Polyprenylated benzophenone derivatives with a novel tetracyclo[8.3.1.03,11.05,10]tetradecane core skeleton from Clusia burle-marxii exhibited cytotoxicity against GL-15 glioblastoma-derived human cell line

Article abstract of DOI:10.1016/j.fitote.2019.104346

Three new polyprenylated benzophenone derivatives (1–3) were identified in the hexane extract of Clusia burle-marxii trunks, through the isolation and structural elucidation of their methyl derivatives, along with two known polyprenylated benzophenone derivatives sampsonine N (4) and obdeltifolione C (5). Burlemarxiones A (1) and B (2) show an unprecedent tetracyclo[8.3.1.0^3,11.0^5,10]tetradecane core skeleton. These compounds are a pair of β-diketones in tautomeric equilibrium, whereas isonemorosonol (3) is the respective β-diketone pair in tautomeric equilibrium with nemorosonol. Burlemarxione A methyl derivative (1a) and sampsonine N exhibited strong in vitro cytotoxic activity against GL-15 glioblastoma-derived human cell line.

Full text of DOI:10.1016/j.fitote.2019.104346

Fitoterapia138(2019)104346
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Polyprenylated benzophenone derivatives with a novel
tetracyclo[8.3.1.03,11.05,10]tetradecane core skeleton from
Clusia burle-marxii exhibited cytotoxicity against GL-15 glioblastoma-
derived human cell line
⁎
Caline G. Ferraza,b,c, , Paulo R. Ribeiroa,c, Édson J. Marquesa, Renata Mendonçad,
Maria Lenise S. Guedese, Edilberto R. Silveiraf, Ramon El-Bachág, Frederico G. Cruza
a Grupo de Estudos de Substâncias Naturais Orgânicas (GESNAT), Instituto de Química, Universidade Federal da Bahia, Rua Barão de Jeremoabo s/n, 40170-115
Salvador, Brazil
b Centro de Ciências Exatas e Tecnológicas, CETEC, Universidade Federal do Recôncavo da Bahia, Rua Rui Barbosa, no710, 44.380-000 Cruz das Almas, Brazil
c Metabolomics Research Group, Departamento de Química Orgânica, Instituto de Química, Universidade Federal da Bahia, Rua Barão de Jeremoabo s/n, 40170-115
Salvador, Brazil
d Instituto de Química, Universidade Federal do Rio Grande do Norte, CP 1524, 59072-970 Natal, RN, Brazil
e Instituto de Biologia, Universidade Federal da Bahia, 40.170-290 Salvador, BA, Brazil
f Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60021-940 Fortaleza, CE, Brazil
g Laboratório de Neuroquímica e Biologia Celular, Departamento de Bioquímica e Biofísica, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Bahia,
Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three new polyprenylated benzophenone derivatives (1–3) were identified in the hexane extract of Clusia burle-
marxii trunks, through the isolation and structural elucidation of their methyl derivatives, along with two known
polyprenylated benzophenone derivatives sampsonine N (4) and obdeltifolione C (5). Burlemarxiones A (1) and
B (2) show an unprecedent tetracyclo[8.3.1.03,11.05,10]tetradecane core skeleton. These compounds are a pair of
β-diketones in tautomeric equilibrium, whereas isonemorosonol (3) is the respective β-diketone pair in tauto-
meric equilibrium with nemorosonol. Burlemarxione A methyl derivative (1a) and sampsonine N exhibited
strong in vitro cytotoxic activity against GL-15 glioblastoma-derived human cell line.
Clusiaceae
Cytotoxicity
Human glioblastoma
Polyprenylated benzophenones
1. Introduction
polyprenylated benzophenone derivatives, whereas more polar extracts
lack this class of compounds [5–7]. In this work, we report the identi-
Benzophenone synthases catalyzes the formation of 2,4,6-trihy-
droxybenzophenone from one benzoyl-CoA and three malonyl-CoA
units via intramolecular cyclization reactions. Extensive prenylation of
the phloroglucinol moiety of 2,4,6-trihydroxybenzophenone leads to
the production of polyprenylated benzophenone derivatives [1]. Intri-
guing caged benzophenone derivatives can be produced through spe-
cific and highly coordinated set of reactions and rearrangements.
Moreover, many of these compounds show promising biological prop-
erties such as antiprotozoal, [2] anti-HIV [3] and antitumoral [4].
