Discovery of Orally Efficacious Phosphoinositide 3-Kinase Î´ Inhibitors

Article pubs.acs.org/jmc

Discovery of Orally Efficacious Phosphoinositide 3‑Kinase δ Inhibitors with Improved Metabolic Stability Leena Patel,* Jayaraman Chandrasekhar, Jerry Evarts,† Kristen Forseth, Aaron C. Haran, Carmen Ip, Adam Kashishian, Musong Kim, David Koditek, Sandy Koppenol, Latesh Lad, Eve-Irene Lepist, Mary E. McGrath, Stephane Perreault, Kamal D. Puri,‡ Armando G. Villaseñor, John R. Somoza, Bart H. Steiner, Joseph Therrien, Jennifer Treiberg, and Gary Phillips Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, California 94404, United States S Supporting Information *

ABSTRACT: Aberrant signaling of phosphoinositide 3-kinase δ (PI3Kδ) has been implicated in numerous pathologies including hematological malignancies and rheumatoid arthritis. Described in this manuscript are the discovery, optimization, and in vivo evaluation of a novel series of pyridine-containing PI3Kδ inhibitors. This work led to the discovery of 35, a highly selective inhibitor of PI3Kδ which displays an excellent pharmacokinetic profile and is efficacious in a rodent model of rheumatoid arthritis.

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INTRODUCTION Phosphoinositide 3-kinases (PI3Ks) are involved in the signal transduction of numerous cellular functions via the formation of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3).1 Dysfunctional regulation of the PI3K pathway results in a variety of disease states including several cancers, autoimmune disorders, and inflammation.2 Class 1 PI3Ks comprise four isoforms: PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ. PI3Kα and -β are ubiquitously expressed in all tissues, while PI3Kδ and -γ are confined to leukocytes.3 Pan PI3K inhibitors have been shown to exhibit clinical efficacy in oncology.1a,4 However, the utility of the pan inhibitors has been limited by side effects such as hyperglycemia, maculopapular rash, and gastrointestinal intolerance.4c,5 In an attempt to mitigate dose-limiting toxicity, recent efforts have focused upon isoform-selective PI3K inhibitors.1a,6 Idelalisib, a selective inhibitor of the δ isoform, has provided clinical benefits to patients with relapsed chronic lymphocytic lymphoma, follicular lymphoma, and small lymphocytic lymphoma.7 Furthermore, PI3Kδ inhibitors have been shown to demonstrate promising efficacy in inflammatory diseases.8 We recently disclosed a series of propeller-shaped PI3Kδ inhibitors anchored by a novel 2,4,6-triaminopyrimidine hinge binder (Figure 1).9 Discovery of the 2,4,6-triaminopyrimidine5-carbonitrile hinge binding motif led to the elimination of aldehyde oxidase mediated metabolism which had afflicted many of our previous compounds. Efforts to improve the isoform selectivity profile of 1 resulted in the discovery of compound 2, a highly selective PI3Kδ inhibitor. Additionally, 2 © 2016 American Chemical Society

demonstrated significant and dose-dependent efficacy in a rat established collagen-induced arthritis (CIA) model. We did not develop this compound further due to its twice-daily human dose projection. Instead we focused our efforts on discovering novel molecules that (a) showed potent inhibition of PI3Kδ (HWB PI3Kδ EC50 < 10 nM), (b) displayed isoform selectivity of greater than 100-fold over PI3Kα, PI3Kβ, and PI3Kγ, to enable high target coverage at trough concentrations, and (c) exhibited a low predicted clearance of 0.2 L/h/kg or less in human hepatocytes to allow for a once-daily dosing regimen. Herein, we describe the discovery of compound 35, a selective and orally efficacious inhibitor of PI3Kδ suitable for q.d. dosing in humans.

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CHEMISTRY The quinazolinone derivatives 11−19 and 23−35 were prepared in a manner analogous to that described in Scheme 1. The quinazolinone intermediates 7 were synthesized in one pot by treatment of 2-aminobenzoic acid 3 and the BOCprotected amino acid 4 with diphenyl phosphite and pyridine, followed by addition of the appropriate aniline 6. 10 Deprotection of 7 with trifluoroacetic acid furnished the primary amines 8. Final compounds of the general structure 10 were prepared by reacting 2,4-diamino-6-chloropyrimidine-5carbonitrile 9 with intermediate 8 in the presence of potassium fluoride and N,N-diisopropylethylamine. Received: August 4, 2016 Published: September 23, 2016 9228

DOI: 10.1021/acs.jmedchem.6b01169 J. Med. Chem. 2016, 59, 9228−9242

Journal of Medicinal Chemistry

Article

Figure 1. Structure and properties of lead PI3Kδ molecules.

