Discovery of NVP-BYL719 a potent and selective phosphatidylinositol-3 kinase alpha inhibitor selected for clinical evaluation

Article abstract of DOI:10.1016/j.bmcl.2013.05.007

Phosphatidylinositol-3-kinase α (PI3Kα) is a therapeutic target of high interest in anticancer drug research. On the basis of a binding model rationalizing the high selectivity and potency of a particular series of 2-aminothiazole compounds in inhibiting PI3Kα, a medicinal chemistry program has led to the discovery of the clinical candidate NVP-BYL719.

Full text of DOI:10.1016/j.bmcl.2013.05.007

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3741–3748
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of NVP-BYL719 a potent and selective
phosphatidylinositol-3 kinase alpha inhibitor selected
for clinical evaluation
Pascal Furet, Vito Guagnano, Robin A. Fairhurst, Patricia Imbach-Weese, Ian Bruce, Mark Knapp,
Christine Fritsch, Francesca Blasco, Joachim Blanz, Reiner Aichholz, Jacques Hamon, Doriano Fabbro,
⇑
Giorgio Caravatti
Novartis Institutes for BioMedical Research, WKL-136.4.12, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphatidylinositol-3-kinase
research. On the basis of a binding model rationalizing the high selectivity and potency of a particular
series of 2-aminothiazole compounds in inhibiting PI3K
the discovery of the clinical candidate NVP-BYL719.
a (PI3Ka) is a therapeutic target of high interest in anticancer drug
Received 25 March 2013
Revised 30 April 2013
Accepted 4 May 2013
Available online 14 May 2013
a, a medicinal chemistry program has led to
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
PI3K inhibitors
Antitumor agent
Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that are
important in controlling signaling pathways involved in cell prolif-
eration, motility, cell death and cell invasion.1,2 Human cells con-
tain three genes (PIK3CA, PIK3CB and PIK3CD) encoding the
Indeed, several low molecular weight compounds are under active
clinical development, including pan-PI3K inhibitors such as GDC-
0941,17 XL-147,18 BKM120,19 ZSTK-474,20 and CH-513279921 and
p110
form-specific inhibitors may exhibit anti-cancer activity in PI3Ka
mutant tumors without causing the potential side effects that
could be expected from interference with the other isoforms.
Here we report about the medicinal chemistry aspects of the
a a iso-
isoform-specific inhibitors such as INK-1117.18 P110
catalytic subunits of class IA PI3K enzymes, termed p110
and p110d. P110 and p110b are expressed in most tissues,
whereas p110d is expressed primarily in leukocytes. The class IB
PI3K consists of only one enzyme, PI3K . Its catalytic subunit,
p110 , is encoded by PIK3CG and is also expressed primarily in
leukocytes.
a
, p110b
a
c
c
discovery of NVP-BYL719, an a-specific PI3K inhibitor from the
2-aminothiazole class which entered clinical trials in 2010.
It has previously been reported that the 2-aminothiazole scaf-
fold is a valuable template to obtain PI3K inhibitors showing iso-
form selectivity.22 In particular, it was found that attaching a (S)-
pyrrolidine carboxamide moiety to the 2-amino group through an
Dysregulation of the PI3K signaling pathway is implicated in
many human cancers3,4 and includes the inactivation of the PTEN
tumor suppressor gene,5 amplification/overexpression or activat-
ing mutations of some receptor tyrosine kinases (e.g., erbB3, erbB2,
EGFR), and amplification of genomic regions containing AKT or
PIK3CA genes.6–8 In addition, it was found that PIK3CA is somati-
cally mutated in many human cancers, for example, in 32% of colo-
rectal cancers,9 27% of glioblastomas,9,10 25% of gastric cancers,9–11
36% of hepatocellular carcinomas,12 and 18–40% of breast can-
cers.13–16 From these mutation frequencies, PIK3CA is one of the
two most commonly mutated genes identified in human cancers.
No mutations of PIK3CB, PIK3CD, and PIK3CG have been identified.
urea linkage confers selectivity for the
PI3K inhibitors as exemplified by compound 1 (Fig. 1).22 We were
interested to optimize this type of selective PI3K inhibitors to-
a isoform in this class of
a
wards compounds suitable for pharmacological studies in animal
tumor models. Among the different potent analogs of 1 available
at the onset of the program, compound 2 (Table 1) was selected
as the starting point of this effort. To guide the optimization pro-
cess, we decided to construct a binding model of 2 in the ATP
As most p110
a
mutations constitutively activate its kinase
pocket of the kinase domain of PI3K
crystal structure of the human p110
by Huang et al.,23 the only PI3K
structure that was available at
a
a
. To this end we used the
activity, PI3K
a
appears to be an ideal target for drug development.
