Discovery of 4-morpholino-pyrimidin-6-one and 4-morpholino-pyrimidin-2-one- containing Phosphoinositide 3-kinase (PI3K) p110β isoform inhibitors through structure-based fragment optimisation

Article abstract of DOI:10.1016/j.bmcl.2012.08.101

The discovery of 4-morpholino-pyrimidin-6-one and 4-morpholino-pyrimidin-2- one-containing inhibitors of Phosphoinositide 3-kinases (PI3K) p110β isoform is reported. Structure-based optimisation of the original fragment hit resulted in lead compounds with improvements in ligand efficiency, lipophilicity efficiency, p110β potency and selectivity over p110α.

Full text of DOI:10.1016/j.bmcl.2012.08.101

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6665–6670
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 4-morpholino-pyrimidin-6-one and 4-morpholino-pyrimidin-2-o-
ne-containing Phosphoinositide 3-kinase (PI3K) p110b isoform inhibitors thr-
ough structure-based fragment optimisation
Fabrizio Giordanetto a, , Andreas Wållberg a, Johan Cassel a, Saswati Ghosal a, Michael Kossenjans a,
⇑
Zhong-Qing Yuan a, Xiaoping Wang b, Lifeng Liang b
a Medicinal Chemistry, AstraZeneca R&D, CVGI iMed, Mölndal, Pepparedsleden 1, SE-431 83 Mölndal, Sweden
b WuXi AppTech, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 7 September 2012
The discovery of 4-morpholino-pyrimidin-6-one and 4-morpholino-pyrimidin-2-one-containing inhibi-
tors of Phosphoinositide 3-kinases (PI3K) p110b isoform is reported. Structure-based optimisation of
the original fragment hit resulted in lead compounds with improvements in ligand efficiency, lipophilic-
Keywords:
ity efficiency, p110b potency and selectivity over p110a.
Fragment-based
Phosphoinositide 3-kinase
PI3K
Ó 2012 Elsevier Ltd. All rights reserved.
p110b
Inhibitor
Phosphoinositide 3-kinases (PI3K) are lipid kinases that have
been involved in a variety of pathological conditions including can-
cer,1–5 thromboembolism,6 immunodeficiency and inflamma-
tion.7,8 Class 1 PI3Ks phosphorylate the 30-hydroxy position of
the inositol ring of Phosphoinositide-4,5-biphosphate (PIP2) to
produce Phosphoinositide-3,4,5-triphosphate (PIP3).9 Class 1A
function in platelets22,23 and mediates ADP-induced platelet aggre-
gation.24 As such, p110b plays a key role in promoting platelet acti-
vation and thrombus formation, and the use of a selective p110b
inhibitor, TGX-221, has resulted in significant anti-thrombotic ef-
fects in different in vivo models.6,25 In the same studies, treatment
with TGX-221 did not prolong bleeding time, contrarily to other
commonly prescribed antiplatelet agents (aspirin and clopidogrel),6
and did not alter heart rate, blood pressure or vascular
conductance.25
PI3Ks comprise the p110
1B includes the p110 catalytic subunit. The p110
genetically linked to the occurrence of cancer,1–5 and a number
of p110
inhibitors have reached clinical trials for cancer.10 Addi-
tionally, p110 is activated by the insulin receptor tyrosine kinase,
a, b and d catalytic subunits whereas class
c
a
isoform was
a
Because of these results, we were interested in developing
chemical series inhibiting p110b. In addition to potent p110b inhi-
a
and was shown to be involved in insulin signalling and glucose
bition, lack of p110
a inhibition or a high degree of selectivity to-
metabolism.11,12
wards p110 was desired. This is because selective p110
a
a
p110b has also been implicated in the development of phospha-
tase and TENsin homologue- (PTEN) deficient tumours using p110b
conditional knock-out mice13 and shRNAs in cells.14 Based on these
findings, a number of groups have recently reported selective p110b
inhibitors with in vitro and in vivo antiproliferative effects including
benzimidazole- and benzoxazole-pyrimidones,15 1,2,4-triazolo[1,5-
a]pyrimidin-7(3H)-ones,16 imidazo[1,2-a]pyrimidin-5(1H)-ones,17
thiazolopyrimidinones18 and 2-amino-pyrimidines.19 Furthermore,
the p110b isoform has been shown to regulate platelet functional
responses. For instance, p110b controls glycoprotein VI-mediated
inhibitors have been shown to block the acute effects of insulin
treatment in vitro and in vivo,11,12 and these effects would not be
compatible with an anti-thrombotic therapy. Selectivity criteria
against other p110 isoforms were not strictly defined, whilst ade-
quate selectivity (>50ꢀ) versus protein kinases in general was
desirable to minimise the risk of side effects.
