Enhancing macrocyclic diterpenes as multidrug-resistance reversers: Structure-activity studies on jolkinol D derivatives

Article abstract of DOI:10.1021/jm301441w

The phytochemical study of Euphorbia piscatoria yielded jolkinol D (1) in a large amount, whose derivatization gave rise to 12 ester derivatives (2-13) and hydrolysis to compound 14. The in vitro modulation of P-gp of compounds 1-14 was evaluated through a combination of transport and chemosensitivity assays, using the L5178 mouse T lymphoma cell line transfected with the human MDR1 gene. Apart from jolkinol D, all derivatives (2-14) showed potential as MDR reversal agents. In this small library of novel bioactive macrocyclic lathyrane diterpene derivatives, designed to evaluate structure-activity relationships essential in overcoming multidrug resistance (MDR), some correlations between MDR reversal and molecular weight, accessible solvent areas, and octanol/water partition coefficient were identified that can contribute to the development of new selective P-gp reversal agents.

Full text of DOI:10.1021/jm301441w

Article
pubs.acs.org/jmc
Enhancing Macrocyclic Diterpenes as Multidrug-Resistance
Reversers: Structure−Activity Studies on Jolkinol D Derivatives
Mariana Reis,† Ricardo J. Ferreira,† Maria M. M. Santos,† Daniel J. V. A. dos Santos,† Joseph Molnar,‡
́
and Maria-Jose U. Ferreira*,†
́
†Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade de Farmac
Gama Pinto, 1649-019, Lisboa, Portugal
‡Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Dom
́
ia, Universidade de Lisboa, Av. Prof.
́
ter 10, H-6720 Szeged,
́
Hungary
S
* Supporting Information
ABSTRACT: The phytochemical study of Euphorbia pisca-
toria yielded jolkinol D (1) in a large amount, whose
derivatization gave rise to 12 ester derivatives (2−13) and
hydrolysis to compound 14. The in vitro modulation of P-gp
of compounds 1−14 was evaluated through a combination of
transport and chemosensitivity assays, using the L5178 mouse
T lymphoma cell line transfected with the human MDR1 gene.
Apart from jolkinol D, all derivatives (2−14) showed potential
as MDR reversal agents. In this small library of novel bioactive
macrocyclic lathyrane diterpene derivatives, designed to
evaluate structure−activity relationships essential in over-
coming multidrug resistance (MDR), some correlations between MDR reversal and molecular weight, accessible solvent
areas, and octanol/water partition coefficient were identified that can contribute to the development of new selective P-gp
reversal agents.
gp reversal agents. Nevertheless, despite encouraging results in
in vitro assays, to date, there are no reversal agents clinically
available.10−12 For instance, first-generation drugs like
verapamil13 and cyclosporine A,14 besides revealing low affinity
for P-gp, had distinct pharmacological actions on the
cardiovascular (calcium channel blocker) and immune
(immunosuppressant) systems. As the high serum concen-
trations required for P-gp inhibition severely increased their
adverse effects, analogues of these drugs (without intrinsic
pharmacological activity) were designed as a way to reduce
their side effects (second-generation modulators); however,
they also showed increased toxicity15 and alterations in the
pharmacokinetics of cytotoxic drugs.16 Third generation MDR
modulators, like tariquidar (XR9576)17 and zosuquidar
(LY335979),18 inhibit P-gp at the nanomolar level and were
until recently under development but have not been approved
for clinical use so far. The hampering of progress in this area
can be attributable to the lack of knowledge concerning drug−
transporter interactions to a certain extent because of the
absence of detailed structural information of human P-gp.
Meanwhile, in 2009 the structure of the first mammalian ABC
transporter (murine, with 87% sequence identity to human P-
gp) was published, showing for the first time P-gp complexed
with a ligand.19 Despite its low resolution (3.8 Å), it represents
1. INTRODUCTION
Multidrug resistance (MDR) designates a phenomenon where
resistance to one drug is accompanied by resistance to drugs
that are structurally and functionally unrelated. The develop-
ment of MDR and subsequent failure of chemotherapy are
widespread problems in many types of tumors. One of the most
significant mechanisms of MDR results from overexpression of
membrane efflux pumps belonging to the evolutionarily
conserved family of ATP binding cassette (ABC) proteins,
which transports anticancer drugs out of the cells, preventing
them from reaching their cellular targets. A very important
member of this superfamily is P-glycoprotein (P-gp).1−3 This
protein, with 170 kDa, is the product of the human multidrug
resistance (MDR1) gene. It is classified as a pseudosymmetrical
heterodimer, where each monomer is composed of six
membrane spanning segments (transmembrane domain,
TMD) and one nucleotide-binding domain (NBD). The
TMDs mediate the recognition and transport of substrates,
and the NBDs are responsible for the ATP-binding and
hydrolysis and consequently for the generation of conforma-
tional changes on the protein.4−7
One of the most accepted strategies to overcome MDR
mediated by P-gp is based on the development of reversal
agents that when coadministered with an anticancer drug will
circumvent its efflux and consequently will avoid chemotherapy
failure.8,9 In this manner, a considerable number of natural and
synthetic compounds have been described in the literature as P-
Received: July 12, 2012
Published: January 21, 2013
© 2013 American Chemical Society
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Figure 1. Scheme showing how jolkinol D aliphatic esters were prepared (2−8).
a huge step toward allowing structure-based drug design
approaches. Aller et al. characterized a large hydrophobic
chamber of approximately 6.000 Å3 as the presumptive drug-
binding pocket (DBP), comprising mostly of hydrophobic and
aromatic residues near the periplasmatic lipid bilayer interface
and, on the opposite side, more polar and possibly charged
residues. These authors also identified three possible over-
lapping drug-binding sites in which residues Phe724 and
Val978 seem to display a central role in the ligand-protein
interactions, being common to the three binding sites.19,20
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Figure 2. Scheme showing how jolkinol D aromatic (9−13) and hydrolysis products (14) were prepared.
In our previous studies, many macrocyclic diterpenes and
some policyclic derivatives have been isolated from several
species of Euphorbia genus. Most of them were found to be
very strong P-gp modulators, exhibiting as well synergistic
interactions with cytotoxic drugs.21−25 In addition, the acylation
pattern of these compounds also seems to play a significant role
in the reversal of MDR.
To identify more potent P-gp modulators, a new
phytochemical study of Euphorbia piscatoria Ait. has been
carried out. Since a large amount of the lathyrane diterpene
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jolkinol D26 was isolated, 13 derivatives could be prepared by
esterification of the hydroxyl group at C-3 with different groups
and by hydrolysis of the ester function at C-15. Therefore, a
new set of homologous bioactive macrocyclic lathyrane
diterpenes was created for which the MDR reversal potency
could be assessed. These chemical modifications were evaluated
in vitro through the rhodamine-123 exclusion assay (for the
modulatory effect) and a chemosensitivity assay over a
checkerboard microplate method (drug interactions between
compounds and doxorubicin) in human MDR1 gene trans-
fected mouse lymphoma cells.
Table 1. Antiproliferative Activity of Jolkinol D (1) and
Derivatives (2−14) on L5178Y Mouse Lymphoma Cells
(PAR Cells) and in Human MDR1-Gene Transfected Mouse
Lymphoma Cells (MDR Cells)
a
IC50 (μM)
b
compd
PAR cell
MDR cell
selectivity index
jolkinol D (1)
89.87 0.80
38.21 5.52
18.33 4.00
23.99 0.45
38.66 6.30
17.20 2.55
14.34 2.64
20.58 7.11
34.70 6.93
41.16 3.03
21.67 6.60
43.99 3.67
12.13 0.46
36.42 5.77
1.21 0.51
99.67 4.53
60.35 1.25
22.16 3.94
26.84 5.32
60.27 6.43
18.76 3.11
14.74 0.73
48.92 4.16
33.08 5.44
45.07 3.33
32.71 5.17
52.60 3.84
14.95 4.33
48.92 4.16
1.46 0.11
0.9
0.6
0.8
0.9
0.6
0.9
1
jolkinoate A (2)
jolkinoate B (3)
jolkinoate C (4)
jolkinoate D (5)
jolkinoate E (6)
jolkinoate F (7)
jolkinoate G (8)
jolkinoate I (9)
jolkinoate J (10)
jolkinoate K (11)
jolkinoate L (12)
jolkinoate M (13)
jolkinodiol (14)
DMSO (2%)
The combination of the functional biological results and
some physicochemical descriptors allowed a better character-
ization of some structure−activity relationships correlating
chemical derivatization and anti-MDR activity.
