Discovery and Pharmacological Characterization of Novel Quinazoline-Based PI3K Delta-Selective Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.6b00119

Inhibition of the lipid kinase PI3Kδ is a promising principle to treat B and T cell driven inflammatory diseases. Using a scaffold deconstruction-reconstruction strategy, we identified 4-aryl quinazolines that were optimized into potent PI3Kδ isoform selective analogues with good pharmacokinetic properties. With compound 11, we illustrate that biochemical PI3Kδ inhibition translates into modulation of isoform-dependent immune cell function (human, rat, and mouse). After oral administration of compound 11 to rats, proximal PD markers are inhibited, and dose-dependent efficacy in a mechanistic plaque forming cell assay could be demonstrated.

Full text of DOI:10.1021/acsmedchemlett.6b00119

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Letter
Discovery and Pharmacological Characterization of
Novel Quinazoline-based PI3K delta-selective Inhibitors
Klemens Hoegenauer, Nicolas Soldermann, Frederic Stauffer, Pascal Furet, Nadege Graveleau, Alexander
B. Smith, Christina Hebach, Gregory J. Hollingworth, Ian Lewis, Sascha Gutmann, Gabriele Rummel,
Mark Knapp, Romain M Wolf, Joachim Blanz, Roland Feifel, Christoph Burkhart, and Frederic Zecri
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00119 • Publication Date (Web): 02 Jun 2016
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Discovery and Pharmacological Characterization of Novel
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Klemens Hoegenauer*a, Nicolas Soldermann*a, Frédéric Stauffera, Pascal Fureta, Nadege Graveleaua,
Alexander B. Smitha, Christina Hebacha, Gregory J. Hollingwortha, Ian Lewisa, Sascha Gutmannb, Ga-
briele Rummelb, Mark Knappe, Romain M. Wolfa, Joachim Blanzc, Roland Feifelc, Christoph Burkhartd
and Frédéric Zécria
aGlobal Discovery Chemistry, bCenter for Proteomic Chemistry, cMetabolism and Pharmacokinetics, dAutoimmunity, Transplantation and
Inflammation, Novartis Institutes for BioMedical Research, Novartis Campus, CH-4002 Basel, Switzerland; eGlobal Discovery Chemistry,
5300 Chiron Way, Emeryville, California 94608, USA.
system has been demonstrated in genetically modified mice
ABSTRACT: Inhibition of the lipid kinase PI3Kδ is a promising
(PI3KδD910A/D910A knock-in and PI3Kδ-/-)12-14 and through the use
principle to treat B- and T-cell driven inflammatory diseases.
of specific tool compounds in in vitro and in vivo preclinical
Using a scaffold deconstruction–reconstruction strategy, we iden-
models.15-17 The strong biological rationale led us and others18-21
tified 4-aryl quinazolines that were optimized into potent
PI3Kδ isoform selective analogues with good pharmacokinetic
properties. With compound 11, we illustrate that biochemical
to initiate a medicinal chemistry program aimed at the discovery
of PI3Kδ−selective inhibitors with suitable properties and efficacy
to allow for development as an anti-inflammatory therapeutic.
