Discovery of phosphoinositide 3-kinases (PI3K) p110β isoform inhibitor 4-[2-hydroxyethyl(1-naphthylmethyl)amino]-6-[(2S)-2-methylmorpholin-4-yl]-1H- pyrimidin-2-one, an effective antithrombotic agent without associated bleeding and insulin resistance

Article abstract of DOI:10.1016/j.bmcl.2012.08.102

Structure-based evolution of the original fragment leads resulted in the identification of 4-[2-hydroxyethyl(1-naphthylmethyl)amino]-6-[(2S)-2- methylmorpholin-4-yl]-1H-pyrimidin-2-one, (S)-21, a potent, selective phosphoinositide 3-kinases (PI3K) p110β isoform inhibitor with favourable in vivo antiplatelet effect. Despite its antiplatelet action, (S)-21 did not significantly increase bleeding time in dogs. Additionally, due to its enhanced selectivity over p110α, (S)-21 did not induce any insulin resistance in rats.

Full text of DOI:10.1016/j.bmcl.2012.08.102

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6671–6676
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of phosphoinositide 3-kinases (PI3K) p110b isoform inhibitor
4-[2-hydroxyethyl(1-naphthylmethyl)amino]-6-[(2S)-2-methylmorpholin-4-yl]-
1H-pyrimidin-2-one, an effective antithrombotic agent without associated
bleeding and insulin resistance
Fabrizio Giordanetto a, , Andreas Wållberg a, Saswati Ghosal a, Tommy Iliefski a, Johan Cassel a,
⇑
Zhong-Qing Yuan a, Henrik von Wachenfeldt a, Søren M. Andersen a, Tord Inghardt a, Anders Tunek b,
Sven Nylander c
a Medicinal Chemistry, AstraZeneca R&D, CVGI iMed, Pepparedsleden 1, SE-431 83 Mölndal, Sweden
b DMPK, AstraZeneca R&D, CVGI iMed, Pepparedsleden 1, SE-431 83 Mölndal, Sweden
c Bioscience, AstraZeneca R&D, CVGI iMed, Pepparedsleden 1, SE-431 83 Mölndal, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 September 2012
Structure-based evolution of the original fragment leads resulted in the identification of 4-[2-hydroxy-
ethyl(1-naphthylmethyl)amino]-6-[(2S)-2-methylmorpholin-4-yl]-1H-pyrimidin-2-one, (S)-21, a potent,
selective phosphoinositide 3-kinases (PI3K) p110b isoform inhibitor with favourable in vivo antiplatelet
effect. Despite its antiplatelet action, (S)-21 did not significantly increase bleeding time in dogs. Addition-
Keywords:
Fragment-based
Phosphoinositide 3-kinase
PI3K
ally, due to its enhanced selectivity over p110
a
, (S)-21 did not induce any insulin resistance in rats.
Ó 2012 Elsevier Ltd. All rights reserved.
p110b
Inhibitor
Thromboembolism
Anti-thrombotic agent
Insulin resistance
Phosphoinositide 3-kinases (PI3K) are a family of lipid kinases
that regulate signal transduction mechanisms involved in cell
regulation and function. Three different classes of PI3Ks (Class I,
II and III) have been reported to date. Class I PI3Ks phosphorylate
the 30-hydroxy position of the inositol ring of phosphatidylinosi-
tol-4,5-biphosphate (PIP2) to produce phosphatidylinositol-3,4,
5-triphosphate (PIP3).1 Class I PI3Ks have been subsequently
arterial thrombosis without a corresponding increase in bleeding
time, as observed for aspirin and clopidogrel.20
A program with the aim to develop selective p110b inhibitors
for the treatment of arterial thrombosis was thus initiated in our
laboratories. Here, selectivity against the p110a isoform was of
special importance, because of its implication in insulin signalling
and risk to cause insulin resistance.7,8 The program revolved
classified into Class IA, comprising the p110
a, b and d catalytic sub-
around the hypothesis that selectivity over p110a could be
units and Class IB (p110
c
catalytic subunit). p110
a
was shown to
achieved by exploiting differences in the conformational plasticity
of the isoforms, as already demonstrated for p110c7 and p110d.21
In particular, it was hypothesised that the selectivity pocket in-
be implicated in cancer by genetic analysis2–6 and also to regulate
insulin signalling and glucose metabolism.7,8
A role for p110b in the development of phosphatase and TENsin
homologue-(PTEN) deficient tumors has been established9,10 and
several chemical classes of p110b inhibitors have been recently re-
ported as anti-proliferative agents.11–15 Additionally, p110b regu-
lates platelet activation via different mechanisms, including
duced by a conformational arrangement in p110c and p110d could
be accessible on p110b as well, based on sequence similarities.
