Structure Overhaul Affords a Potent Purine PI3KδInhibitor with Improved Tolerability

Article abstract of DOI:10.1021/acs.jmedchem.8b01818

PI3Kδcatalytic activity is required for immune cell activation, and has been implicated in inflammatory diseases as well as hematological malignancies in which the AKT pathway is overactive. A purine PI3Kδinhibitor bearing a benzimidazolone-piperidine motif was found to be poorly tolerated in dog, which was attributed to diffuse vascular injury. Several strategies were implemented to mitigate this finding, including reconstruction of the benzimidazolone-piperidine selectivity motif. Structure-based design led to the identification of O- and N-linked heterocycloalkyls, with pyrrolidines being particularly ligand efficient and kinome selective, and having an improved safety pharmacology profile. A representative was advanced into a dog tolerability study where it was found to be well tolerated, with no histopathological evidence of vascular injury.

Full text of DOI:10.1021/acs.jmedchem.8b01818

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Article
Structure Overhaul Affords a Potent Purine
PI3K# Inhibitor with Improved Tolerability.
Joey L Methot, Hua Zhou, Sam Kattar, Meredeth Mcgowan, Kevin Wilson, Yudith Garcia, Yongi
Deng, Michael D Altman, Xavier Fradera, Charles A Lesburg, Thierry Fischmann, Chaomin Li,
Steve Alves, Sanjiv Shah, Rafael Fernandez, Peter Goldenblatt, Armetta Hill, Lynsey Shaffer,
Dapeng Chen, Vince Tong, Robbie L McLeod, Hongshi Yu, Alan Bass, Ray Kemper, Nicholas
T Gatto, Lisa LaFranco-Scheuch, Benjamin Wesley Trotter, Timothy Guzi, and Jason Katz
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01818 • Publication Date (Web): 15 Apr 2019
Downloaded from http://pubs.acs.org on April 16, 2019
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Journal of Medicinal Chemistry
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Structure Overhaul Affords a Potent Purine PI3K Inhibitor
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with Improved Tolerability.
Joey L. Methot,*a Hua Zhou,a Sam D. Kattar,a Meredeth A. McGowan,a Kevin Wilson,a Yudith Garcia,a Yongi
Deng,a Michael Altman,b Xavier Fradera,b Charles Lesburg,c Thierry Fischmann,c Chaomin Li,d Steve Alves,e
Sanjiv Shah,f Rafael Fernandez,f Peter Goldenblatt,f Armetta Hill,f Lynsey Shaffer,f Dapeng Chen,g Vince Tong,g
Robbie L. McLeod,h Hongshi Yu,i Alan Bass,j Ray Kemper,j Nicholas T. Gatto,j Lisa LaFranco-Scheuch,j
Benjamin Wesley Trotter,a Timothy Guzi,a Jason D. Katza
Departments of (a) Discovery Chemistry, (b) Modeling and Informatics, (c) Structural Chemistry, (d) Process Chemistry, (e)
Discovery Biology, (f) In Vitro Pharmacology, (g) Preclinical Pharmacokinetics and Drug Metabolism, (h) In Vivo
Pharmacology, (i) Discovery Pharmaceutical Science, and (j) Safety Assessment and Laboratory Animal Resurces.
Merck & Co., Inc., 33 Avenue Louis Pasteur, Boston, MA 02115 USA.
Keywords: PI3K inhibitor, vascular injury, adenosine uptake
ABSTRACT: PI3K catalytic activity is required for immune cell activation, and has been implicated in inflammatory diseases as
well as hematological malignancies in which the AKT pathway is overactive. A purine PI3K inhibitor bearing a benzimidazolone-
piperidine motif was found to be poorly tolerated in dog, which was attributed to diffuse vascular injury. Several strategies were
implemented to mitigate this finding, including reconstruction of the benzimidazolone-piperidine selectivity motif. Structure-based
design led to the identification of O- and N-linked heterocycloalkyls, with pyrrolidines being particularly ligand efficient and kinome
selective, and having an improved safety pharmacology profile. A representative was advanced into a dog tolerability study where it
was found to be well tolerated, with no histopathologic evidence of vascular injury.
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INTRODUCTION
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Phosphoinositide 3-kinase delta (PI3K) is expressed primarily in leukocytes, where it phosphorylates the 3′ position of the
inositol ring of phosphatidylinositol, a messenger which serves as a membrane anchor for several protein kinases (e.g. AKT) to
regulate a broad range of cellular activities such as cell survival, proliferation, differentiation, and migration.1 PI3K knock-out or
kinase dead mice are viable and fertile, and resistance to induced inflammatory disease in these genetically modified mice is consistent
with involvement of PI3K activity in dysregulated or over-active inflammatory response. Furthermore, the restricted expression in
immune cells suggest a potentially safer therapeutic benefit of PI3K inhibitors versus other modulators of AKT for the treatment of
inflammatory conditions such as asthma, rheumatoid arthritis, COPD, systemic lupus erythematosus, or activated PI3K syndrome.2
The role of PI3K in B-cell activation has been implicated in hematologic malignancies, and clinical proof of concept in patients with
chronic lymphocytic leukemia was achieved by the selective PI3K inhibitor idelalisib.3 Idelalisib is also approved for the treatment
of follicular B-cell non-Hodgkin lymphoma relapsed small lymphocytic lymphoma.4 Recently, Ali and colleagues have reported that
inhibition of PI3K in murine immunocompetent syngeneic models protects against a spectrum of cancers via an adaptive immune-
mediated anti-tumor response, including solid tumors with low expression of PI3K.5 Consequently, numerous pharmaceutical and
academic organizations have invested in PI3K inhibitors to treat asthma and other inflammatory diseases, as well as liquid and solid
tumors.6
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BACKGROUND
As part of our PI3K inhibitor program, we characterized potent and selective inhibitor 1 (Figure 1)7 which features a 6,8-
disubstituted purine hinge binding motif. The inhibitor had good PI3K potency (IC50 = 6.3 nM; Ligand Binding Efficiency 0.33)
and good selectivity versus other class I PI3K isoforms (70x vs PI3K, 90x vs PI3K, 1,000x vs PI3K), as well as excellent kinome
selectivity (>100x vs 260 kinases). Kinases PI3K and PI3K are ubiquitously expressed with roles in cell proliferation and
metabolism, and genetic knockouts are embryonically lethal.8 We therefore sought high selectivity versus PI3K and PI3K,
however the highly conserved ATP-binding site of the class I PI3Ks presented a significant challenge for medicinal chemistry. The
phosphorylation status of serine-473 of AKT in the Ramos Burkitt’s lymphoma-derived B cell line is driven by PI3K, and is a good
measure of cellular activity of PI3K inhibition (compound 1 had AKT-pSer473 IC50 = 63 nM).
Potency evaluation in human whole blood facilitated clinical human dose projections, in which we targeted the B-cell activation
,
surface biomarker CD69 IC50 or basophil activation biomarker CD63 IC50 at trough concentration.9 10 B-cells play critical roles in
the humoral response by recognizing antigens and antibody production, and are implicated in many diseases of the immune system
such as rheumatoid arthritis & lupus. CD69 is expressed in several hemopoietic cells, is an early activation marker in chronic
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Journal of Medicinal Chemistry
lymphocytic leukemia, and is correlated with poor clinical prognosis.11 In human whole blood, 1 inhibited anti-human CD79b-
1
2
induced expression of the B-cell biomarker CD69 with an IC50 value of 810 nM.
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9
The plasma clearance for 1 in male Wistar Han rat was low (5.8 mL/min/kg), and the bioavailability good (76%), however the
low unbound volume led to a moderate half-life (1.2 h), see Table 2. In contrast, the plasma clearance in male beagle dogs was very
high (Clp > Qhep). Consistent with this, compound 1 was stable to NADPH-supplemented rat and human microsome incubations.
However in dog microsome incubations, two metabolites were observed; N-dealkylation of the pyrazole as well as oxidation of the
top imidazopyridinone ring. Compound 1 was clean in CYP and ion channel panels, nor an inducer of PXR.
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Compound 1 was generally well-tolerated when dosed in a rat toxicology study up to 750 mg/kg/day, with increased blood pressure
(+20 mmHg) and heart rate (+100 bpm) observed at exposures (uAUC 123 M•h, uCmax 8.0 M) exceeding predicted clinical
efficacy at 75 mg bid human dose (uAUC0-24h 2.8 M•h, uCmax 0.34 M) based on the human whole blood CD69 IC50 at trough
concentration.12 In contrast, 1 was poorly tolerated in dog as low as 30 mpk (uAUC 4.4 M•hr, uCmax 0.33 M) which is near the
exposure predicted for clinical efficacy based on the CD69 IC50. Histologic analysis demonstrated diffuse vascular injury with medial
necrosis of the arteries in multiple organs that was not found in a repeat dose study in rat (images in Figure 4).13 The cause of this
observation was unknown, however we speculated it might have been associated with hemodynamic changes. We extensively
profiled 1 in available off-target panels, but found few hits that would explain the toxicity in dogs. The compound was found to be
a modest inhibitor of several phosphodiesterases; PDE1 (IC50 = 2,350 nM), PDE2 (IC50 = 4,900 nM), PDE3 (IC50 = 1,030 nM), PDE4
(IC50 = 1,190 nM) and PDE10 (IC50 = 4,010 nM).14 It was also an inhibitor of adenosine transporter15 (AdT) binding (IC50 = 130 nM)
and cellular uptake (AdU) (IC50 = 72 nM) in HeLa cells.
The PDE and adenosine uptake off-targets may have been associated with effects on hemodynamic changes, and so we screened
available close analogs of 1 in these assays. Inhibitors bearing alternative five- and six-membered heteroaromatics at the purine C(8)
position, or amides at purine C(8) instead of the 1-ethyl-5-methylpyrazole, offered only modest improvements in selectivity versus
the phosphodiesterase panel or adenosine transporter cellular uptake. Pyrazole N-substituent SAR compounds that were tolerated as
inhibitors did not improve the off-target profile. Neither did close analogs of the benzimidazolone moiety, or piperidine isosteres
such as spirocycles, lead to improvements in the off-target profile. Efforts are summarized in Figure 2, with some examples studied
within this series shown in the supporting information with PDE and AdU inhibition data. We recognized that the benzimidazolone
piperidine moiety of 1 is a privileged scaffold for GPCR inhibitors,16 potentially binding to an off-target not present in screening
panels.
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1
2
N
NH
O
3
4
5
6
7
8
N
N
N
PI3K IC50 = 6.3 nM
PI3K IC50 = 450 nM
pAKT IC50 = 63 nM
AdU IC50 = 72 nM
N
N
N
N
N
9
1
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Figure 1. Purine 1 and crystal structure of 1 with PI3K, resolved to 3.10 Å. The purine makes hydrogen bond contacts to
the kinase hinge region while the benzimidazolone moiety fills pocket lined by Trp760 and Met752 residues. PDB ID: 6MUL.
Benzimidazolone
Substitution
N
NH
O
N
Alternative
Piperidine
Heterocycles,
Amides
Isosteres,
Spirocycles
N
N
N
N
N
N
N
Figure 2. SAR conducted to mitigate both HeLa cell adenosine uptake and phosphodiesterase off-target activities.
In the absence of a better understanding of the root cause of the vascular injury observed in dogs, we elected to invest in more
substantive structural modifications to the scaffold while monitoring selectivity versus PI3K family and other known off-targets.
Ultimately, the absence of the vascular injury phenotype in rat dictated that a resource-intensive safety study in dog would need to
be conducted to demonstrate that the risk of the series had been mitigated. To justify this resource commitment, we sought a potent
structurally distinct inhibitor with high selectivity versus all known off-targets. In the following section, we outline the discovery of
a new pyrrolidine inhibitor by structure-based design.
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Journal of Medicinal Chemistry
Table 1. Selectivity motif screen with the 9-methyl-8-pyrazolopurine hinge-binding core.
1
2
3
4
Ramos HeLa
pAKT AdU
Ramos HeLa
pAKT AdU
PI3K PI3K
PI3K PI3K
N
N
N
N
N
N
N
N
N
N
IC50
IC50
IC50
IC50
(nM)
IC50
IC50
IC50
IC50
(nM)
(nM)
(nM)
N
N
(nM)
(nM)
(nM)
(nM)
5
6
7
8
O
O
O
O
O
N
N
N
N
2a
2b
24
406
719
6,550
2l
800
42
1,190
1,310
>10,000
2,560
HN
HN
9
10
11
12
13
14
15
16
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18
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22
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O
N
1,085 >10,000
>10,000 2m
1,290
13
HN
HN
HN
O
O
N
2c
5.2
2.8
274
262
106
20
2n
0.9
5.3
35
1,590
7,480
HN
O
O
N
O
N
2d
3,130
2o
169
190
O
HN
O
O
O
N
O
N
2e
2f
34
11
57
16
26
1.6
9.2
1,070
90
1,470
153
>10,000 2p
0.5
3.9
15
35
16
9,190
HN
HN
O
O
O
O
O
O
N
N
5,520
2q
270
597
62
>10,000
1,400
HN
HN
O
O
O
N
N
2g
2h
2i
904
453
540
94
2,210
2,770
187
>10,000 2r
>10,000 2s
>10,000 2t
572
S
O
O
N
N
294
68
>10,000 8,330
>10,000
4,500
O
HN
rac
O
O
N
N
2,950
970
8,330
45
S
O
HN
HN
O
O
N
N
2j
24
7,250
3,910
2u
2v
1,350 1,785
7,150
HN
HN
rac
O
N
N
2k
338
309
4.3
121
2,250
O
HN
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Table 2. Additional properties of selected early lead compounds.
