Discovery of 9-(1-anilinoethyl)-2-morpholino-4-oxo-pyrido[1,2-a]pyrimidine- 7-carboxamides as PI3Kβ/δ inhibitors for the treatment of PTEN-deficient tumours

Article abstract of DOI:10.1016/j.bmcl.2014.06.040

Starting from TGX-221, we designed a series of 9-(1-anilinoethyl)-2- morpholino-4-oxo-pyrido[1,2-a]pyrimidine-7-carboxamides as potent and selective PI3Kβ/δ inhibitors. Structure-activity relationships and structure-property relationships around the aniline and the amide substituents are discussed. We identified compounds 17 and 18, which showed profound pharmacodynamic modulation of phosphorylated Akt in the PC3 prostate tumour xenograft, after a single oral dose. Compound 17 also gave significant inhibition of tumour growth in the PC3 prostate tumour xenograft model after chronic oral dosing.

Full text of DOI:10.1016/j.bmcl.2014.06.040

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3928–3935
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 9-(1-anilinoethyl)-2-morpholino-4-oxo-pyrido[1,
2-a]pyrimidine-7-carboxamides as PI3Kb/d inhibitors for the
treatment of PTEN-deficient tumours
Bernard Barlaam b, , Sabina Cosulich , Sébastien Degorce , Martina Fitzek ,
⇑
b,
b
b
Fabrizio Giordanetto , Stephen Green , Tord Inghardt , Laurent Hennequin a,§,
Urs Hancox , Christine Lambert-van der Brempt , Rémy Morgentin , Sarah Pass ,
Patrick Plé , Twana Saleh , Lara Ward
c,à
b
c
b
a
a,–
b
a
a
b
a
AstraZeneca, Centre de Recherches, Z.I. La Pompelle, B.P. 1050, Chemin de Vrilly, 51689 Reims, Cedex 2, France
AstraZeneca Pharmaceuticals, Oncology iMed, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom
AstraZeneca R&D, CVMD iMed, Pepparedsleden 1, SE-431 83 Mölndal, Sweden
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting from TGX-221, we designed a series of 9-(1-anilinoethyl)-2-morpholino-4-oxo-pyrido[1,2-a]
pyrimidine-7-carboxamides as potent and selective PI3Kb/d inhibitors. Structure–activity relationships
and structure–property relationships around the aniline and the amide substituents are discussed. We
identified compounds 17 and 18, which showed profound pharmacodynamic modulation of phosphory-
lated Akt in the PC3 prostate tumour xenograft, after a single oral dose. Compound 17 also gave signifi-
cant inhibition of tumour growth in the PC3 prostate tumour xenograft model after chronic oral dosing.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 3 June 2014
Revised 11 June 2014
Accepted 13 June 2014
Available online 21 June 2014
Keywords:
PI3Kb inhibitor
Rational design
PTEN-deficient tumours
Phosphoinositide-3-kinases (PI3Ks) are a class of enzymes that
catalyse the phosphorylation of phosphoinositides at the 3-hydro-
xyl position. PI3Ks are grouped into three classes, based on their
tolerability profile, especially given the fact that the first having
entered clinical trials are not highly selective for PI3Ks. On the
other hand, isoform-selective PI3K inhibitors would have the
potential to completely block the relevant target while limiting
1
substrate specificity and structural features. Class I PI3Ks are fur-
3
ther divided into class IA enzymes (PI3K
a
, PI3Kb and PI3Kd) and
toxicities associated with broader inhibition profiles. Moreover,
class IB enzymes (PI3K ). Class I PI3Ks primarily generate phos-
c
the generation of isoform selective inhibitors has started to
elucidate individual distinct functions for these lipid kinases. For
instance, PI3Kd has been shown to play a critical role in B-cell
signalling in response to a number of cytokines and chemokines.
Specific inhibition of PI3Kd has been shown to have activity in
human B-cell cancers such as chronic lymphocytic leukemia
phatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3), which acts as a
second messenger to trigger a diverse set of signalling cascades,
whilst the tumour suppressor PTEN (phosphatase and tensin
homologue) reverses this process.2
The different class I isoforms of PI3K were originally thought to
be redundant in function, and as such, most of the PI3K inhibitors
currently in clinical trials are pan-PI3K inhibiting all four PI3K
isoforms. One concern associated with pan-PI3K inhibitors is their
4
(CLL) and indolent non-Hodgkin lymphoma (iNHL).
Specific catalytic inhibition of PI3Kb has been reported to block
platelet responses to shear stress and to regulate the formation of
5
arterial thrombi. In human cancer models, inhibition of PI3Kb is
thought to be important in tumours which have lost PTEN, as dele-
⇑
Corresponding author. Tel.: +44 1625 231853.
E-mail address: bernard.barlaam2@astrazeneca.com (B. Barlaam).
Current address: Novartis Institutes for Biomedical Research, WKL.125.13.17,
tion of PI3Kb, but not PI3Ka was shown to markedly impair tumor-
6
igenesis driven by the loss of PTEN. These results have prompted
drug discovery efforts to find selective orally active PI3Kb inhibi-
tors and, as a result, many PI3Kb selective inhibitors have been
Klybeckstrasse 141, CH-4002 Basel, Switzerland.
à
Current address: Taros Chemicals GmbH & Co. KG, Emil-Figge-Str. 76a, 44227
Dortmund, Germany.
7
disclosed recently. However, at the start of this work, only few
§
Current address: Galderma R&D, les Templiers, 2400 route des Colles, 06410
isoform selective inhibitors had been reported, mainly from the
Sophia-Antipolis, France.
8
–
TGX series as exemplified by TGX-221. Subsequent efforts at
Current address: Edelris, 115 Avenue Lacassagne, 69003 Lyon, France.
http://dx.doi.org/10.1016/j.bmcl.2014.06.040
0
960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

B. Barlaam et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3928–3935
3929
9
AstraZeneca resulted in the discovery of AZD6482, a PI3Kb inhib-
Table 1
Structure, PI3Kb cellular activity of 1, 2, 2a–d
itor showing inhibition of platelet aggregation suitable for short
intravenous infusion in humans (see Fig. 1).
