Twenty-Nine New Limonoids with Skeletal Diversity from the Mangrove Plant, Xylocarpus moluccensis

Abstract

Twenty-nine new limonoids—named xylomolins A₁–A₇, B₁–B₂, C₁–C₂, D–F, G₁–G₅, H–I, J₁–J₂, K₁–K₂, L₁–L₂, and M–N, were isolated from the seeds of the mangrove plant, Xylocarpus moluccensis. Compounds 1–13 are mexicanolides with one double bond or two conjugated double bonds, while 14 belongs to a small group of mexicanolides with an oxygen bridge between C1 and C8. Compounds 15–19 are khayanolides containing a Δ^8,14 double bond, whereas 20 and 21 are rare khayanolides containing a Δ^14,15 double bond and Δ^8,9, Δ^14,15 conjugated double bonds, respectively. Compounds 22 and 23 are unusual limonoids possessing a (Z)-bicyclo[5.2.1]dec-3-en-8-one motif, while 24 and 25 are 30-ketophragmalins with Δ^8,9, Δ^14,15 conjugated double bonds. Compounds 26 and 27 are phragmalin 8,9,30-ortho esters, whereas 28 and 29 are azadirone and andirobin derivatives, respectively. The structures of these compounds, including absolute configurations of 15–19, 21–23, and 26, were established by HRESIMS, extensive 1D and 2D NMR investigations, and the comparison of experimental electronic circular dichroism (ECD) spectra. The absolute configuration of 1 was unequivocally established by single-crystal X-ray diffraction analysis, obtained with Cu Kα radiation. The diverse cyclization patterns of 1–29 reveal the strong flexibility of skeletal plasticity in the limonoid biosynthesis of X. moluccensis. Compound 23 exhibited weak antitumor activity against human triple-negative breast MD-MBA-231 cancer cells with an IC₅₀ value of 37.7 μM. Anti-HIV activities of 1, 3, 8, 10, 11, 14, 20, 23–25, and 27 were tested in vitro. However, no compounds showed potent inhibitory activity.

1. Introduction

Limonoids are highly oxidized tetranortriterpenoids from a biosynthetic precursor with a 4,4,8-trimethyl-17-furanylsteroid skeleton. This group of natural products has attracted considerable attention because of its abundance, fascinating structural diversity, and various biological activities [1,2,3,4]. Xylocarpus is a well-known genus of mangrove plants which has been found to produce various types of limonoids with a broad range of bioactivities, such as insect antifeedant, antitumor, neuroprotective, gastroprotective, and antidepressant-like activities [5,6,7,8,9,10,11]. X. moluccensis, a true mangrove tree, is mainly distributed in Bangladesh, India, Indochina, Malesia, and tropical Australia. Previous chemical investigations of X. moluccensis resulted in the isolation of more than 100 limonoids with diverse carbon skeletons, such as mexicanolide, phragmalin, gedunin, andirobin, and khayanolide compounds [12,13,14,15,16,17,18]. Limonoids with diverse skeletons from X. moluccensis revealed structural plasticity in the limonoid biosynthesis of this mangrove species. This inference drove us to obtain and identify more novel and bioactive limonoids from this mangrove plant.

Further investigation of the seeds of X. moluccensis afforded fourteen mexicanolides (1–14), seven khayanolides (15–21), two unusual limonoids with a (Z)-bicyclo[5.2.1]dec-3-en-8-one substructure (22, 23), two 30-ketophragmalins (24, 25), two phragmalin 8,9,30-ortho esters (26, 27), an azadirone derivative (28), and an andirobin derivative (29) (Figure 1). Herein, we report the isolation, structural identification, antitumor, and anti-HIV activities of these new limonoids.

2. Results and Discussion

In this paper, 16 compounds (viz. 1, 3–6, 8, 9, 12–14, 20, 21, 24, 26, 27, and 29) were obtained from seeds of the Thai X. moluccensis, whereas 13 compounds (viz. 2, 7, 10, 11, 15–19, 22, 23, 25, and 28) were isolated from those of the Indian X. moluccensis.

Compound 1 was obtained as a colorless crystal. The molecular formula of 1 was established from the positive HRESIMS ion peak at m/z 615.2785 (calcd. for [M + H]⁺, 615.2800) to be C₃₃H₄₂O₁₁, implying thirteen degrees of unsaturation. According to the ¹H and ¹³C NMR spectroscopic data (

Table 1. ¹H NMR spectroscopic data (400 MHz, in CDCl₃) of compounds 1–7 (δ in ppm, J in Hz).

Position	1	2	3	4	5	6	7
3	4.97 s	4.93 s	5.04 s	5.12 s	5.06 s	5.19 s	5.52 s
5	3.35 s	3.13 s	3.28 s	3.47 s	3.45 s	3.09 s	3.10 br d (10.8)
6α	5.47 s	4.52 br s	4.54 s	5.44 s	5.47 s	4.52 s	2.32 dd (16.5, 1.6)
6β							2.42 dd (16.5, 10.7)
9	2.10 br s	2.14 br s	2.46 br s	2.49 br d (6.0)	2.42 br s	2.17 br s	2.08 m
11α	1.80 m	1.84 m	1.85 m	1.84 m	1.84 m	1.82 m	1.74 m
11β	1.92m	1.84 m	1.91 m	1.99 m	1.94 m	1.82 1	1.81 m
12β	1.81 m	1.72 m	1.74 m	1.86 m	1.85 m	1.71 m	1.75 m
12α	1.18 m	1.24 m	1.23 m	1.13 m	1.22 m	1.17 m	1.13 m
15α	3.46 dt (20.8, 2.8)	3.47 dt (20.8, 3.2)	3.64 dd (20.8, 3.2)	5.23 d (2.4)	3.65 dd (20.8, 3.2)	3.51 dt (20.8, 3.2)	3.52 dt (20.8, 2.8)
15β	3.79 d (20.8)	3.77 d (20.8)	3.85 br d (20.8)		3.81 br d (20.8)	3.88 br d (20.8)	3.88 d (20.8)
17	5.58 s	5.53 s	5.50 s	5.50 s	5.57 s	5.56 s	5.73 s
18	1.05 s	1.03 s	1.09 s	1.06 s	1.12 s	1.05 s	1.09 s
19	1.27 s	1.52 s	1.51 s	1.26 s	1.26 s	1.49 s	1.23 s
21	7.52 br d (0.8)	7.47 br d (0.8)	7.50 br s	7.59 t (0.8)	7.57 br s	7.47 br d (0.8)	7.54 br s
22	6.45 br d (1.2)	6.41 br d (0.8)	6.42 br d (1.2)	6.50 br d (1.2)	6.48 br d (1.2)	6.41 br t (0.8)	6.47 br d (1.2)
23	7.44 t (1.6)	7.43 t (1.6)	7.44 t (1.6)	7.45 t (1.6)	7.44 t (1.6)	7.43 t (1.6)	7.42 br d (1.6)
28	0.84 s	0.78 s	0.78 s	0.82 s	0.82 s	0.79 s	0.71 s
29	1.04 s	1.05 s	1.06 s	1.09 s	1.05 s	1.21 s	0.97 s
30β	3.23 d (14.4)	3.21 d (14.8)	4.69 s	5.00 s	4.69 s	3.72 d (14.4)	3.47 d (14.8)
30α	1.79 1	1.78 m				2.06 br d (14.4)	2.07 1
7-OMe-31	3.73 s	3.83 s	3.83 br s	3.75 s	3.76 s	3.84 s	3.70 s
3-OAcyl
33	2.64 m	2.45 m	2.38 m	2.27 m	2.17 s	2.20 s	2.22 s
		2.43 m
34	1.22 d (7.2)	1.20 t (7.6)	1.50 m	1.49 m	6-Acyl	2-Acyl	2-Acyl
			1.80 m	1.80 m
35	1.24 d (7.2)		0.99 t (7.2)	0.95 t (7.2)	2.18 s	2.13 s	2.12 s
36	6-Acyl		1.19 d (7.2)	1.20 d (7.2)
37	2.19 s			6-Acyl
38				2.18 s
2-OH	4.12 br s	4.16 s	4.62 s	4.22 s	4.57 br s
6-OH		2.80 br s	2.85 s			2.80 br s
15-OH				3.66 br s
30-OH			2.77 br s	2.58 br s	2.67 br s

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

and

Table 2. ¹³C NMR spectroscopic data (100 MHz, in CDCl₃) of compounds 1–7 (δ in ppm).

Position	1	2	3	4	5	6	7
1	216.7 qC	217.2 qC	213.8 qC	212.7 qC	213.1 qC	209.4 qC	209.6 qC
2	78.0 qC	77.8 qC	79.4 qC	79.3 qC	79.5 qC	85.6 qC	85.8 qC
3	86.1 CH	86.7 CH	86.3 CH	85.2 CH	86.4 CH	83.4 CH	81.3 CH
4	39.7 qC	39.5 qC	39.9 qC	39.9 qC	39.8 qC	40.4 qC	40.1 qC
5	44.2 CH	45.1 CH	45.7 CH	44.7 CH	44.8 CH	44.7 CH	40.7 CH
6	72.7 CH	73.1 CH	73.0 CH	72.6 CH	72.7 CH	73.2 CH	33.3 CH2
7	171.1 qC	175.2 qC	175.1 qC	171.5 qC	171.2 qC	175.2 qC	174.1 qC
8	125.7 qC	126.6 qC	128.6 qC	135.4 qC	127.6 qC	126.2 qC	125.5 qC
9	52.9 CH	53.3 CH	47.4 CH	46.0 CH	46.9 CH	53.1 CH	52.3 CH
10	52.4 qC	52.4 qC	51.8 qC	52.5 qC	51.9 qC	53.4 qC	53.2 qC
11	18.7 CH2	18.9 CH2	18.4 CH2	17.9 CH2	18.2 CH2	18.9 CH2	18.7 CH2
12	29.4 CH2	29.6 CH2	29.1 CH2	27.8 CH2	28.8 CH2	29.5 CH2	29.1 CH2
13	38.3 qC	38.2 qC	38.5 qC	39.4 qC	38.6 qC	38.2 qC	38.3 qC
14	133.5 qC	132.6 qC	137.8 qC	140.1 qC	138.5 qC	133.2 qC	133.4 qC
15	33.4 CH2	33.6 CH2	32.8 CH2	65.4 CH	32.8 CH2	33.6 CH2	33.5 CH2
16	169.2 qC	169.3 qC	168.9 qC	173.6 qC	169.2 qC	169.3 qC	169.9 qC
17	80.8 CH	80.8 CH	80.7 CH	81.1 CH	80.6 CH	80.6 CH	80.3 CH
18	18.0 CH3	18.3 CH3	17.7 CH3	16.1 CH3	17.6 CH3	18.4 CH3	18.1 CH3
19	16.7 CH3	17.6 CH3	17.6 CH3	17.0 CH3	16.8 CH3	17.8 CH3	16.9 CH3
20	120.5 qC	120.6 qC	120.5 qC	119.8 qC	120.3 qC	120.6 qC	120.5 qC
21	141.5 CH	141.1 CH	141.2 CH	141.9 CH	141.7 CH	141.2 CH	141.8 CH
22	109.8 CH	109.7 CH	109.7 CH	109.8 CH	109.9 CH	109.7 CH	109.9 CH
23	143.1 CH	143.2 CH	143.2 CH	143.2 CH	143.1 CH	143.2 CH	142.9 CH
28	22.5 CH3	22.6 CH3	22.8 CH3	22.5 CH3	22.7 CH3	22.6 CH3	22.8 CH3
29	22.7 CH3	22.6 CH3	22.7 CH3	22.7 CH3	22.6 CH3	23.5 CH3	21.1 CH3
30	44.4 CH2	44.6 CH2	73.0 CH	73.6 CH	72.9 CH	40.1 CH2	40.3 CH2
31	53.2 CH3	53.2 CH3	53.3 CH3	53.3 CH3	53.3 CH3	53.3 CH3	52.2 CH3
	3-Acyl	3-Acyl	3-Acyl	3-Acyl	3-Acyl	3-Acyl	3-Acyl
32	175.9 qC	173.3 qC	175.5 qC	175.3 qC	169.7 qC	169.6 qC	169.6 qC
33	34.5 CH	28.0 CH2	41.2 CH	41.8 CH	21.2 CH3	21.2 CH3	21.3 CH3
					6-Acyl	2-Acyl	2-Acyl
34	19.9 CH3	9.4 CH3	26.1 CH2	26.5 CH2	169.7 qC	169.1 qC	169.0 qC
35	18.4 CH3		11.6 CH3	11.8 CH3	21.0 CH3	21.8 CH3	21.7 CH3
36			17.5 CH3	17.5 CH3
	6-Acyl			6-Acyl
37	169.7 qC			169.8 qC
38	21.0 CH3			21.0 CH3

), five elements of unsaturation were due to four ester groups, a keto carbonyl function, and three carbon-carbon double bonds; thus, the molecule was pentacyclic. The ¹H and ¹³C NMR spectroscopic data (Table 1 and Table 2) showed the presence of a β-substituted furan ring [δ_H 7.52 br d (J = 0.8 Hz H-21), 6.45 br d (J = 1.2 Hz H-22), 7.44 t (J = 1.6 Hz H-23)], four tertiary methyl groups (δ_H 1.27 s H₃-19, 1.04 s H₃-29, 1.05 s H₃-18, 0.84 s H₃-28), a methoxy (δ_H 3.73 s H₃-31), and a keto function (δ_C 216.7 qC), indicating a mexicanolide-type limonoid for 1.

