Design and evaluation of a series of pyrazolopyrimidines as p70S6K inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.01.105

The 70-kDa ribosomal protein S6 kinase (p70S6K) is part of the PI3K/AKT/mTOR pathway and has been implicated in cancer. High throughput screening versus p70S6K led to the identification of aminopyrimidine 3a as active inhibitor. Lead optimization of 3a resulted in highly potent, selective, and orally bioavailable pyrazolopyrimidines. In this manuscript we report the structure-activity relationship of this series and pharmacokinetic, pharmacodynamic, and efficacy data of the lead compound 13c.

Full text of DOI:10.1016/j.bmcl.2012.01.105

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2283–2286
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and evaluation of a series of pyrazolopyrimidines as p70S6K inhibitors
⇑
Joerg Bussenius , Neel K. Anand, Charles M. Blazey, Owen J. Bowles, Lynne Canne Bannen,
Diva S.-M. Chan, Baili Chen, Erick W. Co, Simona Costanzo, Steven C. DeFina, Larisa Dubenko,
Stefan Engst, Maurizio Franzini, Ping Huang, Vasu Jammalamadaka, Richard G. Khoury, Moon H. Kim,
Rhett R. Klein, Douglas Laird, Donna T. Le, Morrison B. Mac, David J. Matthews, David Markby,
Nicole Miller, John M. Nuss, Jason J. Parks, Tsze H. Tsang, Amy L. Tsuhako, Yong Wang, Wei Xu,
Kenneth D. Rice
Exelixis, 210 E. Grand Avenue, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The 70-kDa ribosomal protein S6 kinase (p70S6K) is part of the PI3K/AKT/mTOR pathway and has been
implicated in cancer. High throughput screening versus p70S6K led to the identification of aminopyrim-
idine 3a as active inhibitor. Lead optimization of 3a resulted in highly potent, selective, and orally bio-
available pyrazolopyrimidines. In this manuscript we report the structure–activity relationship of this
series and pharmacokinetic, pharmacodynamic, and efficacy data of the lead compound 13c.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 20 December 2011
Revised 17 January 2012
Accepted 20 January 2012
Available online 2 February 2012
Keywords:
p70S6K inhibitor
Pyrazolopyrimidine
PI3K pathway
Cancer
Hyperactivated signaling due to dysregulation of the phosphoin-
ositide 3-kinase (PI3K)/phosphatase and tensin homolog on
chromosome ten (PTEN) pathway is observed in many cancers
and correlates with tumor growth and survival.1 These observations
suggest that inhibitors targeting components of the PI3K/PTEN
kinase cascade may have utility for therapeutic intervention in
cancer, and several excellent reviews describing this strategy have
been published.2–5 The last kinase of the PI3K/AKT/mammalian
target of rapamycin (mTOR) pathway is the 70-kDa ribosomal pro-
tein S6 kinase (p70S6K). p70S6K is a serine/threonine kinase that
belongs to the AGC kinase family.6 In response to activation by a
complex of mTOR with two proteins, Raptor and GbL (this complex
is commonly referred to as TORC1), p70S6K phosphorylates the S6
protein of the 40S ribosomal subunit. Phosphorylation of S6 is in-
volved in translational control of 50 oligopyrimidine tract mRNAs
and ultimately results in upregulation of cellular protein synthesis.7
As part of our overall strategy to develop kinase inhibitors with
different inhibition profiles within the PI3K/Akt/mTOR pathway we
were interested in investigating the selective inhibition of p70S6K,
which has been targeted by other groups as well.8
against p70S6K. This campaign led to the identification of the
4-amino-5-nitropyrimidine-containing inhibitor 3a with an IC50
of 85 nM (no activity below 2000 nM against any other kinase
of our in-house panel), which was considered acceptable for
further structure–activity optimization efforts (Table 1).9 These
efforts consisted of two separate activities which were carried
out in parallel. First, to improve the biochemical activity of 3a
Table 1
Biochemical activity of compounds 3a–h
Y
Cl
X
N
N
H
N
N
N
R
ID
X
Y
R
p70S6K IC50 (nM)
3a
3b
3c
3d
3e
3f
NO2
NO2
H
H
H
H
H
H
Me
H
H
H
H
H
Me
85
3
Our p70S6K research program began with a high throughput
screening campaign of our internal small molecule library
>2000
>2000
196
162
107
13
Me
N = CH
CH = CH
CH = N
CH = N
3g
3h
⇑
Corresponding author. Tel.: +1 6508377000; fax: +1 6508378177.
