Complete assignments of NMR data and assessment of trypanocidal activity of new eremantholide C derivatives

Chemical transformations of eremantholide C (1), a sesquiterpene lactone that was isolated from Lychnophora trichocarpha Spreng. led to five new derivatives: 1’,2’epoxyeremantholide C (2), 5-n-propylamine-4,5-dihydro-1’,2’-epoxyeremantholide C (3), 5-n-propylammonium-4,5-dihydro-1’,2’epoxyeremantholide C chloride (4), 5-n-propylammonium-4,5-dihydroeremantolide C chloride (5) and 16-O-ethyleremantholide C (6). The structures of all these derivatives were assigned on the basis of IR, MS, 1H and 13C NMR data by 1D and 2D techniques. Eremantholide C and the derivatives 2, 4 and 5 were evaluated against trypomastigotes Y and CL strains of Trypanosoma cruzi. Eremantholide C completely inhibited the growth of both the parasites strains while all derivatives were partially active against the CL strain and inactive against the Y strain.


INTRODUCTION
Sesquiterpene lactones are chemical markers of certain plant families such as Asteraceae.The large number of biological activities experimentally described up to now, raised great interest on this group of substances.The sesquiterpene lactones of the furanheliangolide type are biogenetically derived from heliangolides and are often found in species of the genus Lychnophora, that is native from Brazil (Bohlmann and Jakupovic 1990).In previous studies, eremantholide C and its oxidized derivatives showed activity against Trypanosoma cruzi (Oliveira et al. 1996, Saúde-Guimarães et al. 2007).Other biological/pharmacological activities were reported for eremantholide C, such as antibacterial, anti-hyperuricemic, anti-gouty arthritis, anti-inflammatory and antitumor (Barrero et al. 2000, Saúde et al. 2002, de Souza et al. 2012, DÊNIA A. SAÚDE-GUIMARÃES, DÉLIO S. RASLAN, EGLER CHIARI and ALAÍDE B. DE OLIVEIRA Ferrari et al. 2013, Saúde-Guimarães et al. 2014).Aiming to obtain new bioactive derivatives of eremantholide C (Figure 1), this sesquiterpene lactone was submitted to chemical modifications.

GENERAL PROCEDURES
Mass spectra were obtained with VG Autospec (electron impact) and HP 5988A (chemical ionization with methane) spectrometers by direct injection (ionization chamber at 200 °C).IR spectra were taken at Galaxy 3000-FTIR spectrophotometer (Mattson Instruments).NMR spectra were taken at Bruker Avance DPX (4.7T), DRX (9.4T), equipped with a 5mm dual probe, at 300 K, with TMS as internal reference.One-dimensional 1 H and 13 C NMR spectra were acquired under standard conditions, with 90° pulse widths of 8.00 µs and 8.50 µs for 1 H and 13 C, respectively. 1H NMR spectra were obtained using a sweep width of 3 kHz over 32k data points. 13C NMR spectra were obtained using a sweep width of 31 kHz.DEPT, 1 H, 1 H COSY and 1 H, 13 C HETCOR techniques were performed using standard pulse sequences supplied by the spectrometer manufacturer.
5-n-Propylamine -4,5-dihydro-1',2'-epoxyeremantholide C (3) was obtained by reaction of 2 (0.55 mmol) with n-propylamine (5.94 mmol), at -18 °C for 16 h (Kirk 1973).After this period, the excess of the amine was removed by evaporation under reduced pressure, at room temperature.The residue obtained was purified by PTLC on silica gel (0.5 mm thickness, eluent -hexane: EtOAc 1:1) resulting in 82 mg (35% yield) of 3 as a white solid.This compound was dissolved in dry THF and treated with gaseous HCl leading to the formation of 4 as a white hydrosoluble solid (quantitative yield).
