Synthesis and structure-activity relationships of 1,2,4-triazolo[1,5-a] pyrimidin-7(3H)-ones as novel series of potent β isoform selective phosphatidylinositol 3-kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.03.039

A series of 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones with excellent enzyme inhibition, improved isoform selectivity, and excellent inhibition of downstream phosphorylation of AKT has been identified. Several compounds in the series demonstrated potent (～ 0.100 μM IC₅₀) growth inhibition in a PTEN deficient cancer cell line.

Full text of DOI:10.1016/j.bmcl.2012.03.039

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3198–3202
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of 1,2,4-triazolo[1,5-a]pyrimidin-
7(3H)-ones as novel series of potent b isoform selective phosphatidylinositol
3-kinase inhibitors
Robert M. Sanchez a, , Karl Erhard a, Mary Ann Hardwicke b, Hong Lin a, Jeanelle McSurdy-Freed c,
⇑
Ramona Plant b, Kaushik Raha d, Cynthia M. Rominger b, Michael D. Schaber e, Michael D. Spengler c,
Michael L. Moore a, Hongyi Yu a, Juan I. Luengo a, Rosanna Tedesco a, Ralph A. Rivero a
a Cancer Metabolism Chemistry, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
b Cancer Metabolism Biology, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
c Cancer Metabolism DMPK, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
d Platform Technology Sciences, Computational Chemistry, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
e Platform Technology Sciences, Screening and Compound Profiling, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, 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 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones with excellent enzyme inhibition, improved iso-
form selectivity, and excellent inhibition of downstream phosphorylation of AKT has been identified. Sev-
Received 8 February 2012
Accepted 8 March 2012
Available online 16 March 2012
eral compounds in the series demonstrated potent (ꢀ 0.100
lM IC50) growth inhibition in a PTEN
deficient cancer cell line.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
PI3K-b inhibitor
PTEN-deficient
Phosphatidylinositol 3-kinase
Triazolopyrimidinone
Structure–activity relationship
The phosphatidylinositol 3-kinases (PI3K) are a family of en-
zymes involved in the regulation of cell survival, metabolism and
proliferation.1 Disruption of the PI3K signaling pathway plays a
key role in the formation of cancerous cells from normal cells,
and inactivation of the tumor suppressor protein PTEN is often
seen through aberrant pathway activation.2 This lipid-phosphatase
is one of the most commonly mutated proteins in human cancers
and loss of PTEN protein expression as measured by immunohisto-
chemical staining of PTEN has been observed in approximately 40%
of glioblastoma, 50% of prostate, 57% of endometrial cancers, in
addition to a number of other tumor types, including melanoma
and breast cancers.3 Preclinical studies have shown that tissue-
specific deletion of the PI3K-b isoform in the prostate specifically
reduces PI3K signaling and blocks the formation of aggressive pros-
tate tumors.4 Based on the premise that the PI3K-b isoform is nec-
essary for cancer proliferation, a program was initiated to identify
PI3K-b selective inhibitors for the treatment of cancers driven by
loss of PTEN.
potent and selective, the high clearance observed in rodent PK
analysis precluded further in vivo studies.5 In an effort to improve
on the overall profile of the imidazo[1,2-a]-pyrimidin-5(1H)-ones,
the isosteric 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones illustrated
in Figure 1 were explored. This modest change allowed for the
opportunity to leverage our knowledge of the structure–activity
relationships to quickly identify potent and selective analogs.
The synthetic route used to prepare the 1,2,4-triazolo[1,5-
a]pyrimidin-7(3H)-ones is shown in Scheme 1. The triazole I6
was treated with diethyl malonate and sodium ethoxide in ethanol
A series of novel imidazopyrimidinones, exemplified by com-
pound 1, were previously designed in our laboratories. Although
⇑
Corresponding author.
E-mail address: robert.m.sanchez@gsk.com (R.M. Sanchez).
Figure 1. Structures of imadazopyrimidines and triazolopyrimidinones.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.039

