Discovery of CDZ173 (Leniolisib), Representing a Structurally Novel Class of PI3K Delta-Selective Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.7b00293

The predominant expression of phosphoinositide 3-kinase δ (PI3Kδ) in leukocytes and its critical role in B and T cell functions led to the hypothesis that selective inhibitors of this isoform would have potential as therapeutics for the treatment of aller

Full text of DOI:10.1021/acsmedchemlett.7b00293

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Letter
Discovery of CDZ173 (leniolisib), Representing a
Structurally Novel Class of PI3K Delta-Selective Inhibitors
Klemens Hoegenauer, Nicolas Soldermann, Frederic Zecri, Ross S. Strang, Nadege Graveleau,
Romain M Wolf, Nigel G. Cooke, Alexander B. Smith, Gregory J. Hollingworth, Joachim Blanz,
Sascha Gutmann, Gabriele Rummel, Amanda Littlewood-Evans, and Christoph Burkhart
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00293 • Publication Date (Web): 25 Aug 2017
Downloaded from http://pubs.acs.org on August 29, 2017
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Discovery of CDZ173 (leniolisib), Representing a Structurally
Novel Class of PI3K Delta-Selective Inhibitors
Klemens Hoegenauer*a, Nicolas Soldermann*a, Frédéric Zécria, Ross S. Stranga, Nadege Graveleaua, Romain M. Wolfa, Nigel G. Cookea,
Alexander B. Smitha, Gregory J. Hollingwortha, Joachim Blanzb, Sascha Gutmannc, Gabriele Rummelc, Amanda LittlewoodꢀEvansd, Chrisꢀ
toph Burkhartd
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aGlobal Discovery Chemistry, bPK Sciences, cChemical Biology and Therapeutics, dAutoimmunity, Transplantation and Inflammation,
Novartis Institutes for BioMedical Research, Novartis Campus, CHꢀ4002 Basel, Switzerland
way is hyperactive.4,5 The importance of PI3Kδ for the function of
ABSTRACT: The predominant expression of Phosphoinositide
cells involved in inflammatory respiratory diseases (T cells, mast
3ꢀkinase δ (PI3Kδ) in leukocytes and its critical role in B and T
cell functions led to the hypothesis that selective inhibitors of this
cells, neutrophils)6–8 has guided the development of inhaled and
oral PI3Kδ inhibitors for the treatment of asthma or chronic obꢀ
structive pulmonary disease.9–11 Moreover, PI3Kδ is overactive in
isoform would have potential as therapeutics for the treatment of
allergic and inflammatory disease. Targeting specifically PI3Kδ
should avoid potential side effects associated with the ubiquitousꢀ
ly expressed PI3Kα and β isoforms. We disclose how morphing
the heterocyclic core of previously discovered 4,6ꢀdiaryl
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patients with autoimmune diseases such as rheumatoid arthritis
and systemic lupus erythematosus,13,14 where PI3Kδ inhibitors
have been effective in respective murine models.15,16 In patients
with Activated PI3Kδ Syndrome (APDS), mutations in the
PIK3CD gene encoding p110δ lead to an immunodeficiency charꢀ
acterized by recurrent infections and prominent lymphoproliferaꢀ
tion.17,18 In summary, there is strong evidence that PI3Kδ inhibiꢀ
tion is a promising therapeutic principle for the treatment of
APDS as well as inflammatory and autoimmune diseases,19–21
prompting numerous companies to engage in programs geared
towards the identification of potent and selective PI3Kδ inhibiꢀ
tors.11,22–29
quinazolines to
a
significantly less lipophilic 5,6,7,8ꢀ
tetrahydropyrido[4,3ꢀd]pyrimidine, followed by replacement of
one of the phenyl groups with a pyrrolidineꢀ3ꢀamine, led to a
compound series with optimal onꢀtarget profile and good ADME
properties. A final lipophilicity adjustment led to the discovery of
CDZ173 (leniolisib), a potent PI3Kδ selective inhibitor with suitꢀ
able properties and efficacy for clinical development as an antiꢀ
inflammatory therapeutic. In vitro, CDZ173 inhibits a large specꢀ
trum of immune cell functions, as demonstrated in B and T cells,
neutrophils, monocytes, basophils, plasmocytoid dendritic cells
and mast cells. In vivo, CDZ173 inhibits B cell activation in rats
and monkeys in a concentrationꢀ and timeꢀdependent manner.
After prophylactic or therapeutic dosing, CDZ173 potently inhibꢀ
ited antigenꢀspecific antibody production and reduced disease
symptoms in a rat collagenꢀinduced arthritis model. Structurally,
CDZ173 differs significantly from the first generation of PI3Kδ
and PI3Kγδꢀselective clinical compounds. Therefore, CDZ173
could differentiate by a more favorable safety profile. CDZ173 is
currently in clinical studies in patients suffering from primary
Sjögren’s syndrome and in APDS/PASLI, a disease caused by
gainꢀofꢀfunction mutations of PI3Kδ.
