Discovery of a potent, selective, and orally available PI3Kδ inhibitor for the treatment of inflammatory diseases

Article abstract of DOI:10.1021/acsmedchemlett.6b00438

The delta isoform of the phosphatidylinositol 3-kinase (PI3Kδ) has been shown to have an essential role in specific immune cell functions and thus represents a potential therapeutic target for autoimmune and inflammatory diseases. Herein, the optimization of a series of pyrrolotriazinones as potent and selective PI3Kδ inhibitors is described. The main challenge of the optimization process was to identify an orally available compound with a good pharmacokinetic profile in preclinical species that predicted a suitable dosing regimen in humans. Structure?activity relationships and structure?property relationships are discussed. This medicinal chemistry exercise led to the identification of LAS191954 as a candidate for clinical development.

Full text of DOI:10.1021/acsmedchemlett.6b00438

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Letter
Discovery of a potent, selective, and orally available
PI3Kd inhibitor for the treatment of inflammatory diseases
Montse Erra, Joan Taltavull, Angelique Gréco, Francisco Javier Bernal, Juan Francisco Caturla, Jordi
Gracia, María Domínguez, Mar Sabaté, Stéphane Paris, Salomé Soria, Begoña Hernández, Clara
Armengol, Judit Cabedo, Mònica Bravo, Elena Calama, Montserrat Miralpeix, and Martin D. Lehner
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00438 • Publication Date (Web): 30 Nov 2016
Downloaded from http://pubs.acs.org on November 30, 2016
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ACS Medicinal Chemistry Letters
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Discovery of a potent, selective, and orally available PI3K
inhibitor (LAS191954) for the treatment of inflammatory
diseases.
Montse Erra£*, Joan Taltavull£, Angelique Gréco£€, Francisco Javier Bernal£, Juan Francisco Caturla£,
Jordi Gràcia£, María Domínguezˤ, Mar Sabatéˤ, Stéphane Paris£, Salomé Soria£, Begoña Hernández£□,
Clara Armengol#, Judit Cabedo∆, Mónica Bravo∆, Elena Calama∆, Montserrat Miralpeix∆, and Martin D.
Lehner∆≠.
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£Medicinal Chemistry and Screening, ˤPharmacokinetics and Metabolism, # Systems Biology, ∆Respiratory Therapeutic
Area, Almirall R&D, Barcelona, Spain.
KEYWORDS: Phosphoinositide-3-kinase delta inhibitor, PI3Kinhibitor, structure-activity relationship, autoimmune dis-
eases, inflammatory diseases, lead optimization.
ABSTRACT: The delta isoform of the Phosphatidylinositol 3-kinase (PI3K) has been shown to have an essential role in specific
immune cell functions and thus represents a potential therapeutic target for autoimmune and inflammatory diseases. Herein, the
optimization of a series of pyrrolotriazinones as potent and selective PI3K inhibitors is described. The main challenge of the opti-
mization process was to identify an orally available compound with a good pharmacokinetic profile in pre-clinical species that
predicted a suitable dosing regimen in humans. Structure-activity relationships and structure-property relationships are discussed.
This medicinal chemistry exercise led the identification of LAS191954 as a candidate for clinical development.
The phosphatidylinositol-3-kinase (PI3K) signal transduction
pathway is central to a plethora of different cellular processes
involving metabolism, proliferation, differentiation or activa-
tion. Hence, manipulation of the PI3K pathway represents an
interesting approach for treatment of a number of different
pathological conditions such as cancer, where inhibitors of
class IA PI3K with varying isoform selectivity profiles have
already been established as treatment options for different
indications.
Scheme 1. Our initial lead 2 showed similar PI3K inhibitor
potency to IC87114 (110 nM versus 130 nM) and also high
selectivity against the other class I PI3K isoforms. Moving the
methyl group from the phenyl ring in compound 2 to the linker
(compound 3), slightly improved the PI3K inhibitor potency
to 75 nM and removed the potential for atropisomerism12
present in ortho-substituted compounds 1 and 2. Taking into
account the stereochemistry of known PI3K selective inhibi-
tors in the clinic such as Idelalisib13 and Duvelisib14, the S-
enantiomers were initially targeted.
