Discovery of a Selective Phosphoinositide-3-Kinase (PI3K)-γ Inhibitor (IPI-549) as an Immuno-Oncology Clinical Candidate

Article abstract of DOI:10.1021/acsmedchemlett.6b00238

Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3K-γ (26 or IPI-549) with >100-fold selectivity over other lipid and protein kinases. IPI-549 demonstrates favorable pharmacokinetic properties and robust inhibition of PI3K-γ mediated neutrophil migration in vivo and is currently in Phase 1 clinical evaluation in subjects with advanced solid tumors.

Full text of DOI:10.1021/acsmedchemlett.6b00238

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Letter
Discovery of a Selective Phosphoinositide-3-Kinase (PI3K)-
# Inhibitor (IPI-549) as an Immuno-Oncology Clinical Candidate
Catherine Anne Evans, Tao Liu, André Lescarbeau, Somarajan J. Nair, Louis Grenier, Johan
André Pradeilles, Quentin Glenadel, Thomas Tibbitts, Ann M. Rowley, Jonathan P DiNitto,
Erin E. Brophy, Erin L. O'Hearn, Janid A. Ali, David G. Winkler, Stanley I. Goldstein, Patrick
O'Hearn, Christian M. Martin, Jennifer G. Hoyt, John R. Soglia, Culver Cheung, Melissa
M. Pink, Jennifer L. Proctor, Vito J. Palombella, Martin R. Tremblay, and Alfredo C. Castro
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00238 • Publication Date (Web): 22 Jul 2016
Downloaded from http://pubs.acs.org on July 25, 2016
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Discovery of a Selective Phosphoinositideꢀ3ꢀKinase (PI3K)ꢀγ
Inhibitor (IPIꢀ549) as an ImmunoꢀOncology Clinical Candidate
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Catherine A. Evans,* Tao Liu, André Lescarbeau, Somarajan J. Nair, Louis Grenier, Johan A. Praꢀ
deilles, Quentin Glenadel, Thomas Tibbitts, Ann M. Rowley,‡ Jonathan P. DiNitto, Erin E. Brophy, Erin
L. O’Hearn,† Janid A. Ali, David G. Winkler, Stanley I. Goldstein, Patrick O’Hearn, Christian M. Marꢀ
tin, Jennifer G. Hoyt, John R. Soglia, Culver Cheung, Melissa M. Pink, Jennifer L. Proctor, Vito J. Palꢀ
ombella,§ Martin R. Tremblay, Alfredo C. Castro*
Infinity Pharmaceuticals, Inc. 784 Memorial Drive, Cambridge, Massachusetts 02139, United States
KEYWORDS: IPIꢀ549, PI3Kꢀgamma inhibitor, phosphoinositideꢀ3ꢀkinase, isoform selectivity, neutrophil migration, immunoꢀoncology
ABSTRACT: Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3Kꢀγ (26 or IPIꢀ549)
with >100ꢀfold selectivity over other lipid and protein kinases. IPIꢀ549 demonstrates favorable pharmacokinetic properties and
robust inhibition of PI3Kꢀγ mediated neutrophil migration in vivo and is currently in Phase 1 clinical evaluation in subjects with
advanced solid tumors.
Phosphoinositideꢀ3ꢀkinases (PI3Ks) belong to a family of
signal transducing enzymes that mediate key cellular functions
in cancer and immunity. The primary role of these lipid kinasꢀ
es entails catalysis of the phosphorylation of the inositol ring
of membrane phosphoinositides, which then serve as sites for
the activation of secondary messengers involved in the regulaꢀ
tion of multiple biological processes, including cell growth,
survival, differentiation, proliferation and migration. PI3Ks
are divided into three classes (I, II, and III) based on substrate
specificity, sequence homology, and types of regulatory subuꢀ
nits. Class I PI3Ks are further divided into two groups: Class
IA, which includes PI3Kꢀα, β, and δ, and Class IB, which conꢀ
tains only one member, PI3Kꢀgamma (γ). While Class IA
PI3Ks are predominantly activated by receptor tyrosine kinase
signaling, PI3Kꢀγ is activated primarily by Gꢀprotein coupled
receptors.1ꢀ6
known to promote an immuneꢀsuppressive tumor microenviꢀ
ronment that permits tumor growth.9ꢀ12 In addition, tuꢀ
morꢀassociated myeloid cells are postulated to support tumor
regrowth after radiation or chemotherapy, and to enable metaꢀ
static spread.13 These preclinical studies highlight a critical
role for PI3Kꢀγ in myeloid cell biology, and suggest that
PI3Kꢀγ inhibition in tumorꢀassociated myeloid cells may be
effective at preventing tumor growth in a variety of settings.
