Fluorinated amino-derivatives of the sesquiterpene lactone, parthenolide, as 19F NMR probes in deuterium-free environments

Article abstract of DOI:10.1021/jm201114t

The design, synthesis, and biological activity of fluorinated amino-derivatives of the sesquiterpene lactone, parthenolide, are described. A fluorinated aminoparthenolide analogue with biological activity similar to the parent natural product was discovered, and its X-ray structure was obtained. This lead compound was then studied using ¹⁹F NMR in the presence and absence of glutathione to obtain additional mechanism of action data, and it was found that the aminoparthenolide eliminates amine faster in the presence of glutathione than in the absence of glutathione. The exact changes in concentrations of fluorinated compound and amine were quantified by a concentration-reference method using ¹⁹F NMR; a major benefit of applying this strategy is that no deuterated solvents or internal standards are required to obtain accurate concentrations. These mechanistic data with glutathione may contribute to the conversion of the amino-derivative to parthenolide, the active pharmacological agent, in glutathione-rich cancer cells.

Full text of DOI:10.1021/jm201114t

Article
pubs.acs.org/jmc
Fluorinated Amino-Derivatives of the Sesquiterpene Lactone,
19
Parthenolide, as F NMR Probes in Deuterium-Free Environments
James R. Woods,† Huaping Mo,†,‡ Andrew A. Bieberich,† Tanja Alavanja,§ and David A. Colby*,†,§
†Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907, United States
‡Interdepartmental NMR Facility, Purdue University, West Lafayette, Indiana 47907, United States
§Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States
S
* Supporting Information
ABSTRACT: The design, synthesis, and biological activity of
fluorinated amino-derivatives of the sesquiterpene lactone,
parthenolide, are described. A fluorinated aminoparthenolide
analogue with biological activity similar to the parent natural
product was discovered, and its X-ray structure was obtained.
This lead compound was then studied using 19F NMR in the
presence and absence of glutathione to obtain additional
mechanism of action data, and it was found that the
aminoparthenolide eliminates amine faster in the presence of
glutathione than in the absence of glutathione. The exact
changes in concentrations of fluorinated compound and amine
were quantified by a concentration-reference method using 19
F
NMR; a major benefit of applying this strategy is that no deuterated solvents or internal standards are required to obtain accurate
concentrations. These mechanistic data with glutathione may contribute to the conversion of the amino-derivative to
parthenolide, the active pharmacological agent, in glutathione-rich cancer cells.
INTRODUCTION
■
The sesquiterpene lactones are a class of bioactive natural
products that commonly have an α-methylene-γ-lactone
substructure that reacts with nucleophilic sulfhydryl groups
present in enzymes, proteins, and glutathione.1 These thiol
additions may be reversible or irreversible, and they define the
mechanism of action of these natural products.1,2 However,
many of these compounds are poorly soluble in water, and this
issue has limited their potential therapeutic use in humans. To
address this shortcoming, amines have been added to the α-
methylene-γ-lactone substructure as an efficient way to enhance
the water solubility of the parent molecules and to retain their
biological activity. Amino-adducts have been prepared from the
sesquiterpene lactones, alantolactone,3 costunolide,4 partheno-
lide (1),5−7 α-santonin,8 helenalin,9 and ambrosin (Figure 1).10
A key amino-adduct of parthenolide (i.e., DMAPT or LC-1)
has advanced into clinical studies in humans11−13 despite a
long-standing bias in medicinal chemistry against using
molecules with a potential covalent mechanism of action in
clinical trials.2 Also, the sesquiterpene lactones, sausseramines
A−E, have a naturally occurring α-methylamino lactone
substructure rather than an α-methylene-γ-lactone.14,15
Figure 1. Structures of selected sesquiterpene lactones and the amino
derivative, DMAPT.
temperature within 12 h.15 Subsequently, Hsieh and co-workers
analyzed cell lysates by LC/MS/MS after treatment with an
amino-parthenolide derivative and found only the presence of
parthenolide.7 On the other hand, Crooks and co-workers have
It has been hypothesized that these α-methylamino lactone
derivatives serve as prodrugs in which the amine is released and
the enone is regenerated in the presence of the biological
nucleophiles (Figure 2).3,7,10,14,15 Yoshikawa and co-workers
have demonstrated that this retro-Michael reaction occurs with
the sausseramines in 1% aqueous HCl solution at room
Received: August 19, 2011
Published: October 27, 2011
© 2011 American Chemical Society
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fluorinated aminoparthenolide and parthenolide and the 19F
NMR data quantitatively demonstrate that elimination of the
fluorinated amine from the parthenolide derivative occurs more
rapidly in the presence of the biological thiol, glutathione, than
without glutathione.
Figure 2. Conversion of α-methylamino-γ-butyrolactones to α-
methylene-γ-butyrolactones and trapping with biological thiols.
RESULTS AND DISCUSSION
■
Chemistry. Our initial strategy was to prepare a series of
fluorinated amino-derivatives of the sesquiterpene lactone,
parthenolide (1), and to identify an analogue with biological
activity most similar to the parent compound. Interest in
parthenolide (1) has surged beyond other members of the
sesquiterpene lactones, because it was shown in 2005 to
selectively kill cancer stem cells isolated from patients with
myelogenous leukemia.29 We have previously synthesized
fluorinated derivatives of this key natural product30 and
demonstrated the ability of the parent compound and its
derivatives to selectively initiate apoptosis in multiple myeloma
cancer stem cell populations.31 Using the prior report of
Crooks to prepare amino-adducts,5 we focused our attention on
pyrrolidine and piperidine derivatives of parthenolide and
identified respective fluorinated amines that are commercially
available. Our choice of fluorinated amines corresponds well
with the recent studies from scientists at Merck.32 Additionally,
we selected amines that contain a trifluoromethyl group
because this functional group can be easily visualized by 19F
NMR. In other words, using 19F NMR with a low concentration
of fluorinated compound, the sensitivity for a trifluoromethyl
group is 3-fold higher than that of a single fluorine atom. Using
pyrrolidine, fluoro-substituted pyrrolidines, piperidine, and
fluoro-substituted piperidines, the parthenolide derivatives 2−
12 were prepared using the literature protocol (Table 1).5
shown that less than 3% of the aminoparthenolide, DMAPT,
degrades to parthenolide in cell culture buffer after a 24 h
incubation.5 On the basis of these literature precedents, we
aimed to design a derivative of parthenolide that could serve as
a probe for this retro-Michael reaction in the presence of the
biological thiol, glutathione. Such a mechanistic probe would
have potential use with other sesquiterpene lactones and
molecules with a covalent binding mechanism. A key requisite
to accomplish this objective would be to selectively observe
release of the amine from the parent molecule or prodrug.