Hexane extracts obtained from Clusia species are prominent sources of
fication of three new polyprenylated benzophenone derivatives, along
with sampsonine N and obdeltifolione C in the hexane extract of Clusia
burle-marxii trunks.
The glioblastoma is the most aggressive and lethal brain tumor that
affects the human central nervous system. Its treatment faces several
setbacks such as rapid cell proliferation, invasive growth, angiogenesis,
and immunosuppression. Surgical removal of the tumor is first line of
therapy, followed by radiation and chemotherapy [8–10]. Few che-
motherapy agents are able to cross the blood brain barrier, which is
strongly linked to the drug-resistant phenotype of glioblastoma [11].
⁎ Corresponding author at: Centro de Ciências Exatas e Tecnológicas, CETEC, Universidade Federal do Recôncavo da Bahia, Rua Rui Barbosa, no710, 44.380-000
Cruz das Almas, Brazil.
E-mail addresses: califerraz@gmail.com, calineferraz@ufrb.edu.br (C.G. Ferraz).
https://doi.org/10.1016/j.fitote.2019.104346
Received 2 July 2019; Received in revised form 21 August 2019; Accepted 25 August 2019
Availableonline26August2019
0367-326X/©2019ElsevierB.V.Allrightsreserved.

C.G. Ferraz, et al.
Fitoterapia138(2019)104346
Compound 1a (31.96 μM) and sampsonine N (26.00 μM) exhibited
strong in vitro cytotoxic activity against GL-15 glioblastoma-derived
human cell line as compared to temozolomide, the current drug used
for the treatment of some brain tumors. Therefore, these compounds
might be considered as potential chemotherapy agent candidates to
treat glioblastoma.
diazomethane and purified by HPLC to afford 1a (0.0026%), 2a
(0.00043%), and 3a (0.00034%), along with sampsonine
N (4,
0.00019%), and obdeltifolione C (5, 0.00007%) (Fig. 1). Methylation
with diazomethane was necessary to avoid the interconversion of the
pair of β-diketones in tautomeric equilibrium and, therefore to allow
their separation.
Burlemarxiones A (1) and B (2) are a pair of β-diketones in tauto-
meric equilibrium. Their enol ethers 1a and 2a were obtained as a
yellow gum and are levorotatory presenting [α]2D0 = −113.0 (c 1.0,
CHCl3) and [α]2D0 = − 78.0 (c 1.08, CHCl3), respectively. The HRESIMS
data of 1a exhibited a pseudomolecular ion [M + H]+ at m/z 517.3310
(calcd for 517.3312) corresponding to a molecular formula of C34H44O4
(Supplementary Fig. 54).
2. Results and discussion
Dried powdered trunk of Clusia burle-marxii (10.0 Kg) was extracted
with n-hexane by maceration at room temperature for 72 h. The n-
hexane extract (128 g) was subjected to silica gel column chromato-
graphy and some of the resulting fractions were methylated with
Fig. 1. Chemical structures of polyprenylated benzophenones derivatives from C. burle-marxii.
2

C.G. Ferraz, et al.
Fitoterapia138(2019)104346
Detailed examination of 1H and 13C NMR spectra of 1a (Table 1)
C-6 and C-13, allowing us to establish the precise connection among C-
22, C-4, C-6 and C-13 to C-5. The methylene hydrogens of the second
prenyl group, H-17a and H-17b, correlated with C-2, C-3, C-4, C-14 and
C-18 allowing us to establish the precise connection among C-17, C-2,
C-4 and C-14 to C-3. The correlations of the hydroxyl hydrogen with C-
1, C-5, C-6 located this group at C-6, which is also linked to C-5 and C-1.
Correlations of H-29 and H-34 (OMe) with C-27 permitted to establish
the linkage of the phenyl and the methoxyl groups with C-27. Corre-
lations of H-13a and H-13b with C-4, C-5, C-6, C-7, C-12 and C-22
confirmed the connection of C-13 with C-5. Additionally, the correla-
tions of H-14a and H-14b with C-2, C-3, C-4, C-6, C-7 and C-12 allowed
us to deduce the presence of a seven-membered ring formed among C-5,
C-4, C-3, C-14, C-7, C-12 and C-13 and to confirm the linkage between
indicated the presence of one phenyl, two prenyl and one methoxyl
groups along with two methyl groups of a gem-dimethyl system
[6,12–16]. 13C NMR spectrum showed one signal at δ 210.4 that was
attributed to a non-conjugated carbonyl group. Additionally, a 13C NMR
signal at δ 192.4, along with signals at δ 163.9 and δ 118.3 suggested
the presence of an α,β-unsaturated carbonyl system with an oxygena-
tion at the β-carbon [6,12,16,17]. It was still noted a signal at δ 85.5
attributed to a non-hydrogenated carbon (tertiary alcohol). The ana-
lysis of 13C and DEPT spectra indicated the presence of seven methyl,
seven methylene, eight methine and twelve non-hydrogenated carbons.
