Synthesis and biological evaluation of 3-aminoisoquinolin-1(2H)-one based inhibitors of the dual-specificity phosphatase Cdc25B

Article abstract of DOI:10.1016/j.bmc.2015.01.043

The cell division cycle 25B dual specificity phosphatase (Cdc25B) regulates the normal progression of the mammalian cell cycle by dephosphorylating and activating cyclin-dependent kinase (Cdk) complexes, particularly in response to DNA damage. Elevated Cdc25B levels enable a bypass of normal cell cycle checkpoints, and the overexpression of Cdc25B has been linked to a variety of human cancers. Thus, Cdc25B is an attractive target for the development of anticancer therapeutics. Herein we describe the synthesis and biological evaluation of a series of non-quinoid inhibitors of Cdc25B containing the 3-aminoisoquinolin-1(2H)-one pharmacophore. In addition to several strategies that address specific substitution patterns on isoquinolines, we have applied a regioselective Pd-catalyzed cross-coupling methodology to synthesize a new lead structure, 6-(3-aminophenyl)-3-(phenylamino)isoquinolin-1(2H)-one (13), which proved to be a reversible, competitive Cdc25B inhibitor with a K_i of 1.9 μM. Compound 13 prevented human cancer cell growth and blocked Cdc25B-mediated mitotic checkpoint bypass. Molecular docking studies support binding near the catalytic site.

Full text of DOI:10.1016/j.bmc.2015.01.043

Bioorganic & Medicinal Chemistry 23 (2015) 2810–2818
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 3-aminoisoquinolin-1(2H)-one
based inhibitors of the dual-specificity phosphatase Cdc25B
Kara M. George Rosenker a, William D. Paquette a, Paul A. Johnston b, Elizabeth R. Sharlow c,
Andreas Vogt d, Ahmet Bakan d, , John S. Lazo c, , Peter Wipf a,b,e,
⇑
⇑
a Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA 15260, USA
b Department of Pharmaceutical Sciences, University of Pittsburgh, 3501 5th Avenue, Pittsburgh, PA 15260, USA
c Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA
d Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA
e Department of Bioengineering, 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:
The cell division cycle 25B dual specificity phosphatase (Cdc25B) regulates the normal progression of the
mammalian cell cycle by dephosphorylating and activating cyclin-dependent kinase (Cdk) complexes,
particularly in response to DNA damage. Elevated Cdc25B levels enable a bypass of normal cell cycle
checkpoints, and the overexpression of Cdc25B has been linked to a variety of human cancers. Thus,
Cdc25B is an attractive target for the development of anticancer therapeutics. Herein we describe the
synthesis and biological evaluation of a series of non-quinoid inhibitors of Cdc25B containing the
3-aminoisoquinolin-1(2H)-one pharmacophore. In addition to several strategies that address specific
substitution patterns on isoquinolines, we have applied a regioselective Pd-catalyzed cross-coupling
Received 1 November 2014
Revised 20 January 2015
Accepted 23 January 2015
Available online 31 January 2015
Keywords:
Cdc25B
3-Amino-1,2-dihydro-1-isoquinolinone
Palladium cross-coupling
Checkpoint bypass
Molecular docking
methodology to synthesize
1(2H)-one (13), which proved to be a reversible, competitive Cdc25B inhibitor with a Ki of 1.9
a
new lead structure, 6-(3-aminophenyl)-3-(phenylamino)isoquinolin-
M.
l
Compound 13 prevented human cancer cell growth and blocked Cdc25B-mediated mitotic checkpoint
bypass. Molecular docking studies support binding near the catalytic site.
Ó 2015 Published by Elsevier Ltd.
1. Introduction
encouraged the pursuit of specific small molecule inhibitors.3–5
Structurally, Cdc25B contains a shallow, solvent-exposed active
The cell division cycle 25B phosphatase (Cdc25B), a member of
a subclass of protein tyrosine phosphatases that dephosphorylates
tyrosine, serine, and threonine residues, fulfills a critical role in the
regulation of the cell cycle by activating cyclin-dependent kinase
(Cdk) complexes. The oncogenic phosphatase Cdc25B also serves
as a key component in cell cycle checkpoints that are activated
in response to DNA damage.1,2 Deregulation of these checkpoint
mechanisms can contribute to genetic instability and, ultimately,
tumorigenesis. The overexpression of Cdc25B phosphatase in many
human cancers1 further supports its clinical significance and has
site cleft with no distinct features for substrate recognition or
inhibitor binding, which presents a formidable challenge for
designing inhibitors and formulating informative structure-activ-
ity relationships.4,5 As a target for small molecules, the phospha-
tase is highly susceptible to detergent-like agents displacing
water molecules from a large cavity (the ‘swimming pool’) next
to the active site.4b Furthermore, redox-active and electrophilic
compounds are frequent hits in high-throughput screening cam-
paigns because of the presence and high reactivity of a cysteine
residue in the HCX5R motif in the active site loop,3 thus generally
frustrating screening efforts to identify high-quality hits resulting
in leads that are suitable for further medicinal chemistry refine-
ment. While some promising advancements have been made, a
clinically active candidate in this field has yet to emerge and,
accordingly, discerning new chemotypes remains highly desirable.
While non-quinoid compounds have attracted attention as
Cdc25 inhibitors,5 quinoid-based compounds represent the major-
ity of the currently known potent Cdc25 inhibitors and are likely to
Abbreviations: Cdc25B, cell division cycle 25B phosphatase; Cdk, cyclin-depen-
dent kinase; IC50, concentration that inhibits enzyme activity by 50% of the
maximum activity; OMFP, O-methyl fluorescein phosphate.
⇑
Corresponding authors. Fax: +1 (434) 982 0735 (J.S.L.), +1 (412) 624 0787
(P.W.).
E-mail addresses: lazo@virginia.edu (J.S. Lazo), pwipf@pitt.edu (P. Wipf).
Present address: Kabbage, Inc., 505 Sansome St, Suite 1900, San Francisco, CA
94111, USA.
http://dx.doi.org/10.1016/j.bmc.2015.01.043
0968-0896/Ó 2015 Published by Elsevier Ltd.

