Novel polyprenylated benzophenone derivatives from Clusia burle-marxii

Article abstract of DOI:10.1016/j.fitote.2020.104760

Three new caged polyprenylated benzophenone derivatives named burlemarxiones D-F (1–3) were isolated from the hexane extract of Clusia burle-marxii trunks. Burlemarxione D (1) contains the tetracyclo[8.3.1.0^3,11.0^5,10]tetradecane core skeleton also observed for burlemarxione A, its probable immediate precursor. However, two additional rings are formed to produce an unprecedented complex-caged core skeleton. These additional rings could be formed by a radical cyclization reaction of one prenyl group at C-5 with C-1 and C-33, followed by oxidative dehydrogenation (rearomatization) or by an intramolecular [4 + 2] radical cycloaddition (Diels-Alder reaction), followed by an enolization reaction (rearomatization). Burlemarxiones E and F were isolated after methylation with diazomethane that was necessary to avoid the interconversion of the pair of β-diketones in tautomeric equilibrium. The proposed biosynthetic pathway for burlemarxiones D-F involves the condensation of either lavandulyl pyrophosphate or 2-(1-methylvinyl)-hexa-5-enyl pyrophosphate with the acylphloroglucinol derivative 6-benzoyl-5-hydroxy-5-cyclohexen-1,3-dione, followed by consecutive prenylation reactions. Therefore, Clusia burle-marxii reinforces the claim that the genus Clusia is an important source of sophisticated caged polyprenylated benzophenone derivatives.

Full text of DOI:10.1016/j.fitote.2020.104760

Fitoterapia xxx (xxxx) xxx
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Novel polyprenylated benzophenone derivatives from Clusia burle-marxii
a
,
b
,
*
,
1
a,
b
c
d
Caline G. Ferraz
, Paulo R. Ribeiro , Renata Mendonça , Edilberto R. Silveira ,
a
Frederico G. Cruz
a
Grupo de Estudos de Subst aˆ ncias Naturais Org aˆ nicas, Instituto de Química, Universidade Federal da Bahia, Rua Bar a˜ o de Jeremoabo s/n, 40170-115 Salvador, Brazil
Metabolomics Research Group, Departamento de Química Org aˆ nica, Instituto de Química, Universidade Federal da Bahia, Rua Bar a˜ o de Jeremoabo s/n, 40170-115
b
Salvador, Brazil
c
Instituto de Química, Universidade Federal do Rio Grande do Norte, CP 1524, 59072-970 Natal, RN, Brazil
Departamento de Química Org aˆ nica e Inorg aˆ nica, Universidade Federal do Cear a´ , 60021-940 Fortaleza, CE, Brazil
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three new caged polyprenylated benzophenone derivatives named burlemarxiones D-F (1–3) were isolated from
Clusiaceae
the hexane extract of Clusia burle-marxii trunks. Burlemarxione D (1) contains the tetracyclo[8.3.1.03,11.05,10
]
Burlemarxione
Nemorosonol
tetradecane core skeleton also observed for burlemarxione A, its probable immediate precursor. However, two
additional rings are formed to produce an unprecedented complex-caged core skeleton. These additional rings
could be formed by a radical cyclization reaction of one prenyl group at C-5 with C-1 and C-33, followed by
oxidative dehydrogenation (rearomatization) or by an intramolecular [4 + 2] radical cycloaddition (Diels-Alder
reaction), followed by an enolization reaction (rearomatization). Burlemarxiones E and F were isolated after
methylation with diazomethane that was necessary to avoid the interconversion of the pair of β-diketones in
tautomeric equilibrium. The proposed biosynthetic pathway for burlemarxiones D-F involves the condensation of
either lavandulyl pyrophosphate or 2-(1-methylvinyl)-hexa-5-enyl pyrophosphate with the acylphloroglucinol
derivative 6-benzoyl-5-hydroxy-5-cyclohexen-1,3-dione, followed by consecutive prenylation reactions. There-
fore, Clusia burle-marxii reinforces the claim that the genus Clusia is an important source of sophisticated caged
polyprenylated benzophenone derivatives.
Polyprenylated benzophenones
1
. Introduction
Caged polyprenylated benzophenone are polycyclic polyprenylated
presents an adamantyl skeleton [27]. Several polyisoprenylated benzo-
phenones have been isolated from Clusia floral resins, such as spiritone
isolated from C. burchellii and C. fluminensis, insignone isolated from
C. insignis, and scrobiculatone A and B isolated from C. scrobiculat [24].
Three prenylated benzophenone derivatives named as 28,29-epoxyplu-
kenetione A, 33-hydroperoxyisoplukenetione C, and 15,16-dihydro-16-
hydroperoxyplukenetione F have been isolated from C. havetiodes var.