Scheme 1a

Reagents and conditions: (i) pyridine, HOP(OPh)2, 50 °C, 2 h; (ii) 50 °C, 3−12 h, 50−100% yield; (iii) CF3COOH, CH2Cl2, rt, 0.5−4 h, 80− 100% yield; (iv) KF, i-Pr2NEt, DMSO, 90 °C, 8−14 h, 30−95% yield.

a

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RESULTS AND DISCUSSION To accomplish our goal of discovering a potent and selective PI3Kδ inhibitor with a low predicted clearance in human hepatocytes (hHep CLpr), we began by further modifying 1. We had previously observed a correlation between a reduction in calculated log P and lower predicted clearance determined in human hepatocytes.9 Attenuation of lipophilicity by replacing the phenyl group present in 1 with a pyridine ring gave 11, which exhibited excellent PI3Kδ potency, better isoform selectivity, and a lower predicted clearance in comparison to 1 (Table 1). Previous SAR data revealed that modifications at the 3- and 5-positions of the phenyl ring of 1 led to an improved isoform selectivity profile. 5-Fluoropyridine 12 was more selective over the β isoform (β/δ = 430 (12) vs 160 (11)). Additionally, the substituted pyridines 13−15 demonstrated superior β/δ and γ/δ ratios, compared to 11. Unfortunately, the subtle increase in lipophilicity resulted in higher predicted clearances for 12−15. The addition of groups such as the trifluoromethyl (16) and the cyano (17)

compromised PI3Kδ potency and selectivity against PI3Kγ. We speculated the loss in potency could be due to electrostatic repulsion in the hydrophobic II (H2) binding region of PI3Kδ. Since 11 met the requirements for our target product profile, we evaluated this compound in PK studies across a number of preclinical species. When dosed in rats, 11 demonstrated intermediate total clearance in comparison to hepatic blood flow, a high volume of distribution, and good oral bioavailability (Table 2). Low clearance was observed in dog and cynomolgus monkey; however, both species presented low volumes of distribution. Allometric scaling predicted a human volume of 0.7 L/kg. This low projected volume, combined with a predicted clearance of 0.08 L/h/kg, afforded an approximate half-life of 6 h, inadequate for q.d. dosing in humans. The chemical stability of 11 was measured in aqueous solutions as a function of pH and temperature. The solution stability of 11 was determined to be pH-dependent. At pH 2.0 and 40 °C, 11 displayed hydrolytic degradation, with a half-life 9229



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Table 1. Pyridine Optimization and SAR

a Average of ≥2 determinations. Run in quadruplicate. bAverage of ≥2 determinations. Run in triplicate. cDetermined in human hepatocytes. ND = value not determined.

of 2.7 h (Table 3). The pyridine-containing compounds 12, 18, and 19 also degraded at low pH at rates comparable to 11. In contrast, the half-lives of compounds 1 and 2 were 358 and 34 h, respectively, indicating that the phenyl-containing compounds were chemically stable under aqueous acidic conditions. We postulate that the mechanism of acid-mediated hydrolytic degradation of 11 commences with protonation of the pyridine ring (Figure 2). The resulting electron-deficient quinazolinone (20) is susceptible to ring-opening to form the nitrillium ion (21). Subsequent hydrolysis followed by

tautomerization furnishes the bis-amide (22), the structure of which was confirmed by independent synthesis. The proposed mechanism of chemical degradation at low pH suggests that formation of 21 may be favored by charge delocalization into the pyridine ring. Therefore, we hypothesized that disruption of the delocalization between the pyridine ring and the quinazolinone would disfavor formation of ring-opened intermediate 21. To test this hypothesis, we designed compounds in which the pyridine possesses an ortho-methyl substituent and thus 9230