The data also suggest that 1a contains one aromatic and four aliphatic
rings.
Table 1
1H and 13C NMR spectral data of 1a and 2a recorded at 500 MHz in benzene-d6.
1a
2a
Position
δH (J in Hz)
δC
HMBC
δH (J in Hz)
δC
HMBC
1
118.3 (C)
192.4 (aC=O)
64.6 (C)
116.2
165.3
58.2
2
3
4
210.4 (aC=O)
66.4 (C)
211.4
59.9
5
6
85.5 (C)
85.9
7
49.8 (C)
49.6
8a
1.20 (d,14.0)
2.32 (d,14.0)
43.4 (CH2)
6, 7, 9, 10, 12, 14, 15, 16
6, 7, 9, 10, 12, 14, 15, 16
1.07 (d, 14.0)
2,39 (d, 14.0)
44.9
7, 9, 10, 12, 14,15
7, 9, 10, 12, 14,15
8b
9
29.7 (C)
30.2
38.0
10a
10b
11a
11b
12
13a
13b
14a
14b
15
16
17a
17b
18
19
20
21
22a
22b
23
24
25
26
27
28
29
30
31
32
33
OMe
OH
1.11 (dd, 5.0, 13.5)
1.50 (ddd, 3.0, 5.0, 13.5)
1.73 (dd, 5.0, 13.0)
1.42 (dd, 2.5, 5.0)
1.37 (m)
37.7 (CH2)
9, 12, 15,16
9, 12, 15,16
10, 13
1.13 (dd, 5.5, 13.5)
1.46 (m)
9, 16
7, 10, 13
7
29.5 (CH2)
1.83 (dd, 7.3, 12.8)
1.38 (m)
29.8
7
45.4 (CH)
42.3 (CH2)
14
1.46 (m)
45.2
40.9
1.83 (dd, 7.0, 12.5)
2.21 (dd, 8.0, 12.5)
1.68 (m)
4,5, 12, 22
4, 5, 6, 7, 12
2, 3, 4, 6, 7, 12
2, 3, 4, 7, 8, 12
8, 9, 10, 16
8, 9, 10, 15
2, 3, 4, 14, 18
2, 3, 4, 14, 18
20, 21
1.68 (dd, 8.0, 13.5)
2.11 (dd, 9.0, 13.0)
1.70 (ov)
4, 5, 11, 12, 22
4, 5, 7, 11
48.2 (CH2)
54.2
2, 3, 4, 7, 8, 12
2, 3, 4, 7, 8
1.78 (d, 13.5)
2.04 (d, 13.0)
0.94 (s)
1.09 (s)
32.9 (CH3)
33.7 (CH3)
26.2 (CH2)
34.3
33.3
27.4
8, 9, 10, 16
0.95 (s)
0.96 (s)
8, 9, 10, 15
2.62 (dd, 7.5, 15.0)
2.72 (dd, 7.5, 15.0)
5.37 (t)
2.72 (dd, 7.0, 15.5)
2.79 (dd, 8.0, 15.0)
5.48 (t, 7.8)
2, 3, 4, 14, 18
2, 3, 4, 14, 18
120.8 (CH)
133.08 (C)
17.8 (CH3)
25.9 (CH3)
30.5 (CH2)
121.6
133.2
18.3
1.45 (s)
18, 21
1.53 (s)
1.54(s)
18, 20
1.73 (s)
26.6
2.78 (dd; 8.5; 13.5)
2.94 (dd; 7.0; 14.0)
5.40 (m)
4, 5, 6, 13, 23
4, 5, 6, 13, 23
24
2,66 (dd, 7.8, 14.3)
2.92 (dd, 7.0, 14.5)
5.58 (t, 7.3)
31.7
4, 5, 6
4, 5, 6
121.4 (CH)
133.06 (C)
17.9 (CH3)
26,1(CH3)
163.9
121.5
133.7
18.4
1.66 (s)
1.62 (s)
24
24
1.61 (s)
1.71 (s)
26.6
196.3
139.6
129.8
129.2
133.7
129.2
129.8
63.0
132.9 (C)
128.3 (CH)
129.4 (CH)
129.6 (CH)
129.4 (CH)
128.3 (CH)
56.5 (CH3)
7.10 (m)
7.10 (m)
7.10 (m)
7.10 (m)
7.10 (m)
2.67 (s)
5.75 (s)
27
7.98 (d, 7.3)
7.03 (t, 7.3)
7.07 (t, 4.8)
7.03 (t, 7.3)
7.98 (d, 7.3)
2.90 (s)
27, 31
28, 29, 31
28, 29, 31
27, 31
2
27
27
1, 5, 6
5.70 (s)
1, 5, 6, 7
a
Carbonyl groups were assigned based on their δC.
