Bis-Michael Acceptors as Novel Probes to Study the Keap1/Nrf2/ARE Pathway

Article abstract of DOI:10.1021/acs.jmedchem.6b01132

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator that promotes the transcription of cytoprotective genes in response to oxidative/electrophilic stress. Various Michael-type compounds were designed and synthesized, and their potency

Full text of DOI:10.1021/acs.jmedchem.6b01132

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Article
Bis-Michael Acceptors as Novel Probes to Study the Keap1/Nrf2/ARE Pathway
Ludovic J. Deny, Hussein Traboulsi, André M. Cantin,
Eric Marsault, Martin V. Richter, and Guillaume Bélanger
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 28 Sep 2016
Downloaded from http://pubs.acs.org on September 28, 2016
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Bis-Michael Acceptors as Novel Probes to Study the
Keap1/Nrf2/ARE Pathway
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†§
§‡
§‡
§||
Ludovic J. Deny , Hussein Traboulsi , André M. Cantin , Éric Marsault , Martin V.
§‡
Richter , and Guillaume Bélanger†§*
†Département de chimie, Université de Sherbrooke, Sherbrooke, Québec J1K 2R1, Canada
§Institut de pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec J1H
5
N4, Canada
‡Département de médecine, service de pneumologie, et Centre de recherche clinique du Centre
hospitalier de l’Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada
||Département de pharmacologie, Université de Sherbrooke, Sherbrooke, Québec J1K 2R1,
Canada
KEYWORDS. Nrf2, Keap1, Michael acceptor, Nazarov acceptor, antioxidant response element,
bis-electrophile, luciferase assay, Oxidative/Electrophilic stress.
ABSTRACT. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator that promotes
the transcription of cytoprotective genes in response to oxidative/electrophilic stress. Various
Michael–type compounds were designed and synthesized and their potency to activate the
Keap1/Nrf2/ARE pathway was evaluated. Compounds bearing two Michael–type acceptors
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proved to be the most active. Tether length and rigidity between the acceptors was crucial. This
study will help to understand how this feature disrupts the interaction between Keap1 and Nrf2.
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INTRODUCTION
Oxidative/electrophilic stress (OES) is the consequence of an abnormal production of reactive
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oxygen and nitrogen species and/or electrophiles in the cells. OES stress is linked to over 200
diseases and conditions such as cancers, diabetes, pulmonary infections and neurodegenerative
disorders. The Keap1/Nrf2/ARE pathway plays a central role in the cellular response to OES since
the Antioxidant Response Element (ARE) is an essential component of the cellular antioxidant
defense and is under the transcriptional control of Nuclear factor erythroid 2-related factor 2
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Nrf2).2–4
Because Kelch like ECH Associated Protein 1 (Keap1) regulates Nrf2-mediated transcription,
the former is recognized as a therapeutic target with high potential. Indeed, the genes targeted by
the Keap1/Nrf2/ARE pathway are involved in the synthesis and conjugation of glutathione and
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antioxidant proteins and enzymes. Understanding the Keap1/Nrf2/ARE pathway has thus become
extremely important and the focus of drug discovery programs recently initiated by several
research groups.6–18
In the basal state, Nrf2 is sequestered in the cytosol via its interaction with a Keap1 dimer. When
this Keap1-Nrf2 complex binds to the Keap1-dependent E3-ubiquitin ligase (Cullin 3), Nrf2 is
then degraded by the proteasome and the Keap1 dimer is free to bind to a newly translated Nrf2.
This maintains low concentrations of free Nrf2 in the nucleus and transcription of genes related to
ARE at basal levels. When Keap1 is targeted by small molecules, the conformation of the Keap1
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dimer is modified and its interactions with Nrf2 are tighter. Accordingly, the ubiquitination of Nrf2
is stopped and newly synthesized Nrf2 can accumulate in the nucleus and turn on the expression
of cytoprotective genes. According to Dinkova-Kostova et al., this mode of action is proper to
electrophilic inducers.19
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Keap1 comprises five domains: 1) the N-terminal region, 2) the Broad complex, Tramtrack,
Bric-a-Brac (BTB) domain (exposing Cys151, responsible for the formation of the Keap1 dimer),
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) the Intervening region (IVR, exposing Cys273, Cys288, and 6 others), 4) the Kelch repeat
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domain (responsible for the recognition of Nrf2), and 5) the C-terminal region. Among strategies
used to disrupt the Keap1-Nrf2 interaction, non-covalent protein-protein interaction inhibitors
have recently been reported.6–10 Several crystal structures reveal that these molecules target the
binding site of Nrf2 on Keap1 (Kelch repeat domain).7–9 Another strategy consists of using
electrophiles from synthetic or natural sources.11–18 The latter proved particularly successful
(Figure 1) and are believed to target some of the cysteine (Cys) residues of Keap1. Among them,
Cys151 (BTB domain) as well as Cys273 and Cys288 (IVR domain) appear to be the most
sensitive to electrophiles.21
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Figure 1. Electrophilic modulators of the Keap1/Nrf2/ARE pathway.
Despite potential toxicity issues associated with off-target interactions, covalent drugs
nonetheless offer certain advantages over traditional drugs such as low administration doses and
frequency, selectivity, and potentially longer action times.22,23 Most of the time, covalent drugs are
designed in a way that a warhead reacts irreversibly with the targeted host. This renders off-target
interactions hard to circumvent except by modulating the reactivity of the warhead. We decided to
tackle this issue in a completely different way: since Keap1 offers a unique profile with its multi-
cysteine domains, we rather opted to design new agonists bearing two moderately reactive
reversible cysteine acceptors. Dual cysteine trapping should significantly increase the target
residence time: once the first cysteine is anchored, trapping of a second cysteine becomes a
kinetically favored intramolecular process that should also be thermodynamically beneficial due
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to enthalpy gain and weak entropy variation. Proper distance and/or nature of the acceptors,
combined with traditional secondary non-covalent interactions, should provide selectivity to either
the BTB or the IVR domains for these new agonists. Herein, we report the synthesis of series of
single and double Michael-type acceptor compounds and their biological evaluation toward the
Keap1/Nrf2/ARE pathway. For the first time, a systematic study of the effect of the length and
rigidity of the tether between the Michael acceptors, as well as the effect of the electrophilicity of
these acceptors offers new insight into how this therapeutic target reacts to multi-electrophile
compounds.24
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SYNTHESIS
Single-electrophilic compounds. ,’-Unsaturated β-dicarbonyle compounds were
synthesized in three to four steps from the corresponding ketones. First, the methoxycarbonylation
of monoprotected cyclohexane-1,4-dione 1 gave the β-ketoester 2 (Scheme 1). Upon deprotonation
with two equivalents of LDA, the resulting dianion was reacted with a series of electrophiles.25
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Finally, selenation and oxidative elimination generated the Michael acceptors 3 and 4.