/p85 complex reported
a
a
the time we initiated the work reported in this Letter. We
docked24 2 in the unliganded ATP pocket of the kinase domain
⇑
Corresponding author. Tel.: +41 61 696 5844; fax: +41 61 696 2455.
E-mail address: giorgio.caravatti@novartis.com (G. Caravatti).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.007

3742
P. Furet et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3741–3748
co-crystal structure with PI3Kc 25
which the thiazole nitrogen and the 2-NH group of 2 form biden-
tate hydrogen bonds with the backbone NH and carbonyl of PI3K
residue V851 while its pyrimidine moiety sits in the less solvent
accessible part of the cavity, the so called affinity pocket, formed
by residues Y836, I932, I848, I800, D933, K802, P778 and M772.
Placing the inhibitor in the cavity in such a way immediately pro-
.
This implied an orientation in
H
H
N
N
N
N
N
O
S
S
a
O
O
NH2
Cl
N
N
S
O
NH
1
PIK-93
O
vided a hypothesis for the structural basis of the
a isoform selec-
tivity of the 2-aminothiazole inhibitors carrying the (S)-
pyrrolidine carboxamide urea moiety. As shown in Figure 2 this
binding mode allows the amide group of the latter to form three
HO
a
selective 2-aminothiazole derivative 122
Figure 1. Chemical structures of PI3K
and PIK-93.
hydrogen bonds with PI3Ka, exploiting the full potential of a pri-
mary amide group for such interactions. In one of them the inhib-
itor amide nitrogen is a donor for the backbone carbonyl of
residue S854 while the two others involve both the amide car-
bonyl and nitrogen that engage in donor–acceptor interactions
of this structure with the same binding mode as that of a related
unselective 2-aminothiazole PI3K inhibitor, PIK-93, observed in a
Table 1
Inhibition of p110
a
, p110b, p110d and p110
c
activity (biochemical assay), in vitro metabolic clearance using rat liver microsomes and CYP450 3A4 inhibition
H
N
N
R2
5
R3
S
O
4
6
3
X
N
2
1
R1
a
Compound
R1
X
R2
R3
IC50
(lM)
Microsomal CL, rat (
l
L minÀ1 mgÀ1
)
CYP450 3A4 IC50 (lM)
P110a
P110b
P110d
P110c
N
2
3
2-tert-Bu
2-tert-Bu
N
CH3
CH3
0.0075
0.014
1.8
0.35
0.21
62b
77b
>10
>10
O
N
NH2
NH2
NH
CH
4.4
0.33
4.2
0.43
4.6
O
N
4
2-tert-Bu
CH
CH3
2.7
>9.1
104b
n.d.
O
5
6
2-tert-Bu
2-tert-Bu
CH
CH
CH3
CH3
0.62
>9.1
4.9
8.2
0.59
3.3
0.93
>10
186b
36b
n.d.
n.d.
N
N
O
N
NH2
7
2-tert-Bu
N
CH3
CH3
CH3
CH3
0.019
0.005
0.019
0.016
1.2
1.2
4.1
4.0
0.29
0.29
2.0
0.32
0.25
1.1
44
29
41
36
n.d.
>10
>10
>10
NH2
O
N
2-
8
CH
CH
N
CF3
O
N
NH2
NH2
NH2
9
2-
CN
O
N
10
2-iso-Pr
0.96
1.4
O

P. Furet et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3741–3748
3743
M)
Table 1 (continued)
a
Compound
R1
X
R2
R3
IC50
(lM)
Microsomal CL, rat (
l
L minÀ1 mgÀ1
)
CYP450 3A4 IC50 (l
P110
a
P110b
P110d
P110c
N
11
12
21
22
27
28
2-cyclo-Bu
6-iso-Pr
N
CH3
CH3
H
0.020
0.32
1.8
>9.1
3.9
5.5
1.2
3.1
0.69
0.61
53
n.d.
27
17
96
47
>10
n.d.
>10
n.d.
n.d.
>10
O
N
NH2
NH2
NH2
NH2
NH2
NH2
N
n.d.
1.1
n.d.