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disrupts charge delocalization into the pyridine ring and decelerates chemical degradation at low pH. To unambiguously assign the absolute stereochemistry of the atropisomers and to understand the variation on PI3Kδ potency, we obtained a crystal structure of 27 bound to PI3Kδ (Figure 3). The X-ray cocrystal structure reveals that 27 binds in a propeller-like fashion to the ATP binding site of the p110δ catalytic subunit of PI3Kδ. Hydrogen bonding interactions are observed from the triaminopyrimidine hinge binder to Val828 and Glu826. The chloroquinazolinone moiety adopts an orthogonal conformation to the hinge binder and projects into a specificity pocket that is not accessible in the apo protein.5c,12 The pyridine ring is situated perpendicular to the quinazolinone and extends beyond the ATP-binding pocket into the H2 region. The structure shows the more active atropisomer has P configuration (see Supporting Information, SI2).11a,13 The biochemical potency difference of the two atropisomers can also be rationalized by the structural characterization. Hydrogen bonding interactions between the 2-amino of the pyrimidine hinge binder, the pyridyl nitrogen of the P atropisomer, and Ser831 are mediated by a key water molecule. This water is likely displaced in the binding of the M atropisomer. Additionally, the orientation of the pyridyl nitrogen of 28 precludes interaction with Ser831. The contribution of these interactions is consistent with the observed potency difference between 27 and 28. To evaluate the thermal stability of interconversion, a solution of 27 in THF was maintained at 40 °C, and epimerization was monitored by HPLC over the course of 72 h. Using the Eyring equation, we estimated the Gibbs energy of activation of 27 to be 28.3 kcal/mol (Figure 4).14 On the basis of the estimated activation energy, the half-life of 27 at 40 °C was calculated to be 72.9 days. According to LaPlante et al.,11c,d these results suggest that 27 would be categorized as a class 2 atropisomer. Development of class 2 atropisomers can be challenging given that the stereochemical integrity of these compounds can be compromised during production, storage, and administration of the drug. For these reasons, 27 was not progressed further. An alternative strategy to mitigate the acid-mediated degradation was to disfavor the formation of the nitrillium ion shown in 21, via attenuation of the basicity of the N1 on the quinazolinone ring. ACD/Labs pKa calculations on the quinazolinone suggested that addition of an EWG on C8 of the

Table 2. DMPK Profile Compound 11 CL (L/h/kg) species

in vitroa

in vivob

Vssb,c (L/kg)

t1/2 (h)d

F (%)e

rat dog cyno human

0.45 0.18 0.18 0.08

1.5 ± 0.13 0.2 ± 0.02 0.1 ± 0.02

1.9 ± 0.23 0.9 ± 0.5 0.5 ± 0.07

2.5 ± 1.4 5.6 ± 3.0 4.9 ± 0.3

42

a Predicted clearance determined by incubating 3H-labeled compound in hepatocytes. bIntravenous dose of 1 mg/kg. cVolume of distribution at steady state. dHalf-life iv. eOral dose of 5 mg/kg. 11 was formulated in 12% w/v Captisol in water for dosing in rats and dog; 10% Solutol HS-15 (v/v), 15% N-methylpyrrolidone (NMP) (v/v), and 75% normal saline at 0.5 mg/mL in cynomolgus monkeys. Mean ± SD, n = 3.

cannot adopt a planar conformation with the quinazolinone. The pyridine-containing compounds shown thus far possess low rotational energy barriers. It is well-known that orthosubstituents on biaryl compounds may impose restricted rotation due to steric repulsion and increased barriers to rotation.11 Furthermore, energy minimizations with the Merck molecular force field (MMFF) suggest a rotational barrier of 17 kcal/mol for the unsubstituted pyridines and >30 kcal/mol for the ortho-methylpyridines. Consistent with the suspected higher rotational energy barriers was our ability to separate the pairs shown in Table 4 by column chromatography on silica gel. One atropisomer was more potent than the other in our PI3K inhibitory assays; the 2-methylpyridyl 23 exhibited potent and selective PI3Kδ inhibitory activity, while its diastereisomer (24) was 10-fold less potent. Relocation of the methyl from the 2-position to the 4-position of the pyridine generated compounds 25 and 26, with the former being significantly more potent. While 25 displayed a low predicted clearance in hHeps, it suffered from inadequate selectivity over PI3Kβ. As anticipated, the addition of a fluorine to the 5-position of the pyridine yielded analogs 27 and 28 which exhibited an improved β-selectivity profile compared to 25. We found 27 to have an optimum balance of PI3Kδ potency, selectivity, and hHep stability. Intrigued by these results, we subjected the single atropisomers 23, 25, and 27 to chemical stability testing in aqueous acidic media. These analogs exhibited minimal hydrolytic degradation at pH 2 (t90 of >3 h for 23, 25, and 27), confirming that introduction of conformational constraint Table 3. Chemical Stability of PI3Kδ Analogs

a

Determined in human hepatocytes. bn = 1. ND = value not determined. 9231



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Figure 2. Postulated mechanism of acid mediated chemical degradation of 11.

