Discovery of 2-arylthieno[3,2-d]pyrimidines containing 8-oxa-3-azabi-cyclo[3.2.1]octane in the 4-position as potent inhibitors of mTOR with selectivity over PI3K

Article abstract of DOI:10.1016/j.bmcl.2009.10.075

2-Aryl-4-morpholinothieno[3,2-d]pyrimidines are known PI3K inhibitors. This class of compounds also potently inhibited the homologous enzyme mTOR. Replacement of the morpholine group in these compounds with an 8-oxa-3-azabicyclo[3.2.1]octane group led to mTOR inhibitors with selectivity over PI3K. Optimization of the 2-aryl substituent led to the discovery of 2-(4-ureidophenyl)-thienopyrimidines as highly potent (IC₅₀ <1 nM) mTOR inhibitors with excellent selectivity (up to >1000-fold) over PI3K and good potency in a cellular proliferation assay (IC₅₀ <50 nM).

Full text of DOI:10.1016/j.bmcl.2009.10.075

Bioorganic & Medicinal Chemistry Letters 20 (2010) 375–379
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 2-arylthieno[3,2-d]pyrimidines containing 8-oxa-3-azabi-
cyclo[3.2.1]octane in the 4-position as potent inhibitors
of mTOR with selectivity over PI3K
b
b
b
a
Jeroen C. Verheijen a, , Ker Yu , Lourdes Toral-Barza , Irwin Hollander , Arie Zask
*
a Chemical Sciences, Wyeth Research, 401 N. Middletown Rd, Pearl River, NY 10965, USA
b Oncology Research, Wyeth Research, 401 N. Middletown Rd, 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:
2-Aryl-4-morpholinothieno[3,2-d]pyrimidines are known PI3K inhibitors. This class of compounds also
potently inhibited the homologous enzyme mTOR. Replacement of the morpholine group in these com-
pounds with an 8-oxa-3-azabicyclo[3.2.1]octane group led to mTOR inhibitors with selectivity over PI3K.
Optimization of the 2-aryl substituent led to the discovery of 2-(4-ureidophenyl)-thienopyrimidines as
highly potent (IC50 <1 nM) mTOR inhibitors with excellent selectivity (up to >1000-fold) over PI3K and
good potency in a cellular proliferation assay (IC50 <50 nM).
Received 28 September 2009
Revised 15 October 2009
Accepted 19 October 2009
Available online 22 October 2009
Keywords:
mTOR
Ó 2009 Elsevier Ltd. All rights reserved.
PI3K
Cancer
Oncology
Kinase
PI3K and mTOR are key components of the PI3K-Akt-mTOR sig-
naling cascade, one of the most frequently upregulated pathways
in tumor biology. Consequently, inhibitors of mTOR and/or PI3K
have recently been the focus of an intense effort towards the devel-
opment of novel anticancer medicines.1,2 2-Aryl-4-morpholinothi-
eno[3,2-d]pyrimidines (e.g., 1, 2 and GDC-0941, Fig. 1) have been
described as potent inhibitors of the various PI3K-isoforms.3–6
We recently reported that pyrazolopyrimidines with a similar sub-
stitution pattern as 1 and 2 potently inhibited the homologous en-
zyme, mTOR. On the pyrazolopyrimidines, introduction of isosteres
for the morpholine and phenol/aminopyrimidine substituents
could lead to increased mTOR potency and selectivity.7–11 Here,
we report the application of those findings to the thienopyrimidine
scaffold, to give novel, highly potent mTOR inhibitors with selectiv-
ity over PI3K.
Chlorination of 4 with phosphorus oxychloride proceeded smoothly
to give 5 in near-quantitative yield. Regioselective nucleophilic dis-
placement of the 4-chloride with 6, followed by Suzuki coupling
gave access to target compounds 8a–h.
