Synthesis and in vitro and in vivo evaluation of phosphoinositide-3-kinase inhibitors

Article abstract of DOI:10.1021/ml1001932

Phospoinositide-3-kinases (PI3K) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. A series of 2-morpholino, 4-substituted, 6-(3-hydroxyphenyl) pyrimidines have been reported as potent inh

Full text of DOI:10.1021/ml1001932

pubs.acs.org/acsmedchemlett
Synthesis and in Vitro and in Vivo Evaluation
of Phosphoinositide-3-kinase Inhibitors
Matthew T. Burger,* Mark Knapp, Allan Wagman, Zhi-Jie Ni, Thomas Hendrickson,
Gordana Atallah, Yanchen Zhang, Kelly Frazier, Joelle Verhagen, Keith Pfister, Simon Ng,
Aaron Smith, Sarah Bartulis, Hanne Merrit, Marion Weismann, Xiaohua Xin,
Joshua Haznedar, Charles F. Voliva, Ed Iwanowicz, and Sabina Pecchi
Global Discovery Chemistry/Oncology & Exploratory Chemistry, Novartis Institutes for Biomedical Research,
4560 Horton Street, Emeryville, California 94608, United States
ABSTRACT Phospoinositide-3-kinases (PI3K)are important oncology targets due
to the deregulation of this signaling pathway in a wide variety of human cancers.
A series of 2-morpholino, 4-substituted, 6-(3-hydroxyphenyl) pyrimidines have been
reported as potent inhibitors of PI3Ks. Herein, we describe the structure-guided
optimization of these pyrimidines with a focus on replacing the phenol moiety,
while maintaining potent target inhibition and improving in vivo properties. A series of
2-morpholino, 4-substituted, 6-heterocyclic pyrimidines, which potently inhibit
PI3K, were discovered. Within this series a compound, 17, was identified with
suitable pharmacokinetic (PK) properties, which allowed for the establishment of
a PI3K PK/pharmacodynamic-efficacy relationship as determined by in vivo
inhibition of AKTSer473 phosphorylation and tumor growth inhibition in a mouse
A2780 tumor xenograft model.
KEYWORDS phosphoinositide 3-kinase alpha, PI3K/AKT pathway
he phospoinositide-3-kinase (PI3K) family of lipid kinases
is involved in a diverse set of cellular functions, includ-
T
ing cell growth, proliferation, motility, differentiation,
glucose transport, survival intracellular trafficking, and memb-
rane ruffling.1 PI3Ks can be categorized in class I, II, or III,
depending on their subunit structure, regulation, and
substrate selectivity.2 Class IA PI3Ks are activated by
receptor tyrosine kinases and consist of a regulatory
subunit (p85) and a catalytic subunit (p110). There are
three catalytic isoforms: p110 R, β, and δ. A single class IB
PI3K, activated by G protein-coupled receptor, consists of
only one member: a p110 γ catalytic subunit and a p101
regulatory subunit. The primary in vivo substrate of the
class I PI3Ks is phosphatidylinositol (4,5) diphosphate,
which, upon phosphorylation at the 3-position of the
inositol ring to form phosphatidylinositol triphosphate
(3,4,5)P3, serves as a second messenger by activating a series
of downstream effectors that mediate the cellular functions
mentioned above. The PI3K isoforms have different distribu-
tions and share similar cellular functions, which are context
dependent. In particular, p110R pathway deregulation has
been demonstrated in ovarian, breast, colon, and brain
cancers.3,4 Inhibitors of PI3KR represent an intriguing ther-
apeutic modality for these indications, and as such, there is
much interest in generating suitable molecules to test this
hypothesis in the clinic.5-9
Figure 1. PI3KR enzymatic potency and rat PK properties of
6-substituted, 4-(aminopyrid-3-yl), 2-morpholino pyrimidines.
that exhibit high selectivity toward other serine/threonine as
well as tyrosine kinases. While exhibiting potent in vitro
properties, the in vivo potential of such compounds may be
limited due to the presence of the phenol moiety. Described
herein are our efforts to identify potent morpholino pyrimi-
dinyl inhibitors of PI3K that do not require a phenol group
and exhibit PK properties suitable for achieving in vivo target
modulation and efficacy.
