 (11) Å, b = 18.2616 (3) Å, c = 21.8783 (3) Å, α = 90°, β = 90°, γ = 90°, V = 2644.36 (7) ų, Z = 4, D_calcd = 1.147 g cm⁻³, μ = 0.551 mm⁻¹, crystal size: 0.14 × 0.12 × 0.11 mm³, 13,817 measured reflections, 5179 (R_int = 0.0367) independent reflections, 307 parameters, 0 restraints, F(000) = 1008.0, R₁ = 0.0467, wR₂ = 0.1178 (all data), R₁ = 0.0443, wR₂ = 0.1147 (I > 2σ(I)), and goodness-of-fit (F²) = 1.025. The absolute structural parameter was 0.08 (13), the Flack x parameter was 0.09(14), and the Hooft y was 0.02(13).

CCDC-1872275 (1) and 1872277 (2) contained the supplementary crystallographic data for this paper (excluding structure factors). These data were provided free of charge by The Cambridge Crystallographic Data Centre.

3.7. FXR Activation Bioassay

By cloning genes encoding FXR into pCI-neomammalian expression vector, the human FXR expression plasmid was constructed. By cloning a genomic DNA fragment upstream of the transcription start site into the luciferase vector pGL4.14 (luc2/Hygro) based on the previous method [29], bile salt export pump (BSEP) promoter reporter was constructed. Human hepatoma HepG2 cells were transiently transfected with the expression plasmid of FXR (100.0 ng), the reporter vector of BSEP promoter luciferase (100.0 ng), and the null-Renilla luciferase plasmid (10.0 ng) as an internal control. After the incubation for 24 h, cells were treated with vehicle DMSO (0.1%), compounds 1, and 4–9, respectively, at different concentrations (10.0, 100.0 nM, 1.0, or 10.0 µM) for 24 h. Then, cells were harvested for the determination of luciferase activity. Chenodeoxycholic acid (CDCA) was used as the positive control at the final concentration of 80.0 μM.

3.8. PXR Activation Bioassay

As described previously [30], the expression vector of human PXR and the human PXR XREM-driven luciferase reporter plasmid (CYP3A4XREM-luciferase) were constructed. By using the lipofectamine 3000 (Invitrogen, Carlsbad, CA., USA), human hepatoma HepG2 cells were transfected with expression and reporter plasmids, along with pGL4.74 (Promega, Beijing, China) as an internal standard. Then, cells were treated with vehicle DMSO (0.1%), compounds 1, and 4–10, respectively, at different concentrations (10.0, 100.0 nM, 1.0, or 10.0 µM) for 24 h. By using the Dual-luciferase^® Reporter Assay System (Promega, Beijing, China), the luciferase activities of the above compounds were recorded. By dividing the Firefly luciferase signal by the Renilla luciferase signal, the co-transfected plasmid results were normalized. Rifampicin, the well-known human PXR agonist, was used as the positive control at the final concentration of 10.0 μM.

4. Conclusions

In summary, 10 new triterpenoid compounds, including nine tirucallanes (1–9) and one apotirucallane (10), were obtained from mangrove plants X. granatum, X. moluccensis, and E. agallocha. The absolute configurations of 1, 2, 4, and 6–8 were unequivocally determined by single-crystal X-ray diffraction analysis (Cu Kα), modified Mosher’s method, and the comparison of experimental ECD spectra. Compounds 5, 6, 7, and 9 displayed potent activation effects on FXR at the concentration of 10.0 μM; whereas 10 exhibited very significant agonistic effects on PXR at nanomolar concentration. This work demonstrated that mangrove tirucallane- and apotirucallane-type triterpenoid compounds are valuable natural products for the discovery of leading compounds with potent activation effects on human FXR and PXR.