Novel purine and pyrazolo[3,4-d]pyrimidine inhibitors of PI3 kinase-α: Hit to lead studies

Article abstract of DOI:10.1016/j.bmcl.2009.11.051

Series of purine and pyrazolo[3,4-d]pyrimidine inhibitors of phosphatidylinositol-3-kinases (PI3K) have been prepared. The optimized purine inhibitors show good potency in a PI3K p110α (PI3K-α) fluorescence polarization assay with good selectivity versus PI3K p110γ (PI3K-γ) and the mammalian target of rapamycin (mTOR). The related pyrazolo[3,4-d]pyrimidines show potent PI3K-α and mTOR inhibition with good selectivity versus PI3K-γ. Representative compounds showed activity in a cellular proliferation assay against Caco-2 colorectal, LoVo colorectal and PC3MM2 prostate adenocarcinoma cancer cells. Signaling through the PI3K pathway was confirmed via inhibition of phospho-AKT in MDA-361 cells.

Full text of DOI:10.1016/j.bmcl.2009.11.051

Bioorganic & Medicinal Chemistry Letters 20 (2010) 636–639
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel purine and pyrazolo[3,4-d]pyrimidine inhibitors of PI3 kinase-a: Hit
to lead studies
a
b
a
a
Adam M. Gilbert a, , Pawel Nowak , Natasja Brooijmans , Matthew G. Bursavich , Christoph Dehnhardt ,
Efren Delos Santos a, Larry R. Feldberg c, Irwin Hollander c, Stephen Kim c, Sabrina Lombardi a,
Kaapjoo Park a, Aranapakam M. Venkatesan a, Robert Mallon c
*
a Medicinal Chemistry, Chemical Sciences, Wyeth Research, Pearl River, NY 10965, USA
b Computational Chemistry, Chemical Sciences, Wyeth Research, Pearl River, NY 10965, USA
c Discovery Oncology, Wyeth Research, 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:
Series of purine and pyrazolo[3,4-d]pyrimidine inhibitors of phosphatidylinositol-3-kinases (PI3K) have
Received 15 October 2009
Revised 9 November 2009
Accepted 16 November 2009
Available online 5 December 2009
been prepared. The optimized purine inhibitors show good potency in a PI3K p110
cence polarization assay with good selectivity versus PI3K p110 (PI3K- ) and the mammalian target
of rapamycin (mTOR). The related pyrazolo[3,4-d]pyrimidines show potent PI3K- and mTOR inhibition
with good selectivity versus PI3K- . Representative compounds showed activity in a cellular proliferation
a (PI3K-a) fluores-
c
c
a
c
assay against Caco-2 colorectal, LoVo colorectal and PC3MM2 prostate adenocarcinoma cancer cells.
Signaling through the PI3K pathway was confirmed via inhibition of phospho-AKT in MDA-361 cells.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Phosphatidylinositol-3-kinase
Phosphatidylinositol-3-kinases alpha
Mammalian target of rapamycin
Cellular proliferation
Oncology
Phosphatidylinositol-3-kinases (PI3K) are lipid kinases that
associate with Receptor Tyrosine Kinases (RTKs) and Ras. Eight
PI3Ks have thus far been identified and they have been separated
into 4 classes (IA, IB, II and III) based on substrate specificity and se-
quence homology. The class IA PI3Ks function by phosphorylating
the 3-hydroxyl position of phosphatidylinositol 4,5-diphosphate
(PIP2) to produce phosphatidylinositol 3,4,5-triphosphate (PIP3)
which influences cell shape, motility, growth, differentiation, and
survival via activation of Akt kinase.1,2 Of the 4 subtypes of class
related kinase of interest in cancer chemotherapy that signals
downstream of Akt.7 We herein describe the initial hit to lead opti-
mization of these purines to potent PI3K-a inhibitors with good
selectivity versus PI3K-c and mTOR. Work on related PI3K-a scaf-
folds has been previously published by Astellas8 and Piramed.9 We
also describe a related series of pyrazolo[3,4-d]pyrimidines which
show potent PI3K-a and mTOR activity with good selectivity ver-
sus PI3K- . We have recently published additional work on the
c
pyrazolo[3,4-d]pyrimidines as a series of selective inhibitors of
Ia PI3Ks, p110
cell growth and inhibit apoptosis in epithelial tumor cells. Addition-
ally, PI3K-
a (PI3K-a) deregulation has been shown to stimulate
mTOR.10,11
Purines (Tables 1 and 2) were prepared via the route shown in
Scheme 1.12 Thus commercially available 1 when heated with cyc-
lic amines selectively produces the 6-amino analogs 2.13 Suzuki
cross-coupling under standard conditions produces the corre-
sponding 2-aryl compounds 3.14 Mitsunobu reaction of 2 with
1-benzylpiperidin-4-ol produces piperidine analogs 4.15 Debenzy-
lation (R = Bn to R = H) is accomplished by standard hydrogenation.
