N-Phenyl-4-hydroxy-2-quinolone-3-carboxamides as selective inhibitors of mutant H1047R phosphoinositide-3-kinase (PI3Kα)

Article abstract of DOI:10.1016/j.bmc.2012.09.059

This work describes our efforts to optimize the lead PI3Kα inhibitor N-benzyl 4-hydroxy-2-quinolone-3-carboxamide using structure-based design and molecular docking. We identified a series of N-phenyl 4-hydroxy-2-quinolone-3- carboxamides as selective inhibitors of mutant H1047R versus wild-type PI3Kα and we also showed that the cell growth inhibition by these compounds likely occurs by inhibiting the formation of pAKT and induction of apoptosis.

Full text of DOI:10.1016/j.bmc.2012.09.059

Bioorganic & Medicinal Chemistry 20 (2012) 7175–7183
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N-Phenyl-4-hydroxy-2-quinolone-3-carboxamides as selective inhibitors
of mutant H1047R phosphoinositide-3-kinase (PI3Ka)
Dima A. Sabbah a, Neka A. Simms b, Wang Wang b, Yuxiang Dong a, Edward L. Ezell c, Michael G. Brattain b,
Jonathan L. Vennerstrom a, Haizhen A. Zhong d,
⇑
a College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA
b Eppley Cancer Institute, University of Nebraska Medical Center, 985920 Nebraska Medical Center, Omaha, NE 68198-5950, USA
c Eppley Cancer Institute, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA
d Department of Chemistry, University of Nebraska at Omaha, DSC 362, 6001 Dodge Street, Omaha, NE 68182, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 June 2012
Revised 18 September 2012
Accepted 25 September 2012
Available online 12 October 2012
This work describes our efforts to optimize the lead PI3Ka inhibitor N-benzyl 4-hydroxy-2-quinolone-3-
carboxamide using structure-based design and molecular docking. We identified a series of N-phenyl 4-
hydroxy-2-quinolone-3-carboxamides as selective inhibitors of mutant H1047R versus wild-type PI3K
a
and we also showed that the cell growth inhibition by these compounds likely occurs by inhibiting the
formation of pAKT and induction of apoptosis.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
PI3K
Selectivity
Colon cancer
AKT phosphorylation
Apoptosis
1. Introduction
By means of pharmacophore modeling and database searching,
we identified N-benzyl 4-hydroxy-2-quinolone-3-carboxamide (1)
Phosphatidylinositol 3-kinases (PI3Ks) phosphorylate phospha-
tidylinositol 4,5-biphosphate (PIP2) to generate 3,4,5-triphosphate
(PIP3), which phosphorylates downstream signaling proteins such
as AKT, leading to increase cell growth and proliferation. PI3Ks
are negatively regulated by the phosphatase and tensin homolog
protein (PTEN), which dephosphorylates PIP3.1–3 PI3Ks are divided
into three classes based on their substrate specificity and primary
as a lead compound with IC50 values of 1.1 and 0.73
l
M against the
wild-type (WT) PI3K
a and mutant (MUT) H1047R PI3Ka, respec-
tively (Fig. 1).18 Compound 1 suppressed the formation of pAKT
and induced apoptosis in a human colon carcinoma cell line. We
previously suggested that it might be possible to identify selective
inhibitors of the H1047R mutant PI3K
a guided by the structural
differences between the mutant and wild-type enzyme.19 This
work describes our efforts in optimizing 1, which led to the discov-
ery of a series of N-phenyl-4-hydroxy-2-quinolone-3-carboxam-
structures. Class IA comprises the PI3K
a, b, and d isoforms encoded
by their respective genes PIK3CA, PIK3CB, and PIK3CD.4 PI3K
a
mutations (E545K and E542K in the helical domain, and H1047R
in the kinase domain) are observed in breast (27%), endometrial
(24%), colon (15–32%), brain (27%), stomach, and upper digestive
ides with selectivity for the H1047R mutant PI3Ka.
2. Chemistry
tract (19–25%) tumors.5–7 The PI3K
a
H1047R mutant has a 2-fold
increase in lipid kinase activity, and thus poses a unique potential
to decrease the efficacy of anticancer drugs targeting PI3K
.8 Thus,
selective inhibition of PI3K mutants may provide additional ther-
apeutic benefits. Numerous PI3K
inhibitors have been reported9–14
and some of these have progressed to clinical trials.15–17 Yet few of
Target compounds were designed to probe the contribution of
the lactam and phenolic oxygen atoms (2–4), benzylic carbon (5),
and the lactam hydrogen (6) to the activity of 1. Compounds 2
and 420–22 were prepared by HOBt/EDCl couplings of the corre-
sponding quinoline-3-carboxylic acids with benzylamine. Herein
we reported for the first time, 1H and 13C NMR spectra of 2 and
4. Target compounds 5 and 7–11 were prepared in neat one-pot
reactions (Scheme 1) between triethyl methanetricarboxylate
(12) and the corresponding anilines (13) at 170–195 °C for 6 h.
a
a
a
these are PI3Ka mutant-selective.
⇑
Corresponding author. Tel.: +1 402 554 3145; fax: +1 402 554 3888.
E-mail address: hzhong@unomaha.edu (H.A. Zhong).
Target compound 623,24 was prepared by methylation of
5
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.059

7176
D. A. Sabbah et al. / Bioorg. Med. Chem. 20 (2012) 7175–7183
X
O
O
X
N
O
Y
OH
O
N
H
N
H
N
H
N
H
N
R
O
1
2
3
5
6
, X = OH
, X = H
, X = OH, Y = H
, R = H
4, X, Y = H
, R = Me
Figure 1. Analogues of lead compound (1).
X
Y
OH
O
O
EtO
EtO
O
X
Y
X
Y
a
N
H
+
OEt
Z
H2N
N
H
O
O
Z
Z
12
13
5, X, Y, Z = H, 35%
7, X = OMe Y, Z = H, 58% 10, X, Y = H, Z = F, 10%
11
9, X = F, Y, Z = H, 29%
8
, X = Me Y, Z = H, 47%
, X = H, Y = F, Z = H, 39%
Scheme 1. Conditions: (a) 170–195 °C, 6 h.
