Abstract
Quinone-methide triterpenes of the tingenone series are effective cytotoxic and antimicrobial agents. Under an acidic condition, they can rearrange to the phenolic analogues. In the current study, six new compounds (7–12) along with three known products (4–6) were obtained from the acid-rearranged reaction of tingenone derivatives (1–3). Their structures were determined on the basis of spectroscopic evidence. Antiviral and antimicrobial activities of all compounds were evaluated. Data associated to their structures were suggested. The quinone-methide chromophore at rings A/B was necessary for antimicrobial activity. The acid-rearranged phenolic analogues were less toxic but lost antimicrobial potential. However, compounds with the phenolic ring conjugated to two vinyl groups (compounds 4–6) had an inhibitory effect on Herpes simplex virus with a selective index greater than 5.
Introduction
Tingenone is a quinone-methide triterpene isolated from plants of the family Celastraceae. This compound and other quinone-methide triterpenes have been reported for several interesting biological activities, such as cytotoxic (Kutney et al., 1981; Ngassapa et al., 1994), antibacterial (Moujir et al., 1990; Sotanaphun et al., 1999), antifungal (Sotanaphun et al., 1999) and antiparasitic (Goijman et al., 1985) activities. The quinone-methide system at rings A/B of these compounds is important for their bioactivities (Gonzalez et al., 1988; Hideji et al., 1991; Osamu et al., 1994), whereas the oxygenated substitution on ring E is associated with potency (Gonzalez et al., 1988; Moujir et al., 1990). Under an acidic condition, the quinone-methide chromophore can easily rearrange to phenolic systems (Nakanishi et al., 1965; Delle Monache et al., 1973, 1979; Pomponi et al., 1974; Sotanaphun et al., 1999). Three isomers, isomers I, II, and III, have been determined as their products. Isomers III of tingenone derivatives (compounds 4–6; ) were demonstrated to lose antimicrobial activity (Sotanaphun et al., 1999). However, in our continuing investigation, these compounds were found to have an inhibitory effect on Herpes simplex virus. Moreover, six new compounds (7–12) were isolated from the reaction products of the acid-catalyzed reaction of tingenone derivatives (1–3). Their antiviral and antimicrobial activities were evaluated.
Figure 1 Structures of compounds 1–12.
Materials and Methods
General experimental procedures
UV and IR spectra were recorded on a Hitachi U-2000 spectrophotometer (Tokyo, Japan) and a Nicolet Magna-IR spectrometer 750 (USA). Optical rotations were measured with a Perkin Elmer 341 polarimeter (USA). Electron impact mass spectrometry (EI-MS) was obtained on a Hewlett Packard 5989B mass spectrometer (USA). NMR spectra were recorded at 500 MHz for 1H and 125 MHz for 13C on a JEOL-A500 (Alpha series) spectrometer (Japan) in CDCl3 with trimethylsilane (TMS) as internal standard. Heteronuclear multiple bonded spectometry (HMBC) experiments were optimized for J. = 8 Hz. Thin-layer chromatography (TLC) was developed on a precoated silica gel 60 F254 (0.2 mm thick, Merck, Darmstadt, Germany) and sprayed with diluted H2SO4 in ethanol and heated at 110°C for 15 min. Flash chromatography was performed on a silica gel 60 (15–40 µm, Merck). Sephadex LH20 (Pharmacia) was used as adsorbent for gel chromatography.
Compounds 1–3, used as substrates of the reaction, were isolated from the stem bark of Glyptopetalum sclerocarpum. Laws (Sotanaphun et al., 1998).
Preparation of acid-rearranged compounds (4–8) in methanol
Isotingenone III (4), 22β-hydroxy-isotingenone III (5), and 20-hydroxy-20-epi.-isotingenone III (6) were prepared from tingenone III (1), 22β-hydroxytingenone (2), and 20-hydroxy-20-epi.-tingenone (3), respectively, using the method modified from Sotanaphun et al. (1999). Each parent compound (500 mg) was dissolved in 1% v/v of concentrated HCl in methanol and left at 5°C for 1 h. The product solution was then diluted with water and extracted with EtOAc. The EtOAc extract was washed with water and dried over anhydrous sodium sulfate. After concentration, the main resulting product, isomer III, was crystallized. A flash chromatography eluted with hexane/acetone (3:1) was carried out to get more yield of the isomer III from the mother liquor. Compounds 4 (206 mg), 5 (420 mg), and 6 (314 mg) were obtained.