K. M. George Rosenker et al. / Bioorg. Med. Chem. 23 (2015) 2810–2818
2811
We evaluated the requirements of this scaffold for the inhibi-
tion of the in vitro phosphatase activity of recombinant human
Cdc25B. In general, we observed only modest gains in potency rel-
ative to the 3.3
l
M IC50 of compound 1. Substitution on the aniline
ring gave analogs with IC50 values ranging from 0.8
lM to 13.6 lM
(Table 1, entries 1–9). Specifically, the 4-methyl and 4-fluoroani-
lines were more potent inhibitors compared to ortho-substituted
anilines. Interestingly, the sterically most demanding para-substi-
tuted anilines 4e and 4h (Table 1, entries 5 and 8) were the least
potent analogs tested in this series. The aniline moiety could be
replaced by a benzyl amine, with the parent analog (4j) and the
methoxy-substituted derivative (4k) showing inhibitory activities
superior to those observed with the N-alkylated analogs (Table 1,
entries 12 and 13). Heterocyclic amines were also explored; both
the triazole (4n) and the pyridine (4o) showed inhibitory activities
that were comparable to those seen with the piperidine (4p) and
morpholine (4q) analogs. The linear aminohexanol-substituted
analog (4r) showed the most potent inhibitory activity
(IC50 = 640 nM), suggesting that this compound should be explored
further, but solubility considerations and the presence of the free
alcohol in the freely rotatable side chain, which can readily be
metabolized in vivo by oxidation or glucuronidation, discouraged
us from pursuing 4r at this time.
Figure 1. Chemical structure of the lead compound, 3-amino-1,2-dihydro-1-
isoquinolinone, identified by high throughput screening.
act through irreversible oxidation of the catalytic cysteine resi-
due.5–7 As part of an initial high-throughput screening campaign,
we identified the inhibitory properties of the 3-amino-1,2-dihy-
dro-1-isoquinolinone scaffold 1 (Fig. 1), which exhibited 50%
in vitro inhibition (IC50) of recombinant Cdc25B catalytic activity
at a concentration of 3.3 l
M.8 Previously, these isoquinolinones
have not been extensively characterized chemically or biologi-
cally.9 Therefore, we initiated a medicinal chemistry program to
investigate the chemical topography for analogs in this series and
established robust synthetic pathways toward the target mole-
cules. We subsequently used biological and computational meth-
ods to characterize the lead aminoisoquinolinone 13.
Given the conformational flexibility of the 3-amino substituent
in 1 and to further evaluate the structural requirements for Cdc25B
inhibition, we also synthesized a fused tetracyclic analog 7 to
restrict the available rotamers of the arene ring. The synthesis of
7 was accomplished in 71% yield by heating the homophthalic
anhydride 5 at reflux with o-phenylenediamine 6 in acetic acid
2. Results and discussion
2.1. Chemical synthesis and phosphatase inhibition
The synthesis of analogs of compound 1 began with the prepa-
ration of 2-cyanomethylbenzoic acid 3, which was readily available
from the nucleophilic opening of phthalide 2 with sodium cyanide
under solvent-less conditions at 185 °C (Table 1).10 Subsequent
treatment of the acid with aniline in chlorobenzene at reflux tem-
peratures afforded the target compound 1 in 58% yield.11 The rapid
construction of the isoquinolinone core allowed us to synthesize a
variety of compounds (4a–r) by modifying the amine used in the
condensation reaction, with yields ranging from 25% to 82%
(Table 1, entries 1–18). The low yields obtained in some cases were
mainly attributed to the insufficient solubility of the products in 2-
propanol and/or methanol, which were used to streamline the
purification process.
(Scheme 1).12 The Cdc25B inhibition observed with 7 (IC50 = 5.3
lM)
is consistent with a shallow active site, that is, relatively insensi-
tive to the steric orientation of the 3-amino substituent.
A unique characteristic of the isoquinolinone core is the nucle-
ophilic nature of the 4-position. This ‘enamine-like’ position can
react with suitable electrophiles,11,13 providing an additional area
for diversification. While we did not exploit this position exten-
sively, we synthesized the acylated analog 8 in 45% yield by the
treatment of isoquinolinone 4b with benzoyl chloride in dioxane
(Scheme 2). Substitution at this position, however, proved to be
detrimental to the inhibitory activity (IC50 >50 lM).
Table 1
Synthesis of 3-amino-1,2-dihydro-1-isoquinolinones 4a–r
R1 = amine
Yield (%)
IC50 (lM)
a
Entry
Compound
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
2-Chloroaniline
4-Methylaniline
2-Fluoroaniline
4-Fluoroaniline
para-Anisidine
2,5-Dimethoxyaniline
2-Aminophenol
Ethyl 4-aminobenzoate
2-Phenylaniline
55
69
48
81
45
59
35
62
45
60
70
76
82
74
53
60
75
25
4.19 0.49
0.77 0.17
1.85 0.07
0.83 0.11
13.63 2.79
5.25 0.62
1.21 0.20
10.75 0.75
2.57 0.32
0.85 0.17
0.77 0.11
1.55 0.32
3.80 0.68
4.48 1.37
3.31 0.55
2.31 0.39
3.37 0.30
0.64 0.16
9
10
11
12
13
14
15
16
17
18
Benzyl amine
4-Methoxylbenzyl amine
N-Methylbenzyl amine
2-Bromo-N-methylbenzyl amine
1,2,4-Triazole-3-amino
2-Aminopyridine
Piperidine
Morpholine
6-Amino-1-hexanol
a
Mean value for 50% inhibition of Cdc25B SD; n = 3.

2812
K. M. George Rosenker et al. / Bioorg. Med. Chem. 23 (2015) 2810–2818
zoic acid 10, which was then exposed to Suzuki cross-coupling
conditions. Unfortunately, this approach was not an effective
method for incorporating functionalization due to the difficulties
in monitoring the reaction progress and purifying the crude prod-
uct mixture. We also explored the use of 11 and 12 as coupling
partners, and although the Suzuki reaction was successful in the
case of 11 to provide 13, this strategy was not generally employed
due to limited scope and the potential contamination of the prod-
ucts with trace amounts of palladium species. Instead, a modified
protocol was implemented to introduce various substituents at
the 5-position prior to lactone opening (Scheme 4). Suzuki cou-
pling of 5-bromoisobenzofuran-1(3H)-one 9 with commercially
available electron-rich and electron-poor boronic acids proved to
be a more successful strategy and afforded the desired substituted
phthalides 14a–d in high yields (Scheme 4). Subsequent exposure
to NaCN in DMSO resulted in the desired benzoic acids 15a–d. Iso-
lation of the crude benzoic acids was accomplished by diluting the
reaction mixture with water and precipitating the product by acid-
ification with concentrated HCl. Analysis of the crude products by
1H NMR clearly indicated the desired compounds were the major
constituents, and no further purification was performed. The
resulting benzoic acid derivatives were then exposed to aniline,
and, in one case, o-anisidine, in chlorobenzene at reflux to afford
isoquinolinones 16a–d.
Scheme 1.
Scheme 2.
