Limonoids Containing a C1–O–C29 Moiety: Isolation, Structural Modification, and Antiviral Activity

Five new limonoids named thaigranatins A–E (1–5), containing a C1–O–C29 moiety, were isolated from seeds of the Thai Xylocarpus granatum, collected at the mangrove swamp of Trang Province, together with the known limonoid, granatumin L (6). The structures of these compounds were established by HR-ESIMS and extensive NMR spectroscopic data. The absolute configuration of 1 was unequivocally determined by single-crystal X-ray diffraction analysis, conducted with Cu Kα radiation; whereas that of 2 or 6 was established to be the same as that of 1 by the similarity of their electronic circular dichroism (ECD) spectra. In view of the marked antiviral activity of 6, its structure was modified via hydrolysis with alkaline KOH, esterification with diazomethane and various organic acids, and oximization with hydroxyamine. Finally, 18 derivatives, viz. 7–10, 8a–8i, 9a–9b, and 10a–10c, were obtained. In vitro antiviral activities of these derivatives against human immunodeficiency virus 1 (HIV-1) and influenza A virus (IAV) were evaluated. Most notably, 8i exhibited marked inhibitory activity against HIV-1 with an IC50 value of 15.98 ± 6.87 μM and a CC50 value greater than 100.0 μM; whereas 10b showed significant inhibitory activity against IAV with an IC50 value of 14.02 ± 3.54 μM and a CC50 value greater than 100.0 μM.


Introduction
Xylocarpus granatum, a true mangrove plant, belongs to the family Meliaceae, of which the main secondary metabolites are limonoids [1,2]. Krishnolide A, a khayanolide-type of limonoid isolated from the Indian mangrove, Xylocarpus moluccensis, showed moderate antiviral activity against human immunodeficiency virus 1 (HIV-1) with an IC 50 value of 17.45 ± 1.65 µM and a CC 50 value of 78.45 ± 1.69 µM, respectively [3]. Three other khayanolide-type of limonoids obtained from the Thai Xylocarpus moluccensis, viz. thaixylomolins I, K, and M, exhibited moderate antiviral activity against influenza A virus (IAV). The most potent one is thaixylomolin I with an IC 50 value of 77.1 ± 8.7 µM [4].
To search for new antiviral natural compounds from mangrove plants, seeds of the Thai X. granatum, collected in the mangrove swamp of Trang Province, were investigated to afford five new limonoids containing a C 1 -O-C 29 moiety, named thaigranatins A-E (1-5), along with the known one, granatumin L (6) (Figure 1), which exhibited antiviral activity against HIV-1. To find promising antiviral drug leads, structural modification was applied to granatumin L (6). Herein, we report the isolation and structural elucidation of thaigranatins A-E (1-5), the structural modification of granatumin L (6), and antiviral activities of the modified derivatives of 6 against HIV-1 and IAV. To search for new antiviral natural compounds from mangrove plants, seeds of the Thai X. granatum, collected in the mangrove swamp of Trang Province, were investigated to afford five new limonoids containing a C1-O-C29 moiety, named thaigranatins A-E (1-5), along with the known one, granatumin L (6) (Figure 1), which exhibited antiviral activity against HIV-1. To find promising antiviral drug leads, structural modification was applied to granatumin L (6). Herein, we report the isolation and structural elucidation of thaigranatins A-E (1-5), the structural modification of granatumin L (6), and antiviral activities of the modified derivatives of 6 against HIV-1 and IAV.

Results and Discussion
2.1. Structure Identification of Natural Limonoids 1-5 Compound 1 was obtained as colorless crystals. Its molecular formula C32H40O10, indicating 13 degrees of unsaturation, was established by the positive HR-ESIMS ion peak at m/z 607.2509 (calcd. for [M + Na] + 607.2514). According to the 1 H and 13 C NMR spectroscopic data (Tables 1 and 2), 7 degrees of unsaturation were due to 3 carbonyl groups and 4 carbon-carbon double bonds; thus, the molecule was hexacyclic. The 13 C NMR spectroscopic data and DEPT experiments indicated the presence of 6 methyl groups (a methoxy group, a methyl group linked to a secondary carbon and 4 methyl groups linked to tertiary carbons), 4 methylene groups, 12 methine groups (including 5 olefinic), and 10 quaternary carbons (including 3 carbonyl groups) in 1.