Building on our previously reported virtual screening results26
and the available PI3K structural framework,27–29 fragment hit 1
(Fig. 1) was evolved with the aim to improve p110b potency and
p110a selectivity, as herein reported.
platelet activation,20,21 potentiates integrin
aIIbb3 adhesive
As outlined in Fig. 2, based on the predicted interaction of 1
with the ATP-binding pocket of p110b, modelled based on homol-
ogy with p110V,26 and the possible substitution vectors on 1, 5
main variation elements could be conceptually identified: (a) the
pyrimidinone scaffold, (b) the carbonyl function, (c,d) the two
⇑
Corresponding author. Tel.: +46 31 7065723; fax: +46 31 7763710.
E-mail address: fabrizio.giordanetto@astrazeneca.com (F. Giordanetto).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.101

6666
F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6665–6670
O
N
Pyrimidin-6-one was the most potent and efficient (LE and LLE)
of the pyrimidinone isomers (cf. 2, p110b IC50 of 1.9 M and 3, 4
l
HN
Table 1). Despite its decreased potency, pyrimidin-2-one still rep-
resented an interesting scaffold owing to its reduced LogD and
adequate LLE (cf. 3 and 2, 4 Table 1). Scaffold hopping from pyri-
midinone to pyridinone increased lipophilicity but didn’t result
in any potency or efficiency advantage (cf. 5 and 2, 4 Table 1).
We then turned our attention to the carbonyl group of our
initial fragment 1. Incidentally, this can also be represented by a
tautomeric hydroxyl group on a fully aromatic ring, but since pyri-
midinones are overall the most abundant tautomers,31,32 mole-
cules will be depicted as lactams in the remainder of this letter.
The carbonyl group of 1 was predicted to interact with K883
(Fig. 2), a residue of catalytic importance due to its interaction with
the triphosphate backbone of ATP.11 We therefore designed a set of
compounds (6–15) to probe such interaction and its effect on
p110b potency, as outlined in Table 2.
Compounds 6–8 were synthesized from methyl 2,6-dichloropy-
rimidine-4-carboxylate by displacement of the most reactive chlo-
rine atom with morpholine (Scheme 2) followed by hydrolysis of
the less reactive one with sodium hydroxide. The ester functional-
ity was then transformed to amide by converting the ester to acid
chloride and then reacting it with amines in DCM (40%). Compound
9 was commercially available.33 Compound 10 was made by heat-
ing 4-chloro-6-morpholinopyrimidin-2-amine34 in benzylamine
(neat) in a microwave oven at 150 °C for 3 h (33%). Compounds
11 and 12 were prepared from N-benzyl-2-chloro-6-morpholino-
pyrimidin-4-amine.35 Heating with KCN in DMSO gave compound
11 (43%). Compound 12 was made from 11 by hydrogenation in a
H-cubeÒ at 1 ml/min, 50 °C, Full H2, Ra/Ni (CatCart 30 mm) in
saturated NH3 in MeOH (52%). Compound 13 was synthesized as
previously described by Lou et al.36 Compounds 14 and 15 were
made from methyl 2,6-dichloroisonicotinate (Scheme 2). Heating
with morpholine followed by palladium catalyzed amination gave
compound 14 (29%), while reduction of the methyl ester with
LiAlH4 gave compound 15 (65%).
