Novel imidazolopyrimidines as dual PI3-Kinase/mTOR inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.11.057

This article describes the syntheses and SAR of a series of imidazolopyrimidine derivatives, which are evaluated as inhibitors of PI3-Kinase (PI3 K) and mTOR. These compounds were found to be ATP competitive with good tumor cell growth inhibition, and suppression of pathway specific biomakers such as phosphorylation of Akt at T308.

Full text of DOI:10.1016/j.bmcl.2009.11.057

Bioorganic & Medicinal Chemistry Letters 20 (2010) 653–656
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel imidazolopyrimidines as dual PI3-Kinase/mTOR inhibitors
a
a
a
Aranapakam M. Venkatesan a, , Christoph M. Dehnhardt , Zecheng Chen , Efren Delos Santos ,
Osvaldo Dos Santos a, Matthew Bursavich a, Adam M. Gilbert a, John W. Ellingboe a,
Semiramis Ayral-Kaloustian a, Gulnaz Khafizova a, Natasja Brooijmans a, Robert Mallon b,
Irwin Hollander b, Larry Feldberg b, Judy Lucas b, Ker Yu b, Jay Gibbons b, Robert Abraham b,
Tarek S. Mansour a
*
a Chemical Sciences, Wyeth Research, Pearl River, NY 10965, United States
b Oncology, Wyeth Research, Pearl River, NY 10965, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
This article describes the syntheses and SAR of a series of imidazolopyrimidine derivatives, which are
evaluated as inhibitors of PI3-Kinase (PI3 K) and mTOR. These compounds were found to be ATP compet-
itive with good tumor cell growth inhibition, and suppression of pathway specific biomakers such as
phosphorylation of Akt at T308.
Received 8 October 2009
Revised 10 November 2009
Accepted 16 November 2009
Available online 1 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keyword:
PI3-Kinase
In recent years, the phosphatidylinositide-3-kinase (PI3 K/Akt)
signaling pathway has been recognized as a key pathway in cell pro-
liferation, growth, survival, protein synthesis and glucose metabo-
lism.1–6 Phosphatidylinositol-3-kinases (PI3 K) are lipid kinases,
that phosphorylate the phosphatidylinositol-diphosphate (PIP-2)
to its corresponding phosphatidylinositol-triphosphate (PIP-3).
PI3 Ks belong to the super family of PI3K related Kinases (PIKKs)
which includes mTOR, ATM, ATR and DNA-PK. The product of PI3 K
activity, PIP-3, acts as a second messenger that is responsible for
the activation of the downstream kinase Akt.1
is frequently mutated in numerous late stage tumors causing ele-
vated levels of PIP-3 which might contribute to oncogenesis.7,8
Although only the p110a isoform is mutated in human cancers,
the other PI3 K class I isoforms also have oncogenic potential.
p110b PIK3CB isoform gene overexpression has been observed in
breast and ovarian cancers.1b,12 Hence, inhibiting PI3 K
a is an
attractive target for cancer therapy. Additionally, it has been dem-
onstrated that the PI3 K pathway is activated following mTOR inhi-
bition, thus providing a strong rationale for developing dual PI3 K
and mTOR kinase inhibitors.13,14
Eight PI3-kinases have been identified and categorized as class
IA, class IB, class II, and class III enzymes. This classification is
based on the sequence homology and substrate preference(s).
