Isolation, synthesis, and biological activity of tomentosenol A from the leaves of Rhodomyrtus tomentosa

Abstract

Tomentosenol A (1), along with a pair of epimers, 4S-focifolidione (2) and 4R-focifolidione (3), were isolated from the leaves of Rhodomyrtus tomentosa. 1 was the first example of a new meroterpenoid class with a unique skeleton that contained a free syncarpic acid coupled with a terpenoid unit, and showed excellent antimicrobial and cytotoxic activities. The absolute configuration of 1 was unambiguously determined by a chemical conversion between 1 and the predetermined 2. In contrast to the common hetero Diels–Alder cycloaddition embodied in previously reported meroterpenoid biosynthesis, an Alder-ene reaction was proposed as the key transformation to account for the biosynthesis of 1, which was confirmed by a biomimetic total synthesis.

---

Introduction

Plants of the Myrtaceae family are rich in structurally varied and biologically meaningful acylphloroglucinols and their meroterpenoids, and thus have stimulated considerable phytochemical efforts aimed at exploring new antibiotics attributed to their remarkable antibacterial activities reported in recent years.¹ Rhodomyrtus tomentosa is an evergreen plant native to southern and southeastern Asia, and is particularly widely distributed in southern China.² The extract from this plant possesses strong inhibitory activities against Gram-positive bacteria.^3–5 The chemical constituents of R. tomentensa have been reported to include flavones,⁶ triterpenes,^7–9 steroids,⁷ hydrolysable tannins,¹⁰ and acylphloroglucinols.^6,11–13 In our ongoing effort to search for new natural antibacterial products derived from medicinal plants, an extensive bioactivity-guided fractionation resulted in the isolation of a potent antibacterial acylphloroglucinol-meroterpenoid possessing a unique skeleton that contained a free syncarpic acid with a terpenoid unit, named tomentosenol A (1) (Fig. 1), along with a pair of meroterpenoids 4S-focifolidione (2) and 4R-focifolidione (3). Herein, the details of isolation, structural elucidation, chemical transformation to determine absolute configuration, as well as biological evaluation of those isolates were described. And a hypothetical pathway with Alder ene reaction as the key transformation was proposed to account for the biogenesis of 1 and rationalized by a biomimetic total synthesis.

Fig. 1 The chemical structures of compounds 1–3.

Results and discussion

Compound 1 was obtained as needle crystal and its molecular formula was determined as C₂₅H₃₈O₃ based on a HRESIMS measurement (positive mode; [M + H]⁺ m/z 387.2895, calcd 387.2821), which indicated the presence of eight indices of hydrogen deficiency in the molecule. Consideration of the ¹³C NMR data showed a set of 10 peaks characteristic of a syncarpic acid unit commonly observed in many other natural products from Rhodomyrtus.^6,11–13 This assumption was also confirmed by HMBC correlations of the methyl singlets at δ_H 1.33 and 1.32 to C-5 (δ_C 198.0), C-6 (δ_C 55.7), and C-7 (δ_C 212.9) as well as the methyl singlets at δ_H 1.43 and 1.46 to C-7, C-8 (δ_C 47.7), and C-8a (δ_C 170.1) (Fig. 2). The ¹H NMR data of 1 (Table 1) displayed eight methyl group signals at δ_H 0.75, 0.84, 0.98, 0.99, 1.06, 1.32, 1.33, 1.35, and 1.39, as well as a number of overlapping aliphatic signals ranging from 1.40 to 3.02 ppm. The ¹³C NMR spectrum of 1 exhibited 25 carbon signals, including eight methyls, four methylenes, five methines, and eight quaternary carbons. Moreover, the ¹H–¹H COSY correlations indicated that the following fragment a (C-4/C-1′′/C-2′′/C-3′′, C-2'′′/C-4′′), along with the HMBC correlations from H₃-3′′ to C-1′′, and C-2′′, H₃-4′′ to C-1′′ and C-2′′, and H₂-1′′ to C-4a suggested the presence of an isobutyl group located at the C-4a of the syncarpic acid. Further, a monoterpene unit was deduced to account for the remaining 10 carbon resonances. The ¹H–¹H COSY fragment b (C-3′/C-4′/C-5′/C-7′/C-1′) and the HMBC correlations from H₂-3 to C-1′ and C-3′ as well as H₃-9′ and H₃-8′ to C-1′, C-5′ and C-6′, and H₂-7′ to C-2 and C-4′ suggested that 1 contained the basic structural unit of a pinene. Furthermore, the two constituents were conjugated, as determined via the HMBC correlations from 8a-OH to C-8a and H₂-3 to C-4a to give the planar structure of 1 as shown in Fig. 1.