a
deregulation is involved ꢀ26% of breast cancers in all
stages, as well as being associated with lymph node metastasis.
Similarly, PTEN (the phosphatase that regulates PIP3 levels) dereg-
ulation is associated with resistance to chemotherapeutic agents
that target the EGFR/Her2 signaling pathway.3,4 Thus PI3K-
a has
emerged as one of the premier targets for cancer treatment.5
A high-throughput screen of our compound library was run and
a series of purines were identified as having activity against
PI3K-a, p110c (PIK3-c), a related PI3K subtype (class IB) which is
Compounds were assayed using PI3K-a and PI3K-c fluorescence
polarization assays monitoring PIP3 production.16 Compounds
were assayed for mTOR potency using the DELFIA format assaying
for phosphorylated His6-SK6.17 Cellular proliferation activity was
assayed by monitoring cell growth densities in Caco2, LoVo, and
PC3MM2 cell cultures.18 Inhibition of PI3K signaling in MDA-361
cells was detected by immunoblotting protein lysates for phos-
phorylated-Akt (p-Akt).
a potential therapeutic target for inflammatory and autoimmune
diseases,6 and the mammalian target of rapamycin (mTOR), a
* Corresponding author.
E-mail address: gilbera@wyeth.com (A.M. Gilbert).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.051

A. M. Gilbert et al. / Bioorg. Med. Chem. Lett. 20 (2010) 636–639
637
Table 1
X
N
X
N
Structure–activity relationships of purine 2-(R2) and 4-(R3) positions
R3
Cl
a
b
N
N
N
N
N
N
N
N
N
Cl
N
R2
N
N
H
N
H
N
Cl
N
Cl
N
H
6-21
4
1
2
N
Compd R2
R3
PI3K-
a
PI3K-
c
mTOR
Bn
IC50 (nM)a IC50 (nM)a IC50 (nM)a
c
c
6
7
8
9
10
11
(4-OH)Ph
N-Morpholine 2538
N-Morpholine 2894
N-Morpholine 6700
N-Morpholine 1760
N-Morpholine 2330
8558
7972
10,000
5830
7371
7582
450
4500
nt
nt
150
nt
X
X
N
(4-HOCH2)Ph
(4-CONH2)Ph
(4-SO2Me)Ph
6-Indole
6-Dihydrobenzo- N-Morpholine 3477
dioxane
6-Benzodioxole N-Morpholine 1211
3-Thiophene
3-Furan
N
N
N
N
N
N
N
H
N
Ar
N
Ar
12
13
14
15
16
17
18
19
20
21
6907
8000
8900
3900
nt
2383
nt
nt
nt
nt
2500
2800
N-Morpholine 2381
N-Morpholine 3787
N-Morpholine 232
N-Piperidine 3769
N-Morpholine 2915
N-Morpholine 3902
11,000
2343
10,322
8297
7907
7909
5135
6742
5
3
(3-OH)Ph
(3-OH)Ph
(3-CN)Ph
N
R
R = Bn
R = H
d
(3-SO2Me)Ph
(3-NHSO2Me)Ph N-Morpholine 4379
(2-OH)Ph
2-Thiophene
Scheme 1. Reagents and conditions: (a) Amine, EtOH, reflux; (b) 1-benzylpiperidin-
4-ol, DEAD, PhP3, 0–23 °C; (c) ArB(OH)2, cat. Pd(PPh3)4, Na2CO3, DMF, H2O, 100 °C;
(d) H2, Pd/C , MeOH, 23 °C.
