Krishnolides A-D: New 2-Ketokhayanolides from the Krishna Mangrove, Xylocarpus moluccensis.

Four new khayanolide-type limonoids with a 2-carbonyl group, named krishnolides A-D (1-4), were isolated from the seeds of an Indian mangrove, Xylocarpus moluccensis, collected in the mangrove swamp of Krishna estuary, Andhra Pradesh. The relative and absolute configurations of these compounds were established by HR-ESIMS, extensive NMR investigations, single-crystal X-ray diffraction analysis with CuKα radiation, and experimental electronic circular dichroism (ECD) spectra. Krishnolides A-D are unusual khayanolides containing two large ester substituents of five or four carbon atoms at the C-3 and C-30 positions, respectively. Krishnolide A, containing an 8,14-epoxy group, exhibited moderate anti-Human Immunodeficiency Virus (HIV) activity with an IC50 value of 17.45 ± 1.65 μM and a CC50 value of 78.45 ± 1.69 μM, respectively. This is not only the first report of natural khayanolides from Indian mangroves of the genus Xylocarpus, but also the first report of the anti-HIV activity of khayanolide.

1. Introduction

Plants of the genus Xylocarpus, belonging to the family Meliaceae, are mangroves mainly found in East Africa, India, Bangladesh, Southeast Asia, Southern China, and Northern Australia. Limonoids are the main secondary metabolites of these mangroves [1,2,3]. Previously, a variety of limonoids with abundant structural diversity and various bioactivities, such as antiviral and antitumor activities, were identified from seeds of the mangrove X. moluccensis [4,5,6,7]. Thaixylomolin I, a khayanolide-type limonoid obtained from the Thai X. moluccensis, exhibited stronger inhibitory activity against pandemic influenza A virus (subtype H1N1) than that of the antiviral drug, ribavirin [6]. Khayanolides are a small group of limonoids containing an octahydro-1H-1,6-methanoindene moiety as the ring A/B-fused carbotricyclic core. To date, only 36 khayanolides have been reported [8,9,10,11,12,13,14,15,16,17,18,19,20,21]. In order to search for new antiviral limonoids, seeds of the Indian X. moluccensis, collected in the mangrove swamp of Krishna estuary, Andhra Pradesh, were further investigated to afford four new khayanolides, named krishnolides A–D (1–4) (Figure 1). Herein, we report the isolation, structural elucidation, and anti-HIV activities of these khayanolides.

Figure 1

Structures of compounds 1–4.

2. Results and Discussion

Compound 1 was obtained as a colorless crystal. Its molecular formula was established as C36H46O12 by the positive HR-ESIMS ion peak at m/z 693.2871 (calcd. for [M + Na]+ 671.2881), indicating 14 degrees of unsaturation. According to the 1H and 13C NMR spectroscopic data of 1 (Table 1 and Table 2), seven degrees of unsaturation were due to a keto carbonyl group, two carbon-carbon double bonds, and four ester functionalities; thus, the molecule was heptacyclic. The NMR spectroscopic data of 1 resembled those of thaixylomolin K [6], except for the presence of an 8,14-epoxy group (δC 73.6 (C-8), 65.1 (C-14)), a 3-(2-methyl)butyryloxy function (δH 2.35 (m), 1.46 (m), 1.55 (m), 0.80 (t, J = 7.2 Hz), 1.04 (d, J = 7.2 Hz); δC 175.2 (qC), 40.8 (CH), 26.7 (CH2), 11.6 (CH3), 16.3 (CH3)), and a 30-isobutyryloxy moiety (δH 2.63 (m), 1.09 (d, J = 7.2 Hz), 1.10 (d, J = 7.2 Hz); δC 175.6 (qC), 33.2 (CH3), 19.0 (CH3), 18.7 (CH)) (Table 1 and Table 2, recorded in DMSO-d6), and the absence of the Δ8,14 double bond, the 3-acetoxy group, and the C-30 methine moiety. HMBC cross-peaks between H-9/C-8, H-9/C-14, H2-15/C-8, H2-15/C-14, and H3-18/C-14 confirmed the existence of the 8,14-epoxy group, being corroborated by the degrees of unsaturation of 1. Those from H-3 (δ 5.44 (s)) to the carbonyl carbon (δC 175.2 (C-32)) of the 2-methylbutyryloxy function placed it at C-3, whereas the location of an isobutyroxy moiety at C-30 was supported by the deshielded quaternary C-30 signal (δ 87.2) in 1, as compared to that (δ 63.1) in thaixylomolin K. HMBC correlations from 1-OH (δ 5.86 (br s)) and H2-29 (δ 2.43 (1H, d, J = 12.6 Hz), 2.09 (1H, d, J = 12.6 Hz)) to C-30 (Figure 2a) demonstrated the above deduction.