The NMR spectroscopic data of 1 (Table 1 and Table 2) were similar to those of khayalenoid H [19], except for the replacement of the 3-O-acetyl group in khayalenoid H by an isobutyryloxy group [δ_H 2.64 m 1H, 1.22 d (J = 7.2 Hz 3H), 1.24 d (J = 7.2 Hz 3H); δ_C 175.9 qC, 34.5 CH, 19.9 CH₃, 18.4 CH₃] in 1. The presence of the isobutyryloxy group was corroborated by ¹H-¹H COSY correlations between H₃-34/H-33 and H₃-35/H-33. The significant HMBC correlation from H-3 (δ_H 4.97 s) to the carbonyl carbon (δ_C 175.9 qC) of the isobutyryloxy group placed it at C-3 (Figure 2a).

The relative configuration of 1 was established by diagnostic NOE interactions (Figure 2b). Those between H-17/H-11β, H-17/H-12β, H-17/H-5, and H-5/H₃-28 revealed their cofacial relationship, and were arbitrarily assigned as the β-oriented H-17 and H-5. NOE interactions between H-9/H-12α, H-9/H₃-19, H₃-18/H-12α, H-3/H₃-29, and 2-OH/H₃-29 assigned the α-orientation for H-9, H₃-18, H₃-19, H-3, and 2-OH.

In order to establish the absolute configuration of 1, single-crystal X-ray diffraction analysis with Cu Kα radiation (Flack parameter of −0.06 (10), Flack x of −0.14 (11), and Hooft y of −0.04 (3); suitable crystals of 1 were obtained from acetone/methanol (1:2) at room temperature) was employed, which unequivocally assigned the absolute configuration of 1 as 2R,3S,5S,6R,9S,10R,13R,17R. The computer-generated perspective drawing of the X-ray structure of 1 is shown in Figure 3. Therefore, the absolute configuration of 1 named xylomolin A₁ was assigned as shown (Figure 2a).

Compound 2 was obtained as an amorphous white powder. The molecular formula was determined to be C₃₀H₃₈O₁₀ by the negative HRESIMS ion peak at m/z 593.2143 (calcd. for [M + Cl]⁻, 593.2159). The NMR spectroscopic data of 2 resembled those of 1 (Table 1 and Table 2), except for the replacement of the 3-O-isobutyryl moiety and the 6-O-acetyl group in 1 by a 3-O-propionyl moiety (δ_H 2.45 m 1H, 2.43 m 1H, 1.20 t (J = 7.6 Hz 3H); δ_C 173.3 qC, 28.0 CH₂, 9.4 CH₃) and a 6-OH group in 2, respectively. The presence of a 3-O-propionyl moiety was confirmed by ¹H-¹H COSY correlations between H₃-34/H₂-33 and HMBC cross-peaks between H₃-34/C-33, H₃-34/C-32, H₂-33/C-32, and H-3/C-32. The relative configuration of 2 (except for that of C-6) was determined to be the same as that of 1 by NOE interactions between H-17/H-15β, H-15β/H-30β, H-17/H-12β, H-17/H-11β, H-11β/H-5, H-17/H-5, and H-5/H₃-28, and those between H-9/H₃-19, H₃-19/H₃-29, H₃-29/H-3, and H₃-18/H-15α. Thus, the structure of 2—named xylomolin A₂—was assigned as shown in Figure 1.

Compound 3 gave the molecular formula C₃₂H₄₂O₁₁ as established by the HRESIMS ion peak at m/z 625.2620 (calcd. for [M + Na]⁺, 625.2619). The NMR spectroscopic data of 3 (Table 1 and Table 2) were similar to those of moluccensin S [20], except for the presence of an additional 30-OH group, which was supported by the downshifted C-30 signal (δ_C 73.0 CH in 3, whereas δ_C 44.6 CH₂ in moluccensin S) and HMBC correlations from the proton of 30-OH to C-30, C-2, and C-8. NOE interactions between H-17/H-12β, H-17/H-11β, H-5/H-12β, H-5/H-17, H-5/H₃-28, and H-5/H-30 revealed the β-orientation for H-5, H-17, H-30, and H₃-28, and the corresponding 30α-OH. Similarly, those between H-3/2-OH, H₃-29/2-OH, H-9/H₃-19, H₃-29/H₃-19, and H₃-18/H-11α assigned the α-orientation for H-3, H-9, H₃-18, H₃-19, H₃-29, and 2-OH. Thus, the structure of 3—named xylomolin A₃—was determined to be 30α-hydroxy-moluccensin S.

Compound 4 provided the molecular formula of C₃₄H₄₄O₁₃ as determined from the positive HRESIMS ion peak at m/z 683.2675 (calcd. for [M + Na]⁺, 683.2674). The ¹H and ¹³C NMR spectroscopic data of 4 resembled those of 3 (Table 1 and Table 2), the difference being the presence of a 15-OH function (δ_H 3.66 br s) and a 6-acetoxy group (δ_H 2.18 s 3H; δ_C 169.8 qC, 21.0 CH₃) in 4. The existence of a 15-OH function was corroborated by the downshifted C-15 signal (δ_C 65.4 CH in 4, whereas δ_C 32.8 CH₂ in 3) and HMBC correlations from the proton of 15-OH to C-14, C-15, and C-16. The HMBC correlation from H-6 to the carbonyl carbon (C-37) of the acetoxy group placed it at C-6. The relative configuration of 4 (except that of C-15) was assigned as the same as that of 3 on the basis of NOE interactions. Those between H-17/H-15 and H-5/H-30 assigned the β-oriented H-15 and H-30, and the corresponding 15α-OH group. Consequently, the structure of 4—named xylomolin A₄—was identified as 15α-hydroxy-6-acetoxy-xylomolin A₃.

The molecular formula of 5 was determined to be C₃₁H₃₈O₁₂ by the positive HRESIMS ion peak at m/z 625.2252 (calcd. for [M + Na]⁺, 625.2255). The ¹H and ¹³C NMR spectroscopic data of 5 (Table 1 and Table 2) were closely related to those of khayalenoid H, the difference being the existence of a 30-OH function, which was supported by the downshifted C-30 signal (δ_C 72.9 CH in 5, whereas δ_C 44.4 CH₂ in khayalenoid H). HMBC correlations from the proton of 30-OH (δ_H 2.67 br s) to C-30 and C-8 demonstrated the above deduction. The NOE interaction between H-5/H-30 assigned the β-oriented H-30 and the corresponding 30α-OH. Thus, the structure of 5—named xylomolin A₅—was assigned as 30α-hydroxy-khayalenoid H.

Compound 6 was isolated as a white and amorphous powder. Its molecular formula was determined to be C₃₁H₃₈O₁₁ from the positive HRESIMS ion peak at m/z 609.2311 (calcd. for [M + Na]⁺, 609.2306). The ¹H and ¹³C NMR spectroscopic data of 6 (Table 1 and Table 2) closely resembled those of khayalenoid H [19], except for the replacement of the 2-OH function and the 6-O-acetyl group in khayalenoid H by a 2-O-acetyl (δ_H 2.13 s 3H; δ_C 169.1 qC, 21.8 CH₃) and a 6-OH group (δ_H 2.80 br s) in 6, respectively. HMBC correlations from the proton of the 6-OH group to C-5, C-6,and C-7 placed it at C-6. The presence of the 2-O-acetyl group in 6 was corroborated by the downshifted C-2 in 6 (δ_C 86.5 qC in 6, whereas δ_C 77.9 qC in khayalenoid H). HMBC correlations from H₂-30 and H-3 to C-2 confirmed the above deduction. The relative configuration of 6 was determined to be the same as that of khayalenoid H based on diagnostic NOE interactions between H-17/H-15β, H-17/H-12β, H-17/H-5, and H-5/H₃-28 and those between H-9/H₃-19, H₃-19/H₃-29, and H₃-18/H-15α. Therefore, the structure of 6—named xylomolin A₆—was determined to be 2-O-acetyl-6-O-deacetyl-khayalenoid H.

The molecular formula of 7 was determined to be C₃₁H₃₈O₁₀ by the positive HRESIMS ion peak at m/z 593.2358 (calcd. for [M + Na]⁺, 593.2357). The ¹H and ¹³C NMR spectroscopic data of 7 (Table 1 and Table 2) were closely related to those of 6, except for the absence of the 6-OH group, which was corroborated by the upshifted CH₂-6 signal (δ_H 2.32 dd (J = 16.5, 1.6 Hz), 2.42 dd (J = 16.5, 10.7 Hz), δ_C 33.3) in 7. ¹H-¹H COSY correlations between H₂-6/H-5 and HMBC cross-peaks from H₂-6 and C-5 and C-7 confirmed the above result. The analysis of diagnostic NOE interactions revealed that 7 possessed the same relative configuration as that of 6. Therefore, the structure of 7—named xylomolin A₇—was assigned as 6-dehydroxy-xylomolin A₆.

Compound 8 was isolated as a white and amorphous powder. It had a molecular formula of C₃₁H₃₆O₁₁ as deduced from the positive HRESIMS ion peak at m/z 607.2151 (calcd. for [M + Na]⁺, 607.2150). The ¹H and ¹³C NMR spectroscopic data of 8 (

Table 3. ¹H NMR spectroscopic data (400 MHz) of compounds 8–14 (δ in ppm, J in Hz).

Position	8 1	9 1	10 1	11 1	12 2	13 1	14 1
2		3.02 t (6.0)
3	5.51 s	4.26 d (6.0)	4.83 s	4.99 s	4.55 s	4.76 s	4.55 s
5	2.98 s	2.94 br s	3.12 s	3.20 dd (9.2, 3.2)	3.22 s	3.49 s	2.67 s
6	5.51 s	5.47 s	4.38 s	2.32 m	4.40 d (4.8)	5.55 s	4.20 br s
				2.35 m
9			2.29 dt (12.4, 2.8)	2.29 m	1.71 t (13.2)	2.81 m	2.30 3
11α	2.38 m	2.35 m	1.84 m	1.77 m	1.68 m	1.78 m	1.83 m
11β	2.38 3	2.35 3	1.38 qd (12.8, 4.4)	1.56 m	1.33 m	2.18 m	2.30 m
12β	1.46 m	1.45 m	2.00 m	1.93 m	1.85 d (14.0)	1.44 m	1.95 m
12α	1.65 m	1.61 m	1.24 td (13.6, 4.4)	1.29 m	1.19 m	2.05 m	1.41 m
15α	5.90 s	5.89 s	6.28 s	6.32 s	5.99 s	2.93 d (18.0)	6.08 s
15β						3.05 d (18.0)
17	5.10 s	5.09 s	5.12 s	5.16 s	5.25 s	5.61 s	4.84 s
18	1.01 s	1.00 s	1.06 s	1.06 s	1.16 s	1.05 s	1.24 s
19	1.30 s	1.17 s	1.54 s	1.28 s	1.29 s	1.28 s	1.43 s
21	7.50 br s	7.49 br s	7.52 br s	7.51 br s	7.85 br s	7.76 br s	7.48 br t (0.8)
22	6.45 br d (1.2)	6.45 br d (1.2)	6.50 br d (1.2)	6.49 br d (1.2)	6.60 br d (1.2)	6.48 br s	6.42 br d (1.2)
23	7.46 t (1.6)	7.45 t (1.6)	7.45 t (1.6)	7.44 t (1.6)	7.70 t (1.6)	7.45 br s	7.42 t (1.6)
28	1.20 s	1.17 s	0.81 s	0.74 s	0.69 s	0.92 s	0.84 s
29	1.01 s	1.13 s	1.09 s	0.82 s	1.04 s	1.07 s	1.59 s
30β	3.55 d (17.6)	3.29 d (17.6)	6.29 d (4.4)	6.31 d (4.4)	3.38 d (15.6)	5.60 d (2.0)	5.49 s
30α	2.42 br d (17.6)	2.52 br d (17.6)			2.31 d (15.6)
31	3.74 s	3.75 s	3.84 s	3.69 s	3.65 s	3.73 s	3.88 s
	3-Acyl	6-Acyl	3-Acyl	3-Acyl	3-Acyl	3-Acyl	3-Acyl
33	2.21 s	2.07 s	2.22 s	2.57 m	2.54 m	2.11 s	2.02 s
34	6-Acyl			1.61 m	1.60 m	6-Acyl	30-Acyl
				1.81 m	1.54 m
35	2.11 s			1.04 t (7.2)	0.93 t (7.2)	2.19 s	2.59 m
36				1.25 d (6.8)	1.12 d (6.8)		1.13 d (6.8)
37							1.17 d (6.8)
1-OH							4.55 3
2-OH			4.07 br s		5.08 s	4.17 br s	4.11 br s
6-OH			2.97 br s		5.58 d (4.8)		2.93 s
8-OH					5.05 s

¹ Recorded in CDCl₃; ² Recorded in DMSO-d₆; ³ Overlapped signals assigned by ¹H-¹H COSY, HSQC, and HMBC spectra without designating multiplicity.

and

Table 4. ¹³C NMR spectroscopic data (100 MHz) of compounds 8–14 (δ in ppm).

