Synthesis of piperlogs and analysis of their effects on cells

Article abstract of DOI:10.1016/j.tet.2013.05.080

Piperlongumine (PL) is a naturally occurring small molecule previously shown to induce cell death preferentially in cancer cells relative to non-cancer cells. An initial effort to synthesize analogs highlighted the reactivities of both of piperlongumine's α,β-unsaturated imide functionalities as key features determining PL's cellular effects. In this study, a second-generation of analogs was synthesized and evaluated in cells to gain further insight into how the reactivity, number, and orientation of PL's reactive olefins contribute to its ability to alter the physiology of cells.

Full text of DOI:10.1016/j.tet.2013.05.080

Tetrahedron 69 (2013) 7559e7567
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Synthesis of piperlogs and analysis of their effects on cells
,
b
,
,
b
,
,
,
b
,
z
Zarko V. Boskovic a y, Mahmud M. Hussain a y, Drew J. Adams a y, Mingji Dai a
,
Stuart L. Schreiber a
,b,
*
a Howard Hughes Medical Institute, Broad Institute, 7 Cambridge Center, Cambridge, MA 02142, USA
b Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Piperlongumine (PL) is a naturally occurring small molecule previously shown to induce cell death
preferentially in cancer cells relative to non-cancer cells. An initial effort to synthesize analogs high-
lighted the reactivities of both of piperlongumine’s a,b-unsaturated imide functionalities as key features
determining PL’s cellular effects. In this study, a second-generation of analogs was synthesized and
evaluated in cells to gain further insight into how the reactivity, number, and orientation of PL’s reactive
olefins contribute to its ability to alter the physiology of cells.
Received 28 February 2013
Received in revised form 19 May 2013
Accepted 20 May 2013
Available online 28 May 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Michael acceptors
Reactive oxygen species
Piperlongumine
Toxicity
1. Introduction
The recent finding that the naturally occurring small molecule
piperlongumine (PL, Fig. 1)9,10 can selectively kill cancer cells and
In nature, nucleophiles present in proteins and nucleic acids
encounter a wealth of biologically active electrophilic small mole-
cules.1 Several drug discovery efforts have explored the potential of
targeted covalent inhibitorsdelectrophilic small molecules whose
reactivity is limited to a small fraction of the proteomedto circum-
vent the off-target effects and potential toxicity of indiscriminate
covalent inhibitors.2,3 Many biologically active electrophilic small
molecules contain Michael acceptors that can undergo hetero-
conjugate addition with the nucleophilic thiols in cysteine residues
in irreversible4 or reversible fashion.5 One such thiol-containing
shrink tumors in xenograft models prompted an in-depth evalua-
tion of the structure/activity relationships and chemical reactivity
of this simple molecule.11 This initial study suggested that the
electrophilicity of the C2eC3 olefin is critical for many if not all of
PLs effects on cells (viability, ROS levels, protein glutathionylation),
and demonstrated that this functionality reacts with simple thiols
in DMSO solution in vitro. In contrast, modifying or ablating the
potential reactivity of the presumably less reactive, C7eC8, olefin
(e.g., by hydrogenation, as in PL-H2, or introducing steric hindrance,
as in PL-DHN; Fig. 1) yielded compounds with reduced effects on
viability that in some cases retained the ability to elevate ROS
levels, challenging the notion that oxidative stress was sufficient for
cell death. In addition, it was found that tethering two PL molecules
together through an ether linkage at the C12 alkoxy group en-
hanced cellular potency with respect to viability by roughly 20-fold
compared to PL itself (PL-DI, Fig. 1).
Building on these observations, we have subsequently designed
and synthesized an additional collection of 29 PL analogsd
piperlogsdand tested them in assays measuring cell viability, ROS
levels, and protein glutathionylation. These analogs vary the nature
and the number of electrophilic centers in the PL scaffold and
provide new insight into the interdependence of the C2eC3 and
C7eC8 olefins, the minimum pharmacophore of PL, and the effect
of linker length and its chemical properties on the cellular effects of
a series of tethered PL ‘dimers’. This investigation has demonstrated
that subtle differences in the chemical properties of Michael
tripeptide, glutathione (g-L-glutamyl-L-cysteinylglycine), serves as
a primary source of cellular reducing equivalents (along with
NADPH), protects cells from oxidative stress and xenobiotics, and
regulates redox-sensitive signaling pathways.6,7 From a chemical
standpoint, it is therefore not surprising that small molecules capa-
ble of hetero-conjugate addition with thiols could have an impact on
cellular redox homeostasis. In fact, several electrophilic small mol-
ecules have been shown to perturb the redox state of cancer cells by
increasing levels of reactive oxygen species (ROS), which may be the
cause of their cytotoxic effects.8
* Corresponding author. E-mail address: stuart_schreiber@harvard.edu (S.L.
Schreiber).
y
These authors contributed equally.
Present address: Department of Chemistry, Purdue University, West Lafayette,
z
IN 47907, USA.
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.05.080

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Z.V. Boskovic et al. / Tetrahedron 69 (2013) 7559e7567
O
O
O
O
(a)
(b)
(c)
enhances toxicity
PL's EC50
MeO
MeO
MeO
MeO
needed for protein glutathionylation
not needed for ROS elevation
H1703: 2.8 µM
HeLa: 12 µM
N
N
O
O
OMe
OMe
MeO
critical for toxicity
ROS elevation
protein glutathionylation
1
8
N
2
3
6
9
PL-DHN
PL-H2
7
10
5
MeO
12
11
OMe
O
O
O
O
4
PL-DI EC50
PL
OMe
MeO
O
N
N
H1703: 0.16 µM
HeLa: 0.45 µM
oligomerization greatly
enhances potency
N
O
OMe
OMe
PL-DI
Fig. 1. (a) Previously established SAR of piperlongumine, PL, with carbon numbering used in this paper; (b) EC50 of PL in H1703 and HeLa cells determined from three independent
assays; (c) structures of dihydronaphthalene PL analog, PL-DHN; C7eC8 dihydro PL, PL-H2; and tethered PL ‘dimer’, PL-DI.
acceptors can have profound implications for their effects on oxi-
dative stress and viability and suggests new testable hypotheses
regarding the mechanism of action of these analogs for evaluation
in further cellular experiments.
levels (measuring conversion of a nonfluorescent dichlorodihy-
drofluorescein to fluorescent dichlorofluorescein);13 and (3) pro-
tein glutathionylation detected by immunofluorescence using
a glutathione-recognizing antibody.14 Assay performance of PL and
piperlogs can be found in Supplementary data.