O
N
In addition to previously reported approaches at AstraZeneca to
find orally active PI3Kb inhibitors,11 one option we considered was
further optimisation of TGX-221 and AZD6482. From the start, we
decided to remove the carboxylic acid present in AZD6482, as we
considered it as the probable cause of poor exposure following oral
administration (see preceding paper15). On the other hand,
TGX-221 is a potent PI3Kb inhibitor with similar cellular potency
N
N
O
NH
R
as AZD6482 (IC50 0.022 lM for TGX-221 vs IC50 0.040 lM for
Compd
R
PI3Kb cell IC50
(
l
M)a
Enantiomeric status
AZD6482) and is highly lipophilic (measured LogD7.4 3.6), and, as
a consequence, has poor physical and metabolic properties.
Internal data showed that biological activity in this series resides
essentially in one enantiomer: for AZD6482, the (R)-enantiomer
1
2
2
2
(AZD6482)
(TGX-221)
a
2-CO
H
3-Me
4-F
3-Cl
3-F, 5-F
2
H
0.040
0.022
0.081b
0.19
0.017
0.009
(R)
Racemic
Racemic
Racemic
b
9
c
is over 200 times more active than the (S)-enantiomer and the
2c
2d
Note
Note
c
(
R)-enantiomer of TGX-221 is more potent than the (S) one, as
7e
recently published by another group. In addition, our internal
work provided evidence that substitution of the aniline with small
substituents could modulate PI3Kb cellular potency (as illustrated
in Table 1). An homology model of the PI3Kb enzyme, based on
available public and un-published in-house crystal structures of
a
b
c
Numbers are a geometric mean of 2 or more values.
n = 1.
Single enantiomer, unknown absolute configuration.
PI3Kc, docked with TGX-221 (R)-enantiomer (see Fig. 2) helped
12
rationalise the observed SAR. This homology model recapitulated
key interactions typical of PI3Ks, as seen in the crystal structure of
Thr308 in PIK3CA mutant human breast ductal carcinoma BT474
cells sensitive to PI3K inhibition and at Ser473 in PTEN-null
breast adenocarcinoma MDA-MB-468 cells sensitive to PI3Kb
AZD6482 bound in PI3K
ogy models of PI3Kb reported by other groups.
c
(see preceding paper15) and other homol-
a
7,11
As anticipated,
1
0
the oxygen of the morpholine makes a key H-bond interaction with
Val854 in the hinge region and the pyridopyrimidone occupies the
central part of the ATP binding site. TGX-221 adopts a T-shape
conformation and induces a conformational change in the P-loop
with the movement of a conserved methionine Met779, creating
a small hydrophobic pocket limited by Met779 and Trp787 where
the aniline of TGX-221 lies. This conformational change was first
inhibition).
Compounds 3–24 were evaluated as racemates or pure enanti-
1
6
omers as indicated in Tables 2 and 3 and selectivity among PI3K
kinases is reported for selected compounds in Table 4. When iso-
lated as single enantiomers, compounds 3–21 were obtained after
separation of the two enantiomers by chiral preparative HPLC at
the final step (see supplementary material for the conditions and
characterisation used). Compounds 3–17 were synthesised accord-
ing to Scheme 1. As described in the preceding paper,15 the key
ester 27, a common intermediate for all the different routes is
readily available in a few steps from aminopyridine 25. The first
route relied on methyl ketone 29, again described previously.15
From 29, reductive amination with aniline followed by amide cou-
1
3
described by Knight to explain degrees of selectivity of some
inhibitors among the PI3K class I family. A similar explanation
was also published for ‘propeller shape’ selective PI3Kd inhibi-
1
4
tors. This hypothesis is currently the most widely accepted
explanation for gain of selectivity for PI3Kb and PI3Kd inhibitors
versus PI3Ka and PI3Kc.
1
5
17
Previously, we showed that amides at the 7-position of the
pyridopyrimidone core were tolerated and we disclosed initial
SAR of the 9-(1-anilinoethyl)-2-morpholino-4-oxo-pyrido[1,2-
a]pyrimidine-7-carboxamides series. The 7-position of the pyrido-
pyrimidone points towards a hydrophilic region containing several
aspartic acids in the PI3Kb isoform (i.e., Asp923 and Asp937), with
access to the solvent. In this Letter, we describe further optimisa-
tion of this series as orally active PI3Kb/d selective inhibitors and
their potential application as anticancer agents in PTEN-deficient
tumour models. Selectivity among the PI3K isoforms was routinely
pling gave 4. However, for less nucleophilic anilines, the reduc-
tive amination step was too sluggish. So compounds 5–13 were
made from 29 as follows: selective reduction of the methyl ketone
with Luche reagent, followed by amide coupling with N,N-dimeth-
ylethylenediamine gave alcohol 30. Bromination and aniline
displacement, either one pot or in two separate steps, gave
compounds 5–13. However, the Heck reaction to make 29 was
found troublesome in some instances. An alternative route was
devised to circumvent this potentially problematic step. Again,
starting from the key ester 27, Stille coupling with tributyl
(1-ethoxyvinyl)stannane gave methyl ketone 31 after hydrolysis.
Formation of the imine with 4-fluoroaniline and its reduction,
followed by saponification of the ester and amide coupling affor-
ded 14. Because of the reduced reactivity of 4-fluoroaniline, imine
formation required activation with titanium tetrachloride.
Therefore, an alternative route via the alcohol 32 was also used
for 3, 15–17. Compound 31 was subjected to reduction with Luche
reagent to give alcohol 32. Bromination and displacement with the
corresponding aniline, followed by saponification of the ester and
measured with enzyme assays and the
a/b selectivity was also
monitored in cell assays (inhibition of Akt phosphorylation at
O
N
O
N
N
N
N
N
O
O
17
amide coupling gave 3, 15–17.