Fig. 2 ¹H–¹H COSY and key HMBCs of compound 1.

Table 1 ¹H and ¹³C NMR data of 1 in CDCl₃ (δ in ppm, J in Hz)

No.	1
	δH	δC
1′	2.08, (m)	45.3, CH
2′(2)		146.3, C
3′	5.12, (s)	118.3, CH
4′	2.13, (m)	31.3, CH2
5′	2.04, (m)	40.7, CH
6′		38.2, C
7′a	2.33, (dt, 5.6, 8.5)	31.8, CH2
7′b	0.93, (d, 8.5)
8′	1.26, (s)	21.0, CH3
9′	0.83, (s)	26.3, CH3
3a	2.49, (m)	41.2, CH2
3b	2.06, (m)
4	3.02, (br s)	31.1, CH
5		198.0, C
4a		114.3, C
6		55.7, C
7		212.9, C
8		47.7, C
8a		170.1, C
9	1.46, (s)	25.2, CH3
10	1.43, (s)	24.9, CH3
11	1.33, (s)	25.6, CH3
12	1.32, (s)	23.6, CH3
1′′a	1.69, (m)	42.9, CH2
1′′b	1.41, (overlapped)
2′′	1.41, (overlapped)	26.5, CH
3′′	0.89, (d, 6.0)	23.4, CH3
4′′	0.87, (d, 6.0)	22.3, CH3
8a-OH	5.72, s

---

The confirmation of the C-4 stereogenic centre and the absolute configuration of 1 was rationalized by the chemical transformation from 1 to (4S)-focifolidione (2) via a cationic cyclization methodology as shown in Scheme 1. Upon treatment with p-toluenesulfonic acid under reflux toluene for 1 h, 1 smoothly underwent the 6-exo cationic cyclization as expected to form one single product. The product was proved to be identical in both optical rotation and NMR spectrum to those of 2. Thus, the final configuration of 1 was ambiguously determined to be 4-((S)-1-((1R,5S)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-4-methylpentan-2-yl)-5-hydroxy-2,2,6,6-tetramethylcyclohex-4-ene-1,3-dione as depicted.

Scheme 1 The chemical transformation from 1 to 2.

4S-Focifolidione (2) was first isolated from the leaves, stems and essential oil of K. ericoides.^14,15 However, its C-4 epimer was never found in nature. In our investigation, both the 4S- and 4R-focifolidiones were isolated in the leaves of R. tomentosa using silica gel column chromatography followed by semi-preparative HPLC.

Compound 1 was the first example of a new meroterpenoid class with a unique skeleton that contained a free syncarpic acid moiety conjugated with a terpenoid unit. Regarding the structure novelty of 1, a unique pathway was proposed (as depicted in Scheme 2) to account for the biogenesis of 1. Shortly, 1 was assumed to originate from (−)-β-pinene and syncarpic acid derivatives i, which was better-known as the key intermediate in the biosynthesis of meroterpenoids. Notably, although hetero Diels–Alder cycloaddition was usually believed to be the key biosynthesis transformation of natural meroterpenoids, the concerted Alder ene reaction was rarely reported in natural product biosynthesis. In this study, 1 was disclosed as the natural meroterpenoid biogenetically generated by a concerted Alder ene reaction, in contrast to the hetero Diels–Alder cycloaddition embodied in the formation of focifolidiones 2 and 3.