Table 1

1H (400 MHz) NMR spectroscopic data of compounds 1–4 (δ in ppm, J in Hz).

Position	1 a	1 b	2 a	3 a	4 a
3	5.68 s	5.44 s	5.51 s	5.22 s	5.21 s
5	2.37 m	2.16 br d (8.0)	2.63 m	2.49 c	2.48 c
6a	2.22 br d (14.0)	2.36 c	2.41 c	2.30 br d (12.8)	2.32 br d (12.8)
6b	2.43 br d (14.0)	2.42 m	2.41 c	2.47 c	2.48 c
9	2.07 m	1.97 m		2.58 m	2.59 m
11α	1.36 c	1.41 c	2.20 m	1.77 m	1.78 m
11β	1.36 c	1.19 m	2.50 m	1.50 c	1.50 c
12α	1.28 m	1.13 m	1.59 m	1.44 m	1.44 m
12β	1.34 c	1.17 c	1.45 m	1.49 m	1.50 c
15α	3.06 d (19.0)	3.15 d (19.0)	6.45 br s	3.62 dd (20.0, 3.8)	3.62 dd (20.0, 3.8)
15β	3.28 d (19.0)	3.00 d (19.0)		3.73 dd (20.0, 3.2)	3.74 dd (20.0, 3.2)
17	5.25 s	5.06 s	5.08 s	5.10 s	5.13 s
18	1.13 s	1.02 s	1.05 s	1.05 s	1.05 s
19	1.08 s	0.98 s	1.04 s	1.07 s	1.07 s
21	7.59 br s	7.69 br s	7.47 br s	7.48 br s	7.47 br s
22	6.45 br s	6.52 br s	6.45 br s	6.43 br s	6.42 br s
23	7.43 br s	7.75 br s	7.43 br s	7.43 br s	7.42 br s
28	1.05 s	0.97 s	1.04 s	0.98 s	0.98 s
29pro-S	2.13 d (12.6)	2.09 d (12.6)	1.97 d (12.6)	2.03 d (12.8)	2.03 d (12.8)
29pro-R	2.62 d (12.6)	2.43 d (12.6)	2.65 d (12.6)	2.50 d (12.8)	2.50 d (12.8)
7-OMe-31	3.70 s	3.63 s	3.70 s	3.66 s	3.66 s
3-OAcyl
33	2.43, m	2.35 c	2.53 m	2.49 c	2.65 m
34	1.52 m	1.46 m	1.51 m	1.51 c	1.19 d (7.2)
	1.70 m	1.55 m	1.73 m	1.68 m
35	0.93 t (7.2)	0.80 t (7.2)	0.95 t (7.2)	0.91 t (7.2)	1.20 d (7.2)
36	1.15 d (7.2)	1.04 d (7.2)	1.23 d (7.2)	1.15 d (7.2)
30-OAcyl
38	2.66 m	2.63 m	2.66 m	2.71 m	2.72 m
39	1.21 d (7.0)	1.09 d (7.2)	1.20 d (7.0)	1.20 d (7.0)	1.20 d (7.0)
40	1.21 d (7.0)	1.10 d (7.2)	1.20 d (7.0)	1.26 d (7.0)	1.26 d (7.0)
1-OH		5.86 s

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

Table 2

13C (100 MHz) NMR spectroscopic data of compounds 1–4 (δ in ppm).

