Phytochemistry 163 (2019) 38–45

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Picraviane A and B: Nortriterpenes with limonoid-like skeletons containing a T

heptanolide E-ring system from Picramnia glazioviana
Leila Gimenesa,b, Joao Marcos Batista Juniora,c, Fernando Martins dos Santos Juniora,d,
Matheus da Silva Souzae, Liany Luna-Dulceyf, Javier Esteves Ellenae, Marcia Regina Cominettif,
Maria Fatima das Graças Fernandes da Silvaa, Paulo Cezar Vieiraa,g, Joao Batista Fernandesa,1,
Dan Staerkb,∗,1
a
Department of Chemistry, Federal University of Sao Carlos (UFSCar), 13565-905, Sao Carlos, SP, Brazil
b
Department of Drug Design and Pharmacology, University of Copenhagen, DK-2100, Copenhagen, Denmark
c
Institute of Science and Technology, Federal University of Sao Paulo, 12231-280, Sao Jose dos Campos, SP, Brazil
d
Department of Organic Chemistry, Chemistry Institute, Fluminense Federal University (UFF), Niterói, RJ, Brazil
e
São Carlos Institute of Physics, University of São Paulo, 13566-590, São Carlos, SP, Brazil
f
Department of Gerontology, Federal University of Sao Carlos, 13565-905, Sao Carlos, SP, Brazil
g
School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, 14040-903, Ribeirao Preto, SP, Brazil

ARTICLE INFO ABSTRACT

Keywords: Two highly oxygenated nortriterpenes, picraviane A and B, were isolated from the ethanolic extract of Picramnia
Picramnia glazioviana Engl. (Picramniaceae) glazioviana Engl. The structures were determined by analysis of HRMS and 2D NMR spectroscopic data. Single-
Nortriterpenes crystal X-ray diffraction data was also obtained for picraviane B. The absolute configuration of both compounds
Picravianes A and B were assigned by comparison of experimental and calculated vibrational and electronic circular dichroism
Vibrational circular dichroism
spectroscopy, respectively. Picraviane A showed a moderate cytotoxic activity against the triple negative MDA-
Electronic circular dichroism
Triple negative breast cancer
MB-231 breast cancer cell line. These compounds represent an undescribed class of natural products with li-
monoid-like skeletons containing a heptanolide as the E-ring.

1. Introduction their continued importance as research subject within chemical, bio-

The biosynthetic machinery in plants produces a plethora of spe-
cialized metabolites, and natural products are characterized by complex
chemical scaffolds that are highly oxygenated, contains multiple ring
systems, and have specific stereochemical configurations. Thus, nature
has provided us with a large pool of specialized metabolites with unique
and specific properties that have driven the pharmaceutical industry's
discoveries over the past century (Harvey et al., 2015; Kingston, 2011;
Newman and Cragg, 2016). Thus, according to Newman and Cragg
(2016), 49 percent of all small-molecule drug entities approved for
cancer treatment from the 1940s to 2014 were natural products or
natural products derivatives. However, nature as a source of drug-like
chemicals is still largely unexplored, and from an estimated 250,000 to
500,000 species of higher plants, only 5–15% have already been in-
vestigated phytochemically. In addition, the increasing number of un-
described chemical plant constituents reported every year emphasize
logical, and medicinal research (Cragg and Newman, 2005; Pérez et al.,
2014; Yuan et al., 2016). The isolation of natural products is therefore,
besides contributing to the understanding of natural selection and
coevolution in plants, an important source of bioactive leads for drug
discovery and development.
The genus Picramnia, historically placed into the Simaroubaceae
family, was together with Alvaradoa, and recently Nothotalisia, moved
into the Picramniaceae family due to the absence of quassinoids, which
are characteristic taxonomical markers of Simaroubaceae (Diaz et al.,
2004; Fernando and Quinn, 1995; Jiwajinda et al., 2001; Thomas,
2011). Picramnia comprises approximately 40 species distributed ex-
clusively in America tropics, i.e., from United States, Mexico and Cen-
tral America to Colombia, Venezuela, Peru, Brazil, Paraguay, and Ar-
gentina in South America (Fernando and Quinn, 1995; Pirani and
Devecchi, 2016). Some Picramnia species have traditionally been used
as vermicides (Solis et al., 1995) and for treatment of skin diseases

Corresponding author. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2,
∗

DK-2100, Copenhagen, Denmark.