E-mail address: jbusseni@exelixis.com (J. Bussenius).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.105

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J. Bussenius et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2283–2286
we embarked on a survey of different substitution patterns on
the phenylpiperazine part of the molecule. A total of 55 com-
pounds were evaluated, and from this set the 2-methyl-5-chlor-
ophenylpiperazine 3b had the lowest IC50 of 3 nM (Table 1).
Second, we were concerned about the potential for multistep
reductive bioactivation forming hazardous metabolites from the
nitro group contained in 3a10 and therefore directed our efforts
to find a replacement for this substituent. Complete removal
(compound 3c) or substitution with other small groups (e.g.,
3d) resulted in significant loss of activity. More successful was
the incorporation of both the nitro and the amino nitrogens in
an imidazole heterocycle. The resulting imidazopyrimidine 3e
exhibited only a twofold loss of activity compared to 3a. Encour-
aged by this result we also prepared the pyrrolopyrimidine 3f
and pyrazolopyrimidine 3g. In particular the pyrazolopyrimidine
3g was almost as active as the original HTS hit 3a. We then syn-
thesized compound 3h to determine if the SAR from the phenyl-
piperazine pattern translated from the nitropyrimidines to the
new pyrazolopyrimidine. Indeed, by making the small change
from 3g to 3h the biochemical activity was increased almost
tenfold. Compound 3h was viewed as lead compound for further
SAR development, considering the improvement of biochemical
activity and reduction of potential metabolic liabilities over
HTS hit 3a. The compounds in Table 1 were prepared by reaction
of commercially available chloropyrimidine analogs or the corre-
sponding bicyclic systems with either 3-chlorophenylpiperazine
or 5-chloro-2-methylphenylpiperazine as illustrated in Scheme 1.
Despite good biochemical activity, compound 3h only had an
cells (Table 2).11 We therefore planned on increasing the lipophil-
icity of the compounds to improve permeability. To get a better
understanding of which modifications could be beneficial, homol-
ogy models of p70S6K were constructed based on the crystal
structure of active (PDB code 1O6L) and inactive (PDB code
1GZK) Akt (PKB). The model proposed binding of the pyrazole
NH with the carbonyl of Glu173 and the pyrimidine N-7 with the
backbone NH of Leu175 in the kinase hinge region. The model also
predicted that substitution in the 6-position of the pyrazolopyrim-
idine would not be tolerated, since it was too close to the C@O of
the backbone of Leu175. This was confirmed experimentally by
compound 4 (Table 2). On the other side, substitution on C-3 with
a hydrophobic group seemed to be favorable. The synthesis of
compounds 5a and 5b with increased steric bulk in the 3-position
suggested that this proposal was also correct. In addition to a po-
sitive effect on biochemical activity, the structural change resulted
in compounds with increased lipophilicity as indicated by their
NC
H2N
R2
NC
NC
R2
R2
O
a, b
c
Cl
N
OMe
N
H
6a: R2 = iPr
6b: R2 = nPr
7a,b
8a,b
Cl
N
R2
R2
N
OH
N
d
e
N
N
N
IC50 of 1.1
lM in the cellular S6 phosphorylation assay in A549
N
H
N
H
10a,b
9a,b
Cl
Scheme 2. Synthesis of non-commercial chloropyrazolopyrimidines. Reagents and
conditions: (a) malononitrile, THF, NaH, rt, 16; (b) TMS-diazomethane, Et2O, MeOH,
0 °C, 3 h; (c) hydrazine monohydrate, EtOH, 80 °C, 3 h; (d) formic acid, 110 °C, 4 h;
(e) SOCl2, DMF, reflux, 2 h.
