Discovery of a series of 8-(1-phenylpyrrolidin-2-yl)-6-carboxamide-2-morpholino-4H-chromen-4-one as PI3Kβ/δ inhibitors for the treatment of PTEN-deficient tumours

Article abstract of DOI:10.1016/j.bmcl.2017.03.027

Attempts to lock the active conformation of compound 4, a PI3Kβ/δ inhibitor (PI3Kβ cell IC₅₀ 0.015?μM), led to the discovery of a series of 8-(1-phenylpyrrolidin-2-yl)-6-carboxamide-2-morpholino-4H-chromen-4-ones, which showed high levels of potency and selectivity as PI3Kβ/δ inhibitors. Compound 10 proved exquisitely potent and selective: PI3Kβ cell IC₅₀ 0.0011?μM in PTEN null MDA-MB-468 cell and PI3Kδ cell IC₅₀ 0.014?μM in Jeko-1 B-cell, and exhibited suitable physical properties for oral administration. In vivo, compound 10 showed profound pharmacodynamic modulation of AKT phosphorylation in a mouse PTEN-null PC3 prostate tumour xenograft after a single oral dose and gave excellent tumour growth inhibition in the same model after chronic oral dosing. Based on these results, compound 10 was selected as one of our PI3Kβ/δ preclinical candidates.

Full text of DOI:10.1016/j.bmcl.2017.03.027

Accepted Manuscript
Discovery of a series of 8-(1-phenylpyrrolidin-2-yl)-6-carboxamide-2-morpho-
lino-4H-chromen-4-one as PI3Kβ/δ inhibitors for the treatment of PTEN-
deficient tumours
Bernard Barlaam, Sabina Cosulich, Sébastien Degorce, Rebecca Ellston,
Martina Fitzek, Stephen Green, Urs Hancox, Christine Lambert-van der Brempt,
Jean-Jacques Lohmann, Mickaël Maudet, Rémy Morgentin, Patrick Plé, Lara
Ward, Nicolas Warin
PII:
S0960-894X(17)30262-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.027
BMCL 24779
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 February 2017
9 March 2017
12 March 2017
Please cite this article as: Barlaam, B., Cosulich, S., Degorce, S., Ellston, R., Fitzek, M., Green, S., Hancox, U.,
Lambert-van der Brempt, C., Lohmann, J-J., Maudet, M., Morgentin, R., Plé, P., Ward, L., Warin, N., Discovery
of a series of 8-(1-phenylpyrrolidin-2-yl)-6-carboxamide-2-morpholino-4H-chromen-4-one as PI3Kβ/δ inhibitors
for the treatment of PTEN-deficient tumours, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://
dx.doi.org/10.1016/j.bmcl.2017.03.027
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Discovery of a series of 8-(1-phenylpyrrolidin-2-yl)-6-carboxamide-2-morpholino-4H-
chromen-4-one as PI3Kβ/δ inhibitors for the treatment of PTEN-deficient tumours.
Bernard Barlaam,*,a Sabina Cosulich,a Sébastien Degorce,a Rebecca Ellston,b Martina Fitzek,b Stephen
Green,b Urs Hancox,b Christine Lambert-van der Brempt,c Jean-Jacques Lohmann,c Mickaël Maudet,c
Rémy Morgentin,c Patrick Plé,c Lara Ward,b Nicolas Warinc
a IMED Oncology, AstraZeneca, Darwin Building, Cambridge Science Park, 319 Milton Road, Cambridge CB4 0WG, United Kingdom.
b IMED Oncology, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom.
c AstraZeneca, Centre de Recherches, Z.I. La Pompelle, B.P. 1050, Chemin de Vrilly, 51689 Reims, Cedex 2, France
ARTICLE INFO
ABSTRACT
Article history:
Received
Attempts to lock the active conformation of compound 4, a PI3Kβ/δ inhibitor (PI3Kβ cell IC50 0.015
µM), led to the discovery of a series of 8-(1-phenylpyrrolidin-2-yl)-6-carboxamide-2-morpholino-4H-
chromen-4-ones, which showed high levels of potency and selectivity as PI3Kβ/δ inhibitors.