R. M. Sanchez et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3198–3202
3199
Scheme 1. Synthesis of 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones. (a) Diethyl malonate, NaOEt, EtOH, reflux, 18 h (49–84%); (b) POCl3, 80 °C, 2 h, (7–73%); (c) NaOH, 100 °C,
1 h, (20–50%); (d) R1–Br, K2CO3, DMF, microwave, 120 °C, 10 min, (25–54%); (e) morpholine, EtOH, microwave, 150 °C, 1 h (25–88%).
at reflux to yield dihydroxy cyclized product II. This was chlori-
nated with phosphorus oxychloride to yield di-chlorinated product
III. The dichlorinated compound was hydrolyzed to IV with sodium
hydroxide at 100 °C. Selective alkylation of the triazole ring was
accomplished by treatment with substituted benzyl halides and
potassium carbonate under microwave irradiation at 120 °C to pro-
vide V, followed by treatment with morpholine under microwave
irradiation at 150 °C to give the desired product VI.
The structure–activity relationships previously established
within the imidazopyrimidinone series allowed for the focus of
efforts to be centered on 2,3 disubstituted phenyl groups at R1.
Specifically, Table 1 contains the isoform enzyme data for repre-
sentative compounds where R1 is 2,3-dichloro phenyl. These com-
pounds exhibited excellent PI3K-b activities ranging from IC50
but provided no real advantage over the unsubstituted analog 9.
Importantly, excellent selectivity versus all the other enzyme iso-
forms was maintained.
An important step in the progression of intracellular enzyme
inhibitors is the ability to illustrate activity in a cellular context.
In this case, mechanistic cell-based activity was demonstrated by
measuring the inhibition of phosphorylation of AKT at serine 473
in a PTEN deficient breast cancer cell line, MDA-MB-468.10 The plot
of PI3K-b-enzyme pIC50 values of compounds 2–15 versus the
pIC50 values of pAKT inhibition seen in Chart 1 shows a 1.5-fold
log shift but is in acceptable agreement between PI3K-b enzyme
and the cellular inhibition of AKT phosphorylation.
Table 2
Biochemical activity8 in PI3K isoformsa
values of 0.025–0.0013 lM. The addition of a 2-methyl group to
the morpholine was well tolerated. Chiral separation of the enan-
tiomers (compounds 6 and 7) resulted in a more active enantiomer
7, but in this case provided no real advantage over the unsubstitut-
ed analog 2.7 Furthermore, the series displayed improved selectiv-
ity versus the delta enzyme (6 to 50-fold).
As can be seen in Table 2, a modest increase in activity was ob-
served when R1 was the 2-methyl, 3-trifluoromethyl phenyl com-
pared with the 2,3-dichlorophenyl moiety. In this instance,
several analogs (9, 10, 14, 15) were very potent inhibitors of the
No.
R2
R3
PI3K enzyme IC50 (lM)
PI3K-b enzyme with IC50 values of 0.001 lM or less. A preference
a
b
d
c
for one stereoisomer of 2-methyl morpholine (14) was observed,
9
Me
SMe
cPr
Me
Me
4-Morpholinyl
4-Morpholinyl
4-Morpholinyl
(2-Me)-4-Morph ( )
(2-Me)-4-Morph (E1)
(2-Me)-4-Morph (E2)
(2-Me)-4-Morph
0.63
0.32
2.50
0.40
1.60
0.32
1.30
0.0006
0.0004
0.0013
0.0013
0.0031
0.0003
0.0010
0.020
0.025
0.040
0.010
0.063
0.004
0.025
0.79
1.30
1.60
0.16
0.79
0.06
0.20
10
11
12
13
14
15
Table 1
Biochemical activityb in PI3K isoformsa
Me
SMe
a
IC50 values of b and d activities are means of at least 2 experiments.
No.
R2
R3
PI3K enzyme IC50 (lM)
a
b
d
c
2
3
4
5
6
7
8
Me
SMe
cPr
Me
Me
Me
SMe
4-Morpholinyl
4-Morpholinyl
4-Morpholinyl
(2-Me)-4-Morph ( )
(2-Me)-4-Morph (E1)
(2-Me)-4-Morph (E2)
4-Pyridylc
0.63
1.60
2.50
0.32
2.50
1.00
0.40
0.0013
0.0016
0.0025
0.0020
0.0250
0.0013
0.0130
0.050
0.031
0.130
0.025
0.500
0.040
0.079
0.50
0.50
2.50
0.25
7.90
0.50
2.00
a
b
c
IC50 values of b and d activities are means of at least 2 experiments.
Ref.8.
Ref.9.
Chart 1. Enzyme versus cell activity compounds 2–15.