N
N
O
p110β IC50 34 μM
LE: 0.38
Figure 1. Virtual screening-derived fragment hit.
methyl groups substituting the amine and (e) the morpholine ring.
We hypothesised that these elements could serve different pur-
poses and designed variations sets accordingly: (a) nitrogen-walk
on the pyrimidinone scaffold to identify the best possible core
structure according to ligand efficiency and ligand lipophilicity
efficiency (LE and LLE, respectively), (b) replacement of the car-
bonyl group to identify optimal interactions with the affinity pock-
et, (c) growth towards the selectivity pocket to increase p110b
potency and selectivity over p110a, (d) growth towards the ribose
binding region to target p110b selective residues and (e) modifica-
tion of the morpholine ring to optimise the interaction with the
hinge region.
Firstly, the 2-(dimethylamino) group of 1 was replaced with a
bulkier 2-(benzylamino) side chain to extend towards the selectiv-
ity pocket and gain potency via increased van der Waals contacts.
This afforded a reference system for the pyrimidinone scaffold
exploration, as summarised in Table 1. In order to prepare all three
2,4,6-trisubstituted pyrimidinone isomers, we decided to start our
synthesis with 2,4,6-trichloro-pyrimidine and then perform a
chromatographic separation. The regiochemical assignments were
confimed with 1D and 2D-NMR experiments. Alkylation with 4-
methoxy benzyl alcohol (Scheme 1) followed by reaction with mor-
pholine gave the three intermediate isomers which were separated
by flash chromatography. Palladium catalyzed amination followed
by deprotection of the 4-methoxy benzylethers yielded compounds
2–4 (20–33%) as well as 16–25 (16–27%). Compound 5 was pre-
pared from 4-(4,6-dichloropyridin-2-yl)morpholine30 following
the methods described in Scheme 1 (step (a) followed directly by
(d) and (e) (29%)).
In the pyrimidin-6-one scaffold, replacement of the carbonyl
group with an unsubstituted carboxamide retained micromolar
potency (6, p110b IC50: 2.5 lM) but didn’t offer an advantage in
terms of LE, while substituted amides (7, 8) resulted in a greater
than 10-fold potency loss, possibly indicating spatial constraints
in the p110b affinity pocket. Substitution of the carbonyl function-
ality with a methyl group significantly decreased affinity (9, p110b
IC50: 130
the carbonyl group with an amino function reduced potency (10,
p110b IC50: 110 M), as did the aminomethyl substituent (12).
lM). In the pyrimidin-2-one ring system, replacement of
l
Complete removal of the carbonyl retained potency (cf. 13 and 3,
Table 1), while substitution to nitrile increased p110b affinity (cf.
11 and 3, Table 1). However, both modifications deteriorated LLE
compared to the carbonyl group and were not considered further.
Introduction of methyl ester (14) and hydroxymethyl (15) groups
in lieu of the carbonyl did not improve p110b potency, as shown
in Table 1. Taken together, the results from this design set con-
firmed the original carbonyl group as the best substituent in terms
of p110b potency, LE and LLE. This information coupled with the re-
sults at a scaffold level, prompted us to further optimise the pyrim-
idin-2-one and pyrimidin-6-one scaffolds. Specifically, we asked
whether a p110b potency improvement as well as selectivity over
p110
a
could be achieved by optimally filling the selectivity pocket,
previously described for p110
c
11 and p110d.29 The occurrence of a
similar conformational rearrangement in p110b has not been veri-
fied yet, due to the lack of appropriate experimental structures of
p110b co-crystallised with inihbitors. However, based on amino
acid sequence similarity across isoforms, we hypothesised that a
similar pocket could be accessible on p110b, if induced by an appro-
priate inhibitor. A designed set expanding the benzyl substituent
Figure 2. Predicted interactions between 1 and a homology model of p110b26 and
postulated variation opportunities.