Careful analysis of the HMBC data (Table 1 and Fig. 2) allowed the
construction of the core skeleton of the molecules and the precise as-
signment of the two prenyl group positions. The methylene hydrogens,
H-22a and H-22b, from the prenyl group are correlating with C-4, C-5,
C-14 and C-3. The correlation of H-15 and H-16 with each other and
with C-8, C-9 and C-10, the correlation of H-8a and H-8b with C-7, C-9,
C-10, C-12, C-14 and C-15, the correlation of H-10b with C-9 and C-16,
of H-11a with C-7, C-10 and C-13 and the correlation of H-11b with C-7
3

C.G. Ferraz, et al.
Fitoterapia138(2019)104346
Fig. 3. Selected HMBC correlations (1H → 13C) of 2a.
Fig. 2. Selected HMBC correlations (1H → 13C) of 1a.
allowed us to define the 9,9-dimethyl-six-membered ring formed by C-
7, C-8, C-9, C-10, C-11, C-12, and the attachment between C-7 and C-6
and between C-12 and C-13.
The proposed core skeleton of compounds 1a and 2a occur with
two possible stereochemistries at C-12. Unfortunately, the 1H NMR
spectrum of 2a showed that H-12 and H-10b have superimposed
chemical shifts (δ 1.46) and, therefore a careful analysis of the
NOESY data was necessary to unquestionably establish the relative
stereochemistry and the spatial position of methylene hydrogens.
The correlation observed between H-13a (δ 1.68) and H-22a (δ 2.66)
determines the relative positions of H-13a and H-13b (δ 2.11)
(Fig. 4a).
Compound 2a is an isomer of 1a and presented very similar NMR
spectral pattern (Table 1). The analysis of the HMBC data of 2a allowed
us to deduce its carbon skeleton as that presented by 1a, however the
correlation of H-14 and H-17 with δ 165.3 (C-2) and the correlation
between H-29 with δ 196.3 (C-27) allowed us to locate the conjugated
carbonyl in C-27 and the double bond between C-1 and C-2 (Fig. 3).
Fig. 4. Tridimensional stick representations of 2a to allow visualization of selected NOESY correlations.
4

C.G. Ferraz, et al.
Fitoterapia138(2019)104346
H-13b correlated with the signal at δ 1.46. This correlation was
NMR signals very similar to nemorosonol, except for the 13C NMR
signals at the positions 1, 2 and 27 (Supplementary Table 1). [12] This
observation suggests that 3a might exist as a pair of β-diketones in
tautomeric equilibrium with nemorosonol. It is worth noticing that
nemorosonol was previously isolated from Clusia burle-marxii. [18].
The correlation of H-10, H-34, and H-17 with δ 164.6 (C-2) and the
correlation of H-29 with at δ 196.0 (C-27) allowed us to locate the
conjugated carbonyl in C-2 and the double bond between C-1 and C-27
(Fig. 5).