In the immune system, the PI3K delta isoform plays a central
role in both innate and adaptive immune cell functions. Taking
advantage of the strong dependency of B cells on functional
PI3K1, the oral PI3K inhibitor Idelalisib has been success-
fully developed as a novel treatment for different types of B-
cell malignancies2. In addition, the involvement of PI3K in
central immune functions suggests a therapeutic potential of
PI3K inhibitors as novel broad-acting anti-inflammatory
agents for autoimmune diseases3 and pathologies with an
allergic or inflammatory component such as allergic rhinitis4,
asthma5, or COPD6. Apart from inhibiting B cell activation,
pharmacological inhibition of PI3K delta has been demon-
strated to attenuate T cell receptor induced cytokine produc-
tion7, degranulation of basophils and mast cells4,8 as well as
induction of oxidative burst by neutrophils9. In addition,
PI3K seems to be involved in corticosteroid resistance in-
duced by oxidative stress in macrophages10, thus indicating an
activity profile complementary to other currently treatments
such as corticosteroids.
Scheme 1. Pyrrolotriazinone scaffold
Thus, an extensive structure-activity relationship (SAR) study
was then performed around compound 3 to improve the poten-
cy and modulate ADME properties.
Initially, an exploration of the hinge binder region was under-
taken and PI3K inhibitory potency in both enzymatic and
cellular assays was assessed together with in vitro metabo-
lism15 for each compound (Table 1). Introduction of other
purine-like hinge binders (4, 5) increased the in vitro potency
for PI3K considerably and the compounds showed low in
vitro metabolism. Other bicyclic rings (6-8) demonstrated
lower potency and decreased metabolic stability when com-
The discovery of the propeller-shaped inhibitor 1 (IC87114,
ICOS) and the mechanisms by which  isoform selectivity can
be accomplished focused our attention11. Following this strat-
egy, our efforts toward the identification of new PI3K inhibi-
tors were based on a pyrrolotriazinone scaffold as shown in
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pared to compounds 4 and 5. Monocyclic rings were then and 16 resulted in derivative 18 and 17 respectively, but only
1
2
3
4
5
6
7
8
studied and cyanopyrimidine 11 showed the best overall bal-
ance between potency and in vitro metabolism.
compound 17 showed an improved microsomal stability whilst
maintaining good PI3K potency. In contrast, introduction of a
trifluoroethyl group (compound 19) was less tolerated. Cyclic
analogue 20 kept in vitro potency but was metabolically less
stable. Introduction of fluorine atoms in the cyclopentyl ring
of 20 with the aim of increasing metabolic stability led to a
less potent compound 21. Introduction of a spirocyclic ring
was also studied (compound 22) but a big drop off in potency
was observed.
Table 1. SAR exploration of the hinge binder
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
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31
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53
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56
57
58
59
60
Table 2. Linker SAR exploration
Compd
R1
M-CSF p-
%
Metabo-
PI3K
Aktb IC50 lism
(HTRF)a
IC50 (nM)
(nM)
7
(rat/human)c
18 / 30
4
5
6
7
8
9
10
3.1
60
52
9
Compd
14
R2
M-CSF p-
%
Metabo-
PI3K
6.5
-
30 / 26
100 / 86
44 / 49
48 / 58
-
Aktb IC50 lism
(HTRF)a
IC50 (nM)
(nM)
-
(rat/human)c
38 / 49
25
150
47
15
16
1800
11
-
32 / 30
32 / 32
18
73
210
17
18
19
20
21
22
4.8
13
16
ND / 7
24 / 31
-
10
11
12
13
12
-
51 / 43
15 / 24
25/ 33
-
4.3
91
4.7
76
-
51
610
4.6
31
1600
6.6
370
-
1200
46 / 48
30 / 35
-
aPI3K activities were measured with an ATP concentration fixed at
the Km of PI3K by HTRF, where the PIP3 product is detected by
displacement of biotin-PIP3 from an energy transfer complex. bTHP-1
cells were treated with compounds for 30 min., stimulated with M-
CSF at EC80 for 3 minutes and then lysed to measure (by ELISA)
pAkt (Thr308) produced through PI3K. c % metabolism expressed as
disappearance of parent compound after microsomal incubation for 30
min (1 mg/ml protein and 5 µM compound at 37 ºC).