stenocarpa fruits. The benzophenone derivative 28,29-epoxyplukene-
tione A shows a tricyclo[3.3.1.13 ]decane-2,4,9-trione core skeleton,
whereas the benzophenone derivative 33-hydroperoxyisoplukenetione
acylphloroglucinols (PPAPs) derivatives characteristic of the Clusiaceae
and Hypericaceae families [7,10,13,25,27,30,33]. Clusia is a prominent
genus belonging to Clusiaceae family and it is known to produce
benzophenone derivatives with great complexity and structural variety
[
2,6,8,9,10,11,16,17,22,24,27]. Benzophenone derivatives found in
,7
Clusia species usually showed high prenylation pattern, but low oxida-
tion indices [2]. So far, burlemarxione A-C, sampsonine N, and obdel-
tifolione C have been isolated from Clusia burle-marxii. Burlemarxiones A
,13
1,7
C
shows unusual 4,5-dioxatetracyclo[9.3.1.19 .0 ]hexadecane-
and
B
were reported to possess an unprecedented tetracyclo
12,14,15-trione caged moiety [6]. Biogenetically, PPAPs are derived
from a mixed” mevalonate/methylerythritol phosphate and polyketide
biosynthetic pathway. Their acylphloroglucinol cores are produced by a
characteristic polyketide-type biosynthesis involving the condensation
of one acyl-CoA and three malonyl-CoA units. Prenylation of this core
8.3.1.03 .05,10]tetradecane core skeleton [10]. Sampsonine N was
,11
[
first isolated from Hypericum sampsonii [13] and it presents a unique
caged tetracyclo[7.3.1.13 .0 ]tetradecane-2,12,14-trione skeleton,
,11
3,7
whereas obdeltifolione C was first isolated from C. obdeltifolia and it
*
Corresponding author at: Centro de Ci ˆe ncias Exatas e Tecnol o´ gicas – CETEC, Universidade Federal do Rec oˆ ncavo da Bahia, Rua Rui Barbosa, no710, 44.380-000
Cruz das Almas, Brazil.
E-mail address: califerraz@gmail.com (C.G. Ferraz).
Centro de Ci ˆe ncias Exatas e Tecnol o´ gicas – CETEC, Universidade Federal do Rec oˆ ncavo da Bahia, Rua Rui Barbosa, n 710, 44.380-000, Cruz das Almas, Brazil.
1
o
https://doi.org/10.1016/j.fitote.2020.104760
Received 15 August 2020; Received in revised form 7 October 2020; Accepted 12 October 2020
Available online 16 October 2020
0
367-326X/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: Caline G. Ferraz, Fitoterapia, https://doi.org/10.1016/j.fitote.2020.104760

C.G. Ferraz et al.
Fitoterapia xxx (xxxx) xxx
Fig. 1. Chemical structures of polyprenylated benzophenones derivatives from
C. burle-marxii.
1
13
Fig. 2. Key HMBC correlations ( H → C) of burlemarxione D (1).
Table 1
NMR spectral data of burlemaxione D recorded at 500 MHz in benzene-d
6
.
from Clusia burle-marxii. They were named as burlemarxiones D (1), E
(2), and F (3) (Fig. 1). The structure of burlemarxione D was proposed
o
1
13
N
H
C
HMBC
1
13
after careful analysis of H and C NMR, DEPT 135, gHMBC, gHMQC,
1
2
3
4
5
6
7
71.1 (C)
NOESY, IR, and HRESIMS spectra. Burlemarxione D was obtained as a
203.3 (C=O)
64.3 (C)
2
0
yellow gum, [
α]
D
= ꢀ 8.0 (c 1.0, CHCl
3
), and its molecular formula
212.7 (C=O)
70.8 (C)
(C33
H
40
O
4
) was established by HRESIMS (positive mode) on the basis of
+
ꢀ 1
the ion peak at m/z 501.2751 [M + H] . The IR spectrum showed ab-
92.2 (C)
sorption bands for a hydroxyl (3522 cm ) and carbonyl groups (1735,
43.0 (C)
ꢀ 1
1
705 and 1675 cm ), suggesting the presence of at least one hydroxyl
8
a
1.95 (d, 14.0)
1.74 (d,14.0)
42.2 (CH
2
)
1,5, 6,7,9,14,16
7,9,10,12,14, 16
1
and three carbonyl groups. The H NMR spectrum showed the presence
of four aromatic protons: δ 7.97 (1H, d, J = 7.5 Hz), δ 7.11 (1H, t, J =
.0 Hz), δ 7.00 (1H, t, J = 7.5 Hz) and δ 6.94 (1H, d, J = 8.0 Hz)
Table 1). Additional signals at δ 5.72 (1H, t, J = 7.5 Hz) and two
methyl doublets at δ 1.57 (J = 1.0 Hz) and δ 1.58 (J = 1.0 Hz) indi-
cated the presence of prenyl group (Table 1, Supplementary
Figs. 1–46). Four additional singlets at δ 0.82, 0.89, 0.93 and 1.03
8
b
9
1
29.8 (C)
H
H
0a
0.99 (ov)
1.41 (ov)
1.31 (ov)
1.41(ov)
38.0 (CH
2
)
)
8
H
H
1
0b
12
(
H
1
1a
29.0 (CH
2
6,7,8
H
H
1
1b
12
1
1
2
1.53(ov)
54.2 (CH)
3,6
a
3a
1.60 (ov)
2.11 (m)
32.7 (CH
2
)
)
1,4,5,12
1,4,5,6,7,12
2,3,4,7,8,12
2,3,6,7,8,12,17
8, 9,10,16
9,10,15
H
1
3b
confirmed the presence of the four methyl groups (Table 1, Supple-
mentary Figs. 1–46).