The importance of the phenol moiety in 1 for PI3K
binding as well as the phenols effect on in vivo properties
can be seen in contrasting the properties of phenol 1 with
Received Date: August 16, 2010
Accepted Date: October 6, 2010
Published on Web Date: October 13, 2010
We have reported phenolic mopholino pyrimidines,10 such
as compound 1 (Figure 1), as potent pan class I PI3K inhibitors
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the trifluoromethylphenyl analogue 2. The trifluoromethyl-
phenyl analogue 2 is 60-fold less active against PI3KR,
while its rat PK is improved relative to phenol 1 when
considering the % F, area under the curve (AUC), and iv t1/
2. Thus, the challenge for compound optimization that we
faced was to mimic the phenol binding interaction with a
group that would not adversely affect the pharmacokinetic
(PK) properties.
To approach this challenge, we turned to the cocrystal
structure of compound 1 in PI3Kγ to gain an understanding
of the phenol OH's binding interactions.11 Given the high
homology between the R and the γ isoforms and approxi-
mately the same potency of 1 against the two isoforms, p110γ
was used as a surrogate for p110R. The cocrystal structure of
1 in the ATP binding site of PI3Kγ, Figure 2, indicates the key
binding contacts being made by the phenol as well as the
morpholine group. The morpholine oxygen forms a hydro-
gen bond to the hinge Val882 NH. The phenol hydroxyl makes
hydrogen bonds with Asp841 and Tyr867. The C4 aminopyr-
idyl substituent extends out toward solvent and does not
appear to make any specific hydrogen bonds.
Figure 2. Structure of 1 in PI3Kγ.
Scheme 1. Synthesis of 4,6-Substituted 2-Morpholino Pyrimidinesa
With this structural insight, our strategy to identify phenol
replacements was to survey a variety of heterocycles at the
pyrimidine C6 position that would have the ability to make
hydrogen-bonding interactions with the Asp841 and Tyr867
residues, identify such groups, and then profile their PK
properties in rat. In this C6 survey, the morpholine at C2
and a 3-aminoquinoline at C4 were held constant. Upon
identification of a suitable C6 phenol replacement, further
optimization at the C4 position to modulate druglike proper-
ties and maintain potency was envisioned since the cocrys-
tal structure indicated that this position extends out toward
solvent and would tolerate a range of substituents.
a Reagents: (a) H2NR1R2, DIEA, MeCN, 45 °C. (b) Morpholine, 45 °C.
(c) HetB(OR)2, Pd(dppf)Cl2, DME, 2 M Na2CO3, 110 °C. (d) NaOEt, diethyl
malonate, reflux. (e) POCl3, reflux. (f) Amine, Pd(OAc)2, BINAP, Cs2CO3,
THF, 110 °C.
Table 1. Biochemical Inhibition of PI3KR, Inhibition of AKTSer473 Phosphorylation, and Antiproliferative Effect in A2780 Cells by
6-Substituted 2-Morpholinopyrimidin-4-yl)quinolin-3-amines
no.
R
PI3KR IC50 (μM)
pAKTSer473 A2780 EC50 (μM)
A2780 EC50 (μM)
3
4
3-phenol
0.061
0.135
0.055
0.066
0.253
0.231
0.044
0.008
0.774
<0.002
0.007
0.247
0.37
0.65
0.31
0.33
0.33
0.45
3.73
0.31
0.59
0.36
4.84
pyridin-3-yl
5
6-aminopyridin-3-yl
6
1H-pyrazolo[3,4-b]pyridin-5-yl
2-hydroxypyridin-4-yl
pyrazin-2-yl
7
8
0.39
3.30
0.45
9
5-aminopyrazin-2-yl
10
11
12
13
14
pyrimidin-5-yl
2-hydroxypyrimidin-5-yl
2-aminopyrimidin-5-yl
2-methylaminopyrimidin-5-yl
2-dimethylaminopyrimidin-5-yl
0.04
0.11
0.15
0.14
0.67
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C6-modified 2-morpholino pyrimidines III were synthe-
sized byseveral routes as depicted in Scheme 1.12 Sequential
nucleophilic substitution of 2,4,6-tribromopyrimidine, I,
at the 4-position with a range of amines, then at the
2-position by morpholine yielded bromopyrimidine II. Sub-
seqeunt Suzuki reaction yielded target compounds III. Alter-
natively, condensation of morpholine formamidine hydro-
bromide IVand diethylmalonate followed by refluxing in POCl3
yielded 2-morpholino 4,6-dichloropyrimidine V, which could
undergo amination and Suzuki reaction to access target com-
pounds. For preparing a series of C4 analogues with a fixed
C6 heterocycle, the Suzuki step was performed first to yield
chloropyrimidine VI prior to the Buchwald step yielding
target compounds III. When necessary, the heterocyclic
boronate esters used to install the C6 heteroaryl groups were
prepared from the corresponding heteroarylbromides.
