Bioactivities of simplified adociaquinone B and naphthoquinone derivatives against Cdc25B, MKP-1, and MKP-3 phosphatases

Article abstract of DOI:10.1016/j.bmc.2008.10.090

Some simplified adociaquinone B analogs and a series of 1,4-naphthoquinone derivatives were synthesized and tested against the three enzymes Cdc25B, MKP-1, and MKP-3. Cdc25B and MKP-1 in particular are enzymes overexpressed in human cancer cells, and they

Full text of DOI:10.1016/j.bmc.2008.10.090

Bioorganic & Medicinal Chemistry 17 (2009) 2276–2281
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bioactivities of simplified adociaquinone B and naphthoquinone derivatives
against Cdc25B, MKP-1, and MKP-3 phosphatases
Shugeng Cao a, , Brian T. Murphy a, , Caleb Foster b, John S. Lazo b, David G. I. Kingston a,
*
a Department of Chemistry, Virginia Polytechnic Institute and State University, 3111 Hahn Hall, M/C 0212, Blacksburg, VA 24061, USA
b Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Some simplified adociaquinone B analogs and a series of 1,4-naphthoquinone derivatives were synthe-
sized and tested against the three enzymes Cdc25B, MKP-1, and MKP-3. Cdc25B and MKP-1 in particular
are enzymes overexpressed in human cancer cells, and they represent potential molecular targets for
novel cancer chemotherapeutic treatments. A number of analogs exhibited significant inhibitory activity
against these enzymes, and the bioassay data in addition to structure–activity relationships of these com-
pounds will be discussed.
Received 15 May 2008
Revised 3 October 2008
Accepted 31 October 2008
Available online 8 November 2008
Keywords:
Naphthoquinone
Cdc25B
Ó 2008 Elsevier Ltd. All rights reserved.
MKP-1
Adociaquinone B
Structure–activity relationships
1. Introduction
At a chemical level, promotion of the transition between G2–M
by CDK1-cyclin A and B is catalyzed via dephosphorylation by a
The cell cycle is a highly regulated process that controls eukary-
otic growth and proliferation. Transition through the four phases of
the cell cycle (G1, S, G2, M) is regulated by cyclin-dependant kinase
(CDK)-cyclin complexes, which are activated by a subclass of dual-
specificity protein tyrosine phosphatases, namely Cdc25A, B, and
C.1 Studies have linked the oncogenesis of numerous human tu-
mors with the overexpression of Cdc25A and B, thus suggesting
that the inhibition of these dual-specificity phosphatases may be
a viable and attractive method of cancer treatment.1–4
Cdc25B was initially shown to primarily activate CDK1-cyclin A
and CDK1-cyclin B at the G2–M transition of the cell cycle via
dephosphorylation of Thr14 and Tyr15 residues,4–8 although more
recent studies have revealed considerable functional overlap
among the three Cdc25s in the G1–S and G2–M transitions.9–11
For example, Cdc25B can activate Cdk2-cyclin A.12 These findings
and others demonstrate the Cdc25 enzymes and their correspond-
ing CDK-cyclin complexes have multiple cellular roles.13 Expres-
sion of Cdc25B is uniquely increased after DNA-damage induced
by carcinogens, which may reflect a casual role in the genetic insta-
bility associated with cancer.3,14
specific cysteine thiolate anion found in a shallow pocket of
Cdc25B.15–17 Binding to or oxidation of this thiolate anion prevents
activation of the CDK1-cyclin complex, hence triggering cell cycle
arrest.18,19 Further cellular effects of these enzymes can be found
in a recent study by Cazales et al. They reported that inhibiting
Cdc25 phosphatase activity alters microtubule dynamics and im-
pairs mitotic spindle assembly, resulting in disruption of the mito-
tic process.20 Furthermore, they observed with human HT29 colon
cancer cells an enhancement of the antiproliferative activity of the
microtubule-targeting paclitaxel when it was combined with a
Cdc25B inhibitor. This emphasizes the complex and critical role
of the Cdc25 family in cell cycle regulation and supports further
studies on the mechanism of action of small molecule inhibitors
of these protein tyrosine phosphatases.