attributed to H-12 and not to H-10a because in neither of the two
possible stereochemistries H-10a would correlate with H-13b. This
correlation unambiguously established that H-12 was in the same side
of H-13b and in the opposite side of 6-OH. This assumption was con-
firmed by the observation of the correlation between H-12 and H-14a (δ
1.70) which in its turn establishing the relative position of H-14a and H-
14b (δ 2.04) (Fig. 4a). Additionally, the observation of correlations
between H-14b with H-29/H-33 (δ 7.98) supports this assumption
(Fig. 4b). The correlation between H-29/H-33 and H-8a (δ 2.39) along
with the correlation between H-8b (δ 1.07) and H-14b defined the re-
lative positions of H-8a and H-8b (Fig. 4a and b). The proposed relative
position of H-8a and H-8b is supported by the correlation observed
between H-8a and 6-OH (δ 5.70) (Fig. 4a). H-15 (δ 0.94) correlated
with H-14a (δ 1.70) and with H-8b (δ 1.07) (Fig. 4), whereas H-16 (δ
0.96) correlated with H-8a (δ 2.39) (Fig. 4). These correlations defined
the relative positions of the gem-dimethyl groups at C-9. The detailed
analysis of the NMR data allowed us to conclude that burlemarxiones A
and B present an unprecedent tetracyclo [8.3.1.03,11.05,10]tetradecane
core skeleton (Fig. 1).
Compound 3a was obtained as a yellow gum and presented an
[α]2D0 = −48.0 (c − 1.35, CHCl3). The HRESIMS data of 3a exhibited a
pseudomolecular ion [M + H]+ at m/z 517.3054 (calcd for 517.3312)
corresponding to a molecular formula of C34H44O4 (Supplementary
Fig. 55). Its molecular formula (C34H44O4) was deduced from the
pseudo molecular ion peak (m/z 517.3054 [M + H]+) obtained from
the HRESIMS (positive mode), along with the analysis of the 13C NMR,
and DEPT data (Table 2). Compound 3a showed a pattern for the 13C
Table 2
1H and 13C NMR spectral data of 3a recorded at 500 MHz in benzene-d6.
Position
δH (J in Hz)
δC (DEPT 135o)
HMBC
1
116.8 (C)
164.6 (C)
57.6 (C)
2
3
Fig. 5. Selected HMBC correlations (1H → 13C) of 3a.
4
211.1 (aC]O)
59.6 (C)
5
6
86.4 (C)
7a
2.10 (dd; 9.0; 13.5)
1.68 (dd; 5.5; 13.5)
1.67 (ov)
40.9 (CH2)
3, 5, 6, 9
3, 5, 8
6, 9
7b
Benzophenones within the genus Clusia have been classified ac-
cording to their core skeleton into six groups: type I to type VI. Type I
benzophenones encompass simple 2,4,6-trihydroxybenzophenone de-
rivatives, whereas polyprenylated caged benzophenones are mainly
grouped into types III-V. [19] A more general classification has divided
benzophenones into two categories: basic benzophenone skeletons
(BBS) and polyisoprenylated benzophenones skeletons (PPBS). PPBS
benzophenone derivatives with unique core skeletons containing bi-,
tri-, and/or tetracyclic ring systems have been further categorized into
four groups: type A to D. [20] Type-A PPBS encompass the most so-
phisticated and complex polyprenylated benzophenone derivatives,
which are mainly produced by Clusia species. [7,13–16,19,20] A search
using burlemarxiones A and B as queries has indicated that these mo-
lecules present a novel caged carbon skeleton. As a matter of fact, the
closest hit in terms of similarity was to nemorosonol (91% of simi-
larity). [21,22] Therefore, burlemarxiones A and B present a new tet-
racyclo[8.3.1.03,11.05,10]tetradecane core skeleton.
8
49.4 (CH)
49.0 (C)
9
10a
10b
11a
11b
12
1.85 (d; 9.0)
1.58 (d; 9.0)
2.54 (m)
50.2 (CH2)
2, 3, 9
2, 3, 6, 9
8, 12, 13
8, 12, 13
33.9 (CH2)
2.28 (m)
5.10 (t; 7.0)
125.0 (CH)
132.2 (C)
13
14
1.65 (s)
18.6 (CH3)
26.4 (CH3)
20.8 (CH3)
27.1 (CH2)
12, 13
15
1.71 (s)
12, 13
16
1.18 (s)
6, 9
17a
17b
18
2.80 (m)
2.71 (m)
5.55 (t; 7.0)
2, 3, 4, 10, 18, 19
2, 3, 4, 10, 18, 19
121.5 (CH)
133.9 (C)
19
20
1.60 (s)
18.4 (CH3)
26.6 (CH3)
31.8 (CH2)
21
1.72 (s)
22a
22b
23
2.95 (dd; 7.5; 14.5)
2.68 (m)
4, 5, 6, 23, 24
4, 5, 6, 23, 24
5.48 (t;7.5)
121.6 (CH)
133.3 (C)
Burlemarxiones A and B are more likely biosynthesized through the
condensation of one lavandulyl pyrophosphate molecule and precursor
I, in a step that resembles a SN2 reaction, followed by an oxidation of
the lavandulyl moiety to produce intermediate II. Several compounds
with the lavandulyl moiety have been isolated from Clusia species:
xantochimol and guttiferone E from C. rosea [23], nemorosinic acid
from C. nemorosa [24], weddellianone from C. weddeliana [17], and 13-
benzoyl-6,6,8,14,14-pentamethyl-11,15-di(3-methyl-2-butenyl)-9-ox-
atetracyclo[11.3.1.01,10.03,8]heptadec-10-ene-12,17-dione from C. ob-
deltifolia [7].