Consequently, an appropriate inhibitor could induce such a pocket
and selectively discriminate between p110b and p110a isoforms.
Based on our previously reported hit identification22 and evolu-
tion23 efforts, the discovery and in vivo characterisation of 4-[2-
hydroxyethyl(1-naphthylmethyl)amino]-6-[(2S)-2-methylmorph-
glycoprotein VI16,17 and integrin IIbb3.18,19 Importantly, treatment
a
with the selective p110b inhibitor TGX-221 significantly reduced
olin-4-yl]-1H-pyrimidin-2-one,
a
novel p110b inhibitor with
significant selectivity over p110
and minimised bleeding and insulin resistance is herein described.
a, promising antithrombotic effect
⇑
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.2012.08.102

6672
F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6671–6676
O
O
O
N
N
HN
HN
HN
N
N
N
N
N
H
N
N
H
O
O
O
3
1
2
p110β IC50 2600 nM
p110β IC50 34 μM
p110β IC50 93 nM
LE: 0.3; LLE: 4.09
LE: 0.38
LE: 0.38; LLE: 4.63
LogD7.4: 1.5
LogD7.4: 2.4
Solubility: >100 μM
Solubility: 4.5 μM
HLM T1/2: >115 mins
HLM T1/2: 41 mins
Reactive metabolites: No
Reactive metabolites: Yes
Figure 1. Original fragment hit (1)22 and derived pyrimidin-6-one (2) and pyrimidin-2-one (3) fragment leads.23 p110b potency measured using AlphaScreen35 LE: ligand
efficiency (ꢀRTln(IC50)/number of heavy atoms). LLE: ligand lipophilicity efficiency (pIC50 – LogD7.4).26,27 LogD7.4 measured using HPLC.36 DMSO/HBSS solubility measured at
pH = 7.4.36 HLM T1/2: compound half-life in human liver microsomes preparations (0.5 mg/mL). Reactive metabolites formed after 300 incubation with human liver
microsomes (1 mg/mL) in the presence of glutathione.41
Table 1
p110b potency and ligand efficiencies for compounds in the ‘hinge binder’ variation
set
O
HN
R1
N
N
H
d
Compound R1
p110b
IC50
p110
IC50
a
LEb
LLEc LogD7.4
a
a
(l
M)
(lM)
*
N
N
N
2
0.093
0.034
4.3
2.2
0.38 4.63 2.4
0.39 4.57 2.9
O
O
*
4
*
5
24
82.6
0.25 1.92 2.7
O
*
N
Figure 2. Predicted interactions between 2 and a homology model of p110b22 and
postulated variation opportunities. Compound 2 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
6
31
160
0.25 2.91 1.6
0.43 5.55 2.6
OH
*
7
0.007
0.84
N
p110c
–PIK-39 complex (PDB: 2CHW).7
*
N
8
11
62.3
0.28 3.07 1.9
N
H
Structure-based evolution of the initial fragment hit 1, identi-
fied via structure-based virtual screening of the AstraZeneca frag-
ment collection22 resulted in lead compounds 2 and 3 with
complementary in vitro profiles.23 While the pyrimidin-6-one
derivative 2 possessed adequate p110b potency and suboptimal
solubility and metabolic properties, the pyrimidin-2-one isomer
3 showed decreased p110b affinity but a more favourable profile,
as shown in Figure 1. According to the predicted interaction
between 2 and a homology model of p110b22 and the structure–
activity relationships derived for the affinity and selectivity pock-
ets23 two areas for further chemical explorations remained
(Fig. 2). Because of time constraints, it was decided to (a) employ
the pyrimidin-6-one core for variations targeting the hinge region
and, in parallel, (b) use the pyrimidin-2-one scaffold bearing the
standard 4-morpholino hinge binder motif to target the ribose
pocket (Fig. 2). The aim was then to combine the best structural
variations obtained from these two tracks in the final lead
compound.