2. Results and discussion
PL contains two a,b-unsaturated imide functionalities in a con-
tinuous conjugated system, with the C2eC3 olefin more reactive
toward thiol addition in vitro.11 To explore the necessity of
the imide functionality, a variety of analogs containing multiple
PL analogs were prepared by acylation of the amide nitrogen of 1
with an appropriate acid chloride (Scheme 1A). Deprotonation of 1
Scheme 1. Synthetic procedures for the preparation of piperlogs.
was achieved with n-BuLi, at ꢀ78 ꢁC. Subsequent reaction with
a variety of acyl chlorides at low temperatures led to imides
designed to test hypotheses about PL reactivity. One of the more
a,b-unsaturated amides were prepared (Table 1). Strikingly, these
analogs did not diminish cell growth and viability in two cancer cell
lines and did not increase ROS or protein glutathionylation levels.
Amide analogs retaining or lacking the trimethoxycinnamoyl group
found in PL were equally inactive. The diminished reactivity toward
commonly used amides of type 1, a,b-unsaturated d-lactam, 2, was
prepared through ring-closing metathesis of homoallyl crotona-
mide with catalytic amounts of Grubbs’ second-generation ruthe-
nium catalyst.12 Notably, this cyclization failed when a homoallyl
acrylamide was used as a substrate. PL ‘dimers’ in which two PL
units are tethered by an ether linkage were generally made from
phenol 39 and various diols by a Mitsunobu dehydrative coupling
using azodicarboxylate and triphenylphosphine (Scheme 1B). Al-
ternatively, two PL units could be connected through an ester
linkage by acylation of phenol 3 with bis acid chlorides in the
presence of triethylamine (Scheme 1C). Non-commercially available
mono and bis acid chlorides were prepared from the corresponding
acids by using oxalyl chloride and catalytic DMF. The purity of
compounds isolated following silica chromatography was evaluated
using 1H and 13C NMR; additionally, purity at the time of cellular
testing was evaluated using LCeMS analysis. Compounds that had
substantively degraded were excluded from further analysis.
The resulting collection of small molecules containing multiple
Michael acceptors was evaluated in the following cell-based assays:
(1) cell viability (ATP coupled luciferase activity assay); (2) ROS
hetero-conjugate addition of
a,b-unsaturated amides relative to
a,b-unsaturated imides appears in this setting sufficient to ablate
completely a variety of cellular effects.x Notably, this finding is in
accord with previous reports of low background reactivity for
acrylamide-containing targeted kinase inhibitors5 and suggests
that the limit of reactivity for cellular electrophiles lies between
a,
b-unsaturated imides and amides.
BRD2285, an analog bearing an
a
-cyano amide functionality,
obtained through Knoevenagel condensation, had surprisingly lit-
tle effect on cell viability in spite of its more reactive double bond
(Table 1). Curiously, this compound retained the ability to increase
ROS levels, suggesting that, as with previous PL analogs PL-H2 and
PL-DHN and many additional small molecule enhancers of ROS
x
The superior ability of imides (compared to amides) to stabilize developing
negative charge on
a-carbon in conjugate addition reactions was used in a number
of developed synthetic organic methods.15

Z.V. Boskovic et al. / Tetrahedron 69 (2013) 7559e7567
7561
Table 1
Bis- -unsaturated amides are inactive in viability, ROS increase, and protein glutathionylation assays
a,b
Structure
ID
Structure
ID
O
O
N
MeO
MeO
O
H
N
O
N
H
BRD2724
BRD2285
BRD6008
N
H
O
OMe
CF3
O
O
N
O
MeO
H
N
O
N
H
BRD1764
BRD1088
N
H
MeO
NMe2
O
OMe
OMe
OMe
OMe
OMe
OMe
O
O
H
N
Me
N
MeO
MeO
MeO
BRD2537
N
H
N
Me
OMe
O
O
MeO
OMe
OMe
O
O
MeO
MeO
OMe
N
H
N
H
BRD9521
OMe
OMe
OMe
(Fig. 1), ROS increase does not necessarily lead to cell death.23
BRD6008, a vinylogous urea analog of BRD2285 presumed to be
far less reactive toward hetero-conjugate addition did not show any
activity in the performed cellular assays (Table 1).
Previously, the presence of both the C2eC3 and C7eC8 olefins of
PL was demonstrated to be essential for its cellular activities. An
additional series of analogs were prepared that varied both of these
electrophilic functionalities, and the spacing between them, to gain
additional perspective into the minimum requirements for the cel-
lular activity of this scaffold. The trimethoxycinnamoyl moiety could
be replaced by simpler acryloyl (Table 2, BRD4809), or methacryloyl
substituents (Table 2, BRD3152) to produce analogs with slightly
enhanced or diminished potency relative to PL, respectively. De-
this highly reactive compound undergoes hetero-conjugate addition
with other intracellular nucleophiles prior to reacting with the cel-
lular targets that give rise to cell death following treatment with PL
or BRD4809. The C1eC5 ring could also be simplified without loss of
activity. Replacing it with an acyclic crotonyl imide had a minimal
effect on potency, as long as Michael acceptors at the C2eC3 and
C7eC8 positions were present (Table 2, BRD6245). Excising both the
lactam and the aromatic ring found in PL led to BRD7991, which
although less potent than PL, killed cancer cells (H1703 line) and
elevated ROS and protein glutathionylation. The efficacy of this an-
alog, whose molecular weight is less than half that of PL (153 Da vs
317 Da), further supports the central role of electrophilicity in de-
termining cellular efficacy in this compound collection.
creased potency as a consequence of
a
-substitution (fluoro in
Replacing the C7eC8 styryl olefin with a phenylacetylene
derivative (Table 2, BRD5481) provided a more potent PL analog
BRD2847, methyl in BRD2501, phenyl in BRD5545) was also recog-
nized in the previous study.11 Substitution of an ethoxycarbonyl
group at C8, which would be predicted to give rise to a more reactive
C7eC8 Michael acceptor, led to a less active compound (Table 2,
BRD8143) compared to acryloyl analog BRD4809. It is possible that
than des-trimethoxy PL (BRD4524) reported previously (0.91
mM vs
3.5 M EC50 in H1703). This outcome may be a function of the
m
enhanced reactivity toward hetero-conjugate addition of such
alkynyl Michael acceptors, or may derive from the altered steric or
Table 2
EC50 values of imide-containing piperlogs in H1703 and HeLa cell lines determined from three independent assays
Structure ID EC50 H1703 ( M) EC50 HeLa ( M) Structure
m
m
ID
EC50 H1703 (
m
M) EC50 HeLa (mM)
O
O
O
O
BRD4809 2.2
BRD3152 4.7
8.1
BRD7991 8.0
BRD5481 0.91
>20
N
N
H
O
O
O
O
N
19
16
14
4.1
N
O
O
O
O
O
N
BRD8143 3.5
BRD6245 4.0
BRD6312 2.3
BRD8091 1.0
11
N
O
N
O
O
O
O
O
O
OMe
OMe
OMe
O
N
H
N
4.3
OEt
O
OMe
OMe
O

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Z.V. Boskovic et al. / Tetrahedron 69 (2013) 7559e7567
stereoelectronic constraints on reactivity.16 Additionally, although
PL was shown to have a variety of protein targets in cell extracts as
assessed by quantitative proteomics, the precise range of targets
accessed by each analog has not been determined. As such, varia-
tion in performance between analogs may reflect either differing
affinities for various cellular protein and non-protein targets or
differing physicochemical properties, including solubility, perme-
ability, and subcellular localization.