NH
NH
Compounds 18–21 were made according to Scheme 2: from 29,
selective reduction of the methyl ketone with Luche reagent,
followed by amide coupling with dimethylamine gave alcohol 33.
Bromination followed by displacement with the corresponding
aniline gave compounds 18–21. Finally, compounds 18 and 22–
24 were made from carboxylic acid 34, which derived from 32
CO H
2
1
(TGX-221)
2 (AZD6482)
Figure 1. Structures of TGX-221 and AZD6482.

3
930
B. Barlaam et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3928–3935
Figure 2. TGX-221, (R)-enantiomer docked into an homology model of PI3Kb (a) Ribbon diagram showing key residues and interactions (b) Surface (in red) showing the
hydrophobic pocket occupied by the aniline moiety and the solvent accessible hydrophilic channel off the 7-position of pyridopyrimidone.
Table 2
Structure, PI3Kb cellular activity, permeability and lipophilicity of compounds 3–14
O
O
N
R1
N
R2
N
N
O
NH
R
Compd
NR1R2
R
PI3Kb cell IC50
(l
M)a
Caco-2
LogD7.4c
Enantiomeric statusd
b
P
app
3
4
5
6
7
8
9
NMe
2
—
—
0.156
0.008
0.075
0.010
0.007
0.012
0.002
0.023
0.074
0.530
0.004
2.4
1.7
(À)
NHCH
NHCH
NHCH
NHCH
NHCH
NHCH
NHCH
NHCH
NHCH
NHCH
2
2
2
2
2
2
2
2
2
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
2
2
2
2
2
2
2
2
2
2
0.2
(À)
4-Cl
3-Cl
2-Cl
4-F
3-F
2-F
3-CF
Racemic
Racemic
Racemic
Racemic
Racemic
Racemic
Racemic
Racemic
(À)
0.2
0.3
0.8
1.8
2
1.9
10
11
12
13
3
4-Me
2-F, 3-Cl
0.9
2.7
a
b
c
Numbers are a geometric mean of 2 or more values.
À6
Apparent permeability from A (pH 6.5) to B (pH 7.4) in Caco-2 cell line, 10 cm/s.
18
LogD7.4 obtained by shake-flask methodology.
d
Optical rotation sign for compounds isolated as a pure enantiomer.
using the route described in Scheme 3. Chiral separation of the
enantiomers was performed at the carboxylic acid 3416 stage for
compounds isolated as a single enantiomer (e.g., 23–24).
allowed easy separation of the two diastereoisomers by chroma-
tography on silica gel. Oxidative cleavage of the major one with
lead tetraacetate gave the enantiopure amine 37. The exact
configuration of 37 has not been rigorously proven at this stage.
But coupling of 37 with 1-bromo-3,5-difluorobenzene under
Buchwald conditions gave 18 (without evidence of racemisation).
The best coupling conditions found used conditions with a
An enantioselective route to 18 was also investigated (see
Scheme 4): it was based on the enantioselective reduction of an
imine to give enantiopure amine 37, followed by coupling with 1-
bromo-3,5-difluorobenzene via a Buchwald reaction. Several chiral
auxiliaries were tried including tert-butylsulfinamide and phenyl-
glycinol. The best results were obtained with (R)-phenylglycinol
tert-butyldimethyl silyl ether: reaction of 29 with (R)-phenylgly-
cinol tert-butyldimethyl silyl ether in the presence of titanium
tetraisopropoxide afforded imine 35 as a mixture of Z and E
isomers. Reduction of this mixture of imines with sodium
cyanoborohydride at À30 °C in THF gave a 4:1 mixture of diastere-
oisomers. Deprotection of the silyl protecting group with TBAF
1
9
catalyst made from xant-Phos, Pd
2 3
dba and 1-Br-3,5-F-Ph, which
allowed completion of the reaction.
1
5
As previously reported, an amide substituent at the C-7
position of the pyridopyrimidone was tolerated as illustrated by
3 and 4: the dimethyl amide 3 showed strong inhibition of PI3Kb
(IC50 11 nM) in the enzyme assay, with excellent degree of selectiv-
ity versus PI3Ka (50 fold) and PI3Kc (550 fold) while keeping some
PI3Kd activity (IC50 110 nM). At the cellular level, 3 retained

B. Barlaam et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3928–3935
3931
Table 3
Structure, PI3Kb cellular activity, human hepatocyte Clint, lipophilicity, solubility and permeability of compounds 14–24
O
O
R1
N
N
R2
N
N
O
NH
R
Compd
NR1R2
R
PI3Kb cell IC50
(
l
M)a
Human hep. Clintb
LogD7.4c
Solub.
l
Md
Caco-2
Enantiomeric statusf
e
P
app
14
15
16
17
18
19
20
21
22
23
24
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
2
2
2
2
2
2
2
2
4-F
3-F
0.062
0.039
0.073
0.034
0.003
0.011
0.011
0.025
0.008
0.025
0.029
3
45
9
2.3
ND
2.7
2.7
3
3.5
—
2.9
2.1
2.7
2.6
130
23
28
20
15
—
10
46
930
316
34
39
—
44
50
—
—
—
—
—
(À)
Racemic
Racemic
(À)
2-F, 3-F
3-F, 4-F
3-F, 5-F
3-Cl, 5-F
3-F, 4-F, 5-F
2-F, 3-F, 5-F
3-F, 5-F
3-F, 5-F
3-F, 5-F
9
11
19
13
15
10
6
(R)
(À)
(À)
(À)
N(Me)CH
4-Morpholine
4-Me-1-piperazine
2
CH
2
OH
Racemic
(R)
6
>1500
9
(R)
a
b
c
d
e
f
Numbers are a geometric mean of 2 or more values.
6
Human hepatocyte intrinsic clearance,
LogD7.4 obtained by shake-flask methodology.
