Eucalmaidials A and B, phloroglucinol-coupled sesquiterpenoids from the juvenile leaves of Eucalyptus maideni

Abstract

Two new phloroglucinol-coupled sesquiterpenoids, eucalmaidials A and B (1 and 2), were isolated from the juvenile leaves of Eucalyptus maideni, along with eight known macrocarpals (3–10), eucalyptone (11), and three known triterpenoids (12–14). Eucalmaidials A and B represent a new skeleton of phloroglucinol-coupled iphionane. Their structures were elucidated by extensive NMR spectroscopic analysis and theoretical calculation of the ¹³C NMR chemical shifts. The biosynthetic pathway of 1 and 2 was also postulated. Compounds 1, 3, 5, 7, 8, and 10–14 were evaluated for their antifungal and antibacterial activities. Compound 1 exhibited antifungal activity against Candida glabrata with an IC₅₀ value of 0.75 μg mL⁻¹.

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Introduction

Phloroglucinol-coupled sesquiterpenoids, e.g. macrocarpals,^1–5 euglobals,^6,7 eucalyptone,⁸ eucalyptals,⁹ and eucalyptin,¹⁰ are a group of typical secondary metabolites from the genus Eucalyptus (Myrtaceae).^6,11,12 They display a wide spectrum of significant bioactivities, e.g. antimicrobial,^1,3 antitumour,^9,10 and granulation^6,7 and HIV-Rase inhibitory effects.² These unusual types of natural products are considered to be formed through [4 + 2] cycloaddition of a O-quinone methide to a sesquiterpenoid double bond¹¹ or by nucleophilic addition of a sesquiterpenoid unit to an isoprenylated phloroglucinol.^9,11 Only a limited number of sesquiterpenoids, e.g. eudesmane, aromadendrene, guaiane and cadinane, are involved in the formation of these compounds, in comparison to the diversity of sesquiterpenoids found in the Eucalyptus volatile oil.¹³

E. maideni F. Muell is an introduced tall timber tree growing widely in the southern part of China. Preliminary TLC and HPLC analysis indicated that the juvenile leaves were rich in phloroglucinol derivatives. As part of a continuing study and search for new phloroglucinol-coupled sesquiterpenoids, the juvenile leaves of this plant were further chemically investigated.^14,15 Since macrocarpals are common components in genus Eucalyptus,^11,16 MS was employed for dereplication purposes. This led to the isolation of two new compounds (1 and 2) bearing an iphionane moiety, biogenetically related to eudesmane. Compounds 1 and 2 represent a new skeleton of 3,5-diformyl-isopentylphloroglucinol coupled sesquiterpenoids. In addition, nine known phloroglucinol-coupled sesquiterpenoids, macrocarpals A (3), B (4),² H (5),³ K (6),⁵ I (7), J (8),³ E (9), G (10)² as well as eucalyptone (11),⁸ and three known triterpenoids, 3β-hydroxy-11α-methoxyurs-12-en-28-oic acid (12),¹⁷ 3β-hydroxyurs-9(11),12-dien-28-oic acid (13),¹⁸ and 3β,13β-dihydroxyurs-11-en-28-oic acid (14)¹⁹ were also identified during the process. We herein describe the isolation and structure elucidation of these compounds and their antifungal or antibacterial activities.

Results and discussion

The fresh juvenile leaves of E. maideni (7.8 kg) were extracted with 80% aq. acetone three times. The extracts were combined and concentrated to small volume (5 L), which was partitioned with CHCl₃. The CHCl₃ extract (50 g) was subjected to column chromatography on silica gel, MCI CHP20P, RP-8, and semipreparative HPLC to yield 11 phloroglucinol-coupled sesquiterpenoids (1–11) and three triterpenoids (12–14).