a
Results are mean of at least two experiments. Experimental errors within 20% of
value.35
b
Calculated as ꢀRTln(IC50 p110b)/HAC.
c
Calculated as pIC50 (p110b)—LogD7.4
.
d
Experimental LogD at pH = 7.4 using HPLC.36
followed by hydrolysis with sodium hydroxide (Scheme 1). The
mono chloro pyrimidine intermediate was used in both reactions
with amines (4–6) and in Suzuki couplings with boronic acids
(7–8). Final step yields were generally poor for both reactions
(4–23% and 5–11%, respectively).
Introduction of a methyl group on morpholine to yield 2-meth-
ylmorpholine derivative 4 improved p110b potency to 34 nM and
selectivity over p110a from 46- to 65-fold without deteriorating li-
gand efficiencies (cf. 4 and 2, Table 1). 3-Methoxyazetidine (4) and
piperidin-4-ol (5) analogs drastically reduced p110b affinity to
micromolar levels. While a 4-pyridyl substituent was predicted
to be an effective hinge binder, CYP450 inhibition was likely to
result from such chemical modification. Therefore, a 2-methyl-4-
pyridyl substituent (7) was directly evaluated to sterically hinder
the pyridine’s nitrogen and minimize any potential CYP450
Compounds 4–8 were designed to modulate the supposed
hydrogen-bond interaction with the hinge region of p110b
(Fig. 2), as shown in Table 1. Synthesis started by alkylating 4,6-
dichloropyrimidin-2-amine with 1-(bromomethyl)naphthalene,

F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6671–6676
6673
Cl
Cl
Cl
O
R
O
N
N
N
NH
NH
N
NH
NH
Cl
Cl
a
b
c
NH
N
N
NH2
Scheme 1. Preparations of compounds 2 and 4–8. Reagents and conditions: (a) 1-(bromomethyl)naphthalene, K2CO3, MeCN, microwave 150 °C, 20 min, 27%. (c) NaOH,
DMSO, water microwave 120 °C, 20 min, 51%. (c) For amines: DCM, microwave 120 °C, 20 min 4–23%. For arylations: boronic acid, Na2CO3, (PPh3)2PdCl2, dioxane/water 5:1,
80 °C 6 h, 5–11%.
Table 2
p110b potency and ligand efficiencies for the ‘ribose pocket’ exploration set
O
N
HN
N
N
R1
O
M)a
p110
a
IC50
(
l
M)a
p110a/p110b selectivity
LEb
LLEc
LogD7.4
d
Compound
R1
p110b IC50
(l
3
9
H
Me
Et
cPr
2.6
0.6
0.13
0.97
0.09
0.1
>83.3
212
82.8
76
30.8
51.5
73.7
112
149
56.9
205
>83.3
>83.3
>32x
355x
628x
78x
340x
524x
373x
808x
690x
58X
0.30
0.33
0.35
0.29
0.34
0.33
0.30
0.32
0.30
0.26
0.26
0.25
0.26
4.09
3.86
4.38
3.41
5.49
5.26
4.90
4.86
4.17
3.01
2.69
4.11
4.40
1.5
2.37
2.5
2.6
1.55
1.75
1.8
2
2.5
3
3.15
1.4
1.5
10
11
12
13
14
15
16
17
18
19
20
(CH2)2OH
(CH2)3OH
(CH2)4OH
CH2CH(Me)OH
CH2CH(Et)OH
CH2CH(CF3)OH
CH2C(Me2)CH2OH
(CH2)2NMe2
(CH2)3NMe2
0.2
0.14
0.22
0.98
1.4
3.1
1.2
143x
>26x
>64x
a
b
c
Results are mean of at least two experiments. Experimental errors within 20% of value.35
Calculated as ꢀRTln(p110b IC50)/HAC.