3200
R. M. Sanchez et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3198–3202
Table 3
Cellular and proliferation activity in MDA-MB-468 cell linea
No.
R1
R2
R3
MDA-MB-468 IC50
pAKT
(l
M) EC50
Growth
2
3
4
7
(2,3-diCl)Ph
(2,3-diCl)Ph
(2,3-diCl)Ph
(2,3-diCl)Ph
(2-Me,3-CF3)Ph
(2-Me,3-CF3)Ph
(2-Me,3-CF3)Ph
(2-Me,3-CF3)Ph
(2-Me,3-CF3)Ph
(2-Me,3-CF3)Ph
Me
SMe
cPr
Me
Me
SMe
cPr
Me
Me
Me
4-Morpholinyl
4-Morpholinyl
4-Morpholinyl
(2-Me)-4-Morph(E2)
4-Morpholinyl
4-Morpholinyl
4-Morpholinyl
(2-Me)-4-Morph( )
(2-Me)-4-Morph(E1)
(2-Me)-4-Morph(E2)
0.0340
0.0130
0.0200
0.0034
0.0015
0.0051
0.0041
0.0027
0.0079
0.0005
0.170
0.240
0.260
0.110
0.110
NA
0.040
0.055
0.310
0.013
9
10
11
12
13
14
a
IC50 values given are means of at least 2 experiments.
Table 4
Comparison of compounds 1 and 14a
Selective PI3K-b inhibitor comparison (lM)
PI3K-b IC50
0.0013
0.0079
32
0.0003
0.0040
6.3
0.16
0.005
2.5
0.013
2.2
PI3K-d IC50
mTORb IC50
DNA-PKb IC50
0.80
MDA-MB-468 pAKT IC50
HCC1954 pAKT IC50
MDA-MB-468 Growth EC50
HCC1954 Growth EC50
0.078
> 29
0.14
9.7
a
IC50 values given are means of at least 2 experiments.
Member of the phosphatidylinositol 3-kinase related kinase family.
b
In an effort to understand the downstream consequences of
Importantly, compound 14 was significantly more potent at inhib-
iting the phosphorylation of AKT in MDA-MB-468 cells as well as
inhibiting proliferation in the same cell line. Both compounds 1
and 14 were significantly less active in the cellular pAKT and pro-
liferation assays in a PTEN wild-type cell line (HCC1954), support-
ing our hypothesis of the selectivity of PI3K-b inhibitors for PTEN
deficient tumor cell lines compared to PTEN wild-type.
While a second novel PI3K-b inhibitor chemotype with good
in vitro activity had been identified, the compounds in this series
suffered from very high rat clearance rates, similar to the isosteric
imidazopyrimidinone series,12 and thus were not appropriate for
in vivo studies. A hypothesis that the high clearance might be re-
lated to the potential reactivity of the carbonyl, led to the prepara-
tion of the isomeric compound 1613 where the carbonyl might be
less electrophilic. This compound, shown in Table 5, had slightly
diminished enzyme and cell activity with rat clearance rates that
were still too high for the molecules to be considered for in vivo
analysis.
inhibiting the phosphorylation of AKT, the PTEN deficient breast
cancer cell line, MDA-MB-468, was evaluated to determine the
effect of the compounds on tumor cell growth. A relevant cell line
would be one that is PTEN deficient, as our hypothesis is that growth
of PTEN deficient cell lines will be sensitive to PI3K-b inhibitors,
while growth of wild-type PTEN cell-lines should be unaffected by
selective PI3K-b inhibitors. As can be seen in Table 3, most of the
more potent enzyme and cellular inhibitors also inhibited prolifera-
tion in MDA-MB-468 cell lines at sub-micromolar concentrations.11
Compound 14 was a particularly potent inhibitor of phosphoryla-
tion of AKT and cell proliferation with IC50 values of 0.0005 and
0.0133
lM, respectively, whereas the less preferred isomer 13 dem-
onstrated reduced IC50’s of 0.079 and 0.031
lM, respectively.
As shown in Table 4, triazolopyrimidinone 14 compared very
favorably with previously reported selective PI3K-b inhibitor com-
pound 1. The selectivity versus the delta isoform improved to
greater than 10-fold, and the selectivity versus two other members
of the phophatidylinositol 3-kinase related family of proteins
mTOR and DNA-PK was 21,000 and 500-fold, respectively.
In summary, a novel series of triazolopyrimidinones as potent
and selective inhibitors of PI3K-b has been described. Compounds