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1
Wistar Han Rat PK Profile
Clp / Clint Vd / Vdu
(mL/min/kg) (L/kg)
Beagle Dog PK Profile
PPB Clp / Clint Vd / Vdu
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3
4
5
6
7
8
9
Human CD69 CD63
PPB IC50 IC50
(nM) (nM)
LogD
PPB
(%)
t1/2 (h) F (%)
t1/2 (h) F (%)
(%)
79
(%)
90
58
51
72
67
48
27
(mL/min/kg) (L/kg)
b
1
5.8 / 60a
2d 50 / 290c
2j
58 / 370c
2n 49 / 410c
2o 23 / 98a
2p 95 / >Qhep
2q 41 / 126a
0.4 / 4
1.1 / 3
1.8 / 4
1.1 / 4
0.8 / 2
1.7 / 3
1.2 / 2
1.2
0.3
0.4
0.3
0.4
0.2
0.8
76
46 / >Qhep
7.9 / 25c
4.6 / 90 1.1
61
95
58
810
790
130
1.4
1.7
0.8
1.0
1.2
0.8
1.1
0.9 / 2
1.2
55
35
330
52
55
4.1 / 8c
0.9 / 2
0.9 / 3
2.7
1.5
41
26
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c
5.8 / 10b
100
17
360
415
a
b
Notes: 0.5 mg/kg iv, 1 mg/kg po in DMSO/PEG400/H2O-20/60/20, n=2; 0.25 mg/kg iv, 0.5 mg/kg po in DMSO/PEG400/H2O-
20/60/20, n=2; c0.05 mg/kg in DMSO/PEG400/H2O-20/60/20, n=2. Clp is plasma clearance, Qhep is hepatic blood flow, Clint is the in
vivo intrinsic clearance; Clint = [100Clp/(100-PPB)]x[(84–Clp)/84] in rat; Clint = [100Clp/(100-PPB)]x[(30–Clp)/30] in dog.
RESULTS AND DISCUSSION
To aid in the design of new inhibitors based on the purine 1, we obtained a co-crystal structure of 1 bound to the PI3K complex,
which was resolved to 3.10 Å (Figure 1). The inhibitor was bound to the catalytic kinase domain and anchored by hinge interactions
between the purine N(1) and C(2)-H to Val828-NH and CO of PI3K, respectively, and filled pockets typical of many type-I PI3K
inhibitors targeting the active form.17 The purine N(9)-methyl group partially occupied a hydrophobic pocket lined by the gatekeeper
Ile825 as well as Tyr813 of PI3K. The positioning of the gatekeeper Ile825 (conserved in PI3K family) above the plane of the
inhibitor, part of the roof of the active site and creating space for Tyr813, is a distinct feature of the lipid kinases versus protein
kinases.18 The purine C(8)-pyrazole heterocycle occupied the polar pocket lined by Lys779, Asp787, Asp911, and Ile910 of PI3K
with a possible direct or water-mediated contact to Tyr813, a pocket commonly referred to as the affinity pocket for PI3K inhibitors.
The purine core was also sandwiched by an edge-to-face interaction with the indole of Trp760 and a hydrophobic interaction with
Met900 of PI3K.
The piperidine ring of 1 mainly served as scaffolding to orient the benzimidazolone moiety towards the specificity or selectivity
pocket, lined by residues Trp760 and Met752, while the carbonyl of the benzimidazolone interacts with the carboxamide of Asn836
of PI3K. This unique binding mode was distinct from that observed for the idelalisib structural class, in which a larger specificity
pocket is created between Trp760 and Met752 as the putative basis for isoform selectivity.19 The binding of 1 suggested that inducing
a larger pocket is not necessary for good selectivity. Interestingly, this mode was also reported for morpholine PI3K inhibitors by
Genentech, exemplified by GNE-293, in which the protein conformation was similar to the apo form with the selectivity piece resting
on what was termed the “tryptophan shelf”.20 It is proposed that interaction with the “tryptophan shelf” is critical for isoform
selectivity, and inhibitors in this class exploit differences in the dynamics of the G-loop-equivalent between isoforms (e.g. Thr802
versus Lys/Arg802).
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Given that modifications to the piperidine benzimidazolone, or alternative substitution at the purine C(8)/C(9) positions did not
2
offer significant improvements in selectivity versus the phosphodiesterase panel or adenosine transporter cellular uptake, we elected
to replace the piperidine benzimidazolone moiety utilizing the structure of 1 bound to PI3K. We envisioned a library assembled by
structure-based design and a virtual screen, disconnecting at the purine via nucleophilic displacement or metal-mediated coupling of
various amines and alcohols to the corresponding 6-chloropurine (see Scheme 1).
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5
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8
9
The virtual screen of commercial and proprietary amines and alcohols was based on 3D overlays with reported PI3K inhibitors
as well as 3D similarity to compound 1. Hence the library was enriched with alcohols and amines that would be capable of reaching
the “tryptophan shelf” nearly perpendicular to the plane of the purine hinge-binding motif, with the understanding that constraints
may be needed to bias toward the active conformation.
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Over the course of screening diverse amines and alcohols (see Table 1), we discovered potent (S)--amino amides derived from
simple L-amino acids. For example, the early lead alanine-derived morpholine amide 2a gave promising potency (PI3K IC50 = 24
nM) but only modest selectivity (17x versus PI3K). For 8-aryl-purine inhibitors of this type, selectivity versus PI3K was generally
lower than observed with other isoforms (e.g. PI3K, PI3K), hence PI3K became a benchmark kinase to assess selectivity.
Importantly, compound 2a was 100x less potent that 1 in the adenosine uptake assay (AdU IC50 = 6550 nM), indicating that significant
changes to the selectivity motif can lead to purines largely void of this off-target. (S)-2-Amino-2-ethylamides derived from L-ethyl
glycine were optimal among the L-amino acids explores (e.g. 2c), with larger -substitution not further enhancing potency. The
enantiomers derived from D-amino acids (e.g. 2b) were significantly less potent (>100x) than those from L-amino acids. A cis-2,6-
dimethylmorpholine amide gave a slight boost in potency (2d; PI3K IC50 = 2.8 nM; Ligand Binding Efficiency 0.37; selectivity 94x
versus PI3K) with ligand efficiency comparable to 1. Truncation to a simple dimethyl amide (2e) was 7x less potent than 2c,
suggesting a minimum amide size needed to interact with the tryptophan shelf. While the clearance in rat for 2d was high, we were
encouraged by the low clearance in dog and the particularly low intrinsic clearance (25 mL/min/kg). The low unbound volumes in
rat and dog, however, led to short half-lives (0.3-1.2 h).
In addition to acyclic -amino amides such as 2d, we also identified cyclic analogs such as cyclobutanes and cyclopentanes in
various configurations. For example, cyclopentane 2f bearing 3(R)-amino, 1(S)-amide groups did offer promising potency, however
the selectivity versus PI3K was low (9x). Transposing the amide to azetidines 2g or 2h provided new opportunities with promising
potency but still modest PI3K selectivity (16-28x) was obtained. Notably, 2g and 2h were not inhibitors of adenosine uptake. Ring
expansion of the azetidine to the 3-aminopyrrolidine gave a remarkable boost in potency, ligand efficiency and selectivity.
Furthermore, the (S) configuration analog 2j was found to be more potent and selective (PI3K IC50 = 1.6 nM; Ligand Binding
Efficiency 0.43; 57x selectivity versus PI3K than the corresponding (R) configuration analog 2i, with excellent selectivity over
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adenosine uptake inhibition maintained.21 Furthermore, compound 2j had better selectivity in the phosphodiesterase panel (PDE4
IC50 = 8,400 nM; PDE1-3 and 5-11 IC50 = >20,000 nM) versus 1.
1
2
3
4
5
6
7
8
9
Ring expansion to the six-membered rings 3-aminopiperidine amide (e.g. 2k) or 4-aminopiperidine amide (e.g. 2l) gave losses
in potency, suggesting a unique bioactive conformation achieved by the (S)-3-aminopyrrolidine amide. The choice of the capping
group at the pyrrolidine N(1) was critical for potency, with amides, ureas and carbamates most preferred.22 The absence of a capping
group resulted in near complete potency loss, while alkyl caps (e.g. propyl 2m) were much less potent than the corresponding amides.
Growing to larger amides (e.g. cyclopropylamide 2n and tetrahydropyranylamide 2p) maintained PI3K potency. The rat clearance
for amides such as 2j and 2n (Table 2) were generally high (Clp 49-58 mL/min/kg), contributing to short half-lives (0.3-0.4 h).
Importantly, the excellent selectivity versus the PDE1-11 family was maintained with amide 2p (PDE1-11 IC50 = >20,000 nM).
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In parallel with the NH-linked selectivity motifs, we explored O-linked motifs as well. Direct O-linked analogs were typically
5x less potent than the corresponding NH-linked analogs, however Clp/Clint were slightly improved in some cases. For example, O-
linked cyclopropyl and tetahydropyranyl amides 2o and 2q are less potent than 2n and 2p, respectively. However, the rat PK profiles
for 2o and 2q was improved versus 2j, 2n or 2p, with a lower intrinsic clearance and a longer rat half-life (up to 0.8 h). The dog PK
was also promising with low plasma clearance (4.1-5.8 mL/min/kg), moderate half-life (1.5-2.7 h) and excellent bioavailability
(100%). Though intrinsic clearances were low, the generally low volumes with this series limit half-life. As found with the NH-
linker, the (S) configuration of O-linked 2q was >300x more potent than the (R) configuration, which had PI3K IC50 = 1,450 nM.
Tetrahydropyran 2q (PI3K IC50 = 3.9 nM; Ligand Binding Efficiency 0.36) had good to excellent selectivity versus PI3K
(69x), PI3K (1400x) and PI3K (970x), and >1,000x selectivity versus a 265-kinase panel. The human whole blood CD69 biomarker
assay potency had an IC50 of 360 nM, which was our Ctrough target. The pyrrolidine amides, with 2q included, did not have CYP
inhibition or CYP time-dependent inhibition, nor cardiac ion channel or PXR receptor induction activities. Solubility was generally
good for the pyrrolidine amides, reaching >10 mg/mL for 2q for example. Unlike 1, compound 2q was inactive in the adenosine
uptake HeLa cell assay (AdU IC50 = >10,000 nM) and inactive in the phosphodiesterase PDE1-11 assay panel (IC50 = >50,000 nM
all isoforms).
The S-linked pyrrolidine 2r was only 4x less active than the O-linked 2q analog, while C-linked analog 2s was 75x less potent.
The latter could be due to the preference for benzylic alkyl groups to adopt a conformation perpendicular to the plane of the aromatic
ring, not coplanar as is typical for amino or hydroxy groups. Given the high preference for the (S) enantiomer, it was not surprising
that thiophene 2t suffered a potency loss. Being flat, the achiral thiophene was unable to adopt the preferred conformation of 2q.
While carbonyl deletion to N-propyl 2m led to a potency loss, lactam 2v recovered the loss potency, either through attenuating the
amine basicity or a conformational control; representing an alternative lead compound for further optimization. The absolute
stereochemistry of 2v was not determined, however it is 50x more potent than it’s enantiomer (enantiomer PI3K IC50 = 210 nM).
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We obtained a crystal structure of 2q with PI3K (Figure 3), confirming the (S) configuration, which was consistent with the
commercially sourced chiral pyrrolidine. The purine core binds to the hinge as it did with 1, however the tetrahydropyran was
projected towards the “tryptophan shelf” by the gearing effect of the pyrrolidine amide. Our data suggested filling the space between
the Trp760 and Met752 was required for good PI3K isoform selectivity, but not necessarily sufficient alone. A conformational
analysis of truncated N- and O-linked pyrrolidines using QM methods (M06-2X/6-31G* with PCM solvent model) indicated that the
bioactive conformation and its amide rotamer is the lowest energy conformation. The oxygen or nitrogen linker is pseudo-axial and
flat with respect to the purine.
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Given that tetrahydropyran 2q combined good bioavailability in dog with superb selectivity in safety pharmacology assays
(AdU IC50 = >10,000 nM, PDE1-11 IC50 = >20,000 nM), it was advanced as a tool compound to evaluate tolerability in repeat dose
toxicology studies in rat and dog, and especially the potential to induce vascular injury. In telemetered rat, no hemodynamic effects
were observed on arterial blood pressure or heart rate at 60 mg/kg (uAUC 61 M•hr, uCmax 44 M). Nor was there evidence of
vascular injury in rat administered 750 mpk for four days (uAUC 990 M•hr, uCmax 200 M). In an 8-day repeat-dose toxicology
study in two male beagle dogs dosed at 25 mpk (uAUC 222 M•h, uCmax 46 M), no adverse effects were observed, including no
body weight loss and no histomorphologic evidence of vascular injury or hemodynamic effects (vascular tissue image Figure 4).
There was a very slight decrease in red blood cell count and basophils, however this was not deemed toxicologically significant. This
exposure exceeded the predicted human clinical efficacy at 350 mg bid (uAUC0-24h 14 M•h, uCmax 2.5 M) by a 16x margin, while
with inhibitor 1, vascular injury was found at the therapeutic dose (using CD69 IC50 trough in both cases). This exploratory safety
assessment study in dog suggested we could mitigate the vascular injury risk of the purine series, establishing a path to further lead
optimization with the pyrrolidine amide as the selectivity motif.
The short rat and dog half-lives observed with 2q (0.8-1.5 h) contributed to the high predicted human dose (350 mg bid) and
we turned to MetID analysis as a guide to address the half-life. While we did not detect significant turnover in microsome or
hepatocyte incubations (Clint of <110 and <40 mL/min/kg, respectively), a rat bile duct cannulation study indicated tetrahydropyran
oxidation, pyrazole N-dealkylation and cleavage of the ether bond. In addition, 17% of parent compound 2q was recovered in urine
out to 24 hours post-dose, with another 2% found in bile; suggesting a only minor excretion contribution to the plasma clearance.
