Chemical components and pharmacological properties of secretions from parotoid glands of Bufonidae family members have been studied for a long time.1 This secretions are a fascinating source of active compounds, such bufadienolides and alkaloids.2 In the search for new molecules with important pharmacological properties, the 3β-OH groups of bufadienolides are commonly derivatized with an acetyl group along a synthetic route, to protect them from side reactions.3 This work has as objective to isolate and purify chemical constituents from Rhinella marina venom and to derivatize some of them to biological activity.
The fraction MB (56.1 mg), was subjected to separation on a HPLC with isocratic progamation and mobile fase of 60% of CH3CN. The other fraction CRV-6 of the methanolic extract of R. marina was subjected to fractionation by cromatographic columns, resulting in the isolation of compounds: 3, 4, 5 and 6. The compounds 1 (marinobufagin - 10.1 mg) and 2 (telocinobufagin - 5.3 mg), previously isolated from R. marina venom, were submited to acetylation with acetic anydride in the presence of piridine, resulting in the compounds 7 (10.2 mg) and 8 (4.6 mg), respectively. The compound 6 (5.3 mg) was submited to methylation with diazomethane, obtaining the substance 9 (5.1 mg). The isolated and derivatized compounds were identified by 1H and 13C NMR 1D and 2D, and ESI-MS-MS mass spectrometry being identified as bufalin (3), dehydrobufotenine (4), suberoyl arginine (5), marinobufotoxin (6), 3-acetyl-marinobufogenin (7), 3-acetyl-telocinobufogenin (8) and 3(N-methyl suberoyl arginate) marinobufogenin (9) (Figure 1).
Figure 1 – Isolated compounds of the venom from R. marina and their derivatives

In conclusion, were isolated and identified six compounds, a bufotoxin marinobufotoxin, isolated for the first time from R. marina and obtained three derivatives 3-acetyl-telocinobufagenin, 3-acetyl-marinobufogenin and 3(N- methyl suberoyl arginate) marinobufogenin, derivate for the first time, which were referred to biological activities.
1Sousa, L.Q.; Machado, K.C.; Oliveira, S.F.C.; Araújo, L.S.; Monção-Filho, E.S.; Cavalcante, A.A.C.M.; Vieira-Junior, G.M. Ferreira, P.M.P. Bufadienolides from amphibians: A promising source of anticancer prototypes for radical innovation, apoptosis triggering and Na+ /K+ -ATPase inhibition. Toxicon. v. 127, p. 63-76, 2017.
2Ferreira, P.M.P.; Lima, D.J.B.; Debiase, B.W.; Soares, B.M.; Machado, K.C.; Noronha, J.C.; Rodrigues, D.J.; Sinhorin, A.P.; Pessoa, C.; Vieira-Junior, G. M. Antiproliferative activity of Rhinella marina and Rhaebo gutatus venom extracts fom Southern Amazon. Toxicon. v.72, p.43-51, 2013.
3Cunha-Filho, G.A., Resck, I.S., Cavalcanti, B.C., Pessoa, C.O., Moraes, M.O., Ferreira, J.R., Rodrigues, F.A., Dos Santos, M.L. Cytotoxic profile of natural and some modified bufadienolides from toad Rhinella schneideri parotoid gland secretion. Toxicon. v. 56, p. 339–348, 2010