Discovery of 3,6-dihydro-2H-pyran as a morpholine replacement in 6-aryl-1H-pyrazolo[3,4-d]pyrimidines and 2-arylthieno[3,2-d]pyrimidines: ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR)

Article abstract of DOI:10.1016/j.bmcl.2009.11.050

The morpholine hinge-region binding group on a series of pyrazolopyrimidine and thienopyrimidine mammalian target of rapamycin (mTOR) inhibitors was replaced with 3,6-dihydro-2H-pyran (DHP), giving compounds of equivalent potency and selectivity versus PI3K. These results establish the DHP group as a hinge-region binding motif for the preparation of highly potent and selective mTOR inhibitors.

Full text of DOI:10.1016/j.bmcl.2009.11.050

Bioorganic & Medicinal Chemistry Letters 20 (2010) 640–643
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 3,6-dihydro-2H-pyran as a morpholine replacement in
6-aryl-1H-pyrazolo[3,4-d]pyrimidines and 2-arylthieno[3,2-d]pyrimidines:
ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR)
a
a
b
b
Joshua Kaplan a, , Jeroen C. Verheijen , Natasja Brooijmans , Lourdes Toral-Barza , Irwin Hollander ,
*
Ker Yu b, Arie Zask a
a Chemical Sciences, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, United States
b Oncology Research, Wyeth Research, 401 N. Middletown Road, 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:
The morpholine hinge-region binding group on a series of pyrazolopyrimidine and thienopyrimidine
mammalian target of rapamycin (mTOR) inhibitors was replaced with 3,6-dihydro-2H-pyran (DHP), giv-
ing compounds of equivalent potency and selectivity versus PI3K. These results establish the DHP group
as a hinge-region binding motif for the preparation of highly potent and selective mTOR inhibitors.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 14 October 2009
Revised 9 November 2009
Accepted 16 November 2009
Available online 4 December 2009
The mammalian target of rapamycim (mTOR), a serine/threonine
protein kinase, is a member of the phosphoinositide-3-kinase-re-
lated kinase (PIKK) family and is a key mediator of signaling through
the PI3K–AKT pathway. The deregulation of PI3K–AKT–mTOR sig-
naling is one of the most common genetic alterations in proliferative
diseases and as such has garnered intense interest from industry and
academia.1 mTOR exists in at least two functionalcomplexes, mTOR/
raptor (mTORC1) and mTOR/rictor (mTORC2), which play critical
roles in the transduction of environmental stimuli into cellular pro-
cesses, including cellulargrowthand survival, metabolism, and ribo-
somal synthesis. mTORC1, which is inhibited by rapamycin analogs,
phosphorylatesS6K and4E-BP1.2 mTORC2, unaffectedby rapamycin
analogs, activates AKT via phosphorylation of serine 473.3 Rapamy-
cin inhibition of mTORC1 has been shown to result in activation of
AKT via a feedback mechanism.4 An ATP-competitive inhibitor is ex-
pected to affect both mTOR complexes and thus lead to decreased
AKT activity. Since AKT activation has anti-apoptotic, pro-survival
effects, ATP-competitive mTOR inhibitors may have increased anti-
tumor effects compared to rapamycin analogs.
We have identified a series of 4-morpholino-6-aryl-1H-pyrazol-
o-[3,4-d]pyrimidines that are highly potent and selective
ATP-competitive inhibitors of mTOR enzymatic activity, cellular
proliferation, and in vivo tumor growth.5–8 The morpholine group
in these compounds forms the crucial hinge region interaction
with mTOR via a hydrogen bond between the morpholine oxygen
and Val2240.5–7 In efforts to further improve the potency, selectiv-
ity and/or PK/PD properties of our inhibitors we have investigated
derivatives of the morpholine group.9 We here report the success-
ful use of the 3,6-dihydro-2H-pyran (DHP) group as an alternative
to morpholine as a hinge region binding motif for the design of po-
tent and selective mTOR inhibitors.
Pyrazolopyrimidines incorporating the DHP group were pre-
pared by the route outlined in Scheme 1.10 The synthetic procedures
described herein were not optimized for yield; however, products of
sufficient yield and purity for medicinal chemistry purposes were
commonly obtained. Compound 1, 5-amino-1-(2,2,2-trifluoro-
ethyl)-1H-pyrazole-4-carbonitrile, was prepared by cyclization of
2,2,2-trifluoroethyl hydrazine with 2-(ethoxymethylene)malono-
nitrile. Benzoylation of the amino group of 1 gave 2, which upon
treatment with refluxing base and peroxide provided 3, containing
the pyrazolopyrimidine core. Heating a neat suspension of 3 with
phosphorus oxychloride in a sealed tube gave chloride 4. Hydroge-
nation under one atmosphere of hydrogen in the presence of di-
tert-butyldicarbonate gave 5. Subjection of 5 to Stille conditions in
the presence of tributyl(3,6-dihydro-2H-pyran-4-yl)stannane11
gave coupling product 6. Following removal of the BOC protecting
group with trifluoroacetic acid, work-up with aqueous sodium
bicarbonate gave aniline intermediate 7. Treatment of 7 with tri-
phosgene provided the putative intermediate isocyanate to which
the respective amines were added, furnishing final ureas 11–13.
As shown in Table 1, the replacement of morpholine in ureid-
ophenyl-bearing 1H-pyrazolo[3,4-d]pyrimidines 8–10 with DHP
led to highly potent12 and selective13 mTOR inhibitors 11–13. Flu-
oroethyl urea 12, particularly, demonstrated substantially weaker
activity against PI3Ka, thereby providing a compound with
>2000-fold mTOR selectivity. In addition to their enzyme activity
and selectivity, DHP containing compounds also generally had
comparable antiproliferative potency in LNCap cells14 and stability
in nude mouse microsomes.15
Molecular modeling provided an explanation for the similar
activities of the morpholine and DHP containing inhibitors. When
* Corresponding author.
E-mail addresses: kaplanj1@wyeth.com, kaplanj1@pfizer.com (J. Kaplan).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.050

