Structure Overhaul Affords a Potent Purine PI3KÎ´ Inhibitor with

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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 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 benzimidazolonepiperidine 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 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 immunemediated 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

BACKGROUND As part of our PI3K inhibitor program, we characterized potent and selective inhibitor 1 (Figure 1)7 which features a 6,8disubstituted 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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lymphocytic leukemia, and is correlated with poor clinical prognosis.11 In human whole blood, 1 inhibited anti-human CD79binduced expression of the B-cell biomarker CD69 with an IC50 value of 810 nM. 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. 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.



N

NH O

N

N N

N

N

N

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N

PI3K IC50 = 6.3 nM PI3K IC50 = 450 nM pAKT IC50 = 63 nM AdU IC50 = 72 nM

N

1

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 Heterocycles, Amides

Piperidine 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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Table 1. Selectivity motif screen with the 9-methyl-8-pyrazolopurine hinge-binding core. N

N N

N

N

N

PI3K IC50 (nM)

PI3K IC50 (nM)

Ramos pAKT IC50 (nM)

HeLa AdU IC50 (nM)

N

N N

N

N

N

2a

HeLa AdU IC50 (nM)

PI3K IC50 (nM)

800

1,190

42

1,310

1,290

2,560

0.9

35

13

1,590

5.3

169

190

7,480

0.5

35

16

9,190

3.9

270

62

>10,000

15

597

572

1,400

294

>10,000

8,330

>10,000

68

2,950

970

4,500

1,350

1,785

8,330

7,150

4.3

121

45

2,250

O

O O

Ramos pAKT IC50 (nM)

PI3K IC50 (nM)

N

24

406

719

6,550

N

2l

>10,000

HN

HN O

2b

N

O

1,085

>10,000

>10,000

N

2m

HN

HN O

O

2c

N

O

5.2

274

106

N

2n

HN

HN

O O

2d

O

N

2.8

262

20

3,130

N

2o O

HN

O

O

N

2e

34

1,070

1,470

>10,000

2p

HN

O

N HN

O

N

O

O

2f

11

90

153

5,520

O

N

2q O

HN

O

O N

2g

57

904

2,210

>10,000

2r

HN

O

N S O

O N

2h

O

HN

16

453

2,770

>10,000

O

N

2s

rac

O

O N

2i

26

540

187

>10,000

2t

N S

O

O

HN O N

2j

O

1.6

94

24

7,250

2u HN

HN

N rac

O N

2k HN

9.2

338

309

3,910

N

2v HN


O


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Table 2. Additional properties of selected early lead compounds.

1 2d 2j 2n 2o 2p 2q

Wistar Han Rat PK Profile

Beagle Dog PK Profile

Clp / Clint Vd / Vdu PPB t1/2 (h) F (%) (mL/min/kg) (L/kg) (%)

Clp / Clint Vd / Vdu PPB t1/2 (h) F (%) (mL/min/kg) (L/kg) (%)

5.8 / 60a 50 / 290c 58 / 370c 49 / 410c 23 / 98a 95 / >Qhepc 41 / 126a

46 / >Qhepb 7.9 / 25c

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

52 55

90 58 51 72 67 48 27

4.1 /

8c

5.8 / 10b

4.6 / 90 0.9 / 2

1.1 1.2

0.9 / 2

2.7

0.9 / 3

1.5

61

95 58

Human CD69 CD63 LogD PPB IC50 IC50 (%) (nM) (nM) 79

810

790 130

55

35

330

17

360

415

41 100

26

1.4 1.7 0.8 1.0 1.2 0.8 1.1

Notes: a0.5 mg/kg iv, 1 mg/kg po in DMSO/PEG400/H2O-20/60/20, n=2; b0.25 mg/kg iv, 0.5 mg/kg po in DMSO/PEG400/H2O20/60/20, 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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Journal of Medicinal Chemistry Given that modifications to the piperidine benzimidazolone, or alternative substitution at the purine C(8)/C(9) positions did not

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). 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. 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-2,6dimethylmorpholine 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. 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). 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, Olinked 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 NHlinker, 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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Journal of Medicinal Chemistry 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. 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

Structure Overhaul Affords a Potent Purine PI3KÎ´ Inhibitor with

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