2-Arylureidophenyl-4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)triazines as highly potent and selective ATP competitive mTOR inhibitors: Optimization of human microsomal stability

Article abstract of DOI:10.1016/j.bmcl.2010.02.031

Isosteric replacement of one of the 3,5-ethylene-bridged morpholines in 2-arylureidophenyl-4,6-di(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)triazines led to significant improvements in human microsomal stability. 3-R-Me-morpholine and tetrahydropyran were identified as preferred isosteres for the bridged morpholine. Combination of tetrahydropyran substitution with an N-Me-piperazinophenylureido group led to 27, that selectively suppressed mTOR biomarkers in vivo and possessed excellent efficacy in a murine xenograft model.

Full text of DOI:10.1016/j.bmcl.2010.02.031

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2648–2653
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Arylureidophenyl-4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)triazines as
highly potent and selective ATP competitive mTOR inhibitors: Optimization
of human microsomal stability
a
a
a
b
a
Jeroen C. Verheijen a, , David J. Richard , Kevin Curran , Joshua Kaplan , Ker Yu , 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:
Isosteric replacement of one of the 3,5-ethylene-bridged morpholines in 2-arylureidophenyl-4,6-di(3-oxa-
8-azabicyclo[3.2.1]octan-8-yl)triazines led to significant improvements in human microsomal stability.
3-R-Me-morpholine and tetrahydropyran were identified as preferred isosteresfor thebridged morpholine.
Combination of tetrahydropyran substitution with an N-Me-piperazinophenylureido group led to 27, that
selectively suppressed mTOR biomarkers in vivo and possessed excellent efficacy in a murine xenograft
model.
Received 20 January 2010
Revised 4 February 2010
Accepted 8 February 2010
Available online 11 February 2010
Keywords:
mTOR
Ó 2010 Elsevier Ltd. All rights reserved.
PI3K
Kinase inhibitors
Cancer
Oncology
Microsomes
Rapamycin and its analogs (‘rapalogs’) have shown clinical effi-
cacy in the treatment of cancer. These compounds are allosteric
inhibitors of a complex between the mammalian Target Of Rapa-
mycin (mTOR) and raptor (mTOR complex 1, mTORC1).1 Inhibition
of mTORC1 signaling leads to increased Akt activity through a feed-
back mechanism, resulting in potential anti-apoptotic effects.2 In
addition, although the rapalogs strongly inhibit mTORC1 signaling
to S6K, they only have marginal effects on mTORC1 signaling to
4EBP.3,4 It is expected that increased anti-tumor efficacy may be
obtained with ATP competitive active site inhibitors of mTOR. Such
inhibitors would disrupt mTORC1 signaling to both S6K as well as
4EBP. Moreover, a second functional complex of mTOR, mTORC2,
which is not affected by rapalogs,5 contributes to Akt activation
by phosphorylation of Akt at serine 473.6 ATP competitive mTOR
inhibitors would be expected to disrupt mTORC2-mediated phos-
phorylation of serine 473 and thus prevent the feedback activation
of Akt observed for the rapalogs. Therefore, several groups,7–11
including our own,12–18 have recently embarked on an intense
search for ATP competitive mTOR inhibitors.
Compound 1 is a highly potent and selective inhibitor with excellent
activity in murine models of human tumor xenografts. We generally
observed excellent PK parameters (high AUC, low clearance) for
compounds with good microsomal stability. Although 1 displayed
excellent stability in nude mouse microsomes (t1/2 >30 min), its clin-
ical development was precluded by its rapid degradation by human
microsomes (t1/2 <5 min). The discrepancy between microsomal sta-
bility in the pre-clinical efficacy species (nude mouse) and the target
species (human) suggests that the CYP isoforms responsible for the
major metabolic pathways differ between these two species. In this
Letter, we describe our efforts aimed at increasing the human micro-
somal stability of derivatives of 1, while maintaining the excellent
potency and selectivity characteristics.
In the case of pyrazolopyrimidine mTOR inhibitors, we had
shown that the human microsomal stability could be enhanced by
variation of the polar substituent at the 4-position of the phen-
ylureidophenyl group.12,15,17 Initial efforts on the triazine series
therefore centered on this group as well. The compounds listed in
Table 1 were prepared according to the schemes in our preceding
Letter.19 A variety of different polar groups were examined, resulting
in all cases in highly potent inhibitors of both mTOR.20 All
compounds also potently inhibited proliferation of a prostate tumor
cell line (LNCaP) that is characterized by hyperactive PI3K-Akt-
mTOR signaling.21 We have previously demonstrated that cellular
proliferation inhibition in this model correlates with inhibition of
mTOR signaling.15 Replacement of the N-methyl piperazine with a
In the preceding Letter19 we presented the design and synthesis
of di-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-arylureidophenyl-tria-
zines as potent and selective mTOR inhibitors (e.g., 1, Table 1).
* Corresponding author.
E-mail addresses: verheij@wyeth.com, jv.verheijen@gmail.com (J.C. Verheijen).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.031