0.4
1
2. RESULTS AND DISCUSSION
0.9
0.7
0.8
0.8
0.7
2.1. Chemistry. Fractionation of the ethyl acetate soluble
fraction of the methanol extract of the aerial parts of E.
piscatoria by chromatographic methods and crystallization
provided the known macrocyclic diterpene jolkinol D (1)
(Figure 1). This compound, previously isolated from Euphorbia
a
1
Values of IC50 are the mean standard error of the mean of three
jolkini Boiss, was identified by comparison of its H NMR
b
spectrum with that reported in the literature.26 The
combination of 13C NMR, DEPT, and 2D-NMR spectra
(COSY, HMQC, and HMBC) allowed the assignment of the
13C resonances, which are reported here for the first time. The
chemical derivatization of the free hydroxyl group at C-3 with
alkanoyl anhydrides and alkanoyl/aroyl/sulfonyl chlorides
yielded 12 derivatives in 50−97% yields (2−13, Figures 1
and 2). Hydrolysis of the ester function at C-15 yielded
compound 14 in 81% yield (Figure 2). The new compounds
were characterized by comparison of their spectroscopic data
with those of jolkinol D (1). The main differences between the
NMR data of compound 1 and the data of the ester derivatives
(2−13) were observed for the carbon and proton chemical
independent experiments. Selectivity index = IC50(PAR cells)/
IC50(MDR cells).
not show a significant discrimination between cell lines, instead
indicating that the compounds are equally active against both
PAR cells (chemo-sensitive) and MDR cells (chemoresistant by
overexpression of P-gp).
Inhibition of Rhodamine-123 Efflux P-gp-Mediated. The
rhodamine-123 exclusion assay was used to access the potential
P-glycoprotein-mediated MDR reversing activity of this set of
compounds (1−14) (Table 2). In this assay, the fluorescence
activity ratio (FAR) was evaluated through the accumulation
ratio of rhodamine-123 (substrate of P-gp) between MDR and
PAR cells and considering that when the FAR values are higher
than 1, MDR reversal has occurred. As such, compounds that
exhibit FAR values higher than 10 can be classified as strong
MDR modulators.27 Verapamil, a well-known modulator,13 was
used as positive control. To study whether MDR reversal
activity had dose−effect dependence, compounds 1−14 were
tested in several concentrations (2, 20, 40, and 80 μM).
However, under the assay conditions, some of the compounds
(3, 4, 6, 7, and 8) revealed toxicity above 40 μM (observed in
the variations of the forward scatter and side scatter light
parameters, data not shown). For this reason, only the MDR-
reversal activities at 2 and 20 μM were chosen to be
summarized in Table 1. At 20 μM, except for jolkinol D (1)
and jolkinodiol (14), all the compounds showed a strong effect
on P-gp modulation, with FAR values ranging from 12.13 to
109.17, suggesting that a modification at position C-3 greatly
contributed to increase P-gp inhibitory activity. At 2 μM,
compounds 8, 9, 10, 11 and 13 showed FAR values higher than
10 (10.55 − 28.09), leading us to classify them as strong MDR
modulators (Figure 3). These results are of particular interest,
since the basal MDR reversal activity of verapamil at 22 μM is
approximately 20.
1
shifts of the pentacyclic ring. In the H NMR spectra of the
compounds 2−13, a significant paramagnetic effect was
observed for the signal of H-3, which appeared at approximately
1.0−1.6 ppm downfield. As expected, in the 13C NMR spectra,
similar paramagnetic effects at α-carbons were also observed,
displaying the highest shifts of C-3 of compounds 12 and 13
(ΔδC = +2.9 and +11.3 ppm, respectively). The β-carbons C-2
and C-4 of the esters also showed predictable diamagnetic
effects (ΔδC ≅ 0.9−1.3 and 1.1−1.9 ppm, respectively).
On the contrary, for the hydrolyzed derivative of 1,
jolkinodiol (14), the α-carbon C-15 appeared upfield by 2.7
from its position in 1 and C-1, C-4, and C-14 (β-carbons) were
shifted downfield (ΔδC = +2.0, + 0.5, + 2.6 ppm, respectively).
The marked effect at C-14 (β-carbon) could also be explained
by an intramolecular hydrogen bond between the correspond-
ing ketone function and the free hydroxyl group at C-15. This
effect could also be observed at both H-12 and C-12 of the
enone system because of the mesomeric effect (ΔδH = +0.8
ppm and ΔδC = +5.1 ppm, respectively).
2.2. Biological Activities. In Vitro Antiproliferative Assay.
The antiproliferative activity of jolkinol D (1) and derivatives
(2−14) was evaluated using the MTT assay in L5178Y mouse
lymphoma cells (PAR cells) and in human MDR1-gene
transfected L5178Y mouse lymphoma cells (MDR cells). The
IC50 values are presented in Table 1. All the derivatives showed
a higher antiproliferative activity when compared with jolkinol
D (1). The determination of the selectivity index (Table 1) did
The Overton cumulative histogram subtraction algorithm
was calculated for compounds 9, 10, 11, and 13. This algorithm
subtracts histograms on a channel-by-channel basis to provide a
percentage of positive cells,28 allowing calculation of the
percentage of the population that reverted to a chemosensitive
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Table 2. Multidrug Resistance Reversal Activities of Jolkinol D (1) and Derivatives (2−14) on Human MDR1-Gene Transfected
Mouse Lymphoma Cells (MDR Cells)
a
b
c
d
e
compd
PAR cells
concn (μM)
FSC
SSC
FL-1
SD
peak
FAR
1592
1718
1680
1686
1738
1791
1746
1667
1766
1798
1756
1751
1772
1573
1210
1834
1708
1825
1765
1698
1785
1844
1736
1776
1790
1756
1758
1756
1753
1742
1689
680
751
673
673
718
877
717
762
731
819
711
831
725
533
104
963
721
857
702
776
716
886
708
839
719
777
706
773
725
798
754
48.44
0.67
17.91
0.64
45.09
0.53
MDR cells
jolkinol D (1)
2
20
2
0.44
0.60
0.37
0.79
0.92
0.47
0.57
0.35
jolkinoate A (2)
jolkinoate B (3)
jolkinoate C (4)
jolkinoate D (5)
jolkinoate E (6)
jolkinoate F (7)
jolkinoate G (8)
jolkinoate I (9)
jolkinoate J (10)
jolkinoate K (11)
jolkinoate L (12)
jolkinoate M (13)
jolkinodiol (14)
verapamil
0.65
0.54
0.53
1.16
20
2
18.40
1.65
16.30
2.86
11.50
0.63
33.76
2.95
20
2
16.70
2.88
11.30
4.24
16.00
1.00
30.64
5.15
20
2
58.80
0.97
26.60
1.09
47.00
0.58
107.89
1.74
20
2
14.00
3.99
10.20
4.98
15.40
1.07
25.69
7.14
20
2
6.14
8.06
3.79
12.13
4.99
2.79
3.18
2.29
20
2
42.10
5.90
21.10
6.99
42.20
3.40
77.25
10.55
52.66
24.15
84.22
21.11
56.88
28.09
109.17
9.32
20
2
28.70
13.50
45.90
11.80
31.00
15.70
59.50
5.21
11.50
10.40
21.40
8.85
26.40
13.80
39.20
9.31
20
2
20
2
11.40
10.90
21.50
5.08
31.60
16.00
56.20
3.28
20
2
20
2
14.50
8.98
10.20
9.29
15.40
7.77
28.66
16.06
52.77
0.84
20
2
26.70
0.47
14.80
0.34
29.40
0.42
20
22
1.71
1.94
0.65
3.38
5.70
5.08
4.00
20.00
a
b
c
FSC: forward scatter count of cells in the samples. SSC: side scatter count of cells in the samples. FL-1: mean fluorescence intensity of the cells.
d
e
SD: standard deviation. FAR (fluorescence activity ratio) values were calculated by using the equation given in the Experimental Section.
phenotype. The data are presented in Figure 4 and show that
jolkinoate K (11) and jolkinoate I (9) reverted 95% and 70.2%
of the population of MDR cells to a chemosensitive phenotype,
respectively (at 20 μM). Moreover, the direct correlation
between the increase of concentration and number of cells with
a chemosensitive phenotype is an indicator of a dose-dependent
inhibitory effect on the MDR reversal activity (Figure 4).
Drug Combination Assays. Since the studied compounds
showed a good MDR reversal activity, the following step was
the investigation of the combined effect of these compounds
and an antineoplastic drug, such as doxorubicin. This
chemotherapeutic agent not only is transported by P-gp but
also induces its expression in cancer cells, and therefore, its
cytostatic efficacy is limited by P-gp activity.29 As can be
observed in Table 3, all tested compounds but jolkinoate D (5)
synergistically enhanced the cytotoxicity of doxorubicin
(combination index, CI = 0.18−0.65). These results are in
accordance with the assumption that a promising P-gp inhibitor
would consequently be able to increase the action of the
cytotoxic drug because of greater efflux impairment. Jolkinol D
(1) did not display a good MDR reversal ability but showed a
synergistic interaction with doxorubicin comparable to the most
active inhibitors (10, 12), suggesting that other distinct targets/
pathways besides the P-gp inhibition can be involved and
should be the subject of further investigation. Jolkinoate D (5)
was a strong inhibitor at 20 μM but in the combination assay
had an antagonistic effect.
2.3. Structure−Activity Relationships. Physicochemical
Properties: K-Means Clustering and Linear Regression
Analysis. The monoacylated derivatives at C-3 are a good set
of homologous compounds to determine structure−activity
relationships. Therefore, some physicochemical properties of
compounds 1−14 were determined (Table 4) and subdivided
by the K-means cluster analysis into five main clusters in which
a high degree of similarity was found (Table 4).