Lead optimization would involve blocking these metabolic sites as well as improving the potency in order to lower the predicted
human dose.
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2q
Figure 3. Crystal structure of 2q with PI3K, resolved to 3.05, respectively. The purine makes hydrogen bond contacts to
the kinase hinge region while the pyrrolidine amide gears the tetrahydropyran towards Trp760 residue. PDB ID: 6MUM.
A
B
C
Figure 4. Histology of coronary artery from untreated (A), compound 1 treated (B) and compound 2q treated (C) dogs. Note
that both untreated (A) and compound 2q treated (C) coronary arteries appear normal. The coronary artery from dog treated
with compound 1 (B) shows medial degeneration characterized by disruption of the tunica media by hemorrhage, fibrin and
cellular debris (arrows). Bar=100 m.
SYNTHESIS
Compounds were prepared via the route outlined in Scheme 1. Intermediate chloropyrazolopurine 5 was prepared by an
oxidative cyclocondensation of N-ethyl-1H-5-methyl-pyrazole-4-carboxaldehyde 3 with 6-chloro-N4-methylpyrimidine-4,5-diamine
4 using FeCl3 and air. Ether-linked products were prepared by treatment of chloropurine 5 with an alcohol and NaH, while amine-
linked products were prepared from the corresponding primary or secondary amine with iPr2NEt or K2CO3 at 80 °C. In some cases,
a selectivity motif was introduced bearing a protective group (e.g. an N-Boc group), and then after nucleophilic displacement, the
protective group was removed using standard conditions (e.g. TFA or HCl) and the functional group capped (e.g. amide formation
with acid chloride or acid and HATU). Details are specified in the experimental section for all compounds.
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Scheme 1.a Synthesis of key 6-aminoalkyl and 6-hydroxyalkyl purines.
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Cl
N
Cl
H2N
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i
N
N
N
N
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CHO
+
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N
H
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Cl
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H
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iv, v
ii (L = O) or
iii (L = NH)
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Boc
Cl
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H
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Boc
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iii (L = NH)
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aReagents and conditions: (i) FeCl3•6H2O, air, DMF, 85 oC, 75%; (ii) NaH, THF, 0 ºC to ambient temperature; (iii) iPr2NEt or K2CO3,
DMF or t-BuOH, 80 ºC; (iv) TFA, DCM or HCl, dioxane; (v) RCOCl, iPr2NEt, DMF or RCO2H, HATU, DMF.
CONCLUSION
Profiling of lead benzimidazolone purine 1 in toxicology studies uncovered poor tolerability in dog at the predicted efficacious
exposure that was attributed to diffuse vascular injury. While the pharmacological root cause was not established, adenosine uptake
and phosphodiesterase inhibition were found as off-targets of concern. Reconstruction of the selectivity motif with structure-inspired
screening led to the identification of novel -amino amides as well as hydroxy and aminocycloalkyl amides with excellent potency
and selectivity versus the kinome, cellular adenosine uptake and phosphodiesterases; with pyrrolidines being particularly efficient.
A co-crystal structure of representative 2q revealed a binding mode similar to 1. Given the excellent selectivity in safety
pharmacology assays and the acceptable bioavailability in dog, 2q was selected to derisk the purine series with respect to vascular
injury. In a pivotal 8-day dog repeat-dose safety experiment, no histomorphometric evidence of vascular injury was found at 16-fold
over the efficacious exposure, based on achieving a CD69 IC50 at trough, which was substantially improved versus the margin
observed with 1. This result chartered a lead optimization effort to lower the predicted human dose by improving both the potency
in human whole blood as well as the half-life, utilizing the MetID analysis with 2q. Results of this optimization campaign will be
the focus of a forthcoming communication.
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EXPERIMENTAL SECTION
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General Synthetic Chemistry Methods. Reactions were performed in dried round bottom flasks or capped vials with stirring under
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an inert atmosphere of nitrogen unless stated otherwise. Solvents in septum-sealed bottles and reagents were obtained from
o
commercial suppliers and used as. All temperatures are in degrees Celsius (oC), and ambient temperature is 20 C. Microwave
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reactions were performed with a Biotage Initiator Series microwave. Most compounds were purified by reverse-phase preparative
HPLC or MPLC on silica gel. The course of the reactions was followed by LC/MS (30 mm x 2 mm, 2 m column; 3% to 98%
MeCN/water with 0.05% TFA gradient over 2.3 minutes; 0.9 mL/min flow rate; ESI; UV detection at 254 nm). Final compounds
were analyzed by NMR, LC/MS and HRMS. NMR spectra were recorded on either a 400, 500 or a 600 MHz Varian spectrometer,
chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane and referenced to residual solvent. Coupling
constants are reported in Hz. High-resolution LCMS was obtained on a Waters Acquity UPLC with a Acquity UPLC BEH C18
1.7um column with 5-100% MeCN/water with 0.1% formic acid coupled to a Waters Xevo G2 QTof with an ESI+ ionization mode.
The purity of all compounds screened in the biological assays was examined by LC/MS analysis and was found to be ≥95% unless
otherwise specified.
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(S)-8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-N-(1-methyl-2-morpholin-4-yl-2-oxoethyl)-9H-purin-6-amine
(2a).
1
Prepared in a fashion similar to that used for the synthesis of 2d. [α]D +105° (c = 0.19, MeOH); H-NMR (400 MHz, CD3OD): δ
8.16 (s, 1 H), 7.79 (s, 1 H), 5.19 (s, 1 H), 4.14-4.16 (m, 2 H), 3.50-3.70 (m, 11 H), 2.44 (s, 3 H), 1.33-1.39 (m, 6 H); 13C NMR (100
MHz, CD3OD)  172.2, 152.8, 151.7, 150.0, 146.0, 139.8, 137.8, 118.8, 108.6, 66.3 (2C), 46.0, 43.9 (2C), 42.5, 29.2, 17.0, 14.0, 9.1;
HRMS (ESI) calc’d for C19H27N8O2 [M+H]+, 399.2257; found, 399.2263.
(R)-8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-N-(1-methyl-2-morpholin-4-yl-2-oxoethyl)-9H-purin-6-amine
(2b).
Prepared in a fashion similar to that used for the synthesis of 2d. [α]D -123° (c = 0.31, MeOH); 1H-NMR (400 MHz, CD3OD) δ 8.16
(s, 1 H), 7.79 (s, 1 H), 5.20 (s, 1 H), 4.12-4.18 (m, 2 H), 3.56-3.70 (m, 11 H), 2.44 (s, 3 H), 1.33-1.39 (m, 6 H); 13C NMR (100 MHz,
CD3OD)  172.2, 152.8, 151.7, 150.1, 146.0, 139.7, 137.8, 118.9, 108.6, 66.3 (2C), 46.0, 44.0 (2C), 42.4, 29.2, 16.9, 14.0, 9.0; HRMS
(ESI) calc’d for C19H27N8O2 [M+H]+, 399.2257; found, 399.2267.
(S)-2-(8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-ylamino)-1-morpholinobutan-1-one (2c). Prepared in a
fashion similar to that used for the synthesis of 2d, giving 43% yield. [α]D +66° (c = 0.080, MeOH); 1H-NMR (400 MHz, CD3OD):
δ 8.16 (s, 1 H), 7.79 (s, 1 H), 4.18-4.12 (m, 2 H), 3.71-3.45 (m, 11 H), 2.44 (s, 3 H), 3.47-3.39 (m, 2 H), 1.87-1.73 (m, 2 H), 1.37-
1.33 (m, 3 H), 0.96-0.92 (m, 3 H); 13C NMR (100 MHz, CD3OD)  171.8, 153.2, 151.5, 150.0, 145.9, 139.7, 137.8, 118.8, 108.5,
51.3, 44.0, 42.4, 39.2, 34.1, 33.4, 29.2, 25.2, 13.9, 9.0, 8.9; MS (ESI) calc’d for C20H29N8O2 [M+H]+, 413; found, 413.
(S)-1-((2S,6R)-2,6-Dimethylmorpholino)-2-((8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)amino)butan-1-
one (2d).
A
solution of (9H-fluoren-9-yl)methyl ((S)-1-((2S,6R)-2,6-dimethylmorpholino)-1-oxobutan-2-yl)carbamate
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(US2008/171783 A1, 100 mg, 0.23 mmol) in piperidine (0.6 mL) and DCM (3 mL) was stirred at ambient temperature for 3 h. The
mixture was evaporated under reduced pressure and the residue purified by reverse phase chromatography (gradient of water/ MeCN
with 0.05% TFA) to afford (S)-2-amino-1-((2S,6R)-2,6-dimethylmorpholino)butan-1-one intermediate (40 mg, 53 %). MS (ESI)
calc’d for C10H21N2O2 [M+H]+, 201; found, 201.
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A solution of (S)-2-amino-1-((2S,6R)-2,6-dimethylmorpholino)butan-1-one (40 mg, 0.2 mmol) in t-BuOH (1 mL) and iPr2NEt (1
mL) was treated with 6-chloro-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purine (5, 100 mg, 0.36 mmol). The solution was
stirred at 100 °C for 48 h, then water (10 mL) was added and the mixture was extracted with EtOAc. The combined organic layers
was evaporated under reduced pressure and the residue purified by reverse phase chromatography (gradient of water/MeCN with 10
mM NH4HCO3) to afford (S)-1-((2S,6R)-2,6-dimethylmorpholino)-2-((8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-
yl)amino)butan-1-one (2d, 10 mg, 11 %) as a white solid. [α]D +64° (c = 1.1, MeOH); 1H NMR (400 MHz, CD3OD) δ 8.33–8.21 (m,
1 H), 7.92 (s, 1 H), 5.32 (s, 1 H), 4.44–4.33 (m, 1 H), 4.31–4.07 (m, 3 H), 3.83 (s, 3 H), 3.73–3.46 (m, 2 H), 3.02–2.86 (m, 1 H), 2.57
(s, 3 H), 2.49–2.35 (m, 1 H), 2.04–1.80 (m, 2 H), 1.52–1.43 (m, 3 H), 1.31–1.13 (m, 6 H), 1.10–1.00 (m, 3 H); 13C NMR (100 MHz,
CD3OD)  173.4, 153.5, 153.2, 151.6, 150.1, 139.7, 137.9, 118.7, 108.6, 71.7 (2C), 54.7, 51.1 (2C), 44.0, 29.2, 25.0, 17.6 (2C), 14.0,
9.1, 8.9; HRMS (ESI) calc’d for C22H33N8O2 [M+H]+, 441.2726; found, 441.2734.
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(2S)-2-(8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-ylamino)-N,N-dimethylbutanamide (2e). Prepared in a
fashion similar to that used for the synthesis of 2d. [α]D +69° (c = 1.1, MeOH); 1H NMR (400 MHz, CD3OD) δ 8.26 (s, 1 H), 7.90
(s, 1 H), 5.28 (br, 1 H), 4.24 (q, J = 7.2 Hz, 2 H), 3.81 (s, 3 H), 3.27 (s, 3 H), 2.97 (s, 3 H), 2.55 (s, 3 H), 1.95 (m, 1 H), 1.83 (m, 1
H), 1.45 (t, J = 8 Hz, 3 H), 1.04 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CD3OD)  173.0, 153.3, 151.7, 149.9, 145.9, 139.8, 137.9,
118.7, 108.6, 51.3, 43.9, 36.3 (2C), 34.8, 29.2, 14.0, 9.1, 8.9; HRMS (ESI) calc’d for C18H27N8O [M+H]+, 371.2308; found, 371.2325.
((1R,3S)-3-((8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)amino)cyclopentyl)-(morpholino)methanone (2f).
A solution of 6-chloro-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purine (5, 320 mg, 1.16 mmol) and (1R,3S)-methyl 3-
aminocyclopentanecarboxylate (166 mg, 1.16 mmol) in a mixture of t-BuOH/iPr2NEt (1:1, 5 mL) was stirred at 80 ºC for 72 h. The
solvent was removed under reduced pressure then dissolved in H2O (2 mL) and THF (5 mL), treated with LiOH (73 mg, 1.7 mmol)
and stirred for 16 h. The mixture was diluted with AcOEt and washed with 1 N aqueous HCl. The combined organic layers were
washed with brine, dried (Na2SO4), filtered and concentrated to provide (1R,3S)-3-((8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-
9H-purin-6-yl)amino)cyclopentane-1-carboxylic acid (250 mg, 100%). MS (ESI) calc’d for C18H24N7O2 [M+H]+, 370; found, 370.
The residue was dissolved in DMF (4 mL), treated with morpholine (62 mg, 0.71 mmol), HATU (305 mg, 0.80 mmol), N-
methylmorpholine (135 mg, 1.34 mmol) and stirred for 16 h. The mixture was purified by reverse phase chromatography (gradient
of water/MeCN with 10 mM NH4HCO3) to provide 150 mg (51%) of 2f. [α]D +17° (c = 1.3, MeOH); 1H NMR (400 MHz, DMSO-
d6) δ 8.15 (s, 1 H), 7.94 (s, 1 H), 7.54 (d, J = 8.0 Hz, 1 H), 4.18 (q, J = 7.2 Hz, 2 H), 3.90 (s, 3 H), 3.48-3.56 (m, 8 H), 3.19-3.30 (m,
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1 H), 2.59 (s, 3 H), 1.84-2.20 (m, 7 H), 1.37 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CD3OD)  175.2, 153.4, 151.6, 149.7, 145.5,
139.8, 137.7, 118.8, 108.8, 66.6, 66.4, 52.0, 46.0, 43.9, 42.4, 39.1, 35.8, 32.8, 29.1, 27.5, 14.0, 9.1; HRMS (ESI) calc’d for C22H31N8O2
[M+H]+, 439.2570; found, 439.2569.
