Pyrazolopyrimidines as highly potent and selective, ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR): Optimization of the 1-substituent

Article abstract of DOI:10.1016/j.bmcl.2009.12.086

A series of pyrazolopyrimidine mammalian Target Of Rapamycin (mTOR) inhibitors with various substituents at the 1-position have been prepared, resulting in compounds with excellent potency, selectivity and microsomal stability. Combination of a 1-cyclohexyl ketal group with a 2,6-ethylene bridged morpholine in the 4-position and a ureidophenyl group in the 6-positon resulted in compound 8a, that selectively suppressed key mTOR biomarkers in vivo for at least 8 h following iv administration and showed excellent oral activity in a xenograft tumor model.

Full text of DOI:10.1016/j.bmcl.2009.12.086

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1440–1444
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazolopyrimidines as highly potent and selective, ATP-competitive
inhibitors of the mammalian target of rapamycin (mTOR):
Optimization of the 1-substituent
a
a
a
b
Kevin J. Curran a, , Jeroen C. Verheijen , Joshua Kaplan , David J. Richard , Lourdes Toral-Barza ,
*
Irwin Hollander b, Judy Lucas b, Semiramis Ayral-Kaloustian a, Ker Yu b, Arie Zask a
a Chemical Sciences, Wyeth Research, 401 N. Middletown Rd, Pearl River, NY 10965, United States
b Oncology Research, Wyeth Research, 401 N. Middletown Rd, 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:
A series of pyrazolopyrimidine mammalian Target Of Rapamycin (mTOR) inhibitors with various substit-
uents at the 1-position have been prepared, resulting in compounds with excellent potency, selectivity
and microsomal stability. Combination of a 1-cyclohexyl ketal group with a 2,6-ethylene bridged mor-
pholine in the 4-position and a ureidophenyl group in the 6-positon resulted in compound 8a, that selec-
tively suppressed key mTOR biomarkers in vivo for at least 8 h following iv administration and showed
excellent oral activity in a xenograft tumor model.
Received 6 October 2009
Revised 18 December 2009
Accepted 22 December 2009
Available online 4 January 2010
Keywords:
mTOR
Ó 2010 Elsevier Ltd. All rights reserved.
P13K
Kinase
Rapamycin and its analogs (e.g., Torisel™) inhibit the serine–
threonine protein kinase mammalian Target Of Rapamycin (mTOR)
and as such play an important role in the disruption of key cell-
signaling pathways in a variety of tumors.1 mTOR forms two
functional complexes in cells: mTORC1 and mTORC2.2 While rapa-
mycin and its analogs allosterically bind to mTORC1 and inhibit
cell signaling mediated by mTORC1, mTORC2 activates Akt by
phosphorylation of Ser473, resulting in potentially anti-apoptotic
effects.3,4 Treatment with rapamycin leads to upregulation of Akt
signaling in certain cells.5 Since a direct inhibitor of mTOR would
inhibit the function of mTORC1 and mTORC2, a reduction in Akt
signaling would be expected. Thus, inhibition of mTOR as opposed
to mTORC1 alone may lead to more efficacious therapeutic agents.
We have previously demonstrated that highly potent and selec-
tive ATP-competitive inhibitors of mTOR can be prepared from a
pyrazolopyrimidine scaffold, equipped at the 4-position with a
morpholine derivative, and the 6-position with a ureidophenyl
group.6–9 In this Letter, we describe a detailed overview of the
structure–activity relationship (SAR) in the 1-position of the pyraz-
olopyrimidines culminating in the identification of a compound
with excellent in vivo efficacy.
afforded the 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine, which
was treated with 8-oxa-3-azabicyclo[3.2.1]octane (2,6-ethylene
bridged morpholine)8,13 to give 2. Suzuki coupling of 2 with the
pinacol ester of 4-aminophenylboronic acid gave the correspond-
ing aniline that was converted to the ureidophenyl analog 3 by
treatment with triphosgene and an amine. When R1 is benzylpi-
peridine, debenzylation of 3 with
a-chloroethyl chloroformate
was followed by treatment with the appropriate chloroformate
to give carbamates 4.
The compounds in Tables 3 and 4 were prepared according to
Schemes 2 and 3. As shown in Scheme 2, 1,4-cyclohexanedione
mono-ethyl ketal 5 was treated with t-butyl carbazate followed
by removal of the t-Boc group in refluxing water to give hydrazine
6. Condensation of hydrazine 6 with trichloroaldehyde 1, and con-
version of resulting 7 to ureas 8 was done as described above.
