SAR studies of epoxycurcuphenol derivatives on leukemia CT-CD4 cells

Article abstract of DOI:10.1016/j.bmc.2012.09.042

Bioactive natural products are a potential source of new pharmaceuticals since they offer new modes of action and more specific activities. The use of derivatization also enables the optimal structure for their biological activity to be determined. In thi

Full text of DOI:10.1016/j.bmc.2012.09.042

Bioorganic & Medicinal Chemistry 20 (2012) 6662–6668
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
SAR studies of epoxycurcuphenol derivatives on leukemia CT-CD4 cells
José L. G. Galindo a, Mariola Macías b, José M. G. Molinillo a, Alba Muñoz-Suano b, Ascensión Torres a,
Rosa M. Varela a, Francisco García-Cozar b, Francisco A. Macías a,
⇑
a Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, C/República Saharaui, s/n, 11510-Puerto Real (Cádiz), Spain
b Departamento de Biomedicina, Biotecnología y Salud Pública (Inmunología) y Unidad de Investigación del Hospital Universitario de Puerto Real, Servicio Central
de Investigación en Ciencias de la Salud, Edificio Andrés Segovia, C/Dr. Marañón 3, 11002–Cádiz (Cádiz), Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 June 2012
Revised 5 September 2012
Accepted 11 September 2012
Available online 25 September 2012
Bioactive natural products are a potential source of new pharmaceuticals since they offer new modes of
action and more specific activities. The use of derivatization also enables the optimal structure for their
biological activity to be determined. In this study several epoxycurcuphenol derivatives were synthe-
sized. The substitution pattern on the aromatic and oxirane rings was varied along with that at the ben-
zylic position and the length of the side chain was altered. These changes were made in order to gain a
deeper understanding of the structural requirements for activity. The biological activities of these com-
pounds were evaluated on the human leukemia cell line Jurkat using an antiproliferative assay. The activ-
ity results and structural requirements are discussed.
Keywords:
Antiproliferative bioassay
Leukemia cells
Ó 2012 Elsevier Ltd. All rights reserved.
Sunflower
Helianthus annuus
Epoxycurcuphenol derivatives
SAR studies
1. Introduction
oxirane group (2a, 2b) (Fig. 1). They showed interesting biological
activities when evaluated in animal cells. Compounds 2a and 2b
Bioactive natural products are a potential source of new phar-
maceuticals1,2 since they offer new modes of action and more spe-
cific interactions. Among the plants and microorganisms from
which allelopathic agents have been isolated, Helianthus annuus
L. (sunflower) is very interesting because of its high production
of secondary metabolites3 especially in sesquiterpene lactones,4,5
heliespirones,6,7 annuionones,8 helibisabonols,9 heliannuols10,11
and phenolic compounds.12
were evaluated for their effects on cytokine production by human
primary CD4+ T cells and on cell division on the human tumoral
cell line Jurkat, murine antigen specific CD4+ T cell line D5, murine
Primary CD4 T Cells from TcR (T cell receptor) Transgenic DO11.10
mice, human primary naïve PBMC and human primary CD4+ T
cells. In the case of the lymphoid human cell line Jurkat, addition
of sesquiterpenes at a final concentration of 1
lM impaired cell
division in almost 100% of cells.17
Heliannuols, a new class of compounds isolated from Helianthus
annuus, constitute a family of new compounds with a novel
heterocyclic sesquiterpene structural backbone named heliann-
ane.13 This skeleton is characterized by a benzenoid moiety fused
to a five- to eight-membered heterocyclic ring. Among these com-
pounds, heliannuol D (1) (Fig. 1) has special relevance due to the
high phytotoxic activity it has shown.14 The fact that these allelo-
chemicals may be useful for developing new active compounds
makes their synthesis at large scale an attractive goal.15 Besides
the synthetic analogous and intermediates obtained during their
synthesis are also of interest, since their bioactivity offers hints
about the structural requirements needed.
In order to gain a deeper understanding of the structural
requirements for bioactivity in this family of compounds we
carried out a Structure–Activity Relationship (SAR) study and
HO
O
OH
Heliannuol D (1)
HO
HO
O
O
In the course of our research toward the synthesis of heliannuol
D (1)16 we obtained two aromatic diasteroisomers that present an
OH
OH
7R*,10R*-10,11-epoxsycurcuhydroqunone (2a) 7S*,10R*-10,11-epoxsycurcuhydroqunone (2b)
⇑
Corresponding author.
E-mail address: famacias@uca.es (F.A. Macías).
Figure 1. Structures of heliannuol D and its intermediates with an oxirane ring 2a
and 2b.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.042

J. L. G. Galindo et al. / Bioorg. Med. Chem. 20 (2012) 6662–6668
6663
R1
R2 R3
R4
HO
BnO
2a + 2b
3a + 3b
4
5a + 5b
6a + 6b
7a + 7b
Me OH Me Me
H
H
Me
H
OH Me Me
a
OH
H
Me
Me
Me
Me
C6 Series
C5 Series
R3
14, 14a
R4
13
OBn
70%
H
Me
11
19
R2
OH Et
6
3
7
9
R4
12
5
4
H
H Et
1
8
10
O
2
8a + 8b
9a + 9b
10
11a + 11b Me
12a + 12b
Me OH Me
H
H
H
H
H
OH
R1
15
BnO
BnO
H
H
OH Me
b
OH
H
H
Me
O
84%
OBn
HO
OBn
H
OH Et
20
21
CH3
14a
CH3
14
14
O
HO
11
6
7
10
CH3
11
6
7
9
5
4
1
8
O
5
10
OH
1
8
2
4
2
3
3
OH
H3C
OH
OH
OH
15
2a + 2b
+
c
13a + 13b (C5 Series)
14a + 14b (C4 Series)
HO
48%
O
Figure 2. Compounds synthesized. Numbering of compounds are assigned as
derivatives of curcuphenol or curcuhydroquinone, in agreement with IUPAC
recommendation in section F (see Supplementary data), so for 14a/14b 9,12,13-
trinor-curcuhydroquinone is assigned.