Compound 1 was obtained as a yellowish amorphous powder. The HRESIMS displayed a quasi-molecular ion peak at m/z 485.2531 [M − H]⁻ (calcd 485.2539), corresponding to a molecular formula of C₂₈H₃₇O₇ with 10 degrees of unsaturation. The UV absorption bands at 272 and 387 nm suggested the presence of a 3,5-diformyl-phloroglucinol chromophore.⁹ The ¹³C NMR and DEPT spectra of 1 showed 28 carbon signals (Table 1), including six methyls, five methylenes, six methines, and 11 quaternary carbons. Of which, eight carbons [δ_C 192.0 (CH), 191.9 (CH), 172.9 (C), 171.7 (C), 169.4 (C), 107.8 (C), 107.6 (C), and 105.8 (C)] arose from a typical 3,5-diformylphloroglucinol unit, and one ketone (δ_C 198.9) and one tetrasubstituted double bond (δ_C 163.2 and 132.6) were observed. These accounted for 8 out of the 10 degrees of unsaturation. The remaining two degrees of unsaturation therefore required that 1 possessed two additional rings.

Table 1 ¹³C and ¹H NMR data of 1 and 2 in pyridine-d₅

	1		2
No.	δC	δH (mult., JHz)	δC	δH (mult., JHz)
1	52.4	2.93 (dd, 3.1, 7.8)	51.9	3.12 (ddd, 3.4, 7.6, 11.6)
2α	38.4	2.73 (dd, 7.8, 15.0)	38.9	3.01 (dd, 7.6, 14.5)
2β		2.65 (dd, 3.1, 15.0)		2.52 (dd, 3.4, 14.5)
3	132.6		132.1
4	198.9		198.7
5	163.2		164.5
6α	27.1	3.96 (brd, 11.7)	26.9	4.01 (brd, 12.5)
6β		2.12 (dd, 6.4, 11.7)		2.09 (dd, 8.0, 12.5)
7	49.8	1.67 (m)	49.6	1.55 (m)
8α	23.9	1.97 (m)	23.8	1.46 (m)
8β		1.67 (m)		1.36 (m)
9α	43.0	1.67 (m)	40.1	1.46 (m)
9β		2.34 (m)		1.82 (m)
10	49.9		50.3
11	71.4		71.4
12	27.8	1.41 (s)	28.1	1.33 (s)
13	27.6	1.41 (s)	27.3	1.32 (s)
14	16.9	1.26 (s)	17.1	1.20 (s)
15	30.7	2.09 (s)	30.9	2.31 (s)
1′	172.9		173.7
2′	107.6		107.8
3′	171.7		173.0
4′	107.8		107.8
5′	169.4		171.6
6′	105.8		105.0
7′	192.0	10.58 (s)	191.9	10.59 (s)
8′	191.9	10.57 (s)	191.6	10.59 (s)
9′	32.9	3.90 (dd, 3.3, 11.3)	33.0	3.66 (ddd, 3.0, 11.6, 11.6)
10′a	41.7	2.49 (dd, 11.3, 11.3)	42.3	2.39 (dd, 11.6, 11.6)
10′b		1.53 (ddd, 3.3, 11.3, 11.3)		1.38 (m)
11′	27.2	1.67 (m)	26.5	1.67 (m)
12′	24.9	1.10 (d, 6.1)	25.0	1.11 (d, 6.3)
13′	22.2	0.94 (d, 6.1)	22.5	0.93 (d, 6.3)

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First, the protonated C–C connectivity in the molecule were established from C-2 to C-13′ and C-6 to C-9 by the ¹H-¹H COSY spectrum (bold lines in Fig. 1). The structural fragments were then connected with each other and other functional groups by the HMBC experiment (Fig. 1). The correlations of H-1 with C-3 and H-2 with C-3 and C-5 linked a double bond Δ^3,5 to C-2. The correlations of Me-14 with C-1, C-5, C-9, and C-10 allowed the connections of C-1, C-5, and C-9 to the quaternary carbon C-10. Thus, a five-membered A-ring was constructed. Me-15 correlated with C-3 and C-4, indicating an α,β-unsaturated ketone. The HMBC correlations of H-6 to C-3 and C-5, together with the correlation of H-9 with C-10 established the six-membered B-ring. A hydroxyisopropyl group was fixed at C-7 by the correlations of Me-12(13) with C-11 and C-7. An iphionane type sesquiterpenoid moiety thus emerged in 1.^20,21 Finally, the HMBC correlations of H-9′ with C-1′, C-5′, and C-6′ confirmed the presence of 3,5-diformyl-isopentylphloroglucinol unit, which was attached to the iphionane sesquiterpenoid moiety via the C-1–C-9′ bond. The planar structure of 1 was therefore constructed.