Position	8 1	9 1	10 1	11 1	12 2	13 1	14 1
1	211.1 qC	212.5 qC	212.0 qC	212.7 qC	215.9 qC	213.9 qC	108.4 qC
2	76.9 qC	50.5 CH	77.5 qC	77.4 qC	76.2 qC	76.9 qC	81.1 qC
3	81.6 CH	76.7 CH	87.2 CH	85.5 CH	86.5 CH	85.8 CH	85.6 CH
4	39.6 qC	39.7 qC	39.9 qC	39.7 qC	39.5 qC	39.4 qC	38.8 qC
5	55.0 CH	55.4 CH	44.8 CH	40.8 CH	44.6 CH	44.8 CH	44.6 CH
6	69.8 CH	70.2 CH	72.0 CH	32.7 CH2	71.2 CH	72.4 CH	71.7 CH
7	170.6 qC	170.9 qC	175.5 qC	173.5 qC	175.7 qC	171.0 qC	175.9 qC
8	126.1 qC	127.1 qC	134.7 qC	134.1 qC	71.3 qC	138.8 qC	80.0 qC
9	148.4 qC	150.3 qC	55.3 CH	53.8 CH	61.6 CH	53.3 CH	51.5 CH
10	51.9 qC	51.2 qC	53.2 qC	52.6 qC	48.4 qC	49.8 qC	43.1 qC
11	22.3 CH2	22.2 CH2	22.5 CH2	21.5 CH2	20.7 CH2	20.3 CH2	15.6 CH2
12	29.7 CH2	29.7 CH2	33.3 CH2	32.4 CH2	32.5 CH2	28.3 CH2	25.2 CH2
13	36.8 qC	36.8 qC	37.6 qC	37.5 qC	37.8 qC	41.0 qC	38.8 qC
14	156.4 qC	157.4 qC	160.7 qC	160.0 qC	168.3 qC	72.9 qC	158.3 qC
15	111.6 CH	110.6 CH	113.4 CH	113.2 CH	114.6 CH2	38.9 CH2	118.4 CH
16	165.2 qC	165.7 qC	164.8 qC	164.6 qC	164.3 qC	168.4 qC	163.1 qC
17	80.6 CH	80.7 CH	79.7 CH	79.7 CH	78.5 CH	77.2 CH	81.4 CH
18	16.2 CH3	16.1 CH3	22.5 CH3	21.9 CH3	22.6 CH3	15.7 CH3	19.5 CH3
19	18.2 CH3	18.3 CH3	16.2 CH3	15.6 CH3	18.5 CH3	15.7 CH3	22.1 CH3
20	119.8 qC	119.9 qC	120.0 qC	120.1 qC	120.0 qC	120.1 qC	119.9 qC
21	141.2 CH	141.2 CH	141.4 CH	141.5 CH	142.3 CH	141.7 CH	141.3 CH
22	109.9 CH	110.0 CH	110.2 CH	110.2 CH	110.6 CH	109.8 CH	109.9 CH
23	143.2 CH	143.1 CH	143.3 CH	143.2 CH	143.2 CH	143.1 CH	143.0 CH
28	26.3 CH3	25.6 CH3	21.4 CH3	21.5 CH3	21.7 CH3	22.0 CH3	24.0 CH3
29	28.0 CH3	28.3 CH3	23.0 CH3	20.8 CH3	24.8 CH3	22.1 CH3	24.5 CH3
30	39.2 CH2	28.8 CH2	133.5 CH	133.0 CH	45.9 CH2	130.2 CH	74.9 CH
31	53.0 CH3	52.8 CH3	53.4 CH3	52.1 CH3	52.0 CH3	53.4 CH3	53.4 CH3
	3-Acyl	6-Acyl	3-Acyl	3-Acyl	3-Acyl	3-Acyl	3-Acyl
32	170.2 qC	169.2 qC	169.9 qC	176.0 qC	174.7 qC	170.7 qC	171.5 qC
33	20.9 CH3	20.5 CH3	20.7 CH3	41.4 CH	40.4 CH	20.4 CH	20.7 CH3
	6-Acyl					6-Acyl	30-Acyl
34	169.1 qC			26.9 CH2	26.4 CH	169.6 qC	176.0 qC
35	20.5 CH3			11.9 CH3	11.2 CH3	21.0 CH3	34.2 CH
36				17.0 CH3	16.5 CH3		19.0 CH3
37							19.1 CH3

¹ Recorded in CDCl₃; ² Recorded in DMSO-d₆.

) were similar to those of heytrijunolide E [21] with Δ^8,9 and Δ^14,15 conjugated double bonds, except for the absence of the 15-OH group and the presence of an acetoxy group at C-3 in 8. The absence of the 15-OH group was confirmed by the upshifted C-15 signal (δ_C 111.6 qC in 8, whereas δ_C 134.7 qC in heytrijunolide E) and HMBC correlations between H-15/C-14, H-15/C-16, H-15/C-8, and H-15/C-13. The HMBC correlation from H-3 (δ_H 5.51 s) to the carbonyl carbon (δ_C 170.2 qC) of the acetoxy group placed it at C-3. The relative configuration of 8 was established to be the same as that of heytrijunolide E based on diagnostic NOE interactions between H₃-28/H-5, H-5/H-11β, H-12β/H-17, H₃-29/H-3, H-12α/H₃-18, and H-11α/H₃-19. Therefore, the structure of 8—named xylomolin B₁—was assigned as 15-dehydroxy-3β-acetoxy-heytrijunolide E.

Compound 9 provided the molecular formula C₂₉H₃₄O₉ as established by the positive HRESIMS ion peak at m/z 527.2273 (calcd. for [M + H]⁺, 527.2276). The ¹H and ¹³C NMR spectroscopic data of 9 (Table 3 and Table 4) were similar to those of heytrijunolide E [21], the difference being the absence of the 2-OH and 15-OH groups in 9, which was corroborated by the upshifted C-15 (δ_C 110.6 CH in 9, whereas δ_C 134.7 qC in heytrijunolide E) and C-2 (δ_C 50.5 CH in 9, whereas δ_C 78.2 qC in heytrijunolide E) signals. A proton (δ_H 3.02 t (J = 6.0 Hz)) exhibiting ¹H-¹H COSY correlations to H-3 and H-30 and HMBC cross-peaks to C-1, C-3, C-4, and C-30 was assigned as H-2. The existence of H-15 (δ_H 5.89 s) was further confirmed by its HMBC correlations to C-8, C-13, C-14, and C-16. The relative configuration of 9 was determined to be the same as that of heytrijunolide E based on diagnostic NOE interactions between H₃-28/H-5, H-5/H-11β, H-12β/H-17, H₃-29/H-3, H-12α/H₃-18, and H-11α/H₃-19. Thus, the structure of 9—named xylomolin B₂—was assigned as 2,15-dedihydroxy-heytrijunolide E.

Compound 10 had a molecular formula of C₂₉H₃₄O₁₀ as determined from the positive HRESIMS ion peak at m/z 543.2238 (calcd. for [M + H]⁺, 543.2230). The NMR data of 10 (Table 3 and Table 4) closely resembled those of moluccensin U [20], except for the replacement of the 3-O-(2-methyl)butyryl group in moluccensin U by an acetoxy group (δ_H 2.22 s 3H; δ_C 169.9 qC, 20.7 CH₃) in 10. The significant HMBC cross-peak from H-3 (δ_H 4.83 s) to the carbonyl carbon of the above acetoxy group placed it at C-3. NOE interactions between H-11β/H-17, H-12β/H-17, H-11β/H-5, H-5/H₃-28, H-9/H₃-18, H-9/H₃-19, and H₃-29/H-3 revealed the same relative configuration of 10 as that of moluccensin U. Therefore, the structure of 10—named xylomolin C₁—was assigned as 3-O-acetyl-3-de(2-methyl) butyryloxy-moluccensin U.

Compound 11 afforded the molecular formula C₃₂H₄₀O₉ as established by the positive HRESIMS ion peak at m/z 591.2570 (calcd. for [M + Na]⁺, 591.2565). The ¹H and ¹³C NMR spectroscopic data of 11 (Table 3 and Table 4) closely resembled those of swietmanin G [22], the difference being the replacement of the 3-isobutyryloxy group in swietmanin G by a 2-methylbutyryloxy group (δ_H 2.57 m 1H, 1.61 m 1H, 1.81 m 1H, 1.04 t (J = 7.2 Hz 3H), 1.25 d (J = 6.8 Hz 3H); δ_C 176.0 qC, 41.4 CH, 26.9 CH₂, 11.9 CH₃, 17.0 CH₃) in 11. The deduction was confirmed by ¹H-¹H COSY correlations between H₃-36/H-33, H₃-35/H₂-34, and H₂-34/H-33, and HMBC correlations between H-3/C-32, H₃-35/C-34, H₃-35/C-33, H₃-36/C-33, H₃-36/C-32, and H₃-36/C-34. NOE interactions between H-12β/H-17, H-17/H-11β, H-5/H-11β, H-5/H₃-28, H-9/H₃-18, H-9/H₃-19, and H₃-29/H-3 assigned the α-orientation for H-9, H₃-18, H₃-19, and H-3. Thus, the structure of 11—named xylomolin C₂—was assigned as 3-O-(2-methyl)butyryl-3-deisobutyryloxy-swietmanin G.

Compound 12 had the molecular formula C₃₂H₄₂O₁₁ as determined from the positive HRESIMS ion peak at m/z 625.2612 (calcd. for [M + Na]⁺, 625.2619). The ¹H and ¹³C NMR spectroscopic data of 12 (Table 3 and Table 4) were similar to those of moluccensin U [20], except for the absence of the Δ^8,30 double bond and the presence of an 8-OH group. This finding was verified by the upshifted C-8 (δ_C 71.3 qC) and C-30 (δ_C 45.9 CH₂) signals as compared with those (δ_C 134.5 qC and 133.6 CH) of moluccensin U, respectively. The presence of the 8-OH group was confirmed by HMBC correlations from its proton to C-8, C-9, and C-30. NOE interactions between 8-OH/H-9 and 8-OH/H₃-18 assigned the α-orientation for the 8-OH group. The relative configuration of 12 (except that of C-8) was established to be identical to that of moluccensin U based on diagnostic NOE interactions between H-12β/H-17, H-5/H₃-28, H-9/H₃-19, and H₃-29/H-3. Thus, the structure of 12—named xylomolin D—was assigned as 8,30-dihydrogen-8α-hydroxy-moluccensin U.

Compound 13 was obtained as an amorphous white power. The molecular formula was determined to be C₃₁H₃₈O₁₂ by the positive HRESIMS ion peak at m/z 625.2250 (calcd. for [M + Na]⁺, 625.2255). The ¹H and ¹³C NMR spectroscopic data of 13 (Table 3 and Table 4) were similar to those of 8, except for the replacement of Δ^8,9 and Δ^14,15 conjugated double bonds in 8 by a Δ^8,30 double bond (δ_C 138.8 qC C-8, 130.2 CH C-30) in 13, and the presence of an additional 14-OH group. HMBC correlations between H₂-15/C-14, H₂-15/C-16, H₂-15/C-13, H-30/C-14, H-30/C-9, and H-9/C-8 demonstrated the above deduction. In order to establish the relative configuration of the 14-OH group in 13, two possible 3D structures with a 14α-OH and 14β-OH groups, respectively, were simulated by using the ChemBio3D software (Figure 4). When the 14-OH group occupies α-orientation, the space distance between H-5/H-17 is around 2.4 Å (Figure 4a), implying the presence of a strong NOE interaction between these protons. On the contrary, when the 14-OH group occupies the β-orientation, the space distance between H-5/H-17 is around 5.8 Å (Figure 4b), indicating the absence of a NOE interaction between these protons. Quite evidently, the NOE interaction between H-5/H-17 could be utilized as an effective criterion to resolve the relative configuration of the 14-OH group. Thus, the orientation of the 14-OH group in 13 was assigned as α based on the strong NOE interaction between H-5/H-17. Furthermore, the relative configuration of the whole molecule of 13 (except that of C-14) was determined to be the same as that of 8 on the basis of NOE interactions between H₃-28/H-5, H-5/H-11β, H-12β/H-17, H₃-29/H-3, H-12α/H₃-18, and H-11α/H₃-19. Thus, the structure of 13—named xylomolin E—was assigned as depicted.

Compounds 2–13 are analogues of 1. From the point of view of biogenetic origins, these mexicanolides should possess the same absolute configurations of carbon skeletons as that of 1. The absolute sterostructures of 2–13 are shown as in Figure 1.

The molecular formula of 14 was determined to be C₃₃H₄₂O₁₃ by the positive HRESIMS ion peak at m/z 669.2521 (calcd. for [M + Na]⁺, 669.2518). The NMR spectroscopic data of 14 (Table 3 and Table 4) were similar to those of xylorumphiin H [23], being a mexicanolide containing a C1-O-C8 bridge, except for the presence of an additional Δ^14,15 double bond (δ_H 6.08 s 1H; δ_C 158.3 qC, 118.4 CH) and an additional 6-OH function (δ_H 2.93 s) in 14. The existence of the Δ^14,15 double bond was corroborated by HMBC correlations between H₃-18/C-14, H-15/C-8, and H-15/C-16. The downshifted C-6 signal (δ_C 71.7 CH in 14, whereas δ_C 32.3 CH₂ in xylorumphiin H), along with HMBC cross-peaks from H-6 to C-5 and C-7, supported the location of a hydroxy group at C-6. Similar NOE interactions of 14 as those of xylorumphiin H suggested that both mexicanolides possessed the same relative configuration. Thus, the structure of 14—named xylomolin F—was assigned as 6-hydroxy-14,15-dedihydrogen-xylorumphiin H.

The molecular formula of 15 was established by the positive HRESIMS ion peak at m/z 587.2494 (calcd. for [M + H]⁺, 587.2492) to be C₃₁H₃₈O₁₁, implying thirteen degrees of unsaturation. According to the NMR spectroscopic data of 15 (

Table 5. ¹H NMR spectroscopic data (400 MHz) of compounds 15–21 (δ in ppm, J in Hz).