0.83
1.4
O
N
2-
CH
CH
N
0.0095
0.039
0.016
0.012
CF3
O
N
2-
Cl
0.43
0.34
0.81
CF3
O
N
2-Diethylamino
CH3
CH3
0.41
0.69
O
N
2-
N
O
a
The values are averages of at least two separate determinations.27
Includes the cofactor for glucuronyl transferases UDPGA.
b
Figure 2. Proposed binding mode of compound 2 in the ATP pocket of PI3Ka. Hydrogen bonds are represented as dashed lines.
with the side chain amide group of residue Q859. The former
hydrogen bond, being made with the backbone of the protein,
can also exist in the other PI3K isoforms. In contrast, residue
Q859 is not conserved within the PI3K family. Isoforms b, d and
mary amide group of the inhibitor as a glutamine. In principle,
the side chain of the d isoform asparagine could form such inter-
actions. However, a simple modeling experiment in which Q859
was mutated into an asparagine indicated that the shorter side
chain of this amino acid did not allow the donor–acceptor hydro-
gen bonds to be formed without compromising the other favor-
able interactions of the inhibitor with the ATP pocket. The
c
have an aspartic acid, an asparagine and a lysine residue,
respectively, at this position. The aspartic acid and lysine residues
of the b and isoforms are obviously not able to establish the
same hydrogen bond donor–acceptor interactions with the pri-
c
docking model thus strongly suggested that the PI3K
a selectivity

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P. Furet et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3741–3748
of 2 and its analogs originated in the formation of two stabilizing
specific hydrogen bonds with the side chain of Q859, interactions
that are not possible with the other isoforms.
the iso-propyl or cyclo-butyl group (compounds 10 and 11, respec-
tively) at this position were slightly less active than the latter in
inhibiting PI3Ka. Replacement of the tert-butyl group in 2 by the
The model was used to design modifications of 2 aimed at mod-
ulating the compound physico-chemical properties while preserv-
ing its high potency. For instance, following the observation that
in the model, the pyrimidine N3 nitrogen of the inhibitor did not
make any polar interaction with the ATP pocket, we expected no sig-
nificant loss of activity by replacing it by a carbon atom. Indeed, the
resulting pyridine analog 3 turned out to be as potent in inhibiting
slightly larger diethylamino group (compound 27) did not lead to
an improvement in activity likely due to the different shape of this
substituent.28
The model could also explain the significant loss of activity ob-
served with the 6-iso-propyl pyrimidine isomer (compound 12) of
10. Assuming the same binding mode for this compound orients its
pyrimidine N3 nitrogen towards the hydrophobic wall of the cavity
in the region corresponding to the side chain of Y836 where it is
unable to form a hydrogen bond compensating for the solvation
energy lost upon binding.
PI3K
alterations in the pyrrolidine carboxamide moiety were then envis-
aged to probe our PI3K selectivity concept. Methylation of the
a
as 2 with the same selectivity profile.26 Analogues of 3 with
a
amide group (compound 4), its removal (compound 5) or inversion
of the stereochemistry (compound 6) resulted in unselective micro-
molar inhibitors, a consequence of dramatic losses of PI3Ka inhibi-
tory activity. These results gave strong support to the postulated
The synthetic routes to prepare the 2-aminothiazole derivatives
are outlined in Schemes 1 and 2. In the synthesis of the 5-(4-pyrid-
inyl) substituted derivatives the key step was the palladium-cata-
lyzed direct arylation of 4-methyl-2-acetaminothiazole with a 4-
bromopyridine following a method developed in the group of
Miura.29 This is exemplified in Scheme 1 for the synthesis of 8.
The required 4-bromo-2-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-pyri-
bidentate hydrogen bonds with Q859 as the structural determinant
of PI3K
ment of the prolineamide moiety in 2 by the corresponding azeti-
dine derivative led to a slight decrease of PI3K inhibition while
the level of PI3Kb, PI3Kd and PI3K inhibition was maintained (com-
a selectivity in this class of inhibitors. Interestingly, replace-
a
dine 17 was prepared in two steps from the c-pyrone 15 which
c
in turn was prepared following an analogous procedure developed
independently by Koreeda and Morgan.30,31 After the direct aryla-
tion reaction, deprotection of the acetaminothiazole under acidic
conditions was followed by the introduction of the prolineamide
urea function in two steps via the imidazolide 20 to give the de-
sired compound 8. Treatment of 4-methyl-2-acetaminothiazole
with appropriate 4-bromopyridine derivatives followed by prolin-
eamide urea formation provided compounds 3, 6, and 9 (Table 1).
Similarly, reaction of corresponding imidazolides with proline N-
methylcarboxamide or pyrrolidine gave rise to the products 4
and 5, respectively. Compounds 21 and 22 were prepared by using
the reaction sequence shown in Scheme 1 but coupling 4-bromo-
pyridine derivative 17 with 2-acetaminothiazole or 4-chloro-2-
acetaminothiazole, respectively.
In the 5-(4-pyrimidinyl)-substituted aminothiazole series the
key step was the build-up of the pyrimidine ring by reacting an
appropriate amidine or guanidine derivative with the dimethyl-
amino-vinyl ketone 24. This is exemplified in Scheme 2 for the syn-
thesis of compound 10. As in the pyridine series, the prolineamide
urea function was introduced in two steps to produce compounds
2, 11, 12, 27 and 28. The azetidine analog 7 was prepared by react-
ing the corresponding imidazolide with azetidine 2-carboxamide.
pound 7). According to the binding model, this effect could be as-
cribed to a loss of one favorable van der Waals contact with the
imidazole ring of the side chain of PI3K
reducing the ring size to four atoms. In contrast, PI3Kb, PI3Kd and
PI3K , having respectively, a glutamic acid, an aspartic acid and a
a residue H855 caused by
c
threonine residue at the corresponding position in their sequence,
cannot form such an interaction with the prolineamide moiety.
Another example of modification inspired by the binding model
was the replacement of one of the methyls of the tert-butyl group
of 2 or 3 by a trifluoromethyl or a cyano substituent. As shown in
Figure 3, the model suggested that the tert-butyl group did not
fully occupy the space available in a small cavity formed by the
side chains of residues I800, I848, P778 and K802. In the direction
of one of the methyls there was some space left allowing the
accommodation of a slightly larger group, such as a trifluoromethyl
one, in the small cavity. Another of the tert-butyl methyls was
pointing towards the amino group of the side chain of K802. This
led to the idea of replacing it by a cyano group targeting K802
for hydrogen bonding. Consistent with these notions, the resulting
analogs of 3, 8 and 9, showed potent and selective inhibition of
PI3Ka while the analogs of 2 having smaller substituents such as
Figure 3. Detailed view of the binding model of 2 showing a small cavity formed by residues P778, I800, K802, and I848 proximal to the tert-butyl group of the compound.

P. Furet et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3741–3748
3745
O
O
Br
N
CF3
CF3
c
d
a
b
O
O
CF3
CF3
CF3
O
N
H
OH
13
Cl
14
15
S
16
17
N
H
N
H
N
H
N
N
N
N
NH2
N
N
N
O
S
S
S
O
O
O
e
g
f
h
NH2
N
N
N
N
CF3
19
CF3
18
CF3
CF3
20
8
Scheme 1. Reagents and conditions: (a) Oxalyl chloride, chloroform, reflux, 4 h, quant; (b) (i) (E)-4-methoxy-3-buten-2-one and LiHMDS, THF, À78 °C, 1 h, then solution of
the acid chloride in THF was added at À78 °C then in 2.5 h to rt, (ii) TFA, toluene, À10 °C to rt, 55%; (c) aqueous ammonia, 65 °C, 1 h, 65%. (d) POBr3, 1,2-DCE, 85 °C, 1 h, 51%;
(e) 4-methyl-2-acetamidothiazole, Pd(OAc)2/tBu3PÁHBF4, Cs2CO3, DMF, 120 °C, 2 h, 88%; (f) 6 N HCl, EtOH, 85 °C, 1 h, 95%; (g) CDI, DCM, reflux, 15 h, 83%; (h)
L-prolineamide,
Et3N, DMF, rt, 15 h, 87%.
O
N
N
N
N
S
O
N
N
N
NH2
a
b
c
N
NH2
N
H
S
S
S
N
N
O
N
N
N
23
24
25
26
O
N
N
d
N
H
S
N
N
O
NH2
10
Scheme 2. Reagents and conditions: (a) DMF–DMA, reflux, 15 h, 32%; (b) iso-butyramidine hydrochloride, NaOH, 2-methoxyethanol, 125 °C, 1 h, 50%; (c) CDI, DMF, 80 °C,
15 h, 39%; (d) -prolineamide, Et3N, DMF, 40 °C, 15 h, 84%.
L
Table 2
Encouraged by the biochemical results, the compounds were
also tested in cellular assays measuring their ability to block the
PI3K/Akt signaling pathway (Table 2).32 The same trends in po-
tency and selectivity as in the biochemical assays were observed.
In particular, compounds 2, 3 and 8 were able to produce a potent
Inhibition of Akt phosphorylation in Rat1-myr-p110x cells
Compound
IC50 (lM)
Rat1-myr-
Rat1-myr-
p110b
Rat1-myr-
p110d
p110a
double digit nanomolar inhibition of PI3K
activation.
a-dependent Akt
1
2
3
4
5
6
7
8
0.13
2.1
3.1
8.0
>10
>10
n.d.
6.4
2.2
>10
5.9
7.3
>10
2.4
>10
4.4
9.6
0.62
1.5
0.72
5.8
0.77
n.d.
1.6
1.2
>10
1.3
>10
>10
2.9
0.039
0.061
>10
2.5
n.d
0.23
0.074
0.64
0.10
0.44
4.6
0.14
0.34
0.17
0.15
An assessment of the metabolic stability of compounds 2 and 3
was performed by incubation of these compounds with rat liver
microsomes. This revealed two main metabolic pathways: hydro-
lysis of the primary amide to the carboxylic acid and aliphatic
hydroxylation of either the tert-butyl group or the methyl substitu-
ent in 4-position of the thiazole ring (Fig. 4). With the aim to gain
potency as previously mentioned but also to block one of the iden-
tified metabolic pathways, one of the methyls of the tert-butyl
group was replaced by a trifluoromethyl substituent as exemplified
by compounds 8 and 21. This led to a significantly reduced in vitro
clearance compared to compound 3 (Table 1). When in addition
the 4-methyl group of the amino-thiazole in 8 was replaced by a
chlorine atom, the clearance dropped even further (compound
22). These findings indicate that both groups, the tert-butyl and
the 4-methyl group, are major sites of metabolism in vitro for 3.
Replacement of the tert-butyl group by a 1-methyl-cyclopropyl
one (compound 28 vs compound 2) also gave a protective effect,
although slightly lower than the trifluoromethyl substitution.
9
10
11
12
21
22
27
28
4.9
2.5
3.0
However, when incubated with rat liver microsomes, compound
28 led to the formation of reactive intermediates that could be
trapped by glutathione.

3746
P. Furet et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3741–3748
H
N
H
H
N
HO
N
N
N
N
N
O
N
N
O
S
S
S
O
O
O
NH2
NH2
OH
O
X
X
X
N
N
N
Figure 4. Main metabolic pathways of compounds 2 and 3 (X = N and X = CH, respectively) using rat liver microsomes.
Table 3
Pharmacokinetic parameters in female Sprague Dawley rats at 1 mg/kg iv, solution formulation NMP/PEG200 (30:70)
Compound
t½ (h)
CL in vivo/in vitro (mL minÀ1 kgÀ1
)
Vss (L/kg)
PPBb (%)
N
H
N
S
N
O
2
O
1.2 0.1
21 2/62a
1.2 0.1
94.0
N
N
N
NH2
N
H
N
S
N
O
28
1.7 0.0
3.4 1.6
2.9 0.2
8
1/47
0.9 0.1
1.4 0.2
1.9 0.1
97.1
N
N
O
NH2
N
H
N
S
N
3
O
39 9/77a
n.d.
O
NH2
N
H
N
S
N
O
8
10 1/29
94.3
N
O
NH2
CF3
a
Includes the cofactor for glucuronyl transferases UDPGA.
Plasma protein binding (%) in rat plasma.
b
Table 4
Bioavailability of 8 (NVP-BYL719) in mice, rats, and dogs
In a next step, the pharmacokinetic parameters of selected com-
pounds were assessed in rats (Table 3). As shown by the data in Ta-
bles 1 and 3 there was a reasonable correlation between in vitro
and in vivo clearance. For both sets of compounds (2 vs 28 and 3
vs 8) the modification of the tert-butyl group led to a significantly
reduced in vivo clearance. The half-life and volume of distribution
of the compounds with the lower clearance 8 and 28 were moder-
ate. In addition, compound 8 displayed excellent oral bioavailabil-
ity in rats, mice and dogs (Table 4) and did not show any significant
inhibition of the CYP450 enzymes. Moreover, it had no activity
against the class III lipid kinase family member Vps34 and the re-
lated class IV PIKK protein kinases mTOR, DNA-PK and ATR in bio-
Species
Doses
BAV (%)
CL in vivo
Cmax p.o. dose
(mL minÀ1 kgÀ1
)
normal (lM)
Mouse
Rat
1 mg/kg iva
106
58
8
10
8
0.52
3 mg/kg p.o.b
3.4 mg/kg ivc
15 mg/kg p.o.d
0.1 mg/kg iva
0.6 mg/kg p.o.b
0.19
1.07
Dog
140
a
b
c
Solution in NMP:PEG200 (30:70).
Solution in NMP:PEG300:SolutolHS15:water (10:30:20:40).
Solution in PEG200/phosphate buffer pH 7.4 (2/1, v/v).
Suspension in methylcellulose.
d
chemical assays (IC50 >9.1
involved in DNA-damage processes in cell-based assays (IC50
>10 M on S15P-p53 and S1981P-ATM). The selectivity of com-
lM) and did not interfere with PIKKs
l
pound 8 was also assessed against 442 kinases in different kinase
panels (in-house, Invitrogen, Ambit). Overall, there was a higher
tested and most of them were not inhibited at all at concentrations
up to 10
M.33
than 50-fold selectivity window for p110
a against all kinases
l

P. Furet et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3741–3748
3747
Figure 5. Crystal structure of PI3K
a
in complex with 8. Interactions of the compound with the ATP binding pocket. Hydrogen bonds are represented as dashed lines.
5. Sansal, I.; Sellers, W. R. J. Clin. Oncol. 2004, 22, 2954.
Based on its overall favorable profile compound 8 was selected
for in vivo antitumor efficacy studies in nude mice where it
showed dose-dependent inhibition of tumor growth. Its anti-tu-
mor response in PIK3CA-dependent tumor models ranged from tu-
mor stasis to tumor regression and the treatments were well
tolerated by the animals.33
6. Cheng, J. Q.; Godwin, A. K.; Bellacosa, A.; Taguchi, T.; Franke, T. F.; Hamilton, T.
C.; Tsichlis, P. N.; Testa, J. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9267.
7. Cheng, J. Q.; Ruggeri, B.; Klein, W. M.; Sonoda, G.; Altomare, D. A.; Watson, D.
K.; Testa, J. R. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3636.
8. Shayesteh, L.; Lu, Y.; Kuo, W. L.; Baldocchi, R.; Godfrey, T.; Collins, C.; Pinkel, D.;
Powell, B.; Mills, G. B.; Gray, J. W. Nat. Genet. 1999, 21, 99.
9. Samuels, Y.; Velculescu, V. E. Cell Cycle 2004, 3, 1221.
10. Hartmann, C.; Bartels, G.; Gehlaar, C.; Hotkamp, N.; von Deimling, A. Acta
Neuropathol. 2005, 109, 639.
11. Li, V. S.; Wong, C. W.; Chan, T. L.; Chan, A. S.; Zhao, W.; Chu, K. M.; So, S.; Chen,
X.; Yuen, S. T.; Leung, S. Y. BMC Cancer 2005, 5, 29.
The binding model could be validated by the determination of
the crystal structure of PI3Ka in complex with compound 8 at
2.2 Å resolution.34 The co-crystal structure confirmed the existence
of all the interactions inferred from docking 2 in the ATP pocket of
the apo structure of PI3Ka. In particular, the pair of donor–acceptor
hydrogen bonds between the inhibitor amide group and the side
chain of Q859 was observed, fully backing the proposed structural
12. Lee, J. W.; Soung, Y. H.; Kim, S. Y.; Lee, H. W.; Park, W. S.; Nam, S. W.; Kim, S. H.;
Lee, J. Y.; Yoo, N. J.; Lee, S. H. Oncogene 2005, 24, 1477.
13. Bachman, K. E.; Argani, P.; Samuels, Y.; Silliman, N.; Ptak, J.; Szabo, S.; Konishi,
H.; Karabas, B.; Blair, B. G.; Lin, C.; Peters, B. A.; Velculescu, V. E.; Park, B. H.
Cancer Biol. Ther. 2004, 3, 772.
14. Campbell, I. G.; Russell, S. E.; Choong, D. Y.; Motgomery, K. G.; Ciavarella, M. L.;
Hooi, C. S.; Cristiano, B. E.; Pearson, R. B.; Phillips, W. A. Cancer Res. 2004, 64,
7678.