N-Morpholine 667
N-Morpholine 640
a
Values are means of two experiments, standard deviation is 10%.
idine); R2 = (3-HOCH2)Ph) shows potent PI3K-
45 nM), 25-fold selectivity versus PI3K- and 15-fold selectivity
versus mTOR. The corresponding R2 = (3-OH)Ph analog 25 shows
even greater selectivity versus PI3K- and similar mTOR potency
compared to 24. The R1 = 4-piperidine analog 26 also shows good
PI3K- potency, selectivity versus PI3K- (11-fold) and mTOR
(>100-fold) while the corresponding R2 = (3-OH)Ph analog 27
shows reduced selectivity over PI3K- and mTOR compared to 26.
Interested in how the corresponding pyrazolo[3,4-d]pyrimidine
analogs of Table 2 would profile, these compounds were prepared
according to the route in Scheme 2. Thus barbituic acid 28 is con-
verted to pyrimidine 29 via a known procedure.19 Formation of the
corresponding 1-piperidinyl-1H-pyrazolo[3,4-d]pyrimidine 30 is
accomplished via reaction with 1-benzyl-4-hydrazinylpiperidine
a inhibition (IC50:
c
Table 2
Structure–activity relationships of purine 9-(R1) and 2-(R2) positions
c
O
a
c
N
N
N
c
R2
N
N
R1
22-27
Compd R1
R2
PI3K-
a
PI3K-
c
mTOR
IC50 (nM)a IC50 (nM)a IC50 (nM)a
22
23
4-(N-Bn-piperi-
dine)-CH2
4-(N-Bn-piperi-
dine)-CH2
(3-HOCH2)Ph 248
(3-OH)Ph 199
1650
2378
10,500
600
Cl
O
Cl
N
24
25
26
27
4-(N-Bn-piperidine) (3-HOCH2)Ph 45
4-(N-Bn-piperidine) (3-OH)Ph 80
(3-HOCH2)Ph 58
(3-OH)Ph 65
1134
10,815
683
691
140
7200
870
OHC
Cl
N
b
NH
N
a
N
4-Piperidine
4-Piperidine
N
131
N
Cl
O
N
H
O
Cl
a
Values are means of two experiments, standard deviation is 10%.
30
28
N
29
Bn
Initially the 2-aryl (R2) moiety of the purine scaffold was varied
(Table 1). The (4-OH)Ph compound 6 shows
lM PI3K-a and PI3K-c
O
N
O
N
inhibition and sub- mTOR activity. Replacements for the
lM
(4-OH)Ph moiety (7–14) give compounds with similar PI3K-
a
potency. The R2 = 6-indole moiety can be used as an isosteric
d
c
N
N
replacement for the (4-OH)Ph group as shown in 10. An improve-
N
N
N
ment in PI3K-a potency as well as selectivity versus PI3K-c and
N
N
Ar
N
Cl
mTOR is seen with the R2 = (3-OH)Ph analogs 15. The importance
of morpholine as the 4-purine substituent is seen in 16 which
32
31
N
R
has reduced PI3K-
(3-OH)Ph moiety in 15 give less potent compounds (17–19). The
(2-OH)Ph and 2-thiophene compounds (20, 21) show sub-
PI3K- potency, but are not as potent as the 3-OH analog 15.