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8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-N-(1-propanoylazetidin-3-yl)-9H-purin-6-amine (2g). Prepared in a fashion
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similar to that used for the synthesis of 2j. H NMR (500 MHz, DMSO-d6)  8.24 (br s, 1 H), 8.21 (br s, 1 H), 7.95 (br s, 1 H), 4.94
9
(br m, 1 H), 4.46 (t, 1 H), 4.19 (m, 3 H), 4.13 (m, 1 H), 3.94 (m, 1 H), 3.77 (s, 3 H), 2.60 (s, 3 H), 2.08 (m, 2 H), 1.38 (m, 3 H), 0.98
(m, 3 H); 13C NMR (100 MHz, DMSO-d6)  173.4, 153.4, 153.3, 152.0, 145.9, 139.4, 137.9, 119.3, 109.2, 57.7, 55.0, 44.2 (d), 30.3,
24.4, 15.5, 10.6, 9.3; HRMS (ESI) calc’d for C18H25N8O [M+H]+, 369.2151; found, 369.2157.
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8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-N-[1-(tetrahydro-2H-pyran-4-ylcarbonyl)azetidin-3-yl]-9H-purin-6-amine
(2h). Prepared in a fashion similar to that used for the synthesis of 2j. 1H NMR (500 MHz, DMSO-d6)  8.24 (br, 2 H), 7.95 (br, 1
H), 4.95 (m, 1 H), 4.55 (m, 1 H), 4.15-4.23 (m, 4 H), 3.95 (m, 1 H), 3.82-3.88 (m, 2 H), 3.77 (m, 3 H), 3.35 (m, 2 H), 2.60 (s, 3 H),
1.50-1.58 (m, 5 H), 1.38 (m, 3 H); 13C NMR (100 MHz, DMSO-d6)  174.1, 153.2 (2C), 152.0, 145.9, 139.4, 137.9, 119.5, 109.2,
66.6 (2C), 57.7, 55.0, 44.2 (2C), 36.5, 30.3, 28.6 (2C), 15.5, 10.6; HRMS (ESI) calc’d for C21H29N8O2 [M+H]+, 425.2413; found,
425.2421.
(R)-1-(3-((8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)amino)pyrrolidin-1-yl)propan-1-one (2i). Prepared
in a fashion similar to that used for the synthesis of 2j. [α]D -57° (c = 0.13, MeOH); 1H NMR (600 MHz, DMSO-d6) δ 8.35 (s, 1 H),
7.94 (s, 1 H), 4.15 (q, J = 7 Hz, 2 H), 3.77 (s, 3 H), 3.67 (m, 1 H), 3.60 (m, 1 H), 3.55-3.45 (m, 2 H), 3.37 (m, 1 H), 2.52 (s, 3 H),
2.15-2.25 (m, 2 H), 2.09 (m, 1 H), 2.01 (m, 1 H), 1.32 (t, J = 7 Hz, 3 H), 0.95 (m, 3 H); 13C NMR (100 MHz, DMSO-d6)  171.8,
152.0, 150.3, 150.0, 146.4, 139.7, 138.0, 118.6, 108.6, 51.3, 49.9, 44.7, 44.2, 30.4, 30.2, 27.4, 15.3, 10.6, 9.3; HRMS (EI) calc’d for
C19H27N8O [M+H]+, 383.2308; found 383.2309.
(S)-1-(3-((8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)amino)pyrrolidin-1-yl)propan-1-one (2j). A solution
of 6-chloro-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purine (5, 500 mg, 1.81 mmol) in DMF (18 mL) was treated with
iPr2NEt (0.80 mL, 4.6 mmol) and (S)-tert-butyl 3-aminopyrrolidine-1-carboxylate (0.55 mL, 3.1 mmol). The mixture was stirred at
80 °C for 72 h. The mixture was diluted with EtOAc and washed with water, brine, dried (Na2SO4) and concentrated. The residue
was purified by chromatography on SiO2 (gradient of 0-10% MeOH/DCM) to afford (S)-tert-butyl 3-((8-(1-ethyl-5-methyl-1H-
pyrazol-4-yl)-9-methyl-9H-purin-6-yl)amino)pyrrolidine-1-carboxylate (345 mg, 45%). [α]D +46° (c = 0.67, MeOH); 1H NMR (600
MHz, DMSO-d6) δ 8.19 (s, 1 H), 7.89 (s, 1 H), 7.73 (s, 1 H), 4.13 (q, J = 7.2 Hz, 2 H), 3.72 (s, 3 H), 3.65-3.54 (m, 1 H), 3.50-3.40
(m, 1 H), 3.35-3.18 (m, 3 H), 2.52 (s, 3 H), 2.20-2.05 (m, 1 H), 2.04-1.90 (m, 1 H), 1.38-1.33 (2 s, 9 H), 1.32 (t, J = 7.3 Hz, 3 H); MS
(EI) calc’d for C21H31N8O2 [M+H]+, 427; found 427.
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Journal of Medicinal Chemistry
A solution of (S)-tert-butyl 3-((8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)amino)pyrrolidine-1-carboxylate (345
mg, 0.809 mmol) in dioxane (2 mL) was treated with 4 M HCl in dioxane (1 mL, 4 mmol). The mixture was stirred at ambient
temperature for 2 days and concentrated giving (S)-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-N-(pyrrolidin-3-yl)-9H-purin-6-
amine, HCl (323 mg, 100 % yield). MS (EI) calc’d for C16H23N8 [M+H]+, 327; found 327.
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A solution of (S)-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-N-(pyrrolidin-3-yl)-9H-purin-6-amine, HCl (20 mg, 0.046 mmol)
in DMF (0.5 mL) was treated with propionyl chloride (5 µL, 0.06 mmol) and NEt3 (0.05 mL, 0.36 mmol). The mixture was stirred
at ambient temperature for 16 h, filtered and purified on reverse phase chromatography (gradient of water/MeCN with 0.1% TFA) to
afford (S)-1-(3-((8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)amino)pyrrolidin-1-yl)propan-1-one 2j (21 mg, 90 %
yield). 1H NMR (600 MHz, DMSO-d6) δ 8.32 (s, 1 H), 7.93 (s, 1 H), 4.14 (q, J = 7.2 Hz, 2 H), 3.76 (s, 3 H), 3.70-3.64 (m, 1 H),
3.64-3.56 (m, 1 H), 3.54-3.44 (m, 2 H), 3.42-3.30 (m, 1 H), 2.52 (s, 3 H), 2.30-1.95 (m, 4 H), 1.32 (t, J = 7.2 Hz, 3 H), 0.94 (t, J =
7.4 Hz, 3 H); 13C NMR (100 MHz, CD3OD)  173.9, 153.4, 151.7, 150.2, 145.9, 139.7, 137.8, 118.8, 108.7, 52.0, 50.9, 44.6, 44.0,
31.4, 29.2, 27.2, 13.9, 9.0, 7.9; HRMS (EI) calc’d for C19H27N8O [M+H]+, 383.2308; found 383.2317.
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8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-N-[(3S)-1-propanoylpiperidin-3-yl]-9H-purin-6-amine (2k). A solution of 6-
chloro-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purine 5 (770 mg, 2.8 mmol) and (S)-tert-butyl 3-aminopiperidine-1-
carboxylate (500 mg, 2.5 mmol) in t-BuOH (5 mL) and NEt3 (10 mL) was stirred in a sealed tube for 72 h at 110 °C. The reaction
mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL). The combined organic layers were dried (Na2SO4),
filtered and concentrated. The residue was purified by chromatography on silica (1:1 EtOAc/DCM) to afford the synthetic
intermediate (3S)-tert-butyl-3-(8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-ylamino)piperidine-1-carboxylate (750
mg, 68%) as a white solid. MS (EI) calc’d for C22H33N8O2 [M+H]+, 441; found, 441.
A solution of (3S)-tert-butyl3-(8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-ylamino)piperidine-1-carboxylate (300
mg, 0.68 mmol) in DCM (4 mL) was treated with TFA (1 mL) and stirred for 2 h at room temperature. The reaction was diluted with
water (10 mL) and extracted with EtOAc (10 mL). The combined organic layers were dried (Na2SO4), filtered and concentrated to
afford the free base as a white solid (200 mg, 87 %). MS (EI) calc’d for C17H25N8 [M+H]+, 341; found, 341.
A solution of the free base (50 mg, 0.15 mmol) in DCM (3 mL) was treated with propionyl chloride (28 mg, 0.30 mmol) and NEt3
(46 mg, 0.45 mmol). The resulting mixture was stirred at ambient temperature for 15 h. The reaction was washed with water (10
mL) and extracted with EtOAc (10 mL). The combined organic layers were dried (Na2SO4), filtered and concentrated. The residue
was purified by reverse phase chromatography (gradient of water/MeCN with 10 mM NH4HCO3) to afford 2k as a white solid (36
mg, 61 %). [α]D +60° (c = 1.1, MeOH); 1H NMR (400 MHz, DMSO-d6) δ 8.28-8.31 (m, 1 H), 7.91 (s, 1 H), 4.72-3.37 (m, 8 H), 3.27-
3.42 (m, 2 H), 2.16-2.55 (m, 6 H), 1.49-1.92 (m, 6 H), 1.08-1.14 (m, 3 H); 13C NMR (100 MHz, CD3OD)  173.8, 153.3, 151.8,
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145.8, 145.7, 139.7, 137.8, 118.7, 108.7, 49.9, 46.0, 44.0, 41.8, 29.2, 26.0, 25.9, 23.4, 14.0, 9.0, 8.6; HRMS (EI) calc’d for C20H29N8O
[M+H]+, 397.2464; found, 397.2467.
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1-(4-((8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)amino)piperidin-1-yl)propan-1-one (2l). Prepared in a
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fashion similar to that used for the synthesis of 2k. H NMR (500 MHz, DMSO-d6)  8.49 (s, 1 H), 8.01 (s, 1 H), 5.58 (m, 1 H), 4.19
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(m, 2 H), 4.08 (m, 1 H), 3.85 (s, 3 H), 3.84 (m, 2 H), 3.15 (m, 2 H), 2.59 (s, 3 H), 2.37 (m, 2 H), 2.10 (m, 2 H), 1.76 (m, 1 H), 1.63
(m, 1 H), 1.37 (m, 3 H), 1.01 (m, 3 H); 13C NMR (100 MHz, DMSO-d6)  171.7, 158.8, 154.2, 151.0, 148.5, 139.9, 138.1, 120.8,
108.8, 72.6, 44.2 (2C), 42.8, 31.7, 31.0, 30.7, 26.0, 15.5, 10.7, 9.9; HRMS (EI) calc’d for C20H29N7O2 [M+H]+, 398.2304; found
398.2318.
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8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-N-[(3S)-1-propylpyrrolidin-3-yl]-9H-purin-6-amine (2m). A solution of (S)-8-
(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-N-(pyrrolidin-3-yl)-9H-purin-6-amine, HCl (100 mg, 0.300 mmol; see intermediate
used for preparation of 2j) in DCM (3 mL) was treated with propionaldehyde (35 mg, 0.60 mmol) and AcOH (0.2 mL). The mixture
was stirred for 30 min at ambient temperature, then treated with sodium triacetoxyborohydride (130 mg, 0.60 mmol) and the mixture
stirred for 15 h. The reaction was diluted with water and extracted with EtOAc. The combined organic layers were dried (Na2SO4),
filtered and concentrated. The residue was purified by reverse phase chromatography (gradient of water/MeCN with 10 mM
NH4HCO3) to afford 2m as a white solid (24 mg, 22 %). [α]D +16° (c = 1.1, MeOH); 1H NMR (400 MHz, DMSO-d6) δ 8.14 (s, 1
H), 7.79 (s, 1 H), 4.14 (q, J = 7.6 Hz, 2 H), 3.70 (s, 3 H), 3.20-3.21 (m, 1 H), 2.95-3.05 (m, 1 H), 2.80-2.85 (m, 1 H), 2.57-2.67 (m, 2
H), 2.32-2.48 (m, 6 H), 1.78-1.81 (m, 1 H), 1.40-1.52 (m, 2 H), 1.34 (t, J = 7.6 Hz, 3H), 0.85 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz,
CD3OD)  155.1, 153.0, 151.6, 151.4, 146.1, 139.8, 137.9, 119.0, 108.6, 59.0, 57.0, 52.9, 43.9, 30.2, 29.2, 19.7, 14.0, 10.1, 9.1;
HRMS (ESI) calc’d for C19H29N8 [M+H]+, 369.2525; found, 369.2522.
N-[(3S)-1-(Cyclopropylcarbonyl)pyrrolidin-3-yl]-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-amine
(2n).
Prepared in a fashion similar to that used for the synthesis of 2i. [α]D +70° (c = 0.4, MeOH); 1H NMR (600 MHz, DMSO-d6) δ 8.33
(s, 1 H), 7.94 (s, 1 H), 4.15 (q, J = 7.2 Hz, 2 H), 4.10-3.80 (m, 2 H), 3.67 (s, 3 H), 3.60-3.25 (m, 3 H), 2.53 (s, 3 H), 2.35-1.60 (m, 3
H), 1.32 (t, J = 7.2 Hz, 3 H), 0.80-0.60 (m, 4 H); 13C NMR (100 MHz, CD3OD)  173.4, 153.3, 151.7, 150.2, 145.9, 139.7, 137.9,
118.8, 108.6, 73.3, 52.1, 44.7, 44.1, 31.4, 29.2, 14.0, 12.0, 9.1, 6.7, 6.6; HRMS (EI) calc'd for C20H27N8O [M+H]+, 395.2308; found
395.2302.
(S)-Cyclopropyl(3-((8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)oxy)pyrrolidin-1-yl)methanone
(2o).