24
25
1.75 (s)
1.50 (s)
18.3 (CH3)
26.6 (CH3)
196.0 (aC=O)
139.4 (C)
26
27
28
29–33
30–32
31
7.98 (d;7.3)
7.02 (t;7.3)
7.08 (t;4.8)
5.53 (s)
129.9 (CH)
129.2 (CH)
133.7 (CH)
27, 31
27, 28, 29
27, 28
1, 5, 6
2
6-OH
OMe
2.90 (s)
63.1 (CH3)
a
Carbonyl groups were assigned based on their δC.
5

C.G. Ferraz, et al.
Fitoterapia138(2019)104346
A sequence of reactions as delineated in (Fig. 6) produce produces
The mixture was then diluted with 12.50 mL of distilled water and
cooled in an ice bath. Then, ice cold NaNO2 solution (43%) was added
to the mixture. After vacuum filtration, nine grams of nitro-
somethylurea were obtained. The samples were methylated in an
four- and five-membered rings as shown in intermediate IV. Further
prenylations and intramolecular cyclizations lead to the formation of
burlemarxione A, which is a tautomer of burlemarxione B (Fig. 6).
Fig. 6. Putative biosynthesis pathway of burlemarxione A.
Glioblastoma is an extremely resistant brain tumor due to the in-
herent hurdle for the delivery of chemotherapy agents across the blood
brain barrier into the brain. For these reasons, temozolomide is one of
the few chemotherapy agents applied to treat glioblastoma brain tu-
mors. Nevertheless, patients survival expectations for this type of tumor
is approximately 11 months if they undergo surgery, radiotherapy and
chemotherapy with temozolomide. Cytotoxicity of 1a and sampsonine
N was assessed against GL-15 multiforme glioblastoma-derived human
cell line. Compound 1a showed EC50 of 31.96 μM, whereas EC50 for
sampsonine N was 26.00 μM. Temozolomide as positive control had an
EC50 of 716 μM.
ethereal diazomethane solution recently prepared by a reaction invol-
ving the slow addition of nitrosomethylurea to an aqueous KOH solu-
tion (50%) in sulfur ether in an ice bath The sample to be methylated
was solubilized in CHCl3 and the ethereal ice-cold diazomethane solu-
tion was added gradually until no further gas evolution was observed.
The solvent was evaporated at room temperature and the product was
examined by 1H NMR to ensure completion of the methylation reaction.
3.3. Plant material
C. burle-marxii trunk was collected at Mucugê city (Bahia state,
Brazil). The specimens were identified by Maria Lenise da Silva Guedes,
PhD from the Biology Institute of the Federal University of Bahia,
3. Experimental section
Brazil.
A voucher specimen (ALCB-61584) was deposited in the
3.1. General experimental procedures
Herbarium Alexandre Leal Costa at Federal University of Bahia.
Optical rotations were recorded on a PerkinElmer model 343 po-
larimeter. IR spectra were recorded on Perkin Elmer (FT-IR Spectrum
1000) spectrophotometer. NMR spectra were measured on Varian
Gemini 2000 (INOVA-500) and Bruker spectrometers. The resonances
of residual CHCl3 (7.26 and 77.0 ppm) and benzene (7.20 and
128.0 ppm) were used as internal references for 1H and 13C NMR
spectra acquisition. HRESIMS were recorded on a SHIMADZU LCMS-IT-
TOF.
3.4. Extraction and isolation
Dried powdered trunk (10.0 kg) was extracted with n-hexane three
times by maceration. The extract (121.17 g) was fractionated with
hexane and hexane/EtOAc mixtures in a silica gel column. Fractions 3
and 4 were combined and methylated with diazomethane. The me-
thylation was necessary to improve the chromatographic separations of
mixture whose 1H NMR spectrum showed signals between δ 15 and δ 16
indicating the probable presence of tautomers in keto-enol equilibrium.