Fig. 1 Key ¹H-¹H COSY ([thick line, graph caption]) and HMBC (→) correlations of 1.

The relative configuration of 1 was established on the basis of the ROESY experiment (Fig. 2). The strong NOE correlations of Me-14 with H-6β, H-8β and H-9β indicated that these protons were co-facially oriented on the B-ring with a chair conformation, assuming Me-14 adopted a β-configuration as shown in the previously reported iphionanes.²¹ In addition, Me-14 correlated with H-2β, while H-1 showed strong correlation with H-9α, indicating that H-1 was α-oriented. The obvious NOE correlations of H-7 with H-6α, and Me-12(13) with both H-6α and H-6β indicated that the hydroxyisopropyl group at C-7 adopted the β-equatorial orientation, which was supported by the chemical shift of C-7 at δ 49.8.²² Thus, the relative configurations of C-1, C-7 and C-10 in 1 were assigned as (1S*,7R*,10S*).

Fig. 2 Key ROESY (H ↔ H) correlations of 1.

Compound 2, a yellowish amorphous powder, has the same molecular formula C₂₈H₃₇O₇ as that of 1, which was determined by HRESIMS at m/z 485.2544 [M − H]⁻ (calcd 485.2539) and its ¹³C NMR data. The ¹H and ¹³C NMR spectra of 2 closely resemble those of 1. Detailed 2D NMR analysis (HSQC, ¹H-¹H COSY, and HMBC) suggested compound 2 was a C-9′ epimer of 1, resulting from the coupling of an iphionane type sesquiterpenoid moiety with an isopentylphloroglucinol moiety in an opposite stereochemical fashion. It is evident that the chemical shift of C-9 and the carbons of the phloroglucinol moiety for 2 differ from those for 1, which may be attributed to the different orientations of the aromatic ring system. Different shielding effects of the phloroglucinol ring to H-1, H-2 and H-9 in 1 and 2 were also noticeable by their chemical shifts (Table 1).

The relative configuration of C-9′ in 1 and 2 was assigned by comparison of their ROESY spectra. First, two isomers with the same configurations at C-1, C-7 and C-10, but a opposite configuration at C-9′, that is, 1S*,7R*,10S*,9′R* and 1S*,7R*,10S*,9′S* isomers, were optimized by the MM2 energy minimization. In the optimized models, only H-10′ from the 1S*,7R*,10S*,9′R* isomer showed NOE correlations with H-9α and H-9β. While H-10′ from the 1S*,7R*,10S*,9′S* isomer showed NOE correlations with H-2α and H-2β (Fig. 3). Careful analysis of the ROESY spectral data of 1 and 2 revealed that H-10′ showed correlations with H-9α and H-9β, but no correlation with H-2α or H-2β in 1; while correlations of H-10′ with H-2α or H-2β were observed in 2. Thus, the relative configurations of 1 and 2 were determined as (1S*,7R*,10S*,9′R*)-1 and (1S*,7R*, 10S*,9′S*)-2, respectively. These should cause H-1 and H-2 in 1 being upfield-shifted in comparison with those of in 2, due to the shielding effects of the phloroglucinol ring; while H-9 in 2 should be upfield-shifted for the same reason. This was consistent with the ¹H NMR data (Table 1) of H-1, H-2 and H-9 for 1 and 2.

Fig. 3 Energy-minimized (MM2) structures of 1 and 2 showing key ROESY (H ↔ H) correlations.