We have recently disclosed the optimization of novel 4,6ꢀdiaryl
quinazolines as PI3Kδꢀselective inhibitors.30 These efforts led to
discovery of quinazoline 1a as a potent and isoform selective
inhibitor of PI3Kδ (Table 1). We could demonstrate that comꢀ
pound 1a is effective in downꢀregulating PI3Kδꢀmediated signalꢀ
ing in vitro and in vivo, ultimately resulting in doseꢀdependent
efficacy in a mechanistic rodent model after oral dosing. Howevꢀ
er, when amorphous material of compound 1a was exposed to an
aqueous environment, a crystalline trihydrate polymorph formed
over time. This trihydrate had low aqueous solubility, and we
hypothesized that solubilityꢀlimited absorption was the reason for
the underproportional exposure increase which we observed with
compound 1a in higher dose rat PK studies.30 When we directly
compared trihydrate vs amorphous material in rat PK studies, we
indeed observed a significant drop in exposure (ca. 5ꢀfold, data
not shown). A polymorph with poor aqueous solubility that is
formed under an aqueous environment poses a serious developꢀ
ment hurdle as inconsistent or erratic exposure levels become
more likely. This not only impacts preclinical efficacy or toxicoꢀ
logical studies, but also poses a risk in later clinical development
(safety risk if exposure is too high, lacking efficacy risk if too
low). It became the project goal to identify a replacement for
compound 1a with an improved solubility of crystalline material.
KEYWORDS Phosphoinositide-3-kinase delta inhibitor,
PI3Kδ inhibitor, lead optimization, structure-activity rela-
tionship, PK/PD studies, B cell inhibition
Phosphoinositideꢀ3ꢀkinase δ (PI3Kδ) is a lipid kinase composed
of an enzymatic p110δ subunit and a regulatory p85 subunit exꢀ
pressed predominantly in hematopoietic cells. PI3Kδ catalyzes the
intracellular conversion of phosphatidylinositol 4,5ꢀbisphosphate
(PIP2) to phosphatidylinositol 3,4,5ꢀtrisphosphate (PIP3) downꢀ
stream of a number of receptor tyrosine kinases (eg. BCR, TCR,
FcεR1)1,2 as well as some G proteinꢀcoupled receptors (GPCR;
e.g. CXCR5).3
We recently disclosed that the 4ꢀaryl spacer could be substituted
by a pyrrolidineoxy substituent which led to an improved memꢀ
brane permeability profile and increased ligand efficiency.31
When the core quinazoline was changed to other heterocycles that
still contained the hinge binding nitrogen, namely quinolines, 1,5ꢀ
naphthyridines and cinnolines, we observed that a substantial
modulation of the lipophilicity was the consequence. Not every
Inhibition of PI3Kδ has been shown to be beneficial for the treatꢀ
ment of hematological malignancies where the PI3K/AKT pathꢀ
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derivative, including the compounds that are presented in this
selectivity, we optimized PI3K activity empirically using bioꢀ
chemical and cellular assay results.31 The first structures of selecꢀ
tive ligands complexed with PI3Kδ became available only after
CDZ173 had been identified. It was only at this point that a solid
selectivity rational on the molecular level could be developed
(Figure 1). A comparison of the structures of quinazoline 1 bound
to PI3Kα and PI3Kδ revealed differences in the binding modes of
the inhibitor to the ATP sites of both isoforms.30 Whereas the
acetylꢀpiperazine group of the ligand stacks to the side chain of
W760 in PI3Kδ, the corresponding interaction to W780 in
PI3Kαis prevented by R770 in PI3Kα and the ligand is bound in
an extended conformation to this isoform (Figure 1, left panel).
This loss of Van der Waals interactions between the inhibitor and
W780 in PI3Kα correlates with considerable loss in potency of
the inhibitor for PI3Kα compared to PI3Kδ. We hypothesized that
maintaining the stacking interaction with W760 (PI3Kδ) while
preventing the ligand from adopting an extended conformation
would offer a design principle for PI3Kδ inhibitors with exquisite
selectivity over PI3Kα. To test this hypothesis, we obtained the
coꢀcrystal structure of CDZ173 bound to the ATP site of PI3Kδ
(Figure 1, right panel). This structure reveals that the propioꢀ
namide group of CDZ173 stacks with W760 in a similar way to
the acetylꢀpiperazine group of compound 1a. This binding mode
of CDZ173 confirms our docking results,31 and strengthens our
hypothesis that the (S)ꢀenantiomer is preferred regarding PI3Kδ
potency and isoform selectivity. Supplementary Figure 10 shows
that the binding modes observed for compounds 1a and CDZ173
clearly differ from previously discovered PI3Kδ inhibitors. “Proꢀ
pellerꢀshaped” inhibitors such as GSꢀ643624 bind to a pocket that
is induced by insertion of the quinazoline group of GSꢀ643624
between W760 (PI3Kδ) and M752 (PI3Kδ) (‘WMꢀpocket’).32 In
the coꢀcrystal structures of compounds 1 and CDZ173, M752
(PI3Kδ) occupies the area where the quinazoline of GSꢀ643624 is
bound. The WMꢀpocket is not induced for our inhibitor series.
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manuscript, showed a tight correlation of log D with other paramꢀ
eters that are known to be modulated by lipophilicity, such as
solubility, membrane permeability and microsomal stability. Nevꢀ
ertheless we noticed some consistent impact by lipophilicity
changes which we outline in the following section.