Scheme 2 also shows the synthesis of the cyclohexyl methyl
ether intermediates. The ketal 7 was hydrolyzed to the ketone
and reduced with lithium aluminum hydride to yield 9, as a 3:1
mixture of trans to cis isomers, respectively, that were separated
by silica gel column chromatography. Alkylation with methyl io-
dide and sodium hydride afforded methyl ethers 10 (trans) and
11 (cis), that were then converted into the methylureidophenyl
compounds (12 and 13, respectively).
The analogs in Tables 1 and 2 were synthesized as previously
described7,10–12 or as shown in Scheme 1. Cyclization of an appro-
priate hydrazine with 2,4,6-trichloropyrimidine-5-carbaldehyde 1
The cis and trans cyclohexanols, as well as additional ketals
could be prepared from ketone 14, obtained after hydrolysis of
8a with HCl (Scheme 3). Treatment of 14 with either L-Selectride
or lithium aluminum hydride gave the cis isomer 16 or the trans
* Corresponding author.
E-mail address: currank@wyeth.com (K.J. Curran).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.086

K. J. Curran et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1440–1444
1441
Table 1
Table 2
1-Piperidine carbamates
1-Alkyl substituted pyrazolopyrimidines
O
N
O
N
4
N
N
N
N
N
N1
O
N
O
N
6
R
N
N
N
N
H
H
H
H
N
mTORa PI3Kaa Sel.b
LNCapa Micros.c
R
Compd
R
3a
3b
3c
3d
3e
3f
–CH3
–Et
–iPr
–CH2CF3
–CH2CH2OH
1.34
0.74
0.12
0.50
2.85
247
270
234
1616
375
966
184
364
1950
3232 38
132
66
170
70
5
>30
>30
>30
>30
NT
Compd
R
mTORa
0.22
PI3Kaa Sel.b
LNCapa
26
Micros.c
>30
O
O
O
4a
1803
2021
1475
8195
O
O
O
650
400
–CH2CH2N(CH3)2 14.5
5
4b
4c
0.19
0.26
10,636
5673
6
21
11
NT = not tested.
a
Average IC50 (nM). The average error for IC50 determinations was <25%.
PI3K
Nude mouse microsomes T1/2 (min).
b
c
a/mTOR.
27
a
b
c
Average IC50 (nM). The average error for IC50 determinations was <25%.
PI3K /mTOR.
Nude mouse microsomes T1/2 (min).
a
4a was selected as the starting point for further SAR studies, aimed
at identifying the optimal substituent in the 1-position. Initial ef-
forts focused on the preparation of other carbamoylpiperidines
(e.g., 4b and 4c, Table 1). Although the resulting inhibitors re-
mained highly potent and selective, their microsomal stability
was significantly reduced. We had previously shown that amides
and ureas were also inferior to carbamates in this position.7
Next, a number of 1-alkyl substituted pyrazolopyrimidines
were investigated (Table 2). The presence of alkyl groups at this
site generally led to inhibitors with reduced selectivity and moder-
ate cellular activity (e.g., 3a and 3b). Two compounds that were
highly selective and possessed good cellular activity (isopropyl
analog 3c and trifluoroethyl analog 3d) were stable in nude mouse
microsomes but suffered from low human microsomal stability
(T1/2 = 12 and 5 min, respectively). Introduction of polar substitu-
ents (primary alcohol 3e or tertiary amine 3f) reduced both
enzyme and cellular potency.
isomer 15, respectively. Treatment of ketone 14 with the appropri-
ate alcohol in the presence of trimethyl or tripropyl orthoformate,
molecular sieves and p-toluenesulfonic acid afforded the ketals 17
and 18.
SAR studies of 4-morpholino-pyrazolopyrimidines revealed that
a ureidophenyl group was the optimal substituent in the 6-position
and resulted in the identification of potent and selective inhibitors
with a methylcarbamoyl piperidine in the 1-position.7,9 The selec-
tivity of these inhibitors could be further enhanced by introduction
of a 2,6-ethylene bridged morpholine in the 4-position,8 leading to
compounds such as 4a (Table 1), which combined high potency
and selectivity14–16 with excellent stability in a nude mouse micro-
some assay.17 Molecular modeling suggests that a single amino
acid difference between PI3K and mTOR (Phe961Leu) accounts
for the profound selectivity seen by creating a deeper pocket in
mTOR that can accommodate bridged morpholines.8 Compound
As substitution at the 1-position with acyclic alkyl groups did
not result in analogs which met our requirements for cellular
O
N
Cl
a, b
c,d
NH2
N
N
HN
R1
O
N
+
N
Cl
N
Cl
N
Cl
R1
1
2
O
N
O
N
Bn N
e, f
R1
=
N
N
N
N
N
N
O
N
O
N
R1
R2
R2
N
N
N
N
H
H
H
H
R3
N
4
3
O
O
Scheme 1. R1, R2, and R3 are as defined in tables. Reagents and conditions: (a) ethanol, triethylamine; (b) 2,6-ethylene bridged morpholine; (c) 4-aminophenylboronic acid
pinacol ester, palladium (0), sodium carbonate, toluene, ethanol; (d) dichloromethane, triethylamine, triphosgene, then R2NH2; (e) dichloroethane,
chloroformate; (f) dichloromethane, R3OCOCl.