Cluster I comprises compounds 1 and 14 (Table 4),
molecules that have a molecular weight below 400 g·mol−1
and log P lower than 4.0 and that are often considered poor P-
gp substrates.30 Cluster II includes compounds 2, 3, and 4
(Table 4), only differing in the size of the ester side chain (one
or two methylene units). In this group and despite the low
number of molecules, it was possible to correlate the increase in
P-gp inhibitory activity with the increase of the molecular
weight (MW, r2 = 0.96) and solvent accessible area (ASA, r2 =
0.94 at 2 μM and at 20 μM, with ASA, r2 = 0.77). Cluster III
comprises compounds 6, 7, 9, and 13 (Table 4). Within this
group, a single correlation could be determined with ASA (r2 =
0.84, 20 μM). However, compounds bearing an aromatic
moiety (9 and 13) displayed higher MDR reversal activity at 2
μM when compared with all the alkanoyl derivatives. This
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Figure 3. Reversal of the MDR phenotype by jolkinoates G (A) and K (B) at 2 and 20 μM, in comparison with jolkinol D (20 μM): MDR,
chemoresistant cell by overexpression of P-gp; PAR, parental chemosensitive cell.
Figure 4. Relation between fluorescence activity ratio (FAR, lines) and percentage of MDR cells that reverted to a PAR phenotype (bars) for
compounds 9−11 and 13. This analysis clearly shows the dose-dependent inhibitory effect on P-gp modulation.
observation is in agreement with previous studies by our
the establishment of additional hydrophobic interactions within
the DBP. Cluster IV includes the most active molecules
group31−33 that identified aromatic moieties to be important for
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Table 3. Evaluation of the Interaction between Compounds
1−14 and Doxorubicin on Human MDR1-Gene Transfected
Mouse Lymphoma Cells (MDR Cells)
4. EXPERIMENTAL SECTION
4.1. Chemistry. General Procedures. Pyridine of HPLC grade was
dried with solid KOH followed by distillation and stored with Linde
4A molecular sieves. All the other reagents and solvents were obtained
from commercial suppliers and were used without further purification.
The infrared spectra were collected on an Affinity-1 (Shimadzu) FTIR
spectrophotometer. High resolution mass spectra were recorded on a
drug combination with
doxorubicin
CI SE for
a
b
ratio
IC50
interaction
jolkinol D (1)
40:1
38:1
8:1
0.26 0.05
0.18 0.03
0.34 0.04
0.39 0.04
2.02 0.26
0.23 0.07
0.25 0.04
0.31 0.05
0.35 0.06
0.26 0.03
0.30 0.04
0.27 0.04
0.21 0.05
0.65 0.11
synergy
synergy
synergy
synergy
antagonism
synergy
synergy
synergy
synergy
synergy
synergy
synergy
synergy
synergy
jolkinoate A (2)
jolkinoate B (3)
jolkinoate C (4)
jolkinoate D (5)
jolkinoate E (6)
jolkinoate F (7)
jolkinoate G (8)
jolkinoate I (9)
jolknoate J (10)
jolkinoate K (11)
jolkinoate L (12)
jolkinoate M (13)
jolkinodiol (14)
FTICR-MS Apex Ultra (Bruker Daltonics) 7 T instrument. Column
̈
chromatography was performed on silica gel (Merck 9385). Merck
silica gel 60 F254 plates were used in analytical TLC, with visualization
under UV light and by spraying with sulfuric acid/methanol (1:1),
followed by heating. NMR spectra were recorded on a Bruker 400
20:1
5:1
4:1
Ultra-Shield instrument (1H 400 MHz, 13C 100.61 MHz). 1H and 13
C
30:1
65:1
40:1
33:1
10:1
18:1
10:1
23:1
chemical shifts are expressed in δ (ppm) referenced to the solvent used
and the proton coupling constants J in hertz (Hz). Spectra were
assigned using appropriate COSY, DEPT, HMQC, and HMBC
sequences. HPLC was performed on a Merck-Hitachi HPLC system,
with UV detection. All tested compounds were purified to ≥95%
purity as determined by HPLC (Merck LiChrospher 100 RP-18; 5 μm,
125 mm × 4 mm column; MeOH/H2O as the mobile phase).
Plant Material. Euphorbia piscatoria Ait. was collected in the Garcia
de Orta garden, Lisbon, Portugal (April 2011), and was identified by
Dr. Teresa Vasconcelos (plant taxonomist) of Instituto Superior de
Agronomia, Technical University of Lisbon, Portugal. A voucher
specimen (no. 276) has been deposited at the herbarium of Instituto
Superior de Agronomia.
Extraction and Isolation. The air-dried powdered plant (8.4 kg)
was exhaustively extracted with methanol (9 × 6 L) at room
temperature. Evaporation of the solvent (under vacuum, 40 °C) from
the crude extract yielded a residue of 1.3 kg. This residue was
resuspended in MeOH/H2O solution (1:2, 4 L) and extracted with
EtOAc (8 × 3 L). The ethyl acetate soluble fraction was dried
(Na2SO4) and evaporated under vacuum, yielding a residue (462.4 g)
that was chromatographed over SiO2 (2 kg), using mixtures of n-
hexane−EtOAc (1:0 to 0:1) and EtOAc−MeOH (9:1 to 3:7) as
eluents. According to differences in composition as indicated by TLC,
14 crude fractions were obtained (fractions A−O). Jolkinol D (1) was
obtained (2.9 g) through crystallization (EtOAc/n-hexane) from the
crude fraction G (17.58 g).
a
Data are shown as the best combination ratio of the tested
b
compounds and doxorubicin. Combination index (CI) values listed
in the table are the mean standard error (SE) for an inhibitory
concentration of 50% (IC50). CI < 1: synergy. CI = 1: additivity. CI >
1: antagonism. Values were determined experimentally and calculated
using the Chou and Talalay method and CalcuSyn software.
identified in this particular study, namely, compounds 8 and
10−12 (Table 4). The only strong correlation found within this
group was between octanol/water partition coefficient (log P, r2
= 0.96 and r2 = 0.86 at 2 and 20 μM, respectively) and the
biological activity (these two properties are inversely propor-
tional). Jolkinoate D (5) remained alone in cluster V because
its physicochemical features are very dissimilar to those of the
remaining compounds (Table 4).
Jolkinol D, 15β-Acetoxy-3β-hydroxylathyra-5E,12E-dien-14-one
3. CONCLUSIONS
1
(1). H NMR (400 MHz, CDCl3) δ 6.65 (1H, d, J = 11.4 Hz, H-12),
The common scaffold of this particular set of compounds and
the limited variation achieved through the derivatization at C-3
provide an ideal model for structure−activity relationship
studies. Biological studies clearly demonstrated the effect of C-3
pattern modification in the MDR reversal activity. As the
combination index between these compounds and doxorubicin
revealed a synergistic effect, the combination of cytotoxic and
MDR reversal effects would hold promise in the treatment of
MDR.
K-means analysis obtained five main clusters for which strong
correlations between activity and log P, MW, or ASA could be
specifically identified . However, as each group had its own
behavior regarding physicochemical descriptors (no correlation
or correlation in different descriptors), the hypothesis of a
multifactorial inhibition mechanism is reinforced, thus con-
tributing to a better clarification of the polispecificity in the P-
gp efflux reversion.
It has become clear in several studies the importance of
aromatic moieties for P-gp efflux modulation. This new SAR
analysis, in a different perspective, corroborated the importance
of the aromatic ring for binding inside the DBP, namely,
through its electronic and steric effects. Hydrophobicity
remains a critical factor, but the new data acquired in this
study provided a different insight on how the acylation of a
single hydroxyl group, with different moieties, can affect the
modulation ability of P-gp efflux.
5.67 (1H, d, J = 10.9 Hz, H-5), 3.91 (1H, dd, J = 8.2, 3.6 Hz, H-3),
3.49 (1H, dd, J = 14.0, 8.1 Hz, H-1α), 2.57 (1H, d, J = 13.3 Hz, H-7α),
2.38 (1H, dd, J = 10.9, 3.6 Hz, H-4), 2.21 (1H, ddd, J = 14.5, 6.8, 3.3
Hz, H-8α), 2.04 (1H, m, H-2), 2.01 (3H, s, Me-22), 1.83 (3H, s, Me-
20), 1.77 (1H, td, J = 13.1, 6.5 Hz, H-7β), 1.57−1.48 (2H, m, H-1β,
H-8 β), 1.46 (3H, s, Me-17), 1.40 (1H, dd, J = 11.4, 8.2 Hz, H-11),
1.17 (3H, s, Me-18), 1.08 (3H, d, J = 6.7 Hz, Me-16), 1.06 (1H, m, H-
9), 1.04 (3H, s, Me-19) ppm; 13C NMR (101 MHz, CDCl3) δ 195.5
(C-14), 170.0 (C-21), 146.7 (C-12), 143.2 (C-6), 132.3 (C-13), 119.6
(C-5), 95.4 (C-15), 80.1 (C-3), 52.8 (C-4), 44.1 (C-1), 39.4 (C-2),
36.9 (C-7), 34.4 (C-9), 29.8 (C-11), 29.4 (C-18), 28.5 (C-8), 24.6 (C-
10), 21.8 (C-22), 21.1 (C-17), 16.6 (C-19), 13.9 (C-16), 12.5 (C-20)
ppm.
