Discovery of 2-ureidophenyltriazines bearing bridged morpholines as potent and selective ATP-competitive mTOR inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.02.045

Incorporation of bridged morpholines in monocyclic triazine PI3K/mTOR inhibitors gave compounds with increased mTOR selectivity relative to the corresponding morpholine analogs. Compounds with ureidophenyl groups gave highly potent and selective mTOR inhi

Full text of DOI:10.1016/j.bmcl.2010.02.045

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2644–2647
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 2-ureidophenyltriazines bearing bridged morpholines
as potent and selective ATP-competitive mTOR inhibitors
a
a
a
a
b
Arie Zask a, , Jeroen C. Verheijen , David J. Richard , Joshua Kaplan , Kevin Curran , Lourdes Toral-Barza ,
*
Judy Lucas b, Irwin Hollander b, Ker Yu b
a Chemical Sciences, Wyeth Research, 401 N. Middletown Rd, Pearl River, NY 10965, USA
b Oncology Research, Wyeth Research, 401 N. Middletown Rd, 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:
Incorporation of bridged morpholines in monocyclic triazine PI3K/mTOR inhibitors gave compounds with
increased mTOR selectivity relative to the corresponding morpholine analogs. Compounds with ureid-
ophenyl groups gave highly potent and selective mTOR inhibitors. Potency and selectivity was demon-
strated both in vitro and in vivo through biomarker suppression studies. Select compounds exhibited
potent inhibition of tumor growth in nude mouse xenograft assays upon PO and IV administration.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 21 January 2010
Revised 5 February 2010
Accepted 8 February 2010
Available online 13 February 2010
Keywords:
mTOR
PI3K
Kinase
Inhibitor
Cancer
Oncology
Antiproliferative
The mammalian target of rapamycin (mTOR) is a member of the
phosphoinositide-3-kinase (PI3K)-related kinases (PIKKs), high
molecular mass serine/threonine protein kinases. mTOR is fre-
quently upregulated in cancer and is a clinically validated cancer
treatment target.1–4 Development of dual-pan PI3K/mTOR inhibi-
tors has also been an area of intense research activity.4–6 However,
as PI3K is upstream of mTOR in the signaling pathway and its iso-
forms are involved in a number of biological processes, highly po-
tent and specific ATP-competitive mTOR inhibitors may be better
tolerated, and exhibit a higher therapeutic index for enhanced
clinical efficacy. The extensive conservation of the ATP-binding
pockets of mTOR and PI3K makes the development of specific mTOR
inhibitors particularly challenging. Recently, we7–13 and others14–18
reported on selective mTOR inhibitors.
We previously reported that bridged morpholines on pyrazolo-
pyrimidine (1)10,11 and thienopyrimidine (2)13 scaffolds with a
para-ureidophenyl substituent led to potent mTOR inhibitors with
greater selectivity for mTOR versus PI3K than the corresponding
morpholine containing analogs. Molecular modeling suggests that
a single amino acid difference (Phe961Leu)19 in the hinge regions
of PI3K and mTOR leads to a deeper pocket in mTOR relative to
PI3K that can accommodate the additional steric bulk of the mor-
pholine bridge.10 We have extended these discoveries to other
scaffolds and report here and in the accompanying papers20,21
our findings with mTOR inhibitors based on a triazine scaffold.
O
O
N
N
S
N
N
N
R
R
N
O
N
O
N
R
R
1
2
N
N
N
N
H
H
H
H
Analogs were synthesized as shown in Schemes 1 and 2. Treat-
ment of cyanuric chloride with one equivalent of morpholine or a
bridged morpholine (3–5) gave mono-substituted dichlorotria-
zines (Scheme 1). Addition of another equivalent of a different
morpholine gave the di-substituted triazine. Alternatively, two
equivalents of a morpholine could be added to give the di-substi-
tuted chlorotriazine. Coupling with 4-aminophenylboronic acid
under Suzuki coupling conditions followed by reaction with tri-
phosgene and an amine gave the corresponding ureas. Analogs
6–8 were prepared by reaction of the di-morpholine chlorotriazine
intermediate with 2-(difluoromethyl)-1H-benzo[d]imidazole.
Analogs could also be synthesized from 4-nitrophenyltriazine
dichloride (Scheme 2). Reaction of the dichloride with a morpho-
line derivative gave displacement of chloride to provide the
* Corresponding author. Tel.: +1 646 709 7801.
E-mail address: ariez@aol.com (A. Zask).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.045