(dimethyl sulfate/cesium carbonate/DMF). Target compound 3 was
commercially available. Structural elucidation of the target com-
pounds was determined by 1H and 13C NMR and elemental analy-
sis; in some cases, they were further characterized by 2D NMR
(HMBC, HSQC, and COSY) (Tables S1 and S2). Assigning the 13C
NMR chemical shifts of 9–11 was assisted by calculation and com-
parison of the chemical shifts to those of 5 and known fluorinated
substituted benzenes.20 Our 2D-NMR data also confirm that 1 (2-
keto and 4-enol), 2 (2-keto), and 3 (4-enol) are the observed tau-
tomers for these target compounds.
The remaining docked pose adopted a conformation different from
those observed in modes A and B. These docking results suggest
that the lactam and the phenolic oxygen (4-OH) atoms form
important H-bonds in the PI3Ka binding pocket; in binding mode
A, the 4-OH and the amide carbonyl groups form two H-bonds with
Glu849 and Val851, whereas in binding mode B, the lactam func-
tional group forms H-bonds with the main chain NH and carbonyl
groups of Val851. Compound 1, with both lactam and the phenolic
functional groups, maximizes the H-bond interactions with PI3K,
and therefore binds more tightly than compounds 2, 3, and 4,
which have only one or none of these two essential functional
groups. The growth inhibition data (Table 1) support the impor-
tance of these interactions. The significantly diminished inhibition
of both the WT and MUT colon cancer cell lines by 6, the N-methyl
derivative of 5, confirms the important contribution of the lactam
3. Results and discussion
The growth inhibition of the HCT116 colon cancer cell lines
(both WT and H1047R MUT) by compounds 1–6 is shown in
Table 1. HCT116 is a highly malignant colon carcinoma cell line
NH to interaction with PI3K
The data for 5 shows that the benzylic carbon atom of 1 plays a
inhibition; although 5 is slightly less
a.
harboring both wild-type and mutant (H1047R) PI3Ka, and it
was produced from a primary tumor tissue culture.25 The colon
carcinoma cell lines containing only wild-type (WT) or mutant
less important role in PI3K
a
potent than 1, it is similar to LY29400218 in potency with some
selectivity for inhibition of the HCT116-MUT (H1047R) cell line. In-
trigued by this result, we envisioned that simple derivatives of 5
might have increased selectivity for inhibition of the HCT116-
MUT (H1047R) cell line.
H1047R (MUT) PI3K
a were prepared by asymmetrical knockout
of the MUT and WT genes, and are denoted as HCT116-WT and
HCT116-MUT, respectively.26
The one to two-order of magnitude loss of potency for 2–4 ver-
sus 1 reveal that the lactam and the phenolic oxygen atoms of 1 are
Glide docking studies of 5 and its derivatives 7–11 into WT (PDB
a
id:2RD0)4 and MUT (H1047R) PI3K (PDB id:3HHM)28 were used
required for good PI3Ka inhibition (Table 1), an outcome sup-
to guide molecule design. These data showed that target com-
pounds 7–11 show comparable docking scores to that of 5, sug-
gesting that they might have similar binding affinities to 5
(Table 2). Moreover, 1 and 5 match the same pharmacophoric
points (Fig. 3), suggesting that although 5 does not have the meth-
ylene group of 1, it occupies a similar binding pocket, in good
agreement with what we observed experimentally (Table 1). The
ported by our docking experiments (Fig. 2). To investigate the
binding mode of 1 to H1047R mutant and to investigate the neces-
sity of both the lactam and the phenolic groups, we applied in-
duced-fit docking27 of 1 to the H1047R mutant model. The top
ten docked poses from the induced-fit docking were clustered
and classified as either mode A or mode B. Four out of 10 docked
poses adopted mode A, and 5 out of 10 exhibited mode B (Fig. 2).
more negative docking scores for 5, 7, 9–11 toward the MUT PI3K
a
isoform suggested that these compounds might be mutant-selec-
tive. These computational data suggest that 7–11 may have similar
potencies yet better selectivities than those of the lead compound
1. Figure S1 showed that the calculated docking scores (kcal/mol)
are positively correlated with the experimentally obtained IC50s,
suggesting that docking scores could be used to predict ligand
binding affinities.
Table 1
Growth inhibition IC50
(lM) of the HCT116-WT and HCT116-MUT (H1047R) cell lines
Compounds
HCT116-WT
HCT116-MUT (H1047R)
LY294002a
6.7
1.1
420
24
5.3
1a
2
3
4
5
0.73
330
19
65
9.4
110
120
2.5
100
Table 3 shows that compounds 5, and 7–11 inhibit both WT and
MUT (H1047R) PI3Ka isoforms. As measured by the selectivity
6
index, they were generally more potent against the H1047R mu-
tant with IC50s in the same range of that of LY294002 (especially
a
IC50 data from Sabbah et al.18

D. A. Sabbah et al. / Bioorg. Med. Chem. 20 (2012) 7175–7183
7177
Figure 2. Two binding modes of 1 in the PI3Ka kinase catalytic site. (A) The first binding conformation: the C4 phenol and the amide carbonyl group bind to E849 and V851,
respectively. (B) The second binding conformation: the C2 (CO and NH) of the lactam bind to V851. The C atoms of 1 are depicted in green; the H atoms in white; the O atoms
in red; and the N atoms in blue.
Table 2
Table 3
Glide docking scores (kcal/mol) against the WT and MUT PI3K
a
Growth inhibition (IC50, lM) by N-phenyl-4-hydroxy-2-quinolone-3-carboxamides
Compound
Docking score (Kcal/mol)
Compounds HCT116-WT HCT116-MUT (H1047R) Selectivity index
(IC50 WT/IC50 MUT)
WT PI3K
a
(2RD0)
MUT (H1047R) PI3Ka (3HHM)
LY294002a
6.7
1.1
9.4
64
5.3
0.73
2.5
1.3
1.5
3.8
10
1
5
7
8
9
À12.0
À9.20
À7.30
À11.3
À10.1
À9.24
À9.63
À10.4
À9.97
À9.68
À10.6
À10.9
À10.3
À10.6
1a
5
7
6.3
8
9
10
11
27
15
22
6.5
5.6
6.9
3.6
0.28
4.8
2.2
6.0
23
10
11
a
IC50 data from Sabbah et al.18
For the HCT116-WT cell lines (both WT and MUT), all com-
pounds inhibited PI3K with activities comparable to that of
a
LY294002. Normalization of kinase inhibition effect (Fig. 4C and
D, and Table 4) was performed with LY294002 as a reference.