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Scheme 1α
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αReagents and conditions: (a) NaH, Me
CH Cl , 0 °C; (d) H , CH Cl , 0 °C.
CO , reflux; (b) LDA, R-I, rt; (c) PhSeBr, pyridine,
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In order to vary the lipophilicity of the compounds, the same sequence was applied to the gem-
dimethyl analog 5 (Scheme 2). For β-ketoesters bearing an unsaturation (cf. compounds 10 and
12), final oxidation gave best results using DDQ.
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Scheme 2α
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αReagents and conditions: (a) NaH, KH, Me
pyridine, CH Cl , 0 °C; (d) H , CH Cl , 0 °C; (e) DDQ, 1,4-dioxane, rt.
CO , THF, reflux; (b) LDA, R-X, rt; (c) PhSeBr,
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To study the influence of the ester portion, transesterification of 6 using allyl alcohol or tert-
butanol as the solvent gave mixtures of esters. After oxidation, 7 and 18 or 7 and 19, respectively,
could be isolated (Scheme 3). We also prepared the corresponding acid 20 by hydrolysis of
unsaturated keto-ester 7.13
To test the influence of the electrophilicity of the Michael acceptor, several variations were made
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(Scheme 4). First, a reduction of 7 in Luche conditions afforded the alcohol 21. Chloro- and
iodoenones 23 and 24 were prepared using known procedures.31,32 Morita-Baylis-Hillman reaction
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on 22 with paraformaldehyde gave the allylic alcohol 25. Further transesterification afforded
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6.34 Classic methoxycarbonylation of enone 22 provided keto-ester 27 which, upon α-
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methylation, afforded 28 as a racemic mixture. On the other hand, oxidation of keto-ester 27 by
selenium dioxide gave dienone 29.36
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Scheme 3α
αReagents and conditions: (a) DMAP, 4 Å MS, ROH, reflux; (b) PhSeBr, Pyridine, CH
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Cl ,
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Scheme 4α
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αReagents and conditions: (a) CeCl
rt; (c) I , pyridine, CCl , rt; (d) Paraformaldehyde, NaHCO
, NaBH
, MeOH, rt; (b) PhI(OAc)
, pyridine·HCl, CH
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, imidazole, THF, rt; (e) 6, Et
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toluene, Dean-Stark; (f) NaH, Me
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Bis-electrophilic compounds. In order to attach two Michael acceptors on the same molecule
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and assess the influence of rigidity on activity, we used tethers of different length (1-4 carbons,
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1-32) and rigidity (alkane, alkene 33, and alkyne 34, Scheme 5). These tethers were incorporated
by bis-alkylation with the dianion of 6. No significant amount of single alkylation bi-products was
observed. The ensuing tandem oxidations afforded the desired products 30-34, albeit in low yield.
A series of compounds bearing a dioxolane instead of the gem dimethyl was also prepared for
comparison (35, 36). Finally, unsymmetrical bis-electrophilic compound 37 was obtained by
oxidation of 26 with DDQ.
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Scheme 5α
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αReagents and conditions: (a) LDA, R-X, rt; (b) PhSeBr, pyridine, CH
CH Cl , 0 °C; (d) DDQ, 1,4-dioxane, rt; (e) DDQ, K CO , 1,4-dioxane, rt.
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RESULTS AND DISCUSSION
ARE inducing activity and cytotoxicity. To evaluate the ARE-inducing activity, all
compounds, at concentrations ranging from 0 to 100 µM, were incubated with ARE-Luc-
transfected human embryonic kidney cells (HEK-293T) for 24 h. Suforaphane (10 M) was used
as a positive control. Also, NAD(P)H:quinone oxidoreductase (NQO1) induction was measured
by quantitative polymerase chain reaction (qPCR) in RAW 264.7 cells using sulforaphane as a
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positive control. In addition, cytotoxicity was evaluated for each molecule in a lactate
dehydrogenase (LDH) assay in HEK-293T cells. The data are reported as Mean ± SEM based on
three separated experiments.
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Lipophilicity as an off-target indicator. Compounds lacking any substitution at the C4 position
(see 7, with two hydrogens instead of gem-dimethyl) were too electrophilic. Difficulties in their
isolation and purification compromised the validity of their evaluation.
To counterbalance for the level of reactivity of these electrophiles, steric hindrance around C4
was increased. We chose commercially available ketone 1 as a starting point for comparison.
Analogues with a dioxolane moiety (3, 4, 35 and 36) showed moderate activity and significant
toxicity at the higher concentrations. (Figure 2).
We hypothesized that toxicity was due to the hydrophilicity associated to the dioxolane
moiety.14,37 To test this hypothesis, we increased the compounds’ lipophilicity. Replacing the
dioxolane with a gem-dimethyl provided an increased activity with no toxicity even at high
concentrations (e.g., 7). Further exploration of the substitution at C6 was essential to generate
unambiguous biological data (Table 1). The single electrophiles showed better activities at 100
µM (compounds 7–12), with no apparent toxicity. They were much more lipophilic than their
dioxolane analogues according to their clogP values, which tends to support our hypothesis.
Importance of electrophilicity. In order to induce the transcription of genes controlled by ARE,
a first cysteine nucleophilic addition to the electrophilic compounds has to occur. Analogs 7 – 12
suggest that unsaturated keto esters are indeed suitable electrophiles to study Keap1 reactivity.
Replacement of the methyl ester by larger groups (allyl 18 or t-Bu 19) gave a slight increase in
activity, although accompanied with increased toxicity, whereas the corresponding acid (20)
retained most of the activity. Replacement of the methyl ester by a chloride (23) had no significant
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effect, but the iodide derivative (24) was not reactive enough. This effect was also observed with
other poor electrophiles, such as those lacking the ketone (21) or the ester (25, 26, and 28), with
the exception of compound 27. This comes as a surprise because it is hard to evaluate the combined
participation of the carbonyls in 27 while we expected 29 to be much more potent.38
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A
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Figure 2. (A) Fold induction of ARE-luciferase gene expression; (B) % cell mortality induced by
compounds 3, 4, 35, 36 and 7 at 10, 20, 50 and 100 M
Boost in activity with a second Michael acceptor. In order to take advantage of the numerous
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cysteine residues of Keap1, we tested compounds with two Michael-type acceptors. Right away,
this second class of molecules turned out to be very potent (30, 31, and 38), with an astonishing
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67-fold increase from 7 to 31 at 10 M. Even though solubility and toxicity became an issue at
higher concentrations, the study of similar compounds provided new insights about the target.