Towards this end, several pharmaceutical companies and aca-
demic institutions have been concentrating their efforts on develop-
ment of small molecule PI3 K/mTOR inhibitors. The first generation
of pan-PI3 K inhibitors, LY294002 1 and Wortmannin 2 (Fig. 1),
which inhibit the catalytic activity of all class I PI3 Ks, were not
developed clinically because of toxicity and poor pharmaceutical
properties.2,15 More recently, a bismorpholino-triazine based com-
pound, ZSTK-474 3, was reported by the Japanese cancer founda-
tion.16,17 ZSTK-474 is an ATP competitive pan-PI3 K class I enzyme
inhibitor. Another compound that inhibits all class I PI3 K isoforms,
but has significantly less potency versus mTOR is GDC-0941. This
compound4 was reported by Genentech, and recentlyentered phase
I clinical studies.18
The class IA subgroup consists of p110a, p110b and p110d iso-
forms. Among these three isoforms, the gene encoding the p110
a
subunit, PIK3CA, is over expressed in ovarian, colorectal, brain
(glioblastoma), and gastric cancers.6,9–11 Additionally, mutations
in PIKC3A have been documented to cause elevated PI3 K-a kinase
activity in breast, endometrial, colon, and other cancers.1b There is
significant evidence suggesting that the PI3 K/Akt pathway is
deregulated in many human cancers.1–6 PI3 K overexpression or
mutation causes activation of Akt kinase, which leads to tumor
progression, proliferation, survival, growth, invasion, angiogenesis
and metastasis. Additionally, the PIP3 lipid phosphatase PTEN
(Phosphatase and tensin homologue deleted on chromosome ten)
Among the various small molecule PI3 K inhibitors, BEZ-235
(Novartis) 6 is the most advanced clinical candidate. It inhibits
PI3 K and mTOR kinases in an ATP competitive manner. BEZ235
also reported to inhibit cancer cell proliferation, causing cell cycle
arrest at the GI stage.19,20 Other novel small molecules that inhibit
* Corresponding author.
E-mail address: venkata@wyeth.com (A.M. Venkatesan).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.057

654
A. M. Venkatesan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 653–656
O
N
O
O
O
O
O
O
O
N
N
N
O
H
O
N
N
N
O
O
N
O
1
2
CF3
3
LY294002
Wortmannin
ZSTK-474
O
N
O
N
CN
O
N
N
O
S
N
NH
N
N
N
N
OH
N
N
N
N
N
4
MeO2S
5
6
GDC-0941
PI-103
BEZ-235
Figure 1. Known PI3-K inhibitors.
PI3 K/mTOR are on the horizon, but their structures have not yet
been revealed.21 Here in, we describe the syntheses and biological
evaluation of a series of 4-morpholino-2-aryl-purine derivatives
arylboronic acids under thermal or microwave assisted Suzuki reac-
tion condition. The piperidino group was introduced in the 9-posi-
tion of compound 8 using the N-BOC protected piperidine-4-ol,
Ph3P and DEAD at room temperature to yield 10. Different, R groups
onthepiperidinenitrogenincompound12wereintroducedbyatwo
step process. Initially the BOC-group in compound 11 was removed
using trifluoro acetic acid in dichloromethane to yield 12, in almost
quantitative yields and the R groups were introduced by a ZnCl2
mediated reductive amination protocol.
which are potent PI3 Ka/mTOR inhibitors.
Therequired2-aryl-substituted-4-morpholino-imidazolopyrim-
idine derivatives 9 and 13 were conveniently prepared from the
commercially available 2,4-dichloropurine 7 as indicated in Scheme
1. The 2,4-dichloropurine 7 was reacted with 2 equiv of morpholine
in ethanol at room temperature to yield the monomorpholino deriv-
ative 8 in good yield. The 2nd chlorine in intermediate 8 was substi-
tuted with aryl or heteroaryl groups using their respective
It was reported from several sources18,22 as well confirmed by
Wyeth workers23(based on PI3 K- homology model,23 Fig. 2) that
c
O
N
O
N
N
Cl
N
N
N
N
b
N
a
c
N
N
N
N
H
N
H
N
H
Ar
N
9
Cl
Cl
Cl
8
7
O
O
N
O
N
N
N
N
N
N
N
N
N
N
b
N
d
Ar
Ar
N
N
N
N
N
H
O
O
O
O
12
10
11
O
N
e
Ar is substituted phenyl
or pyridyl
N
N
N
Ar
N
N
R
13
Scheme 1. General method to synthesize 14–34. Reagents and conditions: (a) 2 equiv of morpholine, ethanol, rt/2 h; (b) ArB(OH)2, DMF, Pd(PPh3)4,
(c) tert-butyl 4-hydroxy-1-piperidinecarboxylate, PPh3, DEAD, THF, rt; (d) dioxane, TFA; (e) R–CHO, ZnCl2, NaBH3CN, MeOH, rt, 12 h.