A. Zask et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2644–2647
Cl
2645
R1
N
HN
HN
O
HN
4(SS)
O
N
N
a
N
N
O
HN
4(RR)
O
N
Cl
Cl
3
5
O2N
O2N
a
b
b
R1
N
R1
N
N
N
N
N
R1
N
c, d
Cl
R1
N
R2
e
O
R2
a
a
N
N
6 - 8
N
N
N
N
R3
O2N
N
N
Cl
R2
Cl
N
Cl
Cl
Cl
H
H
Scheme 2. R1, R2 and R3 are as defined in the tables: (a) 1 equiv morpholine or 3–5,
Et3N; (b) 2 equiv morpholine or 3–5, Et3N; (c) H2, Pd/C; (d) triphosgene, Et3N, then
R3NH2.
c, d
c, d
R1
R1
N
a
N
N
N
N
Table 1
O
N
R2
O
Cl
R1
R3
R3
N
N
F
N
H
N
F
N
N
H
H
H
N
N
R2
Scheme 1. R1, R2 and R3 are as defined in the tables. (a) One equiv morpholine or 3–
5, Et3N; (b) two equiv morpholine or 3–5, Et3N; (c) 4-aminophenylboronic acid,
Pd(0), sodium carbonate; (d) triphosgene, triethylamine, then R3NH2; (e) 2-
(difluoromethyl)-1Hbenzo[d]imidazole.
N
Compd R1
R2
mTORa
(nM)
PI3Ka
(nM)
Selectivityb LNCapa
(nM)
mono-substituted triazine. The remaining chloride could be dis-
placed by a different morpholine derivative. Alternatively, both
chlorines could be displaced simultaneously by treatment with
two equivalents of a morpholine derivative. Hydrogenation of the
nitro group followed by conversion of the resulting aniline to the
urea as described above gave the desired analogs.
6
7
8
Morph. Morph. 45 19
6.5 0.5 0.15
210
680
180
3
3
3
44 5.5
60 15
124 40
24
2.8
0.41
Morph.
2
a
Average IC50 SEM.
PI3K IC50/mTOR IC50
b
.
ZSTK474 (6), a dual PI3K/mTOR inhibitor, is a triazine with two
morpholines and a difluoromethylbenzimidazole group (Table 1).22
Molecular modeling studies have shown that a single amino acid
difference between PI3K and mTOR (Phe961Leu)19 leads to a differ-
ence in the depth of the binding pocket that can accommodate a
bridged morpholine in mTOR but not in PI3K.10 Based on this
observation, we predicted that replacement of the morpholines
in 6 with bridged morpholines would lead to increased mTOR
selectivity due to the deeper binding pocket in mTOR relative to
PI3K. Replacement of both morpholines with a 2,6-bridged mor-
pholine (3) gave 7 with a 19-fold increase in selectivity for mTOR
over PI3K (Table 1). Consistent with the molecular modeling,
mTOR was able to accommodate the bridged morpholine in its
deeper binding pocket. In contrast, the PI3K potency decreased sig-
nificantly. Replacement of only one of the morpholines with a
bridged morpholine (3) gave 8 with a substantially lesser degree
Table 2
O
N
N
N
O
N
N
R3
O
N
N
H
H
Compd
R3
mTORa (nM)
PI3Ka (nM)
Sel.b
LNCapa (nM)
9
10
11
Me
Et
2.5 0.5
2.8 0.6
2.6 0.5
680
272
961
802
190
180
90
2692
2125
12
13
0.50 0.01
1.7 0.4
142
282
51
45
N
87
9
2.3
N
N
a
Average IC50 SEM.
PI3K IC50/mTOR IC50
b
.