1
4a
1.88 (d, 13.5)
1.76 (d, 13.5)
1.03(s)
50.2 (CH
2
1
4b
Analysis of 13C and DEPT NMR spectra indicated six methyl groups,
seven methylene, seven methine and thirteen non-hydrogenated carbon,
including three carbonyl groups. The 13C NMR spectrum showed signals
1
1
1
5
34.8 (CH
33.4 (CH
26.8 (CH
3
3
2
)
)
)
6
0.82(s)
7a
2.57 (ov)
2.57 (ov)
5.72 (t, 7.5)
2,3,14,18,19
2,3,14,18,19
1
7b
of two non-conjugated (δ
00.5) carbonyl groups. Two olefinic carbons at δ
confirmed the presence of the prenyl groups (Table 1). The presence of
the hydroxyl group suggested by IR was confirmed by the signal at δ
2.2 that was attributed to a non–hydrogenated oxygenated carbon
C
212.7 and δ
C
203.3) and one conjugated (δ
C
1
1
2
2
2
8
120.2 (CH)
134.8 (C)
2
C
120.2 and at δ 134.8
C
9
0
1.57 (d, 1.0)
1.58 (d, 1.0)
1.99 (dd, 14.0,18.5)
2.11 (m)
18.3 (CH
26.4 (CH
29.5 (CH
3
3
2
)
)
)
19,21
1
19,20
C
2a
1,4,5,6,23
1,4,5,6,23,24
2, 5,6, 24
9
2
2b
13
(
tertiary alcohol). Analysis of C NMR and DEPT 135 data suggested a
2
2
2
2
2
2
2
3
3
3
3
3
2.73 (sl)
57.1 (CH)
37.7 (C)
certain degree of similarity between Burlemarxione D and Burle-
marxione A [10]. Distinguished differences, however, were observed for
C-1, C-2, C-23, C-24, C-25, C-27, and C-33 (Table 1). Key correlations in
the HMBC analysis allowed us to undoubtedly establish the structure of
compound 1 (Fig. 2, Table 1, Supplementary Figs. 1–46). Two additional
rings, in relation to burlemarxione A, were formed by the cyclization of
the prenyl group at C-5 with C-1 and C-33 was confirmed by the
4
5
6
7
8
9
0
1
2
3
0.93 (s)
0.89 (s)
30.3 (CH
26.2 (CH
3
)
)
26,33
3
24,25,33
200.5 (*C=O)
137.3 (C)
7.97(d, 7.5)
7.00 (t, 7.5)
7.11(t, 8.0)
6.94 (d, 8.0)
127.3 (CH)
127.5 (CH)
133.8 (CH)
124.2 (CH)
151.0 (C)
27,31,33
30,32
30
24,28,29
following correlations: a) H-25 (δ
151.0); b) H-32 (δ 6.94) with C-24 (δ
H-22b (δ 2.11) with C-1 (δ 71.1) and C-5 (δ
with C-2 (δ 203.3), C-5 (δ 70.8), and C-6 (δ
H
0.93) and H-26 (δ
37.7); c) H-22a (δ
70.8); d) H-23 (δ
92.2) (Fig. 2a). These
H
0.89) with C-33
1.99) and
2.73)
(
δ
C
H
C
H
J values in Hz.
H
C
C
H
C
C
C
moiety affords monocyclic polyprenylated acylphloroglucinols
MPAPs), which may be further cyclized to PPAP-type metabolites with
spectral features were similar to those of garcibracteatone, a poly-
prenylated benzophenone isolated from Garcinia bracteata (Clusiaceae)
[28].