l Wave, 175 °C, 15 min;

A. M. Venkatesan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 653–656
655
O
O
N
N
N
N
N
N
N
N
R1
R1
N
N
H
X
X
N
R
14 X =CH; R1 = -OH
30 X =N; R1 = -OH
15-20 X = CH; R1 = -OH; R =H
22 X =CH; R1 = -CH2OH; R =H
23-29 X =CH; R1 = -CH2OH
31-34 X =N; R1 = -OH
Figure 3. Novel imidazolopyrimidines.
metabolism studies in microsomes indicated that glucuronidation
of the parent compound was common to all the phenol derivatives
(data not shown). This indicated that the phenolic group is a
metabolic liability. Therefore, the phenolic –OH was replaced by a
–CH2OH group. This modification, led to examples 21–29. As can
be seen from Table 1, replacing the phenolic –OH group with –
CH2OH did not dramatically alter the potency against PI3 Ka, but
potency against mTOR decreased significantly for derivatives 22
and 23. Comparison of compound 16 with 21 illustrates that potency
Figure 2. Docking of 14 in a PI3Kc homology model.
the morpholino oxygen of 14 forms a hinge region H-bond interac-
tion with Val851, that is crucial for the inhibition of PI3 K enzyme
activity. The phenolic–OH group in the aryl entity formed an H-bond
interaction with the Asp810. The initial compound 14 prepared in
this series, exhibited an IC50 value of 150 nM against PI3 Ka, and
molecular modeling of 14 (Fig. 2) suggested that by introducing po-
lar groups at N-9 position of 14, potency against PI3 K and mTOR
could be increased, through interaction of the N-9 substituent with
a
for PI3 K
compound 21 was 15-fold more selective for PI3 K
The most potent compound in this series was 29, which had growth
inhibition IC50 values of 0.685 M versus MDA-361 (breast) and
0.512 M versus PC3MM3 (prostate) human tumor cell lines. This
a
remains nearly the same for the both compounds; but
a
than mTOR.
l
l
Ser919 in PI3 Ka, Ser2342 in mTOR and Asp950 in PI3 Kc. In the
compound also had good microsomal stabilities.
present study, modifications were not only made at the N-9 position
as of the purine scaffold of compound 14, but also on the aryl moiety
to improve potency.
When a piperidine ring was introduced at the N-9 position of the
purine core of 14, the resulting compound 15 was two-fold more po-
tent than the N-9 unsubstituted derivative 14. Compound 15 also
had an improved solubility profile over 14. Substitution of the piper-
idine nitrogen with various groups yielded analogues 16–20. Among
Next, the phenyl moiety in the 2-position of the purine was re-
placed with a 5-hydroxy-3-pyridyl group (e.g., 30–34). This modi-
fication gave potent analogues such as 30, 31 and 32.
The pyridyl substituted compound 30 was found to be 10-fold
more potent than the corresponding phenyl substituted derivative
14. This could be due to an extra hydrogen bond interaction that
could possibly occur with Lys 802 with the 3-pyridyl nitrogen
atom (see Fig. 3).
these five compounds, analogues 18 and 19 displayeda better PI3 K
potency compared to 15 (twofold). However, the microsomal stabil-
ity of these phenolic derivatives was very poor. In vitro (phase II)
a
Compound 29 was assayed in vitro for its ability to suppress
appropriate cellular biomarkers. PI3 Ka inhibition should result
Table 1
Biological data for imidazolopyrimidines 14–34
Compd #
X
R1
R
IC50 values in nM
PI3 K
PI3 Ka
c
mTOR
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
–OH
–OH
–OH
–OH
–OH
–OH
–OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
–OH
150
64
75
66
40
37
55
63
57
189
65
42
70
23
44
16
11
14
16
42
94
2343
133
10,000
583
368
341
306
1134
683
1378
776
1102
594
223
640
265
47
1650
950
140
170
152
200
51
1000
7200
2100
310
160
1700
720
2050
>4000
620
970
160
590
570
H
Benzyl
–CH2-3-pyridyl
–CH2-(6-morpholino)-3-pyridyl
–CH2-(5-chloro)-3-pyridyl
–CH2-(6-methoxy)-3-pyridyl
Benzyl
CH3CO–
–CH2-2-pyridyl
2-Flourobenzyl
4-Fluorobenzyl
–CH2-{4-(4-pyridyl)-phenyl}
2,4-Difluoro-phenyl
–CH2-(4-O(CH2)3NMe2)phenyl
N
N
N
N
–OH
–OH
–OH
–OH
H
44
–CH2-4-chlorophenyl
–CH2-4-tolyl
–CH2-6-flouoro-3-pyridyl
119
204
61

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A. M. Venkatesan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 653–656
30
10
3
1
0.30
0
[µM]
probed with antibodies (Ab) specific to the protein and phospho-
protein components of the PI3 K/Akt/mTOR signaling pathway.