The incorporation of fluorine onto molecules is a powerful
strategy in medicinal chemistry to modulate hydrophobic
character and decrease metabolism,16 and the derivatization of
bioactive natural products with fluorine has been attracting
considerable attention.17−19 Another powerful, yet somewhat
less-utilized role for fluorine is as a tag for 19F NMR. Indeed,
19F NMR is an analytical tool that is ideal for probing biological
1
systems with fluorinated compounds because, unlike the H
and 13C nuclei, there is no background signal for 19F.20−27
Fluorinated molecules have served as valuable 19F NMR probes
in high-throughput screening,20,26,27 drug metabolism,21 protein
binding,20,22,24,26,27 and assessing gene expression.23,25 We
hypothesized that the incorporation of fluorine onto the amine
on an aminoparthenolide would not adversely affect biological
activity. Additionally, we envisioned that the release of the
fluorinated amine from the parent compound would be
observed by 19F NMR, even at low concentrations, and
additional mechanism of action data could be obtained with
these 19F NMR probes (Figure 3). Recently, we have described
Table 1. Synthesis of Fluorinated Amino-Derivatives of
Parthenolide
Figure 3. Design strategy for 19F NMR probes using fluorinated α-
methylamino-γ-butyrolactones and the respective elimination of
fluorinated amines.
a concentration-reference NMR method to determine the
concentration of pharmaceutical agents without the use of
deuterated solvents or internal standards.28 This method
determines the concentration of analyte from solvent as a
reference and applies the data to the calculation of partition
coefficient, or log P. We now aim to extend the utility of this
NMR technique to 19F NMR because it will enable the direct
determination of concentration of a fluorinated pharmaceutical
agent in biological conditions where standards and deuterated
solvents are not normally compatible. Herein, we report the
design, synthesis, and biological activity of fluorinated amino-
derivatives of the sesquiterpene lactone, parthenolide, and the
subsequent application of the no-D concentration-reference
NMR technique to determine the concentration of fluorinated
compound and production of fluorinated amine using 19F
NMR. Our biological data establish similarities between the
a
Isolated yields.
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a
Good yields of the products were routinely obtained and each
compound was a single diastereomer, except compounds 3 and
5 that are inseparable 1:1 mixtures of diastereomers due to the
use of racemic 3-fluoro and 3-trifluoromethylpiperidine,
respectively. To unambiguously assign the stereochemistry of
the newly formed center on the lactone ring in compounds 2−
12, we obtained an X-ray structure of compound 8 (Figure 4).
Table 2. Antiproliferative Assay in HL-60 Cells
compd GI50 (95% Cl) μM
TGI (95% Cl) μM
LC50 (95% Cl) μM
1
0.52(0.19−1.42)
0.84(0.19−3.79)
1.13(0.64−20.1)
16.9(4.68−61.3)
1.69(0.46−6.20)
1.77(0.52−6.05)
0.69(0.07−7.12)
1.00(0.57−1.75)
0.75(0.41−1.40)
0.58(0.33−1.01)
24.7(11.9−51.1)
8.32 (4.21−16.5)
1.36 (0.65−2.85)
3.99(1.03−15.4)
12.2(1.98−75.7)
nd
1.88 (0.72−4.90)
10.1 (0.78−130)
nd
2
3
4
nd
5
7.29(1.64−32.5)
5.70 (2.24−14.5)
5.58(1.18−27.3)
2.14(1.10−4.15)
1.74(0.97−3.12)
1.18(0.68−2.03)
46.2(31.3−68.1)
18.3(8.17−41.1)
21.3(0.33−1400)
8.07(2.70−24.1)
17.7(0.45−700)
3.11 (1.19−8.15)
2.54(1.13−5.70)
1.66(0.76−3.62)
nd
6
7
8
9
10
11
12
23.8(9.17−61.7)
Figure 4. X-ray structures of aminoparthenolide derivatives: (A)
(11R)-13-dimethylammonio-11,13-dihydro-parthenolide salt (semi-
fumarate counterion removed for clarity),33 (B) compound 8.
a
Antiproliferative assays were conducted in the HL-60 (human
promyelocytic leukemia) cell line. nd is not determined.
As expected, the R-isomer at the C11 position is formed
exclusively, and this result is similar to literature precedent.6
The identity of the stereocenter and conformation of the
macrocycle 8 correlate well with prior X-ray data from the
aminoparthenolide, DMAPT.33
for any two compounds, an extra sum-of-squares F test is used
to determine whether a better fit is achieved via a single model
or separate models for the two compounds, p value <0.05.42,43
Parthenolide (1) displays activity in our assay (GI50 = 0.52
μM) similar to previous reports in HL-60 cells.34 Amino-
derivatives 2, 3, and 5−10 manifest similar GI50 values in the
assay compared with 1. Indeed, most amino-derivatives of the
sesquiterpene lactones produced biological activity very similar
to the parent compound across many types of assays.3−10
Notable exceptions in this series of molecules are the 3,3-
difluoropiperidine analogue 4, the (S)-3-fluoropyrrolidine
analogue 11, and the 3,3-difluoropyrrolidine analogue 12. A
rationale for the comparison of the more potent activity of 4,4-
difluoropiperidine 7 (GI50 = 0.69 μM) to the less potent 3,3-
difluoropiperidine 4 (GI50 = 16.9 μM) may be the attenuated
basicity of the piperidine nitrogen on the later compound. This
relationship between fluorination and pKa for 3,3-difluoropiper-
idine and 4,4-difluoropiperidine has been elegantly described by
Antiproliferative Activity. The biological activity ex-
hibited by each of the parthenolide derivatives 2−12, as well
as parthenolide (1), was determined using an antiproliferative
assay with HL-60 (human promyelocytic leukemia) cells,
because the major application of parthenolide and its
derivatives is as an antileukemic agent.34 To ensure that the
fluorinated lead compound most resembled the biological
activity of the parent compound in our assays, a fine resolution
cancer cell growth inhibition assay was developed and
implemented. Typically, the NCI60 human tumor cell line
anticancer drug screen is a significant tool in cancer research; it
has solved technical barriers to in vitro drug screening35 and has
generated orthogonal benefits in the form of multitissue
systems biology studies that would not have been possible prior
to its existence.36−40 However, the NCI60 screen was designed
for applications in a high throughput manner for hundreds of
thousands of compounds that manifest varying activity.34
Therefore, each compound is screened with only five 10-fold
dilution steps, and a linear interpolation analytical method is
used that avoids fitting a model to estimate GI50, TGI, and
LC50. While this strategy reduces the time and resources
needed to characterize many compounds, fitting a parametric
model to dose−response data allows the benefit of pairwise
statistical comparison of GI50, TGI, and LC50 across
compounds.41 The design of the HL-60 assay employed for
compounds 1−12 has its explicit goal as the ability to
characterize the antiproliferative properties of sets of synthetic
analogues to explore structural variation of a parent molecule.
In this case, quantitative shifts in GI50, TGI, or LC50 should be
discernible among structural variants even though such shifts
may occur within a relatively narrow dosage range considered
to be pharmaceutically relevant (the nanomolar to low
micromolar range). Individual compounds 1−12 were tested
in quadruplicate across 72 h, and GI50 and TGI values were
calculated along with the 95% confidence interval (Table 2).
The LC50 values for 1, 2, 5−10, and 12 were also determined.
Specifically, a sigmoid inhibitory dose−response model was fit
to the data for each compound using nonlinear regression via a
least-squares fit with propagation of error throughout normal-
ization calculations. For the best-fit values of GI50, TGI, or LC50
Diederich, Kansy, and Muller.44 The biological activity (i.e.,
̈
GI50 values) across compounds 10−12 is intriguing, because
the (S)-3-fluoropyrrolidine analogue 11 is almost 50-fold less
potent than the (R)-3-fluoropyrrolidine analogue 10. Fur-
thermore, the 3,3-difluoropyrrolidine 12 displays activity that
can be envisioned to be a 1:1 mixture of both (R)-10 and (S)-
11. Analysis of the LC50 values distinguishes aminoparthenolide
analogues 2, 3, and 5−7 from parthenolide (1) and 8−10. Even
though many of the parthenolide derivatives inhibit cell growth,
only a subset of compounds (i.e., 8−10) eliminate the HL-60
cell population at a level similar to the parent compound, and
only two fluorinated compounds (i.e., 8 and 10) exhibit this
effect. After this analysis of the biological data, we selected
aminoparthenolide 8 for further studies with 19F NMR because
this compound has the requisite trifluoromethyl group and
maintains activity similar to the parent compound (1). Side-by-
side plots of the growth inhibition curves for parthenolide 1
and lead compound 8 show the high level of similarity between
the biological activities in treated HL-60 cells (Figure 5). The
slight divergence between the GI50 values was not found to be
statistically significant.