Scheme 2. (a) Et3SiH, BF3ꢀEt2, DCM, –55 °C ? 0 °C, 12 h (b) m-CPBA, EtOAc, DCM,
2 h, rt; (c) H2/Pd/C, DMF, 2 h, rt.
synthesized 24 derivatives with different side chains and substitu-
tion patterns in the aromatic ring and the alkylic side chain (Fig. 2).
We changed the number and position of hydroxyl groups, the pres-
ence of additional methyl or ethyl groups in the aromatic ring and/
or in the side chain and the pattern of substitution in the aromatic
ring. The compounds were classified into three families depending
on the length of the side chain and the substitution pattern at the
oxirane ring: C6 (oxirane ring with two methyl groups in the last
carbon, and a side chain that is six carbons in length; compounds
2–7), C5 (with a side chain five carbons long and the oxirane ring
without any terminal methyl groups; compounds 8–13) and C4
(with a four-carbon side chain and an oxirane ring without any
methyl group; compound 14) (Fig. 2). All compounds were
obtained as a pair of diastereoisomeric products apart from com-
pounds 4 and 10.
and the correct side chain obtained according the procedures re-
ported by our group (Scheme 1).16 The second stage of the synthe-
sis (Scheme 2) hasthree steps and these are slightly different to
those reported previously. The first step was the reduction of the
hydroxyl group at C-7 (20) and this was achieved using triethylsi-
lane in dichloromethane and BF3ꢀOEt2 as catalyst to give the
desired product in 70% yield. Subsequent epoxidation of 20 gave
compound 21 in 84% yield. Finally, palladium-catalyzed (Pd/C)
hydrogenation of 21 led to selective cleavage of the benzylic ethers
to yield the pair of diastereoisomers 2a and 2b in 45% yield each.
These compounds could be separated by HPLC.
Depending on the nature of the starting material, this synthetic
methodology led to the corresponding pairs of diastereoisomers
shown in Figure 2. The intermediates are shown in the Supplemen-
tary data, although some reactions needed to be slightly modified
depending on the specific reactivity requirements of the interme-
diates. For example, the dehydroxylation reaction to give the pre-
cursors of 4 and 10 required the use a large excess (10 equiv) of
triethylsilane and the reaction was carried out at 0 °C. The epoxida-
tion in the C5 series was performed with an increased amount of
m-CPBA and a longer reaction time. All pairs of diastereoisomers
were separated and purified by HPLC techniques, with the excep-
tion of 14a and 14b, which could not be isolated. In the hydrogena-
tion reactionto obtain 13a and 13b it was impossible to remove the
aromatic hydroxyl group without affecting the oxirane ring. In this
case, the final compounds were those resulting from reductive
cleavage of the oxirane ring. The absence of terminal methyl
groups in the oxirane ring of compounds in C5 markedly changed
the reactivity of the system and this is the probable cause of this
behaviour.
2. Chemistry
A total of 24 compounds were synthesized (Fig. 2); 18 of them
were tested for antiproliferative potential (Jurkart cell line from
leukemia CT-CD4) to evaluate different modifications on the basic
skeleton of 2a and 2b. The synthesis is based on that of heliannuol
D and resembles the biogenetic route proposed for heliannuols and
helibisabonols, where compounds 2a and 2b are intermediates
proposed in this biogenetic route.10,11,18 Thus, the synthetic proce-
dure used herein has two main stages. The first stage involves the
preparation of the appropriate aromatic bisabolene skeleton 19
from a benzenoid precursor (2-methylhydroquinone 15 as starting
material) with the desired substitution pattern in the aromatic ring
O
HO
AcO
HO
a
b
c
3. Results and discussion
3.1. Synthesis
100%
OH
100%
OAc
79%
OH
15
16
17
O
HO
The synthetic procedure used for the pair of diastereoisomers
2a and 2b has been used and optimized to obtain the complete
family of compounds presented. Only slight modifications were
necessary in the synthesis of some compounds, with changes to
the amount of reagent or the reaction temperature, as described
in the Experimental section, to improve yields. All reactions pro-
ceeded with good yields (between 40 and 95%) (Supplementary
data page S6).
BnO
BnO
d
89%
OBn
OBn
18
19
Scheme 1. (a) (CH3CO)2O, Py, 24 h, rt; (b) BF3ꢀEt2O, 3 h, 120 °C; (c) Benzyl Bromide,
K2CO3, Dimethoxyethane, 12 h, 80 °C; (d) 5-Br-2-methyl-pentene, Mg, I2, THF, 1 h,
65 °C.

6664
J. L. G. Galindo et al. / Bioorg. Med. Chem. 20 (2012) 6662–6668
3.2. Relative stereochemistry
obtained, allowing us to propose the relative stereochemistries
shown in Table 2.
The final compounds 2a/2b to 14a/14b have two chiral centres
at C-7 and C-10 (in the C6 and C5 series) or C-7 and C-9 (in the C4
series) that provide two pairs of diastereoisomers each (except for
4 and 10, which have one asymmetric centre). The relative stereo-
chemistry of each diastereoisomer was elucidated by NMR and
theoretical conformational studies that will be discussed below.