Position	1 a	1 b	2 a	3 a	4 a
1	85.5 qC	85.0 qC	88.6 qC	86.2 qC	86.2 qC
2	203.2 qC	205.9 qC	197.9 qC	199.2 qC	199.4 qC
3	83.1 CH	83.4 CH	79.1 CH	79.9 CH	80.2 CH
4	40.6 qC	40.8 qC	41.9 qC	40.5 qC	40.6 qC
5	42.0 CH	42.4 CH	46.4 CH	39.4 CH	39.4 CH
6	33.5 CH2	33.4 CH2	34.1 CH2	34.3 CH2	34.3 CH2
7	172.7 qC	173.6 qC	173.4 qC	172.9 qC	172.9 qC
8	73.5 qC	73.6 qC	124.6 qC	132.2 qC	132.3 qC
9	51.3 qC	51.2 qC	161.9 qC	46.8 CH	46.7 CH
10	53.1 qC	53.5 qC	60.5 qC	56.4 qC	56.4 qC
11	16.7 CH2	16.5 CH2	20.6 CH2	18.5 CH2	18.6 CH2
12	30.1 CH2	30.3 CH2	30.7 CH2	31.3 CH2	31.3 CH2
13	38.1 qC	37.9 qC	37.8 qC	41.1 qC	41.1 qC
14	65.4 qC	65.1 qC	151.7 qC	139.4 qC	139.3 qC
15	35.2 CH2	35.5 CH2	113.8 CH	32.8 CH2	32.9 CH2
16	169.1 qC	169.0 qC	165.4 qC	169.5 qC	169.5 qC
17	76.9 CH	76.9 CH	80.2 CH	80.3 CH	80.3 CH
18	16.2 CH3	16.6 CH3	16.3 CH3	16.6 CH3	16.7 CH3
19	16.2 CH3	16.0 CH3	13.7 CH3	15.6 CH3	15.6 CH3
20	119.8 qC	120.3 qC	120.3 qC	120.4 qC	120.4 qC
21	141.7 CH	141.7 CH	141.2 CH	141.3 CH	141.3 CH
22	109.5 CH	110.0 CH	110.1 CH	110.0 CH	110.0 CH
23	143.4 CH	144.6 CH	143.0 CH	143.1 CH	143.0 CH
28	19.1 CH3	18.8 CH3	21.2 CH3	19.9 CH3	19.9 CH3
29	42.9 CH2	43.3 CH2	41.2 CH2	42.9 CH2	42.9 CH2
30	86.3 qC	87.2 qC	92.6 qC	91.4 qC	91.4 qC
7-OMe-31	51.9 CH3	52.1 CH3	52.0 CH3	51.7 CH3	51.8 CH3
3-OAcyl
32	175.3 qC	175.2 qC	175.7 qC	175.6 qC	176.0 qC
33	40.9 CH	40.8 CH	40.5 CH	40.7 CH	33.9 CH
34	26.8 CH2	26.7 CH2	26.6 CH2	27.0 CH2	18.5 CH3
35	11.3 CH3	11.6 CH3	11.6 CH3	11.5 CH3	18.9 CH3
36	16.1 CH3	16.3 CH3	16.5 CH3	16.3 CH3
30-OAcyl
37	175.4 qC	175.6 qC	175.9 qC	177.3 qC	177.4 qC
38	33.5 CH	33.2 CH	33.8 CH	33.6 CH	33.6 CH
39	18.7 CH3	19.0 CH3	18.8 CH3	19.1 CH3	19.1 CH3
40	18.8 CH3	18.7 CH3	19.1 CH3	18.9 CH3	19.1 CH3

a Recorded in CDCl3; b Recorded in DMSO-d6.

Figure 2

(a) Selected 1H-1H COSY and HMBC correlations for compound 1 (measured in DMSO-d6); (b) Diagnostic Nuclear Overhauser Effect (NOE) interactions for compound 1 (measured in DMSO-d6).

The relative configuration of 1 was assigned by analysis of the Nuclear Overhauser Effect (NOE) interactions (Figure 2b), of which those between H-17/H-11β, H-11β/H-5, H-17/H-5, and H-5/H3-28 revealed their cofacial relationship and were arbitrarily assigned as the β-oriented H-5, H-17, and H3-28. In turn, those between H3-18/H-15α, H3-19/H-9, H3-19/1-OH, and H-9/1-OH assigned the α-orientation for H3-18, H3-19, H-9, and 1-OH. The significant NOE interaction from H-3 to H-29 (δ 2.43 (d, J = 12.6 Hz)), but not from H-3 to H-5, established 3α-H and the corresponding 3β-(2-methyl)butyryloxy function. However, the orientation of the 8,14-epoxy group could not be determined by NOE interactions.

After considerable effort, suitable crystals of 1 were obtained in acetone/n-hexane (4:1) at room temperature. Thus, single-crystal X-ray diffraction analysis, conducted with CuKα radiation, was carried out to establish the relative and absolute configurations of 1. The absolute configuration of 1, named krishnolide A, was unequivocally established as 1R,3S,4R,5S,8R,9R,10S,13S,14S,17S,30R,33S (Flack parameter of −0.06 (10), Flack x of −0.00(9), and Hooft y of −0.00(4)) [22]. Computer-generated perspective drawings of the X-ray structure of 1 are shown in Figure 3 and Figure S1.