3400
Optimization of the linker was then explored as shown in
Table 2. An ethyl group was well tolerated giving a compound
(14) of similar potency to the methyl derivative 4 but poorer
microsomal stability. The corresponding (R)-enantiomer of 14
was synthesized (compound 15) and the resultant drop off in
potency confirmed the preference of PI3K for the (S)-
enantiomer. Replacing the hinge binder of compound 14 for
the cyanopyrimide (16) slightly improved potency and in vitro
metabolism. Appending a hydroxyl group onto compounds 11
aPI3K activities were measured with an ATP concentration fixed at
the Km of PI3K by HTRF, where the PIP3 product is detected by
displacement of biotin-PIP3 from an energy transfer complex. bTHP-1
cells were treated with compounds for 30 min., stimulated with M-
CSF at EC80 for 3 minutes and then lysed to measure (by ELISA)
pAkt (Thr308) produced through PI3K. c % metabolism expressed as
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ACS Medicinal Chemistry Letters
disappearance of parent compound after microsomal incubation for
30min (1 mg/ml protein and 5 µM compound at 37 ºC).
Appending a hydroxyl group onto the methyl group of 23
resulted in a compound 24 with reasonable PI3K inhibitor
potency and improved metabolic stability. After further study
it was found that placement of other substituents at position 5
(25, 26 and 27) gave rise to compounds with excellent poten-
cies and good metabolic stability. Further characterization of
compound 25 showed the formation of GSH adducts following
incubation of 25 with microsomes in presence of glutathione
(GSH) and as such was not further profiled. Finally, the imid-
azotriazinone 32 showed a substantially diminished PI3K
inhibitor potency in comparison to the corresponding pyr-
rolotriazinone 11.
1
2
3
4
5
6
7
8
Compounds 11 and 17 were then profiled in a rat in vivo
pharmacokinetic (PK) experiment. However, for both com-
pounds, in vivo clearance in rat was higher than expected from
the microsomal stability data, resulting in moderate systemic
exposure and short half-lives (Table 3).
Table 3. Rat PK profiles of compounds 11 and 17
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10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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Compd
t1/2 (h)a AUC
(ng*h/ml)a
Cl
Vss
(ml/min/kg)a
(l/kg)a
Table 4. Distal core SAR exploration
11
17
0.9
1.1
386
450
43.5
36.8
1.9
1.6
aMean values (n=2) in Wistar rat after an administration of 1mg/kg
i.v.. Parameters calculated from plasma samples: t1/2 = half-life, Cl =
clearance, AUC = area under the curve, Vss = volume of distribution
at steady state.
Compd
R3
M-CSF p-
%
Metabo-
PI3K
Aktb IC50 lism
(HTRF)a
IC50 (nM)
(nM)
1.6
(rat/human)c
44 / 80
To better understand the binding mode of the ligands an X-ray
co-crystal of human PI3K in complex with compound 11 was
performed. The structure was solved at a resolution of 2.85Å,
revealing the detailed binding mode of the ligand. Like
IC87114, compound 11 adopted a propeller-shaped confor-
mation where the pyrrolotriazinone moiety was sandwiched
into the induced hydrophobic specificity pocket between
Trp760 and Met752. The cyanopyrimidine ring of 11 served as
the hinge binder and formed two specific hydrogen bonds to
the main chain atoms of Val828 and Glu826. The following
residues were found in the vicinity of the ligand with a maxi-
mum distance of 3.9Å: Met752, Pro758, Trp 760, Ile777,
Tyr813, Ile825, Glu826, Val827, Val828, Ser831, Asp832,
Met900, Ile910, and Asp911. A closer look at the binding
pocket surface (Figure 1) suggested that the pyrrolotriazinone
core ring could be substituted at the 5,6 or 7-position to further
modulate potency.