Calculated as pIC50 (p110b)—LogD7.4
.
d
Experimental LogD at pH = 7.4 using HPLC.36
O
atoms by heating with the amine in a microwave oven followed
by adding an excess of morpholine and heating again gave the final
products in one step (Scheme 2). The amines were purchased from
commercial sources or synthesized via reductive amination of
1-naphthaldehyde, as described in the Supplementary data.
Although the yields are moderate at best (3–31%) this provided a
practical and very efficient route to the desired compounds and
their preliminary in vitro evaluation.
OH
HN
N
a, b
N
N
+
NH
R1
N
R1
N
Cl
Cl
O
Scheme 2. Preparations of compounds 9–20. Reagents and conditions: (a) NMP,
microwave oven (120 °C, 15 min). (b) morpholine (30 equiv) microwave oven
(120 °C, 30 min), 3–31%.
Progressive increase of size and lipophilicity of the substituent
inhibition. Unfortunately, while 7 offered the most potent p110b
inhibitor in the set (IC50: 7 nM, Table 1), it was also an effective
improved p110b potency and selectivity over p110
representing the best compromise (10, p110b IC50
p110 /p110b: 628-fold, Table 1), as the bulkier cyclopropyl deriv-
a
with ethyl
:
130 nM,
CYP450 inhibitor (CYP3A4 IC50: 0.7
l
M and CYP2C9: 0.4
lM) and
a
was not pursued further. The pyrazole analog 8 had only moderate
potency, as displayed in Table 1.
Substitution on the amine to target the pocket surrounding the
ribose moiety of ATP, based on superimposition of the homology
ative (11) displayed decreased affinity. Introduction of a hydroxyl
group (12–14) was tolerated and positively contributed to
ligand-lipophilicity efficiency (LLE26,27(12, p110b IC50: 130 nM,
p110a/p110b: 628-fold, LLE: 5.49, Table 1). The steric bulk of the
model of p110b with previously reported p110
c
crystal structures
side chain was accepted to some extent (15–16) but potency dete-
riorated when the group became too large (17–18). Replacement of
the alcohol with a basic amino group (19–20) markedly reduced
potency, as shown in Table 2.
Overall the alcohol side chains provided ligands with adequate
potency, excellent selectivity and low lipophilicity. A decision to
combine the best LLE side chain (12) with 2-methylmorpholine,
previously identified as an effective hinge region binder, was then
taken and the corresponding enantiomeric pair, exemplified by (S)-
21, was synthesized according to Scheme 3. Here, 2,4,6-trichloro-
pyrimidine was partially hydrolyzed with solid sodium hydroxide
(PDB: 1E8X and 2CHW)7,24 was then explored. Based on conforma-
tional analysis of the ligands, substitution on the naphtylmethan-
amine’s nitrogen atom was predicted to stabilize the propeller-like
ligand conformation required for interaction between the naphtha-
lene ring and the selectivity pocket (Fig. 2). Additionally, the pres-
ence of polar residues offered opportunities for additional
protein–ligand interactions. Design of compounds 9–20 served to
verify those assumptions, as shown in Table 2.
Compounds 9–20 were synthesized in a one pot procedure from
4,6-dichloropyrimidin-2-ol.25 Displacement of one of the chlorine

6674
F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6671–6676
H
N
O
OH
a
O
O
Cl
OH
HN
N
HN
N
d
c
b
N
N
N
N
N
N
N
Cl
O
Cl
Cl
Cl
Cl
OH
(S)-21
OH
Scheme 3. Preparation of (S)-21. Reagents and conditions: (a) H2NCH2CH2OH, NaBH(OAc)3, DCM, 70%; (b) NaOH, THF, 41%; (c) naphthalene aminoalcohol, DIPEA, ACN, reflux,
38%; (d) (S)-2-methylmorpholine, DIPEA, ACN, 70 °C, 69%.