Solubility from solid material, phosphate buffer (pH 7.4).
Apparent permeability from A (pH 6.5) to B (pH 7.4) in Caco-2 cell line, 10 cm/s.
For compounds isolated as a pure enantiomer, absolute configuration if known, otherwise optical rotation sign.
lL/min/10 cells.
18
À6
Table 4
potency was reduced with larger groups (chloro 6 IC50 10 nM,
trifluoromethyl 11 74 nM). ortho Substitution with a fluorine also
drew our attention: although 10 had slightly reduced cellular
activity (IC50 23 nM), its permeability was significantly increased
Biological activity of compounds 3, 4, 13, 14, 17 and 18 in different PI3K enzyme and
cell assays
Compd PI3K
a
PI3Kb
enz IC50
PI3K
enz IC50
c
PI3Kd
enz IC50
PI3K
cell IC50
a
PI3Kb
cell IC50
a
a
a
a
a
a
enz IC50
À6
compared to its regioisomers (Caco-2 Papp 0.8Á10 cm/s for 10 vs
À6
3
4
1
1
1
1
0.56
0.37
0.092
1.3
0.34
0.075
0.011
0.008
0.007
0.037
0.007
0.005
6.0
6.9
0.67
13.5
3.0
0.11
9.6
2.2
0.156
0.008
0.004
0.062
0.034
0.003
0.2Á10 cm/s for the corresponding para-fluoro 8 and 0.3Á10
b
0.085
0.051
0.23
0.045
0.032
À6
cm/s for the meta fluoro 9), despite no significant change of lipo-
3
4
7
8
0.97
11.6
4.7
philicity. A possible explanation for the improvement of perme-
ability is the shielding of the aniline NH by the fluorine in the
ortho position, lowering the polar surface area. The 2-fluoro
0.51
1.2
3
-chloro compound 13 combined the properties we were looking
a
l
M, numbers are a geometric mean of 3 or more values for enzyme assays and 2
for: good potency at the cellular level (IC50 4 nM) with an exquisite
selectivity versus PI3K
or more values for cell assays.
b
n = 1.
a
(240 fold at the cellular level) and
À6
improved permeability (Caco-2 Papp 0.9Á10 cm/s). Compound 13
displayed good pharmacokinetics properties in mouse, low clear-
ance and good bioavailability, and moderate plasma protein bind-
ing (see Table 5). At this stage, from limited modifications of the
aniline from 3, we had also identified a few neutral compounds
of interest, 14 (dimethylamide, para-fluoro aniline) being the most
prominent example: despite being significantly less potent at the
cellular level (IC50 62 nM), 14 showed excellent selectivity versus
activity on PI3Kb (IC50 156 nM) and good selectivity over PI3Ka (60
fold), albeit potency was slightly reduced (7 fold) compared to
TGX-221. The introduction of the dimethylamide group signifi-
cantly reduced lipophilicity (
interesting starting point. At the cellular level, the dimethylamino-
ethyl amide 4 was significantly more potent against PI3Kb and
D
LogD7.4 = À1.2), making 3 a very
more selective over PI3K
a
than the dimethyl amide 3 (IC50 8 nM,
PI3K
a
(190 fold at the cellular level), improved permeability
À6
with a selectivity ratio of 270). One hypothesis for the improve-
ment of activity and selectivity is a potential ionic interaction of
the basic dimethylamino group with one of the aspartic acid in
the region, most likely Asp923 in the PI3Kb isoform modified as
(Caco-2 Papp 34.10 cm/s), low plasma protein binding and
acceptable pharmacokinetics properties in mouse with good
bioavailability despite a short half life (t1/2 = 1.1 h).
Compounds 13 and 14 were evaluated in an acute pharmacody-
namic experiment following a single oral dose (50 mg/kg for 13
and 100 mg/kg for 14) in SCID mice bearing PTEN-null PC3 prostate
tumour xenografts. Target modulation was assessed by measuring
Akt phosphorylation levels at Ser473 at 30 min and 4 h. We saw
only modest inhibition of p-Akt with 13, despite the massive free
exposure. On the other hand, 14 delivered profound inhibition
of p-Akt at both time points (see Table 6).
These results prompted us to concentrate our efforts on the
neutral series derived from 3 and the substitution on the aniline
was further investigated. As previously shown for the basic
Ser919 in the PI3K
in Caco-2 (Papp 0.2Á10 cm/s).