5-n-Propylammonium-4,5-dihydroeremantolide C chloride (5) was obtained from the reaction of 1 (0.58 mmol) with n-propylamine (5.94 mmol), at -18 °C for 10 h (Kirk 1973).After this period, the excess of amine was removed by evaporation under reduced pressure, at room temperature.The residue obtained was purified by PTLC on silica gel (0.5 mm thickness, eluent -hexane: EtOAc 1:1) affording 120 mg of a yellowish pasty material.Silica TLC of this material showed three spots when revealed by iodine.After development with ninhydrin, the TLC showed only a rosy spot, characteristic of amines.This material was then dissolved in dry THF and treated with gaseous HCl to give the hydrochloride 5 as a white hydrosoluble solid (70 mg).
16-O-Ethyleremantholide C (6) was obtained, according to the methodology described by Partwardhan et al. 1974, by reacting 1 (0.28 mmol) with 0.08 mL of triethyl orthoformate and 25 mg of Amberlyst resin 15.The reaction mixture was stirred at room temperature for 3 days.Then it was neutralized with aqueous K 2 CO 3 solution and filtered.The product was extracted with diethyl ether.The organic layer was washed with water, filtered on anhydrous sodium sulfate and concentrated under reduced pressure, yielding 72 mg of ether 6 as a white solid (66% yield).

RESULTS AND DISCUSSION
Data of the 1 H and 13 C NMR spectra of derivatives 2-6 are given at on Tables I and II, respectively.The chemical shifts were assigned by consideration of known substituent effects of the groups concerned and with the aid of both 1 H, 1 H COSY and 1 H, 13 C HETCOR contour maps.The stereochemistry of the carbons were determined based on coupling constants and 1 H, 1 H NOESY contour maps.
From the reaction of 1 with m-chloroperbenzoic acid, the main product 2 was obtained and it was characterized by 1 H NMR data.The presence of an epoxy group in 2 was highlighted in the 1 H NMR spectrum by the two doublets at δ3.11 (J = 5.5 Hz) and δ2.70 (J = 5.5 Hz), attributed to H-2'a and H-2'b, which appear in the 1 H NMR spectrum of 1 at δ5.31 (br s) and δ5.07 (t, J = 1.6 Hz), respectively.
The signals at δ130.00 and δ115.80,attributed to the olefinic carbons C-1' and C-2', respectively, in the 13 C NMR spectrum of 1, are replaced in the spectrum of 2 by the signals at δ59.50 and δ58.47, typical chemical shifts of oxygenated carbons with hybridization sp 3 .In addition, the signal attributed to C-3 in the spectrum of 1 (δ19.00) is shifted to δ11.44 in the spectrum of 2, confirming the epoxidation of the ∆ 1 double bond of 1.
The mass spectrum of 2 showed the molecular ion peak at m/z 362, which represents an increment of 16 mass units to the molar mass of 1, corresponding to the addition of an oxygen atom.This mass is consistent with the molecular formula C 19 H 22 O 7 .
Data of IR, MS, 1 H NMR and 13 C NMR of 2, are in agreement with the new derivative 1',2'-epoxyeremantholide C.
Epoxide 2 was reacted with methylamine, cyclohexylamine, diethylamine and n-propylamine.Reactions with the first three amines led to mixtures of many products, in such a way that no derivative with suitable purity was obtained.The reaction of 2 with n-propylamine afforded a less complex mixture of products which, after separation by column chromatography on silica gel, led to the amino product 3 with a 35% yield.The amine 3 was transformed into its hydrochloride salt by reaction with gaseous HCl in dry THF, leading to the hydrosoluble compound 4.
The multiplets in δ5.02-4.98 and δ6.03-6.00attributed, respectively, to H-6 and H-5, in the 1 H NMR spectrum of 2 appear in the spectrum of 3 at δ4.26 (d, J = 6.5 Hz) and at δ2.99 (brs), respectively.The quartet at δ2.89 (J = 7.2 Hz), that appears in the 1 H NMR spectrum of 2, was assigned to H-4.A correlation of this signal with the doublets at δ1.37 (J = 7.2 Hz), assigned to H-15, is indicated by the values of the coupling constants and by the COSY 1 H-1 H contour map.The signals at δ134.27 and δ130.24 which are attributed to the olefinic carbons C-5 and C-4, respectively, as well as the signal attributed to C-15 (δ20.39) in the 13 C NMR spectrum of 2, are shifted in the 13 C NMR spectrum of 3 to δ65.24, δ38.20 and δ16.70, respectively, allowing the proposition that the amine group was linked to the C-5 of compound 2.