Since no suitable crystals could be obtained for X-ray analysis in the study, a computational calculation using the gauge-independent atomic orbital (GIAO) method was carried out to confirm the relative configuration of 1 and 2. Conformational searches were performed for (1S,7R,10S,9′R)-1 and (1S,7R,10S, 9′S)-2 using MMFF94S force field and the stable geometries were optimized at the B3LYP/6-31G (d) level using the reported methods.^23–27 The lower energy conformations were used for ¹³C NMR calculations at the B3LYP/6-311++G (2d, p) level. After Boltzmann statistics, the obtained ¹³C NMR data were compared with corresponding experimental data (ESI†). The experimental ¹³C NMR of C-9 in 1 and 2 showed major difference (δ_C 43.0 in 1; δ_C 40.1 in 2), which matched with the calculated ¹³C NMR (δ_C 44.2 in 1; δ_C 41.1 in 2) of C-9. In addition, The calculated ¹³C NMR of C-10, C-14, and C-9′ in (1S,7R,10S,9′R)-1 were slighter upfield-shifted than those in calculated (1S,7R,10S,9′S)-2, more than 1.0 ppm difference. These were further supported by the experimental ¹³C NMR data (Table 1).

Eucalmaidials A (1) and B (2) represent a novel chemo-type of phloroglucinol-coupled sesquiterpenoids. Iphionane-type sesquiterpenoid have only reported from family Asteraceae and Cyperaceae.²¹ Their biogenetic precursor was considered to be eudesmane-type sesquiterpenoids.²¹ The pathway to account for the plausible formation of 1 and 2 was postulated (Scheme 1). 4,6-Diformyl-2-isopentanoyl-phloroglucinol (15), the most abundant compound in the titled genus, was reported to biosynthetically produce the carbocationic intermediate 16.²⁸ Then, the intermediate 16 attacks the germacrol (17) to generate the intermediate 18. Macrocarpals I and J (7 and 8) are generated by ensuing hydration. Alternatively, deprotonation of intermediate 18 led to the macrocarpal H (5), K (6), and intermediate 19, from which compounds 1 and 2 are generated by subsequent oxidation, Claisen condensation and dehydration.²¹

Scheme 1 Hypothetical biogenetic route of 1 and 2.

The known compounds, macrocarpals A (3), B (4), H (5), K (6), I (7), J (8), E (9), G (10), eucalyptone (11), 3β-hydroxy-11α-methoxyurs-12-en-28-oic acid (12), 3β-hydroxyurs-9(11),12-dien-28-oic acid (13), and 3β,13β-dihydroxyurs-11-en-28-oic acid (14), were determined on the basis of ESI-MS and ¹H, ¹³C and 2D NMR data, as well as by comparison with data reported in the literature. Compounds 10–12 were reported from the genus Eucalyptus for the first time.

Compounds 1, 3, 5, 7, 8 and 10–14 were tested for their anti-fungal and antibacterial activity against Candida glabrata, Staphylococcus aureus, and methicillin-resistant S. aureus (Table 2). Eucalmaidial A (1), macrocarpal I (7), and eucalyptone (11) showed antifungal activity against C. glabrata with an IC₅₀ of 0.75, less than 0.8, 0.62 μg mL⁻¹, respectively; while macrocarpals A (3), H (5), J (8), and G (10) showed moderate antifungal activity, in comparison with corresponding positive controls, amphotericin B (IC₅₀ = 0.14 μg mL⁻¹). Triterpenoids (11–12) showed no antifungal activity against C. glabrata. Of all the tested compounds, only macrocarpal G (10) showed moderate antibacterial activity against S. aureus and methicillin-resistant S. aureus, in comparison with corresponding positive controls, Ciprofloxacin (IC₅₀ = 0.08 μg mL⁻¹).