As part of our core scaffold modifications we extended our invesꢀ
tigations to partially saturated bicyclic systems, and in this context
we discovered 5,6,7,8ꢀtetrahydropyrido[4,3ꢀd]pyrimidine (THPP)
as a suitable core that decreased lipophilicity significantly. This is
illustrated by comparing quinazoline 1b (HTꢀlogP = 2.7) to the
similarly substituted THPP analogue 2a (HTꢀlogP = 0.7); the
lipophilicity drop of two orders of magnitude resulted in a marked
decrease of membrane permeability (PAMPA log Pe = ꢀ4.9∙10ꢀ6
→ 6.7∙10ꢀ6 cm∙secꢀ1 at pH = 6.8), and a loss in cellular activity
(PI3Kδ IC50 = 2.63 ꢀM). Replacing one of the amide bonds by an
ether bridge (2a→2b) increased lipophilicity (HTꢀlogP = 2.2),
reduced topological polar surface area (tPSA= 91 → 80 Å2), imꢀ
proved membrane permeability, and the biochemical activity
translates better into cellular assays. When we introduced the
pyrrolidineoxyꢀsubstituents on the THPP core we found that the
overall biochemical and cellular PI3Kδ activities for our first
analogues such as 3a were moderate. At the same time, the lipoꢀ
philicity space appeared highly promising, and ADME properties
such as membrane permeability, solubility and stability in liver
microsomes were favourable. By SAR expansion we learned that
introducing a CF3 group in the 3ꢀposition of the methoxypyridine
(3a→3b) increased PI3Kδ potency by a factor of five. This modiꢀ
fication was accompanied by a lipophilicity increase by almost
one order of magnitude, and solubility and microsomal stability
were negatively impacted. For oxygenꢀlinked derivatives (3a-d),
the optimal balance was found combining a methyl group in the 3ꢀ
position of the pyridine with a tetrahydropyran group as R2 subꢀ
stituent (compound 3c). Similar to the pair 3a/3b, an introduction
of a CF3 group (3c→3d) led to an increased in vitro clearance and
reduced solubility.
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As outlined in Table 1, the inhibitory effect on PI3Kα, β and δ
isoforms on the cellular level was routinely assessed in Ratꢀ1 cells
transfected with human myrꢀp110 PI3K isoforms using the PI3Kꢀ
dependent phosphorylation of AKT (pAKT) as a readout. The
PI3Kδ activity of CDZ173 in this artificial assay system translated
well into rodent Bꢀcell activation assays (murine splenocytes and
rat whole blood), indicating sufficient fraction unbound and minꢀ
imal unspecific binding (Table 1). CDZ173 potently inhibits forꢀ
mation of proximal pathway markers (pAKT) and also antiꢀ
IgM±ILꢀ4ꢀinduced expression of distal markers of B cell activaꢀ
tion (CD69) and coꢀstimulation (CD86) across species using
whole blood or isolated cells (Table 2). In mouse, antiꢀIgMꢀ
induced B cell proliferation is also potently blocked. These results
confirm and extend published findings with PI3Kδ deficient mice
and PI3Kδ selective tool compounds.33,34 The effects of CDZ173
on T cell responses are shown in Table 2. The mixed lymphocyte
reaction (MLR) is a model for allogeneic T cell activation in vitro.
The MLR with both human peripheral blood mononuclear cells
(PBMCs) and mouse splenocytes is potently inhibited by
CDZ173. The aCD3ꢀstimulated proliferation of T cells from huꢀ
man PBMC is also inhibited, and the reduced dependency on
PI3Kδ in the presence of aCD28 coꢀstimulation could be conꢀ
firmed.33 The differentiation of murine and human aCD3/aCD28
mAbꢀstimulated T cells into Th2 or Th17 cells is significantly
blocked. In summary, CDZ173 potently blocks PI3Kδ dependent
signaling in B and T cells across species and matrices.
When we switched the ether linker to an amine (3e-h), two
ADME parameters were markedly affected: reduced in vitro
clearance and reduced PAMPA permeability (compare comꢀ
pounds 3b vs 3h, 3c vs 3e and 3d vs 3f). In general, amineꢀlinked
analogues were also chemically more stable towards acid cataꢀ
lyzed hydrolysis compared to their oxygenꢀlinked counterparts. In
contrast to the oxygenꢀlinked derivatives, the optimal overall
property space for amineꢀlinked analogues was achieved by a
more lipophilic R1/R2 substitution pattern, such as that shown for
propionamide derivatives 3g and 3h. Compound 3h was found to
have the optimal profile, hydrophilic enough to ensure good soluꢀ
bility and metabolic stability, yet not too polar to allow for favorꢀ
able membrane permeability. This compound was selected for
further profiling and later became our clinical candidate now
known as CDZ173 (leniolisib).
Compared to compound 1a, CDZ173 is similar with respect to the
overall onꢀtarget/selectivity profile, moderate rat liver microsomal
turnover and solubility of amorphous material. In contrast to
compound 1a, CDZ173 shows good PAMPA permeability, enaꢀ
bling favourable absorption as we will discuss later. In addition,
the solubility of crystalline CDZ173 (free base) was sufficient to
support all pharmacological and early toxicological experiments.
At a later stage, a crystalline phosphate salt with an >500ꢀfold
solubility improvement was identified in a salt screen (see Supꢀ
plementary Table 1) and chosen as clinical service form in order
to reduce the risk for exposure variability and/or food effects.
CDZ173 shows a 30ꢀfold cellular selectivity over PI3Kα (Table 1,
cellular assay section in Supporting Information) which translates
to a preservation of insulin homeostasis in mice up to a dose as
high as 100 mg/kg bid (Supplementary Figure 9). While the celluꢀ
lar isoform selectivity against PI3Kα and β was determined in
Ratꢀ1 cells as already described, PI3Kγ activity has to be tested
At the time when the optimization work was conducted, we did
not have any xꢀray coꢀcrystal structures for PI3Kδ selective ligꢀ
ands. As a surrogate, we used a homology model based on PI3Kγ.