Figure 3

Oak Ridge Thermal-Ellipsoid Plot Program (ORTEP) illustration of the X-ray structure of compound 1. Ellipsoids are given at the 30% probability level.

Compound 2, a white amorphous powder, had the molecular formula C36H44O11 as established by the positive HR-ESIMS ion peak at m/z 653.2956 (calcd. for [M + H]+ 653.2956). The 1H and 13C NMR spectroscopic data of 2 (Table 1 and Table 2) were similar to those of 1 except for the replacement of the 8,14-epoxy group in 1 by conjugated Δ8,9 and Δ14,15 double bonds (δC 124.6 (C-8, qC), 161.9 (C-9, qC), 151.7 (C-14, qC), 113.8 (C-15, CH)), which was corroborated by HMBC correlations between H-15/C-8, H-15/C-13, H-15/C-14, H-15/C-16, H3-18/C-14, and H3-19/C-9. The relative configuration of 2 was determined to be the same as that of 1 by NOE interactions between H-17/H-12β, H-5/H-11β, H-5/H3-28, H-19/H-29, and those between H3-18/H-11α, H-3/H-29. The absolute configuration of 2, named krishnolide B, was established to be the same as that of thaixylomolin N (1R,3S,4R,5S,10S,13R,17R,30S-configured) by the similarity of their electronic circular dichroism (ECD) spectra (Figure 4a) [6].

Figure 4

(a) Comparison of the experimental circular dichroism (CD) spectra of compound 2 and the known compound, thaixylomolin N, containing conjugated Δ8,9 and Δ14,15 double bonds; (b) Comparison of the experimental CD spectra of compounds 3, 4, and the known compound, thaixylomolin L, containing a Δ8,9 double bond.

Compound 3 provided the molecular formula C36H46O11, as established by the positive HR-ESIMS ion peak at m/z 677.2917 (calcd. for [M + Na]+ 677.2932). The NMR spectroscopic data of 3 (Table 1 and Table 2) displayed structural similarity to that of 1, except for the replacement of the 8,14-epoxy group in 1 by a Δ8,14 double bond (δC 132.2 (C-8, qC), 139.4 (C-14, qC)) in 3. 1H-1H COSY homoallylic couplings between H2-15/H-9 and HMBC correlations between H2-15/C-8, H2-15/C-14, H3-18/C-14, H-17/C-14, and H-9/C-8 confirmed the existence of the Δ8,14 double bond. The relative configuration of 3 was deduced to be identical to that of 1 by NOE interactions between H-17/H-12β, H-11β/H-5, H-17/H-5, H-5/H3-28, and those between H3-18/H-15α, H3-18/H-9, H3-19/H-9, H-3/H -29. The absolute configuration of 3, named krishnolide C, was established to be the same as that of thaixylomolin L (1R,3S,4R,5S,9R,10S,13R,17R,30S-configured) by the similarity of their ECD spectra (Figure 4b) [6].

The molecular formula of 4 was assigned as C35H44O11 by the positive HR-ESIMS ion peak at m/z 663.2771 (calcd. for [M + Na]+ 663.2776). The NMR spectroscopic data of 4 (Table 1 and Table 2) were similar to those of 3, except for the replacement of the 3-(2-methyl)butyryloxy group in 3 by an isobutyryloxy moiety (δH 2.65 (1H, m, H-33), 1.19 (3H, d, J = 7.2 Hz, H3-34), 1.20 (3H, d, J = 7.2 Hz, H3-35); δC 176.0 (C-32, qC), 33.8 (C-33, CH), 18.5 (C-34, CH3), 18.9 (C-35, CH3)) in 4. The existence of the isobutyryloxy group was corroborated by 1H-1H COSY cross-peaks between H-33/H3-34 and H-33/H3-35, and HMBC correlations between H-33/C-32, H3-34/C-32, and H3-35/C-32. The strong HMBC correlation from H-3 (δ 5.21, s) to the carbonyl carbon (δ 176.0 (C-32)) of the isobutyryloxy group confirmed its location at C-3. The relative configuration of 4 was determined to be the same as that of 3 based on NOE interactions between H-17/H-12β, H-11β/H-5, H-5/H3-28, and those between H3-18/H-15α, H3-18/H-9, H3-19/H-9, H-3/H-29. The absolute configuration of 4, named krishnolide D, was established to be the same as that of 3 (1R,3S,4R,5S,9R,10S,13R,17R,30S-configured) by the similarity of their ECD spectra (Figure 4b) [6].

