J. Sosa-Rueda et al.
Phytochemistry xxx (xxxx) xxx
4
.7. Cell growth inhibition assay
Acknowledgments
The effect of each compound on the proliferation of human tumor
This work was supported by the Government of the State of Veracruz
de Ignacio de la Llave, Veracruz Council for Scientific Research and
Technological Development [COVEICyDET, grant number 14 1953/
2021], and by the Spanish Ministry of Science and Innovation (PID2019-
109476RB-C21). This study made use of the CESGA and SGAI-CSIC
supercomputing facilities. We thank G. Jones for English text edition,
funded by Cabildo de Tenerife, TFinnova Program supported by MEDI
and FDCAN.
cell lines was determined using the XTT (sodium 3’-[1-(phenyl-
aminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sul-
fonic acid hydrate) cell proliferation kit (Roche Molecular Biochemicals,
Mannheim, Germany), as previously described (Cen-Pacheco et al.,
3
2
012). Cells (1.5–5.0 × 10 in 100
μ
L) were incubated in DMEM culture
medium containing 10% heat-inactivated FBS, in the absence and
ꢀ 4
presence of the indicated compounds at a concentration range of 10 to
ꢀ 9
1
0
M, in 96-well flat-bottomed microtiter plates. Following 72 h of
◦
incubation at 37 C in a humidified atmosphere of air/CO
2
(19/1), the
Appendix A. Supplementary data
XTT assay was performed. Measurements were made in triplicate, and
each experiment was also repeated three times. The IC50 (50% inhibitory
concentration) value, defined as the drug concentration required to
cause 50% inhibition of cell proliferation with respect to the untreated
controls, was determined for each compound. Data are shown as
means ± standard deviation (SD) of three independent experiments,
each performed in triplicate.
References
Anaya-Esparza, L.M., García-Maga n˜ a, M.L., Domínguez-Avila, J.A., Yahia, E.M., Salazar-
L o´ pez, N.J., Gonz a´ lez-Aguilar, G.A., Montalvo-Gonz a´ lez, E., 2020. Annonas:
´
1
4
.8. Computational details
QM-NMR calculations followed the approach previously described
Capon, R.J., 2020. Extracting value: mechanistic insights into the formation of natural
Cen-Pacheco, F., Mollinedo, F., Villa-Pulgarín, J.A., Norte, M., Fern ´a ndez, J.J.,
Hern a´ ndez Daranas, A., 2012. Saiyacenols A and B: the key to solve the controversy
Cen-Pacheco, F., Ortiz-Celiseo, A., Peniche-Carde n˜ a, A., Bravo-Ruiz, O., L o´ pez-
Fentanes, F.C., Valerio-Alfaro, G., Fern a´ ndez, J.J., 2019a. Studies on the bioactive
Cen-Pacheco, F., Valerio-Alfaro, G., Santos-Luna, D., Fern ´a ndez, J.J., 2019b. Sclerin, a
by Lauro et al. This relies on establishing the statistical MAEΔΔδ
parameter, defined as
∑
(
ΔΔδ)
MAEΔΔδ
=
n
ΔΔδ
obtained from the summation (Σ) of the n computed absolute δ error
values (Δδ), normalized to the number of Δδ errors considered (n).
The crystallographic structures for squamins A (CCDC 196170) and B
(
CCDC 196169) were optimized at the B3LYP/6-31G (d) level of theory.
These optimized structures were then used to obtain the calculated
magnetic shielding constants (
) at the mPW1PW91/6-31 + G(d,p) level
of theory. Subsequently, unscaled chemical shifts (δ ) were calculated
using TMS as reference standard according to the expression δ
where is the shielding tensor and is the shielding tensor of TMS
computed at the same level of theory used to calculate . The structures
Daranas, A.H., Sarotti, A.M., 2021. Are computational methods useful for structure
σ
u
u
=
σ
0
σ
x
- ,
σ
x
σ
0
(
σ
x
Domínguez, H.J., Napolitano, J.G., Fern ´a ndez-S ´a nchez, M.T., Cabrera-García, D.,
Novelli, A., Norte, M., Fern
a´ ndez, J.J., Daranas, A.H., 2014. Belizentrin, a highly
of squamins C–F (1–4) were built using the crystallographic coordinates
of compounds 5 and 6 as templates, making the necessary modifications
in selected amino acids. Afterward, the resulting structures were
geometrically optimized at the B3LYP/6-31G (d) level of theory. Due to
the large size of the molecules, availability of crystallographic structures
for 1–2 and the resemblance of their NMR data with those of 3–6 (which
suggests that all of them adopt very similar conformations) computa-
tional studies were undertaken using only single conformations for each
compound.
1
Escobedo-Lopez, D., Campos-Rojas, E., Rodríguez-Núnez, J.R., Alia-Tejacal, I., Núnez-
Colin, C.A., 2018. Priority areas to collect germplasm of Annona (annonaceae) in
´
˜
˜
Grimblat, N., Gavín, J.A., Daranas, A.H., Sarotti, A.M., 2019. Combining the power of J
Grimblat, N., Zanardi, M.M., Sarotti, A.M., 2015. Beyond DP4: an improved probability
for the stereochemical assignment of isomeric compounds using quantum chemical
As all comparisons are between two sets of calculated data (A, B) and
two sets of experimental data (1, 2), the number of possible comparisons
for each pair of data is two (AB/12 and AB/21). For each comparison,
Δδcalc and Δδexp parameters are calculated using the equation ΔδcalcA-
Hern ´a ndez Vidal, L., L o´ pez Moctezuma, H., Vidal Martínez, N.A., Ruiz Bello, R., Castillo
Rocha, D.G., Chiquito Contreras, R.G., 2014. La situaci o´ n de las Annonaceae en
B
= δcalcA- δcalcB, where δcalcA and δcalcB are chemical shifts calculated for
Kutateladze, A.G., Holt, T., 2019. Structure validation of complex natural products: time
A and B data sets. This procedure is also followed to calculate Δδexp
.
Finally, the specific Δδcalc and Δδexp values are compared atom by atom,
using the ΔΔδ parameter defined as ΔΔδ = | Δδcalc - Δδexp |. The ΔΔδ
parameter detects similarities between calculated and experimental data
sets and is used for calculating the MAEΔΔδ parameter (Table 3 and S17-
S19).
Lauro, G., Das, P., Riccio, R., Reddy, D.S., Bifulco, G., 2020. DFT/NMR approach for the
configuration assignment of groups of stereoisomers by the combination and
3
Leite, D.O.D., Nonato, C.de F.A., Camilo, C.J., de Carvalho, N.K.G., da Nobrega, M.G.L.
A., Pereira, R.C., da Costa, J.G.M., 2020. Annona genus: traditional uses,
Declaration of competing interest
4
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Li, S.W., Cuadrado, C., Yao, L.G., Daranas, A.H., Guo, Y.W., 2020. Quantum mechanical-
NMR-aided configuration and conformation of two unreported macrocycles isolated
6