The methylated fraction was fractioned with hexane/EtOAc mixtures in
a silica gel CC. From the fraction 5 of this column, after successive
chromatography on silica gel column, and preparative TLC (silica gel;
hexane-EtOAc 9:1) compound 1a (260 mg) was isolated. Fraction 7 was
also submitted to successive chromatography on silica gel column and
one of the fractions was purified with HPLC on silica gel (hexane/AcOEt
85:15) giving the compounds 2a (43.0 mg) and 3a (34 mg). Sampsonine
N (19.0 mg) and obdeltifolione C (7.0 mg) were isolated from fractions
3.2. Methylation reaction
Nitrosomethylurea was initially prepared by slowly stirring an
aqueous solution of NaOH (38.5%), Br2 (22.0 g) and acetamide
(14.75 g). This mixture was heated to 100 °C and then cooled in an ice
bath for two hours. Acetyl methyl urea crystals (12.25 g) were obtained
after vacuum filtration. The acetyl methyl urea crystals were dissolved
in 12.50 mL of concentrated HCl and heated to 100 °C for about 12 min.
6

C.G. Ferraz, et al.
Fitoterapia138(2019)104346
10 and 11 of this column, after successive chromatography on silica gel
column, and preparative TLC (silica gel; hexane-EtOAc 9:1).
[5] P.R. Ribeiro, C.G. Ferraz, M.L.S. Guedes, D. Martins, F.G. Cruz, A new biphenyl and
antimicrobial activity of extracts and compounds from Clusia burlemarxii,
Fitoterapia 82 (8) (2011) 1237–1240.
Burlemarxione A (1a): [α]2D0 = −113.0 (c 1.35, CHCl3); 1H and 13
C
[6] F.G. Cruz, J.S.R. Teixeira, Polyprenylated benzophenones with a tricyclo
[4.3.1.13,8] undecane skeleton from Clusia obdeltifolia, J. Braz. Chem. Soc. 15 (4)
(2004) 504–508.
NMR, see Table 1; HRESIMS m/z 517.3319 [M + H]+. IR (KBr) νmax
3480, 3418, 2920, 2857, 1724, 1692, 1611, 1589, 1449 cm−1
.
[7] J.S.R. Teixeira, F.G. Cruz, Polyisoprenylated benzophenone derivatives from Clusia
obdeltifolia, Tetrahedron Lett. 46 (16) (2005) 2813–2816.
Burlemarxione B (2a): [α]2D0 = −78.0 (c 1.35, CHCl3); 1H and 13C
NMR, see Table 1; HRESIMS m/z 517.3319 [M + H]+
.
[8] P.L.C. Coelho, S.R.V.B. de Freitas, B.P.S. Pitanga, V.D.A. da Silva, M.N. Oliveira,
M.S. Grangeiro, C.S. Souza, R.S. El-Bachá, M.F.D. Costa, P.R. Barbosa,
I.L.O. Nascimento, S.L. Costa, Flavonoids from the Brazilian plant croton betulaster
inhibit the growth of human glioblastoma cells and induce apoptosis, Brazil. J.
Pharm. 26 (1) (2016) 34–43.
Isonemorosonol (3a): [α]2D0 = −48 (c 1.35, CHCl3); 1H and 13C
NMR, see Table 2; HRESIMS m/z 517.3054 [M + H]+. IR (KBr) νmax
3418, 2926, 1721, 1599, 1446, 1341 cm−1
.
[9] B.L. Santos, M.N. Oliveira, P.L.C. Coelho, B.P.S. Pitanga, A.B. Da Silva, T. Adelita,
V.D.A. Silva, M.D.F.D. Costa, R.S. El-Bachá, M. Tardy, H. Chneiweiss, M.P. Junier,
V. Moura-Neto, S.L. Costa, Flavonoids suppress human glioblastoma cell growth by
inhibiting cell metabolism, migration, and by regulating extracellular matrix pro-
teins and metalloproteinases expression, Chem. Biol. Interact. 242 (2015) 123–138.
[10] T.C.C. Silva, G.P.F. Lopes, N.J.M. Filho, D.M. de Oliveira, E. Pereira, B.P.S. Pitanga,
R.S. Costa, E.S. Velozo, S.M. Freire, J. Clarêncio, H.L. Borges, S.K. Rehen, V. Moura-
Neto, S.L. Costa, Specific cytostatic and cytotoxic effect of dihydrochelerythrine in
glioblastoma cells: role of NF-κB/β-catenin and STAT3/IL-6 pathways, Anticancer
Agents Med Chem. 18 (10) (2018) 1386–1393.
