Discovery of Clinical Development Candidate GDC-0084, a Brain Penetrant Inhibitor of PI3K and mTOR

Article abstract of DOI:10.1021/acsmedchemlett.6b00005

Inhibition of phosphoinositide 3-kinase (PI3K) signaling is an appealing approach to treat brain tumors, especially glioblastoma multiforme (GBM). We previously disclosed our successful approach to prospectively design potent and blood-brain barrier (BBB) penetrating PI3K inhibitors. The previously disclosed molecules were ultimately deemed not suitable for clinical development due to projected poor metabolic stability in humans. We, therefore, extended our studies to identify a BBB penetrating inhibitor of PI3K that was also projected to be metabolically stable in human. These efforts required identification of a distinct scaffold for PI3K inhibitors relative to our previous efforts and ultimately resulted in the identification of GDC-0084 (16). The discovery and preclinical characterization of this molecule are described within.

Full text of DOI:10.1021/acsmedchemlett.6b00005

Subscriber access provided by GAZI UNIV
Letter
The Discovery of Clinical Development Candidate
GDC-0084, a Brain Penetrant Inhibitor of PI3K and mTOR
Timothy P Heffron, Chudi O. Ndubaku, Laurent Salphati, Bruno Alicke, Jonathan Cheong, Joy Drobnick,
Kyle Edgar, Stephen E. Gould, Leslie B. Lee, John D. Lesnick, Cristina Lewis, Jim Nonomiya, Jodie
Pang, Emile G. Plise, Steve Sideris, Jeffrey Wallin, Lan Wang, Xiaolin Zhang, and Alan G Olivero
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00005 • Publication Date (Web): 16 Feb 2016
Downloaded from http://pubs.acs.org on February 16, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Medicinal Chemistry Letters is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
The Discovery of Clinical Development Candidate GDC-0084,
a Brain Penetrant Inhibitor of PI3K and mTOR
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Timothy P. Heffron,* Chudi O. Ndubaku, Laurent Salphati, Bruno Alicke, Jonathan Cheong, Joy Drob-
nick, Kyle Edgar, Stephen E. Gould, Leslie B. Lee, John D. Lesnick, Cristina Lewis, Jim Nonomiya,
Jodie Pang, Emile G. Plise, Steve Sideris, Jeffrey Wallin, Lan Wang, Xiaolin Zhang, Alan G. Olivero.
Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080
PI3K, kinase inhibitor, blood-brain-barrier penetration, CNS, oncology.
ABSTRACT: Inhibition of phosphoinositide 3-kinase (PI3K) signaling is an appealing approach to treat brain tumors, especially
glioblastoma multiforme (GBM). We previously disclosed our successful approach to prospectively design potent and blood-brain
barrier (BBB) penetrating PI3K inhibitors. The previously disclosed molecules were ultimately deemed not suitable for clinical
development due to projected poor metabolic stability in humans. We, therefore, extended our studies to identify a BBB penetrat-
ing inhibitor of PI3K that was also projected to be metabolically stable in human. These efforts required identification of a distinct
scaffold for PI3K inhibitors relative to our previous efforts and ultimately resulted in the identification of GDC-0084 (16). The
discovery
and
preclinical
characterization
of
this
molecule
are
described
within.
Owing to an associated poor prognosis and limited treatment
options, glioblastoma multiforme (GBM) represents a signifi-
cant unmet medical need.1 In this particular disease, aberrant
PI3K signaling is associated with >80% of cases.2 While nu-
merous PI3K inhibitors have advanced to clinical study,3 to
treat GBM effectively it is anticipated that the inhibitor would
need to freely cross the blood-brain barrier (BBB). To this
end, we have previously reported on our initial efforts to iden-
tify BBB penetrating PI3K inhibitors.4 In those efforts mole-
cules 1 and 2 (Table 1) were identified which freely crossed
the BBB and demonstrated a PD effect in normal mouse brain
tissue as well as efficacy in intracranial mouse tumor models
of GBM. Despite the evident free brain penetration of 1 and
2, they were not selected for clinical development due to poor
projected clearance in humans (based on both human liver
microsomal stability, Table 1, and allometric scaling, data not
shown). We, therefore, extended our efforts to identify potent,
BBB penetrating inhibitors of PI3K with more desirable meta-
bolic stability that would be suitable for clinical study.
