6-(1,3-Dihydroxy-3-phenylpropylidene)-5-hydroxy-2,2,4-trimethylcyclohex-4-ene-1,3-dione

Abstract

A novel compound involved in the aroma of the fruit Campomanesia lineatifolia was isolated; the structure was determined by spectroscopic methods, mainly 1D and 2D NMR.

Introduction

Champanones A, B and C are compounds isolated from the fruit of Campomanesia lineatifolia R. & P. (Myrtaceae) [1,2]. These compounds are characterized by the presence of several methyl groups in the A ring of a flavonoid or chalcone. Here, we report the structure of the new champanone D on the basis of NMR, mainly HMBC experiment; in addition, the substitution pattern can explain the biosynthesis of the other compounds.

Results and Discussion

Champanone D, 2 was isolated as a yellow powder and its structure was assigned as follows. NMR spectra displayed the presence of three methyl groups due to the singlets at δ 1.43, 1.46 and 1.89 (3H each one); in addition, a dt (2H) was detected at δ 3.02 for a methylene group methylene, and a d (J = 5.1 Hz) at δ 5.36 (1H). Finally, two more singlets were observed at δ 7.47 (5H, phenyl group) and at δ 13.84 (chelated hydroxyl group). COSY ¹H-¹H indicates a coupling between methylene at δ 3.02 with the singlet at δ 5.36; both signals correspond to a methylene and a methine group according to HMQC experiment.

Additionally ¹³C JMOD experiment revealed the presence of three methyl groups at δ 7.94, 23.11 and 25.53, which according to HMQC experiment correlates with methyl groups located at δ 1.89, 1.46 and 1.43, respectively, then, the last two signals were assigned to gem-dimethyl group. Other detectable signals were a methylene group at δ 38.26, and three methine carbon atoms at δ 76.12, 125.85 and 128.97 coupling to the intense singlet at δ 7.47 in ¹H-NMR. Also six quaternary carbon atoms were displayed in ¹³C JMOD at δ 52.40, 101.48, 107.35, 138.13, 161.32 and 182.91, as well as two carbonyl signals at δ 201.79 and 198.11. The existence of an oxygenated methine at δ 76.12 indicates the presence of an alcohol. A formula C₁₈H₂₀O₅ can be assigned to compound 2 based on these data.

When the spectroscopic properties of compound 2 were compared to those reported for other compounds isolated from Campomanesia lineatifolia, a high similarity was appreciated specifically with champanone B, since both possess three methyl groups (including a gem-dimethyl group), two carbonyl groups and a side phenyl ring (Figure 1).

Nevertheless, the possibility that both compound have the same structure was excluded since ¹H and ¹³C-NMR of champanone B were significantly dissimilar to those obtained in this work; in addition, HMBC experiment showed several anomalies. Thus, a carbonyl group at δ 198.11 ppm displayed long-range correlation with the methyl group and the gem-dimethyl group. However, the other carbonyl group at δ 201.79 only coupled to gem-dimethyl, which means coupling correlations involving five bonds (Figure 2, left). To meet all requirements of an HMB experiment, the new compound 2 should have several positions interchanged in relation to champanone B. If methyl and carbonyl groups were relocated as displayed in Figure 2 (right), all observed J³ and J² long-range correlations would be correct.

Aside from champanone D, champanone C, whose chemical shifts are shown in

Table 1. ¹H and ¹³C-NMR (CDCl₃) of several champanones, including champanone D 2.

No.	Champanone C 1		Champanone C [1]		Champanone D 2		Champanone B [1]
	δ1H	δ13C	δ1H	δ13C	δ1H	δ13C	δ1H	δ13C
1		186.4		186.0		161.32		197.4
2		48.84		48.4		52.40		48.2
2-Me	1.43 s	25.23	1.41 s	24.8	1.41 s	23.11	1.45 s	24.6
2-Me′	1.46 s	25.18	1.45 s	24.7	1.43 s	25.53	1.45 s	24.6
3		197.05		196.3		201.79		172.1
4		106.04		105.6		107.35		104.5
4-Me	1.82 s	7.08	1.80 s	6.6	1.87 s	7.94	1.92 s	6.7
5		164.50		164.0		182.91		191.0
6		103.53		103.1		101.48		105.8
1′		194.97		194.5		198.11		186.8
2′	2.85 dd	42.04	2.88 dd	41.6	3.00 m	38.26	7.92 d	123.3
	3.12 dd		3.10 dd
3′	5.60 dd	81.62	5.58 dd	81.2	5.35 d	76.12	8.30 d	144.5
1″		126.45		129.6		138.13		135.3
2″ y 6″	7.44 m	129.56	7.41 m	126.0	7.45 s	128.97	7.66 m	128.8
3″ y 5″	7.48 m	130.03	7.47 m	129.1	7.45 s	128.97	7.39 m	129.0
4″	7.48 m		7.47 m	129.6	7.45 s	128.97	7.39 m	130.5
-OH	11.65 s		11.61 s		15.86 s		19.18 s

, was also isolated.