19F NMR and Quantitative Studies. With the amino-
parthenolide 8 in hand, we now aimed to use the compound as
a
19F NMR probe to elucidate the role of its structural
alteration in the presence of glutathione and to acquire
additional mechanism of action data for aminoparthenolides.
Higher levels of the key biological thiol, glutathione, have been
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elimination of the amine from 8. Ultimately, 80 °C was selected
because some elimination of the amine occurred after 24 h
(Figure 6A) and no other byproducts were observed in the full
Figure 5. Comparison of growth inhibition of HL-60 cells between
parthenolide (1) and compound 8 after three days. Data for
parthenolide (1) are colored in black and compound 8 are colored
in red. GI50 values for 1 and 8 are not statistically significantly
different. The horizontal dotted line marks 100% day 0 cell density
(i.e., the original population size of the cells when they were first
seeded into the wells).
observed in cancer cells over nonmalignant cells,45,46 and these
data have been recently exploited by Boger and co-workers for
the preferential activation of prodrugs of duocarmycin-based
anticancer agents in cancer cells.47 Glutathione is especially
important in the environment of cancer cell populations, and
Chmielewski,48 Finn,49 and others50 have described fluores-
cence probes for imaging cellular thiols such as glutathione. 19F
NMR probes for measuring the presence of glutathione are rare
in the literature;51 however, an advantage for 19F labeling over
fluorescent labeling is that structural data for the molecule and
its derivatives can be gathered by 19F NMR. Moreover, our
studies were planned to extend the use of fluorinated agents in
gathering a mechanism of action by measuring changes to the
compound itself rather than the presence/reactivity of the
biological thiol. The key feature in compound 8 is that the
trifluoromethyl group is present on the parent compound but
will also be present on the amine upon elimination (see Figure
3). The change in signal will be recorded in the 19F NMR
spectrum, and the change in concentration will be recorded
using the no-D concentration-reference method (see below).28
Indeed, previous 19F NMR methods routinely require the
addition of deuterated solvent,20−22,51 and the significance of
our plan is that deuterated solvents are not needed. Also, 19F
NMR data can be acquired rapidly with high sensitivity. Our
initial studies were conducted at pH 7.4 (biological pH) using
compound 8 (1 mM) in the presence of glutathione (13.5 mM)
and in the absence of glutathione; common biological
concentrations of glutathione are 1−15 mM.48 To ensure
complete solubility of these components at biological pH, a 7:3
DMSO/H2O (v/v) solvent mixture was chosen; the major
limiting factor was the solubility of 8 in aqueous solution. Even
though the aqueous solubility of DMAPT was significantly
enhanced by conversion to its fumarate salt,5 our attempts to
convert 8 to its fumarate salt using this method were not
successful because the amine salt would not crystallize from
organic solvents (e.g., Et2O and EtOH). Although a high
percentage of DMSO limits the direct translation for our results
to aqueous biological environments, it ensured complete
solubility throughout the experiments and has been routinely
used as a cosolvent for observing Michael additions.2,49 Next,
preliminary studies were conducted at 37 °C, 60 °C, 80 °C, and
100 °C to identify temperatures that would promote the
Figure 6. 19F NMR spectrum across three days depicting the
production of 4-trifluoromethylpiperidine from aminoparthenolide 8
in the absence and presence of glutathione. (A) Time course of 1 mM
compd 8 in 7:3 DMSO/H2O (pH 7.4) at 80 °C. (B) Time course of 1
mM compd 8 in the presence of 13.5 mM glutathione in 7:3 DMSO/
H2O (pH 7.4) at 80 °C.
19F NMR spectrum even after extended reaction times, such as
three days (data not shown). Therefore, studies were
conducted across a three day time-course (similar to the
antiproliferative assay) and 19F NMR spectra were obtained at
24 h for samples at 80 °C with and without glutathione (Figure
6). Only 128 scans were required, and thus, the total acquisition
time for each 19F NMR spectrum was 3 min 24 s with a typical
sensitivity of 40:1 or better. The data clearly display the
production of 4-trifluoromethylpiperidine from compound 8 in
the presence and absence of glutathione; however, the
production of the free amine is nearly complete in the presence
of glutathione whereas a much smaller amount is generated
when glutathione is not present.
To quantify the concentration changes in the parent
compound 8 and the 4-trifluoromethylpiperidine, we applied
our concentration-reference method28 and carried out the two
reactions side-by-side, each in triplicate (Figure 7). The
concentrations were calculated as the average of the three
trials, plotted across the 72 h time-course, and referenced to the
first measurement (t = 0 h, 1 mM compound 8). Again, neither
deuterated solvents nor internal standards were used. In the
absence of glutathione, the production of 4-trifluoromethylpi-
peridine rose to nearly 167 μM and leveled off across three
days. However, in the presence of glutathione, the concen-
tration of 4-trifluoromethylpiperidine rose to nearly 0.7 mM,
while the concentration of compound 8 continually decreased
to nearly 170 μM (starting from 1.0 mM). This concentration-
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mechanism of action data, and it was found that a higher
concentration of amine was observed from the amino-
parthenolide in the presence of glutathione. The exact changes
in concentrations were quantified by 19F NMR without the
necessity of deuterated solvents or the addition of standards
using a concentration-reference technique. We assert that the
implications of the mechanistic data obtained from these
studies are that if the amine is eliminated in the presence of a
nucleophilic thiol, the addition of the thiol to the compound
(now a Michael acceptor) will out-complete the readdition of
the amine. This action may contribute to the overall
mechanism of action and cellular selectivity of the compound.
For example, in populations of cancer cells with higher levels of
glutathione than nonmalignant cells, the conversion of an
aminoparthenolide to parthenolide may be more prevalent. The
main limitation of our 19F NMR studies is that a large portion
of DMSO was required to maintain the solubility of the
aminoparthenolide derivative, and this fact may limit the
translational potential of these data. The preparation of highly
water-soluble fluorinated amino-compounds is clearly the next
objective as well as the direct observation and quantification of
concentration changes of fluorinated compounds in cell culture
and other biological systems. Also, the studies detailed here
contribute to developing a general method of creating
fluorinated amino-derivatives of compounds with covalent-
binding modes for quantitative mechanism of action studies in
living systems by 19F NMR. Additional studies to identify
fluorinated amines with hydrophilic substituents to enhance the
aqueous solubility of subsequent amino-adducts are currently
underway.
Figure 7. Graph illustrating the NMR quantification of the production
of 4-trifluoromethylpiperidine from aminoparthenolide 8 during three
days in the absence and presence of glutathione. The concentrations of
4-trifluoromethylpiperidine and 8 are given as the average values
across three trials with error depicted as two standard deviations.
Concentrations are referenced to the first measurement (t = 0 h, 1
mM). Concentrations for 4-trifluoromethylpiperidine are highlighted
in red, and concentrations for 8 are highlighted in blue. (A)
Concentrations from a 72 h time course starting with 1 mM 8 in
7:3 DMSO/H2O (pH 7.4) at 80 °C. (B) Concentrations from a 72 h
time course starting with 1 mM 8 in the presence of 13.5 mM
glutathione in 7:3 DMSO/H2O (pH 7.4) at 80 °C.
EXPERIMENTAL SECTION
■
Chemistry. The synthesis of compounds 2−12 was conducted in a
manner similar to the literature procedure.5 Characterization data for
the new compounds 3−8 and 10−12 are listed below. Combustion
analysis was conducted to establish a level of ≥95% purity.