Prepared in a fashion similar to that used for the synthesis of 2q. [α]D +150° (c = 0.67, MeOH); 1H NMR (600 MHz, DMSO-d6) δ
8.49 (s, 1 H), 8.00 (s, 1 H), 5.83-5.95 (m, 1 H), 4.17 (q, J = 7 Hz, 2 H), 3.84 and 3.85 (2s, 3 H), 3.85-4.10 (m, 2 H), 3.40-3.80 (m, 2
H), 2.56 (s, 3 H), 1.17-2.44 (m, 2 H), 1.71-1.85 (m, 1 H), 1.35 (t, J = 7.2 Hz, 3 H), 0.66-0.76 (m, 4 H); 13C NMR (100 MHz, CD3OD)
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Journal of Medicinal Chemistry
 173.5, 158.6, 153.7, 150.9, 148.7, 140.3, 138.0, 120.5, 108.1, 76.6, 52.4, 44.8, 44.0, 31.4, 29.4, 13.9, 11.9, 9.1, 6.8, 6.6; HRMS (EI)
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calc'd for C20H26N7O2 [M+H]+, 396.2148; found 396.2150.
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8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-N-[(3S)-1-(tetrahydro-2H-pyran-4-ylcarbonyl)pyrrolidin-3-yl]-9H-purin-6-
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amine (2p). Prepared in a fashion similar to that used for the synthesis of 2j. [α]D +72° (c = 0.67, MeOH); H NMR (600 MHz,
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DMSO-d6) 8.34 (s, 1 H), 7.93 (br, 1 H), 4.13–4.17 (m, 2 H), 3.66-3.92 (m, 6 H), 3.47-3.61 (m, 2 H), 3.17-3.40 (m, 4 H), 2.56-2.70
(m, 2 H), 2.52 (s, 3 H), 2.14-2.24 (m, 1 H), 1.97-2.10 (m, 2 H), 1.50-1.60 (m, 3 H), 1.32 (t, J = 6 Hz, 3 H); 13C NMR (100 MHz,
CD3OD)  175.4, 153.3, 151.6, 150.8, 145.8, 139.6, 137.0, 118.7, 108.7, 66.7 (2C), 51.8, 51.3, 44.4, 39.4, 31.4, 29.6, 29.2, 28.3, 28.2,
14.0, 9.0; HRMS (EI) calc'd for C22H31N8O2 [M+H]+, 439.2570; found 439.2579.
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(S)-(3-((8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)oxy)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-
yl)methanone (2q). A solution of (S)-pyrrolidin-3-ol (15.0 g, 172 mmol) and triethylamine (19.2 g, 189 mmol) in 100 mL of CH2Cl2
was treated dropwise at 0 °C with a solution of tetrahydro-2H-pyran-4-carbonyl chloride (26.4 g, 177 mmol) in 75 mL of CH2Cl2.
The mixture was stirred for 2 h and concentrated. The residue was taken up in 1 L of EtOAc, stirred overnight, then filtered to remove
amine salts. The mother liquor was concentrated to provide 36 g (100%) of (S)-(3-hydroxypyrrolidin-1-yl)(tetrahydro-2H-pyran-4-
1
yl)methanone. H NMR (600 MHz, CDCl3)  4.52 (d, J = 6.0 Hz, 1 H), 4.03-3.99 (m, 2 H), 3.70-3.38 (m, 6 H), 2.64-2.50 (m, 1 H),
2.06-1.98 (m, 2 H), 1.93-1.86 (m, 2 H), 1.71-1.60 (m, 2 H).
A solution of (S)-(3-hydroxypyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone (4.32 g, 21.7 mmol) in 60 mL of THF was treated
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at 0 C with a 60% mineral oil suspension of sodium hydride (1.01 g, 25.2 mmol) followed by 6-chloro-8-(1-ethyl-5-methyl-1H-
pyrazol-4-yl)-9-methyl-9H-purine (5, 5.00 g, 18.1 mmol). The reaction mixture was stirred for 2 h and allowed to warm to ambient
temperature. The crude reaction mixture was concentrated and the residue purified by chromatography on SiO2 (gradient of 0-20%
MeOH/DCM) to give 5.0 g (63%) of 2q. []D +29 (c 0.58, CHCl3); 1H NMR (600 MHz, CDCl3)  8.48 (d, J = 7.0 Hz, 1 H), 7.75 (d,
J = 6.3 Hz, 1 H), 5.93 (s, 1 H), 4.20–4.16 (m, 2 H), 4.04–3.93 (m, 2 H), 3.89, 3.87 (2 s, 3 H), 3.86-3.70 (m, 3 H), 3.45-3.35 (m, 2 H),
2.65-2.52 (m, 1 H), 2.60, 2.56 (2 s, 3 H), 2.50-2.45 (m, 1 H), 2.40-2.23 (m, 2 H), 1.94-1.86 (m, 2 H), 1.66-1.60 (m, 2 H), 1.45 (t, J =
6 Hz, 3 H); 13C NMR (125 MHz, DMSO-d6)  172.9, 158.6, 154.2, 150.9, 148.6, 139.9, 138.0, 120.8, 108.7, 76.7, 75.2, 66.7, 52.0,
44.1, 39.0, 38.9, 31.9, 30.7, 30.1, 28.8, 15.4, 10.6; HRMS (EI) calc'd for C22H30N7O3 [M+H]+, 440.2410; found, 440.2414.
((S)-3-(8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-ylthio)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-
yl)methanone (2r). A solution of tert-butyl (S)-3-mercaptopyrrolidine-1-carboxylate (1.1 g, 5.4 mmol) in THF (18 mL) was treated
with a 60% suspension of NaH (0.42 g, 10.5 mmol) portionwise at 0 ºC. The resulting mixture was allowed to warm to ambient
temperature, stirred for 15 min, then treated with 5 (1.15 g, 4.2 mmol) and stirred for 12 h. The reaction was quenched with methanol
(10 mL) and concentrated. The crude mixture was purified by chromatography on SiO2 (EOAc/hexane = 1:9 to 1:4) to give (3S)-
tert-butyl 3-(8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-ylthio)pyrrolidine-1-carboxylate (1.0 g, 55 %) as a white
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1
solid. H NMR (400Hz, CDCl3) δ 8.70 (s, 1 H), 7.84 (s, 1 H), 4.65-4.75 (m, 1 H), 4.23 (q, J = 6.8 Hz, 2 H), 3.98-4.10 (m, 1 H), 3.94
(s, 3 H), 3.40-3.60 (m, 3 H), 2.68 (s, 3 H), 2.48-2.51 (m, 1 H), 2.10-2.20 (m, 1 H), 1.48-1.55 (m, 12 H); MS (EI) calc’d for C21H30N7O2S
[M+H]+, 444; found, 444.
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A solution of (3S)-tert-butyl 3-(8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-ylthio)pyrrolidine-1-carboxylate (300
mg, 0.67 mmol) in CH2Cl2 (3 mL) was treated with TFA (1 mL) slowly at 0 ºC. The reaction was stirred at ambient temperature for
1 h, and concentrated to give 8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-6-((S)-pyrrolidin-3-ylthio)-9H-purine (200 mg, 87 %)
as a yellow oil. MS (EI) calc’d for C16H22N7S [M+H]+, 344; found, 344.
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This pyrrolidine was converted to the title compound, ((S)-3-(8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-
ylthio)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone (2r) via the method described for the synthesis of 2q. [α]D +19° (c =
1.4, MeOH); 1H NMR (400Hz, DMSO-d6) δ 8.70 (s, 1 H), 8.07 (s, 1 H), 5.90 (m, 1 H), 4.60-4.75 (m, 1 H), 4.20 (m, 2 H), 3.84 (s, 3
H), 3.60-3.90 (m, 3 H), 3.30-3.50 (m, 4 H), 2.60-2.75 (m, 1 H), 2.62 (s, 3 H), 2.40-2.50 (m, 1 H), 1.95-2.10 (m, 1 H), 1.50-1.60 (m,
4 H), 1.37 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CD3OD)  174.2, 158.5, 157.1, 150.9, 150.0, 140.9, 138.0, 129.8, 107.7, 66.8,
66.7, 52.5, 45.1, 41.3, 39.8, 39.3, 31.7, 29.9, 29.4, 28.2, 14.0, 9.2; HRMS (EI) calc’d for C22H30N7O2S [M+H]+, 456.2181; found,
456.2189.
(3-((8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)methyl)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-
yl)methanone (2s). 9-BBN (0.5 M in THF; 2.4 mL, 1.2 mmol) was added to a solution of tert-butyl-3-methylenepyrrolidine-1-
carboxylate (200 mg, 1.09 mmol) in dry THF (1 mL) under a nitrogen atmosphere at 0 °C. The mixture was heated at 70 ºC for 2.5
h and was allowed to cool to ambient temperature to afford a solution of boronate reagent which was directly used in the next step.
A microwave reaction tube was charged with 5 (300 mg, 1.09 mmol), PdCl2(dppf) (80 mg, 0.11 mmol), K2CO3 (300 mg, 2.19 mmol),
the boronate reagent prepared in the previous step, water (1 mL) and DMSO (2 mL). The mixture was heated under microwave
irradiation at 100 °C for 30 min. The mixture was purified by chromatography on SiO2 (EtOAc-Petroleum ether; 1:20) to afford tert-
butyl 3-((8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)methyl)pyrrolidine-1-carboxylate (220 mg, 67%) as a white
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solid. H-NMR (400 MHz, CD3OD) δ 8.79 (s, 1 H), 8.02 (s, 1 H), 4.30 (q, J = 7.6 Hz, 2 H), 3.94 (s, 3 H), 3.40-3.55 (m, 2 H), 3.30-
3.40 (m, 2H), 3.20-3.30 (m, 1 H), 2.80-2.90 (m, 1 H), 2.67 (s, 3 H), 1.95-2.05 (m, 1 H), 1.74-1.77 (m, 1 H), 1.32-1.48 (m, 11 H); MS
(EI) calc’d for C22H32N7O2 [M+H]+, 426; found, 426.
A mixture of tert-butyl 3-((8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl)methyl)pyrrolidine-1-carboxylate (200
mg, 0.47 mmol) and HCl (3 mL, 4.0 M in dioxane, 12 mmol) was stirred at ambient temperature for 1 h. The solvent was evaporated
to afford 8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-6-(pyrrolidin-3-ylmethyl)-9H-purine-HCl salt (147 mg, 86 %) as a white
solid and the product was directly used in the next step.
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A mixture of 8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-6-(pyrrolidin-3-ylmethyl)-9H-purine-HCl salt (67 mg, 0.185 mmol),
tetrahydro-2H-pyran-4-carboxylic acid (26 mg, 0.20 mmol), HATU (100 mg, 0.28 mmol) and iPr2NEt (72 mg, 0.56 mmol) in DMF
(2 mL) was stirred at ambient temperature for 20 h. The mixture was purified by reverse phase chromatography (gradient of
water/MeCN with 10 mmol NH4HCO3) to afford 2s (15 mg, 19 %) as a white solid. 1H-NMR (400 MHz, CDCl3) δ 8.82 (s, 1 H),
8.03 (s, 1 H), 4.29 (q, J = 7.6 Hz, 2 H), 3.76-3.95 (m, 5 H), 3.62-3.75 (m, 1 H), 3.39-3.47 (m, 2 H), 3.32-3.34 (m, 4 H), 2.70-2.97 (m,
2 H), 2.64 (s, 3 H), 2.05-2.17 (m, 1 H), 1.64-1.80 (m, 6 H), 1.49 (t, J = 7.6 Hz, 3 H); 13C NMR (100 MHz, CD3OD)  174.1, 157.8,
152.5, 151.0, 150.9, 140.8, 138.0, 132.4, 108.3, 66.8, 66.7, 51.0, 45.4, 44.0, 39.3, 38.8, 35.1, 31.3, 29.6, 28.2, 28.1, 14.0, 9.3; HRMS
(EI) calc’d for C23H32N7O2 [M+H]+, 438.2617; found, 438.2619.
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(4-(8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yloxy)thiophen-2-yl)(morpholino)methanone (2t). Prepared in
a fashion similar to that used for the synthesis of 2d. 1H NMR (400 MHz, CD3OD) δ 8.50 (s, 1 H), 8.0 (s, 1 H), 7.50-7.61 (m, 2 H),
4.27-4.29 (s, 2 H), 3.95 (s, 3 H), 3.71-3.82 (m, 8 H), 2.58 (s, 3 H), 1.46-1.50 (m, 3 H); 13C NMR (100 MHz, CD3OD)  162.3, 157.9,
155.1, 150.7, 149.8, 147.5, 140.4, 138.2, 135.7, 124.9, 120.7, 116.3, 108.6, 66.6 (2C), 44.3 (2C), 40.5, 31.0, 15.4, 10.8; HRMS (ESI)
calc’d for C21H24N7O3S [M+H]+, 454.1661; found, 454.1657.
3-{[8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-yl]amino}-1-(2-methylpropyl)pyrrolidin-2-one
(2u).
Prepared in a fashion similar to that used for the synthesis of 2d, then assayed in racemic form. 1H NMR (500 MHz, DMSO-d6) 
8.19 (s, 1 H), 7.95 (s, 1 H), 5.02 (m, 1 H), 4.18 (m, 2 H), 3.78 (s, 3 H), 3.35 (m, 2 H), 3.04 (m, 2 H), 2.59 (m, 3 H), 2.39 (m, 1 H),
2.12 (m, 1 H), 1.93 (m, 1 H), 1.37 (m, 3 H), 0.83-0.93 (m, 7 H); 13C NMR (100 MHz, DMSO-d6)  172.6, 153.9, 151.8 (2C), 139.3,
137.8 (2C), 119.5, 109.2, 50.5, 44.1, 44.0, 30.2, 26.5, 26.2, 20.5, 20.4, 15.4, 10.6; HRMS (EI) calc’d for C20H29N8O [M+H]+,
397.2464; found, 397.2465.