Scheme 2 Proposed biogenetic pathways of 1–3.

To further confirm the absolute configuration of compounds 1–3, and shed more light on the biogenesis of 1, a biomimetic asymmetric synthesis of 1–3 was conducted using the route illustrated in Scheme 3. To summarize, the synthesis of 1–3 was initiated with commercially available phloroglucinol 4 and (−)-β-pinene 8 as starting materials. The syncarpic acid 6 could be readily derived from phloroglucinol 4 through simple tetramethylation and acid-mediated retro-Claisen condensation, and in turn became the key intermediate i, through a simple modified Knoevenagel condensation. The intermediate i was then subjected to the neat (−)-β-pinene 8 intimately in a nitrogenous environment to afford the desired product 1 in moderate reaction efficiency (21% yield) and focifolidione epimers 2 and 3 with a total yield of 57%. As expected, the synthetic products 1–3 were afterwards proved to be identical in all respects to the corresponding natural products, also providing supportive evidence for the proposed biosynthetic pathways embedding an Alder ene reaction as the key transformation.

Scheme 3 The biomimetic asymmetric synthesis of 1.

Tomentosenol A (1) and R/S-focifolidiones were biologically evaluated for antibacterial activities against microbes Escherichia coli and Staphylococcus aureus, as well as growth inhibitory activities of 1 towards human cancer cell lines MCF-7 (human breast adenocarcinoma cell line), NCI-H460 (human non-small cell lung cancer cell line), SF-268 (human glioma cell line) and HepG-2 (human hepatoma carcinoma cell line). As a result, 1 exhibited potent antibacterial activity with a MIC value of 4.74 μM against S. aureus, comparable to the positive control substance vancomycin (MIC = 1.23 μM). Besides, 1 showed moderate antitumor activities to all the four cancer cell lines with IC₅₀ values of 8.66 ± 0.24 μM, 8.62 ± 0.31 μM, 10.01 ± 0.41 μM, and 9.44 ± 0.36 μM.

Conclusions

In summary, we described herein the isolation and structural elucidation of a new acylphloroglucinol named tomentosenol A (1), which represented new type of meroterpenoid with an unprecedented monoterpenoid-based skeleton, along with a pair of epimers, 4S-focifolidione and 4R-focifolidione, from the leaves of Rhodomyrtus tomentosa. 4R-Focifolidione was obtained from a natural source for the first time. The first total synthesis of 1 was achieved with absolute configuration confirmed by a chemical conversion. The Alder ene reaction was also established as a viable synthetic strategy to efficiently synthesize such a class of meroterpenoids. The antibacterial and antitumor activities of compounds 1–3 were also evaluated wherein 1 showed potent biological activities against all the test organisms.

Experiment section

General experimental procedures

Optical rotations were measured on a Perkin-Elmer 341 polarimeter (Perkin-Elmer, Boston, MA, USA); UV spectra were recorded in MeOH on a Perkin-Elmer Lambda 35 UV-vis spectrophotometer (Perkin-Elmer, Boston, MA, USA); 1D and 2D NMR spectra were recorded on a Bruker Advance-500 spectrometer with TMS as internal standard (Bruker BioSpin AG, Fällanden, Switzerland); ESIMS data were obtained on a MDS SCIEX API 2000 LC/MS/MS mass spectrometer (AB MDS Sciex, USA). HRESIMS data were obtained on a Bruker Bio TOF IIIQ mass spectrometer (Bruker Daltonics, MA, USA). Solvents were analytical grade (Shanghai Chemical Plant, Shanghai, China). Silica gel (200–300 mesh) was used for column chromatography, and precoated silica gel GF₂₅₄ plates (Qingdao Marine Chemical Inc., Qingdao, China) were used for TLC. C₁₈ reversed-phase silica gel (150–200 mesh, Merck), MCI gel (CHP20P, 75–150 μm, Mitsubishi Chemical Industries Ltd.), and Sephadex LH-20 gel (Amersham Biosciences) were also used for column chromatography. TLC spots were visualized under UV light and by dipping into 5% H₂SO₄ in alcohol followed by heating.