PI3Kδ inhibition translates into modulation of isoform dependent
Our efforts towards the identification of PI3K inhibitors which
are selective for the delta isoform started with imidazoquinoline 1
(BEZ235, Table 1), a pan-PI3K/mTOR inhibitor with potent anti-
tumor activity.22 We had a good understanding of the binding
mode of imidazoquinoline 1 based on docking studies of numer-
ous compounds of this scaffold into a homology model of PI3Kα
which was built based on the reported x-ray structure of PI3Kγ
kinase domain.23 Using a similar homology model for PI3Kδ, we
hypothesized that we would gain selectivity by reaching out to the
non-conserved amino acid residues that line the entrance of the
ATP binding site (see Supplementary Figure 3). To this end, we
first deconstructed the imidazoquinoline to the quinazoline frag-
ment 2a (Table 1) to remove the structural elements which inter-
act in the selectivity pocket and contribute significantly to the
strong affinity for mTOR. Quinazoline 2a contains some crucial
core elements for PI3K binding affinity, namely the position-1
nitrogen acting as a hinge binder and the quinoline which fills the
affinity-pocket (conserved over all PI3K isoforms). We detected
submicromolar activity for PI3Kα, only, making this compound a
very ligand efficient PI3Kα inhibitor. Some marginal activity on
PI3Kδ was retained whilst the affinity towards all other PI3K
isoforms as well as mTOR was above the highest test concentra-
tion. Introduction of a 4-phenyl substituent (2b) increased the
activity for PI3Kα and δ considerably with some emerging selec-
tivity over the β and γ isoforms. Modification of the substitution
pattern of the phenyl group led to some additional favorable mod-
ulation of the PI3Kδ (higher) and mTOR (lower) activity, exem-
plified by dimethoxyphenyl analogue 2c. Modelling suggested
that the best exit vector to selectively interfere with the non-
conserved residues at the entrance of the PI3K ATP binding site
(selectivity pocket) would be from the meta-position of the newly
introduced phenyl group. In particular, our intention was to form a
salt bridge with the non-conserved D832 side chain of the PI3Kδ
isoform with a hydrogen bond donor (e.g. a protonated amine) on
the ligand (see Supplementary Figure 3). We probed this concept
with a small library and in that way identified piperazine amide
immune cell function (human, rat and mouse). After oral admin-
istration of compound 11 to rats, proximal PD markers are inhib-
ited, and dose-dependent efficacy in a mechanistic plaque forming
cell assay could be demonstrated.
KEYWORDS Phosphoinositide-3-kinase delta inhibitor,
PI3Kδ inhibitor, lead optimization, structure-activity rela-
tionship, PK/PD studies, B cell inhibition
Phosphoinositide-3-kinases (PI3K) are lipid kinases responsible
for the generation of the second messenger phosphatidylinositol
(3,4,5) triphosphate (PIP3) from the substrate phosphatidylinositol
(4,5) biphosphate (PIP2) leading to phosphorylation of Akt and
subsequent progression of cell differentiation, proliferation, motil-
ity and survival.1,2 PI3K have been classified according to their
structural and functional properties. The class I enzymes can be
divided into IA and IB PI3K. In mammals, class IA PI3K includes
three different isoforms: PI3Kα, PI3Kβ, and PI3Kδ while class IB
includes PI3Kγ. These enzymes are heterodimers consisting of the
p110 catalytic subunits (p110α, p110β, p110γ, and p110δ) and a
regulatory subunit (most commonly p85α).3
During the past decade there has been a great deal of interest in
the development of inhibitors of the class I PI3K family. Initially,
broad-spectrum inhibitors of the α, β, γ and δ isoforms were tar-
geted as potential oncology therapeutics.4–6 However, the recogni-
tion that the γ and δ isoforms are predominantly expressed in
leukocytes and play a critical role in T and B cell function7 led to
speculation that selective inhibitors of these isoforms could offer
potential as therapeutics for the treatment of allergic and inflam-
matory diseases.8 Moreover, specifically targeting PI3Kδ should
avoid potential side effects associated with the ubiquitously ex-
pressed PI3Kα and β isoforms.9-11 PI3Kδ is expressed primarily
in hematopoietic cells and its functional relevance in the activa-
tion of leukocytes of the adaptive as well as the innate immune
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between 2 and 3 to increase our chances for success. Bis-amides
2d as a lead compound, demonstrating potency on PI3Kδ and
significant selectivity over all other Class I PI3K isoforms as well
as mTOR. Based on these encouraging results, we began to opti-
mize substituents of the quinazoline in positions 4 and 6 (for syn-
thetic details, see supporting information).
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7-11 are neutral and do not show any activity on the hERG chan-
nel up to 30 µM. For many compounds, the piperazine acylation
also led to significant metabolic stability increase in microsomal
preparations.