pAkt (T308)
cPARP
Abs obtained from Cell Signaling Technology were: anti( )-Akt,
a
29
a
-phospho(p)-Akt at T308, -Akt, -cleaved PARP, and a-actin.
a
a
Actin
Specific antigen/antibody interactions were identified by a horse-
radish peroxidase (HRP) conjugate secondary Ab that enabled
chemiluminescent signal detection.
Figure 4. Inhibition of pAkt at T308, and induction of cleaved PARP by 29.
Acknowledgment
in suppression of the phosphorylation of Akt particularly at the
threonine 308 site (T308, Fig. 4). As can be seen in Fig. 4, compound
29 inhibits Akt T308 phosphorylation in MDA361 breast tumor
cells, with an IC50 value of about ꢀ300 nM, after a 4 h exposure
The authors thank the members of the Wyeth Chemical Tech-
nologies group for analytical and spectral determination.
time. Compound 29 also induced cleaved PARP at 10 and 30 lM.
References and notes
Cleaved PARP is a marker for cell apoptosis (programmed cell
death). Actin (control protein) signal was unaffected by 29.
In this Letter, we have disclosed a series of novel 2-aryl or hetero-
aryl substituted-4-morpholino imidazolopyrimidine derivatives
14–34 possessing moderate to excellent PI3K/mTOR inhibitory
activity. Most interestingly, from the selectivity point of view
(against mTor) analogues such as 21, 22 and 29 are all of great inter-
est. Introduction of the 5-hydroxy-3-pyridyl appendage at the 2-po-
sition of the imidazolopyrimidine scaffold yielded potent analogues
such as 30 and 32. This could be due to an extra hydrogen bond inter-
action that could possibly occur between the pyridyl nitrogen and
Lys802. Analog 29 showed a good correlation between cell growth
inhibition and biomarker (pAkt at T308) suppression. Further stud-
ies concerning agents that target the PI3-K and mTor will be re-
ported in due course.
Cell growth inhibition assay: The MDA361 and PC3mm2 cell lines
were obtained from ATCC. Cell lines were grown at 37 oC in 5% CO2
incubators in growth media supplemented with penicillin/strepto-
mycin and 10% fetal calf serum. Cell growth inhibition was
determined using the CellTiter 96 aqueous non-radioactive cell
proliferation assay from Promega. This homogeneous colorimetric
method determined the number of viable cells in proliferation as-
says. The assay was carried out in 96 well format following manufac-
turer’s instructions, with cell number per well being adjusted based
on growth characteristics of the various cell lines used. Assay end-
point data was quantitated after 72 h compound exposure using a
Victor2 V (Wallac) model 1420 multilabel HTS counter.
Cell lysis and Western blotting: Cell lysis enabled biochemical
analysis of PI3 K/mTOR signaling pathway proteins after exposure
of cells to compounds. Cells (3 Â 105) were seeded onto 6-well
microtiter plates (Nunc) 24 h prior to being exposed to compound
in complete growth media. Cells were exposed to these compounds
for 4 h. After exposure to compounds cell growth media was re-
moved and cells were washed twice with cold (4 °C) PBS. Cell lysis
buffer (0.2 ml) was then added to each microtiter plate well with
sufficient mixing to insure complete cell lysis. Cell lysis buffer
consisted of: 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM
Na2EDTA 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate,
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1 mM b-glycerophosphate, 1 mM Na3VO4, and 1
Cell lysates were then spun for 30 s at 14,000 rpm. Supernatant
(75 l) was combined with 30
lg/ml leupeptin.
l
l
l of 3Â protein gel loading buffer
[187.5 mM Tris–HCl (pH 6.8), 6% (w/v) SDS, 30% glycerol, 0.03%
(w/v) bromophenol blue, and 125 mM DTT]. Samples were boiled
(5 min) separated by SDS–PAGE, transferred to nitrocellulose and