Table 3
p110b potency and ligand efficiencies for the enantiomeric pair 21
M)a
p110
27.2
a
IC50
(l
M)a
p110
97.4
c
IC50
(l
M)a
p110d IC50
(l
M)a
LEb
LLEc
5.52
LogD7.4
d
Compound
R1
p110b IC50
(l
O
N
N
HN
(S)-21
0.038
0.168
0.35
1.9
N
N
N
O
O
OH
O
HN
(R)-21
1
349
0.28
4.1
1.9
N
OH
a
b
c
Results are mean of at least two experiments. Experimental errors within 20% of value.35
Calculated as ꢀRTln(p110b IC50)/HAC.
Calculated as pIC50 (p110b)—LogD7.4
.
d
Experimental LogD at pH = 7.4 using HPLC.36
in THF and 4,6-dichloropyrimidin-2-ol precipitated upon addition
of water. In two steps the chlorides were substituted with naph-
thalene aminoalcohol and subsequently with commercially avail-
able (S)-2-methylmorpholine to afford the final product (S)-21.
The naphthalene aminoalcohol was prepared by reductive amina-
tion of 1-naphthaldehyde with 2-aminoethanol in a good yield,
as detailed in the Supplementary data.
Table 4
In vitro/vivo profile of (S)-21
(S)-21
195
400
2.1
a
pAKT IC50 (nM)
Hu collagen-induced PRP aggregation IC50 (nM)
b
c
Hu ADP-induced whole blood platelet aggregation IC50
Dog ADP-induced whole blood platelet aggregation IC50
(
l
M)
M)
c
(l
3.8
Solubilityd
(
lM)
100
>173
No (0.0)
30
1/2
18
e
Profiling of the two isomers indicated that p110b potency
resided in the S enantiomer of 21, delivering a potent p110b inhib-
hHEP T1/2 (min)
RMf
g
Dog PPB Fu (%)
itor with favourable selectivity towards p110a (>700-fold), as
Dog dose iv/poh
(lmol/kg)
shown in Table 3. (S)-21 was also very selective towards p110
c
Dog CLh (mL/min/kg)
Dog Fh (%)
25
(>2500-fold), whilst only 4.4-fold selective against p110d. These
findings are in line with previous observations from different
chemical series of p110b inhibitors, showing clear selectivity over
a
b
c
N = 2, SD = 0.04.37
N = 4. SD = 0.1.38
a and p110c
but not towards p110d.11–15 It is interesting to
N > 3, SD=0.3.39
p110
note that the so-called ‘selectivity pocket’ could be induced on
both p110
and p110d7,24 and possibly on p110b.11 However, the
DMSO/HBSS solubility measured at pH = 7.4.36
d
e
Compound half-life as measured from 300 incubation with human liver
c
microsomes (1 mg/mL).40
conformational and energetic properties regulating the formation
of those pockets could vary across isoforms, with p110b and
p110d having similar characteristics and adapting to the present
f
Ratio of reactive metabolite (RM) formation measured from 300 incubation with
human liver microsomes (1 mg/mL) in the presence of glutathione.41
g
% Fraction unbound in plasma measured by equilibrium dialysis (18 h at 37 °C).
Pharmacokinetic parameters calculated from noncompartmental analysis con-
and p110c 11
.
The thera-
h
ligands in a distinct manner than p110
a
centrations in fasted Beagle male dogs (iv/po N = 2).