A majority of the known small molecule Cdc25B inhibitors are
quinones or quinone-type compounds. Naphthoquinone derivative
NSC 672121 (Fig. 1, 2 lM inhibition of Cdc25B) has received con-
O
O
O O
O
S
OH
O
S
N
H
O
O
* Corresponding author. Tel.: +1 540 231 6570; fax: +1 540 231 7702.
E-mail address: dkingston@vt.edu (D.G.I. Kingston).
URL: http://www.kingston.chem.vt.edu/ (D.G.I. Kingston).
These authors contributed equally to this work.
NSC 672121
Adociaquinone B
Figure 1. Structures of potent Cdc25B inhibitors.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.090

S. Cao et al. / Bioorg. Med. Chem. 17 (2009) 2276–2281
2277
siderable attention after emerging from an activity-based screen-
ing of a National Cancer Institute (NCI) Chemical Repository of
10,070 compounds.21 Since then several studies have attempted
to improve this scaffold through analog synthesis.22–27 A very re-
cent article presents the synthesis and biological evaluation of sev-
eral new quinolinedione and naphthoquinone derivatives,
containing carboxylic or malonic acids groups introduced to mimic
the role of the phosphate moieties of cyclin-dependent kinase
complexes. The most efficient compounds showed inhibitory activ-
dione (1) by conversion to diethyl cyclohexane-1,2-dicarboxylate
(2), reduction to 1,2-cyclohexanedimethanol (3), tosylation to the
bis-tosylate 4, and treatment with base to give 1,2-dimethylenecy-
clohexane (5) as previously described.37,38 1,2-dimethylenecyclo-
hexane (5) was then added to benzoquinone in a Diels–Alder
reaction, followed by treatment with potassium carbonate to give
the hydroquinol 6.39 Air oxidation of 6 gave naphthoquinone 7,
as well as a small amount of epoxide 8 as an oxidation by-product.
Compound 7 was coupled with hypotaurine to yield 9.40
ity against Cdc25B with IC50 values in the 10
l
M range, and were
Compounds 10–15 [1,4-benzoquinone (10), 1,4-naphthoqui-
none (11), 9,10-anthraquinone (12), 2-methyl-1,4-naphthoqui-
none (13), 5-hydroxy-1,4-naphthoquinone (14), and 5,8-
dihydroxy-1,4-naphthoquinone (15)] were purchased from com-
mercial sources. Compounds 16–20 were prepared via coupling
reactions of 1,4-naphthoquinone derivatives with hypotaurine,
ethanolamine, and ethanol (Scheme 2). The synthesis of 18 from
2-methylnaphthoquinone involved the loss of the C2 methyl group
by an interesting previously described mechanism involving conju-
gate addition of ethanolamime to the C2 methyl group followed by
the loss of methyleneimino-ethanol in a reverse-Mannich type
reaction.41,42
cytotoxic against HeLa cells.28 Furthermore, we have previously re-
ported a number of isolates from the Indonesian sponge Xestospon-
gia sp., and among them identified what is believed to be the most
potent known inhibitor of Cdc25B, adociaquinone B (80 nM).29
Herein, we report the design and synthesis of simplified adociaqui-
none B analogs in addition to several naphthoquinone derivatives,
and their subsequent ability to inhibit Cdc25B dual-specificity
phosphatase. Our basic approach of evaluating why adociaquinone
B might exert such potent Cdc25B activity was to systematically
modify both the west and the east hemispheres of the molecule.
To assess the necessity of the fused tricyclic benzofuranone moiety,
chemical synthesis led to analog 9 containing a simplified west
hemisphere, while analogs 10–20 were either synthesized or pur-
chased to evaluate the importance of a basic naphthoquinone moi-
ety in addition to an adjacent heterocyclic ring system.