(
diverse carbon skeletons [32]. Furthermore, polyprenylated benzophe-
none derivatives possess a great variety of biological activities such as
cytotoxic [3,10,14,21,23,31], antimicrobial [1,19,20], antioxidant
The correlations of the methylene protons at δ
2.57 (H-17b) with C-2 (δ 203.3), C-3 (δ 64.3), C-14 (δ
120.2), C-19 (δ 134.8) confirmed the connection of a prenyl group to C-
3 (Fig. 2a). The correlations of the methylene protons at δ 1.60 (H-13a)
and δ 2.11 and (H-13b) with C-4 (δ 212.7), C-5 (δ 70.8), C-12 (δ
54.2), along with the correlations of H-13b with C-6 (δ 92.2) and C-7
43.0) confirmed the connection of C-13 with C-5 (Fig. 2a). The
correlations of the methylene protons at δ 1.88 (H-14a) and δ 1.76 (H-
4b) with C-2 (δ 203.3), C-3 (δ 64.3), C-4 (δ 212.7), C-7 (δ 43.0), and
C-8 (δ 42.2), along with the correlations of H-14b with δ C-6 (δ 92.2),
H
2.57 (H-17a) and
C
C
C
50.2), C-18 (δ
C
[
29], and antiparasitic [15,26]. In the present work, we report the
C
identification of three new caged polyprenylated benzophenone de-
rivatives named burlemarxiones D-F from the Clusia burle-marxii and
discuss their putative biosynthesis pathways.
H
H
C
C
C
C
(δ
C
2
. Results and discussion
H
H
1
C
C
C
C
Three novel polyprenylated benzophenone derivatives were isolated
C
C
C
2

C.G. Ferraz et al.
Fitoterapia xxx (xxxx) xxx
and H-22a determines the relative stereochemistry of H-13a and H-13b,
as well as the relative stereochemistry of H-22a and H-22b (Fig. 3). The
correlation between H-13b and H-12 unambiguously established that H-
1
2 was in the same side of H-13b and in the opposite side of 6-OH, just as
observed for its biosynthetic precursor burlemarxione A [10]. This
assumption was confirmed by the observation of the correlation be-
tween H-12 and H-8b, and the correlation of H-8b with H-15, which in
its turn establishing the relative position of H-8a and H-8b (Fig. 3).
These correlations also defined the relative positions of the gem-
dimethyl groups at C-9. The correlation between H-13a and H-14a
unambiguously established the relative position of H-14a and H-14b.
The correlation between H-23 and H-22a, along with the correlation
between H-23 and H-25 unambiguously established that H-23 was in the
same side of H-22a and H-25 and in the opposite side of 6-OH (Fig. 3).
These correlations support the suggestion of the two additional rings
formed by the cyclization of one prenyl group at C-5 with C-1 and C-33
(
Scheme 1).
Burlemarxione D could be produced from its probable immediate
biosynthetic precursor by two distinct pathways (Scheme 1). In the first
putative pathway, burlemarxione A could undergo a radical cyclization
reaction, followed by oxidative dehydrogenation (rearomatization)
(
Scheme 1a). In the second putative pathway, burlemarxione A could
undergo an intramolecular [4 + 2] radical cycloaddition (Diels-Alder
reaction), followed by an enolization reaction (rearomatization)
(
Scheme 1b). Both pathways are plausible since similar biosynthetic
reactions have been suggested for complex caged benzophenones with
1
1
skeleton of the type PPAPs isolated from genus Garcinia and Hypericum
Fig. 3. Key NOESY correlations ( H → H) of burlemarxione D (1).
[
5,28].
The structure of burlemarxione E (2) was proposed after careful
confirmed the connection of C-14 with C-3 allowing us to deduce the
presence of a seven-membered ring formed amongst C-3, C-4, C-5, C-7,
C-12, C-13 and C-14 (Fig. 2b).
analysis of the spectroscopic data of its methyl derivative (2a).
Methylation with diazomethane was necessary to avoid the intercon-
version of the pair of β-diketones in tautomeric equilibrium [10]. Bur-
Burlemarxione D (1) could occur with two possible configurations at
C-12. A careful analysis of the NOESY data, along with the proposed
biosynthesis pathway of burlemarxione A [10] was necessary to un-
questionably establish the relative stereochemistry and the spatial po-
sition of methylene hydrogens. The correlation observed between H-13b
2
0
lemaxione E methyl derivative (2a) was isolated as a yellow gum, [
+11,0 (c 0,5; CHCl ), and its molecular formula (C34 ) was
established by HRESIMS (positive mode) on the basis of the ion peak at
α
]
D
=
3
44 4
H O
+
1
m/z 517.3012 [M + H] . The H NMR spectrum of 2a indicated the
Scheme 1. Putative biosynthesis pathway of burlemarxione D from burlemarxione A.
3

C.G. Ferraz et al.