Prepared compounds were initially screened in biochem-
ical PI3K assays, and compounds with PI3KR IC50 values
< 100 nM were tested in the A2780 ovarian carcinoma cell
line (where the PI3K pathway is deregulated due to PTEN
deletion) for inhibition of cell proliferation and phosphoryla-
tion of AKTSer473 as a target modulation readout. The results
of the C6 substituent survey, Table 1, indicate that a 3-pyridyl
group 4 is potent, being only 2-fold less active against PI3KR
than the phenol starting point 3. Substituted pyridines that
place hydrogen bond donors ortho to the ring nitrogen were
evaluated, and the aminopyridine 5 exhibits increased potency
relative to the 3-pyridyl compound 4, being equipotent to the
phenol. Introduction of the 5-pyrimidyl increases potency
relative to the 3-pyridyl, with compound 10 being more
potent than the phenol 3. In contrast, pyrazine 8 exhibits
comparable potency to the 3-pyridyl 4. When the 2-amino group
was introduced into the C6 pyrimidine, further potency enhan-
cements were observed with the aminopyrimidine 12 being
g30-fold more active than the starting phenol 3 against PI3KR.
Interestingly, the pyridone 7, which mimics the meta orien-
tation of phenol 3, exhibited reduced activity relative to 3, high-
lighting the subtleties of trying to mimic a phenolic interaction.
Upon identification of phenol replacements with main-
tained (aminopyridine) or improved (aminopyrimidine) in
vitro potency, the aminopyridine- and aminopyrimidine-
containing PI3K inhibitors 4, 5, and 12 were assessed in
rat PK. As indicated in Table 2, the PK properties were
improved relative to the phenol 3. The % F was increased
from 1% to acceptable levels (23-56%), and the clearance
value was reduced substantially. The improvement was great-
est for aminopyridine 5 with 56% F, CL=5 mL/min/kg, and
iv t1/2=103 min. The comparison of PK properties between
the phenolic compounds 1 (Figure 1) and 3 (Table 2), where
the % F went from 9 to 1%, indicated that PK properties with
a fixed C6 group could be modulated by the C4 substituent
and suggested that the PK properties of the C6 aminopyr-
idine or aminopyrimidines could be further improved by
optimization of the C4 substituent.
With a phenol replacement that improved both in vitro
and in vivo properties identified, a C4 survey where the
2-morpholinyl and 6-(5-substituted-2-aminopyrimididyl)
groups of the central pyrimidine were held fixed was con-
ducted. As may be expected from the cocrystal structure of
compound 1 with PI3Kγ (Figure 2), a variety of groups are
tolerated at the C4 position (Table 3). The aminoquinoline 12
as well as the 6-substituted 3-aminopyridyl-substituted com-
pounds 15-17 were extremely potent, being active at the limit
of detection of the enzymatic assay. Additionally, of note is
the potency and ligand efficiency13 of the 4-H-substituted 20
(ΔG = -0.56 kcal mol-1 per non-H atom!), which inhibited
PI3KRwith an IC50=14 nm, A2780 pAKT473 EC50=132 nM,
and A2780 EC50 = 5 μM. Additionally, the C2 symmetric
bis aminopyrimidine-substituted morpholino pyrimidine 18
was potent, IC50 = 6 nm and A2780 EC50 = 0.23 μM.
Table 2. Rat PK Propertiesa of Phenol Replacements
iv t1/2
(min)
CL
oral AUC
(μM h)
no.
(mL/min/kg)
V
ss (L/kg)
po % F
3
4
30
55
179
26
5
0.03
16
6.9
2.4
0.8
1.9
1
46
56
23
5
103
43
93
12
37
6
a Amounts: 5 mpk iv and 20 mpk po.
Table 3. Biochemical Inhibition of PI3KR, Inhibition of AKTSer473 Phosphorylation, and Antiproliferative Effect in A2780 Cells by
4-Substituted, 2-Morpholino, 6-(2-Aminopyrimid-5-yl) Pyrimidines
no.