Table 4. SAR of Ortho-Methyl Pyridine Atropisomers

a Average of ≥2 determinations. Run in quadruplicate. bDetermined in human hepatocytes. ND = value not determined. cn = 1. ND = value not determined.

strated resilience to acid-mediated hydrolysis, requiring almost 3 h at 40 °C to exhibit 10% degradation. This was consistent with our hypothesis that attenuation of the pKa of N1 dramatically reduces hydrolytic degradation rates at low pH. Additionally, 32 attracted our attention for its striking combination of potent PI3Kδ binding and low predicted clearance in hHeps. For these reasons 32 was progressed to in vivo pharmacokinetic assessment. The in vivo rat PK of 32 was promising (Table 6); there was good correlation between the in vitro and in vivo clearance and a high volume of distribution accompanied by moderate oral bioavailability. Unfortunately, like 11, compound 32 presented

quinazolinone would decrease the pKa of N1 (Table 5). To test this theory, a small series of compounds 29−32 was investigated. Interestingly, the calculated N1 pKa of the 8chloro analog 29 was similar to that of 11. Furthermore, 29 displayed reduced PI3Kδ inhibitory activity and selectivity over PI3Kβ in comparison to 11. Reduction in pKa was achieved with the dichloro analog 30, which exhibited equipotent activity to 11. Gratifyingly, 30 demonstrated superior chemical stability in aqueous acidic media in comparison to 11. The fluorine atoms on quinazolinone 31 significantly reduced the pKa of N1. Unfortunately, this compound was 10-fold less potent relative to 30. Finally, the 8-chloro-5-fluoroquinazolinone 32 demon9232



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isoform-specific in vitro cell-based assays utilizing primary human B-cells in whole blood, primary human basophils in 25% serum, and isolated leukocytes (WBCs) in low serum, cultured murine embryonic fibroblasts (MEFs) in low serum, and a cultured human cancer prostate cell line (PC3) in low serum (Table 7). In these cell-based assays, 35 demonstrated high selectivity for p110δ over the other class I PI3K isoforms. Furthermore, 35 was evaluated for its potential to interact with 456 kinases, including 11 lipid and 56 mutant kinases, at a compound concentration of 10 μM. 35 did not significantly interact with any of the kinases other than the PI3K isoforms (see Supporting Information, SI2 and SI4). The excellent kinome selectivity may be explained by the cocrystal structure of 35 bound to the ATP binding site of the kinase domain of PI3Kδ (Figure 5). The substituted pyrimidine of 35 serves as the hinge binder and forms a three-point interaction to Val828 and Glu826. Water mediated hydrogen bonds are formed from the pyridine nitrogen atom to Ser831 and the pyrimidine hinge binder. Similar to other propellershaped inhibitors, the quinazolinone of 35 induces a conformational change in the P-loop at the top of the ATP binding site to form a lipophilic pocket enclosed by Trp760 and Met752.5c,12a,15 This binding pose may imbue propeller-shaped inhibitors with improved selectivity over related lipid kinases that show reduced conformational flexibility in this region.12a Advanced characterization of 35 revealed that it did not inhibit CYPs or hERG at a concentration of 25 μM. Moreover, 35 was nonmutagenic in an Ames micronucleus test and no substantial activity was noted when tested for off-target activity in a CEREP panel screen at 10 μM. The summary of pharmacokinetic parameters in all species after intravenous and oral administration of 35 is presented in Table 8. The in vivo data suggest that 35 has low to intermediate total clearance (CL) in comparison to hepatic blood flow and a volume of distribution (Vss) approximately equal to (rat) or higher than (all other species) total body water (0.7 L/kg). The apparent elimination half-life and mean residence time (MRT) were 2−3 h across species. The high oral bioavailability in rat and dog is consistent with low observed clearance and predicted hepatic extraction. The human dose estimation assumes an oral bioavailability of 25%, a predicted volume of distribution of 1.2 L/kg, and a clearance rate of 0.05 L/h/kg to predict an approximate half-life of 16 h in humans. The target trough concentration at steady state is 55 nM, which allows target coverage of at least 2-fold EC90 (21 nM) as measured in 96% serum in the B-cell assay. The projected human dose of 35 is 15 mg, dosed once-daily.