Results and Discussion
2.1. Structure Identification of Natural Limonoids 1-5 Compound 1 was obtained as colorless crystals. Its molecular formula C 32 H 40 O 10 , indicating 13 degrees of unsaturation, was established by the positive HR-ESIMS ion peak at m/z 607.2509 (calcd. for [M + Na] + 607.2514). According to the 1 H and 13 C NMR spectroscopic data (Tables 1 and 2), 7 degrees of unsaturation were due to 3 carbonyl groups and 4 carbon-carbon double bonds; thus, the molecule was hexacyclic. The 13 C NMR spectroscopic data and DEPT experiments indicated the presence of 6 methyl groups (a methoxy group, a methyl group linked to a secondary carbon and 4 methyl groups linked to tertiary carbons), 4 methylene groups, 12 methine groups (including 5 olefinic), and 10 quaternary carbons (including 3 carbonyl groups) in 1.

Structural Modifications of Granatumin L (6)
In our previous bioassay, granatumin L (6) showed an inhibitory rate of 67.10 ± 3.04% against HIV-1 at the concentration of 20 µM. Thus, structural modification of the natural compound 6, particularly the bioequivalent substitution of the C-3 natural substituent, was investigated to find its potent and selective antiviral analogs. To achieve this purpose, 3 different protocols, viz. hydrolysis with alkaline KOH, esterification with diazomethane and various organic acids, and oximization with hydroxyamine, were employed (Scheme 1). Compound 7 was obtained by the hydrolysis of 6 with an alkaline KOH. The ester functions at the C-3 and C-7 positions of 7 were hydrolyzed to give a hydroxy group at the C-3 position and a carboxyl group at the C-7 position, respectively, in the yield of 96.8%. Owing to the formation of the stable six-membered ring, the δ-lactone function of ring-D was not hydrolyzed. Then, 7 was treated with diazomethane to afford 8 as expected. Afterwards, the hydroxy group at the C-3 position of 8 was oxidized with IBX to give 9 with a carbonyl group at the C-3 position. Fortunately, the hemiacetal function of 8 was retained under the reaction condition.

Mar. Drugs
Alternatively, 7 was first oxidized and then methylated to prepare 9. An intermediate compound 10 was obtained in the high yield of 98.8%. However, the final yield of 9 was 76.2%, which made this strategy inadvisable. Finally, the hydroxy group at the C-3 position of 8 was esterified with various organic acids to afford a series of compounds 8a-8i. The bioisosteric substitution of the ketone function at the C-3 position of 9 and 10 with different oximes led to the generation of derivatives 9a-9b and 10a-10c, respectively.

Antiviral Activity Bioassay
The inhibitory activities of natural limonoid 6 and all the modified derivatives 7-10, 8a-8i, 9a-9b, and 10a-10c against HIV-1 were tested [14]. Efavirenz was used as the positive control. The results were summarized in Table 3. The lack of substituent at the C-3 position of 8, 9, and 10 combined with the disappearance of their anti-HIV-1 activities indicated that the substituent at the C-3 position is Compound 7 was obtained by the hydrolysis of 6 with an alkaline KOH. The ester functions at the C-3 and C-7 positions of 7 were hydrolyzed to give a hydroxy group at the C-3 position and a carboxyl group at the C-7 position, respectively, in the yield of 96.8%. Owing to the formation of the stable six-membered ring, the δ-lactone function of ring-D was not hydrolyzed. Then, 7 was treated with diazomethane to afford 8 as expected. Afterwards, the hydroxy group at the C-3 position of 8 was oxidized with IBX to give 9 with a carbonyl group at the C-3 position. Fortunately, the hemiacetal function of 8 was retained under the reaction condition.
Alternatively, 7 was first oxidized and then methylated to prepare 9. An intermediate compound 10 was obtained in the high yield of 98.8%. However, the final yield of 9 was 76.2%, which made this strategy inadvisable. Finally, the hydroxy group at the C-3 position of 8 was esterified with various organic acids to afford a series of compounds 8a-8i. The bioisosteric substitution of the ketone function at the C-3 position of 9 and 10 with different oximes led to the generation of derivatives 9a-9b and 10a-10c, respectively.