With the ability to construct compounds by varying the 3-
amino side chain, our next focus was on the functionalization of
The biological activity of analogs 16a–d is summarized together
with 13 and 11 in Table 2. Interestingly, this core structure toler-
ated considerable arene modifications with some slight improve-
ment in the IC50 being seen for the halogenated analog 11
(Table 2, entry 6). Collectively, however, the structure–activity
the arene ring using 5-bromoisobenzofuran-1(3H)-one
9
(Scheme 3). Opening of the lactone of 9 following the aforemen-
tioned solvent-less protocol with NaCN resulted in decomposition
of the starting material. Gratifyingly, adding DMSO as the solvent
resulted in a high yield of the desired 5-bromo-2-cyanomethylben-
Scheme 3.
Scheme 4.

K. M. George Rosenker et al. / Bioorg. Med. Chem. 23 (2015) 2810–2818
2813
Table 2
Inhibitory activity of isoquinolinones 11, 13, and 16a–d
R1
R2
Yielda (%)
IC50 (lM)
b
Entry
Compound
1
2
3
4
5
6
16a
16b
16c
16d
13
4-Trifluoromethoxybenzene
2-Fluorobenzene
3-Acetamidobenzene
3-Acetylbenzene
3-Aminobenzene
Br
OMe
H
H
H
H
50
59
42
51
65
60
2.48 0.33
1.77 0.41
2.00 0.05
3.19 0.14
3.18 0.72
1.16 0.44
11
H
a
Yields from Schemes 3 and 4.
Mean value for 50% inhibition of Cdc25B SD, n = 3.
b
Scheme 5.
relationship was relatively flat, consistent with the shallow nature
of the Cdc25B active site. Because of the similarity of the IC50 val-
ues across the entire isoquinoline series, we focused on compound
13 as the prototypic representative of this inhibitor chemotype for
further investigation of its biochemical properties, in part guided
by the relative compactness, adequate aqueous solubility, and
structural rigidity of this structure. We found 13 did not generate
reactive oxygen species in the assay conditions, a common unde-
sirable artifact for phosphatase inhibitors.14 Compound 13 also
of Pd2(dba)3 and P(t-Bu)3 in a sealed tube at 120 °C. After obtaining
compound 20, the final demethylation proceeded smoothly with
4 M HCl in 1,4-dioxane at 80 °C to afford the desired product 13,
whose spectroscopic properties matched material previously syn-
thesized according to Scheme 3.
2.3. Biological evaluation
Because of the extreme sensitivity of Cdc25B to molecules that
either are electrophilic or generate reactive oxygen species,5,8 we
examined the reversibility of phosphatase inhibition by compound
13 as well as by the acetylated amide analog 16c, using a previ-
ously described dilution method.7 An irreversible inhibitor would
be predicted to cause a time-dependent inhibition of enzyme activ-
ity when preincubated in the absence of the substrate. As antici-
pated, preincubation of purified Cdc25B enzyme with the
was ꢀ5-fold less active against human PTP1B in vitro (IC50 = 15.3
lM).
Therefore, we synthesized compound 13 in larger quantities by uti-
lizing a regioselective cross-coupling strategy that we previously
developed for another series of enzyme inhibitors.15
2.2. Synthesis scale-up
The scale-up route for 13 began with the protection of the most
electrophilic C(1)-position of 1,3,6-trichloroisoquinoline 17 with a
methoxy group (Scheme 5). Regioselective palladium-catalyzed
Buchwald–Hartwig amination at the 3-position required the
screening of several catalyst systems. The best ratio of the desired
to undesired coupled products was achieved using Pd(OAc)2 and
Xantphos. Under these conditions, the desired compound 19 was
isolated in 49% yield (57% based on recovered starting material),
and only 23% (27% based on recovered starting material) of the
undesired C-6 coupled product was observed. The Suzuki cross-
coupling reaction at the 6-position was successful in the presence
irreversible naphthoquinone inhibitor DA3001-1 (10 lM) for
5 min produced a >60% inhibition in enzyme activity compared
to vehicle control, with further loss of activity with longer incuba-
tion times (Fig. 2A), consistent with previous results.7 In contrast,
preincubation with concentrations that exceed or equal IC50 values
for Cdc25B inhibition, namely 4 lM for 13 and 2 lM for 16c, for 5,
10, or 20 min, produced no significant decrease in enzyme activity.
These results suggested both 13 and 16c are reversible inhibitors of
Cdc25B. We next evaluated 13 for in vitro generation of H2O2,
using our previously described method.8 The quinolinedione
DA3003-1 was a robust generator of H2O2 in contrast to 13, which

2814
K. M. George Rosenker et al. / Bioorg. Med. Chem. 23 (2015) 2810–2818
Figure 2. Reversibility of Cdc25B in vitro inhibition and generation of H2O2 by 13.
(A) Cdc25B was preincubated with 4 M 13, 2 M 16c or 10 M DA3003-1 for 0, 5,
l
l
l
10 or 20 min and diluted 10-fold before an in vitro assay for phosphatase activity
with OMFP was conducted. All values are based on the 0 min value. N = 3
independent experiments, bars = SEM. (B) Concentration–response of 13 and
DA3003-1 to generate H2O2 using
15 min incubation, H2O2 production as absorbance was measured with
a
previously described assay.8,14,24 After
a
a
phenol-horseradish peroxidase detection reagent. N = 3 independent experiments.
Bars = SEM unless smaller than the symbols.
Figure 3. Kinetics of Cdc25B inhibition. (A) Inhibition by various concentrations of
13 of the dephosphorylation of the artificial substrate OMFP. The indicated
concentrations of OMFP were incubated for 20 min with different concentrations
of 13. These are representative of 3 independent experiments. (B) A Lineweaver–
Burk plot was generated with concentrations of 13 ranging from 0 lM to 20 lM and
various OMFP concentrations. Dephosphorylation of OMFP was measured for up to
produced no detectable reactive oxygen species at concentrations
well above those required for Cdc25B inhibition (Fig. 2B). A kinetic
analysis of Cdc25B inhibition by 13 was consistent with a compet-
20 min to determine the initial rate.
itive inhibitor having a Ki of 1.9 lM, making it one of the most
potent reversible in vitro Cdc25B inhibitors reported to date
(Fig. 3). Dephosphorylation of OMFP, while being a convenient
method to estimate intrinsic phosphatase activity, may not fully
reproduce the dephosphorylation of the endogenous protein sub-
strates.16 Therefore, we examined the ability of 13 to dephosphor-
ylate in vitro an established protein substrate, cyclin-dependent
kinase 1 (Cdk1). Incubation of Cdc25B with hyperphosphorylated
Cdk1 resulted in a complete loss of covalent phosphate on Cdk1
Tyr15 (Fig. 4, lane 7). Compound 13 caused a concentration-depen-
dent inhibition of the ability of Cdc25B to dephosphorylate Cdk1
(Fig. 4, lanes 1–5). As anticipated for a putative inhibitor of Cdc25B,
13 blocked the proliferation of several human tumor cell types that
express Cdc25B17, namely cervical cancer (HeLa), prostate (PC-3),
breast (MDA-MB-231), lung (A549) and colorectal (HCT116)
(Table 3).
Figure 4. Inhibition of Cdk1 dephosphorylation by 13. Tyr15 phosphorylated Cdk1
bound to cyclin B was incubated with recombinant Cdc25B in the presence or
absence of 8 mM Na3VO4 or various concentrations of 13. The extent of dephos-
phorylation was determined by Western blotting for phosphorylation of Tyr15 on
Cdk1 as described elsewhere.25,26
Table 3
Cancer cells treated with DNA damaging agents frequently
arrest in the G2 phase of the cell cycle due to activation of a DNA
damage checkpoint. Ectopic expression of Cdc25B can override
the checkpoint, allowing cells to ‘escape’ and resume cell cycle pro-
gression after DNA damage.18 The mitotic bypass effect of Cdc25
overexpression is dependent on its phosphatase activity and inhi-
bition of Cdc25B restores the defective checkpoint.18 This forms
the basis of a quantitative mitotic arrest assay using U2OS cells,
which have some endogenous Cdc25B but have been engineered
to conditionally express high levels of Cdc25B. These cells have
previously been successfully exploited to study potential small
molecule inhibitors of Cdc25B.18,19 U2OS cells that have been trea-
Antiproliferative effects of 13
a
Cell line
IC50
(
lM)
HeLa
PC-3
MDA-MB-231
A549
HCT116
12.3 6.9 (6)b
32.9 3.8 (2)
6.8 0.9 (2)
6.3 2.8 (2)
5.0 0.8 (2)
a
Mean SEM or range.
Number of independent experiments performed in triplicate.
b
repression, failed to enter mitosis as measured by Ser10 histone
3 phosphorylation after treatment with nocodazole (500 nM),
which traps cells in mitosis (Fig. 5, dotted lines). In contrast,
ted with the DNA damaging agent etoposide (40
lM), but have not
been induced to over-express Cdc25B because of tetracycline

K. M. George Rosenker et al. / Bioorg. Med. Chem. 23 (2015) 2810–2818
2815
Figure 6. Proposed binding of 13 with Cdc25B. (A) Potential binding motif for 13
with the active site of Cdc25B. Compound 13 forms hydrogen bonds with the side-
chain of Thr547 and the backbone carbonyl oxygen atoms of Cys426 and Arg544.
(B) The comparison of the predicted binding motif of 13 (blue) and OMFP (red and
cyan) shows that both OMFP and 13 overlap significantly in the swimming pool in
their energetically most favorable docking poses.
Figure 5. Inhibition of Cdc25B-dependent mitotic arrest by 13. (A) U2OS cells
transfected with Cdc25B were treated for 1 h with etoposide (40 M) followed by a
23 h incubation with nocodazole (500 nM) in the presence of vehicle (DMSO) or 13.
Representative fluorescence micrographs of phospho-histone H3-positive cells
(PHH3) and Hoechst-stained nuclei (nuclei) are shown. Bar = 150 lm. (B) Quanti-
l
fication of mitotic bypass by high-content analysis. Etoposide-treated cells U2OS
cells have low levels of phospho-histone H3 positive cells (dotted line) that increase
with Cdc25B overexpression (closed triangle). Increasing concentrations of 13
gradually reduce mitotic cells, indicative of Cdc25B inhibition.
presence of water molecules.4b In this site, 13 formed hydrogen
bonds with the side-chains of Thr547 and the backbone carbonyl
oxygen atoms of Cys426 and Arg544. While this interaction mode
did not completely occlude the catalytic cavity, it potentially over-
lapped with bulky substrates and was distinct from our previous
model for 5169131.7b This is a binding pose that has not previously
been favored by other known small molecule inhibitors of
Cdc25B.5 To probe the extent of potential overlap that could lead
to inhibition, we docked OMFP at the same site and found its best
docking pose (Fig. 6B). The fluorescein moiety of OMFP also pre-
ferred to occupy the swimming pool region and overlapped signif-
icantly with 13, consistent with the observed competitive nature of
the inhibition.
U2OS cells over-expressing Cdc25B readily entered mitosis after
etoposide exposure, with approximately 20% of the nocodazole
treated population being in mitosis. Treatment with compound
13, however, inhibited the Cdc25B-mediated entry into mitosis
with an IC50 value of 33.6 lM. These results are consistent with
intracellular inhibition of Cdc25B by compound 13. It should be
noted our studies do not exclude inhibition of other Cdc25 family
members by compound 13 and future studies with cells devoid
of Cdc25B would be valuable.
2.4. Molecular modeling
2.5. Conclusions
To gain insights into the potential inhibitory mechanism of 13,
we performed docking simulations using the Cdc25B crystal struc-
ture 1qb0.20 The docking search space included the catalytic site
and the nearby cavities in which 13 could possibly bind and over-
lap with the artificial substrate OMFP. The most favorable docking
pose of 13 (Fig. 6A) was found in a cavity formed by highly hydro-
philic moieties in the C-terminal residues of Cdc25B, which has
been referred to as the ‘swimming pool’ due to the abundant
Previous investigators have examined the 3-aminoisoquinoline
structure for anticonvulsant and antidepression activity,21 but not
as an anticancer agent. Interestingly, neither the synthesis nor the
pharmacology of 3-aminoisoquinolinones has been extensively
studied. Our results suggest that the 3-aminoisoquinolinone chem-
otype will tolerate considerable modifications while retaining
inhibitory effects for Cdc25B. The synthetic methods describe

2816
K. M. George Rosenker et al. / Bioorg. Med. Chem. 23 (2015) 2810–2818
herein enable a facile analog generation. We identified a new
lead compound, 6-(3-aminophenyl)-3-(phenylamino)isoquinolin-
1[2H]-one 13, which was a reversible and competitive in vitro
inhibitor of Cdc25B, prevented tumor cell growth and reinforced
a cell cycle checkpoint abrogated by Cdc25B ectopic expression.
Thus, the 3-aminoisoquinoline series provides an attractive new
core structure for additional analysis and optimization.
3.1.4. 3,6-Dichloro-1-methoxyisoquinoline (18)
To solution of 1,3,6-trichloroisoquinoline 17 (1.00 g,
a
4.3 mmol) in anhyd MeOH (43 mL) was added NaOMe (1.39 g,
25.7 mmol). The solution was stirred at reflux under an atmo-
sphere of N2 for 4 h. The mixture was cooled to room temperature,
diluted with EtOAc, quenched with 1 M HCl, and stirred for 5 min.
The reaction mixture was poured into H2O and extracted with
EtOAc. The combined organic layers were washed with brine, dried
(MgSO4), and concentrated under reduced pressure to provide 18
(0.950 g, 97%) as a white solid: mp 106.2–107.1 °C; IR (ATR, neat)
3077, 3085, 3027, 2152, 1616, 1564, 1448, 1376, 1357, 1322,
3. Experimental section
;
1195, 1089, 902, 874, 822, 662 cmÀ1 1H NMR (DMSO-d6,
3.1. Chemistry
400 MHz) d 8.10 (d, 1H, J = 8.9 Hz), 7.98 (d, 1H, J = 2.0 Hz), 7.60
(dd, 1H, J = 8.9, 2.1 Hz), 7.50 (s, 1H), 4.06 (s, 3H); 13C NMR
(DMSO-d6, 100 MHz) d 160.1, 142.5, 140.0, 136.8, 127.8, 126.0,
124.7, 115.8, 112.4, 54.6; HRMS (ESI) m/z calcd for C10H8NOCl2
([M+H]+) 227.9983, found 227.9985.
3.1.1. 3-Phenylamino-2H-isoquinolin-1-one (1)
To
a
solution of 2-cyanomethylbenzoic acid10 (714 mg,
4.43 mmol) in chlorobenzene (8.0 mL) was added aniline
(0.41 mL, 4.43 mmol). The reaction mixture was heated at reflux
for 5 h and allowed to cool to room temperature. The solvent
was removed in vacuo and the resulting solid residue was tritu-
rated with 2-propanol, filtered, and washed with 2-propanol
and EtOH to provide 1 (603 mg, 58%) as an off-white solid: mp
193.8–195.3 °C; IR (ATR, neat) 3310, 2982, 2839, 1664, 1635,
3.1.5. 6-Chloro-1-methoxy-N-phenylisoquinolin-3-amine (19)
To a reaction vial containing 18 (0.500 g, 2.19 mmol), Pd(OAc)2
(0.098 g, 0.438 mmol), Xantphos (0.317 g, 0.548 mmol), and
Cs2CO3 (1.44 g, 4.38 mmol) under an atmosphere of Ar was added
toluene (73 mL) and aniline (0.22 mL, 2.41 mmol). The reaction
mixture was heated to 80 °C under an atmosphere of Ar for 2.5 h,
cooled to room temperature, filtered through CeliteÒwith EtOAc
washings, and concentrated under reduced pressure to give a
brown–orange residue. The residue was adsorbed onto SiO2 and
purified by chromatography on SiO2 (100% hexanes, 2% EtOAc/hex-
anes, 10% EtOAc/hexanes) to provide 19 (0.303 g, 49%, 57% BRSM)
as a yellow solid: mp 98.3–100.1 °C; IR (ATR, neat) 3353, 3049,
1595, 1541, 1333 cmÀ1 1H NMR (DMSO-d6, 600 MHz) d 10.76
;
(s, 1H), 8.00 (d, 1H, J = 7.8 Hz), 7.95 (s, 1H), 7.50 (ddd, 1H,
J = 8.4, 7.2, 1.8 Hz), 7.39 (d, 1H, J = 7.8 Hz), 7.33 (ddd, 2H, J = 8.4,
7.8, 1.8 Hz), 7.19–7.16 (m, 3H), 7.00 (dd, 1H, J = 7.2, 7.2 Hz),
6.04 (s, 1H); 13C NMR (DMSO-d6, 150 MHz) d 162.7, 142.0,
141.2, 140.5, 132.9, 129.9, 127.0, 125.4, 123.3, 122.4, 121.6,
119.8, 85.2; HRMS (EI) m/z calcd for C15H12N2O 236.0950, found
236.0950.
3031, 2952, 1622, 1596, 1558, 1374, 1316, 1303, 1169 cmÀ1 1H
;
3.1.2. 5H-Benzo[4,5]imidazo[1,2-b]isoquinolin-1-one (7)
NMR (DMSO-d6, 400 MHz) d 9.02 (s, 1H), 7.95 (d, 1H, J = 8.8 Hz),
7.73 (d, 1H, J = 1.9 Hz), 7.55 (d, 2H, J = 7.7 Hz), 7.29 (t, 2H,
J = 7.5 Hz), 7.18 (dd, 1H, J = 8.8, 2.0 Hz), 6.91 (t, 1H, J = 7.3 Hz),
6.65 (s, 1H), 4.07 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) d 159.8,
150.7, 141.5, 141.4, 135.8, 128.8 (2 C), 126.0, 123.3, 122.7, 120.7,
118.51 (2 C), 111.6, 92.5, 53.9; HRMS (ESI) m/z calcd for
To a reaction vial was added homophthalic anhydride (200 mg,
0.93 mmol), o-phenylenediamine (100 mg, 0.93 mmol), and glacial
AcOH (1 mL). The mixture was heated at reflux for 4 h. Upon cool-
ing to room temperature, a yellow solid formed and was collected
by suction filtration and subsequently washed with AcOH. The
solid was dried under high vacuum overnight to yield 7 (153 mg,
71%) as a yellow solid: mp 305 °C (dec); IR (ATR, neat) 3101,
C
16H14N2OCl ([M+H]+) 285.0795, found 285.0782.
3048, 1662, 1618, 1573, 1455 cmÀ1
;
1H NMR (DMSO-d6,
3.1.6. 6-(3-Aminophenyl)-1-methoxy-N-phenylisoquinolin-3-
amine (20)
600 MHz) d 11.88 (s, 1H), 8.63 (d, 1H, J = 7.8 Hz), 8.28 (d, 1H,
J = 7.8 Hz), 7.64 (d, 1H, J = 7.8 Hz), 7.61 (ddd, 1H, J = 7.8, 6.6,
1.2 Hz), 7.41 (ddd, 1H, J = 8.4, 7.8, 1.2 Hz), 7.35 (d, 1H, J = 7.8 Hz),
7.24 (ddd, 1H, J = 8.4, 6.6, 1.2 Hz), 7.21 (ddd, 1H, J = 8.4, 8.4,
1.2 Hz), 6.37 (s, 1H); 13C NMR (DMSO-d6, 150 MHz) d 159.5,
141.9, 139.2, 133.7, 132.4, 128.4, 127.4, 126.6, 125.3, 121.9,
120.5, 117.9, 116.3, 109.7, 79.1; HRMS (EI) m/z calcd for
To a reaction vial containing 19 (0.419 g, 1.47 mmol), KF
(0.256 g, 4.41 mmol), Pd2(dba)3 (0.273 g, 0.294 mmol), and 3-
aminophenylboronic acid (0.470 g, 2.94 mmol) under an atmo-
sphere of Ar was added P(t-Bu)3 (0.124 g, 0.588 mmol) in 2.0 mL
of freshly distilled and degassed 1,4-dioxane followed by an addi-
tional 7.5 mL of 1,4-dioxane. The reaction vial was sealed and
heated to 120 °C for 3 h. The reaction mixture was cooled to room
temperature, and filtered through CeliteÒwith EtOAc washings. The
combined organic fractions were concentrated under reduced
pressure to give an orange-yellow residue. The polarity of the
impurities required two chromatographic separations for optimal
purification. The residue was adsorbed onto SiO2 and purified by
chromatography on SiO2 (100% CH2Cl2, 2% CH2Cl2/MeOH) to pro-
vide the desired product as a mixture with a more apolar impurity.