Figure 3. 2.6 Å X-ray crystal structure of 27 bound in the PI3Kδ ATPbinding site (PDB code 5T7F). 27 adopts a propeller shape with the quinazolinone ring occupying a specificity pocket formed by Trp760 and Met752. The triaminopyrimidine establishes hydrogen bonding interactions to the hinge residues, Val828 and Glu826. An optimally placed water molecule forms multiple interactions to the ligand and the protein. Blue dashed lines represent H-bond contacts between the inhibitor and protein. For clarity, all other residues have been removed.

a low Vss in dogs which would compromise its suitability as a drug with a once-daily dosing regimen. In an effort to try and improve the volume of distribution across species, a small subset of compounds was synthesized while keeping the 8chloro-5-fluoroquinazolinone constant (Table 6). The 5fluoropyridine compound 33 displayed a high affinity for PI3Kδ and, as expected, was selective across the other PI3K isoforms. Although 33 exhibited an improved Vss in the dog, the predicted clearance based on incubation of tritiated compound in hHeps was 2-fold higher than 32. Despite having a remarkable balance of PI3Kδ potency and selectivity, ethyl derivative 34 exhibited lower volumes in both rat and dog than methyl analog 32. Finally, we found the cyclopropyl compound 35 to display high PI3Kδ potency, isoform selectivity, and chemical stability at low pH. Furthermore, when dosed in vivo, 35 displayed excellent in vitro/in vivo correlation, good oral availability in rat, and a higher volume of distribution in the dog relative to the other molecules in the series. With these promising data in hand we sought to better understand the isoform selectivity of 35. The cellular potency of 35 against all four class I PI3K isoforms, PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ, was determined using a series of PI3K

Figure 4. Model showing the minima of 27 and 28 and the transition state for the interconversion of the atropisomers. Experimentally, the free energy of activation was estimated to be 28.3 kcal/mol. 9233



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Table 5. PI3Kδ inhibition and Modulation of N1 Basicity

IC50 (nM)b compd

R

R

ACD/pK, N1

PI3Kδ

PI3Kα

PI3Kβ

PI3Kγ

EC50 (nM),c PI3Kδ HWB

CLpr d (L/h/kg)

stability, 40 °C, pH 2, t90/t50 e

11 29 30 31 32

Cl H Cl F Cl

H Cl Cl F F

1.97 1.72 0.48 0.12 0.28

0.6 6 0.7 8 0.4

1030 3100 1300 4020 770

100 390 76 770 46

72 1100 330 940 51

0.9 5 0.9 11 0.5

0.12 0.25 0.16 ND 0.09

0.2/2.7 ND 2.3/15 ND 2.8/19

1

2

a

ACD/ Labs pKapredictor. bAverage of ≥2 determination. Run in quadruplicate. cHuman whole blood basophil assay of ≥2 determination. Run in triplicate. dDetermined in human hepatocytes. ND = value not determined. en = 1. ND = value not determined.

a

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CONCLUSION To summarize, we have identified a novel series of potent and highly selective pyridine-containing PI3Kδ inhibitors. We successfully improved the metabolic stability of compound 1 by replacing the phenyl ring with a pyridine ring (11) (Figure 10). Although 11 exhibited low predicted clearance in human hepatocytes, it was projected to be suitable for a b.i.d. dosing regimen in humans based on the low volume of distribution observed in preclinical species. Furthermore, 11 was susceptible to hydrolytic degradation at low pH. The addition of an EWG to C8 of 35 attenuated the basicity of N1 thereby mitigating the acid-mediated hydrolytic ring opening. Compound 35 demonstrates efficacy in a rat model of arthritis and is predicted to be suitable for q.d. dosing in humans. The favorable in vitro and in vivo attributes of 35 resulted in its selection as a development candidate, GS-9901.17