a
isoform. However, 4 had modest permeability
À6
First, we explored the structure–activity relationships of the
aniline: whereas ortho or meta substitution with a chlorine was
tolerated, para-substitution with a chlorine was poorly tolerated
2
0
(
see 5, 6 and 7 vs 4). Overall, para-substitution on the aniline
was poorly tolerated, a fluorine being the only group maintaining
similar potency: see fluoro 8 versus chloro 5 or methyl 12. meta
Substitution was more rewarding, a fluorine improving even
cellular activity further (fluoro 9 IC50 2 nM). It is worth noting that

3
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B. Barlaam et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3928–3935
Cl
Cl
O
Cl
Cl
O
O
O
O
N
MeO C
2
O
MeO C
HO C
N
2
2
Cl
Cl
N
N
2
6
NH2
N
7
N
N
b-c
d
Br
5
Br
O
Br
O
2
2
28
m
e
O
N
O
O
O
N
MeO C
2
N
N
HO C
2
N
H
N
N
O
N
N
N
N
O
h-i
O
O
3
O
OH
0
1
3
29
n-p
q
j (for 5-12)
(
for 14)
f-g
for 4)
or k-l (for 13)
(
O
O
N
O
N
O
O
MeO C
2
N
N
N
N
N
H
N
r-u
for 3,15-17
N
N
N
N
O
O
NH
Ar
, 14-17
OH
2
O
NH
Ar
3
3
4
-13
Scheme 1. Synthesis of compounds 3–17. Reagents and conditions: (a) malonic acid, 2,4,6-trichlorophenol (2 equiv), POCl
evolvement), to make 26 (crude); (b) 26 (1.2 equiv), toluene, 90 °C, 18 h, 93%; (c) MsCl (1.1 equiv), NEt (1.2 equiv), THF, 10–20 °C then morpholine (3 equiv), THF, 50 °C, 82%;
d) NaOH (1.5 equiv), water, 70 °C, 1 h, 92%; (e) 4-(vinyloxy)butan-1-ol (5 equiv), 1,3-bis(diphenylphosphino)propane (0.1 equiv), Pd(OAc) (0.025 equiv), K CO (2.5 equiv),
(5 equiv), AcOH, polystyrene supported trimethylammonium cyanoborohydride, DMF–water
Cl , rt, 95%; chiral preparative HPLC (for 4); (h) CeCl O (1.05 equiv), MeOH–
3
(2.5 equiv), 20 °C to 110 °C, 2 h (caution: gas
3
(
2
2
3
DMF–water (9:1), 80 °C then 28, 135 °C, 3 h; HCl (aq) quantitative; (f) PhNH
2
i
(
3:1), rt, 18 h, 48%; (g) NH
CH Cl (2:1), then NaBH
6 equiv), CH Cl or NMP, no base or K
m) tributyl(1-ethoxyvinyl)stannane (1.1 equiv), (PPh
3 equiv), CH Cl , 0 °C to rt, 16 h; AcOH, NaBH CN (1.7 equiv), CH
for 14); (q) CeCl O (1 equiv), MeOH–CH Cl
t) NaOH, THF–MeOH–water, rt; (u) Me
2
CH
2
CH
2
NMe
2
(2 equiv), NEt Pr
2
(5 equiv), HATU (1.5 equiv), CH
CH CH NMe (1.05 equiv), NEt Pr
(1 equiv), CH Cl , quantitative; (l) ArNH
(0.04 equiv), dioxane, 100 °C, 18 h; 3 M HCl, 10 min; quantitative; (n) TiCl
2
2
3
Á7H
2
i
2
2
4
(1.3 equiv), 15 °C, 15 min, 84%; (i) NH
CO (for 5–12); (k) PBr
PdCl
2
2
2
2
2
(1.1 equiv), TSTU (1.2 equiv), CH
2
Cl
2
, rt, 1 h, 61%; (j) PBr
3
(1 equiv), ArNH
2
(
(
(
(
(
2
2
2
3
3
2
2
2
(4 equiv), NMP, 40 °C, 2 days, 66%; chiral preparative HPLC (for 13);
3
)
2
2
4
(0.6 equiv), PhNH
NH, TSTU, NEt Pr
2
(1.5 equiv), NEt
3
i
2
2
3
2
Cl
2
–MeOH (8:1), rt, 2 h, 61%; (o) NaOH, THF–MeOH–water, rt, 87%; (p) Me
2
2
, CH
Cl
2
Cl
2
, rt, 83%
3
Á7H
2
2
2
(4:1), then NaBH
4
(1 equiv), rt, 5 min, 64%; (r) CH Cl , PBr (1 equiv), 0 °C, 3 days, 93%; (s) ArNH
2
2
3
2
, MeOH–CH
2
2
, 25–50 °C;
i
2
NH, TSTU, NEt Pr
2
, CH
2
Cl
2
, rt (for 3 and 15–17; chiral preparative HPLC for 3 and 17).
O
O
N
O
O
O
N
N
N
HO C
2
N
N
N
O
a-b
c-d
N
N
N
N
O
O
NH
Ar
O
OH
33
18-21
29
Scheme 2. Synthesis of compounds 18–21. Reagents and conditions: (a) CeCl
3
Á7H
2
O (1.05 equiv), MeOH–CH
2 2 4 2
Cl (2:1), then NaBH (1.3 equiv), 15 °C, 15 min, 84%; (b) Me NH,
i
TSTU, NEt Pr
2
, CH
2
Cl
2
, rt, 10 h, 71%; (c) PBr
3
(1 equiv), CH
2
Cl
2
, rt, 18 h, 59%; (d) ArNH
2
(1.5 equiv), PhNEt (3 equiv), DMF, 50 °C; chiral preparative HPLC.
2
sub-series, a fluorine in the 3-position (compound 15) gave a slight
increase in cell potency, but also lowered metabolic stability.
Next, we looked at bis-substitution of the aniline: both the
stability further: the hydroxyethyl methyl amide 22 showed the
expected solubility improvement (probably linked to the reduction
of lipophilicity) and medium/low metabolic stability; other amides
such morpholine 23 and N-methylpiperazine 24 had better
metabolic stability but reduced cellular potency.
Representative compounds of the series and their activity on
the different PI3K isoforms is reported in Table 4: at the enzyme
level, all compounds showed potent activity against PI3Kb and
2
,3- and 3,4-difluoro (resp. 16 and 17) showed a similar pattern:
similar cell potency as for 15, with improved metabolic stability.
However, the 3,5-difluoro 18 was 10-fold more active in cell (IC50
3
nM), with low/medium metabolic stability. Unfortunately this
compound had low solubility, probably as a consequence of
increased lipophilicity. Further modifications of the 3,5-difluoro-
substitution of 18 were evaluated (compounds 19–21), but all
suffered from a combined reduction of both potency and metabolic
stability. Modifications of the dimethylamide portion were evalu-
ated, with the aim of improving solubility and reducing metabolic
PI3Kd isoforms, with excellent selectivity against PI3K
a and PI3Kc.