a-chloroethyl

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K. J. Curran et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1440–1444
Table 3
1-Cyclohexyl analogs
O
N
N
N
N
O
N
R
N
N
H
H
Compd
R
mTORa
PI3Kaa
Sel.b
LNCapa
Micros.c
Mouse
20
Human
10
14
15
16
12
13
0.22
0.24
0.30
0.38
0.33
1301
882
5913
3975
757
32
20
20
2
O
>30
>30
>30
17
>30
>30
15
OH
OH
O
227
1170
705
3079
2136
1
NT
O
O
17
8a
18
0.74
0.21
1.19
648
875
2
2
8
15
30
O
O
1180
305
5619
305
>30
>30
26
O
O
>30
O
NT = not tested.
a
Average IC50 (nM). The average error for IC50 determinations was <25%.
b
c
PI3K
a/mTOR.
Nude mouse or human microsomes T1/2 (min).
Table 4
Ureidophenyl analogs of 8a
O
N
N
N
N
O
N
R
N
N
H
H
O
O
Compd
R
mTORa
P13kaa
Sel.b
LNCapa
Micros.c
Mouse
Human
8b
8c
–Et
–CH2CH2F
0.26
0.22
4577
5129
17,604
23,313
1
1
27
>30
10
16
8d
0.22
12,000
54,545
2
25
9
8e
0.16
353
2206
4
>30
13
8f
0.12
0.12
220
270
1833
2250
1
1
>30
>30
13
13
N
8g
N
a
b
c
Average IC50 (nM). The average error for IC50 determinations was <25%.
PI3K /mTOR.
Nude mouse or human microsomes T1/2 (min).
a

K. J. Curran et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1440–1444
1443
O
N
Cl
NH2
N
O
O
HN
Cl
N
1
Cl
a,b
d,e
N
N
c
O
N
O
Cl
N
O
O
5
6
O
O
O
N
O
N
7
h,i
O
N
f,g
N
N
N
N
N
N
Cl
N
Cl
N
N
N
N
O
N
9
10 trans
11 cis
R2
N
N
H
O
O
8
H
H
O
O
h,i
12, 13
Scheme 2. R2 is as defined in tables. Reagents and conditions: (a) hexanes, t-butyl carbazate; (b) water, heat; (c) water, ethanol, triethylamine, 2,6-ethylene bridged
morpholine; (d) concd hydrochloric acid; (e) tetrahydrofuran, lithium aluminum hydride; (f) chromatography; (g) tetrahydrofuran, sodium hydride, iodomethane; (h) 4-
aminophenylboronic acid pinacol ester, palladium (0), sodium carbonate, toluene, ethanol; (i) dichloromethane, triethylamine, triphosgene, then RNH2.
Vehicle
25 mg/kg
P-S6K (T389)
P-AKT (S473)
P-AKT (T308)
P-S6 (S240/244)
O
N
O
O
O
O
b
17
18
c
a
N
N
8a
N
O
N
d
16 cis
OH
N
N
β-Actin
H
H
O
14
Figure 1. Biomarker inhibition following dosing of compound 8a. Nude mice
bearing MDA361 tumors were dosed intravenously (IV) with vehicle or 25 mg/kg of
8a. Biomarkers were measured after 8 h. The dosing vehicle was 5% ethanol, 2%
tween-80, 5% polyethelene glycol (PEG-400).