PI3K and mammalian target of rapamycin (mTOR) inhibitor
from the same thienopyrimidine scaffold as 1 and 2, we had
also studied a related purine scaffold (eg. 4, Table 1). Com-
pounds within this purine series of PI3K inhibitors that had
good human microsomal stability were readily identified. As
an example, while it did not advance to clinical development,
compound 4 was identified as a pan-PI3K inhibitor on a purine
scaffold that had appealing projected human clearance based
on liver microsomal stability (Table 1).
With our knowledge that desirable metabolic stability was
attainable on a purine scaffold, we resolved to evaluate purine-
based inhibitors for their potential to achieve the desired bal-
ance of potency, metabolic stability and low transporter medi-
ated efflux. The previous purine PI3K inhibitors that we had
made, such as 4, were not designed to have low P-gp and Bcrp
mediated efflux. Indeed, compound 4 was not expected to be
freely BBB penetrating given the high number of hydrogen
bond donors in the molecule. In fact, compound 4 is a signifi-
cant substrate of both P-gp and Bcrp as determined by the
efflux ratios in cell permeability assays (Table 1). It remained
to be seen whether or not we could realize purine PI3K inhibi-
tors that were capable of avoiding transport by P-gp and Bcrp
while also maintaining good human metabolic stability.
We continued our efforts to identify brain penetrating inhibi-
tors of PI3K by attempting to identify analogs of thienopyrim-
idines 1 and 2 that attained an acceptable combination of low
transporter mediated efflux, good potency and low projected
human clearance. In our attempts, we identified potent mole-
cules that achieved good metabolic stability in human liver
microsomes and many potent molecules that had low efflux
ratios in MDR1 (gene coding for P-glycloprotein, P-gp) and
breast cancer resistance protein (Bcrp) transfected MDCK cell
permeability assays (data not shown). The combination of
good human metabolic stability and low efflux ratios in this
thienopyrimidine series of analogs, however, remained elu-
sive. As a result we sought an alternative series of molecules
that might allow for the desired combination of properties.
At first we were concerned that the additional polarity of a
purine scaffold, relative to a thienopyrimidine, might render
such molecules more likely to be transporter substrates. To
evaluate the feasibility of a purine core for brain penetration,
we first studied compound 5 (Table 2). Despite a topological
polar surface area (TPSA) of 107 Å2, 5 has a low efflux ratio
in a P-gp transfected MDCK cell permeability assay. Addi-
tionally, compound 5 showed encouraging human microsomal
stability. Unfortunately, this analog lacked potency in a PC3
cell proliferation assay. Nevertheless, we were encouraged
that the combination of low efflux and good human microso-
mal stability that was unattainable in the thienopyrimidine
series of molecules could be obtained on a purine-based PI3K
inhibitor. We were then pleased to find that substitution at R1
Prior to our efforts to identify BBB penetrating PI3K inhibi-
tors, we had identified clinical inhibitors of PI3K that were
suited for the treatment of peripheral disease. In our studies
leading to the discovery of GDC-0980 (3, Table 1),5 a pan-
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 7
with either ethyl (6) or cyclobutyl (7) groups improved the the annulated product 13. Finally, Suzuki coupling of 13 with
1
2
3
4
5
6
7
8
cellular potency and maintained low efflux ratios (Table 2).
However, the ethyl and cyclobutyl substitutions each led to
reduced human liver microsomal stability. Compound 8 was
even more potent than the previous purine analogs and had
excellent human liver microsomal stability (Table 2). The
introduction of a new hydrogen bond donor relative to com-
pounds 5-7, however, led to very high efflux ratios for 8. This
result was consistent with our experience with thienopyrimi-
dine PI3K inhibitors and led us to synthesize compound 9, the
methyl ether analog of 8 and a direct analog of thienopyrimi-
dine 2. Compound 9 retained the excellent cellular potency of
8 and, as we had suspected, the conversion of the tertiary al-
cohol of 8 to the methyl ether of 9 ablated P-gp and Bcrp me-
diated efflux. While the human liver microsomal stability of 9
was moderate, we sought to further improve upon this result.
boronate ester 14 afforded the final compound 16.