The mass spectrum of 3 presented the molecular ion peak at m/z 421 u, corresponding to 59 mass units higher than the molecular ion of 2, and it corresponds to n-propylamine group (C 3 H 7 NH 2 ).The molar mass of 3 was compatible with the molecular formula C 22 H 31 O 7 N.
Based on data retrieved from IR, MS, 1 H and 13 C NMR it was concluded that the product obtained was that resulting from the amine addition to carbon-5 of the epoxide 2, through a Michael's reaction, generating the novel derivative 5-n-propylamine-1',2'-epoxyeremantholide (3).This might be com pared to the proposed mechanism for the antitumor activity of the eremantholides by reaction with biological nucleophiles (Mc Dougal et al. 1989).
The shifting of the signals due to the hydrogen and carbon atoms 4, 5, 15, 4'a, 4'b, 5' e 6' of compounds 3 and 4 could be observed by comparing their 1 H and 13 C NMR spectra.
The signal corresponding to H-6 in the 1 H NMR spectra of 4 overlapped that of the solvent (D 2 O) at δ4.74.This was confirmed by the 1 H-1 H COSY contour map of 4, evidencing correlations between the signal at δ4.74 (brs), and the signals attributed to H-7 (δ3.09, dd, J = 4.2 and 7.8 Hz),   for 1, 2, 3, 4, 5 and 6.The two doublets at δ2.71 (J = 5.5 Hz) and δ3.12 (J = 5.5 Hz) assigned to H-2'a and b, respectively, and the signals at δ59.71 and δ53.80 on the 13 C NMR spectrum, due to carbons 1' and 2', respectively, showed that the epoxy group remained unchanged in the molecule of 3.
The assignments made to the signals in the 1 H NMR spectrum of 3 were confirmed by the values of the coupling constants and by the 1 H-1 H COSY contour map which showed the following correlations: H-4 and H-15; H-4'a, H-4'b and H-5'; H-5' and H-6'; H-6 and H-7, and H-2'a and b.Chemical shifts for 13 C NMR spectrum of 3 were assigned by comparison with the spectrum of 2 and based on the DEPT spectrum.NEW TRYPANOCIDAL EREMANTHOLIDE C DERIVATIVES and to H-5 (δ3.97, brs).The 1 H-1 H COSY also evidenced correlations between the following hydrogen atoms: H-4 and H-15; H-8, H-9a and H-9b; H-9a and H-9b; H-4'a, H-4'b and H-5'; H-5'and H-6'; H-2'a, H-2'b and H-3'.
The relative configuration of the n-propylamine and the C-15-methyl group was defined on the basis of the 1 H, 1 H NOESY (Figure 2), that indicated correlation between the H-5 signal at δ3.97 (brs) , with the signals at δ4.74 and δ3.56 (q, J = 7.2 Hz), attributed to H-6 and H-4, respectively.This correlation showed a -cis relationship between the H-4 and H-5 atoms with a β configuration.Consequently, the C-15-methyl group and the n-propylamine group should have an α configuration.Based on data from IR, MS, 1 H and 13 C NMR, COSY and NOESY it was concluded that compound 4 is 5-n-propylammonium-4,5-dihydro-1',2'-epoxyeremantholide C chloride, a novel compound that is firstly described in this paper.
In order to confirm the positioning of the n-propylamine group at the C-5 of compound 2, the reaction of 1 with n-propylamine was carried out.Three spots were observed when a silica gel TLC plate of the reaction mixture was sprayed with iodine.Under ninhydrin, only a rosy spot typical of amine, was observed.Then, the mixture was treated with gaseous HCl in dry THF yielding 5 as a white hydrosoluble solid.