2646
A. Zask et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2644–2647
1800
1400
1000
600
Vehicle
µM
15 mg/kg PO
10 mg/kg PO
5 mg/kg PO
P-S6K T389
P-AKT S473
P-AKT T308
AKT
Cyclin D1
c-Myc
200
Actin
-2
0
2
4
6
8
10
Days
Figure 2. In vitro signaling inhibition of 16 in MDA361 cells.
Figure 1. Efficacy of 13 in the U87MG xenograft model.
Control
25 mg/kg
10 mg/kg
of increase in mTOR selectivity due to the ability of either morpho-
line or 3 to access the hinge region.
p-S6K (TORC1)
P-473 (TORC2)
P-308 (PI3K/PDK1)
p-S6
We were encouraged by these results and reasoned that
replacement of the benzimidazole in 7 with a 4-ureidophenyl
group, which molecular modeling based on the X-ray structure of
Actin
PI3Kc has shown to form 3 hydrogen bonds to mTOR on a pyrazol-
opyrimidine scaffold,7 would lead to an increase in mTOR potency.
Attachment of a 4-methylureidophenyl group to the triazine, in
addition to the 2,6-ethylene bridged morpholines, led to a potent
and selective inhibitor 9 with good cell activity (Table 2). As seen
previously,7 further increases in selectivity were obtained with
analogs containing the ethyl and cyclopropyl ureas (10 and 11,
respectively). A 4-pyridyl urea gave a subnanomolar mTOR inhibi-
tor 12 while a 4-methylpiperazine arylurea gave 13 with single di-
git nanomolar inhibition of proliferation of a prostate tumor cell
line (LNCap) that is characterized by hyperactive PI3K-Akt-mTOR
signaling.23 We have previously demonstrated that cellular prolif-
eration inhibition in this model correlates with inhibition of mTOR
signaling.8 The data in Table 2 reveal that compounds with similar
enzyme IC50s can possess drastically different cellular proliferation
IC50s (cf. 9–11, 13), possibly reflecting differences in cellular
permeability and intracellular distribution. On the basis of its
Figure 3. In vivo signaling inhibition of 16 in MDA361 xenografts, 8 h after IV dose.
At 10 mpk a strong inhibition of TORC1 and TORC2 and negligible inhibition of
PI3K/PDK1 was seen. At 25 mpk near complete inhibition of TORC1 and TORC2 and
little inhibition of PI3K signaling was seen.
in vitro potency, 13 was assayed in a nude mouse U87MG xeno-
graft model and showed potent inhibition of tumor growth upon
oral dosing daily X 5 (Fig. 1). On day 8, tumor/control was 44%
for the 10 mpk group.
In an effort to synthesize analogs that maintained the potent
activity of 13 but were more selective for mTOR we investigated
other bridged morpholine systems. Incorporation of chiral 2,5-
methylene bridged morpholines 4(RR) and 4(SS) gave analogs 14
and 15, respectively. As seen previously with the pyrazolopyrimi-
dine scaffold,10 the analog incorporating the SS enantiomer was
Table 3
R1
N
N
R2
O
N
R3
N
N
H
H
Compd
14
R1
R2
R3
mTORa (nM)
130
PI3Ka (nM)
1819
Sel.b
14
LNCapa (nM)
4(RR)
4(SS)
5
4(RR)
4(SS)
5
550
80
N
N
N
N
N
N
N
N
15
35
3438
98
16
1.0 0.1
2.0
899 107
150
899
75
1.3
17
3
5
<0.8
a
Average IC50 SEM.
PI3K IC50/mTOR IC50
b
.

A. Zask et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2644–2647
2647
1400
1200
1000
800
600
400
200
0
Testing of 16 in nude mice bearing U87MG xenografts gave potent
activity upon IV or PO dosing.
Veh
25 mg/kg PPOO
12.5 mg/kkggPPOO
5 mg/kg IIVV
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Figure 4. Efficacy of 16 in theU87MG xenograft model.
more potent for mTOR than the analog with the RR enantiomer and
displayed higher selectivity versus PI3K (Table 3). However, 15 did
not meet our potency or selectivity requirements and was not eval-
uated further. We had seen previously10 with the pyrazolopyrimi-
dine scaffold that the 3,5-ethylene bridged morpholine 5 gave
analogs that had comparable potency and selectivity to those with
the 2,6-bridged morpholine 3. In contrast, in the case of the tri-
azine ring system, when the 3,5-ethylene bridged morpholine 5
was incorporated, an analog 16 with a 10-fold increase in selectiv-
ity was obtained (Table 3). As expected, the analog 17 with one of
each bridged morpholine 3 and 5 did not see an increase in selec-
tivity due to the accessibility of both 3 and 5 to the hinge region
(Table 3). The mTOR selectivity of 16 translated into in vitro cellu-
lar selectivity with the mTOR biomarker S473 being preferentially
suppressed with respect to the PI3K T308 biomarker (Fig. 2). In
vivo suppression of the S473 biomarker in preference to the T308
biomarker was also seen after 8 h upon dosing nude mice bearing
MDA361 tumors with 10 or 25 mpk of 16 (Fig. 3). In the U87MG
nude mouse xenograft assay, 16 showed potent inhibition of tumor
growth with daily X5 oral dosing (Fig. 4). At 5 mpk, on day 9 a tu-
mor/control ratio of 31% was achieved.
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19. Phe961 in PI3K corresponds to Leu in mTOR. PI3Kc amino acid sequence
numbering is used.
In summary, incorporation of bridged morpholines onto the
monocyclic triazine scaffold bearing a ueridophenyl group gave po-
tent and selective mTOR inhibitors. The 3,5-ethylene bridged mor-
pholine gave more selective inhibitors than the 2,6-ethylene
bridged morpholines. Potent and selective analog 16 was shown
to selectively suppress mTOR biomarkers both in vitro in
MDA361 cells and in vivo in nude mice bearing U87MG tumors.
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