Fitoterapia xxx (xxxx) xxx
Table 2
6
NMR spectral data of burlemaxione E (2a) recorded at 500 MHz in benzene-d .
o
1
13
N
H
C
HMBC
1
2
3
4
5
6
7
124.6
173.6
58.5
207.0
73.6
196.2
46.4
8
a
1.88 (d, 14.0)
1.21 (d, 14.0)
44.9
5,7,9,12,15,16
8
b
7,9,10, 12, 15,16
9
1
31.1
39.9
0a
1.06 (m)
12
1
0b
1.16 (m)
9,12
12,13
1
1a
0.92 (m)
24.9
1
1
1b
1.06 (m)
2
2.02 (m)
39.7
40.2
1
1
3a
3b
1.45 (t, 13.0)
1.69 (ov)
2,3,7, 11,12,17
2,3,4,7,12
9, 10
1
1
1
4
0.75 (s)
35.7
28.7
15.9
31.7
5
6
0.74 (s)
8,9, 10,14
5,7,12
0.79 (s)
1
7a
7b
2.62 (dd, 8.0, 14.5)
2.74 (dd, 7.0, 14.5)
5.48 (t, 7,0)
3,4,18,19
2,3,18
1
1
1
2
2
2
8
121.6
133.8
18.4
9
0
1.62 (s)
19,21
19
1
1.63 (s)
26.9
2a
2.57 (dd, 6.0, 13.5)
2.84 (dd, 8.5, 13,5)
5.58 (t, 7,0)
24,9
5,6,23
4,5,23,24
2
2b
2
2
2
2
2
2
2
3
3
3
3
3
121.6
134.5
18;8
4
5
6
7
8
9
0
1
2
3
1.70 (s)
1.65 (s)
24,26
24
1
1
Fig. 5. Key NOESY correlations ( H → H) of 2a.
26.4
195.8
139.1
129.3
130.0
133.5
130.0
129.3
61.0
similar to burlemarxione B [10]. The correlations of the methylene
8.01 (m)
7.10 (m)
7.10 (m)
7.10(m)
8.01 (m)
3.26 (s)
27
protons at δ
121.6), along with the correlation of H-17a with C-4 (δ
C-19 (δ 133.8) and the correlation of H-17b with C-2 (δ
confirmed the connection of one prenyl group to C-3 (Fig. 4a).
The correlations of the methylene protons at δ 2.57 (H-22a) and δ
2.84 (H-22b) with C-5 (δ 73.6) and C-23 (δ 121.6), along with the
correlation of H-22a with C-6 (δ 196.2) and the correlation of H-22b
with C-4 (δ 207.0) and C-24 (δ 134.5) confirmed the connection of the
other prenyl group to C-5 (Fig. 4a). The correlation of the methyl pro-
tons at δ 3.26 (H-34) with C-2 (δ 173.6) and the correlation of the
aromatic proton at δ 8.01 (H-29/33) with C-27 (δ 195.8) suggested
that the methoxyl group was linked to C-2 and the phenyl group was
linked to C-27 (Fig. 4a). The correlations of both methylene protons at δ
H
2.62 (H-17a) and 2.74 (H-17b) with C-3 (δ
C
58.5) and C-18
207.0) and
173.6)
(
δ
C
C
C
C
27
2
OMe
H
H
J values in Hz.
C
C
C
C
C
H
C
H
C
H
1
.45 (H-13a) and 1.69 (H-13b) with C-2 (δ
6.4), and C-12 (δ 39.7), along with the correlation of H-13a with C-11
24.9) and C-17 (δ 31.7) and the correlation of H-13b with C-4 (δ
07.0) confirmed the connection of C-13 with C-3 (Fig. 4a). Addition-
0.79 (H-16) with C-5 (δ
39.7) confirmed the connection of C-7
C
173.6), C-3 (δ
C
58.5), C-7 (δ
C
4
C
(
δ
C
C
C
2
ally, the correlation of the methyl protons at δ
3.6), C-7 (δ 46.4), and C-12 (δ
H
C
7
C
C
with C-5 and allowed us to deduce the presence of a six-membered ring
formed by C-3, C-4, C-5, C-7, C-12, and C-13 (Fig. 4a). 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-5 and C-7 was confirmed by the following cor-
1
13
Fig. 4. Key HMBC correlations ( H → C) of 2a.
presence of a monosubstituted benzene ring [δ
H
7.10 (3H, m), δ 8.01
relations: a) of both methylene protons at δ
8b) with C-7 (δ 46.4), C-9 (δ 31.1), C-15 (δ
along with the correlation of H-8a with C-5 (δ
of H-8b with C-10 (δ 39.9.0) and C-12 (δ
methyl protons at δ 0.75 (H-14) and δ 0.74 (H-15) with C-9 (δ
and C-10 (δ 39.9) (Fig. 4b).
A careful analysis of the NOESY data was necessary to unquestion-
ably establish the relative stereochemistry and the spatial position of
methylene hydrogens and methyl groups of 2a. The correlation observed
between H-13a and H-17, along with the correlation between H-13b and
H-22 determine the relative spatial position of H-13a and H-13b (Fig. 5).