OH 15 trans
Scheme 3. Reagents and conditions: (a) concd hydrochloric acid; (b) molecular
sieves, p-toluenesulfonic acid, corresponding alcohol and alkyl orthoformate; (c)
tetrahydrofuran, L-Selectride; (d) tetrahydrofuran, lithium aluminum hydride.
sustained suppression of the mTORC2 biomarker P-AKT (S473), as
well as complete inhibition of the downstream biomarker P-S6
(S240/244) for at least 8 h after an intravenous dose of 25
mg/kg.18 Excellent mTOR selectivity was shown by lack of suppres-
potency and microsomal stability, we examined cyclic analogs
which were more structurally related to piperidines. To this end,
a series of cyclohexyl analogs were prepared (Table 3). Ketone 14
was potent and selective in the enzyme assays, but was only mod-
erately stable in nude mouse and human microsomes. Alcohols 15
and 16 exhibited sub-nanomolar activity in the enzyme assay and
were stable in both nude mouse and human microsomes (T1/2
>30 min) and potent in the LNCap cell assay (IC50 = 20 nM). Intro-
duction of a methyl ether, as in 12 and 13, resulted in compounds
with improved cellular activity relative to alcohols 15 and 16.
However, microsomal stability remained modest. The inclusion of
cyclohexyl ketals at the 1-position (8a, 17 and 18) gave mTOR
inhibitors with excellent cellular potency. Analog 8a18 also dis-
sion of the PI3Ka biomarker P-AKT (T308).
The anti-tumor efficacy of analog 8a in a nude mouse xenograft
model with MDA361 tumor cells is shown in Figure 2.18 A dose of
10 mg/kg given once daily resulted in near complete tumor stasis,
whereas a daily dose of 20 mg/kg led to complete inhibition of tu-
mor growth. No significant body weight changes were observed in
the mice.
As 8a contains a ketal functional group which could be poten-
tially labile in vivo, PK studies in the nude mouse were conducted
to verify the structure of the active compound. These studies re-
vealed that ketone 14 was not present at a detectible level. Alco-
hols 15 and 16 were detected at low levels (less than 10% based
upon AUC).
played greater than 5000-fold selectivity over PI3Ka and good sta-
bility in both mouse and human microsomes. Biomarker studies
with 8a in a nude mouse MDA361 xenograft model (Fig. 1) showed
complete suppression of the mTORC1 biomarker P-S6K (T389) and
Due to the promising properties exhibited by ketal 8a, a number
of analogs were prepared which contained modification at the

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K. J. Curran et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1440–1444
600
500
400
300
200
100
Acknowledgments
control
20 mg/kg
10 mg/kg
The authors thank Dr. Li Di and Susan Li for human microsome
assays, Dr. Joe Marini and Angela Bretz for mouse microsome as-
says, and Wei-Guo Zhang for mTOR assay development.
References and notes
1. For a recent review of mTOR inhibitors in oncology see: Verheijen, J.C.; Yu, K.;
Zask, A. Ann. Rep. Med. Chem., John E. Macor, editor, 2008, 43, 189.
2. Sarbassov, D. D.; Ali, S. M.; Kim, D.-H.; Guertin, D. A.; Latek, R. R.; Erdjument-
Bromage, H.; Tempst, P.; Sabatini, D. M. Curr. Biol. 2004, 14, 1296.
3. Sarbassov, D. D.; Guertin, D. A.; Ali, S. M.; Sabatini, D. M. Science 2005, 307,
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4. Hresko, R. C.; Mueckler, M. J. Biol. Chem. 2005, 280, 40406.
5. Shi, Y.; Yan, H.; Frost, P.; Gera, J.; Lichtensein, A. Mol. Cancer Ther. 2005, 4, 1533.
6. Yu, K.; Toral-Barza, L.; Shi, C.; Zhang, W.-G.; Lucas, J.; Shor, B.; Kim, J.;
Verheijen, J.; Curran, K.; Malwitz, D. J.; Cole, D. C.; Ellingboe, J.; Ayral-
Kaloustian, S.; Mansour, T. S.; Gibbons, J. J.; Abraham, R. T.; Nowak, P.; Zask, A.
Cancer Res. 2009, 69, 6232.
7. Zask, A.; Verheijen, J. C.; Curran, K.; Kaplan, J.; Richard, D. J.; Nowak, P.;
Malwitz, D. J.; Brooijmans, N.; Bard, J.; Svenson, K.; Lucas, J.; Toral-Barza, L.;
Zhang, W.-G.; Hollander, I.; Gibbons, J. J.; Abraham, R. T.; Ayral-Kaloustian, S.;
Mansour, T. S.; Yu, K. J. Med. Chem. 2009, 52, 5013.
8. Zask, A.; Kaplan, J.; Verheijen, J.; Richard, D. J.; Curran, K.; Brooijmans, N.;
Bennett, E.; Toral-Barza, L.; Hollander, I.; Ayral-Kaloustian, S.; Yu, K. J. Med.
Chem. 2009. doi:10.1021/jm901415x.