(11R)-13-(3-Fluoropiperdine)-11,13-dihydroparthenolide
1
(3). Yield 79%; mp = 132−136 °C. H NMR (500 MHz, CDCl3) δ
5.20 (d, J = 12.0 Hz, 1H), 4.69−4.52 (m, 1H), 3.84 (t, J = 9.1 Hz, 1H),
3.83* (t, J = 9.1 Hz, 1H), 2.84* (t, J = 5.0 Hz, 1H), 2.82 (t, J = 5.0 Hz,
1H), 2.78−2.63 (m, 3H), 2.55−2.02 (m, 11H), 1.82 (m, 2H), 1.70 (s,
3H), 1.62 (m, 2H), 1.50 (m, 1H), 1.29 (s, 3H), 1.25 (m, 1H). 13C
reference method can quickly quantify low micromolar concen-
NMR (125 MHz, CDCl3) δ 176.6, 176.5*, 134.7, 124.8, 87.8 (d, JCF
=
trations of analyte in solution with high sensitivity. Overall, our 19
F
171 Hz, 1C), 82.4, 66.7, 61.5, 58.4* (d, JCF = 22.1 Hz, 1C), 58.1 (d, JCF
= 22.5 Hz, 1C), 56.8*, 56.0, 53.7*, 53.6, 48.1*, 47.4, 46.4*, 46.2, 41.1,
36.6, 30.2, 30.1*, 29.7 (d, JCF = 19.5 Hz, 1C), 29.6* (d, JCF = 19.8 Hz,
1C), 24.0, 22.0 (d, JCF = 6.4 Hz, 1C), 21.9 (d, JCF = 6.1 Hz, 1C), 17.2,
17.1. 19F NMR (282 MHz, CDCl3) δ −181.56 (m, 1F), −182.08* (m,
1F). IR (film) νmax 2928, 1769, 1200, 1112, 1073, 989 cm−1. HRMS
(EI) m/z calcd for C20H30FNO3 (M + H)+ 352.2288, found 352.2284.
Anal. Calcd for C20H30FNO3: C, 68.35; H, 8.60; N, 3.99. Found: C,
68.46; H, 8.48; N, 3.91. * denotes peak of diastereomer.
NMR data shows that elimination of the fluorinated amine
from the parent aminoparthenolide derivative occurs more
rapidly in the presence of glutathione than in the absence of
glutathione. The implications of these findings are that if the
amine is eliminated in the presence of a biological thiol, the
parent compound (now with a Michael acceptor) will
preferentially react with the thiol rather than recombine with
the amine. These data and assertions correlate well with the
previous work of Finn and co-workers, which describes that
biological thiols can easily outcompete amines in binding to
Michael acceptors.49
(11R)-13-(3,3-Difluoropiperdine)-11,13-dihydroparthenolide
1
(4). Yield 89%; mp =182−186 °C. H NMR (300 MHz, CDCl3) δ
5.19 (d, J = 11.8 Hz, 1H), 3.84 (t, J = 9.0 Hz, 1H), 2.84 (s, 1H), 2.82
(s, 1H), 2.80−2.00 (m, 13H), 1.95−1.72 (m, 4H), 1.69 (s, 3H), 1.60
(m, 1H), 1.28 (s, 1H), 1.24 (m, 1H). 13C NMR (125 MHz, CDCl3) δ
176.4, 134.7, 124.7, 120.1 (t, JCF = 241 Hz), 82.5, 66.6, 61.5, 59.2 (t,
JCF = 27.5 Hz), 55.3, 52.8, 47.4, 46.4, 41.0, 36.6, 32.0 (t, JCF = 22.9
Hz), 30.0, 24.0, 22.1 (t, JCF = 4.4 Hz, 1C), 17.2, 17.0. 19F NMR (282
MHz, CDCl3) δ −100.5 (d, JFF = 236 Hz, 1F), −101.8 (d, JFF = 236
Hz, 1F). IR(film) νmax 2925, 1766, 1172, 1117, 1003 cm−1. HRMS
(ESI) m/z calcd for C20H29F2NO3 (M + H)+ 370.2194, found
CONCLUSIONS
■
The design, synthesis, and biological activity of fluorinated
amino-derivatives of the sesquiterpene lactone, parthenolide,
were described. During the course of this work, we have
identified an aminoparthenolide with biological activity similar
to the parent natural product and obtained its X-ray structure.
This lead compound was then studied using 19F NMR in the
presence and absence of glutathione to obtain additional
370.12194; [α]22 −6.2° (c 1.39, CHCl3). Anal. Calcd for
D
C20H29F2NO3·0.5H2O: C, 63.47 H, 7.99; N, 3.70. Found: C, 63.50;
H, 7.61; N, 3.52.
7938
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Journal of Medicinal Chemistry
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(11R)-13-(3-(Trifluoromethyl)piperdine)-11,13-dihydropar-
CDCl3) δ 5.22 (m, 1H), 5.18−5.05 (m, 1H), 3.83 (t, J = 8.7 Hz, 1H),
2.99−2.72 (m, 6H), 2.53−1.99 (m, 11H), 1.69 (s, 3H), 1.63 (m, 1H),
1.29 (s, 3H), 1.25 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 176.3,
1
thenolide (5). Yield 64%. H NMR (300 MHz, CDCl3) δ 5.19 (d, J
= 12.0 Hz, 1H), 3.84 (t, J = 9.1 Hz, 1H), 3.82* (t, J = 9.0 Hz, 1H),
3.03−2.67 (m, 5H), 2.48−1.89 (m, 12H), 1.78 (m, 1H), 1.70 (s, 3H),
1.65−1.42 (m, 3H), 1.30 (s, 3H), 1.25 (m, 1H). 13C NMR (125 MHz,
CDCl3) δ 176.3, 176.2*, 134.6, 126.8 (q, J = 278 Hz, 1C), 124.9, 82.3,
66.6, 61.5, 57.5*, 56.3, 54.5, 53.7*, 53.5*, 52.7, 48.7*, 47.6, 46.4,
46.3*, 41.2, 41.1 (q, JCF = 26 Hz, 1C), 36.6, 30.1, 30.0*, 24.3, 24.1,
23.9*, 22.9, 17.2, 17.0. 19F NMR (282 MHz, CDCl3) δ −72.15 (d, JHF
= 27.9 Hz, 3F); IR (film) νmax 2927, 1770, 1257, 1124, 1093, 992
cm−1. HRMS (ESI) m/z calcd for C21H30F3NO3 (M + H)+ 402.2256,
found 402.2260. Anal. Calcd for C21H30F3NO3·1.0EtOAc: C, 61.33; H,
7.82; N, 2.86. Found: C, 63.15; H, 7.82; N, 2.90. * denotes peak of
diastereomer.
134.6, 125.0, 93.5 (d, JCF = 175 Hz, 1C), 82.2, 66.5, 61.5, 61.3 (d, JCF
=
22.6 Hz, 1C), 53.1, 52.9, 47.3, 47.2, 41.1, 36.6, 32.7 (d, JCF = 22.0 Hz,
1C), 30.0, 24.1, 17.2, 16.9. 19F NMR (282 MHz, CDCl3) δ −168.80
(dqu, JHF = 55.8, 28.8 Hz, 1F). IR (film) νmax 2919, 1771, 1252, 1176,
1073, 988 cm−1. HRMS (EI) m/z calcd for C19H28FNO3 (M + H)+
338.2131, found 338.2133; [α]27D −19.6° (c 0.48, CHCl3). Anal. Calcd
for C19H28FNO3: C, 67.63; H, 8.36; N, 4.15. Found: C, 67.45; H, 8.50;
N, 3.98.