As this model was not able to explain the reasons for the observed
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differently due to its distinct signaling downstream of GPCR. We
a dose of 10 mg/kg b.i.d. significantly ameliorated disease paramꢀ
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eters and strongly reduced the degree of established autoantibody
responses (Figure 3 and Supplementary Figure 7). The PK profile
and the dose normalized AUC observed in the multiple dose exꢀ
periments was comparable to data obtained from single dose PK
studies in naive animals. In an ozoneꢀinduced lung inflammation
model in mice, CDZ173 doseꢀdependently inhibited the increase
in bronchoalveolar lavage (BAL) neutrophil and macrophage
numbers with ED50 values of 16 mg/kg and 40 mg/kg, respectiveꢀ
ly (Supplementary Figure 8). There was no effect on the ozoneꢀ
induced plasma leakage, as measured by the increased BAL seꢀ
rum albumin levels (data not shown). In all presented preclinical
multiꢀdose pharmacology studies, CDZ173 was well tolerated and
there were no apparent signs of toxicity.
measured the PI3Kγꢀdependent activation of U937 monocytes by
the GPCRꢀligand MIPꢀ1α, and CDZ173 showed no activity up to
the highest test concentration (IC50 > 7.4 ꢀM). In addition, the
cellular activity on PI3Kγ was assessed via the activation of
mouse bone marrowꢀderived mast cells (BMMCs) stimulated with
adenosine where CDZ173 was a very weak inhibitor (IC50 = 7.8
ꢀM). When tested in biochemical assays against the Class III
PI3K Vps34, and enzymes of the wider PI3K family (mTOR and
PI4K), CDZ173 was inactive up to the highest test concentration
(IC50 > 9.1 ꢀM). Whilst DNAꢀPK was moderately inhibited by
CDZ173 (biochemical IC50 = 0.88 ꢀM), this activity did not transꢀ
late to an inhibition of p53 in a stressed p53REꢀbla HCT116 reꢀ
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porter cell line further down in the pathway of DNAꢀrepair (IC50
>
Taken together, the in vitro properties and the results in precliniꢀ
cal models of autoimmune and inflammatory diseases demonꢀ
strate the potential of CDZ173 to act as a therapeutic for immuneꢀ
mediated diseases. Because of the big structural differences to the
firstꢀgeneration of clinical PI3Kδ and/or PI3Kγδꢀselective inhibiꢀ
tors, CDZ173 could favourably differentiate in terms of its safety
profile. After supportive preclinical toxicology studies and a sucꢀ
cessful phase I clinical campaign, CDZ173 (leniolisib) is currently
being explored in clinical trials for APDS (ClinicalTrials.gov
identifier: NCT02435173) and primary Sjögren’s Syndrome
(NCT02775916).
3 ꢀM).
CDZ173 showed no activity up to the highest test concentration
when tested against CYP isoform assays (3A3, 2D9, 2D6, 2C9),35
a panel of ion channels (including hERG)36 and a protease panel.
In a panel of 50 safety related targets (GPCRs, ion channels,
transporters), CDZ173 only showed measurable activity for
hPDE4D (IC50=4.7 ꢀM) and 5HT2B (IC50=7.7 ꢀM). Furthermore,
CDZ173 was tested against a panel of 442 diverse serꢀ
ine/threonine, tyrosineꢀ and lipid kinases utilizing DiscoverX’
KINOMEscan® technology at 10 ꢀM. The only kinase that was
inhibited by CDZ173 (in addition to the expected Class I PI3Ks)
was RPS6KA5 (76% inhibition, no cellular followꢀup as no negaꢀ
tive impact on safety/tolerability was anticipated).
ASSOCIATED CONTENT
Supporting Information
The pharmacokinetic profile of CDZ173 is shown in Table 3. In
general, the compound is absorbed very quickly across species as
can be seen by an early Tmax of the oral profiles. Whereas clearꢀ
ance is low to moderate in rats and monkeys, it was found that
clearance in dogs is very low resulting in a very high exposure in
blood. As dog plasma protein binding is very high (>99%) and
restricting distribution of the compound into tissue (Vss = 0.3
L·kgꢀ1), dogs were excluded as a potential species for toxicologiꢀ
cal studies. In contrast to compound 1a,30 CDZ173 did not show
underproportional exposure increase comparing 3 and 30 mg/kg
doses in rats.
Full descriptions of all biological assays and in vivo studies. Charꢀ
acterization of all compounds. Full experimental procedures for
the sequence leading to CDZ173. Crystallographic data collection
and refinement statistics for crystal structures. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
PDB code for the Xꢀray crystal structure described in this study
has been deposited in the Protein Data Bank under the accession
code 5O83.