H
1.88 (H-8a) and 1.21 (H-
28.7), and C-16 (δ 15.9),
73.6) and the correlation
39.7) (Fig. 3b); b) of the
31.1)
(
2H, m)] and one methoxyl group at δ
Figs. 47–62). The existence of two prenyl groups were inferred by the
presence of two olefinic protons at δ 5.58
1.63,
1.62. Three additional methyl groups singlets were observed at δ
0.75 and δ
0.74. The 13C NMR spectrum suggested the pres-
ence of three carbonyl groups at δ 207.0, δ 196.2 and δ 195.8 (Table 2
Supplementary Figs. 47–62), indicating the presence of two conjugated
and one nonconjugated carbonyl groups. Signals at δ 173.6 and δ
24.6 indicated the presence of a conjugated β-oxygenated double bond
H
3.26 (Table 2, Supplementary
C
C
C
C
C
H
5.48 (1H, t, J = 7.0 Hz) and δ
H
C
C
(
1H, t, J = 7.0 Hz) and four methyl groups at δ
H
1.70, δ 1.65, δ
H
H
H
H
C
and δ
H
H
C
0
.79, δ
H
H
C
C
C
C
C
1
4

C.G. Ferraz et al.
Fitoterapia xxx (xxxx) xxx
Scheme 2. Putative biosynthesis pathway of burlemarxione E.
The correlation between H-13b and H-11b, along with the correlation
between H-11b and H-10a, unambiguously establishing the relative
position of H-10a and H-10b, as well as the relative position of H-11a
and H-11b. These attributions are supported by the correlation of H-10b
and H-8a. Finally, the correlation between H-8a and H-15, along with
showed a highly similar pattern to isonemorosonol [10], except for the
signals at the positions 12, 13, 14, and 15 (Table 3, Supplementary
Figs. 63–74).
Nevertheless, the most remarkable changes were observed at the
positions 12 and 14 (Table 3, Supplementary Figs. 63–74). These dif-
ferences are most likely caused by the changes in the substitution
pattern of the double bond. While isonemorosonol has a dimethylallyl
group attached to C-8, we suggest that compound 3a has an isopentenyl
group attached to C-8 instead. The correlations of the geminal olefinic
3
the correlation of H-15 and 2-OCH also defined the relative positions of
the gem-dimethyl groups at C-9.
We suggest that Burlemarxione E is biosynthesized through the
condensation of one lavandulyl pyrophosphate molecule and precursor
I, in two consecutive steps, which include the oxidation of the lavandulyl
moiety to produce intermediate III. Then, intermediate III undergoes
two consecutive prenylation reactions, once again in two consecutive
protons protons at δ
along with the correlation of H-15 (δ
13 (δ 145.7), the correlation of H-7b (δ
C
11 (δ 33.8) allowed us to establish the connection of the isopentenyl
H
4.81 (H-14) with C-12 (δ
1.67) with C-12 (δ 36.6) and C-
C
1.74) with C-8 (δ 48.5) and C-
C C
36.6) and C-15 (δ 22.5),
H
C
C
H
steps that resemble a S
N
2 reaction, to produce burlemarxione E
(
Fig. Scheme 2). Lavandulyl pyrophosphate is commonly used to pro-
group (Fig. 6).
duce benzophenone derivatives in Clusia species ([4,7,9,10,18]),
including burlemarxiones A, B, C, and E isolated from Clusia burle-
marxii.
Here we suggest that 2-(1-methylvinyl)-hexa-5-enyl pyrophosphate
is used for the biosynthesis of burlemarxione F instead of lavandulyl
pyrophosphate. This is reasonable since 2-(1-methylvinyl)-hexa-5-enyl
pyrophosphate is commonly used to produce benzophenone derivatives
in Clusia species, such as guttiferone C and lanceolatone [12,24].
Initially, 2-(1-methylvinyl)-hexa-5-enyl pyrophosphate is condensed
with precursor I to produce intermediate IV. Then, intermediate IV
undergoes two consecutive prenylation reactions to produce burle-
marxione F (Fig. Scheme 3).
The structure of burlemarxione F (3) was proposed after careful
analysis of the spectroscopic data of its methyl derivative (3a).
Methylation with diazomethane was necessary to avoid the intercon-
version of the pair of β-diketones in tautomeric equilibrium [10]. Bur-
2
0
lemaxione F methyl derivative (3a) was isolated as a yellow gum, [
ꢀ 26,0 (c 0,82; CHCl ), and its molecular formula (C34 ) was
established by HRESIMS (positive mode) on the basis of the ion peak at
α
]
D
=
3
44 4
H O
+
1
m/z 517.3047 [M + H] . The H NMR spectrum of 3a indicated the
presence of a monosubstituted benzene ring [δ 7.08 (1H, t, J = 7.0 Hz),
7.02 (2H, t, J = 7.5 Hz), δ 7.98 (2H, d, J = 8.0 Hz)]. The existence of
two prenyl groups were inferred by the presence of two olefinic protons
at δ 5.48 (1H, t, J = 7.5 Hz) and δ 5.54 (1H, t, J = 7.5 Hz) and four
methyl groups at δ 1.77, δ 1.72, δ 1.59, and δ 1.52. Two additional
singlets of methyl groups were observed at δ 1.67 and δ 1.15. The
presence of a terminal double bond was inferred by the geminal protons
at δ 4.82 (1H, s) and δ
4.81 (1H, s). The 13C NMR spectrum of 3a
3. Experimental section
H
δ
H
3.1. General experimental procedures
H
H
Optical rotations were recorded on a PerkinElmer model 343
polarimeter. 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
H
H
H
H
H
H
H
H
3
residual CHCl (7.26 and 77.0 ppm) and benzene (7.20 and 128.0 ppm)
5

C.G. Ferraz et al.