Position	15 1	16 1	17 1	18 1	19 1	20 2	21 1
2							5.77 s
3	4.87 s	4.86 s	4.94 s	5.01 s	5.02 s	4.97 s
5	2.24 br s	2.22 s	2.13 3	2.24 dd (11.2, 2.4)	2.26 3	2.87 s	2.19 br s
6	4.36 br s	4.37 br d (1.6)	4.33 s	2.27 dd (16.0, 2.8)	2.28 dd (16.0, 2.8)	4.83 br d (3.6)	4.42 s
				2.43 dd (16.0, 11.2)	2.43 dd (16.0, 11.2)
9	2.52 m	2.51 m	2.51 m	2.45 m	2.46 m	2.49 dd (6.4, 2.8)
11α	1.81 m	1.80 m	1.84 m	1.77 m	1.79 m	2.20 3	2.25 m
11β	1.40 m	1.40 m	1.40 m	1.46 m	1.48 m	2.07 m	2.03 dd (18.8, 4.0)
12β	1.58 m	1.53 m	1.60 m	1.54 m	1.55 m	1.76 m	1.36 m
12α	1.43 m	1.43 m	1.47 m	1.43 m	1.44 m	1.27 m	1.51 overlapped
15α	3.71 3	3.73 dd (20.0,3.2)	3.43 dd (19.2, 1.6)	3.43 br d (18.4)	3.43 dd (19.6, 1.2)	5.72 br s	6.55 s
15β	4.07 dd (20.0, 2.8)	4.09 dd (20.0,3.2)	3.98 dd (19.2, 3.2)	3.94 ddd (19.6, 3.6, 2.0)	3.93 ddd (19.6, 3.6, 2.0)
17	5.13 s	5.06 s	5.18 s	5.17 s	5.14 s	5.66 s	5.09 s
18	1.05 s	1.04 s	1.04 s	1.01 s	1.01 s	1.38 s	1.00 s
19	1.34 s	1.34 s	1.29 s	1.01 s	1.01 s	1.45 s	1.33 s
21	7.45 br s	7.43 br s	7.46 s	7.47 br s	7.47 br s	7.64 br s	7.49 br s
22	6.41 br d (1.2)	6.41 br d (0.8)	6.43 br d (0.8)	6.43 br d (1.2)	6.43 br d (0.8)	6.58 br d (1.2)	6.45 br d (1.2)
23	7.43 t (1.6)	7.42 t (1.6)	7.43 t (1.6)	7.42 t (1.6)	7.42 t (1.6)	7.60 t (1.6)	7.44 t (1.6)
28	1.19 s	1.20 s	1.20 s	1.00 s	0.99 s	1.01 s	1.13 s
29pro-R	1.77 d (13.2)	1.80 d (12.8)	1.97 d (12.6)	1.99 d (12.8)	1.98 d (12.4)	2.21 d (11.2)	2.35 d (12.8)
29pro-S	2.43 d (13.2)	2.45 d (12.8)	2.31 d (12.6)	2.15 d (12.8)	2.15 d (12.4)	2.88 d (11.2)	2.70 d (12.4)
30			3.46 br s	3.44 br s	3.45 br s
31	3.86 s	3.85 s	3.87 s	3.67 s	3.67 s	3.80 s	3.86 s
	3-Acyl	3-Acyl	3-Acyl	3-Acyl	3-Acyl	3-Acyl	2-Acyl
33	2.15 s	2.57 m	2.13 s	2.66 m	2.47 m	1.94 s	2.42 m
	30-Acyl
34	3.50 m	1.20 d (7.2)		1.21 d (6.8)	1.73 m	1-Acyl	1.48 m
	3.69 m				1.52 m		1.73 m
35	1.27 t (6.8)	1.22 d (7.2)		1.19 d (7.2)	0.94 t (7.2)	2.55 m	0.97 t (7.6)
		30-Acyl
36		3.51 m			1.17 d (7.2)	1.11 d (7.2)	1.22 d (7.2)
		3.67 m
37		1.27 t (6.8)			5.08 s	1.10 d (7.2)
1-OH	2.91 br s	2.85 s					2.74 s
6-OH	2.92 d (3.2)	2.88 d (3.2)	2.94 br d (2.7)		5.58 d (4.8)	4.69 br d (3.6)	3.19 s
8-OH					5.05 s	5.00 s
30-OH						4.87 s	3.43 s

¹ Recorded in CDCl₃; ² Recorded in acetone-d₆; ³ Overlapped signals assigned by ¹H-¹H COSY, HSQC, and HMBC spectra without designating multiplicity.

and

Table 6. ¹³C NMR spectroscopic data (100 MHz) of compounds 15–21 (δ in ppm).

Position	15 1	16 1	17 1	18 1	19 2	20 1	21 1
1	84.5 qC	84.3 qC	85.2 qC	84.8 qC	84.8 qC	92.6 qC	85.5 qC
2	204.3 qC	204.1 qC	204.3 qC	204.1 qC	203.9 qC	209.1 qC	82.4 CH
3	84.8 CH	84.7 CH	85.0 CH	83.3 CH	83.0 CH	90.6 CH	207.8 qC
4	40.6 qC	40.6 qC	41.2 qC	40.6 qC	40.5 qC	45.1 qC	52.8 qC
5	45.4 CH	45.5 CH	46.0 CH	40.7 CH	40.5 CH	43.4 CH	51.4 CH
6	72.0 CH	72.1 CH	71.9 CH	34.2 CH2	34.2 CH2	71.9 CH	71.0 CH
7	175.3 qC	175.3 qC	175.4 qC	172.7 qC	172.7 qC	176.2 qC	175.2 qC
8	134.9 qC	135.0 qC	135.2 qC	134.5 qC	134.4 qC	76.8 qC	130.4 qC
9	48.3 CH	48.1 CH	49.4 CH	47.9 CH	48.0 CH	48.4 CH	156.6 qC
10	55.9 qC	56.0 qC	57.0 qC	56.4 qC	56.4 qC	48.0 qC	58.8 qC
11	19.5 CH2	19.5 CH2	20.2 CH2	19.4 CH2	19.4 CH2	17.8 CH2	20.2 CH2
12	31.9 CH2	32.0 CH2	32.2 CH2	31.9 CH2	31.9 CH2	29.5 CH2	30.0 CH2
13	40.7 qC	40.7 qC	40.4 qC	40.5 qC	40.6 qC	38.7 qC	37.8 qC
14	138.4 qC	138.5 qC	132.5 qC	133.0 qC	133.0 qC	160.0 qC	154.9 qC
15	33.1 CH2	33.0 CH2	35.4 CH2	35.4 CH2	35.3 CH2	120.2 CH	113.4 CH
16	169.6 qC	169.9 qC	169.8 qC	170.3 qC	170.3 qC	164.9 qC	165.7 qC
17	80.5 CH	80.5 CH	80.8 CH	80.7 CH	80.7 CH	81.2 CH	80.4 CH
18	17.7 CH3	17.4 CH3	17.5 CH3	16.8 CH3	16.8 CH3	21.4 CH3	16.2 CH3
19	17.0 CH3	17.1 CH3	16.7 CH3	14.9 CH3	14.9 CH3	22.2 CH3	15.7 CH3
20	120.6 qC	120.6 qC	120.6 qC	120.5 qC	120.5 qC	121.1 qC	120.3 qC
21	141.1 CH	141.1 CH	141.1 CH	141.2 CH	141.1 CH	142.7 CH	141.2 CH
22	110.0 CH	110.0 CH	110.1 CH	110.1 CH	110.1 CH	111.1 CH	110.1 CH
23	143.1 CH	143.1 CH	143.0 CH	142.9 CH	142.9 CH	144.0 CH	143.0 CH
28	19.8 CH3	19.7 CH3	19.5 CH3	19.2 CH3	19.2 CH3	15.7 CH3	16.3 CH3
29	44.7 CH2	44.9 CH2	45.4 CH2	44.6 CH2	44.5 CH2	41.5 CH2	39.3 CH2
30	92.2 qC	92.3 qC	63.0 CH	63.4CH	63.5 CH	83.6 qC	82.4 qC
31	53.2 CH3	53.1 CH3	53.3 CH3	51.8 CH3	51.7 CH3	53.0 CH3	53.3 CH3
	3-Acyl	3-Acyl	3-Acyl	3-Acyl	3-Acyl	3-Acyl	2-Acyl
32	169.9 qC	176.0 qC	170.1 qC	176.5 qC	176.0 qC	170.1 qC	176.0 qC
33	20.6 CH3	33.8 CH	20.7 CH3	33.9 CH	40.8 CH	21.3 CH3	41.3 CH
	30-Acyl					1-Acyl
34	63.3 CH2	19.0 CH3		18.9 CH3	26.7 CH2	176.0 qC	26.5 CH2
35	15.7 CH3	19.1 CH3		18.9CH3	11.5 CH3	35.1 CH	11.7 CH3
		30-Acyl
36		63.4 CH2		17.0 CH3	16.5 CH3	19.4 CH3	17.2 CH3
37		15.7 CH3				19.3 CH3

¹ Recorded in CDCl₃; ² Recorded in acetone-d₆.

), seven elements of unsaturation were due to three carbon-carbon double bonds, one carbonyl group, and three ester functionalities; thus, 15 should be hexacyclic. The NMR spectroscopic data of 15 resembled those of thaixylomolin L [18], being a khayanolide isolated from seeds of the Thai X. moluccensis, except for the presence of an additional 6-OH group in 15. Strong ³J HMBC correlations from H₂-29 to C-30 further confirmed a khayanolide for 15 instead of a phragmalin, which should exhibit weak ⁴J HMBC correlations between H₂-29/C-30. HMBC cross-peaks from an active proton (δ_H 2.91 d (J = 3.4 Hz)) to C-5 (δ_C 45.4, CH), C-6 (δ_C 72.0, CH), and C-7 (δ_C 175.3, qC) (Figure 5a) revealed the existence of a 6-OH group in 15.

The relative configuration of 15 was assigned by analysis of NOE interactions (Figure 5b). Those between H-17/H-12β, H-17/H-15β, H-17/H-11β, and H-11β/H-5 revealed their cofacial relationship and were assigned as β-oriented. In turn, NOE interactions between H₃-18/H-15α, H-9/H₃-19, H₃-19/1-OH, and H-34/H_pro-R-29 indicated the α-orientation for H-9, H₃-18, H₃-19, 1-OH, and 30-OEt. The NOE interaction between H-3/H_pro-R-29 established the 3α-H and the corresponding 3β-acetoxy function. Therefore, the relative configuration of 15 was determined. Comparison of the electronic circular dichroism (ECD) spectrum of 15 with that of thaixylomolin L [18] showed that 15 had the same 1R,3S,4R,5S,9R,10S,13R,17R,30S-absolute configuration as that of thaixylomolin L (Figure 6a). Thus, the structure of 15—named xylomolin G₁—was assigned as depicted.

Compound 16 had a molecular formula of C₃₃H₄₂O₁₁ as determined from the positive HRESIMS ion peak at m/z 615.2809 (calcd. for [M + H]⁺, 615.2805). The NMR spectroscopic data of 16 (Table 5 and Table 6) were closely related to those of 15, the difference being the replacement of 3-acetoxy group in 15 by an isobutyryloxy group (δ_H 2.57 m, 1.20 d (J = 7.2 Hz), 1.22 d (J = 7.2 Hz); δ_C 176.0 qC, 33.8 CH, 19.0 CH₃, 19.1 CH₃) in 16. The presence of the above isobutyryloxy group was further supported by ¹H-¹H COSY correlations between H₃-34/H-33 and H₃-35/H-33, and HMBC correlations between H-33/C-32, H₃-34/C-32, and H₃-35/C-32. The significant HMBC cross-peak from H-3 to the carbonyl carbon (C-32) of the isobutyryloxy group confirmed its location at C-3. NOE interactions between H-17/H-12β, H-17/H-15β, H-17/H-11β, H-11β/H-5, H-17/H-5, H-5/H₃-28, H-3/H_pro-_R-29, H-15α/H₃-18, H₃-18/H-9, H-9/H₃-19, and 1-OH/H-9 indicated the same relative configuration of 16 as that of 15. Comparison of the ECD spectrum of 16 with that of thaixylomolin L concluded that 16 had the same 1R,3S,4R,5S,9R,10S,13R,17R,30S-absolute configuration as that of thaixylomolin L (Figure 6a). Thus, the structure of 16—named xylomolin G₂—was assigned as 3-O-isobutyryl-3-deacetoxy-xylomolin G₁.

Compound 17 was isolated as an amorphous yellow solid. Its molecular formula was determined to be C₂₉H₃₄O₁₀ by the positive HRESIMS ion peak at m/z 543.2246 (calcd. for [M + H]⁺, 543.2230). The similarities between the NMR spectroscopic data of 17 (Table 5 and Table 6) and 15 revealed their close structural resemblance, except for the absence of the ethoxyl group at C-30, which was confirmed by the upshifted C-30 signal (δ_C 63.0 CH in 17, whereas δ_C 92.2 qC in 15) and HMBC cross-peaks between H-30/C-1, H-30/C-2, H-30/C-8, and H-30/C-10. The relative configuration of 17 was determined to be identical to that of 15 by analysis of NOE interactions. Comparison of ECD spectra of 17 and 15 concluded that both compounds had the same 1R,3S,4R,5S,9R,10S,13R,17R,30S-absolute configuration (Figure 6a). Thus, the structure of 17—named xylomolin G₃—was assigned as 30-deethoxyl-xylomolin G₁.

Compound 18 provided the molecular formula C₃₁H₃₈O₉ as established by the positive HRESIMS ion peak at m/z 555.2593 (calcd. for [M + H]⁺, 555.2594). The NMR spectroscopic data of 18 (Table 5 and Table 6) were similar to those of 15, the difference being the absence of the 30-ethoxyl group and the 6-OH function in 18. The upshifted C-30 (δ_C 63.4 CH in 18, whereas δ_C 92.2 qC in 15) and C-6 (δ_C 34.2 CH₂ in 18, whereas δ_C 72.1 CH in 15) signals and HMBC cross-peaks between H-30/C-1, H-30/C-2, H-30/C-8, H-30/C-10, H-6/C-5, and H-6/C-7 supported the above deduction. The relative and absolute configurations of 18 were determined to be the same as that of 15 by analysis of their NOE interactions and ECD spectra (Figure 6a). Thus, the structure of 18—named xylomolin G₄—was concluded to be 30-deethoxyl-6-dehydroxy-xylomolin G₁.