C2eC3 olefins. Again, linker length did not greatly affect perfor-
mance in our cellular assays. Owing to their multivalent electro-
philicity but strikingly reduced ability to induce cell death relative
to other ‘dimers’, these reagents will be helpful control compounds
during further exploration of hypotheses regarding cellular mech-
anisms by which ‘dimers’ induce cell death, including alkylation of
DNA or cytoskeletal components as described above.
Like PL-H2, BRD3930 and BRD2522 also elevated ROS at nontoxic
doses; however, these analogs also potently elevated protein
glutathionylation even at nontoxic doses (Supplementary data).
These analogs represent the first for which protein glutathionylation
has been observed independently of cell death. Together with
BRD85988, BRD3930 and BRD2522 reinforce the conclusion that
multivalent electrophilicity is insufficient to observe PL’s cellular
effects; rather, the particular spacing and/or reactivity of the C2eC3
and C7eC8 olefins are critical.
Interestingly, a significant potency increase has been observed
when the PL skeleton is duplicated within one molecule (e.g., PL-DI,
,
Fig. 111). To explore this effect further, an initial set of analogs was
synthesized to determine whether the potency of these compounds
depends on the length and chemical nature of the linker joining
two PL segments.17,18 Changing the chemical linkage from an ether
to an ester bond led to a dramatic loss of potency compared to other
‘dimers’, likely due to facile hydrolytic cleavage of phenyl esters in
cellular environment.19 Compared to PL, ester-linked dimer
BRD1781 was only slightly more active, in line with the expected
effective doubling of PL concentration in cells.
3. Conclusions
A set of analogs with polyethylene glycol ether linkers spanning
By assembling a second-generation collection of piperlongu-
mine analogs, we have gained new insight into how the structure
and chemical reactivity of this scaffold relate to its cellular effects.
lengths from 4 to 15
s
bonds were also prepared (Fig. 2). These
M H1703, 0.45 M HeLa; Fig. 1)
analogs, as well as PL-DI (EC50 0.16
m
m
showed nearly identical EC50 values within each of two cell lines
investigated. BRD4547, whose electrophilic functionalities are fixed
in close proximity by a phenylene linker, retained the greatly en-
hanced cellular potency of this set of PL ‘dimers’ without greatly
exceeding the molecular weight of PL. Furthermore, few significant
differences in potency among these compounds were observed in
ROS and glutathionylation assays. Apparently, the gain in efficacy
observed from tethering two PL units is independent of the exact
spacing between those units.
We note that the
essential for all cellular effects and cannot be recapitulated by
-unsaturated amides, presumably due to amides’ lower re-
a,b-unsaturated imide functionality appears
a,b
activity. Several analogs retain PL-like properties while removing
the trimethoxyphenyl ring or simplifying the C1eC5 ring to a cro-
tonyl substituent, further highlighting the key role of the electro-
philic functionalities of the PL scaffold in determining its cellular
effects. Moreover, a collection of ‘dimeric’ analogs in which two PL
units are tethered by linkers of varying length indicates that the
high potency of these compounds in cellular assays is largely in-
dependent of linker length, an observation that may provide new
hypotheses for future cellular studies of the mechanism of action of
these compounds. Notably, tethering of PL units lacking a reactive
olefin at C7eC8 led to ‘dimeric’ analogs that were no more potent
than PL itself and far less potent than various analogs that tether
two PL units, further highlighting the critical contribution of the
C7eC8 olefin to PL-induced cell death. Together, these observations
clarify that not all electrophilic functionalities are created equal and
This observation suggests new hypotheses regarding the en-
hanced potency of PL ‘dimers’ relative to PL in our assays. Although
these ‘dimers’ can likely cross-link distinct sets of cellular nucleo-
philes as a result of their unique spacing of PL units, their uniform
performance in cellular assays suggests a common mode of in-
ducing cell death. Particularly attractive candidates include the
cross-linking of DNA or cytoskeletal components.20 These assem-
blies of repeating alkylatable subunits might be equally reactive
with piperlogs of widely varying linker length and are well-
established targets for chemotherapeutic agents, including multi-
valent electrophiles like nitrogen mustards and busulfan.21,22
Additionally, a series of ‘dimers’ were synthesized from PL
subunits in which reactivity at C8 was reduced or eliminated
(Fig. 3). BRD85988 was prepared by tethering two molecules of
PL-DHN, an analog previously shown to elevate ROS levels and
deplete glutathione, but with diminished effects on cell death rel-
ative to PL, using a propanediol linker in direct analogy to BRD4605
(Fig. 3). An intermediate analog containing one unit of PL and one
unit of PL-DHN with the same linker was also prepared (BRD5525).
Notably, BRD85988, containing two PL-DHN units, was found to
lower cell viability with potency comparable to PL. In contrast,
BRD4605, which includes two PL units, displayed 15e25-fold
greater potency, and BRD5525 displayed intermediate potency.
Despite its relatively small effect on cell viability, BRD85988 po-
tently elevated ROS even at nontoxic doses without elevating
protein glutathionylation, much like PL-DHN itself.
further demonstrate the essential role of the a,b-unsaturated imide
electrophiles in determining PL’s cellular effects.
4. Experimental section
4.1. General information
Dry solvents were purchased from SigmaeAldrich. Unless oth-
erwise stated, all reagents were obtained from commercial sources
and used without further purification. 1H NMR spectra were
recorded on Varian Unity/Inova 500 (500 MHz), or Bruker Ultra-
shield 300 (300 MHz) spectrometers. 1H NMR data are reported as
follows: chemical shift in parts per million (ppm) relative to CHCl3
(7.27 ppm), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet;
quin, quintet; m, multiplet; br, broadened), coupling constant (Hz),
and integration. 13C NMR spectra were recorded on Varian Unity/
Inova 500 (126 MHz) or Bruker Ultrashield 300 (75 MHz) spec-
trometers. 13C NMR chemical shifts are reported in parts per million
(ppm) relative to solvent. All 13C NMR spectra were determined
with broadband decoupling. High-resolution mass spectra (HRMS)
were obtained through the Broad Institute Chemical Biology Ana-
lytical Chemistry facility. All reactions were magnetically stirred
and monitored by thin-layer chromatography (TLC) using E. Merck
silica gel 60 F254 precoated plates (0.25 mm). Flash chromatography
was performed using a CombiFlash companion system (Teledyne
ISCO, Inc.) with pre-packed FLASH silica gel columns (Biotage, Inc.).