Two other main compounds were detected in the less polar fractions (F1 and F2) during the isolation of 6. F1 (36 mg) was subjected to a flash chromatography eluted with hexane/acetone gradient (9:1 to 4:1) to yield compound 7 (15 mg). F2 (55 mg) was consequently purified by using a gel chromatography eluted with EtOAc/MeOH (4:1) and a flash chromatography using hexane/diethyl ether (1:1) solvent system to obtain compound 8 (16 mg).
Preparation of acid-rearranged compounds (9–12) in acetone
Compounds 1–3 (each 50 mg) were examined under the same procedure as the method described above, except that acetone was used instead of methanol as solvent. The total crude products were purified by flash chromatography using hexane/acetone (4:1) as an eluent to yield compounds 9 (44 mg), 10 (45 mg), and 11 (42 mg), respectively.
The more polar chromatographic fraction during the purification of compound 10 was further passed through a gel chromatograph eluted with EtOAc/MeOH (4:1) to obtain compound 12 (2.5 mg).
Antiherpetic assay
The antiherpetic activity was determined by a plaque reduction assay (Abou-Karam & Shier, 1990). A stock solution of 10 mg/ml in dimethyl sulfoxide (DMSO) for substance tested was prepared and two-fold dilution in complete media of the test substance, starting at the maximum noncytotoxic concentration, was made (concentration of DMSO in tested concentration was less than 2%). Each concentration of substance was mixed with an equal volume (25 µl) of HSV-1 (KOS) and HSV-2 (Baylor186) containing 30 plaque forming units (PFU) in 96-well microtiter plate. The mixture was incubated at room temperature for 1 h before 50 µl of Vero cell suspension (6 × 105 cells/ml) were added and further incubated at 37°C in 5% CO2 humidified incubator for another 1 h. The overlay medium containing corresponding concentration of substance was added, and the cells were incubated for 2 days. The concentration that produces 50% inhibition of plaque formation (ED50) was determined. Acyclovir was used as a positive control.
Antimicrobial assay
The antimicrobial assay was performed according to the broth dilution technique (Cleeland & Squires, 1991) and reported as the minimal inhibitory concentration (MIC). Gram-positive bacteria (Staphylococcus aureus. ATCC 6538P and Bacillus subtilis. ATCC 6633) and Gram-negative bacterium (Escherichia coli. ATCC 25922) were maintained on Muller Hinton media (Difco, USA). Yeast (Candida albicans. ATCC 10230) and fungus (Microsporum gypseum. pathological stain) were cultured on Sabouraud dextrose media (Difco, USA). The inocula of all microorganisms were adjusted to MacFarland no. 0.5 turbidity. Two-fold serial dilution of each test compound was prepared in DMSO, and 0.25 ml of each dilution was mixed with 4.5 ml of broth media and 0.25 ml of the microorganism inoculum. After incubation at 37°C for 1 day for bacteria, 3 days at 30°C for yeast, and 5 days at 30°C for fungus, the MIC values were determined as the lowest concentration that visibly inhibited the microorganism growth.
20-Hydroxy-20-epi-isotingenone IV (7)
Pale yellow powder (EtOAc); [α]D−176° (c 0.15, MeOH); UV (MeOH) λmax (log ∈): 235 (4.29), 260 (3.92), 333 (3.55) nm; IR (film) νmax: 3600–3100, 2930, 1715, 1652, 1631, 1455, 1394, 1270, 756 cm−1; 1 HNMR (CDCl3, 500 MHz):; 13C NMR (125 MHz, CDCl3): ; EI-MS(70 eV) m./z. (rel. int.): 436 [M]+ (17), 418 [M − H2O]+ (3), 256 (3), 241 (10), 215 (19), 202 (16), 201 (100), 200 (69).