R
PI3KR IC50 (μM)
pAKTSer473 A2780 EC50 (μM)
A2780 EC50 (μM)
12
15
16
17
18
19
20
quinolin-3-yl-amino
<0.002
<0.002
<0.002
<0.002
0.006
0.04
0.11
0.01
0.09
0.15
1.13
0.14
0.73
0.23
1.96
4.98
6-phenoxypyridin-3-yl-amino
6-(1-methylpiperidin-4-yloxy)pyridin-3-yl-amino
6-methoxypyridin-3-yl-amino
2-amino-pyrimid-5-yl
tetrahydro-2H-pyran-4-yl-amino
H
0.013
0.22
0.13
0.014
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PI3K activity, as evidenced by the modulation of phoshpory-
lation of AKTSer473 and tumor growth inhibition in A2780
tumor-bearing mice, has been described. Compounds from
this series that inhibit PI3K in vitro, have a more pronounced
effect on the phosphorylation of AKTSer473 in vivo and show
enhanced efficacy in PI3K-driven tumor models will be
reported in due course.
SUPPORTING INFORMATION AVAILABLE Experimental
details for the synthesis and characterization of all compounds,
biological assay, and pharmacology model procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
Figure 3. PKPD relationship of compound 17 in the A2780
xenograft model.
AUTHOR INFORMATION
Corresponding Author: *Tel: 510-923-3537. Fax: 510-923-
3360. E-mail: matthew.burger@novartis.com.
REFERENCES
(1)
Engelman, J. A.; Luo, J.; Cantley, L. C. The evolution of
phosphatidylinositol 3-kinases as regulators of growth and
metabolism. Nat. Rev. Genet. 2006, 7, 606–619.
(2)
Katso, R.; Okkenhaug, K.; Ahmandi, K; White, S.; Timms, J.;
et al. Cellular function of phosphoinositide 3-kinases: Impli-
cations for development, homeostasis and cancer. Annu. Rev.
Cell Dev. Biol. 2000, 17, 615–675.
(3)
Samuels, Y.; Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.;
Szabo, S.; Yan, H.; Gasdar, A.; Powell, S. M.; Riggins, G. J.;
Willson, J. K.; Markowitz, S.; Kinzler, K. W.; Vogelstein, B.;
Velculescu, V. E. High frequency of mutations of the PIK3CA
gene in human cancers. Science 2004, 304, 554.
Leslie, N. R.; Downes, C. P. PTEN function: how normal cells
control it and tumour cells lose it. Biochem. J. 2004, 382, 1–11 .
Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Targeting the phos-
phoinositide 3-kinase pathway in cancer. Nat. Rev. Drug
Discovery 2009, 8, 627–644.
Figure 4. Efficacy of compound 17 in the A2780 xenograft model.
Compounds with a positive combination of enzyme inhi-
bition, cell target modulation, antiproliferative activity, and
solubility were profiled further in rat PK studies. One such
compound, 17,14 exhibited reasonable rat PK (5 mpk iv,
20 mpk oral, oral t1/2 =77 min, % F=89, CL=79 mL/min/kg,
Vss = 2.6 L/kg, and oral AUC = 9 μM h) and was studied
further in mouse PK/PD and efficacy studies.15 Modulation
of AKTThr308 and AKTSer473 phosphorylation was examined
in A2780 xenograft tumors at time points ranging from
30 min to 24 h after a single 100 mg/kg dose of compound 17.
As can be seen in Figure 3, at 8 h, >50% of target inhibition
was achieved. The target modulation decreased as the com-
pound exposure decreased, with the modulation approach-
ing the vehicle level at 24 h.
Efficacy experiments were then conducted in the A2780
tumor xenograft model, where tumor-bearing mice were
administered compound 17 twice daily at 10 and 60 mg/kg.
Tumor growth inhibition (50%) was observed at the 60 mg/kg
dose level, while at 10 mg/kg, no inhibitory activity was obser-
ved (Figure 4).
While the in vivo antiproliferative effect of compound 17
did not result in complete stasis or regression, the data
support the notion that inhibition of PI3K and phosporyla-
tion of AKTSer473 in vivo with a compound from this series
has an effect on tumor growth.