peutic consequences of the reported p110b/p110d selectivity
profiles will need to be assessed further, due to p110d’s known
involvement in regulating the immune system.28
gen-induced platelet aggregation in human platelet rich plasma
(IC50: 400 nM). (S)-21 also inhibited ADP-induced platelet aggrega-
tion in human and beagle dogs whole blood (IC50: 2.1 and 3.8 lM,
As listed in Table 4, (S)-21 showed functional inhibitory activity
in cell-based systems (IC50: 195 nM) and was able to inhibit colla-

F. Giordanetto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6671–6676
6675
an increase in bleeding time only apparent at the highest concen-
tration (ECBT:2: 14.8 1.2
M)33These results are indicative of a
l
favourable anti-thrombotic—bleeding separation, when compared
to previously published antiplatelet agents.31
From the onset of the program, minimisation of potency against
p110a
, due to its demonstrated role in insulin signalling7,8 was of
special importance. Having established its promising pharmacody-
namic and safety profile in dogs, (S)-21 was evaluated for its effects
on insulin-mediated pharmacology. Interestingly, when pre-
incubated with human adipocytes, (S)-21 did not inhibit insulin
induced glucose uptake in the tested concentration interval (3–
40
selective p110b/
previously reported by our group, which significantly inhibited
glucose uptake with an IC50 of 4.4
M.32 Building on this promising
in vitro findings, (S)-21 was then dosed to rats to monitor in vivo
effects on Glucose, Insulin and the relative Homeostatis Model
Analysis (HOMA) insulin resistance index.34 Here, when four con-
l
M). This represented a step improvement over AZD6482, a less
a
inhibitor (IC50: 0.01 and 0.87 M, respectively)
l
Figure 3. PKPD relationship between the plasma concentration of (S)-21 (lM),
l
ex vivo inhibition of whole blood ADP-induced platelet aggregation (j, solid line)
and fold increase in bleeding time (N, dashed line), following administration to
Beagle dogs (N = 2).33
secutive, increasing doses of (S)-21 (0.2, 0.4, 0.8 and 1.6 lg/kg/
min) were administered to rats24the relative HOMA index did
not change from baseline, while a significant, concentration-
dependent increase was observed with AZD6482, as displayed in
Figure 4. When considering the plasma concentrations of the two
compounds in such a study (Fig. 4), these results neatly reflected
the differences in p110
As hypothesised earlier, the improved p110b/
a
potency between (S)-21 and AZD6482.
selectivity profile
a
of (S)-21 reduced the likelihood of interference with insulin-
related pathways and represents a significant advantage from a
safety and developability perspective.
In summary, structure-guided evolution of the initial fragment
leads resulted in the identification of 4-[2-hydroxyethyl(1-naph-
thylmethyl)amino]-6-[(2S)-2-methylmorpholin-4-yl]-1H-pyrimidin-
2-one (S)-21, the first example of a potent p110b isoform inhibitor
with exquisite selectivity over p110a, favourable in vivo
antithrombotic effect, minimised risk for bleeding and insulin
resistance.
Figure 4. PKPD relationship between compound plasma concentrations (lM) and
the relative homeostasis model analysis (HOMA) insulin resistance index (the
product of plasma glucose and plasma insulin) in SD rats. Data represents
means SEM (N = 6)34AZD6482 is depicted as red squares, (S)-21 as black squares.
A relative HOMA index of 3 (dashed line) represents a statistically significant
change from baseline.
Acknowledgments
We thank the following people: Jan-Arne Björkman and Helen
Zachrisson for Folts dog experiments; Elham Nikookhesal and
Göran Wahlund for rat HOMA index experiments; Malin Enerbäck,
Britt-Marie Wissing, Agnes Leffler, Åke Jägervall, Fredrik Wågberg,
Anna-Karin Asztély, and Helena Lindmark for in vitro platelet func-
tion experiments. Eva-Marie Andersson, Johanna Ehnebom and
Tony Clementz for insulin induced glucose uptake experiments.
We also thank the anonymous referees for providing constructive
comments and helpful suggestions.
respectively), as shown in Table 4. Furthermore, (S)-21 showed no
inhibition of ion channels implicated in cardiovascular function
(hERG, Nav1.5, IKs, Cav1.2, Kv4.3) at the highest tested concentra-
tion (31.6
a selectivity panel consisting of 98 different proteins when tested
at 10 M. The CYP450 2C9 isoform was the only CYP isoform inhib-
ited by (S)-21 (IC50 = 4 M) when tested up to 20 M. In line with
l
M).29 (S)-21 did not display any significant binding in
l
l
l
previously reported pyrimidin-2-one derivatives from the same
series, (S)-21 possessed adequate solubility and in vitro metabolic
stability and did not form any reactive metabolites (Table 4).Owing
to the favourable in vitro profile, (S)-21 was evaluated in terms of
its in vivo antithrombotic effect, with special emphasis on the sep-
aration between antithrombotic effect and bleeding risk, using a
modified Folt’s model, as previously described30–32
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.08.102.