Table 1.. 1H NMR data of compounds 7–12 (δ in ppm, 500 MHz, CDCl3).
19,20-Didehydro-isotingenone III (8)
Pale yellow powder (EtOAc); [α]D 87° (c 0.1, MeOH); UV (MeOH) λmax (log ∈): 250 (4.50), 305 (3.55) nm; IR (film) νmax: 3600 − 3100, 2930, 1718, 1658, 1621, 1454, 1379, 1298, 753 cm−1;1 H NMR (CDCl3, 500 MHz): ; 13C NMR (125 MHz, CDCl3): ; EI-MS (70 eV) m./z. (rel. int.): 418 [M]+ (2), 403 [M − CH3]+ (2), 253 (2), 241 (8), 214 (4), 201 (6).
Table 2.. 13C NMR data of compounds 7–12 (δ in ppm, 125 MHz, CDCl3).
6β-Acetonyltingenol (9)
Pale yellow powder (EtOAc); [α]D:−102° (c 0.15, MeOH); UV (MeOH), λmax (log ε): 218 (4.07), 250 (3.71), 280 (3.58) nm; IR (film) νmax: 3700 − 3100, 2955, 1702, 1453, 1384, 1292, 754 cm−1; 1H NMR (CDCl3, 500 MHz): ; 13C NMR (125 MHz, CDCl3): ; EI-MS (70 eV) m./z. (rel. int.): 478[M]+ (23), 419 (2), 241 (100), 215 (16), 202 (17), 201 (75).
6β-Acetonyl-22β-hydroxytingenol (10)
Pale yellow powder (EtOAc); [α]D: −107° (c 0.15, MeOH); UV (MeOH), λmax (log ε): 218 (4.04), 284 (3.47) nm; IR (film) νmax: 3600 − 3100, 2931, 1704, 1456, 1379, 1292, 755 cm−1; 1H NMR (CDCl3, 500 MHz): ; 13C NMR (125 MHz, CDCl3): ; EI-MS (70 eV) m./z. (rel. int.): 494 [M]+ (5), 437 (8), 436 (8), 243 (16), 241 (16), 215 (13), 202 (24), 201 (100).
6β-Acetonyl-20-hydroxy-20-epi-tingenol (11)
Pale yellow powder (EtOAc); [α]D:−8° (c 0.15, MeOH); UV (MeOH) λmax (log ε): 219 (4.21), 286 (3.73) nm; IR (film) νmax: 3600 − 3200, 2955, 1709, 1458, 1378, 1364, 1291, 754 cm−1; 1 H NMR (CDCl3 500 MHz): ; 13C NMR (125 MHz, CDCl3): ; EI-MS (70 eV) m./z. (rel. int.): 494 [M]+ (4), 437 (4), 436 (4), 243 (19), 241 (14), 215 (19), 202 (22), 201 (100).
6-(E-2′-Oxo-propylidene)-22β-hydroxytingenol (12)
Pale yellow powder (EtOAc); [α]D:−131° (c 0.06, MeOH); UV (MeOH) λmax (log ε): 216 (3.96), 268 (3.44), 322 (3.75), 366 (3.65) nm; IR (film) νmax: 3600 − 3100, 2960, 2930, 1705, 1652, 1620, 1543, 1458, 1378, 1296, 755 cm−1; 1 H NMR (CDCl3, 500 MHz): ; 13C NMR (125 MHz, CDCl3): ; EI-MS (70 eV) m./z. (rel. int.): 492 [M]+ (4), 477 (18), 436 (8), 241 (87), 215 (21), 202 (31), 201 (100).
Results
Compounds 4–6 were identified as isotingenone III, 22β-hydroxy-isotingenone III, and 20-hydroxy-20-epi.-isotingenone III, respectively, by comparing their IR and TLC Rf. values with the authentic substances.