The structure of champanone D could explain the biosynthesis of several types of compounds, since retro-enolization of the C-5 carbonyl group leads to two type of molecules. One of them with a gem-dimethyl at C-4 or free rotation around C-4, C-5 bond produces a molecule with a C-2-gem-dimethyl group, alike champanone C (Figure 3).

Phloroglucinol derivatives have mainly been reported in the species of the Myrtaceae and Hypericaceae families [3,4] and exhibit several biological activities including degenerative diseases and antibiotics, especially antivirus [5].

Experimental Section

¹H and ¹³C-NMR spectra were recorded on a Bruker AMX ((Karlsruhe, Germany) operating at 300 and 75.0 MHz respectively, chemical shifts (δ) were reported in ppm and coupling constants (J) in Hz, CDCl₃ was used as solvent and internal standard. Infrared spectra were measured in KBr with a Thermonicolet Avatar 330 (Madison, WI, USA), mass spectra was recorded using a TQD triple quadrupole mass spectrometer with an orthogonal electrospray ionization source Z-spray (Waters, Milford, MA, USA) was used. Cone gas as well as desolvation gas was dry nitrogen. The cone gas and the desolvation gas flows were optimized at approximately 60 L/h and 1100 L/h, respectively. Other parameters optimized were: capillary voltage, 4.2 kV in positive ionization mode; lens voltage 0.3 V; source temperature, 105 °C and desolvation temperature, 400 °C. Dwell times of 0.01 s/transition were selected.

Silica gel 60 (200–300 mesh) (Merck, Darmstadt, Germany) and Sephadex LH-20 (Sigma, St. Louis, MO, USA) were used for all separations, while silica gel 60 F₂₅₄ (Merck) was used for analytical thin layer chromatography (TLC). The compounds were detected under UV light (254, 366 nm) and by spraying with H₂SO₄ (10%) followed by heating.

Dry seeds of Campomanesia lineatifolia (50 g) obtained in the local market were milled and then extracted by percolation (0.5 L) with ethanol. After evaporation, the residue (3.5 g) was dissolved in 100 mL of a mixture methanol/water (3:1 v/v), extracted with hexane (3 × 100 mL) and EtOAc (3 × 100 mL); the last extract yield a yellow powder (450 mg).

The compounds were purified by flash chromatography in a medium pressure chromatographic system, using a Biotage^® SNAP Cartridge, KP-SIL, packed with 50 g of silica (50 μm), and run with Hex:EtOAc 95:5 (v/v) until EtOAc100%, flux rate 10 mL/min. Then, seventy fractions of 50 mL each were collected and monitored by tlc in silica gel chromatoplates 60 F₂₅₄, eluted with Hex:EtOAc (7:3, v/v), revealed with AcOH-H₂SO₄ spray and heated at 100 °C. Fractions with similar composition were mixed together to obtain only 7 chromatographic fractions. Fraction 3 (75 mg) was purified by repeated column chromatography until compound 1 (10 mg) was obtained, while from fraction 5 (65 mg) compound 2 was obtained (35 mg).

Champanone C, 5-hydroxy-6,8,8-trimethyl-2-phenyl-2H-chromene-4,7(3H,8H)-dione (1), was isolated as yellow needles, m.p. = 152.3 °C–153.2 °C. MS (TQD EI+): m/z 321.27 (40) [M+Na]⁺, 299.22 (70) [M+H]⁺, 227.29 (100).

Champanone D, 6-(1,3-dihydroxy-3-phenylpropylidene)-5-hydroxy-2,2,4-trimethylcyclohex-4-ene-1,3-dione (2). Was isolated as amorphous yellow powder, m.p. = 139.5 °C–141.5 °C, FT-IR ν^KBr_max = 3045, 2989, 2930, 1734, 1645, 1630, 1440, 965. MS (TQD EI⁺): m/z 339.25 (25) [M+Na]⁺, 316.43 (70) [M+H]⁺, 284.54 (100).