Introduction of the ethylene-bridged morpholine 6 on the thie-
nopyrimidine core led to increased selectivity for mTOR over
PI3K-a (Table 2). Compounds 8a and 8b inhibited mTOR with sim-
ilar potency compared to the corresponding 4-morpholinothieno-
pyrimidines 1 and 2. In contrast, compounds 8a and 8b were
significantly less potent against PI3K-
sevenfold (8a compared to 1) or 31-fold (8b compared to 2) in-
creased selectivity over PI3K- . We have previously described sim-
a than 1 and 2, resulting in
a
ilar decreases in PI3K potency upon introduction of ethylene-
bridged morpholine 6 on pyrazolopyrimidines, which was ascribed
to a steric clash with Phe961. This residue is smaller (Leu) in mTOR,
thus avoiding the steric clash.11 When the meta-phenol (8a) was re-
placed with a para-phenol (8c), a modest increase in potency
against mTOR was observed. In addition, the potency against
Compounds 1 and 2 were prepared following published proce-
a
dures3,4 and assayed against PI3K- and mTOR,12 revealing that
these leads are dual inhibitors of PI3K and mTOR, with moderate
potency (Table 1).
PI3K-a was decreased, resulting in significantly increased selectiv-
Scheme 1 describes the synthesis of thienopyrimidines carrying
bridged morpholine analog 6.13 3-Aminothiophene-2-carboxamide
3 was condensed with triphosgene to give thienopyrimidine 4.
ity for mTOR. Next, we addressed whether the pyrimidine ring
nitrogens in 8b contributed to mTOR activity. Removal of one of
the nitrogens, resulting in aminopyridine 8d, led to a small increase
in mTOR potency and a pronounced increase in selectivity over
PI3K-
a. In contrast, removal of both nitrogens (to give aniline 8e)
* Corresponding author. Tel.: +1 845 602 2800; fax: +1 845 602 5561.
E-mail address: verheij@wyeth.com (J.C. Verheijen).
resulted in decreased mTOR inhibitory activity, while maintaining
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.075

376
J. C. Verheijen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 375–379
O
N
O
N
O
N
O
S
O
N
S
N
N
HN
N
S
S
N
N
OH
N
N
N
N
1
GDC-0941
2
N
NH2
Figure 1. Representative examples of 2-arylthieno[3,2-d]pyrimidine PI3K inhibitors.
O
O
N
selectivity over PI3K-a. A further increase in selectivity was ob-
served for indole 8f and acetamide 8g (392-fold and 165-fold selec-
tive over PI3K- , respectively). Replacement of the acetamide in 8g
with a methyl carbamate (8h) resulted in further enhanced potency
and selectivity over PI3K-
N
N
a
a
S
S
N
N
a.
N
O
In case of the pyrazolopyrimidine scaffold, we have shown
that the mTOR activity could be increased by replacement of
the carbamoylphenyl group with a ureidophenyl group, which
was able to form three hydrogen bonds to the enzyme (two
bonds between urea NHs and an aspartate, one bond between
the urea carbonyl and a lysine).7,10 Based on these observations,
the ureidophenyl group was explored as an isostere for the carba-
moylphenyl moiety in 8h. As illustrated in Scheme 2, treatment of
aniline 8e with triphosgene followed by addition of excess amine
gave target ureidophenyl compounds 9a–h. A dramatic increase
in potency was observed for methyl ureidophenyl 9a (Table 3)
compared to methyl carbamoylphenyl 8h (Table 2). The ureid-
ophenyl compound inhibited the mTOR enzyme at sub-nanomo-
lar concentrations while maintaining excellent selectivity over
8e
R
9a-h
NH2
N
N
H
H
Scheme 2. Reagents: (a) (1) triphosgene, CH2Cl2, NEt3; (2) RNH2, 22–89%.
concentrations than with carbamoylphenyl 8h. The dramatic in-
crease in potency observed when the ureidophenyl group was
introduced on the thienopyrimidine core suggests that these com-
pounds interact with their target enzymes in a manner similar to
the pyrazolopyrimidines.
Based on the promising activity of methyl ureido compound 9a,
additional ureido compounds were investigated (Table 3). Alky-
lureido compounds (e.g., 9b–9e) were very potent (<1 nM) and
PI3K-a. Cellular proliferation was also inhibited at much lower
highly selective (>1000-fold over PI3K-
a) mTOR inhibitors with
good to excellent potency in a cellular proliferation assay (IC50
:
12–180 nM). Aryl ureido compounds (9f–9h) were also potent
mTOR inhibitors with somewhat decreased selectivity over PI3K-
Table 1
Inhibitory concentrations (IC50’s) of thienopyrimidines 1 and 2 against mTOR, PI3K-
a, although pyridinyl ureido compounds 9g and 9h both retained
a
high selectivity (>100-fold) over PI3K-a and displayed excellent
and LNCaP cellular proliferation
potency against cellular proliferation.