J. C. Verheijen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2648–2653
2649
Table 1
The effect of different substituents at the 4-position of the phenylureidophenyl group on activity, selectivity and microsomal stability
O
N
N
N
O
N
N
R
O
N
N
H
H
a
Compd
R
mTOR IC50
(nM)
PI3K-
a
Sel.b
LNCaP cell
IC50 (nM)
Mouse microsome
T1/2 (min)
Human microsome
T1/2 (min)
a
c
c
IC50 (nM)
899 107
947
1
2
1.0 0.1
892
1.3
>30
>30
2.5
4
N
N
0.57 0.01
1.0 0.2
1661
<0.8
HN
N
N
3
4
5
1127
499
1127
460
4.0
5.0
3.0
>30
>30
>30
3
6
6
N
1.1 0.3
N
0.89 0.09
226
255
H
N
6
7
8
9
1.6 0.2
0.9 0.3
0.8 0.2
955
483
389
579
537
458
3.0
0.8
0.8
>30
13
2.5
3
N
N
O
N
O
>30
4
N
O
0.26 0.06
0.18 0.02
961
253
3768
1406
0.8
>30
>30
<1
<1
HO
HO
O
10
<0.8
a
b
c
Mean SEM.
Selectivity (IC50 PI3K-
Nude mouse or human microsomal stability.
a/IC50 mTOR).
secondary amine (2) resulted in an equipotent compound with
excellent selectivity over PI3K-a 22
Introduction of a second basic
amine, as in benzylic piperidine 3, was well tolerated. Smaller ben-
zylic amines (4 and 5) were slightly less selective. Aminoethoxy sub-
stituents (6–8) also showed slightly less selectivity than the
piperazinophenyl compounds. Primary alcohols led to extremely
Metabolite identification studies revealed that the 3,5-ethylene
.
bridge on the morpholine group was the primary site of metabo-
lism. This observation prompted us to explore compounds where
one of the 3,5-bridged morpholines had been replaced with an-
other substituent. The synthesis of these compounds is depicted
in Scheme 1.24 Chloride 11 (prepared as described in the preceding
Letter19) was treated with an amine in the presence of base (for
compounds 15–19) or subjected to Stille coupling conditions with
tributyl(3,6-dihydro-2H-pyran-4-yl)stannane (for compounds 20
potent compounds with excellent selectivity over PI3K-
a (9 and
10). Unfortunately, all compounds in Table 1 still suffered from poor
stability in human microsomes.23
O
N
O
N
O
N
O
N
a or b
c or d
e
N
N
N
N
N
N
N
N
35-80%
80-100%
40-90%
R1
O
R1
N
N
R1
N
Cl
N
R2
11
12
13
14
O2N
O2N
H2N
N
N
H
H
Scheme 1. R1 and R2 are as defined in the tables. Reagents and conditions: (a) amine, NEt3; (b) tributyl(3,6-dihydro-2H-pyran-4-yl)stannane, Pd(PPh3)4, toluene; (c) H2, Pd/C;
(d) Fe, AcOH; (e) triphosgene, NEt3, then R2NH2.