The immunoblot assay of pAKT-S473 and total AKT (Fig. 5)
showed that all compounds inhibited the formation of phosphory-
lated AKT at Ser473 (pAKT-S473) in the MUT cell lines compared
to DMSO. However, when compared to the reference compound
LY294002, which most potently inhibited pAKT-S473 formation in
HCT116-WT cells, only 2 and 5 inhibited the formation of pAKT-
S473 in both WT and MUT with activities comparable to those of
LY294002. It is intriguing to observe that 7–11 showed much weak-
er or no inhibition of pAKT-S473 formation in the WT cells com-
pared to that of LY294002. Compounds 7 and 8 showed similar
inhibition on pAKT-S473 in the MUT cells, whereas compounds 9–
11 had much weaker activities. However, 9–11 inhibited MUT cell
growth (Table 3) comparable to that of LY294002. This suggests that
growth inhibitory activities of 9–11 in both the HCT116 WT and
MUT cells might be mediated through alternative pathways not
involving AKT activation. Excluding 9–11, the other compounds at
the indicated concentrations inhibited the formation of pAKT-
S473 in the MUT cell lines comparable to that of LY294002. Similar
to LY294002, 5 inhibited pAKT-S473 formation in both WT and MUT
Figure 3. PI3Ka pharmacophore model with 1 (C atoms in green) and 5 (C atoms in
yellow).
for the H1047R mutant-expressing cells), a reference compound
used for PI3K inhibition studies. Of these, 7 and 11 showed the
highest selectivities.
In order to determine whether growth inhibition of the colon
cancer cell lines by 2–5 and 7–11 was mediated by the PI3K /
AKT pathway, we performed PI3Ka kinase assays and immunoblot
cells. The inhibition of WT PI3K
pAKT-S473 by 2 in the WT cells are surprising, because the IC50s of
cell growth inhibition (Table 1) of 2 were 420 and 330 M. In addi-
tion, none of the compounds had any effect on the expression of to-
tal AKT protein in either the WT or MUT cells indicating that they
would not be expected to interrupt the normal functions of AKT.
We also assessed whether growth inhibition of the colon cancer
cells by these compounds were mediated by an apoptotic
a and the inhibition of formation of
analyses of AKT activation in the HCT116-WT and HCT116-MUT
cell lines. Compounds were tested at a single concentration that
approximately corresponded to the range of the previously deter-
mined cell growth inhibition IC50 data (Tables 1 and 3). For in-
stance, larger concentrations were used in the pAKT and
apoptosis assays for compounds 2, 4, and 7 because of their higher
IC50s in growth inhibition assays.
l

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D. A. Sabbah et al. / Bioorg. Med. Chem. 20 (2012) 7175–7183
Figure 4. Direct inhibition of PI3K kinase activity by 2–5 and 7–11 in the WT (A) and H1047R MUT (B)1047R mutant (bottom). The areas of each band were calculated using
Image J software and compared to LY294002 and the band quantification for kinase inhibition is labeled for the WT (C) and MUT (D).
Table 4
Normalized activities against PI3K kinase and the apoptosis (cleaved PARP) using LY294002 (1.0-fold) as a reference, and the normalized activities against pAKT-S473 using DMSO
(1.0-fold) as a reference
.
PI3K Kinase
pAKT-S473
Cleaved PARP
HCT116-MUT
HCT116-WT
HCT116-MUT
HCT116-WT
HCT116-MUT
HCT116-WT
LY294002
5
7
8
9
10
11
3
4
2
1.00
0.89
0.82
0.75
0.65
0.69
0.75
0.56
0.70
0.14
1.00
0.71
0.64
0.79
0.59
0.75
0.90
0.88
0.87
0.80
0.01
0.11
1.66
1.66
0.95
1.60
1.01
1.00
1.59
0.09
0.41
0.40
0.44
0.41
0.86
1.23
0.51
0.20
0.30
0.13
1.00
0.01
0.01
1.00
1.01
0.88
0.40
0.71
0.81
0.92
1.00
4.99
3.90
1.14
1.37
5.05
1.35
0.20
0.06
0.05
Note: In PI3K kinase and pAKT-S473 assays, the smaller the number, the less PIP3 that was generated, the more PI3K
a that was inhibited, and thus the less AKT that was
phosphorylated. For the apoptosis assay, the larger the number, the more cleaved PARP that was generated, the more apoptosis that was induced.
mechanism by using a poly-(ADP-ribose) polymerase (PARP) cleav-
age detection assay in both HCT116-WT and MUT cells (Fig. 6). The
appearance of cleaved products of PARP (88 kDa) is widely used as
an indicator of apoptosis.29,30 Assessing PARP cleavage in the
HCT116-WT cells showed that, except for 5 and 7, all compounds
induced some apoptosis in the WT cell line. Interestingly,
LY294002 (10 lM) induced little apoptosis in HCT116-MUT cells,
whereas compounds 5, 7, and 10 were much more effective in
inducing apoptosis. However, compounds 8, 9 and 11 did not in-
duce much apoptosis in the MUT cells (Fig. 6).
To quantify the activities of tested compounds, we used Image J
software to calculate the areas of the bands in Figures 4–6 and

D. A. Sabbah et al. / Bioorg. Med. Chem. 20 (2012) 7175–7183
7179
Figure 5. Inhibition of total AKT and phosphorylated AKT at Ser473 (pAKT-S473) by 2–5 and 7–11 in the WT (A) and the H1047R MUT (B) 1047R mutant (bottom). The areas
of each band were calculated using Image J software and compared to LY294002 and the band quantification for pAKT formation is labeled for the WT (C) and MUT (D). The
compounds in X-axis in C and D are the same.
Figure 6. PARP cleavage by 2–5 and 7–11 in WT (top) and the H1047R MUT (bottom) 1047R mutant (bottom). The areas of each band were calculated using Image J software
and compared to LY294002 and the band quantification for cleaved PARP formation is labeled for the WT (C) and MUT (D). The compounds in X-axis in C and D are the same.
quantified each band in comparison to LY294002 for the PI3K
kinase and PARP cleavage tests, and DMSO for the AKT assay
(Table 4). Table 4 showed that 5 inhibited PI3K kinase and
formation of pAKT in both WT and MUT cells, but it induced apop-
tosis only in MUT cells. Table 4 also indicated that 7 and 10 in-
duced more apoptosis in the MUT cells than LY294002. All three
assays indicated that 7–11 were weaker inhibitors of PI3K kinase
and pAKT formation than LY294002, suggesting that mechanisms
other than the PI3K /AKT pathway may be involved to account
for the observed apoptosis and/or growth inhibition.