E-mail address: ds@sund.ku.dk (D. Staerk).
1
These senior authors contributed equally to this work.

https://doi.org/10.1016/j.phytochem.2019.03.024
Received 10 January 2019; Received in revised form 25 March 2019; Accepted 26 March 2019
0031-9422/ © 2019 Elsevier Ltd. All rights reserved.
L. Gimenes, et al.
(Balderrama et al., 2001; Robledo et al., 2015). Earlier phytochemical
investigations of Picramnia species showed the presence of anthraqui-
nones, anthrones, oxanthrones glycosides, and triterpenoids (Diaz et al.,
2004; Jacobs, 2003; Rodríguez-Gamboa et al., 2001, 1999), but no
undescribed natural products have been reported from Picramnia spe-
cies in recent years (Martínez et al., 2013; Robledo et al., 2015).
Triple-negative breast cancer (TNBC) is a highly aggressive subtype
of breast cancer, with a low survival rate for patients (Zhang et al.,
2012). This cancer form is characterized by the lack of expression of
estrogen receptors, progesterone receptors, and human epidermal
growth factor receptor-2 (Gluz et al., 2009). The treatment against this
subtype of breast cancer is performed through conventional che-
motherapy - but the treatment is causing several adverse reactions.
Thus, there is an urgent need for discovery of natural product-based
drug leads against TNBC.
As part of our phytochemical studies of Picramnia species, and
contributions to its chemosystematical classification, two undescribed
nortriterpenes, picraviane A (1) and B (2), with an undescribed skeleton
were isolated from Picramnia glazioviana Engl. (Picramniaceae). In this
paper, the isolation and full structure elucidation of compounds 1 and 2
by 2D NMR spectroscopy, single-crystal X-ray crystallography, elec-
tronic circular dichroism and vibrational circular dichroism are de-
scribed, and a possible biogenetic pathway is proposed. The cytotoxic
activity of 1 and 2 were also evaluated against the triple-negative breast
cancer (TNBC) cell line MDA-MB-231.
2. Results and discussion
Crude ethanol extract of P. glazioviana was redissolved in methanol-
water (1:3) and successively extracted with hexane, dichloromethane
and ethyl acetate. The dichloromethane phase was subjected to re-
versed-phase HPLC, leading to isolation of two undescribed nor-
triterpenes, picraviane A (1) and B (2) containing a heptanolide E-ring
(Fig. 1A). The structure elucidation of 1 and 2 are described below.
2.1. Structure elucidation of picravianes A (1) and B (2)
26
The dextrorotatory nortriterpene 1 ([α] D : +43.53 (c. 0.76,
MeOH)) was assigned the molecular formula C38H46O9 as determined
by HRESIMS ([M+H]+ 647.3210, calcd. 647.3220, ΔM + 0.7 ppm),
indicating an index of hydrogen deficiency of 16. The structure was
established based on spectroscopic data from 1D 1H NMR, 13C NMR,
and DEPT135 as well as 2D COSY, HSQC, HMBC, H2BC, and NOESY
experiments.
The 1H NMR spectrum of 1 displayed two methyl groups at δ 1.11
and 1.15 (3H, s, H-24 and H-23, respectively), an oxymethine at δ 4.21
(1H, dd, J = 3.7 and 1.6 Hz, H-1), a methine at δ 2.48 (1H, dd, J = 13.8
and 2.0 Hz, H-5), and two diastereotopic proton pairs corresponding to
a methylene (δ 2.64, 1H, dd, J = 16.8 and 1.6 Hz, H-2α and δ 2.95, 1H,
dd, J = 16.8 and 3.7 Hz, H-2β) and an oxymethylene (δ 4.59, 1H, d,
J = 13.3 Hz, H-25α and δ 4.63, 1H, d, J = 13.3 Hz, H-25β). These
Fig. 1. A: Structure of picraviane A (1) and picraviane B (2). B: Key COSY (green) and HMBC (blue arrows pointing from H to C) correlations observed for 1. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
39
Phytochemistry 163 (2019) 38–45