R. M. Sanchez et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3198–3202
3201
Table 5
Comparison of compounds 9 and 16a
b,c
No.
X
Y
PI3K-b IC50
(l
M)
pAKT IC50
(
lM)
Growth Inhib. EC50
(lM)
Rat CL
(mL/min/kg)
9
16
N
C
C
N
0.0006
0.0020
0.015
0.092
0.11
0.21
130
83
a
b
c
IC50 and clearance values given are means of at least 2 experiments.
Ref. 14.
Ref. 15.
9. Compound 8 was prepared as shown in the following scheme.
in this series are the most potent and selective PI3K-b inhibitors re-
ported to date with excellent anti-proliferation activity in a PTEN
deficient cell line. While the overall profile of these new inhibitors
represents an improvement over previously reported PI3K-b selec-
tive inhibitors, the observed high rodent clearance rates precluded
their utility for in vivo analysis.
Acknowledgment
Reagents and conditions: (a) ethyl 3-oxo-3-(4-pyridinyl)propanoate, AcOH,
120 °C, overnight, 18%; (b) 1,2-dichlorobenzyl bromide, K2CO3, DMF, 125 °C,
microwave, 30 min, 18%.
The authors thank Dr. Minghui Wang for NMR structural confir-
10. Breast cancer cells were plated, incubated for approximately 16-20 hours
and then treated with compound for 30 minutes. Final DMSO concentration
on all cells was 0.15%. The cells were washed with Tris buffer and lysed in
MesoScale Discovery (MSD) lysis buffer containing protease and phosphatase
mation of compounds 2 and 8.
Supplementary data
inhibitors (included in MSD kit). MSD Ser473-AKT duplex plates (Cat
#
MS6000) were used according to the manufacturer’s instructions and plates
were read on a SECTOR™ Imager 6000 using MSD Workbench software. For
analysis of the Ser473-pAKT concentration response curves, the data was
normalized using the corresponding total AKT value (sum of pAKT and AKT
signal) and plotted as the percent of the DMSO-treated control values. The
data was fit in Graphpad Prism version 4 for Windows (Graphpad Software,
San Diego, California).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.039.
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13. Compound 16 was prepared as shown in the following scheme.
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7. Enantiomers were separated by chiral HPLC, absolute configuration unknown.
E1 and E2 are denoted as the compounds relative elution off the column.
Details can be found in Supplementary data.
8. Inhibition of PI3K-isoforms was measured using
a continuous read time-
Reagents and conditions: (a) NH2NH2, THF, rt, overnight, 91%; (b)
triethylorthoformate, THF, AcOH, 135 °C, microwave, 30 min; KOH, MeOH
resolved fluorescence resonance energy transfer displacement assay Gray, A.;
Olsson, H.; Batty, I. H.; Priganica, L.; Downes, C. P. Anal. Biochem. 2003, 313, 234.

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R. M. Sanchez et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3198–3202
70 °C, 2 h, 60%; (Siegel, S.; Int. Patent WO113469, 2008); (c) (3-
trifluoromethyl-2-methyl)benzyl bromide, K2CO3, DMF, rt, overnight; (d)
morpholine, DIEA, MeOH, RT, overnight, 3% (combined steps c and d).
15. All studies were conducted after review by the GSK Institutional Animal Care
and Use Committee and in accordance with the GSK Policy on the Care, Welfare
and Treatment of Laboratory Animals.
14. Clearance is calculated following IV administration. The formulation for an IV
dose is 1% DMSO, 20% Encapsin in saline at a drug concentration of 0.25 mg/kg.