Compound 7 was a new acid-rearranged product of 3. It possessed two carbonyl groups, supported by two IR bands at 1631 and 1715 cm−1 and two 13C NMR signals at δ 197.1 and 215.2 ppm. These carbonyl groups were correspondingly assigned to those at positions 2 and 21, normally found in tingenone derivatives. Also, in the downfield region of 1H NMR, a singlet at δ 4.25 ppm, two correlated doublet (J. = 7.9 Hz) at δ 7.18 and 7.10 ppm, and a broad singlet at δ 6.31 ppm could be clearly assigned to H-1, H-6, H-7, and 3-OH, respectively. By means of a HMBC experiment, a structure of compound 7 could be determined as shown in . The conjugation between an α,β-unsaturated carbonyl group of ring A and the aromatic ring B was demonstrated by the long-ranged correlations between their protons and carbons. Three-bond couplings from H3-25 to C-7 and C-9 suggested the presence of a methyl group on C-8. The most interesting long-ranged correlations were those from H-1 to C-13 and C-14, which suggested the formation of an unusually 7-membered ring between C-1 and C-14. The highly upfield signals of H3-26 (δ 0.54 ppm) and H-15α (δ 0.65 ppm) were observed due to the shielding effect of π-electrons of rings A/B at these protons. These data indicated the stereochemistry of H-1 and CH3-26 to be at β- and α-sides, respectively. Compound 7 possessed a novel skeleton among the acid-rearranged products of quinone-methide triterpenes that have previously been reported (isomers I, II, and III). Thus, isomer IV was named for this isomer, and compound 7 was elucidated to be 20-hydroxy-20-epi.-isotingenone IV.
Figure 2 Some important HMBC correlations within rings A/B/C of compound 7.
Compound 8 was the other acid-rearranged product of compound 3. It showed similar spectroscopic data to those of compound 4. However, it revealed only one signal of H-19 in NMR spectrum at δ 6.70 ppm. Based on an HMBC experiment, this proton showed long-range couplings to C-17, C-21, and C-29, confirming its position at C-19. The downfield shift of H3-29 (δ 1.83 ppm), which exhibited three-bond correlations to C-19 and C-21, suggested a double bond between C-19 and C-20. The IR band at 1658−1 also supported the conjugated carbonyl function group. From these data, the structure of 8 was concluded as 19,20-didehydro-isotingenone III.
Compounds 9–11 were acid-catalyzed products in acetone of compounds 1–3. In MS, their molecular ions indicated that the molecular weights of these compounds were 58 a.m.u. more than those of compounds 1–3. Thus, an additive reaction of an acetonyl group was proposed. Compounds 9–11 exhibited comparable NMR data with those of compounds 1–3, except for the replacement of quinone-methide characteristic with the phenolic signals. The singlet at δ 6.77–6.79 ppm was assigned as that of H-1, whereas two correlated methine proton signals ( J. = 10.7 to 10.9 Hz) at δ 4.00–4.02 ppm and 5.88–5.91 ppm were those of H-6 and H-7, respectively. H-6 also exhibited more vicinal couplings to the two methylene protons at δ 2.72–2.73 (H-1′a) and 2.46–2.48 (H-1′b). Based on extensive NMR experiments, a series of long-range connectivity from the methylene protons (H2-1′) to a carbonyl carbon (C-2′, δ 207.9–208.0 ppm) and from this carbonyl carbon to the methyl protons (H3-3′, δ 2.16–2.17 ppm) could be observed. These data concluded the presence of an acetonyl group at position 6. From the NOEs observed between H3-25 and H2-1′, the acetonyl group was placed on β-side. Therefore, the structures of compounds 9–11 were determined as 6β-acetonyltingenol, 6β-acetonyl-22β-hydroxytingenol, and 6β-acetonyl-20-hydroxy-20-epi.-tingenol, respectively.
The last product, compound 12, showed similar NMR data to those of compound 10. But in the 1H NMR, a methine proton at H-7 was downfield shifted to δ 7.75 ppm, and only one proton at position 1′ was observed as a downfield singlet at δ 6.07 ppm. This suggested that a double bond was present between C-6 and C-1′. The molecular ion of this compound, which was 2 a.m.u. less than that of compound 10, also supported the presence of this double bond. By means of a HMBC experiment, long-range correlations from H-1′ to C-5, C-7, and C-2′ and from H-7 to C-6 and C-1′ confirmed the position of this functional group. This double bond caused a conjugated system within rings A/B and the side chain, the reasonable IR band at 1543 cm−1 and the downfield shift of H-7. Its stereochemistry was assigned as entegegen. based on the NOE observed between H-1′ and H3-23. From all of these data, compound 12 was elucidated as 6-(E.-2′-oxo-propylidene)-22β-hydroxytingenol.