signals resembled the 1H NMR signals observed for the A and A′ rings of
the well-known limonoid limonin (Tavares et al., 2016), and this sub-
structure was further supported by HMBC correlations from H-1 to C-10
at δ 45.4, from H-1, H-2, and H-25 to the carbonyl group C-3 at δ 169.7,
and from H-23 and H-24 to each other's carbon atoms as well as to C-4
at δ 80.8 and C-5 at δ 53.7.
The B, C and D-rings were subsequently identified based on com-
bined use of COSY and HMBC spectra. Thus, the COSY spectrum
showed three spin systems corresponding to the C-5 – C-6 – C-7, the C-9
– C-11 – C-12, and the C-15 – C-16 backbones. These spin systems were
assembled based on HMBC correlations from H-7, H-9 and H-15β to the
quaternary C-8 (δ 43.4), from H-7, H-12 and H-15β to the quaternary C-
14 (δ 41.0), from H-11, H-12 and H-15α to the olefinic C-13 (δ 138.9),
from H-12 and H-16 to C-18 (δ 118.2), and from H-15α and H-16 to C-
17 (δ 149.0) – as can be seen from the HMBC correlations provided in
Fig. 1B. HMBC correlations from H-26 and H-27 to C-8 and C-14, re-
spectively, supported the position of these methyl groups, and the up-
field shift of H-7 and HMBC correlations from H-7 and H-2ʺ/H-6ʺ to the
carbonyl group at δ 165.0 (C-7ʺ) showed the presence of a benzoyl
group attached to C-7. The highly substituted heptanolide ring system
(E-ring), was established based on HMBC correlations from H-19 to the
C-17 – C-18 double bond, to the quaternary C-20 (δ 44.0) and the two
methyl groups attached to C-20 (δ 24.7 and 28.1, C-28 and C-29 re-
spectively), and to C-2' (δ 169.9) of the acetyl group. Furthermore, the
H-21 methylene group was identified as intervening C-20 and C-22
based on HMBC correlation to these two carbon atoms (Fig. 1). The
complete assignments of all 1H and 13C NMR resonances are given in
Table 1, and all 2D correlations as well as 2D NMR spectra are given in
Supplementary data Table S1 and Figs. S1–S17, respectively.
The relative stereochemical configuration at C-1, C-5, C-7, C-8, C-9, C-
10, and C-14 of 1 was established using the NOESY spectrum. Thus,
NOESY cross peaks between H-1 and H-5, H-9 and H-23 as well as be-
tween H-27 and H-9, H-15α, H-16, and H2''/H-6'', confirmed these hy-
drogens to be on the same side of the molecule, i.e., tentatively assigned as
α. Likewise, NOESY cross peaks between H-26 and H-6β, H-7, H-11β, H-
15β and H-25 confirmed these hydrogens to be on the other side of the
molecule, i.e. tentatively assigned as β. These NOEs established the re-
lative stereochemistry of 1 as 1S*,5R*,7R*,8R*,9S*,10R*,14R*. However,
the flexibility of the E-ring did not allow unambiguous conclusions of the
configuration at C-19 based on the observed NOEs. Thus, density func-
tional theory calculations of IR and VCD spectra were performed for the
lowest-energy conformations of (1S,5R,7R,8R,9S,10R,14R,19S)-1 (Fig. 2A)
and its 19-epimer (1S,5R,7R,8R,9S,10R,14R,19R)-1 (Fig. 2B).
The calculated [wB97XD/PCM(CHCl3)/6-311G**] IR and VCD
spectra of these two epimers (solid red lines) - as well as VCD of their
respective enantiomers (dotted lines) - are shown in Fig. 3A and Fig. 3B,
respectively. The spatial arrangement of the A′- and E-rings combined
with the opposite configuration for the chiral center at C-19 resulted in
some VCD bands with reversed signs, especially fundamentals 132 and
171, which could be safely used to discriminate the two diastereomers.
These bands arise mainly from the CeO stretching vibration of the
acetyl group at C-19 coupled with CH3 deformation modes of ring E
L. Gimenes, et al. Phytochemistry 163 (2019) 38–45