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Table 2 shows that the biochemical activity of piperazine amide
2d translates well to Rat-1 cells that have been transfected with
myrisoylated, and therefore constitutively active, PI3Kα, β and δ
isoforms without significant activity drop. Isoform selectivity
observed for biochemical assays is maintained in the cellular as-
says. The cellular activity on PI3Kδ was also replicated in murine
splenocytes (measured as inhibition of anti-IgM-induced expres-
sion of the costimulatory molecule CD86 on B cells), yet a ca. 5-
fold activity drop was observed. Due to its moderate lipophilicity
and positive charge, piperazine amide 2d is very soluble at neutral
pH. Areas identified for improvement were interference with the
hERG channel as well as metabolic instability in rat liver micro-
somes.
As already mentioned, our initial design hypothesis to achieve
isoform selectivity was based on forming a salt bridge between
the ligand and D832. Neutral derivatives like acetylated pipera-
zines 7-11 are not able to offer a hydrogen bond donor, and we
were initially surprised by the observed selectivity. In order to
elucidate the molecular reason, we co-crystallized compound 11
with PI3Kα and PI3Kδ, and these structures have been determined
to 3 Å and 2.9 Å, respectively (see supporting information). In
general, the structures reveal an overall fold similar to previous
structures solved in complex with other inhibitors28. The ligand is
bound in the ATP site of the kinase domains, and forms an identi-
cal, direct hydrogen bond to V828 (δ) or V850 (α) in the hinge
and with K779 (δ) or K802 (α) in the affinity-pocket of the active
site (see Supplementary Figure 2). Water-mediated hydrogen
bonds are formed with the side chain oxygens of D787 and Y813
in the affinity pocket and with the backbone carbonyl oxygen of
S813 at the ATP binding site cleft, as observed in the slightly
better resolved PI3Kδ structure. The acetyl-piperazine group of
the ligand stacks to W760 in the PI3Kδ structure (Figure 1, right
panel), which leads to strong attractive dispersion forces. In
PI3Kα, this interaction is not possible because R770- in contrast
to the smaller T750 in the δ isoform - blocks the access to the
corresponding tryptophan W780 and the ligand protrudes from the
ATP binding pocket in an extended conformation (Figure 1, left
panel). The solvent-exposed, electrostatic interaction between
R770 and compound 11 in PI3Kα cannot compensate the van der
Waals interactions between the acetyl-piperazine ring and the
protein in the PI3Kδ complex resulting in a preference of com-
pound 11 for the PI3Kδ isoform. Force field refinements of the
original coordinates29,30 induced only minor local changes and
provide further support for these observations. Binding free ener-
gies that were calculated by MM/GBSA computations on the
energy-refined crystal structures of compound 11, using the Am-
ber Generalized-Born (GB) options igb = 2 or 5 confirm a prefer-
ence of PI3Kδ by 1.7 (igb=5) to 2.1 (igb=2) kcal/mol.31
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We found that the activity on PI3Kδ could be preserved to a large
extent by replacing the position-6 quinoline motif of 2d with less
lipophilic monocyclic features. A selection of these analogues is
shown in Table 2 (compounds 3d-6d). Whereas compounds 3d,
4d and 6d still displayed significant selectivity towards the other
PI3K isoforms, introduction of a meta-CF3 substituent (5d) led to
a potency increase of PI3Kα and β in addition to a substantial rise
in log P. Further translation of the cellular response to the spleno-
cyte assay was generally better for less lipophilic derivatives,
perhaps due to less unspecific binding to plasma proteins. The
combination of the basic N-methyl piperazine and aromatic rings
led to an overlap with the well-known hERG pharmacophore and
thus to a substantial inhibition in a dofetilide binding assay.24
Decreasing the activity on the hERG channel was possible by
lowering lipophilicity (e.g. compound 4d and 6d) assessed by a
high-throughput (HT-) logP assay,25 but a complete suppression, a
clear project requirement, could not be achieved for these basic
piperazine amides. Modulation of the microsomal turnover26 for
these derivatives also turned out to be possible to some degree,
although overall clearance still remained high. In a rat PK exper-
iment, compound 6d showed substantial clearance (CL = 33
mL·min-1·kg-1, see Supplementary Table 1). Metabolite identifica-
tion studies with 6d suggested major metabolism around the pi-
perazine moiety leading us to conclude that a major clearance
reduction would not be possible without modifying this metabolic
weak spot, ultimately leading to the introduction of a second am-
ide functionality (Table 3). Pyridine 6e and piperidine 6f illustrate
that the bridging phenyl ring can also be replaced by saturated or
unsaturated heterocyclic rings, but as for all other analogues the
impact on hERG and metabolism remained moderate.