Having fulfilled all of the criteria we initially set, we investigated the therapeutic potential of 35 for the treatment of rheumatoid arthritis (RA) by evaluating the ability of 35 to inhibit cellular functions that contribute to the pathology of the disease. PI3Kδ is highly expressed in RA synovium. Reduction of B-cell and T-cell production of inflammatory cytokines can be achieved by inhibition of PI3Kδ.2b,d The ability of 35 to inhibit activation of B-cell receptor (BCR) stimulated Blymphocytes in vitro was assessed in human whole blood. BCR signaling is PI3Kδ-dependent and was stimulated in the assay with anti-IgD antibody. Compound 35 inhibited BCR-induced B-cell activation with an EC50 of 2.4 nM and an EC90 of 21 nM. In order to build a correlation between target coverage and efficacy, we tested 35 in a rat whole blood ex vivo anti-IgD stimulated B-cell assay. B-cell activation was monitored by upregulation of surface CD86 expression as measured by flow cytometry. We found 35 to exhibit EC50 and EC90 values of 1.6 nM and 31.5 nM, respectively. Results from a rat PK study at an oral dose of 1 and 5 mg/kg suggest that plasma levels of 35 at 12 h exceed 32 nM (Figure 6). Therefore, 35 was dosed b.i.d. to maintain plasma concentrations greater than EC90 to establish efficacy and proof of concept of PI3Kδ inhibition in a rodent model of inflammation. Female Lewis rats with established collagen-induced arthritis (CIA) were dosed orally with 35 at 0.3 mg/kg, 1 mg/kg, and 3 mg/kg twice-daily for 7 days starting on the 10th day after the second collagen immunization.16 Compound 35 demonstrated significant reduction in ankle swelling in rats treated with 0.3 mg/kg 35 (31% reduction), 1 mg/kg 35 (67%), and 3 mg/kg 35 (65%) (Figure 7). Plasma concentrations measured on day 7 (12 h after last dose) were consistent with maintaining approximately 60%, 80%, and >90% inhibition of PI3Kδ at 0.3, 1.0, and 3 mg/kg doses, respectively, at trough levels (Figure 8). Evaluation of histopathology data confirmed animals treated with 35 at 1 mg/kg demonstrated reduced disease scores in multiple histological measurements, including inhibition of pannus formation (58%), cartilage damage (50%), bone resorption (50%), and periosteal bone formation (48%) with ED50 values ranging from 0.6 to 2.3 mg/kg (Figure 9). These data suggest that maintaining PI3Kδ coverage between EC50 and EC90 over the dosing interval was disease modifying in this rat established model of RA.

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EXPERIMENTAL SECTION

General Procedures. Starting materials and reagents were used as purchased. All chromatography solvents were HPLC grade and used without further purification. Flash column chromatography was carried out using prepacked silica gel (15−45 μM) cartridges. Microwaveassisted reactions were executed using a CEM microwave system. Atropisomer compounds 23−28 were purified by column chromatography on silica gel eluting with EtOAc in hexanes (0−100%). All other final compounds were purified by preparative HPLC on a Gilson 271 system, as described previously.9 NMR spectra were recorded on a Varian 400-MR 400 MHz spectrometer. NMR data are reported as follows: chemical shifts (δ) in ppm from an internal standard or residual solvent, multiplicity, coupling constants (J) in Hz, and integration. The purity of the tested compounds was assessed by an Agilent LCMS instrument (as previously described).9 The purity of final compounds was determined to be ≥95% by HPLC and 1H NMR. (S)-2,4-Diamino-6-((1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile (11). The title compound was obtained according the general procedure described in Scheme 1 (see Supporting Information SI19 or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 2.4 Hz, 0.5H), 8.58 (dd, J = 4.8, 1.5 Hz, 0.5H), 8.51 (dd, J = 4.9, 1.7 Hz, 1.5H), 8.06 (dm, J = 8.5 Hz, 0.5H), 7.85 (dm, J = 8.0 Hz, 0.5H), 7.8 (td, J = 8.1, 1.4 Hz, 1H), 7.68 (ddd, J = 8.2, 3.9, 1.2 Hz, 1H), 7.60 (dt, J = 7.9, 1.3 Hz, 1H), 7.55 (ddd, J = 8.1, 4.8, 0.7 Hz, 0.5H), 7.48 (ddt, J = 8.1, 4.8, 0.7 Hz, 0.5H), 4.85 (m, 1H), 1.35 (d, J = 6.8 Hz, 3H). ES/ MS m/z = 434.1 (M + H+). 9234



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Table 6. SAR of 8-Chloro, 5-Fluoro Derivatives

a Average of ≥2 determinations. Run in quadruplicate. bHuman whole blood basophil assay of ≥2 determinations. Run in triplicate. cDetermined in hepatocytes. dIntravenous dose of 1 mg/kg. eVolume of distribution at steady state. fOral dose of 5 mg/kg. Number of animals per dosing route is 3.