Table 1 and calculated IR and VCD data for compound (+)-1 (Fig. 3A, solid red
1
H NMR (600 MHz) and13C NMR (150 MHz) data of compounds 1 and 2 in lines), established the absolute stereochemical configuration of 1 as
chloroform-d. 1S,5R,7R,8R,9S,10R,14R,19S. In order to further support this assign-
Position 1 2 ment, a quantitative analysis was performed using the confidence level
algorithm (Debie et al., 2011) by assessing the neighbourhood simila-
δCa δH (J in Hz) a
δC δH (J in Hz) rities between the experimental spectrum and the computed spectra for
both epimers at C-19. The results indicated similarity values (Σ) for the
1 80.6 4.21 dd (3.7, 1.6) 80.3 4.05 dd (3.7, 1.6)
2 35.5 β: 2.95 dd (16.8, 3.7) 35.9 β: 2.93 dd (16.7, 3.7) IR spectra of 96.6 for 19S and 95.2 for 19R. The similarity measure-
α: 2.64 dd (16.8, 1.6) α: 2.61 dd (16.7, 1.5) ments of the VCD spectra confirmed compound (+)-1 as
3 169.7 170.2 1S,5R,7R,8R,9S,10R,14R,19S (ΣVCD = 74.5 for 19S and ΣVCD = 32.4 for
4 80.8 81.1
19R).
5 53.7 2.48 dd (14.4, 2.0) 60.7 1.71 br d (11.7, 1.8) 26
6 25.5 β: 1.97 td (14.4, 2.0) 18.3 1.62 m The structure of the dextrorotatory nortriterpene 2 ([α] D : +63.93
α: 1.72 dt (14.4, 2.6) (c. 0.41, MeOH)) was elucidated on the basis of spectroscopic data from
7 74.7 5.36 t (2.6) 33.6 1.64 m
1D 1H NMR, 13C NMR, and DEPT135 as well as 2D COSY, HSQC,
8 43.4 39.5
9 42.4 2.82 dd (10.3, 7.8) 44.7 2.14 dd (11.4, 5.8) HMBC, H2BC, and NOESY experiments. Compound 2 had a molecular
10 45.4 45.3 formula of C31H42O7 as determined by HRESIMS ([M
11 26.1 β: 2.55 ddd (19.4, 10.3, 4.1) 26.2 β: 2.40 m +H]+ = 527.2998, calcd. 527.3008, ΔM + 1.0 ppm) indicating an
α: 2.43 ddd (19.4, 7.8, 4.1) α: 2.19 m index of hydrogen deficiency of 11. The 13C NMR spectrum was iden-
12 116.8 5.77 br t (4.1) 118.4 5.95 br t (3.8)
13 138.9 136.9
tical to that observed for 1, except for the absence of signals for the
14 41.0 40.7 benzoyl group as well as the disappearance of the oxymethine observed
15 25.8 β: 1.87 m 26.8 β: 1.72 m for C-7 in 1 and the appearance of an additional methylene signal for C-
α: 1.38 dt (12.8, 3.8) α: 1.45 dt (12.8, 3.6) 7 in 2. This indicated that 2 has the same structure as 1, except for the
16 24.9 2.23 m 25.4 2.31 m
absence of the benzoyl group, which is in agreement with the 2J and 3J
17 149.0 148.8
18 118.2 119.2 HMBC correlations observed from H-5 at δ 1.71 to C-6 at δ 18.3 and C-7
19 73.3 5.28 s 73.6 5.36 s at δ 33.6.
20 44.0 44.2 A single-crystal X-ray diffraction study confirmed the molecular
21 43.8 β: 2.15 d (12.0) 43.8 β: 2.21 d (12.1) structure of 2 and allowed us to establish the same relative configura-
α: 2.31 d (12.0) α: 2.42 d (12.1)
22 169.1 169.3
tion as that observed for 1. Picraviane B crystallized in the non-cen-
23 30.6 1.15 s 31.0 1.28 s trosymmetric monoclinic space group P 21. The asymmetric unit com-
24 21.6 1.11 s 21.6 1.14 s prises one molecule of picraviane B and one water molecule from the
25 65.9 β: 4.63 d (13.3) 66.1 β: 4.60 d (13.1) crystallization medium (Fig. 4). A water molecule mediated the junc-
α: 4.59 d (13.3) α: 4.52 d (13.1)
tion of two picraviane B molecules through classical OeH•••O hydrogen
26 17.6 0.88 s 17.3 0.80 s
27 24.8 1.23 s 21.0 1.13 s
bonds [OW–HWA•••O1: 2.924(9) Å, ∠ = 172.9(2)° and OW–HWB•••O2:
28 24.7 1.14 s 24.7 1.15 s 2.930(9) Å, ∠ = 166.2(2)°]. As a consequence, two adjacent chains are
29 28.1 0.93 s 28.0 0.93 s placed together (Fig. 5A). These chains grow along the a-axis and are
1ʹ 20.8 2.01 s 20.8 2.04 s responsible to give rise to a C22 (8) graph set highlighted as an orange
2ʹ 169.9 170.0 dashed line in Fig. 5B.
1ʺ 130.1
2ʺ/6ʺ 129.6 8.02 XX′-system
Molecules are inserted on both sides of this chain forming a plane.
3ʺ/5ʺ 128.7 7.46 AA′-system As expected from the planar conformation of the raw material, a
4ʺ 133.6 7.59 tt (8.4, 1.2) layered structure could be observed. The dominant driving force in the
7ʺ 165.0 3D network assemblies are the non-classical CeH•••O hydrogen bonds
b between nearby picraviane B molecules functional groups [C2eH2A
Apparent splittings, s = singlet, d = doublet, t = triplet, br = broad.
a •••O6: 3.282(8) Å, ∠ = 146.1(2)°; C16eH16A•••O5: 3.327(1) Å,
Referenced relative to chloroform signal at δ 77.0 and 7.26 for13C and 1H,
∠ = 139.1(2)° and C21eH21A•••O5: 3.563(9) Å, ∠ = 168.8(2)°] and
respectively.
π•••π interactions connecting the aromatic centers of picraviane B from
adjacent planes, forming supramolecular wave-like layers (Fig. 5C).
(fundamental 132), as well as from CH2 twisting of ring A′ and the in-
This structure is in agreement with analysis of 2D NMR experiments,
plane CeH bending of H-19 coupled with the CH2 wagging of ring E
and all 2D correlations as well as 2D NMR spectra are given in
(fundamental 171). The excellent correlation between experimental