Compound 19 gave the molecular formula C₃₂H₄₀O₉ as determined from the positive HRESIMS ion peak at m/z 569.2753 (calcd. for [M + H]⁺, 569.2751). The NMR data of 19 (Table 5 and Table 6) were closely related to those of 18, except for the replacement of the 3-isobutyryloxy group in 18 by a 2-methylbutyryloxy group (δ_H 2.47 m 1H, 1.73 m 1H, 1.52 m 1H, 0.94 t (J = 7.2 Hz 3H), 1.19 d (J = 7.2 Hz 3H); δ_C 176.0 qC, 40.8 CH, 26.7 CH₂, 11.5 CH₃, 16.5 CH₃) in 19. The presence of the 2-methylbutyryloxy group was supported by the ¹H-¹H COSY correlations between H-33/H-34, H-33/H₃-36, and H-34/H₃-35 and HMBC cross-peaks between H-33/C-32, H₂-34/C-32, H₃-36/C-32, H₃-36/C-34, and H₃-35/C-33. The significant HMBC cross-peak from H-3 to the carbonyl carbon (C-32) of the above 2-methylbutyryloxy group assigned its location at C-3. The relative and absolute configurations of 19 were determined to be the same as those of 18 by analysis of their NOE interactions and ECD spectra (Figure 6a). Thus, the structure of 19—named xylomolin G₅—was assigned as 3-O-(2-methyl)butyryl-3-deisobutyryloxy-xylomolin G₄.

Compound 20 was isolated as an amorphous white powder. Its molecular formula was determined to be C₃₃H₄₀O₁₃ by the positive HRESIMS ion peak at m/z 667.2359 (calcd. for [M + Na]⁺, 667.2361). The NMR spectroscopic data of 20 (Table 5 and Table 6) resembled those of 15, except for the presence of an additional 8-OH group (δ_H 5.00 s) and the replacement of the Δ^8,14 double bond, 1-OH function, and 30-ethoxyl group in 15 by a Δ^14,15 double bond (δ_H 5.72 br s H-15; δ_C 160.0 qC C-14, 120.2 CH C-15), a 1-O-isobutyryl moiety (δ_H 2.55 m H-35, 1.11 d (J = 7.2 Hz H₃-36), 1.10 d (J = 7.2 Hz H₃-37); δ_C 176.0 qC C-34, 35.1 CH C-35, 19.4 CH₃ C-36, 19.3 CH₃ C-37), and a 30-OH group (δ_H 4.87 s) in 20, respectively (Table 5 and Table 6, recorded in CDCl₃). HMBC correlations between H-15/C-14, H-15/C-16, H-15/C-13, H-15/C-8, 8-OH/C-8, and 30-OH/C-30 confirmed the presence of a Δ^14,15 double bond and the existence of two hydroxy groups at C-8 and C-30, respectively. The presence of the isobutyryloxy group was supported by ¹H-¹H COSY cross-peaks between H-35/H₃-36 and H-35/H₃-37 and HMBC correlations between H-35/C-34, H₃-36/C-34, and H₃-37/C-34. Its location at C-1 was corroborated by the downshifted C-1 signal (δ_C 92.6 qC in 20, whereas δ_C 84.5 qC in 15). The relative configuration of 20 was established by NOE interactions, in which those between H-17/H-12β, H-12β/H-5, H-5/H₃-28 assigned the β-orientation for H-17, H-5, H₃-28, whereas those between H-11α/H₃-18, H₃-18/8-OH, 8-OH/H-9, H_pro-R-29/H-3, H-9/H₃-19, and H-3/30-OH concluded the α-orientation for H₃-18, 8-OH, H-9, H-3, H₃-19, and 30-OH. Thus, the structure of 20—named xylomolin H—was assigned as depicted.

Compound 21 afforded the molecular formula C₃₂H₃₈O₁₁ as deduced from the positive HRESIMS ion peak at m/z 621.2304 (calcd. for [M + Na]⁺, 621.2306). The NMR spectroscopic data of 21 (Table 5 and Table 6) were similar to those of thaixylomolin H [18], except for the presence of an additional 6-OH group (δ_H 3.19 s) and the replacement of the 2-acetoxy group by a 2-methylbutyryloxy moiety (δ_H 2.42 m 1H, 1.73 m 1H, 1.48 m 1H, 0.97 t (J = 7.6 Hz 3H), 1.22 d (J = 7.2 Hz 3H); δ_C 176.0 qC, 41.3 CH, 26.5 CH₂, 11.7 CH₃, 17.2 CH₃) in 21. The presence of the 6-OH group was supported by the downshifted C-6 signal (δ_C 71.0 CH in 21, whereas δ_C 31.6 CH₂ in thaixylomolin H), the ¹H-¹H COSY cross-peak between H-5/H-6, and HMBC correlations between H-6/C-5 and H-6/C-7. The existence of the 2-methylbutyryloxy group was corroborated by ¹H-¹H COSY cross-peaks between H-33/H₃-36, H-33/H-34, and H₂-34/H₃-35 and HMBC correlations between H-33/C-32, H-34/C-32, H₃-36/C-32, H-33/C-34, H₃-36/C-34, H₃-35/C-34, H-34/C-33, H₃-35/C-33, and H₃-36/C-33. The significant HMBC correlation from H-2 to the carbonyl carbon (C-32) of the 2-methylbutyryloxy group placed it at C-2. NOE interactions between H-17/H-12β, H-11α/H₃-19, H-11α/H₃-18, H-11β/H-5, H-5/H₃-28, H₃-19/1-OH, H_pro-R-29/H-2, and H-2/30-OH assigned the β-orientation for H-17, H₃-28, and H-5, and the α-orientation for H-2, H₃-18, H₃-19, 30-OH, and 1-OH. The ECD spectrum of 21 was identical to that of thaixylomolin H (Figure 6b), concluding that 21 had the same 1R,2R,4R,5R,10S,13R,17R,30R-absolute configuration as that of thaixylomolin H. Thus, the structure of 21—named xylomolin I—was identified as 6-hydroxy-2-O-(2-methyl)butyryl-2-deacetoxy-thaixylomolin H.

Compound 22 had the molecular formula C₂₉H₃₂O₁₀ as determined from the positive HRESIMS ion peak at m/z 541.2077 (calcd. for [M + H]⁺, 541.2074). The similarities between the NMR spectroscopic data of 22 (

Table 7. ¹H NMR spectroscopic data (400 MHz, in CDCl₃) of compounds 22–29 (δ in ppm, J in Hz).

Position	22	23	24	25	26	27	28	29
1							7.14 d (10.4)	3.52 m
2β							5.90 d (10.4)	2.92 dd (14.4,6.4)
2α								2.46 dd (14.4,3.6)
3	5.54 s	5.50 s	4.88 s	4.86 s	5.17 s	4.82 s
5	2.92 s	2.94 s	2.80 br s	2.82 s	2.40 s	2.37 d (13.6)	2.22 dd (12.4, 2.8)	2.82 d (10.4)
6a	4.26 br s	4.27 br s	4.58 s	4.58 s	4.51 s	2.40 br d (23.2)	1.93 m (α)	2.24 d (16.4)
6b						2.42 dd (23.2, 13.6)	1.99 m (β)	2.62 dd (16.4, 10.4)
7							5.30 t (2.8)
9	3.23 m	3.25 m					2.54 m	2.22 m
11α	1.89 m	1.88 m	2.63 m	2.62 m	1.94 m	1.98 t (14.4)	2.10 m	1.65 m
11β	1.75 m	1.74 m	2.37 m	2.37 dd (20.0, 3.6)	2.26 m	2.19 dd (14.4, 4.0)	1.83 m	2.30 m
12β	1.83 m	1.83 m	1.39 m	1.40 td (12.8, 4.8)	1.56 m	3.87 br d (13.6)	1.70 m	1.96 m
12α	1.25 m	1.24 m	1.72 m	1.72 dd (12.8, 4.0)	1.25 m		2.20 m	1.35 dd (17.2, 4.4)
15α	6.00 s	5.99 s	7.23 s	7.18 s	6.56 s	6.00 s	5.87 s	2.86 d (18.0)
15β								2.60 d (18.0)
17	5.33 s	5.34 s	5.01 s	5.01 s	5.61 s	5.80 s	2.53 overlapped	5.66 s
18	1.08 s	1.07 s	1.04 s	1.04 s	1.38 s	1.43 s	1.43 s	0.93 s
19	1.21 s	1.22 s	1.54 s	1.54 s	1.54 s	1.33 s	1.24 s	0.97 s
20							2.97 ddd (12.0, 9.2, 2.8)
21α	7.53 br s	7.53 br s	7.51 br s	7.51 s	7.45 br s	7.67 br s		4.85 br d (18.3)
21β								5.03 dd (18.3, 2.0)
22α	6.49 d (1.2)	6.49 d (1.2)	6.48 br d (1.2)	6.47 br d (1.2)	6.41 br s	6.61 br s	2.44 m	6.08 br s
22β							2.49 m
23α	7.47 t (1.6)	7.47 t (1.6)	7.46 t (1.6)	7.45 t (1.6)	7.43 br s	7.53 br s	4.23 ddd (10.4, 9.2, 6.8)
23β							4.46 td (8.8, 2.0)
28	1.21 s	1.22 s	1.00 s	1.00 s	0.87 s	0.82 s	1.10 s	1.03 s
29pro-R	2.49 d (19.2)	2.46 br d (18.8)	1.81 dd (10.8, 2.0)	1.81 dd (10.4, 2.0)	1.67 br d (11.0)	1.80 d (11.2)	1.09 s	1.20 s
29pro-S	2.56 d (19.2)	2.58 d (18.8)	2.71 d (10.8)	2.70 d (10.4)	2.36 d (11.0)	1.86 d (11.2)
30α	6.59 br d (1.6)	6.56 br d (1.6)			5.34 s	4.49 s	1.29 s	4.92 s (30a)
30β								5.22 s (30b)
31	3.81 s	3.81 s	3.81 s	3.82 s	3.80 s	3.76 s	7-Acyl	3.73 s
32	3-Acyl	3-Acyl	3-Acyl	3-Acyl			1.97 s
33	2.17 s	2.51 m	2.27 m	2.45 m	1.70 s	1.68 s
34		1.53 m	1.42 m		3-Acyl	3-Acyl
		1.74 m	1.58 m
35		0.96 t (7.2)	0.87 t (7.6)	1.11 d (6.8)	2.07 s
36		1.18 d (7.2)	1.07 d (6.8)	1.11 d (6.8)	2-Acyl	6.88 q (6.8)
37					2.17 s	1.73 br d (6.8)
38						1.84 s
1-OH			2.81 s	2.84 br s		3.45 s
2-OH			4.93 s	4.93 br s		3.57 s
6-OH	3.01 s	3.06 br s	3.12 br s	3.13 s

and

Table 8. ¹³C NMR spectroscopic data (100 MHz, in CDCl₃.) of compounds 22–29 (δ in ppm).

Position	22	23	24	25	26	27	28	29
1	215.1 qC	215.4 qC	85.8 qC	85.9 qC	84.4 qC	84.4 qC	156.7 CH	77.5 CH
2	193.6 qC	193.5 qC	80.7 qC	80.8 qC	84.0 qC	75.7 qC	126.0 CH	39.2 CH2
3	82.9 CH	82.4 CH	87.2 CH	87.2 CH	85.6 CH	86.6 CH	204.0 qC	212.4 qC
4	41.1 qC	41.2 qC	45.6 qC	45.6 qC	44.4 qC	43.7 qC	44.1 qC	48.1 qC
5	49.2 CH	49.2 CH	48.2 CH	48.1 CH	44.8 CH	40.5 CH	46.2 CH	42.9 CH
6	70.9 CH	70.9 CH	71.5 CH	71.5 CH	71.5 CH	33.8 CH2	23.5 CH2	32.6 CH2
7	175.1 qC	175.2 qC	175.0 qC	174.9 qC	174.1 qC	174.1 qC	73.9 CH	173.7 qC
8	149.7 qC	149.6 qC	121.0 qC	121.1 qC	84.0 qC	83.7 qC	44.8 qC	144.8 qC
9	43.7 CH	44.1 CH	169.8 qC	169.7 qC	86.8 qC	87.1 qC	37.8 CH	49.6 CH
10	52.7 qC	52.6 qC	48.8 qC	48.8 qC	49.0 qC	47.4 qC	39.9 qC	44.0 qC
11	21.0 CH2	21.2 CH2	25.6 CH2	25.6 CH2	26.3 CH2	34.3 CH2	15.8 CH2	23.7 CH2
12	30.8 CH2	30.8 CH2	30.1 CH2	30.1 CH2	29.5 CH2	66.5 CH	31.5 CH2	29.2 CH2
13	40.0 qC	40.0 qC	36.5 qC	36.5 qC	38.1 qC	44.7 qC	47.9 qC	41.8 qC
14	165.9 qC	166.2 qC	152.1 qC	152.2 qC	154.6 qC	152.3 qC	193.6 qC	80.1 qC
15	115.1 CH	115.0 CH	115.8 CH	115.7 CH	122.3 CH	123.7 CH	123.8 CH	33.5 CH2
16	163.8 qC	163.8 qC	165.4 qC	165.5 qC	163.9 qC	162.4 qC	205.1 qC	168.2 qC
17	79.2 CH	79.1 CH	80.3 CH	80.3 CH	80.6 CH	78.2 qC	61.8 CH	81.2 CH
18	19.6 CH3	19.6 CH3	15.8 CH3	15.8 CH3	19.6 CH3	13.2 CH3	24.2 CH3	14.1 CH3
19	15.3 CH3	15.4 CH3	16.1 CH3	16.0 CH3	17.4 CH3	15.6 CH3	19.1 CH3	21.7 CH3
20	119.6 qC	119.7 qC	119.9 qC	119.9 qC	119.6 qC	121.5 qC	36.8 CH	164.3 qC
21	141.5 CH	141.5 CH	141.3 qC	141.3 CH	141.5 CH	142.3 CH	177.5 qC	72.4 CH2
22	110.0 CH	110.1 CH	110.0 CH	110.0 CH	109.8 CH	109.7 CH	29.2 CH2	117.8 CH
23	143.4 CH	143.4 CH	143.2 CH	143.2 CH	143.2 CH	144.9 CH	66.5 CH2	172.5 qC
28	16.7 CH3	16.5 CH3	16.6 CH3	16.6 CH3	15.4 CH3	14.5 CH3	27.0 CH3	25.5 CH3
29	49.7 CH2	49.6 CH2	43.1 CH2	43.1 CH2	40.5 CH3	39.0 CH3	21.3 CH3	21.4 CH3
30	134.7 CH	134.8 CH	194.9 qC	195.0 qC	74.0 CH	78.2 CH	26.8 CH3	112.5 CH2
							7-Acyl
31	53.2 CH3	53.2 CH3	53.3 CH3	53.3 CH3	53.2 CH3	52.3 CH3	169.8 qC	52.2 CH3
	3-Acyl	3-Acyl	3-Acyl	3-Acyl
32	170.4 qC	176.2 qC	174.3 qC	174.8 qC	119.5 qC	119.4 qC	21.0 CH3
33	20.6 CH3	40.7 CH	41.3 CH	34.2 CH	16.6 CH3	16.5 CH3
					3-Acyl	3-Acyl
34		26.6 CH2	26.5 CH2	18.8 CH3	169.0 qC	167.8 qC
35		11.5 CH3	11.8 CH3	19.0 CH3	21.7 CH3	130.1 qC
					2-Acyl
36		16.4 CH3	16.7 CH3		170.7 qC	139.7 CH
37					21.9 CH3	14.4 CH3
38						12.5 CH3