23
24
1.1
4.3
35
7 / 23
25
1.1
2.6
3.0
17
3.8
7.8
3.0
51
9 / 13
26
20 / 16
16 / 37
23 / 34
54 / 82
LAS191954
27
28
29
Figure 1. X-ray co-crystal structure of the binding pocket
surface of human PI3K containing compound 11
8.8
11
30
31
32
4.0
71
57
11
24 / 22
380
210
-
14 / 23
aPI3K activities were measured with an ATP concentration fixed at
the Km of PI3K by HTRF, where the PIP3 product is detected by
displacement of biotin-PIP3 from an energy transfer complex. bTHP-1
cells were treated with compounds for 30 min., stimulated with M-
CSF at EC80 for 3 minutes and then lysed to measure (by ELISA)
pAkt (Thr308) produced through PI3K. c % metabolism expressed as
disappearance of parent compound after microsomal incubation for 30
min (1 mg/ml protein and 5 µM compound at 37 ºC).
Thus, several analogues with different substitutions on the
pyrrolo ring were synthesized and tested. A methyl group was
well tolerated in different positions (compounds 23 and 29)
but the resultant derivatives were metabolically unstable.
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primary PI3Kδ-dependent cellular assay based on M-CSF-
1
2
3
4
5
6
7
8
induced AKT phosphorylation, a downstream effector of
PI3Kδ, in the human monocytic cell line THP-1. An IC50 of
7.8 nM was obtained indicating that the compound had excel-
lent permeability and cellular activity. To evaluate the cellular
inhibition of PI3Kβ, an assay based on stimulation of HUVEC
From this set of compounds, 26 and 27 were further profiled in
a rat in vivo PK experiment (Table 5) to determine if the in
vitro metabolism results were, in these cases, predictive of the
in vivo clearance values. These results were compared with
Idelalisib, the most advanced compound in clinical develop-
ment at that moment.
cells with sphingosine-1-P was employed16. The results (IC50
=
295 nM) indicated that the enzymatic selectivity between δ
and β isoforms (36-fold) was maintained at the cell-based
level (38-fold). The compound was also selective against an
extensive panel of protein and lipid kinases and GPCRs.
Table 5. Rat PK profiles of compounds 26 and 27 in com-
parison with Idelalisib
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10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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54
55
56
57
58
59
60
Compd
t1/2
AUC
Cl
Vz
F
PI3Kδ kinase is involved in the activation of B cells upon
antigen binding to the B cell receptor (BCR)17 and thus inhibi-
tors of PI3K are expected to inhibit BCR activation. The
effect of LAS191954 on the function of human B cells was
assessed in vitro by crosslinking the B-cell receptor with anti-
IgD antibodies and assessing the early activation marker CD69
in the CD19+ B cell subset by flow cytometry. On isolated
PBMC, compound 26 showed an IC50 of 4.6 nM. Similar assay
performed in human whole blood yielded an IC50 of 47 nM.
(h)a
(ng*h/ml)a
(ml/min/kg)a
(l/kg)a
(%)b
Idelalisi
b
0.8
512
32.8
2.2
68
26
27
3.1
3.3
1729
2040
9.6
8.6
2.6
2.4
101
113
aMean values (n=2) in Wistar rat after an administration of 1mg/kg
i.v. bMean values (n=2) in Wistar rat after an administration of
1mg/kg p.o.
From an ADME perspective, further in vitro characterization
of compound 26 was performed as summarized in Table 7.
Plasma protein binding18 was low in preclinical species (60-
65% in mouse/rat/dog) in contrast to human (95%). Plasma
protein binding may be the major factor accounting for the
difference in potency between the isolated human PBMC and
whole blood assays previously described. The compound
showed a good PAMPA19 value as expected by the cellular
potencies obtained. Moreover, compound 26 did not present
any signs of instability in plasma. The potential capacity of
compound 26 to inhibit the main human CYP450 isoforms
(CYP1A2, 3A4, 2D6, 2C9 and 2C19) was investigated in
human liver microsomes. Compound 26 displayed IC50 > 25
µM for the five CYP isoforms assayed. No differences were
observed after 15 min of preincubation time, suggesting that
compound 26 is not a mechanism-based inhibitor. In addition,
the potential capacity of the compound to induce CYP1A and
3A4 isoforms was assessed in cultured human cryopreserved
hepatocytes and compound 26 did not increase neither CYP1A
nor 3A4 activity at concentrations up to 50 µM. Taking into
account the results of these studies, drug-drug interactions due
to induction or inhibition of human CYP450 isoforms are not
expected in a clinical setting. Furthermore, no formation of
GSH-adducts was observed following incubation of compound
26 with rat and human liver microsomes in the presence of
glutathione (GSH).