15. Levine, D. A.; Bogomolniy, F.; Yee, C. J.; Lash, A.; Barakat, R. R.; Borgen, P. I.;
Boyd, J. Clin. Cancer Res. 2005, 11, 2875.
16. Wu, G.; Xing, M.; Mambo, E.; Huang, X.; Liu, X.; Guo, Z.; Chatterjee, A.;
Goldenberg, D.; Gollin, S. M.; Sukumar, S.; Trink, B.; Sidransky, D. Breast Cancer
Res. 2005, 7, R609.
PI3Ka selectivity concept. In addition, the X-ray structure revealed
that the pyridine nitrogen atom of 8 is part of a hydrogen bond net-
work involving three water molecules and the side chains of resi-
dues Y836, D810, D933 and K802, the latter residue also making
a hydrogen bond with one of the fluorine atoms of the trifluoro-
methyl group. This is illustrated in Figure 5.
17. Folkes, A. J.; Ahmadi, K.; Alderton, W. K.; Alix, S.; Baker, S. J.; Box, G.;
Chuckowree, I. S., et al J. Med. Chem. 2008, 51, 5522.
In summary, with compound 8 (NVP-BYL719) we have discov-
ered a potent and selective PI3Ka inhibitor having a suitable ADME
18. Emerling, B. M.; Akcakanat, A. Cancer Res. 2011, 71, 7351.
19. Burger, M. T.; Pecchi, S.; Wagman, A.; Ni, Z.-J.; Knapp, M.; Hendrickson, T.;
Atallah, G.; Pfister, K.; Zhang, Y.; Bartulis, S.; Frazier, K.; Ng, S.; Smith, A.;
Verhagen, J.; Haznedar, J.; Huh, K.; Iwanowicz, E.; Xin, X.; Menezes, D.; Merritt,
H.; Lee, I.; Wiesmann, M.; Kaufman, S.; Crawford, K.; Chin, M.; Bussiere, D.;
Shoemaker, K.; Zaror, I.; Maira, S.-M.; Voliva, C. F. ACS Med. Chem. Lett. 2011, 2,
774.
20. Yaguchi, S.; Fukui, Y.; Koshimizu, I.; Yoshimi, H.; Matsuno, T.; Gouda,
H.; Hirono, S.; Yamazaki, K.; Yamori, T. J. Natl. Cancer Inst. 2006, 98,
545.
profile for pharmacological evaluation. The compound has shown
good efficacy in inhibiting the growth of PI3K
a driven tumors in
animal xenograft models as well as good tolerability.33 It is now
in clinical evaluation to assess its therapeutic potential for treating
cancers in which the PIK3CA gene is mutated or amplified.
Acknowledgments
21. Ohwada, J.; Ebiike, H.; Kawada, H.; Tsukazaki, M.; Nakamura, M.; Miyazaki, T.;
Morikami, K.; Yoshinari, K.; Yoshida, M.; Kondoh, O.; Kuramoto, S.; Ogawa, K.;
Aoki, Y.; Shimma, N. Bioorg. Med. Chem. Lett. 2011, 21, 1767.
22. Bruce, I.; Akhlaq, M.; Bloomfield, G. C.; Budd, E.; Cox, B.; Cuenoud, B.; Finan, P.;
Gedeck, P.; Hatto, J.; Hayler, J. F.; Head, D.; Keller, T.; Kirman, L.; Leblanc, C.; Le
Grand, D.; McCarthy, C.; O’Connor, D.; Owen, C.; Oza, M. S.; Pilgrim, G.; Press, N.
E.; Sviridenko, L.; Whitehead, L. Bioorg. Med. Chem. Lett. 2012, 22, 5445.
23. Huang, C.-H.; Mandelker, D.; Schmidt-Kittler, O.; Samuels, Y.; Velculescu, V. E.;
Kinzler, K. W.; Vogelstein, B.; Gabelli, S. B.; Amzel, L. M. Science 2007, 318, 1744.
PDB code 2RD0.
The authors would like to thank Mickael Le Douget, Vincent
Bordas, Aurore Roustan, Dorothee Arz, Van Huy Luu, Jasmin Wirth,
Susanne Vollmer, Philippe Ramstein and Werner Gertsch for their
excellent technical assistance.
