A new antitrypanosomal cerebroside isolated from Piper lepturum Kunth (Piperaceae)

Natural products isolated from plants could be important sources of new prototypes for development of new drugs against tropical neglected disease, including Chagas. Caused by the parasite Trypanosoma cruzi, it is a potentially fatal disease, and it is estimated that about 6-7 million people are infected worldwide, mostly in Latin America.[1-2] As part of our continuous studies concerning the identification of antitrypanosomal metabolites from Brazilian flora, the MeOH extract from leaves of Piper lepturum displayed activity against trypomastigote forms of T. cruzi (100% of parasite death at 300 ug/mL) and was subjected to a bioactivity-guided fractionation. After several chromatographic steps including SiO2, Sephadex LH-20 and RP-HPLC, one new cerebroside (Figure 1) was isolated whose structure was elucidated by spectral analysis. 13C and DEPT NMR spectra displayed six signals at δc 74.6 (C-5”), 71.6 (C-3”), 70.2 (C-2”), 69.5 (C-4”), 62.8 (C-6”), and 103.9 (C-1”) characteristic of a glycoside unit. Also, signals at δc 68.3 and 73.4 were attributed, respectively, to carbinolic carbons C-1 and C-3 of a sphingolipid unit while that at δc 57.2, characteristic of a carbon linked to a nitrogen atom, was attributed to C-2. One signal at δc 173.0 was associated to carbonyl carbon C-1’. These spectra showed also several peaks at the range of δc 29.6-29.1, which were assigned to methylene side chains, one intense signal at δc 14.1, assigned to terminal methyl carbons C-22 and C-20’. Finally, two sp2 carbons at δc 130.0 and
128.3 were indicatives of a double bond with cis configuration. The 1H NMR spectrum displayed one broad triplet at δH 0.97 (J = 7.5 Hz, H-20’/H-22), one broad singlet at δH 1.25 (H-12 to H-20, H- 5 to H-7 and H-3’ to H-19’) and multiplets at δH 2.12 – 2.03 (H-8 and H-11), at δH 4.41 - 3.51 (H-1” to H6”) and at δH 5.37-5.33 (H-9 and H-10). HRESIMS (positive mode) spectrum showed the [M+H]+ at m/z 812.6976 corresponding to the molecular formula C48H94NO8. Moreover, the MS/MS spectrum showed a signal at m/z 633.6421 concerning to a bonding cleavage of carbon C-1, confirming the presence of a glycoside unit. Additionally, the presence of peaks at m/z 295.2999, 267.3050, 195.2110 and 169.1955 in the MS/MS spectrum, allow us to infer the presence of a C20 saturated alkyl side chain, a nitrogen atom linked at C-2 and another C22 alkyl side chain with one double bond at C-9/C-10, whose characterization is herein described for the first time. This compound displayed activity against trypomastigote forms of T. cruzi with an EC50 value of 15.3 ± 5.9 μM (benznidazole EC50 = 5.5 ± 2.1 μM). Furthermore, it did not display cytotoxicity against NCTC cells (CC50 > 200 μM) and gave SI >13. Considering the properties of this compound it could be used as a starting point to develop new leads for treatment of Chagas disease.

References:
1. Pecoul, B. et al., PLoS Negl. Trop. Dis. 2016, 10, e0004343;
2. World Health Organization, 2019.

Acknowledgments: CAPES, CNPq and FAPESP.