As seen in Table 5, compounds 26 and 27, with substituents at
position 5 of the pyrrolo ring, showed increased plasma expo-
sure compared to unsubstituted compounds 11 and 17, excel-
lent oral bioavailability and reduced clearance in rat, which
was more consistent with the observed microsomal stability
values.
Additionally, the PK properties of both compounds 26 and 27
were studied in dog as a second preclinical species to allow for
prediction of the probable posology in humans based on al-
lometric scaling. The dog in vivo PK data are shown in Table
6. Both compounds 26 and 27 displayed a superior PK profile
to Idelalisib in the two preclinical species. Compound 26
(LAS191954), with excellent oral bioavailability and low
clearance in the two species, gave a superior predicted half-life
in humans and was further profiled.
Table 6. Dog PK profiles of compounds 26 and 27 in com-
parison with Idelalisib
Compd
t1/2
AUC
Cl
Vz
F
(h)a
(ng*h/ml)a
(ml/min/kg)a
(l/kg)a
(%)b
Idelalisi
b
1.6
924
21.5
2.5
-
26
27
10.2
4.2
9441
3937
1.4
4.2
1.2
1.5
98
-
Table 7. In vitro ADME profile of compound 26
Human
PPB PAMPA Papp Plasma stabil- CYP450
(% bound)a
(*10-6 cm/s)b
2.8
ity (24h)c
100%
inhibitiond
aMean values (n=2) in Beagle dog after an administration of 1mg/kg
i.v. bMean values (n=3) in Beagle dog after an administration of
1mg/kg p.o.
95.4
> 25M
aMean PPB values determined by equilibrium dialysis at a plasma
concentration of 1 µM at 37ºC. bPermeability values at RT in PBS
(containing 2% DMSO), compound concentration in donor com-
For the assessment of selectivity, the enzymatic potency of
compound 26 on the four class I PI3K recombinant human
isoforms was determined by HTRF with a compound pre-
incubation time of 30 min. Compound 26 showed a potency
on the target of 2.6 nM, with the highest selectivity versus
PI3Kα (8.2 M) and the lowest versus PI3Kγ and PI3Kβ (72
and 94 nM respectively). The compound was then tested in a
c
partment: 20 µM. Compound 26 stability at 37ºC at 2 µg/ml in
d
plasma (anticoagulant: sodium heparin) CYP450 in HLM with
and without preincubation for CYP1A2, 3A4, 2D6, 2C9 and 2C19
isoforms.
In terms of cardiac safety, compound 26 did not show any
activity on the hERG channel up to 10 M.
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ACS Medicinal Chemistry Letters
As a consequence of these encouraging results, the compound
was further characterized in different in vivo models.
1
2
PI3Kδ has been implicated in T-cell proliferation in vitro and
in vivo. Cytokine production induced by concanavalin A (con
A) or anti-CD3 antibody activation of naïve CD4+ T cells in
mice is blocked by PI3Kδ inhibition20. To assess the effect of
LAS191954 on T cell cytokine production in vivo, a rat model
of con A induced IL2 production was employed in which the
compound was administered orally one hour prior to an intra-
venous con A challenge (10 mg/kg) and IL2 levels were
measured 90 minutes later. LAS191954 inhibited IL2 produc-
tion with an ID50 of 0.13 mg/kg as compared to an ID50 of 1.6
mg/kg of Idelalisib as shown in Figure 2.
3
4
5
6
7
8
9
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
Figure 2. Inhibition of ConA induced increase in IL-2
levels in rat plasma of LAS191954 vs reference compound
Idelalisib
In both in vivo models LAS191954 showed efficacy at signifi-
cantly lower doses than Idelalisib. These results could not be
explained by differences in in vitro potency as Idelalisib ex-
hibited a similar cellular PI3K inhibitor potency with an IC50
of 7.6 nM, but they are in accordance with a superior PK pro-
file (higher systemic exposure and longer half-life) of
LAS191954 in rat.