Fitoterapia xxx (xxxx) xxx
Table 3
6
NMR spectral data of burlemaxione F (3a) recorded at 500 MHz in benzene-d .
o
a
N
δ
H
(J in Hz)
δ
C
HMBC
δ
C
o
(
DEPT 135 )
1
2
3
4
5
6
116.0
164.2
57.1
210.6
59.0
85.5
40.7
40.7
48.5
48.3
50.0
50.0
33.8
36.6
36.6
145.7
110.1
116.8 (C)
164.6 (C)
57.6 (C)
211.1 (*C=O)
59.6 (C)
86.4 (C)
7
7
8
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
2
a
2.12 (d, 9.0)
1.74 (d, 9.0)
1.57 (d, 13.5)
3,5,6,9
3,5,8,11
6,9
40.9 (CH
40.9 (CH
2
)
)
b
2
49.4 (CH)
49.0 (C)
0a
0b
1
1.85 (d, 17.5)
1.57 (d, 13,5)
1.78 (m)
2,3,9
2,3,6,9
15
50.2 (CH
50.2 (CH
33.9 (CH
2
2
2
)
)
)
2a
2b
3
2.00 (m)
15
125.0 (CH)
125.0 (CH)
132.2 (C)
1.78 (m)
15
4a
4b
5
4.81(s)
12,15
12,15
12,13
18.6 (CH
18.6 (CH
26.4 (CH
20.8 (CH
27.1 (CH
27.1 (CH
3
3
3
3
2
2
)
)
)
)
)
)
4.82(s)
1.67 (s)
22.5
6
1.15 (s)
20.3
7a
7b
8
2.72 (dd, 7.0, 15.5)
2.79 (dd, 8.0, 15.0)
5.48 (t,7.5)
26.6
2,3,4,10,18,19
2,3,4,10,18,19
26.6
120.9
133.4
17.8
121.5 (CH)
133.9 (C)
9
0
1.59 (s)
18.4 (CH
26.6 (CH
31.8 (CH
31.8 (CH
3
3
2
2
)
)
)
)
1
1.72 (s)
26.0
2a
2b
3
2,66 (dd, 7.8, 14.3)
2.92 (dd, 7.0, 14.5)
5.54 (t,7.5)
31.3
4,5,6,23,24
4,5,6,23,24
31.3
1
13
Fig. 6. Key HMBC correlations ( H → C) of 3a.
120.9
132.8
17.8
121.6 (CH)
133.3 (C)
4
5
1.77 (s)
1.52 (s)
18.3 (CH
26.6 (CH
3
)
)
3.3. Plant material
6
26.0
3
7
195.4
138.8
129.4
128.9
133.1
128.9
129.4
62.5
196.0 (*C=O)
139.4 (C)
C. burle-marxii trunk was collected at Mucug ˆe 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, Brazil.
A voucher specimen (ALCB-61584) was deposited in the Herbarium
Alexandre Leal Costa at Federal University of Bahia.
8
9
7.98 (d, 8.0)
7.02 (t, 7.5)
7.08 (t, 7.0)
7.02 (t, 7.5)
7.98 (d, 8.0)
2.89 (s)
27,31
129.9 (CH)
129.2 (CH)
133.7 (CH)
129.2 (CH)
129.9 (CH)
0
27,28,29
27,28
1
2
3
27,31
2
-OMe
3
63.1 (CH )
OH
5.53 (s)
1,5,6
3.4. Extraction and isolation
J values in Hz.
a
The dried trunk of Clusa burle-marxii (10 kg) were powdered and
extracted by maceration in hexane and methanol at room temperature
for three times each to obtain hexane (121.17 g) and methanol extracts.
The hexane extract (121.17 g) was subjected to a chromatographic
column on silica gel 60 (70–230 mesh), initially using pure hexane as
eluents, then the mixture of hexane/EtOAc in increasing polarity
gradient and finally pure methanol, obtaining it if 25 fractions. Fractions
Isonemorosonol Ferraz et al. [10].
1
were used as internal references for H and 13C NMR spectra acquisition.