The appealing in vitro properties of 16 described in Table 3
led us to evaluate it in in vivo pharmacokinetic studies. Table
4 includes the microsomal stability of 16 as well as key in vivo
pharmacokinetic parameters in rodents. The correlation be-
tween in vivo clearance and predicted clearance based on mi-
crosomal stability gave us greater confidence that human
clearance would be low as predicted in a human metabolic
stability assays (Table 3).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To verify that 16 was indeed capable of penetrating the BBB,
we determined the brain-to-plasma ratio in rats. After a 15
mg/kg dose of 16, the total brain-to-plasma ratio was 1.9-3.3.
While we did not determine brain binding for rats, and there-
fore cannot report Bu/Pu, we determined the concentration of
16 in cerebral spinal fluid (CSF). The CSF concentration is
sometimes employed as a surrogate for unbound brain concen-
tration.9 We found that the CSF-to-free plasma concentration
ratio in rats was 0.7-1.0, indicating that 16 effectively pene-
trates the BBB (Figure 1). In addition to demonstrating that
16 is capable of crossing the BBB in rats, we determined the
unbound brain-to-unbound plasma concentration (Bu/Pu) ratio
in female CD-1 mice. The Bu/Pu ratio of 0.4, at both 1 and 6 h
post 25 mg/kg oral dose of 16 demonstrate the molecule is
capable of substantial free brain penetration (Figure 1).
The excellent metabolic stability of compound 4 had initially
incited our investigation of whether we could realize brain
penetrating purine PI3K inhibitors. Its excellent potency and
metabolic stability were desirable but the efflux ratios sug-
gested the molecule would not freely penetrate the BBB (Ta-
ble 2). We, therefore, sought to eliminate the two hydrogen
bond donors of the alcohols in compound 4 in an effort to re-
duce transporter mediated efflux. Rather than alkylation of the
alcohols of 4, we investigated the effective result of ring clos-
ing condensation.
Figure 1. CNS penetration of 16 in rat and mouse.
The first cyclized analog of 4 using this approach, compound
15 (Table 3), was found to have excellent human metabolic
stability and low efflux ratios but modest cellular potency
(Table 3). We previously found that analogs without R1 sub-
stitution were more potent inhibitors of mTOR and generally
had better cellular potency in cell lines whose proliferation
were driven by aberrant PI3K signaling.6 Compound 16,
where R1 is H, achieved an excellent balance of cellular poten-
cy, metabolic stability and lack of efflux (Table 3). Addition-
ally, compound 16 was found to be highly selective against a
panel of 229 kinases where it inhibited none by >50% (1 µM
16, Supporting Information). Furthermore, 16 maintains inhi-
bition of each of the Class I PI3K isoforms (Supporting Infor-
mation) but with more potent inhibition of mTOR. Compound
16 was also tested in five different GBM cell lines and was
found to have anti-proliferative EC50’s ranging from 0.3-1.1
uM.7
[Brain]/[Plasma] ratios determined after oral dose of 16 to fe-
male CD-1 mice or male Sprague-Dawley rats as an MCT suspen-
sion. *[Brain]u and [Plasma]u refer to the unbound concentration
measured in the brain and plasma respectively. **[CSF] refers to
a
the concentration measured in the cerebral spinal fluid. Deter-
mined to be identical at both 1 h and 6 h after administration of 25
mg/kg 16 to female CD-1 mice. The [Brain]/[Plasma] ratios are
the mean values from 3 animals per time point. bDetermined after
administration of 15 mg/kg 16 to male Sprague-Dawley rats.
[Brain]/[Plasma] determined for 1 animal at each of 0.25 and 2 h
and 3 at 8 h. Data reported are the range across the three
timepoints (average of the 3 animals at 8h). c[CSF] determined for
1 animal at each of 0.25 and 2 h and 3 at 8 h. Data reported are
the range across the three timepoints (average of the 3 animals at
8h).
Scheme 1. Synthetic route to obtain tricyclic purine-based
brain penetrant PI3K inhibitor 16.
To further verify that 16 was indeed capable of penetrating the
BBB to engage its target where intended, we evaluated the
effect of 16 on pAKT in normal brain tissue. After a 25 mg/kg
dose of 16 administered orally, pAKT in normal mouse brain
tissue was significantly inhibited at 1 and 6 h post-dose (Fig-
ure 2). The potent inhibition of pAKT at both time points in
this study demonstrates that 16 inhibits its target behind a fully
intact BBB.