R
Cl
N
N
Cl
N
R
X
N
X
a
N
Table 3
Activity of solubilized analogs
+
Y
Y
N
N
H
N
H
N
N
H
4'
3'
Z
Cl
R4R3N
3
1
2
Scheme 1. Synthesis of compounds 3 (for detailed structures see Table 1). Reagents
and conditions: (a) EtOH, DIEA, 70 °C, 4–15 h, (for 3c: NMP, DIEA, 180 °C, 16 h; for
3d: DMSO, DIEA, 140 °C, 15 h).
N
N
R2
N
Table 2
N
Activity of substituted pyrazolopyrimidines
N
H
N
3
R2
N
N
1
NH
N
Cl
ID
R2
Z
NR3R4
p70S6K IC50 (nM)
A549 Cell IC50 (nM)
11a
12a
13a
Et
Et
Et
O
CH2
NH
NEt2
NEt2
NEt2
11
3
3
339
211
184
N
N
1
6
R
11b
12b
13b
Et
Et
Et
O
N
N
N
5
2
3
243
152
80
ID
R1
R2
p70S6K IC50 (nM)
A549 Cell IC50 (nM)
clogP
CH2
4
Me
H
H
H
H
H
H
H
H
H
>2000
13
9
6
16
6
—
4.0
3.5
3.8
4.3
4.7
4.9
4.4
4.5
3h
5a
5b
5c
5d
5e
5f
1132
500
150
207
275
86
Me
Et
iPr
nPr
Br
NH
11c
13c
14
Et
Et
Br
CF3
O
NMe2
NMe2
NMe2
NMe2
6
2
2
4
143
45
43
NH
NH
NH
7
29
15
50
CF3
41

J. Bussenius et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2283–2286
2285
clogP values, and also significantly increased their cellular poten-
cies (Table 2). However, there are limitations to the overall size
of this group. Larger alkyl substituents as in 5c or 5d showed no
benefit in biochemical activity and also led to a reduction in cell
based potency. The SAR motivated us to focus on the improved
lead compound 5b for ongoing optimization. Also of interest were
the bromo-compound 5e and the trifluoromethyl analog 5f, since
both substituents resulted in further improvements in cellular
potency.
For the synthesis of the compounds shown in Table 2 the same
reaction as described in Scheme 1 was used. The required substi-
tuted 4-chloropyrazolopyrimidines were either commercially
available (R2 = H, Me, Et, Br, CF3); or prepared according to Scheme
2 (R2 = iPr, nPr). Condensation of malononitrile with either butyryl-
or isobutyryl chloride afforded enols, which were treated with
TMS-diazomethane to give the enolethers 7a,b. Cyclization of
7a,b with hydrazine provided the pyrazoles 8a,b. Compounds
8a,b were then transformed into the chloroypyrazolopyrimidines
10a,b by condensation with formic acid followed by chlorination
with thionyl chloride.
to incorporate a basic amino group in order to improve aqueous
solubility. Our homology model predicted substitution at C-30 on
the phenyl group as most likely to avoid negative interactions with
the protein. The 40-position on the other hand was too close to the
G-loop of the enzyme to allow appropriate substitution. Evaluation
of compounds with basic amino groups attached via oxygen,
carbon, and nitrogen on C-30 of the phenyl moiety (11a–c, 12a
and b, 13a–c, Table 3) indeed showed good tolerance for these sub-
stituents. While all the compounds are very potent, two trends can
be observed. First, the biochemical activities of the C and N linked
compounds are comparable, and both are considerable higher than
the O analogs. For the O analogs the reduced biochemical activity is
also reflected in lower cellular potency. Second, despite almost
identical biochemical activities of the C and N analogs, the N linked
compounds show significantly higher cellular potency. In particu-
lar the dimethyl-aminoethyl-amino analog 13c demonstrates
excellent potency against S6 phosphorylation in A549 cells.
