Incorporation of water-solubilizing groups in pyrazolopyrimidine mTOR inhibitors: Discovery of highly potent and selective analogs with improved human microsomal stability

Article abstract of DOI:10.1016/j.bmcl.2009.10.096

A series of highly potent and selective pyrazolopyrimidine mTOR inhibitors which contain water-solubilizing groups attached to the 6-arylureidophenyl moiety have been prepared. Such derivatives displayed superior potency to those in which these appendages

Full text of DOI:10.1016/j.bmcl.2009.10.096

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6830–6835
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Incorporation of water-solubilizing groups in pyrazolopyrimidine mTOR
inhibitors: Discovery of highly potent and selective analogs with
improved human microsomal stability
a
a
a
b
David J. Richard a, , Jeroen C. Verheijen , Kevin Curran , Joshua Kaplan , Lourdes Toral-Barza ,
*
Irwin Hollander b, Judy Lucas b, 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 highly potent and selective pyrazolopyrimidine mTOR inhibitors which contain water-solubi-
lizing groups attached to the 6-arylureidophenyl moiety have been prepared. Such derivatives displayed
superior potency to those in which these appendages were attached to alternative sites. In comparison to
unfunctionalized arylureido compounds, these analogs demonstrated enhanced cellular potency and
significantly improved stability towards human microsomes, resulting in an mTOR inhibitor with impres-
sive efficacy in a xenograft model with an intermittent dosing regimen.
Received 30 September 2009
Revised 20 October 2009
Accepted 21 October 2009
Available online 25 October 2009
Keywords:
mTOR
Ó 2009 Elsevier Ltd. All rights reserved.
PI3K
Kinase
The serine–threonine kinase mTOR (mammalian Target of Rap-
amycin) is a component of the phosphoinositide 3-kinase (PI3K)
signaling pathway which plays a key role in regulation of cellular
growth and has been extensively investigated as an oncology
target.1 In response to increased levels of insulin, nutrients, and
energy supply, mTOR mediates proliferation by activation of a
number of downstream proteins.2,3 The clinical efficacy of deriva-
tives of the natural product rapamycin, such as ToriselTM, has vali-
dated mTOR inhibition as an anti-cancer treatment. mTOR
signaling involves two distinct complexes, mTOR/raptor (mTORC1)
and mTOR/rictor (mTORC2). Signaling through mTORC1 results in
phosphorylation of proteins such as S6K and 4E-BP1, whereas
mTORC2 activates AKT by phosphorylation of serine 473. Rapamy-
cin and its analogs are allosteric inhibitors of mTORC1 but have no
effect on mTORC2. In contrast, a small molecule which binds at the
ATP-binding site of mTOR would inhibit both complexes. Selective
inhibition of mTORC1 by rapamycin results in upregulation of
AKT by a negative feedback mechanism,4 whereas inhibition of
mTORC2 is expected to result in reduced AKT activity. As AKT acti-
vation has been correlated with anti-apoptotic effects,5,6 inhibition
of both mTORC1 and mTORC2 may result in increased anti-prolif-
erative efficacy compared with mTORC1 inhibition alone.
which decrease mTOR activity without affecting PI3K activity
may demonstrate improved tolerability relative to dual mTOR/
PI3K inhibitors, selectivity over PI3K was deemed desirable. Recent
efforts,7–11 including our own,12–14 have resulted in the first exam-
ples of ATP-competitive mTOR inhibitors which show selectivity
over PI3K.
We have recently prepared a series of pyrazolopyrimidines
which contain substituted morpholines at the 4-position of the
pyrazolopyrimidine core.15 This modification resulted in dramati-
cally improved selectivity over PI3K in comparison with analogs
which contained an unsubstituted morpholine ring. The previously
disclosed compounds were efficacious in various xenograft tumor
models following daily oral dosing.16,17 In certain cases, intermit-
tent iv administration of anti-tumor drugs may be preferred over
daily oral administration, requiring adequate solubility for formu-
lation. In this Letter we report a thorough examination of the effect
of introduction of water-solubilizing groups (WSGs) at various
positions of pyrazolopyrimidines that contain substituted mor-
pholines, with the aim of producing potent and selective inhibitors
with improved physicochemical and pharmaceutical properties,
suitable for intermittent iv administration.