1 is rendered as sticks (carbon atoms are
coloured in green, oxygen atoms in red, nitrogen atoms in blue). Hydrogen bonds
are displayed as dashed, black lines. Residues numbering based on the p110
39 complex (PDB:2CHW).11
c–PIK-

F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6665–6670
6667
Table 1
p110b potency and ligand efficiencies for the scaffold variation design set
Table 2
p110b potency and ligand efficiencies for the ‘affinity pocket’ exploration set
d
LEb LLEc LogD7.4
R1
Compound Structure
p110b p110
IC50 IC50
M)a M)a
a
W
Y
(
l
(l
TGX-2216
0.049 5.7
0.37 3.7 3.6
N
X
N
H
O
O
HN
Compound R1
TGX-2216
W
X
Y
p110bIC50 p110
M)a M)a
a
IC50 LEb LLEc
2
3
4
5
1.9
29
63
34
12.2
0.37 4.52 1.2
(l
(l
N
N
N
O
N
0.049
NH 1.9
N
N
N
N
5.7
12.2
0.37 3.7
0.37 4.52
0.33
0.26
0.25
H
O
O
O
O
2
6
@O
C(O)NH2
CH
CH
N
N
N
N
N
2.5
23
25
7
8
C(O)NHMe CH
C(O)NMe2 CH
N
HN
>83.3 0.29 4.04 0.5
9
Me
CH
N
N
N
N
130
0.25
N
H
3
@O
CH NH 29
>83.3
>83.3
41.4
>83.3
>83.3
142
0.29 4.04
0.26 2.78
0.36 1.98
0.25 3.66
0.30 3.21
0.29 2.67
0.26 ꢁ0.11
0.27 1.96
10
11
12
13
4
NH2
CN
CH2NH2
H
@O
CH
CH
CH
CH
N
N
N
N
110
1.6
87
O
NH
N
36
283
142
0.27 3.1 1.1
0.29 2.67 1.8
CH NH CH 34
CH
CH
N
N
N
O
N
H
14
15
C(O)OMe
CH2OH
N
N
CH 32
CH 43
240
119
a
Results are mean of at least two experiments. Experimental errors within 20% of
value.37
b
Calculated as -RTln(p110b IC50)/HAC.
Calculated as p110b pIC50–LogD7.4
c
.
N
H
N
H
a
Results are mean of at least two experiments. Experimental errors within 20% of
value.37
Accordingly, bulkier substituents would have provided a better
fit. In the pyrimidin-6-one scaffold, the naphtyl (16) and 2,3-dichlo-
b
Calculated as -RTln(p110b IC50)/HAC.
c
Calculated as p110b pIC50 – LogD7.4
.
rophenyl (17) moieties dramatically improved p110b potency (IC50
:
d
Experimental LogD at pH = 7.4 using a HPLC method previously described.38
93 and 84 nM, respectively). Gratifyingly, ligand efficiency was also
improved. These substitutions also increased selectivity over
p110a, from sixfold (1) to 45- and 35-fold for compounds 16 and
17, respectively. The chlorine atoms walk on the phenyl group
(17–20) did not result in any affinity or selectivity gains, confirming
that the 2,3-dichloro was the preferred substitution pattern,
as shown in Table 3. Introduction of polarity through hydrophilic
(16–25) was therefore synthesized according to Scheme 1, as sum-
marised in Table 3.
The benzyl group of 2 was predicted to partially fill the pocket
originated by the side chain movements of M804 and W812.
PMB
N
PMB
N
Cl
Cl
O
N
O
a
b, c
OH
N
N
+
N
O
Cl
Cl
Cl
N
Cl
Cl
O
Cl
N
N
O
N
N
O
Cl
Cl
PMB
PMB
PMB
N
N
N
d, e
N
O
O
4
O
1
Ratio: 8
:
:
H
N
H
N
H
H
N
H
R
R
O
R
N
O
N
O
N
NH
N
N
N
O
N
O
N
O
4
3, 22-25
2, 16-21
Scheme 1. Preparation of compounds 2–4 and 16–25. Reagents and conditions: (a) Cs2CO3, MeCN, 16 h, rt; (b) morpholine, Cs2CO3, MeCN, 16 h, rt; (c) Separation of isomers
by flash chromatography on silica gel (petroleum ether: EtOAc 20:1); (d) RNH2, Cs2CO3, Pd(OAc)2 (10 mol %), BINAP (1 mol %), dioxane, microwave oven (130 °C, 30 min, N2-
atmosphere); (e) TFA, DCM, 2 h, rt PMB = 4-methoxy-benzyl.