References and notes
Here, based on in vitro dog anti-platelet potency (whole blood
ADP-induced aggregation, IC50: 3.8 lM), dog plasma protein bind-
ing (Fu: 30%) and the preliminary dog PK profile (Table 4), 5
ascending doses of (S)-21 were administered intravenously (bolus
15.8–591.8 lg/kg and infusion 8.7–378.7 lg/kg/min) to anaesthe-
tized beagle dogs (N = 2), as detailed in Ref. 33. The pharmacody-
namic–pharmacokinetic (PKPD) relationship between the plasma
exposure of (S)-21 and its effects on ex vivo platelet aggregation
and bleeding time is summarized in Figure 3. (S)-21 resulted in a
concentration-dependent inhibition of ex vivo platelet aggregation
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The experiment consisted of a control period of 30 min, followed by five
consecutive 30-min periods with increasing doses of test compound. In the
control period, animals were treated intravenously with vehicle (30%
cyclodextrin). In the subsequent 30-min periods, test compound was
administered as bolus infusions under 1 min, followed by
a continuous
infusion for 29 min. Bleeding time (BT) measurements were obtained
starting at 15 min into each infusion period by making a 12-mm-long and 1-
mm-deep incision in the tongue. Blood was blotted from the incision
continuously with flat swabs 2.5 ꢁ 2.5 cm (Medett, Penafiel, Portugal). BT
was measured from the moment the tongue was incised until no blood
appeared on the swab, and the maximal observation period was 15 min. If
bleeding had not stopped within 15 min, a BT of 15 min was assigned to enable
data analysis. Blood for ex vivo platelet aggregation and for determination of
test compound plasma concentration was taken 20 min into each infusion
period. For each test compound dose, the following data were obtained: test
compound plasma exposure (lM), ex vivo platelet aggregation (% inhibition),
bleeding time (Fold increase). These values have been used to derive the
pharmacodynamic-pharmacokinetic (PKPD) relationships. EC50 for platelet
aggregation was defined as the test compound plasma concentration at which
platelet aggregation was inhibited by 50% and was calculated with GraphPad
Prism software using the sigmoidal dose–response function. ECBT:2 for bleeding
time was the test compound plasma concentration at which bleeding time was
increased by 2-fold and was calculated with GraphPad Prism software using
the exponential growth function.
14. 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.
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34. Overnight fasted Sprague–Dawley (SD) rats (N = 6) were infused with 4
consecutive escalating doses of test compound during 30 min. A blood sample
at the end of each dose interval gave plasma exposure, as well as glucose and
insulin levels, the product of which was used to calculate the relative
homeostasis model analysis (HOMA) insulin resistance index. The HOMA
index reported here has been normalized to the vehicle-treated control group
(N = 4) and to median values before treatment.
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antibody) in breast adenocarcinoma MDA-MB-468 cells (1500 cells/40 ll/well)
following incubation with test compound (2 h), using the AcumenÒ Explorer
laser scanning fluorescence microplate cytometer. MDA-MB-468 cells are
maintained in DMEM supplemented with 10% heat inactivated foetal calf
serum and 1% L-glutamine (200 mM). Media is removed from the flask, cells are
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A.; Prabaharan, H.; Thompson, P. E.; Smith, G. E.; Shepherd, P. R.; Daniele, N.;
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resuspended in media to a final density of 1500 cells/well in a volume of 40
ll
(3.75 ꢁ 104 cells/ml). 40
l
l of cells are added to each well of a plate (Greiner)
using a Wellmate or Multidrop. Plates are incubated overnight at 37 °C, 5% CO2.