Plant material

The green leaves of R. tomentosa were collected from Nankang country of Jiangxi Province, China, in June 2012, identified by Dr Fa-Guo Wang of SCBG and a voucher specimen (No. # SCBG-NPL-120012) was deposited at the Laboratory of Natural Product Chemistry Biology, SCBG.

Extraction and isolation

The air-dried powdered leaves (20 kg) of R. tomentosa were extracted with 95% EtOH (20 L × 3) for 24 h. After removal of the solvent by evaporation, the residue (2.5 kg) was suspended in H₂O and then extracted successively with n-hexane (3 L × 3), ethyl acetate (3 L × 3). Each solvent was evaporated off under reduced pressure to yield extracts of n-hexane soluble (400 g), ethyl acetate soluble (1100 g). The n-hexane part was subjected to column chromatography (silica gel) with a gradient elution (n-hexane/ethyl acetate, 100 : 0 to 0 : 100, v/v) to afford seven fractions (A–G). Fraction B (n-hexane/ethyl acetate, 95 : 5, v/v) was further purified by RP-C₁₈ silica gel (MeOH/H₂O, 95 : 5 to 100 : 0, v/v) to afford fractions B₁ and B₂. Fraction B₁ was further purified by Sephadex LH-20 (MeOH) chromatography, followed by repeated silica gel and semiprep-HPLC (MeCN/H₂O, 100 : 0, v/v) to obtain compound 2 (8.0 mg, t_R 52.5 min) and 3 with litter impurity (5.0 mg, t_R 53.9 min), respectively. Fraction C (n-hexane/ethyl acetate, 90 : 10, v/v) was further fractionated by RP-C₁₈ silica gel (MeOH/H₂O, 95 : 5 to 100 : 0, v/v) to afford fractions C₁ and C₂. Fraction C₁ was further purified by Sephadex LH-20 (MeOH) chromatography and repeated silica gel chromatography (n-hexane/ethyl acetate, 10 : 1 to 5 : 1, v/v) to give 1 (15 mg).

Tomentosenol A (1). Colorless needle crystal; [α]²⁰_D −12.6 (c 0.15, CHCl₃). UV (MeOH) λ_max (log ε) 267.5 (5.01) nm. ¹H (500 MHz) and ¹³C (125 MHz) NMR spectral data, see Table 1; positive ESIMS: m/z 387 [M + H]⁺; HRESIMS: m/z 387.2895 [M + H]⁺ (calcd for C₂₅H₃₉O₃, 387.2821).

Absolute configuration of 4-isobutyl in 1. A sample of 1 (8.0 mg) was dissolved in toluene (2 mL), PTSA (1.0 mg) was then added. The resulting reaction mixture was refluxed for 1 hour until all the start material was consumed completely. Then, the reaction was directly purified through a short column chromatography (silica gel; n-hexane/ethyl acetate, 10 : l) to give the 2 (6.1 mg, 76% yield). All of the characteristic data of the synthetic compound 2 were found to be identical to those of the isolated one.

The synthesis of 4-acetyl-5-hydroxy-2,2,6,6-tetramethylcyclohex-4-ene-1,3-dione 5. A flame-dried 250 mL flask was charged with acetylphloroglucinol 4 (5.50 g, 33.0 mmol) and anhydrous MeOH (120 mL). Sodium methoxide (14.4 g, 266 mmol) was added slowly. After stirring for 10 min, methyl iodide (28.4 mL, 228 mmol) was added dropwise at 0 °C and kept it stirring at this temperature for 30 min. Then, the ice bath was removed, and the reaction mixture was stirred for another 24 h at room temperature until all the starting material was consumed. The mixture was then quenched with 2 N HCl (150 mL). The resulting mixture was extracted with EtOAc (4 × 150 mL), and the combined organic phases were dried with Na₂SO₄ and concentrated in vacuo. The residue was purified by flash chromatography (silica gel; n-hexane/ethyl acetate, 5 : l) to afford product 5 (6.36 g, 86% yield) as a colorless needle crystal. ¹H NMR (500 MHz, CDCl₃): δ 1.33 (s, 6H), 1.42 (s, 6H), 2.57 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 23.8, 24.3, 27.4, 52.0, 56.7, 109.4, 196.7, 199.1, 201.7, 210.0.