Anti-HIV activities of 1–4 were tested in vitro by HIV-I virus-transfected 293 T cells [23]. At the concentration of 20 μM, only 1 showed a strong inhibitory rate of 79.75 ± 0.77%. Efavirenz was used as the positive control, with an inhibitory rate of 88.54 ± 0.45% at the same concentration. Values of IC50 and CC50 for 1 were 17.45 ± 1.65 and 78.45 ± 1.69 μM, respectively. Structure-activity relationship analyses of 1–4 concluded that the 8,14-epoxy group is pivotal for the anti-HIV activity of khayanolides. However, only two other khayanolides with an 8,14-epoxy group, viz. khayanolide A and 1-O-acetylkhayanolide A, have hitherto been reported [9,12].

Previously, 36 khayanolides were reported. These khayanolides exhibited antifeedant, anti-acetylcholinesterase, anti-butyrycholinesterase, anti-lipoxygenase, antitumor, antimicrobial, and anti-H1N1 (antiviral) activities [8,9,10,11,12,13,14,15,16,17,18,19,20,21], but no khayanolide has previously been reported to have anti-HIV activity. In this paper, the anti-HIV activities of khayanolides were reported for the first time.

3. Materials and Methods

3.1. General Methods

Optical rotations were determined with an MCP 200 modular circular polarimeter (Anton Paar Opto Tec GmbH, Seelze, Germany). UV spectra were recorded on a GENESYS 10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Shanghai, China), and HR-ESIMS was obtained on an LC-ESIMS system in the positive-ion mode (Bruker Daltonics, Bremen, Germany). NMR spectra were measured on a Bruker AV-400 NMR spectrometer. Preparative HPLC was carried out on C18 reversed-phase columns (YMC, 250 × 10 mm i.d., 5 μm) using a Waters 2535 pump equipped with a Waters 2489 UV detector (Waters Corporation, Milford, MA, USA). Single-crystal X-ray diffraction analysis of 1 was performed on an Agilent Xcalibur Atlas Gemini Ultra-diffractometer with mirror monochromated CuKα radiation (λ = 1.54184 Å) at 150 K. Silica gel (100–200 mesh) (Qingdao Mar. Chem. Ind. Co. Ltd., Qingdao, China) and C18 reversed-phase silica gel (50 μm, YMC Co. Ltd., Kyoto, Japan) were used for column chromatography.

3.2. Plant Material

Seeds of Xylocarpus moluccensis were collected in September 2007 at the mangrove swamp of Krishna estuary, Andhra Pradesh, India. Identification of the mangrove was done by one of the authors (T.S.). A voucher sample (No. IXM200701) is maintained in Marine Drugs Research Center, College of Pharmacy, Jinan University.

3.3. Extraction and Isolation

The air-dried seeds (6.0 kg) of X. moluccensis were powdered and extracted with 95% EtOH (6 × 15 L) at room temperature to afford the resulting extract (824.6 g), which was suspended in water (2.5 L) and extracted with EtOAc (6 × 7.5 L) to afford the EtOAc portion (299.1 g). The EtOAc portion was chromatographed on a silica gel column (150 × 8.5 cm i.d.), eluted with a gradient mixture of CHCl3/MeOH (100:0 to 5:1, v/v) to afford 223 fractions. Fractions 68–71 (22.9 g) were combined and further purified by C18 reversed-phase silica gel column chromatography (60 × 3 cm i.d.), and eluted with a gradient mixture of acetone/H2O (40:60 to 100:0, v/v) to yield 111 subfractions, among which subfractions 63–65 (351.2 mg) were combined and purified by preparative HPLC (MeCN/H2O, 55:45) to give compounds 1 (t = 51.5 min, 20.2 mg) and 2 (t = 46.6 min, 4.4 mg). Fractions 58–67 (43.0 g) were combined and separated by C18 reversed-phase silica gel column chromatography (58 × 5.5 cm i.d.), and eluted with a gradient mixture of acetone/H2O (40:60 to 100:0, v/v) to give 136 subfractions, among which the subfraction 98 (1.5 g) was subjected to preparative HPLC (MeCN/H2O, 55:45) to yield compound 3 (t = 45.5 min, 7.1 mg), whereas the subfraction 100 (820.2 mg) was purified by preparative HPLC (MeOH/H2O, 65:35) to give compound 4 (t = 48.3 min, 2.5 mg).