(11R)-13-(3−3-Difluoropyrrolidine)-11,13-dihydropartheno-
lide (12). Yield 78%; mp =154−156 °C. 1H NMR (300 MHz,
CDCl3) δ 5.20 (d, J = 11.6 Hz, 1H), 3.84 (t, J = 8.9 Hz, 1H), 3.01−
2.88 (m, 4H), 2.81 (td, J = 7.0, 2.6 Hz, 2H), 2.73 (d, J = 8.9 Hz, 1H),
2.41−1.96 (m, 10H), 1.70 (s, 3H), 1.62 (m, 1H), 1.30 (s, 3H), 1.24
(11R)-13-(4-Fluoropiperdine)-11,13-dihydroparthenolide
1
(6). Yield 89%; mp = 142−144 °C. H NMR (300 MHz, CDCl3) δ
(m, 1H). 13C NMR (125 MHz, CDCl3) δ 176.0, 134.5, 129.6 (t, JCF
=
5.20 (d, J = 5.9 Hz, 1H), 4.66 (d, J = 9.3, 1H), 3.83 (t, J = 5.4 Hz, 1H),
2.81 (dd, J = 8.1, 2.9 Hz, 1H), 2.73−2.64 (m, 3H), 2,72 (d, J = 5.4 Hz,
1H), 2.44−2.35 (m, 4H), 2.29−2.04 (m, 6H), 1.96−1.78 (m, 4H),
1.70 (s, 3H). 1.64 (m, 1H), 1.30 (s, 3H), 1.23 (m, 1H). 13C NMR
(125 MHz, CDCl3) δ 176.4, 134.6, 124.9, 88.3 (d, JCF = 170 Hz, 1C),
82.2, 66.6, 61.5, 56.3, 50.3 (d, JCF = 6.4 Hz, 2C), 48.1, 46.5, 41.2, 36.6,
31.6 (d, JCF = 19.5 Hz, 2C), 30.1, 24.1, 17.2, 17.0.19F NMR (282 MHz,
CDCl3) δ −180.27 (m, 1F). IR (film) νmax 2929, 1770, 1174, 1039,
1006, 987 cm−1. HRMS (ESI) m/z calcd for C20H30FNO3 (M + H)+
352.2288, found 352.2285; [α]28D −5.3° (c 0.60, CHCl3). Anal. Calcd
for C20H30FNO3: C, 68.35; H, 8.60; N, 3.99. Found: C, 68.50; H, 8.70;
N, 3.82.
247 Hz, 1C), 125.2, 82.3, 66.4, 62.4 (t, JCF = 29 Hz, 1C), 61.5, 52.9,
52.8, 47.3, 47.0, 41.1, 36.6, 35.8 (t, JCF = 24 Hz, 1C), 29.9, 24.1, 17.2,
16.9. 19F NMR (282 MHz, CDCl3) δ −92.58 (dqu, JFF = 229 Hz, JHF
=
14.4 Hz, 1F), −93.50 (dqu, JFF = 229 Hz, JHF = 14.4 Hz, 1F). IR (film)
νmax 2927, 1772, 1264, 1109, 1073, 990 cm−1. HRMS (EI) m/z calcd
for C19H27F2NO3 (M + H)+ 356.2037, found 356.2033; [α]24 −9.9°
D
(c 0.86, CHCl3). Anal. Calcd for C19H27F2NO3·0.5H2O: C, 62.62; H,
7.74; N, 3.84. Found: C, 63.55; H, 7.56; N, 3.67.
Cell Culture.34 HL-60 (human promyelocytic leukemia) cells
were obtained from ATCC. Cell culture was maintained in phenol red
free RPMI-1640 supplemented with 10% fetal bovine serum and filter
sterilized (hereafter referred to as “medium”). Cells were cultured at
37 °C in a humidified incubator under 5% CO2 in air.
(11R)-13-(4,4-Difluoropiperdine)-11,13-dihydroparthenolide
1
(7). Yield 75%. mp =148−152 °C. H NMR (300 MHz, CDCl3) δ
Cell Proliferation Assay.34 The GI50, TGI, and LC50 values for
HL-60 cell proliferation in the presence of parthenolide (1) and its
amino-derivatives 2−12 were determined by MTT assay. On day 0,
HL-60 cells were seeded in 96-well plates at 5000 cells per well in 100
μL medium (day 0 plate) and 80 μL medium (day 3 plate). “Day 0”
plates contained alternating columns of wells with medium-only and
medium + cells for the purpose of providing day 0 cell density
measurements and requisite background Ab 570 nm controls during
plate reading. “Day 3” plates contained one column of medium-only
wells as background light absorbance controls (wells A1−H1), one
column of cells to receive vehicle control (0.5% DMSO in medium,
wells A2−H2), and 10 columns of cells to receive dilution series for
two compounds (compound 1 in wells E−H of each column and
compound 2 in wells A−D of each column). Immediately after
assembly, treatment wells of the day 3 plate received 20 μL of a
solution of test compound dissolved in medium + DMSO for a final
concentration of 0.5% DMSO in growth medium. Each compound
was tested at 10 concentrations in quadruplicate. The cells in day 3
plate column 2 received the vehicle control at this time. Immediately
after assembly, the day 0 plate was processed with an MTT protocol:
20 μL MTT solution (2.5 mg/mL) in serum-free RPMI-1640 was
added to each well. The plate was then incubated for 4 h, after which
each well received 100 μL of acidic isopropyl alcohol (10% Triton X-
100 plus 0.1 N HCl in anhydrous isopropyl alcohol). Formazan
crystals were allowed to solubilize for 16 h. After complete
solubilization of formazan crystals, Ab 570 nm was measured using a
Spectramax 384 Plus plate reader (Molecular Devices). An identical
MTT assay was performed with each day 3 plate after 72 h incubation
postassembly. All plates were incubated at 37 °C in a humidified
incubator under 5% CO2 in air. GI50, TGI, and LC50 values for HL-60
cell proliferation were calculated by GraphPad Prism 5 software. A full
description of these calculations is provided in the Supporting
Information.43
5.19 (d, J = 10.0 Hz, 1H), 3.84 (t, J = 9.1 Hz, 1H), 2.81 (qd, J = 13.7,
5.0 Hz, 2H), 2.71 (d, J = 8.9 Hz, 1H), 2.64 (t, J = 5.4 Hz, 4H), 2.47−
1.90 (m, 12H), 1.70 (s, 3H), 1.63 (m, 1H), 1.29 (s, 3H), 1.26 (m,
1H). 13C NMR (125 MHz, CDCl3) δ 176.2, 134.5, 125.1, 121.6 (t, JCF
= 240 Hz, 1C), 82.2, 66.5, 61.5, 55.3, 50.8 (t, JCF = 4.9 Hz, 2C), 47.0,
46.7, 41.2, 36.6, 34.0 (t, JCF = 22.8 Hz, 2C), 30.0, 24.1, 17.2, 17.0. 19F
NMR (282 MHz, CDCl3) δ −98.27 (m, 2F). IR νmax 2923, 1764,
1254, 1132, 1100, 991 cm−1. HRMS (ESI) m/z calcd for
C20H29F2NO3 (M + H)+ 370.2194, found 370.2191; [α]24 −11.6°
D
(c 0.56, CHCl3). Anal. Calcd for C20H29F2NO3: C, 65.02; H, 7.91; N,
3.79. Found: C, 64.66; H, 7.76; N, 3.75.
(11R)-13-(4-(Trifluoromethyl)piperdine)-11,13-dihydropar-
1
thenolide (8). Yield 80%; mp = 182−184 °C. H NMR (300 MHz,
CDCl3) δ 5.20 (d, J = 11.3 Hz, 1H), 3.83 (t, J = 9.0 Hz, 1H), 2.92 (d, J
= 10.5 Hz, 2H), 2.80 (dd, J = 13.6, 4.9 Hz, 1H), 2.72 (d, J = 9.0 Hz,
1H), 2.70 (dd, J = 13.6, 4.9 Hz, 1H), 2.47−1.96 (m, 11H), 1.83 (m,
2H), 1.70 (s, 3H), 1.63−1.49 (m, 3H), 1.29 (s, 3H), 1.25 (m, 1H). 13
C
NMR (125 MHz, CDCl3) δ 176.4, 134.6, 127.1 (q, JCF = 277 Hz, 1C),
125.0, 82.2, 66.6, 61.5, 56.5, 53.6, 52.7, 48.1, 46.4, 41.2, 40.1 (q, JCF
=
27.2 Hz, 1C), 36.6, 30.1, 24.8, 24.7, 24.1, 17.2, 17.0. 19F NMR (282
MHz, CDCl3) δ −73.86 (d, JHF = 7.7 Hz 3F). IR (film) νmax 2930,
1769, 1254, 1137, 1083, 988 cm−1. HRMS (ESI) m/z calcd for
C21H30F3NO3 (M + H)+ 402.2256, found 402.2260; [α]24 −2.6° (c
D
0.26, CHCl3). Anal. Calcd for C21H30F3NO3: C, 62.83; H, 7.53; N,
3.49. Found: C, 62.86; H, 7.45; N, 3.49.
(11R)-13-((R)-(−)-3-Fluoropyrrolidine)-11,13-dihydroparthe-
1
nolide (10). Yield 83%; mp = 114−122 °C. H NMR (300 MHz,
CDCl3) δ 5.23 (m, 1H), 5.18−5.04 (m, 1H), 3.83 (t, J = 8.7 Hz, 1H),
3.03−2.67 (m, 6H), 2.52−2.03 (m, 11H), 1.69 (s, 3H), 1.62 (m, 1H),
1.30 (s, 3H), 1.25 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 176.3,
134.7, 125.0, 93.2 (d, JCF = 175 Hz, 1C), 82.2, 66.5, 61.5, 61.3 (d, JCF
=
22.5 Hz, 1C), 53.2, 53.1, 47.5, 47.4, 41.1, 36.6, 32.9 (d, JCF = 21.9 Hz,
1C), 30.0, 24.1, 17.2, 16.9. 19F NMR (282 MHz, CDCl3) δ −168.59
(dqu, JHF = 56.4, 28.8 Hz, 1F). IR (film) νmax 2923, 1770, 1252, 1153,
1072, 989 cm−1. HRMS (EI) m/z calcd for C19H28FNO3 (M + H)+
338.2131, found 338.2132; [α]25D −9.8° (c 0.41, CHCl3). Anal. Calcd
for C19H28FNO3·1.0MeOH: C, 65.02; H, 8.73; N, 3.79. Found: C,
67.06; H, 8.35; N, 3.60.
19F NMR and Quantitative Studies. A solution of glutathione
(69 mg, 0.22 mmol) in water (4.5 mL) and DMSO (10.5 mL) was
titrated to pH 7.4 (Fisher Scientific, Accumet Basic) using 1.0 M
NaOH (220 μL). Next, glutathione solution (2.7 mL, pH 7.4) was
treated with a 10 mM solution of compound 8 in DMSO (300 μL),
and an aliquot of the resultant solution was immediately transferred to
an NMR tube for 19F NMR analysis. The remaining solution of
(11R)-13-((S)-(+)-3-Fluoropyrrolidine)-11,13-dihydroparthe-
1
nolide (11). Yield 86%; mp = 116−118 °C. H NMR (300 MHz,
7939
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Journal of Medicinal Chemistry
Article
compound 8 and glutathione was heated to 80 °C in an oil bath, and
additional aliquots were obtained at 24, 48, and 72 h for 19F NMR
analysis. Side-by-side control experiments were performed without any
glutathione using the same procedures and reaction conditions, and
aliquots were also taken for 19F NMR analysis. The 1D 19F NMR
spectra were acquired without lock on a Bruker DPX 300 MHz
equipped with a QNP probe operating at rt. The sweep width was 253
ppm, acquisition time was 458 ms and the relaxation delay between
scans was 1 s. For each fluorine spectrum, 128 scans were accumulated
after 8 dummy scans. All FID were line-broadened by 3 Hz, Fourier
transformed, manually phased and baseline corrected. Fluorine
chemical shift was indirectly referenced by solvent (DMSO) proton
at 2.5 ppm.52 Signals representing remaining compound 8 and amine
were integrated, and the total compound amount at time 0 (mainly
compound 8) was normalized to 1.28,53
Synthesis of 13-Amino Costunolide Derivatives as Anticancer Agents.
Bioorg. Med. Chem. Lett. 2006, 16, 4195−4199.
(5) Neelakantan, S.; Nasim, S.; Guzman, M. L.; Jordan, C. T.;
Crooks, P. A. Aminoparthenolides as Novel Anti-leukemic Agents:
Discovery of the NF-κB Inhibitor, DMAPT (LC-1). Bioorg. Med.
Chem. Lett. 2009, 19, 4346−4349.
(6) Nasim, S.; Crooks, P. A. Antileukemic Activity of Amino-
parthenolide Analogs. Bioorg. Med. Chem. Lett. 2008, 18, 3870−3873.
(7) Hwang, D.-R.; Wu, Y.-S.; Chang, C.-W.; Lien, T.-W.; Chen, W.-
C.; Tan, U.-K.; Hsu, J. T. A.; Hsieh, H.-P. Synthesis and Antiviral
Activity of a Series of Sesquiterpene Lactones and Analogues in the
Subgenomic HCV Replicon System. Bioorg. Med. Chem. 2006, 14, 83−
91.
(8) Klochkov, S. G.; Afanas’eva, S. V.; Pushin, A. N.; Gerasimova, G.
K.; Vlasenkova, N. K.; Bulychev, Y. N. Synthesis and Cytotoxic
Activity of α-Santonin Amino-Derivatives. Chem. Nat. Compd. 2009,
46, 817−823.
(9) Lee, K.-H.; Furukawa, H.; Huang, E.-S. Antitumor Agents. 3.
Synthesis and Cytotoxic Activity of Helenalin Amine Adducts and
Related Derivatives. J. Med. Chem. 1972, 15, 609−611.
(10) Hejchman, E.; Haugwitz, R. D.; Cushman, M. Synthesis and
Cytotoxicity of Water-soluble Ambrosin Prodrug Candidates. J. Med.
Chem. 1995, 38, 3407−3410.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra and X-ray data (PDF, CIF). Also, a
full description of the methods for the calculation of GI50, TGI,
and LC50 values. This material is available free of charge via the
Internet at http://pubs.acs.org.
(11) Hassane, D. C.; Sen, S.; Minhajuddin, M.; Rossi, R. M.; Corbett,
C. A.; Balys, M.; Wei, L.; Crooks, P. A.; Guzman, M. L.; Jordan, C. T.
Chemical Genomic Screening Reveals Synergism between Partheno-
lide and Inhibitors of the PI-3 Kinase and mTOR Pathways. Blood
2010, 116, 5983−5990.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (765) 496-3962. Fax: (765) 494-1414. E-mail:
dcolby@purdue.edu. Address: Purdue University, 575 Stadium
Mall Drive, West Lafayette, IN 47907.
(12) Guzman, M. L.; Rossi, R. M.; Neelakantan, S.; Li, X.; Corbett,
C. A.; Hassane, D. C.; Becker, M. W.; Bennett, J. M.; Sullivan, E.;
Lachowicz, J. L.; Vaughan, A.; Sweeney, C. J.; Matthews, W.; Carroll,
M.; Liesveld, J. L.; Crooks, P. A.; Jordan, C. T. An Orally Bioavailable
Parthenolide Analog Selectively Eradicates Acute Myelogenous
Leukemia Stem and Progenitor Cells. Blood 2007, 110, 4427−4435.