6668
F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6665–6670
H
H
N
N
O
O
R1
Cl
R1
O
N
O
O
N
O
O
b
N
N
a
c
R2
N
N
N
H
N
N
Cl
Cl
N
N
N
N
O
O
Cl
Cl
N
N
Cl
Cl
O
O
6-8
OH
OH
O
O
O
e
d
f
R2
Cl
N
N
N
H
N
N
O
N
O
15
f
O
O
R2
N
H
N
O
14
Scheme 2. Preparation of compounds 6–8 and 14–15. Reagents and conditions: (a) Morpholine, MeOH, 0 °C, overnight; (b) LiOH, MeOH:water 3:1, rt, 2 h then oxalyl chloride,
30 °C, 30 min then R1NH2, triethylamine, DCM, 30 °C, 15 h; (c) R2NH2, Cs2CO3, KI (cat.), MeCN or DMSO, 90 °C, overnight, (d) Morpholine, K2CO3, MeCN, 80 °C overnight; (e)
LiAlH4, THF, rt; (f) R2NH2, K2CO3, Pd(OAc)2 (10 mol %), BINAP (10 mol %), dioxane, microwave oven (130 °C, 30 min, N2-atmosphere).
substituents (e.g. cyano, hydroxymethyl) and heterocycles (e.g.
pyridine, quinoline) was not tolerated and resulted in a weak or
no p110b inhibition (data not shown).The pyrimidin-2-one core
structure displayed a comparable SAR to the pyrimidin-6-one iso-
mer, with naphtyl (22) and 2,3-dichlorophenyl (23) as the most
interesting substituents. Although p110b potency decreased from
Table 3
p110b potency and ligand efficiencies for the ‘selectivity pocket’ fine tuning set
O
X
W
HN
R1
N
O
N
H
Compound
2
R1
W
C
X
p110b IC50
(
l
M)a
p110
a
IC50
(l
M)a
LEb
LLEc
4.52
*
N
1.9
12.2
4.3
0.37
1639
C
N
0.093
0.38
4.63
*
Cl
Cl
Cl
17
18
19
C
C
C
N
N
N
0.084
0.47
0.57
3
0.42
0.39
0.39
4.52
4.42
4.35
*
*
7.9
Cl
4.88
*
Cl
*
20
C
N
1.4
1.5
4.52
3.38
0.35
3.46
Cl
Cl
*
*
21
3
C
N
C
0.35
0.29
3.54
4.04
Cl
N
29
>83.3

F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6665–6670
6669
Table 3 (continued)
Compound
R1
W
N
X
C
p110b IC50
(l
M)a
p110
a
IC50
(
l
M)a
LEb
0.3
LLEc
22
23
24
2.6
>83.3
100
4.09
4.03
3.09
2.55
*
Cl
Cl
N
N
N
C
C
C
1.5
10
0.35
0.26
0.23
*
52.8
146
*
*
25
36
a
Results are mean of at least two experiments. Experimental errors within 20% of value.37
Calculated as -RTln(p110b IC50)/HAC.
b
c
Calculated as p110b pIC50 – LogD7.4
.
As previously observed, pyrimidin-2-one derivatives possessed
lower lipophilicity than their pyrimidin-6-one counterparts (2, 3
Table 1). Additional profiling of compounds in the two pyrimidi-
none regioisomer classes, highlighted differences in solubility,
metabolic stability and reactive metabolite formation, as exempli-
fied in Table 4 for 16 and 22. While pyrimidin-2-ones were gener-
ally less potent at p110b than pyrimidin-6-ones, their lower
Table 4
Selected physicochemical and metabolic parameters for exemplified pyrimidin-6-one
and pyrimidin-2-one derivatives
O
W
HN
N
X
N
H
lipophilicity and better selectivity over p110a resulted in a more
O
favourable profile for future optimisation.