General Preparation of Jolkinol D Derivatives 2−13. A solution of
jolkinol D (1 equiv) in dry pyridine (2 mL for 0.039 mol of jolkinol D)
was stirred for 5 min at room temperature before addition of the
suitable anhydride or chloride (2 equiv). The mixture was stirred for
48 h at room temperature. The reaction mixture was concentrated
under vacuum at 40 °C, and the obtained residue was purified by flash
column chromatography.
Jolkinoate A, 3β,15β-Diacetoxylathyra-5E,12E-dien-14-one (2).
was obtained from reaction with acetic anhydride (Merck KGaA,
Darmstradt, Germany; 10.2 mg, 0.1 mol). The residue was purified by
flash column chromatography (silica gel, CH2Cl2/MeOH 100:0 to
99:1) to afford 13 mg (0.031 mol, 56% yield) of an amorphous white
1
powder. IR (NaCl) νmax 1730 (CO), 1270 (C−O) cm−1; H NMR
(400 MHz, CDCl3) δ 6.66 (1H, d, J = 11.4, H-12), 5.34 (1H, dt, J =
10.7, 1.8 Hz, H-5), 5.23 (1H, t, J = 3.6 Hz, H-3), 3.50 (1H, dd, J =
754
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b
Table 4. Cluster Analysis (K-Means Algorithm) of Compounds 1−14
a
b
Physicochemical properties were calculated using MOE, version 2010.10.34 Grouping variables are physicochemical properties: molecular weight
(MW), molecular volume (MV), logarithm of the octanol/water partition coefficient (log P), molar refractivity (MR), topological polar surface area
(TPSA), accessible solvent area (ASA), and FAR (at 2 μM).
13.9, 8.0 Hz, H-1α), 2.50 (2H, m, H-4, H-7α), 2.21−2.17 (2H, m, H-
8α, H-2), 2.01 (3H, s, Me-22), 1.83 (3H, s, Me-20), 1.75 (1H, td, J =
13.1, 2.6 Hz, H-7β), 1.52, (1H, dd, J = 12.5, 2.8 Hz, H-8β), 1.47 (1H,
dd, J = 8.4, 4.9 Hz, H-1β), 1.44 (3H, d, J = 1.3 Hz, Me-17), 1.39 (1H,
dd, J = 7.5, 3.9 Hz, H-11), 1.17 (3H, s, Me-18), 1.06 (1H, m, H-9),
1.05 (3H, s, Me-19), 0.95 (3H, d, J = 6.7 Hz, Me-16), 2.13 (1H, s, Me-
2′) ppm; 13C NMR (100 MHz, CDCl3) δ 195.4 (C-14), 170.8 (C-1′),
169.9 (C-21), 146.80 (C-12), 143.2 (C-6), 132.2 (C-13), 118.7 (C-5),
94.6 (C-15), 81.1 (C-3), 51.2 (C-4), 44.8 (C-1), 38.4 (C-2), 36.9 (C-
7), 34.2 (C-9), 29.8 (C-11), 29.3 (C-18), 28.5 (C-8), 24.6 (C-10),
21.6 (C-22), 21.1 (C-2′), 21.0 (C-17), 16.4 (C-19), 13.9 (C-16), 12.4
(C-20) ppm.
(Sigma-Aldrich Chemie GmbH, Riedstrasse D-89555, Steinhelm,
Germany; 10.8 mg, 0.08 mol). The residue was purified by flash
column chromatography (silica gel, CH2Cl2/MeOH = 100:0 to 99:1)
to afford 10 mg (0.023 mol, 56% yield) of a yellow oil. IR (NaCl) νmax
1737 (CO), 1271 (C−O) cm−1; MS m/z (rel intens) 439 [M +
Na]+ (100), 417 [M]+ (38); 1H NMR (400 MHz, CDCl3) δ 6.65 (1H,
d, J = 11.4 Hz, H-12), 5.33 (1H, dt, J = 10.7, 1.3 Hz, H-5), 5.24 (1H, t,
J = 3.6 Hz, H-3), 3.52 (1H, dd, J = 13.9, 8.0 Hz, H-1α), 2.50 (1H, dd, J
= 10.7, 3.8 Hz, H-4), 2.44 (1H, m, H-7α), 2.20−2.16 (2H, m, H-8α,
H-2), 2.01 (3H, s, Me-22), 1.85 (3H, d, J = 1.0 Hz, Me-20), 1.75 (1H,
td, J = 13.1, 2.6 Hz, H-7β), 1.50 (1H, dd, J = 14.4, 2.5 Hz, H-8β), 1.45
(3H, d, J = 1.4 Hz, Me-17), 1.41 (1H, dd, J = 7.9, 3.5 Hz, H-1β),
1.38−1.32 (1H, m, H-11), 1.17 (3H, s, Me-18), 1.06 (1H, m, H-9),
1.04 (3H, s, Me-19), 0.96 (3H, d, J = 6.7 Hz, Me-16), 2.44 (2H, q, J =
Jolkinoate B, 15β-Acetoxy-3β-propionatelathyra-5E,12E-dien-14-
one (3). 3 was obtained from reaction with propionic anhydride
755
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15.1, 7.6, H-2′), 1.21 (3H, t, J = 7.6, Me-3′) ppm; 13C NMR (100
MHz, CDCl3) δ 195.0 (C-14), 173.7 (C-1′), 169.5 (C-21), 146.5 (C-
12), 142.8 (C-6), 131.9 (C-13), 118.6 (C-5), 94.3 (C-15), 80.6 (C-3),
50.9 (C-4), 44.4 (C-1), 38.1 (C-2), 36.7 (C-7), 33.5 (C-9), 29.0 (C-
11), 28.9 (C-18), 28.3 (C-8), 27.6 (C-2′), 24.3 (C-10), 21.2 (C-22),
20.6 (C-17), 16.0 (C-19), 13.6 (C-16), 12.0 (C-20), 9.2 (C-3′) ppm;
HRMS-ESI-TOF m/z calcd C25H36O5Na (M+ + Na) 439.2455, found
439.2458.
Jolkinoate C, 15β-Acetoxy-3β-butanoatelathyra-5E,12E-dien-14-
one (4). 4 was obtained from reaction with butanoic anhydride
(Sigma-Aldrich Chemie GmbH, Riedstrasse D-89555, Steinhelm,
Germany; 12.6 mg, 0.08 mmol). The residue was purified by flash
column chromatography (silica gel, n-hexane/EtOAc = 100:0 to 9:1)
to afford 10 mg (0.024 mol, yield 61%) of a yellow oil. IR (NaCl) νmax
1743 (CO), 1259 (C−O) cm−1; MS m/z (rel intens) 453 [M +
Na]+ (100), 431 [M]+ (11), 381 [M + Na − CH3CH2CH2CO]+ (35);
1H NMR (400 MHz, CDCl3) δ 6.65 (1H, d, J = 11.4 Hz, H-12), 5.33
(1H, d, J = 10.6 Hz, H-5), 5.24 (1H, t, J = 3.6 Hz, H-3), 3.52 (1H, dd,
J = 13.9, 8.0 Hz, H-1α), 2.49 (1H, dd, J = 10.7, 3.8 Hz, H-4), 2.37 (1H,
td, J = 7.3, 1.9 Hz, H-7α), 2.22−2.12 (2H, m, H-8α, H-2), 2.00 (3H, s,
Me-22), 1.82 (3H, s, Me-20), 1.74 (1H, td, J = 13.1, 2.9 Hz, H-7β),
1.48 (1H, dd, J = 13.5, 2.5 Hz, H-11), 1.48−1.46 (1H, m, H-1β, H-
8β), 1.45 (3H, s, Me-17), 1.39 (1H, dd, J = 7.9, 3.5 Hz, H-1β), 1.16
(3H, s, Me-18), 1.08−1.04 (1H, m, H-9), 1.04 (3H, s, Me-19), 0.96
(3H, d, J = 7.4 Hz, Me-16), 2.30 (2H, t, J = 7.4 Hz, H-2′), 1.65 (2H, q,
J = 7.4 Hz, H-3′), 0.94 (3H, t, J = 3.4 Hz, Me-4′) ppm; 13C NMR (100
MHz, CDCl3) δ 195.4 (C-14), 173.2 (C-1′), 169.8 (C-21), 146.8 (C-
12), 143.0 (C-6), 132.2 (C-13), 118.8 (C-5), 94.6 (C-15), 80.4 (C-3),
50.9 (C-4), 44.5 (C-1), 38.2 (C-2), 35.6 (C-2′), 36.1 (C-7), 34.2 (C-
9), 29.7 (C-11), 29.3 (C-18), 28.4 (C-8), 24.6 (C-10), 21.6 (C-22),
21.0 (C-17), 18.0 (C-3′), 16.0 (C-19), 13.8 (C-16), 13.5 (C-4′), 12.0
(C-20) ppm; HRMS-ESI-TOF m/z calcd C26H38O5Na (M+ + Na)
453.2617, found 453.2606.