Compound 10 proved exquisitely potent and selective: PI3Kβ cell IC50 0.0011 µM in PTEN null
MDA-MB-468 cell and PI3Kδ cell IC50 0.014 µM in Jeko-1 B-cell, and exhibited suitable physical
properties for oral administration. In vivo, compound 10 showed profound pharmacodynamic
modulation of AKT phosphorylation in a mouse PTEN-null PC3 prostate tumour xenograft after a
single oral dose and gave excellent tumour growth inhibition in the same model after chronic oral
dosing. Based on these results, compound 10 was selected as one of our PI3Kβ/δ preclinical
candidates.
Revised
Accepted
Available online
Keywords:
PI3Kβ/δ inhibitor
Rational design
PTEN-deficient tumours
2016 Elsevier Ltd. All rights reserved
The PI3K-AKT signalling pathway plays a critical role in cell growth, proliferation, motility and survival. In human cancer, this
pathway is activated by several mechanisms, including somatic mutations, deletions and amplifications. Within this pathway, Class I
phosphoinositide 3-kinases (PI3Ks) have emerged as attractive targets for the treatment of cancers. This class is composed of 4
members: PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ. All isoforms phosphorylate phosphatidylinositol (4,5)-bisphosphate (PIP2) to generate
phosphatidylinositol (3,4,5)-triphosphate (PIP3). PIP3 acts as a second messenger to trigger a diverse set of signalling cascades, whilst
the tumour suppressor PTEN (phosphatase and tensin homolog) reverses this process.1
While the different members of Class I PI3Ks were originally thought to be redundant in function, the generation of isoform selective
inhibitors has started to elucidate individual distinct functions for these lipid kinases.2 For instance, PI3Kδ has been shown to play a
critical role in B-cell signalling in response to a number of cytokines and chemokines. Specific inhibition of PI3Kδ has been shown to
have activity in human B-cell cancers such as chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin lymphoma (iNHL).3
Similarly, inhibition of PI3Kβ is thought to be important in PTEN-deficient tumours, as deletion of PI3Kβ was shown to markedly
impair tumorigenesis driven by the loss of PTEN.4 These results have prompted drug discovery efforts to find selective orally available
PI3Kβ inhibitors and, as a result, many PI3Kβ selective inhibitors have been disclosed in recent years.5
O
O
O
O
O
N
N
N
N
O
R
N
N
N
N
N
O
O
N
NH
O
NH
R
F
F
F
Y
X
3 AZD8186, R= H
4 R= Me
1 (X= H, Y= F), (R)-enantiomer
2 (X= F, Y= H), (-)-enantiomer
(R)-TGX221 (R= H)
AZD6482 (R= CO2H)
Figure 1. Structures of some pyridopyrimidone- and chromenone-based PI3Kβ/δ inhibitors

Initial work at AstraZeneca resulted in the discovery of AZD6482,6 a PI3Kβ inhibitor showing inhibition of platelet aggregation
suitable for short intravenous infusion in humans. We subsequently reported further optimisation of AZD6482 to potent and orally
available PI3Kβ/δ inhibitors.7-9 An essential aspect of this work was the removal of the carboxylic acid, for which we had to
compensate the increase of lipophilicity in this pyridopyrimidone series (measured logD7.4 of TGX-221: 3.6). Substitution with an
amide at the 7-position of the pyridopyrimidone core afforded orally available compounds, as exemplified by 1 and 2, the latter showing
potent tumour growth inhibition in a PTEN-deficient tumour model.7b Scaffold hopping to the more hydrophilic chromenone increased
human metabolic stability and led to the identification of the clinical candidate 3 (measured logD7.4:2.3), known as AZD8186, currently
in phase I clinical trials.8 Interestingly, during this work, we showed that alkylation of the nitrogen of the aniline by a methyl group (i.e.
4) was tolerated with minimal loss of potency (PI3Kβ cell IC50 0.015 µM for 4 vs. 0.003 µM for 3). We previously reported our
homology model of PI3Kβ kinase, the T-shaped active conformation for this series of PI3Kβ inhibitors and the key interactions with the
PI3Kβ kinase.7b On the basis of the docking of 4 in this model, the methyl on the asymmetric carbon bearing the aniline and the methyl
on the nitrogen of the aniline would be on the same side and, therefore, might offer the possibility for cyclisation. We hypothesised that
cyclisation may limit conformational freedom even further and lock the molecule into its active conformation. Modelling work
suggested that a five membered ring would have the required conformation (see overlay in Figure 2). The pyrrolidine linker was
predicted to overlay very well with that of the acyclic linker, although the phenyl ring could be slightly shifted. This was thought to
potentially change the SAR previously reported around optimal substitution of the aniline.
a
b
Figure 2: Overlay of the docked structures for 4 (cyan) and 8 (orange) into the previously described homology model of PI3Kβ (a) Ribbon diagram showing the
predicted conformation of the pyrolidine linker in relation to the acyclic version. b) Surface (in red) showing the available pocket focused on the 3,5-
difluorophenyl moiety.