Compound
X, W
16
22
In summary, exploration of the initial fragment hit 1 indicated
the importance of the carbonyl group for interaction with p110b
and identified pyrimidin-6-one and pyrimidin-2-one as suitable
scaffolds for further derivatisation. Expansion of the ligands to-
wards the postulated p110b selectivity pocket resulted in improve-
N, CH
2.4
4.5
41
Yes (0.21)
CH, N
1.5
>100
>115.2
No (0.0)
a
logD7.4
Solubility (
l
M)b
HLM T1/2 (min)c
RMd
ments in p110b potency, selectivity over p110a and LLE, thus
a
Experimental LogD measured at pH = 7.4 using a HPLC method previously
supporting the original hypothesis and providing adequate starting
points for the final structural optimisation and in vivo character-
isation, as detailed in the accompanying paper.
described.38
b
DMSO/HBSS solubility measured at pH = 7.4.
Compound half-life as measured from 300 incubation with human hepatic liver
c
microsomes (0.5 mg/mL).
d
Ratio of reactive metabolite (RM) formation measured from 300 incubation with
human hepatic liver microsomes (1 mg/mL) in the presence of glutathione.40
References and notes
1. Samuels, Y.; Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.; Szabo, S.; Yan, H.;
Gazdar, A.; Powell, S. M.; Riggins, G. J.; Willson, J. K.; Markowitz, S.; Kinzler, K.
W.; Vogelstein, B.; Velculescu, V. E. Science 2004, 304, 554.
2. Levine, D. A.; Bogomolniy, F.; Yee, C. J.; Lash, A.; Barakat, R. R.; Borgen, P. I.;
Boyd, J. Clin. Cancer Res. 2005, 11, 2875.
3. Whyte, D. B.; Holbeck, S. L. Biochem. Biophys. Res. Commun. 2006, 340, 469.
4. 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.
5. Campbell, I. G.; Russell, S. E.; Choong, D. Y.; Montgomery, K. G.; Ciavarella, M.
L.; Hooi, C. S.; Cristiano, B. E.; Pearson, R. B.; Phillips, W. A. Cancer Res. 2004, 64,
7678.
6. 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. E.; Shepherd, 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.
7. Hirsch, E.; Katanaev, V. L.; Garlanda, C.; Azzolino, O.; Pirola, L.; Silengo, L.;
Sozzani, S.; Mantovani, A.; Altruda, F.; Wymann, M. P. Science 2000, 287,
1049.
pyrimidin-6-one to pyrimidin-2-one derivatives (cf. 22 and 16, 23
and 17, Table 3), the fold improvements in potency and lipophilicity
efficiency over the phenyl baselines were similar (cf. 22 and 3 vs. 16
and 2, Table 3). More importantly, the best pyrimidin-2-one com-
pounds offered an improvement in selectivity over p110a, as exem-
plified by 22 (no measurable inhibition at the assay top
concentration) and 23 (66-fold) in Table 3. Introduction of a methyl
group on the benzylic position was not tolerated by the enzyme,
regardless of stereochemistry (24, 25 Table 3). Because of its favour-
able p110b potency and higher lipophilicity than its pyrimidin-2-
one isomer (see below), evaluating the general kinase selectivity
of 16 was particularly important. 16 did not show any measurable
8. Puri, K. D.; Doggett, T. A.; Douangpanya, J.; Hou, Y.; Tino, W. T.; Wilson, T.; Graf,
T.; Clayton, E.; Turner, M.; Hayflick, J. S.; Diacovo, T. G. Blood 2004, 103, 3448.
9. Vanhaesebroeck, B.; Waterfield, M. D. Exp. Cell. Res. 1999, 253, 239.
10. Bradbury, R.H. doi: http://dx.doi.org/10.1002/0471266949.bmc214.
11. 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.