Antiviral Activity Bioassay
The inhibitory activities of natural limonoid 6 and all the modified derivatives 7-10, 8a-8i, 9a-9b, and 10a-10c against HIV-1 were tested [14]. Efavirenz was used as the positive control. The results were summarized in Table 3. The lack of substituent at the C-3 position of 8, 9, and 10 combined with the disappearance of their anti-HIV-1 activities indicated that the substituent at the C-3 position is essential for anti-HIV-1 activity of this limonoid skeleton. The oxidation of the hydroxy group at the C-3 position into a carbon-oxygen or carbon-nitrogen double bond did not improve anti-HIV-1 activity. For example, derivatives 9a-9b and 10a-10c containing a C=O or C=N double bond exhibited no activity.
Anti-HIV-1 bioassay (Table 3) revealed that the C-3 esterified products, particularly derivatives with a fatty acid ester at the C-3 position, markedly enhanced anti-HIV-1 activity. The derivative 8i exhibited the highest inhibition rate of 99.95 ± 0.01% at the concentration of 20.0 µM; whereas 8g showed an inhibition rate of 50.84 ± 6.96% at the same concentration. The IC 50 values for 8i and 8g are 15.98 ± 6.87 and 21.98 ± 4.65 µM, respectively, among which the former is better than that of 6 (19.23 ± 0.12 µM). The results indicated that the introduction of alkyl groups at the C-3 position helped to maintain anti-HIV-1 activity. However, compounds with aromatic substitution at the C-3 position showed decreased activity. Antiviral activities of all the modified derivatives of 6 against IAV were also evaluated [14]. Ribavirin was used as the positive control. Derivatives 8i and 8g exhibited no inhibitory activities against IAV at the concentration of 20.0 µM; whereas 8b, 8c, 8d, and 10b showed marked anti-IAV activities with IC 50 values in the range of 14.0-22.8 µM (Table 4). The strongest inhibitory activity of 10b revealed the importance of a C-3-substituted O-methyl oxime group.
The above anti-HIV-1 and anti-IAV activities of modified derivatives shed light on the structural optimization of natural limonoid 6, particularly suitable substituted groups at the C-3 position. In-depth structural modification and structure-activity relationship studies of the limonoid skeleton of 6 will be proceeded by us in the near future.

Plant Material
Seeds of Xylocarpus granatum were collected in August 2012 from the Thai mangrove swamps of the Trang Province. Identification of the mangrove was done by one of the authors (J.W.). A voucher sample (No. ThaiXG-02) is maintained in Marine Drugs Research Center, College of Pharmacy, Jinan University.

Extraction and Isolation
The air-dried and powdered seeds (9.0 kg) of Xylocarpus granatum were extracted 5 times with EtOH (95%, v/v) at room temperature to afford the resulting extract (1363.6 g). After removal of the solvent under vacuum, the residue was partitioned between water and EtOAc to yield the EtOAc portion (307.7 g). The EtOAc portion (150 g) was chromatographed on a silica gel column  Granatumin L (6) (500.0 mg) was stirred in methanol (25 mL) at room temperature for 20 min. Then 10% aqueous KOH solution was slowly added, and the mixture was stirred at room temperature for 48 h. Thin-layer chromatography (TLC) was used to monitor the products. After the reaction was completed, methanol was removed under reduced pressure. The remaining solution was neutralized with 10% HCl to pH 5-6, followed by C 18 reversed-phase (RP 18

General Procedure for the Preparation of 8a-8i
Compound 8 (5.0 mg, 0.01 mmol) and various organic acid (1.5 eq.) were dissolved in dry dichloromethane (2.0 mL). Then the mixture was combined with DCC (1.5 eq.) and DMAP (1.0 eq.), and then stirred at room temperature for 3-5 h [16]. TLC was used to monitor the products. After the reaction was completed, the reaction solvent was evaporated. The resulting residue was dissolved in acetone or acetonitrile; whereas the insoluble matter was precipitated by centrifugation. The supernatant was purified by RP 18 HPLC to afford the pure corresponding esterified products (8a-8i).    of pNL-Luv-E − -Vpu − and 0.4 µg of pHIT/G. After 48 h, the VSV-G pseudo-typed viral supernatant 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 pseudo-typed virus (MOI = 1) at 37 • C for 48 h in the absence or presence of compounds at different concentrations. The inhibition rate was determined by using a firefly luciferase assay system (Promega, Beijing). Efavirenz was used as the positive control for HIV-1; whereas ribavirion was used as the positive control for IAV.

Conclusions
In conclusion, five new limonoids named thaigranatins A-E (1-5), containing a C 1 -O-C 29 moiety, were isolated from seeds of the Thai Xylocarpus granatum, collected at the mangrove swamp of Trang Province, together with the known one, granatumin L (6). The structures of these limonoids, including absolute configurations of 1, 2, and 6 were established by HR-ESIMS, extensive NMR investigations, single-crystal X-ray diffraction analysis (Cu Kα radiation), and comparison of experimental ECD spectra. The structural modification of 6 via hydrolysis, esterification, and oximization, afforded 18 derivatives, viz. 7-10, 8a-8i, 9a-9b, and 10a-10c. The derivative 8i exhibited marked inhibitory activity against HIV-1 with an IC 50 value of 15.98 ± 6.87 µM and a CC 50 value greater than 100.0 µM; whereas 10b showed significant inhibitory activity against IAV with an IC 50 value of 14.02 ± 3.54 µM and a CC 50 value greater than 100.0 µM. These results demonstrate that the mangrove X. granatum continues to be an abundant resource to produce new antiviral limonoids. In-depth structural modification and structure-activity relationship studies of the limonoid skeleton of 6 will be proceeded by us for the discovery of antiviral drug leads.