In order to determine the structural basis for PI3K
these compounds, we employed induced fit docking (IFD) against
the WT PI3K (2RD0) and MUT (H1047R) PI3K
(3HHM).26,27 The
a inhibition by
a
a
IFD approach was proposed by Sherman et al.31,32 and has been suc-
cessfully applied in our study of epidermal growth factor receptor
(EGFR) inhibitors.33 The IFD approach takes into consideration the
conformational changes in proteins by the following method:
ligands are docked to a protein in a rigid docking using softened-
potential docking with the van der Waals radii for protein atoms
at a 0.7 scaling using the Glide program27,34,35 and the top ligand

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D. A. Sabbah et al. / Bioorg. Med. Chem. 20 (2012) 7175–7183
Figure 7. Binding conformations of 7 in (A) the wild-type PI3Ka (PDB ID:2RD0) and (B) mutant (H1047R) PI3Ka (PDB ID:3HHM) binding domains. H-bonds are depicted in
dotted blue lines.
poses are then subjected to a minimization along with protein res-
idues within 5 Å of ligands using the Prime program,27 followed by
a redocking procedure against the minimized protein. In this way,
protein plasticity can be taken into account during the whole dock-
ing process.
Our IFD docking data for 1, 5, and 7–11 show that these com-
pounds bind to the kinase cleft of both WT and MUT PI3Ka, partic-
ularly with the backbones of Tyr836, Glu849, Val851, Gln859, and
Asp933. Other computational19,36 and experimental28 data also
showed the significance of these key binding residues, especially
Asp933. Our previous docking studies suggested the importance
5. Experimental
5.1. Chemistry
1D-NMR spectra were recorded with a Varian INOVA 500 MHz
spectrometer. Chemical shifts are expressed in d units using the
residual DMSO and CDCl3 signals as the standard when measured
in DMSO-d6 and CDCl3, respectively. 2D-NMR spectra were mea-
sured with Bruker Avance III 400-MHz spectrometer. Melting
points were determined using Ez-melt apparatus. Elemental anal-
yses were recorded on a PerkinElmer 2400 II series CHN analyzer
and performed by M-H-W Labs, Phoenix, AZ.
of Gln859 in designing mutant and/or isoform-specific PI3K
inhibitors; the Gln859 in PI3K corresponds to Lys890 in PI3K
Consistent with this hypothesis (Fig. 7), 7 has a 10-fold selec-
tivity against MUT PI3K over WT PI3K and forms a H-bond with
Gln859 of MUT H1047R. The induced-fit docking of 7 to the WT
and MUT PI3K showed that 7 forms three H-bonds with Val851
a
a
c 19
.
5.1.1. N-Benzyl 2-hydroxyquinoline-3-carboxamide (2)
a
a
2-Hydroxyquinoline-3-carboxylic acid (95 mg, 0.5 mmol), ben-
zylamine (64 mg, 0.6 mmol), 1-hydroxybenzotriazole (68 mg,
0.5 mmol), and triethylamine (152 mg, 1.5 mmol) were dissolved
in dichloromethane (5 ml) and acetonitrile (10 ml). After the addi-
tion of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (288 mg, 1.5 mmol), the mixture was stirred for 24 h at
rt. The crude precipitate was filtered and washed by water, then
by acetonitrile to give 2 as a white precipitate (94 mg, 68%); mp
248–250 °C; 1H NMR (500 MHz, DMSO-d6) d 3.94 (s, 2H), 7.27–
7.43 (m, 6H), 7.52 (d, J = 8.3 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.88
(d, J = 7.8 Hz, 1H), 8.64 (s, 1H); 13C NMR (125.7 MHz, DMSO-d6) d
42.60, 118.01, 119.38, 120.73, 123.16, 128.22, 128.56, 128.63,
129.68, 132.50, 142.63, 143.56, 164.62, 166.22.
a
and Gln859 of the MUT whereas it forms two H-bonds with
Glu849 and Val851 in the WT.
4. Conclusions
Using both WT and H1047R MUT HCT116 colon cancer cell
lines, this preliminary investigation of lead PI3K
vealed that the lactam and the phenolic oxygen atoms are required
for good PI3K inhibition, an outcome supported by docking
a inhibitor 1 re-
a
experiments. We also discovered that N-phenyl-4-hydroxy-2-
quinolone-3-carboxamides 5 and 7–11 were selective inhibitors
of the MUT (H1047R) versus WT PI3Ka and also showed that the
5.1.2. N-Benzyl quinoline-3-carboxamide (4)
cell growth inhibition of 5 likely occurs by inhibiting the formation
of pAKT and induction of apoptosis in MUT cells. The cell growth
inhibition by 7–11, however, is not mediated through the PI3K /
AKT pathway. Whether differences in cellular bioavailability im-
pact the activity differences between cell and enzyme assays re-
mains to be determined. Induced fit docking (IFD) studies
identified key residues that contribute to inhibitor binding to
3-Quinolinecarboxylic acid (87 mg, 0.5 mmol), benzylamine
(64 mg, 0.6 mmol), 1-hydroxybenzotriazole (68 mg, 0.5 mmol),
and triethylamine (152 mg, 1.5 mmol) were dissolved in acetoni-
trile (10 ml) for 5 min. After the addition of 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide hydrochloride (288 mg, 1.5 mmol), the
mixture was stirred for 24 h at rt. The acetonitrile was then evap-
orated and water was added. After filtration and drying, 4 was iso-
lated as a white crystalline solid (192 mg, 73%); mp 141–143 °C;
PI3Ka
. Interestingly, 523,24,37–39 was previously identified as an
anti-angiogenic agent24,40,41 and was investigated for the treat-
ment of choroidal neovascularization.39 In addition, compounds
with the core structure of 5 decreased tumor volume, induced
apoptosis, and generated hypoxia-induced death in human pros-
tatic carcinoma cells39 and had in vivo antitumor effects in rat
and human prostate cancers42–44 and in Lewis lung cell carcinoma
cells.45
Lit. mp 139–144°C43–45 1H NMR (500 MHz, CDCl3) d 4.72 (d,
;
J = 5.9 Hz, 2H), 6.79 (s, 1H), 7.31–7.45 (m, 5H), 7.61 (t, J = 7.3 Hz,
1H), 7.78 (d, J = 7.3 Hz, 1H), 7.87 (t, J = 7.8 Hz, 1H), 8.13 (d,
J = 8.8 Hz, 1H), 8.60 (d, J = 1.5 Hz, 1H), 9.27 (d, J = 2 Hz,1H); 13C
NMR (125.7 MHz, CDCl3) d 44.34, 126.86, 126.88, 127.55, 127.85,
128.04, 128.72, 128.90, 129.39, 131.30, 135.61, 137.76, 148.10,
149.28, 165.49.