2.2. Bioactivities
Quinones 7–20, together with the lead compound adociaqui-
none B, were evaluated as inhibitors of Cdc25B, MKP-1, and
MKP-3. The bioassay for inhibition of Cdc25B was conducted with
an epitope-tagged (histidine6) catalytic domain of human recom-
binant Cdc25B, which contained amino acids 275–539 of the full-
length protein and has been previously described.18 The results
are shown in Table 1. The antiproliferative activities of the com-
pounds against the A2780 human ovarian cancer cell line were also
determined, to establish their ability to penetrate cells and demon-
strate in vivo activity.
In addition to Cdc25, several other protein tyrosine phospha-
tases have been identified as potential cancer chemotherapeutic
targets. As examples, the mitogen-activated protein kinase phos-
phatases-130,31 and -332 (MKP-1 and MKP-3), which are distal
effectors for many extracellular growth factors, stress detectors,
and drug sensors, have been suggested as possible targets. MKP-
1 expression is elevated in prostate, breast, gastric, and renal can-
cer33,34 and is correlated with decreased progression-free sur-
vival.35 Moreover, reduction in MKP-1 expression by antisense
restricts tumorigenicity.32 The activities of the target compounds
against these phosphatases were determined as a measure of their
selectivity for Cdc25B.
2.3. Discussion
Several conclusions can be drawn from these results. First, with
the exception of the amino derivatives compounds 18 and 19, the
current data clearly support the notion that naphthoquinone-type
2. Results and discussion
2.1. Synthesis
The compounds evaluated were prepared by the literature
methods. The synthesis of compound 9 has not previously been de-
scribed, although it has been claimed in a patent,36 and so it is de-
scribed briefly here (Scheme 1). Conjugated diene 5 was obtained
from the commercially available hexahydroisobenzofuran-1,3-
R
O
O
O
R
O
S
a
N
H
O
O
16 R = H
11 R = H
17 R = OH
14 R = OH
OH
O
O
H
c
a
N
b
b
c
R
O
OH
OH
5
6
O
13
18 R = H
O
O
O
O
O
19 R = CH3
O
S
e
O
O
X
N
H
1,2-napthoquinone
7 X = bond
8 X = O
9
d
OEt
20
Scheme 1. Reagents and conditions: (a) benzoquinone, 50% DCM/DMSO, N2, rt,
24 h; (b) K2CO3, acetone, rt, overnight; (c) air, acetone, rt; (d) 8 was a by-product of
the reaction of 7 with hydrogen peroxide produced during the auto-oxidation of 6
to 7; (e) 7 + hypotaurine, EtOH/MeCN/H2O (1:1:1), 40 °C, 2 h.
Scheme 2. Reagents and conditions: (a) hypotaurine in H2O:MeCN:EtOH (1:1:1),
40 °C, 4 h; (b) ethanolamine in MeCN, 45 °C, 2 h; (c) 50% MeCN/EtOH, DMAP, 40 °C,
5 h.

2278
S. Cao et al. / Bioorg. Med. Chem. 17 (2009) 2276–2281
Table 1
Since the compounds are structurally similar it is unlikely that this
Biological activities of compounds 7–20 (lM)
difference in activity is solely due to cell permeability factors, sug-
gesting that these compounds may have other mechanisms of ac-
tion. In context of the other protein kinase phosphatase assays,
16 and 17 are moderately selective for Cdc25B as opposed to
MKP-1 and MKP-3, indicating that these compounds have attrac-
tive properties for further development as selective inhibitors of
Cdc25B.