) and those of trangmolin F [16], containing a (Z)-bicyclo[5.2.1]dec-3-en-8-one substructure, revealed their close structural resemblance. However, the 3-O-isobutyryl function in trangmolin F was replaced by an acetoxy group (δ_H 2.17 s 3H; δ_C 170.4 qC, 20.6 CH₃) in 22, being unambiguously confirmed by HMBC cross-peaks between H-3/C-32 and H₃-33/C-32 (Figure 7a). The relative configuration of 22 was assigned by NOE interactions (Figure 7b). Those between H-17/H-12β, H-12β/H₃-19, and H₃-19/H-5 revealed their cofacial relationship and were determined as β-oriented, whereas those between H-3/H-9, H_pro-R-29/H-3, and H-12α/H₃-18 indicated the α-orientation for H-3, H-9, and H₃-18, and the corresponding 3β-acetoxy function. The ECD spectrum of 22 was nicely matched with that of trangmolin F (Figure 8a), concluding that the absolute configuration of 22 was the same as that of trangmolin F. Thus, the structure of 22—named xylomolin J₁—was assigned as 3-O-acetyl-3-deisobutyryloxy-trangmolin F.

Compound 23 provided the molecular formula of C₃₂H₃₈O₁₀ as established by the positive HRESIMS ion peak at m/z 583.2548 (calcd. for [M + H]⁺, 583.2543). The NMR spectroscopic data of 23 (Table 7 and Table 8) resembled those of 22, except for the replacement of the 3-acetoxy group in 22 by a 2-methylbutyryloxy moiety (δ_H 2.51 m 1H, 1.53 m 1H, 1.74 m 1H, 0.96 t (J = 7.2 Hz, 3H), 1.18 d (J = 7.2 Hz, 3H); δ_C 176.2 qC, 40.7 CH, 26.6 CH₂, 11.5 CH₃, 16.4 CH₃) in 23. The presence of the 2-methylbutyryloxy group was further evidenced by ¹H-¹H COSY cross-peaks between H-33/H₃-36, H-33/H₂-34, and H₂-34/H₃-35 and HMBC correlations between H-33/C-32, H-34/C-32, and H₃-36/C-32. The HMBC correlation from H-3 to the carbonyl carbon (C-32) of the above 2-methylbutyryloxy group placed it at C-3. The relative configuration of 23 was confirmed to be the same as that of 22 by analysis of NOE interactions. Comparison of ECD spectra of compounds 23, 22, and trangmolin F (Figure 8a) revealed that these compounds had the same absolute configuration. The absolute configuration of C-6 was further determined by the modified Mosher α-methoxy-α-(trifluoromethyl)phenylacetyl (MTPA) ester method [24]. The ∆δ values of H-5, H₃-19, and H₃-29 were positive, while that of H₃-31 was negative (Figure 8b). This regular arrangement concluded the R-absolute configuration for C-6. Finally, the absolute configuration of 23—named xylomolin J₂—was unequivocally established as 3S,4R,5S,6R,9S,10R,13R,17R.

Compound 24 had the molecular formula C₃₂H₃₈O₁₁ as determined from the positive HRESIMS ion peak at m/z 621.2306 (calcd. for [M + Na]⁺, 621.2306). The NMR spectroscopic data of 24 (Table 7 and Table 8) were similar to those of moluccensin I [25], except for the presence of an additional 6-OH group (δ_H 3.12 br s) and the replacement of the 1-O-isobutyryl group in moluccensin I by a 1-OH function (δ_H 2.81 s) in 24. The downshifted C-6 signal (δ_C 71.5 CH in 24, whereas δ_C 33.2 CH₂ in moluccensin I) and HMBC correlations from the active proton (δ_H 3.12 br s) to C-5, C-6, and C-7 revealed the presence of the 6-OH group (Figure 9a). The existence of the 1-OH function was confirmed by the upshifted C-1 signal (δ_C 85.8 qC in 24, whereas δ_C 90.8 qC in moluccensin I) and strong HMBC cross-peaks from the active proton (δ_H 2.81 s) to C-1, C-2, and C-10 (Figure 9a). The relative configuration of 24 was identified as the same as that of moluccensin I based on NOE interactions between H-17/H-12β, H-5/H-11β, H-5/H-28, H₃-18/H-11α, H₃-19/H_pro-S-29, H-3/H_pro-R-29, and 2-OH/H_pro-R-29 (Figure 9b). Therefore, the structure of 24—named xylomolin K₁—was assigned as 6-hydroxy-1-O-deisobutyryl-moluccensin I.

Compound 25 provided the molecular formula C₃₁H₃₆O₁₁ as determined from the positive HRESIMS ion peak at m/z 607.2164 (calcd. for [M + Na]⁺, 607.2150). The NMR spectroscopic data of 25 (Table 7 and Table 8) were similar to those of 24, except for the replacement of 3-O-(2-methyl)butyryl in 24 by an isobutyryloxy group (δ_H 2.45 m 1H, 1.11 d (J = 6.8 Hz, 3H), 1.11 d (J = 6.8 Hz, 3H); δ_C 174.8 qC, 34.2 CH, 18.8 CH₃, 19.0 CH₃) in 25. The HMBC correlation from H-3 to the carbonyl carbon (C-32) of the isobutyryloxy group placed it at C-3. The relative configuration of 25 was determined to be the same as that of 24 based on NOE interactions between H-17/H-12β, H-11β/H-5, H-5/H₃-28, H-12α/H₃-18, H₃-18/H-11α, H_pro-R-29/H-3, and H_pro-S-29/H₃-19. Thus, the structure of 25—named xylomolin K₂—was identified as 3-O-isobutyryl-3-de(2-methyl)butyryloxy-xylomolin K₁.

Compound 26 was obtained as an amorphous white powder. Its molecular formula was determined to be C₃₃H₃₈O₁₄ by the positive HRESIMS ion peak at m/z 681.2149 (calcd. for [M + Na]⁺, 681.2154). The NMR spectroscopic data of 26 (Table 7 and Table 8) resembled those of 2-O-acetyl-2-dehydroxy-12-deacetylxyloccensin U [18], being a phragmalin 8,9,30-ortho ester isolated from X. moluccensis, except for the presence of an additional 6-OH group and the absence of the 12-OH group in 26. The presence of the 6-OH group was supported by the downshifted C-6 signal (δ_C 71.5 CH in 26, whereas δ_C 33.7 CH₂ in 2-O-acetyl-2-dehydroxy-12-deacetylxyloccensin U), the ¹H-¹H COSY cross-peak between H-5/H-6 and HMBC correlations between H-6/C-5 and H-6/C-7. The upshifted C-12 signal (δ_C 29.5, CH₂ in 26, whereas δ_C 66.6 CH in 2-O-acetyl-2-dehydroxy-12-deacetylxyloccensin U), ¹H-¹H COSY cross-peaks between H₂-11/H₂-12, and HMBC correlations between H₃-18/C-12 (Figure 10a) confirmed the absence of the 12-OH group in 26. NOE correlations between H-17/H-12β, H-12β/H-5, H-17/H-5, H-5/H-30, H₃-18/H-12α, H₃-18/H-11α, H₃-19/H_pro-S-29, and H-3/H_pro-R-29 revealed the same relative configuration of 26 as that of 2-O-acetyl-2-dehydroxy-12-deacetylxyloccensin U (Figure 10b). The ECD spectrum of 26 was nicely matched with that of 2-O-acetyl-2-dehydroxy-12-deacetylxyloccensin U (Figure 10c), revealing the same absolute configuration for the two compounds. Thus, the structure of 26—named xylomolin L₁—was assigned as 6-hydroxy-12-dehydroxy-2-O-acetyl-2-dehydroxy-12-deacetylxyloccensin U.

The molecular formula of 27 was determined to be C₃₄H₄₀O₁₃ by the positive HRESIMS ion peak at m/z 679.2374 (calcd. for [M + Na]⁺, 679.2361). The NMR spectroscopic data of 27 (Table 7 and Table 8) closely resembled those of swietephragmin G [26], being a phragmalin 8,9,30-ortho ester, except for the presence of an additional 12-OH group, which was supported by the downshifted C-12 signal (δ_C 66.5 CH in 27, whereas δ_C 29.2 CH₂ in swietephragmin G), ¹H-¹H COSY cross-peaks between H₂-11/H-12, and HMBC correlations between H₃-18/C-12. The strong NOE interaction between H-17/H-12 assigned the β-oriented H-12 and the corresponding α-orientation for the 12-OH group. The NOE interaction between H₃-37/H₃-38 assigned the E configuration for the double bond of 3-tigloyloxy group. Therefore, the structure of 27—named xylomolin L₂—was identified as 12α-hydroxy-swietephragmin G.

Compound 28 had the molecular formula C₂₈H₃₇O₆ as determined from the positive HRESIMS ion peak at m/z 469.2603 (calcd. for [M + H]⁺, 469.2585). The NMR spectroscopic data of 28 (Table 7 and Table 8) were closely related to those of andirolide Q [27], except for the different positions of the ester carbonyl carbon of the C-17 attached five-membered γ-lactone ring, viz. C-21 in 28 instead of C-23 in andirolide Q. Significant ¹H-¹H COSY correlations between H-17/H-20, H-20/H₂-22, H₂-22/H₂-23 and the HMBC correlation between H-17/C-21 confirmed the above deduction (Figure 11a). NOE interactions between H-17/H-12β, H-12β/H₃-30, H₃-30/H-7, H₃-30/H-11β, H-11β/H₃-19, and H₃-18/H-11α, H₃-18/H-12α, H₃-18/H-20, H₃-18/H-9, and H-9/H-5 indicated the β-orientation for H-7, H-17, H₃-19, and H₃-30, and the α-orientation for H-5, H-9, H₃-18, and H-20 (Figure 11b). Hence, the relative configuration of 28—named xylomolin M—was assigned as depicted.

Compound 29 afforded the molecular formula C₂₇H₃₄O₈ as deduced from the positive HRESIMS ion peak at m/z 509.2147 (calcd. for [M + Na]⁺, 509.2146). The NMR spectroscopic data of 29 (Table 7 and Table 8) closely resembled those of moluccensin O [25], except for the absence of the 21-OH group, which was corroborated by the upshifted C-21 signal (δc 72.4 CH₂ in 29, δc 98.1 CH in moluccensin O) and HMBC correlations from H₂-21 (δ_H 4.85 br d (J = 18.3 Hz), 5.03 dd (J = 18.3, 2.0 Hz)) to C-20 and C-22. The relative configuration of 29 was assigned as the same as that of moluccensin O based on NOE correlations. Thus, the structure of compound 29—named xylomolin N—was assigned as 21-dehydroxy-moluccensin O.

The antitumor activities of 1, 3, 8, 10, 11, 14–16, 20, 23, 25, and 27 were tested by the MTT cytotoxity assay against five human tumor cell lines, including human colorectal HCT-8 and HCT-8/T, human ovarian A2780 and A2780/T, and human breast MD-MBA-231 (Table S1) [28]. Cisplatin was used as the positive control. Compounds 11 and 23 showed weak activities against the tested cancer cell lines, whereas the other ten compounds were inactive at 100 μM. Compound 23 exhibited selective antitumor activity against human breast MD-MBA-231 cancer cells with an IC₅₀ value of 37.7 μM.

Anti-HIV activities of 1, 3, 8, 10, 11, 14, 20, 23–25, and 27 were tested in vitro with the HIV-1 virus transfected 293 T cells [29]. At the concentration of 20 μM, 1, 11, 23, and 24 showed inhibitory rates of 17.49 ± 6.93%, 24.47 ± 5.04%, 14.77 ± 5.91%, and 14.34 ± 3.92%, respectively (Table S2). Efavirenz was used as the positive control with an inhibitory rate of 88.54 ± 0.45% at the same concentration.

3. Materials and Methods

3.1. General Methods

Optical rotations were recorded on a MCP500 modular circular polarimeter (Anton Paar Opto Tec GmbH, Seelze, Germany). UV spectra were obtained on a GENESYS 10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Shanghai, China). HRESIMS were measured on a Bruker maXis ESI-QTOF mass spectrometer (Bruker Daltonics, Bremen, Germany). NMR spectra were recorded on a Bruker AV-400 spectrometer with TMS as the internal standard. Single-crystal X-ray diffraction analyses were made on an Agilent Xcalibur Atlas Gemini Ultra-diffractometer (Agilent Technologies, Santa Clara, CA, USA) with mirror monochromated CuKα radiation (λ = 1.54178 Å) at 150 K. Semi-preparative HPLC (Waters Corporation, Milford, MA, USA) was performed on a Waters 2535 pump equipped with a waters 2998 photodiode array detector and YMC C18 reverse-phased columns (250 mm × 10 mm i.d., 5 μm). For column chromatography, silica gel (100–200 mesh) (Qingdao Mar. Chem. Ind. Co. Ltd., Qingdao, China) and C₁₈ reverse-phased silica gel (ODS-A-HG 12 nm, 50 µm, YMC Co. Ltd., Kyoto, Japan) were used. ECD spectra were measured on a Jasco J-810 spectropolarimeter (JASCO Corporation, Tokyo, Japan) in MeCN.

3.2. Plant Material

A batch of seeds of Xylocarpus moluccensis were collected at the mangrove swamp of Trang Province, Thailand, in June 2013, whereas another batch of seeds of the same mangrove plant were collected in Godavari estuary, Andhra Pradesh, India, in October 2009, respectively. The identification of the plant was performed by one of the authors (J.W.) and Mr. Tirumani Satyanandamurty (Government Degree College at Amadala Valasa, India). Voucher samples (No. ThaiXM-03 and No. IXM200901, respectively) were maintained in the Marine Drugs Research Center, College of Pharmacy, Jinan University.

3.3. Extraction and Isolation

The dried seeds (10.0 kg, ThaiXM-03) of X. moluccensis were extracted with 95% (v/v) EtOH at room temperature (5 × 20 L) to afford the EtOH extract (680.0 g), which was suspended in water and extracted with EtOAc. The resulting EtOAc extract (296.0 g) was chromatographed on silica gel column and eluted with CHCl₃/MeOH (100:0 to 5:1) to yield 160 fractions.

Fractions 26–28 (31.6 g) were combined and further purified with an RP-18 column (acetone/H₂O, 50:50 to 100:0) to afford 57 subfractions. Subfraction 23 was purified by preparative HPLC (YMC-Pack ODS-5-A, 250 mm × 10 mm i.d., MeOH/H₂O, 50:50) to afford compound 1 (45.0 mg).

Fractions 29–40 (111.0 g) were combined and further purified with an RP-18 column (acetone/H₂O, 50:50 to 100:0) to afford 360 subfractions. The combination of subfractions 90–95 was subjected to preparative HPLC (YMC-Pack ODS-5-A, 250 mm × 10 mm i.d., MeCN/H₂O, 38:62) to give compound 8 (72 mg), along with five other subfractions (SFr.90-95-1 to SFr.90-95-5). Recrystallization of SFr.90-95-1 afforded compound 20 (15.3 mg). Further purification of SFr.90-95-2 with preparative HPLC (YMC-Pack ODS-5-A, 250 mm × 10 mm i.d., MeOH/MeCN/H₂O, 10:40:50) yielded compounds 3 (7.1 mg), 5 (4.3 mg), 12 (37 mg), 13 (2.8 mg), and 21 (1.2 mg), whereas that of SFr.90-95-3 with preparative HPLC (YMC-Pack ODS-5-A, 250 mm × 10 mm i.d., MeOH/H₂O, 60:40 or MeCN/H₂O, 35:65) afforded compounds 6 (4.3 mg) and 9 (3.1 mg). SFr.90-95-4 was further purified with preparative HPLC (YMC-Pack ODS-5-A, 250 mm × 10 mm i.d., MeCN/H₂O, 46:64) to afford compound 14 (57.0 mg). Subfractions 78–83 were combined and further purified by preparative HPLC (YMC-Pack ODS-5-A, 250 × 10 mm i.d., MeOH/H₂O, 50:50) to give compounds 24 (3.5 mg), 26 (2.0 mg), 27 (7.0 mg), and 29 (2.5 mg).

Fractions 157–175 (14.5 g) were combined and further purified with an RP-18 column (acetone/H₂O, 40:60 to 100:0) to afford 45 subfractions. Subfraction 27 was purified by preparative HPLC (YMC-Pack 250 mm × 10 mm i.d., MeCN/MeOH/H₂O, 50:15:35) to afford compound 4 (1.2 mg).

The air-dried seeds (15.0 kg, IXM200901) were powdered and extracted with 95% (v/v) EtOH (5 × 20 L) at room temperature to afford the EtOH extract (1.1 kg), which was partitioned between EtOAc and water to afford the EtOAc portion (572.0 g). Then, 252.0 g of the EtOAc extract was further subjected to a silica gel column (105.0 cm × 9.5 cm i.d.) and eluted with a gradient mixture of CHCl₃/MeOH (100:1 to 5:1) to afford 184 fractions.

Fractions 38–43 (26.0 g) were combined and further separated on an RP-18 column (64.0 cm × 6.3 cm i.d.), and eluted with a gradient mixture of acetone/H₂O (40:60 to 100:0) to afford 74 subfractions.

Subfractions 12–16 (1.5 g) were combined and separated by preparative HPLC (YMC-Pack ODS-5-A, 250 mm × 10 mm i.d., MeCN/H₂O, 36:64) to afford seven parts (SFr.12-16-1 to SFr.12-16-7). SFr.12-16-2 was further purified by preparative HPLC (YMC-Pack ODS-5-A, 250 mm × 10 mm i.d., MeOH/H₂O, 49:51) to yield compounds 10 (49.3 mg) and 25 (25.7 mg). SFr.12-16-4 and SFr.12-16-5 were further purified by preparative HPLC (YMC-Pack ODS-5-A, 250 mm × 10 mm i.d., MeOH/H₂O, 52:48) to afford compounds 15 (13 mg) and 22 (3.0 mg), respectively. SFr.12-16-6 was further subjected to preparative HPLC (MeOH/H₂O, 51:49) to yield compound 2 (2.0 mg), whereas SFr.12-16-7 was further purified by preparative HPLC (MeOH/H₂O, 60:40, subsequently with MeCN/H₂O, 60:40, and then MeCN/MeOH/H₂O, 60:20:20) to yield compound 28 (1.5 mg).

Preparative HPLC (YMC-Pack ODS-5-A, 250 mm × 10 mm i.d., MeOH/H₂O, 58:42) was performed on subfraction 27 (2.9 g) to gain compound 16 (4.0 mg). Subfractions 32–35 (1.5 g) were combined and separated by preparative HPLC (YMC-Pack ODS-5-A, 250 mm × 10 mm i.d., MeCN/H₂O, 55:45) to give four parts (SFr.32-35-1 to SFr.32-35-4). SFr.32-35-2 and SFr.32-35-3 were purified by preparative HPLC (MeOH/H₂O, 65:35, MeOH/H₂O, 60:40, respectively) to yield compounds 23 (27.0 mg) and 11 (27.5 mg), respectively, whereas recrystallization (acetone) of SFr.32-35-4 afforded compound 7 (23.8 mg).

Fractions 48–86 (43.9 g) were combined and further performed on an RP-18 column (46.7 cm × 6.4 cm i.d.), and eluted with a gradient mixture of MeCN/H₂O (50:50 to 100:0), to afford 81 subfractions. Subfraction 4 (6.5 g) was purified by preparative HPLC (MeOH/H₂O, 53:47) to yield compound 17 (5.5 mg). Subfractions 8–14 (10.4 g) were combined and further separated on an RP-18 column (62.0 cm × 6.5 cm i.d.) and eluted with a gradient mixture of acetone/H₂O (50:50 to 100:0) to afford 37 subsubfractions, among which subsubfractions 12–15 (2.1 g) were combined and separated on the preparative HPLC (MeOH/MeCN/H₂O, 50:15:35) to give compound 18 (2.1 mg). Subfraction 17 (1.78 g) was subjected to preparative HPLC (MeOH/H₂O, 80:20) to yield compound 19 (1.2 mg).

Xylomolin A₁ (1): Colorless crystal; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −62.0 (c = 0.06, acetone); UV (MeCN) λ_max (log ε) 199.7 (3.7) nm; ECD (c 0.41 mM, MeCN) λ_max (Δε) 190.0 (−12.0), 289.6 (−2.2) nm; ¹H and ¹³C NMR spectroscopic data see Table 1 and Table 2; HRESIMS m/z 615.2785 [M + H]⁺ (calcd. for C₃₃H₄₃O₁₁, 615.2800).

Xylomolin A₂ (2): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −74.7 (c 0.04, acetone); UV (MeCN) λ_max (log ε) 194.0 (4.5), 279.0 (3.3) nm; ¹H and ¹³C NMR spectroscopic data see Table 1 and Table 2; HRESIMS m/z 593.2143 [M + Cl]⁻ (calcd. for C₃₀H₃₈O₁₀Cl, 593.2159).

Xylomolin A₃ (3): White, amorphous solid; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −96.0 (c 0.05, acetone); UV (MeCN) λ_max (log ε) 198.9 (3.9) nm; ECD (c 0.16 mM, MeCN) λ_max (Δε) 191.0 (−14.5) nm; ¹H and ¹³C NMR spectroscopic data see Table 1 and Table 2; HRESIMS m/z 625.2620 [M + Na]⁺ (calcd. for C₃₂H₄₂NaO₁₁, 625.2619).

Xylomolin A₄ (4): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −24.0 (c 0.08, acetone); UV (MeCN) λ_max (log ε) 197.0 (3.9), 201.8 (3.8) nm; ECD (c 0.15 mM, MeCN) λ_max (Δε) 190(−8.1), 200.4 (−5.0), 211.6 (−6.6) nm; ¹H and ¹³C NMR spectroscopic data see Table 1 and Table 2; HRESIMS m/z 683.2675 [M + Na]⁺ (calcd. for C₃₄H₄₄NaO₁₃, 683.2674).

Xylomolin A₅ (5): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −52.0 (c 0.04, acetone); UV (MeCN) λ_max (log ε) 201.0 (3.8) nm; ECD (c 0.16 mM, MeCN) λ_max (Δε) 191.0 (−8.6) nm; ¹H and ¹³C NMR spectroscopic data see Table 1 and Table 2; HRESIMS m/z 625.2252 [M + Na]⁺ (calcd. for C₃₁H₃₈NaO₁₂, 625.2255).

Xylomolin A₆ (6): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −216.0 (c 0.10, acetone); UV (MeCN) λ_max (log ε) 197.8 (4.0) nm; ECD (c 0.16 mM, MeCN) λ_max (Δε) 190.0 (−12.8), 216.4 (+1.5), 294.2 (−2.3) nm; ¹H and ¹³C NMR spectroscopic data see Table 1 and Table 2; HRESIMS m/z 609.2311 [M + Na]⁺ (calcd. for C₃₁H₃₈NaO₁₁, 609.2306).

Xylomolin A₇ (7): White powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −250.7 (c 0.08, acetone); UV (MeCN) λ_max (log ε) 193.0 (4.5) nm; ¹H and ¹³C NMR spectroscopic data see Table 1 and Table 2; HRESIMS m/z 593.2358 [M + Na]⁺ (calcd. for C₃₁H₃₈NaO₁₀, 593.2357).

Xylomolin B₁ (8): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +212.0 (c 0.08, acetone); UV (MeCN) λ_max (log ε) 204.0 (3.9), 285.4 (4.0) nm; ECD (c 0.10 mM, MeCN) λ_max (Δε) 252.1 (−1.6), 278.9 (+8.6) nm; ¹H and ¹³C NMR spectroscopic data see Table 3 and Table 4; HRESIMS m/z 607.2151 [M + Na]⁺ (calcd. for C₃₁H₃₆NaO₁₁, 607.2150).

Xylomolin B₂ (9): White, amorphous solid; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +137.0 (c 0.10, acetone); UV (MeCN) λ_max (log ε) 207.2 (3.7), 285.2 (4.0) nm; ECD (c 0.19 mM, MeCN) λ_max (Δε) 190.0 (+3.6), 213.7 (−1.1), 282.2 (+9.5) nm; ¹H and ¹³C NMR spectroscopic data see Table 3 and Table 4; HRESIMS m/z 527.2273 [M + H]⁺ (calcd. for C₂₉H₃₅O₉, 527.2276).

Xylomolin C₁ (10): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +156.0 (c 0.07, acetone); UV (MeCN) λ_max (log ε) 191.0 (4.3), 275 (4.1) nm; ¹H and ¹³C NMR spectroscopic data see Table 3 and Table 4; HRESIMS m/z 543.2238) [M + H]⁺ (calcd. for C₂₉H₃₅O₁₀, 543.2230).

Xylomolin C₂ (11): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +125.7 (c 0.09, acetone); UV (MeCN) λ_max (log ε) 194.0 (4.7), 277 (4.6) nm; ¹H and ¹³C NMR spectroscopic data see Table 3 and Table 4; HRESIMS m/z 591.2570 [M + Na]⁺ (calcd. for C₃₂H₄₀NaO₉, 591.2565).

Xylomolin D (12): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −34.0 (c 0.05, acetone); UV (MeCN) λ_max (log ε) 208.2 (3.8) nm; ECD (c 0.16 mM, MeCN) λ_max (Δε) 190.0 (−2.6), 217.5 (+13.7), 244.5 (−0.3), 261 (+ 1.5), 291.2 (−3.8) nm; ¹H and ¹³C NMR spectroscopic data see Table 3 and Table 4; HRESIMS m/z 625.2612 [M + Na]⁺ (calcd. for C₃₂H₄₂NaO₁₁, 625.2619).

Xylomolin E (13): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −68.0 (c 0.04, acetone); UV (MeCN) λ_max (log ε) 207.2 (3.9) nm; ECD (c 0.15 mM, MeCN) λ_max (Δε) 190.0 (−9.5), 213.7 (+2.5) nm; ¹H and ¹³C NMR spectroscopic data see Table 3 and Table 4; HRESIMS m/z 625.2250 [M + Na]⁺ (calcd. for C₃₁H₃₈NaO₁₂, 625.2255).