(13) Ghantous, A.; Gali-Muhtasib, H.; Vuorela, H.; Saliba, N. A.;
Darwiche, N. What Made Sesquiterpene Lactones Reach Cancer
Clinical Trials? Drug Discovery Today 2010, 15, 668−678.
(14) Matsuda, H.; Toguchida, I.; Ninomiya, K.; Kageura, T.;
Morikawa, T.; Yoshikawa, M. Effects of Sesquiterpenes and Amino
Acid−Sesquiterpene Conjugates from the Roots of Saussurea lappa on
Inducible Nitric Oxide Synthase and Heat Shock Protein in
Lipopolysaccharide-Activated Macrophages. Bioorg. Med. Chem.
2003, 11, 709−715.
(15) Matsuda, H.; Kageura, T.; Inoue, Y.; Morikawa, T.; Yoshikawa,
M. Absolute Stereostructures and Syntheses of Saussureamines A, B,
C, D and E, Amino Acid-Sesquiterpene Conjugates with Gastro-
protective Effect, from the Roots of Saussurea lappa. Tetrahedron 2000,
56, 7763−7777.
(16) Hagmann, W. K. The Many Roles for Fluorine in Medicinal
Chemistry. J. Med. Chem. 2008, 51, 4359−4369.
ACKNOWLEDGMENTS
■
We are grateful to Purdue University and a grant from the
Indiana Elk Charities through the Purdue University Center for
Cancer Research for financial support. We acknowledge the
CCCB Cell Culture/Flow Cytometry Center, Purdue Uni-
versity, as well as the Molecular Discovery and Evaluation
Shared Resource, Purdue University Center for Cancer
Research. Cancer cell growth inhibition assay development
was performed with support from NCI T32CA09634-18 to
Purdue University Center for Cancer Research, and the
respective biological data were collected in the Purdue
University Center for Cancer Research Molecular Discovery
and Evaluation Shared Resource supported by NCI CCSG
CA23168 to Purdue University Center for Cancer Research.
ABBREVIATIONS USED
■
DMAPT, dimethylaminoparthenolide; HL-60, human promye-
locytic leukemia cells; NCI60, National Cancer Institute 60
human cancer cell lines; (log P), partition coefficient
(17) Eustaquio, A. S.; O’Hagan, D.; Moore, B. S. Engineering
Fluorometabolite Production: Fluorinase Expression in Salinispora
tropica Yields Fluorosalinosporamide. J. Nat. Prod. 2010, 73, 378−382.
(18) Tang, P.; Furuya, T.; Ritter, T. Silver-Catalyzed Late-Stage
Fluorination. J. Am. Chem. Soc. 2010, 132, 12150−12154.
(19) Erb, J.; Alden-Danforth, E.; Kopf, N.; Scerba, M. T.; Lectka, T.
Combining Asymmetric Catalysis with Natural Product Functionaliza-
tion through Enantioselective α-Fluorination. J. Org. Chem. 2010, 75,
969−971.
REFERENCES
■
(1) Kitson, R. R. A.; Millemaggi, A.; Taylor, R. J. K. The Renaissance
of α-Methylene-γ-butyrolactones: New Synthetic Approaches. Angew.
Chem., Int. Ed. 2009, 48, 9426−9451.
(2) Avonto, C.; Taglialatela-Scafati, O.; Pollastro, F.; Minassi, A.; Di
Marzo, V.; De Petrocellis, L.; Appendino, G. An NMR Spectroscopic
Method to Identify and Classify Thiol-trapping Agents: Revival of
Michael Acceptors for Drug Discovery? Angew. Chem., Int. Ed. 2011,
50, 467−471.
(3) Lawrence, N. J.; McGown, A. T.; Nduka, J.; Hadfield, J. A.;
Pritchard, R. G. Cytotoxic Michael-Type Amine Adducts of α-
Methylene Lactones Alantolactone and Isoalantolactone. Bioorg. Med.
Chem. Lett. 2001, 11, 429−431.
(4) Srivastava, S. K.; Abraham, A.; Bhat, B.; Jaggi, M.; Singh, A. T.;
Sanna, V. K.; Singh, G.; Agarwal, S. K.; Mukherjee, R.; Burman, A. C.
(20) Stockman, B. J. 2-Fluoro-ATP as a Versatile Tool for 19F NMR-
Based Activity Screening. J. Am. Chem. Soc. 2008, 130, 5870−5871.
(21) Keun, H. C.; Athersuch, T. J.; Beckonert, O.; Wang, Y.; Saric, J.;
Shockcor, J. P.; Lindon, J. C.; Wilson, I. D.; Holmes, E.; Nicholson, J.
K. Heteronuclear 19F−1H Statistical Total Correlation Spectroscopy as
a Tool in Drug Metabolism: Study of Flucloxacillin Biotransformation.
Anal. Chem. 2008, 80, 1073−1079.
(22) Papeo, G.; Giordano, P.; Brasca, M. G.; Buzzo, F.; Caronni, D.;
Ciprandi, F.; Mongelli, N.; Veronesi, M.; Vulpetti, A.; Dalvit, C.
7940
dx.doi.org/10.1021/jm201114t|J. Med. Chem. 2011, 54, 7934−7941

Journal of Medicinal Chemistry
Article
Polyfluorinated Amino Acids for Sensitive 19F NMR-Based Screening
and Kinetic Measurements. J. Am. Chem. Soc. 2007, 129, 5665−5672.
(23) Yu, J.; Mason, R. P. Synthesis and Characterization of Novel
lacZ Gene Reporter Molecules: Detection of α-Galactosidase Activity
by 19F Nuclear Magnetic Resonance of Polyglycosylated Fluorinated
Vitamin B6. J. Med. Chem. 2006, 49, 1991−1999.
(24) Yu, L.; Hajduk, P.; Mack, J.; Olejniczak, E. Structural Studies of
Bcl-xL/ligand Complexes using 19F NMR. J. Biomol. NMR 2006, 34,
221−227.
(25) Yu, J.; Liu, L.; Kodibagkar, V. D.; Cui, W.; Mason, R. P.
Synthesis and Evaluation of Novel Enhanced Gene Reporter
Molecules: Detection of β-Galactosidase Activity Using 19F NMR of
Trifluoromethylated aryl β-D-Galactopyranosides. Bioorg. Med. Chem.
2006, 14, 326−333.
(26) Dalvit, C.; Ardini, E.; Flocco, M.; Fogliatto, G. P.; Mongelli, N.;
Veronesi, M. A General NMR Method for Rapid, Efficient, and
Reliable Biochemical Screening. J. Am. Chem. Soc. 2003, 125, 14620−
14625.
(27) Dalvit, C.; Fagerness, P. E.; Hadden, D. T. A.; Sarver, R. W.;
Stockman, B. J. Fluorine-NMR Experiments for High-Throughput
Screening: Theoretical Aspects, Practical Considerations, and Range of
Applicability. J. Am. Chem. Soc. 2003, 125, 7696−7703.
(28) Mo, H.; Balko, K. M.; Colby, D. A. A Practical Deuterium-Free
NMR Method for the Rapid Determination of 1-Octanol/Water
Partition Coefficients of Pharmaceutical Agents. Bioorg. Med. Chem.
Lett. 2010, 20, 6712−6715.
(29) Guzman, M. L.; Rossi, R. M.; Karnischky, L.; Li, X.; Peterson,
D. R.; Howard, D. S.; Jordan, C. T. The Sesquiterpene Lactone
Parthenolide Induces Apoptosis of Human Acute Myelogenous
Leukemia Stem and Progenitor Cells. Blood 2005, 105, 4163−4169.
(30) Han, C.; Barrios, F. J.; Riofski, M. V.; Colby, D. A.