Table 2 Antifungal and antibacterial activities of 1, 3, 5, 7, 8, and 10–14

Compounds	C. glabrata^a	S. aureus	MRS^b
a IC50 value of antifungal or antibacterial activities, μg mL^−1.b Methicillin-resistant S. aureus.c No activity at the conc. of 20 μg mL^−1.
1	0.75	—^c	—
3	1.38	1.87	1.16
5	1.71	3.29	2.74
7	<0.8	8.23	4.53
8	1.93	—	—
10	1.59	0.53	<0.8
11	0.62	8.05	6.29
12	—	—	14.88
13	—	5.94	8.44
14	—	—	—
Amphotericin B	0.14
Ciprofloxacin		0.08	0.08

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Conclusions

In summary, two new phloroglucinol-coupled iphionanes, eucalmaidial A and B (1, 2) were isolated from the juvenile leaves of E. maideni, along with nine known phloroglucinol coupled sesquiterpenoids (3–11) and three known triterpenoids (12–14). Iphionane moiety is rare in the family Myrtaceae. They were proposed to be biogenetically derived from phloroglucinol coupled eudesmane. Triterpenoids 10–12 were reported from the genus Eucalyptus for the first time. Eucalmaidial A (1) macrocarpal I (7), and eucalyptone (11) showed antifungal activity against C. glabrata with an IC₅₀ of 0.75, less than 0.8, 0.62 μg mL⁻¹, respectively. And only macrocarpal G (10) showed moderate antibacterial activities against S. aureus and methicillin-resistant S. aureus.

Experimental section

General procedures

Column chromatography was performed on Silica gel, 200–300 mesh (Qingdao Haiyang Chemical Co.), MCI-gel CHP20P, 75–100 μm (Mitsubishi Chemical Co., Ltd.), and RP-8 (40–63 μm, Merck). Preparative HPLC (Waters 600, USA) was performed using a C-18 column (ZORBAX, 9.4 mm i.d. × 250 mm, USA) and developed isocratically at room temperature with MeOH–H₂O (85/15); detection wavelength (276 nm). Thin-layer chromatography (TLC) was performed on precoated silica gel H plates, 0.2–0.25 mm thick (Qingdao Haiyang Chemical Co.), with CHCl₃–MeOH–H₂O [9 : 1 : 0.1 (a) or 8 : 2 : 0.2 (b), v/v/v], and spots were detected by spraying with anisaldehyde–H₂SO₄ reagent followed by heating. Optical rotations were obtained on a JASCO P-1020 polarimeter. UV spectroscopic data were measured on a Shimadzu-210A double-beam spectrophotometer. IR spectra of samples in KBr discs were recorded on a Bruker-Tensor-27 spectrometer with KBr pellets. NMR spectra were carried out on either a Bruker DRX-500 or an Avance 600 MHz spectrometer. ESI-MS or HR-ESI-MS was recorded on an API QSTAR Pular-1 mass spectrometer.

Plant materials

The juvenile leaves of E. maideni were collected in the Botanical Garden of Kunming Institute of Botany, Chinese Academy of Sciences, Yunnan, China, in June 2009, and identified by Prof. Xiao Cheng (Botanical Garden, Kunming Institute of Botany, Chinese Academy of Sciences). A voucher specimen (KIB-ZL-200901) has been deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and isolation