The PI3Kb selectivity over PI3K was confirmed at the cellular
a
level. We next turned our attention to the in vivo pharmacokinetic
properties of these molecules: representative compounds were
evaluated in mouse pharmacokinetics: 17 and 18 had acceptable

B. Barlaam et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3928–3935
3933
O
O
N
O
O
N
R1
HO C
N
R2
N
2
MeO C
N
2
N
d or
a-c
N
e-f
N
N
N
O
O
NH
O
NH
OH
32
F
F
F
F
34
18, 22-24
Scheme 3. Synthesis of compounds 18, 22–24. Reagents and conditions: (a) PBr
3%; (c) NaOH, THF–MeOH–water, rt, 100%; (d) HN(Me)CH CH O-TBDPS, TSTU, NEt Pr
carboxylic acid stage); (f) n-Pr-phosphonic anhydride trimer or TSTU, R1R2NH, NEt Pr
3
(1 equiv), CH
2
Cl
2
, 0 °C, 3 days, 93%; (b) 3,5-F-PhNH
2
(4 equiv), MeOH–CH
2
Cl
2
50 °C, 2 days,
i
8
2
2
2 2
, CH Cl
2
, rt; TBAF, THF, rt, 42% over 2 steps (for 22); (e) Chiral preparative HPLC (at the
i
2
, CH
2
Cl
2
, rt.
O
O
N
O
O
N
O
N
N
N
N
HO C
N
N
2
a-b
c
N
O
N
N
N
O
O
O
O
NH
Ph
Ph
29
OTBS
OTBS
35
36
O
N
d-e
O
N
N
N
O
f
O
NH
N
N
N
N
O
F
F
NH2
1
8
37
2 2 4
, rt, 65%; (b) (R)-PhCH(NH )CH O-TBDMS, Ti(O Pr) , THF, 70 °C,
i
i
i
Scheme 4. Enantioselective synthesis of 18. Reagents and conditions: (a) Me
2 h (c) NaBH
CN (0.9 equiv), AcOH (3 equiv), THF, À30 °C, 2 h, diastereoisomeric mixture (syn/anti: 4:1); (d) TBAF (2 equiv), THF, rt; purification on silica gel (0–20% PrOH in
CH Cl ), 2nd eluted isomer: 42% (over 3 steps); (d) Pb(OAc) , (1.3 equiv), MeOH, 0 °C, 69%; (e) Pd-catalyst (1 equiv; made from: xant-Phos (2.2 equiv), Pd dba (1 equiv), 1-Br-
,5-F-Ph (9 equiv), benzene; precipitation in ether, 74%), Cs CO (3.5 equiv), 1-Br-3,5-F-Ph (4 equiv), dioxane, 90 °C, 24 h, 29%.
2
NHÁHCl, TBTU, NEt Pr
2
2
, CH Cl
2
1
3
2
2
4
2
3
3
2
3
antitumour activity of 17 at the 200 mg/kg b.i.d. oral chronic dose
was evaluated in the PTEN-null PC3 prostate tumour xenograft
model in SCID mice: 17 showed 40% inhibition of tumour growth
at the end of the experiment (see Fig. 3).
In summary, based on the docking on TGX-221 in a PI3Kb
homology model, we have designed a series of 9-(1-anilinoeth-
yl)-2-morpholino-4-oxo-pyrido[1,2-a]pyrimidine-7-carboxamides
Table 5
Pharmacokinetic parameters in mice and protein binding of compounds 13, 14, 17
and 18 in mouse and human plasma
Compd
CLa
F%a
Mo/Hu ppb %freeb
13
14
17
18
10
45
74
82
39
70
35
31
1.6/3.3
12/21
8.7/14
4.7/12
a
Mouse pharmacokinetic parameters; hairy mice dosed at 5
30 lmol/kg p.o.; clearance (mL/min/kg) and bioavailability (%).
b
lmol/kg i.v. and
Table 6
Pharmacokinetic exposure, free cover over PI3Kb cellular IC50 for compounds 13, 14,
Protein binding in nude mouse and human plasma, expressed as fraction
17 and 18 and effect on p-Akt in a SCID mice PC3 xenograft model
unbound (%).
Compd
Time point (h)
% Inhib
p-Akt
Total plasma
concn (lM)
Free coverb
a
pharmacokinetic parameters, including bioavailability (see Table
1
1
14
1
1
1
1
18
18
3
3
0.5
4
0.5
4
0.5
4
8
1
4
8
26
40
85
60
81
81
71
92
80
56
21
12
71
8.1
3.9
2.3
0.3
13
83
48
140
16
10
5.9
2.5
204
106
41
5). Compounds 17 and 18 were subsequently administered at
higher doses, respectively 200 mg/kg and 100 mg/kg and, despite
their modest solubility, delivered sustained good plasma levels
up to 8 h, possibly because of prolonged absorption in the gut. They
were selected for evaluation in our acute pharmacodynamic model
4
7
7
7
(
single oral dose in SCID mice bearing PC3 prostate tumour
xenografts). As shown in Table 6, both compounds gave very strong
inhibition of p-Akt (>80% inhibition) up to 4 h with significant
inhibition retained at 8 h (71% and 56% resp. for 17 and 18).
Based on its strong and sustained pharmacodynamic inhibition
of p-Akt up to 8 h following a single oral administration, the
6.8
2.6
1
8
a
Compounds 13, 14, 17 and 18 were administered orally as a suspension (single
dose of 50 mg/kg for 13, 100 mg/kg for 14 and 18, 200 mg/kg for 17).
b
Free cover over PI3Kb cellular IC50
.

3
934
B. Barlaam et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3928–3935
7.
(a) Certal, V.; Halley, F.; Virone-Oddos, A.; Thompson, F.; Filoche-Romme, B.; El-
Ahmad, Y.; Carry, J.-C.; Delorme, C.; Karlsson, A.; Abecassis, P.-Y., et al. Bioorg.