D. A. Sabbah et al. / Bioorg. Med. Chem. 20 (2012) 7175–7183
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5.1.3. N-Phenyl 4-hydroxy-2-quinolone-3-carboxamide (5)
Triethyl methanetricarboxylate (232 mg, 1 mmol) and aniline
(93 mg, 1 mmol) were mixed and heated at 170–195 °C for six
hours. The reaction was cooled to rt and treated with ethanol, fil-
tered and washed with ethanol to give 5 as a yellow sandy com-
170–190 °C. The reaction was cooled to rt and treated with aceto-
nitrile, filtered and washed with acetonitrile to afford 10 as a yel-
low solid (114 mg, 29%); mp 334–336 °C; 1H NMR (500 MHz,
DMSO-d6) d 7.27 (t, J = 8.5 Hz, 2 H), 7.46–7.51 (m, 1H), 7.62–7.76
(m, 4H), 12.18 (s, 1H), 12.60 (s,1H), 16.43 (s,1H); 13C NMR
2
pound (98 mg, 35%); mp 303–305 °C; Lit. Mp 298–305°C32,34
;
1H
(125.7 MHz, DMSO-d6) d 97.03, 108.69 (d, JCF = 24.0 Hz), 114.85
NMR (500 MHz, DMSO-d6) d 7.21 (t, J = 7.3 Hz, 1H), 7.34 (t,
J = 7.8 Hz, 1H), 7.43–7.45 (m, 3H), 7.66 (t, J = 7.8 Hz, 2H), 7.74 (t,
J = 8.1 Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), 12.10 (s, 1H), 12.66 (s, 1H),
16.48 (s, 1H); 13C NMR (125.7 MHz, DMSO-d6) d 97.29, 114.89,
116.71, 121.49, 123.41, 124.76, 125.58, 129.92, 134.94, 137.51,
139.42, 163.51, 169.75, 173.06.
(d, 4JCF = 8.6 Hz), 115.82 (d, 2JCF = 24.2 Hz), 115.82 (d,
3
2
2JCF = 24.2 Hz), 118.36 (d, JCF = 8.2 Hz), 122.71 (d, JCF = 25 Hz),
122.86 (d, 3JCF = 8.2 Hz), 122.89 (d, 3JCF = 8.2 Hz), 132.97
(4JCF = 3 Hz), 135.46 (4JCF <2 Hz), 157.37(d, JCF = 240.4 Hz), 159.0
1
1
4
(d, JCF = 241.8 Hz), 162.45, 168.69, 171.39 (d, JCF = 5.0 Hz). Anal.
for C16H10 F2N2O3: Calcd C, 60.76; H, 3.19; N, 8.86. Found C,
61.00; H, 3.28; N, 8.79.
5.1.4. N-Phenyl 4-hydroxy-1-methyl-2-quinolone-3-
carboxamide (6)
5.1.8. N-(2-Fluorophenyl) 8-fluoro-4-hydroxy-2-quinolone-3-
carboxamide (10)
A mixture of 5 (180 mg, 0.64 mmol), Cs2CO3 (651 mg, 2 mmol)
and Me2SO4 (253 mg, 2 mmol) in DMF (5 mL) under Ar was heated
at 120 °C for 48 h. After cooling to rt, the reaction mixture was di-
luted with water (50 mL). The resulting precipitate was collected
by filtration and dried to give 6 as a white solid (118 mg, 63%).
2-Fluoroaniline (111 mg, 1 mmol) and triethyl methanetricarb-
oxylate (232 mg, 1 mmol) were mixed and heated for six hours at
170–190 °C. The reaction was cooled to rt and treated with ethanol,
filtered and washed with chloroform to afford 11 as a yellow solid
(20.3 mg, 10%); mp 288–290 °C; 1H NMR (500 MHz, DMSO-d6) d
7.21–7.46 (m, 4H), 7.68 (t, J = 8.5 Hz,1H), 7.87 (d, J = 8.1 Hz, 1H),
8.30 (t, J = 7.26 Hz,1H), 12.24 (s, 1H), 12.86 (s, 1H), 16.33 (s, 1H);
;
Mp 194–196 °C; Lit. Mp 196–198 °C34,35 1H NMR (500 MHz,
DMSO-d6) d 3.70 (s, 3H), 7.21 (t, J = 7.5 Hz, 1H), 7.42 (t, J = 8.0 Hz,
2H), 7.44 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 7.7 Hz, 2H), 7.70 (t,
J = 7.9 Hz, 1H), 7.86 (t, J = 7.8 Hz, 1H), 8.15 (d, J = 7.6 Hz, 1H),
12.69 (s, 1H), 16.60 (s, 1H); 1H NMR (500 MHz, CDCl3) d 3.74 (s,
3H), 7.17 (t, J = 7.4 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 7.37–7.42 (m,
3H), 7.69 (d, J = 7.9 Hz, 2H), 7.72 (t, J = 8.3 Hz, 1H), 8.26 (d,
J = 8.3 Hz, 1H), 12.52 (s, 1H), 16.75 (s, 1H); 13C NMR (125.7 MHz,
CDCl3) d 29.34, 97.18, 114.31, 116.25, 121.28, 122.63, 124.83,
125.63, 129.02, 134.00, 137.20, 139.88, 162.84, 169.38, 172.24.
2
13C NMR (125.7 MHz, DMSO-d6) d 97.66, 115.66 (d, JCF = 18.7 Hz),
3
2
116.44 (d, JCF = 3.8 Hz), 119.44 (d, JCF = 16.8 Hz), 120.14 (d,
4JCF = 3.8 Hz), 122.78 (3JCF < 2 Hz), 122.85 (d, JCF = 6.9 Hz), 125.07
3
4
2
3
(d, JCF = 3.4 Hz), 125.20 (d, JCF = 10.6 Hz), 125.96 (d, JCF = 7.7 Hz),
2
1
127.95 (d, JCF = 14.8 Hz), 149.27 (d, JCF = 247.1 Hz), 153.01 (d,
1JCF = 244.7 Hz), 162.97, 169.29, 172.14 (d, JCF = 3.4 Hz). Anal. for
4
C16H10F2N2O3: Calcd C, 60.76; H, 3.19; N, 8.86. Found C, 60.92; H,
3.39; N, 8.64.