The fresh juvenile leaves of E. maideni (7.8 kg) were extracted with 80% aqueous acetone at room temperature (30 L × 3, each 1 week). After removal of organic solvents, the extracts were concentrated to small volume (5 L) and partitioned with CHCl₃ (2.5 L × 5) after filtration of the precipitate. The CHCl₃ fraction (50 g) was subjected to silica gel column chromatography (CC), eluting with CHCl₃–MeOH (0 : 1–7 : 3) to afford six fractions. TLC analysis in neutral (CHCl₃–MeOH–H₂O 8 : 2 : 1) and acidic (benzene/HCOOEt/HCOOH 4/5/0.5) system revealed that compounds in fractions 3–6 contained phenolic hydroxyl group or carboxyl groups. Further HPLC analysis [separation and detection modules: Waters 2695, Waters 2996; column: agilent C18 (4.6 i.d. × 250 mm); gradient: 0–5 min: 10% MeOH, 5–35 min 10–100%, 35–40 min, 100%; flow rate: 1.0 min mL⁻¹ these fractions revealed that in late eluted parts, around 30 min, peaks contained characteristic UV profile of acylphloroglucinol group, 270 nm and 380 nm. Fractions 3–6 were further monitored by ESIMS. Fraction 3 (ESIMS⁻ m/z 453, 485), fraction 4 (ESIMS⁻ m/z 471), fraction 5 (ESIMS⁻ m/z 471, 485), and fraction 6 (ESIMS⁻ m/z 471, 489) contained the molecular ion of possible phloroglucinol sesquiterpenoids. Further purification on fraction 3 (2.5 g) over silica gel (CHCl₃–MeOH–H₂O, 9 : 1 : 0.1–7 : 3 : 0.5), MCI gel CHP20P (70–100% MeOH), and RP-8 (70–90% MeOH) to yield 10 (60 mg, MW 454), 11 (60 mg, MW 486), and 12 (40 mg), 13 (9 mg), and 14 (8 mg). Fraction 5 (5.1 g) was subjected to chromatography on MCI gel CHP20P (60–100% MeOH), silica gel (CHCl₃–MeOH–H₂O, 9 : 1 : 0.1–7 : 3 : 0.5), and semipreparative HPLC (80% MeOH–H₂O, water was acidified with 0.1% CF₃COOH) to yield 1 (9.0 mg), and 2 (2.0 mg), together with 3 (20 mg, MW 472), 4 (18 mg, MW 472), 5 (20 mg, MW 472), and 6 (8 mg, MW 472). Fraction 6 (2.5 g) was applied to MCI gel CHP20P (70–100% MeOH), silica gel (CHCl₃–MeOH–H₂O, 9 : 1 : 0.1–7 : 3 : 0.5), and RP-8 (70–90% MeOH) to yield 7 (16 mg), and 8 (25 mg).

Eucalmaidial A (1): yellow amorphous powder; [α]²⁰_D + 8.3 (c 0.2, MeOH); UV (MeOH), λ_max (log ε) 272 (3.86), 387 (3.32) nm; IR (KBr) ν_max 3431, 2923, 2581, 1630, 1511, 1384, 1273, 1128, 1033 cm⁻¹; ¹H NMR (pyridine-d₅, 500 MHz), see Table 1; ¹³C NMR (pyridine-d₅, 125 MHz), see Table 1; ESIMS (negative ion mode) m/z 485 [M − H]⁻; HRESIMS m/z 485.2531 [M − H]⁻ (calcd for C₂₈H₃₇O₇, 485.2539).

Eucalmaidial B (2): yellow amorphous powder; [α]²⁰_D − 21.1 (c 0.3, MeOH); UV (MeOH), λ_max (log ε) 274 (4.20), 388 (3.81) nm; IR (KBr) ν_max 3432, 2926, 2865, 1632, 1454, 1311, 1148, 1097 cm⁻¹; ¹H NMR (pyridine-d₅, 500 MHz), see Table 1; ¹³C NMR (pyridine-d₅, 125 MHz), see Table 1; ESIMS (negative ion mode) m/z 485 [M − H]⁻; HRESIMS m/z 485.2544 [M − H]⁻ (calcd for C₂₈H₃₇O₇, 485.2539).

Antifungal and antibacterial assay

Susceptibility testing was performed using a modified version of the NCCLS method using organisms obtained from the American Type Culture Collection (Manassas, VA) including Candida glabrata ATCC 90030, Staphylococcus aureus ATCC 29213, and methicillin-resistant Staphylococcus aureus ATCC 33591. Detailed procedures have been described in a previous paper.²⁹

Acknowledgements

We are grateful to the members of the Analytical Group in the West China State Key Laboratory of Phytochemistry and Plant Resources in Kunming Institute of Botany, for the measurements of all spectra. This work was supported by the 973 Program (2011CB915503) of Ministry of Science and Technology of China and the Twelfth 5-Year National Science & Technology Support Program (2012BAI29B06).

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Footnote

† Electronic supplementary information (ESI) available: UV, IR, MS, HRESIMS, and NMR spectra, as well as the calculated ¹³C NMR data for new compounds 1 and 2. See DOI: 10.1039/c4ra01078g

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