Additional PI3K- potency can be obtained by functionalization
a and PI3K-c activity. Replacements of the
N
Bn
R = Bn
R = H
e
lM
a
Scheme 2. Reagents and conditions: (a) POCl3, DMF, reflux, 60%; (b) 1-benzyl-4-
hydrazinylpiperidine dihydrochloride, Et3N, EtOH, À78 °C to 0 °C, 82%; (c) mor-
pholine, EtOH, 61%; (d) ArB(OH)2, Pd(PPh3)4, Na2CO3, dioxane, water, 100 °C; (e) H2,
Pd(OH)2/C (10%), MeOH.
a
of the 9-purine position R1 (Table 2). While 22 and 23 show a sim-
ilar profile to compounds in Table 1, analog 24 (R1 = 4-(N-Bn-piper-

638
A. M. Gilbert et al. / Bioorg. Med. Chem. Lett. 20 (2010) 636–639
dihydrochloride.10 Morpholine is then added selectively at the 4-
position to produce 31. The 6-aryl moiety is incorporated via stan-
dard Suzuki cross-coupling conditions to give 32, (R = Bn to R = H)
is accomplished by standard hydrogenation.
Table 3
Structure–activity relationships of pyrazolo[3,4-d]pyrimidine 1-(R1) and 6-(R2)
positions
O
The pyrazolo[3,4-d]pyrimidines (Table 2) show potent PI3K-
a
N
inhibition, but a different selectivity profile compared to the pur-
ines. While the purine analogs tend to show modest mTOR po-
tency, the pyrazolo[3,4-d]pyrimidines are more potent. Thus 33
(R1 = 4-(N-Bn-piperidine); R2 = (3-HOCH2)Ph)) shows potent PI3K-
N
N
R2
N
N
R1
33-36
a
and mTOR potency (compare with 24). The corresponding
R2 = (3-HOCH2)Ph analog 34 shows improved selectivity versus
PI3K- but maintains good mTOR potency (compare with 25).
The R1 = 4-piperidine analogs 35 and 36 show improved PI3K-
Compd R1
R2
PI3K-
a
PI3K-
c
IC50 mTOR IC50
(nM)a
IC50 (nM)a (nM)a
c
33
34
4-(N-Bn-
piperidine)
4-(N-Bn-
(3-HOCH2)Ph 25
(3-OH)Ph 32
975
90
83
a
selectivity versus mTOR compared to 33 and 34 but these com-
pounds have more mTOR potency compared to purines 26 and 27.
Rationalization of the potency and selectivity of the purine ser-
2367
piperidine)
35
36
4-Piperidine (3-HOCH2)Ph 26
4-Piperidine (3-OH)Ph 36
510
993
290
215
ies of inhibitors is shown by analyzing a PI3K-a homology model,
based on an X-ray structure of PI3K- , and compound 26 (Fig. 1).
c
a
Values are means of two experiments, standard deviation is 10%.
The critical H-bond between Val851 and the oxygen of the R2 mor-
pholine explains the reduction in potency seen for 16 (R2 = piperi-
dine) compared to the morpholine analogs. The R2 = (3-HOCH2)Ph
moiety makes a hydrogen bond with Asp810. Selectivity of 26 for
PI3-a versus PI3K-c and mTOR is not readily apparent, but the ob-
served selectivity profile of the compounds in Tables 1 and 2 leads
Comparing this data with PI3K-
reveals two possible trends. First, improved cellular activity ap-
pears to track with improving PI3K- and mTOR potency. Secondly,
the compound with the most potent cellular profile is pyrazol-
o[3,4-d]pyrimidine 33 which has a more desired p log D (1.46)
for compounds with good cellular permeability and stability com-
a, mTOR and p log D (pH 7.4 data)
a
us to hypothesize that the Asp810 H-bonding is more favorable in
PI3K-
ity versus PI3K-
protonatable piperidine nitrogen being better tolerated by Asp950
in PI3K- versus Ser919 and Ser2342 residues in PI3K- and mTOR,
a
resulting in more potent compounds. The reduced selectiv-
c
seen in 26 versus 24 may be rationalized by the
pared to other compounds with similar PI3K-a and mTOR potency
c
a
profiles (compare with 25 (3.00) and 34 (1.95)).