Figure 2. Inhibition of p-AKT by 16 in normal mouse brain
tissue along with corresponding brain and unbound brain con-
The synthesis of 16 (Scheme 1) began with lithiation of purine
108 followed by alkylation with acetone to provide tertiary
alcohol 11. The THP group was next deprotected to provide
12. Subsequent alkylation with 1,2-dibromoethane afforded
ACS Paragon Plus Environment

Page 3 of 7
ACS Medicinal Chemistry Letters
concentration dependent inhibition of pAKT was observed at
centrations.
1
both 1 and 4 h post-dose, indicating that the tumor growth
inhibition is the result of on-target inhibition (Figure 4).
2
3
4
5
6
7
8
9
The U87 subcutaneous tumor model can be considered a sur-
rogate for the intracranial U87 model because the intracranial
model has a compromised blood brain barrier following en-
graftment.11 We previously studied a different BBB penetrat-
ing PI3K/mTOR inhibitor in intracranial GBM models and
would expect comparable efficacy with 16 given its free brain
penetration.12
*Significantly different from untreated control. p<0.05, t-test.
[Brain] determined after 25 mg/kg oral dose of 16 female CD-1
mice as an MCT suspension. [Brain]u refers to the unbound con-
centration measured in the brain. Data are reported as mean val-
ues ± S.D. from 3 animals per time point.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Effect of 16 on the PD marker pAKT in the U87
MG/M human glioblastoma xenograft model after 24 days of
continuous dosing. Tumors were excised from animals 1 and 4 h
after the last administered dose on day 24 and processed for anal-
ysis of pAKT as described in Supporting Information. Indicated
values are the means for groups of 3 animals and error bars indi-
cate  standard error of the mean. Levels of pAkt (Ser473) and
total Akt were measured by electrochemiluminescence using Me-
so Scale Discovery according to manufacturer’s instructions
(Gaithersburg, MD).
In addition to the pharmacodynamic effect in normal brain
tissue, 16 was studied in a subcutaneous U87 tumor xenograft
model of glioblastoma in mice.10 In this study, 16 achieved
significant and dose-dependent tumor growth inhibition (Fig-
ure 3). Tumor growth inhibition was first observed at a 2.2
mg/kg dose level. Higher doses led to greater tumor growth
inhibition, including tumor regressions at the 17.9 mg/kg dose
level. Each of these doses was well tolerated for the duration
of the study.
Figure 3. In vivo efficacy of 16 versus U87 MG/M human
glioblastoma xenografts. Female NCr nude mice bearing sub-
cutaneous tumors were administered escalating doses of 16
orally as a suspension in vehicle (0.5% methylcellulose/0.2%
Tween-80) or vehicle once daily (QD) for 23 days. Changes
in tumor volumes over time by dose for each compound are
depicted as cubic spline fits generated via Linear Mixed Ef-
fects analysis of log-transformed volumes.
T
o summarize, we have identified 16, a potent purine-based
inhibitor of PI3K and mTOR that is capable of penetrating the
BBB. Additionally, 16 has excellent human metabolic stabil-
ity in microsomal and hepatocyte incubations. Compound 16
demonstrated inhibition of pAKT, a key signal within the
PI3K pathway, in both normal brain tissue and in U87 glio-
blastoma xenograft tumors in mice. Along with pAKT inhibi-
tion in U87 tumors, significant tumor growth inhibition was
achieved. The promising preclinical profile of 16, along with
the significant unmet medical need for glioblastoma treat-
ments, led to the advancement of 16 to clinical development.
Details of additional preclinical and clinical studies of 16 will
be reported elsewhere.