We describe therein the discovery of a series of 8-(1-phenylpyrrolidin-2-yl)-6-carboxamide-2-morpholino-4H-chromen-4-one as orally
available PI3Kβ/δ selective inhibitors and their potential application as anticancer agents in PTEN-deficient tumours. Compounds 5-15
were evaluated as racemates or pure enantiomers as indicated in Table 1, and selectivity among PI3K kinases is reported for selected
compounds in Table 2.10
Compounds 5-15 were made according to Schemes 1-4. We previously reported the synthesis of dimethyl amide 179 from 16,8 via a
one-pot saponification / amide coupling with dimethyl amine via the activated 4,6-methoxytriazine-2-oxy ester. We anticipated
accessing compound 5 from 19, which would be obtained from 17 by palladium-catalyzed arylation of N-Boc pyrrolidine with the
option of high enantioselectivity (Scheme 1, dotted arrow).11 However, despite significant optimisation, 19 was obtained only in a very
modest yield.12
We turned to another strategy (Scheme 1): Suzuki coupling of 17 with N-Boc-pyrrole-2-boronic acid proceeded with good yield to give
18. Selective hydrogenation of the Boc-pyrrole 18 gave pyrrolidine 19. Boc deprotection followed by Buchwald reaction with the
corresponding aryl bromide afforded 5 as a racemate, and 6 as a pure enantiomer after preparative chiral chromatography. A similar
sequence was developed from ester 16, allowing amide variation at the last step (e.g. compounds 8-9 and 13, Scheme 2).
Separation of the enantiomers at the N-Boc pyrrolidine 22 stage was carried out and provided the (R)-N-Boc-pyrrolidine 25 and (S)-N-
Boc-pyrrolidine 26. Configuration of each enantiomer was assigned by X-ray structure determination of 29, the (S,S) tartrate salt of 28,
obtained from 26 after Boc deprotection.13 The Boc deprotection of 25 provided enantiomerically pure 27 having the (R) configuration.
A similar sequence as in Scheme 2 was carried out: Buchwald coupling with the appropriate aryl bromide provided esters 30-32 which
were converted into the corresponding amides 6-7, 10-12 and 15 (Scheme 3).
An alternative route was developed in Scheme 4. The one-pot saponification / amide coupling with morpholine via the activated 4,6-
methoxytriazine-2-oxy ester gave 33. Heck coupling with N-Boc-2,3-dihydropyrrole afforded the 2,5-dihydropyrrole 34 (contaminated
with the 2,3-dihydropyrrole analogue). Boc deprotection with trifluoroacetic acid and hydrogenation afforded pyrrolidine 35, which was
then coupled with 1-bromo-3,5-fluorobenzene or 1-bromo-4-fluorobenzene under Buchwald conditions to give 10 or 14 after chiral
chromatography.13
Initial compounds 5 and 6 showed exquisitely strong PI3Kβ inhibition (cellular IC50 around 0.003 µM, Table 1). This level of potency
was unprecedented for unsubstituted or 4-fluoro phenyl in earlier series (e.g. PI3Kβ cellular IC50 0.128 µM for the racemic 4-
fluorophenyl analogue of 3).7b,8 Selectivity over PI3Kα and PI3Kγ was maintained as illustrated by 6 (PI3Kα cellular IC50 0.18 µM;
PI3Kα/β cellular selectivity ratio: 50; PI3Kγ enzyme IC50 0.33 µM), while PI3Kδ activity was conserved (PI3Kδ enzyme IC50 0.0082
µM). We had previously shown that 3-mono- or 3,5-di-substitution of the phenyl with fluorine was preferred in the earlier series: indeed
compounds 7 and 8 showed increased PI3Kβ potency (cellular IC50 0.0012 µM for both), but not to the same extent, presumably owing

to slight differences in the way the phenyl ring fits in the active site. High permeability and evidence of oral exposure was seen in this
series as exemplified by 5 and 8, but these early compounds 5-8 showed moderate to high human hepatocyte turnover. We turned our
attention to the amide part of the molecule, with the intention to reduce lipophilicity as a way of increasing metabolic stability of our
initial molecules. Many amides (i.e. 9-13) were tolerated and maintained high levels of potency. Gratifyingly, less lipophilic compounds
9 and 13 had improved metabolic stability, but introduction of an additional H-bond donor (hydroxyl) compromised permeability (i.e.