Fig. 2. Structures of the lowest-energy conformers of (+)-picraviane A (1) calculated for (1S,5R,7R,8R,9S,10R,14R,19S)-1 (A) and its 19-epimer
(1S,5R,7R,8R,9S,10R,14R,19R)-1 (B) at the wB97XD/PCM(CHCl3)/6-311G** level.

40
L. Gimenes, et al. Phytochemistry 163 (2019) 38–45

Fig. 3. Comparison of the observed IR and

VCD spectra of (+)-picraviane A (1) with
the calculated [wB97XD/PCM(CHCl3)/6-
311G**] IR and VCD spectra. A: The
(1S,5R,7R,8R,9S,10R,14R,19S)-diaster-
eomer (solid lines) and the corresponding
enantiomer (1R,5S,7S,8S,9R,10S,14S,19R)
(dotted line). B: The (1S,5R,7R,8R,9S,
10R,14R,19R)-diastereomer (solid lines)
and the corresponding enantiomer
(1R,5S,7S,8S,9R,10S,14S,19S) (dotted
lines). Numbers represent selected vibra-
tional modes.

Supplementary data Table S2 and Figs. S19–S34, respectively. The

Fig. 4. ORTEP type diagram of the asymmetric unit of the monohydrate of 2.
Ellipsoids are drawn as 50% probability levels.
complete assignment of all 1H and 13C resonances are given in Table 1.
Crystal data, structure refinement and crystallographic parameters are
shown in Supplementary data Table S3.
The relative stereochemical configuration of 2 was unambiguously
established to be 1S*,5R*,8R*,9S*,10R*,14S*,19S* by combined use of
2D NOESY and X-ray crystallography. The absolute stereochemical
configuration was determined by comparing experimental UV and ECD
data acquired in methanol with calculated UV and ECD spectra using
time-dependent density functional theory for the lowest-energy con-
formation of (1S,5R,8R,9S,10R,14S,19S)-2. The good agreement be-
tween experimental and calculated data, i.e., positive and negative
Cotton effects at 250 and 220 nm, respectively (Fig. 6), confirmed this
as the absolute stereochemical configuration of (+)-2. The ECD data
calculated for the 19R epimer of compound 2 is presented in the
Supplementary data Fig. S40. It is noteworthy that compound 2, despite
having the same spatial arrangement of atoms as that in compound 1,
has a change in the stereodescriptor for C-14 due to removal of the

Fig. 5. A: One water molecule mediates the junction of two

picraviane B (2) molecules giving rise to parallel chains. B:
The 1D chains grow along the a-axis in which multiple
water and picraviane B molecules are forming an ac-plane
via OeH•••O hydrogen bonds. C: 3D arrangement of crys-
talline picraviane B monohydrate showing the supramole-
cular wave-like layers.

41
L. Gimenes, et al. Phytochemistry 163 (2019) 38–45

Fig. 6. A: Comparison of the observed UV and ECD spectra of (+)-picraviane B (2) with the calculated [CAM-B3LYP/PCM(MeOH)/TZVP//B3LYP/6-31G*] UV and
ECD spectra of the (1S,5R,8R,9S,10R,14S,19S) stereo isomer. B: Structure of lowest-energy conformer identified for (1S,5R,8R,9S,10R,14S,19S).

benzoyl group at C-7 in compound 2.
2.2. Biogenesis
Although the presence of the A and A′ rings in 1 and 2 are common
in some types of limonoids in Rutaceae family, this is the first work
reporting the presence of these rings in nortriterpenes, which suggests
that similar enzymes as those in the Sapindales order can be involved in
the formation of these compounds. Besides, limonoids and quassinoids
are biosynthetically related to euphol/tirucallol type precursors after
losing 4 and 10 carbons from the side-chain, respectively (Dewick,
2002). However, the formation of Picraviane A and B is very well
supported by the precursor oleanolic acid, a triterpene already isolated
from some Picramnia species (Balderrama et al., 2001; Martínez et al.,
2013), and also present in the crude ethanol extract of P. glazioviana
(C30H47O3, [M-H]- = 455.3518, calcd. 435.3531, ΔM = + 2.8 ppm,
Supplementary data, Fig. S36) suggesting a plausible biogenetic
pathway for 1 and 2 as illustrated in Fig. 7.
Initially, the A and A′ rings can be formed by oxidation of C-1 and C-
25 of the shown intermediate A, followed by two intramolecular cy-
clization reactions and consequently loss of two water molecules to
form C containing the limonoid-type A and A′ rings. Allyllic oxidation
of C followed by decarboxylation and dehydration in ring D
(intermediate D) results in E with the 29 carbon atoms as seen for
nortriterpenes. An allylic oxidation of E would lead to the formation of
intermediate F that would convert to G by a Baeyer-Villiger oxidation,
which presents the main skeleton of both 1 and 2. Finally, picraviane A
(1) and B (2) can be formed by further oxidations at C-19 and C-7,
which keeps the same stereochemistry at C-5, C-9, C-25, C-26 and C-27
as their suggested precursor. HRESIMS spectra of intermediates D, E
and F are shown in Supplementary data Figs S37–S39.
2.3. Cytotoxic and morphology evaluations
Terpenoids are well-known for their cytotoxic activity (Chudzik
et al., 2015; Thoppil and Bishayee, 2011), and compounds 1 and 2 were
therefore evaluated for their cytotoxic activity against the MDA-MB-
231 breast cancer cell line. Picraviane A exhibited moderate cytotoxic
activity with IC50 of 72.62 ± 0.18 μM, while picraviane B did not show
significant activity within the tested range (IC50 > 100 μM). These
results indicate the benzoate unit present in 1 may be essential for the
observed activity.
The moderate cytotoxicity of 1 on MDA-MB-231 cells is comparable
to the IC50 value reported for the natural triterpenoid 3-epimaslinic acid
isolated from Dillenia suffruticosa (Tor et al., 2015). These results em-
phasize the continued need for a deeper understanding of the effect of

Fig. 7. Biogenetic pathway proposed for 1 and 2.