Similarly, two analogs were prepared by tethering PL units that
both lack a C7eC8 olefin (BRD3930 and BRD2522), a scaffold
analogous to PL-H2 (Fig. 1). These analogs lack the trimethox-
yphenyl moiety of PL, which appears to contribute only modestly to
the cellular effects of PL (Fig. 3). Use of a variable length aliphatic
linker enabled synthesis of ‘dimers’ of substantially reduced mo-
lecular weight relative to PL-DI and related structures. In analogy to
BRD85988 discussed above, BRD3930 and BRD2522 reduced cell
viability less potently than PL despite containing two reactive

Z.V. Boskovic et al. / Tetrahedron 69 (2013) 7559e7567
7563
Fig. 2. EC50 values of tethered ‘dimeric’ imide-containing piperlogs in H1703 and HeLa cell lines. All data are expressed as meanꢂstandard deviation for three independent
experiments.
4.2. Compound synthesis and characterization
(1 equiv), triphenylphosphine (5 equiv), and diisopropyldiazadi-
carboxylate (5 equiv). The mixture was stirred for 24 h, volatiles
evaporated, and residue was separated by silica gel column chro-
matography with hexanes/EtOAc 50:50 to 10:90 gradient.
4.2.1. Acylation of amides. To solution of amide in THF (0.2 M,
1.1 equiv for mono acyl chlorides, 2.2 equiv for bis acyl chlorides) at
ꢀ78 ꢁC was slowly added cold solution of n-BuLi in hexanes (2.5 M,
1 equiv) and stirred for 15 min. Appropriate acyl chloride (1 equiv)
was added dropwise and the reaction mixture was stirred for 30 min
and then warmed to room temperature slowly. Reaction mixture
was diluted with ethyl acetate, partitioned with NH4Cl(aq, satd), dried
with brine and Na2SO4, and purified by column chromatography.
4.2.4. Acylation of amine. To solution of diamine (1 equiv), trie-
thylamine (2.2 equiv), and DMAP (0.1 equiv) in THF (0.215 M) was
added acid chloride (2.2 equiv) and the reaction mixture was stir-
red overnight. Volatiles were evaporated in vacuo and residue was
fractionated by silica gel column chromatography using methanol
(0e20%) in DCM.
4.2.2. Acylation of phenol. To solution of phenol 3 (2 equiv) and
triethylamine (6 equiv) in dichloromethane (0.1 M) was added bis
acid chloride (1 equiv) dropwise. Reaction mixture was stirred at
room temperature for 24 h. Reaction mixture was evaporated in
vacuo and purified by silica gel column chromatography using
gradient of methanol (0e5%) in DCM.
4.2.5. Synthesis of BRD6245. To a suspension of (E)-but-2-enamide
(59.9 mg, 0.70 mmol, 1.0 equiv) in 3.5 mL THF was added n-BuLi in
hexanes (2.5 M, 0.34 mL, 0.85 mmol, 1.2 equiv) slowly at ꢀ78 ꢁC.
The reaction mixture was warmed up to room temperature and
stirred for 8 h before a solution of (E)-3-(3,4,5-trimethoxyphenyl)
acryloyl chloride (361 mg, 1.41 mmol, 2.0 equiv) in 2 mL of THF was
added dropwise at ꢀ78 ꢁC. The reaction mixture was allowed to
warm to rt overnight. The crude reaction mixture was diluted with
4.2.3. Etherification of phenol. To solution of phenol 3 (2.2 equiv) in
a 1:1 mixture of toluene and THF (0.023 M) were added diol

7564
Z.V. Boskovic et al. / Tetrahedron 69 (2013) 7559e7567
Fig. 3. Viability (top left) and ROS increase (top right) of ‘dimeric’ piperlogs with C7eC8 olefin modifications determined from three independent experiments. All data are ex-
pressed as meanꢂstandard deviation for three independent experiments.
ethyl acetate, and quenched with cold NH4Cl(aq, satd), and the or-
ganic layer separated. The aqueous layer was extracted twice with
ethyl acetate. The combined organic layers were washed with brine
and dried over MgSO4. After filtration and evaporation under re-
duced pressure, the residue was purified by column chromatogra-
phy to yield the pure compound. BRD7991 was prepared in
a similar procedure using methacryloyl chloride.
(m, 4H), 1.78 (dd, J¼6.8, 1.0 Hz, 6H), 1.36e1.46 (m, 4H); 13C NMR
(126 MHz, DMSO-d6)
d 16.8, 26.5, 37.9, 125.9, 136.7, 164.6. Exact
mass (MþNa)þ calcd: 247.1422; found 247.1414.
4.3.3. BRD1781: bis(2,6-dimethoxy-4-((E)-3-oxo-3-(2-oxo-5,6-
dihydropyridin-1(2H)-yl)prop-1-en-1-yl)phenyl)dodecanedioate. 1H
NMR (500 MHz, chloroform-d)
d
7.69 (d, J¼15.1 Hz, 2H), 7.45
(d, J¼15.6 Hz, 2H), 6.96 (dt, J¼9.5, 4.0 Hz, 2H), 6.82 (s, 4H), 6.06
(dt, J¼9.6, 1.8 Hz, 2H), 4.02e4.08 (m, 4H), 3.85 (s, 12H), 2.62
(t, J¼7.6 Hz, 4H), 2.46e2.53 (m, 4H), 1.78 (quin, J¼7.4 Hz, 4H),
1.40e1.49 (m, 4H), 1.34 (br, 8H); 13C NMR (126 MHz, chloroform-d)
4.3. Compound characterization
4.3.1. BRD1088: (2E,20E)-N,N0-(ethane-1,2-diyl)bis(3-(3,4,5-
trimethoxyphenyl)acrylamide). 1H NMR (300 MHz, chloroform-d)
d 24.8, 25.1, 29.0, 29.3, 29.5, 33.9, 41.6, 56.2, 104.9, 121.9, 125.8,
d
7.42 (d, J¼15.5 Hz, 2H), 6.69 (s, 4H), 6.34 (d, J¼15.6 Hz, 2H), 3.82
133.2, 143.5, 145.6, 152.3, 165.8, 168.8, 171.4. Exact mass (MþH)þ
(s, 12H), 3.79 (s, 6H), 3.46e3.39 (m, 2H), 3.35e3.24 (m, 4H). Exact
mass (MþH)þ calcd: 501.2237, found 501.2223.
calcd: 801.3599; found 801.3581.