12. Chaussade, C.; Rewcastle, G. W.; Kendall, J. D.; Denny, W. A.; Cho, K.; Grønning,
L. M.; Chong, M. L.; Anagnostou, S. H.; Jackson, S. P.; Daniele, N.; Shepherd, P. R.
Biochem. J. 2007, 404, 449.
inhibition when tested at 1 lM against a large selectivity panel
including 65 different tyrosine, serine/threonine and DNA kinases.
Among the lipid kinases, 16 inhibited p110d and p110V with IC50
values of 0.14 and 18.5 lM, respectively. As the residues predicted
to interact with 16 are virtually conserved across the p110 isoforms,
the observed selectivity profile probably indicates that p110b and
p110d have similar conformational properties, and adapt to the
present ligands in a different manner than p110a and p110V.

6670
F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6665–6670
13. Jia, S.; Liu, Z.; Zhang, S.; Liu, P.; Zhang, L.; Hyun, S.; Zhang, L. J.; Signoretti, S.;
Loda, M.; Roberts, T. M.; Zhao, J. J. Nature 2008, 45, 776.
24. Garcia, A.; Kim, S.; Bhavaraju, K.; Schoenwaelder, S. M.; Kunapuli, S. P. Biochem.
J. 2010, 429, 369.
14. 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.
15. Certal, V.; Halley, F.; Virone-Oddos, A.; Delorme, C.; Karlsson, A.; Rak, A.;
Thompson, F.; Filoche-Rommé, B.; El-Ahmad, Y.; Carry, J. C.; Abecassis, P. Y.;
Lejeune, P.; Vincent, L.; Bonnevaux, H.; Nicolas, J. P.; Bertrand, T.; Marquette, J.
P.; Michot, N.; Benard, T.; Below, P.; Vade, I.; Chatreaux, F.; Lebourg, G.; Pilorge,
F.; Angouillant-Boniface, O.; Louboutin, A.; Lengauer, C.; Schio, L. J. Med. Chem.
2012, 55, 4788.
16. Sanchez, R. M.; Erhard, K.; Hardwicke, M. A.; Lin, H.; McSurdy-Freed, J.; Plant,
R.; Raha, K.; Rominger, C. M.; Schaber, M. D.; Spengler, M. D.; Moore, M. L.; Yu,
H.; Luengo, J. I.; Tedesco, R.; Rivero, R. A. Bioorg. Med. Chem. Lett. 2012, 22, 3198.
17. 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.; Schaber, M. D.; Schulz, M. J.;
Spengler, M. D.; Tedesco, R.; Xie, R.; Zeng, J. J.; Rivero, R. A. Bioorg. Med. Chem.
Lett. 2012, 22, 2230.
25. Sturgeon, S. A.; Jones, C.; Angus, J. A.; Wright, C. E. Eur. J. Pharmacol. 2008, 587,
209.
26. Giordanetto, F.; Kull, B.; Dellsén, A. Bioorg. Med. Chem. Lett. 2011, 21, 829.
27. Walker, E. H.; Perisic, O.; Ried, C.; Stephens, L.; Williams, R. L. Nature 1999, 402,
313.
28. Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.; Hawkins, P. T.; Wymann,
M. P.; Williams, R. L. Mol. Cell. 2000, 6, 909.
29. Berndt, A.; Miller, S.; Williams, O.; Le, D. D.; Houseman, B. T.; Pacold, J. I.;
Gorrec, F.; Hon, W. C.; Liu, Y.; Rommel, C.; Gaillard, P.; Ruckle, T.;
Schwarz, M. K.; Shokat, K. M.; Shaw, J. P.; Williams, R. L. Nat. Chem. Biol.
2010, 6, 117.
30. Tornoee, C.W.; Khanzhin, N.; Rottlaender, M.; Watson, W.P.; Greve, D.R.
WO2006092143, 2006.