respectively. It’s also possible that large hydrophobic groups on the
piperidine (R = –Bn) in 22–25 are better tolerated by hydrophobic
To ensure that compounds were inhibiting PI3K signaling in
cells, two analogs were tested for p-Akt (at threonine 308) inhibi-
tion in MDA-361 human breast adenocarcinoma cells. Immuno-
residues in PI3K-
hydrophilic Lys890 in PI3K-
(Tables 1 and 2) show better PI3K-
a
(Met858) and mTOR (Leu2264) versus the more
. In general, the purine inhibitors
selectivity versus mTOR than
c
staining for cleaved-PARP (cPARP) was also conducted as
a
a
marker of apoptosis. Results for purine 24 and pyrazolo[3,4-
d]pyrimidine 35 are shown in Figure 2. p-Akt inhibition is seen
the corresponding pyrazolo[3,4-d]pyrimidines (Table 3) , but as the
nitrogens of the purine and pyrazolo[3,4-d]pyrimidine cores are
not involved in direct interactions with the enzymes, it is not clear
how the increased selectivity for the purines is achieved.
starting at 3
shows p-Akt reduction starting at 3
at 10 M. Suppression of p-Akt by both compounds is indicative
l
M for 24 and is complete at 30
lM. Similarly 35
lM and is almost complete
l
Several of the more potent purine and pyrazolo[3,4-d]pyrimi-
dine analogs were tested for cellular proliferation activity against
Caco-2 epithelial colorectal adenocarcinoma cells, LoVo human co-
lon carcinoma cells, and PC3 human prostate cancer cells (Table 4).
of inhibition of cellular PI3Ks which are upstream of Akt in the
PI3K signaling pathway.
In conclusion, we have disclosed series of purine and pyrazol-
o[3,4-d]pyrimidines PI3K inhibitors which show activity in adeno-
carcinoma cellular proliferation assays and inhibit p-Akt formation
in MDA-361 cells. Purine inhibitors have been identified which
show potent PI3K-a activity, and good selectivity (>10-fold) versus
PI3K- and mTOR. The related pyrazolo[3,4-d]pyrimidines main-
c
tain good PI3K-a potency and show good potency against mTOR
Table 4
Cellular proliferation inhibitory activity of selected purine and pyrazolo[3,4-
d]pyrimidines
Compd PI3K-
a
mTOR
Caco IC50 LoVo IC50 PC3 IC50 p log D
IC50 (nM)a IC50 (nM)a (nM)a
(nM)a
(nM)a
(pH 7.4)
20
21
22
23
24
25
26
27
33
34
35
36
667
640
248
199
45
80
58
65
25
2500
2800
10,500
600
691
140
7200
870
90
83
290
>10,000
>10,000
5787
6489
5168
>10,000
4496
4037
1342
5969
7307
>10,000
>10,000
9269
7347
1282
2.42
2.72
3.11
3.52
2.59
3.00
>10,000
3077
3508
1047
1947
1870
2022
511
1111
2364 À0.82
1521 À1.27
345
940
1.46
1.95
32
26
36
1608
1433
1576
5300
5584
690 À1.83
Figure 1. Binding mode of compound 26 in a PI3K-
X-ray structure of PI3K- . Residues of PI3K- (green), PI3K-
(magenta) are included to help rationalize selectivity.
a
homology model based on an
215
922 À1.27
c
a
c
(orange) and mTOR
a
Values are means of two experiments, standard deviation is 10%.

A. M. Gilbert et al. / Bioorg. Med. Chem. Lett. 20 (2010) 636–639
639
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a homology model derived from an X-
c
a
c
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