-5
0
5
10
15
20
Days
9. Verheijen, J. C.; Richard, D. J.; Curran, K.; Kaplan, J.; Lefever, M.; Nowak, P.;
Malwitz, D. J.; Brooijmans, N.; Toral-Barza, L.; Zhang, W.-G.; Hollander, I.;
Ayral-Kaloustian, S.; Mansour, T. S.; Yu, K.; Zask, A. J. Med. Chem. 2009.
doi:10.1021/jm9013828.
10. Richard, D. J.; Verheijen, J. C.; Curran, K.; Kaplan, J.; Brooijmans, N.; Toral-Barza,
L.; Hollander, I.; Yu, K.; Zask, A. Bioorg. Med. Chem. Lett. 2009, 19, 6830.
11. Kaplan, J.; Verheijen, J. C.; Brooijmans, N.; Toral-Barza, L.; Hollander, I.; Yu, K.;
Zask, A. Bioorg. Med. Chem. Lett. doi: 10.1016/j.bmcl.2009.11.050.
12. Zask, A.; Kaplan, J. A.; Verheijen, J. C.; Curran, K. J.; Richard, D. J.; Ayral-
Kaloustian, S. WO Patent Appl. 2009052145A1, 2009.
Figure 2. In vivo efficacy study: Nude mouse xenograft model using MDA361
tumors. Compound 8a was dosed PO qd  5 per cycle, (ends day 17). The dosing
vehicle was 5% ethanol, 2% tween-80, 5% polyethelene glycol (PEG-400).
ureidophenyl moiety (Table 4). A variety of alkyl and aryl ureas dis-
played excellent potency and selectivity. Although these inhibitors
were only moderately stable in human microsomes, they displayed
13. Braun, J.; Seemann, J. Chem. Ber. 1923, 56, 1840.
unprecedented selectivity over PI3K
a (cf. 8d: >50,000-fold
14. Biological methods for determination of mTOR inhibition are described in:
Toral-Barza, L.; Zhang, W. G.; Lamison, C.; Larocque, J.; Gibbons, J.; Yu, K.
Biochem. Biophys. Res. Commun. 2005, 332, 304.
15. Biological methods for determination of inhibition of cellular proliferation are
described in: Yu, K.; Toral-Barza, L.; Discafani, C.; Zhang, W. G.; Skotnicki, J.;
Frost, P.; Gibbons, J. Endocr. Relat. Cancer 2001, 8, 249.
16. Biological methods for determination of PI3K inhibition are described in: Zask,
A.; Kaplan, J.; Toral-Barza, L.; Hollander, I.; Young, M.; Tischler, M.; Gaydos, C.;
Cinque, M.; Lucas, J.; Yu, K. J. Med. Chem. 2008, 51, 1319.
17. Biological methods for determination of microsomal stability are described in:
Tsou, H.-R.; Liu, X.; Birnberg, G.; Kaplan, J.; Otteng, M.; Tran, T.; Kutterer, K.;
Tang, Z.; Suayan, R.; Zask, A.; Ravi, M.; Bretz, A.; Grillo, M.; McGinnis, J. P.;
Rabindran, S. K.; Ayral-Kaloustian, S.; Mansour, T. S. J. Med. Chem. 2009, 52,
2289.
selective).
In summary, a thorough examination of substituents at the 1-po-
sition of pyrazolopyrimidine mTOR inhibitors led to the discovery of
cyclohexylketal derivatives. The combination of this group with a
2,6-ethylene bridged morpholine in the 4-position resulted in com-
pound 8a which exhibited improved potency, selectivity, and micro-
somal stability. Administration of this compound to nude mice
resulted in in vivo biomarker suppression that was sustained for at
least 8 h. Excellent in vivo efficacy was obtained with 8a in a nude
mouse xenograft model using MDA361 tumor cells. Ureidophenyl
analogs of 8a resulted in unprecedented selectivity (>50,000-fold)
18. For full biological characterization of 8a see: Yu, K.; Shi C.; Toral-Barza, L.;
Lucas, J.; Shor, B.; Kim, J.; Zhang, W.-G.; Mahoney, R.; Gaydos, C.; Tardio, L.;
Kim, K.; Curran, K. J.; Ayral-Kaloustian, S.; Mansour, T.S.; Abraham, R. T.; Zask,
A.; Gibbons, J. J. Cancer Res., unpublished results.
over PI3Ka. The availability of such highly selective mTOR inhibitors
makes these compounds valuable pharmacological tools to help
facilitate further elucidation of the mTOR/PI3K pathway.