Pyrazolopyrimidines containing the 1-ethyl substituent were
prepared by modification of our previously described route
(Scheme 1).13,14 Trichloropyrimidine aldehyde 1 was converted to
pyrazolopyrimidine 2 by treatment with ethyl hydrazine followed
by bridged morpholine in a two-step, one-pot process. Suzuki
coupling provided an aniline (3) which was subsequently
Phosphatidylinositol 3-kinase (PI3K) is a membrane-bound
lipid kinase which is found upstream of mTOR. As compounds
* Corresponding author. Tel.: +1 845 602 2143; fax: +1 845 602 5561.
E-mail address: richard4@wyeth.com (D.J. Richard).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.096

D. J. Richard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6830–6835
6831
ated by the reaction of a malonitrile derivative (7) with trifluoro-
ethyl hydrazine. Acylation of the resulting amine, followed by
cyclization, provided the pyrazolopyrimidine core (10). A sequence
involving chlorination, amine displacement, reduction, and urea
formation was then utilized to provide the final products (13).
To determine the optimal site for the addition of water-solubi-
lizing groups, pyrazolopyrimidine analogs were prepared in which
a basic amine was incorporated at two different positions. The
choice of sites was influenced by molecular modeling in an mTOR
homology model, derived from X-ray crystallographic data of pyr-
O
N
O
N
Cl
(a)
(c)
N
O
N
N
N
N
N
Cl
Cl
N
N
N
Cl
N
R1
R1
1
H2N
3
2 ; R1 = Et
5 ; R1 = H;
azolopyrimidines bound to PI3Kc, that suggested that solvent could
(b)
be accessed by groups extending from either the 1-position or the
6-ureidophenyl position.13 The effects of introduction of basic
amines at these positions were explored for alkyl- and arylureido
compounds. When a basic amine was introduced at the 1-position
of alkyl ureido 14, the resulting analog 15 displayed significantly
decreased mTOR activity (see Table 1). The mTOR inhibitors in this
study were also examined for their growth inhibitory properties in
two PTEN deficient cell lines (resulting in overactive PI3K-AKT-
mTOR signaling): LNCAP prostate cancer cells and MDA-468 breast
cancer cells. We have previously shown that mTOR inhibitors of this
class lead to potent inhibition of proliferation of cell lines driven by
hyperactive PI3K-AKT-mTOR signaling, whereas cell lines that do
not rely on hyperactive PI3K-AKT-mTOR signaling are less sensi-
tive.14 In comparison with analog 14, dimethylamino-substituted
analog 15 showed reduced cellular activity. Similar results were ob-
tained for introduction of basic amines at the 6-ureidophenyl posi-
tion; dimethylaminoethyl urea 16 possessed poor mTOR and
cellular activity compared to ethyl urea 14.
6 ; R1 = (CH2)2NMe2
O
N
N
(d)
N
N
N
O
R1
R2
N
H
N
H
4
Scheme 1. Reagents and conditions: (a) ethyl hydrazine, triethylamine, ethanol,
CH2Cl2, then 8-oxa-3-azabicyclo[3.2.1]octane hydrochloride (R1 = Et); hydrazine,
triethylamine, ethanol, CH2Cl2, then 8-oxa-3-azabicyclo[3.2.1]octane hydrochloride
(R1 = H); (b) 2-(dimethylamino)ethanol, PPh3, DIAD, CH2Cl2; (c) 4-aminophenylbo-
ronic acid pinacol ester, Pd(PPh3)4, Na2CO3, toluene, ethanol; (d) triphosgene,
CH2Cl2, then R2NH2.
transformed to the desired urea (4) by reaction with triphosgene
and the appropriate amine. The synthesis of an N,N-dimethylethyl
analog was accomplished in a similar fashion. However, in this case
a pyrazolopyrimidine lacking substitution at the 1-position (5),
prepared by reaction of 1 with hydrazine, was functionalized under
Mitsunobu conditions to install the alkylamine, yielding interme-
diate 6. Suzuki coupling and urea formation then provided the final
compound (4).
In the case of arylureido compounds, introduction of a basic
amine at the 1-position also resulted in significantly reduced activ-
ity (compare 17 and 18). In sharp contrast, incorporation of a basic
amine at the para position of the arylureidophenyl group (19) led
to an inhibitor with sub-nanomolar mTOR and cellular potency.
Interestingly, PI3Ka activity was significantly less affected by
the addition of basic amines than was mTOR activity, particularly
in the case of dimethylaminoethyl urea 16 (compare to 14). This
Trifluoroethyl pyrazolopyrimidines were synthesized by the
route depicted in Scheme 2. Trifluoroethylpyrazole 8 was gener-
suggests utility for aminoalkyl substituents in the design of PI3K
a
inhibitors.
The potency of functionalized 3-pyridyl urea 19 prompted a
more thorough investigation of analogs containing various groups
at this position. Substitution by small amines (as in 20 and 21, Ta-
ble 1) resulted in retention of potency and selectivity but a decrease
in cellular activity. Cell activity was restored by the inclusion of a
larger group, as shown by morpholine derivative 22. This analog
NC
(a)
(b)
NC
NC
O
OEt
N
N
N
CN
N
N
H
H2N
CF3
CF3
(d)
O2N
7
9
8
was also notable for its extraordinary selectivity over PI3Ka. Incor-
OH
poration of a basic amine in the form of an ether-linked morpholine
resulted in 23 which was equipotent to piperazine derivative 19.
Analogs with basic, aliphatic amines (19 and 23) displayed
Cl
N
(c)
N
N
N
N
N
N
N
excellent solubility at pH 3 (>100 lg/ml), thereby facilitating for-
CF3
CF3
mulation of these compounds for in vivo iv administration. In addi-
tion, these compounds displayed marked improvement in human
microsomal stability. Whereas 3-pyridyl urea 17 exhibited a half-
life of only 4 min in this assay, piperazine-substituted pyridyl urea
19 showed a half-life of greater than 30 min. Previous studies of
this class of mTOR inhibitors have demonstrated a good correlation
between increased microsomal stability and reduced in vivo clear-
ance.13–15 This finding suggested that incorporation of appropriate
substituents at this site could have significant effects on the phar-
macokinetic properties of this class of compounds.
Building upon the success observed with 4-piperazine substi-
tuted pyridinyl urea 19, an investigation of the effect of substitu-
tion on the 4-position of phenyl ureas was conducted (Table 2).
Piperazinophenyl urea 25 was comparable to unsubstituted phenyl
urea derivative 24 with respect to mTOR activity. However, the
piperazine group provided an advantage over the unsubstituted
O2N
O2N
10
11
O
N
O
(e), (f)
H2N
(g)
N
N
N
N
N
N
N
N
N
O
CF3
R
CF3
N
N
H
H
12
13
Scheme 2. Reagents and conditions: (a) trifluoroethyl hydrazine, ethanol, water;
(b) p-NO2PhCOCl, CH2Cl2, CH3CN; (c) 30% H2O2, 2.5 N aq NaOH, ethanol; (d) POCl3,
cat. DMF; (e) 8-oxa-3-azabicyclo[3.2.1]octane hydrochloride, triethylamine, etha-
nol; (f) Pd/C, ethyl acetate, THF; (g) triphosgene, CH2Cl2, then selected amine.

6832
D. J. Richard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6830–6835
Table 1
Introduction of water-solubilizing groups at the 1- and 6-arylureidophenyl positions
O
N
N
N
N
O
N
R2
R1
N
H
N
H
Compd R1
R2
mTOR IC50 (nM) PI3K
a
IC50 (nM) Selectivity (over PI3K
2226
a
)
LNCaP cell IC50 (nM) MDA468 cell IC50 (nM)
a
a
14
0.6
1249
16
80
15
16
13
3125
240
280
1800
N
51
836
148
16
350
1250
1.2
N
17
18
0.1
1115
<0.8
N
N
2.7
0.7
235
196
89
50
220
0.8
N
N
N
19
275
<0.7
N
H
N
20
21
0.9
1.5
270
282
302
282
17
6
200
80
N
N
N
O
O
N
22
23
0.3
1.0
2154
373
7302
383
0.8
8
N
O
N
<0.7
0.8
N
a
Average IC50. The average error for IC50 determinations was <25%.
phenyl analog in terms of both cellular activity and human micro-
somal stability. The presence of a basic, aliphatic amine appeared
to be required for enhanced stability, as shown by the decreased
stability of pyrrolidine 26 relative to 25. An analog containing
the trifluoroethyl substituent at the 1-position as well as a pipera-
zinophenyl urea (27) also displayed excellent activity and good
stability. The secondary amine 28 showed activity equivalent to
that of the N-methyl analog 27.
were somewhat less potent against mTOR than the unsubstituted
analog (24, Table 2), although the cellular activity of 30 was supe-
rior. Amines 29 and 30 also exhibited greater human microsomal
stability (14 min in each case) than 24 (human microsomal stabil-
ity of 5 min). Dimethylbenzylamine 31 showed reduced cellular
activity but increased stability. Modification to a primary amine
(32) led to an analog which was highly potent and stable. The
incorporation of an additional basic nitrogen in the form of a piper-
azine was well-tolerated (33). N,N-Dimethylethyl ether 34 was
equipotent to the unsubstituted phenyl urea 24 (Table 2) but
showed significantly improved human microsomal stability.
The solvent-exposed nature of the 4-aryl position was used to
incorporate other WSGs such as acyclic basic amines. Both di-
and tri-methylethylenediamine derivatives (29 and 30, Table 3)

D. J. Richard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6830–6835
6833
Table 2
Piperazine and pyrrolidine substitution at the 6-phenylureido- position
O
N
N
N
R1
N
O
N
R2
N
H
N
H
R1
R2
mTOR IC50
(nM)
PI3K
(nM)
a
IC50
Selectivity
(over PI3K
LNCaP cell
IC50 (nM)
MDA468 cell
IC50 (nM)
Micros. Stab.
(t1/2, human, min)
a
a
Compd
a
)
H
24
0.3
66
212
0.8
12
5
N
25
26
27
0.3
51
1436
150
188
<0.7
<0.7
>30
5
N
N
6.0
0.6
243
237
55
120
1.5
N
F
F
F
F
N
F
<0.8
24
HN
F
28
0.2
76
318
<0.8
1.0
—
N
a
Average IC50. The average error for IC50 determinations was <25%.
Table 3
Incorporation of other basic or polar substituents
O
N
N
N
R1
N
O
N
N
H
N
H
R1
mTOR IC50
(nM)
PI3K
(nM)
a
IC50
Selectivity
(over PI3K
LNCaP cell
IC50 (nM)
MDA468 cell
IC50 (nM)
Micros. Stab.
(t1/2, human, min)
a
a
Compd
a)
H
N
29
1.7
0.9
207
122
2.8
7
14
14
N
N
30
256
295
0.8
0.8
N
N
31
32
1.1
0.2
263
21
239
95
7
30
29
H2N
<0.8
<0.8
>30
N
33
0.7
320
451
<0.8
0.8
>30
>30
N
N
O
34
35
0.8
0.1
136
40
172
335
<0.7
<0.7
8
<0.7
11
HO
(continued on next page)

6834
D. J. Richard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6830–6835
Table 3 (continued)
R1
mTOR IC50
(nM)
PI3Ka IC50
(nM)
Selectivity
(over PI3K
LNCaP cell
IC50 (nM)
MDA468 cell
IC50 (nM)
Micros. Stab.
(t1/2, human, min)
a
a
Compd
a)
HO
36
37
0.1
68
570
<0.7
<0.7
10
23
O
N
0.3
109
350
<0.7
<0.7
N
H
O
N
38
0.2
0.2
102
175
586
972
<0.7
<0.7
4
>30
6
N
H
O
39
<0.7
N
a
Average IC50. The average error for IC50 determinations was <25%.
In addition to basic amines, the incorporation of other polar
functional groups was investigated. The presence of hydroxy-
methyl and hydroxyethyl substituents (as in 35 and 36, Table 3)
provided analogs which were exceptionally potent in both enzyme
and cell assays but displayed modest human microsomal stability.
Hydrazides 37 and 38 were highly potent and stable. The selectiv-
ity enhancement observed by morpholine substitution with 3-pyr-
idyl urea analogs was also exhibited in the phenyl urea series.
Thus, analog 39 displayed comparable mTOR binding but reduced
PI3K affinity (compare to 24, Table 2).
kg), low Vss (1.2 L/kg), moderate half-life (2.9 h) and high exposure
(12,466 h ng/ml) following an iv dose of 5 mg/kg in nude mice.
Inhibition of mTOR signaling in vivo was studied in an MDA-361
breast adenocarcinoma nude mouse xenograft model.19 Eight
hours after a 25 mg/kg IV dose, 27 showed complete suppression
of TORC1 signaling (p70-S6K), resulting in significant down-regu-
lation of its substrate pS6. In addition, near complete inhibition
of TORC2 was observed (p-AKT S473). The strong inhibition of
mTOR signaling correlated with good plasma exposure at the 8 h
time point (2499 ng/ml).
Piperazine-substituted phenyl urea 27 was notable for its prom-
ising activity and selectivity over PI3Ka. This compound was also
Daily iv dosing of compound 27 in an MDA-361 nude mouse
xenograft model was well-tolerated and resulted in excellent inhi-
bition of tumor growth at low doses (10–25 mg/kg). We also exam-
ined use of this inhibitor on an intermittent basis, as such a
schedule would be more clinically relevant. We were gratified to
find that intermittent (every 5th day) intravenous administration
of a 20 mg/kg dose resulted in complete tumor stasis (p <0.005)
(Fig. 2). To the best of our knowledge, this finding represents the
first example of in vivo anti-tumor efficacy following intermittent
dosing of an ATP-competitive mTOR inhibitor.
highly selective against the other PI3K isoforms, as shown in Table
4. This derivative also displayed excellent selectivity over a variety
of other kinases; of the 242 kinases examined, none showed
greater than 25% inhibition at 2 l
M concentration of 27.18
The selectivity observed at the enzymatic level was also evident
when 27 was examined at the cellular level. As seen in Figure 1,
this compound resulted in complete inhibition of signaling by
mTORC1 (P-S6K T389) and significant inhibition of mTORC2 (P-
AKT-S473) at a concentration of 25 nM.19 Phosphorylation of the
PI3K/PDK1 biomarker AKT-T308 was not affected, illustrating the
highly selective nature of 27. These data correlated well with inhi-
bition of cellular proliferation, as was previously also observed.13–15
Compound 27 showed good microsomal stability in the desired
efficacy species (T1/2 >30 min in nude mouse). The pharmacoki-
netic profile of 27 was characterized by low clearance (7 ml/min/
A
B
C
D
E
F
P-S6K T389
P-AKT S473
P-AKT T308
A=Control, B=2 µM, C=0.667 µM, D=0.222 µM,
µM, F = 0.025 µM.
E=0.074
Figure 2. Tumor growth in a nude mouse xenograft model of MDA-361 cells for
vehicle (circles) and compound 27 (squares). 27 was dosed at 20 mg/kg IV on days
1, 5, 9, 13, 17 and 21.
Figure 1. Inhibition of mTOR signaling following treatment of MDA-361 cells with
27.

D. J. Richard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6830–6835
6835
Table 4
Inhibition of mTOR and PI3K isoforms by 27
a
a
a
a
Compd
mTOR IC50 (nM)
0.6
PI3K
150
a
IC50 (nM)
PI3Kb IC50 (nM))
PI3K
c
IC50 (nM)
PI3Kd IC50 (nM)
27
>10,000
>10,000
945
a
Average IC50. The average error for IC50 determinations was <25%.
5. Songyang, Z.; Baltimore, D.; Cantley, L. C.; Kaplan, D. R.; Franke, T. F. Proc. Natl.
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12. 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-
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In summary, a variety of potent and selective pyrazolopyrimi-
dine mTOR inhibitors have been prepared. These analogs contained
water-solubilizing groups attached to the aryl ring of arylureid-
ophenyl pyrazolopyrimidines. The presence of such moieties re-
sulted in compounds which were generally of equivalent or
greater potency against the mTOR enzyme than the unsubstituted
derivatives and possessed excellent activity in a cellular prolifera-
tion assay. The inclusion of a basic amine significantly enhanced
human microsomal stability, a trend which was observed for a
variety of functional groups regardless of structure (nitrogen, car-
bon, and oxygen containing linkers). The combination of excellent
potency and stability resulted in an inhibitor (27) that demon-
strated impressive efficacy in a xenograft model with an intermit-
tent dosing regimen. The highly selective nature of this analog was
demonstrated by complete abrogation of mTOR signaling in MDA-
361 tumor cells without affecting PI3K signaling. These results
demonstrate that modifications of the arylureido moiety may be
employed to optimize the physicochemical, pharmaceutical and
pharmacokinetic properties of this class of mTOR inhibitors.
13. 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.
14. Verheijen, J.; 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.; Yu, K.; Zask, A. J. Med. Chem. 2009, in press,
doi:10.1021/jm9013828.
15. Zask, A.; Kaplan, J.; Verheijen, J. C.; Richard, D. J.; Curran, K.; Brooijmans, N.;
Bennett, E.; Toral-Barza, L.; Hollander, I.; Ayral-Kaloustian, S.; Yu, K. J. Med.
Chem. 2009, in press, doi:10.1021/jm901415X.
16. Curran, K. J.; Verheijen, J. C.; Kaplan, J.; Richard, D. J.; Toral-Barza, L.; Hollaner,
I.; Lucas, J.; Ayral-Kaloustian, S.; Yu, K.; Zask, A. Unpublished results.
17. Yu, K.; Shi, C.; Toral-Barza, L.; Lucas, J.; Shor, B.; Kim, J. E.; Zhang, W.-G.;
Mahoney, R.; Gaydos, C.; Tardio, L.; Kim, S. Y.; Conant, R.; Curran, K.; Kaplan, J.;
Verheijen, J.; Ayral-Kaloustian, S.; Mansour, T. S.; Abraham, R. T.; Zask, A.;
Gibbons, J. J. Unpublished results.
Acknowledgements
The authors thank Dr. Li Di and Susan Li for human microsome
assays, Angela Bretz for mouse microsome assays, Dr. Inder Chau-
dhary for pharmacokinetic and plasma exposure studies, Wei-Guo
Zhang for mTOR assay development, and Robert Mahoney and
Kenneth Kim for biomarker and xenograft data.
References and notes
18. An assay of the activity of 27 against a panel of 242 protein kinases was
performed by SelectScreenTM profiling (Invitrogen).
19. Biological methods for determination of cellular mTOR signaling inhibition and
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13
efficacy in nude mouse xenograft models have been described in Ref.
Supplementary data therein.
and