31. Kashima, C.; Katoh, A.; Shimizu, M.; Omote, Y. Heterocycles 1984, 22, 2591.
32. Brown, D. J. In Comprehensive Heterocyclic Chemistry; Katritzky, A. R., Rees, C.
W., Eds.; Pergamon: Oxford, 1984; Vol. 3, p 57.
18. Lin, H.; Schulz, M. J.; Xie, R.; Zeng, J.; Luengo, J. I.; Squire, M. D.; Tedesco, R.; Qu,
J.; Erhard, K.; Mack, J. F.; Raha, K.; Plant, R.; Rominger, C. M.; Ariazi, J. L.; Sherk,
C. S.; Schaber, M. D.; McSurdy-Freed, J.; Spengler, M. D.; Davis, C. B.; Hardwicke,
M. A.; Rivero, R. A. ACS Med. Chem. Lett. 2012, 3, 524.
19. Kim, J.; Hong, S.; Hong, S. Bioorg. Med. Chem. Lett. 2011, 21, 6977.
20. Kim, S.; Mangin, P.; Dangelmaier, C.; Lillian, R.; Jackson, S. P.; Daniel, J. L.;
Kunapuli, S. P. J. Biol. Chem. 2009, 284, 33763.
21. Gilio, K.; Munnix, I. C.; Mangin, P.; Cosemans, J. M.; Feijge, M. A.; van der
Meijden, P. E.; Olieslagers, S.; Chrzanowska-Wodnicka, M. B.; Lillian, R.;
Schoenwaelder, S.; Koyasu, S.; Sage, S. O.; Jackson, S. P.; Heemskerk, J. W. J. Biol.
Chem. 2009, 284, 33750.
22. Schoenwaelder, S. M.; Ono, A.; Sturgeon, S.; Chan, S. M.; Mangin, P.; Maxwell,
M. J.; Turnbull, S.; Mulchandani, M.; Anderson, K.; Kauffenstein, G.; Rewcastle,
G. W.; Kendall, J.; Gachet, C.; Salem, H. H.; Jackson, S. P. J. Biol. Chem. 2007, 282,
28648.
33. Interbioscreen Ltd., PO Box 218, Moscow, 119019, Russia.
34. Aurora Fine Chemicals Ltd, Reininghausstrasse 49, Graz, A-8020, Austria.
35. Montebugni, D.; Bravo, P.; Brenna, E.; Mioskowski, C.; Panzeri, W.; Viani, F.;
Volonterio, A.; Wagner, A.; Zanda, M. Tetrahedron 2003, 59, 7147.
36. Lou, G.; Chen, L.; Poindexter, G. S. Tetrahedron Lett. 2002, 43, 5739.
37. Bailey, J.P.; Giles, M.B.; Pass, M. WO2006005914, 2006.
38. Wan, H.; Holmén, A. G. Comb. Chem. High Throughput Screen 2009, 12, 315.
39. NMR and HRMS for compound 16 1H NMR (500 MHz, cdcl3) d 8.05 (d, 1H), 7.81
(dd, 2H), 7.60–7.33 (m, 4H), 7.12 (s, 1H), 4.96 (m, 2H), 4.25 (d, 1H), 3.71–3.55
(m, 4H), 3.31 (s, 4H), 2.60 (s, 1H). 13C NMR (126 MHz, cdcl3) d 165.8, 164.5,
153.6, 134.1, 133.9, 131.7, 128.8, 128.3, 126.6, 126.4, 126.1, 125.7, 123.8, 66.7,
44.8, 43.0. HRMS for C19 H20 N4 O2 337.1670, calcd 337.1664.
40. Ratio obtained by dividing the total integrated peak area of glutathione
adducts of the test compound by the integrated peak area of the major
glutathione adduct of the control compound clozapine (average value, N = 3).
RM formation is ranked as follows: ratio >1 = ‘High’; Ratio between 0.25–
1 = ‘Medium’; Ratio < 0.25 = ‘Low’.
23. Canobbio, I.; Stefanini, L.; Cipolla, L.; Ciraolo, E.; Gruppi, C.; Balduini, C.; Hirsch,
E.; Torti, M. Blood 2009, 114, 2193.