J. Kaplan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 640–643
641
OH
N
a
b
NC
NC
c
N
NC
O
N
OEt
N
N
N
N
N
CN
H2N
N
H
O2N
F3C
F3C
O2N
F3C
1
2
3
Cl
Cl
O
e
N
N
d
f
N
N
+
CF3
N
N
N
N
SnBu3
O2N
F3C
BocHN
4
5
O
O
O
g
h
N
N
N
N
N
N
N
O
N
N
N
N
N
R
CF3
CF3
N
N
F3C
BocHN
H2N
H
H
11 - 13
6
7
Scheme 1. Reagents and conditions: (a) 70 wt % aq trifluoroethyl hydrazine, ethanol; (b) p-NO2BzCl, CH2Cl2, CH3CN; (c) 30% H2O2, 2.5 N aq NaOH, ethanol; (d) POCl3; (e)
BOC2O, H2, 10% Pd/C, THF; (f) PdCl2 (PPh3)2, DMF; (g) TFA, CH2Cl2, followed by aq NaHCO3 work-up; (h) triphosgene, Et3N, CH2Cl2, then RNH2.
Table 1
6-Ureidophenyl-l-1H-pyrazolo[3,4-d]pyrimidines
R1
N
N
N
O
N
CF3
R2
N
H
Compd
R1
R2
mTORa
P13Kaa
Sel.b
LNCapa
Micros.c
11
8
DHP
morpholine
–NHCH3
2
1
200
194
100
194
260
230
13
26
12
9
DHP
morpholine
–NHCH2CH2F
1
0.6
2300
315
2300
525
72
60
>30
22
13
10
DHP
morpholine
1
0.2
66
10
66
50
380
<27
25
>30
H
N
N
a
b
c
Average IC50 (nM). The average error for IC50 determinations was <25%.
P13K IC50/mTOR IC50
Nude mouse microsomes T1/2 (min).
a
.
the inhibitors were docked in an mTOR homology model derived
from a PI3K
crystal structure,5 similar binding modes were
gave 16 which upon demethylation with boron tribromide gave
the bromide 17. Introduction of the DHP group as a morpholine
replacement, by Stille coupling of 17 with tributyl(3,6-dihydro-
2H-pyran-4-yl)stannane, gave 18. Reduction of the double bond in
18 by hydrogenation gave THP 19.
c
observed for inhibitors containing morpholine and DHP groups.
The oxygen atoms were well overlaid, and similar H-bonds to the
hinge region valine were seen for both isosteres. Docking studies
also suggested a similar binding mode for compounds in which the
double bond in the DHP group had been reduced to give a tetrahy-
dro-2H-pyran (THP). Therefore, we explored whether the THP group
could also function as a morpholine isostere, by preparation of DHP
and THP containing inhibitors on a test system. As outlined in
Scheme 2, reaction of 5-amino-1-phenyl-1H-pyrazole-4-carboni-
trile 14 with 3-anisoyl chloride followed by base-catalyzed conden-
sation provided 6-(3-methoxyphenyl)-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4-ol 15. Chlorination using phosphorus oxychloride
Remarkably, reduction of the olefin in 18 to give the THP con-
taining pyrazolopyrimidine 19 led to a profound decrease in mTOR
and PI3K
a potency (Table 2). Consideration of the minimum
energy conformation of the two inhibitors in solution, however,
clarifies the cause of the decreased activity of the THP analog. As
shown in Figure 1A, a conformational search of 18 revealed that
in the minimum energy conformation the DHP group is co-planar
with the pyrazolopyrimidine core. A similar conformation was ob-
served for morpholino-pyrazolopyrimidines (not shown). In con-