Med. Chem. Lett. 2012, 22, 6381; (b) Certal, V.; Halley, F.; Virone-Oddos, A.;
Delorme, C.; Karlsson, A.; Rak, A.; Thompson, F.; Filoche-Romme, B.; El-Ahmad,
Y.; Carry, J.-C., et al. J. Med. Chem. 2012, 55, 4788; (c) Lin, H.; Schulz, M. J.; Xie,
R.; Zeng, J.; Luengo, J. I.; Squire, M. D.; Tedesco, R.; Qu, J.; Erhard, K.; Mack, J. F.,
et al. ACS Med. Chem. Lett. 2012, 3, 524; (d) Yu, H.; Moore, M. L.; Erhard, K.;
Hardwicke, M. A.; Lin, H.; Luengo, J. I.; McSurdy-Freed, J.; Plant, R.; Qu, J.; Raha,
K., et al. ACS Med. Chem. Lett. 2013, 4, 230; (e) Lin, H.; Erhard, K.; Hardwicke, M.
A.; Luengo, J. I.; Mack, J. F.; McSurdy-Freed, J.; Plant, R.; Raha, K.; Rominger, C.
M.; Sanchez, R. M., et al. Bioorg. Med. Chem. Lett. 2012, 22, 2230; (f) Hardwicke,
M. A.; Ariazi, J.; Bushdid, P.; Erhard, K.; McSurdy-Freed, J.; Lin, H.; Luengo, J.;
Mack, J.; Pietrak, B.; Plant, R.; Qu, J.; Raha, K.; Rominger, C.; Sanchez, R. M.;
Schaber, M. D.; Schulz, M. J.; Sherk, C.; Sinnamon, R.; Skordos, K.; Spengler, M.;
Squire, M.; Tedesco, R.; Xie, R.; Yu, H.; Zeng, J.; Rivero, R. A. Abstract MEDI 21,
243rd ACS National Meeting, San Diego, CA, March 25–29, 2012.
8
9
.
.
Jackson, S. P.; Robertson, A. D.; Kenche, V.; Thompson, P.; Prabaharan, H.;
Anderson, K.; Abbott, B.; Goncalves, I.; Nesbitt, W.; Schoenwaelder, S.; Saylik D.
PCT Int. Appl. 2004, WO2004016607.
Nylander, S.; Kull, B.; Björkman, J. A.; Ulvinge, J. C.; Oakes, N.; Emanuelsson, B.
M.; Andersson, M.; Skäby, T.; Inghardt, T.; Fjellström, O.; Gustafsson, D. J.
Thromb. Haemost. 2012, 10, 2127.
1
0. For description of the PI3K enzyme assays (a, b, c and d isoforms) and the
Figure 3. Tumour growth inhibition of 17 in a PTEN-null PC3 prostate tumour
xenograft model in SCID mice.
PI3Kb cell assay (inhibition of Ser473 Akt phosphorylation endpoint in breast
adenocarcinoma MDA-MB-468 cells), see: Barlaam, B. C.; Degorce, S. L.;
Lambert-Van der Brempt, C. M. P.; Morgentin, R.; Ple, P. U.S. Pat. Appl. Publ.
2
011, US20110098271.
as PI3Kb/d inhibitors. Optimisation of the aniline and the amide
substituents led to identification of potent PI3Kb/d inhibitors with
The PI3Kb enzyme assay, as configured, reaches the tight binding limit for
some inhibitors in Table 4. Copeland R. A., Enzymes, 2nd ed.; Wiley-VCH: New
York, 2000; pp 305–317. In order to determine the tight binding limit for the
PI3Kb enzyme assay, the concentration of functional enzyme for the PI3Kb
isoform was determined experimentally. The tight binding limit is half the
concentration of functional enzyme, which gives a tight binding limit of 10 nM
for PI3Kb.
excellent selectivity versus PI3Ka and PI3Kc such as 17 and 18.
Both compounds show profound pharmacodynamic modulation
of p-Akt in PTEN-null PC3 prostate tumour bearing mice after oral
administration and 17 gave significant inhibition of tumour growth
in the same xenograft model.
PI3Ka cell assay (inhibition of Tyr308 Akt phosphorylation endpoint in human
breast ductal carcinoma BT474 cells): BT474 cells were seeded into black 384
well plates at a density of 5600 cells/well in DMEM containing 10% FCS and 1%
glutamine and allowed to adhere overnight. The following morning
compounds in 100% DMSO were added to assay plates by acoustic
Acknowledgments
dispensing. After a 2 h incubation at 37 °C and 5% CO
aspirated and the cells were lysed with a buffer containing 25 mM Tris–HCl,
mM EDTA, 3 mM EGTA, 50 mM sodium fluoride, 2 mM sodium
2
the medium was
We would like to acknowledge the following scientists for
their contribution in the synthesis or characterization of the
compounds: Christian Delvare, Delphine Dorison Duval, Myriam
Didelot, Olle Karlsson, Patrice Koza, Jean-Jacques Lohmann, Mick-
aël Maudet, Marie-Jeanne Pasquet, Aurélien Péru, Michel Vautier,
Nicolas Warin and the members of the SAR Screening Groups for
generating the cell and biochemical data. We would like to thank
Marie Rydén Landergren (AstraZeneca Mölndal) for determining
the absolute configuration of 18 by VCD.
3
orthovanadate, 0.27 M sucrose, 10 mM b-glycerophosphate, 5 mM sodium
pyrophosphate, 0.5% Triton X-100 and complete protease inhibitor cocktail
tablets (1 tab per 50 mL lysis buffer). After 20 min, the cell lysates were
transferred into ELISA plates which had been pre-coated with an anti total Akt
antibody in PBS buffer and non-specific binding blocked with 1% BSA in PBS
buffer containing 0.05% Tween 20. Plates were incubated overnight at 4 °C. The
next day the plates were washed with PBS buffer containing 0.05% Tween 20
and further incubated with a mouse monoclonal anti-phospho Akt Thr308 for
2 h. Plates were washed again as above before addition of a horse anti-mouse-
HRP conjugated secondary antibody. Following a 2 h incubation at room
temperature, plates were washed and QuantaBlu substrate working solution
was added to each well. The developed fluorescent product was stopped after
Supplementary data
60 min by addition of Stop solution to the wells. Plates were read using a Tecan
Safire plate reader using 325 nm excitation and 420 nm emission wavelengths,
respectively.