To test the hypothesis that pharmacological inhibition of
PI3Kꢀγ in tumorꢀassociated myeloid cells could block their
immuneꢀsuppressive function and lead to enhanced immune
attack on tumor cells, a potent and selective synthetic
smallꢀmolecule inhibitor of the PI3Kꢀγ isoform was needed.
Although inhibitors of PI3Kꢀγ have been reported over the
past decade, their selectivity for PI3Kꢀγ over other PI3K
isoforms or pharmacologic properties were until recently not
suitable for assessment of inhibiting only PI3Kꢀγ in vivo.14ꢀ23
Hence, we set out to identify potent and selective inhibitors of
The PI3Kꢀγ isoform is expressed in immune cells and has
limited, if any, expression in normal or malignant epithelial
cells and connective tissue cells.7 Studies of PI3Kꢀγ knockout
mice show that PI3Kꢀγ is important for cellular activation and
migration in response to certain chemokines.7ꢀ8 PI3Kꢀγ signalꢀ
ing is particularly important for the function of myeloid cells,
where it is downstream of Gꢀprotein coupled receptors
(GPCRs) (e.g., chemokine receptors) and RAS.7ꢀ9 In addition,
PI3Kꢀγ can be activated in response to tissue hypoxia in these
cells.10 A critical role for PI3Kꢀγ in the unique myeloidꢀ
derived cells that constitute a key component of the imꢀ
muneꢀsuppressive tumor microenvironment has also been
demonstrated in PI3Kꢀγ deletion and kinaseꢀdead knockꢀin
studies. For example, murine syngeneic tumors grow slower
when transplanted into immuneꢀcompetent mice where PI3Kꢀγ
is genetically inactivated.9,10 This growth reduction is due to
the abrogation of tumorꢀassociated myeloid cells that are
PI3Kꢀγ with desirable drugꢀlike properties
.
The Nꢀphenyl 8ꢀchloroisoquinolinone motif served as a posꢀ
sible scaffold to explore alternative hinge binding motifs that
may provide unique interactions with PI3Kꢀγ.24 To this end,
we assembled a focused collection of 8ꢀchloroisoquinolinone
analogs that could be readily synthesized having hinge binding
motifs containing at least one hydrogen bond acceptor and 0ꢀ2
hydrogen bond donors. We screened this focused collection
for their ability to inhibit PI3Kꢀγ and PI3Kꢀδ at physiological
ATP concentrations (3 mM) and were pleasantly surprised to
see that compounds 1 and 2 showed moderate selectivity for
PI3Kꢀγ over PI3Kꢀδ (Table 1).
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Table 1. Structure-activity relationship from hinge (R1)
and linker modifications (R2).
tivity. The enantiomer of 1 and the desꢀmethyl achiral analog
1
2
3
4
5
6
7
7 were largely inactive against both PI3K isoforms. Interestꢀ
ingly, the selectivity was reduced when the chiral methyl is
extended to an ethyl substituent (8 vs 1). These data highlight
key hydrogen bonding features on the hinge binding motifs
and the preferred chiral linker of Nꢀphenyl 8ꢀ
chloroisoquinolinones 1 and 2 that make them bona fide leads
for the development of selective PI3Kꢀγ inhibitors
.
8
9
We evaluated leads 1 and 2 for selectivity across other PI3K
isoforms and found 1 to be remarkably selective across other
Class I and Class II PI3K isoforms (Table S1). Therefore, we
focused our attention on further optimization of compound 1.
Taking the published coꢀcrystal structures of propellerꢀshaped
ligand PIKꢀ39 with PI3Kꢀδ and PI3Kꢀγ into consideration, we
evaluated C8 substitution on compound 1 to access a nonꢀ
conserved region within PI3Ks with the aim of improving the
PI3Kꢀγ potency and selectivity (Table 2).25, 26 We found small
C8 substitutions such as methyl (9) and fluoro (10) did not
have a profound impact on PI3Kꢀγ potency or selectivity.
However, a significant loss in PI3Kꢀγ potency was seen with
an electronꢀwithdrawing cyano group (11) or electronꢀ
donating groups (12-14) at the 8 position. Vinyl substitution at
the 8 position (15) also had a negative impact on PI3Kꢀγ poꢀ
tency. However, 8ꢀalkyne substitutions (16 and 17) unexpectꢀ
edly provided modest improvements in potency for PI3Kꢀγ
over the 8ꢀchloro substitution in 1. Importantly, the methyl
alkyne analog 17 also demonstrated weaker activity against
PI3Kꢀδ compared to 1 and 16, and thus 17 had >40ꢀfold selecꢀ
tivity for PI3Kꢀγ over PI3Kꢀδ, suggesting that the alkyne subꢀ
stitution makes significant nonꢀfavorable interactions with
PI3Kꢀδ at the nonꢀconserved residues adjacent to the specificiꢀ
ty pocket (Lys802 for PI3Kꢀγ versus Thr750 for PI3Kꢀδ) as
does the aminopyrazolopyrimidine at the hinge binding reꢀ
gion21,22 (see Supporting Information, Figure S1).