2650
J. C. Verheijen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2648–2653
Table 2
The effect of morpholine replacements on activity, selectivity and microsomal stability
O
N
N
N
N
N
O
N
R
N
N
H
H
mTORa IC50 (nM) PI3K-
a
IC50 (nM) Sel.b
LNCaP cell IC50 (nM) Mouse microsome T1/2 (min) Human microsome T1/2 (min)
a
c
c
Compd
R
OH
15
0.7 0.1
1028
1546 <0.8
>30
5.5
N
HO
16
17
18
19
20
21
5.4 0.9
6.6 0.7
8.8 0.9
1.2 0.2
0.7 0.2
0.52 0.05
2506
4766
592
460
728
67
50
>30
—
>30
—
N
H
N
N
240
800
6
—
—
S
N
665 106
578
78
>30
—
18
—
(R)
O
51
8
25
O
O
777 129
1494 0.8
>30
27
a
b
c
Mean SEM.
Selectivity (IC50 PI3K-
Nude mouse or human microsomal stability.
a
/IC50 mTOR).
and 21). The resulting nitro compound 12 was reduced to the ani-
line (13) by hydrogenation (for 15–19, 21) or by treatment with
iron and acetic acid (20). Aniline 13 was converted into urea 14
by treatment with triphosgene and an amine.
Replacement of the 3,5-bridged morpholine with a ring-opened
analog (15, Table 2) resulted in a potent inhibitor that was selec-
result in decreased selectivity. Therefore, we selected morpholine
derivatives that were not expected to potently bind to PI3K-a 26,27
Indeed, thiomorpholine 18 did not result in potent inhibition of
PI3K. Unfortunately, the potency of 18 against mTOR was not in-
creased compared to 16 and 17. In contrast, 3-R-Me-morpholine, an-
other poor binder of PI3K,26 led to good mTOR and cellular potency
.
tive over PI3K-
a
but still suffered from poor stability in human
while remaining selective over PI3K-
even more potent against mTOR. As expected,27 20 also possessed
significantly increased potency against PI3K- , resulting in de-
creased selectivity. We have previously demonstrated that reduc-
a (19). Dihydropyran 20 was
microsomes. In contrast, replacement of the bridged morpholine
with isopropylamine (16) led to excellent stability in this assay.
Unfortunately, the latter substitution also resulted in decreased
potency. Attempts to enhance potency with other small alkylamine
substituents (e.g., 17) were not met with success. It should be
noted that compound 16 still contains one bridged morpholine, a
potential source for microsomal degradation. The data in Table 2
suggests that replacement of a single bridged morpholine may be
sufficient to provide increased microsomal stability. A possible
explanation for this observation may be that one of the bridged
morpholines plays a key role in presenting the second bridged
morpholine to CYP-450 in a spatial orientation that leads to rapid
oxidation. A related series of compounds in which both bridged
morpholines have been replaced with isosteres is discusses in
our accompanying Letter.25
Since compounds 1 and 15 both displayed excellent potency,
and both were equipped with a morpholine-like substituent, we
focused our efforts on substituents that resembled morpholine.
In our accompanying Letter,19 we have shown that the selectivity
of di-morpholino-triazines depends on the nature of both morpho-
line groups. Thus, because of the C2 symmetrical nature of the tri-
azine inhibitors, the presence of a single morpholine derivative
a
tion of the olefin in the dihydropyran group leads to poor PI3K-
a
binding.27 When the same strategy was applied to 20, resulting tet-
rahydropyran 21 maintained mTOR potency but lost the ability to
potently inhibit PI3K-a. The data in Table 2 reveal that compounds
with similar enzyme IC50s can possess drastically different cellular
proliferation IC50s (cf. 15 and 20), presumably reflecting differences
in cellular permeability and intracellular distribution. Both 3-R-Me-
morpholine derivative 19 and tetrahydropyran 21 combined excel-
lent mTOR and LNCaP potency and selectivity over PI3K-a with sig-
nificantly increased human microsomal stability. Therefore,
additional analogs of 19 and 21 were explored.
Table 3 lists analogs containing the tetrahydropyran group.
Replacement of the tertiary amine in 21 with a secondary amine
(22) resulted in decreased selectivity and cellular potency. Ethyl-
piperazine 23 was equipotent to the methyl analog (21) and dis-
played excellent stability in human microsomes. The cellular
potency of isopropyl analog 24 was somewhat decreased, which
could be restored by moving the amine to an exocyclic position
(25). The latter substitution resulted in a moderate loss of micro-
somal stability. As observed previously,28 inhibitors containing
capable of forming the hinge region hydrogen bond to PI3K-a will