3.5. Cytotoxic testing
Cytotoxicity was assessed against GL-15 multiforme glioblastoma-
derived human cell line as described previously [25,26]. The effect of
the benzophenones derivatives on GL-15 cell viability was tested using
the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT; Sigma, St. Louis, MO) assay after 24 and 72 h exposure in 96-well
plates. The optic absorbency was measured at a wavelength of 560 nm
using a BIO-RAD 550PLUS spectrophotometer (Hercules, CA). Four
replicate wells were used for each analysis. The results were expressed
as EC50 (μM) [25,26].
[11] M. Da Ros, V. De Gregorio, A.L. Iorio, L. Giunti, M. Guidi, M. de Martino,
L. Genitori, I. Sardi, Glioblastoma chemoresistance: the double play by micro-
environment and blood-brain barrier, Int. J. Mol. Sci. 19 (10) (2018).
[12] S. Cerrini, D. Lamba, F.D. Monache, R.M. Pinherio, Nemorosonol, a derivative of
tricyclo-[4.3.1.03,7]-Decane-7-hydroxy-2,9-dione from Clusia nemorosa,
Phytochemistry 32 (4) (1993) 1023–1028.
[13] C.M.A. De Oliveira, A.L.M. Porto, V. Bittrich, A.J. Marsaioli, Two polyisoprenylated
benzophenones from the floral resins of three Clusia species, Phytochemistry 50 (6)
(1999) 1073–1079.
Author contributions
[14] J. Lokvam, J.F. Braddock, P.B. Reichardt, T.P. Clausen, Two polyisoprenylated
benzophenones from the trunk latex of Clusia grandiflora (Clusiaceae),
Phytochemistry 55 (1) (2000) 29–34.
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
[15] A.L.M. Porto, S.M.F. Machado, C.M.A. De Oliveira, V. Bittrich, M.D.C.E. Amaral,
A.J. Marsaioli, Polyisoprenylated benzophenones from Clusia floral resins,
Phytochemistry 55 (7) (2000) 755–768.
Funding sources
[16] R. Mangas Marín, A. Bello Alarcón, O. Cuesta Rubio, A.L. Piccinelli, L. Rastrelli,
Polyprenylated benzophenones derivatives from Cluisa minor fruits, Lat. Am. J.
Pharm. 27 (5) (2008) 762–765.
Financial support was provided by FINEP, CAPES, CNPq, and
FAPESB.
[17] A.L.M. Porto, S.s.M.F. Machado, C.l.M.A. de Oliveira, V. Bittrich, M.d.C.E. Amaral,
A.J. Marsaioli, Polyisoprenylated benzophenones from Clusia floral resins,
Phytochemistry 55 (7) (2000) 755–768.
Declaration of Competing Interest
[18] C.G. Ferraz, Benzofenonas, Triterpenos e Esteróides de Clusia burle-marxii, Federal
University of Bahia, Salvador, Brazil, 2005.
The authors declare no competing financial interest.
Appendix A. Supplementary data
[19] M.C. Anholeti, S.R. De Paiva, M.R. Figueiredo, M.A.C. Kaplan, Chemosystematic
aspects of polyisoprenylated benzophenones from the genus Clusia, Anais Acad.
Brasil. Ciencias 87 (1) (2015) 289–301.
[20] S.B. Wu, C. Long, E.J. Kennelly, Structural diversity and bioactivities of natural
benzophenones, Nat. Prod. Rep. 31 (9) (2014) 1158–1174.
[21] F.D. Monache, G.D. Monache, R. Moura Pinheiro, L. Radics, Nemorosonol, a deri-
vative of tricyclo-[4.3.1.03,7]-Decane-7-hydroxy-2,9-dione from Clusia nemorosa,
Phytochemistry 27 (7) (1988) 2305–2308.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fitote.2019.104346.
[22] A. Oya, N. Tanaka, T. Kusama, S.Y. Kim, S. Hayashi, M. Kojoma, A. Hishida,
N. Kawahara, K. Sakai, T. Gonoi, J. Kobayashi, Prenylated benzophenones from
Triadenum japonicum, J. Nat. Prod. 78 (2) (2015) 258–264.
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