In summary, the structure-guided evolution of a series of in
vitro potent 6-phenolic, 4-substituted, 2-morpholinopyrimi-
dinyl PI3K inhibitors lacking suitable properties for in vivo
activity into a series of 6-heterocyclic, 4-substituted, 2-mor-
pholino pyrimidines with properties sufficient for in vivo
(4)
(5)
(6)
(7)
Nuss, J. M.; Tsuhako, A. L.; Anand, N. K. In Annual Reports in
Medicinal Chemistry; Macor, J. E., Ed.; Academic Press: Ox-
ford, 2009; Vol. 44, pp 339-351.
Maira, S.-M.; Stauffer, F.; Brueggen, J.; Furet, P.; Schnell, C.;
Fritsch, C.; Brachmann, S.; Chene, P.; De Pover, A.; Schoemaker,
K.; Fabbro, D.; Gabriel, D.; Simonen, M.; Murphy, L.; Finan, P.;
Sellers, W.; Garcia-Echeverria, C. Identification and character-
ization of NVP-BEZ235, a new orally available dual phosphati-
dylinositol 3-kinase/mammalian target of rapamycin inhibitor
with potent in vivo antitumor activity. Mol. Cancer Ther. 2008, 7,
1851–1863.
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. The Identification of
2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-
4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a
Potent, Selective, Orally Bioavailable Inhibitor of Class I PI3
Kinase for the Treatment of Cancer. J. Med. Chem. 2008, 51,
5522–5532.
(8)
(9)
Knight, S. D.; Adams, N. D.; Burgess, J. L.; Chaudhari, A. M.;
Darcy, M. G.; Donatelli, C. A.; Luengo, J. E.; Newlander, K. A.;
r
2010 American Chemical Society
37
DOI: 10.1021/ml1001932 ACS Med. Chem. Lett. 2011, 2, 34–38
|

pubs.acs.org/acsmedchemlett
Parrish, C. A.; Ridgers, L. H.; Sarpong, M. A.; Schmidt, S. J.;
Van Aller, G. S.; Carson, J. D.; Diamond, M. A.; Elkins, P. A.;
Gardiner, C. M.; Garver, E.; Gilbert, S. A.; Gontarek, R. R.;
Jackson, J. R.; Kershner, K. L.; Luo, L.; Raha, K.; Sherk, C. S.;
Sung, C. M.; Sutton, D.; Tummino, P. J.; Wegrzyn, R. J.; Auger,
K. R.; Dhanak, D. Discovery of GSK2126458, a Highly Potent
Inhibitor of PI3K and the Mammalian Target of Rapamycin.
ACS Med. Chem. Lett. 2010, 1, 39–43.
(10) Pecchi, S.; Renhowe, P. A.; Taylor, C.; Kaufman, S.; Merrit, H.;
Wiesmann, M.; Shoemaker, K.; Knapp, M. S.; Hendrickson,
T. F.; Fantl, W.; Voliva, C. F. Identification and structure-
activity relationship of 2-morpholino 6-(3-hydroxyphenyl)-
pyrimidines, a class of potent and selective PI3-kinase inhib-
itors. Bioorg. Med. Chem. Lett. 2010, accepted for publication.
(11) The structure of 1 in PI3Kγ has been deposited in the RCSB
Protein Data Bank under the accession code 3P2B.
(12) Burger, M.; Ni, Z.-J.; Pecchi, S. Atallah, G.; Bartulis, S.; Frazier,
K.; Smith, A.; Verhagen, J.; Zhang, Y.; Wagman, A.; Ng, S.;
Pfister, K.; Poon, D.; Louie, A.; Pick, Y.; Barsanti, P.; Iwanowicz,
E.; Fantl, W.; Hendrickson, T.; Knapp, M.; Merritt, H.; Voliva, C.;
Wiesmann, M.; Xin, X. Pyrimidine derivatives used as PI3K
inhibitors. WO2007/084786 A1.
(13) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency; a
useful metric for lead selection. Drug Discovery Today 2004,
9, 430–431.
(14) Compound 17 is a pan class 1 PI3K inhibitor (PI3K R, β, γ,
and δ IC50 values = 0.001, 0.092, 0.009, and 0.020 μM;
PI4Kβ, mTOR, and VPS34 IC50 values = 5, 4, and >9 μM;
pAKTThr308 A2780 EC50 =0.6 μM.
(15) Mouse PK parameters at 5 mpk iv and 10 mpk po; oral t1/2
=
282 min, % F=87, and CL=99 mL/min/kg. Vss =1.1 L/kg,
and oral AUC=2 μM h.
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