42
L. Gimenes, et al.
inhibitors of cell lines from the aggressive triple negative breast cancer.
Hence, the effects on cell morphology of picraviane A (1) were eval-
uated. As shown in Fig. 8A, incubation of compound 1 induced mor-
phological changes on the tumor breast cells that are better visualized
after 24 h of treatment at the concentrations of 80 and 100 μM, where
cells developed circular shapes accompanied by loss of cell density (red
arrows). Moreover, in Fig. 8B after 48 h of treatment, compound 1 in-
duced large morphological changes at 60, 80 and 100 μM, causing a
total lack of adherence and appearance of round cells and cellular
debris, indicating cell death.
3. Conclusions
In conclusion, the isolation of an undescribed class of nortriterpenes
with a limonoid-like skeleton containing a heptanolide E ring supports
the classification of Picramnia sp. into the Picramniaceae family. In
addition to shear light on the chemodiversity in Picramnia spp., these
findings hold promises for identifying further nortriterpenes with po-
tential as lead compounds against triple negative breast cancer.
4. Material and methods
4.1. General experimental procedures
NMR spectra were acquired using a Bruker Avance III 600 MHz
spectrometer equipped with a Bruker SampleJet sample changer and a
cryogenically cooled gradient inverse triple-resonance 1.7-mm TCI
probe-head (Bruker Biospin, Rheinstetten, Germany) and on a 600 MHz
Bruker Avance III spectrometer equipped with a cryogenically cooled 5-
mm DCH probe optimized for 13C and 1H, and a 400 MHz Bruker
AvanceTM III NanoBay 9,4 T spectrometer. CDCl3 (δH = 7.26 ppm,
δC = 77.0 ppm) was used as reference signal. Mass spectra were re-
corded on a micrOTOF-Q II mass spectrometer (Bruker Daltonik GmbH,
Bremen, Germany) equipped with an electrospray ionization (ESI) in-
terface. Mass spectra were acquired in positive ionization mode for pure
43
Phytochemistry 163 (2019) 38–45
Fig. 8. Effects of picraviane A (1) on cell
morphology of the triple negative MDA-MB-
231 breast cancer cell line. A: Cellular
morphology of MDA-MB-231 after 2, 4, 24
and 48 h of treatment with indicated con-
centrations of 1 [100 × amplification, scale
bar = 100 μm]. B: MDA-MB-231 cells
treated for 48 h with 1 at the three highest
concentration resulting in a decrease in cell
density compared to untreated cells (con-
trol) [200 × amplification, scale
bar = 50 μm].
compounds and negative ionization mode for the crude ethanol extract.
HPLC analyses were performed on a Shimadzu LC-10AD system con-
sisting of a LC- 10AD pump (Kyoto, Japan), a SPD 10AD UV–Vis de-
tector working with two wavelengths, and a SCL 10A VP system con-
troller. This equipment was connected to a CBM – 10A and a Shimadzu
Class – VP software was used for data acquisition.
Optical rotations were measured at 589 nm using a
Bellingham + Stanley ADP 410 polarimeter (Fischer Scientific,
Hampton, NH, USA). IR and VCD spectra of compound 1 were recorded
with a Single-PEM ChiralIR-2X FT-VCD spectrometer (BioTools, Inc.,
Jupiter, FL, USA) using a resolution of 4 cm−1 and a collection time of
4 h. The optimum retardation of the ZnSe photoelastic modulator
(PEM) was set at 1400 cm−1. Minor instrumental baseline offsets were
eliminated from the final VCD spectrum by subtracting the VCD spec-
trum of compound 1 from that obtained for the solvent under the same
conditions. The IR and VCD spectra were recorded in a BaF2 cell with
100 μm path length using CDCl3 as solvent. The sample was prepared
using 5 mg of compound 1 dissolved in 120 μL of solvent. The IR
spectrum of compound 2 was recorded on a Perkin Elmer Spectrum One
FT-IR spectrometer (Perkin Elmer, Waltham, MA, USA) using MeOH as
solvent. UV and ECD spectra of 2 were recorded with a Jasco J-815
spectrometer (Jasco, Tokyo, Japan) in the 190–350 nm region using the
following parameters: bandwidth 1 nm; response 1 s; scanning speed
100 nm/min; 3 accumulations; room temperature; sample in methanol
solution; 0.1 cm cell path length; concentration 0.32 mg/mL.
4.2. Plant material and extraction
Leaves of Picramnia glazioviana Engl. (Picramniaceae) were col-
lected in Piuma and Marataizes, Espirito Santo, 3 km from Morro do
Aghá, Rodovia ES-60, 20°52′S, 40°46′W, Atlantic Rainforest, Brazil in
November 1988 and were identified by Dr. Jose Rubens Pirani. A
voucher specimen has been deposited at the Botanical Department, Sao
Paulo University, Sao Paulo, Brazil (Herbal SPF, collection number J. R.