Further structure activity analysis showed that simplification of
16 and 17 to basic naphthoquinone derivatives (compounds 11,
13, 14, 15; IC50 = 2.76, 3.38, 1.98, 1.00 lM, respectively) resulted
in a 2- to 3-fold decrease in activity (with the exception of 15), sug-
gesting that the heterocyclic ring of 16 and 17 does play a role in
either binding within the active pocket, or some other aspect of
the mechanism of action. This hypothesis is strengthened by the
observed 10-fold loss in activity when the heterocyclic ring is
Compound
Cdc25Ba
MKP-1a
MKP-3a
A2780b
Adociaquinone B
7
8
9
0.08/0.11
9.53
22.3
2.3
1.10
21.8
>50
38.7
> 50
8.45
>50
24.0
13.0
9.37
17.8
33.8
>50
1.53
>50
49.9
>50
>50
19.8
>50
20.5
12.4
6.90
42.6
>50
>50
>50
1.35
26 0.06
0.75 0.03
1.4 0.07
6.9 0.1
10
11
12
13
14
15
16
17
18
19
20
>50
7.8 0.4
2.76
9.51
3.38
1.98
1.00
0.94
0.88
>50
2.3 0.03
0.58 0.45
2.6 0.06
1.4 0.05
0.08 0.001
4.3 2.3
2.1 0.02
64 1.2
*
>50
0.27
>50
0.82
0.37 0.01
a
substituted by a six-membered aromatic ring (12, IC50 = 9.51 lM).
IC50 values (lM) for activity against the catalytic domain of Cdc25B. The data
for adociaquinone B are for activity against the catalytic domain (0.11) and full-
Interestingly, in the cases of 18 and 19 a complete loss of activity is
observed, presumably due to either the substitution of the sulfox-
ide moiety with that of an amine, or the absence of cyclization to
the naphthoquinone, or both. The complete loss of enzyme activity
is also observed with compound 10, thus suggesting that the basic
naphthoquinone skeleton is a primary requirement for bioactivity.
The aforementioned data contribute to the quest of determining
exactly how such naphthoquinone derivatives exert their specific
Cdc25B enzymatic bioactivity, and may highlight the presence of
mechanistic complexities of potent Cdc25B inhibitors such as ado-
ciaquinone B.
length Cdc25B (0.08).
b
Antiproliferative activity (IC50
lM); growth of the A2780 human ovarian cell
line according to the procedure described,27,43,44 with paclitaxel (IC50 23.4 nM) as
the positive control. All compounds were tested in triplicate in this assay.
*
The fluorescence reading was not indicative of the true biological activity of 19.
The fluorescence reading indicated that 19 was not active, but microscopic analysis
clearly showed that significant inhibition of the growth of A2780 cells occurred.
molecules have the potential to be effective therapeutic agents,
though their exact mechanism of Cdc25 inhibition is still a topic
of discussion. A few computational studies have attempted to uti-
lize modeling to predict various binding interactions of the Cdc25
dual-specificity phosphatases. The docking orientation of Cdc25B
with its Cdk2-cyclin A protein substrate has previously been re-
ported, and therein the possibility was recognized of targeting sev-
eral potential small molecule binding pockets to achieve disruption
of protein substrate recognition.12 Additionally, in an effort to pos-
tulate a mechanism of enzyme inhibition, attempts were made to
dock known small molecule inhibitors of Cdc25B within its shallow
active site.45 Using a naphthoquinone derivative, one docking pro-
gram showed hydrogen bonding between the quinone carbonyl
oxygens with Arg482 and Arg544 of the shallow pocket. As previ-
ously suggested,20 this implies that the quinone functionality is
necessary for specific enzyme–ligand binding that propagates the
observed activity. An alternate docking program bound the naph-
thoquinone inhibitors in such a way that the quinone moiety
was in close proximity to the Cys473 thiol residue.33 Indeed, the
structurally similar para-quinolinediones DA3003-1 and JUN1111
were shown to engage in redox cycling, thus irreversibly oxidizing
the Cys473 thiol to its sulfonic product via production of reactive
oxygen species (ROS).46
In regard to our study the most active compound was the ortho-
quinone 20. However, its activity is relatively non-selective, sug-
gesting that it operates by a non-selective mechanism and is thus
unlikely to have development potential.
The simplification of adociaquinone B by the deletion of two
rings on the west hemisphere gave compound 9, with activity
against Cdc25B of 2.3
compound (0.08 or 0.11
diminished its ability to bind effectively with residues in the active
pocket of Cdc25B, thus reducing its potency. Quinones 16 and 17,
lacking three rings on the west hemisphere of adociaquinone B,
nevertheless were more potent than 9 toward Cdc25B, with IC50
Experimental evidence from our previous study27 allowed us to
hypothesize that the mechanism of action of adociaquinone B is
oxidation of the catalytic cysteine of Cdc25B. If this is correct, the
current bioassay data suggest that the fused tricyclic benzofura-
none moiety may play a pivotal role in effectively positioning the
quinone for Cys473 thiolate oxidation. Comparison of 9 with 16
and 17 suggests that the absence of three rings on the west hemi-
sphere of adociaquinone B may grant the resulting small molecule
more versatility by allowing increased access to the binding site,
though further studies are needed to prove such a hypothesis.
3. Experimental
3.1. General experimental methods
All of the reagents and solvents received from commercial
sources were used as purchased, at greater than 98% purity. 1H
and 13C NMR spectra were obtained on Varian Unity 400 MHz,
Inova 400 MHz, and JEOL Eclipse 500 MHz spectrometers. High
resolution FAB and low resolution EI mass spectra were obtained
on a JEOL HX-110 instrument, and a Finnigan LTQ LC/MS, respec-
tively. Reaction mixtures were worked up by the standard proce-
dure of quenching the reaction, extracting the resulting aqueous
mixture with EtOAc or Et2O or dichloromethane, washing the or-
ganic solution with water and brine, drying over Na2SO4, filtering,
and concentrating to give crude product. Final compounds were
purified to homogeneity by preparative TLC, and their purity
was assessed by 1H NMR spectroscopy. All the reported com-
pounds had purities of 95% or better as estimated from analysis
of their NMR spectra.
l
M, at least 20-fold less than that of the lead
M). The deletion of these rings may have
l
3.2. Synthesis
values of 0.94 and 0.88 lM, respectively, so the simple idea that
deletion of the western rings reduces activity is clearly inadequate
to explain the observed effects. It should also be noted that the
antiproliferative activities of 16 and 17 differ by a factor of over
20, even though their inhibitory effects on Cdc25B are very similar.
3.2.1. Synthesis of compound 9
A solution of compound 5 (12 mg, 111.1
dichloromethane (1 mL) was added to the stirred solution of
benzoquinone (72 mg, 666.7 mol) in dry DMSO (2 mL) under a
l
mol)35,36 in dry
l

S. Cao et al. / Bioorg. Med. Chem. 17 (2009) 2276–2281
2279
nitrogen atmosphere.37 The mixture was stirred at rt for 24 h,
which was then dried under nitrogen stream. Complete removal
of the DMSO was achieved by washing the film with water and
drying under a nitrogen stream.47 The dried residue was dissolved
in dry acetone (2 mL). A spatula tip of potassium carbonate was
added, and the mixture was stirred at rt overnight under a nitrogen
atmosphere.37 The reaction mixture was worked up by the stan-
dard procedure of quenching the reaction, extracting the resulting
aqueous mixture with EtOAc, washing the organic solution with
water and brine, drying over Na2SO4, filtering, and concentrating
to give crude product. The residue was purified by silica gel TLC
(EtOAc/hexanes 1:6) to give 6 (Rf 0.50, 21.6 mg, 90%, and 8 (Rf
0.30, 12.5 mg, 10%). Compound 7 (silica gel TLC, EtOAc/hexanes
1:6, Rf 0.40, 5 mg, 30%) was an automatically oxidized product of
6 (5,6,7,8,9,10-hexahydroanthracene-1,4-diol) in acetone under
data with those in the literature48: 1H NMR (DMSO-d6) d 12.83 (1H,
s), 9.50 (1H, br s), 7.64 (1H, t, J = 7.2 Hz), 7.55 (1H, dd, J = 7.6,
0.8 Hz), 7.34 (1H, dd, J = 7.6, 0.8 Hz), 3.87 (2H, m), 3.41 (2H, m);
13C NMR (DMSO-d6) d 181.5, 177.9, 160.8, 147.8, 135.0, 130.2,
125.8, 119.1, 113.7, 110.0, 48.2, 39.8; EIMS m/z 280.1 [M+H]+ (calcd
for C12H10O5NS, 280.02).
3.2.4. Synthesis of compound 18
Ethanolamine (80.5
lL, 1.34 mmol) was added to a stirred solu-
tion of 2-methyl-1,4-naphthoquinone (13) (230 mg, 134
lmol) in
5 mL acetonitrile, and the mixture was stirred at 45 °C for 2 h.
Unexpectedly, loss of the C-2 methyl resonance was observed in
the 1H NMR spectrum. The structure was thus elucidated by one
and two-dimensional NMR methods and subsequently confirmed
by X-ray crystallography. It is postulated that two molecules of
ethanolamine are involved in a reverse-Mannich type reaction,
ultimately resulting in loss of methyleneimino-ethanol at the C-2
position. Loss of a methyl group from quinones has been reported
some time ago, and mechanistic details were described.41,42 The
crude product was purified by silica gel TLC (EtOAc) to afford or-
ange crystals, compound 18 (Rf 0.44, 9.6 mg, 3.3%): 1H NMR
(DMSO-d6) d 7.98 (1H, dd, J = 8.0, 1.2 Hz), 7.93 (1H, dd, J = 7.6,
1.2 Hz), 7.82 (1H, dt, J = 8.0, 1.2 Hz), 7.72 (1H, dt, J = 7.6, 1.2 Hz),
7.34 (1H, br t, J = 5.6 Hz), 5.72 (1H, s), 4.87 (1H, t, J = 5.6 Hz),
3.58 (2H, q, J = 5.6 Hz), 3.23 (2H, q, J = 5.6 Hz); 13C NMR (DMSO-
d6) d 181.6, 181.4, 148.7, 134.9, 133.1, 132.2, 130.3, 125.9, 125.3,
99.6, 58.4, 44.6; FABMS m/z 199.4 [MÀH2O] (calcd for C12H9O2N,
199.21).
an air atmosphere. To a solution of 7 (4 mg, 18.9
lmol) in 1.5 mL
ethanol/acetonitrile/water (1:1:1) was added hypotaurine
(6.2 mg, 56.9 lmol), and the reaction mixture was stirred at
40 °C for 2 h.38 The crude product obtained was purified by silica
gel TLC (DCM/EtOAc 1:1) yielding compound 9 (Rf 0.25, 3 mg, 50%).
3.2.1.1. 5,6,7,8,9,10-Hexahydroanthracene-1,4-diol (6). 1H NMR
(C5D5N) d 7.02 (2H, s), 3.63 (4H, s), 2.01 (4H, m), 1.58 (4H, m); 13C
NMR (C5D5N) d 149.1, 126.0, 124.5, 112.9, 31.9, 30.2, 23.1; EIMS
m/z 217.1 [M+H]+ (calcd for C14H17O2, 217.1).
3.2.1.2. Compound 7. 1H NMR (CDCl3) d 7.73 (2H, s), 6.87 (2H, s),
2.86 (4H, m), 1.81 (4H, m); 13C NMR (CDCl3) d 185.3, 144.3, 138.6,
129.4, 127.2, 29.8, 22.5; EIMS m/z 213.2 [M+H]+ (calcd for
C14H13O2, 213.1).
3.2.5. Synthesis of compound 19
Ethanolamine (80.5
tion of 2-methyl-1,4-naphthoquinone (13) (230 mg, 134
l
L, 1.34 mmol) was added to a stirred solu-
3.2.1.3. Compound 8. 1H NMR (CDCl3) d 7.68 (4H, s), 3.95 (4H, s),
2.87 (8H, m), 1.82 (8H, m); 13C NMR (CDCl3) d 191.0, 145.5, 129.2,
128.0, 55.4, 29.9, 22.5; CIMS m/z 229.0 [M+H]+ (calcd for C14H13O3,
229.1).
lmol) in
5 mL acetonitrile, and the mixture was stirred at 45 °C for 2 h.
The crude product was purified by silica gel TLC (EtOAc) to afford
compound 19 as red, needle-like crystals (Rf 0.75, 62.0 mg, 20%).
Compound 19 was identified on the basis of one- and two-dimen-
sional NMR data, including 1H, 13C, g-COSY, g-HSQC, and g-HMBC
experiments in addition to MS analyses: 1H NMR (DMSO-d6) d
7.87 (2H, t, J = 6.8 Hz), 7.73 (1H, t, J = 7.6 Hz), 7.64 (1H, t,
J = 7.6 Hz), 6.47 (1H, br s), 4.92 (1H, br t, J = 5.2 Hz), 3.57 (4H, m),
2.06 (3H, s); 13C NMR (DMSO-d6) d 182.1, 181.7, 146.8, 134.3,
132.7, 132.0, 130.2, 125.6, 125.4, 110.8, 60.5, 46.8, 10.7; FABMS
m/z 232.1 [M+H]+ (calcd for C13H14O3N, 231.10).
3.2.1.4. Compound 9. 1H NMR (CDCl3) d 7.71 (1H, s), 7.68 (1H, s),
3.84 (1H, t, J = 6.3 Hz), 3.35 (1H, t, J = 6.3 Hz), 2.87 (4H, m), 1.76
(4H, m); 13C NMR (CDCl3) d 178.6, 174.8, 146.7, 146.0, 142.5,
130.0, 127.3, 127.1, 126.4, 111.0, 48.3, 40.0, 29.3, 28.7, 22.0, 22.0;
EIMS m/z 318.1 [M+H]+ (calcd for C16H16NO4S, 318.1).
3.2.2. Synthesis of compound 16
A solution of hypotaurine (60 mg, 550.0
was added to a stirred solution of 1,4-naphthoquinone (11) (60 mg,
379.0 mol) in 20 mL acetonitrile/ethanol (1:1), and the mixture
lmol) in water (10 mL)
3.2.6. Synthesis of compound 20
A solution of 1,2-naphthoquinone (30 mg, 190
acetonitrile/ethanol (1:1) and in the presence of
l
l
mol) in 20 mL
catalytic
was stirred at 40 °C for 4 h. The crude product solution was refrig-
erated to yield a yellow precipitate, and filtered to afford com-
pound 16 (62.6 mg, 63%). Compound 16 was identified on the
basis of one- and two-dimensional NMR data, including 1H, 13C,
g-COSY, g-HSQC, and g-HMBC experiments in addition to MS anal-
yses: 1H NMR (DMSO-d6) d 9.16 (1H, br s), 7.99 (2H, dd, J = 4.0,
1.6 Hz), 7.89 (1H, t, J = 7.6 Hz), 7.77 (1H, t, J = 7.6 Hz), 3.85 (2H, br
t, J = 5.5 Hz), 3.38 (2H, br t, J = 5.5 Hz); 13C NMR (DMSO-d6) d
178.7, 174.6, 146.8, 135.7, 132.9, 132.5, 130.0, 126.4, 125.8,
111.1, 48.26, 39.18; EIMS m/z 264.1 [M+H]+ (calcd for C12H10O4NS,
264.03).
a
amount of DMAP, was stirred at 40 °C for 5 h. The crude product
was purified by silica gel column chromatography (CHCl3/EtOAc
1:1) followed by an open reversed phase C18 column (MeOH/
H2O 3:2) to afford compound 20 as yellow needle-like crystals
(3.8 mg, 10%). Compound 20 was identified on the basis of one-
and two-dimensional NMR data, including 1H, 13C, g-COSY, g-
HSQC, and g-HMBC experiments in addition to MS analyses: 1H
NMR (CD3CN) d 8.00 (1H, d, J = 8.4 Hz), 7.91 (1H, d, J = 8.0 Hz),
7.75 (1H, t, J = 8.0 Hz), 7.63 (1H, t, J = 8.4 Hz), 5.94 (1H, s), 4.24
(2 H, q, J = 6.8 Hz), 1.49 (3H, t, J = 6.8 Hz); 13C NMR (CD3CN) d
180.6, 180.2, 168.5, 135.9, 133.2, 132.3, 131.6, 129.1, 125.4,
104.3, 66.9, 14.2; FABMS m/z 203.7 [M+H]+ (calcd for C12H10O3,
203.21).
3.2.3. Synthesis of compound 17
A solution of hypotaurine (9.3 mg, 85.0
was added to a stirred solution of 5-hydroxy-1,4-naphthoquinone
(14) (10 mg, 57.0 mol) in 20 mL acetonitrile/ethanol (1:1), and
lmol) in water (10 mL)
l
3.3. Antiproliferative assay
the mixture was stirred at 40 °C for 4 h. The crude product was
purified over a flash silica column (CHCl3/EtOAc 1:1) to afford yel-
low needle-like crystals, compound 17 (3.6 mg, 23%). This specific
regioisomer was identified upon comparison of its 1H and 13C NMR
Determination of antiproliferative activity was performed at
Virginia Polytechnic Institute and State University against the

2280
S. Cao et al. / Bioorg. Med. Chem. 17 (2009) 2276–2281
A2780 ovarian cancer cell line as previously described.43 The
A2780 cell line is a drug-sensitive human ovarian cancer cell line.44
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.10.090.
3.4. In vitro phosphatase assays
3.4.1. Protein preparation
References and notes
As previously noted, the bioassay for inhibitors of Cdc25B was
conducted with an epitope-tagged (histidine6) catalytic domain
of human recombinant Cdc25B, which contained amino acids
275–539 of the full-length protein and has been previously de-
scribed.18 The histidine6-tagged catalytic domain and full-length
Cdc25B were isolated and purified from Escherichia coli with Ni–
NTA resin as described previously.18 Human recombinant VHR
and PTP1B phosphatases were purchased from BIOMOL (Plymouth
Meeting, PA). The expression and purification of an epitope-tagged
(histidine6) recombinant MKP-1 has been described previously.49
Briefly, His-tagged MKP-1 was expressed in BL21(DE3) competent
bacterial cells transformed with the pET21a-MKP-1 expression
vector and induced with IPTG. Biologically active MKP-1 was par-
tially purified from IPTG-induced bacterial cell lysates by Cobalt-
column chromatography eluted with imidazole, frozen, aliquoted
and stored at À80 °C. The relative purity of the MKP-1 enzyme
preparation was visualized by separating the protein components
by SDS–PAGE and staining with Coomassie blue. The identity of
the MKP-1 enzyme was confirmed by the presence of the antici-
pated 42 kDa band together with immuno-reactivity with antibod-
ies against MKP-1 (Santa-Cruz, M-18, Catalog No. SC-1102) and the
His-Tag epitope (Cell Signaling, Catalog No. 2365) on Western blots
(data not shown). The expression of recombinant MKP-3 protein
used a bacterial expression plasmid. A Rattus norvegicus dual-spec-
ificity phosphatase6 (MKP-3)50 and pET21a-MKP-3 plasmid51 gen-
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centrations to determine the concentration required to inhibit
enzyme activity by 50% (IC50). To test for sensitivity to redox regu-
lation, in some studies with full-length Cdc25B, we adjusted the fi-
nal concentration of DTT in the enzyme buffer, which contained
30 mM Tris (pH 8.5), 75 mM NaCl, 1 mM EDTA, and 0.033% bovine
serum albumin, from the standard of 1 mM to a range from 0 to
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Acknowledgments
This work was supported in part by a National Cooperative Drug
Discovery Group grant awarded to the University of Virginia by the
National Cancer Institute (U19 CA50771), and this support is grate-
fully acknowledged. We thank Mr. B. Bebout for obtaining the mass
spectra and Mr. T. Glass for assistance obtaining the NMR spectra.

S. Cao et al. / Bioorg. Med. Chem. 17 (2009) 2276–2281
2281
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