Design and synthesis of novel quinone inhibitors targeted to the redox function of apurinic/apyrimidinic endonuclease 1/redox enhancing factor-1 (Ape1/Ref-1)

Article abstract of DOI:10.1021/jm9014857

The multifunctional enzyme apurinic endonuclease 1/redox enhancing factor 1 (Ape1/ref-1) maintains genetic fidelity through the repair of apurinic sites and regulates transcription through redox-dependent activation of transcription factors. Ape1 can therefore serve as a therapeutic target in either a DNA repair or transcriptional context. Inhibitors of the redox function can be used as either therapeutics or novel tools for separating the two functions for in vitro study. Presently there exist only a few compounds that have been reported to inhibit Ape1 redox activity; here we describe a series of quinones that exhibit micromolar inhibition of the redox function of Ape1. Benzoquinone and naphthoquinone analogues of the Ape1-inhibitor E3330 were designed and synthesized to explore structural effects on redox function and inhibition of cell growth. Most of the naphthoquinones were low micromolar inhibitors of Ape1 redox activity, and the most potent analogues inhibited tumor cell growth with IC₅₀ values in the 10-20 μM range.

Full text of DOI:10.1021/jm9014857

1200 J. Med. Chem. 2010, 53, 1200–1210
DOI: 10.1021/jm9014857
Design and Synthesis of Novel Quinone Inhibitors Targeted to the Redox Function of
Apurinic/Apyrimidinic Endonuclease 1/Redox Enhancing Factor-1 (Ape1/Ref-1)
Rodney L. Nyland II,† Meihua Luo,‡ Mark R. Kelley,‡,§ and Richard F. Borch*,†
†Department of Medicinal Chemistry and Molecular Pharmacology and the Cancer Center, Purdue University, West Lafayette, Indiana 47907,
‡Department of Pediatrics (Section of Hematology/Oncology), Herman B.Wells Center for Pediatric Research, and §Departments of Biochemistry
and Molecular Biology, and Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202
Received October 8, 2009
The multifunctional enzyme apurinic endonuclease 1/redox enhancing factor 1 (Ape1/ref-1) maintains
genetic fidelity through the repair of apurinic sites and regulates transcription through redox-dependent
activation of transcription factors. Ape1 can therefore serve as a therapeutic target in either a DNA
repair or transcriptional context. Inhibitors of the redox function can be used as either therapeutics or
novel tools for separating the two functions for in vitro study. Presently there exist only a few
compounds that have been reported to inhibit Ape1 redox activity; here we describe a series of quinones
that exhibit micromolar inhibition of the redox function of Ape1. Benzoquinone and naphthoquinone
analogues of the Ape1-inhibitor E3330 were designed and synthesized to explore structural effects on
redox function and inhibition of cell growth. Most of the naphthoquinones were low micromolar
inhibitors of Ape1 redox activity, and the most potent analogues inhibited tumor cell growth with IC50
values in the 10-20 μM range.
Introduction
determined that these were two distinct functions of the same
protein, initially thought to reside in two nonoverlapping
domains but later determined to have a minor degree of
overlap.1-5 However, redox or repair can be silenced indepen-
dently using specific point mutations for each activity, indicat-
ing that each function can act independently.
Two fundamental concerns present in managing cellular
homeostasis are maintaining DNA fidelity and regulating the
expression of the genetic information contained therein. With
regard to DNA fidelity, a major recurring event cells must
overcome is the formation of apurinic or apyrimidinic (APa)
sites, formation of which takes place on the order of 104 times
per cell per day by spontaneous glycosidic hydrolysis.1-3 AP
sites in DNA have numerous deleterious ramifications, in-
cluding prohibiting DNA replication, cytotoxicity, and muta-
genicity. Spontaneous glycosidic hydrolysis is not the only
route to forming AP sites; glycosylases as well as DNA
damaging agents can induce AP site formation in DNA.1
The ubiquitous enzyme apurinic/apyrimidic endonuclease 1
(Ape1) is a major component of the base excision repair
(BER) pathway and has the responsibility of repairing AP
sites throughout the genome. Ape1 possesses multiple enzy-
matic functions; the most relevant to BER is the 50AP-
endonuclease activity that initiates the removal of AP sites.
Gene expression is also controlled in part by Ape1. At the
same time the BER function of Ape1 was being explored,
another enzyme was identified that performed redox-depen-
dent regulation of numerous transcription factors. This en-
zyme was named redox enhancing factor 1 (ref-1) and was
linked to the regulation of transcription factors such as acti-
vator protein 1 (AP-1), hypoxia inducing factor 1alpha (HIF-
1R), and nuclear factor kappa B (NFκB). It was subsequently
Through both the redox and DNA repair functions, Ape1
supports cancer cell proliferation, and elevated expression levels
have been shown to correlate to poor patient prognosis.1-3
Ape1 is overexpressed in a number of cancers, where increased
levels of DNA repair leads to resistance against DNA damaging
agents, and increased redox activity is expected to enhance
replication through redox cycling of transcription factors.
Therefore Ape1 represents an interesting therapeutic target in
different mechanistic contexts. Inhibitors of the BER function
of Ape1 can be utilized as a complementary treatment option for
those encountering resistance to DNA-damaging agents. Alter-
natively, inhibition of the redox function of Ape1 might interfere
with regulation of transcription and alter a number of stress-
induced responses of cancer cells. Recent data indicate that
blocking the repair function of Ape1 leads to cell death, while
redox activity inhibition leads to decreased cell growth and
cytostatic effects.6 Additionally, recent data indicate that
blocking Ape1 redox function blocks angiogenesis.6-8 Small
molecule inhibitors of the redox function can also serve as tools
to separate the two functions of Ape1 without the lethality of
knocking out Ape1 completely.9
The design of inhibitors targeting the redox function of
Ape1 is hindered by a lack of information regarding the redox
active site. Mutation analysis has shown that cysteine 65
is necessary for redox activity; however, in every crystal
structure C65 is buried, suggesting that a conformational
change might be required to present the relevant redox-active
*To whom correspondence should be addressed. Phone: (765)494-
1403. Fax: (765)494-1414. E-mail: borch@purdue.edu.
a Abbreviations: AP, apurinic/apyrimidinic; Ape1, apurinic/apyrimi-
dinic endonuclease 1; BER, base excision repair; EMSA, electrophoretic
mobility shift assay; ref-1, redox-enhancing factor 1.
r
pubs.acs.org/jmc
Published on Web 01/12/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1201
Scheme 1a
Figure 1. E3330, a reported inhibitor of Ape1 redox activity.1
structure.10 Furthermore, there is only one known compound
in the literature that has been shown to inhibit the redox
function of Ape1.1 To provide structural insight into potential
inhibitor specificity for the redox active site, a series of
benzoquinones and naphthoquinones has been synthesized
based on the structure of (E)-3-(5,6-dimethoxy-3-methyl-1,
4-dioxocyclohexa-2,5-dienyl)-2-nonylpropenoic acid (E3330, 1),
a known inhibitor of the redox function of Ape1.1 (Figure 1)
Analogues with improved physicochemical and binding profiles
also have the potential to provide crystallographic data when
complexed with the protein to elucidate the structure of the redox
active site. The structure of 1 provides five regions for potential
exploration in preliminary SAR studies, namely (1) the substi-
tuent on the 3-position of the quinone ring, (2) the benzoquinone
structure, (3) the substituent R to the carboxyl group, (4) the
carboxylate moiety, and (5) the saturation of the substituent at
the quinone 2-position.
a Reagents and conditions: (a) H2O2, H2SO4, MeOH, reflux 3 h.
(b) K2CO3, MeI, acetone, reflux 2 d. (c) (i) nBuLi, THF, 0 °C 1 h;
(ii) MeI, 0 °C, 2 h. (d) CHCI2OCH3, TiCl4, CH2Cl2, 0 °C to rt 4 h.
(e) NaH, (EtO)2P(O)CHRCO2Et, THF, rt 12 h. (f) KOH, EtOH, reflux
30 min. (g) HNO3, AcOH, EtOAc, rt 4 h. (h) NMFA, POCl3, CH2Cl2, rt
2 d. (i) SO2Cl2, CH2Cl2, rt, 1 h. (j) CAN, MeCN, H2O, 1 h. (k) R-Bromo-
γ-butyrolactone, (EtO)3P, then NaH, toluene, reflux 8 h. (l) H2SO4,
CH(OCH3)3, MeOH, reflux 12 h. (m) KOH, EtOH, reflux 30 min.
Results and Discussion
Chemistry. The synthesis of benzoquinone 1 (E3330) has
been reported in a patent, and key transformations have
appeared separately in print.11,12 The patent synthesis begins
with the tribromination of p-cresol followed by a Cu(I)-
mediated Ullmann reaction to generate 4-methyl-2,3,6-
trimethoxyphenol that is alkylated in situ to provide 2,3,4,
5-tetramethoxytoluene.12 In our hands, the methanolysis of
4-methyl-2,3,6-tribromophenol resulted in a complex mix-
ture that included products derived from reduction of the
bromo substituents; these side products were very difficult to
remove, and the resulting tetramethoxytoluene could not be
obtained pure. To overcome these difficulties, we developed
an alternative synthesis of 1 (Scheme 1) that was also used for
the synthesis of several analogues.10 Bayer-Villiger oxida-
tion of 2,3,4-trimethoxybenzaldehyde 2 and subsequent
hydrolysis in situ gave 2,3,4-trimethoxyphenol, which was
then alkylated to provide 1,2,3,4-tetramethoxybenzene 3
in excellent yield.13 Ring methylation was then performed
to provide 2,3,4,5-tetramethoxytoluene in high yield and
purity,14 and the aldehyde moiety was introduced using R,
R-dichloromethyl methyl ether and titanium(IV) chloride15
to give 4 in 95% yield. The chloroaldehyde intermediate 6
was prepared by formylation of 3 using N-methyl formani-
lide and POCl3, followed by chlorination with sulfuryl
chloride.16 The unsaturated ester moiety was introduced by
Emmons condensation of aldehyde 4 or 6 and the appro-
priate phosphonate. All of the Emmons products derived
from aldehyde 4 were formed exclusively as the E isomer.
When using the chloro-substituted aldehyde 6, however, the
E:Z selectivity is 5:1 when the reaction is run at room
temperature; performing the reaction in refluxing toluene
provides 100% E isomer. The esters were hydrolyzed and the
intermediate acids were oxidized to the benzoquinones 1,
5a-e and 7. The 3-methyl analogues were easily oxidized
with either nitric acid or ceric ammonium nitrate (CAN);
however, successful oxidation of the 3-chloro analogue could
only be accomplished using CAN. Finally, the methoxyethyl
substituent was introduced by condensation of 4 with the
Emmons reagent prepared from R-bromo-γ-butyrolactone
to give lactone 8. Treatment of 8 with trimethylorthoformate
in acidic methanol generated the ring-opened methoxyethyl
analogue;17 hydrolysis of the methyl ester and oxidation
afforded the quinone 9.
Carboxamide analogues of the free acids were also of
interest both for SAR and for the potential development of
coupled products. However, all efforts to introduce a
hydroxyethylamide moiety by reaction with the benzoqui-
none acids (e.g., 5a) were unsuccessful. Therefore, acid 10
was converted to amide 11 and subsequently oxidized to
give the desired quinone amide 12 (Scheme 2). The synthesis
of 11 was initially attempted using DCC as a reagent;
surprisingly, amide 11 was obtained as a 1:1 mixture of
E and Z products. In contrast, the E stereochemistry was
completely retained when 11 was synthesized using PyBOP
in the coupling reaction. Further exploration of coupling
conditions using a variety of unsaturated acids and amines
showed that activation with either DCC or DIC leads to
mixtures of E and Z products, but activation with PyBOP,
methyl chloroformate, or oxalyl chloride do not. It is
hypothesized that a reversible intramolecular cyclization
of the activated carbodiimide intermediate is taking place
that leads to loss of the stereochemical integrity of the
double bond (Scheme 2). The amidation product E-11
was then readily oxidized using nitric acid to provide
hydroxyethylamide 12.