J. C. Verheijen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2648–2653
2651
Table 3
Inhibitors containing the tetrahydropyran substituent
O
N
N
N
R
O
N
O
N
N
H
H
IC50 (nM) Sel.b
LNCaP cell IC50 (nM) Mouse microsome T1/2 (min) Human microsome T1/2 (min)
a
a
c
c
Compd
R
mTOR IC50 (nM) PI3K-
a
N
21
0.52 0.05
0.535 0.005
0.56 0.02
777 129
294
1494 0.8
>30
>30
>30
27
N
HN
22
23
550
20
27
N
N
N
574 26
1025 <0.8
>30
N
N
24
0.88 0.04
660 16
751
18
>30
>30
N
25
26
0.56 0.04
0.81 0.07
382 14
79 18
682
97
1.0
>30
>30
20
N
O
<0.8
>30
N
N
O
O
O
N
N
27
28
0.46 0.02
0.34 0.06
148 17
325
226
0.9
>30
>30
>30
>30
N
77
7
<0.8
N
N
N
29
30
0.14 0.02
0.7 0.1
54 6.0
876
377
<0.8
9
>30
>30
>30
20
N
H
O
1208
a
b
c
Mean SEM.
Selectivity (IC50 PI3K-
Nude mouse or human microsomal stability.
a
/IC50 mTOR).
benzamido water solubilizing groups were extremely potent
against cellular proliferation and possessed excellent microsomal
stability (26–29). Although methylpiperazino benzamide 26 suf-
fered from a 15-fold loss in selectivity compared to 21, other
for further evaluation. First, we explored whether 27 was selective
over other kinases in addition to PI3K- . The IC50 against another
PI3K isoform, PI3K- , was 1256 nM (2730-fold selective). In addi-
tion, when 27 was screened against an in-house panel of 21 kinases,
none of them were inhibited at IC50s below 50 M (>100,000-fold
a
c
amides maintained high selectivity over PI3K-
a
(27–29). Dimeth-
l
ylaminoethoxy analog 30 was less potent in the cellular assay
and was only moderately stable in the microsomal assay.
Similar trends were observed for analogs incorporating 3-R-Me-
morpholine (Table 4). Thus, N-linked water solubilizing groups led
selectivity).29 In addition to its excellent stability in human micro-
somes, 27 was highly stable in human hepatocytes as well, as evi-
denced by low clearance (0.0083 mL/min/million cells) and a high
half-life (>60 min). Finally, the potential for cardiac effects and
drug–drug interactions was explored. Inhibitory concentrations of
27 against hERG and various cytochrome P450 isoforms (1A2, 2A6,
to potent compounds with good selectivity over PI3K-
a (19, 31–
34). Amide-linked substituents resulted in increased potency but
decreased selectivity (35–38). Compounds 36 and 37 combined
excellent potency and stability with good selectivity (>200-fold)
2B6, 2C8, 2C9, 2C19, 2D6, 3A4) were greater than 30
fold higher than the mTOR IC50). The only CYP isozyme that was
inhibited below 30 M was CYP 2C8, with an IC50 of 3 M. The latter
value is still more than 6,500-fold higher than the mTOR IC50
lM (>65,000-
over PI3K-a. The dimethylaminoethoxy group again resulted in
l
l
the least potent analog (39).
.
Based on its excellent potency, selectivity over PI3K-
somal stability across different species, compound 27 was selected
a
and micro-
Next, we evaluated the effects of 27 in vivo. The pharmacokinet-
ics of 27 following intravenous administration (5 mg/kg) were

2652
J. C. Verheijen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2648–2653
Table 4
Inhibitors containing the 3-R-Me-morpholine substituent
O
N
N
N
R
O
N
N
(R)
O
N
N
H
H
IC50 (nM) Sel.b LNCaP cell IC50 (nM) Mouse microsome T1/2 (min) Human microsome T1/2 (min)
a
a
c
c
Compd
R
mTOR IC50 (nM) PI3K-
a
N
19
1.2 0.2
0.40 0.04
1.2 0.1
665 106
286 <1
1006
578
706
838
6
>30
>30
>30
18
17
23
N
HN
31
32
<0.8
1.0
N
N
N
N
N
33
34
35
1.25 0.05
0.90 0.06
0.57 0.05
970
776
494
105
6.0
>30
>30
>30
>30
20
N
443 68
8.0
N
O
60
7
<0.8
>30
N
N
O
O
O
N
N
36
37
0.42 0.07
0.25 0.03
102 18
245
212
<0.8
<0.8
21
>30
>30
N
52
1
3
>30
N
N
N
38
39
0.10 0.02
1.7 0.1
43
407
359
<0.8
12
>30
>30
20
12
N
H
O
610
a
b
c
Mean SEM.
Selectivity (IC50 PI3K-
Nude mouse or human microsomal stability.
a
/IC50 mTOR).
characterized by low clearance (5 mL/min/kg), a moderate volume
of distribution (1.3 L/kg) and a moderate to long half-life (3.4 h),
resulting in high exposure (AUC0–inf: 16,654 h ng/mL). Intravenous
Control
27
Ã
P-S6K (T389)
P-Akt (S473)
P-Akt (T308)
P-S6
administration of 27 (25 mg/kg) to nude mice bearing MDA361 tu-
mor xenografts30 resulted in complete inhibition of mTORC1 sig-
naling, lasting at least 8 h, as evidenced by the lack of
phosphorylation of S6K and its downstream substrate S6 (Fig. 1).
Phosphorylation of the mTORC2 substrate, Akt (S473) was also
significantly inhibited. The selective nature of inhibitor 27 was
exemplified by the lack of suppression of phosphorylation of
the PI3K/PDK1 biomarker Akt (T308). Mean plasma and tumor
concentrations of 27 at the 8 h time point were 2161 ng/mL and
2474 ng/mL, respectively.
Actin
Figure 1. Inhibition of mTOR signaling in vivo following a single dose of mTOR
kinase inhibitor 27 (25 mg/kg, iv). Nude mice bearing sc MDA361 tumors were
injected iv with vehicle or 25 mg/kg of 27. Tumor lysates were prepared 8 h after
injection and were immunoblotted with antibodies against P-S6K (T389), P-Akt
(S473), P-Akt (T308), P-S6 and b-actin.
Administration of low intravenous doses of 27 (3 mg/kg, qd) for
five days to nude mice bearing human glioblastoma U87MG xeno-
grafts resulted in pronounced tumor growth arrest (Fig. 2). To the

