Two new polyamine alkaloids from the Bufo viridis toad venom

Abstract Two new polyamine alkaloids (bufonines A-B), together with four known alkaloids, bufotenidine (3), bufotenine (4), 1-(β-d-ribofuranosyl)-lH-1,2,4-triazone (5) and proline (6) were isolated from the Bufo viridis toad venom. Their structures were identified by UV, HR-ESI-MS, NMR spectral analyses, and comparison of theoretical and experimental ECD data. All compounds were tested in vitro cytotoxicity against three human cancer cell lines (HT-29, A549 and Hela). None of the compounds showed cytotoxicity towards all tested cell lines. To the best of our knowledge, this is the first report of alkaloid components from Bufo viridis toad venom. Graphic Abstract


Introduction
The Bufonidae family, which contains approximately 350 species of toads, is an important source of natural medicine. Bufo viridis is the most common amphibian in farmland and grassland in Xinjiang Province, China. Many kinds of toad products, such as toad venom and toad skin, have been used for hundreds of years and are widely applied in Asia, especially in China, Korea, and Japan. (Chen et al. 2017;Ying et al. 2021).
The chemical constituents of toad venom are complex, including bufadienolides (Meng et al. 2021;Meng et al. 2016), alkaloids , proteins (Kowalski et al. 2018;Wang et al. 2017;) and peptides (Xia et al. 2019;Yang et al. 2016). Recent clinical studies have suggested that toad venom had significant analgesic (Zhou et al. 2004), anti-inflammatory (Qi et al. 2014), as well as significant cardiotonic (Krylov 2002) and anti-cancer activities Qi et al. 2014). However, the chemical constituents of Bufo viridis toad venom has seldom been studied (Soliev et al. 2007;Tashmukhamedov et al. 1994), as compared with the venoms of toads living in other regions. And the Bufo viridis toad venom showed the highest diversity of constituents in terms of substance classes among all the other toad venoms . The isolation of the physiologically active components of the Bufo viridis toad venom and the study of their structures and biological activities are of considerable interest.
In this paper, we describe the isolation and structural elucidation of two new polyamine alkaloids, along with four known alkaloids ( Figure 1). Their structures were established by extensive spectroscopic data analysis and comparison with literature values. Furthermore, the cytotoxic activities of all the isolated compounds were evaluated.

Results and discussion
Compound 1 had the appearance of a pale yellow amorphous powder. Its molecular formula was determined to be C 13 H 24 N 4 O 5 by HR-ESI-MS from the ion at m/z 317.1810 þ , 317.1811) with 4 degrees of unsaturation, and the molecular formula was supported by 1 H-NMR and 13 C-NMR spectral data in D 2 O (Table  S1). Its 13 C NMR and HSQC spectra represented the signals of 13 carbon resonances, including four carbonyl signals (d C 183.0, 181.2, 179.5, and 159.7), eight methylenes (d C 43.5, 38.5, 37.4, 31.6, 30.7, 28.0, 27.5 and 27.2) and one methine group at d C 57.1.
And the 1 H-NMR spectral data revealed 17 hydrogen signals, so the remaining hydrogen atoms were reasonably contributed to a hydroxyl group, two imido groups and two amino groups. The 1 HÀ 1 H COSY correlations of H-2/H-3/H-4/H-5, H-2 0 /H-3 0 /H-4 0 /H-5 0 /H-6 0 along with HSQC spectra established corresponding segments ( Figure S1). The HMBC correlations of H-2 0 (d H 2.29) to C-3 0 (d C 28.0) and C-4 0 (d C 30.7); H-4 0 (d H 1.32) to C-2 0 (d C 38.5), C-3 0 (d C 28.0), C-5 0 (d C 27.2) and C-6 0 (d C 37.4); H-6 0 (d H 2.33) to C-4 0 (d C 30.7), C-5 0 (d C 27.2) and C-7 0 (d C 183.0) led to the interpretation of a hexanoic acid moiety. And the HMBC correlations of H-2 (d H 4.22) to C-1(d C 181.2), C-3(d C 31.6) and C-4 (d C 27.5); H-5 (d H 3.20) to C-3(d C 31.6), C-4 (d C 27.5) and C-7 (d C 159.7) established the presence of a ureidopentanamide moiety. The attachment of C-1 0 to the methine group C-2 was deduced by the HMBC correlations between H-2 (d H 4.22) and C-1 0 (d C 179.5). The NOESY correlations were observed between H-2 and H-3/H-4, H-5 and H-3/H-4, H-6 0 and H-3 0 /H-4 0 /H-5 0 . The absolute configuration of 1 was established by comparison of its experimental electronic circular dichroism (ECD) spectra with that obtained by quantum chemical ECD calculation. The geometrical optimization and energy calculation of conformers for ECD calculation were performed by TmoleX 4.3 software, using the DFT/m4 method at the def-TZVP level. The stable conformer obtained was subjected to ECD calculation by the DFT/m4 method at the def-TZVP level. The overall pattern of the calculated ECD spectrum for 2S was in good agreement with the experimental ECD spectrum of 1 ( Figure S2). Thus, the structure of 1 was established as (S)-5 and 13 C-NMR spectral data in CD 3 OD (Table S1) showed that the structure of compound 2 was closely similar to that of compound 1, and the only structural difference was the existence of an additional methylene group in compound 2. This conclusion was confirmed by the significant HSQC correlation between protons. And the HMBC correlations ( Figure S1) were observed H-4 0 (d H 1.30) to C-2 0 (d C 37.1) and C-3 0 (d C 26.8); H-5 0 (d H 1.30) to C-6 0 (d C 26.2) and C-7 0 (d C 35.6); H-7 0 (d H 2.20) to C-6 0 (d C 26.2) and C-8 0 (d C 178.6) led to the interpretation of a heptanoic acid moiety. The NOESY correlations were observed between H-2 and H-3/H-4, H-5 and H-3/H-4, H-7 0 and H-5 0 /H-6 0 . The same absolute configuration of 2S for compound 2 as that of compound 1 was identified according to the comparison of their ECD spectra ( Figure S2). Thus, the structure of 2 was established as (S)-5-[(1 0 -amide) heptanoic acid-2-yl] ureidopentanamide, named bufonine B.
Compounds 1-6 were evaluated for their cytotoxic activities against three human cancer cell lines (HT-29, A549 and Hela). Unfortunately, they showed no obvious cytotoxicity potential at the concentration of 50 lM (Table S2).

Conclusions
In summary, two new polyamine alkaloids, together with four known alkaloids were isolated from the Bufo viridis toad venom. The absolute configurations of new compounds 1-2 were established by ECD spectra. All compounds were tested in vitro cytotoxicity, but the inhibition activity was not observed. This research provided important scientific support for the sustainable utilization and development of characteristic toad venom resources.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Scientific Report of Foreign Expert by the PIFI Fund of the Chinese Academy of Sciences for invited scientists (No. 2019VBA0013). Thanks for scientific data by National Basic Science Data Center " CAM Resources Data Base" (NO. NBSDC-DB-19).