Compound 11 showed selectivity against 3 lipid kinases, 40 pro-
tein kinases and 36 targets of an internal safety panel (full list, see
Supporting Information). As it was the compound with the most
promising overall in vitro profile, it was selected for further
pharmacological characterization including its effects on isoform-
dependent immune cell function. Since PI3Kδ has been described
as essential for B cell activation and function,14,32,33 we measured
the effect of compound 11 on human and rodent B cell activation.
Our data demonstrate a consistent and potent inhibition of PI3Kδ-
mediated B cell functions across different species (Table 4). Prox-
imal readout parameters such as pAkt were equally well inhibited
as further downstream events such as surface activation markers
CD69 /CD86 or proliferation.
We then discovered that the terminal piperazine N-methyl group
could be substituted by short acyl groups. Table 3 outlines that N-
acetyl piperazines 7, 8, 9 and 11 show an excellent potency profile
on PI3Kδ and selectivity profile over other PI3K isoforms. As for
the N-methyl piperazine 5d, addition of a meta-CF3 group in the
position-6 aromatic ring of the quinazoline was detrimental to
isoform selectivity (compound 10), presumably due to an H-bond
between one of the fluorine atoms and the catalytic lysine. Due to
the increased PSA of bis-amides compared to their mono-amide
counterparts we noticed that PAMPA effective permeability
(logPe in 10-6 cm∙sec-1)27 at neutral pH dropped, and that transla-
tion into cellular assays needed to be closely monitored. Com-
pound 7 shows an >5-fold potency drop in the mouse splenocyte
assay, a trend we noticed also for other compounds with HT-logP
around 3 or above (data not shown), presumably due to an in-
creased likelihood for unspecific binding. These observations led
us to preferentially place compounds into the HT-logP window
To assess whether compound 11 was suitable for in vivo studies,
we tested its pharmacokinetic properties in rats and dogs (Table
5). At an oral dose of 3 mg/kg, compound 11 showed a moderate
22% oral bioavailability in rats. While a higher total exposure
could be reached with increasing the dose to 30 mg/kg, this over-
all gain was not dose-linear, and bioavailability dropped to 10%,
likely due to solubility limited absorption. In dogs, overall phar-
macokinetic properties for compound 11 were similar (low clear-
ance and low volume of distribution), with a somewhat better
42% oral bioavailability at 0.3 mg/kg. Overall, we concluded that
compound 11 had adequate pharmacokinetic properties to assess
the translation of inhibiting B cell activation in vitro to the in vivo
situation. To this end, PK/PD studies were performed in rodents
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following a single oral dose of compound 11 (30 mg/kg) in male
*(K.H.)
Tel:
+41
79
6181814.
E-mail:
klem-
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Lewis rats. Blood was collected at various time points, and
changes for two PD biomarker expression profiles as well as drug
levels were determined (Figure 2). In agreement with the PK ex-
periment in Sprague Dawley (SD) rats, compound concentration
in Lewis rat blood reached a maximum after 2 hours with blood
levels around 5 µM. A clear relationship of drug exposure and
inhibition of Akt phosphorylation and anti-IgM/rIL-4-induced
CD86 expression was observed. Within the test samples, the high-
est pAkt inhibition (88%) was observed 6 hours after compound
administration, whereas inhibition of CD86 expression was at its
maximum of 71% already after 2 hours.
ens.hoegenauer@novartis.com.
