5-Ureidobenzofuranone indoles as potent and efficacious inhibitors of PI3 kinase-α and mTOR for the treatment of breast cancer

Article abstract of DOI:10.1016/j.bmcl.2010.04.139

A series of 5-ureidobenzofuran-3-one indoles as potent inhibitors of PI3Kα and mTOR has been developed. The best potency in cells was obtained when the urea group was extended to a 4-[2-(dimethylamino)ethyl]methylamino amidophenyl group. A 7-fluoro group

Full text of DOI:10.1016/j.bmcl.2010.04.139

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3526–3529
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Ureidobenzofuranone indoles as potent and efficacious inhibitors of PI3
kinase-a and mTOR for the treatment of breast cancer
a,
a
a
a
Nan Zhang a, , Semiramis Ayral-Kaloustian , James T. Anderson , Thai Nguyen , Sasmita Das ,
Aranapakam M. Venkatesan a, Natasja Brooijmans b, Judy Lucas c, Ker Yu c, Irwin Hollander c,
Robert Mallon c
*
*
a Discovery Medicinal Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
b Computational Chemistry, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
c Oncology Research, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 5-ureidobenzofuran-3-one indoles as potent inhibitors of PI3K
a and mTOR has been devel-
Received 9 February 2010
Revised 26 April 2010
Accepted 28 April 2010
Available online 17 May 2010
oped. The best potency in cells was obtained when the urea group was extended to a 4-[2-(dimethyl-
amino)ethyl]methylamino amidophenyl group. A 7-fluoro group on the indole ring also enhanced
cellular potency. Compound 18i, incorporating the optimal functional groups, showed high potency in
cellular lines and was further studied in vivo. It was able to inhibit the biomarker phosphorylation for
8 h when dosed at 25 mg/kg iv. In the MDA-MB-361 breast cancer model, it shrank the tumor size
remarkably when dosed at 25 mg/kg iv on days 1, 5, and 9.
Keywords:
PI3K inhibitor
mTOR inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
The phosphoinositol 3-kinase (PI3K) signaling pathway plays an
important role in mitogenic signaling, cell survival, cell growth, pro-
liferation, and metabolic control. PI3Ks (a, b, c, d) are a family of en-
mTOR. As indicated by modeling based on the PI3Kc crystal struc-
ture, the ureido moiety can mimic the hydroxyl binding interactions
in the active site and capture further interaction common to both
PI3K and mTOR binding. It has the advantage that it is not subject
to glucuronidation as observed in the hydroxyl series, thus affording
the compound higher metabolic stability. We now report a series of
zymes, which phosphorylate the 30-OH position of the inositol ring
of phosphatidylinositol-4,5-bisphosphate (PIP2).1–3 Activation of
the PI3K signaling cascade has a positive effect on cell growth, sur-
vival, and proliferation. Constitutive up-regulation of PI3K signaling
is observed in several types of cancers, and can have a deleterious
effect on cells, leading to uncontrolled proliferation, enhanced
migration, and adhesion-independent growth. These events favor
not only the formation of malignant tumors, but also development
of inflammatory and autoimmune diseases.1–3 The mammalian tar-
get of rapamycin (mTOR) is a key component of the PI3K signaling
pathway.4,5 It shares high sequence similarity with PI3K at the ATP
binding site. Therefore, blocking the PI3K/AKT signaling pathway
5-ureidobenzofuran-3-one indoles as potent inhibitors of PI3K
aand
mTOR.
The synthesis of 1-(3-oxo-2,3-dihydro-1-benzofuran-5-yl)urea
(5) is shown in Scheme 1. Starting from 2-hydroxy-5-nitroaceto-
O
O
NO2
Br
NO2
b
a
c
HO
O
by inhibiting the lipid kinase PI3Ka (which phosphorylates PIP2 to
HO
1
2
PIP3) and/or mTOR provides a promising new strategy for cancer
therapy. Active-site dual inhibitors of PI3K/mTOR have the potential
for enhanced efficacy.5,6
O
NO2
d
NH2
We recently reported a series of 4,6-dihydroxybenzofuran-3-one
O
O
a
and mTOR.7 This work was further
O
indoles as inhibitors of PI3K
3
4
extended to hydroxybenzofuran-3-one 7-azaindoles8 and
ureidobenzofuran-3-one 7-azaindoles9 as selective inhibitors of
H
N
H
N
R
O
O
5
* Corresponding authors. Tel.: +1 845 602 3425; fax: +1 845 602 5561 (S.A.-K.).
E-mail addresses: zhangn@wyeth.com (N. Zhang), ayralks@wyeth.com, ayr-
alks@verizon.net (S. Ayral-Kaloustian).
Scheme 1. Synthesis of 1-(3-oxo-2,3-dihydro-1-benzofuran-5-yl)urea (5). Reagents
and conditions: (a) CuBr2; (b) Et3N; (c) H2/Pd–C; (d) RN = C@O.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.139

N. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3526–3529
3527
NO2
phenone (1), bromination with CuBr2 followed by cyclization led to
NH2
NO2
F
F
5-nitro-1-benzofuran-3(2H)-one (3). Reduction of the nitro group
gave 5-amino-1-benzofuran-3(2H)-one (4).9 Treatment with
methylisocyanate or 3-pyridylisocyanate gave the desired inter-
mediate 5.
F
c
a
b
OH
O
O
12
10
11
For more complex ureas where corresponding isocyanates are
unavailable, the synthesis is exemplified in Scheme 2. Starting from
4-nitrobenzoyl chloride, amidation followed by hydrogenation gave
4-amino-N-[2-(dimethylamino)ethyl]-N-methylbenzamide (8),
which was coupled with 5-amino-1-benzofuran-3(2H)-one (4)
using triphosgene to give the desired 5-ureidobenzofuranone 9.
Synthesis of the indole portion of the 7-fluoro compounds and
final coupling is shown in Scheme 3. Des-fluoro compounds were
prepared in a similar manner. Methylation of 3-fluoro-4-nitrophe-
nol, followed by reduction of the nitro group gave compound 12. A
Gassman oxindole synthesis10 provided compound 14. The thio-
methyl group was removed by Zn–Cu couple. Treatment with
POBr3 in DMF introduced both the formyl group and the bromo
group. Suzuki coupling gave the desired indole aldehyde 17, which
was condensed with 5-ureidobenzofuranones 5 or 9 to give the de-
sired final compound 18. A single (Z)-isomer was obtained exclu-
sively for the final products.
EtO
O
S
NH2
O
e
d
F
O
S
N
H
F
13
14
O
CHO
O
g
O
f
O
Br
N
N
H
H
F
F
15
16
O
Urea
CHO
O
O
O
h
N
N
N
N
N
H
H
N
F
As shown in Table 1, compounds 18a–d exhibited high potency
in inhibiting both PI3Ka and mTOR. In the cellular assays, the 7-flu-
F
17
18
oro group on the indole ring afforded about twofold higher po-
tency. Ureas 18b and 18d, bearing the 3-pyridyl group, were
much less potent than the corresponding methyl ureas 18a and
18c, in the PC3-mm2 cells. Neither of these observations could
be fully explained with solubility, or permeability data.
Scheme 3. Synthesis of 1-[(2Z)-2-{[7-fluoro-5-methoxy-2-(1,3,5-trimethyl-1H-
pyrazol-4-yl)-1H-indol-3-yl]methylidene}-3-oxo-2,3-dihydro-1-benzofuran-5-
yl]urea (18). Reagents and conditions: (a) Me2SO4; (b) H2/Pd–C; (c) ethyl 2-
(methylthio)acetate, sulfuryl chloride; iPr2NEt; (d) 0.1 N HCl; (e) Zn–Cu couple,
70 °C; (f) POBr3/DMF; (g) 1,3,5-trimethyl-1H-pyrazole-4-boronic acid pinacol ester,
Pd(0); (h) 5 or 9, HCl, EtOH, 60 °C.
Excellent enzyme potency was retained with analogues 18e–i
(Table 2). Compound 18i was shown to be a pan-PI3K inhibitor
with IC50 values of 0.7, 2.4, and 0.6 nM versus PI3K b,
tively and was just as potent against two of the most frequently
occurring mutant forms of PI3K (E545K and H1047R, 0.3 and
0.5 nM, respectively), as seen with our earlier morpholino-triazine
based series bearing ureas.11 Furthermore, these analogues with
extended benzamido ureas achieved enhanced cellular potencies
relative to 18a–d, also as previously observed.11,12
c, d, respec-
Table 1
In vitro data for 5-ureidobenzofuranones 18a–18d
a
H
H
O
N
N
R
O
O
O
N
H
N
Compound 19, an analog of compound 18f with a hydrogen in-
stead of the 1,3,5-trimethyl-1H-pyrazol-4-yl group at the 2-position
N
X
18a-d
of the indole, achieved high selectivity for PI3Ka (IC50 = 0.9 nM) ver-
a
Compd
X
R
IC50 (nM)
PC3-mm2
sus mTOR (IC50 = >4000 nM). However, its cellular potency was
much lower than that of compound 18f, underscoring the impor-
tance of the 1,3,5-trimethyl-1H-pyrazol-4-yl group at the 2-position
for cellular activity. While lower cellular potency could be in part
PI3K
a
mTOR
MDA-MB-361
18a
18b
18c
18d
H
H
F
Methyl
3-Pyridyl
Methyl
0.4
0.5
0.4
1.0
1.3
5.2
2.4
1.5
130
>10,000
62
36
76
20
39
F
3-Pyridyl
>1000
a
Determinations were made at eight concentrations, in triplicate, and repeat
values agreed within 630%, on average.
N
Cl
a
b
N
O
O2N
O2N
O
due to lack of mTOR activity, this does not explain the total lack of
activity observed in the two cell lines. Among various amino groups
introduced, the highest cellular potency was obtained with [2-
(dimethylamino)ethyl]methylamino group (compounds 18g and
18i). Compound 18i, bearing the 7-fluoro group on the indole ring,
provided the highest potency in the PC3-mm2 breast cell line and
excellent potency in the MDA-MB-361 breast line, in part owing to
the combined effect of enhanced mTOR and PI3K isoform potency.
In vitro biomarker studies (Western blots) at 4 h post dosing in cells
corroborated the potenciesobserved in the cell growth assays. Select
compounds that were poor in PC3-mm2 growth inhibition were also
tested for downstream effectors of PI3K. It appeared that the com-
pounds could not reach the target in sufficient concentrations to in-
hibit AKT phosphorylation, owing in part to differences in
permeability or efflux mechanisms in this cell line. As above, the
6
7
N
c
N
H2N
O
8
O
O
H
H
N
N
N
O
N
9
O
Scheme 2. Synthesis of N-[2-(dimethylamino)ethyl]-N-methyl-4-{[(3-oxo-2,3-
dihydro-1-benzofuran-5-yl)carbamoyl]amino}benzamide (9). Reagents and condi-
tions: (a) N,N,N0-Trimethylethylenediamine; (b) H2/Pd–C; (c) triphosgene, then 4.