1 0 0
7 5
5 0
2 5
0
In summary, a new series of potent and selective PI3K in-
hibitors has been identified. The SAR exercise focused on
optimizing both the in vitro potency and the ADME profile of
the compounds in order to find a potentially once-a-day drug.
This culminated in the identification of LAS191954 as a can-
didate for clinical development.
0 .0 3
0 .1
0 .3
1
3
1 0
3 0
1 0 0
d o s e [ m g / k g ] p . o .
I d
L
e
l a l i s i b ( I D
=
5
1
. 6
0
m
g
/ k
g
g
)
5
0
A
S
1
9
1
9 5
4
( I D
=
. 1
3
m
/ k g )
0
ASSOCIATED CONTENT
Supporting Information
PI3Kδ participates in the proliferation, differentiation, migra-
tion and cytokine production of T lymphocytes; in the migra-
tion and release of ROS and proteases by neutrophils as well
as in the migration, alternative M2 activation and loss of ster-
oid sensitivity of macrophages. PI3Kδ additionally regulates
Fcε mediated mast cell and basophil degranulation. Thus, the
anti-inflammatory efficacy of LAS191954 was assessed in an
airway allergic inflammation model characterized by infiltra-
tion of eosinophils to the alveolar compartment in response to
ovalbumin challenge in previously sensitized Brown Norway
rats.
Characterization of all compounds. Experimental procedures for
the sequence leading to key compound 26. Description of all
biological assays and in vivo studies. Crystallographic data collec-
tion and refinement statistics for crystal structure of compound
11. This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
PDB code for X-ray crystal structure described in this study has
been deposited in the Protein Data Bank under the following
accession codes: 5M6U (compound 11 in complex with PI3K)
A twice daily administration of LAS191954 1h prior to and
6h post ovalbumin (OVA) challenge dose-dependently re-
duced the number of eosinophils in the bronchoalveolar lavage
(BAL) at 24h in rat with an ID50 of 0.16 mg/kg. The refer-
ence compound Idelalisib, administered also twice daily, dis-
played a considerably lower potency with an ID50 of 15 mg/kg.
AUTHOR INFORMATION
Corresponding Author
* Phone: + 34 93 312 87 14. E-mail:
montse.erra@almirall.com
Figure 3. Inhibition of OVA BAL cell accumulation in BN
rats at 24h by twice daily doses of LAS191954 and
Idelalisib
Author Contributions
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of
the manuscript.
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€Pharmaxis Ltd, 20 Rodborough Road, Frenchs Forest, NSW
2086, Australia
≠Bionorica SE, Kerschensteinerstraße 11-15, 92318 Neumarkt,
Germany.
□Fundació ACE, Marqués de Sentmenat, 57, 08029 Barcelona,
(11) Berndt, A.; Miller S.; Williams O.; Le D.D.; Houseman B.T.;
Pacold, J.I.; Gorrec, F.; Hon, W.-C.; Liu, Y.; Rommel, C.;
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Spain.
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The authors declare no competing financial interest.
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Wong M. H.; Liclican A.; Xing W.; Lagpacan L.; Wang R.;
Shultz B. E.; Papalia G. A.; Samuel D.; Lad L.; McGrath M.
E. Structural, Biochemical, and Biophysical Characterization
of Idelalisib Binding to Phosphoinositide 3-Kinase . J. Biol.
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F.; Brophy E. E.; Pink M. M.; Proctor J. L.; Lussier J.; Martin
C. M.; Hoyt J. G.; Tillotson B.; Murphy E. L.; Lim A. R.;
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K. A.; Fritz C. C.; Dunbar J. L.; Poter J. R.; Rommel C.; Pal-
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ACKNOWLEDGMENT
The authors wish to thank Proteros Biostructures GmbH for
X-ray structure determination of human PI3K in complex
with compound 11 (PDB code: 5M6U). We also acknowledge
the significant technical support provided by Jordi Sanahuja,
Gaspar Casals and Antonia Molina. We thank Sonia Espinosa
and Josep M. Huerta for their support on physicochemical
properties analysis and structural characterization of com-
pounds.
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