Caco-2 Papp 2.6x10-6 cm/s for 9) and, as a consequence, oral exposure in mouse was low. Similarly, the slightly basic N-methyl
piperazine 11 had only modest permeability (i.e. Caco-2 Papp 3.3x10-6 cm/s). Overall, the most interesting amide was morpholine 10,
displaying high permeability (i.e. Caco-2 Papp 18.10-6 cm/s), low human hepatocyte turnover (Clint 5 µL/min/106 cell) and acceptable
mouse oral exposure (AUC 2.8 µM.h following a 30 µmol/kg oral dose). Pharmacokinetics of 10 and 15 were evaluated in mouse
(see Table 3). The observed in vivo clearance was well predicted from in vitro mouse hepatocytes, suggesting that clearance was
essentially metabolic in the liver.
Based on its overall profile and properties, compound 10 was selected for further evaluation. Compound 10 was profiled in dog
pharmacokinetics, confirming that in vivo clearance could essentially be predicted from in vitro hepatocytes. Compound 10 (from
crystalline material, melting point 180 °C) had excellent aqueous solubility: 430 µg/mL in FASSIF (Fasted Simulated Intestinal Fluid)
and 230 µg/mL in aqueous buffer (pH 6.5) and did not show any significant hERG inhibition (hERG IC50 45 µM) or CYP P450
inhibition (IC50 >30 µM against CYP 1A2, 2C9, 2C19, 2D6 and 3A4).
The broader kinase selectivity of 10 was evaluated in several kinase panels. 10 showed excellent selectivity in a panel of 71 protein
kinases (‘Dundee University protein kinase’ panel) at 1 µM, showing less than 25% of inhibition against all kinases, except MNK1 and
MARK4 respectively 36% and 32% of inhibition. Selectivity among lipid kinases was assessed in a panel of 15 kinases (‘Dundee
University lipid kinase’ panel), where no significant activity (IC50 >50 µM for all kinases, except SphK1 IC50 29 µM) was seen outside
of PI3K class I enzymes. Bespoke kinase assays confirmed selectivity against selected kinases from the closely related PIKK family
(e.g. m-TOR IC50 42 µM). The selectivity profile of 10 among the PI3K isoforms was evaluated in cell based assays: 10 is a potent
inhibitor of PI3Kβ [IC50 0.0011 µM (+/- SEM: 0.0002)] and PI3Kδ [IC50 0.014 µM (+/- SEM: 0.001)], with good selectivity over
PI3Kα [IC50 0.14 µM (+/- SEM: 0.06)] and PI3Kγ (IC50 1.5 µM)10
Compound 10 was selected for in vivo evaluation in PTEN-deficient tumour models. In an acute pharmacodynamic experiment
following a single oral dose (100 mg/kg or 30 mg/kg) of 10 in nude mice bearing PTEN-deficient PC3 prostate tumour xenografts,
target modulation was assessed by measuring AKT phosphorylation levels at Ser473 at 30 minutes, 2 and 8 hours. At both doses, strong
inhibition of AKT phosphorylation was observed at early time points, with still significant inhibition at 8 hours (see Figure 3).
The antitumour activity of 10 was evaluated in the PTEN-deficient PC3 prostate tumour xenograft model in nude mice at 100 mg/kg
b.i.d. and 50 mg/kg b.i.d. Strong inhibition of tumour growth was seen at both doses, resp. 83% and 71% tumour growth inhibition at
100 mg/kg and 50 mg/kg b.i.d. at the end of the study (Figure 4).
In summary, based on the docking of 4 in our PI3Kβ homology model and the analysis of its active conformation, we have designed a
series of 8-(1-phenylpyrrolidin-2-yl)-6-carboxamide-2-morpholino-4H-chromen-4-one as PI3Kβ/δ inhibitors. Optimisation of this novel
series led to compound 10, a potent PI3Kβ/δ inhibitors with excellent selectivity vs. PI3Kα and PI3Kγ, and suitable physical properties
for oral administration. Compound 10 showed profound pharmacodynamic modulation of AKT phosphorylation in PTEN-deficient
PC3 prostate tumour bearing mice after oral administration and gave significant inhibition of tumour growth in the same xenograft
model. Compound 10 was selected as a PI3Kβ/δ preclinical candidate alongside AZD8186.
Figure 3. Inhibition of AKT phosphorylation in the PC3 xenograft model in nude mice following oral administration of compound 10 (100 mg/kg and 30 mg/kg)
and associated free plasma concentration at 30 minutes, 2 hours and 8 hours.
Figure 4. Tumour growth inhibition in the PC3 xenograft model in nude mice following chronic oral administration of compound 10 (100 mg/kg b.i.d. and 50
mg/kg b.i.d.).

b
16 (Y= OMe)
17 (Y= NMe2)
a
18
c
e
5-6
19 (R= Boc)
20 (Y= H)
d
Scheme 1. Synthesis of compounds 5-6. (a) KOH (2 eq.), H2O, rt, 4 h; Me2NH.HCl (3 eq.), 2-Cl-4,6-MeO-1,3,5-triazine (4.3 eq.), N-Me-morpholine (7 eq.),
H2O, rt to 70 °C, 78%; (b) N-Boc-pyrrole-2-boronic acid (1.2 eq), (PPh3)2PdCl2 (0.02 eq.), Na2CO3, DME-water, 80 °C, 8 h, 62%; (c) 5% Rh/Al2O3, MeOH, H2, 10
atm, 50 °C, 3.5 h, H-Cube®, then 10% Pd/C, MeOH, H2, 60 atm, 60 °C, 6 h, H-Cube®, 50%; (d) 4M HCl in dioxane, CH2Cl2, rt, 4 h, quantitative; (e) xant-Phos
(0.09 eq.), Pd(OAc)2 (0.06 eq.), Ar-Br (1.3 eq.) Cs2CO3 (1.5 eq.), dioxane, 100 °C, 16 h, 19-38%; chiral chromatography (for 6).
O
O
O
O
O
O
O
b
O
N
N
O
X
O
Boc
N
16 (X= Br)
21 (X= N-Boc-pyrrole-2-yl)
22
a
c
O
O
O
O
O
R1
N
R2
O
e-f
F
N
O
N
O
O
N
R
N
F
8-9, 13
23 (R= H)
24 (R= 3,5-di-F Ph)
d
Scheme 2. Synthesis of compounds 8-9, 13. (a) N-Boc-pyrrole-2-boronic acid (1.1 eq), (PPh3)2PdCl2 (0.02 eq.), Na2CO3, DME-water, 80 °C, 7 h, 70%; (b) 5%
Rh/Al O , MeOH, H , 5 atm, 65 °C, 7 h, H-Cube®, 83%; (c) 4M HCl in dioxane, CH2Cl2, rt, 4 h, 83%; (d) Pd-catalyst (0.05 eq. ; made from: xant-Phos (2.2 eq.),
Pd2dba3 (1 eq.), 1-bromo-3,5-difluorobenzene (9 eq.), benzene; precipitation in ether, 39%), Cs2CO3 (1.5 eq.), 1-bromo-3,5-difluorobenzene (1.1 eq.), dioxane, 80
2
3
2
°C, 23 h, 58%; (e) NaOH, MeOH, 40 °C, 23 h, 35%; (f) R1R2NH, TSTU or TBTU, NEtiPr2 or N-methylmorpholine, DMF or NMP, rt, 66-71%.

a
26
25
22
b
b
e-f
6-7,10-12,15
28 free base
S S
, )-tartrate salt
27 (R= H)
30-32 (R= 3-F Ph, 4-F Ph, 3,5-di-F Ph)
d
c
29 (
Scheme 3. Synthesis of compounds 6-7, 10-12, 15. (a) chiral chromatography; (b) 4M HCl in dioxane, CH2Cl2, rt, 8 h, 88%; (c) (S,S)-tartaric acid, MeOH and
isopropanol vapour diffusion crystallization; (d) Pd(OAc)2 (0.05 eq.), xant-Phos (0.1 eq.), Cs2CO3 (1.5 eq.), aryl bromide (1.3 eq.), dioxane, 100 °C, 15 h; (e)
NaOH, MeOH (f) R1R2NH, TSTU or TBTU or nPr-phosphonic anhydride trimer, NEtiPr2, CH2Cl2 orCHCl3, rt, 44-77%.
b
16 (Y= OMe)
33 (Y= morpholine)
a
34
c-d
e
10, 14
35
Scheme 4. Synthesis of compounds 10, 14. (a) KOH (2 eq.), H2O, rt, 4 h; morpholine (3 eq.), 2-Cl-4,6-MeO-1,3,5-triazine (4.3 eq.), N-methylmorpholine (0.5
eq.), H2O, rt, 18 h, 78%; (b) N-Boc-2,3-dihydropyrrole (1.4 eq), PPh3 (0.1 eq.), Pd(OAc)2 (0.05 eq.), K2CO3, DMF, 100 °C, 16 h, 88%; (c) TFA, CH2Cl2,
quantitative. (d) 5% Pd/C, MeOH, H2, 5 atm, 45 °C, 3 h, H-Cube®, 53%; (e) xant-Phos (0.10 eq.), Pd(OAc)2 (0.05 eq.), 1-bromo-3,5-difluorobenzene (1.3 eq.)
Cs2CO3 (3 eq.), dioxane, 100 °C, 2 h, 75% (for 10) or biphenyl-2-yldicyclohexylphosphine (0.10 eq.), Pd(OAc)2 (0.05 eq.), 1-bromo-4-fluorobenzene (1.3 eq.),
Cs2CO3 (1.5 eq.), dioxane, 100 °C, 15 h, 46% (for 14); chiral chromatography.
Table 2. Biological activity of compounds 6, 10, 15 in different PI3K enzyme and cell assays.
PI3Kα
enz IC50
0.024
PI3Kβ
enz IC50
0.0067
0.0071
0.0061
PI3Kγ
enz IC50
0.33b
PI3Kδ
enz IC50
0.0082
0.0086
0.0051
PI3Kα
cell IC50
0.18
PI3Kβ
cell IC50
0.0034
0.0011
0.0028
PI3Kγ
cell IC50
PI3Kδ
a
cell IC50
cmpd
a
a
a
a
a
a
a
6
10
-
1.5b
-
-
0.014
-
0.013
0.019
0.19
0.34
0.14
0.30
15
a µM, numbers are a geometric mean of 2 or more values
b n=1
Table 3. Pharmacokinetic properties of compounds 10 and 15.
Mo/Dog
CL a
Mo/Dog
Vdssa
Mo/Dog
F%a
Mo/Dog/Hu
b
Mo/Dog/Hu
c
heps CLint
Cpd
fu
10
15
60 /23
47 / -
1 / 1.2
0.9 / -
26 / 29
18 / -
19 / 28 / 17
- / - / 11
68 / 23 / 2.7
134 / - / -

a
Mouse and dog pharmacokinetic parameters from blood concentrations; hairy mice dosed at 5 µmol/kg i.v. and 30 µmol/kg p.o., dogs dosed at 4 µmol/kg i.v.
and 10 µmol/kg p.o.; Clearance (mL/min/kg), Vdss (L/kg) and bioavailability (%).
b protein binding in mouse, dog or human plasma, expressed as fraction unbound (%).
c
intrinsic clearance from mouse, dog or human hepatocytes, CLint (mL/min/106 cell)
Acknowledgments
We would like to acknowledge the following scientists for their contribution to the synthesis, purification or characterization of the
compounds: Christian Delvare, Delphine Dorison Duval, Nancy De Grace and Gordon Currie, and the members of the SAR Screening
Groups for generating the cell and biochemical data. We would like to thank Anne Ertan (AstraZeneca Södertälje) for solving the
structure of 29 by single crystal X-ray diffraction.
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Lengauer, C.; Schio, L. J. Med. Chem., 2012, 55, 4788. c) Yu, H; Michael L. Moore, M. L.; Erhard, K.; Hardwicke, M. A.; Lin, H.; Luengo, J. I.; McSurdy-
Freed, J.; Plant, R.; Qu, J.; Raha, K.; Rominger, C. M.; Schaber, M. D.; Michael D. Spengler, M. D.; Rivero, R. A. ACS Med. Chem. Lett., 2013, 4, 230.
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.
6
7
a) Giordanetto, F.; Barlaam, B.; Berglund, S.; Edman, K.; Karlsson, O.; Lindberg, J.A.; Nylander, S.; Inghardt, T. Bioorg. Med. Chem. Lett. 2014, 24, 3936
b) Barlaam, B.; Cosulich, S.; Degorce, S.; Fitzek, M.; Giordanetto, F.; Green, S.; Inghardt, T.; Hennequin, L.; Hancox, U.; Lambert-van der Brempt, C.;
Morgentin, R.; Pass, S.; Plé,P.; Saleh, T.; Ward, L. Bioorg. Med. Chem. Lett. 2014, 24, 3928.
8
9
Barlaam, B.; Cosulich, S.; Degorce, S.; Fitzek, M.; Green, S.; Hancox, U.; Lambert-van der Brempt, C.; Lohmann, J.-J.; Maudet, M.; Morgentin, R.;
Pasquet, M.-J.; Péru, A.; Plé, P.; Saleh, T.; Vautier, M.; Walker, M.; Ward, L.; Warin, N. J. Med. Chem., 2015, 58, 943.
Barlaam, B.; Cosulich, S.; Degorce, S.; Fitzek, M.; Green, S.; Hancox, U.; Lambert-van der Brempt, C.; Lohmann, J.-J.; Maudet, M.; Morgentin, R.; Péru,
A.; Plé, P.; Saleh, T.; Ward, L.; Warin, N. Bioorg. Med. Chem. Lett. 2016, 26, 2318.
10 Selected representative compounds were evaluated against the four PI3K isoforms (enzyme and/or cell assays, see Table 2). Biological protocols for PI3K
enzymes are available in Ref 8. Protocols for PI3Kα, PI3Kβ, PI3Kδ cellular assays are available in Ref 8 and for PI3Kγ cellular assay in Ref 14): PI3Kα,
inhibition of AKT phosphorylation at Thr308 in PIK3CA mutant human breast ductal carcinoma BT474 cells; PI3Kβ, inhibition of AKT phosphorylation at
Ser473 in PTEN null breast adenocarcinoma MDA-MB-468 cells; PI3Kδ, inhibition of AKT phosphorylation at Ser473 in Jeko-1 B cells; PI3Kγ, inhibition
of AKT phosphorylation at Ser473 in mouse monocytic RAW264 cells.
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 TGX-221, AZD6482 and 1-4.
It is worth noting that we assessed PI3K selectivity based on cellular rather than enzyme assays, some of the PI3K enzyme assays reached “tight binding
conditions” for the most potent inhibitors. Copeland, R. A. Enzymes; 2nd Ed.; Wiley-VCH: New York, 2000; pp 305−317. Tight binding conditions: the
concentration of functional enzyme for each PI3K isoform was determined as described in Ref. 8. The tight binding limit is half the concentration of
functional enzyme which gave a tight binding limit of 10 nM for α/β and 15 nM for δ IC50 enzyme assays. The tight binding limit for γ was not reached and
so could not be experimentally determined.
11 Barker, G.; McGrath, J.L.; Klapars, A.; Stead, D.; Zhou, G.; Campos, K. R.; O’Brien, P. J. Org. Chem. 2011, 76, 5936 and associated references.
12 The best conditions for this reaction involved deprotonation of N-Boc pyrrolidine, transmetallation with ZnCl2 and Negishi coupling with 17: 10 mol%
Pd(OAc)2, 12.5 mol% PtBu3.HBF4 with the organozinc species (3 eq., made from deprotonation with (−)-sparteine / sec-butyllithium and transmetallation
−70 °C)
assessed. The major side product of the reaction was the des-bromo compound from reduction of 17.
with ZnCl2 in dry methyl t-butyl ether at
at room temperature gave the expected pyrrolidine 19 in 25% yield, the enantiomeric excess was not
13 The crystal structure of 29 has been determined by single-crystal X-ray diffraction technique at 200K. 29 was crystallised from MeOH - isopropanol. The
absolute configuration around the chiral atom was determined as S by relating to the known stereo centre of the counter ion, (S,S)-tartaric acid. As a
consequence, the configuration of compounds 6,7,10-12,15 was determined to be R. All experimental details and procedures are available in: Barlaam, B.C.;
Degorce, S. L.; Lambert-Van der Brempt, C. M. P.; Lohmann, J.-J. M.; Ple, P. Preparation of phenylpyrrolidinylmorpholinooxochromenecarboxamide
derivatives for use as PI3-kinase inhibitors PCT Int. Appl. (2012), WO 2012140419.
Characterization of compound 10: 1H NMR (CDCl3) 1.96-2.17 (m, 3H), 2.45-2.57 (m, 1H), 3.07-3.83 (m, 14H), 3.83-3.95 (m, 4H), 5.07 (d, 1H), 5.58 (s,
1H), 5.92 (dd, 2H), 6.11 (ddt, 1H), 7.24 (d, 1H), 8.15 (s, 1H); MS-ESI m/z 526 [MH]+; [α]D
: - 4.5° (14.8 mg in 2 mL of acetonitrile); Analytical HPLC,
20°
flow: 1 mL/min, Chiralpak ID, 4.6mm x 250mm, 5µm, eluent: EtOH/MeOH 50:50), tR 17.73 min (other enantiomer tR 13.52 min).
14 Barlaam, B. Cosulich, S.; Delouvrie, B.; Ellston, R.; Fitzek, M.; Germain, H.; Green, S.; Hancox, U.; Harris, C. S.; Hudson, K.; Lambert-van der Brempt, C.;
Lebraud, H.; Magnien, F.; Lamorlette, M.; Le Griffon, A.; Morgentin, R.; Ouvry, G.; Page, K.; Pasquet, G.; Polanska, U.; Ruston, L.; Saleh, T.; Vautier, M.;
Ward, L. Bioorg. Med. Chem. Lett. 2015, 25, 5155.

Table 1. Structure, PI3K cellular activity, human hepatocyte Clint, lipophilicity, solubility and permeability of compounds 5-15.
Caco-2
d
Papp
Mouse p.o.
AUCe
Enantiomeric
statusf
PI3Kβ cell
IC50 (µM)a
0.0032
0.0034
0.0012
0.0012
0.0020
0.0011
0.0023
0.0014
0.0030
0.0045
0.0028
PI3Kα cell
IC50 (µM)a
0.48
Human
hep. Clint
Cmpd
NR1R2
Ar
Log D7.4
b
5
NMe2
NMe2
NMe2
Ph
4-F Ph
3-F Ph
3,5-di-F Ph
3,5-di-F Ph
3,5-di-F Ph
3,5-di-F Ph
3,5-di-F Ph
3,5-di-F Ph
4-F Ph
2.4
-
11
14
15
17
4
19
-
6.0
1.0
-
2.4
0.3
2.8
-
racemic
R
6
0.18
7
8
0.091
0.15
0.87
2.5
2.7
2.0
2.5
2.3
2.9
2.3
2.2
2.2
-
R
racemic
racemic
R
NMe2
33
2.6
18
3.3
-
9
N(Me)CH2CH2OH
4-morpholine
4-Me-1-piperazine
azetidine
10
11
12
13
14
15
0.14
0.25
0.058g
0.63
1.2
2.7
11
9
R
-
R
4-OH-1-piperidine
4-morpholine
4-morpholine
<3
8
6
-
-
racemic
note h
R
-
14
-
1.7
3-F Ph
0.30
a numbers are a geometric mean of 2 or more values.
b human hepatocyte intrinsic clearance, µL/min/106 cells.
c solubility from solid material, phosphate buffer (pH 7.4).
d apparent permeability from A (pH 6.5) to B (pH 7.4) in Caco-2 cell line, 10-6 cm/s.
e AUC after oral dosing (30 µmol/kg): hairy mice
f for compounds isolated as a pure enantiomer, absolute configuration.
g n=1
h Single enantiomer; absolute configuration not determined.

Graphical Abstract
Cyclisation
Conformational lock
PI3K cell IC 0.015
β
M
µ
Improved potency
(PI3K cell IC 0.0011 M)
50
β
µ
50
10
4
Excellent in vivo efficacy
(oral administration)