5.1.5. N-(4-Methoxyphenyl) 4-hydroxy-6-methoxy-2-quinolone-
3-carboxamide (7)
5.1.9. N-(3-Fluorophenyl) 7-fluoro-4-hydroxy-2-quinolone-3-
carboxamide (11)
p-Methoxyaniline (123 mg, 1 mmol) was dissolved in dimethyl-
formamide (1 ml). Then, triethyl methanetricarboxylate (232 mg,
1 mmol) was added. The mixture was heated for six hours at
170–190 °C. The reaction was cooled to rt and treated with ethanol,
filtered and washed with ethanol to yield 8 as a yellow solid
(143 mg, 58%); mp 308–310 °C; 1H NMR (500 MHz, DMSO-d6) d
3.79 (s, 3H), 3.86 (s, 3H), 7.01 (d, J = 8.9 Hz, 2H), 7.40 (br s, 3H),
7.59 (d, J = 8.9 Hz, 2H), 12.01 (s, 1H), 12.63 (s, 1H), 16.67 (s, 1H);
13C NMR (125.7 MHz, DMSO-d6) d 55.09, 55.38, 96.39, 104.22,
114.13, 114.51, 117.33, 122.13, 123.65, 129.58, 133.11, 154.53,
156.26, 161.98, 168.39, 171.37; Anal. for C18H16N2O5: Calcd C,
63.52; H, 4.74; N, 8.23. Found C, 63.66; H, 5.00; N, 8.09.
3-Fluoroaniline (111 mg, 1 mmol) and triethyl methanetricarb-
oxylate (232 mg, 1 mmol) were mixed and heated for six hours at
170–190 °C. The reaction was cooled to rt and treated with ethanol,
filtered and washed with ethanol to yield 12 as a yellow solid
(123 mg, 39%); mp 329–331 °C; 1H NMR (500 MHz, DMSO-d6) d
7.05 (t, J = 8.1 Hz, 1H), 7.15 (d, J = 9.8 Hz, 1H), 7.22 (t, J = 8.5 Hz,
1H), 7.39 (d, J = 7.7 Hz, 1H), 7.41–7.48 (m, 1H), 7.67 (d, J = 11 Hz,
1H), 8.07 (t, J = 8.1 Hz, 1H), 12.18 (s,1H), 12.60 (s, 1H), 16.23
(s,1H); 13C NMR (125.7 MHz, DMSO-d6)
d 96.24, 102.06 (d,
2
2JCF = 25.0 Hz), 107.98 (d, JCF = 25.4 Hz), 111.37, 111.66 (d,
2
2JCF = 21.3 Hz), 111.66 (d, JCF = 23.4 Hz), 116.87 (4JCF = 2.4 Hz),
3
3
127.66 (d, JCF = 11.9 Hz), 131.03 (d, JCF = 10.2 Hz), 138.54 (d,
3JCF = 11.1 Hz), 140.79 (d, 3JCF = 12.5 Hz), 162.39 (d, 1JCF = 244.0 Hz),
163.19, 165.54 (d, 1JCF = 250.0 Hz), 169.36, 172.18. Anal. for C16H10
F2N2O3: Calcd C, 60.76; H, 3.19; N, 8.86. Found C, 61.00; H, 3.31; N,
8.88.
5.1.6. N-p-Tolyl 4-hydroxy-6-methyl-2-quinolone-3-
carboxamide (8)
p-Toluidine (107 mg, 1 mmol) was dissolved in dimethylform-
amide (1 ml). Then, triethyl methanetricarboxylate (232 mg,
1 mmol) was added. The mixture was heated for six hours at
170–190 °C. The reaction was cooled to rt and treated with etha-
nol. The precipitate was washed with chloroform to give 9 as a yel-
low solid (137 mg, 47%); mp 324–326 °C; 1H NMR (500 MHz,
DMSO-d6) d 2.33 (s, 3H), 2.42 (s, 3H), 7.24 (d, J = 8.1 Hz, 2H), 7.36
(d, J = 8.1 Hz, 1H), 7.5–7.6 (m, 3H), 7.82 (s, 1H), 11.86 (s, 1H),
12.59 (s, 1H), 16.48 (s, 1H); 13C NMR (125.7 MHz, DMSO-d6) d
20.14, 96.27, 113.89, 115.64, 120.50, 122.90, 129.22, 131.60,
133.72, 134.07, 135.12, 136.52, 162.38, 168.65, 171.87. Anal. for
5.2. Computational methods
5.2.1. Preparation of protein structures
The coordinates of native PI3K
(H1047R) PI3K
/wortmannin complex (PDB code:3HHM),28 and
PI3K
/Ly294002 complex (PDB id:IE7V)46 were retrieved from
a
(PDB id:2RD0),4 mutant
a
c
the RCSB Protein Data Bank. The homology modeling module in
MOE47 was recruited to build the missing residues of 2RD0 and
1E7V. 3HHM has the same missing regions of 2RD0. Thus, 2RD0
was used as a template to fill up the missing gaps of 3HHM apply-
ing a previously described approach.19 First, structural alignment
of 3HHM to 2RD0 was performed using DaliLite48 module of EBI
tools. Second, the homology modeling generated residues of
2RD0 were adopted for 3HHM. Third, each inserted missing se-
quence and its surrounding residues (within 4.5 Å of the modeled
C18H16N2O3: Calcd C, 70.12; H, 5.23; N, 9.09. Found C, 70.44; H,
5.24; N, 9.00.
5.1.7. N-(4-Fluorophenyl) 6-fluoro-4-hydroxy-2-quinolone-3-
carboxamide (9)
4-Fluoroaniline (111 mg, 1 mmol) and triethyl methanetricarb-
oxylate (232 mg, 1 mmol) were mixed and heated for six hours at

7182
D. A. Sabbah et al. / Bioorg. Med. Chem. 20 (2012) 7175–7183
section) were energetically minimized to decrease steric clash.
Fourthly, the treated proteins were prepared using the Protein
Preparation27 wizard in the Schrödinger software suite to maxi-
mize H-bond interactions.
temperature. After further washing in TBST for 30 min, the proteins
were detected by the enhanced chemiluminescence (ECL) system
(Amersham).