Semisynthetic Derivatives of Sesquiterpene Lactones by Palladium-
Catalyzed Arylation of the α-Methylene-γ-lactone Substructure. J. Org.
Chem. 2009, 74, 7176−7179.
(31) Gunn, E. J.; Williams, J. T.; Huynh, D. T.; Iannotti, M. J.; Han,
C.; Barrios, F. J.; Kendall, S.; Glackins, C. A.; Colby, D. A.; Kirshner, J.
The Natural Products Parthenolide and Andrographolide Exhibit
Anticancer Stem Cell Activity in Multiple Myeloma. Leuk. Lymphoma
2011, 52, 1085−1097.
T.; Scudiero, D. A.; Eisen, M. B.; Sausville, E. A.; Pommier, Y.;
Botstein, D.; Brown, P. O.; Weinstein, J. N. A Gene Expression
Database for the Molecular Pharmacology of Cancer. Nature Genet.
2000, 24, 236−244.
(39) Staunton, J. E.; Slonim, D. K.; Coller, H. A.; Tamayo, P.;
Angelo, M. J.; Park, J.; Scherf, U.; Lee, J. K.; Reinhold, W. O.;
Weinstein, J. N.; Mesirov, J. P.; Lander, E. S.; Golub, T. R.
Chemosensitivity Prediction by Transcriptional Profiling. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 10787−10792.
(40) Su, G.; Burant, C. F.; Beecher, C. W.; Athey, B. D.; Meng, F.
Integrated Metabolome and Transcriptome Analysis of the NCI60
Dataset. BMC Bioinf. 2011, 12 (Suppl 1), S36.
(41) Baharith, L. A.; Al-Khouli, A.; Raab, G. A. Cytotoxic Assays for
Screening Anticancer Agents. Stat. Med. 2006, 25, 2323−2339.
(42) GraphPad Prism, 5.04; GraphPad Software: San Diego,
California, U.S.A., 2010.
(43) See Supporting Information for details.
(44) Morgenthaler, M.; Schweizer, E.; Hoffmann-Roder, A.; Benini,
̈
F.; Martin, R. E.; Jaeschke, G.; Wagner, B.; Fischer, H.; Bendels, S.;
Zimmerli, D.; Schneider, J.; Diederich, F.; Kansy, M.; Muller, K.
̈
Predicting and Tuning Physicochemical Properties in Lead Opti-
mization: Amine Basicities. ChemMedChem 2007, 2, 1100−1115.
(45) Meng, Q.; Peng, Z.; Chen, L.; Si, J.; Dong, Z.; Xia, Y. Nuclear
Factor-κB Modulates Cellular Glutathione and Prevents Oxidative
Stress in Cancer Cells. Cancer Lett. 2010, 299, 45−53.
(46) Balendiran, G. K.; Dabur, R.; Fraser, D. The Role of
Glutathione in Cancer. Cell Biochem. Funct. 2004, 22, 343−352.
(47) Lajiness, J. P.; Robertson, W. M.; Dunwiddie, I.; Broward, M.
A.; Vielhauer, G. A.; Weir, S. J.; Boger, D. L. Design, Synthesis, and
Evaluation of Duocarmycin O-Amino Phenol Prodrugs Subject to
Tunable Reductive Activation. J. Med. Chem. 2010, 53, 7731−7738.
(48) Pires, M. M.; Chmielewski, J. Fluorescence Imaging of Cellular
Glutathione Using a Latent Rhodamine. Org. Lett. 2008, 10, 837−840.
(49) Hong, V.; Kislukhin, A. A.; Finn, M. G. Thiol-Selective
Fluorogenic Probes for Labeling and Release. J. Am. Chem. Soc. 2009,
131, 9986−9994.
(50) Kim, G.-J.; Lee, K.; Kwon, H.; Kim, H.-J. Ratiometric
Fluorescence Imaging of Cellular Glutathione. Org. Lett. 2011, 13,
2799−2801.
(51) Potapenko, D. I.; Bagryanskaya, E. G.; Grigoriev, I. A.;
Maksimov, A. M.; Reznikov, V. A.; Platonov, V. E.; Clanton, T. L.;
Khramtsov, V. V. Quantitative Determination of SH Groups using 19F
NMR Spectroscopy and Disulfide of 2,3,5,6-Tetrafluoro-4-mercapto-
benzoic acid. Magn. Reson. Chem. 2005, 43, 902−909.
(32) Kerekes, A. D.; Esposite, S. J.; Doll, R. J.; Tagat, J. R.; Yu, T.;
Xiao, Y.; Zhang, Y.; Prelusky, D. B.; Tevar, S.; Gray, K.; Terracina, G.
A.; Lee, S.; Jones, J.; Liu, M.; Basso, A. D.; Smith, E. B. Aurora Kinase
Inhibitors Based on the Imidazo[1,2-a]pyrazine Core: Fluorine and
Deuterium Incorporation Improve Oral Absorption and Exposure. J.
Med. Chem. 2011, 54, 201−210.
(33) Neelakantan, S.; Parkin, S.; Crooks, P. A. (11R)-13-
Dimethylammonio-11,13-Dihydro-4,5-Epoxycostunolide Semifuma-
rate. Acta Crystallogr., Section E 2009, 65E, o1565.
(34) Nakagawa, Y.; Iinuma, M.; Matsuura, N.; Yi, K.; Naoi, M.;
Nakayama, T.; Nozawa, Y.; Akao, Y. A Potent Apoptosis-inducing
Activity of a Sesquiterpene Lactone, Arucanolide, in HL60 Cells: A
Crucial Role of Apoptosis-inducing Factor. J. Pharmacol. Sci. 2005, 97,
242−252.
(52) Harris, R. K.; Becker, E. D.; Cabral de Menezes, S. M.;
Goodfellow, R.; Granger, P. NMR Nomenclature. Nuclear Spin
Properties and Conventions for Chemical Shifts. Pure Appl. Chem.
2001, 73, 1795−1818.
(53) Mo, H.; Raftery, D. Solvent Signal as an NMR Concentration
Reference. Anal. Chem. 2008, 80, 9835−9839.
(35) Shoemaker, R. H. The NCI60 Human Tumour Cell Line
Anticancer Drug Screen. Nature Rev. Cancer 2006, 6, 813−823.
(36) Park, E. S.; Rabinovsky, R.; Carey, M.; Hennessy, B. T.;
Agarwal, R.; Liu, W. B.; Ju, Z. L.; Deng, W. L.; Lu, Y. L.; Woo, H. G.;
Kim, S. B.; Cheong, J. H.; Garraway, L. A.; Weinstein, J. N.; Mills, G.
B.; Lee, J. S.; Davies, M. A. Integrative Analysis of Proteomic
Signatures, Mutations, and Drug Responsiveness in the NCI 60
Cancer Cell Line Set. Mol. Cancer Ther. 2010, 9, 257−267.
(37) Ross, D. T.; Scherf, U.; Eisen, M. B.; Perou, C. M.; Rees, C.;
Spellman, P.; Iyer, V.; Jeffrey, S. S.; Van de Rijn, M.; Waltham, M.;
Pergamenschikov, A.; Lee, J. C. E.; Lashkari, D.; Shalon, D.; Myers, T.
G.; Weinstein, J. N.; Botstein, D.; Brown, P. O. Systematic Variation in
Gene Expression Patterns in Human Cancer Cell Lines. Nature Genet.
2000, 24, 227−235.
(38) Scherf, U.; Ross, D. T.; Waltham, M.; Smith, L. H.; Lee, J. K.;
Tanabe, L.; Kohn, K. W.; Reinhold, W. C.; Myers, T. G.; Andrews, D.
7941
dx.doi.org/10.1021/jm201114t|J. Med. Chem. 2011, 54, 7934−7941