Supplementary data (synthetic procedure for 17 and 18, chiral
purification conditions and characterization of compounds 3–4,
1
1
1. (a) Giordanetto, F.; Kull, B.; Dellsen, A. Bioorg. Med. Chem. Lett. 2011, 21, 829;
(b) Giordanetto, F.; Waallberg, A.; Cassel, J.; Ghosal, S.; Kossenjans, M.; Yuan,
Z.-Q.; Wang, X.; Liang, L. Bioorg. Med. Chem. Lett. 2012, 22, 6665; (c)
Giordanetto, F.; Waallberg, A.; Ghosal, S.; Iliefski, T.; Cassel, J.; Yuan, Z.-Q.;
von Wachenfeldt, H.; Andersen, S. M.; Inghardt, T.; Tunek, A., et al. Bioorg. Med.
Chem. Lett. 2012, 22, 6671.
1
3–14, 17–21 and 34, and VCD protocol for determining the abso-
lute configuration of 18) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
0
6.040.
2. The PI3Kb homology model was built from a PI3K
cstructure co-crystallised with
2
-morpholin-4-yl-7-phenyl-4H-chromen-4-one (pdb code 1E7V: Walker, E. H.;
References and notes
Pacold, M. E.; Perisic, O.; Stephens, L.; Hawkins, P. T.; Wymann, M. P.; Williams,
R. L. Mol. Cell 2000, 6, 909) using the PRIME homology modelling program. Prime,
1.0 ed.; Schrodinger, Inc. Compound (R)-1 ((R)-TGX-221) was docked manually
by analogy to the binding mode of available public and unpublished in-house
1
.
(a) Vanhaesebroeck, B.; Leevers, S. J.; Ahmadi, K.; Timms, J.; Katso, R.; Driscoll,
P. C.; Woscholski, R.; Parker, P. J.; Waterfield, M. D. Ann. Rev. Biochem. 2001, 70,
5
35; (b) Vanhaesebroeck, B.; Guillermet-Guibert, J.; Graupera, M.; Bilanges, B.
crystal of PI3K
c. Images in have been created in VIDA v4 from OpenEye Scientific
Nat. Rev. Mol. Cell. Biol. 2010, 11, 329.
Software, 3600 Cerrillos Rd., Suite 1107, Santa Fe, NM87507.
2
3
4
.
.
.
Georgescu, M.-M. Genes Cancer 2010, 1, 1170.
13. Knight, Z. S.; 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. A. Cell 2006, 125, 733.
14. Berndt, A.; Miller, S.; Williams, O.; Le, D. D.; Houseman, B. T.; Pacold, J. I.;
Gorrec, F.; Hon, W.-C.; Ren, P.; Rommel, C.; Gaillard, P.; Rückle, T.; Schwartz, K.
M.; Shokat, K. M.; Shaw, J. P.; Williams, R. L. Nat. Chem. Biol. 2010, 6, 117.
15. Giordanetto, F.; Barlaam, B.; Berglund, S.; Edman, K.; Karlsson, O.; Lindberg,
J. A.; Nylander, S.; Inghardt, T. Bioorg. Med. Chem. Lett. 2014, preceding
paper.
16. When the enantiomers were separated, activity resided mainly in one
enantiomer, with the other enantiomer being significantly less active (data
not reported), as previously described for 1 and 2. The absolute configuration
of 18 was determined to be (R) by vibrational circular dichroism (VCD).
Coupling of the (À) enantiomer of carboxylic acid 34 with dimethylamine gave
Fruman, D. A.; Rommel, C. Nat. Rev. Drug Discovery 2014, 13, 140.
(a) Meadows, S. A.; Vega, F.; Kashishian, A.; Johnson, D.; Diehl, V.; Miller, L. L.;
Younes, A.; Lannutti, B. J. Blood 1897, 2012, 119; (b) Norman, P. Expert Opin.
Ther. Pat. 2011, 11, 1773.
Jackson, S. P.; Schoenwaelder, S. M.; Goncalves, I.; Nesbitt, W. S.; Yap, C. L.;
Wright, C. E.; Kenche, V.; Anderson, K. E.; Dopheide, S. M.; Yuan, Y.; Sturgeon, S.
A.; Prabaharan, H.; Thompson, P. E.; Smith, G. D.; Sheperd, P. R.; Daniele, N.;
Kulkarni, S.; Abbott, B.; Saylik, D.; Jones, C.; Lu, L.; Giuliano, S.; Hughan, S. C.;
Angus, J. A.; Robertson, A. D.; Salem, H. H. Nat. Med. 2005, 11, 507.
(a) Jia, S.; Liu, Z.; Zhang, S.; Liu, P.; Zhang, L.; Lee, S. H.; Zhang, J.; Signoretti, S.;
Loda, M.; Roberts, T. M.; Zhao, J. J. Nature 2008, 454, 776; (b) Wee, S.;
Wiederschain, D.; Maira, S.-M.; Loo, A.; Miller, C.; DeBeaumont, R.; Stegmeier,
F.; Yao, Y. M.; Lengauer, C. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 13057.
5
6
.
.

B. Barlaam et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3928–3935
19. Yin, J.; Buchwald, S. L. J. Am. Chem. Soc. 2002, 124, 6043.
3935
1
8 as a single enantiomer. This same (À) enantiomer was used to make 23
and 24.
20. Although tumour levels have not been assessed for 13, we have seen high total
tumour levels for related compounds carrying the same basic side chain and
with similar permeability. However, we suspect that high efflux potential
(MDR-mdck efflux ratio: 15) associated the moderate permeability seen with
13 may limit engagement of the target in the tumour.
1
1
7. Compounds 3 and 4 are the (À) enantiomers of compounds 10 and 14 from the
preceding paper (Ref. 15).
8. LogD7.4 was measured using shake-flask methodology with a buffer/octanol
volume ratio of 100:1. The concentration of compound in the aqueous phase
before and after partitioning with octanol was determined by generic HPLC
analysis.