In order to separate this mixture further, the material was
absorbed onto SiO2 and purified by chromatography on SiO2
(100% hexanes, 5% EtOAc/hexanes, 20% EtOAc/hexanes, 100%
EtOAc) to provide 20 (0.359 g, 72%) as a red–brown solid: mp
165.7–167.5 °C (CHCl3); IR (ATR, neat) 3375, 3029, 2393, 1623,
1594, 1573, 1493, 1446, 1422, 1374, 1336, 1299, 1158, 1105,
C15H10N2O 234.0793, found 234.0792.
3.1.3. 4-Benzoyl-3-p-tolylamino-2H-isoquinolin-1-one (8)
To a reaction vial was added 3-p-tolylamino-2H-isoquinolin-1-
one (50 mg, 0.2 mmol), benzoyl chloride (30 lL, 0.26 mmol), and
1,4-dioxane (4.0 mL). The mixture was heated at reflux for 3 h
and at room temperature for 12 h. The solvent was then removed
in vacuo and the resulting solid residue was triturated with 2-pro-
panol, filtered, and washed with 2-propanol and MeOH to yield 8
(32 mg, 45%) as a yellow/green solid: mp 268–270 °C; IR (ATR,
neat) 3136, 3081, 3019, 1655, 1602, 1555, 1514, 1320,
1303 cmÀ1 1H NMR (DMSO-d6, 600 MHz) d 11.27 (s, 1H), 9.18 (s,
;
1H), 8.13 (d, 1H, J = 7.8 Hz), 7.60 (d, 2H, J = 8.4 Hz), 7.46 (d, 1H,
J = 7.2 Hz), 7.44 (d, 1H, J = 7.2 Hz), 7.36–7.31 (m, 3H), 7.27 (dd,
1H, J = 7.8, 7.2 Hz), 7.01 (d, 2H, J = 7.8 Hz), 6.86 (d, 2H, J = 7.2 Hz),
2.23 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) d 194.2, 162.6, 140.4,
138.2, 138.0, 132.9, 132.5, 132.1, 130.0, 130.0, 129.2, 129.2,
128.7, 127.43, 124.47, 124.3, 120.5, 99.7, 20.9; HRMS (EI) m/z calcd
for C23H18N2O2 354.1368, found 354.1361.
688 cmÀ1 1H NMR (DMSO-d6, 400 MHz) d 8.89 (s, 1H), 8.01 (d,
;
1H, J = 8.6 Hz), 7.75 (s, 1H), 7.57 (d, 2H, J = 8.1 Hz), 7.43 (d, 1H,
J = 8.4 Hz), 7.28 (t, 2H, J = 7.7 Hz), 7.13 (t, 1H, J = 7.7 Hz), 6.99–
6.85 (m, 3H), 6.75 (s, 1H), 6.61 (d, 1H, J = 7.9 Hz), 5.20 (s, 2H),
4.09 (s, 3H); 13C NMR (DMSO-d6, 125 MHz) d 159.6, 149.9, 149.2,

K. M. George Rosenker et al. / Bioorg. Med. Chem. 23 (2015) 2810–2818
2817
143.1, 142.0, 140.7, 140.3, 129.4, 128.7, 124.2, 121.70, 121.65,
120.2, 118.1, 114.7, 113.7, 112.42, 112.39, 93.8, 53.8; HRMS (ESI)
m/z calcd for C22H20N3O ([M+H]+) 342.1606, found 342.1623.
3 mM DTT). All reactions and controls were optimized as
described previously23 and were performed at 1% DMSO final
concentration and with 10–20 ng of enzyme. Fluorescence emis-
sion from the product was measured after a 30 min (20 min for
PTP1B) incubation period at room temperature with a Spectra-
MaxM5 (Excitation 485 nM/Emission 521 nM). IC50 concentra-
tions were determined using Prism 6.0 (GraphPad Software Inc.,
San Diego, CA). For Michaelis–Menten studies with inhibitors,
we used multiple concentrations of OMFP concentrations (10–
3.1.7. 6-(3-Aminophenyl)-3-(phenylamino)isoquinolin-1(2H)-
one (13)
To a flask containing 20 (0.241 g, 0.705 mmol) under an atmo-
sphere of Ar was added 4 M HCl in 1,4-dioxane (24.1 mL). The
reaction vial was sealed and heated to 80 °C for 28 h (Note: The
reaction progress was monitored by 1H NMR of an aliquot of
the crude reaction mixture). The reaction mixture was cooled to
room temperature and concentrated under reduced pressure to
provide a yellow-brown solid. This solid was suspended in H2O
and neutralized (pH 7) with satd aq NaHCO3. The suspension
was filtered and washed with H2O and ether to provide 13
(0.190 g, 82%) as a light yellow-brown solid: mp 184.7 °C (dec);
IR (ATR, neat) 3316, 1635, 1594, 1551, 1495, 1484, 1437, 1340,
160 lM) and incubated the samples for 5–20 min at room tem-
perature. Results were compared with enzyme incubated with
the DMSO vehicle and analyzed on Prism 6.0 Michaelis–Menten
kinetics and Lineweaver–Burk Plot. For reversibility studies, we
used a dilution method described previously7 with minor modifi-
cations. The purified enzyme (750 ng) was preincubated with
concentrations near or above the IC50 (4 lM for 13, 2 lM for
16e) for 0, 5, 10, or 20 min at room temperature in 1.5Â assay
buffer with 0.1% bovine serum albumin and 3 mM DTT. Then
the reaction was diluted 50-fold to determine remaining enzyme
activity using OMFP and the results were compared with enzyme
incubated with a known irreversible inhibitor DA3003-17,22 using
1288 cmÀ1 1H NMR (DMSO-d6, 400 MHz) d 10.72 (s, 1H), 8.04
;
(d, 1H, J = 8.3 Hz), 7.99 (s, 1H), 7.58 (d, 1H, J = 1.5 Hz), 7.40–7.31
(m, 3H), 7.20 (d, 2H, J = 7.6 Hz), 7.12 (t, 1H, J = 7.8 Hz), 7.00 (t,
1H, J = 7.4 Hz), 6.94, t, 1H, J = 1.8 Hz), 6.87 (d, 1H, J = 7.6 Hz),
6.61 (dd, 1H, J = 8.0, 1.4 Hz), 6.13 (s, 1H), 5.39 (bs, 2H); 13C
NMR (DMSO-d6, 100 MHz) d 162.0, 148.7, 144.7, 141.7, 140.7,
140.4, 140.2, 129.4, 129.3, 127.1, 122.3, 121.9, 121.5, 120.0,
119.2, 114.8, 113.9, 112.6, 85.0; HRMS (ESI) m/z calcd for
10 lM for preincubation. Generation of H2O2 was determined as
previously described.24
3.2.4. Cdk1/cyclin B dephosphorylation assay
C
21H18N3O ([M+H]+) 328.1450, found 328.1478.