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2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
First, the addition of a second unsaturated keto-ester enhanced the fold-induction drastically.
Second, the spatial position between the two electrophilic parts is important to modulate activity.
Indeed, going from a simple methylene (30) to a propane linker (31) resulted in a ca. 70-fold
increase (at 20 M), whereas an additional factor 2 was gained with a butane tether (32).
So far, it was not clear whether the tether length optimization reflected an optimal distance
between the two Michael acceptors for reaction with two cysteine residues in the binding site, or
the result of a gain in hydrophobic interactions. To answer this, we decided to rigidify the tether.
From butane (32) to E-butene (33) to butyne (34) tethers, increased rigidity resulted in a significant
increase in activity, with compound 34 being the best compound so far (50-fold-induction at only
1 M). These results strongly suggest an optimal distance and conformation between the Michael
acceptors for interaction with Keap1.
0
1
2
3
4
5
6
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8
9
0
1
2
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9
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2
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2
3
4
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9
0
Table 1. Fold-induction of ARE-luciferase gene expressionα

Fold-induction at: 
clogP
Compound
1
M
20 M
100 M
1
3
4
R = Hex
1.40 ± 0.25 2.19 ± 0.19
0.98 ± 0.04 2.49 ± 0.08
20.19 ± 1.10
2.49
1
R = Me
N/A
-0.16
1
7
R = H
1.36 ± 0.34 6.46 ± 4.26
1.15 ± 0.04 2.00 ± 0.43
1.17 ± 0.11 2.75 ± 0.22
1.34 ± 0.51 2.05 ± 0.06
0.95 ± 0.12 0.92 ± 0.04
1.14 ± 0.12 1.48 ± 0.44
50.58 ± 12.39
64.65 ± 14.31
4.60 ± 0.10
1.82
4.98
2.34
2.91
3.91
2.39
1
8
R = Hex
1
9
R = Me
1
10
11
12
R = allyl
25.32 ± 4.03
1
R = Bn
48.53 ± 14.74
34.16 ± 2.62 (7%)
1
R = propargyl
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R = CH
2
2
2
2
CF
3
0.54 ± 0.07 1.54 ± 0.22
t-Bu 0.65 ± 0.09 2.00 ± 0.17
1.03 ± 0.39 2.22 ± 1.13
17.90 ± 6.18 (11%)
1.75 ± 0.18
2.58
3.26
1.40
1.72
2.30
1
R = CH
CO
2
1
R = CH
CN
17.13 ± 2.52
8.71 ± 1.01
1
R = CH
C(O)Me 0.49 ± 0.06 2.20 ± 0.07
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1
R = (CH
2
2
) CO
2
Me 0.68 ± 0.08 1.24 ± 0.09
1.39 ± 0.27 4.36 ± 0.50
1.05 ± 0.05 3.89 ± 0.39
2.00 ± 0.19 3.11 ± 0.73
1.33 ± 0.18 4.42 ± 0.69
1.22 ± 0.16 1.16 ± 0.23
0.72 ± 0.05 0.77 ± 0.17
2.98 ± 0.36 3.79 ± 1.10
5.96 ± 1.17
2
R = CO
2
2
2
All
t-Bu
H
94.79 ± 22.67 (17%) 2.59
106.40 ± 20.32 (16%) 3.06
2
R = CO
2
R = CO
29.56 ± 1.28
43.41 ± 3.32
17.35 ± 0.47
1.46 ± 0.13
4.02 ± 0.18
1.39
2.34
2.71
0.87
3.40
2
R = Cl
2
24
25
26
R = I
2
R = CH
2
OH
2
R =
2
1
1.00 ± 0.07 1.85 ± 0.37
1.29 ± 0.34
1.91
3
27
R = H
1.05 ± 0.11 3.20 ± 0.35
1.47 ± 0.13 2.43 ± 0.09
41.93 ± 7.94
4.56 ± 0.41
1.43
1.95
3
2
8
R = Me
2
9
1.35 ± 0.21 3.39 ± 0.41
4.49 ± 0.43 3.60 ± 0.48
10.32 ± 0.72
1.96
3
3
0
1
101.10 ± 15.76 (24%) 3.79
4.71 ± 1.11 247.70 ± 17.80 Not tested
4.51
5.04
32
8.23 ± 0.38 Not tested
Not tested
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15.20 ± 0.79 Not tested
52.46 ± 3.01 Not tested
Not tested
Not tested
4.56
4.38
34
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n = 1 0.94 ± 0.03 2.76 ± 0.15
n = 3 1.00 ± 0.04 1.05 ± 0.15
15.94 ± 0.67 (12 %) -1.20
30.91 ± 2.14 (9%) -0.48
3
7
2.02 ± 0.40 3.78 ± 0.56
121.1 ± 20.60 (15%) 3.28


Sulforaphane has a fold-induction of 9.80 ± 1.00 at 10 µM. % Mortality evaluated in the LDH
assay are reported when over 5 %. Predicted values by ChemDraw 12.0.

These results raise the question whether the second Michael acceptor on 30-34 acts as a cysteine
trap. It turns out that the Michael acceptors on these molecules are similar to the ,’-unsaturated
2
0

-cyanoketone of CDDO, which was co-crystallized in the BTB domain of Keap1. We have thus
docked our molecules in the crystal structure and set the distance between the nucleophilic sulfur
and the electrophilic carbon to 1.8 Å as in CDDO. Interestingly, there is no other cysteine residue
in the active site of the BTB domain that could act as a partner for a second nucleophilic addition.
In return, the ketone and the ester functions were able to make hydrogen bonds with other residues
in the active site and within the electronic density area. Hence, in the absence of a crystal of Keap1
with any of compounds 30-34, we cannot answer the question on the nature of the interaction of
the second Michael acceptor with Keap1. As suggested by docking, it could be a H-bond in the
BTB domain, but it could also be that these molecules rather target the IVR region. Unfortunately,
this region of Keap1 has not been crystallized yet and we cannot answer this hypothesis clearly at
this point.
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Figure 3. Docking of 31 in BTB domain of Keap1 using PyMol and NRGsuite
Added functionality reduces activity. In order to prove whether only an H-bond acceptor is
needed in addition to the electrophile, we installed different chemical functions (13 – 17). None of
these compounds showed activity. Even 17 is particularly inactive, although it presents a distance
between the ester side chain and the Michael-type acceptor that resembles the distance between
Michael-type acceptors in 30. Having H-bond acceptors on the side chain (14-17) is even more
detrimental than simple alkyl side chains (8, 10-12), suggesting that this part of the molecule also
plays a role in lipophilic interactions with the active site.