Biochemical IC50
(nM)a
10
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Cmpd
R1
R2
PI3Kꢀγ
PI3Kꢀδ
Me
40
400
1
Me
Me
Me
Me
Me
H
60
400
320
800
2
3
4
5
6
7
8
1000
300
900
2600
>10000
>10000
600
>10000
>7000
150
Various substituted alkyne analogs at the 8ꢀposition that exꢀ
tended further into the nonꢀconserved pocket were then preꢀ
pared and evaluated for PI3Kꢀγ potency and selectivity (Table
3). Additionally, as alkyl substituted alkyne analogs 16 and 17
suffered from poor in vitro metabolic stability, we also
screened these substituted alkyne analogs for improved stabilꢀ
ity in mouse hepatocytes. Interestingly, hydrophobic substituꢀ
ents that extended deeper into the pocket (e.g. phenyl analog
18 vs. methyl
Et
a Data reported as the average of at least two runs. See Supporting
Information for description of assay conditions and SEM values.
The hinge binding motifs on compounds 1 and 2 shared the
following common features: an amide bond to isoquinolinone
core with a chiral linker, an exocyclic amine as part of a hyꢀ
drogen bond donor/acceptor pair, and a heterocyclic nitrogen
oriented towards the core. The importance of these structural
features on potency and selectivity was confirmed by a set of
direct analogs of 1 and 2 (Table 1). Analogs without the exoꢀ
cyclic amine (3 and 4) were significantly less potent and selecꢀ
tive than 1 and 2, respectively. The carboꢀanalog of the 6ꢀ5
bicyclic system (5) was less active than the azaꢀanalog (1),
whereas the same modification on the 6ꢀ6 bicyclic system (6
vs 2) rendered the compound largely inactive against both
PI3K isoforms. These data suggest that the dihedral angle beꢀ
tween the carbonyl amide and the heterocyclic hinge binding
motif 1 and 2 provides a preferred conformation for binding to
both PI3Kꢀγ and PI3Kꢀδ. Changes to the chiral linker between
the hinge binding motif and Nꢀphenyl 8ꢀchloroisoquinolinone
core also proved to be deleterious to PI3K potency and selecꢀ
Table 2. Structure activity relationship of C8 substitution.
Biochemical IC50 (nM)a
Cmpd
R
PI3Kꢀγ
110
PI3Kꢀδ
2600
Me
9
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530
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1
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CN
240
3100
OMe
NHMe
NMe2
1000
1100
1100
>9000
>10000
>10000
9
10
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12
13
14
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16
17
18
19
330b
14
>10000
15
16
Biochemical IC50
(nM)a
Mouse
Hepatocyte
Stability t1/2
(min)
Cmpd
18
R
PI3Kꢀγ PI3Kꢀδ
700
280
110
350
170
110
75
140
ND
102
51
25
>1000
17
>6000
>9000
>9600
>8000
4800
19
a Data reported as the average of at least two runs. bn=1. See Supꢀ
porting Information for description of assay conditions and SEM
values.
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20
analog 17) were found to have eroded PI3Kꢀγ potency and
selectivity. Less hydrophobic groups (e.g. pyridyl analogs 19-
21) maintained good selectivity for PI3Kꢀγ and were found to
be more metabolically stable than methyl alkyne analog 17.
With this in mind, we synthesized and evaluated various anaꢀ
logs with smaller heterocycles on the end of the 8ꢀalkyne (e.g.
22-26). Amongst various 5ꢀmembered ring heterocycle anaꢀ
logs, Nꢀmethylpyrazole analog 26 proved to have excellent
potency and selectivity for PI3Kꢀγ over PI3Kꢀδ in enzymatic
assays and excellent in vitro metabolic stability. Having
achieved this remarkable selectivity in biochemical assays and
in vitro metabolic stability, we further profiled compound 26
in various in vitro and in vivo settings.