References and notes
24. Using a version of MacroModel enhanced for graphics by A. Dietrich, the
compound was manually constructed and docked in the ATP pocket and the
resulting ligand–protein complex energy-minimized using the AMBER⁄/H2O/
GBSA force field. MacroModel: Mohamadi, F.; Richards, N. G. J.; Guida, W. C.;
Liskamp, R.; Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J.
Comput. Chem. 1990, 11, 440.
1. Cantley, L. C. Science 2002, 296, 1655.
2. Katso, R.; Okkenhaug, K.; Ahmadi, K.; White, S.; Timms, J.; Waterfield, M. D.
Annu. Rev. Cell Dev. Biol. 2001, 17, 615.
3. Samuels, Y.; Ericson, K. Curr. Opin. Oncol. 2006, 18, 77.
4. Hennessy, B. T.; Smith, D. L.; Ram, P. T.; Lu, Y.; Mills, G. B. Nat. Rev. Drug Disc.
2005, 4, 988.

3748
P. Furet et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3741–3748
25. Knight, Z. A.; Gonzalez, B.; Feldman, M. E.; Zunder, E. R.; Goldenberg, D. D.;
Williams, O.; Loewith, R.; Stokoe, D.; Balla, A.; Toth, B.; Balla, T.; Weiss, W. A.;
Williams, R. L.; Shokat, K. M. Cell 2006, 125, 733.
reference compound was included in all assay plates in the form of 16 dilution
points in duplicate.
27. The accuracy of our bioassays is exemplified by compound 28 (IC50 SEM (n)):
26. The biochemical inhibitory activities of the compounds were measured using
p110
0.69 0.21 (5); Rat1
tert-butyl group has a
a
0.012 0.001 (16); p110b 3.1 0.30 (15); p110d 0.81 0.16 (5); p110
0.15 0.027 (5); Rat1b >10 (4); Rat1d 3.0 0.65 (5).
volume of around 80 Å3 against 90 Å3 for the
c
Kinase-GloÒ assays for the
a
and b isoforms. AdaptaÒ assays were used for the
a
d and
c
isoforms. For a detailed description of the Kinase-GloÒ assays used see:
28.
A
Caravatti, G., Fairhurst, R. A.; Furet, P.; Guagnano, V.; Imbach, P. PCT Int. Pat.
Appl. WO 2010/029082, March 18, 2010. The AdaptaÒ assays were run as
follows: 50 nL of compound dilutions were dispensed onto white 384-well
diethylamino group. The dimethylamino group is significantly smaller with a
volume of around 60 Å3.
29. Yokooji, A.; Okazawa, T.; Satoh, T.; Miura, M.; Nomura, M. Tetrahedron 2003, 59,
5685.
small volume polystyreneplate. Then 5 lL of PI3K and lipid substrate (PI or
PIP2:PS) followed by 5
l
L of ATP (final assay volume 10
l
L) are incubated at
30. Koreeda, M.; Akagi, H. Tetrahedron Lett. 1980, 21, 1197.
31. Morgan, T. A.; Ganem, B. Tetrahedron Lett. 1980, 21, 2773.
32. The compounds were tested for their ability to inhibit the phosphorylation of
Akt serine 473 in Rat1 cell lines overexpressing activated versions of each class
IA PI3K isoform. For a description of these assays see: Maira, S. M. et al Mol.
Cancer Ther. 2012, 11, 317.
RT. The standard reaction buffer for the AdaptaÒ TR-FRET assay contained
10 mM Tris–HCl pH 7.5, 3 mM MgCl2, 50 mM NaCl, 1 mM DTT, 0.05% CHAPS (v/
v). Reactions were stopped with 5 l -
L of a mixture of EDTA containing the Eu3+
labeled anti-ADP antibody and the Alexa FluorÒ 647-labeled ADP tracer in TR-
FRET dilution buffer (proprietary to IVG). Plates were read 15–60 min later in a
Synergy2 reader using an integration time of 0.4 s and a delay of 0.05 s. Control
for the 100% inhibition of the kinase reaction was performed by replacing the
PI3K by the standard reaction buffer. The control for the 0% inhibition was
given by the solvent vehicle of the compounds (90% DMSO in H2O, v/v). A
33. Fritsch, C. et al. in preparation.
34. The details of this X-ray structure determination will be published elsewhere:
Knapp, M. et al. in preparation. The coordinates have been deposited with PDB
ID code 4JPS.