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best of our knowledge, efficacy at these low doses has not been
demonstrated previously for ATP competitive mTOR inhibitors.
In conclusion, isosteric replacement of the 3-oxa-8-azabicy-
clo[3.2.1]octane group in triazine mTOR inhibitors led to com-
pounds that combined potent and selective inhibition of mTOR
with excellent microsomal stability. A representative inhibitor,
compound 27, displayed potent and selective in vivo inhibition of
mTOR leading to efficacy in murine tumor xenograft models at
unprecedentedly low doses.
17. 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.
18. Curran, K. J.; Verheijen, J. C.; Kaplan, J.; Richard, D. J.; Toral-Barza, L.; Hollander,
I.; Lucas, J.; Ayral-Kaloustian, S.; Yu, K.; Zask, A. Bioorg. Med. Chem. Lett. 2010,
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20. 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.
21. 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.
Acknowledgments
22. 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.
23. 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.
24. The purity and identity of novel compounds was confirmed by HPLC, mass
spectrometry and 1H NMR.
25. Richard, D. J.; Verheijen, J. C.; Yu, K.; Zask, A. Bioorg. Med. Chem. Lett. 2010, 20,
2654.
26. 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, 52, 7942.
The authors thank Wei-Guo Zhang and Lourdes Toral-Barza for
mTOR assays, Dr. Irwin Hollander for PI3K assays, Dr. Joseph Marini
and Angela Bretz for nude mouse microsome assays, Dr. Li Di and
Susan Li for human microsome assays, Dr. Richard Harrison, Dr.
Ann Aulabaugh, Jenny Togias and Kenneth Roberts for kinase panel
assays and Judy Lucas, Rob Mahoney and Kenny Kim for in vivo as-
says. We also thank Drs. Tarek Mansour, Semiramis Ayral-Kalous-
tian, John Ellingboe, Robert Abraham and James Gibbons for
program support and contributions.
27. Kaplan, J.; Verheijen, J. C.; Brooijmans, N.; Toral-Barza, L.; Hollander, I.; Yu, K.;
Zask, A. Bioorg. Med. Chem. Lett. 2010, 20, 640.
References and notes
28. Dehnhardt, C. M.; Venkatesan, A. M.; Delos Santos, E.; Chen, Z.; Santos, O.;
Ayral-Kaloustian, S.; Brooijmans, N.; Mallon, R.; Hollander, I.; Feldberg, L.;
Lucas, J.; Yu, K.; Gibbons, J.; Abraham, R.; Mansour, T. S. J. Med. Chem. 2009.
doi:10.1021/jm9014982.
29. The in-house kinase panel existed of the following kinases: ABL, Aurora B,
CDK1/Cyclin B, CDK2/Cyclin A, CHK1, CK1 gamma1, ERK2, Fyn, GCK, HCK, LYN
A, MK2, P38 alpha, PDGFR alpha, PKA, PKC alpha, PKC beta, ROCK 1, RSK1, SRC,
VEGFR2.
1. For a recent review on rapamycin, the rapalogs and other mTOR inhibitors, see:
Verheijen, J.; Yu, K.; Zask, A. in John E. Macor, editor: Ann. Rep. Med. Chem. 2008,
43, 189.
2. Carracedo, A.; Pandolfi, P. P. Oncogene 2008, 27, 5527.
3. Choo, A. Y.; Yoon, S. O.; Kim, S. G.; Roux, P. P.; Blenis, J. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 17414.
4. Guertin, D. A.; Sabatini, D. M. Sci. Signalling 2009, 2, 1.
5. 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.
30. In vivo methods for determination of biomarker inhibition and efficacy in nude
mouse xenograft models have been described before; see Ref. 12.