*(N.S.) Tel: +41 79 8451506. nicolas.soldermann@novartis.com.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
We thank I. Adam, J. Andre, I. Jeulin, D. Rageot, R. Strang, B.
Thai and V. Tucconi for their synthetic contributions, D. Fabbro,
D. Haasen, A. Hinz, U. McKeever and P. Schmutz for their efforts
towards developing and running biochemical and cellular assays,
C. Beerli for pharmacokinetic assay work, B. Shrestha for protein
expression, R. Elling for crystallography support as well as N. G.
Cooke and G. Weckbecker for scientific guidance. We are grate-
ful to the MX beamline team at the Swiss Light Source (PSI Vil-
ligen, Switzerland) for outstanding support at the beamline and
Expose GmbH (Switzerland) for diffraction data collection.
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As a consequence of these encouraging results on the proximal
PD markers, compound 11 was profiled in an ex vivo plaque
forming cell (PFC) assay.34 This mechanistic assay quantitatively
determines the antibody-producing B cell response following
immunization with sheep red blood cells (SRBC). After four days
of compound treatment, rats were sacrificed and inhibition of
plaques formed due to the SRBC specific B cell antibody re-
sponse was assessed via the PFC assay (figure 3). At a dose of 10
mg/kg bid, plaque formation (vehicle mean 4.18.105 PFC/spleen)
was inhibited by 79% (95% confidence interval [CI] = 56 to 92
percentage points, p<0.001). At 30 mg/kg bid, an inhibition of
94% (95% CI = 89 to 97 percentage points, p<0.001) was
achieved comparable to the inhibition seen with the positive con-
trol CsA (Cyclosporin A, 95% CI = 84 to 98 percentage points,
p<0.001). Full statistics can be found in the supporting infor-
mation.
ABBREVIATIONS
BAV, absolute oral bioavailability; hERG, human ether-à-go-go-
related gene; MM/GBSA, molecular mechanics energies com-
bined with the generalized Born and surface area continuum solv-
ation method; mTOR, mechanistic target of rapamycin; PFC,
plaque forming cell(s), PI3K, Phosphoinositide-3-kinase; SD rats,
Sprague Dawley rats; SRBC, sheep red blood cell(s).
In summary, we have identified novel quinazoline based PI3Kδ
selective inhibitors that we could optimize into cellularly potent
derivatives. With compound 11 we profiled a candidate with good
basic pharmacokinetic properties, however we observed an un-
derproportional exposure increase at higher doses very likely
caused by limited solubility. Compound 11 demonstrated that the
PI3Kδ-dependent inhibition of B cell activation observed in vitro
and in rodent PK/PD studies translates into the full inhibition of B
cell function, namely the T-dependent specific antibody response
in vivo. These results confirmed our initial target hypothesis and
encouraged us to continue our search for a suitable development
candidate.
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Corresponding Authors
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Silengo, L.; Altruda, F.; Wetzker, R.; Wymann, M. P.; Lembo, G.; Hirsch,
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Figure 1. Co-crystal structures of PI3Kα (left panel) and
PI3Kδ (right panel) in complex with compound 11. The pro-
tein is shown as grey surface and the inhibitor is represented
as green sticks. Whereas compound 11 stacks onto W760 in
PI3Kδ, R770 in PI3Kα forces the inhibitor to adopt an extend-
ed conformation. Key amino acid residues are highlighted in
color.