Because of its favourable pharmacokinetic profile in rodents and
monkeys, we tested CDZ173 in various in vivo settings, starting
with PK/PD experiments. In rats, CDZ173 inhibits ex vivo antiꢀ
IgM/ILꢀ4 ꢀinduced proximal pAKT formation in rats in a concenꢀ
trationꢀ and timeꢀdependent manner (see Supplementary Figure
AUTHOR INFORMATION
Corresponding Authors
1). Plotting total blood exposure against the PD effect, an EC50
=
83 nM (EC50 free = 5 nM, rat PPB = 94%) was calculated which
is in agreement with the in vitro rat whole blood B cell activation
assay result (IC50 = 99 nM; Table 1). In cynomolgus monkeys, the
ex vivo activation of CD20+ B cells (antiꢀIgM/ILꢀ4 stimulated
pAKT) was inhibited with an EC50 of ca. 140 nM (see Suppleꢀ
mentary Figure 2). As for rat, the exposure/concentrationꢀ
response profiles are in agreement with the in vitro whole blood
assay result (IC50 = 84 nM, Table 2). The concentrationꢀdependent
PD effect of CDZ173 translated into a dose dependent pharmacoꢀ
logical inhibition of PI3Kδ as demonstrated by inhibition of the
generation of antigenꢀspecific antibody responses to sheep red
blood cells (SRBC) in rat (Figure 2). Encouraged by these results,
CDZ173 was tested in a rat model of collagenꢀinduced arthritis
(rCIA) to assess the correlation of autoantibody levels with reducꢀ
tion of clinical symptoms and histological parameters. CDZ173
significantly inhibited pathogenic antiꢀrat collagen antibodies,
paw swelling, inflammatory cell infiltration, proteoglycan loss
and joint erosion when dosing started before disease onset. Full
efficacy was seen with doses as low as 3 mg/kg b.i.d (Supplemenꢀ
tary Figures 3ꢀ6). In a therapeutic setting, where CDZ173 was
administered when significant paw swelling was already evident,
*(K.H.) Tel: +41 79 6181814. Eꢀmail: klemens.hoegenauer@
novartis.com.
*(N.S.) Tel: +41 79 8451506. Eꢀmail: nicoꢀ
las.soldermann@novartis.com.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
We thank C. Hebach, I. Lewis, A. von Matt for their synthetic and
medicinal chemistry contributions, I. Adam, I. Jeulin, D. Rageot,
B. Thai and V. Tucconi for their contributions in compound synꢀ
thesis, J. Trappe, D. d’Orazio, D. Kempf, C. Guntermann, F.
Ecoeur, D. Fabbro, D. Haasen, A. Hinz and P. Schmutz for their
efforts towards developing and performing biochemical and celluꢀ
lar assays, A Trifilieff, R. Puljic, C. Schnell, C. Fritsch, U.
McKeever, C. Gfell, D. Buffet, S. Bolliger, R. Keller, S. Bay for
in vivo pharmacology, G. Wieczorek, and N. Mamber for histoloꢀ
gy, C. Beerli and B. Urban, P. Ramstein, W. Gertsch, S.
Desrayaud, J. Hengy, S. Widmer, I.R. Mueller, J. Kamholz, R.
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tion and Kinome Profiling Reveal Altered Regulation of Multiple Signalꢀ
ing Pathways in B Lymphocytes from Patients with Systemic Lupus Eryꢀ
thematosus. Arthritis Rheum. 2010, 62 (8), 2412–2423.
Endres, A. Geisser, F.P. Cordoba, A. Kunkler, G. Laue, C. Textor,
and A. Cattini for pharmacokinetic assay work, B. Shrestha for
protein expression, R. Elling for crystallography support, K. Beltz
and P. Santos for CDZ173 developability assessment as well as J.
Wagner and G. Weckbecker for scientific guidance. We are grateꢀ
ful to the MX beamline team at the Swiss Light Source (PSI Vilꢀ
ligen, Switzerland) for outstanding support at the beamline and
Expose GmbH (Switzerland) for diffraction data collection.
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Systemic Lupus in the Mouse. J. Immunol. 2014, 193 (2), 544–554.
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APDS, activated PI3Kδ syndrome; BAL, bronchoalveolar lavage;
BAV, absolute oral bioavailability; BMMC, bone marrowꢀderived
mast cells; CIA, collagenꢀinduced arthritis; CYP, cytochrome
P450; GPCR, G proteinꢀcoupled receptor; hERG, human etherꢀàꢀ
goꢀgoꢀrelated gene; MLR, mixed lymphocyte reaction; THPP,
5,6,7,8ꢀtetrahydropyrido[4,3ꢀd]pyrimidine; mTOR, mechanistic
Target Of Rapamycin; PAMPA, parallel artificial membrane perꢀ
meability assay; PASLI, p110 delta activating mutation causing
senescent T cells, lymphadenopathy, and immunodeficiency;
PBMC, peripheral blood mononuclear cells; PI3K, Phosphoinosiꢀ
tideꢀ3ꢀkinase; SAR, structureꢀactivity relationship; SRBC, sheep
red blood cell(s); tPSA, topological polar surface area.
(17)
Angulo, I.; Vadas, O.; Garçon, F.; BanhamꢀHall, E.; Plagnol,
V.; Leahy, T. R.; Baxendale, H.; Coulter, T.; Curtis, J.; Wu, C.; Blakeꢀ
Palmer, K.; Perisic, O.; Smyth, D.; Maes, M.; Fiddler, C.; Juss, J.; Cilliers,
D.; Markelj, G.; Chandra, A.; Farmer, G.; Kielkowska, A.; Clark, J.;
Kracker, S.; Debré, M.; Picard, C.; Pellier, I.; Jabado, N.; Morris, J. A.;
BarcenasꢀMorales, G.; Fischer, A.; Stephens, L.; Hawkins, P.; Barrett, J.