The Tyr15 phosphorylated Cdk1/cyclinB substrate was isolated
from etoposide-treated HeLa cells as described previously by Dar-
bon et al.25 The in vitro phosphatase inhibition by compound 13
of recombinant Cdc25B (50 ng/mL), isolated as described previ-
ously7, was determined by co-incubation of Cdc25B with or with-
out compound and phosphorylated Cdk1/cyclinB substrate. After
protein separation on a 12% Tris–glycine SDS gel, we used Wes-
tern blotting26 with an antibody against phospho-Tyr15 Cdk1
(Cell Signaling Technology, Inc. Danvers, MA) to detect phospha-
tase activity.
3.2. Biology
3.2.1. Materials
All reagents unless otherwise noted were purchased from
Sigma–Aldrich (St. Louis, MO). The synthesis of DA3003-1 was
reported previously.22
3.2.2. Expression and purification of Cdc25B catalytic domain
Previous methods were used to express the catalytic domain
of Cdc25B.14 Briefly, His6-tagged Cdc25B catalytic domain (350–
566) was expressed in BL21 (D3) competent bacterial cells trans-
formed with the pET28a Cdc25B-Catalytic Domain expression
vector that were grown in Miller broth containing 34 mg/L kana-
mycin at 30 °C as described previously14 with the following
3.2.5. Mitotic bypass assay
U2OS cells overexpressing Cdc25B under the control of the
tetracycline-repressible promoter were the generous gift of Prof.
B. Ducommun (Université de Toulouse, Toulouse, France). The
assay conditions were based on those previously described,7,18,19
but were conducted in 384 well plates using a Biomek2000
(Beckman Coulter, Brea, CA) automated liquid handler for com-
pound and solution dispensing and an ArrayScan imaging system
(Thermo Scientific, Waltham, MA) for detection of phosphohi-
stone H3 in individual cells. Briefly, cells were grown for 24 h
in the absence of tetracycline to allow Cdc25B expression, and
modifications: For induction, isopropyl b-D-1-thiogalactopyrano-
side was added (1 mM final) at 0.4 OD600, and incubated at
30 °C until the solution achieved an 0.8 OD600 value. The solu-
tion was centrifuged at 5g for 20 min and the bacterial pellet
was lysed in the presence of lysozyme (0.75 mg/mL) in the wash
buffer (50 mM Tris–HCl pH 7.0, 300 mM NaCl, 1 mM tris-(2-car-
boxyethyl)phosphine, 0.2% Tween-20, 10% glycerol) containing
then treated for 1 h with etoposide (40 lM). The cells were
8.5 mg/mL of leupeptin, 86
l
g/mL of 4-(2-aminoethyl)benzene-
washed and further incubated in the presence of nocodazole
(500 nM) together with compound 13 or DA3003-1 at the indi-
cated concentrations. After 16 h, cells were fixed, stained with
Hoechst 33342 and anti-phosphohistone H3 (Ser10) antibody
(Cell Signaling Technology, Inc.) and the mitotic index was deter-
mined with an ArrayScan II.
sulfonyl fluoride hydrochloride, and an additional Tris-(2-car-
boxyethyl)phosphine (100 mM). After addition of cobalt TALON
resin, the lysates were washed three times with wash buffer,
and then twice with the wash buffer containing 15 mM imidaz-
ole. The protein was eluted with 150 mM imidazole in the wash
buffer and immediately stored at À80 °C. PTP1B was purchased
from
a
commercial vendor (BIOMOL Research Laboratories
3.2.6. Molecular docking studies
(Plymouth Meeting, PA).
Simulations were conducted with the crystal structure 1qb020
of the active catalytic domain of Cdc25B and OpenEye software
application for docking of 13 and OMFP following our approach
detailed previously for studies of other dual-specificity phospha-
tases.27,28 Briefly, biologically active conformations of the small
molecules were generated using Omega.29 The Cdc25B crystal
structure was prepared for docking using the FRED receptor com-
ponent of the OEDocking application and docking simulations were
performed using FRED.30 The chemgauss scoring method was used
to guide docking and selecting poses in Figure 6.
3.2.3. In vitro enzyme assays
Enzyme activities in the absence and presence of inhibitors
were measured using the artificial substrate O-methyl fluorescein
phosphate (OMFP) at the Km 40 lM concentration (unless other-
wise noted) in 384-well microtiter plates as described previ-
ously22 with some minor modifications. The final incubation
mixture (15
l
L) comprised 3Â assay buffer (90 mM Tris pH 8.0,
255 mM NaCl and 3 mM EDTA, 0.1% bovine serum albumin and

2818
K. M. George Rosenker et al. / Bioorg. Med. Chem. 23 (2015) 2810–2818
7. (a) Brisson, M.; Nguyen, T.; Wipf, P.; Joo, B.; Day, B. W.; Skoko, J. S.; Schreiber, E.
Authorship
M.; Foster, C.; Bansal, P.; Lazo, J. S. Mol. Pharmacol. 1810, 2005, 68; (b) Brisson,
M.; Nguyen, T.; Vogt, A.; Yalowich, J.; Giorgianni, A.; Tobi, D.; Bahar, I.;
Stephenson, C. R. J.; Wipf, P.; Lazo, J. S. Mol. Pharmacol. 2004, 66, 824.
8. Johnston, P. A.; Foster, C. A.; Tierno, M. B.; Shun, T. Y.; Shinde, S. N.; Paquette,
W. D.; Brummond, K. M.; Wipf, P.; Lazo, J. S. Assay Drug Dev. Technol. 2009, 7,
250.
9. Kucherenko, T.; Kysil, V.; Kovtunenko, V. Synth. Commun. 2003, 33, 1163. and
references cited within.
10. Price, C. C.; Rogers, R. G. Org. Synth. 1942, 22, 30.
Conceived and designed the experiments: P.A.J., E.R.S., A.V.,
J.S.L., P.W.
Performed the experiments: K.M.G.R., W.D.P., P.A.J., E.R.S.
Analyzed the data: P.A.J., E.R.S., A.V., J.S.L., P.W.
Computational modeling: A.B.
Wrote the paper: P.A.J., E.R.S., A.V., J.S.L., P.W.