Hz, H-4), 2.50 (1H, ddd, J = 10.6, 3.7, 1.7 Hz, H-7α), 2.21−2.18 (2H,
m, H-8α, H-2), 2.01 (3H, d, J = 2.5, Me-22), 1.83 (3H, s, Me-20), 1.75
(1H, td, J = 13.1, 2.5 Hz, H-7β), 1.48 (1H, dd, J = 5.7, 2.8 Hz, H-8β),
1.46 (1H, dd, J = 8.6, 2.3 Hz, H-1β), 1.44 (3H, d, J = 1.3 Hz, Me-17),
1.38 (1H, dd, J = 7.6, 3.6 Hz, H-11), 1.17 (3H, s, Me-18), 1.09 (1H, m,
H-9), 1.04 (3H, d, J = 1.2 Hz, Me-19), 0.95 (3H, d, J = 6.6 Hz, Me-
16), 2.54 (1H, m, H-2′), 1.21 (2H, dd, J = 7.0, 2.9 Hz, H-3′), 1.12
(2H, m, H-4′), 0.95 (3H, t, J = 7.1 Hz, Me-5′), 1.17 (3H, d, J = 6.7 Hz,
Me-6′) ppm; 13C NMR (100 MHz, CDCl3) δ 195.2 (C-14), 175.9 (C-
1′), 169.6 (C-21), 146.7 (C-12), 142.8 (C-6), 132.1 (C-13), 118.8 (C-
5), 94.6 (C-15), 80.5 (C-3), 51.3 (C-4), 44.8 (C-1), 39.8 (C-2′), 38.3
(C-2), 36.07 (C-7), 34.12 (C-9), 29.59 (C-11), 29.39 (C-3′), 29.15
(C-18), 28.88 (C-4′), 28.22 (C-8), 24.49 (C-10), 21.39 (C-22), 20.8
(C-17), 20.31 (C-5′), 16.6 (C-6′), 16.3 (C-19), 13.8 (C-16), 12.3 (C-
20) ppm; HRMS-ESI-TOF m/z calcd C28H42O5Na (M+ + Na)
481.2924, found 481.2933.
Jolkinoate F, 15β-Acetoxy-3β-(2-ethylbutyrate)lathyra-5E,12E-
dien-14-one (7). 7 was obtained from reaction with 2-ethylbutyryl
chloride (Sigma-Aldrich Chemie GmbH, Riedstrasse D-89555,
Steinhelm, Germany; 11 mg, 0.08 mol). The residue was purified by
column chromatography (silica gel, CH2Cl2) followed by preparative
TLC with n-hexane/EtOAc (9:1) to afford 19 mg (0.042 mol, yield
84%) of a yellow oil. IR (NaCl) νmax 1734 (CO), 1269 (C−O)
cm−1; MS m/z (rel intens) 481 [M + Na]+ (100), 399 [M −
1
CH3CO2]+ (12); H NMR (400 MHz, CDCl3) δ 6.65 (1H, dd, J =
11.4, 0.9 Hz, H-12), 5.36 (1H, d, J = 10.7 Hz, H-5), 5.26 (1H, t, J = 3.5
Hz, H-3), 3.53 (1H, dd, J = 13.9, 8.0 Hz, H-1α), 2.50 (1H, dd, J =
10.7, 3.7 Hz, H-4), 2.44 (1H, dd, J = 13.2, 3.9 Hz, H-7α), 2.20−2.16
(2H, m, H-8α, H-2), 2.00 (3H, s, Me-22), 1.83 (3H, d, J = 0.9 Hz, Me-
20), 1.74 (1H, td, J = 12.0, 2.4 Hz, H-7β), 1.49 (1H, dd, J = 7.2, 1.5
Hz, H-8β), 1.44 (1H, dd, J = 12.8, 7.3 Hz, H-1β), 1.44 (3H, d, J = 1.2
Hz, Me-17), 1.39 (1H, dd, J = 11.5, 8.1 Hz, H-11), 1.16 (3H, s, Me-
18), 1.06 (1H, m, H-9), 1.04 (3H, s, Me-19), 0.89 (3H, d, J = 6.9 Hz,
Me-16), 2.28 (1H, m, H-2′), 1.68−1.58 (4H, m, H-3′, H-5′), 0.93
(6H, t, J = 7.5 Hz, Me-4′, Me-6′) ppm; 13C NMR (100 MHz, CDCl3)
δ 195.6 (C-14), 175.7 (C-1′), 169.8 (C-21), 146.9 (C-12), 142.8 (C-
6), 132.2 (C-13), 119.1 (C-5), 94.7 (C-15), 80.8 (C-3), 51.4 (C-4),
48.4 (C-2′), 44.9 (C-1), 38.4 (C-2), 36.8 (C-7), 34.2 (C-9), 29.6 (C-
18), 29.3 (C-11), 28.3 (C-8), 24.6 (C-10), 22.7 (C-3′/C-5′), 21.5 (C-
22), 21.0 (C-17), 16.4 (C-19), 14.0 (C-16), 12.4 (C-4′/C-6′), 12.1
(C-20) ppm; HRMS-ESI-TOF m/z calcd C28H42O5Na (M+ + Na)
481.2924, found 481.2934.
Jolkinoate D, 15β-Acetoxy-3β-lauroyloxylathyra-5E,12E-dien-14-
one (5). 5 was obtained from reaction with lauroyl chloride (Sigma-
Aldrich Chemie GmbH, Riedstrasse D-89555, Steinhelm, Germany;
17.4 mg, 0.08 mmol). The residue was purified by flash column
chromatography (silica gel, n-hexane/EtOAc = 100:0 to 9:1) to afford
20 mg (0.036 mol, yield 91%) of an amorphous dark yellow powder.
IR (NaCl) νmax 1740 (CO), 1280 (C−O) cm−1; MS m/z (rel
intens) 565 [M + Na]+ (54), 543 [M]+ (3), 381 [M + Na −
1
CH3(CH2)10CO]+ (100); H NMR (400 MHz, CDCl3), δ 6.66 (1H,
d, J = 11.3 Hz, H-12), 5.33 (1H, d, J = 10.7 Hz, H-5), 5.25 (1H, t, J =
3.5 Hz, H-3), 3.51 (1H, dd, J = 13.8, 8.0 Hz, H-1α), 2.50 (1H, dd, J =
10.6, 3.7 Hz, H-4), 2.39 (1H, td, J = 12.0, 2.4 Hz, H-7α), 2.21−2.17
(2H, m, H-8α, H-2), 2.01 (3H, s, Me-22), 1.83 (3H, s, Me-20), 1.75
(1H, td, J = 12.5, 3.0 Hz, H-7β), 1.47 (1H, dd, J = 7.4, 4.3 Hz, H-11),
1.46 (1H, dd, J = 12.4, 2.9 Hz, H-1β), 1.44 (3H, s, Me-17), 1.41 (1H,
dd, J = 7.3, 4.1 Hz, H-8β), 1.17 (3H, s, Me-18), 1.08 (1H, m, H-9),
1.05 (3H, s, Me-19), 0.94 (3H, d, J = 6.7 Hz, Me-16), 2.34 (2H, t, J =
7.5 Hz, H-2′), 1.67−1.58 (18H, m, H-3′−H-11′), 0.87 (3H, t, J = 6.8
Hz, Me-12′) ppm; 13C NMR (100 MHz, CDCl3) δ 195.3 (C-14),
173.3 (C-1′), 169.7 (C-21), 146.7 (C-12), 142.9 (C-6), 132.1 (C-13),
118.7 (C-5), 94.5 (C-15), 80.7 (C-3), 51.2 (C-4), 44.8 (C-1), 38.3 (C-
2), 36.8 (C-7), 34.6 (C-2′), 34.1 (C-9), 34.1−29.3 (C-3′−C-11′), 29.2
(C-11), 29.1 (C-18), 28.3 (C-8), 24.7 (C-10), 22.7 (C-22), 20.8 (C-
17), 16.3 (C-19), 14.1 (C-16), 13.9 (C-12′), 12.3 (C-20) ppm;
HRMS-ESI-TOF m/z calcd C34H54O5Na (M+ + Na) 565.3863, found
565.3875.