The antiviral activities against HSV-1 and HSV-2 of all compounds were evaluated by using the plaque reduction assay, and the results are summarized in . Compounds 1–3 were very toxic to the uninfected Vero cells. Therefore, determining their antiviral activity was unfeasible. All of their acid-catalyzed products (4–12) were less toxic. Compounds 4–6 and 8 exhibited antiviral activity with EC50 2–10 µg/ml. However, only compounds 4–6 presented a selective index greater than 5. Compounds 7–12 were tested for antimicrobial activity, but none of them was active (MIC more than 32 µg/ml).
Table 3.. Antiviral and cytotoxic activities of tingenone derivatives.
Discussion
Based on the acid-labile property of the quinone-methide triterpenes, nine products (compounds 4–12) were obtained from the acid-rearrangement reaction of tingenone derivatives 1–3. The occurrence of compounds 7–12 was reported herein for the first time. Compound 7 possessed a novel skeleton, named isomer IV. A possible mechanism of the formation of this compound is shown in Scheme 1. The reaction mechanism was similar to that of isomer I (Nakanishi et al., 1965). However, after the bond between C-8 and C-14 was broken, the C-9–C-11 bond was rotated and a new bond was formed between C-1 and C-14 with the loss of a proton from 2-OH. Besides compound 7, compound 3 also acid-rearranged to compound 6, and the dehydration of compound 6 was proposed as the origin of the new compound 8. Compounds 9–12 were generated in acidic acetone. Under this condition, acetone was enolized and acted as a nucleophilic intermediate, which could attack at position 6 of tingenone derivatives. The quinone-methide chromophore could further rearrange to an allyl-phenol system (Brown et al., 1973) as seen in compounds 9–11. Compound 12 might be the dehydrogenation product of compound 10.
Scheme 1 Proposed mechanism of the formation of compound 7.
In order to establish some structure-activity considerations, all compounds in this study were classified into two groups based on their ring A/B/C structures: the quinone-methide (compounds 1–3) and the phenolic analogues. The latter group was further divided into two subgroups: the divinyl-phenolic (compounds 4–6 and 8) and the allyl-phenolic compounds (compounds 9–11). In our previous report, compounds 1–3 were potent antimicrobial agents, whereas the divinyl-phenolic compounds (4–6) were inactive (Sotanaphun et al., 1999). Also, in this investigation, the phenolic compounds (8–12) were found to be inactive. However, in contrast to their phenolic analogues, the compounds with the quinone-methide chromophore (1-3) were highly toxic. This indicated that the quinone-methide system was important for antimicrobial activity and toxicity of tingenone derivatives. Bioactivities of compound 7 also supported this suggestion. Though compound 7 still possessed a carbonyl group at position 2 and a hydroxyl group at position 3, it lacked a quinone-methide character. This compound lost antimicrobial activity and its toxicity was lower.
The phenolic analogues of tingenone derivatives were found to exhibit antiviral activity. Among them, compounds with the divinyl-phenolic chromophore (4–6) were significantly more active (EC50 2–6 µg/ml, selective index > 5). In agreement with other activities (Gonzalez et al., 1988; Moujir et al., 1990), oxygenated substitution on ring E influenced on their potency. Introduction of a double bond on ring E (compound 8) decreased the activity (EC50 5–10 µg/ml). Compounds with the allyl-phenolic system (9-11) exhibited very weak antiviral activity against HSV-1 (EC50 12–17 µg/ml) and had no inhibitory effect on HSV-2. Moreover, if the conjugated system of the allyl-vinyl phenolic was present (compound 12), antiviral activity was absolutely lost and toxicity was slightly increased.
Acknowledgment
The authors are grateful to Thailand Research Fund for financial support.
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