4.3.4. BRD2285: (E)-3-((E)-2-cyano-3-(4-(trifluoromethyl)phenyl)
acrylamido)propyl 3-(3,4,5-trimethoxyphenyl)acrylate. 1H NMR
4.3.2. BRD1764: (2E,20E)-N,N0-(butane-1,4-diyl)bis(but-2-
enamide). 1H NMR (500 MHz, DMSO-d6)
d
7.67 (br s, 2H), 6.58
(500 MHz, chloroform-d)
(d, J¼8.3 Hz, 2H), 7.66 (d, J¼15.6 Hz, 1H), 6.96e7.03 (m, 1H), 6.82
d
8.39 (s, 1H), 8.01 (d, J¼7.8 Hz, 2H), 7.75
(dq, J¼15.1, 6.8 Hz, 2H), 5.88 (dd, J¼15.4, 1.2 Hz, 2 H), 3.05e3.14

Z.V. Boskovic et al. / Tetrahedron 69 (2013) 7559e7567
7565
(s, 2H), 6.56 (d, J¼15.1 Hz, 1H), 4.39 (t, J¼5.6 Hz, 2H), 3.90 (d,
J¼1.0 Hz, 9H), 3.63 (q, J¼6.0 Hz, 2H), 2.06 (quin, J¼5.9 Hz, 2H); 13C
4H), 3.73 (s, 4H), 2.53e2.42 (m, 4H); 13C NMR (75 MHz, chloroform-
d)
d 169.1, 166.0, 153.7, 145.7, 144.0, 139.3, 130.8, 126.0, 121.2, 105.7,
NMR (126 MHz, chloroform-d)
d
28.3, 38.8, 56.1 (q, J¼2.6 Hz), 61.0
72.5, 70.9, 70.6, 56.4, 41.8, 25.0. Exact mass (MþNa)þ calcd:
(q, J¼2.9 Hz), 63.1, 105.5, 116.4, 116.7, 126.1 (q, J¼3.8 Hz), 129.7,
130.5, 135.0 (d, J¼1.4 Hz), 145.7, 151.1, 153.4, 159.4, 167.0. Exact mass
(MþNa)þ calcd: 541.1562, found 541.1552.
743.2792, found 743.2786.
4.3.13. BRD4698: bis(2,6-dimethoxy-4-((E)-3-oxo-3-(2-oxo-5,6-
dihydropyridin-1(2H)-yl)prop-1-en-1-yl)phenyl)fumarate. 1H NMR
4.3.5. BRD2522: 1,12-bis(2-oxo-5,6-dihydropyridin-1(2H)-yl)dodec-
(300 MHz, pyridine-d5)
d
8.02 (d, J¼15.6 Hz, 2H), 7.83 (d, J¼15.6 Hz,
ane-1,12-dione. 1H NMR (500 MHz, chloroform-d)
d
6.89 (dt, J¼9.8,
2H), 7.61 (s, 2H), 7.11 (s, 4H), 6.85e6.73 (m, 2H), 6.08 (d, J¼9.7 Hz,
3.9 Hz, 2H), 6.00 (dt, J¼9.8, 2.0 Hz, 2H), 3.98 (t, J¼6.3 Hz, 4H), 2.92 (t,
2H), 4.01 (t, J¼6.4 Hz, 4H), 3.76 (s,12H), 2.29e2.19 (m, 4H); 13C NMR
J¼7.3 Hz, 4H), 2.38e2.45 (m, 4H), 1.56e1.70 (m, 4H), 1.16e1.38 (m,
(75 MHz, pyridine-d5) d 169.2, 166.3,163.0, 153.3, 146.8, 142.7,135.2,
12H); 13C NMR (126 MHz, chloroform-d)
d
24.7, 25.0, 29.2, 29.4,
135.1, 134.8, 130.6, 125.9, 105.8, 56.7, 42.4, 25.3. Exact mass
29.5, 39.3, 40.9, 126.0, 145.0, 165.4, 176.6. Exact mass (MþNa)þ
(MþNa)þ calcd: 709.2009, found 709.1967.
calcd: 411.2260; found 411.2265.
4.3.14. BRD4809: 1-acryloyl-5,6-dihydropyridin-2(1H)-one. 1H NMR
(500 MHz, chloroform-d)
4.3.6. BRD2537:
(3,4,5-trimethoxyphenyl)acrylamide). 1H NMR (300 MHz, chloro-
form-d)
(2E,20E)-N,N0-(ethane-1,2-diyl)bis(N-methyl-3-
d
2.47 (tdd, J¼6.3, 6.3, 4.4, 2.0 Hz, 2H),
4.00 (t, J¼6.6 Hz, 2H), 5.76 (dd, J¼10.3, 1.5 Hz, 1H), 6.02 (dt, J¼9.8,
1.7 Hz, 1H), 6.40 (dd, J¼17.1, 2.0 Hz, 1H), 6.87e6.98 (m, 1H), 7.06 (dd,
d
7.42 (d, J¼15.3 Hz, 2H), 7.10 (d, J¼15.4 Hz, 2H), 6.71 (s, 4H),
3.85 (s, 12H), 3.79 (s, 6H), 3.33e3.23 (m, 4H), 3.17 (s, 6H). Exact
mass (MþH)þ calcd: 529.2550, found 529.2549.
J¼16.8, 10.5 Hz, 1H); 13C NMR (126 MHz, chloroform-d)
d 24.7, 41.4,
125.6, 128.5, 131.6, 145.7, 165.5, 168.8. Exact mass (MþH)þ calcd:
152.0712; found: 152.0699.
4.3.7. BRD2724:
N,N0-(butane-1,4-diyl)diacrylamide. 1H
NMR
(500 MHz, DMSO-d6)
d
1.42 (br, 4H), 3.12 (d, J¼5.4 Hz, 4H), 5.56 (d,
4.3.15. BRD5481: 1-(3-phenylpropioloyl)-5,6-dihydropyridin-2(1H)-
J¼10.3 Hz, 2H), 6.07 (s, 2H), 6.13e6.24 (m, 2H), 8.10 (br, 2H); 13C
one. 1H NMR (500 MHz, chloroform-d)
d 2.43e2.53 (m, 2H), 4.06 (t,
NMR (126 MHz, DMSO-d6)
d
26.7, 38.3, 125.0, 131.9, 164.6. Exact
J¼6.3 Hz, 2H), 6.07 (dt, J¼9.8, 1.5 Hz, 1H), 6.93e6.99 (m, 1H),
mass (MþNa)þ calcd: 219.1109; found 219.1111.
7.33e7.40 (m, 2H), 7.41e7.46 (m, 2H), 7.65 (d, J¼7.8 Hz, 1H); 13C
NMR (126 MHz, chloroform-d)
d 24.4, 40.8, 83.7, 93.3, 120.5, 125.1,
4.3.8. BRD3152: 1-methacryloyl-5,6-dihydropyridin-2(1H)-one. 1H
NMR (300 MHz, chloroform-d)
128.4, 130.4, 133.0, 145.6, 153.0, 163.7. Exact mass (MþNa)þ calcd:
d
2.01 (t, J¼1.2 Hz, 3H), 2.50 (tdd,
248.0687; found 248.0685.