Supporting Information. Assessment of inhibition of 229 kinas-
es by 16, Class I PI3K Kiapp’s for 16, Western data showing inhi-
bition of pAKT and pS6 in U87 cells by 16, synthetic details and
associated analytical data for all reported compounds, experi-
mental details for biochemical and cellular assays, in vitro
transport assays, brain and plasma protein binding, PK studies in
mice, pAKT/tAKT PD evaluation in mouse brain and in vivo
xenograft studies are supplied as Supporting Information. This
material is available free of charge via the Internet at
http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
*Phone: (650) 467-3214. Fax: (650) 225-2061. E-mail: thef-
fron@gene.com
Consistent with the efficacy observed in the U87 xenograft
tumor study, at similar dose levels compound 16 was found to
have a significant PD effect in the U87 tumors. Dose and
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 7
Table 1. MDR1 and Bcrp1 transfected MDCK cell permeability efflux ratios and human liver microsomal stability for previously
identified PI3K inhibitors 1-4.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aDetermined 6 h after administration of 25 mg/kg orally to female CD-1 mice as an MCT suspension. bDetermined 6 h after admin-
istration of 50 mg/kg orally to female CD-1 mice as an MCT suspension. Determined 1 h after administration of 20 mg/kg to fe-
male CD-1 mice as an MCT suspension.
c
Table 2. Cellular potency, TPSA, efflux ratios and human metabolic stability for select purine PI3K inhibitors.
ACS Paragon Plus Environment

Page 5 of 7
ACS Medicinal Chemistry Letters
1
Table 3. Potency, efflux ratios and human metabolic stability of tricyclic purine-based PI3K inhibitors 15 and 16.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Preclinical species hepatocyte stability and in vivo pharmacokinetic data for 16.
aHepatic clearance was predicted from liver microsomes incubations using the “in vitro t1/2 method.”13 bMale Sprague-Dawley rats or fe-
male CD-1 mice were dosed intravenously with 1 mg/kg of 16 prepared in 60% PEG400/10% Ethanol. cCompound 16 was administered
PO at the indicated dose in 0.5% methylcellulose with 0.2% Tween 80 (MCT).
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 7
ABBREVIATIONS
1
ACKNOWLEDGMENT
PI3K, phosphoinositide 3-kinase; GBM, glioblastoma multiforme;
2
3
4
5
6
7
8
The authors wish to thank Mengling Wong, Chris Hamman,
Michael Hayes and Steve Huhn for compound purification and
determination of purity by HPLC, mass spectroscopy, and H
NMR. We thank Krista K. Bowman, Alberto Estevez, Kyle
Mortara, and Jiansheng Wu for technical assistance of protein
expression and purification.
BBB, blood-brain barrier; P-gp, P-glycoprotein; Bcrp, breast can-
cer resistance protein; mTOR, mammalian target of rapamycin;
TPSA, topological polar surface area; dppf, 1,1'-
bis(diphenylphosphino)ferrocene.
1
REFERENCES
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1 Rich, J. N.; Bigner, D. D. Development of Novel Targeted Therapies in the Treament of Malignant Glioma. Nat. Rev. Drug
Discov. 2004, 3, 430-446 and references therein.
2 The Cancer Genome Atlas Network. Comprehensive genomic characterization defines human glioblastoma genes and core
pathways. Nature, 2008, 455, 1061-1068.
3
Yap, T. A.; Bjerke, L.; Clarke, P. A.; Workman, P. Drugging PI3K in cancer: refining targets and therapeutic strategies.
Curr. Opin. Pharmacol. 2015, 23, 98-107.
4 Heffron, T. P.; Salphati, L.; Alicke, B.; Cheong, J.; Dotson, J.; Edgar, J.; Goldsmith, R.; Gould, S. E.; Lee, L. B.; Lesnick,
J. D.; Lewis, C.; Ndubaku, C.; Nonomiya, J.; Olivero, A. G.; Pang, J.; Plise, E. G.; Sideris, S.; Trapp, S.; Wallin, J.; Zhang,
X. The Design and Identification of Brain Penetrant Inhibitors of Phosphoinositide 3-Kinase . J. Med. Chem. 2012, 55,
8007-8020.
5
Sutherlin, D. P.; Bao, L.; Berry, M.; Castanedo, G.; Chuckowree, I.; Dotson, J.; Folkes, A.; Friedman, L.; Goldsmith, R.;
Gunzner, J.; Heffron, T.; Lesnick, J.; Lewis, C.; Mathieu, S.; Murray, J.; Nonomiya, J.; Pang, J.; Pegg, N.; Prior, W. W.;
Rouge, L.; Salphati, L.; Sampath, D.; Tian, Q.; Tsui, V.; Wan, N. C.; Wang, S.; Wei, B.; Wiesmann, C.; Wu, P.; Zhu, B.-Y.;
Olivero, A. Discovery of a Potent, Selective, and Orally Available Class I Phosphatidylinositol 3-Kinase (PI3K)/Mammalian
Target of Rapamycin (mTOR) Kinase Inhbitor (GDC-0980) for the Treatment of Cancer. J. Med. Chem. 2011, 54, 7579-
7587.