The synthesis of 5-hydroxy-2,2,6,6-tetramethylcyclohex-4-ene-1,3-dione (syncarpic acid) 6. Ketone 5 (6.36 g, 28.5 mmol) was added to a solution of 6 N HCl aqueous (120 mL). Reflux was then continued for 24 hours. Following this, the reaction was cooled, and the aqueous were extracted with EtOAc (4 × 150 mL). The combined organic phases were washed with brine, dried over Na₂SO₄ and concentrated in vacuo. The residue was further purified by flash chromatography (silica gel; n-hexane/ethyl acetate, 1 : 1) to afford syncarpic acid 6 (4.05 g, 78% yield) as a slight yellowish solid. ¹H NMR (500 MHz, CDCl₃): ketone: δ 1.31 (s, 12H), 3.61 (s, 2H); enol: δ 1.40 (s, 12H), 5.74 (br d, J = 2.3 Hz, 1H), 8.00 (br s, 1H); ¹³C NMR (125 MHz, CDCl₃): ketone: δ 21.8, 50.2, 59.1, 204.3, 208.9; enol: δ 24.5, 51.2, 59.1, 101.7, 191.9, 204.3, 212.6.

The synthesis of 2,2,4,4-tetramethyl-6-(3-methylbutylidene) cyclohexane-1,3,5-trione i. To a solution of syncarpic acid 6 (182 mg, 1.0 mmol) and isovaleric aldehyde 7 (172 mg, 2.0 mmol) in CH₂Cl₂ (10 mL), proline (11.5 mg, 0.1 mmol) was added in one portion. The resulting reaction mixture was stirred for 30 min at room temperature. Then, it was quickly passed through a short pad (3 cm) of flash chromatography (silica gel; CHCl₃) to afford α,β-unsaturated ketone i (238 mg, 95% yield) as a colorless oil, which was pure enough to be used directly in the next step without further purification. ¹H NMR (500 MHz, CDCl₃): δ 0.95 (d, J = 6.7 Hz, 6H), 1.30 (s, 6H), 1.31 (s, 6H), 1.89 (m, J = 6.7 Hz, 1H), 2.59 (dd, J = 3.0, 3.0 Hz, 2H), 7.51 (dd, J = 3.0 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 21.9, 22.3, 22.6, 28.7, 38.9, 57.9, 58.6, 133.1, 159.1, 196.4, 199.5, 208.8.

The synthesis of tomentosenol A (1). The α,β-unsaturated ketone i (250 mg, 1.0 mmol) and β-pinene (1 mL) were neatly mixed. The mixture was stood at room temperature for 24 h under nitrogen atmosphere. After the start material was disappeared while checking by TLC, the reaction mixture was purified directly by flash chromatography (silica gel; n-hexane/ethyl acetate, from 100 : 1 to 5 : 1) (n-hexane/ethyl acetate, 50 : 1, v/v), and the product 1 (81 mg, 21% yield) (n-hexane/EtOAc, 5 : 1, v/v). The mixture was further purified by flash chromatography (silica gel; n-hexane/ethyl acetate, from 100 : 1 to 20 : 1) to give colorless oil 2 (123 mg, 32% yield) and 3 (97 mg, 25% yield), respectively. All the products were identical to all aspects of the natural isolates.