J¼6.4, 6.4, 4.3, 1.9 Hz, 2H), 3.89 (t, J¼6.5 Hz, 2H), 5.24 (quin,
J¼1.4 Hz, 1H), 5.29 (t, J¼0.9 Hz, 1H), 5.98 (dt, J¼9.7, 1.9 Hz, 1H), 6.93
4.3.16. BRD5525:
(E)-1-(6-(3-(2,6-dimethoxy-4-(3-oxo-3-(2-oxo-
(dt, J¼9.7, 4.1 Hz, 1H); 13C NMR (75 MHz, chloroform-d)
d
18.9, 24.7,
5,6-dihydropyridin-1(2H)-yl)prop-1-en-1-yl)phenoxy)propoxy)-5,7-
dimethoxy-3,4-dihydronaphthalene-2-carbonyl)-5,6-dihydropyridin-
42.3, 117.6, 125.2, 142.6, 145.3, 165.1, 175.0. Exact mass (MþH)þ
calcd: 166.0868; found: 166.0862.
2(1H)-one. 1H NMR (300 MHz, chloroform-d)
d
2.19 (quin, J¼6.4 Hz,
2H), 2.41e2.60 (m, 6H), 2.76e2.91 (m, 2H), 3.78 (s, 3H), 3.78e3.80
(m, 3H), 3.84 (s, 6H), 3.86e3.93 (m, 2H), 4.05 (t, J¼6.4 Hz, 2H),
4.18e4.32 (m, 4H), 6.00 (d, J¼9.8 Hz, 1H), 6.06 (d, J¼10.0 Hz, 1H),
6.53 (s, 1H), 6.79 (s, 2H), 6.88e7.03 (m, 3H), 7.42 (d, J¼15.3 Hz, 1H),
4.3.9. BRD3930: 1,4-bis(2-oxo-5,6-dihydropyridin-1(2H)-yl)butane-
1,4-dione. 1H NMR (500 MHz, chloroform-d)
d
2.41 (dtd, J¼8.5, 4.3,
4.3, 2.0 Hz, 4H), 3.26 (s, 4H), 3.95 (t, J¼6.6 Hz, 4H), 5.98 (dt, J¼9.8,
2.0 Hz, 2H), 6.76e6.95 (m, 2H); 13C NMR (126 MHz, chloroform-d)
7.68 (d, J¼15.8 Hz, 1H); 13C NMR (75 MHz, chloroform-d)
d 174.5,
d
24.5, 34.3, 40.9, 125.8, 145.1, 165.2, 175.3. Exact mass (MþNa)þ
169.1, 166.1, 165.6, 153.8, 152.1, 151.1, 145.6, 145.3, 144.2, 142.7, 139.7,
135.0, 133.5, 130.7, 128.7, 126.1, 125.6, 123.1, 121.1, 108.5, 105.9, 71.1,
71.1, 60.9, 56.4, 56.3, 43.4, 41.9, 31.3, 29.9, 25.0, 23.9, 20.9. Exact
mass (MþNa)þ calcd: 695.2581; found 695.2571.
calcd: 299.1008; found 299.1013.
4.3.10. BRD4547:
1,10-((2E,20E)-3,30-(1,4-phenylene)bis(acryloyl))
bis(5,6-dihydropyridin-2(1H)-one). 1H NMR (500 MHz, chloroform-
d)
d
2.49 (tdd, J¼6.5, 6.5, 4.4, 1.7 Hz, 4H), 4.05 (t, J¼6.3 Hz, 4H), 6.06
4.3.17. BRD6008: (E)-3-((E)-2-cyano-3-(dimethylamino)acrylamido)
(dt, J¼9.6, 1.8 Hz, 2H), 6.95 (dt, J¼9.3, 4.4 Hz, 2H), 7.54 (d, J¼15.6 Hz,
propyl 3-(3,4,5-trimethoxyphenyl)acrylate. 1H NMR (500 MHz,
2H), 7.59 (s, 4H), 7.73 (d, J¼15.6 Hz, 2H); 13C NMR (126 MHz,
chloroform-d)
d
7.79 (s,1H), 7.61 (d, J¼15.9 Hz,1H), 6.82 (s, 2H), 6.54
chloroform-d)
d
24.8, 41.6, 122.7, 125.8, 128.7, 136.6, 142.5, 145.5,
(d, J¼15.9 Hz, 1H), 6.37e6.30 (m, 1H), 4.30 (t, J¼5.8 Hz, 2H), 3.88 (s,
6H), 3.87 (s, 3H), 3.53e3.45 (m, 2H), 3.30 (s, 3H), 3.17 (s, 3H),
165.7, 168.8. Exact mass (MþNa)þ calcd: 399.1321; found 399.1319.
1.99e1.90 (m, 2H); 13C NMR (126 MHz, chloroform-d)
d 167.2, 165.1,
4.3.11. BRD4605:
1,10-((2E,20E)-3,30-((propane-1,3-diylbis(oxy))
156.4, 153.5, 145.3, 140.1, 130.2, 120.2, 117.4, 105.5, 63.4, 61.1, 56.3,
47.5, 38.5, 38.1, 28.8. Exact mass (MþH)þ calcd: 418.1978, found
418.1983.
bis(3,5-dimethoxy-4,1-phenylene))bis(acryloyl))bis(5,6-
dihydropyridin-2(1H)-one). 1H NMR (300 MHz, chloroform-d)
d
7.68 (d, J¼15.6 Hz, 2H), 7.41 (d, J¼15.5 Hz, 2H), 7.06e6.88 (m, 2H),
6.78 (s, 4H), 6.05 (d, J¼9.7 Hz, 2H), 4.25 (t, J¼6.3 Hz, 4H), 4.04 (t,
J¼6.4 Hz, 4H), 3.82 (s, 12H), 2.57e2.41 (m, 4H), 2.18 (quin, J¼6.2 Hz,
4.3.18. BRD6245:
(E)-N-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)
8.93 (s, 1H),
but-2-enamide. 1H NMR (300 MHz, chloroform-d)
d
2H); 13C NMR (75 MHz, chloroform-d)
d
169.1, 166.0, 153.7, 145.6,
7.76 (d, J¼15.6 Hz, 1H), 7.33e7.09 (m, 2H), 6.82 (s, 2H), 6.51 (d,
144.1, 139.7, 130.6, 126.1, 121.1, 105.8, 71.1, 56.3, 41.8, 31.2, 25.0. Exact
J¼15.3 Hz, 1H), 3.90 (s, 6H), 3.89 (s, 3H), 1.98 (dd, J¼6.9, 1.4 Hz, 3H);
mass (MþNa)þ calcd: 669.2424, found 669.2413.