NQO1 expression. Michael-type acceptors are listed as pan assay interference compounds
4
0
(
PAINs). In order to address this eventuality in the case of the present class of molecules, we
measured the expression of NQO1, a Nrf2 positively regulated gene, by qPCR in RAW 264.7 cells
for sulforaphane and 14 compounds of interest at 10 M (supporting information, Table
S1).5,11,13,15,18 We observed the same tendencies as those observed in the luciferase ARE reported
assay (Figure 4). Indeed, an aliphatic side chain (8) is preferred over a functionalized one (17), the
addition of a Michael acceptor enhances activity (cf. 8 and 32), and an increased rigidity between
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Michael acceptors is beneficial (cf. 32, 33, and 34). This confirms that the observed activity is
indeed dependent on the Nrf2 pathway, and not on a promiscuous activity. Moreover, these
Michael acceptors are much more potent than the naturally occurring sulforaphane.
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0
µM
µM
2 µM
S F N
8
17
32
33
34
Figure 4. Fold induction of NQO1 expression by sulforaphane (SFN), compounds 8, 17, 27, 32,
3
3 and 34 at 2, 5 and 10 M
CONCLUSION
In this study, we report herein the synthesis and biological evaluations of 32 Michael acceptors
toward the Keap1/Nrf2/ARE pathway for the transcription of antioxidant cellular responses. This
study gives several insights on how reversible covalent modulators interact with Keap1. The
electrophilic part of the molecule has to be sufficiently lipophilic to show some activity.
Furthermore, the electrophile has to be powerful enough to react with the targeted cysteine, an
essential element in the design of Keap1 ligands to stop the ubiquitination and subsequent
degradation of Nrf2. The 1-methoxycarbonyl-3,3-dimethylcyclohex-2-enone motif answered
these two criteria. Addition of a second Michael acceptor had a crucial impact on activity, up to a
5
0-fold-induction at only 1 M. The distance between the Michael acceptors as well as the rigidity
of the tether were optimized to generate our best compound (34) in this family.
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Our results suggest that the role of the second Michael acceptor is unlikely to be a hydrogen
bond acceptor as none of the compounds with heteroatoms at the end of the side chain showed
activity. We could hypothesize that the second Michael acceptor rather serves as a second cysteine
trap. This remains to be proven, but the consequences of such a hypothesis, should it turn out to
be true, are of high importance. Indeed, trapping of several cysteine residues would imply that the
BTB domain is not the target, but probably the IVR region of Keap1.
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We believe our study will serve to increase the understanding of this target toward the generation
of new drug leads. Our data also highlight the importance to design appropriate electrophiles
toward covalent drug. Investigations on the nature and exact role of the second electrophile are in
progress and the results will be published in due course.
EXPERIMENTAL SECTION
Biology. Cell lines. Human Embryonic Kidney 293 cells (HEK-293T; ATCC CRL-1573) and
the murine macrophage cell line (RAW 264.7; ATCC TIB-71) were obtained from American Type
Culture Collection (Manassas, VA). Cells were cultured using standard methods at 37°C in 5%
CO
with 10% fetal bovine serum (FBS), antibiotics (streptomycin at 100 mg/ml and penicillin at
00 U/ml), 5% sodium pyruvate, 5% nonessential amino acids, and 2 mM L-glutamine (complete
2
– 95% air in Dulbecco's modified eagle medium (DMEM) (Wisent, Canada) supplemented
1
DMEM).
Luciferase assays. The day before transfection, Human Embryonic Kidney HEK 293T cells
5
(
1 × 10 ) were plated in 24-well tissue culture-treated plates. Cells were transiently transfected
with 0.1 g of ARE-luc (Nrf2 response element) plasmid using FuGENE6 transfection reagent
(Promega, Madison, WI; E2691). The day after transfection, cells were treated with Keap1 ligands
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at different concentrations or with sulforaphane at 10 M. After 24 h, cell lysates were than
assayed for luciferase activity using the luciferase assay system from Promega (E1500).
Real-time PCR analyses. RAW 264.7 cells were treated with 2, 5 and 10 M of different
compounds or with 10 M of sulforaphane for 5 hours. Total RNA was then extracted using
Ribozol reagent according to the manufacturer’s protocol. A total of 500 ng of the resulting RNA
was then reverse transcribed using iScript reverse transcription supermix for the reverse
transcriptase quantitative PCR (RTqPCR) kit (Bio-Rad). Real-time PCRs were performed with the
iQ SYBR green supermix (Bio-Rad). Amplification plots were generated using the Rotorgene
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000 Application software version 1.7 (Corbett Research), and fold induction was calculated using
the threshold cycle (2∆∆Ct) method and using 18S expression for normalization. Primer sequences
are:
Human/Mouse 18S:
FW: 5-AGGAATTGACGGAAGGGCAC-3
RV: 5-GGACATCTAAGGGCATCACA-3
Mouse NQO1:
FW: 5-TCACAGGTGAGCTGAAGGAC-3
RV: 5-CTTCCAGCTTCTTGTGTTCG -3
Cell viability assay. The cytotoxicity of Keap1 ligands on cells was determined using the LDH
assay. Briefly, HEK 293T cells were treated with increasing concentrations of Keap1 ligands.
After 24 hours, supernatants were collected and cells were lysed in triton 1%. To measure lactate
dehydrogenase (LDH) activity, buffer containing L-(+)-Lactic acid and Nicotinamide adenine
dinucleotide (NAD) (Sigma-Aldrich; L-1750 and N7004 respectively) was added to the
supernatant and the cells lysate, followed by measurement of absorbance at 340 nm.