642
J. Kaplan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 640–643
OH
Cl
N
N
N
c
a,b
d
N
N
N
N
N
N
N
H2N
14
O
O
15
16
N
O
O
Br
N
e
f
N
N
N
N
N
N
N
N
N
N
N
OH
17
18
19
OH
OH
Scheme 2. Reagents and conditions: (a) 3-Anisoyl chloride, Et3N, CH2Cl2; (b) 30% H2O2, 2.5 N aq NaOH, ethanol; (c) POCl3; (d) BBr3, CH2Cl2; (e) tributyl(3,6-dihydro-2H-
pyran-4-yl)stannane, PdCl2(PPh3)2, DMF; (f) H2, 10% Pd/C, CH2Cl2.
trast, the minimum energy conformation for the THP group is ro-
tated ca. 90° out-of-plane with the core (Fig. 1B). An overlay of
the minimum energy conformation of the THP analog (19) with
the docked binding mode of the DHP compound (18) (Fig. 2), high-
lights a number of clashes of the THP ring with active site residues,
specifically Ile2356 and Trp2239. Hence, steric crowding precludes
the binding of the THP analog 19 in its minimum energy conforma-
tion, forcing the adoption of a co-planar conformation or requiring
a conformational shift of the enzyme to accommodate an out-of-
plane conformation of the THP group in order to bind mTOR.
We also investigated whether the DHP could function as a mor-
pholine surrogate on other scaffolds. We recently showed that
2-ureidophenyl-4-morpholinothieno[3,2-d]pyrimidines also po-
tently inhibited mTOR.17 DHP containing thienopyrimidines were
Figure 1. Comparison of minimum energy conformations. (A) DHP 18 (B) THP 19.
prepared as outlined in Scheme 3. Thus, treatment of 3-aminothi-
ophene-2-carboxamide 20 with triphosgene gave thienopyrimidine
21. Chlorination with phosphorus oxychloride gave 22, which was
ConfGen16 was used to explore the conformational space of each molecule in water.
Redundant conformers were eliminated using an RMSD cutoff of 0.5 Å. The
‘thorough’ search mode was used and a maximum of 128 ring conformations were
reacted with tributyl(3,6-dihydro-2H-pyran-4-yl)stannane under
Stille coupling conditions to furnish 23. Suzuki coupling with the
pinacol ester of 4-aminophenylboronic acid was followed by
treatmentof resulting 24with triphosgeneand aminetogive thetar-
get urea compounds.
generated. The minimum energy conformation from ConfGen for the THP analog
was used to overlay with the docked conformation of the DHP analog.
The biological results of the thienopyrimidine series corrobo-
rate those of the pyrazolopyrimidine core: the DHP group, as mor-
pholine replacement, gives potent in vitro inhibitors of mTOR (25–
29, Table 3). The mTOR potency of inhibitors derived from this
scaffold was slightly lower than that of the corresponding pyrazol-
opyrimidines (compare 26 to 12; 28 to 13) whereas the PI3K activ-
Table 2
Comparison of hinge region binding groups
R
N
N
N
N
OH
Compd
R
mTORa
PI3Kaa
Sel.b
18
19
DHP
THP
18
500
59
1013
3.3
2
Figure 2. Overlay of docked DHP analog 18 (in magenta) and the minimum energy
conformation of THP analog 19 (in orange) in an mTOR homology model based on
a
Average IC50 (nM). The average error for IC50 determinations was <25%.
PI3Ka IC50/mTOR IC50.
the PI3K
c crystal structure. The hydrogen bond between the hinge region and the
b
DHP ring is shown in yellow; steric clashes for the THP analog are shown in cyan.