13C NMR (75 MHz, chloroform-d)
d 166.9, 166.0, 153.7, 146.3, 146.2,
140.9, 130.1, 124.8, 119.1, 106.0, 61.2, 56.4, 18.5. Exact mass (MþH)þ
4.3.12. BRD4691: 1,10-((2E,20E)-3,30-((((ethane-1,2-diylbis(oxy))bis(-
ethane-2,1-diyl))bis(oxy))bis(3,5-dimethoxy-4,1-phenylene))bis(a-
cryloyl))bis(5,6-dihydropyridin-2(1H)-one). 1H NMR (300 MHz,
calcd: 306.1341, found 306.1347.
4.3.19. BRD6312: (E)-1,4-bis(2-oxo-5,6-dihydropyridin-1(2H)-yl)but-
chloroform-d)
d
7.67 (d, J¼15.6 Hz, 2H), 7.40 (d, J¼15.5 Hz, 2H),
2-ene-1,4-dione. 1H NMR (500 MHz, chloroform-dþfew drops of
7.00e6.89 (m, 2H), 6.78 (s, 4H), 6.04 (d, J¼9.7 Hz, 2H), 4.17 (t,
J¼5.1 Hz, 4H), 4.04 (t, J¼6.5 Hz, 4H), 3.86 (s, 12H), 3.80 (t, J¼5.1 Hz,
methanol-d4)
d
7.56 (s, 2H), 6.97e6.87 (m, 2H), 5.96 (d, J¼9.8 Hz,
2H), 3.94 (t, J¼6.5 Hz, 4H), 2.48e2.38 (m, 4H); 13C NMR (126 MHz,

7566
Z.V. Boskovic et al. / Tetrahedron 69 (2013) 7559e7567
chloroform-d)
d
168.1, 165.6, 146.6, 134.1, 125.1, 41.6, 24.7. Exact
(75 MHz, chloroform-d) d 24.8, 41.6, 56.1, 70.3, 72.3, 105.5, 121.0,
mass (MþH)þ calcd: 275.1032, found 275.1039.
125.8, 130.6, 139.1, 143.8, 145.4, 153.5, 165.8, 168.9. Exact mass
(MþNa)þ calcd: 699.2530; found 699.2524.
4.3.20. BRD6335: bis(2,6-dimethoxy-4-((E)-3-oxo-3-(2-oxo-5,6-
dihydropyridin-1(2H)-yl)prop-1-en-1-yl)phenyl) succinate. 1H NMR
4.3.27. BRD9427: 1,10-((2E,20E)-3,30-((3,6,9,12-tetraoxatetradecane-
1,14-diylbis(oxy))bis(3,5-dimethoxy-4,1-phenylene))bis(acryloyl))
bis(5,6-dihydropyridin-2(1H)-one). 1H NMR (300 MHz, chloroform-
(500 MHz, chloroform-d)
d
2.50 (dt, J¼6.3, 4.4 Hz, 4H), 3.12 (s, 4H),
3.85 (s, 12H), 4.06 (t, J¼6.3 Hz, 4H), 6.06 (dt, J¼9.5, 1.6 Hz, 2H), 6.82
(s, 4H), 6.94e7.01 (m, 2H), 7.45 (d, J¼15.6 Hz, 2H), 7.69 (d, J¼15.1 Hz,
d)
d
7.67 (d, J¼15.6 Hz, 2H), 7.40 (d, J¼15.5 Hz, 2H), 7.00e6.88
2H); 13C NMR (126 MHz, chloroform-d)
d
24.8, 29.0, 41.6, 56.2,
(m, 2H), 6.78 (s, 4H), 6.04 (d, J¼9.7 Hz, 2H), 4.16 (t, J¼5.1 Hz, 4H),
4.03 (t, J¼6.4 Hz, 4H), 3.86 (s, 12H), 3.79 (t, J¼5.0 Hz, 4H), 3.75e3.57
(m, 12H), 2.56e2.40 (m, 4H); 13C NMR (75 MHz, chloroform-d)
104.9, 122.0, 125.7, 130.1, 133.4, 143.4, 145.6, 152.3, 165.8, 168.7,
169.6. Exact mass (MþNa)þ calcd: 711.2166; found 711.2135.
d
169.1, 166.0, 153.7, 145.7, 144.0, 139.3, 130.8, 126.0, 121.2, 105.7,
4.3.21. BRD7782: 1,10-((2E,20E)-3,30-(((((oxybis(ethane-2,1-diyl))bi-
s(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(3,5-dimethoxy-4,1-
phenylene))bis(acryloyl))bis(5,6-dihydropyridin-2(1H)-one). 1H
72.5, 70.8, 70.8, 70.6, 56.4, 41.8, 25.0. Exact mass (MþNa)þ calcd:
831.3316, found 831.3306.
NMR (300 MHz, chloroform-d)
d
2.40e2.54 (m, 4H), 3.70 (br, 8H),
4.3.28. BRD9521: (2E,2’E)-N,N0-(propane-1,3-diyl)bis(3-(3,4,5-
trimethoxyphenyl)acrylamide). 1H NMR (500 MHz, chloroform-
3.75e3.82 (m, 4H), 3.86 (s, 12H), 4.04 (t, J¼6.5 Hz, 4H), 4.16 (t,
J¼4.9 Hz, 4H), 6.04 (d, J¼9.8 Hz, 2H), 6.76 (br, 4H), 6.95 (dt, J¼9.5,
4.3 Hz, 2H), 7.40 (d, J¼15.4 Hz, 2H), 7.66 (d, J¼15.4 Hz, 2H); 13C NMR
dþfew drops of methanol-d4) 7.45 (d, J¼15.6 Hz, 2H), 6.71 (s, 4H),
d
6.39 (d, J¼15.6 Hz, 2H), 3.82 (s,12H), 3.80 (s, 6H), 3.40e3.31 (m, 6H),
(75 MHz, chloroform-d)
d 24.8, 41.6, 56.1, 70.3, 70.6, 72.3, 77.2,
1.71 (dt, J¼12.2, 6.3 Hz, 2H); 13C NMR (126 MHz, chloroform-d)
105.4,121.0,125.8,130.6,143.8,145.4,153.4,165.8,168.8. Exact mass
d 167.3, 153.4, 141.0, 139.5, 130.6, 120.0, 105.1, 61.0, 56.1, 36.4, 29.3.
(MþNa)þ calcd: 787.3054; found 787.3047.
Exact mass (MþH)þ calcd: 515.2393, found 515.2394.
4.3.22. BRD7991:
(300 MHz, chloroform-d)
(E)-N-methacryloylbut-2-enamide. 1H
8.35 (s, 1H), 7.25e6.90 (m, 2H), 5.79 (d,
NMR
4.4. Cell culture
d
J¼0.6 Hz, 1H), 5.56 (dd, J¼3.0, 1.4 Hz, 1H), 1.94 (s, 3H), 1.89 (dd,
HeLa and H1703 were obtained from ATCC and cultured in
recommended media in a 37 ꢁC incubator (5% CO2).