Pirani et al., 2468). The leaves of P. glazioviana were dried in a
L. Gimenes, et al.
circulation oven at 40 °C and subsequently milled to yield 513 g of dry
powdered plant material. This material was extracted with 2 l of
ethanol, filtered, and concentrated on a rotary evaporator under re-
duced pressure to yield 41.1 g of crude extract.
4.3. Isolation and characterization data
The crude ethanol extract was submitted to liquid-liquid parti-
tioning by dissolving the extract in MeOH:H2O (1:3) and subsequently
extracting sequentially with hexane, dichloromethane and ethyl acetate
(3 × 400 mL, for each solvent) - and then dried under reduced pressure.
This procedure resulted in 1.53 g of dichloromethane extract. The di-
chloromethane extract was separated using semi-preparative reversed-
phase HPLC on a Phenomenex Luna C18 column, 10 μm, 10 × 250 mm
(Phenomenex, Torrance, CA, USA) using isocratic elution with
ACN:H2O (40:60) at a flow rate of 7 mL/min. Four injections of 100 μL
each of a solution of 370 mg/mL and collection based on chromato-
grams at 254 and 220 nm afford 24.4 mg of 1 and 8.2 mg of 2.
26
Picraviane A (1): white wax, [α] D : +43.53 (c. 0.76, MeOH). UV
obtained from HPLC-PDA-HRESIMS analysis: λmax: 198, 232 and
255 nm. IR: (Fig. 2). 13C and 1H NMR data in Table 1. HRESIMS m/z [M
+H]+ = 647.3210 (calculated for C38H47O9, 647.3220,
ΔM = +0.7 ppm), [M + H-CH3CO2H]+ = 587.3003 (calculated for
C36H43O7, 587.3008, ΔM = +0.1 ppm).
26
Picraviane B (2): white wax, [α] D : +63.93 (c. 0.41, MeOH). UV
obtained from HPLC-PDA-HRESIMS analysis: λmax: 229 and 252 nm. IR
νmax: 2969, 1749, 1379, 1231, 1065 cm−1. 13C and 1H NMR data in
Table 1. HRESIMS m/z [M+H]+ = 527.2998 (calculated for C31H43O7,
527.3008, ΔM = +1.0 ppm), [M + H-CH3CO2H]+ = 467.2791 (cal-
culated for C29H39O5, 467.2797, ΔM = +0.2 ppm).
4.4. Computational methods, VCD and ECD spectra
All density functional theory (DFT) and time-dependent DFT
(TDDFT) calculations were carried out at 298 K either in the gas phase,
in chloroform or methanol solutions using the polarizable continuum
model (PCM) in its integral equation formalism version (IEFPCM) in-
corporated in Gaussian 09 software. Calculations for compound 1 were
performed for the arbitrarily chosen (1S,5R,7R,8R,9S,10R,14R,19S)
and (1S,5R,7R,8R,9S,10R,14R,19R) epimers. All conformational sear-
ches were carried out at the molecular mechanics level of theory with
the Monte Carlo algorithm employing the MM + force field in-
corporated in the HyperChem 8.0.10 software package. The puckering
of rings A and E were also probed manually. For both epimers, three
conformers with relative energy within 6 kcal mol−1 of the lowest-
energy conformer were selected and further geometry optimized at the
B3LYP/6-31G* level. In both cases, a single conformer was identified
with relative energy < 2.2 kcal mol−1, corresponding to more than
97% of the total Boltzmann distribution. The lowest-energy conformer
for each configuration was selected for IR and VCD spectral calculations
at the B3LYP/6-31G* level. These conformers were also further geo-
metry optimized and had their IR and VCD spectra calculated at the
wB97XD/PCM(CHCl3)/6-311G** level. IR and VCD spectra were cre-
ated using dipole and rotational strengths from Gaussian, which were
calculated at the same level used during geometry optimization steps
and converted into molar absorptivities (M−1 cm−1). Each spectrum
was plotted as a sum of Lorentzian bands with half-widths at half-
maximum of 6 cm−1. The calculated wavenumbers were multiplied
with a scaling factor of 0.97 and the final spectra were plotted using
Origin 8 software.
Calculations for compound 2 were performed for the arbitrarily
chosen (1S,5R,8R,9S,10R,14S,19S) configuration, based on the relative
configuration assigned by X-ray crystallography. The conformational
search was carried out at the molecular mechanics level of theory with
the Monte Carlo algorithm employing the MM + force field
44
Phytochemistry 163 (2019) 38–45
incorporated in HyperChem 8.0.10 software package. The puckering of
rings A and E were also probed manually. A single conformer with
relative energy within 6 kcal mol−1 was selected and further geometry
optimized at the B3LYP/6-31G* level. This optimized conformer was
used for UV and ECD spectral calculations. TDDFT was employed to
calculate the excitation energy (in nm) and rotatory strength R in the
dipole velocity (Rvel in cgs units: 10−40 esu2 cm2) form, at the CAM-