Krishnolide A (1). Colorless crystal, = −48 (c 0.1, acetone); UV (MeCN) λmax (logε) 208 (3.80) nm; ECD (c 1.49 mM, MeCN) λmax (Δε) 195 (−0.4), 208 (−0.5), 229 (−0.8), 326 (+0.5) nm; HR-ESIMS m/z 693.2871 [M + Na]+ (calcd. for C36H46NaO12, 693.2887); 1H and 13C NMR spectroscopic data see Table 1 and Table 2.

Krishnolide B (2). White, amorphous solid, = +36 (c 0.1, acetone); UV (MeCN) λmax (logε) 207 (3.81), 282 (3.99) nm; ECD (c 0.19 mM, MeCN) λmax (Δε) 222 (+3.0), 286 (+14.8), 324 (−7.7) nm; HR-ESIMS m/z 653.2956 [M + H]+ (calcd. for C36H45O11, 653.2962); 1H and 13C NMR spectroscopic data see Table 1 and Table 2.

Krishnolide C (3). White, amorphous solid, = −38 (c 0.1, acetone); UV (MeCN) λmax (logε) 206 (3.84) nm; ECD (c 0.38 mM, MeCN) λmax (Δε) 201 (+4.2), 225 (−5.0), 325 (+0.7) nm; HR-ESIMS m/z 677.2917 [M + Na]+ (calcd. for C36H46NaO11, 677.2932); 1H and 13C NMR spectroscopic data see Table 1 and Table 2.

Krishnolide D (4). White, amorphous solid, = −54 (c 0.1, acetone); UV (MeCN) λmax (logε) 204 (4.35) nm; ECD (c 0.39 mM, MeCN) λmax (Δε) 204 (+0.7), 224 (−4.2), 286 (+1.5) nm; HR-ESIMS m/z 663.2771 [M + Na]+ (calcd. for C35H44NaO11, 663.2776); 1H and 13C NMR spectroscopic data see Table 1 and Table 2.

3.4. X-ray Crystal Data for Krishnolide A (

Monoclinic, C36H46O12, space group P2(1), a = 9.33350 (10) Å, b = 18.00600 (10) Å, c = 10.73330 (10) Å, α = 90°, β = 113.9990 (10)°, γ = 90°, V = 1647.89 (3) Å3, Z = 2, Dcalcd. = 1.352 Mg/m3, μ = 0.839 mm−1. Crystal size: 0.40 × 0.34 × 0.30 mm3. 5929 measured reflections, 5810 (Rint = 0.0350) independent reflections, 442 parameters, one restraints, F(000) = 716, R1 = 0.0312, wR2 = 0.0800 (all data), R1 = 0.0304, wR2 = 0.0789 (I > 2σ(I)), and goodness-of-fit (F2) = 1.048. The absolute structural parameter is −0.06(10), the Flack x parameter is −0.00(9), and the value of Hooft y is −0.00(4).

CCDC-1573579 (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. HIV-Inhibitory Bioassay

A total of 293 T cells (2 × 105) were co-transfected with 0.6 μg of pNL-Luv-E−-Vpu− and 0.4 μg of 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). SupT1 cells were exposed to VSV-G pseudotyped HIV-1 (MOI = 1) at 37 °C for 48 h in the absence or presence of the test compounds 1–4 (Efavirenz was used as the positive control). The inhibition rates were determined by using a firefly Luciferase Assay System (Promega) [23].

4. Conclusions

In conclusion, four new khayanolides, containing a 2-methylbutyryloxy or an isobutyroxy substituent at the C-3 and C-30 positions, respectively, were first obtained from the seeds of an Indian mangrove, Xylocarpus moluccensis. The relative and absolute configurations of these compounds were established by HR-ESIMS, extensive NMR investigations, single-crystal X-ray diffraction analysis with CuKα radiation, and experimental electronic circular dichroism (ECD) spectra. Krishnolide A exhibited moderate anti-HIV activity. The 8,14-epoxy group is pivotal for its anti-HIV activity. This is the first anti-HIV report of khayanolides.