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Trunzer, M.; Faller, B.; Zimmerlin, A. Metabolic Soft Spot
Identification and Compound Optimization in Early Discovery Phases
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ACS Medicinal Chemistry Letters
Table 4. Cellular inhibition of PI3Kδ -dependent B cell
1
2
3
4
activation in different species by compound 11.
biochemical IC50 [µM]a
humanb
pAkt
ratc
moused
5
CD86 CD69 pAkt
CD86 Prolif. CD86
6
7
8
9
10
11
12
13
0.029
0.107
0.125
0.089
0.044
0.011
0.070
aMean of a minimum of two independent experiments; standard
b
deviation for pIC50 values <0.3; measured in 90% whole blood;
cmeasured in 50% whole blood; dmeasured in splenocytes
Table 5. PK parametersa for compound 11.
Figure 2. Rat PK/PD for compound 11 at 30 mg/kg p.o. single
Species
Ratb
Ratb
3 (po)
Ratb
Dogc
Dogc
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
dose: inhibition of ex vivo induced B cell activation in 50% whole
blood; PK legend at the left, PD legend at the right; error bars
indicate standard deviation.
Dose [mg·kg-1] 1 (iv)
30 (po) 0.1 (iv) 0.3 (po)
CL
15 (3)
13 (3)
[mL·min-1·kg-1]
VSS [L·kg-1]
t1/2 term. [h]
AUC d.n.d
[nM·h]
2.0 (0.3)
2.0 (0.5)
2315
(537)
1.0 (0.2)
0.9 (0.1)
2798
(715)
500
(168)
22 (7)
237
(24)
10 (1)
1188
(251)
42 (9)
440
(112)
0.8 (0.3)
BAV [%]
Cmax d.n.d
[nM]
48 (14) 20 (4)
Tmax [h]
3.5 (3.1) 5.3 (2.2)
amean values (rat: n = 4, dog: n= 3) with standard deviation in
brackets, bfemale, cmale, dd.n. = dose normalized to 1 mg·kg-1
TOC graphic
N
OMe
O
MeO
N
Figure 3. Dose-dependent inhibition of SRBC-specific IgM re-
sponse by compound 11; error bars indicate standard deviation;
***: p<0.001 compared to vehicle (student’s t-test, two-tailed
distribution).
O
N
N
N
N
N
MeO
NC
N
O
N
N
N
N
N
11
1
2c
PI3K
(BEZ235)
α
δ
δ
inhibitor
PI3K inhibitor
efficacious in vivo
/
inhibitor
pan-PI3K/mTOR
Table 1. Influence of position-4 substituents of the
quinazoline on overall potency, PI3K isoform selectivity
and selectivity towards mTOR
R
O
N
R
N
N
H
O
N
N
N
N
:
:
=
=
2a
2b
2c
2d
R
R
H
OMe
N
N
N
1
biochemical IC50 [µM]a
mTORd
PI3Kαb
PI3Kβb
PI3Kγc
PI3Kδc
1
0.017
0.192
>10
3.2
1.9
>10
0.313
>10
5.6
2.6
>10
0.025
8.1
0.320
0.061
0.049
0.006
>10
0.66
1.8
2a 0.650
2b 0.090
2c 0.095
2d 0.633
5.3
aMean of a minimum of two independent experiments; standard
deviation for pIC50 values <0.3; bKGlo format; cADAPTA format
(values comparable to KGlo); dradiometric protein kinase assay.