4-(8-(1-Ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purin-6-ylamino)-1-isobutylpyrrolidin-2-one (2v). Prepared in a fashion
similar to that used for the synthesis of 2d, then the racemic material (40 mg) was resolved using chiral column chromatography (AD-
H column 30x250 mm, 70 mL/min 60/40/0.1 CO2/MeOH/Et2NH) to give 2v (10 mg) as the first eluted enantiomer followed by the
less active enantiomer next (10 mg). [α]D -19° (c = 0.8, MeOH); 1H NMR (400 MHz, CD3OD) δ 8.30 (s, 1 H), 7.91 (s, 1 H), 4.98-
5.12 (m, 1 H), 4.25 (q, J = 7.2 Hz, 2 H), 3.93-3.95 (m, 1 H), 3.83 (s, 3 H), 3.51-3.52 (m, 1 H), 3.14-3.16 (m, 2 H), 2.90-2.95 (m, 1
H), 2.56-2.65 (m, 4 H), 1.89-1.99 (m, 1 H), 1.46 (t, J= 7.2 Hz, 3 H), 0.90-0.93 (m, 6 H); 13C NMR (100 MHz, CD3OD)  174.0,
153.3, 151.7, 150.3, 146.0, 139.8, 137.8, 118.8, 108.7, 54.5, 49.8, 44.6, 44.0, 37.6, 29.1, 26.5, 18.9 (2C), 14.0, 9.1; HRMS (EI) calc’d
for C20H29N8O [M+H]+, 397.2464; found, 397.2476.
6-Chloro-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purine (5). A 1000-mL 3-necked flask fitted with a thermometer
was charged with N-ethyl-1H-5-methyl-pyrazole-4-carboxaldehyde (2, 68.5 g, 496 mmol), anhydrous N,N-dimethylformamide (400
mL), and 6-chloro-N-methylpyrimidine-4,5-diamine (3, 71.5 g, 451 mmol). The reaction mixture was stirred at ambient temperature
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for 10 minutes to ensure dissolution of all solid material. Iron (III) chloride hexahydrate (122 g, 451 mmol) was then added over the
course of 5 minutes, resulting in the formation of a dark brown solution. A stream of air was continuously bubbled through the stirred
reaction mixture, which was heated to 85 °C and stirred for 14 h. The mixture was poured into 2500 mL of crushed ice and water,
and the resulting pale orange precipitate collected by vacuum filtration and dried on the filter. The solid was triturated with ethanol
(500 mL) at 50 °C for 2 h and at ambient temperature overnight. The residue was collected by vacuum filtration and dried on the
filter to give 93.5 g (75% yield) of intermediate 6-chloro-8-(1-ethyl-5-methyl-1H-pyrazol-4-yl)-9-methyl-9H-purine (5) as a pale
yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1 H), 8.09 (s 1 H), 4.18 (q, 2 H), 3.89 (s, 3 H), 2.63 (s, 3 H), 1.35 (t, 3 H);
MS (EI) calc’d for C12H14ClN6 [M+H]+, 277; found 277.
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PI3K Biochemical HTRF Assay. PI3K family biochemical potencies in the phosphorylation of PIP2 (phosphatidylinositol (4,5)-
bisphosphate) to PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) were measured using an HTRF assay. PI3K biochemical assays
were optimized from an Upstate (Millipore) HTRF kit. Briefly, compounds were serially diluted (3-fold in 100% DMSO) for a 10-
concentration dose response. PI3K reaction buffer was prepared by dilution of stock with DI water, then treated with DTT, PIP2 and
Biotin-PIP3 at a final concentration of 5 M, 5 M and 25 nM, respectively. Enzyme and the compounds were added at ambient
temperature for a 15 min preincubation. Reactions were initiated by addition of substrate solution (PIP2 and ATP) and incubated at
ambient temperature for 1 h before quenching with EDTA. The detection solution (streptavidin-APC with Eu-labeled anti-GST plus
GST-tagged PH-domain) was added and incubated in the dark for 1 h, followed by measurement of the HTRF signal with an Envision
plate reader (330 nm excitation and dual emission detection at 620 nm (Eu) and 665 nm (APC)). The individual kinases were
purchased from Upstate (PI3K 14-602, PI3K 14-603, PI3K 14-558 and PI3K 14-604). The assay format was the same for all
four isoforms, the differences lie in the concentration of enzyme and ATP used. The PI3K, PI3K, PI3K, PI3K assays were run
with 0.5, 1, 0.3, 5 nM of enzyme, respectively. The ATP concentration was 100 M in the PI3K, PI3K, and PI3K assays and 50
M in the PI3K assay. Assay run in duplicate.
Ramos AKT-pSer473 Assay. The phosphorylation status of serine 473 of AKT in the Ramos lymphoma-derived B cell line is driven
by PI3K, hence a good measure of cellular activity of PI3K inhibition. The Ramos cell line expresses cell surface IgM and responds
to IgM crosslinking by activating PI3K-dependent signaling. The AlphaScreen SureFire Akt p‐Ser473 assay was used to measure
the phosphorylation of endogenous AKT in cellular lysates. B Lymphocyte Ramos cells (ATCC catalog #CRL-1596) were split 1:3
every 3 to 4 days and maintained between 100,000-1,000,000 cells/mL. Cells were diluted in assay media (DMEM, high glucose,
HEPES, No phenol red supplemented with sodium pyruvate) to a concentration of 10,000,000 cells/mL and plated (6 L, 60,000 cells
per well) using a BioRaptor. Compounds were serially diluted (3-fold in 100% DMSO) for a 10-concentration dose response and 10
nL added to the wells. The mixture was preincubated for 20 min at ambient temperature. Anti-IgM (2 L of 1 g/mL) was then
added and the plates incubated at 37 oC for 30 min. The cells were then treated with Lysis buffer (2 L of 5x) and incubated for 30
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min at ambient temperature. In the dark, acceptor beads (8 L of 0.039 mg/mL) were added with Ser473 reaction buffer and the
plates incubated for 2 h. Donor beads (3 L of 0.036 mg/mL) were added in the dark and again incubated for 2 h. The plates were
then read on Envision plate reader. Assay run in duplicate.
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SKBr3 AKT-pThr308 Assay. The phosphorylation status of threonine 308 of AKT in the breast cancer cell line SKBR3 is driven
by PI3K, hence a good measure of cellular activity of PI3K inhibition. The protocol is identical to that used in the Ramos AKT-
pSer473 assay, with the following modifications. SKBR3 cells are used, and are stimulated instead by heregulin (2 L of 0.05
g/mL). In addition, Thr308 reaction buffer was used with the acceptor beads. Assay run in duplicate.
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Human Whole Blood B-Cell Activation CD69 Biomarker Assay. B cell activation is linked to a broad number of diseases
including oncology, arthritis, and lupus. Activation of B-cells can be induced ex vivo by stimulation with antibodies that recognize
components of the B-cell receptor. These antibodies crosslink receptors on the surface of the B-cell, inducing receptor signaling
cascade that drives cell activation. The B-cell receptor is composed of three subunits: a transmembrane IgM for antigen recognition,
and CD79a and CD79b, with small cell surface epitopes and prominent intracellular domains containing ITAM signaling subunits.
Human whole blood was obtained from healthy volunteer donors at Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc.,
Kenilworth, NJ, USA. Using an Echo liquid handler, 120 nL of compound in DMSO at varying concentrations in a 96-well plate
was then treated with 100 μL of blood and incubated for 60 min at 37 ºC. To each well was added 11 μL of anti-CD76b antibody
(BD Biosciences) and the mixture incubated for 3 h at 37 ºC. The reaction was stopped by placing on wet ice for 5 min. Each well
was treated with 50 μL of staining cocktail (CD45-V450, 5 μL; CD3-APC, 5 μL; CD20-PerCP-Cy5-5, 7.5 μL; CD69-FITC, 20 μL in
FACS Buffer, 12.5 μL; all from BD Biosciences). The mixture was incubated for 30 min at 4 ºC. RBCs were lysed by adding 1.8
mL FACS lysis buffer to each well, incubated for 20 min at 20 ºC. Spin down plate at 1000xg for 5 min, remove supernatant, and
tap plate on an absorbent pad to remove excess liquid. Resuspend pellet in each well in 250 μL of FACS reading buffer (1X BD stain
buffer, 0.5% Pluronic F68, 0.2 mg/mL human IgG), and transfer cells to a clean 384 well u-bottom greiner plate. Fixed and stained
cells are kept at 4 ºC before analyzing fluorescence with Fortessa A FACS machine. Gating: Gate1 = Lymphocyte, based on CD45
and side scatter; Gate2 = Singlet (from gate1), based on forward scatter A&H; Gate3 = B cell (from gate2), which are CD3-APC
negative and CD20-PerCP Cy5.5 positive; Gate4 = Activated B cell (from gate3), which are CD69-FITC positive. Assay run in
duplicate.
Human Whole Blood Basophil Activation CD63 Biomarker Assay. Basophils are the least common leukocyte but are an important
effector cell population in allergy, with CD63 being one of the best described biomarkers of basophil activation. Human whole blood
was obtained from healthy volunteer donors at Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ,
USA. Using an Echo liquid handler, 120 nL of compound in DMSO at varying concentrations was then treated with 100 μL of blood
and incubated for 30 min at 37 ºC. To each well was added 20 μL of reagent B (Basotest kit; Gylcotope Biotechnology, cat#10-0500)
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and the mixture incubated for 30 min at 37 ºC. Separately, basophil cell activating goat anti-human IgE antibody (0.7 μL; Bethyl
Laboratories) was prepared to a final concentration of 50 ng/mL in reagent A (200 μL; also from Basotest kit). Each well was treated
with 10 μL of the activating anti-IgE, and the plates incubated for 20 min at 37 ºC. The reaction was stopped by placing on wet ice
for 5 min. Each well was treated with 30 μL of reagent F stain (anti-human CD63-FITC and anti-human IgE-PE reagent solution; 10
mL, anti-human CD203c-APC; 1500 μL, anti-human CD45-V450; 313 μL, in FACS dilution buffer; 5 mL). The mixture was
incubated for 20 min at 4 ºC. RBCs were lysed by adding 1.9 mL lysis/fix buffer/reagent G to each well, incubated for 15 min at 20
ºC. Pellet cells at 1500 rpm for 5 min at 4 ºC, aspirate supernatant, resuspend in 70 μL of cold FACS reading buffer (1X phosphate-
buffered saline, 0.2% bovine serum albumin, 0.5% pluronic F-68, 0.2 mg/mL human IgG) and maintain at 4 °C. Plates were read
using LSR Fortessa. Set single white blood cells in anti-CD45-V450 gate, count basophil cells in anti-human IgE gate and analyze
the activated basophil cells in CD63+ gate in Diva software. The IC50 values were calculated from percent inhibition values using
ADA software to fit to a sigmoidal dose response. Assay run in duplicate.
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HeLa Cell Adenosine Uptake (AdU) Inhibition Assay. Dilution plates with compound in DMSO were prepared as a 10-point
titration and diluted with HBSS with 5% FBS to reach concentrations of 25000 nM to 0.8 nM of compound. HeLa cells (ATCC)
were thawed and seeded 25,000 cells/well in Cytostar T plates overnight in MEM and 10% FBS. Remove growth media by flicking,
add 40 μL of HBSS with 5% FBS, then add 40 μL of compound in buffer onto the cells. Incubate for 30 min. Add 20 μL of
radiolabeled 100 nM 3H-adenosine (ARC; 40 Ci/mmol specific activity and 1 mCi/mL concentration from 25000 nM stock
concentration) in HBSS with 5% FBS, and incubate for 60 min. Total volume 100 μL, with compound concentrations of 10000 nM
to 0.32 nM. To measure uptake of radioactive adenosine, plates were read with PerkinElmer TopCount NXT HTS plate reader. Data
was analyzed using ADA Logic to fit to a 4-parameter fit to provide IC50 values. Assay run in duplicate.
PI3K Isolation for Crystallization. The protein used for crystallization was produced via coexpression in Sf9 cells of murine His8-
TEV-p110δ(2-105)-TEV-(106-1044) with His6-p85α(431-600), where TEV indicates a site susceptible to cleavage by TEV protease.
Cell pellet from a 10 L expression was suspended in lysis buffer comprised of 20 mM Tris, pH 8.0, 100 mM NaCl, 5% glycerol, 5
mM BME, 25 U/mL benzonase, and Complete-EDTA free protease inhibitors. The pellet was Dounce homogenized, sonicated, and
clarified by centrifugation. The supernatant was subjected to metal-chelate chromatography using a Histrap Crude FF column and
heparin chromatography, prior to cleavage using His-tagged TEV protease. The resulting solution was run over a Histrap column
using 40 mM imidazole to retain the cleaved purification tag, uncleaved material, and the protease. The flowthrough was pooled,
diluted into buffer containing 50 mM Tris, pH 8.0, and 5 mM DTT and then subjected to anion exchange chromatography using a Q
HP column. Fractions containing the desired protein, as judged by SDS-PAGE, were pooled and adjusted to pH 8.5 using NaOH and
resubjected to anion exchange chromatography using a Q HP column. Selected fractions were pooled, concentrated to 12.5 mg/mL,
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and run over a Superdex column using a running buffer containing 20 mM Tris pH 7.5, 50 mM (NH4)2SO4, 1% ethylene glycol, 1%
betaine, 0.02% CHAPS, and 5 mM DTT. Selected fractions were pooled and concentrated to 10 mg/mL and frozen as 25 µL aliquots.