Acknowledgments
5.2.2. Preparation of ligand structures
Test compounds (ligands) were built based on the coordinates
of wortmannin in 3HHM. The ligands were built using MAESTRO
build panel and subsequently minimized by MacroModel program
using the OPLS2005 force field.
This work was partially supported by a FIRE Grant from the
University of Nebraska at Omaha. DAS acknowledges Al-Zaytoonah
Private University of Jordan, and a Bukey Fellowship from the
University of Nebraska Medical Center for financial support. NAS
and MGB acknowledge NIH (CA72001 and CA38173) for support.
5.2.3. Glide docking
Two grid files for 2RD0 and 3HHM were generated using the
Glide Grid Generation protocol using the bound ligands as
centroids. The scaling factor for van der Waals receptor for the
non-polar atoms was calibrated to 0.8 to furnish some flexibility.
All other parameters were used as defaults.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.09.059.
References and notes
5.2.4. Induced-fit docking (IFD)
The ligand was defined as a centroid in the kinase binding cleft.
Both receptor and ligand Vander Waals scaling factors were set to
0.5 to allow enough flexibility for the best docked ligand pose.
Other settings were determined as default. The ligand pose with
the highest XP Glide score was reported.
1. Vanhaesebroeck, B.; Waterfield, M. D. Exp. Cell Res. 1999, 253, 239.
2. Vanhaesebroeck, B.; Guillermet-Guibert, J.; Graupera, M.; Bilanges, B. Nat. Rev.
Mol. Cell Biol. 2010, 11, 329.
3. Cantley, L. C. Science 2002, 296, 1655.
4. Huang, C.; Mandelker, D.; Schmidt-Kittler, O.; Samuels, Y.; Velculescu, V. E.;
Kinzler, K. W.; Vogelstein, B.; Gabelli, S. B.; Amzel, L. M. Science 2007, 318, 1744.
5. Zhao, L.; Vogt, P. K. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2652.
6. Samuels, Y.; Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.; Szabo, S.; Yan, H.;
Gazdar, A.; Powell, D.; Riggins, G.; Willson, J.; Markowitz, S.; Kinzler, K.;
Vogelstein, B.; Velculescu, V. Science 2004, 304, 554.
5.3. Biological assays
7. Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Nat. Rev. Drug Disc. 2009, 8, 627.
8. Carson, J. D.; Van Aller, G.; Lehr, R.; Sinnamon, R. H.; Kirkpatrick, R. B.; Auger, K.
R.; Dhanak, D.; Copeland, R. A.; Gontarek, R. R.; Tummino, P. J.; Luo, L. Biochem.
J. 2008, 409, 519.
9. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi, T.; Okada, M.;
Ohta, M.; Tsukamoto, S.; Parker, P.; Workman, P.; Waterfield, M. Bioorg. Med.
Chem. 2006, 14, 6847.
10. Hayakawa, M.; Kawaguchi, K.; Kaizawa, H.; Tomonobu, K.; Ohishi, T.; Yamano,
M.; Okada, M.; Ohta, M.; Tsukamoto, S.; Raynaud, F. I.; Parker, P.; Workman, P.;
Waterfield, M. D. Bioorg. Med. Chem. 2007, 15, 5837.
5.3.1. Cell lines and reagents
HCT116, a human colon carcinoma cell line, was established in
tissue culture from a primary tumor as previously described.25 We
prepared cell lines and obtained reagents as described in Refs. 17
(Supplementary data), and 21. Immunoblot analysis antibodies,
poly (ADP-ribose) polymerase (PARP),49 AKT, and phosphorylated
AKT (Ser473), were obtained from cell signaling technology.
11. Hayakawa, M.; Kaizawa, H.; Kawaguchi, K.; Ishikawa, N.; Koizumi, T.; Ohishi,
T.; Yamano, M.; Okada, M.; Ohta, M.; Tsukamoto, S.; Raynaud, F. I.; Waterfield,
M. D.; Parker, P.; Workman, P. Bioorg. Med. Chem. 2007, 15, 403.
12. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi, T.; Yamano, M.;
Okada, M.; Ohta, M.; Tsukamoto, S.; Raynaud, F. I.; Workman, P.; Waterfield, M.
D.; Parker, P. Bioorg. Med. Chem. Lett. 2007, 17, 2438.
13. Knight, S. D.; Adams, N. D.; Burgess, J. L.; Chaudhari, A. M.; Darcy, M. G.;
Donatelli, C. A.; Luengo, J. I.; Newlander, K. A.; Parrish, C. A.; Ridgers, L. H.;
Sarpong, M. A.; Schmidt, S. J.; Van Aller, G. S.; Carson, J. D.; Diamond, M. A.;
Elkins, P. A.; Gardiner, C. M.; Garver, E.; Gilbert, S. A.; Gontarek, R. R.; Jackson, J.
R.; Kershner, K. L.; Luo, L.; Raha, K.; Sherk, C. S.; Sung, C.; Sutton, D.; Tummino,
P. J.; Wegrzyn, R. J.; Auger, K. R.; Dhanak, D. ACS Med. Chem. Lett. 2010, 1, 39.
14. Heffron, T. P.; Wei, B.; Olivero, A.; Staben, S. T.; Tsui, V.; Do, S.; Dotson, J.;
Folkes, A. J.; Goldsmith, P.; Goldsmith, R.; Gunzner, J.; Lesnick, J.; Lewis, C.;
Mathieu, S.; Nonomiya, J.; Shuttleworth, S.; Sutherlin, D. P.; Wan, N. C.; Wang,
S.; Wiesmann, C.; Zhu, B.-Y. J. Med. Chem. 2011, 54, 7815.
5.3.2. Cell growth inhibition
Cell growth was evaluated by an MTT [3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide] (Sigma–Aldrich, St. Louis,
MO) assay using a 96-well plate read at 570 nm absorbance on
microplate reader (Biotek, Winooski, VT) using Gen5 software as
fully described in Ref. 17 (Supplementary data).
5.3.3. PI3K kinase assay
The cells were washed with PBS and lysed in lysis buffer, fol-
lowing the procedures described in Ref. 17 (Supplementary data).
5.3.4. Immunoblot analysis
Cells were lysed in TNESV lysis buffer [50 mmol/L Tris (pH 7.5),
150 mmol/L NaCl, 1% NP40, 50 mmol/L NaF, 1 mmol/L Na3VO4,
15. Kong, D.; Yamori, T. Curr. Med. Chem. 2009, 16, 2839.
16. Wu, P.; Hu, Y. Curr. Med. Chem. 2010, 17, 4326.
17. Sabbah, D. A.; Brattain, M. G.; Zhong, H. Curr. Med. Chem. 2011, 18, 5528.
18. Sabbah, D. A.; Simms, N. A.; Brattain, M. G.; Vennerstrom, J. L.; Zhong, H. Bioorg.
Med. Chem. Lett. 2012, 22, 876.