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Chemistry. General. All reactions requiring anhydrous conditions were conducted in flame-
dried glassware under a dry nitrogen or argon atmosphere. THF was distilled from Na and
benzophenone under nitrogen immediately prior to use. 1,4-dioxane was distilled from Na and
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benzophenone under argon and kept on 4 Å molecular sieves. DCM, MeOH, toluene, i-Pr
2
NH,
Et N, and pyridine were distilled from CaH under nitrogen at atmospheric pressure immediately
3
2
prior to use. All other required fine chemicals were used directly without purification. All reference
to “water” correspond to deionized water. All references to “brine” refer to a saturated aqueous
sodium chloride solution. Melting points were determined on a Thomas Hoover capillary melting
point apparatus and are uncorrected. All new compounds showed chemical purity of ≥95% as
1
1
assessed by H NMR. All NMR spectra were recorded on an AV300 Bruker (300 MHz for H and
13
1
13
75 MHz for C) or an AS400 Variant (400 MHz for H and 100 MHz for C). Chemical shifts
are referenced to  7.26 signal of CHCl
3
(1H NMR) and 77.16 signal of CDCl (13C NMR) as
3
1
internal standards for deuterated chloroform and to  5.30 signal of CH
2
Cl
2
( H NMR) and  53.52
13
signal of CD
reported as follows: chemical shift in ppm (multiplicity [singlet (s), doublet (d), triplet (t), quartet
q), and multiplet (m)], integration, coupling constants [Hz]). Carbon spectra were recorded with
2
Cl
2
( C NMR) as internal standards for deuterated DCM. Data for proton spectra are
(
complete proton decoupling and the chemical shifts are reported in ppm. High resolution mass
spectrometry data were obtained by the ESI-Q-Tof (maXis). TLC was conducted with pre-coated
6
0 Å 250 µm silica gel plates with F-254 indicator and visualized using a combination of UV and
potassium permanganate staining. Flash column chromatography was performed using silica gel
(230–400 mesh). IR spectra were recorded with a FTIR instrument by applying substrates neat.
Usual Reaction Work-up and Purification. After addition of the indicated aqueous solution,
layers were separated. The aqueous phase was extracted with the indicated solvent, and the
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combined organic phases were washed with the indicated aqueous solution (if needed), dried over
anh MgSO , filtered, and concentrated under reduced pressure using a rotary evaporator. The crude
4
material was purified by flash chromatography using silica gel with the indicated eluent.
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Methyl 8-oxo-1,4-dioxaspiro[4.5]decane-7-carboxylate (2). A solution of 1 (3.0 g, 19.2 mmol)
in Me
2
CO
3
(20 mL) was added dropwise to a suspension of NaH (60% in oil, 1.54 g, 38.4 mmol)
(10 mL) at rt. The reaction was heated to reflux for 4 h, then cooled to 0 °C. Saturated
in Me
2
CO
3
aq NH Cl (30 mL) was added and the usual work-up (EtOAc; brine) and purification (5-15%
4
EtOAc in hexanes) afforded compound 2 (3.81 g, 93%) as a pale yellow oil. Spectral data was
consistent with that previously reported.41
Methyl 9-hexyl-8-oxo-1,4-dioxaspiro[4.5]dec-6-ene-7-carboxylate (3). LDA (2.86 mmol)
was prepared by the addition of n-BuLi (2.5 M in hexanes, 1.15 mL, 2.86 mmol) to a precooled (–
7
8 °C) solution of diisopropylamine (402 L, 2.86 mmol) in THF (1.0 mL). The mixture was
strirred for 30 min then a solution of 2 (201.3 mg, 0.94 mmol) in THF (0.5 mL + 0.5 mL for
rinsing) was added dropwise via canula. The mixture was stirred for 1 h at 0 °C then 1-iodohexane
(
0.69 mL, 4.68 mmol) was added. The resulting mixture was allowed to warm up to rt and stirred
overnight. Water (15 mL) was added and the usual work-up (EtOAc) and purification (silica gel
saturated with Et N, 0 to 5% EtOAc in hexanes) gave the corresponding alkylated β-ketoester
158.7 mg confirmed by mass spectrometry) as a yellow oil. A solution of the latter and pyridine
0.12 mL, 1.48 mmol) in CH Cl (2.0 mL) was added to a solution of PhSeCl (269.9 mg, 1.41
Cl (2.0 mL) at rt. The mixture was stirred for 3 h then 1 N aqueous HCl (10 mL)
was added. The usual work-up (CH
3
(
(
2
2
mmol) in CH
2
2
2
Cl
2
) and purification (10% Et
2
O in hexanes) gave the
1
corresponding phenylselanyl-β-ketoester (197.3 mg, 46% over 2 steps) as a yellow oil: H NMR
(CDCl
3
, 400 MHz)  (ppm) 7.57 (d, 2H, J = 7.3 Hz), 7.40 (t, 1H, J = 7.3 Hz), 7.30 (t, 2H, J = 7.4
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Hz), 3.89 (m, 4H), 3.65 (s, 3H), 2.93 (m, 1H), 2.50 (dd, 1H, J = 3.6, 13.7 Hz), 2.17 (d, 1H, J =
1
6
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3.7 Hz), 2.01 (m, 1H), 1.88 (m, 1H), 1.79 (t, 1H, J = 13.5 Hz), 1.28 (br s, 9H), 0.87 (t, 3H, J =
13
.7 Hz). C NMR (CDCl
3
, 100 MHz)  (ppm) 204.72, 170.07, 138.76, 129.68, 128.77, 126.38,
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06.88, 64.93, 64.23, 59.69, 52.74, 46.61, 43.54, 41.75, 31.43, 29.41, 29.20, 26.80, 22.72, 14.22.
SeNa: 477.1152, found: 477.1152. IR (neat)  2952, 2926,
856, 1713, 1436 cm . An aqueous H solution (30 %, 1.0 mL) was added to a solution of
phenylselanyl-β-ketoester (196.1 mg, 0.43 mmol) in CH Cl (3.0 mL) at rt. The mixture was
vigorously stirred for 90 min then water (1 mL) and CH Cl
up (CH Cl
HRMS (ESI-Q-Tof) calcd for C22
H
30
O
5
-1
2
2
O
2
2
2
2
2
(3 mL) were added. The usual work-
1
2
2
) afforded pure 3 (122.8 mg, 95%) as a pale yellow oil: H NMR (CDCl
3
, 300 MHz) 
(ppm) 7.06 (d, 1H, J = 2.0 Hz), 4.07 (m, 4H), 3.81 (s, 3H), 2.72 (m, 1H), 2.21 (m, 1H), 2.05 (dd,
1
3
1
H, J = 12.3, 13.2 Hz), 1.86 (m, 1H), 1.27 (br s, 9H), 0.87 (t, 3H, J = 6.8 Hz). C NMR (CDCl ,
3
75 MHz)  (ppm) 196.20, 164.59, 147.61, 132.55, 103.63, 65.31, 64.92, 52.31, 45.48, 38.37, 31.54,
9.14, 28.91, 26.39, 22.46, 13.96. HRMS (ESI-Q-Tof) calcd for C16 Na+MeOH: 351.1778,
2
H
24
O
5
-1
found: 351.1785. IR (neat)  2926, 1746, 1436, 1256, 1083 cm .
Methyl 9-methyl-8-oxo-1,4-dioxaspiro[4.5]dec-6-ene-7-carboxylate (4). Following the
procedure used to prepare 3, 2 (503.7 mg, 2.35 mmol) was treated with LDA (4.99 mmol) and
iodomethane (0.16 mL, 2.57 mmol) then quenched with saturated aq NH Cl (15 mL) and water (2
4
mL). The usual work-up (EtOAc) and purification (5% EtOAc in hexanes) gave the corresponding
alkylated β-ketoester (278.5 mg confirmed by mass spectrometry) as a pale yellow oil. The latter
was treated with pyridine (0.11 mL, 1.36 mmol) and PhSeBr (319.4 mg, 1.35 mmol) then quenched
with 1 N aq HCl (10 mL). The usual work-up (CH
2
Cl ; brine) and purification (20% EtOAc in
2
hexanes) gave the corresponding phenylselanyl-β-ketoester (109.2 mg, 32% over 2 steps) as a
1
colorless oil: H NMR (CDCl
3
, 400 MHz)  (ppm) 7.58 (d, 2H, J = 7.2 Hz), 7.39 (t, 1H, J = 7.3
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
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Hz), 7.30 (t, 2H, J = 7.5 Hz), 3.85 (m, 4H), 3.64 (s, 3H), 3.06 (m, 1H), 2.50 (dd, 1H, J = 3.8, 13.7
Hz), 2.15 (d, 1H, J = 13.7 Hz), 1.99 (m, 1H), 1.83 (t, 1H, J = 13.4 Hz), 1.13 (d, 3H, J = 6.5 Hz).
13C NMR (CDCl
9.43, 52.78, 43.69, 43.51, 40.75, 14.86. HRMS (ESI-Q-Tof) calcd for C17
, 75 MHz)  (ppm) 170.17, 138.79, 129.71, 128.79, 126.46, 106.79, 64.97, 64.30,
SeNa: 407.0369,
found: 407.0371. IR (neat)  2896, 1736, 1709, 1219 cm . Phenylselanyl-β-ketoester (44.4 mg,
.12 mmol) was treated with aq H (50 wt. %, 0.1 mL) then water (4 mL) and CH Cl (10 mL)
Cl ; saturated aq NaHCO ; brine) afforded pure 4 (27.1 mg,
, 400 MHz)  (ppm) 7.07 (d, 1H, J = 2.2 Hz), 4.05
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
H
20
O
5
-1
0
2
O
2
2
2
were added. The usual work-up (CH
2
2
3
1
quant.) as a pale yellow oil. H NMR (CDCl
3
(m, 4H), 3.80 (s, 3H), 2.86 (m, 1H), 2.17 (ddd, 1H, J = 2.2, 4.8, 13.5 Hz), 2.07 (t, 1H, J = 13.5
13
Hz), 1.16 (d, 3H, J = 6.7 Hz). C NMR (CDCl
3
, 100 MHz)  (ppm) 196.62, 164.77, 148.17,
1
32.62, 103.71, 65.52, 65.16, 52.58, 41.27, 40.83, 14.81. HRMS (ESI-Q-Tof) calcd for
Na: 249.0733, found: 249.0740. IR (neat): 2955, 2934, 2891, 1744, 1690, 1435, 1251
C
11
H
14
O
5
-1
cm .
,4-Dimethylcyclohexanone (5). A suspension of Pd/C (5% wt., 729 mg, 0.4 mol%) in ethanol
4
42
(
20 mL) was added to a solution of 22 (11.6 g, 93.4 mmol) in ethanol (60 mL) at rt. The mixture
was cooled to 0 °C, the flask was purged with hydrogen and the reaction was stirred at rt for 2 h.
1
Complete conversion was assured by H NMR. The resulting mixture was filtered on celite and
washed with CH
2
Cl . The solvent was removed under vacuum to afford 5 (10.7 g, 91%) as a white
2
solid. Spectral data was consistent with that previously reported.43
Methyl 5,5-dimethyl-2-oxocyclohexanecarboxylate (6). Following the procedure used to
prepare 2, 5 (9.6 g, 76 mmol) was treated with NaH (60% in oil, 12.2 g, 305 mmol), Me
2
CO (16.0
3
mL, 190 mmol), and KH (30% in oil, catalytic amount) then quenched with aq 3 M AcOH (120
mL) and poured into brine (100 mL). The usual work-up (EtOAc, 5 × 100 mL) followed by
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distillation (< 1 Torr, 74-76°C) afforded 6 (10.1 g, 72%) as a colorless oil. Spectral data was
consistent with that previously reported.44
Methyl 3,3-dimethyl-6-oxocyclohex-1-enecarboxylate (7).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Following the procedure used to prepare 3, 6 (102.6 mg, 0.56 mmol) was treated with pyridine
(60 µL, 0.74 mmol) and PhSeBr (163.5 mg, 0.69 mmol). The usual work-up (CH
corresponding phenylselanyl-β-ketoester. The latter was treated with aq H (50% wt., 0.5 mL,
.65 mmol) then water (10 mL) was added. The usual work-up (CH Cl2; aqueous saturated
NaHCO ; brine) and purification (10 to 20% EtOAc in hexanes) afforded 7 (58.2 mg, 57%) as a
2
Cl
2
) gave the
2
O
2
8
2
3
yellow oil. Spectral data was consistent with that previously reported.43
Methyl 5-hexyl-3,3-dimethyl-6-oxocyclohex-1-enecarboxylate (8). Following the procedure
used to prepare 3, 6 (204.1 mg, 1.11 mmol) was treated with LDA (2.40 mmol) in THF (1.0 mL)
1
-iodohexane (0.25 mL, 1.69 mmol) then quenched with aq 1 N HCl (10 mL). The usual work-up
(
EtOAc) and purification (5% EtOAc in hexanes) gave the corresponding alkylated β-ketoester
256.8 mg confirmed by mass spectrometry) as a yellow oil. A solution of the latter (49.7 mg, 0.19
(
mmol) in THF (0.3 mL + 0.2 mL for rinsing) was added to a solution of DDQ (57.2 mg, 0.25
mmol) in THF (0.5 mL) via canula at rt. The mixture was stirred for 24 h then water (2 mL) was
added. The usual work-up (EtOAc; brine) and purification (10% EtOAc in hexanes) afforded 8
1
(
19.8 mg, 34% over two steps) as a yellow oil: H NMR (CDCl
3
, 400 MHz)  (ppm) 7.25 (d, 1H,
J = 2.0 Hz), 3.79 (s, 3H), 2.46 (m, 1H), 1.90 (m, 2H), 1.65 (t, 1H, J = 13.7 Hz), 1.29 (m, 9H), 1.24
13
(
s, 3H), 1.20 (s, 3H), 0.87 (t, 3H, J = 6.7 Hz). C NMR (CDCl
163.13, 130.24, 52.35, 43.59, 41.83, 33.91, 31.87, 30.36, 29.53, 29.03, 26.88, 25.40, 22.77, 14.24.
HRMS (ESI-Q-Tof) calcd for C16 Na: 289.1774, found: 289.1784. IR (neat)  2955, 2925,
3
, 100 MHz)  (ppm) 197.09, 165.79,
H
26
O
3
-1
2857, 1745, 1719, 1686 cm .
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5
5
6
Methyl 3,3,5-trimethyl-6-oxocyclohex-1-enecarboxylate (9). Following the procedure used to
prepare 3, 6 (215.8 mg, 1.17 mmol) was treated with LDA (2.34 mmol) and iodomethane (80 µL,
1
.29 mmol) then quenched with aq 1 N HCl (10 mL). The usual work-up (EtOAc) and purification
5% EtOAc in hexanes) gave the corresponding alkylated β-ketoester (150.0 mg, confirmed by
mass spectrometry) as a yellow oil. The latter (100.0 mg, 0.50 mmol) was treated with pyridine
50 µL, 0.62 mmol) and PhSeBr (140.1 mg, 0.59 mmol) then quenched with 1 N aq HCl (5 mL).
The usual work-up (CH Cl ; aq 1 N HCl; brine) gave the corresponding phenylselanyl-β-ketoester
as a yellow oil. The latter was treated with aq H (50 wt. %, 0.5 mL, 8.65 mmol). The usual
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
(
2
2
2
O
2
work-up (CH
2
Cl
2
; saturated aq NaHCO
3
; brine) and purification (5 to 10% EtOAc in hexanes)
1
afforded 9 (58.4 mg, 39% over two steps) as a yellow oil: H NMR (CDCl
3
, 300 MHz)  (ppm)
7
.25 (s, 1H), 3.78 (s, 3H), 2.62 (m, 1H), 1.83 (ddd, 1H, J = 2.3, 4.9, 13.5 Hz), 1.69 (t, 1H), 1.25
1
3
(
s, 3H), 1.18 (s, 3H), 1.12 (d, 3H, J = 6.6 Hz). C NMR (CDCl
3
, 75 MHz)  (ppm) 197.31, 165.72,
1
63.38, 129.98, 52.30, 44.61, 38.67, 33.97, 30.28, 25.43, 14.87. HRMS (ESI-Q-Tof) calcd for
-
1
C
11
H
16
O
3
: 219.992, found: 219.1000. IR (neat)  2962, 1743, 1718, 1686, 1285, 1258 cm .
Methyl 5-allyl-3,3-dimethyl-6-oxocyclohex-1-enecarboxylate (10). Following the procedure
used to prepare 3, 6 (203.0 mg, 1.10 mmol) was treated with LDA (2.40 mmol) and allyl bromide
0.15 mL, 1.73 mmol) then quenched with aq 1 N HCl (10 mL). The usual work-up (EtOAc) and
(
purification (10% EtOAc in hexanes) gave the corresponding alkylated β-ketoester (210.7 mg,
confirmed by mass spectrometry) as a yellow oil. Following the procedure used to prepare 8,
alkylated β-ketoester (101.5 mg, 0.45 mmol) was treated with DDQ (57.2 mg, 0.25 mmol) and
K
2
CO (76.5 mg, 0.55 mmol) then quenched with water (4 mL). The usual work-up (EtOAc; brine)
3
and purification (5 to 10% EtOAc in hexanes) afforded 10 (45.9 mg, 39% over two steps) as a
yellow oil. Spectral data was consistent with that previously reported.45
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Methyl 5-benzyl-3,3-dimethyl-6-oxocyclohex-1-enecarboxylate (11). Following the
procedure used to prepare 3, 6 (203.9 mg, 1.11 mmol) was treated with LDA (2.40 mmol) and
benzyl bromide (0.20 mL, 1.68 mmol) then quenched with aq 1 N HCl (10 mL). The usual work-
up (EtOAc) and purification (5% EtOAc in hexanes) gave the corresponding alkylated β-ketoester
(303.4 mg, confirmed by mass spectrometry) as a yellow oil. Following the procedure used to
prepare 8, alkylated β-ketoester (102.6 mg, 0.37 mmol) was treated with DDQ (97.8 mg, 0.43
mmol) in 1,4-dioxane then hexanes were added, the suspension was filtered and the solvent was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
evaporated. The usual and purification (5 to 10% EtOAc in hexanes) afforded 11 (40.5 mg, 40%
1
over two steps) as a pale brown oil: H NMR (CD
2
Cl , 400 MHz)  (ppm) 7.22 (m, 6H), 3.73 (s,
2
3H), 3.35 (dd, 1H, J = 4.0, 14.0 Hz), 2.78 (m, 1H), 2.43 (dd, 1H, J = 9.0, 14.0 Hz), 1.71 (m, 1H),
13
1
1
2
1
.59 (t, 1H, J = 13.7 Hz), 1.12 (s, 3H), 1.11 (s, 3H). C NMR (CD
2
Cl , 100 MHz)  (ppm) 196.17,
2
65.32, 163.32, 140.22, 130.23, 129.46, 128.86, 126.37, 52.27, 45.45, 41.36, 35.35, 33.99, 30.17,
5.02. HRMS (ESI-Q-Tof) calcd for C17 : 295.1305, found: 295.1314. IR (neat)  2958,
H
20
O
3
-1
742, 1717, 1690, 1271, 1258 cm .
Methyl 3,3-dimethyl-6-oxo-5-(prop-2-yn-1-yl)cyclohex-1-enecarboxylate (12). Following
the procedure used to prepare 3, 6 (199.3 mg, 1.08 mmol) was treated with LDA (2.41 mmol) and
propargyl bromide (80% in toluene, 185 µL, 1.66 mmol) then quenched with aq 1 N HCl (10 mL).