Replacement of the ethyl group on the pyrazolopyrimidine with
a bromo- (14) or trifluoromethyl-substituent (15) did not result
in any improvement of cell based activity over 13c in contrast to
our observations of the unsolubilized analogs 5b, 5e, and 5f.
Ethyl analog 13c also had the best selectivity profile compared
to 14 or 15. When screened against our in-house kinase panel,
compound 13c showed no cross reactivity towards non-AGC
kinases (IC50 >1000 nM). However, some activity towards AGC
With compound 5b being very active in the biochemical assay
and also showing good potency in the cell, we were interested in
its pharmacokinetic profile, which was determined in the rat. As
shown in Table 4, the compound exhibited poor oral absorption
and low oral bioavailability. To overcome these liabilities we chose
Table 4
Comparison of rat PK properties of compound 5b with 13c
ID
Dose (mg/kg)
CL (mL/h/kg)
Vd (L/kg)
T1/2 (h) IV/PO
F (%)
Cmax
(l
M) IV/PO
AUC/dose (lM h kg/mg) IV/PO
5b
13c
2.5
2.5
1549
1044
6.3
3.7
7.1/1.4
2.9/3.3
2
23
6.29/0.03
1.47/0.15
1.74/0.02
1.72/0.45
O
I
Cl
B
Cl
HO
Cl
O
I
Cl
a, b
c
d
N
N
N
NH2
N
N
N
Boc
Boc
Boc
16
18
17
19
g
i, f
e, f
H
N
O
Cl
Cl
Cl
OHC
Cl
R"R'N
R"R'N
R"R'N
N
N
N
N
h, f
N
H
N
H
N
H
N
H
21
22a,b,c
23a,b,c
20a,b,c
Scheme 3. Preparation of phenylpiperazines used for products listed in Table 3. Reagents and conditions: (a) bis(2-chloroethyl)amine, diglyme, K2CO3, reflux, 24 h, 40%; (b)
di-tert-butyldicarbonate, THF, rt, 15 h, 92%; (c) bis(pinacolato)diborane, PdCl2dppf, KOAc, DMSO, 80 °C, 24 h, 48%; (d) H2O2, NaOH, THF, rt, 30 min, quantitative; (e)
substituted chloroethylamine (a: NR0R00 = NEt2, b: NR0R00 = pyrrolidine, c: NR0R00 = NMe2), Cs2CO3, DMF, rt, 16 h; (f) HCl, dioxane, MeOH, reflux, 2 min; (g) allyl alcohol, NaHCO3,
Pd(OAc)2, TBAC, DMF, 50 °C, 4 h, 69%; (h) amine (a: diethylamine, b: pyrrolidine, c: dimethylamine), NaB(OAc)3H, dichloroethane; (i) corresponding aminoethylamine,
NaOtBu, XANTPHOS, Pd2(dba)3, dioxane, reflux, 5 h.

2286
J. Bussenius et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2283–2286
kinases, particularly Rsk2 (68 nM) and PKA (42 nM) were observed.
For the two Akt isoforms Akt1 and Akt2 only weak activity was
measured (371 and 3021 nM, respectively). Compound 14 on the
other hand had stronger activity against AGC kinases (Akt1:
53 nM, Akt2: 311 nM, Rsk2: 248 nM, and PKA: 25 nM). Addition-
ally, this compound showed some cross reactivity with IC50s
<500 nM against several other targets outside the AGC kinase fam-
ily (e.g., PIM1). Further, the trifluoromethyl-analog 15 was also ac-
tive against AGC kinases (Akt1: 74 nM, Akt2: 477 nM, Rsk2: 24 nM,
and particularly PKA with 6 nM).
On the basis of these in vitro data we decided to take compound
13c forward into in vivo studies. First, the pharmacokinetic profile
of 13c was determined in the rat and the results are summarized in
Table 4. Compared to compound 5b, the absorption and oral bio-
availability were significantly improved. This result in combination
with the high potency led to the advancement of 13c into a mouse
pharmacodynamic study in which the inhibition of S6 phosphory-
lation was measured. In this study 13c demonstrated >60% inhibi-
tion at 100 mg/kg PO dosing in the PC3 prostate carcinoma model.