19. Sabbah, D. A.; Vennerstrom, J. L.; Zhong, H. J. Chem. Inf. Model. 2010, 50, 1887.
20. Kametani, T.; Kigasawa, K.; Hiiragi, M. Yakugaku Zasshi 1965, 85, 871.
21. Piechaczek, J.; Bojarska-Dahlig, H. Acta Pol. Pharm. Drug Res. 1966, 23, 7.
22. Billman, J. H.; Rendall, J. L. J. Am. Chem. Soc. 1944, 66, 540.
23. Ukrainets, I. V.; Tkach, A. A.; Sidorenko, L. V.; Gorokhova, O. V. Chem. Heterocycl.
Compd. 2006, 42, 1301.
25 lg/mL h-glycerophosphate, 1 mmol/L phenylmethylsulfonyl
fluoride, one protease inhibitor cocktail tablet (Roche, Indianapolis,
IN) per 10 mL] for 30 min on ice. The supernatants were then col-
lected by centrifugation for 15 min. Protein was determined by the
Pierce BSA method. Protein samples were dissolved in 1x sample
buffer (50 mM Tris, pH6.8, 1% SDS, 10% glycerol, 0.03% bromophe-
24. Shi, J.; Xiao, Z.; Ihnat, M. A.; Kamat, C.; Pandit, B.; Hu, Z.; Li, P.-K. Bioorg. Med.
Chem. Lett. 2003, 13, 1187.
25. Brattain, M. G.; Levine, A. E.; Chakrabarty, S.; Yeoman, L. C.; Willson, J. K. V.;
Long, B. Cancer Metastasis Rev. 1984, 3, 177.
26. Samuels, Y.; Diaz, L. A.; Schmidt-Kittler, O.; Cummins, J. M.; DeLong, L.; Cheong,
I.; Rago, C.; Huso, D. L.; Lengauer, C.; Kinzler, K. W.; Vogelstein, B.; Velculescu,
V. E. Cancer Cell 2005, 7, 561.
27. Protein Preparation Wizard, Maestro, MacroModel, Phase, Induced Fit, Jaguar,
and Glide; Schrödinger, LLC: Portland, OR, 2009.
28. Mandelker, D.; Gabelli, S. B.; Schmidt-Kittler, O.; Zhu, J.; Cheong, I.; Huang, C.;
Kinzler, K. W.; Vogelstein, B.; Amzel, L. M. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
16996.
29. Boulares, A. H.; Yakovlev, A. G.; Ivanova, V.; Stoica, B. A.; Wang, G.; Iyer, S.;
Smulson, M. J. Biol. Chem. 1999, 274, 22932.
nol blue and 1% b-mercaptoethanol). Protein (10–50 lg) was frac-
tionated on a 10% acrylamide denaturing gel and transferred onto a
nitrocellulose membrane (Life Science, Amersham) by electroblot-
ting. The membrane was blocked with 5% nonfat dry milk in TBST
[50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.05% Tween 20] for
1 h at rt or overnight at 4 °C and washed in TBST. The membrane
was then incubated with primary antibodies at 1:1000 dilutions
for 1 h at rt or overnight at 4 °C. After washing with TBST for
30 min, the membranes were then incubated with peroxidase-
conjugated goat anti-mouse or anti-rabbit IgG (Jackson Immuno
Research Laboratories, Inc) at a 1:1000 dilution for 1 h at room

D. A. Sabbah et al. / Bioorg. Med. Chem. 20 (2012) 7175–7183
7183
30. Germain, M.; Affar, E. B.; D’Amours, D.; Dixit, V. M.; Salvesen, G. S.; Poirier, G. G.
J. Biol. Chem. 1999, 274, 28379.
40. Vukanovic, J.; Passaniti, A.; Hirata, T.; Traystman, R. J.; Hartley-Asp, B.; Isaacs, J.
T. Cancer Res. 1993, 53, 1833.
31. Sherman, W.; Beard, H. S.; Farid, R. Chem. Biol. Drug Des. 2006, 67, 83.
32. Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.; Farid, R. J. Med. Chem.
2006, 49, 534.
33. Zhong, H.; Tran, L. M.; Stang, J. L. J. Mol. Graphics Modell. 2009, 28, 336.
34. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.
35. Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.;
Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. J. Med. Chem. 2006, 49, 6177.
36. Li, Y.; Wang, Y.; Zhang, F. J. Mol. Model. 2010, 16, 1449.
37. Richter, R.; Ulrich, H. J. Org. Chem. 1979, 44, 4877.
41. Joseph, I. B. J. K.; Vukanovic, J.; Isaacs, J. T. Cancer Res. 1996, 56, 3404.
42. Vukanovic, J.; Hartley-Asp, B.; Isaacs, J. T. Prostate 1995, 26, 235.
43. Vukanovic, J.; Isaacs, J. T. Cancer Res. 1995, 55, 1499.
44. Vukanovic, J.; Isaacs, J. T. Cancer Res. 1995, 55, 3517.
45. Borgstrom, P. Torres Filho, I. P.; Hartley-Asp,
719.
B Anticancer Res. 1995, 15,
46. Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.; Hawkins, P. T.; Wymann,
M. P.; Williams, R. L. Mol. Cell 2000, 6, 909.
47. The Molecular Operating Environment (MOE); Chemical Computing Group
Inc.: Montreal, QC, Canada, 2009.
48. Holm, L.; Park, J. Bioinformatics 2000, 16, 566.
38. Ukrainets, I. V.; Taran, S. G.; Gorokhova, O. V.; Taran, E. A.; Jaradat, N. A.;
Petukhova, I. Y. Chem. Heterocycl. Compd. 2000, 36, 166.
49. Hayashi, K.; Tanaka, M.; Shimada, T.; Miwa, M.; Sugimura, T. Biochem. Biophys.
Res. Commun. 1983, 112, 102.
39. Abdel-Rahman, M. H.; Yang, Y.; Salem, M. M.; Meadows, S.; Massengill, J. B.; Li,
P.-K.; Davidorf, F. H. Exp. Eye Res. 2010, 91, 837.