C.; Abinun, M.; Clatworthy, M.; Durandy, A.; Doffinger, R.; Chilvers, E.
R.; Cant, A. J.; Kumararatne, D.; Okkenhaug, K.; Williams, R. L.; Conꢀ
dliffe, A.; Nejentsev, S. Phosphoinositide 3ꢀKinase δ Gene Mutation Preꢀ
disposes to Respiratory Infection and Airway Damage. Science 2013, 342
(6160), 866–871.
(18)
Lucas, C. L.; Kuehn, H. S.; Zhao, F.; Niemela, J. E.; Deenick,
E. K.; Palendira, U.; Avery, D. T.; Moens, L.; Cannons, J. L.; Biancalana,
M.; Stoddard, J.; Ouyang, W.; Frucht, D. M.; Rao, V. K.; Atkinson, T. P.;
Agharahimi, A.; Hussey, A. A.; Folio, L. R.; Olivier, K. N.; Fleisher, T.
A.; Pittaluga, S.; Holland, S. M.; Cohen, J. I.; Oliveira, J. B.; Tangye, S.
G.; Schwartzberg, P. L.; Lenardo, M. J.; Uzel, G. DominantꢀActivating
Germline Mutations in the Gene Encoding the PI(3)K Catalytic Subunit
p110δ Result in T Cell Senescence and Human Immunodeficiency. Nat.
Immunol. 2014, 15 (1), 88–97.
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Campuzano, I. D. G.; Cardozo, M.; Dunn, M. C.; Duquette, J.; Fisher, B.;
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Erra, M.; Taltavull, J.; Gréco, A.; Bernal, F. J.; Caturla, J. F.;
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Patel, L.; Chandrasekhar, J.; Evarts, J.; Forseth, K.; Haran, A.
Gràcia, J.; Domínguez, M.; Sabaté, M.; Paris, S.; Soria, S.; Hernández, B.;
Armengol, C.; Cabedo, J.; Bravo, M.; Calama, E.; Miralpeix, M.; Lehner,
M. D. Discovery of a Potent, Selective, and Orally Available PI3Kδ Inhibꢀ
itor for the Treatment of Inflammatory Diseases. ACS Med. Chem. Lett.
2017, 8 (1), 118–123.
C.; Ip, C.; Kashishian, A.; Kim, M.; Koditek, D.; Koppenol, S.; Lad, L.;
Lepist, E.ꢀI.; McGrath, M. E.; Perreault, S.; Puri, K. D.; Villaseñor, A. G.;
Somoza, J. R.; Steiner, B. H.; Therrien, J.; Treiberg, J.; Phillips, G. Disꢀ
covery of Orally Efficacious Phosphoinositide 3ꢀKinase δ Inhibitors with
Improved Metabolic Stability. J. Med. Chem. 2016, 59 (19), 9228–9242.
(12)
Bartok, B.; Boyle, D. L.; Liu, Y.; Ren, P.; Ball, S. T.; Bugbee,
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Patel, L.; Chandrasekhar, J.; Evarts, J.; Haran, A. C.; Ip, C.;
W. D.; Rommel, C.; Firestein, G. S. PI3 Kinase δ Is a Key Regulator of
Synoviocyte Function in Rheumatoid Arthritis. Am. J. Pathol. 2012, 180
(5), 1906–1916.
Kaplan, J. A.; Kim, M.; Koditek, D.; Lad, L.; Lepist, E.ꢀI.; McGrath, M.
E.; Novikov, N.; Perreault, S.; Puri, K. D.; Somoza, J. R.; Steiner, B. H.;
Stevens, K. L.; Therrien, J.; Treiberg, J.; Villaseñor, A. G.; Yeung, A.;
Phillips, G. 2,4,6ꢀTriaminopyrimidine as a Novel Hinge Binder in a Series
of PI3Kδ Selective Inhibitors. J. Med. Chem. 2016, 59 (7), 3532–3548.
(13)
Taher, T. E.; Parikh, K.; FloresꢀBorja, F.; Mletzko, S.; Isenꢀ
berg, D. A.; Peppelenbosch, M. P.; Mageed, R. A. Protein Phosphorylaꢀ
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Qin, L.ꢀY.; Ruan, Z.; Cherney, R. J.; Dhar, T. G. M.; Neels, J.;
Weigelt, C. A.; Sack, J. S.; Srivastava, A. S.; Cornelius, L. A. M.; Tino, J.
A.; Stefanski, K.; Gu, X.; Xie, J.; Susulic, V.; Yang, X.; YardeꢀChinn, M.;
Skala, S.; Bosnius, R.; Goldstein, C.; Davies, P.; Ruepp, S.; SalterꢀCid, L.;
Bhide, R. S.; Poss, M. A. Discovery of 7ꢀ(3ꢀ(Piperazinꢀ1ꢀ
yl)phenyl)pyrrolo[2,1ꢀf][1,2,4]triazinꢀ4ꢀAmine Derivatives as Highly
Potent and Selective PI3Kδ Inhibitors. Bioorg. Med. Chem. Lett. 2017, 27
(4), 855–861.
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Terstiege, I.; Perry, M.; Petersen, J.; Tyrchan, C.; Svensson, T.;
Lindmark, H.; Öster, L. Discovery of Triazole Aminopyrazines as a Highꢀ
ly Potent and Selective Series of PI3Kδ Inhibitors. Bioorg. Med. Chem.