3528
N. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3526–3529
Table 2
In vitro data for 5-ureidobenzofuranones 18e–18i and 19
H
N
H
H
H
O
O
N
N
N
NR1R2
O
NR1R2
O
O
O
O
O
O
O
H
N
H
N
N
H
N
18e-i
19
X
X
NR1R2
IC50 (nM)
a
Compd
X
PI3K
a
mTOR
PC3-mm2
MDA-MB-361
18e
18f
18g
18h
18i
19
H
H
H
F
F
H
4-Morpholinyl
4-Methylpiperazin-1-yl
[2-(Dimethylamino)ethyl]methylamino
4-Morpholinyl
[2-(Dimethylamino)ethyl]methylamino
4-Methylpiperazin-1-yl
0.2
0.2
0.3
0.5
0.2
0.9
4.2
3.8
1.1
2.5
0.3
>4000
94
131
23
57
10
6
8
<3
6
<3
>1000
>1000
a
Determinations were made at eight concentrations, in triplicate, and repeat values agreed within 630%, on average.
enhanced cellular potency of 18g, and especially 18i, could not be
explained based only on permeability, efflux, or solubility since all
the analogues were similar in these properties.
Control
18i
Cleaved PARP
The predicted binding mode of 18g in a PI3Ka homology model
pP70-S6K
pAKT-S473
pAKT-T308
pS6
based on a PI3K crystal structure (PDB access code 3MJW) is
c
shown in Figure 1. The methoxy-group interacts with the hinge re-
gion backbone of Val851 and the urea makes three hydrogen bond-
ing interactions with Asp810 and Lys802, which is the catalytic
lysine. While good homology exists in the active site of PI3K
a,
Actin
PI3K , and mTOR, the rest of the model has poor homology and
c
cannot explain the selectivity observed with 19 versus 18g, or
the other non-selective analogues bearing the bulky pyrazolyl
group. The larger group appears to provide new interactions or a
conformation (not achieved by 19) that is more favorable for bind-
Figure 2. Biomarker inhibition by compound 18i after 8 h, dosed in nude mice at
25 mg/kg iv.
ing to mTOR, without affecting interactions with PI3Ka.
MDA-361 xenograft model
Compound 18i had low water solubility (1 g/mL) at pH 7.4,
however, it could be easily formulated at lower pH. Despite its
low permeability (0.3 ꢀ 10ꢁ6 cm/s in both a–b and b–a directions
in Caco-2 assays), based on its high stability achieved in three spe-
cies (t1/2 >30 min in nude mouse, rat, and human microsomes) and
its excellent in vitro potency versus PI3K and cells, 18i was selected
for further in vivo studies. In biomarker studies (Fig. 2), when
dosed at 25 mg/kg iv in nude mice, it inhibited phosphorylation
1200
1000
800
control
18i 25 iv 1,5,9 ...
600
400
200
-5
0
5
10
15
days
Figure 3. Tumor suppression by 18i, dosed in nude mice at 25 mg/kg iv on days 1,
5, and 9.
of AKT-T308 (PI3K specific marker), AKT-S473, P70S6K, and PS6
(mTOR specific markers) after 8 h. It also induced cleaved poly
ADP-ribose polymerase (PARP), a biomarker for cells undergoing
Figure 1. Close-up of the predicted binding mode of 18g in a PI3K
model based on the PI3K crystal structure. Hydrogen bonding interactions are
shown with magenta dashed lines.
a homology
c

N. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3526–3529
3529
apoptosis. In the MDA-MB-361 xenograft model, 18i was able to
References and notes
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was extended with 4-[2-(dimethylamino)ethyl]methylamino
a and
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amidophenyl group. A 7-fluoro group on the indole ring also en-
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Acknowledgment
We thank Dr. J. Bard for providing the PDB deposition number
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.139.