The structures of all compounds were determined by spectro-
scopic methods (1H and 13C NMR, MS and IR). There are some
trends that are maintained for all diastereoisomers in each series.
The less polar compound 2a shows collapsed signals for protons
H-8 (d 1.67, td, J7–8 = 7.1, J8–9 = 7.9 Hz, 2H) and H-9 (d 1.41, q, J8–
9 = 7.9, J9–10 = 7.6 Hz, 2H). On the other hand, the more polar com-
pound 2b presents separate signals for each proton at C-8; H-8 (d
3.3. Antiproliferative assay on human cells
In this bioassay, we analyse cell division as the end point of cell
proliferation. Cells were exposed to test compounds at a concen-
tration of 10 lM for 24 h, stained with CFSE (Carboxyfluorescein
succinimidyl ester)21–23 and analysed by flow cytometry 4 days la-
ter to determine size distribution and the level of cell division. The
effect of the active compound can be detected from a shift in the
population curve to the left when compared to the control curve.
This change is due to dilution of the CFSE dye between dividing
cells. The effect was quantified using a flow cytometer (Cyan ADP
MLE ™, Bechman ™) to count dividing and non-dividing cells. Sev-
eral parameters have been compared to show the activity of the
compound in comparison with the control value, with the results
expressed as the Proliferation Index (PI, a measure of the increase
in cell numbers in the culture) and Nonproliferative Fraction (NpF,
which represents the fraction in the original culture cells that have
not proliferated during the course of the experiment). The results
are shown in Table 3 and Figure 5.
1.75, m) and H-80 (d 1.59, ddt, J8–8 = 12.8, J7–8 = 9.7, J8 –9 = 6.3 Hz)
(Fig. 3). These facts could suggest an intramolecular bonding in
one of the isomer, which influence its polarity.
0
0
0
Such differences suggest the presence of a rotational barrier
through the C-8/C-9 bond where the two protons are differenti-
ated. A careful study of the structure of compounds 2a/2b shows
that a hydrogen bond can be established between the aromatic hy-
droxyl group and the oxirane ring. Such a bond provides enough
rigidity to differentiate the two H-8 signals.
Of the compounds tested, those with two aromatic hydroxyl
groups are the most active. This effect is clear on comparing com-
pounds that differ only in the number of aromatic hydroxyl
groups; for example, in the C6 series, 2a (NpF: 0.87) and 2b
(NpF: 0.92) are more active than 5a (NpF: 0.41) and 5b (NpF:
0.42), and the same effect can be observed with 8a (NpF: 0.86)
and 8b (NpF: 098) when compared to 11a (NpF: 0.37) and 11b
(NpF: 0.30) in the C5 series (Fig. 5).
The substituent at the benzylic position (C-7) also modifies the
activity. In this case, the optimal group in this position is hydrogen,
as shown by comparison of the bioactivities of compounds 3b, 4
and 6b (Table 3). Thus, 4 does not have a substituent at C-7 and
it is the most active compound (NpF: 0.94), followed by 6b (NpF:
0.62) and 3b (NpF: 0.39), which have an ethyl and a methyl substi-
tuent, respectively. Other structural factors analysed were the sub-
stitution pattern of the oxirane ring, polarity of compounds and the
relation with the stereochemistry and the presence or absence of
an aromatic methyl group at C-4 position.
A theoretical model was developed for each diastereoisomer
using PCMODEL with GMMX (Global-MMX) calculations and
MOPAC (Molecular Orbital PACkage)19,20 to obtain the minimum
energy conformers that contain a hydrogen bond. In the case of
the 7R*10R* diastereoisomer two minimum energy conformers
bearing the hydrogen bridge were found (Table 1), but none of
these had dihedral angles with coupling constants similar to the
experimental values. Two conformers were also found for the
7S*10R* isomer. In this case the theoretical coupling constants
were similar to the experimental values. Consequently, a relative
stereochemistry 7S*10R* is proposed for compound 2b and a rela-
tive stereochemistry 7R*10R* for 2a. The 3D representations of the
selected conformers for each compound are shown in Figure 4,
where the hydrogen bond formed between the aromatic hydroxyl
group and the oxirane ring on 2b can be observed. The same
theoretical study was carried out for the rest of the compounds
The substitution pattern on the oxirane ring affects the activity
by enhancing the effect produced by the factors discussed above. In
most cases, compounds of the C5 series without methyl groups on
the oxirane ring show lower activities than compounds of the C6
series that contain two methyl groups at the end of the side chain
on the oxirane ring as the only difference. Comparison of the activ-
ities of 5a/5b (NpF: 0.42/0.41) with 11a/11b (NpF: 0.37/0.30) or 2a
(NpF: 0.87, PI: 1.62) versus 8a (NpF: 0.86, PI: 1.85)) illustrates this
behaviour (Table 3). This trend is particularly pronounced in the
comparison of the active compounds 6a/6b (NpF: 0.59/0.62) with
the inactive compounds 12a/12b (NpF: 0.40/0.42). This behaviour
can be due to the +I inductive effect of methyls over oxirane ring
increasing the reactivity of this moiety and then their toxicity.24
For active compounds, the most polar one in every pair of diaste-
reoisomers, corresponding to the relative stereochemistry 7S*,10R*
(Table 2), is also the most active. Thus, 8b (PI: 1.09; NpF: 0.98) is
more active than 8a (PI: 1.85; NpF: 0.86) and 2b (PI: 1.36; NpF:
0.92) is more active than 2a (PI: 1.62; NpF: 0.87) (Fig. 5).
The aromatic methyl group is another requirement for activity
in this kind of compound. All compounds that do not bear an aro-
matic methyl at the C-4 position (3b, 4, 6a, 6b, 7a, 7b, 12a, 12b,
13a, 13b) are inactive with the exceptions of 4 (PI: 1.11; NpF:
0.94) and 6a/6b (PI: 2.73/3.04; NpF: 0.59/0.62). These values,
which are very similar to those of the control and have a high pop-
ulation percentage in the 4th and 5th generation, are indicative of
low activity. The activity of 4 is due to the fact that other structural
Figure 3. Detail of the 1H NMR region for the protons at C-9 for 2a (bottom) and 2b
(top).

J. L. G. Galindo et al. / Bioorg. Med. Chem. 20 (2012) 6662–6668
6665
Table 1
Conformers with hydrogen bridge obtained for 7R*10R* and 7S*10R* relative stereochemistry of diastereoisomers 2a and 2b
Conformer
D
Hf
Dihedral angle
Coupling constants
J7,8
dOH–O
ldip
U7,8
U9,10
J9,10
0
0
0
0
U7,8
U9 ,10
J7,8
J9 ,10
7R*10R*#1
7R*10R*#2
7S*10R*#1
7S*10R*#2
–117.85
–117.48
–113.00
–113.25
–66.91°
177.11°
–66.95°
177.06°
161.02°
–83.14°
–51.14°
62.98°
59.68°
175.33°
59.75°
175.42°
154.20°
40.49°
–179.48°
65.80°
2.29
12.32
2.29
12.32
11.12
1.05
2.20
11.66
2.20
11.65
10.63
4.86
11.49
11.6
1.63
7.63
1.63
8.02
1.99
7.93
1.63
7.86
3.648
2.435
4.357
3.687
4.32
2.57
O
H
O
OH
2a
O
H
O
OH
2b
Figure 4. Relative stereochemistry and conformer proposed for the diastereoisomers 2a and 2b.
Table 2
Relative stereochemistry propose for each couple of diastereoisomers
Table 3
Activities in antiproliferative assay with reference to control values
Diastereoisomers Stereochemistry
Diastereoisomers Stereochemistry
2a/2b
3a/3b
5a/5b
6a/6b
7a/7b
7R*10R*/7S*10R* 8a/8b
7R*10R*/7S*10R*
7R*10R*/7S*10R*
7R*10R*/7S*10R*
7S*10R*/7R*10R*
7S*10R*/7R*10R*
PI
NpF
0.87
0.92
0.94
0.59
0.62
0.86
0.98
0.59
0.39
0.41
0.42
0.48
0.46
0.37
0.30
0.40
0.42
0.34
0.32
Parent (%)
53.81
67.44
84.84
21.49
20.30
46.39
89.59
23.70
6.02
8.71
8.09
11.75
10.46
6.23
Gen/%
Active compounds
2a
1.62
1.36
1.11
2.73
3.04
1.85
1.09
2.48
6.47
4.77
5.20
4.10
4.38
5.86
5.71
5.06
5.01
5.52
6.11
4/13.90
3/17.04
2/7.31
3/35.78a
3/21.30b
5/18.91
3/6.73
4/25.99
5/64.37
5/38.84
5/49.30
5/29.37
5/33.67
5/47.87
5/40.06
5/48.54
5/47.67
5/40.57
5/46.79
7R*10R*/7S*10R* 9a/9b
7S*10R*/7R*10R* 11a/11b
7R*10R*/7S*10R* 12a/12b
7S*10R*/7R*10R* 13a/13b
2b
4
6a
6b
8a
8b
requirements outlined below, for example two aromatic hydroxyl
groups and/or the absence of a substituent at the benzylic position
should have more relevance. Thus, the most active diastereoiso-
meric pairs are 2a/2b and 8a/8b, all of which have an aromatic
methyl at C-4. Evermore, if we compare the activity of 2b (NpF:
0.92) and 3b (NpF: 0.399) with a methyl group on C-4 as only dif-
ference in their structure, we can determine that this substitution
has an important influence on the activity of the compounds.
The most effective compound in this bioassay was 7S*,10R*-
12,13-dinor-10,11-epoxy-10,11-dihydrocurcuhydro-quinone (8b),
which fits the requirements described. Its effect can be shown with
the shift in the population curve to the left when compared to the
control curve (Fig. 6).
Control
3b
Non active compounds
5a
5b
7a
7b
11a
11b
12a
12b
13a
13b
5.27
7.95
8.46
6.10
5.19
PI: Proliferation Index.
NpF: Nonproliferative Fraction.
Gen:Most populated generation.
a
The values for the cell population for the 4th and 5th generations are not
insignificant, 13.90 and 11.80, respectively.
Although the 3rd generation is the most popular, the fraction in the 4th has
similar values (20.47) and slightly lower values (18.48) for the 5th generation,
which are very close to the control values (25.99 and 18.43, respectively).
4. Conclusions
b
We have developed a facile synthetic route to epoxycurcuphe-
nol derivatives that contain two chiral centres. These compounds

6666
J. L. G. Galindo et al. / Bioorg. Med. Chem. 20 (2012) 6662–6668
are obtained as pairs of diastereoisomers and their relative stereo-
chemistries were determined by NMR spectroscopy and theoretical
molecular studies.
We have studied the structure–activity relationship (SAR) in
epoxycurcuphenols using the results from the antiproliferative po-
tential in animal cells; it was possible to determine the structural
requirements for optimal activity.
The main requirements for activity are: The compounds should
have two aromatic hydroxyl groups without any substitution at the
C-7 position. Moreover, the activity is improved when the relative
stereochemistry is 7S*,10R*, a methyl group is present in the aro-
matic C-4 position and an monosubstituted oxirane ring is present.
The most active compound synthesized was 8b (7S*, 10R*-12,13-
dinor-10,11-epoxy-10,11-dihydro-curcuhydroquinone) and this
has excellent Proliferation Index and Nonproliferative Fraction val-
ues (1.09 and 0.98, respectively).
In relation to the compounds that showed significant activity
against animal cells, it would be of great interest to carry out
further studies into phenomenon by making slight modifications
to the structure and testing the resulting compounds in other bio-
assays in an effort to identify their mode of action.
THF. 1H and 13C NMR spectra (400 and 100 MHz, respectively)
were recorded on a Varian Unity 400 NMR spectrometer with a
sample temperature of 25 °C using CDCl3 and (CD3)CO. Mass spec-
troscopy was carried out using a GC-MS VG1250 apparatus (ion
trap detector) in EI mode. FTIR (Fourier transform infrared), spectra
were recorded on a Mattson 5020 spectrophotometer. Elemental
analysis was carried out on a LECO CHNS analyser. Purities of syn-
thesized compounds were determined by NMR and HPLC methods,
and corroborated by HRMS with a degree of purity between
95–99%. For flow cytometry analysis, 10,000 live events were col-
lected on a Cyan-ADP-MLE II flow cytometer (DakoCytomation™).
Acquisition and analysis was performed using summit software
(DakoCytomation™).
5.1.2. Synthesis of compounds 2a and 2b
5.1.2.1. 2,5-Diacetoxytoluene (16).
tained in quantitative yield without the need for chromatographic
purification; IR (film, cm–1); 1H NMR (CDCl3, 400 MHz, d ppm); 13
This compound was ob-
C
NMR (CDCl3, 100 MHz, d ppm) and HRMS data are consistent with
that those reported previously.16
5.1.2.2. 2,5-Dihydroxy-4-methylacetophenone (17).
This
compound was obtained in quantitative yield, without the need
for further purification, by the methodology reported previously;16
the IR, 1H NMR,13C NMR and HRMS data are identical to those pub-
lished previously.
5. Experimental
5.1. Chemistry
5.1.1. General
5.1.2.3. 2,5-Dibenzyloxy-4-methylacetophenone (18).
The
Commercially available chemicals were used as received. Dry
THF was obtained by distillation of sodium-treated commercial
crude product was purified by column chromatography (hexane/
EtOAc 20%) to yield 2,5-dibenzyloxy-4-methylacetophenone (18)
(95%). The product has identical spectroscopic data to those
reported previously (IR, 1H and 13C NMR, HRMS).16
5.1.2.4.
(19).
2-O,5-O-Dibenzyl-7-hydroxy-curcuhydroquinone
A catalytic amount of I2 dissolved in dry THF (1 mL)
was added under a N2 atmosphere to a dry flask with calcined
magnesium (2 equiv). A solution of the aromatic compound 18
and 5-bromo-2-methyl-2-pentene (3 equiv) in dry THF (0.075 M
in compound 18) was added slowly with continuous stirring. The
colour of the reaction change from muddy red to clear yellow
and after 10 min the reaction was quenched with NH4Cl (sat)
and extracted with AcOEt (ꢁ5). The combined organic layers were
dried over anhydrous Na2SO4 and the solvent was evaporated un-
der vacuum. The reaction mixture was separated by column chro-
matography (hexane/ethyl acetate 95:5). Compound 19 was
obtained in 89% yield and the spectroscopic data are identical to
those published by Macias et al.16
Compounds arranged according their activity
C: CONTROL
5.1.2.5. 2-O,5-O-Dibenzyl-curcuhydroquinone (20).
Et3Si
(1 equiv) and BF3ꢀEt2 (0.5 equiv, dropwise) were added to
Figure 5. Antiproliferative bioassay results showing NonProliferative Fraction
(NpF) and Proliferative Index (PI), the activity increases from left to right.
500 mg of 19 in DCM (0.02 M) under a N2 atmosphere and the
Figure 6. Population curves for the most active compound (8b) vs control, the cell division with 8b is virtually nonexistent.

J. L. G. Galindo et al. / Bioorg. Med. Chem. 20 (2012) 6662–6668
6667
mixture was stirred at –78 °C. After 15 min the temperature was
raised to –55 °C and the reaction mixture was stirred for 12 h un-
der these conditions. The reaction was quenched with a saturated
solution of NaHCO3 and extracted with DCM (ꢁ3). The organic
layer was dried over anhydrous Na2SO4, filtered and the solvent
was evaporated. The mixture was purified by column chromatog-
raphy (hexane/ethyl ether 95:5). Compound 20 was obtained in
(hexane/EtOAc 40%). Compounds 2a and 2b were obtained in
51% and 45% yield, respectively. The spectroscopic data have been
reported previously for these compounds, with the same values for
IR and HRMS, although the 1H NMR and 13C NMR spectra were ob-
tained with deuterated acetone as solvent.
5.1.2.8.
none (2a).
7R*,10R*-10,11-Epoxy-10,11-dihydrocurcuhydro-qui-
1H NMR (C3D6O, 400 MHz): d 1.13 (s, 3H, H-13),
70% yield. IR (film, cm–1
)
m
max: 1671, 1193, 859; 1H NMR (CDCl3,
0
400 MHz):
d
1.20 (d, 3H, H-14), 1.51 (dq, 1H, J7,8/8 = 7.0,
1.16 (d, 3H, J7,14 = 7 Hz, H-14), 1.19 (s, 3H, H-12) 1.41 (q, 2H, J8,9
= 7.9, J9,10 = 7.6 Hz, H-9), 1.67 (td, 2H, J7,8 = 7.1, J8,9 = 7.9 Hz, H-8),
2.08 (s, 3H, H-15), 2.63 (t, 1H, J9,10 = 7.6 Hz, H-10), 3.09 (tq, 1H,
J7,8 = 7.1, J7,14 = 7.0 Hz, H-7), 6.56 (s, 1H, H-3), 6.60 (s, 1H, H-6);
13C NMR (C3D6O, 100 MHz): d 15.8 (C-15), 18.8 (C-13), 21.7
(C-14), 25.0 (C-12), 27.9 (C-9), 32.6 (C-7), 34.9 (C-8), 58.1 (C-11),
64.7 (C-10), 113.9 (C-3), 118.5 (C-6), 122.6 (C-4), 131.7 (C-1),
148.0 (C-5), 149.3 (C-2).
J8,8 = 6.7, J9,8/8 = 7 Hz, H-80), 1.54 (s, 3H, H-13), 1.62 (m, 1H, H-8),
0
0
0
1.67 (s, 3H, H-12), 1.89 (dt, 1H, J9,10 = 7.0, J9,8/8 = 7.0 Hz, H-9),
0
2.28 (s, 3H, H-15), 3.25 (tq, 1H, J7,8/8 = 7.0, J7,14 = 7.0 Hz, H-7),
4.99 (s, 2H, H-700), 5.02 (s, 2H, H-70), 5.08 (t, 1H, J9,10 = 7.0 Hz, H-
10), 6.75 (s, 1H, H-6), 6.77 (s, 1H, H-3), 7.38 (d, 2H,
J3 ,4 ;3 ,4 = 7.8 Hz, H-40, H-400), 7.40 (ddd, 4H, J3 ,4 ;3 ,4 = 7.8,
0
0
00 00
0
0
00 00
J2 ,3 ;2 ,3 = 7.5, J3 ,5 ;3 ,5 = 1.8 Hz, H-30, H-50, H-300, H-500), 7.45 (d,
0
0
00 00
0
0
00 00
4H, J2 ,3 ;2 ,3 = 7.5 Hz, H-20, H-60, H-200, H-600); 13C NMR (CDCl3,
100 MHz): d 16.2 (C-15), 21.2 (C-14), 25.7 (C-13), 26.2 (C-9),
31.6 (C-7), 37.4 (C-8), 71.0 (C-700), 71.1 (C-70), 111.7 (C-6), 115.6
(C-3), 124.8 (C-10), 124.9 (C-11), 127.1 (C-200, C-600), 127.3 (C-20,
C-60), 127.5 (C-100), 127.6 (C-10), 128.4 (C-30, C-50, C-300, C-500),
131.1 (C-4), 134.4 (C-4), 137.8 (C-40, C-400), 150.2 (C-5), 151.2
(C-2); HRMS calcd for C29H34O2 414.5791, found 414.2578. Ele-
mental analysis calcd for C29H34O3: C, 84.02; H, 8.27; O, 7.71,
found: C, 84.79; H, 7.92; O,7.29.
0
0
00 00
5.1.2.9.
none (2b).
7S*,10R*-10,11-Epoxy-10,11-dihydrocurcuhydro-qui-
1H NMR (C3D6O, 400 MHz): d 1.12 (s, 3H, H-13),
1.15 (d, 3H, J7,14 = 6.8 Hz, H-14), 1.19 (s, 3H, H-12), 1.42 (ddd, 2H,
0
0
J8 ,9 = 6.3, J9,10 = 6.3, J8,9 = 13.4 Hz, H-9), 1.59 (ddt, 1H, J7,8 = 9.7,
J8,8 = 12.8, J8 ,9 = 6.3 Hz, H-80), 1.75 (m, 1H, H-8), 2.07 (s, 3H, H-
15), 2.61 (t,1H, J9,10 = 6.3 Hz, H-10), 3.14 (dqd,1H, J7,14 = 6.8,
0
0
0
J7,8 = 6.5, J7,8 = 9.7 Hz, H-7), 6.56 (s, 1H, H-3), 6.59 (s, 1H, H-6);
13C NMR (C3D6O, 100 MHz): d 15.8 (C-15), 18.8 (C-13), 21.5
(C-14), 25.0 (C-12), 27.7 (C-9), 32.3 (C-7), 34.4 (C-8), 57.9 (C-11),
64.2 (C-10), 113.9 (C-3), 118.2 (C-6), 122.5 (C-4), 131.7 (C-1),
148.0 (C-5), 149.2 (C-2).
5.1.2.6.
2-O,5-O-Dibenzyl-10,11-epoxy-10,11-dihydrocurcu-
A slight excess of m-CPBA (1.12 equiv)
hydroquinone (21).
and sodium acetate (1.2 equiv) were added to a solution of 20
(500 mg) in CH2Cl2 (25 mL). The mixture was stirred for 2 h at
room temperature, washed with NH4Cl aq. (sat) and extracted with
CH2Cl2. The organic layer was washed with NaOH aq (0.5 M) and
dried over anhydrous Na2SO4. The solvent was evaporated under
vacuum and the crude product was purified by column chromatog-
raphy (hexane/ethyl acetate 95:5) to give 21 in 84% yield. IR (film,
5.1.3. Synthesis of compounds 3a and 3b to 14a and 14b
Each pair of diastereoisomers of series’ C6, C5 and C4 (Fig. 2)
was synthesized by the same methodology used to obtain com-
pounds 2a and 2b described in the last section, although several
changes were made to the conditions in certain stages. Reductions
of the hydroxyl group in C-7 to obtain 4 and 10 and epoxidation of
compounds in the C5 and C4 series’ (48, 51, 54, 57, 60, 63 and 66)
were modified slightly. The selective cleavage of the benzyl groups
was carried out under the same conditions for all compounds, but
hydrogenation of 64 led to the reduced product 65 with the loss of
the oxirane ring and only one hydroxyl group in C-10.
cm–1 max 1193, 862; 1H NMR (CDCl3, 400 MHz): d 1.33 (s, 3H, H-
) m
13), 1.26 (s, 3H, H-12), 1.58 (m, 2H, H-9), 2.4 (s, 3H, H-15), 1.81 (m,
1H, H-8), 2.81 (t, 1H, J9,10a/b = 6 Hz, H-10a), 2.76 (t, 1H, J9,10a/
b = 6 Hz, H-10b), 3.46 (dq, 1H, J7a/b,14 = 6.9, J7a/b,8 = 7 Hz, H-7a),
3.40 (dq, 1H, J7a/b,14 = 6.9, J7a/b,8 = 7 Hz, H-7b), 5.15 (s, 3H, H-70),
5.11 (s, 3H, H-700a), 5.10 (s, 3H, H-700b), 1.33 (d, 3H, J7a/
b,14 = 6.9 Hz, H-14), 6.89 (s, 1H, H-6), 6.91 (s, 1H, H-3), 7.41 (d,
All synthetic details, including 1H NMR (CDCl3 or C2D6O,
400 MHz) and 13C NMR (CDCl3 or C2D6O, 100 MHz) data for these
compounds are given in the Supplementary data.
2H, J3 ,4 ;3 ,4 = 7.2 Hz, H40, H400), 7.48 (d, 4H, J2 ,3 ;2 ,3 = 7.4 Hz, H20,
0
0
00 00
0
0
00 00
H-60, H-200, H-600), 7.56 (dd, 4H, J2 ,3 ;2 ,3 = 7.4, J3 ,4 ;3 ,4 = 7.2 Hz, H-
30, H-50, H-300, H-500); 13C NMR (CDCl3, 100 MHz): d 16.1 (C-15),
18.4 (C-13), 21.06 (C-14a), 21.09 (C-14b), 24.7 (C-12), 26.7 (C-
9b), 27.1 (C-9a), 31.5 (C-7b), 31.9 (C-7a), 33.6 (C-8b), 33.9 (C-8a),
57.91 (C-11b), 57.92 (C-11a), 63.9 (C-10b), 64.3 (C-10a), 70.7
(C-700b), 70.7 (C-700a, C-70), 111.4 (C-6), 115.3 (C-3), 127.60 (C-300,
C-500), 127.65 (C-30, C-50), 128.0 (C-40, C-400), 128.78 (C-200, C-600),
128.81 (C-20, C-60), 133.3 (C-4b), 133.4 (C-4a), 137.5 (C-1a, C-1b),
138.15 (C-10, C-100), 150.01 (C-5b), 150.03 (C-5a), 151.0 (C-2b),
0
0
00 00
0
0
00 00
5.2. Bioassay
5.2.1. General
For flow cytometry analysis, 10,000 live events were collected
on
a Cyan-ADP-MLE II flow cytometer (DakoCytomation™).
Acquisition and analysis were performed using summit software
(DakoCytomation™).
151.1 (C-2a); HRMS calcd for
C
29H34O3 430.5785, found
5.2.2. Cell culture and stimulation
430.2516. Elemental analysis calcd for C29H34O3: C, 80.89; H,
7.96; O, 11.15, found: C, 80.90; H, 8.82; O, 10.28.
The cell line Jurkat (American Type Culture Collection,
Manassas, VA, USA) was maintained routinely in RPMI-1640 med-
ium ((Roswell Park Memorial Institute medium) supplemented
5.1.2.7.
none (2a) and 7S*,10R*-10,11-epoxy-10,11-dihydro-curcuhy-
droquinone (2b). Pd/C (50%w) was added to a solution of
21 (50 mg) in dry N,N-dimethylformamide (DMF) and the mixture
was placed under a H2 atmosphere. The reaction mixture was stir-
red for 2 h and was then filtered through a silica column with
EtOAc (25 mL) as the mobile phase. The crude product was washed
with water (ꢁ5) to remove DMF. The organic layer was dried over
anhydrous Na2SO4, filtered and the solvent was evaporated under
vacuum. The mixture was purified by column chromatography
7R*,10R*-10,11-Epoxy-10,11-dihydrocurcuhydro-qui-
with 2 mM L-glutamine, 10 mM HEPES (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid), 10% FBS (Fetal bovine serum), 1%
nonessential amino acids (NEAA), 1% sodium pyruvate, and 1%
penicillin/streptomycin at 37 °C).
5.2.3. CFSE cell division assay
For the CFSE cell division assay, cells were washed and
resuspended in 0.5 mL PBS containing
5
lM
CFDA-SE
[5(and-6)-carboxyfluorescein diacetate succinimidyl ester]
(Molecular Probes, Eugene, OR) and incubated at room temper-

6668
J. L. G. Galindo et al. / Bioorg. Med. Chem. 20 (2012) 6662–6668
ature for 5 min. Labelling was quenched by addition of an equal
volume of FBS, cells were washed twice in PBS, resuspended in
complete medium and allowed to proliferate for four days (cell
number was determined by trypan blue exclusion). Data were
collected on a flow cytometer and analyzed with appropriate
software (DakoCytomation™ and ModFit LT™). Each compound
was assayed in experiments coming at least from four different
labeling. Values are expressed as Proliferation Index (PI) (represents
the sum of cells in all generations divided by the calculated number
of original parent cells present at the start of the experiment). PI is a
measure of the increase in cell number in the culture, values lower
than in control cultures represent inhibition of cellular division
while values higher than in controls represent stimulation; Non-
proliferative Fraction (NpF) (the number of cells in the parent gen-
eration at the time of data collection divided by the calculated
number of cells present in the original culture). NpF represents
the fraction in the original culture that has not proliferated during
the course of the experiment. Values higher than the control indi-
cate an increase in the non-proliferative effect, with the best results
being NpF values close to 1. Parent (percentage of parent cells in the
culture at the time of data collection) values higher than controls
are indicative of an absence of cell division. Most populated gener-
ation (number of the most popular generation and the percentage
of its cells at the moment of data collection), generation values
lower than controls indicate inhibition of the cellular division.
References and notes
1. Ukiya, M.; Akihisa, T.; Yasukawa, K.; Koike, K.; Takahashi, A.; Kimura, Y. J. Nat.
Prod. 2007, 70, 813.
2. Wedge, D. E.; Camper, D. In Connections between Agrochemicals and
Pharmaceuticals in Biologically Active Natural Products: Pharmaceuticals; Cutler,
S. J., Cutler, H. G., Eds.; CRC Press: USA, 2000.
3. Macías, F. A.; Varela, R. M.; Torres, A.; Galindo, J. L. G.; Molinillo, J. M. G. In
Chemical Ecology of Plants: Alleloplathy in Aquatic and Terrestrial Ecosystems;
Inderjit, Mallik. U., Ed.; Birkhäuser Verlag: Suiza, 2002; p 73.
4. Macías, F. A.; Torres, A.; Molinillo, J. M. G.; Varela, R. M.; Castellano, D.
Phytochemistry 1996, 43, 1205.
5. de Luque, A. P.; Galindo, J. C. G.; Macías, F. A.; Jorrín, J. Phytochemistry 2000, 53,
45.
6. Macias, F. A.; Galindo, J. L. G.; Varela, R. M.; Torres, A.; Molinillo, J. M. G.;
Fronczek, F. R. Org. Lett. 2006, 8, 4513.
7. Macías, F. A.; Varela, R. M.; Torres, A.; Molinillo, J. M. G. Tetrahedron Lett. 1998,
39, 427.
8. Macías, F. A.; López, A.; Varela, R. M.; Torres, A.; Molinillo, J. M. G.
Phytochemistry 2004, 65, 3057.
9. Macías, F. A.; Torres, A.; Galindo, J. L. G.; Varela, R. M.; Álvarez, J. A.; Molinillo, J.
M. G. Phytochemistry 2002, 61, 687.
10. Macías, F. A.; Molinillo, J. M. G.; Varela, R. M.; Torres, A. J. Org. Chem. 1994, 59,
8266.
11. Macías, F. A.; Varela, R. M.; Torres, A.; Molinillo, J. M. G. J. Chem. Ecol. 2000, 26,
2173.
12. Kähkönen, M. P.; Hopia, A. I.; Vuorela, H. J.; Rauha, J.-P.; Pihlaja, K.; Kujala, T. S.;
Heinonen, M. J. Agric. Food Chem. 1999, 47, 3954.
13. Macías, F. A.; Varela, R. M.; Torres, A.; Molinillo, J. M. G.; Fronczek, F. R. J. Org.
Chem. 1994, 59, 8261.
14. Macías, F. A.; Varela, R. M.; Torres, A.; Molinillo, J. M. G. J. Nat. Prod. 1999, 62,
1636.
15. Takabatake, K.; Nishi, I.; Shindo, M.; Shishido, K. Enantioselective total
synthesis of Heliannuols D and A J. Chem. Soc. Perkin Trans. 1 2000, 12, 1807.
16. Macías, F. A.; Chinchilla, D.; Molinillo, J. M. G.; Marín, D.; Varela, R. M.; Torres,
A. Tetrahedron 2003, 59, 1679.
Acknowledgments
17. Muñoz-Suano, A.; Chinchilla, A.; Aguilar, S.; Molinillo, J. M. G.; Macías, F. A.;
Rodríguez-Iglesias, M. A.; García-Cozar, F. Transplantation 2009, 88, S24.
18. Harrison, B.; Crews, P. J. Org. Chem. 1996, 62, 2646.
19. PcMODEL 7.50 Serena Software.
20. MOPAC2007, James J. P. Stewart, Stewart Computational Chemistry, Version
7.276W web: http://OpenMOPAC.net.
The authors acknowledge financial support from the Ministerio de
Ciencia e Innovación (MICINN) (Project AGL2009-08864/AGR) and
Consejería de Economía Innovación y Ciencia, Junta de Andalucía
(Project P07-FQM-03031).
21. Hodgkin, P. D.; Lee, J. H.; Lyons, A. B. J. Exp. Med. 1996, 184, 277.
22. Lyons, A. B.; Parish, C. R. J. Immunol. Methods 1994, 171, 131.
23. Lyons, A. B. J. Immunol. Methods 2000, 243, 147.
Supplementary data
24. Ehrenberg, L.; Hussain, S. Mutat. Res. -Rev. Genet. 1981, 1, 1.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.09.042.