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PI3K-Inhibitor Crystallization and Structure Determination. The protein was prepared to a final concentration of 5.3 mg/mL
(as estimated by UV absorbance using an extinction coefficient of 128,230 M-1cm-1 at 280 nm) in the following buffer: 20 mM Tris•Cl
pH 7.5, 0.050 mM ammonium sulfate, 1% betaine, 1% v/v ethylene glycol, 0.02% w/v CHAPS, 5 mM DTT. Crystals of the ligand-
free protein were obtained at 20°C by vapor diffusion in a Nextal plate using a hanging drop set-up by mixing equal volumes of 2.0
L diluted protein and 2.0 L precipitant. The diluted protein was made by mixing 25 L of the stock protein with 6.5 L of distilled
water. The precipitant was 0.1 Na•MES pH 6.8, 30 mM Na2HPO4, 5% v/v MPD, 11 to 17% v/v PEG 600 (optimum of 13% at the
stated protein concentration).
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Crystals of compound 1-bound PI3K were obtained by a combination of micro-seeding and co-crystallization. A stock solution of
100 mM inhibitor in 100% DMSO was prepared then diluted 10-fold in distilled water. Then 1.2 L of this solution was added to
5.0 L of protein sample. The micro-seed stock was prepared using the precipitant solution at optimum PEG 600 concentration (13%
w/v) using Hampton Research’s Seed Bead™ following the manufacturer’s protocol. Then, the seed stock was diluted either 20,
100, 500, or 2000-fold, respectively in the precipitant solution at optimum PEG 600 concentration. Finally 1.5 L of protein –
inhibitor mix was added to 1.5 L of each seed stock for the vapor diffusion experiment. Crystals of compound 2q-bound PI3K
were obtained by the method of soaking, by transferring an apo crystal grown as described previously to a stabilization drop with
ligand, obtained by diluted 50-fold a 100 mM inhibitor stock in DMSO in the precipitant cocktail.
In all cases the precipitant served as cryo-protectant as well. Crystals were harvested using a 0.2 mm mesh loop (Molecular
Dimensions) and flash frozen in liquid nitrogen. The data for the co-crystal with compound 1 were collected at the Canadian Light
Source 08ID-1 beam line on a Rayonic MX300 CCD detector. They were integrated using HKL2000 and scaled using SCALEPACK.
The structure was solved by molecular replacement using the MOLREP program and PDB entry 2WXF as a starting model. For the
co-structure with compound 2q, data were collected at the APS IMCA 17-ID beam line on a Pilatus 6M pixel array detector. The co-
structure with ligand 2q was used as starting model for refinement. For both structures all refinement steps were performed using
alternate runs of the BUSTER software and corrections of the model using the graphical program O.
SUPPORTING INFORMATION
Ancillary HeLa cell adenosine uptake and phosphodiesterase inhibition data for piperidine benzimidazolone analogs of compound 1 as well
as 2j, 2p and 2q. Molecular formula strings for all compounds. Kinase selectivity panel for 2q. The Supporting Information is available
free of charge on the ACS Publications website.
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PDB ID CODES.
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Accession codes have been deposited in the RCSB Protein Data Bank; Compound 1 PDB ID: 6MUL, Compound 2q PDB ID: 6MUM.
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Authors will release the atomic coordinates upon article publication.
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CORRESPONDING AUTHOR
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*E-mail: joey_methot@merck.com; 617-273-0368.
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REFERENCES
1 (a) Di Paolo, G.; De Camilli, P. Phosphoinositides in Cell Regulation and Membrane Dynamics. Nature 2006, 443, 651−657; (b) Hawkins, P.T.;
Anderson, K. E.; Davidson, K.; Stephens, L. R. Signaling Through Class I PI3Ks in Mammalian Cells. Biochem. Soc. Trans. 2006, 34, 647-662.
2 (a) Hawkins, P. T.; Stephens, L.R. PI3K Signalling in Inflammation. Biochim. Biophys. Acta 2015, 1851, 882-897; (b) Stark, A.-K.;
Sriskantharajah, S.; Hessel, E. M.; Okkenhaug, K. PI3K Inhibitors in Inflammation, Autoimmunity and Cancer. Curr. Opin. Pharmacol. 2015, 23,
82-91; (c) Suárez-Fueyo, A.; Rojas, J. M.; Cariaga, A. E.; García, E.; Steiner, B. H.; Barber, D. F.; Puri, K. D.; Carrera, A. C. Inhibition of PI3Kδ
Reduces Kidney Infiltration by Macrophages and Ameliorates Systemic Lupus in the Mouse. J. Immunol. 2014, 193, 544−554; (d)
Sriskantharajah, S.; Hamblin, N.; Worsley, S.; Calver, A. R.; Hessel, E. M.; Amour, A. Targeting Phosphoinositide 3-Kinase δ for the Treatment of
Respiratory Diseases. Ann. N. Y. Acad. Sci. 2013, 1280, 35−39; (e) Suárez-Fueyo, A.; Barber, D. F.; Martínez-Ara, J.; Zea-Mendoza, A. C.;
Carrera, A. C. Enhanced Phosphoinositide 3-Kinase δ Activity is a Frequent Event in Systemic Lupus Erythematosus That Confers Resistance to
Activation-Induced T Cell Death. J. Immunol. 2011, 187, 2376−2385; (f) Conliffe, A. M.; Chandra, A. Respiratory Manifestations of the
Activated Phosphoinositide 3-Kinase delta Syndrome. Frontiers Immunol. 2018, 9, 338; (g) Michalovich, D.; Nejentsev, S. Activated PI3 Kinase
Delta Syndrome: From Genetics to Therapy. Frontiers Immunol. 2018, 9, 369.
3 (a) Macias-Perez, I. M.; Flinn, I. W. GS-1101: A Delta-Specific PI3K Inhibitor in Chronic Lymphocytic Leukemia. Curr. Hematol. Malig. Rep.
2013, 8, 22-27; (b) Fruman, D. A.; Rommel, C. PI3K and Cancer: Lessons, Challenges and Opportunities. Nature Rev. Drug Disc. 2014, 13, 140-
156; (c) Wei, M.; Wang, X.; Song, Z.; Jiao, M.; Ding, J.; Meng, L.-H.; Zhang, A. Targeting PI3Kδ: Emerging Therapy for Chronic Lymphocytic
Leukemia and Beyond. Medical Res. Rev. 2015, 35, 720-752; (d) Thorpe, L. M.; Yuzugullu, H.; Zhao, J. J. PI3K in Cancer: Divergent Roles of
Isoforms, Modes of Activation and Therapeutic Targeting. Nature Rev. Cancer 2015, 15, 7-24.
4 (a) For information regarding idelalisib, see https://www.zydelig.com/index.aspx. Gilead Sciences, Inc.; accessed Jan 12, 2019; (b) Yang,
Q.; Modi, P.; Newcomb, T.; Queva, C.; Gandhi, V. Idelalisib: First-in-Class PI3K Delta Inhibitor for the Treatment of Chronic Lymphocytic
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2
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5
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7
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Leukemia, Small Lymphocytic Leukemia, and Follicular Lymphoma. Clin. Cancer Res. 2015, 21, 1537-1542; (c) Bange, E.; Nabhan, C.; Brander,
D. M.; Lamanna, N.; Ujjani, C. S.; Howlett, C.; Skarbnik, A. P.; Hill, B. T.; Cheson, B. D.; Zent, C. S.; Pu, J. J.; Winter, A. M.; Isaac, K.; Kennard,
K.; Timlin, C.; Dorsey, C.; Dwivedy Nasta, S.; Svoboda, J.; Landsburg, D. J.; Schuster, S. J.; Barr, P. M.; Mato, A. R. Real-World Evidence for
Durable Treatment Responses after Toxicity Related Discontinuation of Idelalisib. Blood 2017, 130, 4325.
10
11
12
13
14
15
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18
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5 (a) Ali, K.; Soond, D. R.; Pineiro, R.; Hagemann, T.; Pearce, W.; Lim, E. L.; Bouabe, H.; Scudamore, C. L.; Hancox, T.; Maecker, H.; Friedman,
L.; Turner, M.; Okkenhaug, K.; Vanhaesebroeck, B. Inactivation of PI(3)K p110δ Breaks Regulatory T-Cell-Mediated Immune Tolerance to
Cancer. Nature 2014, 510, 407-411; (b) Hirsch, E.; Novelli, F. Natural-Born Killers Unleashed. Nature 2014, 510, 342-343.
6 (a) Williams, O.; Houseman, B. T.; Kunkel, E. J.; Aizenstein, B.; Hoffman, R.; Knight, Z. A.; Shokat, K. M. Discovery of Dual Inhibitors of the
Immune Cell PI3Ks p110δ and p110γ: A Prototype for New Anti-Inflammatory Drugs. Chem. Biol. 2010, 17, 123-134; (b) Winkler, D. J.; Faia, K.
L.; DiNitto, J. P.; Ali, J. A.; White, K. 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.; Thomas, B. D.; MacDougall, J. R.; Ren, P.; Liu, Y.; Li, L.-S.; Jessen, K. A.; Fritz, C. C.; Dunbar, J. L.; Porter, J. R.; Rommel,
C.; Palombella, V. J.; Changelian, P. S.; Kutok, J. L. PI3K-d and PI3K-g Inhibition by IPI-145 Abrogates Immune Responses and Suppresses
Activity in Autoimmune and Inflammatory Disease Models. Chem. Biol. 2013, 20, 1364-1374; (c) Cushing, T. D.; Hao, K.; Shin, Y.; Andrews,
K.; Brown, M.; Cardozo, M.; Yi Chen, Y.; Duquette, J.; Fisher, B.; Gonzalez-Lopez de Turiso, F.; He, X.; Henne, K. R.; Hu, Y.-L.; Hungate, R.;
Johnson, M. G.; Kelly, R. C.; Lucas, B.; McCarter, J. D.; McGee, L. R.; Medina, J. C.; Miguel, T. S.; Mohn, D.; Pattaropong, V.; Pettus, L. H.;
Reichelt, A.; Rzasa, R. M.; Seganish, J.; Tasker, A. S.; Wahl, R. C.; Wannberg, S.; Whittington, D. A.; Whoriskey, J.; Yu, G.; Zalameda, L.;
Zhang, D.; Metz, D. P. Discovery and in Vivo Evaluation of (S)‑N‑(1-(7-Fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)‑9H‑purin-6-amine
(AMG319) and Related PI3Kδ Inhibitors for Inflammation and Autoimmune Disease. J. Med. Chem. 2015, 58, 480-511; (d) Ndubaku, C. O.;
Heffron, T. P.; Staben, S. T.; Baumgardner, M.; Blaquiere, N.; Bradley, E.; Bull, R.; Do, S.; Dotson, J.; Dudley, D.; Edgar, K. A.; Friedman, K. S.;
Goldsmith, R.; Heald, R. A.; Kolesnikov, A.; Lee, L.; Lewis, C.; Nannini, M.; Nonomiya, J.; Pang, J.; Price, S.; Prior, W. W.; Salphati, L.; Sideris,
S.; Wallin, J. J.; Wang, L.; Wei, B.; Sampath, D.; Olivero, A. G. Discovery of 2‑{3-[2-(1-Isopropyl-3-methyl‑1H‑1,2−4-triazol-5-yl)-5,6-
dihydrobenzo[f]imidazo[1,2‑d][1,4]oxazepin-9-yl]‑1H‑pyrazol-1-yl}-2-methylpropanamide (GDC-0032): A β‑Sparing Phosphoinositide 3‑
Kinase Inhibitor with High Unbound Exposure and Robust in Vivo Antitumor Activity. J. Med. Chem. 2013, 56, 4597-4610; (e) Down, K.;
Amour, A.; Baldwin, I. R.; Cooper, A. W. J.; Deakin, A. M.; Felton, L. M.; Guntrip, S. B.; Hardy, C.; Harrison, Z. A.; Jones, K. L.; Jones, P.;
Keeling, S. E.; Le, J.; Livia, S.; Lucas, F.; Lunniss, C. J.; Parr, N. J.; Robinson, E.; Rowland, P.; Smith, S.; Thomas, D. A.; Vitulli, G.; Washio, Y.;
Hamblin, J. N. Optimization of Novel Indazoles as Highly Potent and Selective Inhibitors of Phosphoinositide 3‑Kinase δ for the Treatment of
Respiratory Disease. J. Med. Chem. 2015, 58, 7381−7399; (f) Hoegenauer, K.; Soldermann, N.; Stauffer, F.; Furet, P.; Graveleau, N.; Smith, A.
B.; Hebach, C.; Hollingworth, G. J.; Lewis, I.; Gutmann, S.; Rummel, G.; Knapp, M.; Wolf, R. M.; Blanz, J.; Feifel; R.; Burkhart, C.; Zécri, F.
Discovery and Pharmacological Characterization of Novel Quinazoline-Based PI3K Delta-Selective Inhibitors. ACS Med. Chem. Lett. 2016, 7,
762-767; (g) Liu, Q.; Shi, Q.; Marcoux, D.; Batt, D. G.; Cornelius, L.; Qin, L.-Y.; Ruan, Z.; Neels, J.; Beaudoin-Bertrand, M.; Srivastava, A. S.;
Ling Li, L.; Robert J. Cherney, R. J.; Hua Gong, H.; Watterson, S. H.; Weigelt, C.; Gillooly, K. M.; McIntyre, K. W.; Xie, J. H.; Obermeier, M. T.;
Fura, A.; Sleczka, B.; Stefanski, K.; Fancher, R. M.; Padmanabhan, S.; Thatipamula, R. P.; Kundu, I.; Rajareddy, K.; Smith, R.; Hennan, J. K.;
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Xing, D.; Fan, J.; Levesque, P. C.; Ruan, Q.; Pitt, S.; Zhang, R.; Pedicord, D.; Pan, J.; Yarde, M.; Lu, H.; Lippy, J.; Goldstine, C.; Skala, S.;
Rampulla, R. A.; Mathur, A.; Gupta, A.; Arunachalam, P. N.; Sack, J. S.; Muckelbauer, J. K.; Cvijic, M. E.; Salter-Cid, L. M.; Bhide, R. S.; Poss,
M. A.; Hynes, J.; Carter, P. H.; Macor, J. E.; Ruepp, S.; Schieven, G. L.; Tino, J. A. Identification of a Potent, Selective, and Efficacious
8
9
Phosphatidylinositol 3‑Kinase δ (PI3Kδ) Inhibitor for the Treatment of Immunological Disorders. J. Med. Chem. 2017, 60, 5193−5208; (h) Perry,
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M. W. D.; Björhall, K.; Bonn, B.; Carlsson, J.; Chen, Y.; Eriksson, A.; Fredlund, L.; Hao, H.; Holden, N. S.; Karabelas, K.; Lindmark, H.; Liu, F.;
Pemberton, N.; Petersen, J.; Blomqvist, S. R.; Smith, R. W.; Svensson, T.; Terstiege, I.; Tyrchan, C.; Yang, W.; Zhao, S.; Öster, L. Design and
Synthesis of Soluble and Cell-Permeable PI3Kδ Inhibitors for Long-Acting Inhaled Administration. J. Med. Chem. 2017, 60, 5057-5071.