a
b
Compds mTOR IC50
PI3K-
(nM)
a IC50
Sel.c LNCaP cell IC50
(nM)
It had previously been shown3 that substitution at the 2-po-
sition of the thiophene ring (cf. GDC-0941, Fig. 1) could be used
to optimize aqueous solubility and other physicochemical prop-
erties. Based on this report, we explored the effect of these sub-
stitutions on our series. As shown in Scheme 3, the target
compounds were accessible from 2-chlorothienopyrimidine 7.
Formylation of 7 gave aldehyde 10. Reductive amination of 10
(nM)
1
2
49 13
61
41
8.5
0.9
1,300
3
0.14 680
a
Mean SEM.
b
IC50 determinations were done in triplicate at eight (half-log) concentrations.
The average error was <50%.
c
Selectivity (IC50 PI3K-
a
/IC50 mTOR).
O
O
OH
Cl
N
H
S
b
a
6
NH2
NH2
S
S
N
N
c
N
OH
N
Cl
3
5
4
O
N
O
N
d
S
S
N
N
N
8a-h
Ar
N
Cl
7
Scheme 1. Reagents and conditions: (a) triphosgene, dioxane, 80 °C; (b) POCl3, 120 °C, 99% from 3; (c) CH2Cl2, EtOH, NEt3, 93%; (d) ArB(OR)2, Pd(PPh3)4, toluene, EtOH, aq
Na2CO3, microwave, 120 °C, 26–87%.

J. C. Verheijen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 375–379
377
Table 2
Inhibitory concentrations (IC50’s) of thienopyrimidines against mTOR, PI3K-
a
and LNCaP cellular proliferation
O
N
S
N
N
Ar
a
b
Compds
Ar
mTOR IC50 (nM)
PI3K-
399
a
IC50 (nM)
Sel.c
6.9
LNCaP cell IC50 (nM)
OH
8a
58
57
4
8
2800
1200
N
8b
246
4.4
N
NH2
8c
8d
8e
32
4
4
6270
1767
199
51
2800
1250
4300
OH
N
35
NH2
NH2
100
>10,000
8619
>100
8f
22
392
1350
N
H
O
8g
26.5 0.7
4372
6199
165
549
1700
1850
N
H
O
8h
11
4
N
H
O
a
b
c
Mean SEM.
IC50 determinations were done in triplicate at eight (half-log) concentrations. The average error was <50%.
Selectivity (IC50 PI3K- /IC50 mTOR).
a
gave access to 11, which was converted into 2-(4-nitrophenyl)-
thienopyrimidine 12 under Suzuki coupling conditions. Reduc-
tion of the nitro group, followed by urea formation, gave access
to target compounds 13a–d. Alternatively, 2-chlorothienopyrimi-
dine 10 could be reacted first under Suzuki conditions. Reductive
amination of the resulting intermediate 14 gave 15. Conversion
of the nitrophenyl group in 15 into ureidophenyl compounds
16a–d was effected as described for the conversion of 12 into
13a–d.
potency where previously described following introduction of
methylsulfonylpiperazine, and were explained by the formation
of hydrogen bonds between the sulfonyl oxygens and K802 and
A805.3 3-Pyridinyl ureidophenyl 16c was a highly potent dual
inhibitor of mTOR and PI3K with excellent cellular activity,
whereas cyclopropyl ureido compound 16b retained high selectiv-
ity for mTOR over PI3K-a and possessed excellent enzyme and cel-
lular potency.
In conclusion, we have shown that introduction of an 8-oxa-3-
azabicyclo[3.2.1]octane group onto a thienopyrimidine scaffold
greatly increased selectivity for mTOR over PI3K-a. In addition,
The data in Table 4 reveals the effects of substitution on the thi-
ophene ring. Introduction of a methylpiperazinomethyl group led
to compounds with comparable IC50’s against PI3K-
a
(compare
replacement of the aminopyrimidine or phenol substituent in the
Tables 3 and 4). Unfortunately, this particular substitution was
not well tolerated by mTOR. Thus, compounds 13a–d were 10–
20-fold less potent inhibitors of mTOR than the corresponding
unsubstituted thienopyrimidines. Consequently, the selectivity of
compounds 13a–d was significantly decreased. Replacement of
the methylpiperazine with a methylsulfonylpiperazine led to an
approximately 10-fold increase in activity, both against mTOR
6-position with 4-ureidophenyl groups gave access to highly po-
tent (<1 nM) and selective (>1000-fold over PI3K-
inhibitors.
a) mTOR
Acknowledgment
The authors are grateful to Wei-Guo Zhang for mTOR assay
development.
and PI3K-
to good selectivity over PI3K-
a
, resulting in potent mTOR inhibitors with moderate
a
(16a–d). Similar increases in

378
J. C. Verheijen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 375–379
Table 3
Inhibitory concentrations (IC50’s) of thienopyrimidines 9a–h against mTOR, PI3K-
a
and LNCaP cellular proliferation
O
N
S
N
N
O
R
N
H
N
H
a
b
Compds
9a
R
mTOR IC50 (nM)
PI3K-
423
a IC50 (nM)
Sel.c
492
LNCaP cell IC50 (nM)
0.9 0.1
0.7 0.1
85
12
9b
1262
1856
F
9c
0.7 0.2
825
324
1107
982
87
OH
9d
0.34 0.03
180
9e
9f
0.68 0.05
1.25 0.07
1359
82
2013
66
40
140
9g
0.29 0.01
0.44 0.07
119
80
410
182
27
10
N
N
9h
a
b
c
Mean SEM.
IC50 determinations were done in triplicate at eight (half-log) concentrations. The average error was <50%.
Selectivity (IC50 PI3K-
a/IC50 mTOR).
O
N
O
N
O
N
N
c
b
N
a
S
N
N
S
N
7
S
O
N
N
N
Cl
N
Cl
N
12
11
10
d
O2N
O
N
O
N
O
N
R2
c
Ms
b
d
N
N
S
N
N
S
O
S
N
N
N
O
N
N
N
13a-d (R2=Me)
16a-d (R2=Ms)
R1
14
15
N
N
O2N
O2N
H
H
Boc
Boc
HN
N
e
N
f
N
N
NH
Ms
Ms
17
19
18
Scheme 3. Reagents and conditions: (a) BuLi (1.2 equiv), THF, À78 °C; then DMF (1.5 equiv), 91%; (b) NaHB(OAc)3, HOAc, 1,2-dichloroethane, N-Me-piperazine or N-Ms-
piperazine (19), 76% (11), 54% (15); (c) 4,4,5,5-tetramethyl-2-(4-nitrophenyl)-1,3,2-dioxaborolane, Pd(PPh3)4, DME, aq Na2CO3, 90 °C, 83% (12), 51% (14); (d) (1) H2, Pd/C,
CH2Cl2, iPrOH; (2) triphosgene, CH2Cl2, NEt3; (3) R1NH2, 20–55%; (e) MsCl, NEt3, CH2Cl2, 94%; (f) TFA, CH2Cl2, 99%.

J. C. Verheijen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 375–379
379
Table 4
Inhibitory concentrations (IC50’s) of thienopyrimidines 13a–d and 16a–d against mTOR, PI3K-
a
and LNCaP cellular proliferation
O
R2
N
N
N
S
N
O
N
R1
N
N
H
H
R1
R2
mTOR IC50 (nM)
PI3K-a
IC50 (nM)
Sel.c
LNCaP cell IC50 (nM)
a
b
Compds
13a
16a
Me
Ms
20
1.4 0.7
2
322
46
17
33
500
120
13b
16b
Me
Ms
16
1.0 0.1
4
874
179
56
176
400
32
13c
16c
Me
Ms
4.7 0.3
0.6 0.1
148
12
31
21
148
22
N
N
13d
16d
Me
Ms
3.7 0.9
0.45 0.04
86
8.3
23
19
200
120
a
b
c
Mean SEM.
IC50 determinations were done in triplicate at eight (half-log) concentrations. The average error was <50%.
Selectivity (IC50 PI3K- /IC50 mTOR).
a
7. Zask, A.; Verheijen, J. C.; Kaplan, J.; Curran, K.; Richard, D.; Brooijmans, N.;
Lucas, J.; Toral-Barza, L.; Zhang, W.-G.; Hollander, I.; Gibbons, J.; Abraham, R.;
Ayral-Kaloustian, S.; Mansour, T.; Yu, K. J. Med. Chem. 2009, 52, 5013.
8. 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.; Zask, A.; Zhang, W.-G.; Yu, K. J.
Med. Chem., in press. doi:10.1021/jm9012642.
9. Yu, Y.; 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.
10. 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., in press,
doi:10.1021/jm9013828.
11. Zask, A.; Kaplan, J.; Verheijen, J. C.; Richard, D. J.; Curran, K.; Brooijmans, N.;
Bennett, E.; Toral-Barza, L.; Hollander, I.; Ayral-Kaloustian, S.; Mansour, T.; Yu,
K. J. Med. Chem., in press, doi:10.1021/jm901415x.
References and notes
1. Verheijen, J. C.; Yu, K.; Zask, A. Ann. Rep. Med. Chem. 2008, 43, 189.
2. Verheijen, J. C.; Zask, A. Drugs Future 2007, 32, 537.
3. Folkes, A. J.; Ahmadi, K.; Alderton, W. K.; Alix, S.; Baker, S. J.; Box, G.; Chuckowree,
I. S.; Clarke, P. A.; Depledge, P.; Eccles, S. A.; Friedman, L. S.; Hayes, A.; Hancox, T.
C.; Kugendradas, A.; Lensun, L.; Moore, P.; Olivero, A. G.; Pang, J.; Patel, S.; Pergl-
Wilson, G. H.; Raynaud, F. I.; Robson, A.; Saghir, N.; Salphati, L.; Sohal, S.; Ultsch,
M. H.; Valenti, M.; Wallweber, H. J. A.; Wan, N. C.; Wiesmann, C.; Workman, P.;
Zhyvoloup, A.; Zvelebil, M. J.; Shuttleworth, S. J. J. Med. Chem. 2008, 51, 5522.
4. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi, T.; Okada, M.;
Ohta, M.; Tsukamoto, S. i.; Parker, P.; Workman, P.; Waterfield, M. Bioorg. Med.
Chem. 2006, 14, 6847.
5. Olivero, A. G.; Heffron, T.; Berry, L.; Berry, M.; Castanedo, G.; Chang, C.; Dotson,
J.; Friedman, L. S.; Goldsmith, R.; Lesnick, J.; Lewis, C.; Mathieu, S.; Nonomiya,
J.; Pang, J.; Peterson, D.; Prior, W.W.; Salphati, L.; Sampath, D.; Sutherlin, D.;
Tsui, V.; Ultsch, M.; Wang, S.; Wiesmann, C.; Wong, S.; Zhu, B.-Y.; Chuckowree,
I.; Folkes, A.; Shuttleworth, S. In: Proceedings of the 100th Annual Meeting of
the American Association for Cancer Research; 2009 Apr 18–22; Denver, CO.
Philadelphia (PA): AACR; 2009, Abstract nr 2707.
12. Biological test methods have been described before. See: Toral-Barza, L.; Zhang,
W. G.; Lamison, C.; Larocque, J.; Gibbons, J.; Yu, K. Biochem. Biophys. Res.
Commun. 2005, 332, 304; Yu, K.; Toral-Barza, L.; Discafani, C.; Zhang, W. G.;
Skotnicki, J.; Frost, P.; Gibbons, J. Endocr.-Relat. Cancer 2001, 8, 249; 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.
6. Heffron, T. P.; Berry, M.; Castanedo, G.; Chang, C.; Chuckowree, I.; Dotson, J.;
Folkes, A.; Gunzner, J.; Lesnick, J.; Lewis, C.; Malesky, K.; Mathieu, S.; Nonomiya,
J.; Olivero, A.; Pang, J.; Peterson, D.; Salphatis, L.; Sampath, D.; Sutherlin, D.;
Tsui, V.; Ultsch, M.; Wan, N. C.; Wang, S.; Wiesman, C.; Wong, S.; Zhu, B.-Y.
Poster presented at the 237th ACS National Meeting and Exposition March 22–
26, 2009 in Salt Lake City, UT.
13. The purity and identity of the compounds was confirmed by 1H NMR, HPLC and
MS analysis.