J. Kaplan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 640–643
643
O
OH
N
Cl
N
S
S
a
c
b
S
H2N
H2N
N
N
HO
Cl
20
21
22
O
O
O
N
S
S
S
d
e
N
N
N
Cl
N
N
O
23
24
R
H2N
N
N
H
H
Scheme 3. Reagents and conditions: (a) Triphosgene; (b) POCl3; (c) tributyl(3,6-dihydro-2H-pyran-4-yl)stannane, PdCl2(PPh3)2, THF; (d) 4-aminophenylboronic acid pinacol
ester, Pd(PPh3)4, PhCH3/EtOH; (e) Et3N, triphosgene, followed by appropriate amine.
inhibitors. Through the judicious choice of the urea substituent,
scaffold and morpholine replacement, the potency and selectivity
of the inhibitors may be modulated in order to obtain com-
pounds of a desired profile.
Table 3
2-Ureidophenyl-thieno[3,2-d]pyrimidines
O
References and notes
S
N
1. Yuan, T. L.; Cantley, L. C. Oncogene 2008, 27, 5497.
2. Choo, A. Y.; Yoon, S. O.; Kim, S. G.; Roux, P. P.; Blenis, J. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 17414.
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N
R
N
H
3. Sarbassov, D. D.; Guertin, D. A.; Ali, S. M.; Sabatini, D. M. Science 2005, 307,
1098.
4. Carracedo, A.; Pandolfi, P. P. Oncogene 2008, 27, 5527.
5. 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.
Compd
R
mTORa
P13Kaa
Sel.b
LNCapa
Micros.c
25
26
27
–NHCH2CH3
–NHCH2CH2F
–NHPh
3.3
2.5
7.2
1053
1373
28
319
560
4
470
1300
800
3
6
21
6. Nowak, P.; Ayral-Kaloustian, S.; Brooijmans, N.; Cole, D. C.; Curran, K. J.;
Ellingboe, J.; Gibbons, J. J.; Hu, B.; Hollander, I.; Kaplan, J.; Malwitz, D. J.;
Mansour, T. S.; Toral-Barza, L.; Verheijen, J. C.; Zask, A.; Zhang, W.-G.; Yu, K., J.
Med. Chem. doi: 10.1021/jm9012642.
7. 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. doi: 10.1021/
jm9013828.
H
N
28
29
1.1
1.8
35
27
32
15
210
200
>30
>30
N
H
N
8. 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.
9. 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. doi: 10.1021/jm901415x.
N
a
b
c
Average IC50 (nM). The average error for IC50 determinations was <25%.
P13K IC50/mTOR IC50
Nude mouse microsomes T1/2 (min).
a
.
10. The purity and identity of novel compounds was confirmed by HPLC, mass
spectrometry and 1H NMR.
11. Kiely, J. S.; Lesheski, L. E.; Schroeder, M. C. U.S. Patent 4,945,160, 1990.
12. 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.
13. 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.
14. 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.
15. 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.
ity of the thienopyrimidines was similar to or slightly better than
that of the corresponding pyrazolopyrimidines. Consequently, the
thienopyrimidines were somewhat less selective for mTOR than
the pyrazolopyrimidines and may be of particular value for the
development of dual PI3K/mTOR inhibitors. In this respect, pyridi-
nyl ureas 28 and 29 are of interest due to the combination of
potent in vitro inhibition and excellent microsomal stability.
In summary, we have identified the 3,6-dihydro-2H-pyran
(DHP) as a suitable bioisostere for the morpholine group in
pyrazolopyrimidine and thienopyrimidine mTOR inhibitors.
Molecular modeling suggests that the DHP and morpholine
groups form the same essential hydrogen bond to Val2240 in
the hinge region of mTOR. The findings in this paper add to
our expanding repertoire for the design of mTOR and/or PI3K
16. Generated with ConfGen, version 2.1, Schrödinger, LLC, New York, NY, 2009.
17. Verheijen, J. C.; Yu, K.; Toral-Barza, L.; Hollander, I.; Zask, A. Bioorg. Med. Chem.
Lett. doi: 10.1016/j.bmcl.2009.10.075.