Compound 13c was subsequently evaluated in a PC3 xenograft effi-
cacy experiment and dosed orally at 30, 100, and 300 mg/kg twice
a day for 14 days. The compound was also active in this study and
showed a good dose–response relationship with 21%, 49%, and 69%
tumor growth inhibition, respectively
executing cell based assays. Julie Ren and Yeping Zhao generated
the PK data. Paul Keast, Wendy Abulafia, Brian Sutton, and Teresa
Carabeo performed the PD experiment and Jason Chen and F. Mi-
chael Yakes the efficacy study.
References and notes
1. Vivanco, I.; Sawyers, C. L. Nat. Rev. Cancer 2002, 2, 489.
2. Hennessy, B. T.; Smith, D. L.; Ram, P. T.; Lu, Y.; Mills, G. B. Nat. Rev. Drug
Discovery 2005, 4, 988.
3. Engelman, J. A. Nat. Rev. Cancer 2009, 9, 550.
4. Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Nat. Rev. Drug Discovery 2009, 8, 627.
5. Courtney, K. D.; Corcoran, R. B.; Engelman, J. A. J. Clin. Oncol. 2010, 28, 1075.
6. Matthews, D. J.; Gerritsen, M. E. Targeting Protein Kinases for Cancer Therapy;
John Wiley & Sons: Hoboken, NJ, 2010. 274–275 and 282–283.
7. Thomas, G. Biol. Res. 2002, 35, 305.
8. (a) Bandarage, U.; Hare, B.; Parsons, J.; Pham, L.; Marhefka, C.; Bemis, G.; Tang,
Q.; Moody, C. S.; Rodems, S.; Shah, S.; Adams, C.; Bravo, E.; Savic, V.; Come, J. H.;
Green, J. Bioorg. Med. Chem. Lett. 2009, 19, 5191; (b) Ye, P.; Kuhn, C.; Juan, M.;
Sharma, R.; Connolly, B.; Alton, G.; Liu, H.; Stanton, R.; Kablaoui, N. M. Bioorg.
Med. Chem. Lett. 2011, 21, 849.
9. p70S6K assay: kinase activity was measured as the percent of ATP consumed
following
the
kinase
reaction
using
luciferase–luciferin-coupled
chemiluminescence. Reactions were conducted in 384-well white, medium
binding microtiter plates (Greiner). P70S6 Kinase reactions were initiated by
combining test compounds, 500 nM ATP,
12 nM Human p70S6K (residues 1–421, contains D18H and T412E mutations,
Upstate Bio-technology) in 20 L of reaction buffer (20 mM Hepes pH 7.5,
10 mM MgCl2, 0.03% NP40, 0.03% BSA, 1 mM DTT). The reaction mixture was
incubated at ambient temperature for 3 h after which 20 aliquot of
3 lM RRRLSSLRA (Synpep), and
l
lL
Scheme 3 describes the synthesis of highly functionalized phe-
nylpiperazines needed for the preparation of the products shown
in Table 3. Reaction of aniline 16 with bis(2-chloroethyl)amine fol-
lowed by protection with di-tert-butyl dicarbonate yielded iod-
ophenylpiperazine 17. Palladium mediated conversion of 17 into
pinacolester 18 and oxidation with hydrogen peroxide gave phenol
19, which was then alkylated with chloroethylamines followed by
deprotection to afford the phenylpiperazines 20a–c, where the ba-
sic amino group is attached to the phenyl ring via oxygen. The
preparation of the corresponding compounds 22a–c, in which the
basic amine was tethered to the phenyl ring through a propyl lin-
ker, was carried out by Heck reaction of iodophenylpiperazine 17
with allyl alcohol,12 followed by reductive amination and depro-
tection. Finally, the nitrogen linked analogs 23a–c were synthe-
sized by Buchwald–Hartwig amination of 17 followed by
deprotection of the Boc group. The coupling of these phenylpiper-
azines with the respective chloropyrazolopyrimidines was then
carried out again as described in Scheme 1.
luciferase–luciferin mix (50 mM HEPES, pH 7.8, 67 mM oxalic acid (pH 7.8), 5
(or 50) mM DTT, 0.4% Triton X-100, 0.25 mg/mL coenzyme A, 63 mM AMP,
28 mg/mL luciferin and 40,000 units/mL luciferase) was added and the
chemiluminescence signal measured using a Victor2 plate reader (Perkin–
Elmer).