Lett. 2017, 27 (3), 679–687.
9
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Hoegenauer, K.; Soldermann, N.; Stauffer, F.; Furet, P.; Gravꢀ
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eleau, N.; Smith, A. B.; Hebach, C.; Hollingworth, G. J.; Lewis, I.; Gutꢀ
mann, S.; Rummel, G.; Knapp, M.; Wolf, R. M.; Blanz, J.; Feifel, R.;
Burkhart, C.; Zécri, F. Discovery and Pharmacological Characterization of
Novel QuinazolineꢀBased PI3K DeltaꢀSelective Inhibitors. ACS Med.
Chem. Lett. 2016, 7 (8), 762–767.
Figure 2. Doseꢀdependent inhibition of SRBCꢀspecific IgM reꢀ
sponse by CDZ173 (error bars indicate SD); ***: p<0.001 comꢀ
pared to vehicle (student’s tꢀtest, twoꢀtailed distribution).
(31)
Hoegenauer, K.; Soldermann, N.; Hebach, C.; Hollingworth,
G. J.; Lewis, I.; von Matt, A.; Smith, A. B.; Wolf, R. M.; Wilcken, R.;
Haasen, D.; Burkhart, C.; Zécri, F. Discovery of Novel Pyrrolidineoxyꢀ
Substituted Heteroaromatics as Potent and Selective PI3K Delta Inhibitors
with Improved Physicochemical Properties. Bioorg. Med. Chem. Lett.
2016, 26 (23), 5657–5662.
(32)
(33)
http://www.rcsb.org, PDB ID=5T7F.
Okkenhaug, K.; Bilancio, A.; Farjot, G.; Priddle, H.; Sancho,
S.; Peskett, E.; Pearce, W.; Meek, S. E.; Salpekar, A.; Waterfield, M. D.;
Smith, A. J. H.; Vanhaesebroeck, B. Impaired B and T Cell Antigen Reꢀ
ceptor Signaling in p110δ PI 3ꢀKinase Mutant Mice. Science 2002, 297
(5583), 1031–1034.
(34)
Clayton, E.; Bardi, G.; Bell, S. E.; Chantry, D.; Downes, C. P.;
Gray, A.; Humphries, L. A.; Rawlings, D.; Reynolds, H.; Vigorito, E.;
Turner, M. A Crucial Role for the p110δ Subunit of Phosphatidylinositol
3ꢀKinase in B Cell Development and Activation. J. Exp. Med. 2002, 196
(6), 753–763.
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Bell, L.; Bickford, S.; Nguyen, P. H.; Wang, J.; He, T.; Zhang,
B.; Friche, Y.; Zimmerlin, A.; Urban, L.; Bojanic, D. Evaluation of Fluoꢀ
rescenceꢀ and Mass Spectrometry—Based CYP Inhibition Assays for Use
in Drug Discovery. J. Biomol. Screen. 2008, 13 (5), 343–353.
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Finlayson, K.; Turnbull, L.; January, C. T.; Sharkey, J.; Kelly,
J. S. [3H]Dofetilide Binding to HERG Transfected Membranes: A Potenꢀ
tial High Throughput Preclinical Screen. Eur. J. Pharmacol. 2001, 430
(1), 147–148.
Figure 3. Inhibition of paw swelling in a therapeutic rat CIA exꢀ
periment (dosing after disease onset, error bars indicate SEM).
Antigen priming (I°) and boost (2°) indicated with arrows. Treatꢀ
ment period indicated with horizontal line at bottom of graph.
*p<0.01.
FIGURES AND TABLES
Figure 1. Left panel, Structural alignment of PI3Kα and PI3Kδ
with compound 1a bound to the ATP sites. The protein part of
PI3Kδ/compound 1a (PDB 5IS5)30 is depicted as grey surface.
Amino acid side chains T750, M752, W760 and the inhibitor are
represented as blue sticks for PI3Kδ. The corresponding amino
acid and inhibitor structures in PI3Kα (PDB 5ITD)30 are shown in
yellow. Right panel, Comparison of the binding modes of
CDZ173 (green sticks) and compound 1a (blue sticks) to the acꢀ
tive site of PI3Kδ (grey surface with amino acids T750, M752 and
W760 coloured in red, yellow and magenta, respectively.
