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

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 Δ8,14 double bond, whereas 20 and 21 are rare khayanolides containing a Δ14,15 double bond and Δ8,9, Δ14,15 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 Δ8,9, Δ14,15 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.

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 1 H and 13 C NMR spectroscopic data (Tables 1 and 2), 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 1 H and 13 C NMR spectroscopic data (Tables 1 and 2) showed the presence of a β-substituted furan ring [δH 7.52 br d (J
Compound 3 gave the molecular formula C 32 H 42 O 11 as established by the HRESIMS ion peak at m/z 625.2620 (calcd. for [M + Na] + , 625.2619). The NMR spectroscopic data of 3 (Tables 1 and 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 CH 2 in moluccensin S) and HMBC correlations from the proton of 30-OH to    . 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 A 4 -was identified as 15α-hydroxy-6-acetoxy-xylomolin A 3 .
The molecular formula of 5 was determined to be C 31 H 38 O 12 by the positive HRESIMS ion peak at m/z 625.2252 (calcd. for [M + Na] + , 625.2255). The 1 H and 13 C NMR spectroscopic data of 5 (Tables 1 and 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 CH 2 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 A 5 -was assigned as 30α-hydroxy-khayalenoid H.
Compound 6 was isolated as a white and amorphous powder. Its molecular formula was determined to be C 31 H 38 O 11 from the positive HRESIMS ion peak at m/z 609.2311 (calcd. for [M + Na] + , 609.2306). The 1 H and 13 C NMR spectroscopic data of 6 (Tables 1 and 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 CH 3 ) 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 H 2 -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/H 3 -28 and those between H-9/H 3 -19, H 3 -19/H 3 -29, and H 3 -18/H-15α. Therefore, the structure of 6-named xylomolin A 6 -was determined to be 2-O-acetyl-6-O-deacetyl-khayalenoid H.
The molecular formula of 7 was determined to be C 31 H 38 O 10 by the positive HRESIMS ion peak at m/z 593.2358 (calcd. for [M + Na] + , 593.2357). The 1 H and 13 C NMR spectroscopic data of 7 (Tables 1 and 2) were closely related to those of 6, except for the absence of the 6-OH group, which was corroborated by the upshifted CH 2 -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. 1 H-1 H COSY correlations between H 2 -6/H-5 and HMBC cross-peaks from H 2 -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 A 7 -was assigned as 6-dehydroxy-xylomolin A 6 .
Compound 13 was obtained as an amorphous white power. The molecular formula was determined to be C 31 H 38 O 12 by the positive HRESIMS ion peak at m/z 625.2250 (calcd. for [M + Na] + , 625.2255). The 1 H and 13 C NMR spectroscopic data of 13 (Tables 3 and 4) were similar to those of 8, except for the replacement of ∆ 8,9 and ∆ 14,15 conjugated double bonds in 8 by a ∆ 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 H 2 -15/C-14, H 2 -15/C-16, H 2 -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 H 3 -28/H-5, H-5/H-11β, H-12β/H-17, H 3 -29/H-3, H-12α/H 3 -18, and H-11α/H 3 -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 C 33  The downshifted C-6 signal (δ C 71.7 CH in 14, whereas δ C 32.3 CH 2 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.   (Tables 3 and 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 (δH 6.08 s 1H; δC 158.3 qC, 118.4 CH) and an additional 6-OH function (δH 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 (δC 71.7 CH in 14, whereas δC 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-14,15dedihydrogen-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 (Tables 5 and 6), 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 3 J HMBC correlations from H2-29 to C-30 further confirmed a khayanolide for 15 instead of a phragmalin, which should exhibit weak 4 J HMBC correlations between H2-29/C-30. HMBC cross-peaks from an active proton (δH 2.   (Tables 5 and 6), 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 3 J HMBC correlations from H 2 -29 to C-30 further confirmed a khayanolide for 15 instead of a phragmalin, which should exhibit weak 4 J HMBC correlations between H 2 -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 H 3 -18/H-15α, H-9/H 3 -19, H 3 -19/1-OH, and H-34/H pro-R -29 indicated the α-orientation for H-9, H 3 -18, H 3 -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 G 1 -was assigned as depicted.
Compound 18 provided the molecular formula C 31 H 38 O 9 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 (Tables 5  and 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 CH 2 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 G 4 -was concluded to be 30-deethoxyl-6-dehydroxy-xylomolin G 1 .
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 (Tables 7 and 8) 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 (δH 2.17 s 3H; δC 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, Hpro-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.    40.7 CH,26.6 CH 2 ,11.5 CH 3 ,16.4 CH 3 ) in 23. The presence of the 2-methylbutyryloxy group was further evidenced by 1 H-1 H COSY cross-peaks between H-33/H 3 -36, H-33/H 2 -34, and H 2 -34/H 3 -35 and HMBC correlations between H-33/C-32, H-34/C-32, and H 3 -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, H 3 -19, and H 3 -29 were positive, while that of H 3 -31 was negative (Figure 8b). This regular arrangement concluded the R-absolute configuration for C-6. Finally, the absolute configuration of 23-named xylomolin J 2 -was unequivocally established as 3S,4R,5S,6R,9S,10R,13R,17R.  40.7 CH,26.6 CH2,11.5 CH3,16.4 CH3) in 23. The presence of the 2methylbutyryloxy group was further evidenced by . 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 Rabsolute 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 (Tables 7  and 8) were similar to those of moluccensin I [25], except for the presence of an additional 6-OH group (δH 3.12 br s) and the replacement of the 1-O-isobutyryl group in moluccensin I by a 1-OH function (δH 2.81 s) in 24. The downshifted C-6 signal (δC 71.5 CH in 24, whereas δC 33.2 CH2 in moluccensin I) and HMBC correlations from the active proton (δH 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 (δC 85.8 qC in 24, whereas δC 90.8 qC in moluccensin I) and strong HMBC cross-peaks from the active proton (δH 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 ). Therefore, the structure of 24-named xylomolin K1-was assigned as 6-hydroxy-1-O-deisobutyryl-moluccensin I. Compound 24 had the molecular formula C 32 H 38 O 11 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 (Tables 7  and 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 CH 2 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 , and 2-OH/H pro-R -29 ( Figure 9b). Therefore, the structure of 24-named xylomolin K 1 -was assigned as 6-hydroxy-1-O-deisobutyryl-moluccensin I.  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-  Thus, the structure of 25-named xylomolin K2was identified as 3-O-isobutyryl-3-de(2-methyl)butyryloxy-xylomolin K1.
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 (Tables 7  and 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.   (Tables 7  and 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 CH 2 in swietephragmin G), 1 H-1 H COSY cross-peaks between H 2 -11/H-12, and HMBC correlations between H 3 -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 H 3 -37/H 3 -38 assigned the E configuration for the double bond of 3-tigloyloxy group. Therefore, the structure of 27-named xylomolin L 2 -was identified as 12α-hydroxy-swietephragmin G.
Compound 28 had the molecular formula C 28 H 37 O 6 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 (Tables 7 and 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. 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 (Tables 7  and 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 (δH 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.