10. Boelsterli, U. A.; Ho, H. K.; Zhou, S.; Leow, K. Y. Curr. Drug Meta. 2006, 7, 715.
11. A549 cell assay: cells were seeded in 96-well plates (all plates and dishes are
NUNC unless otherwise noted) in DMEM (Cellgro) containing 10% FBS (all FBS
is heat-inactivated and from Hyclone unless otherwise noted), 1% penicillin–
streptomycin (Cellgro), and 1% non-essential amino acids (Cellgro). A549 cells
were seeded at 7.5 ꢀ 103cells/well. Cells were incubated at 37 °C, 5% CO2 for
48 h. Test compound in DMSO was diluted in DMEM. The DMSO concentration
in cell culture medium was maintained at 0.2% in all wells. Cells were treated
with diluted compound and each concentration assayed in triplicate at
concentrations of 1.2–300 nM, 0.004–1 nM rapamycin (Cell Signaling
Technology, 9904) and 0.12–30 nM staurosporine (Calbiochem, 569396)
served as positive controls. Negative control wells were treated with DMSO.
Cells were incubated for 3 h at 37 °C, 5% CO2 for 3 h in the presence of
compound. Post compound treatment cells were fixed as follows: medium was
removed and 100
TBS (Pierce, 28376) was added to each well at room temperature (rt) for
20 min. Cells were quenched with 100 0.6% H2O2 (VWR International,
43038308) in TBS (Pierce, 28376) +0.1%Tween20 (Sigma, P-7949) (TBST) for
l TBS and blocked with 100
ll/well of 4% formaldehyde (Sigma–Aldrich, 08920AD) in
ll
20 min at rt. Plates were washed 3ꢀ with 200
l
ll
In summary, we have outlined the successful optimization of
novel and highly potent p70S6K inhibitors based on the pyrazolo-
pyrimidine structural motif. The resulting lead compound 13c is
selective over other kinases, has a reasonable ADME profile, and
is efficacious in the PC3 xenograft model. Further optimization of
13c leading to clinical candidate XL418 will be reported in due
course.
5% BSA (Jackson ImmunoResearch Laboratory, 001-000-162) in TBST for 1 h at
rt. Anti-phospho-S6 ribosomal protein antibody (Cell Signaling Technology, Ser
240/244 2212L) and anti-total-S6 ribosomal protein antibody (Cell Signaling
Technology, 2215L) were diluted 1:200 in 5% BSA in TBST. Fifty microlitre
primary antibody solution was added to each well and incubated overnight at
4 °C. Plates were washed 3ꢀ with 200
antibody (Chemicon International, 24070101) was diluted at 1:20000 in 5%
non-fat dry milk in TBST. 50 l of antibody solution was added to each well and
l TBST and 2 ꢀ 200
ll TBST. Goat anti-rabbit secondary
l
incubated for 1 h at rt. Plates were washed 3ꢀ with 200
l
ll
TBS. Chemiluminescent substrate (Super Signal Elisa Femto Chemiluminescent
Substrate; Pierce, 37075) was prepared at rt. Hundred microlitre of
chemiluminescent substrate per well was added and then the plate was
shaken for 1 min. Luminescence was read immediately on a Wallac plate
reader.
Acknowledgments
The authors appreciate the contributions of the following
persons. Steven Richards for help with the manuscript. Tracy Lou
for conducting the biochemical assays and Edith Ubannwa for
12. Melpolder, J. B.; Heck, R. F. J. Org. Chem. 1976, 41, 265.