11. Kucherenko, T. T.; Gutsul, R.; Kisel, V. M.; Kovtunenko, V. A. Tetrahedron 2004,
60, 211.
12. Ling, K.; Chen, X.; Fun, H.; Huang, X.; Xu, J. J. Chem. Perkin Trans. 1 1998, 4147.
13. Potikha, L. M.; Shkilna, N. V.; Kisil, V. M.; Kovtunenko, V. A. Chem. Heterocycl.
Comp. 2004, 40, 1052.
14. Soares, K. M.; Blackmon, N.; Shun, T. Y.; Shinde, S. N.; Takyi, H. K.; Wipf, P.;
Lazo, J. S.; Johnston, P. A. Assay Drug Dev. Technol. 2010, 8, 152.
15. Wipf, P.; George, K. M. Synlett 2010, 644; See also: (b) Tikad, A.; Routier, S.;
Akssira, M.; Guillaumet, G. Org. Biomol. Chem. 2009, 7, 5113; (b) Tikad, A.;
Routier, S.; Akssira, M.; Leger, J.-M.; Jarry, C.; Guillaumet, G. Org. Lett. 2007, 9,
4673.
16. De Munter, S.; Köhn, M.; Bollen, M. ACS Chem. Biol. 2013, 8, 36.
17. Daouti, S.; Li, W. H.; Qian, H.; Huang, K. S.; Holmgren, J.; Levin, W.; Reik, L.;
McGady, D. L.; Gillespie, P.; Perrotta, A.; Bian, H.; Reidhaar-Olson, J. F.; Bliss, S.
A.; Olivier, A. R.; Sergi, J. A.; Fry, D.; Danho, W.; Ritland, S.; Fotouhi, N.;
Heimbrook, D.; Niu, H. Cancer Res. 2008, 68, 1162.
18. Bugler, B.; Quaranta, M.; Aressy, B.; Brezak, M. C.; Prevost, G.; Ducommun, B.
Mol. Cancer Ther. 2006, 5, 1446.
19. Kolb, S.; Mondésert, O.; Goddard, M. L.; Jullien, D.; Villoutreix, B. O.;
Ducommun, B.; Garbay, C.; Braud, E. ChemMedChem 2009, 4, 633.
20. Reynolds, R. A.; Yem, A. W.; Wolfe, C. L.; Deibel, M. R., Jr.; Chidester, C. G.;
Watenpaugh, K. D. J. Mol. Biol. 1999, 293, 559.
Acknowledgments
This work was supported by grants from the United States
National Institutes of Health, National Cancer Institute (P01
CA78039), Institute for Mental Health and the Molecular Library
Screening Center Network (U54 MH074411), and the Institute for
General Medical Sciences, Chemical Methodology and Library
Development Centers Program (P50 GM067082), the University
of Pittsburgh Cancer Institute Chemical Biology Facility (P30
CA047904), and generous contributions from the Owens Family
Foundation and the Fiske Drug Discovery Fund. The authors thank
Catherine Corey and Samantha Murray for their technical assis-
tance with the biochemical studies and Prof. B. Ducommun (Uni-
versité de Toulouse, Toulouse, France) for providing the U2OS
cells with inducible Cdc25B expression.
21. Neumeyer, J. L.; Weinhardt, K. K.; Carrano, R. A.; McCurdy, D. H. J. Med. Chem.
1973, 16, 808.
22. Lazo, J. S.; Aslan, D. C.; Southwick, E. C.; Cooley, K. A.; Ducruet, A. P.; Joo, B.;
Vogt, A.; Wipf, P. J. Med. Chem. 2001, 44, 4042.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.01.043.
23. Tierno, M. B.; Johnston, P. A.; Foster, C.; Skoko, J. J.; Shinde, S. N.; Shun, T. Y.;
Lazo, J. S. Nat. Protoc. 2007, 2, 1134.
24. Johnston, P. A.; Soares, K. M.; Shinde, S. N.; Foster, C. A.; Shun, T. Y.; Takyi, H. K.;
Wipf, P.; Lazo, J. S. Assay Drug Dev. Technol. 2008, 6, 505.
25. Darbon, J. M.; Penary, M.; Escalas, N.; Casagrande, F.; Goubin-Gramatica, F.;
Baudouin, C.; Ducommun, B. J. Biol. Chem. 2000, 275, 15363.
26. Brezak, M. C.; Quaranta, M.; Contour-Galcera, M. O.; Lavergne, O.; Mondesert,
O.; Auvray, P.; Kasprzyk, P. G.; Prevost, G. P.; Ducommun, B. Mol Cancer Ther.
2005, 4, 1378.
References and notes
1. (a) Vintonyak, V. V.; Antonchick, A. P.; Rauh, D.; Waldmann, H. Curr. Opin. Chem.
Biol. 2009, 13, 272; (b) Boutros, R.; Lobjois, V.; Ducommun, B. Nat. Rev. Cancer
2007, 7, 495.
27. Korotchenko, V. N.; Saydmohammed, M.; Vollmer, L. L.; Bakan, A.; Sheetz, K.;
Debiec, K. T.; Greene, K. A.; Agliori, C. S.; Bahar, I.; Day, B. W.; Vogt, A.; Tsang, M.
Chembiochem 2014, 15, 436.
28. Sharlow, E. R.; Wipf, P.; McQueeney, K. E.; Bakan, A.; Lazo, J. S. Expert Opin.
Investig. Drugs 2014, 23, 661.
2. Lyon, M. A.; Ducruet, A. P.; Wipf, P.; Lazo, J. S. Nat. Rev. Drug Discov. 2002, 1, 961.
3. Lazo, J. S.; Wipf, P. Anti-Cancer Agents Med. Chem. 2008, 8, 837.
4. (a) Reynolds, R. A.; Yem, A. W.; Wolfe, C. L.; Deibel, M. R.; Chidester, C. G.;
Watenpaugh, K. D. J. Mol. Biol. 1999, 293, 559; (b) Rudolph, J. Mol. Pharmacol.
2004, 66, 780.
29. Hawkins, P. C. D.; Skillman, A. G.; Warren, G. L.; Ellingson, B. A.; Stahl, M. T. J.
Chem. Inf. Model. 2010, 50, 572.
30. McGaughey, G. B. J. Chem. Inf. Model. 2007, 47, 1504.
5. (a) Lavecchia, A.; Di Giovanni, C.; Novellino, E. Mini-Rev. Med. Chem. 2012, 12,
62; (b) Sarkis, M.; Tran, D. N.; Kolb, S.; Miteva, M. A.; Villoutreix, B. O.; Garbay,
C.; Braud, E. Bioorg. Med. Chem. Lett. 2012, 22, 7345.
6. Contour-Galcera, M.; Sidhu, A.; Prévost, G.; Biff, D.; Ducommun, B. Pharmacol.
Ther. 2007, 115, 1.