72
21
43
22
270
27
23
Compound 26 was evaluated for activity across all Class I
PI3K isoforms. The binding affinity of compound 26 for
PI3Kꢀγ was determined by measuring the individual rates conꢀ
stants (koff and kon) and for PI3Kꢀα, β and δ using equilibrium
fluorescent titration. Compound 26 was found to be a remarkꢀ
ably tight binder to PI3Kꢀγ with a Kd of 290 pM and >58ꢀfold
weaker affinity for other Class I PI3K isoforms (Table 4).
Additionally, compound 26 did not significantly inhibit a panꢀ
el of 468 mutant and nonꢀmutant protein and lipid kinases
(including Class II PI3K isoforms) at 1 µM (Tables S1 and
S4). In PI3Kꢀα, ꢀβ, ꢀγ, and ꢀδ dependent cellular phosphoꢀAKT
assays, compound 26 demonstrated excellent PI3Kꢀγ potency
(IC50 = 1.2 nM) and selectivity against other Class I PI3K
isoforms (>146ꢀfold). Furthermore, compound 26 dose deꢀ
pendently inhibited PI3Kꢀγ dependent bone marrowꢀderived
macrophage (BMDM) migration in vitro (Figure 1).28
360
100
16
>10000
>9000
>8400
24
251
>360
25
26
a Data reported as the average of at least two runs. See Supporting
Information for description of assay conditions and SEM values.
Table 4. Class I PI3K selectivity profile for compound 26 in
biochemical and cellular assays.
PI3K Isoform
Assay
Table 3. Structure-activity relationship of C-8 alkynyl sub-
stitution.
α
β
γ
δ
Kd (nM)a
17
82
0.29
23
Biochemical
IC50 (nM)
3200
3500
16
>8400
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tered compound 26 on ILꢀ8 stimulated neutrophil influx into
Cellular IC50
(nM)b
the air pouches on mice.7,8 Compound 26 significantly reduced
neutrophil migration in a dose dependent manner in this model
when administered orally at all of the tested doses (Figure 2a).
The degree of inhibition observed directly correlated with
plasma concentrations of compound 26 in these mice (Figure
2b), clearly demonstrated that orally administered compound
26 can inhibit PI3Kꢀγ function in vivo. In addition, compound
26 (IPIꢀ549) has been shown to inhibit tumor growth in muꢀ
rine syngeneic models through alteration of immune cells in
the tumor microenvironment.28
250
240
1.2
180
1
2
3
4
5
6
7
8
a Binding affinities (Kd) of 26 were determined by methods previꢀ
ously reported.24 See Supporting Information for full details.
b Cellular IC50s for Class I PI3Kꢀα, PI3Kꢀβ, PI3Kꢀγ, PI3Kꢀδ were
determined in SKOVꢀ3, 786ꢀO, RAW 264.7, and RAJI cells, reꢀ
spectively, by monitoring inhibition of pAKT S473 by ELISA.
Compound 26 was also found to be selective against a panel
of 80 GPCRs, ion channels, and transporters at 10 µM (see
Supporting Information, Table S5 for full selectivity details).
Additional in vitro safety assessment of compound 26 demonꢀ
strated that it was negative in Ames mutagenicity assays and
had an IC50 greater than 10 µM in a hERG binding assay (data
not shown).
9
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On the basis of its remarkable PI3Kꢀγ potency and selectiviꢀ
ty, favorable in vitro safety and pharmacokinetic profiles, and
ability to inhibit PI3Kꢀγ in vivo, compound 26 (IPIꢀ549) was
chosen as a development candidate. IPIꢀ549 is currently in
Phase 1 clinical evaluation in subjects with advanced solid
tumors.29
Table 5. In vitro ADME and pharmacokinetics for com-
pound 26.
In vitro ADME
Caco-2 (Papp 10ꢀ6 cm/sec; A to B @ 10 ꢀM)
13
PPB (% free; mouse, rat, dog, monkey, huꢀ
man @ 1 ꢀM)
2.2, 2.8, 2.8,
4.0, 1.1
Metabolic stability (t1/2; min)a
CYP3A4 inhibition (IC50; ꢀM)
Pharmacokinetics
> 360
> 20
Figure 1. Effect of compound 26 on migration of bone marrow
derived macrophages (BMDM) in vitro. BMDMs were stimulatꢀ
ed to migrate toward 100 µg/mL CXCL12 (SDFꢀ1α) for 3 hours
through a 5 micron Boyden Chamber with or without DMSO or a
dose response of compound 26. Migrated BMDMs were counted
and compared to control.