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Table 1. Influence of 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine core introduction on biochemical/cellular potency, PI3K
isoform selectivity and in vitro ADME parameters
1
2
3
4
5
6
7
8
9
biochemical IC50 [ꢀM]a
PI3Kαb PI3Kβb PI3Kγb PI3Kδc PI3Kα PI3Kβ PI3Kδ
cellular IC50 [ꢀM]a, d
mCD86 rCD86 PAMP HTꢀ
RLM HTꢀ
j
IC50
IC50
A
logPh CLint sol
X
R1 R2
g
[ꢀM]a, e [ꢀM]a, f
logPe
[mM]
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
k
CN
H
0.262
0.418
6.94
2.02
6.66
1.65
4.84
>9.1
>9.1
8.94
1.71
4.63
2.70
>9.1
>9.1
>9.1
>9.1
0.009
0.029
0.065
0.083
0.132
0.027
3.44
2.12
>10
9.03
>10
2.60
6.53
3.57
>10
>10
>10
3.11
0.049
0.028
2.63
0.374
5.39
0.074
0.070
nd
nd
nd
0.072
0.066
nd
nd
nd
ꢀ5.5
ꢀ4.9
ꢀ6.7
ꢀ4.1
ꢀ3.7
ꢀ3.6
2.8
2.7
0.7
2.2
2.2
3.1
63
115
91
197
35
160
0.21
>1.0
>1.0
>1.0
>1.0
0.080
1a
1b
2a
2b
3a
3b
O
O
H
Et
CF3 Et
0.792
0.131
nd
nd
O
O
Me
1.94
4.15
1.33
2.02
0.300
6.60
>9.1
3.29
1.06
0.076
0.028
0.030
0.004
6.49
3.55
6.19
2.15
6.79
3.58
9.87
3.99
0.143
0.037
0.358
0.053
0.098
0.084
0.091
0.015
0.092
0.058
0.074
0.016
ꢀ4.2
ꢀ3.7
ꢀ5.9
ꢀ5.5
1.7
2.2
2.6
2.9
60
>1.0
0.27
>1.0
0.24
3c
3d
3e
3f
CF3
0.590
0.373
0.044
149
22
NH Me
NH CF3
41
NH Me Et
NH CF3 Et
0.651
0.244
0.951
0.424
nd
2.23
0.020b 7.41
0.011 1.67
7.91
2.25
0.246
0.056
0.087
0.048
0.073
0.099
ꢀ5.7
ꢀ4.5
2.8
3.2
nd
55
>1.0
0.32
3g
3h*
aMean of a minimum of two independent experiments; standard deviation for pIC50 values <0.3; bKGlo format; cADAPTA format (valꢀ
ues comparable to KGlo); dinhibition of pAkt formation in Ratꢀ1 cells; einhibition of antiꢀIgM induced mCD86 expression on mouse spleꢀ
nocytes; inhibition of antiꢀIgM/ILꢀ4 induced rCD86 expression in 50% rat blood; geffective permeability [10ꢀ6 cm∙secꢀ1] (pH = 6.8), n=1;
f
hHighꢀthroughput logP measurement with immobilized artificial membranes, n=1; intrinsic clearance in incubations of rat liver microꢀ
j
somes [ꢀL∙minꢀ1∙mgꢀ1 protein], n=1; kHighꢀthroughput equilibrium solubility determination [mM] (pH = 6.8); nd = not determined;
*CDZ173 (leniolisib)
Table 2. Cellular inhibition of PI3Kδ -dependent B and T cell functions in different species by CDZ173.
B cellsa
T cells
Species
humanb
aIgM/ILꢀ4
cynob
ratc
moused
aIgM
humane
aCD3/aCD28f
moused
Allo
Allo
Stimulus aIgM
aIgM
aIgM/ILꢀ4
aCD3g
aCD3/aCD28f
MLRf
MLRf
Readout
pAKT CD86 CD69 pAKT pAKT CD86 Prol.g CD86 Prol.g Prol. g Th2 Th17 Prol.g Prol.g Th2 Th17
0.144 0.202 0.193 0.084 0.150 0.099 0.007 0.048 0.079 0.159 0.095 0.073 1.15 0.033 0.010 0.101
IC50 [ꢀM]
aData from at least two independent experiments are shown; standard deviation for pIC50 values <0.3; measured in 90% whole blood;
cmeasured in 50% whole blood; dmeasured in splenocytes; emeasured in PBMCs; fmean of at least four independent experiments; gproliferꢀ
ation (Prol.) measured as 3HꢀThymidine uptake
b
Table 3. PK parametersa for CDZ173 (crystalline free base).
Species
Ratb
1 (iv)
Ratb
3 (po)
Ratb
30 (po)
Dogc
0.1 (iv)
Dogc
0.3 (po)
Monkeyd
0.1 (iv)
17 (2)
Monkeyd
0.3 (po)
Dose [mg·kgꢀ1]
CL [mL·minꢀ1·kgꢀ
28 (3)
2 (1)
1
]
VSS [L·kgꢀ1]
t1/2 term. [h]
3.1 (0.8)
1.8 (0.3)
0.3 (0.1)
1.5 (0.4)
0.6 (0.1)
0.7 (0.4)
AUC d.n.e [nM·h] 1355 (158) 1165 (27) 2107 (655) 20120 (7720) 11690 (6277) 2250 (290) 297 (107)
BAV [%]
86 (2)
391 (161) 334 (105)
0.4 (0.1) <0.25
>100
58 (31)
3410 (1347)
0.7 (0.3)
13 (5)
213 (180)
<0.5
Cmax d.n.e [nM]
Tmax [h]
aMean values with standard deviation in brackets; bfemale, n=4; cmale, n=3; done female, two male; ed.n. = dose normalized to 1 mg·kgꢀ1
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Discovery of CDZ173 (leniolisib), Representing a Structurally
Novel Class of PI3K Delta-Selective Inhibitors
Klemens Hoegenauer*a, Nicolas Soldermann*a, Frédéric Zécria, Ross S. Stranga, Nadege Graveleaua, Romain M. Wolfa, Nigel G. Cookea, Al-
exander B. Smitha, Gregory J. Hollingwortha, Joachim Blanzb, Sascha Gutmannc, Gabriele Rummelc, Amanda Littlewood-Evansd, Christoph
Burkhartd
aGlobal Discovery Chemistry, bPK Sciences, cChemical Biology and Therapeutics, dAutoimmunity, Transplantation and Inflammation, No-
vartis Institutes for BioMedical Research, Novartis Campus, CH-4002 Basel, Switzerland
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