B3LYP level, combined with TZVP basis sets. The calculated rotatory
strengths from the first 30 singlet → singlet electronic transitions were
simulated into an ECD curve using Gaussian bands with a bandwidth of
σ 0.25 eV. Cartesian coordinates of lowest-energy conformers are given
in the last section named ‘CARTESIAN COORDINATES’ in
Supplementary data.
4.5. Single crystal X-ray diffraction of picraviane B (2)
During the extraction of the compound clear light colorless crystal
plates were obtained by slow evaporation of a water:methanol solution
(8:2 v/v) at 5 °C. The crystallographic data for picraviane B (2) were
collected at 293 K on an Bruker Enraf-Nonius diffractometer (Bruker
Karlsruhe, Germany) with CCD detector system equipped with a Mo
source (λ = 0.71073 Å). Data integration and cell determination were
performed using the software Collect and Scalepack (Otwinowski and
Minor, 1997). Data reduction was carried out using the software Denzo-
SMN and Scalepack (Otwinowski and Minor, 1997). Using Olex2
(Dolomanov et al., 2009), the structure was solved (Olex2.solve struc-
ture solution program, Bourhis et al., 2015) using Charge Flipping and
the model obtained was refined by full–matrix least squares on F2 with
the SHELXL refinement package using Least Squares minimization
(Sheldrick, 2015). Non-hydrogen atoms were refined with anisotropic
displacement parameters. The positions of hydrogen atoms were cal-
culated and were included in the structure factor calculations. All the
hydrogen atoms were placed in calculated positions and refined with
fixed individual displacement parameters [Uiso(H) = 1.2Ueq or
1.5Ueq] according to the riding model. Molecular representations, ta-
bles and pictures were generated by ORTEP-3 (Farrugia, 2012), Mer-
cury 3.10.2 (Macrae et al., 2006) and Olex2 softwares. The Crystal-
lographic Information File was deposited in the Cambridge Structural
Database (Groom et al., 2016) under the code CCDC 1862634. Copies of
the data can be obtained, free of charge, via www.ccdc.cam.ac.uk.
4.6. Cytotoxicity assay
MDA-MB-231 cells were obtained from Rio de Janeiro Cell Bank,
maintained in Dulbecco's modified eagle medium supplemented with
10% fetal bovine serum and appropriate antibiotics. Cells were main-
tained at 37 °C in an incubator with 95% O2 and 5% CO2. The effects of
picraviane A (1) and B (2) on the viability of MDA-MB-231 was de-
termined by colorimetric assays (Luna-Dulcey et al., 2018; Mosmann,
1983) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT). MDA-MB-231 cells were seeded (1 × 104 cells/100 μL)
into 96-well plates and incubated until 80% confluence. Subsequently,
cells were exposed to increasing concentrations (3.12–100 μM) of
compounds. Cells were incubated for 48 h under the same conditions as
described above. Cytotoxicity assay was performed in comparison to
the wells where the vehicle solvent (1% DMSO) was added instead of
the tested compounds. Cells were treated with MTT (1 mg/mL) for 4 h.
Medium was removed from the wells and the formed crystals were
dissolved in DMSO. Absorbances were measured using a Labtech LT-
4000 ELISA plate reader (Labtech, Heathfield, UK) at a wavelength of
540 nm.
4.7. Cell morphology of picraviane A (1)
MDA-MB-231 cells (1 × 105/well) were seeded in a 12 well-plate
and after 24 h and exposed to increasing concentrations of the
L. Gimenes, et al.
compound (25–100 μM) for additional 48 h. Cells were examined using
a Nikon T5100 inverted optical microscope (Nikon, Tokyo, Japan).
Photos were taken at 2, 4, 24 and 48 h, and the efficiency of treatment
was compared to control cells (with no treatment).
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgments
L. G. would like to thank São Paulo Research Foundation (FAPESP)
for the doctoral scholarships in Brazil and abroad (2013/20458-1 and
2016/18024-1). The authors would like to thank FAPESP (Grants #
2012/25299-6, 2014/25222-9, 2015/07089-2 and 2016/23794-0) the
National Council for Scientific and Technological Development (CNPq,
J.E. grant #305190/2017-2), Coordination for the Improvement of
Higher Education Personnel (CAPES, Finance Code 001) and the Centre
for Scientific Computing (NCC/GridUNESP). The 600 MHz NMR was
acquired through a grant from ´Apotekerfonden af 1991′, The Carlsberg
Foundation, and the Danish Agency for Science, Technology and
Innovation via the National Research Infrastructure funds.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.03.024.
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