The 1 H NMR spectrum of 5 presented a quartet at δ3.45 (1H, J = 7.2 Hz) that was attributed to H-4.The signals for H-5 and H-15 in this spectrum appeared as a broad singlet at δ3.88 and a doublet at δ1.48 (J=7.2Hz), respectively.In the 1 H NMR spectrum of 1 these atoms are represented by signals at δ6.04−6.03(m) and δ2.05 (t, J = 1.9 Hz), respectively.Values of coupling constants for H-4 and H-15 indicate that these hydrogens are in vicinal positions.
The signal attributed to H-6 in the 1 H NMR spectra of 5 is probably superimposed to that of H 2 O in the solvent (D 2 O), which appear at δ4.74.In order to shift the signal of H 2 O and observe the one from H-6, the 1 H NMR spectra of 5 in D 2 O was run at 330 K, when the signal due to the solvent was shifted to δ4.72 and the doublet attributed to H-6 was seen at δ5.07 (J = 7.9 Hz).The stereochemistry of the n-propylammonium and of the C-15 methyl groups were inferred based on the NOESY technique (Figure 2), which indicated a correlation between the signal at δ3.88 (brs), attributed to H-5, and the signals at δ4.73 (d, J = 7.9 Hz) and δ 3.45 (q, J = 7.2 Hz), corresponding to H-6 and H-4, respectively.These data suggest that H-4 and H-5 have a cis relationship and a β configuration.Consequently, the C-15 methyl and the n-propylammonium groups have a α configuration.
The molecular ion peak m/z 441 u, corresponding to the molecular formula C 22 H 32 O 6 NCl, was not observed at the mass spectrum of 5, but it showed a peak at m/z 405 u that was compatible to the loss of a molecule of HCl from the molecular ion.
Data of IR, MS, 1 H and 13 C NMR spectra, and the COSY and NOESY contour maps are in agreement with the new derivative 5-n-propylammonium-4,5dihydroeremantholide C chloride, a new derivative of eremantholide C.
The 13 C NMR spectrum of 6 shows a signal at δ57.26, characteristic for a sp 3 carbon bonded to oxygen and, therefore, it was attributed to C-4'.The signal at δ15.29 was assigned to the methyl carbon at the 5' position.These data confirmed the presence of an ethoxyl group in the molecule of 6.
In the mass spectrum of 6 the ion [M+1] + appeared at m/z 375 u corresponding to an increase of 29 mass units in comparison with 1, that is compatible with the presence of an ethyl group, and corresponding to the molecular formula C 21 H 26 O 6 .
Assignments of the signals in the 1 H NMR spectrum of 6 were confirmed through the corre-lations observed at the 1 H, 1 H COSY contour map.
Based on the modifications observed in the IR, MS, 1 H and 13 C NMR spectra it was concluded that the compound obtained was the new derivative 16-O-ethyleremantholide C (6).
Eremantholide C derivatives 2, 4 and 5 were tested against trypomastigote forms of Y and CL strains of Trypanosoma cruzi, the infectious agent of Chagas' disease, in comparison with Violet crystal (active at 125 µg.mL -1 ) that was used as reference in the in vitro tests.The results are shown on Table III.Eremantholide C and the derivatives 2, 4 and 5 were evaluated against Y and CL strains of T. cruzi.Eremantholide C completely inhibited the growth of both the parasite strains in the concentrations of 3,600 µg/mL (Y strain) and 1,800 µg/mL (CL strain), respectively, while all the derivatives were partially active against the CL strain and inactive against the Y strain, in the concentrations assayed.
IN VITRO ASSAYS WITH TRYPANOSOMA CRUZI TRYPOMASTIGOTES Albino mice infected with the Y or CL strains of T. cruzi were used to provide trypomastigotes.DÊNIA A. SAÚDE-GUIMARÃES, DÉLIO S. RASLAN, EGLER CHIARI and ALAÍDE B. DE OLIVEIRA