HRESIMS were recorded on a SHIMADZU LCMS-IT-TOF.
3
.2. Methylation reaction
3
and 4 were combined and methylated with diazomethane. The
Nitrosomethylurea was initially prepared by slowly stirring an
methylation was necessary to improve the chromatographic separations
aqueous solution of NaOH (38.5%), Br
2
(22.0 g) and acetamide (14.75
1
◦
of mixture whose H NMR spectrum showed signals between δ 15 and δ
1
g). This mixture was heated to 100 C and then cooled in an ice bath for
6 indicating the probable presence of tautomers in keto-enol equilib-
2
h. Acetyl methyl urea crystals (12.25 g) were obtained after vacuum
rium. The methylated fraction was fractioned with hexane/EtOAc mix-
tures in a silica gel CC to obtain 11 fractions. The Fraction 6 was also
submitted to successive chromatography on silica gel column and pre-
parative TLC (silica gel; hexane/EtOAc 90:10) giving the compounds 1a
filtration. The acetyl methyl urea crystals were dissolved in 12.50 mL of
◦
concentrated HCl and heated to 100 C for about 12 min. The mixture
was then diluted with 12.50 mL of distilled water and cooled in an ice
2
bath. Then, ice cold NaNO solution (43%) was added to the mixture.
(
8.0 mg). Fraction 7 was also submitted to successive chromatography
After vacuum filtration, nine grams of nitrosomethylurea were obtained.
The samples were methylated in an ethereal diazomethane solution
recently prepared by a reaction involving the slow addition of nitro-
somethylurea to an aqueous KOH solution (50%) in sulfur ether in an ice
on silica gel column and one of the fractions was purified with HPLC on
silica gel (hexane/EtOAc 80:20) giving the compounds 2a (7.5 mg).
Fraction 8 was also subjected to successive silica gel column chro-
matography (hexane/EtOAc 75:25) and one of the fractions was dis-
solved in 10.0 mL of hexane/ethyl acetate (98:02) and subjected to
analysis by HPLC, using semi-preparative column X-silica earth (Φ = 4.5
3
bath The sample to be methylated was solubilized in CHCl and the
ethereal ice-cold diazomethane solution 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
cm) and 200
μ
L loop. Thus, the solution injected into the column in 100
μ
L aliquots, being eluted with a flow rate of 4.72 mL/min giving com-
completion of the methylation reaction.
pounds 3a (12.0 mg).
1
Burlemarxione D: [
α
]20
D
= ꢀ 8.0 (c 1.0, CHCl
3
); H and 13C NMR, see
+
Table 1; HRESIMS 501.2751 [M + H] . IR (KBr)
ν
max 3522, 3413, 2926,
6

C.G. Ferraz et al.
Fitoterapia xxx (xxxx) xxx
Scheme 3. Putative biosynthesis pathway of burlemarxione F.
ꢀ
2
857, 1735, 1705, 1675, 1599, 1452 cm 1.
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2
0
); 1H and 13C NMR,
3
benzophenone derivatives from Clusia havetiodes var. stenocarpa, J. Nat. Prod. 64
Burlemarxione E): [
α
]
D
=+11,0 (c 0.5; CHCl
(
2001) 23–25.
+
see Table 2; HRESIMS 517.3012 [M + H] . IR (KBr)
νmax 2888, 1722,
[
[
[
7] F.G. Cruz, J.S.R. Teixeira, Polyprenylated benzophenones with a tricyclo
ꢀ
1
3,8
1
633, 1611, 1489 cm
Burlemarxione F: [
.
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2
0
), 1H and 13C NMR,
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benzophenones from the floral resins of three Clusia species, Phytochemistry 50
Author contributions
(
1999) 1073–1079.
´
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
[10] C.G. Ferraz, P.R. Ribeiro, E.J. Marques, R. Mendonça, M.L.S. Guedes, E.R. Silveira,
R. El-Bach a´ , F.G. Cruz, 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,
Fitoterapia 138 (2019).
Funding sources
[
11] J.G. Gonzalez, E.M. Olivares, F.D. Monache, Citrans and cyclols from Clusia
multiflora, Phytochemistry 38 (1995) 485–489.
Financial support was provided by FINEP, CAPES, CNPq, and
FAPESB.
[
12] K.R. Gustafson, J.W. Blunt, M.H.G. Munro, R.W. Fuller, T.C. McKee, J.H. Cardellina
Ii, J.B. McMahon, G.M. Cragg, M.R. Boyd, The guttiferones, HIV-inhibitory
benzophenones from Symphonia globulifera, Garcinia livingstonei, Garcinia ovalifolia
and Clusia rosea, Tetrahedron 48 (1992) 10093–10102.
Declaration of Competing Interest
We declare no conflict of interest.
A. Supplementary data
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benzophenone derivatives with the novel tetracyclo[7.3.1.13,11.03,7]tetradecane-
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