Jolkinoate G, 15β-Acetoxy-3β-(2-ethylhexanoate)lathyra-5E,12E-
dien-14-one (8). 8 was obtained from reaction with 2-ethylhexanoyl
chloride (Sigma-Aldrich Chemie GmbH, Riedstrasse D-89555,
Steinhelm, Germany; 13.5 mg, 0.08 mol). The residue was purified
by flash column chromatography (silica gel CH2Cl2/MeOH = 99:1) to
afford 33.5 mg (0.069 mol, yield 97%) of a yellow oil. IR (NaCl) νmax
1743 (CO), 1269 (C−O) cm−1; MS m/z (rel intens) 509 [M +
Na]+ (100), 427 [M − CH3CO2]+ (12); 1H NMR (400 MHz, CDCl3)
δ 6.67 (1H, d, J = 11.4 Hz, H-12), 5.39 (1H, d, J = 10.7 Hz, H-5), 5.28
(1H, t, J = 3.5 Hz, H-3), 3.55 (1H, dd, J = 13.9, 8.0 Hz, H-1α), 2.55
(1H, dd, J = 10.7, 3.7 Hz, H-4), 2.44 (1H, d, J = 13.4, H-7α), 2.30−
2.20 (2H, m, H-8α, H-2), 2.00 (3H, s, Me-22), 1.83 (3H, s, Me-20),
1.76 (1H, td, J = 12,8, 2.7 Hz, H-7β), 1.66−1.56 (8H, m, H-1β, H-8β),
1.44 (3H, d, J = 1.0 Hz, Me-17), 1.39 (1H, dd, J = 11.5, 8.3 Hz, H-11),
1.16 (3H, s, Me-18), 1.06 (1H, m, H-9), 1.04 (3H, s, Me-19), 0.92
(3H, d, J = 7.4 Hz, Me-16), 2.26 (1H, m, H-2′), 1.66−1.56 (8H, m, H-
3′, H-4′, H-5′, H-7′), 0.96 (3H, t, J = 7.3 Hz, Me-8′), 0.95 (3H, t, J =
8.8 Hz, Me-6′) ppm; 13C NMR (100 MHz, CDCl3) δ 195.3 (C-14),
175.5 (C-1′), 169.8 (C-21), 146.9 (C-12), 142.8 (C-6), 132.2 (C-13),
119.1 (C-5), 94.6 (C-15), 80.74 (C-3), 51.4 (C-4), 49.8 (C-2′), 44.9
(C-1), 38.5 (C-2), 36.9 (C-7), 34.26 (C-9), 29.7 (C-11), 29.3 (C-18),
28.3 (C-8), 25.3−25.0 (C-3′/C-4′/C-5′/C-7′), 24.6 (C-10), 21.5 (C-
22), 20.9 (C-17), 16.4 (C-19), 14.1 (C-16), 12.4−12.2 (C-6′/C-8′),
Jolkinoate E, 15β-Acetoxy-3β-(2-methylvaleroate)lathyra-
5E,12E-dien-14-one (6). 6 was obtained from reaction with 2-
methylvaleryl chloride (Sigma-Aldrich Chemie GmbH, Riedstrasse D-
89555, Steinhelm, Germany; 10.9 mg, 0.08 mol). The residue was
purified by column chromatography (silica gel, CH2Cl2) followed by a
preparative TLC with n-hexane/EtOAc (6:1) to afford 20 mg (0.044
mol, yield 93.5%) of a yellow oil. IR (NaCl) νmax 1792 (CO), 1271
(C−O) cm−1; MS m/z (rel intens) 481 [M + Na]+ (100), 430 [M −
12.1 (C-20) ppm; HRMS-ESI-TOF m/z calcd C30H46O5Na (M+
+
Na) 509.3237, found 509.3256.
1
CH2CH2]+ (18); H NMR (400 MHz, CDCl3) δ 6.66 (1H, d, J =
Jolkinoate I, 15β-Acetoxy-3β-benzoyloxylathyra-5E,12E-dien-14-
one (9). 9 was obtained from reaction with benzoyl chloride (Merck
KGaA, Darmstradt, Germany; 14 mg, 0.10 mol). The residue was
11.4 Hz, H-12), 5.34 (1H, d, J = 10.7 Hz, H-5), 5.24 (1H, t, J = 4.1 Hz,
H-3), 3.53 (1H, dd, J = 13.9, 8.0 Hz, H-1α), 2.55 (1H, dd, J = 13.7, 6.8
756
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Article
purified by two sequential flash column chromatography (silica gel,
CH2Cl2 and silica gel, CH2Cl2/MeOH = 99:1) to afford 33 mg (0.07
mol, yield 96%) of an amorphous white powder. IR (NaCl) νmax 3030
(C−Hsp2), 1740 (CO), 1273 (C−O) cm−1; MS m/z (rel intens)
487 [M + Na]+ (100), 381 [M + Na − C6H5CO]+ (9), 405 [M −
CH3CO2]+ (7); 1H NMR (400 MHz, CDCl3) δ 6.69 (1H, d, J = 11.4
Hz, H-12), 5.51 (1H, t, J = 3.4 Hz, H-3), 5.39 (1H, d, J = 10.6 Hz, H-
5), 3.64 (1H, dd, J = 14.0, 8.1 Hz, H-1α), 2.63 (1H, dd, J = 10.6, 3.6
Hz, H-4), 2.42 (1H, d, J = 13.4 Hz, H-7α), 2.29 (1H, m, H-2), 2.14
(1H, m, H-8α), 2.08 (3H, s, Me-22), 1.86 (3H, s, Me-20), 1.71 (1H,
td, J = 13.2, 2.5 Hz, H-7β), 1.64 (1H, dd, J = 13.9, 12.4, H-11), 1.47
(3H, d, J = 1.2 Hz, Me-17), 1.45−1.41 (2H, m, H-1β, H-8β), 1.17
(3H, s, Me-18), 1.06 (1H, m, H-9), 1.04 (3H, s, Me-19), 1.00 (3H, d, J
= 6.7 Hz, Me-16), δ 8.11 (2H, t, J = 7.1 Hz, H-3′), 7.62 (1H, t, J = 7.4
Hz, H-5′), 7.48 (2H, t, J = 7.7 Hz, H-4′) ppm; 13C NMR (100 MHz,
CDCl3) δ 195.3 (C-14), 171.2 (C-1′), 169.8 (C-21), 147.0 (C-12),
143.4 (C-6), 133.8 (C-13), 133.2 (C-5′), 130.3 (C-2′), 129.8 (C-3′/C-
7′), 128.6 (C-4′/C-6′), 118.7 (C-5), 94.9 (C-15), 81.9 (C-3), 51.7 (C-
4), 45.2 (C-1), 38.9 (C-2), 36.7 (C-7), 34.3 (C-9), 29.8 (C-11), 29.3
(C-18), 28.5 (C-8), 24.7 (C-10), 21.6 (C-22), 21.1 (C-17), 16.5 (C-
19), 14.2 (C-16), 12.4 (C-20) ppm; HRMS-ESI-TOF m/z calcd
C29H36O5Na (M+ + Na) 487.2455, found 487.2453.
133.3 (C-13), 132.3 (C-5′), 131.8 (C-3′/C-7′) 122.8 (C-2′), 118.9
(C-5), 113.8 (C-4′/C-6′), 94.9 (C-15), 81.7 (C-3), 55.7 (C-8′), 51.7
(C-4), 45.3 (C-1), 38.9 (C-2), 36.7 (C-7), 34.3 (C-9), 29.8 (C-11),
29.3 (C-18), 28.5 (C-8), 24.7 (C-10), 21.6 (C-22), 21.1 (C-17), 16.5
(C-19), 14.1 (C-16), 12.4 (C-20) ppm; HRMS-ESI-TOF m/z calcd
C30H38O6Na (M+ + Na) 517.2560, found 517.2565.
Jolkinoate L, 15β-Acetoxy-3β-(3-trifluormethylbenzoyloxy)-
lathyra-5E,12E-dien-14-one (12). 12 was obtained from reaction
with 3-trifluoromethylbenzoyl chloride (Sigma-Aldrich Chemie
GmbH, Riedstrasse D-89555, Steinhelm, Germany). The residue was
purified by column chromatography (silica gel, CH2Cl2/acetone =
19:1) and preparative TLC (two runs) with n-hexane/acetone (9:1) to
afford 12.6 mg (0.024 mol, yield 50%) of an amorphous white powder.
IR (NaCl) νmax 3020 (C−Hsp2), 1743 (CO), 1257 (C−O) cm−1;
−
MS m/z (rel intens) 555 [M + Na]+ (100), 318 [M − CH3CO2
−
F3CC6H4CO]+ (13); 1H NMR (400 MHz, CDCl3) δ 6.70 (1H, d, J =
12.4, H-12), 5.53 (1H, t, J = 3.4, H-3), 5.37 (1H, d, J = 10.6, H-5),
3.66 (1H, dd, J = 14.1, 8.1, H-1α), 2.65 (1H, dd, J = 10.6, 3.6, H-4),
2.42 (1H, d, J = 13.3, H-7α), 2.31 (1H, m, H-2), 2.14 (1H, m, H-8α),
2.08 (3H, s, Me-22), 1.86 (3H, d, J = 1.0, Me-20), 1.72 (1H, td, J =
13.1, 2.5, H-7β), 1.59 (1H, dd, J = 14.0, 12.3, H-11), 1.47 (3H, d, J =
1.4, Me-17), 1.45−1.41 (2H, m, H-1β, H-8β), 1.17 (3H, s, Me-18),
1.06 (1H, m, H-9), 1.04 (3H, s, Me-19), 1.00 (3H, d, J = 6.7, Me-16),
8.34 (1H, s, H-3′), 8.29 (1H, d, J = 7.8, H-5′), 7.87 (1H, d, J = 7.8, H-
7′), 7.65 (1H, t, J = 7.8, H-6′) ppm; 13C NMR (100 MHz, CDCl3) δ
195.5 (C-14), 170.2 (C-1′), 164.9 (C-21), 147.3 (C-12), 144.2 (C-6),
133.5 (C-13), 132.6 (C-7′), 131.7 (C-4′), 131.6 (C-2′), 130.1 (C-5′),
129.7 (C-6′), 126.7 (C-3′), 125.5 (C-8′), 118.7 (C-5), 95.1 (C-15),
83.0 (C-3), 51.9 (C-4), 45.6 (C-1), 39.2 (C-2), 37.0 (C-7), 34.7 (C-9),
30.1 (C-11), 29.6 (C-18), 28.8 (C-8), 25.1 (C-10), 21.6 (C-22), 21.4
(C-17), 16.74 (C-19), 14.5 (C-16), 12.7 (C-20) ppm; HRMS-ESI-
TOF m/z calcd C30H35F3O5Na (M+ + Na) 555.2334, found 555.2331.