1: colorless oil; [α]²⁰_D −32.3 (c 0.35, CHCl₃); ¹H (500 MHz) and ¹³C (125 MHz) NMR spectral data were the same as the natural compound. 2: colorless oil; [α]²⁰_D +19 (c 0.85, CHCl₃); 3: colorless oil; [α]²⁰_D −78 (c 0.9, CHCl₃); ¹H and ¹³C NMR spectra were consistent with previously reported data.¹⁵ NMR spectra were shown in the ESI.†

Antibacterial activity assay

The MIC values were determined using a broth dilution method (Mueller-Hinton broth) based on the published microdilution method.¹⁶ Vancomycin was used as positive control. Test strains were S. aureus (ATCC6538) and E. coli (ATCC8739) which were purchased from Guangdong Microbiology Culture Centre (Guangzhou).

Antitumor activity assay

Cytotoxicities of compounds 1–3 were assayed against four tumour cell lines, including MCF-7, NCI-H460, SF-268, and HepG-2, with cisplatin (IC₅₀ values of 5.8 ± 0.4 μM, 1.3 ± 0.1 μM, 1.9 ± 0.1 μM, and 1.7 ± 0.1 μM) as positive control. Assays were performed by the SRB method.¹⁷

Acknowledgements

This work was supported by National Science and Technology Major Projects of China (2014ZX10005002-005), and National Natural Science Foundation of China (NSFC) (No. 30973635, 81373293, 81502949).

Notes and references

1. I. P. Singh and S. B. Bharate, Nat. Prod. Rep., 2006, 23, 558–591.
2. J. Chen and L. A. Craven, Flora of China, Science Press/Missouri Botanical Garden Press, Beijing/St. Louis, 2007, vol. 13, p. 330.
3. J. Saising, A. Hiranrat, W. Mahabusarakam, M. Ongsakul and S. P. Voravuthikunchai, J. Health Sci., 2008, 54, 589–595.
4. S. Limsuwan, E. N. Trip, T. R. H. M. Kouwen, S. Piersna, A. Hiranrat, W. Mahabusarakam, S. P. Voravuthikunchai, J. M. van Dijl and O. Kayser, Phytomedicine, 2009, 16, 645–651.
5. G. Lavanya, S. P. Voravuthikunchai and N. H. Towatana, J. Evidence-Based Complementary Altern. Med., 2012, 2012 , article ID 535479.
6. A. Hiranrat and W. Mahabusarakam, Tetrahedron, 2008, 64, 11193–11197.
7. W. H. Hui, M. M. Li and K. Luk, Phytochemistry, 1975, 14, 833–834.
8. W. H. Hui and M. M. Li, Phytochemistry, 1976, 15, 1741–1743.
9. J. Xiong, Y. Huang, Y. Tang, M. You and J. F. Hu, Chin. J. Org. Chem., 2013, 33, 1304–1308.
10. Y. Z. Liu, A. J. Hou, C. R. Ji and Y. J. Wu, Chin. Chem. Lett., 1997, 8, 39–40.
11. A. Hiranrat, W. Chitbankluoi, W. Mahabusarakam, S. Limsuwan and S. P. Voravuthikunchai, Nat. Prod. Res., 2012, 26, 1904–1909.
12. A. Hiranrat, W. Mahabusarakam, A. R. Carroll, S. Duffy and V. M. Avery, J. Org. Chem., 2012, 77, 680–683.
13. D. Salni, M. V. Sargent, B. W. Skelton, I. Soediro, M. Sutisna, A. H. White and E. Yulinah, Aust. J. Chem., 2002, 55, 229–232.
14. B. P. S. Khambay, D. G. Beddie and M. D. J. Simmonds, Phytochemistry, 2002, 59, 69–71.
15. H. Nishiwaki, S. Fujiwara, T. Wukirsari, H. Iwamoto, S. Mori, K. Nishi, T. Sugahara, S. Yamauchi and Y. Shuto, J. Nat. Prod., 2015, 78, 43–49.
16. A. J. Drummond and R. D. Waigh, in Recent Research Developments in Phytochemistry, ed. S. G. Pandalai, Research Signpost, India, 2000, vol. 4, p. 143.
17. P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J. T. Warren, H. Bokesch, S. Kenney and M. R. Boyd, J. Natl. Cancer Inst., 1990, 82, 1107–1112.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra01594h

---