Mouse
3.6
Rat
2.0
Dog
1.9
Monkey
0.56
C
max (µM)b
AUC0ꢀ∞
20568
3.2
8049
4.4
1066
6.7
4030
4.3
(ng*h/mL)b
In vitro absorption, distribution, metabolism and excretion
(ADME) properties and pharmacokinetic parameters of comꢀ
pound 26 were also determined (summarized in Table 5). In
vitro, 26 showed moderate to high cell permeability across
Cacoꢀ2 cell monolayers, was slowly metabolized in cultured
hepatocytes (t1/2 >360 minutes), and demonstrated IC50s greatꢀ
er than 20 ꢀM for the CYP isoforms tested (1A2, 2B6, 2C8,
2C9, 2C19, 2D6, 3A4). In vivo (mice, rats, dog, and monꢀ
keys), compound 26 had excellent oral bioavailability, low
clearance, and distributed into tissues with a mean volume of
distribution of 1.2 L/kg (Table 5). Overall, compound 26 had a
favorable pharmacokinetic profile to allow potent and selecꢀ
tive inhibition of PI3Kꢀγ in vivo.
t1/2 (h)c
CL
3.6
4.4
2.8
4.3
(mL/min/kg)c
Vss (L/kg)c
0.8
88
1.2
57
1.3
65
1.3
31
%F
a Study conducted using cultured human hepatocytes.
b
Determined from oral dosing to male animals; PO dose
levels: 5 mg/kg mouse and rat, 2.5 mg/kg monkey and dog.
Formulation; 0.5% Carboxymethylcellulose, 0.05% Tween80
c
Determined from intravenous dosing to male animals: IV
dose levels: 1 mg/kg mouse and rat, 0.5 mg/kg monkey and
dog. Formulation; 5% NMP, 40% PEG400, 55% PBS
Based on the pharmacokinetic properties in mice and pharꢀ
macological in vitro profile, compound 26 was wellꢀsuited to
investigate the impact of potent and selective PI3Kꢀγ inhibition
in vivo. ILꢀ8 stimulated neutrophil migration into air pouches
in mice has previously been shown to be dependent on PI3Kꢀ
γ. Thus, to demonstrate PI3Kꢀγ dependent activity of comꢀ
pound 26 in vivo, we evaluated the effect of orally adminisꢀ
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Figure 2. (a) Effect of compound 26 on neutrophil migration in the mouse air pouch model. Mice were dosed orally one hour before ILꢀ8
was applied to the air pouch. After 4 hours the air pouch was lavaged and the neutrophils counted using a CELLꢀDYN hematology analyzꢀ
er. *indicates p values <0.05 compared to vehicle. Error bars indicate the standard error of the mean of 10 mice per group. (b) Plasma conꢀ
centration levels of compound 26 at 1 h and 5 h post administration with overlaying cellular IC50 (PI3Kꢀγ and PI3Kꢀδ) and IC25 (PI3Kꢀγ)
values.
CXCL12, chemokine CꢀXꢀC motif 12; CYP, cytochrome P450;
DMSO, dimethysulfoxide; ELISA, enzymeꢀlinked immunosorbent
assay; GPCR, Gꢀproteinꢀcoupled receptor; ILꢀ8, interleukin 8;
NMP, Nꢀmethylꢀ2ꢀpyrrolidone; PBS, phosphateꢀbuffered saline;
PI3K, phosphoinositide 3ꢀkinase; PPB, plasma protein binding;
PEG, polyethylene glycol; SDFꢀ1, stromal cellꢀderived factor 1
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: XXX
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Synthetic procedures and analytical data for 1ꢀ26; PI3K biochemiꢀ
cal and cellular assay conditions; PI3K Class I & II selectivity data
for 1 and 2; full selectivity data for 26; general animal study protoꢀ
cols (PDF)
AUTHOR INFORMATION
Corresponding Authors
* Eꢀmail: catherine.evans@infi.com
* Eꢀmail: alfredo.castro@infi.com
Present Addresses
† Blueprint Medicines, Cambridge, Massachusetts 02139
‡ AbbVie, North Chicago, Illinois 60064
§ Surface Oncology, Cambridge, Massachusetts 02142
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chard, D.; Kozieradzki, I.; Joza, N.; Mak, T. W.; Ohashi, P. S.;
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Dr. Nicole Hurst for coordinating the preclinical safety
evaluation of IPIꢀ549, Dan Snyder and Amael Madec for assisꢀ
tance in preparation of early analogs, Drs. Jeff Kutok and Karen
McGovern for guidance on the pharmacological assessment of IPIꢀ
549, and colleagues at Intellikine, Drs. Pingda Ren and Liansheng
Li for their insights on PI3K inhibitors.
ABBREVIATIONS
ADME, absorption, distribution, metabolism , and excretion;
AKT, protein kinase B; ADP, adenosine diphosphate; ATP, adenoꢀ
sine triphosphate; BMDMs, bone marrowꢀderived macrophages;
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