Jolkinoate M, 15β-Acetoxy-3β-benzenesulfoxylathyra-5E,12E-
dien-14-one (13). 13 was obtained from reaction with benzene-
sulfonyl chloride (Sigma-Aldrich Chemie GmbH, Riedstrasse D-
89555, Steinhelm, Germany). The residue was purified by column
chromatography (silica gel, CH2Cl2/acetone (19:1)) and preparative
TLC with n-hexane/acetone (9:1) to afford 13 mg (0.024 mol, yield
60%) of an amorphous white powder. IR (NaCl) νmax 3015 (C−Hsp2),
1741 (CO), 1378 (SO) 1271 (C−O) cm−1; MS m/z (rel intens)
Jolkinoate J, 15β-Acetoxy-3β-(4-methylbenzoyloxy)lathyra-
5E,12E-dien-14-one (10). 10 was obtained from reaction with 4-
methylbenzoyl chloride (Sigma-Aldrich Chemie GmbH, Riedstrasse
D-89555, Steinhelm, Germany; 16.5 mg, 0.11 mol). The residue was
purified by two sequential flash column chromatography (silica gel, n-
hexane/CH2Cl2 = 2:1 and silica gel, CH2Cl2/MeOH = 99:1) to afford
15 mg (0.030 mol, yield 55%) of an amorphous white powder. IR
(NaCl) νmax 2960 (C−Hsp2), 1735 (CO), 1269 (C−O) cm−1; MS
m/z (rel intens) 501 [M + Na]+ (100), 419 [M − CH3CO2]+ (7), 318
1
[M − CH3CO2 − CH3C6H4CO]+ (6); H NMR (400 MHz, CDCl3)
δ 6.69 (1H, dd, J = 11.4, 0.9, H-12), 5.49 (1H, t, J = 3.3, H-3), 5.38
(1H, d, J = 10.6 Hz, H-5), 3.63 (1H, dd, J = 14.0, 8.1 Hz, H-1α), 2.62
(1H, dd, J = 10.6, 3.5 Hz, H-4), 2.42 (1H, m, H-7α), 2.27 (1H, m, H-
2), 2.13 (1H, m, H-8α), 2.08 (3H, s, Me-22), 1.86 (3H, d, J = 0.9 Hz,
Me-20), 1.70 (1H, td, J = 13.1, 2.5 Hz, H-7β), 1.60 (1H, dd, J = 13.9,
12.4 Hz, H-11), 1.46 (3H, d, J = 1.4 Hz, Me-17), 1.45 (1H, dd, J =
11.3, 8.0 Hz, H-8β), 1.26 (1H, dd, J = 7.6, 6.7 Hz, H-1β), 1.16 (3H, s,
Me-18), 1.06 (1H, m, H-9), 1.03 (3H, s, Me-19), 1.00 (3H, d, J = 6.7
Hz, Me-16), 8.01 (2H, dd, J = 18.1, 8.2 Hz, H-3′/H-7′), 7.30 (2H, dd,
J = 12.1, 8.0 Hz, H-4′/H-6′), 2.45 (3H, s, Me-8′) ppm; 13C NMR (100
MHz, CDCl3) δ 195.3 (C-14), 169.8 (C-21), 166.1 (C-1′), 147.0 (C-
12), 143.9 (C-6), 132.3 (C-13), 130.8 (C-5′), 129.8 (C-4′/C-6′),
129.3 (C-3′/C-7′), 127.7 (C-2′), 118.82 (C-5), 94.9 (C-15), 81.6 (C-
3), 51.7 (C-4), 45.2 (C-1), 38.9 (C-2), 36.7 (C-7), 34.3 (C-9), 29.8
(C-11), 29.3 (C-18), 28.5 (C-8), 24.7 (C-10), 22.0 (C-8′), 21.6 (C-
22), 21.1 (C-17), 16.4 (C-19), 14.1 (C-16), 12.4 (C-20) ppm; HRMS-
ESI-TOF m/z calcd C30H38O5Na (M+ + Na) 501.2611, found
501.2610.
1
523 [M + Na]+ (100), 381 [M + Na − C6H5O3S]+; H NMR (400
MHz, CDCl3) δ 6.57 (1H, d, J = 11.8 Hz, H-12), 5.14 (1H, d, J = 10.5
Hz, H-5), 4.90 (1H, t, J = 3.4 Hz, H-3), 3.47 (1H, dd, J = 14.0, 8.0 Hz,
H-1α), 2.45 (1H, dd, J = 10.5, 3.6 Hz, H-4), 2.19−2.06 (3H, m, H-7α,
H-8α, H-2), 1.98 (3H, s, Me-22), 1.79 (3H, d, J = 0.5 Hz, Me-20),
1.60 (1H, td, J = 12.9, 2.2 Hz, H-7β), 1.45 (1H, dd, J = 13.5, 13.0 Hz,
H-11), 1.36 (3H, d, J = 1.2 Hz, Me-17), 1.35−1.25 (2H, m, H-1β, H-
8β), 1.15 (3H, s, Me-18), 1.05 (1H, m, H-9), 1.00 (3H, s, Me-19),
0.91 (3H, d, J = 6.7 Hz, Me-16), 7.91 (2H, d, J = 8.6 Hz, H-2′/H-6′),
7.63 (1H, t, J = 7.4 Hz, H-4′), 7.54 (2H, t, J = 7.6 Hz, H-3′/H-5′)
ppm; 13C NMR (100 MHz, CDCl3) δ 194.91 (C-14), 169.7 (C-21),
147.0 (C-12), 143.2 (C-6), 137.6 (C-1′), 133.5 (C-13), 131.9 (C-2′/
C-6′), 129.0 (C-4′), 127.8 (C-3′/C-5′), 118.6 (C-5), 93.7 (C-15), 91.4
(C-3), 51.5 (C-4), 44.1 (C-1), 38.7 (C-2), 36.5 (C-7), 34.2 (C-9), 29.6
(C-11), 29.1 (C-18), 28.1 (C-8), 24.6 (C-10), 21.4 (C-22), 20.7 (C-
17), 16.3 (C-19), 14.0 (C-16), 12.2 (C-20) ppm; HRMS-ESI-TOF m/
z calcd C28H36O6SNa (M+ + Na) 523.2125, found 523.2127.
Jolkinoate K, 15β-Acetoxy-3β-(4-methoxybenzoyloxy)lathyra-
5E,12E-dien-14-one (11). 11 was obtained from reaction with 4-
methoxybenzoyl chloride (Sigma-Aldrich Chemie GmbH, Riedstrasse
D-89555, Steinhelm, Germany; 7.5 mg, 0.039 mol). The residue was
purified by two sequential flash column chromatography (silica gel,
CH2Cl2 and silica gel, CH2Cl2/MeOH = 99:1) to afford 14 mg (0.028
mol, yield 73%) of an amorphous white powder. IR (NaCl) νmax 3040
(C−Hsp2), 1740 (CO), 1273 (C−O) cm−1; MS m/z (rel intens)
Preparation of Jolkinodiol 3β,15β-Dihydroxylathyra-5E,12E-dien-
14-one (14). A mixture of Jolkinol D (20 mg, 0.055 mol) in MeOH/
KOH at 10% was stirred for 72 h at room temperature. The residue
was purified by column chromatography (silica gel, CH2Cl2/MeOH =
99:1 to 3:1) to afford 14 mg (0.044 mol, yield 81%) of an amorphous
white powder. IR (NaCl) νmax 3442, 3425, 1650, 1640 cm−1; 1H NMR
(400 MHz, CDCl3) δ 7.44 (1H, dd, J = 11.9, 0.8 Hz, H-12), 5.66 (1H,
d, J = 10.7 Hz, H-5), 3.96 (1H, t, J = 3.0 Hz, H-3), 3.43 (1H, dd, J =
14.0, 9.3 Hz, H-1α), 2.54 (1H, d, J = 13.2 Hz, H-7α), 2.28 (1H, dd, J =
10.7, 3.1 Hz, H-4), 2.17 (1H, br d, J = 14.1 Hz, H-8α), 2.02 (1H, m,
H-2), 1.82 (3H, d, J = 0.9 Hz, Me-20), 1.71 (1H, td, J = 13.1, 2.0 Hz,
H-7β), 1.56 (1H, td, J = 14.3, 2.3 Hz, H-11), 1.47−1.41 (2H, m, H-1β,
H-8β), 1.41 (3H, d, J = 1.2 Hz, Me-17), 1.18 (3H, s, Me-18), 1.11
1
517 [M + Na]+ (100), 435 [M − CH3CO2]+ (9); H NMR (400
MHz, CDCl3) δ 6.69 (1H, d, J = 11.4 Hz, H-12), 5.47 (1H, t, J = 3.3
Hz, H-3), 5.38 (1H, d, J = 10.6 Hz, H-5), 3.63 (1H, dd, J = 14.0, 8.1
Hz, H-1α), 2.61 (1H, dd, J = 10.7, 3.6 Hz, H-4), 2.42 (1H, d, J = 13.6
Hz, H-7α), 2.27 (1H, m, H-2), 2.14 (1H, m, H-8α), 2.08 (3H, s, Me-
22), 1.86 (3H, d, J = 0.9 Hz, Me-20), 1.70 (1H, td, J = 13.0, 2.4 Hz, H-
7β), 1.59 (1H, dd, J = 14.9, 11.2 Hz, H-11), 1.46 (3H, d, J = 1.4 Hz,
Me-17), 1.40 (1H, m, H-8β), 1.25 (1H, m, H-1β), 1.17 (3H, s, Me-
18), 1.06 (1H, m, H-9), 1.04 (3H, s, Me-19), 0.99 (3H, d, J = 6.7 Hz,
Me-16), 8.05 (2H, d, J = 8.9 Hz, H-3′/H-7′), 6.96 (2H, d, J = 8.9 Hz,
H-4′/H-6′), 3.89 (3H, s, H-6′) ppm; 13C NMR (100 MHz, CDCl3) δ
195.3 (C-14), 169.8 (C-21), 163.6 (C-1′), 146.9 (C-12), 143.3 (C-6),
757
dx.doi.org/10.1021/jm301441w | J. Med. Chem. 2013, 56, 748−760

Journal of Medicinal Chemistry
Article
(3H, d, J = 6.9 Hz, Me-16), 1.08 (3H, s, Me-19), 1.05 (1H, m, H-9)
ppm; 13C NMR (100 MHz, CDCl3) δ 198.1 (C-14), 151.8 (C-12),
141.9 (C-6), 132.4 (C-13), 120.2 (C-5), 92.7 (C-15), 81.3 (C-3), 53.3
(C-4), 46.1 (C-1), 38.9 (C-2), 36.7 (C-7), 35.1 (C-9), 29.9 (C-11),
29.3 (C-18), 28.3 (C-8), 24.9 (C-10), 21.0 (C-17), 16.4 (C-19), 14.4
(C-16), 12.5 (C-20) ppm; HRMS-ESI-TOF m/z calcd C20H30O3Na
(M+ + Na) 341.2087, found 341.2095.
4.2. Biological Studies. Cell Lines and Cultures. The L5178Y
mouse T-lymphoma cells (ECACC catalog no. 87111908, U.S. FDA,
Silver Spring, MD, U.S.) were transfected with the pHa MDR1/A
retrovirus as described in the literature.35 The MDR1-expressing cell
line was selected by culturing the infected cells with colchicine (60 ng/
mL), thus maintaining the MDR phenotype expression. L5178Y
(parental, PAR) mouse T-cell lymphoma cells and the human MDR1-
transfected subline (MDR) were cultured in McCoy’s 5A
supplemented with 10% heat-inactivated horse serum, 100 U/L L-
glutamine, and 100 mg/L penicillin−streptomycin mixture, all
obtained from Sigma-Aldrich (Sigma-Aldrich Kft, Budapest, Hungary).
The cell lines were then incubated in a humidified atmosphere (5%
CO2, 95% air) at 37 °C.
Antiproliferative Assay. The antiproliferative effects of the
compounds were tested in a range of decreasing concentrations (2-
fold dilutions) using PAR and MDR mouse lymphoma cell lines as
experimental model. The cells were distributed into 96-well flat
bottom microtiter plates at 1 × 105/mL for a final volume of 100 μL of
medium per well. The different concentrations of each compound
were added into duplicate wells. The plates were initially incubated at
37 °C for 72 h, and at the end of the incubation period, 15 μL of MTT
solution (thiazolyl blue tetrazolium bromide dissolved in PBS to a final
concentration of 5 mg/mL) was added to each well and incubated for
another 4 h. Then 100 μL of 10% SDS solution (sodium dodecyl
sulfate) was added into each well and the plates were further incubated
overnight at 37 °C. Cell growth was determined by measuring the
optical density (OD) at 550 nm (ref, 630 nm) with a Multiscan EX
ELISA reader (Thermo Labsystems, Cheshire, WA, U.S.). The
percentage of inhibition of cell growth was determined according to
eq 1.
Drug Combination Assay. The combination studies were designed
as suggested in the CalcuSyn36 software manual, using a fixed ratio of
the drugs across a concentration gradient. The dilutions of doxorubicin
(14.7−0.1 μM) were made in a horizontal direction and the dilutions
of resistance modifiers (at 2-fold of their IC50 values) vertically in a
microtiter plate to a final volume of 200 μL of medium per well. The
cells were distributed into plates at 2 × 105 cells/mL per well and were
incubated for 48 h under the standard conditions. The cell growth rate
was determined after MTT staining, as previously described. Drug
interactions were evaluated according to Chou using the software
CalcuSyn, version 2.36,37 Each dose−response curve (individual agents
as well as combinations) was fit to a linear model using the median
effect equation in order to obtain the median effect value
(corresponding to the IC50) and slope (m). Goodness-of-fit was
assessed using the linear correlation coefficient r, and only data from
analyses with r > 0.90 were presented. The extent of interaction
between drugs was expressed using the combination index (CI) for
mutually exclusive drugs. A CI close to 1 indicates additivity. CI < 1 is
defined as synergy and CI > 1 as antagonism.
Curve Fitting and Data Analysis. All results were expressed as the
mean SD unless otherwise noted. IC50 values were obtained by best
fitting the dose-dependent inhibition curves in GraphPad Prism 5
program.38,39 K-means clustering was performed with Tanagra, version
1.4.41,40,41 which subdivides the compounds (those with the nearest
mean) into groups that exhibit a high degree of similarity. FAR values
were submitted to statistical analysis, evaluating the coefficient
determination (r2) between each evaluated property and the logarithm
of 1/FAR.41
ASSOCIATED CONTENT
■
S
* Supporting Information
Evaluation of the stability of the esters under in vitro
experimental conditions. This material is available free of
charge via the Internet at http://pubs.acs.org.
⎡
100 − ⎢
⎣
⎤
OD
− ODmedium control
− ODmedium control
AUTHOR INFORMATION
sample
■
⎥ × 100
OD
⎦
(1)
cell control
Corresponding Author
*Phone: +351 217946475. Fax: +351 217946470. E-mail:
mjuferreira@ff.ul.pt.
All the experiments were performed in triplicate. The evaluation of the
stability of the esters, under these experimental conditions, is reported
in the Supporting Information.
Author Contributions
Assay for Rhodamine 123 Accumulation. The cells were adjusted
to a density of 2 × 106 cells/mL, resuspended in serum-free McCoy’s
5A medium, and distributed in 500 μL aliquots. The test compounds
were added at 2 and 20 μM. Verapamil (positive control, EGIS
Pharmaceuticals PLC, Budapest, Hungary) was added at 22 μM and
DMSO at 2% as solvent control. The samples were incubated for 10
min at room temperature, after which 10 μL (5.2 μM final
concentration) of rhodamine-123 was added to the samples. After a
20 min incubation at 37 °C, the samples were washed twice,
resuspended in 500 μL of phosphate buffered saline (PBS), and
analyzed by flow cytometry (Partec CyFlow Space instrument, Partec
All authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by FCT (Fundaca
̧
o para a Cien
̂
cia e a
̃
Tecnologia, Portugal) by Research Projects PTDC/QUI-QUI/
099815/2008 and PEst-OE/SAU/UI4013/2011. M.R. ac-
knowledges FCT for her Ph.D. Grant SFRH/BD/72915/
2010. The authors thank Dr. Teresa Vasconcelos, Instituto
Superior de Agronomia, Universidade de Lisboa, Portugal, for
the taxonomic work on the plant material.
GmbH, Munster, Germany). The resulting histograms were evaluated
̈
regarding mean fluorescence intensity (FL-1), standard deviation, and
peak channel of 20 000 individual cells belonging to the total and gated
populations. The fluorescence activity ratio (FAR) was calculated on
the basis of the quotient between FL-1 of treated/untreated resistant
cell line (MDR mouse lymphoma cells) over treated/untreated
sensitive cell line (PAR mouse lymphoma cells), according to eq 2.
ABBREVIATIONS USED
■
MDR, multidrug resistance; MDR1, multidrug resistance gene
1; TMD, transmembrane domain; NBD, nucleotide-binding
domain; P-gp, P-glycoprotein; DBP, drug-binding pocket; FAR,
fluorescence activity ratio; PAR, parental cell; log P, logarithm
of the octanol/water partition coefficient; MW, molecular
weight; ASA, accessible surface area; TPSA, topological polar
surface area
FL − 1MDR treated
FL − 1MDR untreated
(
(
)
)
FAR =
FL − 1PAR treated
FL − 1PAR untreated
(2)
The evaluation of the stability of the esters, under these experimental
conditions, is reported in the Supporting Information.
758
dx.doi.org/10.1021/jm301441w | J. Med. Chem. 2013, 56, 748−760

Journal of Medicinal Chemistry
Article
(20) Klepsch, F.; Ecker, G. F. Impact of the recent mouse P-
glycoprotein structure for structure-based ligand design. Mol. Inf. 2010,
29, 276−286.
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