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Page 6 of 6
Table 2. Influence of position-4 and -6 substituents of the quinazoline on biochemical/cellular potency, PI3K isoform selec-
tivity, hERG inhibition and in vitro ADME parameters
1
2
3
O
O
O
N
N
N
N
N
MeO
N
N
MeO
N
N
N
4
R
N
N
N
5
6
7
8
N
N
N
2-6d
6e
6f
biochemical IC50 [µM]a
cellular IC50 [µM]a, d
mCD86 PAMPA hERG HT- RLM HT-
f
j
IC50
logPe
IC50 logPh CLint solk
R
PI3Kαb PI3Kβb PI3Kγc PI3Kδc PI3Kα PI3Kβ PI3Kδ
[µM]a, e
[µM]g
9
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2d
3d
4d
5d
6d
0.633
0.410
0.283
0.079
0.259
>10
>10
0.740
9.2
0.049
0.014
0.027
0.009
0.020
1.14
1.09
1.56
0.40
1.56
>12.5
4.89
1.91
0.38
1.91
0.038
0.018
0.037
0.010
0.037
0.190
0.026
0.087
0.074
0.087
-3.9
-4.1
-4.2
-3.6
-3.7
2.6
1.9
11
3.1
2.8
1.9
3.9
2.2
>700 >1.0
MeO
2.57
2.63
0.392
4.15
357
231
213
605
0.85
0.42
0.85
>1.0
MeO
MeO
N
NC
MeO
N
N
9.3
1.6
12
F3C
MeO
7.9
6e
6f
0.462
0.399
6.18
2.04
>10
4.5
0.100m 2.49
7.54
4.62
0.036
0.070
0.112
0.086
-4.4
-3.8
26
1.5
1.9
ndn
201
130
>1.0
>1.0
0.064m 1.82
aMean of a minimum of two independent experiments; standard deviation for pIC50 values <0.3; bKGlo format; cADAPTA format (val-
ues comparable to KGlo); dinhibition of pAkt formation in Rat-1 cells; einhibition of anti-IgM induced mCD86 expression on mouse sple-
nocytes; feffective permeability [10-6 cm∙sec-1] (pH = 6.8), n=1; ginhibition of [3H]dofetilide binding to hERG transfected membranes, n=1;
hHigh-throughput logP measurement with immobilized artificial membranes, n=1; intrinsic clearance in incubations of rat liver micro-
j
somes [µL∙min-1∙mg-1 protein], n=1; kHigh-throughput equilibrium solubility determination [mM] (pH = 6.8); mlow Hill coefficient; nnd =
not determined
Table 3. Influence of position-6 substituents of the quinazoline on biochemical/cellular potency, PI3K isoform selectivity,
hERG inhibition and in vitro ADME parameters
O
N
N
R
O
N
N
7-11
biochemical IC50 [µM]a
PI3Kαb PI3Kβb PI3Kγc PI3Kδc PI3Kα PI3Kβ PI3Kδ
cellular IC50 [µM]a, d
mCD86 PAMPA hERG HT- RLM HT-
f
j
IC50
logPe
IC50 logPh CLint solk
R
[µM]a, e
[µM]g
N
7
0.829
0.127
0.418
0.084
0.262
4.27
1.94
4.84
0.536
1.65
5.6
0.003
0.014
0.029
0.005
0.009
2.10
1.32
2.12
0.690
3.44
4.65
3.38
3.57
2.07
6.53
0.062
0.039
0.028
0.014
0.049
0.393
0.034
0.070
0.093
0.074
-5.3
-5.1
-4.9
-4.1
-5.5
>30
>30
>30
>30
>30
2.8
1.9
2.7
3.1
2.8
525
68
0.61
0.80
>1.0
0.38
0.21
MeO
8
0.220
2.7
MeO
MeO
N
9
115
83
MeO
N
N
10
11
1.4
F3C
MeO
4.63
63
NC
aMean of a minimum of two independent experiments; standard deviation for pIC50 values <0.3; bKGlo format; cADAPTA format (val-
ues comparable to KGlo); dinhibition of pAkt formation in Rat-1 cells; einhibition of anti-IgM induced mCD86 expression on mouse sple-
nocytes; feffective permeability [10-6 cm∙sec-1] (pH = 6.8), n=1; ginhibition of [3H]dofetilide binding to hERG transfected membranes, n=1;
hHigh-throughput logP measurement with immobilized artificial membranes, n=1; intrinsic clearance in incubations of rat liver micro-
j
somes [µL∙min-1∙mg-1 protein], n=1; kHigh-throughput equilibrium solubility determination [mM] (pH = 6.8); nnd = not determined
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