7 Identified as XL-499 at Exelixis, Inc., and as MSD-126796721 at Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth,
NJ, USA. Synthesis described in WO 2012/037226A1.
8 Thomas, M.; Owen, C. Inhibition of PI-3 Kinase for Treating Respiratory Disease: Good Idea or Bad Idea? Cur. Opin. Pharmacol.
2008, 8, 267–274.
9 Calistoga/Gilead reported that PI3K inhibitor GS-1101 reduced upper airway inflammation in allergic rhinitics challenged with an
allergen in a phase Ib study. The group used an ex vivo blood basophil activation assay to correlate inhibition to reduction in allergic
inflammation in vivo. The assay consisted of human blood stimulation with an anti-IgE antibody which triggers a PI3K-dependent
cell activation that can be measured by the up-regulation of surface biomarker CD63. GS-1101 was shown to have an ex vivo IC50
of 150 nM, roughly corresponding to the in vivo concentration associated with a significant reduction in total nasal symptom score
and nasal secretion. See Horak, F.; Puri, K. D.; Steiner, B. H.; Holes, L.; Xing, G.; Zieglmayer, P.; Zieglmayer, R.; Lemell, P.; Yu,
A. Randomized Phase 1 Study of the Phosphatidylinositol-3-kinase delta Inhibitor Idelalisib in Patients with Allergic Rhinitis. J.
Allergy Clin. Immunol. 2016, 137, 1733-1741.
10 Note that while for nononcology indications a CD69 biomarker IC50 trough concentration was targeted, it was recognized that for
oncology indications a CD69 IC90 trough target concentation may be necessary to achieve therapeutic efficacy.
11 (a) Marzio, R.; Mauel, J.; Betz-Corradin, S. CD69 and Regulation of the Immune Function. Immunopharmacol. Immunotoxicol. 1999, 21, 565-
582; (b) Del Poeta, G.; Del Principe, M. I.; Zucchetto, A.; Luciano, F.; Francesco Buccisano, F.; Rossi, F. M.; Bruno, A.; Biagi, A.; Bulian, P.;
Maurillo, L.; Neri, B.; Bomben, R.; Simotti, C.; Coletta, A. M.; Dal Bo, M.; de Fabritiis, P.; Venditti, A.; Gattei, V.; Amadori, S. CD69 is
Independently Prognostic in Chronic Lymphocytic Leukemia: a Comprehensive Clinical and Biological Profiling Study. Haematologica 2012, 97,
279-287; (c) Montraveta, A.; Lee-Vergés, E.; Roldán, J.; Jiménez, L.; Cabezas, S.; Clot, G.; Pinyol, M.; Xargay-Torrent, S.; Rosich, L.; Arimany-
Nardí, C.; Aymerich, M.; Villamor, N.; López-Guillermo, A.; Pérez-Galán, P.; Roué, G.; Pastor-Anglada, M.; Campo, E.; López-Guerra, M.;
Colomer, D. CD69 Expression Potentially Predicts Response to Bendamustine and its Modulation by Ibrutinib or Idelalisib Enhances Cytotoxic
Effect in Chronic Lymphocytic Leukemia. Oncotarget 2016, 7, 5507-5520.
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All animal experiments performed in the manuscript were conducted in compliance with institutional guidelines as defined by
Institutional Animal Care and Use Committee for U.S. institutions.
5
6
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8
9
13 (a) Louden, C.; Brott, D.; Katein, A.; Kelly, T.; Gould, S.; Jones, H.; Betton, G.; Valetin, J.-P.; Richardson, R. J. Biomarkers and Mechanisms
10
11
12
13
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16
17
18
19
20
21
22
23
24
25
26
27
28
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of Drug-Induced Vascular Injury in Non-Rodents. Toxicol. Pathol. 2006, 34, 1; (b) Enerson, B. E.; Lin, A.; Lu, B.; Zhao, H.; Lawton, M. P.;
Floyd, E. Acute Drug-Induced Vascular Injury in Beagle Dogs: Pathology and Correlating Genomic Expression. Toxicol. Pathol. 2006, 34, 27-32.
14 (a) Jeon, Y. H.; Heo, Y. S.; Kim, C. M.; Hyun, Y. L.; Lee, T. G.; Ro, S.; Cho, J. M. Phosphodiesterase: Overview of Protein Structures,
Potential Therapeutic Applications and Recent Progress in Drug Development. Cell. Mol. Life Sci. 2005, 62, 1198-1220; (b) Boswell-Smith, V.;
Spina, D.; Page, C. P. Phosphodiesterase Inhibitors. Bit. J. Pharmacol. 2006, 147, S252-S257.; (c) Feneck, R. Phosphodiesterase inhibitors and
the cardiovascular system. Cont. Ed. Anaesth. Crit. Care 2007, 7, 203-207; (d) Yan, C.; Miller, C. L.; Abe, J. Regulation of Phosphodiesterase 3
and Inducible cAMP Early Repressor in the Heart. Circul. Res. 2007, 100, 489-501; (e) Hantona, G.; Sobry, C.; Dague, N.; Provost, J. P.; Le Net,
J.-L.; Comby, P.; Chevalier, S. Characterisation of the Vascular and Inflammatory Lesions Induced by the PDE4 Inhibitor CI-1044 in the Dog.
Toxicol. Lett. 2008, 179, 15-22.
15 (a) Meester, B. J.; Shankley, N. P.; Welsh, N. J.; Meijler, F. L.; Black, J. W. Pharmacological Analysis of the Activity of the Adenosine Uptake
Inhibitor, Dipyridamole, on the Sinoatrial and Atrioventricular Nodes of the Guinea-pig. Brit. J. Pharmacol. 1998, 124, 729-741; (b) Schwartz, L.
M.; Bukowski, T. R.; Revkin, J. H.; Bassingthwaighte, J. B. Cardiac Endothelial Transport and Metabolism of Adenosine and Inosine. Am. J.
Physiol. Heart. Circ. Physiol. 1999, 277, H1241-H1251; (c) Löffler, M.; Morote-Garcia, J. C.; Eltzschig, S. A.; Coe; I. R.; Eltzschig, H. K.
Physiological Roles of Vascular Nucleoside Transporters. Arterioscler Thromb Vasc Biol. 2007, 27, 1004-1013; (d) van Giezen, J. J. J.; Sidaway,
J.; Glaves, P.; Kirk, I.; Bjorkman, J. Ticagrelor Inhibits Adenosine Uptake In Vitro and Enhances Adenosine-Mediated Hyperemia Responses in a
Canine Model. J. Cardiovasc. Pharmacol. Ther. 2012, 17, 164-172; (e) Ann Wittfeldt, A.; Emanuelsson, H.; Brandrup-Wognsen, G.; van Giezen,
J. J. J.; Jonasson, J.; Nylander, S.; Gan, L. Ticagrelor Enhances Adenosine-Induced Coronary Vasodilatory Responses in Humans. J. Am. Coll.
Cardiol. 2013, 61, 723-727.
16 (a) Bondensgaard, K.; Ankersen, M.; Thøgersen, H.; Hansen, B. S.; Wulff, B. S.; Bywater, R. P. Recognition of Privileged Structures by G-
Protein Coupled Receptors. J. Med. Chem. 2004, 47, 888-899; (b) Paone, D. V.; Shaw, A. W.; Nguyen; D. N.; Burgey, C. S.; Deng, J. Z.; Kane, S.
A.; Koblan, K. S.; Salvatore, C. A.; Mosser, S. D.; Johnston, V. K.; Wong, B. K.; Miller-Stein, C. M.; Hershey, J. C.; Graham, S. L.; Vacca, J. P.;
Williams, T. M. Potent, Orally Bioavailable Calcitonin Gene-Related Peptide Receptor Antagonists for the Treatment of Migraine:ꢀ Discovery of
N-[(3R,6S)-6-(2,3-Difluorophenyl)-2-oxo-1- (2,2,2-trifluoroethyl)azepan-3-yl]-4- (2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin- 1-yl)piperidine-
1-carboxamide (MK-0974). J. Med. Chem. 2007, 50, 5564–5567.
17 (a) Knight, Z. A.; Shokat, K. M. Chemically Targeting the PI3K Family. Biochem. Soc. Trans. 2007, 35, 245-249; (b) Williams, R.; Berndt, A.;
Miller, S.; Hon, W.-C.; Zhang, X. Form and Flexibility in Phosphoinositide 3-Kinases. Biochem. Soc. Trans. 2009, 37, 615–626.
18 Alaimo, P. J.; Knight, Z. A.; Shokata, K. M. Targeting the Gatekeeper Residue in Phosphoinositide 3-Kinases. Bioorg. Med. Chem. 2005, 13,
2825–2836.
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7
8
Ruckle, T.; Schwarz, M. K.; Shokat, K. M.; Shaw, J. P.; Williams, R. L. The p110delta Structure: Mechanisms for Selectivity and Potency of New
PI3K Inhibitors. Nat. Chem. Biol. 2010, 6, 117-124.
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20 (a) Sutherlin, D.P.; Baker, S.; Bisconte, A.; Blaney, P.M.; Brown, A.; Chan, B.K.; Chantry, D.; Castanedo, G.; DePledge, P.; Goldsmith, P.;
Goldstein, D.M.; Hancox, T.; Kaur, J.; Knowles, D.; Kondru, R.; Lesnick, J.; Lucas, M.C.; Lewis, C.; Murray, J.; Nadin, A.J.; Nonomiya, J.; Pang,
J.; Pegg, N.; Price, S.; Reif, K.; Safina, B.S.; Salphati, L.; Staben, S.; Seward, E.M.; Shuttleworth, S.; Sohal, S.; Sweeney, Z.K.; Ultsch, M.;
Waszkowycz, B.; Wei, B. Potent and Selective Inhibitors of PI3Kδ: Obtaining Isoform Selectivity from the Affinity Pocket and Tryptophan Shelf.
Bioorg. Med. Chem. Lett. 2012, 22, 4296-4302; (b) Safina, B. S.; Sweeney, Z. K.; Li, J.; Chan, B. K.; Bisconte, A.; Carrera, D.; Castanedo, G.;
Flagella, M.; Heald, R.; Lewis, C.; Murray, J. M.; Nonomiya, J.; Pang, J.; Price, S.; Reif, K.; Salphati, L.; Seward, E. M.; Wei, B.; Sutherlin, D. P.
Identification of GNE-293, a Potent and Selective PI3Kδ Inhibitor: Navigating in Vitro Genotoxicity While Improving Potency and Selectivity.
Bioorg. Med. Chem. Lett. 2013, 23, 4953-4959.
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21 The S-aminopyrrolidine and S-hydroxypyrrolidine PI3K selectivity motif was independently discovered by a team at Novartis using another
hinge binding core. See (a) 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 Pyrrolidineoxy-Substituted Heteroaromatics as Potent and Selective
PI3Kdelta Inhibitors with Improved Physicochemical Properties. Bioorg. Med. Chem. Lett. 2016, 26, 5657–5662; (b) Hoegenauer, K.;
Soldermann, N.; Zécri, F.; Strang, R. S.; Graveleau, N.; Wolf, R. M.; Cooke, N. G.; Smith, A. B.; Hollingworth, G. J.; Blanz, J.; Gutmann, S.;
Rummel, G.; Littlewood-Evans, A.; Burkhart, C. Discovery of CDZ173 (Leniolisib), Representing a Structurally Novel Class of PI3K Delta-
Selective Inhibitors. ACS Med. Chem. Lett. 2017, 8, 975–980.
22 For the scope of SAR explored with associated PI3K in vitro potency, see a) Achab, A.; Altman, M. D.; Deng, Y.; Guzi, T.; Kattar, S.;
Katz, J. D.; Methot, J. L.; Zhou, H.; McGowan, M.; Christopher, M. P.; Garcia, Y.; Anthony, N. J.; Fradera, X. F.; Mu, C.; Zhang, S.;
Zhang, R.; Fong, K. C.; Leng, X. Preparation of purines as selective inhibitors of human phosphatidylinositol 3-kinase delta. WO
2014/075392 A1, May 22, 2014; b) Achab, A.; Altman, M. D.; Deng, Y.; Kattar, S.; Katz, J. D.; Methot, J. L.; Zhou, H.; McGowan, M.;
Christopher, M. P.; Garcia, Y.; Anthony, N. J.; Fradera, X. F.; Yang, L.; Mu, C.; Wang, X.; Shi, F.; Ye, B.; Zhang, S.; Zhao, X.; Zhang,
R.; Fong, K. C.; Leng, X. Preparation of purines as selective inhibitors of human phosphatidylinositol 3-kinase delta. WO 2014/075393
A1, May 22, 2014.
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