The usual work-up (EtOAc) and purification (5% EtOAc in hexanes) gave the corresponding
alkylated β-ketoester (176.8 mg confirmed by mass spectrometry) as a yellow oil. Following the
procedure used to prepare 8, alkylated β-ketoester (98.0 mg, 0.44 mmol) was treated with DDQ
(
159.7 mg, 0.70 mmol) and K
2
CO (165.7 mg, 1.20 mmol) in 1,4-dioxane then quenched water
3
(10 mL). The usual work-up (EtOAc; brine) and purification (10% EtOAc in hexanes) afforded 12
1
(44.7 mg, 34% over two steps) as a white solid: H NMR (CDCl
3
, 400 MHz)  (ppm) 7.34 (d, 1H,
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
J = 2.2 Hz), 3.80 (s, 3H), 2.81 (ddd, 1H, J = 2.7, 4.0, 17.0 Hz), 2.72 (m, 1H), 2.30 (ddd, 1H, J =
2
.6, 8.7, 17.0 Hz), 2.17 (ddd, 1H, J = 2.3, 4.6, 13.4 Hz), 1.98 (t, 1H, J = 2.7 Hz), 1.78 (t, 1H, J =
13
1
3.8 Hz), 1.29 (s, 3H), 1.24 (s, 3H). C NMR (CDCl
3
, 75 MHz)  (ppm) 194.59, 165.30, 164.37,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
29.58, 81.98, 70.11, 52.43, 42.82, 41.09, 34.03, 30.27, 25.31, 18.86. HRMS (ESI-Q-Tof) calcd
-1
for C13
H
16
O
3
: 243.0992, found: 243.0995. IR (neat)  3262, 2967, 1716, 1684, 1259 cm .
Methyl
3,3-dimethyl-6-oxo-5-(2,2,2-trifluoroethyl)cyclohex-1-enecarboxylate
(13).
Following the procedure used to prepare 3, 6 (203.8 mg, 1.11 mmol) was treated with LDA (2.42
mmol) and 1,1,1-trifluoro-2-iodoethane (130 µL, 1.33 mmol) then quenched with aq 1 N HCl (10
mL). The usual work-up (EtOAc; brine) and purification (5% EtOAc in hexanes) gave the
corresponding alkylated β-ketoester (136.3 mg) as a yellow oil. The latter (136.3 mg) was treated
with pyridine (50 µL, 0.62 mmol) and PhSeBr (138.1 mg, 0.59 mmol) then quenched with 1 N aq
HCl (5 mL). The usual work-up (CH
2
Cl
2
; aq 1 N HCl; brine) afforded the corresponding
(50 wt. %, 0.5 mL,
phenylselanyl-β-ketoester as an orange oil. The latter was treated with aq H
2
O
2
8
.65 mmol). The usual work-up (CH
2
Cl
2
; saturated aq NaHCO
3
; brine) and purification (5%
1
EtOAc in hexanes) afforded 13 (20.1 mg, 7% over two steps) as a yellow oil: H NMR (CDCl
3
,
3
1
00 MHz)  (ppm) 7.35 (d, 1H, J = 2.3 Hz), 3.81 (s, 3H), 3.09 (m, 1H), 2.88 (m, 1H), 2.14 (ddd,
13
H, J = 13.0, 4.2, 1.7 Hz), 1.94 (m, 1H), 1.76 (t, 1H, J = 13.9 Hz), 1.30 (s, 3H), 1.23 (s, 3H). C
, 75 MHz)  (ppm) 193.23, 164.99, 164.10, 129.22, 125.40, 52.51, 41.93, 38.76 (q,
J = 2.2 Hz), 33.97, 32.74 (q, J = 28.9 Hz), 30.18, 24.99. HRMS (ESI-Q-Tof) calcd for C12
NMR (CDCl
3
H
15
F
3
O
3
:
-1
287.0866, found: 287.0870. IR (neat)  2957, 1745, 1717, 1690, 1252 cm .
Methyl 5-(2-(tert-butoxy)-2-oxoethyl)-3,3-dimethyl-6-oxocyclohex-1-enecarboxylate (14).
Following the procedure used to prepare 3, 6 (210.4 mg, 1.14 mmol) was treated with LDA
(2.47 mmol) and tert-butyl bromoacetate (250 µL, 1.69 mmol) then quenched with aq 1 N HCl
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(
10 mL). The usual work-up (EtOAc; brine) and purification (5 to 20% EtOAc in hexanes) gave
the corresponding alkylated β-ketoester (164.0 mg confirmed by mass spectrometry) as a yellow
oil. The latter (93.5 mg) was treated with pyridine (61 µL, 0.75 mmol) and PhSeBr (175.4 mg,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
.74 mmol) then quenched with 1 N aq HCl (5 mL). The usual work-up (CH
brine) afforded the corresponding phenylselanyl-β-ketoester as a yellow oil. The latter was treated
with aq H (50 wt. %, 0.5 mL, 8.65 mmol) then water (5 mL) was added. The usual work-up
CH Cl ; saturated aq NaHCO
2
Cl ; aq 1 N HCl;
2
2
O
2
(
2
2
3
; brine) and purification (10 to 20% EtOAc in hexanes) afforded
1
14 (36.0 mg, 19% over two steps) as a white solid: mp = 47-49°C; H NMR (CDCl
3
, 300 MHz) 
(ppm) 7.30 (d, 1H, J = 2.2 Hz), 3.79 (s, 3H), 3.10 – 2.97 (m, 1H), 2.86 (dd, 1H, J = 16.6, 5.2 Hz),
2.15 (dd, 1H, J = 16.6, 7.4 Hz), 1.90 (ddd, 1H, J = 13.3, 4.8, 2.2 Hz), 1.76 (t, 1H, J = 13.6 Hz),
13
1
1
.46 (s, 9H), 1.29 (s, 3H), 1.21 (s, 3H). C NMR (CDCl
3
, 75 MHz)  (ppm) 195.07, 171.62,
65.37, 163.92, 129.56, 80.82, 52.41, 41.93, 40.84, 35.52, 34.08, 30.26, 28.25, 25.16. HRMS (ESI-
Na: 319.1516, found: 319.1525. IR (neat)  2976, 1724, 1364, 1147,
Q-Tof) calcd for C16
H O
24 5
-1
1
125 cm .
Methyl 5-(cyanomethyl)-3,3-dimethyl-6-oxocyclohex-1-enecarboxylate (15). Following the
procedure used to prepare 3, 6 (828.8 mg, 4.50 mmol) was treated with LDA (9.88 mmol) and
bromoacetonitrile (0.63 mL, 9.04 mmol) then quenched with aq 1 N HCl (10 mL). The usual work-
up (EtOAc; brine) and purification (5 to 20% EtOAc in hexanes) gave the corresponding alkylated
β-ketoester (145.2 mg confirmed by mass spectrometry) as an orange oil. The latter (139.0 mg)
was treated with pyridine (0.29 mL, 3.13 mmol) and PhSeBr (588.4 mg, 2.49 mmol) then quenched
with 1 N aq HCl (5 mL). The usual work-up (CH
2
Cl
2
; aq 1 N HCl; brine) afforded the
(50
; saturated
corresponding phenylselanyl-β-ketoester as a brown oil. The latter was treated with aq H
2
O
2
wt. %, 0.5 mL, 8.65 mmol) then water (5 mL) was added. The usual work-up (CH
2
Cl
2
ACS Paragon Plus Environment
2
9