(S)-2,4-Diamino-6-(1-(5-chloro-3-(5-fluoropyridin-3-yl)-4oxo-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (12). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.66 (m, 0.5H), 8.62 (d, J = 2.8 Hz, 0.5H), 8.55 (d, J = 2.6 Hz, 0.5H), 8.40 (m, 0.5H), 8.16 (dd, J = 2.65, 2.0 Hz, 0.5H), 8.13 (dd, J = 2.6, 2.0 Hz, 0.5H), 7.82 (ddd, J = 8.0, 8.0, 2.2 Hz, 1H), 7.78(m, 0.5H), 7.69 (ddd, 8.2, 3.7, 1.2 Hz, 1H), 7.61 (ddd, J = 7.8, 2.15, 1.2 Hz, 1H), 4.86 (m, 1H), 1.37 (d, J

= 6.7 Hz, 1.5H), 1.36 (d, J = 6.5 Hz, 1.5H). ES/MS m/z = 452.8 (M + H+). (S)-2,4-Diamino-6-(1-(5-chloro-3-(5-chloropyridin-3-yl)-4oxo-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (13). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 5.6 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 7.70 (dd, J = 8.2, 1.0 Hz, 1H), 7.62 (dd, J = 8.0, 1.1 Hz, 1H), 7.55 (dm, J = 5.6 Hz, 1H), 5.03 (m, 1H), 1.38 (d, J = 6.6 Hz, 3H). ES/MS m/z = 469.3 (M + H+). 9235



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Table 7. Summary of 35 Potency in Class I Isoform-Specific Cell-Based Assays PI3K isoform PI3Kδ PI3Kα PI3Kβ PI3Kγ PI3Kδ PI3Kγ a

cell-based assay and stimulus

EC50 a (nM)

cell-based δ selectivity (fold)

human basophil and anti0.31 1 FcεRI murine embryonic fibroblast 1169 3653 and PDGF PC3 and constitutive pAkt 42 131 human basophil and fMLP 403.6 1261 Human Whole Blood Cell Assays (25% Serum) basophil and anti-FcεRI 1.3 1 basophil and fMLP 581 447

Figure 6. Concentration−time profiles of 35 in plasma following a single oral dose at 1 and 5 mg/kg in female Lewis rats (mean ± SD, n = 3). The Cmax at 5 mg/kg was 1780 ± 127 nM at 2 h, and AUCinf was 16 000 ± 1790 nM·h.

Values are geometric mean of ≥1 determination. Run in triplicate.

Hz, 0.5H), 8.31 (d, J = 2.8 Hz, 0.5H), 8.24 (d, J = 2.7 Hz, 0.5H), 8.13 (d, J = 1.9 Hz, 0.5H), 7.84 (t, J = 8.3 Hz, 1H), 7.78 (dd, J = 2.7, 1.9 Hz, 0.5H), 7.72 (ddd, J = 8.2, 1.8, 1.2 Hz, 1H), 7.64 (ddd, J = 7.8, 1.3, 0.6 Hz, 1H), 7.41 (dd, J = 2.8, 2.0 Hz, 1H), 4.98 (m, 1H), 3.85 (s, 1.5H), 3.79 (s, 1.5H), 1.40 (d, 3H). ES/MS m/z = 464.8 (M + H+). (S)-2,4-Diamino-6-(1-(5-chloro-3-(5-cyclopropylpyridin-3yl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine5-carbonitrile (15). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.54− 8.15 (m, 2H), 7.84−7.70 (m, 1H), 7.70−7.44 (m, 3H), 6.88 (dd, J = 6.9, 4.8 Hz, 1H), 6.51 (s, 2H), 6.19 (s, 2H), 4.64 (dt, J = 12.6, 6.7 Hz, 1H), 2.05−1.77 (m, 1H), 1.28 (dd, J = 9.0, 6.7 Hz, 3H), 1.05−0.88 (m, 2H), 0.80−0.56 (m, 2H). ES/MS m/z = 474.1 (M + H+). (S)-2,4-Diamino-6-(1-(5-chloro-4-oxo-3-(5-(trifluoromethyl)pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (16). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 9.24−8.72 (m, 2H), 8.58 (td, J = 2.2, 0.7 Hz, 1H), 7.90− 7.47 (m, 3H), 6.92 (dd, J = 40.0, 7.3 Hz, 1H), 6.47 (d, J = 21.5 Hz, 2H), 6.14 (s, 2H), 4.76 (dp, J = 26.9, 6.6 Hz, 1H), 1.32 (dd, J = 12.3, 6.5 Hz, 3H). ES/MS m/z = 502.1 (M + H+). (S)-2,4-Diamino-6-((1-(5-chloro-3-(5-cyanopyridin-3-yl)-4oxo-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile (17). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 9.05 (dd, J = 4.8, 2.5 Hz, 1H), 9.02−8.92 (m, 1H), 8.62 (dt, J = 4.3, 2.1 Hz, 1H), 8.27 (t, J = 2.1 Hz, 1H), 7.87−7.74 (m, 2H), 7.74−7.55 (m, 4H), 4.83 (dt, J = 23.1, 6.9 Hz, 1H), 1.41−1.31 (m, 3H), ES/MS m/z = 459.2 (M + H+).