6
Sutherlin, D. P.; Sampath, D.; Berry, M.; Castanedo, G.; Chang, Z.; Chuckowree, I.; Dotson, J.; Folkes, A.; Friedman, L.;
Goldsmith, R.; Heffron, T.; Lee, L.; Lesnick, J.; Lewis, C.; Mathieu, S.; Nonomiya, J.; Olivero, A.; Pang, J.; Prior, W. W.;
Salphati, L.; Sideris, S.; Tian, Q.; Tsui, V.; Wan, N. C.; Wang, S.; Wiesmann, C.; Wong, S.; Zhu, B.-Y. Discovery of
(Thienopyrimidin-2-yl)aminopyrimidines as Potent, Selective, and Orally Available Pan-PI3-Kinase and Dual-PI3-
Kinase/mTOR Inhibitors for the Treatment of Cancer. J. Med. Chem. 2010, 53, 1086-1097.
7 In 4-day assays using CellTiter-Glo to monitor proliferation, 16 was studied in the following GBM cell lines: G111, EC50
=
0.27 uM; G96, EC50 = 0.53 uM; G112, EC50 = 0.58 uM; U87, EC50 = 0.74 uM; SF268, EC50 = 1.01 uM; G122, EC50 = 1.01
uM.
8 Murray, J. M.; Sweeney, Z. K.; Chan, B. K.; Balazs, M.; Bradley, E.; Castanedo, G.; Chabot, C.; Chantry, D.; Flagella, M.;
Goldstein, D. M.; Kondru, R.; Lesnick, J.; Li, J.; Lucas, M. C.; Nonomiya, J.; Pang, J.; Price, S.; Salphati, L.; Safina, B.;
Pascal, P. A.; Seward, E. M.; Ultsch, M.; Sutherlin, D. P. Potent and Highly Selective Benzimidazole Inhibitors of PI3-
Kinase Delta. J. Med. Chem. 2012, 55, 7686-7695.
9
Liu, X.; Van Natta, K.; Yeo, H.; Vilenski, O.; Weller, P. E.; Worboys, P. D.; Monshouwer, M. Unbound Drug Concentra-
tion in Brain Homogenate and Cerebral Spinal Fluid at Steady State as a Surrogate for Unbound Concentration in Brain In-
terstitial Fluid. Drug Metab. Dispos. 2009, 37, 787-793.
10 Compound 16 was found to have an anti-proliferation EC50 of 740 nM in U87 cells. Inhibition of pAKT in U87 cells was
demonstrated qualitatively by Western and is included as Supplementary Information.
11
Lee, J.; Kotilarova, S.; Kotliarov, Y.; Li, A.; Su, Q.; Donin, N. M.; Pastorino, S.; Purow, B. W.; Christopher, N.; Zhang,
W.; Park, J. K.; Fine, H. A. Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the
phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 2006, 9, 391-403.
12
Salphati, L.; Heffron, T. P.; Alicke, B.; Nishimura, M.; Barck, K.; Carano, R. A.; Cheong, J.; Edgar, K. A.; Greve, J.;
Kharbanda, S.; Koeppen, H.; Lau, S.; Lee, L. B.; Pang, Plise, E. G.; Pokorny, J. L.; Reslan, H. B.; Sarkaria, J. N.; Wallin, J.
J.; Zhang, X.; Gould, S. E.; Olivero, A. G.; Phillips, H. S. Targeting the PI3K Pathway in the Brain—Efficacy of a PI3K In-
hibitor Optimized to Cross the Blood-Brain Barrier. Clin. Cancer Res. 2012, 18, 6239-6248.
13 Obach, R. S.; Baxter, J. G.; Liston, T. E.; Silber, B. M.; Jones,B. C.; MacIntyre, F.; Rance, D. J.; Wastall, P. The prediction
of humanpharmacokinetic parameters from preclinical and in vitro metabolism data. J. Pharmacol. Exp. Ther. 1997, 283,
46–58.
ACS Paragon Plus Environment

Page 7 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
SYNOPSIS TOC
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment