Preparation and optimization of new 4-(morpholin-4-yl)-(6-oxo-1,6- dihydropyrimidin-2-yl)amide derivatives as PI3Kβ inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.08.072

From a HTS campaign, a new series of pyrimidone anilides exemplified by compound 1 has been identified with good inhibitory activity for the PI3Kβ isoform. The structure of compound 1 in PI3Kγ was solved revealing a binding mode in agreement with the SAR observed on PI3Kβ. These compounds displayed inhibition in the nanomolar range in the biochemical assay and were also potent p-Akt inhibitors in a PTEN-deficient PC3 prostate cancer cell line. Optimization of in vitro pharmocokinetic properties led to compound 25 exhibiting 52% bioavailability in mice and target engagement in an acute PK/PD study.

Full text of DOI:10.1016/j.bmcl.2012.08.072

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6381–6384
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Preparation and optimization of new 4-(morpholin-4-yl)-(6-oxo-1,
6-dihydropyrimidin-2-yl)amide derivatives as PI3Kb inhibitors
Victor Certal a, Frank Halley a, , Angela Virone-Oddos b, Fabienne Thompson a, Bruno Filoche-Rommé a,
⇑
Youssef El-Ahmad a, Jean-Christophe Carry a, Cécile Delorme b, Andreas Karlsson c, Pierre-Yves Abecassis d,
Loic Vincent b, Hélène Bonnevaux b, Jean-Paul Nicolas b, Renaud Morales c, Nadine Michot e,
Isabelle Vade a, Audrey Louboutin a, Sébastien Perron a, Gilles Doerflinger a, Bernadette Tric a,
Sylvie Monget a, Christoph Lengauer b, Laurent Schio a
a Medicinal Chemistry Oncology Drug Discovery, Sanofi, 13 quai Jules Guesde, 94400 Vitry-sur-Seine, France
b Biology Oncology Drug Discovery, Sanofi, 13 quai Jules Guesde, 94400 Vitry-sur-Seine, France
c Structural Biology and Molecular Modeling, Sanofi, 13 quai Jules Guesde, 94400 Vitry-sur-Seine, France
d Drug Safety, Sanofi, 13 quai Jules Guesde, 94400 Vitry-sur-Seine, France
e Protein production, Sanofi, 13 quai Jules Guesde, 94400 Vitry-sur-Seine, France
a r t i c l e i n f o
a b s t r a c t
Article history:
From a HTS campaign, a new series of pyrimidone anilides exemplified by compound 1 has been identi-
Received 28 June 2012
Revised 16 August 2012
Accepted 18 August 2012
Available online 25 August 2012
fied with good inhibitory activity for the PI3Kb isoform. The structure of compound 1 in PI3Kc was solved
revealing a binding mode in agreement with the SAR observed on PI3Kb. These compounds displayed
inhibition in the nanomolar range in the biochemical assay and were also potent p-Akt inhibitors in a
PTEN-deficient PC3 prostate cancer cell line. Optimization of in vitro pharmocokinetic properties led to
compound 25 exhibiting 52% bioavailability in mice and target engagement in an acute PK/PD study.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Pyrimidone anilide
Kinase inhibitor
PI3Kb
pAkt
PTEN
Compelling preclinical results have revealed the key role played
by PI3Kb isoform in the tumorigenicity of PTEN-deficient tumors.1,2
This finding supported a strong rationale for the development of
PI3Kb-selective inhibitors for the treatment of PTEN-deficient can-
cer types. Furthermore, isoform-specific PI3K inhibitors may exhibit
better safety profiles compared to pan-PI3K inhibitors. Recently new
publications on PI3Kb selective inhibitors have emerged such as the
thiazolopyrimidinones3 and a series of aminopyrimidine-based
PI3Kb inhibitors, obtained by a pharmacophore-directed design in
order to investigate whether the risk of potential toxicity and insulin
H
H
N
H
N
Cl
F
O
N
N
O
N
O
N
O
N
O
N
O
25
1
Figure 1. Structure of compounds 1 and 25.
resistance associated with the inhibition of PI3Ka
may be reduced.4
and their characterization in biochemical, cellular and in vivo
settings.
Even more recently new series of imidazo[1,2-a]pyrimidin-5(1H)-
ones,5 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones6 and thiazolopyr-
imi-dinone7 have been published together with the disclosure of a
PI3Kb inhibitor currently in Phase I clinical trials8,9 for the treatment
of PTEN deficient tumors.
Following a PI3K High Throughput Screening (HTS) campaign
performed on 550 K compounds, a chemical series was identified
that was subsequently modified to deliver the pyrimidone anilide
1 (Fig. 1).
This Letter describes the discovery and the optimization of a no-
vel series of PI3Kb isoform selective low molecular mass inhibitors
Compound 110 exhibited potent activity on PI3Kb (IC50 = 42 nM),
high ligand efficiency11 (LE = 0.44) and selectivity over other Class I
PI3K isoforms (IC50 = 2138, 118 nM and >10,000 nM on PI3K
a, d and
⇑
Corresponding author. Tel.: +33 (0)1 58 93 36 14; fax: +33 (0)1 58 93 86 14.
E-mail address: frank.halley@sanofi.com (F. Halley).
c, respectively). Compound 1 was inactive on a panel of 28 protein
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.072

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V. Certal et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6381–6384
kinases including mTOR (IC50 >10 lM). The effect of different aniline
substitutions on the biochemical PI3Kb activity of compound 1 was
investigated. A cellular assay measuring the inhibition of Akt phos-
phorylation (pAkt) in PTEN-deficient PC3 prostate cancer cell line
was used to assess compound cellular activity. Akt phosphorylation
could be inhibited at both S473 (pAkt-S473) and T308 (pAkt-T308)
residues with the same activity, and pAkt-S473 inhibition was used
for further structure–activity relationship. Biochemical and cellular
data obtained on the different pyrimidone anilides are summarized
in Table 1. When plotting the biochemical activity on PI3Kb against
the cellular activity, a trend emerged presenting a rough correlation
between the biochemical and cellular activity (r2 = 0.572, see Fig. 2)
within experimental variability suggesting an overall acceptable cell
penetration for this set of compounds.
Results analysis showed that for the para substitution (R4), the
PI3Kb potency was following the order F > H > Cl, (compounds 6,
1, 9, respectively), resulting in a 3-fold gain in potency when H
was replaced by F.
Substituting at the ortho position (R2) with either methyl
(compound 7) or fluorine (compound 4) for hydrogen resulted in
compounds having 6 times less and 3.5 times less potency, respec-
tively. However, compared to 1 compound 7 had increase delta/beta
selectivity from 2.8 to 18-fold, respectively.
Figure 3. X-ray structure of compound 1 bound to PI3Kc, (pdb code 4G11). For
Met804, the density was null, hence the wire representation.
At the meta position (R3) different substituents were introduced,
including fluorine, chlorine, methyl, methoxy and bromine (com-
pounds 5, 10, 8, 11 and 12, respectively) that led to important activ-
ity modulation. Introduction of bromine led to a 8-fold increase in
activity while methyl and methoxy were 2-fold more active, chlo-
rine maintained activity and fluorine led to a 2-fold drop in activity.
The preceding results were rationalized with the crystal struc-
be flexible since no electron density was observed beyond the Cb
atom.
With the assumption that compound 1 would adopt the same
binding mode in PI3Kb, there would be very limited room for para
substituents (R4 in Table 1) larger than fluorine since they would
clash with the backbone of the p-loop enveloping the phenyl moi-
ety. The detrimental introduction of an ortho substituent (R2 in Ta-
ble 1), could be rationalized by an adverse twist around the
dihedral angle formed between the phenyl and the amide planes,
rotating the phenyl ring away from its optimum position and thus
disrupting the stacking against the indolyl of Trp812. Based on
these analyses, the meta position (R3 in Table 1) appeared to be
the most suited for substitution and thus most significant improve-
ment where found here.
Three of the more active compounds were obtained when com-
bining the para fluorine with different substituents at the R3 posi-
tion such as methyl, chlorine or methoxy (compounds 14, 15 and
16, respectively). These compounds exhibiting similarly remark-
able inhibitory effects on PI3Kb reaching the single digit nanomolar
IC50 value in the biochemical assay. This additive effect was not ob-
served for the bromo derivative compound 17, and electron with-
drawing groups, for instance the methyl ester (compound 18) or
the nitro (compound 19) did not restore potency. Worthy of note,
compound 15 demonstrated strong cellular activity with an IC50 of
19 nM on pAkt-S473 phosphorylation.
ture of compound 1 in PI3Kc (Fig. 3). The structure revealed com-
pound 1 in the ATP binding pocket with the morpholine moiety
making the canonical hinge interaction with the NH backbone of
to be consistent with the NH Line hydrogen bond distance of the
main chain NH of Asp964. As previously observed for PI3Kd specific
inhibitors bound to PI3Kc
12 and more recently to PI3Kd13, the side
chain of Met804 (Met752 in PI3Kd) is displaced to make the so-
called selectivity pocket accessible for ligand binding. The bound
compound 1 adopted a non planar geometry with the phenyl part
occupying the selectivity pocket where it stacked against Trp812.
Despite moderate X-ray resolution (3.4 Å), the electron density
for compound 1 was sufficiently clear to allow for assignment of
the binding mode. However, the Met804 side chain appeared to
Methylation of the pyrimidone N–H (compound 20) led to a 10-
fold drop in activity (469 vs 42 nM on PI3Kb which might be due to
a slight clash between the methyl and the close residue Asp964.
This loss could be compensated, to some extent only, with the
introduction of the para fluorine 21 and the best meta substituents
identified previously as in compounds 22, 23, 24 and 25. The most
active product from this sub-series was compound 25, this mole-
cule being comparable to compound 1 in biochemical and cellular
activity but displaying a more pronounced selectivity versus PI3Kd.
Compounds having PI3Kb IC50 <100 nM and pAkt <500 nM were
then further evaluated for thermodynamic solubility14 and preli-
minary in vitro pharmacokinetic properties including permeability,
microsomal stability and CYP3A4 inhibition (see results in Table 2).
All selected compounds had molecular weight (MW) below 425
and ClogP below 2.6, and none had any significant inhibitory activ-
ity on recombinant human CYP3A4, up to 40 lM. Aqueous solubil-
ity was suboptimal, considering the low MW and ClogP; strong
crystal packing was suspected because of the high melting point
Figure 2. PI3Kb biochemistry pIC50 and pAkt cellular pIC50 correlation in the
pyrimidone anilide series for compounds 1, 4–8, 10, 12–15, 17, 20–22, 24, 25.

V. Certal et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6381–6384
6383
Table 1
Biochemical and cellular activity of pyrimidone anilides
R1
R2
H
N
R3
R4
O
N
N
O
N
O
Compound
R1
R2
R3
R4
MW
PI3Kaa
PI3Kba
PI3Kda
PI3Kca
pAkta,b
1
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
F
H
H
Me
H
Cl
OMe
Br
F
Me
Cl
OMe
Br
CO2Me
NO2
H
H
F
Br
OMe
Cl
H
H
H
F
H
H
Cl
H
H
H
F
F
F
F
F
F
F
H
F
F
314
332
332
332
328
328
328
349
344
393
350
346
367
362
411
390
377
328
346
364
425
376
381
2138
>10,000
6218
2870
>10,000
626
2273
2553
2373
1905
3845
831
1900
3460
4140
8143
4353
42
142
81
13
236
17
398
33
15
5
53
6
2
3
26
175
47
118
>10,000
>10,000
>10,000
>10,000
>10,000
7070
68
349
74
31
586
45
1420
100
47
10
55
71
19
98
38
1745
1255
340
110
100
94
2450
1108
86
4270
164
2783
278
611
189
135
63
137
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5090
>10,000
>10,000
>10,000
>10,000
8010
6710
>10,000
6610
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
8280
148
297
4647
3360
4290
1470
1770
2215
2400
2210
H
Me
Me
Me
Me
Me
Me
>10,000
>10,000
8510
7610
>10,000
>10,000
469
244
262
387
104
42
F
F
F
100
79
a
IC50 values in nM are reported as the mean from at least two independent experiments.
pAkt-S473 inhibition in PC3 cell line.
b
Table 2
In vitro and calculated properties of active compounds 1, 5, 6, 8, 10–17, 25
Compound
MW
PI3Kb IC50 (nM)
pAkt IC50 (nM)
ClogP
PSAa
Solubility pH 7.4
lM
CaCo2 Papp (nm/s)
MLMb%
HLMc
rhCYP3A4e
IC50 lM
Labilityd
%Labilityd
1
5
6
8
10
11
12
13
14
15
16
17
25
314
332
332
328
349
344
393
350
346
367
362
411
381
42
81
13
17
33
15
5
53
6
2
68
74
31
45
100
47
10
55
71
19
98
38
79
1.1
1.6
1.5
1.5
1.8
1.3
2.3
1.9
1.9
2.3
1.6
2.6
2.6
83
83
83
83
83
92
83
83
83
83
92
83
74
73
-
45
3
9
38
6
3
3
3
33
5
5
0
6
14
8
3
14
8
6
8
4
44
55
24
80
43
76
75
25
91
40
71
42
44
40
—
40
—
40
40
40
—
40
40
40
40
40
21
27
54
22
47
38
11
60
14
28
18
12
3
26
42
2
8
66
7
a
b
c
Polar surface area.
MLM = mouse liver microsome.
HLM = human liver microsome.
%Lability = % of compound metabolized after 20 min incubation.
Recombinant human CYP3A4.
d
e
(mp >260 °C for most compounds). Permeability measurements in
CaCo2 cell lines TC7 clone,15 were found to be low (Papp <20 nm/s)
without direct correlation (r2 = 0.04) with polar surface area (PSA)
or ClogP (r2 = 0.32).16 Transport in both apical to basolateral (A–B)
and basolateral to apical (B–A) directions across the cell monolayer
was assessed, enabling the determination of the efflux ratio which
was found to be high (ratio = 26) for the N–H pyrimidone 12 and
low (ratio = 4) for the N–Me pyrimidone 25. Overall, it was ob-
served that for the N–H pyrimidones, low permeability was linked
to strong efflux ratio. Metabolic lability was variable in mouse and
human liver microsomes across compounds. Compound 25 which
exhibited a higher permeability and acceptable mouse liver micro-
some lability was selected for further pharmacological studies.
In vivo administration of compound 25 in female SCID (Severe
Combined Immune Deficient) mice showed a bioavailability of
52% ( Fig. 4). Following iv administration at 3 mg/kg, clearance
was found moderate (Cl = 1.6 L/h/kg), relative to hepatic blood flow
(5.2 L/h/kg), and volume of distribution (Vdss = 1.3 L/kg) suggested

6384
V. Certal et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6381–6384
10000
1000
100
10
activity and selectivity, as well as important cellular potency and
acceptable in vitro pharmacokinetic properties. This high LE chem-
ical series was efficiently prepared in one step from the carboxyl-
3mg/Kg iv.
10mg/Kg po.
ate salts 2 and 3. X-ray structure of compound 1 bound to PI3Kc,
revealed an interaction with Trp812 in a specificity pocket formed
by a shift of Met804. This binding mode is generally consistent
with the SAR obtained in PI3Kb. This series was optimized to com-
pound 25 showing improved selectivity and good pharmacokinetic
properties, with a bioavailability of 52% in SCID mice. This com-
pound induced pAkt inhibition in PTEN-deficient PC3 prostate tu-
mors in SCID mice for at least 1 h but this effect was not
maintained for 6 h. Further optimizations are needed to improve
PK properties and reach sustained target modulation in tumors.
1
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Time (h)
Figure 4. Compound 25 pharmacokinetic at 3 mg/kg iv and 10 mg/kg po admin-
istrations in mice.
Acknowledgment
Table 3
We thank Dr. Carlos Garcia-Echeverria for his guidance and
thorough proof-reading of the manuscript.
PK exposure from compound 25 PK/PD study in PC3 mice xenograft model
Dose
(mg/kg)
Time
(h)
Plasma (
lM)
Tumor
M)
p-Akt (% inh.)
T308/S473
(l
References and notes
30 mg/kg Solution
1
6
1
6
10
11
1
6
86/91
23/0
63/73
0/0
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4.5
1.6
244 mg/kg Suspension
1.7
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R2
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R4
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H2N
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N
O
R4
a
N
O
N
O
Scheme 1. Reagents and conditions: (a) EDCI, DMF, rt, 4–5 h, Yield 30–70%.
significant tissues distribution. Following oral administration at
10 mg/kg, terminal elimination half-life was found to be moderate
(T1/2 = 1.4 h).
Compound 25 was then evaluated in a preliminary acute phar-
macodynamic experiment at two doses and two time points. Com-
pound 25 was administered orally in solution at 30 mg/kg (highest
soluble dose) and in suspension at 244 mg/kg (highest tolerated
dose) in SCID mice bearing PTEN-deficient PC3 prostate tumor
xenografts. Target modulation in tumor tissue was assessed by
measuring Akt phosphorylation levels (on T308 and S473 residues)
at 1 and 6 h after single administration. Results reported in Table 3
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least 1 h, but no effect was detectable at 6 h for both dosages.
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pound exposure in tumor tissue needed to be above 4 lM to reach
more than 50% pAkt inhibition, and (b) the compound was poorly
absorbed in suspension.
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The synthesis for substituted pyrimidinones
presented in Scheme 1.
1 and 25 is
16. Waring, M. L. Expert Opin. Drug Disc. 2010, 5, 235. and references therein..
17. In a typical experiment, to a solution of 1 mmol of 2 or 3 in 4 ml of N,N-
dimethylformamide were added 2 mmol of pyridine, 1.3 mmol of N-[3-
(dimethylamino)propyl]-N0-ethylcarbodiimide hydrochloride (EDCI) and
2 mmol of the corresponding aniline. The reaction mixture was stirred at
ambient temperature for 4–5 h and then evaporated to dryness under reduced
pressure. Water and ethyl acetate were added and the resulting mixture was
stirred for 30 min. The precipitate formed was filtered off and, when necessary,
purified by flash chromatography eluting with a gradient of MeOH in CH2Cl2.
All the final compounds reported herein have been obtained in
only one step synthesis, by reacting the commercially available
substituted anilines with the corresponding sodium carboxylate
salts 2 and 310 in presence of the coupling agent 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) in N,
N-dimethylformamide.17
Following a HTS campaign, a chemical series of pyrimidone ani-
lides was identified, that showed significant PI3Kb biochemical