J¼6.5, 1.2 Hz, 3H); 13C NMR (75 MHz, chloroform-d)
d 167.4, 166.8,
147.1, 140.1, 124.3, 122.7, 18.7, 18.6. Exact mass (MþNa)þ calcd:
176.0687, found 176.0690.
4.5. ROS assay
4.3.23. BRD8091: 2,6-dimethoxy-4-((E)-3-oxo-3-(2-oxo-5,6-
Cells were plated at 4000 cells/well in black 384-well plates
(Corning) and allowed to attach overnight. The next day (ca. 90%
confluency), dilutions of compounds in DMSO were added by pin
transfer (CyBio Vario, 100 nL per well), and incubated for 90 min.
Media was changed using a Thermo Multidrop Combi liquid han-
dihydropyridin-1(2H)-yl)prop-1-en-1-yl)phenylethylfumarate. 1H
NMR (500 MHz, chloroform-d)
d
7.68 (d, J¼15.5 Hz, 1H), 7.46 (d,
J¼15.5 Hz, 1H), 7.14e7.00 (m, 2H), 7.00e6.90 (m, 1H), 6.83 (s, 2H),
6.05 (d, J¼9.7 Hz, 1H), 4.29 (q, J¼7.1 Hz, 2H), 4.05 (t, J¼6.4 Hz, 2H),
3.85 (s, 6H), 2.55e2.40 (m, 2H), 1.34 (t, J¼7.1 Hz, 3H); 13C NMR
dler to phenol red-free DMEM containing 10
mM CM/H2DCF/DA and
(126 MHz, chloroform-d)
d
168.9, 166.0, 164.9, 162.7, 152.4, 145.9,
10 g/mL Hoechst 33342. Following incubation for 15e30 min, cells
m
143.4, 135.6, 134.0, 132.4, 129.8, 125.9, 122.5, 105.0, 61.7, 56.4, 41.9,
were washed twice with PBS. Images were obtained using an
IX_Micro automated fluorescence microscope (Molecular Devices).
Quantitation of pixel intensity was performed using MetaXpress
software and signal intensity was calculated relative to wells in the
same plate treated with DMSO.
25.0, 14.3. Exact mass (MþH)þ calcd: 430.1502, found 430.1503.
4.3.24. BRD8143: (E)-ethyl 4-oxo-4-(2-oxo-5,6-dihydropyridin-
1(2H)-yl)but-2-enoate. 1H NMR (500 MHz, chloroform-d)
d 7.66 (d,
J¼15.4 Hz, 1H), 7.01e6.94 (m, 1H), 6.71 (d, J¼15.4 Hz, 1H), 6.02 (d,
J¼9.8 Hz, 1H), 4.24 (q, J¼7.1 Hz, 2H), 3.99 (t, J¼6.5 Hz, 2H),
2.53e2.42 (m, 2H), 1.30 (t, J¼7.1 Hz, 3H); 13C NMR (126 MHz,
4.6. ATP assay
chloroform-d)
d
167.7, 165.6, 165.5, 146.5, 137.2, 130.5, 125.2, 61.3,
Cells were plated at 1000 per well in white 384-well plates and
allowed to attach overnight. After addition of compounds by pin
transfer, plates were incubated 48 h. At that time, media was re-
moved and replaced with a solution of CellTiter-Glo reagent
(Promega) in PBS. After 10 min, luminescence was read using an
EnVision multilabel plate reader (PerkineElmer) and signal in-
tensity was calculated relative to in-plate DMSO control wells.
41.6, 24.8, 14.3. Exact mass (MþH)þ calcd: 224.0923, found
224.0916.
4.3.25. BRD85988: 1,10-(6,60-(propane-1,3-diylbis(oxy))bis(5,7-
dimethoxy-3,4-dihydronaphthalene-2,20-carbonyl))bis(5,6-
dihydropyridin-2(1H)-one). 1H NMR (300 MHz, chloroform-d)
d
2.20 (quin, J¼6.0 Hz, 2H), 2.48 (m, J¼7.9, 7.9 Hz, 4H), 2.54 (d,
J¼4.9 Hz, 4H), 2.85 (t, J¼8.5 Hz, 4H), 3.79 (s, 6H), 3.80 (s, 6H), 3.89 (t,
J¼6.6 Hz, 4H), 4.26 (t, J¼6.2 Hz, 4H), 6.00 (d, J¼9.8 Hz, 2H), 6.54 (s,
4.7. Immunofluorescence detection of glutathionylated
proteins
2H), 6.87e7.01 (m, 4H); 13C NMR (75 MHz, chloroform-d)
d 174.5,
165.6, 152.1, 151.1, 145.3, 142.7, 135.1, 133.5, 128.7, 125.6, 123.1, 108.5,
71.1, 61.0, 56.3, 43.5, 29.9, 25.1, 23.9, 20.9. Exact mass (MþNa)þ
calcd: 721.2737; found 721.2726.
Cells were plated at 3000 (HeLa) per well and allowed to attach
overnight. After addition of compounds by pin transfer, plates were
incubated for 6 h. Cells were then fixed with 2% paraformaldehyde
in PBS (20 min), permeabilized 30 min with PBSþ0.1% TritonX-100
(‘PBST’), and blocked 30 min with PBSTþ2%BSA. Primary antibody
(Ms anti-glutathione, Abcam Ab19534) was added (1:1250 in
PBSTþ2%BSA) and incubated at 4 ꢁC overnight. Following two
washes with PBST, cells were incubated at rt for 1 h in the dark with
secondary antibody solution (AlexaFluor 488-conjugated goat anti-
mouse, Jackson Immunochemicals, 1:750, plus Hoechst 33342,
4.3.26. BRD9080:
1,10-((2E,20E)-3,30-(((oxybis(ethane-2,1-diyl))bi-
s(oxy))bis(3,5-dimethoxy-4,1-phenylene))bis(acryloyl))bis(5,6-
dihydropyridin-2(1H)-one). 1H NMR (300 MHz, chloroform-d)
d
2.44e2.53 (m, 4H), 3.74e3.94 (m, 16H), 4.04 (t, J¼6.4 Hz, 4H), 4.19
(t, J¼5.1 Hz, 4H), 6.05 (d, J¼9.6 Hz, 2H), 6.78 (s, 4H), 6.95 (dt, J¼9.4,
4.3 Hz, 2H), 7.41 (d, J¼15.4 Hz, 2H), 7.67 (d, J¼15.6 Hz, 2H); 13C NMR

Z.V. Boskovic et al. / Tetrahedron 69 (2013) 7559e7567
7567
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Acknowledgements
We thank Weicheng Zhang for synthesizing compounds
BRD2724 and BRD1764. This research was supported by the Na-
tional Institute of Health (NIGMS-38627). S.L.S is a Howard Hughes
Medical Institute Investigator.
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Supplementary data
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Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2013.05.080.
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