Xylomolin F (14): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +4.0 (c 0.03, acetone); UV (MeCN) λ_max (log ε) 211.0 (4.1) nm; ECD (c 0.21 mM, MeCN) λ_max (Δε) 192.0 (−7.1), 219.0 (+4.0), 265.0 (+3.2) nm; ¹H and ¹³C NMR spectroscopic data see Table 3 and Table 4; HRESIMS m/z 669.2521 [M + Na]⁺ (calcd. for C₃₃H₄₂NaO₁₃, 669.2518).

Xylomolin G₁ (15): White, amorphous solid; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −65.0 (c 0.1, acetone); UV (MeCN) λ_max (log ε) 190.2 (4.20) nm; ECD (c 0.14 mM, MeCN) λ_max (Δε) 207 (+2.3), 238 (−3.8), 290 (+1.4), 321 (−0.32), 344 (+0.32) nm; ¹H and ¹³C NMR spectroscopic data see Table 5 and Table 6; HRESIMS m/z 587.2494 [M + H]⁺ (calcd. for C₃₁H₃₉O₁₁, 587.2492).

Xylomolin G₂ (16): White, amorphous solid; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −72.0 (c 0.05,acetone); UV (MeCN) λ_max (log ε) 190.2 (4.03); ECD (c 0.13 mM, MeCN) λ_max (Δε) 205.0 (+1.7), 239.0 (−4.3), 302.0 (+0.34), 339.0 (+1.1) nm; ¹H and ¹³C NMR spectroscopic data see Table 5 and Table 6; HRESIMS m/z 615.2809 [M + H]⁺ (calcd. for C₃₃H₄₃O₁₁, 615.2805).

Xylomolin G₃ (17): White, amorphous solid; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −43.5 (c 0.04, acetone); UV (MeCN) λ_max (log ε) 196.6 (4.03), 285.0 (2.93) nm; ECD (c 0.15 mM, MeCN) λ_max (Δε) 193.0 (−4.5), 197.0 (−3.4), 205.0 (−7.5), 216.0 (−5.0), 233.0 (−7.9), 298 (+4.3) nm; ¹H and¹³C NMR spectroscopic data see Table 5 and Table 6; HRESIMS m/z 543.2246 [M + H]⁺ (calcd. for C₂₉H₃₅O₁₀, 543.2230).

Xylomolin G₄ (18): White, amorphous solid; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −44.0 (c 0.01,acetone); UV (MeCN) λ_max (log ε) 196.6 (4.08) nm; ECD (c 0.18 mM, MeCN) λ_max (Δε) 203.0 (−8.7), 215.0 (−6.2), 233.0 (−10.3), 301.0 (+5.0) nm; ¹H and¹³C NMR spectroscopic data see Table 5 and Table 6; HRESIMS m/z 555.2593 [M + H]⁺ (calcd. for C₃₁H₃₉O₉, 555.2594).

Xylomolin G₅ (19): White, amorphous solid; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −37.5 (c 0.024, acetone); UV (MeCN) λ_max (log ε) 195.0 (4.27), 284.2 (2.86) nm; ECD (c 0.11 mM, MeCN) λ_max (Δε) 195.0 (+ 0.70), 206.0 (−4.9), 214.0 (−3.5), 231.0 (−6.1), 299.0 (+2.7) nm; ¹H and¹³C NMR spectroscopic data see Table 5 and Table 6; HRESIMS m/z 569.2753 [M + H]⁺ (calcd. for C₃₂H₄₁O₉, 569.2751).

Xylomolin H (20): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +65.0 (c 0.06, acetone); UV (MeCN) λ_max (log ε) 212.3 (4.0) nm; ECD (c 0.16 mM, MeCN) λ_max (Δε) 223 (+10.9), 245 (+0.19),270 (+8.1) nm; ¹H and ¹³C NMR spectroscopic data see Table 5 and Table 6; HRESIMS m/z 667.2359 [M + Na]⁺ (calcd. for C₃₃H₄₀NaO₁₃, 667.2361).

Xylomolin I (21): Light yellow, amorphous gum; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +129.0 (c 0.08, acetone); UV (MeCN) λ_max (log ε) 208.4 (3.8), 287.6 (4.1) nm; ECD (c 0.17 mM, MeCN) λ_max (Δε) 200.0 (+4.6), 213.0 (+2.3), 232.0 (+4.6), 259.0 (−3.9), 291.0 (+10.3) nm; ¹H and ¹³C NMR spectroscopic data see Table 5 and Table 6; HRESIMS m/z 621.2304 [M + Na]⁺ (calcd. for C₃₂H₃₈NaO₁₁, 621.2306)

Xylomolin J₁ (22): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −135.0 (c 0.11, acetone); UV (MeCN) λ_max (log ε) 197.0 (4.9), 213.4 (4.8), 260.8 (4.6) nm; ECD (c 0.039 mM, MeCN) λ_max (Δε) 204.0 (−3.3), 229.0 (+3.3), 266.0 (+6.3), 310.0 (−0.19), 346.0 (+0.95) nm; ¹H and ¹³C NMR spectroscopic data see Table 7 and Table 8; HRESIMS m/z 541.2077 [M + H]⁺ (calcd. for C₂₉H₃₃O₁₀, 541.2074).

Xylomolin J₂ (23): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +232.0 (c 0.1, acetone); UV (MeCN) λ_max (log ε) 196.8 (5.2), 211.0 (5.1), 260.8 (5.0) nm; ECD (c 0.039 mM, MeCN) λ_max (Δε) 190.0 (−3.9), 199.0 (−2.0), 206.0 (−2.9), 225.0 (+3.9), 267.0 (+9.1), 310.0 (−0.17), 335.0 (+1.0) nm; ¹H and ¹³C NMR spectroscopic data see Table 7 and Table 8; HRESIMS m/z 583.2548 [M + H]⁺ (calcd. for C₃₂H₃₉O₁₀, 583.2543).

Xylomolin K₁ (24): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +98.0 (c 0.1, acetone); UV (MeCN) λ_max (log ε) 194.0 (4.3), 271.0 (4.3) nm; ECD (c 0.33 mM, MeCN) λ_max (Δε) 209.0 (+3.0), 211.0 (+2.9), 232.0 (+8.5), 275.0 (−2.8), 300.0 (+6.4) nm; ¹H and ¹³C NMR spectroscopic data see Table 7 and Table 8; HRESIMS m/z 621.2306 [M + Na]⁺ (calcd. for C₃₂H₃₈NaO₁₁, 621.2306).

Xylomolin K₂ (25): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +120.4 (c 0.07, acetone); UV (MeCN) λ_max (log ε) 191.0 (4.3), 272.0 (4.5) nm; ¹H and ¹³C NMR spectroscopic data see Table 7 and Table 8; HRESIMS m/z 607.2164 [M + Na]⁺ (calcd. for C₃₁H₃₆NaO₁₁, 607.2150).

Xylomolin L₁ (26) : White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +64.0 (c 0.09, acetone); UV (MeCN) λ_max (log ε) 213.0 (4.0) nm; ECD (c 0.30 mM, MeCN) λ_max (Δε) 220.0 (+12.5), 249.0 (+ 1.1), 270.0 (+ 0.8), 236.0 (+ 1.9) nm; ¹H and ¹³C NMR spectroscopic data see Table 7 and Table 8; HRESIMS m/z 681.2149 [M + Na]⁺ (calcd. for C₃₃H₃₈NaO₁₄, 681.2154).

Xylomolin L₂ (27): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +42.0 (c 0.06, acetone); UV (MeCN) λ_max (log ε) 214 (4.5) nm; ECD (c 0.15 mM, MeCN) λ_max (Δε) 198.0 (+3.2), 213.0 (−3.6), 234.0 (+11.6) nm; ¹H and ¹³C NMR spectroscopic data see Table 7 and Table 8; HRESIMS m/z 679.2374 [M + Na]⁺ (calcd. for C₃₄H₄₀NaO₁₃, 679.2361).

Xylomolin M (28): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −252.0 (c 0.03, acetone); UV (MeCN) λ_max 200.0 (4.0), 232 (4.1) nm; ¹H and ¹³C NMR spectroscopic data see Table 7 and Table 8; HRESIMS m/z 469.2603 [M + H]⁺ (calcd. for C₂₈H₃₇O₆, 469.2585).

Xylomolin N (29): White, amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −17.0 (c 0.05, acetone); UV (MeCN) λ_max (log ε) 196 (3.7), 211 (3.7) nm; ECD (c 0.21 mM, MeCN) λ_max (Δε) 200.0 (−7.0), 224.0 (+2.1), 252.0 (−0.3) nm; ¹H and ¹³C NMR spectroscopic data see Table 7 and Table 8; HRESIMS m/z 509.2147 [M + Na]⁺ (calcd. for C₂₇H₃₄NaO₈, 509.2146).

3.4. X-ray Crystal Data for Xylomolin A₁ (1)

Orthorhombic, C₃₄H₄₆O₁₂ (C₃₃H₄₂O₁₁·CH₃OH), space group P2(1)2(1)2(1), a = 8.82730 (5) Å, b = 17.93740 (10) Å, c = 20.84108 (13) Å, α = 90°, β = 90°, γ = 90°, V = 3299.95 (3) ų, Z = 4, D_calcd. = 1.302 Mg/m³, μ = 0.816 mm⁻¹. Crystal size: 0.40 × 0.40 × 0.28 mm³. 47,329 measured reflections, 5877 [R_int = 0.0378] independent reflections, 426 parameters, 0 restraints, F(000) = 1384.0, R₁ = 0.0296, wR₂ = 0.0773(all data), R₁ = 0.0286, wR₂ = 0.0763 [I > 2σ(I)], and goodness-of-fit (F²) = 1.065. The absolute structural parameter is −0.06(10), Flack x is −0.14(11), and Hooft y is −0.04(3).

CCDC-1590301 (1) contains the supplementary crystallographic data for this paper (excluding structure factors). These data are provided free of charge by The Cambridge Crystallographic Data Centre.

3.5. MTT Cytotoxicity Assay

Compounds 1, 3, 8, 10, 11, 14–16, 20, 23, 25, and 27 were evaluated by the MTT method for cytotoxicities against human colorectal HCT-8 and HCT-8/T, ovarian A2780 and A2780/T, and breast MD-MBA-231 cancer cell lines. All cell lines were cultured as adherent monolayers in flasks in DMEM culture medium with 10% fetal bovine serum, benzylpenicillin (50 kU/L), and streptomycin (50 mg/L) at 37 °C in a humidified atmosphere of 5% CO₂. Cells were collected with trypsin and resuspended in a final concentration of 5 × 10⁴/mL. One hundred microliter aliquots for each cell suspension were distributed evenly into 96-well multiplates (number of cells per well is 5 × 10³). Different concentrations of the compounds were added into the designated wells. After 72 h, a 10 μL MTT solution (5 mg/mL) was added to each well, and the plate was further incubated for 4 h, allowing viable cells to reduce the yellow MTT into dark-blue formazan crystals which were dissolved in DMSO 100 μL. The absorbance in individual wells was determined at 490 nm by a microplate reader (Biotek, VT, USA) [28]. The concentrations required to inhibit the growth of cancer cells by 50% (IC₅₀ values) were calculated from cytotoxicity curves by Bliss method. The positive control was cisplatin. The IC₅₀ values of cisplatin in human colorectal HCT-8 and HCT-8/T, ovarian A2780 and A2780/T, and breast MD-MBA-231, were 15.43, 21.98, 8.54, 9.26, and 6.25 μM, respectively.

3.6. HIV-Inhibitory Bioassay

For the assay, 293 T cells (2 × 10⁵) were co-transfected with 0.6 μg of pNL-Luc-E⁻-Vpu⁻ and 0.4 μg of vesicular stomatitis virus glycoprotein (VSV-G) expression vector pHIT/G. After 48 h, the VSV-G pseudotyped viral supernatant (HIV-1) was harvested by filtration through a 0.45-μm filter and the concentration of viral capsid protein was determined by p24 antigen capture ELISA (Biomerieux, Shanghai, China). SupT1 cells were exposed to VSV-G pseudotyped HIV-1 (multiplicity of infection (MOI) = 1) at 37 °C for 48 h in the absence or presence of the test compounds (1, 3, 8, 10, 11, 14, 20, 23–25, and 27). Efavirenz was used as the positive control. The inhibition rates were determined by using a firefly Luciferase Assay System (Promega, Madison, WI, USA) [29].

4. Conclusions

Twenty-nine new limonoids were isolated from the seeds of the mangrove plant, Xylocarpus moluccensis, collected in Thailand and India. The structures of these limonoids, including absolute configurations of ten compounds, viz. 1, 15–19, 21–23, and 26, were established by HRESIMS, extensive NMR investigations, single-crystal X-ray diffraction analysis conducted with Cu Kα radiation, and the comparison of experimental ECD spectra. Compounds 1–14 are mexicanolides, whereas 15–21 are khayanolides. Compounds 22 and 23 are unusual limonoids possessing a (Z)-bicyclo[5.2.1]dec-3-en-8-one motif, while 24 and 25 are 30-ketophragmalins. Compounds 26 and 27 are phragmalin 8,9,30-ortho esters, whereas 28 and 29 are azadirone and andirobin derivatives. These results demonstrate that X. moluccensis continues to be an abundant resource for the production of novel limonoids with structural diversity. Compound 23 exhibited selective antitumor activity against human triple-negative breast MD-MBA-231 cancer cells with an IC₅₀ value of 37.7 μM, whereas compounds 1, 11, 23, and 24 showed inhibitory rates of 17.49 ± 6.93%, 24.47 ± 5.04%, 14.77 ± 5.91%, and 14.34 ± 3.92% against HIV-I virus, respectively, at the concentration of 20 μM.