Lychnophoric acid from Lychnophora pinaster: a complete and unequivocal assignment by NMR spectroscopy

The investigation of the hexane extract from aerial parts of Lychnophora pinaster provided, besides others substances, the E-isomer of lychnophoric acid, a sesquiterpene derivative previously isolated from L. affinis.


Introduction
Plant species of the genus Lychnophora (Asteraceae) are known as "candeia", "arnica" and "arnica da serra" and are used in folk medicine as anti-flogistic, anti-rheumatic, and analgesic [1].Typical constituents of Lychnophora species are sesquiterpene lactones [2] of which 15deoxygoyazensolide was shown to be active against Trypanosoma cruzi, the etiological agent of Chagas' disease (American trypanosomiasis) [3].Prompted by this observation we have carried out a screening of Asteraceae plant species in the search of new trypanocidal agents [4] and we have investigated three active Lychnophora species, one of them being L. pinaster Mart.Bioguided fractionation of the hexane and dichloromethane extracts of the aerial parts of this plant [5] led to the isolation of lychnophoric acid(1), previously isolated from L. affinis, that was assayed in vitro against bloodstream forms of T. cruzi and presented 50% growth inhibition in the dose of 12,0mg/mL [6].

General
Melting point was determined on a Mettler FP5 apparatus; [a] D was measured at 25 o C on a Bellincham & Stanley Ltd P-20 polarimeter.IR spectrum was obtained on a Shimadzu/IR-408 spectrometer.EIMS was obtained on a Kratos MS 80 RFA spectrometer. 1 H and 13 C NMR spectra and contour plots were acquired on a Bruker AVANCE DRX400 instrument operating at 400 MHz for 1 H and 100 MHz for 13 C. HPLC analysis was performed with a Shimadzu CR-8, UV detector.CG analysis was performed with a HP5890 gas chromatograph, FID detector, and a VDC3390A integrator.

Plant material
The aerial parts of Lychnophora pinaster Mart.were collected at Serra da Moeda, State of Minas Gerais, Brazil, in March 1992.A voucher specimen has been deposited in the Herbarium of the Instituto de Ciências Biológicas, UFMG, Belo Horizonte, Minas Gerais (BHCB-UFMG 19520).

Extraction procedures
The dried aerial parts (2.0 Kg) were powdered and successively extracted with n-hexane and dichloromethane.The solvents were removed under vacuum, below 40 °C, to give 114.0 g of nhexane and 12.0 g of dichloromethane extracts.

Results and discussion
The hexane extract from the dried aerial parts of L. pinaster was column chromatographed over silica gel affording mixtures of homologue hydrocarbons [7], triterpenes (lupeol, a-and bamyrin, friedelin), fat acids (identified by GLC of their methyl esters), and a caryophyllene derivative, lychnophoric acid (1).The dichloromethane extract afforded a mixture of homologue hydrocarbons.Quercetin and 15-deoxygoyazensolide were detected by HPLC in comparison with authentic samples.
The IR spectrum of compound 1 showed absorption bands due to conjugated carboxylic function group (3600-2400, 1680 cm -1 ), carboncarbon double bonds (1640, 1470, 890 cm -1 ), and gem-dimethyl groups (1370 cm -1 ).Its 1 H NMR spectrum (Table 1) exhibited characteristic signals indicating the presence of a terminal olefinic methylene group (d 4.87 and δ 4.81) and another olefinic hydrogen in an a,b-unsaturated carboxylic group (δ 7.00).Two 3H singlets at d 0.96 and d 1.00 confirmed a gem-dimethyl group.EIMS indicated a [M] + of m/z 254, which in conjunction with 1 H and 13 C NMR data allowed the assignment of the molecular formula C 15 H 22 O 2 to (1).These data are very similar to those reported for lychnophoric acid (3) [8,9]. 1 2 3 However, divergences between 1 and 3 were observed for the 1 H NMR data: the signal of H-5 is shifted to a higher value of d 7.00 in the former, in comparison to that one originally described for lychnophoric acid (δ 6.22) [8].This fact can be explained by the change in the configuration of the double bond from Z-configuration in 3 to Econfiguration in 1, where the closer carbonyl group can contribute with its stereoelectronic deshielding effect.Besides the difference in chemical shifts, a difference in the multiplicity of the H-5 signal in the two compounds is also observed.In the Zisomer (3), this signal is described as a multiplet due to coupling with the two adjacent H-6 and to a long-range coupling with two allylic H-3 [8].The E-isomer (1) 1 H NMR spectrum shows an apparent triplet (δ 7.00, J=7.8 Hz and J=9.3 Hz) for H-5 due to imperfect superposition of the two inner signals of the theoretical double doublet, and the long ran-ge coupling with the two H-3 is not observed.
Despite the use of the Gaussian multiplication with Traficante function altogether in the normal fid, we could not achieve enough improvement of resolution to picture the H-5 theoretical double doublet.Likewise for the compound 2, the signal of H-9 appears as a quartet.All chemical shifts were supported by one and two-dimensional NMR techniques like NOEDIFF, COSY and NOESY.In particular the HMQC experiment was very important to the assignments of the chemical shifts inside the complex envelopes.For example, a strong nOe were observed for the protons H-12a (δ 4.87) with H-10b (δ 1.57), H-10a (δ 1.73), H-9 (δ 2.50) and Me-b group (δ 1.00) and between the protons H-9 (δ 2.50) and Me-a (δ 0.96) as well for the H-12b (δ 4.81) with H-7a,b spin system.The nOe were also observed for H-5 (δ 7.00) and H-6a,b system.The nOe results are summarized in the figure 1.

Conclusions
The 13 C NMR data for compound 1 are very close to those reported for compound 3 [9] (TABLE 1).The authors [9] did not report the 1 H NMR data.
These data led us to consider (1), is in fact the E-isomer of lychnophoric acid, originally described as the Z-isomer (3) in reference 8. Based on the reported 13 C NMR data (Table 1) the compound reported also represents the E-isomer (1) instead of the Z-isomer (3), as previously proposed [9].
The spectral data and nOe results of 1 are in good accord with data reported for aldehyde 2 [10].
as Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil for the assays against Trypanosoma cruzi; T. M. S. Grandi, J. R.