The naphthoquinone analogues were constructed via con-
densation of the appropriate aldehydes and phosphonates

1202 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Nyland et al.
Scheme 2a
Scheme 4
Aldehydes 14a-c were prepared by dichloromethyl methyl
ether formylation of the di- or trimethoxynaphthalenes. The
methylthio substituent was introduced by lithiation of the
hydroxymethylnaphthalene, followed by reaction with di-
methyl disulfide and subsequent oxidation of the hydroxy-
methyl group to the aldehyde (14d). The 3-halo substituents
in 14e-g were introduced by electrophilic halogenation of
14a. Emmons condensation with the naphthaldehydes
showed reduced E selectivity relative to their benzaldehyde
counterparts. The greatest E selectivity was observed with
3-methyl aldehyde 14b, which afforded 100% E in refluxing
toluene and an E:Z ratio of approximately 5:1 at room
temperature. All other analogues produced a signifi-
cant proportion of Z isomer (20-50%) even in refluxing
toluene. The Emmons products 15a-g were converted to the
quinones 16a-g by saponification and subsequent oxida-
tion. Nitric acid was sufficient to oxidize the 3-methyl,
3-methoxy, and 3-methylthio analogues, but efficient oxida-
tion of the 3-halo analogues required treatment with argentic
oxide.
a Reagents and conditions: (a) DCC, ethanolamine, CH2Cl2, rt;
(b) PyBOP, Et3N, rt 30 min, then ethanolamine, rt 4 h; (c) Ag(II)O,
HNO3, AcOH, EtOAc, rt 40 min.
Scheme 3a
An interesting result was observed when a mixture of
3-chloro E- and Z-acids (resulting from hydrolysis of the
crude Emmons product 15f) was oxidized using nitric acid
and argentic oxide; although the E-quinone product 16f
was produced from the E-acid as expected, the Z-quinone
product following oxidation was recovered as a methyl
ester. The oxidation was carried out on pure Z-acid 18,
and the Z-methyl ester 19 was the major product
(Scheme 4). To rationalize the selective esterification of
the Z-carboxylate group, an intramolecular esterification is
hypothesized (Scheme 4) in which the Z-carboxylate at-
tacks the methyl group of the oxonium ion interme-
diate generated in the oxidation reaction. The selective
esterification of the Z-acids is fortuitous in that it provides
a convenient strategy to purify otherwise difficult-to-
separate E and Z products by generating easily separable
E-acid and Z-methyl ester.
Finally, the naphthoquinone series was completed with
the preparation of methoxyethyl side chain analogues
21a-c, the amide 23, and the bromoacid 26 in which
the double bond at the 2-position was fully saturated
(Scheme 5). Syntheses of 21 and 23 were accomplished via
routes analogous to those previously described for the
benzoquinone products. Alcohol 24 (the reduction product
of 14a) provided the starting material for the synthesis
of 26; conversion to the R,3-dibromide followed by dis-
placement of the benzylic bromide afforded the saturated
ester 25. Oxidation and ester hydrolysis gave the desired
product.
a Reagents and conditions: (a) (i) H2, 10% Pd/C, THF, rt, 4 h; (ii) NaH,
Me2SO4, 2 h, rt. (b) R = H, CH3: TiCl4, CHCl2OCH3, CH2Cl2, 0 °C, 4 h;
R = OCH3: (i) nBuLi, THF, -78 °C, 3 h; (ii) DMF, -78 °C. (c) Selec-
tfluor, CH3CN, reflux. (d) SO2Cl2, CH2Cl2, rt, 4 h. (e) Br2, CH2Cl2, rt, 1 h.
(f) R = H: NaH, (EtO)2P(O)CHR’CO2, Et, THF, rt, 12 h; R = CH3,
OCH3, SCH3, F, Cl, Br: NaH, (EtO)2P(O)CHR’CO2Et, PhMe, reflux, 12
h. (g) EtOH, KOH, reflux, 1 h. (h) R = H, CH3, OCH3, SCH3: HNO3,
EtOAc, AcOH, rt, 3 h; R = F, Cl, Br: HNO3, EtOAc, AcOH, Ag(II)O, rt,
2 h. (i) LiAlH4, THF, rt, 12 h. (j) (i) nBuLi, THF, -78 °C; (ii) (SCH3)2,
-78 °C. (k) PCC, CH2Cl2, rt, 8 h.
followed by ester hydrolysis and oxidation to the quinone.
Synthesis of the requisite aldehydes is detailed in Scheme 3.

Article
Redox, Endonuclease, and Cell Growth Inhibition. Com-
pounds were tested for the ability to inhibit the redox
function of Ape1 in a gel shift assay (EMSA), and IC50
values were generated for each compound. Inhibition of
Hey-C2 ovarian cancer cell growth was also assessed. Com-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1203
pounds were also evaluated as inhibitors of Ape1 endonu-
clease activity; all compounds were inactive at concen-
trations up to 100 μM. The redox and cell growth inhibition
results are summarized in Tables 1 and 2; a representative
EMSA assay is shown in Figure 2. Modification of the alkyl
substituent on the double bond of the benzoquinones
(Table 1) had only modest effects on the redox activity;
replacement of the n-nonyl group (1, IC50=10 μM) with a
methyl group (5b, IC50 = 3 μM) enhanced redox activity,
whereas removal of the alkyl group (5a, IC50 = 40 μM)
reduced potency in the redox assay. In contrast, most of
the structural modifications in the benzoquinone series
diminished cell growth inhibitory activity compared to the
lead compound 1. The loss of cell-based activity relative to
redox activity is especially dramatic for compounds 5a, 5b,
and 9. However, these compounds are significantly more
hydrophilic than 1, so they may be too polar to permeate the
cell effectively. By comparison, most of the naphthoqui-
nones (Table 2) were more potent than 1 as inhibitors of
Scheme 5a
a Reagents and conditions: (a) R-bromobutyrolactone, (EtO)3P, then
NaH, toluene, reflux 8 h; (b) H2SO4, CH(OCH3)3, MeOH, reflux 12 h;
(c) KOH, EtOH, reflux 30 min; (d) R = CH3: HNO3, EtOAc, AcOH, rt 3
h; R = Cl or Br: HNO3, EtOAc, AcOH, Ag(II)O, rt 2 h; (e) PyBOP,
Et3N, DMF:CH2Cl2 rt 30 min, then ethanolamine, rt 4 h; (f) Ag(II)O,
HNO3, AcOH, EtOAc, rt 30 min; (g) HBr, CH2Cl2 rt 30 min; (h) Br2,
CH2Cl2, rt 4 h; (i) Li(SiMe3)2N, CH3CO2Et, THF, -78 C to rt 4 h;
(j) HCl, THF, reflux.
Figure 2. Inhibition of Ape1 redox activity measured by electro-
phoretic mobility shift assay. See Experimental Section for details.
Table 1. Redox and Growth Inhibition of Benzoquinone Analogues
compd
R1
R2
R3
redox inhibition IC50, μMa
growth inhibition GI50, μMb
1
CH3
CH3
CH3
CH3
Cl
C9H19
OH
OH
OH
OH
OH
OH
10
40
3
35
250
130
85
5a
5b
5e
7
H
CH3
C4H9
CH3
15
3
45
9
12
CH3
CH3
C2H4OCH3
C9H19
10
10
200
35
NH(CH2)2OH
a Determined in a gel shift EMSA assay; see Experimental Section for details. b Hey-C2 cells treated with drug for 72 h. Values represent the average of
three experiments.
Table 2. Redox and Growth Inhibition of Naphthoquinone Analogues
compd
R1
R2
R3
redox inhibition IC50, μMa
growth inhibition GI50, μMb
16a
16b
16c
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
3
15
1
20
45
40
30
50
15
30
13
30
30
25
8
CH3
OCH3
SCH3
F
16d
16e
1
4
16f(E)
16f(Z)
16g
21a
21b
21c
Cl
Cl
1
2
Br
2
CH3
Cl
C2H4OCH3
C2H4OCH3
C2H4OCH3
CH3
10
3
Br
Cl
1
23
26
NH(CH2)2OH
OH
1
Br
H (saturated)
5
15
a Determined in a gel shift EMSA assay; see Experimental Section for details. b Hey-C2 cells treated with drug for 72 h. Values represent the average of
three experiments.

1204 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Nyland et al.
Ape1 redox activity, with IC50 values in the 1-5 μM range.
Compounds with electronegative substituents in the 3-position
of the naphthoquinone ring generally had the highest
redox inhibitory activity; in contrast, the 3-methyl com-
pounds were the least active in this assay. The best of the
naphthoquinones were 2-4 fold more potent than 1 in
growth inhibitory activity and, although the most potent
compounds were generally 3-chloro or 3-bromo ana-
logues, the structural modifications investigated here had
only modest effects on growth inhibition. It is interesting
to note that functionalization of the carboxylic acid
as the hydroxyethyl amide did not affect either redox or
cell growth inhibitory activity significantly (compounds
E-16f and 23).
reduced with DTT (1.0 mM) at 37 °C for 10 min and then
diluted with PBS buffer to final concentrations of Ape1 and
DTT of 2 mg/mL and 0.2 mM, respectively. A final volume of
18 mL was prepared from EMSA buffer (10 mM Tris [pH 7.5],
50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 5% [vol/vol]
glycerol) to which was added 2 mL of reduced Ape1 protein
and 6 mg of oxidized nuclear extracts (Hey-C2 cells, treated with
0.01 mM diamide for 10 min), and the reaction was incubated at
room temperature for 30 min. One mL of poly(dI-dC) poly(dI-
3
dC) (1 mg/L, Amersham Biosciences, Piscataway, NJ) was
added for 5 min followed by 1 mL of the 50 hexachlorofluore-
scein phosphoramidite (HEX)-labeled double-stranded oligo-
nucleotide DNA (0.1 pmol, The Midland Certified Reagent
Company, Midland, TX) containing the AP-1 consensus
sequence (50CGCTTGATGACTCAGCCGGAA-30), and the
mixture was further incubated for 30 min at room temperature.
The final concentration of DTT in the redox reactions was
0.02 mM. Samples were loaded on a 5% nondenaturing poly-
acrylamide gel and subjected to electrophoresis in 0.5X TBE
buffer (200 V for 1 h at 4 °C) and detected using the Hitachi
FMBio II fluorescence imaging system (Hitachi Genetic Sys-
tems, South San Francisco, CA). The HEX fluorophore is
excited by a solid-state laser at 532 nm (Perkin-Elmer) and emits
a fluorescent light signal at 560 nm, which is then measured
using a 585 nm filter.
Conclusion
The goal of this work was to exploit 1 as a lead to develop
more potent and druggable inhibitors of Ape1 redox activity.
Analogues based on the benzoquinone core showed little
improvement in enzymatic activity, and most were less effec-
tive cell growth inhibitors, presumably because of significant
changes in physicochemical properties. Most of the naphtho-
quinone analogues were highly potent inhibitors of Ape1
redox activity. All of these compounds had favorable physico-
chemical properties, with cLogP values in the 1-3 range. The
most potent compounds have an electron-withdrawing sub-
stituent at the 3-position of the naphthoquinone ring. Several
of these compounds exhibited IC50 values of ∼1 μM in the
Ape1 redox assay and <20 μM in the cell growth inhibition
assay.
Growth Inhibition Assay. Growth inhibition was tested in
vitro using the MTS assay following the procedure of Fishel
et al.19 Hey-C2 ovarian cancer cells were cultured in RPMI 1640
medium containing 10% fetal calf serum. Cells (2-4000) were
aliquoted into each well of a 96-well plate in triplicate and
allowed to adhere overnight. Cells were incubated with drug for
72 h at 37 °C. After 72 h, 0.05 mg/mL MTS reagent was added to
each well and incubated at 37 °C for 4 h, followed by absorbance
measurement at 490 nm. The values were standardized to wells
containing media alone.
Experimental Procedures
Synthetic Procedures. (E)-3-(5,6-Dimethoxy-3-methyl-14-di-
oxocyclohexa-25-dienyl)-2-nonylpropenoic Acid (1). According
to the modified procedure of Murphy et al.,20 NaH (0.135 g,
3.38 mmol) was added to a flame-dried 50 mL 3-neck round-
bottom flask connected to a water-jacketed reflux condenser.
The flask was purged with argon, and THF (20 mL) was added
to the flask followed by triethyl 2-phosphonoundecanoate
(1.08 g, 3.09 mmol) at room temperature. The reaction was
stirred at room temperature for 30 min, and then 4 (0.581 g,
2.42 mmol) dissolved in THF (10 mL) was added rapidly at
room temperature. The reaction was stirred for another 12 h at
room temperature and then acidified with 2 M HCl, diluted with
ethyl acetate, and washed with brine. The organic layer was
dried, filtered, and condensed. The resulting oil was purified
via flash column chromatography (3:17 EtOAc:hexanes) to
provide ethyl (E)-3-(6-methyl-2,3,4,5-trimethoxyphenyl)-2-nonyl-
propenoate (0.603 g, 57%) as a colorless oil. Rf = 0.40 (3:17
EtOAc:hexanes). 1H NMR (CDCl3): δ 0.84 (t, 3H, J=7.2 Hz),
1.08-1.22 (m, 11H), 1.24-1.35 (m, 3H), 1.33 (t, 3H), 2.03 (s,
3H), 2.12 (m, 2H), 3.69 (s, 3H), 3.78 (s, 3H), 3.88 (s, 3H), 3.93 (s,
3H), 4.25 (q, 2H), 7.37 (s, 1H).
The ethyl ester (1.57 g, 3.60 mmol) was dissolved in EtOH
(12.0 mL), and then KOH (0.41 g, 7.3 mmol) was added and the
solution was refluxed for 1 h. The reaction was then acidified
with 2 M HCl, extracted with ethyl acetate, washed two times
with saturated brine, dried, filtered, and condensed. The pro-
duct was obtained as a light-yellow oil (1.43 g, 97%) following
flash chromatography (2:3 Et2O:hexanes 0.5% AcOH). Rf =
0.36 (2:3 Et2O:hexanes 0.5% AcOH). 1H NMR (CDCl3):
δ 0.84 (t, 3H), 1.13 (m, 12H), 1.36 (m, 2H), 2.04 (s,3H), 2.14
(m, 2H), 3.70 (s, 3H), 3.79 (s, 3H), 3.89 (s, 3H), 3.93 (s, 3H),
7.537 (s,1H).
Materials and Methods. All NMR spectra were recorded
using a 300 MHz Bruker spectrometer equipped with a 5 mm
multinuclear probe. 1H chemical shifts are reported in parts per
million using tetramethylsilane as internal standard. 31P NMR
spectra were obtained using broadband 1H decoupling, and
chemical shifts are reported in parts per million using 1%
triphenylphosphine oxide/benzene-d6 as the coaxial reference
(triphenylphosphine oxide/benzene-d6 has a chemical shift of
þ24.7 ppm relative to 85% phosphoric acid). Mass spectral data
were obtained from the Purdue University Mass Spectrometry
Service, and elemental analyses were performed by the Purdue
University Microanalysis Lab. Purity of all tested compounds
was assessed by either combustion analysis or HPLC (C18
reverse phase) and found to be 95% or greater. Silica gel grade
60 (230-400 mesh) was used to carry out all flash chromato-
graphic separations. Thin layer chromatography was performed
using Analtech glass plates precoated with silica gel (250 μm).
Visualization of the plates was accomplished using UV and/or
the following stains: 1% 4-(p-nitrobenzyl)pyridine in acetone
followed by heating and subsequent treatment with 3% KOH in
methanol or p-anisaldehyde dip (1.85% p-anisaldehyde,
20.5% sulfuric acid, 0.75% acetic acid in 95% ethanol)
followed by heating. All anhydrous reactions were carried
out under an atmosphere of argon. Tetrahydrofuran was
distilled prior to use from sodium using benzophenone ketyl
as an indicator. Dichloromethane, triethylamine, pyridine,
and acetonitrile were distilled from calcium hydride prior
to use. Unless otherwise noted, all other solvents were
purchased from Fisher or VWR and used as received. All
chemical reagents were purchased from Aldrich, unless other-
wise noted.
Enzymatic Redox Assay. Inhibition of Ape1 redox activity
was measured using an electrophoretic mobility shift assay
(EMSA).18 Briefly, purified Ape1 protein (10 mg/mL) was
Following modifications to the procedures by Shinkawa et al.
and Flader et al.,11,21 the unsaturated acid (1.13 g, 2.77 mmol)
was dissolved in ethyl acetate (15 mL) at room temperature.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1205
HNO3 (0.75 mL) and AcOH (6 drops) were added at room
temperature, and the reaction was stirred for 4 h. The reaction
was then diluted with EtOAc (20.0 mL), washed with brine,
dried over MgSO4, filtered, and condensed. The red oil was then
purified by flash chromatography (2:3 Et2O:hexanes 0.5%
AcOH), followed by recrystallization from Et2O/hexanes to
afford 1 (0.443 g, 42%) as a red solid. Rf = 0.16 (2:3 Et2O:
hexanes 0.5% AcOH); mp=56-57 °C (lit. 68 °C).5 1H NMR
(CDCl3): δ 0.84 (t, 3H), 1.18 (bs, 14H), 1.39 (bs, 2H), 1.94 (d,
3H), 2.09 (t, 3H,), 3.99 (s, 3H), 4.02 (s, 3H), 7.26 (d, 1H).
(E)-3-(5,6-Dimethoxy-3-methyl-1,4-dioxocyclohexa-2,5-dienyl)-
propenoic Acid (5a). Following the method used for the synthesis
of 1, the Emmons condensation was performed with 4 (0.323 g,
1.34 mmol) to provide ethyl (E)-3-(6-methyl-2,3,4,5-trimethoxy-
phenyl)-propenoate (0.343 g, 83%) as a pale-yellow oil following
chromatography (CH2Cl2 to 1:19 EtOAc:CH2Cl2). Rf = 0.17
(CH2Cl2). H NMR (CDCl3): δ 1.32 (t, 3H), 2.26 (s, 3H), 3.76
(s, 3H), 3.78 (s, 3H), 3.88 (s, 3H), 3.94 (s, 3H), 4.24 (q, 2H), 6.52 (d,
1H), 7.77 (d, 1H).
The ethyl ester (0.066 g, 0.21 mmol) was hydrolyzed (KOH,
EtOH) to provide the unsaturated acid (0.046 g, 78%) as a light-
tan solid following flash chromatography (2:3 Et2O:hexanes
0.5% AcOH) or recrystallization from Et2O/hexanes. Rf=0.16
(2:3 Et2O:hexanes 0.5% AcOH); mp = 96-97 °C. 1H NMR
(CDCl3): δ 3.77 (s, 3H), 3.81 (s, 3H), 3.89 (s, 3H), 3.95 (s, 3H),
6.59 (d, 1H), 7.90 (d, 1H).
The unsaturated acid (0.010 g, 0.033 mmol) was then oxidized
to provide 5c (0.003 g, 32%) as a red solid following flash
chromatography (1:1 Et2O:hexanes 0.5% AcOH) and recrystal-
lization from Et2O/hexanes. Rf =0.10 (2:3 Et2O:hexanes 0.5%
AcOH); mp=124-131 °C. 1H NMR (CDCl3): δ 1.01 (t, 3H), 1.94
(d, 3H), 2.12 (q, 2H), 3.99 (s, 3H), 4.01 (s, 3H), 7.23 (d, 1H).
(E)-3-(4,5-Dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-
2-propylpropenoic Acid (5d). Following the method used for the
synthesis of 1, the Emmons condensation was performed with 4
(0.437 g, 1.82 mmol) to provide ethyl (E)-3-(6-methyl-2,3,4,
5-trimethoxyphenyl)-2-propylpropenoate (0.275 g, 43%) as a
colorless oil following chromatography (1:3 EtOAc:hexanes).
1
Rf = 0.52 (1:3 EtOAc:hexanes). H NMR (CDCl3): δ 0.75 (t,
3H, J=7.2 Hz), 1.33 (t, 3H, J=7.2 Hz), 1.35 (m, 2H), 2.03 (s, 3H),
2.11 (m, 2H), 3.69 (s, 3H), 3.78 (s, 3H), 3.88 (s, 3H), 3.93 (s, 3H),
4.25 (q, 2H, J=7.2 Hz), 7.38 (s, 1H).
1
The ethyl ester (0.034 g, 0.098 mmol) was then hydrolyzed
(KOH, EtOH) to provide the unsaturated acid (0.029 g, 92%) as
a gold oil following flash chromatography (2:3 Et2O:hexanes
1
0.5% AcOH). Rf = 0.18 (2:3 Et2O:hexanes 0.5% AcOH). H
NMR (CDCl3): δ 0.77 (t, 3H), 1.41 (m, 2H), 2.04 (s, 3H), 2.13
(m, 2H), 3.70 (s, 3H), 3.79 (s, 3H), 3.89 (s, 3H), 3.93 (s, 3H),
7.55 (s, 1H).
The unsaturated acid (0.029 g, 0.090 mmol) was then oxidized
to provide 5d (0.016 g, 59%) as a red solid following flash
chromatography (1:1 Et2O:hexanes 0.5% AcOH) and recrys-
tallization from Et2O/hexanes. Rf = 0.12 (2:3 Et2O:hexanes
The unsaturated acid (0.089 g, 0.31 mmol) was then oxidized
to provide 5a (0.024 g, 57%) as a red solid following flash
chromatography (1:1 Et2O:hexanes 0.5% AcOH) and subse-
quent recrystallization from Et2O/hexanes. Rf=0.06 (2:3 Et2O:
1
0.5% AcOH); mp=95-99 °C. H NMR (CDCl3): δ 0.76 (t,
3H), 1.41 (m, 2H), 1.91 (d, 3H), 2.04 (m, 2H), 3.95 (s, 3H), 3.98
(s, 3H), 7.24 (s, 1H).
1
(E)-3-(5,6-Dimethoxy-3-methyl-1,4-dioxocyclohexa-2,5-dienyl)-
2-butylpropenoic Acid (5e). Following the method used for the
synthesis of 1, the Emmons condensation was performed with 4
(0.650 g, 2.71 mmol) to provide ethyl (E)-3-(6-methyl-2,3,4,5-
trimethoxyphenyl)-2-butylpropenoate (0.484 g, 49%) as a yellow
oil following chromatography (1:9 EtOAc:hexanes). Rf=0.54 (1:3
EtOAc:hexanes). 1H NMR (CDCl3): δ 0.73 (t, 3H), 1.12 (m, 2H),
1.29 (m, 2H), 1.32 (t, 3H), 2.03 (s, 3H), 2.13 (m, 2H), 3.69 (s, 3H),
3.78 (s, 3H), 3.88 (s, 3H), 3.92 (s, 3H), 4.25 (q, 2H), 7.37 (s, 1H).
The ethyl ester (0.166 g, 0.453 mmol) was then hydrolyzed
(KOH, EtOH) to provide the unsaturated acid (0.144 g, 94%) as
a yellow amorphous solid following flash chromatography
(2:3 Et2O:hexanes 0.5% AcOH) and recrystallization from
Et2O/hexanes. Rf1= 0.26 (2:3 Et2O:hexanes 0.5% AcOH);
mp = 70-85 °C. H NMR (CDCl3): δ 0.74 (t, 3H), 1.19 (m,
4H), 2.04 (s, 3H), 2.15 (m, 2H), 3.70 (s, 3H), 3.79 (s, 3H), 3.89 (s,
3H), 3.93 (s, 3H), 7.53 (s, 1H).
hexanes 0.5% AcOH); mp=116-125 °C. H NMR (CDCl3):
δ2.19 (s, 3H), 4.00 (s, 6H), 6.76 (d, 1H), 7.60 (d, 1H).
(E)-3-(5,6-Dimethoxy-3-methyl-1,4-dioxocyclohexa-2,5-dienyl)-
2-methylpropenoic Acid (5b). Following the method used for the
synthesis of 1, the Emmons condensation was performed with 4
(0.569 g, 2.37 mmol) to provide ethyl (E)-3-(6-methyl-2,3,4,
5-trimethoxyphenyl)-2-methylpropenoate (0.674 g, 88%) as a
pale-yellow oil following chromatography (1:3 EtOAc:hexanes).
Rf=0.48 (1:3 EtOAc:hexanes). 1H NMR (CDCl3): δ1.31 (t, 3H),
1.71 (d, 3H), 2.01 (s, 3H), 3.67 (s, 3H), 3.77 (s, 3H), 3.87 (s, 3H),
3.91 (s, 3H), 4.24 (q, 2H), 7.47 (d, 1H).
The ethyl ester (0.156 g, 0.481 mmol) was hydrolyzed (KOH,
EtOH) to provide the unsaturated acid (0.121 g, 85%) as a light-
yellow solid following flash chromatography (2:3 Et2O:hexanes
0.5% AcOH) and recrystallization from Et2O/hexanes. Rf=0.16
1
(2:3 Et2O:hexanes 0.5% AcOH); mp=98-102 °C. H NMR
(CDCl3): δ 1.76 (d, 3H, J=1.2 Hz), 2.05 (s, 3H), 3.69 (s, 3H), 3.79
(s, 3H), 3.90 (s, 3H), 3.93 (s, 3H), 7.64 (d, 1H).
The unsaturated acid (0.015 g, 0.044 mmol) was then oxidized
to provide 5e (0.005 g, 40%) as a red solid following flash
chromatography (1:1 Et2O:hexanes 0.5% AcOH) and recrys-
tallization from Et2O/hexanes. Rf = 0.12 (2:3 Et2O:hexanes
The unsaturated acid (0.057 g, 0.19 mmol) was then oxidized
to provide 5b (0.016 g, 31%) as a red solid following flash
chromatography (1:1 Et2O:hexanes 0.5% AcOH) and recrys-
tallization from Et2O/hexanes. Rf = 0.06 (2:3 Et2O:hexanes
0.5% AcOH); mp=134-136 °C. 1H NMR (CDCl3): δ1.77 (d,
3H, J=1.2 Hz), 1.95 (d, 3H, J=1.2 Hz), 4.01 (s, 3H), 4.03 (s, 3H),
7.39 (s, 1H).
(E)-3-(5,6-Dimethoxy-3-methyl-1,4-dioxocyclohexa-2,5-dienyl)-
2-ethylpropenoic acid (5c). Following the method used for the
synthesis of 1, the Emmons condensation was performed with 4
(0.437 g, 1.82 mmol) to provide ethyl (E)-3-(6-methyl-2,3,4,
5-trimethoxyphenyl)-2-ethylpropenoate (0.258 g, 42%) as a color-
less oil following chromatography (1:3 EtOAc:hexanes). Rf=0.50
(1:3 EtOAc:hexanes). 1H NMR (CDCl3): δ 0.94 (t, 3H), 1.33 (t,
3H), 2.02 (s, 3H), 2.15 (q, 2H), 3.69 (s, 3H), 3.78 (s, 3H), 3.89 (s,
3H), 3.92 (s, 3H), 4.26 (q, 2H), 7.36 (s, 1H).
1
0.5% AcOH); mp=54-55 °C. H NMR (CDCl3): δ 0.82 (t,
3H), 1.22 (m, 2H), 1.39 (m, 2H), 1.95 (d, 3H), 2.10 (m, 2H), 3.99
(s, 3H), 4.02 (s, 3H), 7.24 (s, 1H).
2-Chloro-3,4,5,6-tetramethoxybenzaldehyde (6). According to
a general aryl chlorination procedure by Lopez-Alvarado,22
2,3,4,5-tetramethoxybenzaldehyde (0.767 g, 3.39 mmol) was
dissolved in CH2Cl2 (10 mL) at room temperature and then
SO2Cl2 (neat, 0.31 mL, 3.7 mmol) was added at room tempera-
ture. The reaction was stirred for 1 h and monitored by 1H NMR
for completion. The reaction was then diluted with CH2Cl2,
washed with brine, dried over MgSO4, filtered, and condensed.
The resulting oil was then purified by flash column chromato-
graphy (1:4 Et2O:hexanes) to provide 6 (0.861 g, 97%) as a
1
colorless oil. Rf=0.27 (1:4 Et2O:hexanes). H NMR (CDCl3):
δ 3.84 (s, 3H), 3.89 (s, 3H), 3.90 (s, 3H), 4.02 (s, 3H), 10.34
(s, 1H).
The ethyl ester (0.041 g, 0.12 mmol) was then hydrolyzed to
provide the unsaturated acid (0.010 g, 27%) as a gold oil
following flash chromatography (2:3 Et2O:hexanes 0.5%
1
AcOH). Rf =0.16 (2:3 Et2O:hexanes 0.5% AcOH). H NMR
(CDCl3): δ 0.98 (t, 3H), 2.04 (s, 3H), 2.18 (q, 2H), 3.70 (s, 3H),
3.79 (s, 3H), 3.90 (s, 3H), 3.93 (s, 3H), 7.53 (s, 1H).
(E)-3-(3-Chloro-5,6-dimethoxy-1,4-dioxocyclohexa-2,5-dienyl)-
2-methylpropenoic Acid (7). Following the method used for the
synthesis of 1, the Emmons condensation was performed in

1206 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Nyland et al.
refluxing toluene with 6 (0.861 g, 3.30 mmol) to provide ethyl
(E)-3-(2-chloro-3,4,5,6-trimethoxyphenyl)-2-methylpropenoate
(0.559 g, 49%) as a colorless oil following chromatography (1:9
iPr2O:hexanes). Rf=0.29 (1:4 Et2O:hexanes); E:Z=1:0 in reflux-
ing toluene. 1H NMR (CDCl3): δ 1.34 (t, 3H), 1.78 (d, 3H), 3.69
(s, 3H), 3.86 (s, 3H), 3.90 (s, 3H), 3.94 (s, 3H), 4.26 (q, 2H), 7.42
(q, 1H).
as a red solid following flash chromatography (2:3 Et2O:hexanes
0.5% AcOH) and recrystallization from Et2O/hexanes. Rf=0.06
(2:3 Et2O:hexanes 0.5% AcOH); mp = 104-106 °C. H NMR
(CDCl3): δ 1.95 (d, 3H), 2.40 (t, 2H), 3.24 (s, 3H), 3.44 (t, 2H), 3.99
(s, 3H), 4.01 (s, 3H), 7.35 (d, 1H).
1
(E)-N-(2-Hydroxyethyl)-3-(6-methyl-2,3,4,5-tetramethoxy-
phenyl)-2-nonylpropenamide (11). Dicyclohexylcarbodiimide
(0.106 g, 0.514 mmol) was added to a flame-dried 10 mL
round-bottom flask under argon, and then CH2Cl2 (2 mL) was
added at room temperature. In a second flame-dried flask
under argon, a solution of acid (E)-10 (0.167 g, 0.409 mmol)
and 1-hydroxybenztriazole (0.008 g, 0.06 mmol) was prepared
using DMF (3 mL) and CH2Cl2 (1 mL). The acid/HOBt
solution was then added and stirred for 2 h. Ethanol-
amine (0.040 mL, 0.66 mmol) was then added rapidly at room
temperature. The reaction was stirred for an additional 12 h,
diluted with EtOAc, washed with brine, dried over MgSO4,
filtered, and condensed. The resulting white solid, a mixture
of dicyclohexylurea, DCC, and product, was first suspended
in approximately 5 mL of CH2Cl2. The suspension was then
filtered through a pipet containing a cotton plug, and the pipet
was washed two times with 2 mL of CH2Cl2. The combined
fractions were then purified via flash chromatography (1:4
EtOAc:hexanes) to provide two compounds that appeared to
be isomers: (E)-11 (0.088 g, 48%, 2:3 EtOAc:hexanes Rf =
0.36) and (Z)-11 (0.063 g, 33%, 2:3 EtOAc:hexanes Rf=0.71,
coeluted with DCC), both as colorless oils. (E)-11: 1H NMR
(CDCl3): δ 0.83 (t, 3H), 1.13 (m, 12H), 1.32 (m, 2H), 2.03 (s,
3H), 2.13 (m, 2H), 2.70 (t, 1H), 3.55 (m, 2H), 3.69 (s, 3H), 3.78
(s, 3H), 3.80 (m, 2H), 3.88 (s, 3H), 3.92 (s, 3H), 6.31 (bs, 1H),
6.92 (s, 1H). (Z)-11: 1H NMR (CDCl3): δ 3.19 (s, 3H), 2.14 (m,
2H), 2.42 (m, 2H), 3.68 (m, 1H), 3.75 (s, 3H), 3.78 (s, 3H), 3.88
(s, 3H), 3.92 (s, 3H), 4.07 (m, 1H), 6.64 (s, 1H), 8.53 (d, 1H).
(E)-N-(2-Hydroxyethyl)-3-(5,6-dimethoxy-3-methyl-1,4-di-
oxocyclohexa-2,5-dienyl)-2-nonylpropenamide (E-12). Amide
(E)-11 (0.088 g, 0.20 mmol) was added to a flame-dried round-
bottom flask and dissolved in EtOAc (7.0 mL). Then AcOH
(5 drops) and HNO3 (0.5 mL) were added and the reaction was
stirred for 3 h at room temperature. The colorless oil easily
dissolved in EtOAc, and the reaction turned orange upon
addition of HNO3. The reaction was then diluted with EtOAc
(40.0 mL), washed three times with brine, dried over MgSO4,
filtered, and condensed to provide an orange oil. The oil was
then purified using flash chromatography (1:2-1:1 EtOAc:
hexanes) and recrystallized from acetone/hexane to provide
(E)-12 (0.037 g, 45%) as a red oil. Rf=0.24 (2:3 EtOAc:hexanes).
1H NMR (CDCl3): δ 0.84 (t, 3H), 1.17 (m, 12 H), 1.33 (m, 2H),
1.93 (d, 3H), 2.09 (t, 2H), 2.51 (t, 1H), 3.52 (q, 2H), 3.79 (dd, 2H),
3.98 (s, 3H), 4.02 (s, 3H), 6.48 (bs, 1H), 6.53 (s, 1H).
The ethyl ester (0.255 g, 0.740 mmol) was then hydrolyzed
(KOH, EtOH) to provide the unsaturated acid (0.222 g, 95%) as
a red amorphous solid following flash chromatography (2:3
Et2O:hexanes 0.5% AcOH) and recrystallization from Et2O/
1
hexanes. Rf=0.20 (2:3 Et2O:hexanes 0.5% AcOH). H NMR
(CDCl3): δ 1.81 (s, 3H), 3.71 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H),
7.57 (d, 1H).
The unsaturated acid (0.124 g, 0.391 mmol) was dissolved in
acetonitrile (10 mL) at room temperature, then ceric ammonium
nitrate (0.970 g, 1.77 mmol) dissolved in water (8 mL) was added
at room temperature. The reaction was stirred for 30 min and
then extracted with CH2Cl2. The organic layer was washed with
brine, dried over MgSO4, filtered, and condensed. The red oil
was then purified by either flash column chromatography (1:1
Et2O:hexanes 0.5% AcOH) or recrystallization from Et2O/
hexanes to afford 7 (0.026 g, 22%) as a red solid. Rf = 0.32
1
(1:1 Et2O:hexanes 0.5% AcOH); mp=183-185 °C. H NMR
(CDCl3): δ 1.83 (d, 3H), 4.03 (s, 3H), 4.05 (s, 3H), 7.28 (d, 1H).
(E)-40-(6-Methyl-2,3,4,5-tetramethoxyphenyl)-3-ethylidenete-
trahydrofuran-2-one (8). NaH (0.210 g, 5.25 mmol) was added to
a flame-dried 100 mL 3-neck round-bottom flask connected to a
water-jacketed reflux condenser.20 The flask was purged with
argon, and a drying tube was attached to the top. Toluene
(30 mL) was added to the flask, followed by 2-diethylpho-
sphono-γ-butryrolactone (1.83 g, 8.2 mmol) dissolved in toluene
(10 mL) at room temperature. The reaction was heated under
reflux for 30 min, and the aldehyde (4, 0.501 g, 2.09 mmol) was
dissolved in toluene (5 mL) and added slowly at reflux. The
reaction was heated for another 8 h under reflux before being
cooled to room temperature, diluted with ethyl acetate, and
washed with brine. The organic layer was dried, filtered, and
condensed. The resulting oil was purified via flash column
chromatography (1:3 EtOAc:hexanes) to provide 8 (0.429 g,
66%) as a white solid. Rf=0.18 (1:3 EtOAc:hexanes); E:Z=20:1
in refluxing toluene; mp=78-79 °C. 1H NMR (CDCl3): δ 2.12
(s, 3H), 2.85 (dt, 2H), 3.65 (s, 3H), 3.78 (s, 3H), 3.90 (s, 3H), 3.93
(s, 3H), 4.35 (t, 2H), 7.48 (t, 1H).
(E)-3-(5,6-Dimethoxy-3-methyl-1,4-dioxocyclohexa-2,5-dienyl)-
2-methoxyethylpropenoic Acid (9). According to a modified pro-
cedure of King,17 8 (0.084 g, 0.27 mmol) was added to a flame-
dried 10 mL round-bottom flask under argon and a water-jacketed
reflux condenser was attached. Anhydrous MeOH (2 mL) was
then added, followed by H2SO4 (4 drops) and trimethyl ortho-
formate (0.31 mL, 4.0 mmol) at room temperature. The reaction
was then heated under reflux for 12 h, cooled to room temperature,
and the solvent was removed under reduced pressure. The residue
was dissolved in EtOAc, washed with saturated brine, dried over
MgSO4, filtered, and condensed. The resulting crude product was
purifiedviaflashcolumnchromatography (1:3 EtOAc:hexanes)to
provide the methyl ester (0.073 g, 76%) as a pale-yellow solid. Rf=
0.25 (1:3 EtOAc:hexanes); mp=35-37 °C. 1H NMR (CDCl3): δ
2.02 (s, 3H), 2.47 (t, 2H), 3.17 (s, 3H), 3.37 (t, 2H), 3.69 (s, 3H),
3.78 (s, 3H), 3.81 (s, 3H), 3.88 (s, 3H), 3.92 (s, 3H), 7.49 (s, 1H).
The methyl ester (0.074 g, 0.21 mmol) was hydrolyzed follow-
ing the method for 1 to provide the unsaturated acid (0.070 g,
100%) as a tan solid following flash chromatography (2:3 Et2O:
hexanes 0.5% AcOH) and recrystallization from Et2O/hexanes.
Rf = 0.16 (2:3 Et2O:hexanes 0.5% AcOH); mp = 97-99 °C.
1H NMR (CDCl3): δ 2.07 (s, 3H), 2.45 (t, 2H), 3.20 (s, 3H),
3.68 (s, 3H), 3.69 (q, 2H), 3.76 (s, 3H), 3.87 (s, 3H), 3.90 (s, 3H),
7.59 (s, 1H).
1,4-Dimethoxy-2-naphthaldehyde (14a). Following the proce-
dure of Evans et al.,23 1,4-naphthoquinone (13a, 10.00 g, 63.20
mmol) and Pd/C (10 wt %, 0.994 g) were added to a flame-dried
500 mL round-bottom flask at room temperature under argon.
THF (250.0 mL) was then added at room temperature, the
reaction vessel was covered in foil, and then was purged with
hydrogen gas for 10 min. A balloon filled with H2 was attached
and the reaction stirred for 4 h at room temperature. The
reaction was then purged with argon before adding NaH
(5.66 g, 142 mmol) slowly at 0 °C. After 10 min, dimethyl sulfate
(13.0 mL, 137 mmol) was added slowly at 0 °C. The reaction
mixture then became too thick to stir, so THF (50 mL) was
added. The dark-green mixture was allowed to stir at room
temperature for 4 h. The reaction was then filtered through
celite, washed with brine, dried over MgSO4, and condensed.
The resulting solid was then purified by flash chromatography
(EtOAc:hexanes) or recrystallized from Et2O:hexanes to pro-
vide 1,4-dimethoxynaphthalene (11.74 g, 99%) as pink need-
les. Rf =0.76 (CH2Cl2); mp=74-78 °C (lit. 84-86 °C).18 1
H
The resulting unsaturated acid (0.070 g, 0.21 mmol) was oxi-
dized following the procedure for 1 to provide 9 (0.012 g, 19%)
NMR (CDCl3): δ 3.95 (s, 6H), 6.69 (s, 2H), 7.50 (m, 2H),
8.20 (m, 2H).

Article
Following a modified procedure of Ito et al.,24 to a flame-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1207
(1.25 g, 3.37 mmol) was added. The reaction was then heated to
reflux for 8 h, then cooled and extracted with EtOAc. The organic
layer was washed three times with brine, dried over MgSO4, filtered,
and condensed to provide the crude aldehyde as a yellow solid.
Following flash chromatography (3:1 CH2Cl2:hexanes), aldehyde
14e (0.254 g, 45%) was obtained as a yellow solid. Rf = 0.36
(3:1 CH2Cl2:hexanes); mp = 69-71 °C. 1H NMR (CDCl3):
δ 4.07 (s, 3H), 4.09 (d, 3H), 7.54 (t, 1H), 7.66 (t, 1H), 8.18 (m,
2H), 10.54 (s, 1H).
3-Chloro-1,4-dimethoxy-2-naphthaldehyde (14f). Following a
general aromatic chlorination procedure reported by Lopez-
Alvarado,22 aldehyde 14a (2.12 g, 9.81 mmol) was dissolved in
CH2Cl2 (15 mL) at room temperature in a flame-dried 50 mL
round-bottom flask under argon. Neat SO2Cl2 (0.92 mL, 11
mmol) was added at room temperature, and the argon line was
replaced with a drying tube. After 20 h, the reaction was
complete by NMR, and the solution was diluted with CH2Cl2
(50 mL), washed with brine, dried over MgSO4, and filtered. The
resulting solid was purified by flash chromatography (1:1
CH2Cl2:hexanes) to provide 14f (1.63 g, 66%) as white needles.
Rf = 0.21 (1:1 CH2Cl2:hexanes); mp = 94-96 °C. 1H NMR
(CDCl3): δ 3.99 (s, 3H), 4.05 (s, 3H), 7.64 (m, 2H), 8.17 (dd, 2H),
10.61 (s, 1H).
dried 50 mL round-bottom flask was added 1,4-dimethoxy-
naphthalene (1.34 g, 7.12 mmol) dissolved in CH2Cl2 (10.0 mL)
and then cooled to 0 °C under argon. Then TiCl4 (1 M in
CH2Cl2, 8.0 mL, 8.0 mmol) was added slowly at 0 °C followed by
R,R-dichloromethyl methyl ether (0.71 mL, 8.0 mmol) at 0 °C.
The reaction was stirred at 0 °C for 3 h, poured into water, and
stirred for 10 min at room temperature. The reaction was then
extracted with EtOAc, washed with saturated brine, dried over
MgSO4, filtered, and condensed. The resulting oil was purified
using flash chromatography (CH2Cl2) to provide 27 (1.43 g,
93%) as a white solid that was recrystallized from Et2O:hexanes
to provide white needles. Rf=0.62 (CH2Cl2); mp=108-109 °C
(lit. 120-121 °C).22 1H NMR (CDCl3): δ 4.01 (s, 3H), 4.08
(s, 3H), 7.11 (s, 1H), 7.62 (m, 2H), 8.23 (m, 2H), 10.56 (s, 1H).
1,4-Dimethoxy-3-methyl-2-naphthaldehyde (14b). 1,4-Di-
methoxy-2-methylnaphthalene was prepared from 13b (1.90 g,
9.39 mmol) as described above for 14a to give 1.490 g (69%) of
the product as a white solid that was recrystallized from Et2O/
hexanes to provide white needles. Rf = 0.57 (3:7 EtOAc:
hexanes); mp = 78-80 °C (lit. 83.5-83.7 °C).22 1H NMR
(CDCl3): δ 2.63 (s, 3H), 3.85 (s, 3H), 4.05 (s, 3H), 7.59 (dt,
2H), 8.14 (dd, 2H).
Compound 14b was prepared from 1,4-dimethoxy-2-methyl-
naphthalene (3.54 g, 20.6 mmol) as described above for 14a to
give 4.02 g (97%) of the product as a low melting white solid
following flash chromatography (1:19 EtOAc:hexanes). Rf =
0.60 (3:7 EtOAc:hexanes); mp=29-31 °C (lit. 35.9-36.3 °C).22
1H NMR (CDCl3): δ 2.43 (s, 3H), 3.85 (s, 3H), 3.95 (s, 3H), 7.45
(dt, 2H), 8.09 (dd, 2H).
1,3,4-Trimethoxy-2-naphthaldehyde (14c). 1,2,4-Trimethoxy-
naphthalene was prepared from 13h (5.23 g, 33.3 mmol) as
described above for 14a to give 5.19 g (71%) of the product as a
red oil that solidified in the freezer. Rf = 0.17 (1:9 EtOAc:
hexanes), 0.50 (CH2Cl2); mp=oil (lit. 38-40 °C).20 1H NMR
(CDCl3): δ 3.91 (s, 3H), 3.98 (s, 3H), 3.99 (s, 3H), 6.63 (s, 1H),
7.40 (m, 2 H), 8.09 (dd, 2H).
Ethyl (E)-3-(1,4-Dimethoxynaphthalen-2-yl)-2-methylpropeno-
ate (15a). NaH (0.303 g, 7.59 mmol) was added to a flame-dried
50 mL round-bottom flask connected to a water-jacketed reflux
condenser. The flask was purged with argon, and a drying tube
was attached to the top of the condenser. Toluene (20 mL) was
added to the flask followed by triethyl 2-phosphonopropionate
(1.0 mL, 4.6 mmol) at room temperature. The reaction was
heated under reflux for 30 min, and aldehyde 14a (0.542 g,
2.51 mmol) was dissolved in toluene (5 mL) and added slowly at
reflux. The reaction was heated for another 8 h under reflux. The
reaction was then cooled to room temperature, acidified with
2 M HCl, diluted with ethyl acetate, and washed with brine. The
organic layer was dried, filtered, and condensed. The resulting
oil was purified via flash column chromatography (1:19 iPr2O:
hexanes) to provide 15a (0. 554 g, 73%) as a colorless oil. Rf=
0.30 (1:19 iPr2O:hexanes, 4 developments); E:Z=20:1. 1H NMR
(CDCl3): δ 1.36 (t, 3H), 2.10 (d, 3H), 3.91 (s, 3H), 3.96 (s, 3H),
4.29 (q, 2H), 6.70 (s, 1H), 7.51 (m, 2H), 7.96 (d, 1H), 8.15
(m, 2H).
According to the procedure by Syper et al.,25 1,2,4-trimethoxy-
naphthalene (1.07 g, 4.90 mmol) was dissolved in THF
(20.0 mL) at 0 °C under argon, and then nBuLi (1.6 M in
hexanes, 4.0 mL, 10.0 mmol) was added at 0 °C. The reaction
was stirred at 0 °C for 5 h before DMF (0.850 mL, 11.0 mmol)
was added at 0 °C. The reaction was stirred for another 30 min at
room temperature before quenching with water. The reaction
was diluted with ethyl ether and washed with saturated brine.
The organic layer was dried over MgSO4, filtered, and con-
densed. Following purification via column chromatography
(CH2Cl2 to 1:9 EtOAc:CH2Cl2), pure aldehyde 14c (1.07 g,
89%) was obtained as a yellow solid. Rf = 0.25 (1:9 EtOAc:
Ethyl (E)-3-(1,4-Dimethoxy-3-methylnaphthalen-2-yl)-2-methyl-
propenoate (15b). Compound 15b was prepared from 14b (0.506 g,
2.20 mmol) as described above for 15a to give 0.541 g (1.42 mmol,
65%) of the product as a colorless oil following flash chromato-
i
graphy (1:19 Pr2O:hexanes). Rf (E)-15b = 0.22, (Z)-15b = 0.15
i
1
(1:19 Pr2O:hexanes, 4 developments); E:Z = 20:1. (E)-15b. H
NMR (CDCl3): δ 1.36 (t, 3H), 1.79 (s, 3H), 2.28 (s, 3H), 3.73
(s, 3H), 3.87 (s, 3H), 4.30 (q, 2H), 7.49 (m, 2H), 7.70 (s, 1H), 8.07
(m, 2H).
hexanes), 0.20 (CH2Cl2); mp = 43-45 °C (lit. 53 °C d).23 1
H
NMR (CDCl3): δ 3.99 (s, 3H), 4.00 (s, 3H), 4.03 (s, 3H), 7.55 (m,
2H), 8.15 (dd, 2H), 10.57 (s, 1H).
Ethyl (E)-3-(1,3,4-Trimethoxynaphthalen-2-yl)-2-methylpro-
penoate (15c). Compound 15c was prepared from 14c (0.330 g,
1.33 mmol) as described above for 15a to give 0.119 g (27%) of the
product as a yellow oilfollowing flashchromatography(1:9 Et2O:
hexanes). Rf=(E)-15c=0.17, (Z)-15c=0.11 (1:19 iPr2O:hexanes,
4 developments); E:Z = 11:9 in refluxing toluene. 1H NMR
(CDCl3): δ 1.36 (t, 3H), 1.87 (d, 3H), 3.75 (s, 3H), 3.87 (s, 3H),
3.98 (s, 3H), 4.29 (q, 2H), 7.47 (m, 2H), 7.73 (d, 1H), 8.09 (m, 2H).
Ethyl (E)-3-(1,4-Dimethoxy-3-methylsulfanylnaphthalen-2-
yl)-2-methylpropenoate (15d). Compound 15d was prepared
from 14d (0.308 g, 1.17 mmol) as described above for 15a to
give 0.231 g (55%) of the product as an amorphous yellow solid
following flash chromatography (1:9 EtOAc:hexanes) and re-
crystallization from Et2O/hexanes. Rf = 0.26 (1:9 EtOAc:
hexanes); E:Z=7:3. 1H NMR (CDCl3): δ 1.37 (t, 3H), 1.82 (s,
3H), 2.37 (s, 3H), 3.72 (s, 3H), 4.00 (s, 3H), 4.30 (q, 2H), 7.54 (m,
2H), 7.86 (s, 1H), 8.10 (m, 2H).
1,4-Dimethoxy-3-methylthio-2-naphthaldehyde (14d). Pyridi-
nium chlorochromate (PCC, 1.03 g, 4.78 mmol) was added to a
flame-dried 250 mL round-bottom flask followed by dry
CH2Cl2 (90 mL) at room temperature under argon. 1,4-Di-
methoxy-2-hydroxymethyl-3-methylthionaphthalene, synthe-
sized following the procedure of Flader et al.,21 (0.514 g,
1.94 mmol) was dissolved in CH2Cl2 (10 mL) and added slowly
at room temperature. The reaction was stirred for a further 12 h
at room temperature before being poured into a slurry of florisil,
MgSO4, and CH2Cl2. After stirring, the suspension was filtered
through celite and condensed. Purification by flash chromato-
graphy (1:9 EtOAc:hexanes) gave 14d (0.308 g, 1.17 mmol, 62%)
as an amorphous yellow solid. Rf=0.26 (1:9 EtOAc:hexanes).
1H NMR (CDCl3): δ 2.47 (s, 3H), 4.01 (s, 3H), 4.04 (s, 3H), 7.62
(m, 2H), 8.13 (d, 1H), 8.21 (d, 1H), 10.70 (s, 1H).
1,4-Dimethoxy-3-fluoro-2-naphthaldehyde (14e). Aldehyde
14a (0.517 g, 2.39 mmol) and MeCN (20 mL) were added to a
flame-dried 50 mL round-bottom flask under argon and Selectfluor
Ethyl (E)-3-(1,4-Dimethoxy-3-fluoronaphthalen-2-yl)-2-methyl-
propeonoate (15e). Compound 15e was prepared from 14e (0.216 g,

1208 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Nyland et al.
0.922 mmol) as described above for 15a to give 0.226 g (77%) of the
product as a yellow oil following flash chromatography (1:19
Et2O:hexanes). (0.093 g pure E, 0.128 g E/Z mixture). Rf (E)-15e
0.26, (Z)-15e=0.17 (1:19 Et2O:hexanes, 3 developments); E:Z=
5:1. 1H NMR (CDCl3): δ 1.36 (t, 3H), 1.91 (t, 3H), 3.80 (s, 3H),
4.05 (d, 3H), 4.30 (q, 2H), 7.51 (m, 2H), 7.67 (s, 1H), 8.09 (dd, 1H),
8.14 (dd, 1H).
(E)-3-(3-Methoxy-1,4-naphthoquinon-2-yl)-2-methylpropenoic
Acid (16c). (E)-3-(1,3,4-Trimethoxynaphthalen-2-yl)-2-methyl-
propenoic acid was prepared from 15c (0.073 g, 0.22 mmol) as
describedabove for 16a to give 0.067 g (100%) of the product as a
tan solid following flash chromatography (1:3 EtOAc:hexanes
0.5% AcOH) and recrystallization from Et2O/hexanes. Rf=0.13
(1:3 EtOAc:hexanes 0.5% AcOH); mp=119-123 °C. 1H NMR
(CDCl3): δ 1.91 (s, 3H), 3.77 (s, 3H), 3.89 (s, 3H), 3.99 (s, 3H),
7.49 (m, 2H), 7.91 (s, 1H), 8.10 (t, 2H).
Ethyl (E)-3-(3-Chloro-1,4-dimethoxynaphthalen-2-yl)-2-methyl-
propenoate (15f). Compound 15f was prepared from 14f (2.93 g,
11.7 mmol) as described above for 15a to give 2.56 g (65%) of the
product as a yellow solid following flash chromatography (1:19
Et2O:hexanes) and recrystallization from Et2O/hexanes (1.50 g E,
yellow solid; 0.960 g E/Z mixture, gold oil; 0.101 g Z, gold solid).
Compound 16c was prepared from (E)-3-(1,3,4-trimethoxy-
naphthalen-2-yl)-2-methylpropenoic acid (0.060 g, 0.20 mmol)
as described above for 16a to give 0.015 g (27%) of the product
as a yellow solid following flash chromatography (2:3 Et2O:
hexanes 0.5% AcOH) and recrystallization from Et2O/hexanes.
i
Rf (E)-15f = 0.32, (Z)-15f = 0.24 (1:19 Pr2O:hexanes, 4 deve-
lopments); E:Z=15:5; mp=108-109 °C. 1H NMR (CDCl3): δ
1.37 (t, 3H), 1.83 (s, 3H), 3.75 (s, 3H), 3.98 (s, 3H), 4.30 (q, 2H),
7.55 (m, 2H), 7.68 (s, 1H), 8.11 (m, 2H).
Rf=0.43 (2:3 Et2O:hexanes 0.5% AcOH); mp=205 °C d. H
1
NMR (CDCl3): δ 1.83 (d, 3H), 4.15 (s, 3H), 7.53 (d, 1H), 7.73 (m
(5), 2H), 8.08 (m, 2H).
Ethyl (E)-3-(3-Bromo-1,4-dimethoxynaphthalen-2-yl)-2-methyl-
propenoate (15g). Compound 15g was prepared from 14g (0.515 g,
1.75 mmol) as described above for 15a to give 0.373 g (56%) of the
product as a white solid following flash chromatography (1:19
iPr2O:hexanes) and recrystallization from Et2O/hexanes (0.314 g
(E)-3-(3-Methylthio-1,4-naphthoquinon-2-yl)-2-methylprope-
noic Acid (16d). (E)-3-(1,4-Dimethoxy-3-methylthionaphthalen-
2-yl)-2-methylpropenoic acid was prepared from 15d (0.165 g,
0.476 mmol) as described above for 16a to give 0.152 g (100%)
of the product as an orange solid following flash chromatography
(1:3 EtOAc:hexanes 0.5% AcOH) and recrystallization from
Et2O/hexanes. Rf = 0.18 (1:3 EtOAc:hexanes 0.5% AcOH). H
NMR (CDCl3): δ 1.82 (d, 3H), 2.37 (s, 3H), 3.72 (s, 3H), 4.00 (s,
3H), 7.51 (m, 2H), 7.99 (d, 1H), 8.07 (m, 2H).
i
E; 0.059 g Z). Rf (E)-15g = 0.26, (Z)-15g = 0.19 (1:19 Pr2O:
1
1
hexanes, 4 developments); E:Z = 17:3; mp = 102-103 °C. H
NMR (CDCl3): δ 1.37 (t, 3H), 1.81 (d, 3H), 3.74 (s, 3H), 3.97 (s,
3H), 4.31 (q, 2H), 7.56 (m, 2H), 7.65 (d, 1H), 8.11 (m, 2H).
(E)-3-(1,4-Naphthoquinon-2-yl)-2-methylpropenoic acid (16a).
Emmons ester 15a (0.231 g, 0.769 mmol) was dissolved in EtOH
(10 mL), and then KOH (0.40 g, 7.13 mmol) was added to the
reaction. The reaction was heated to reflux and stirred at this
temperature for 30 min. The reaction was then cooled, acidified,
and extracted with ethyl acetate. The organic layer was washed
with brine, dried over MgSO4, filtered, and condensed. The
resulting acid was then used without further purification in the
next step. Alternatively, it can be purified via flash chromatog-
raphy (1:3 EtOAc:hexanes 0.5% AcOH) or by recrystallization
from Et2O/hexanes to provide (E)-3-(1,4-dimethoxynaphtha-
len-2-yl)-2-methylpropenoic acid (0.175 g, 83%) as a white solid.
Rf=0.24 (1:3 EtOAc:hexanes 0.5% AcOH); mp=149-155 °C.
1H NMR (CDCl3): δ 2.16 (d, 3H), 3.86 (s, 3H), 3.99 (s, 3H), 6.75
(s, 1H), 7.53 (m, 2H), 8.15 (d, 1H), 8.17 (m, 2H).
Compound 16d was prepared from (E)-3-(1,4-dimethoxy-3-
methylthionaphthalen-2-yl)-2-methylpropenoic acid (0.180 g,
0.565 mmol) as described above for 16a to give 0.063 g (38%)
of the product as a red fluffy solid following flash chromatog-
raphy (2:3 Et2O:hexanes 0.5% AcOH) and recrystallization
from Et2O/hexanes. Rf=0.45 (2:3 Et2O:hexanes 0.5% AcOH);
1
mp=195-196 °C. H NMR (CDCl3): δ 1.81 (d, 3H), 2.58 (s,
3H), 7.56 (d, 1H), 7.72 (m, 2H), 8.09 (m, 2H).
(E)-3-(3-Fluoro-1,4-naphthoquinon-2-yl)-2-methylpropenoic
Acid (16e). (E)-3-(1,4-Dimethoxy-3-fluoronaphthalen-2-yl)-2-
methylpropenoic acid was prepared from 15e (0.128 g, 0.402
mmol) as described above for 16a to give 0.097 g (83%) of the
product as a white solid following flash chromatography (1:4
acetone:hexanes 0.5% AcOH) and recrystallization from Et2O/
hexanes. Rf = 0.13 (1:4 acetone:hexanes 0.5% AcOH) mp =
157-158.5 °C. 1H NMR (CDCl3): δ 1.93 (d, 3H), 3.82 (s, 3H),
4.06 (d, 3H), 7.52 (m, 2H), 7.83 (d, 1H), 8.10 (d,1H), 8.15 (d, 1H).
19F NMR (CDCl3): δ -137.38.
The crude acid (0.114 g, 0.419 mmol) was dissolved in ethyl
acetate (10 mL) at room temperature and then HNO3 (1 mL)
and AcOH (3 drops) were added at room temperature. The
reaction was stirred at room temperature for 4 h and then
diluted with ethyl acetate and washed with brine. The organic
layer was dried over MgSO4, filtered, and condensed. The
yellow oil was then purified by either flash column chromato-
graphy (2:3 Et2O:hexanes 0.5% AcOH) or recrystallization
from Et2O/hexanes to afford 16a (0.059 g, 58%) as a yellow
solid. Rf=0.43 (2:3 Et2O:hexanes 0.5% AcOH); mp=205 °C d.
1H NMR (CDCl3): δ 2.13 (d, 3H,), 6.99 (s, 1H), 7.79 (m, 3H),
8.11 (m, 2H).
(E)-3-(1,4-Dimethoxy-3-fluoronaphthalen-2-yl)-2-methylpro-
penoic acid (0.097 g, 0.33 mmol) was dissolved in ethyl acetate
(10 mL) at room temperature and then HNO3 (1 mL) and
AcOH (6 drops) are added at room temperature. Silver(II) oxide
(0.309 g, 2.49 mmol) was then added, and the reaction was
stirred vigorously at room temperature for 1 h. The mixture
was filtered through a Pasteur pipet and cotton plug, the plug
was washed with ethyl acetate, and the combined organic layers
were washed with brine. The organic layer was dried over
MgSO4, filtered, and condensed. The yellow oil was then
purified by either flash column chromatography (1:1 Et2O:
hexanes 0.5% AcOH) or recrystallization from Et2O/hexanes
to afford 16e (0.020 g, 23%) as a yellow solid. Rf =0.08 (1:4
(E)-3-(3-Methyl-1,4-naphthoquinon-2-yl)-2-methylpropenoic
Acid (16b). (E)-3-(1,4-Dimethoxy-3-methylnaphthalen-2-yl)-2-
methylpropenoic acid was prepared from 15b (0.176 g, 0.560
mmol) as described above for 16a to give 0.134 g (83%) of the
product as a light-yellow solid following flash chromatography
(3:17 acetone:hexanes 0.5% AcOH) and recrystallization from
Et2O/hexanes. Rf = 0.20 (1:3 EtOAc:hexanes 0.5% AcOH);
1
EtOAc:hexanes 0.5% AcOH); mp=185-190 °C d. H NMR
(CDCl3): δ 1.94 (d, 3H), 7.45 (s, 1H), 7.80 (m, 2H), 8.15 (m, 2H).
1H NMR (DMSO): δ 1.83 (d, 3H), 7.15 (s, 1H), 7.91 (m, 2H),
8.06 (m, 2H).
1
mp=153-155 °C. H NMR (CDCl3): δ 1.80 (s, 3H), 3.75 (s,
3H), 3.89 (s, 3H), 7.51 (m, 2H), 7.72 (s, 1H), 8.09 (m, 2H).
Compound 16b was prepared from (E)-3-(1,4-dimethoxy-3-
methylnaphthalen-2-yl)-2-methylpropenoic acid (0.083 g, 0.29
mmol) as described above for 16a to give 0.048 g (0.19 mmol,
65%) of the product as a yellow solid following flash chromato-
graphy (3:17 acetone:hexanes 0.5% AcOH) and recrystalliza-
tion from Et2O/hexanes. Rf = 0.30 (2:3 Et2O:hexanes); mp =
195-196 °C. 1H NMR (CDCl3): δ 1.81 (bs, 3H), 2.11 (bs, 3H),
7.58 (m, 1H), 7.73 (m, 2H), 8.09 (m, 2H).
(E)-3-(3-Chloro-1,4-naphthoquinon-2-yl)-2-methylpropenoic
Acid (16f). Emmons ester 15f (0.959 g, 2.90 mmol) was hydro-
lyzed as described for 16a to provide (E)-3-(3-chloro-1,4-di-
methoxynaphthalen-2-yl)-2-methylpropenoic acid (0.872 g,
98%) as a tan solid following flash chromatography (3:17
acetone:hexanes 0.5% AcOH) and recrystallization from
Et2O/hexanes. Rf =0.08 (3:17 acetone:hexanes 0.5% AcOH);
1
mp=155-157 °C. H NMR (CDCl3): δ 1.88 (s, 3H), 3.77 (s,
3H), 3.99 (s, 3H), 7.58 (m, 2H), 7.85 (s, 1H), 8.123 (m, 2H).

Article
Following the procedure for 16e, (E)-3-(3-chloro-1,4-di-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1209
0.23 mmol) following the procedure for 9 to provide 0.049 g
(70%) of the product as a yellow solid following flash chromato-
graphy (2:3 Et2O:hexanes 0.5% AcOH) and recrystallization
from Et2O/hexanes. Rf=0.45 (2:3 Et2O:hexanes 0.5% AcOH);
mp=147-148 °C. 1H NMR (CDCl3): δ 2.12 (d, 3H), 2.45 (t, 2H),
3.20 (s, 3H), 3.44 (t, 2H), 7.53 (d, 1H), 7.73 (m, 2H), 8.09 (m, 2H).
(E)-3-(3-Chloro-1,4-naphthoquinon-2-yl)-2-methoxyethylpro-
penoic Acid (1b). The methyl ester was prepared from lactone
20b (0.098 g, 0.30 mmol) following the procedure for 9 to
provide 0.064 g (58%) as tan crystals following flash chromato-
graphy (1:3 EtOAc:hexanes) and recrystallization from Et21O/
hexanes. Rf =0.44 (1:3 EtOAc:hexanes); mp=61-62 °C. H
NMR (CDCl3): δ 2.59 (t, 2H), 3.12 (s, 3H), 3.39 (t, 2H), 3.78 (s,
3H), 3.39 (s, 3H), 3.98 (s, 3H), 7.55 (m, 2H), 7.68 (s, 1H),
8.10 (m, 2H).
The methyl ester (0.064 g, 0.17 mmol) was hydrolyzed follow-
ing the procedure for 9 to provide (E)-3-(3-chloro-1,4-di-
methoxynaphthalen-2-yl)-2-methoxethylpropenoic acid (0.067
g, quantitative) of the product as a tan solid following flash
chromatography (1:3 EtOAc:hexanes 0.5% AcOH) and recry-
stallization from Et2O/hexanes. Rf=0.12 (1:3 EtOAc:hexanes
0.5% AcOH); mp=145-146 °C. 1H NMR (CDCl3): δ 2.60 (t,
2H), 3.21 (s, 3H), 3.45 (t, 2H), 3.80 (s, 3H), 3.98 (s, 3H), 7.56 (m,
2H), 7.80 (s, 1H), 8.11 (m, 2H).
methoxynaphthalen-2-yl)-2-methylpropenoic acid (0.146 g,
0.476 mmol) was oxidized to provide 16f (0.087 g, 0.32 mmol,
67%) as a yellow solid following flash chromatography (2:3
acetone:hexanes 0.5% AcOH) and recrystallization from Et2O/
hexanes. Rf = 0.44 (2:3 acetone:hexanes 0.5% AcOH); mp =
1
229-230 °C. H NMR (CDCl3): δ 1.87 (d, 3H), 7.45 (d, 1H),
7.79 (m, 2H), 8.13 (m, 1H), 8.19 (m, 1H).
(E)-3-(3-Bromo-1,4-naphthoquinon-2-yl)-2-methylpropenoic
Acid (16g). (E)-3-(3-Bromo-1,4-dimethoxynaphthalen-2-yl)-2-
methylpropenoic acid was prepared from 15g (0.314 g, 0.828
mmol) as described above for 16a to give 0.291 g (100%) of the
product as a white solid following recrystallization from Et2O/
hexanes. Rf = 0.32 (1:1 Et2O:hexanes 0.5% AcOH); mp =
1
143-145 °C. H NMR (CDCl3): δ 1.86 (d, 3H), 3.77 (s, 3H),
3.99 (s, 3H), 7.57 (m, 2H), 7.82 (d, 1H), 8.13 (m, 2H).
Following the procedure for 16e, (E)-3-(3-bromo-1,4-di-
methoxynaphthalen-2-yl)-2-methylpropenoic acid (0.291 g,
0.829 mmol) was oxidized to provide 16g (0.122 g, 46%) as a
yellow solid following flash chromatography (1:1 Et2O:hexanes
0.5% AcOH) and recrystallization from Et2O/hexanes. Rf=0.32
1
(1:1 Et2O:hexanes 0.5% AcOH); mp=186-189 °C. H NMR
(CDCl3): δ 1.85 (d, 3H), 7.33 (m, 1H), 7.91 (m, 2H), 8.14
(m, 2H).
(E)-40-(1,4-dimethoxy-2-methylnaphthalen-2-yl)-3-ethylidene-
tetrahydrofuran-2-one (20a). Lactone 20a was prepared follow-
ing the procedure for 8 using aldehyde 14b (0.486 g, 2.11 mmol)
to provide 0.629 g (100%) of product as a white solid following
flash chromatography (1:4 EtOAc:hexanes) and recrystalliza-
tion from Et2O/hexanes. Rf=0.32 (1:3 EtOAc:hexanes); E:Z=
100:0; mp=105-107 °C. 1H NMR (CDCl3): δ 2.37 (s, 3H), 2.92
(dt, 2H), 3.70 (s, 3H), 3.87 (s, 3H), 4.40 (t, 2H), 7.52 (m, 2H), 7.69
(t, 1H), 8.09 (m, 2H).
(E)-40-(3-Chloro-1,4-dimethoxynaphthalen-2-yl)-3-ethylidene-
tetrahydrofuran-2-one (20b). Lactone 20b was prepared follow-
ing the procedure for 8 using aldehyde 14f (0.484 g, 1.94 mmol)
to provide 0.311 g (51%) of the product as a colorless oil
following flash chromatography (1:9 EtOAc:hexanes). Rf =
0.13 (3:37 EtOAc:hexanes, developed 4ꢀ); E:Z = 9:1. 1H
NMR (CDCl3): δ 2.94 (dt, 2H), 3.73 (s, 3H), 3.99 (s, 3H), 4.41
(t, 2H), 7.58 (m, 2H), 7.77 (t, 1H, 3 Hz), 8.12 (m, 2H).
(E)-40-(3-Bromo-1,4-dimethoxynaphthalen-2-yl)-3-ethylidene-
tetrahydrofuran-2-one (20c). Lactone 20c was prepared follow-
ing the procedure for 8 using aldehyde 14g (0.588 g, 1.99 mmol)
to provide 0.307 g (42%) of the product as a colorless oil
following flash chromatography (3:17 EtOAc:hexanes). Rf =
0.09 (1:9 EtOAc:hexanes); E:Z=3:1. 1H NMR (CDCl3): δ 2.92
(dt, 2H, J=3, 7.2 Hz), 3.73 (s, 3H), 3.98 (s, 3H), 4.41 (t, 2H, J=
7.2 Hz), 7.59 (m, 2H), 7.74 (t, 1H), 8.12 (m, 2H).
Compound 21b was prepared from (E)-3-(3-chloro-1,4-di-
methoxynaphthalen-2-yl)-2-methoxethylpropenoic acid (0.067 g,
0.19 mmol) as described for 16e to provide 0.032 g (53%) of the
product as a yellow solid following flash chromatography (2:3
acetone:hexanes 0.5% AcOH) and recrystallization from Et2O/
hexanes. Rf=0.48 (2:3 acetone:hexanes 0.5% AcOH); mp=160 °C
d. 1H NMR (CDCl3): δ 2.52 (t, 2H), 3.17 (s, 3H), 3.45 (t, 2H), 7.47
(s, 1H), 7.78 (m, 2H), 8.12 (m, 1H), 8.18 (m, 1H).
(E)-3-(3-Bromo-1,4-naphthoquinon-2-yl)-2-methoxyethyl-
propenoic Acid (21c). The methyl ester was prepared from
lactone 20c (0.257 g, 0.708 mmol) following the procedure
for 9 to provide 0.290 g (100%) of the product as tan crystals
following flash chromatography (1:3 EtOAc:hexanes) and
recrystallization from Et2O/hexanes. Rf=0.42 (1:3 EtOAc:
hexanes); mp=66-67 °C. 1H NMR (CDCl3): δ 2.57 (t, 2H),
3.12 (s, 3H), 3.39 (t, 2H), 3.77 (s, 3H), 3.86 (s, 3H), 3.97 (s,
3H), 7.42 (m, 2H), 7.65 (s, 1H), 8.10 (m, 2H).
The methyl ester (0.179 g, 0.437 mmol) was hydrolyzed follow-
ing the procedure for 9 toprovide (E)-3-(3-bromo-1,4-dimethoxy-
naphthalen-2-yl)-2-methoxyethylpropenoic acid (0.060 g, 35%)
of the product as a tan solid following flash chromatography (1:3
EtOAc:hexanes 0.5% AcOH) and recrystallization from Et2O/
hexanes. Rf = 0.12 (1:3 EtOAc:hexanes 0.5% AcOH); mp =
1
158-160 °C. H NMR (CDCl3): δ 2.57 (t, 3H), 3.27 (s, 3H),
3.45 (t, 3H), 3.79 (s, 3H), 3.98 (s, 3H), 7.57 (m, 2H), 7.72 (s, 1H),
8.12 (m, 2H).
(E)-3-(3-Methyl-1,4-naphthoquinon-2-yl)-2-methoxyethylpro-
penoic acid (21a). The methyl ester was prepared from lactone
20a (0.243 g, 0.815 mmol) following the procedure for 9 to
provide 0.249 g (59%) as tan crystals following flash chroma-
tography (1:3 EtOAc:hexanes) and recrystallization from Et2O/
Compound 21c was prepared from (E)-3-(3-bromo-1,4-di-
methoxynaphthalen-2-yl)-2-methoxyethylpropenoic acid (0.060 g,
0.15 mmol) as described for 16e to provide 0.029 g (53%) of the
product as a yellow solid following flash chromatography (2:3
acetone:hexanes 0.5% AcOH) and recrystallization from Et2O/
hexanes. Rf = 0.45 (2:3 acetone:hexanes 0.5% AcOH); mp =
168-172 °C. 1H NMR (CDCl3): δ 2.52 (t, 2H), 3.18 (s, 3H), 3.46
(t, 2H), 7.41 (s, 1H), 7.78 (m, 2H), 8.12 (m, 1H), 8.19 (m, 1H).
(E)-N-(2-Hydroxyethyl)-3-(3-chloro-1,4-dioxonaphthoquinon-
2-yl)-2-methylpropenamide (23). Compound 22 (0.117 g, 0.381
mmol) was converted to the hydroxyethylamide prepared as
described for 11 to provide 0.110 g (83%) of the intermediate as a
white solid following flash chromatography (2:3 EtOAc:
hexanes). Rf =0.38 (1:1 acetone:hexanes); mp=165-167 °C.
1H NMR (CDCl3): δ 1.86 (d, 3H), 2.68 (bs, 1H, OH), 3.60 (q,
2H), 3.75 (s, 3H), 3.84 (t, 2H), 3.98 (s, 3H), 6.42 (bs, 1H, NH),
7.43 (s, 1H), 7.55 (m, 2H), 8.10 (m, 2H).
1
hexanes. Rf =0.43 (1:3 EtOAc:hexanes); mp=64-65 °C. H
NMR (CDCl3): δ 2.28 (s, 3H), 2.55 (t, 2H), 3.07 (s, 3H), 3.37 (t,
2H), 3.75 (s, 3H), 3.85 (s, 3H), 3.86 (s, 3H), 7.48 (m, 2H), 7.71 (s,
1H), 8.07 (m, 2H).
The resulting methyl ester (0.104 g, 0.302 mmol) was hydro-
lyzed following the procedure for 9 to provide (E)-3-(1,4-
dimethoxy-3-methylnaphthalen-2-yl)-2-methoxyethylprope-
noic acid (0.076 g, 77%) as a tan solid following flash
chromatography (1:3 EtOAc:hexanes 0.5% AcOH) and re-
crystallization from Et2O/hexanes. Rf = 0.10 (1:3 EtOAc:
hexanes 0.5% AcOH); mp = 145-148 °C. 1H NMR
(CDCl3): δ 2.30 (s, 3H), 2.57 (t, 2H), 3.17 (s, 3H), 3.44 (t,
2H), 3.77 (s, 3H), 3.88 (s, 3H), 7.50 (m, 2H), 7.86 (s, 1H), 8.08
(dt, 2H).
The dimethoxynaphthalene intermediate (0.050 g, 0.14
mmol) was then oxidized following the procedure for 16e to
provide 23 (0.027 g, 60%) as a yellow solid following flash
Compound 21a was prepared from (E)-3-(1,4-dimethoxy-3-
methylnaphthalen-2-yl)-2-methoxyethylpropenoic acid (0.076 g,

1210 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Nyland et al.
chromatography (2:3 EtOAc:hexanes) and recrystallized from
acetone/hexane. Rf=0.25 (1:1 acetone:hexanes); mp=120 °C d.
1H NMR (CDCl3): δ 1.87 (s, 3H), 2.51 (bs, 1H, OH), 3.56 (q,
2H), 3.81 (m, 2H), 6.46 (bs, 1H, NH), 7.03 (s, 1H), 7.77 (m, 2H),
8.10 (m, 1H), 8.17 (m, 1H).
(5) Izumi, T.; Brown, D. B.; Naidu, C. V.; Bhakat, K. K.; Macinnes,
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R. L.; Borch, R. F.; Qiao, X.; Georgiadis, M. M.; Kelley, M. R. Role of
the multifunctional DNA repair and redox signaling protein Ape1/
Ref-1 in cancer and endothelial cells: small-molecule inhibition of the
redox function of Ape1. Antioxid. Redox Signaling 2008,10, 1853–1867.
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Ethyl 3-(3-Bromo-1,4-dimethoxynaphthalen-2-yl)-propionate
(25). Following a modified procedure of Clegg et al.,26 lithium
hexamethyldisilazide (LiHMDS, 1 M in THF, 1.5 mL, 1.5 mmol)
was added to a flame-dried round-bottom flask containing THF
(20 mL) at -78 °C. Ethyl acetate (0.15 mL, 1.5 mmol) was then
added slowly at -78 °C and stirred at -78 °C for 30 min.
2-Bromo-3-bromomethyl-1,4-dimethoxynaphthalene (0.415 g,
1.15 mmol) was then dissolved in THF (10 mL) and added at
-78 °C, at which point the reaction was allowed to warm to room
temperature and then stirred for 4 h. The reaction was monitored
by TLC (CH2Cl2) for disappearance of the less polar starting
material. Additional LiHMDS (2.0 mL, 2.0 mmol) and EtOAc
(0.70 mL, 0.70 mmol) were added and the reaction was monitored
for the disappearance of starting material. When no starting
material was present (ca. 10 h), the reaction was poured into
water, extracted with ethyl acetate, washed with brine, dried over
MgSO4, filtered, and condensed. The product was the purified by
column chromatography (CH2Cl2 to 1:19 EtOAc:CH2Cl2) to
provide 25 (0.361 g, 86%) as a yellow oil. Rf =0.43 (CH2Cl2).
1H NMR (CDCl3): δ 1.26 (t, 3H), 2.62 (m, 2H), 3.29 (m, 2H), 3.91
(s, 3H), 3.95 (s, 3H), 4.17 (q, 2H), 7.51 (m, 2H), 8.02 (m, 1H),
8.07 (m, 1H).
3-(3-Bromo-1,4-naphthoquinon-2-yl)-propionic Acid (26).
Compound 25 (0.361 g, 0.983 mmol) was oxidized following
the procedure for 16e to provide ethyl (3-bromo-1,4-naphtho-
quinon-2-yl)-propionate (0.135 g, 41%) as a yellow solid follow-
ing recrystallization from acetone/hexanes. Rf = 0.44 (1:4
1
acetone:hexanes); mp=78-79 °C. H NMR (CDCl3): δ 1.23
(t, 3H), 2.59 (t, 2H), 3.16 (t, 2H, J=7.8 Hz), 4.12 (q, 2H), 7.74 (m,
2H), 8.13 (m, 2H).
The resulting ethyl ester (0.108, 0.320 mmol) was dissolved in
THF (5 mL) at room temperature and then 2 M HCl (4 mL) was
added at room temperature. The homogeneous solution was
then stirred vigorously and heated under reflux for 24 h, or until
no starting material was observed by TLC (1:4 EtOAc:hexanes).
The reaction was then cooled and diluted with EtOAc (30 mL)
and then washed 2 times with saturated brine, dried over
MgSO4, filtered, and condensed. If any starting ester was still
present, the resulting yellow oil was first purified via flash
column chromatography (1:4 acetone:hexanes 0 to 0.5%
AcOH) followed by recrystallization from Et2O/hexanes to
provide 26 (0.077 g, 78%) as fine yellow needles. Rf = 0.14
(1:3 EtOAc:hexanes 0.5% AcOH); mp = 156-157 °C.
1H NMR (CDCl3): δ 2.65 (t, 2H), 3.15 (m, 2H), 7.74 (m, 2H),
8.13 (m, 2H).
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phosphorodiamidate prodrugs targeted to DT-diaphorase. J. Med.
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for a therapeutic drug for neurodegenerative diseases. Tetrahedron:
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Acknowledgment. Financial support for this work was
provided by the National Cancer Institute (T32 CA09634 to
R.L.N. and CA94025, CA106298, and CA121168 to M.R.
K.), thePurdueUniversity-Indiana UniversityCollaborative
Biomedical Research Program (to R.F.B. and M.R.K.), and
the Riley Children’s Foundation (to M.R.K.).
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