38. Blood was collected by venipuncture from healthy donors into vaccutainer
tubes containing sodium citrate (final concentration in blood 11 mM). Platelet
rich plasma (PRP) light transmission aggregometry (LTA) was evaluated using
the PAP-8 (Biodata, Horsham, PA, USA) aggregometer and in an in house
developed 96 well plate assay.32
39. Blood was collected by venipuncture from healthy donors or beagle dogs into
vaccutainer tubes containing sodium citrate (final concentration in blood
11 mM). Whole blood impedance aggregometry was evaluated using the
Multiplate (Dynabyte, Munich, Germany) impedance aggregometer. Whole
blood shear induced platelet adhesion and aggregation was evaluated using
the cone and plate analyzer (CPA), Impact-R (DiaMed, Yokneaam, Israel).32
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25. Koppel, H. C.; Springer, R. H.; Robins, R. K.; Cheng, C. C. J. Org. Chem. 1961, 26,
792.
40. Test compounds (1
l
l of 10 mM DMSO stock solution) are further diluted with
M and final solvent concentrations
ACN/H2O (1:1) to a final concentration of 2
l
26. Leach, A. R.; Hann, M. M.; Burrows, J. N.; Griffen, E. J. Mol. BioSyst. 2006, 2, 429.
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28. Fruman, D. A.; Bismuth, G. Immunol. Rev. 2009, 228, 253.
29. Patch clamp assay using IONWORKS™ technology in CHO cells expressing the
relevant human ion channel. For Cav1.2: Patch clamp assay using IONWORKS™
technology in CHO cells expressing CACNA1C, CACNB2 and CACNA2D1
(ChanTest, Cleveland, OH), as in Dilbaghi, S.; Abi-Gerges, N.; Morton, M. J.;
Bridgland-Taylor, M. H.; Pollard, C. E.; Valentin, J.-P. J. Pharmacol. Toxicol.
Methods 2010, 62, e1.
in the incubation plates of 0.01% DMSO and 1% ACN. Hepatocyte suspension (2
million cells/ml) is manually added to 96-well plates. Cells are also added to
the blanks, which are placed on a separate plate. Five plates, representing a
single time point, for example, 2, 15, 30, 45 or 60 min, are pre-incubated for
10–23 min at 37 °C. Reactions are started by adding 25
the cells, starting with the longest time point, giving
concentration of 1 M and a cell concentration of 1 million cells/ml. Directly
l
l of 2
l
M substrate to
a
final substrate
l
after compound addition the plate is automatically shaken vertically for 15 s
and horizontally for 15 s (at 500 rpm). The plate is placed in the Cytomat 2C15
30. Folts, J. Circulation 1991, 83, 3.
31. van Giezen, J. J.; Berntsson, P.; Zachrisson, H.; Björkman, J. A. Thromb. Res. 2009,
124, 565.
32. Nylander, S.; Kull, B.; Björkman, J. A.; Ulvinge, J.-C.; Oakes, N.; Emanuelsson, B.
M.; Andersson, M.; Skärby, T.; Inghardt, T.; Fjellström, O.; Gustafsson, D. J.
Thromb. Haem. [Epub ahead of print], http://dx.doi.org/10.1111/j.1538-
7836.2012.04898.x.
incubator. The incubation media used is William’s
25 mM HEPES and 2 mM -glutamine and set to pH 7.4 at 37 °C (5% CO2 and
>80% humidity). The reactions are stopped with three volumes of ice-cold stop
solution (acetonitrile containing 0.8% formic acid and 1 M of a volume marker
(1 5,5-diethyl-1,3-diphenyl-2-iminobarbituric acid). The plates are
E supplemented with
L
l
lM
centrifuged at 4 °C and 3220g for 20 min. The supernatant is diluted 1:1 with
water and analysis is carried out by LCMS.
33. Beagle dogs (N = 2, Rååhöjden, Sweden) were anesthetized, intubated, and
ventilated. The right femoral artery was carefully dissected free, and branches
were ligated and cut. Blood flow was measured continuously with a 1.5-
mmultrasonic transit-time flow probe (Transonic Systems Inc, T206, Ithaca,
NY, USA). The distal part of the vessel intima was damaged, and the flow was
reduced with a variable occluder to obtain a pattern of cyclic flow reductions.
41. 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 and
1 = ‘Medium’; ratio <0.25 = ‘Low’.