Figure 5. 2.6 Å structure of 35 bound to the ATP-binding site of human PI3Kδ:p110δ (PDB code 5T8I). H-bonds between 35 and the hinge residues of PI3Kδ (top to bottom: Glu826:O, Val828:NH, Val828:O) are shown in blue. The quinazolinone adopts a perpendicular conformation to the hinge binder, and the pyridine lies perpendicular to the quinazolinone. Water mediated hydrogen bonds between the pyridine nitrogen to a Ser831 and the pyrimidine NH are shown in blue. (S)-2,4-Diamino-6-(1-(5-chloro-3-(5-methoxypyridin-3-yl)-4oxo-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (14). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.35 (d, J = 1.9

Table 8. Summary Pharmacokinetic Parameters of 35 in Preclinical Species parameter H CLpr (L/h/kg)a CL (L/h/kg)b Vss (L/kg)c terminal t1/2 (h)d MRT (h)e F (%)f

3

Sprague−Dawley rat 0.34 0.43 0.83 2.04 1.95 57

± ± ± ±

0.08 0.10 0.52 0.25

beagle dog 0.42 0.54 1.03 2.01 2.01 49

± ± ± ±

0.17 0.06 0.67 0.45

cynomolgus monkey 0.29 0.41 1.21 2.89 3.05

± ± ± ±

0.15 0.32 0.29 0.46

rhesus monkey 0.35 0.70 1.34 2.45 2.00

± ± ± ±

0.42 0.64 0.28 0.37

a

Predicted clearance determined by incubating 3H-compound in hepatocytes. bIntravenous dose of 1 mg/kg. cVolume of distribution at steady state. Half-life iv. eMean residence time iv. fOral dose of 5 mg/kg. 35 was formulated in 5% ethanol, 55% PEG 400, and 40% water for dosing in rats and dogs; 5% ethanol, 22% PEG 400, and 73% water for dosing in cynomolgus monkeys; and 5% ethanol, 25% PEG 400, and 70% water for dosing in rhesus monkeys. Mean ± SD, n = 3. d

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Figure 7. Compound 35 significantly reduces ankle swelling in rat established collagen-induced arthritis (see Supporting Information for experimental details). Oral dosing was commenced on day 10 or 11 after onset of collagen-induced arthritis and continued for 7 days. Calipers were used to measure right and left ankle diameters daily from day 9 through day18: (∗) p < 0.05 versus arthritis + vehicle. Ankle diameter AUC (calculated from study days 11−18) was considerably reduced toward normal in rats treated with 0.3 mg/kg 35 (31% reduction), 1 mg/kg 35 (67%), and 3 mg/kg 35 (65%). n = 4 normal controls group, n = 8/treatment group, and vehicle (∗) p < 0.05 ANOVA to vehicle (disease), (†) p < 0.05 Student’s t-test to vehicle (disease) (comparison to normal).

Figure 8. Blood/plasma PK of compound 35 from each dosing group was assessed 12 h after the final dose. (S)-2,4-Diamino-6-(1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)propylamino)pyrimidine-5-carbonitrile (18). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.83−8.35 (m, 2H), 8.00 (t, J = 9.9 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.70−7.38 (m, 3H), 6.75 (dd, J = 19.0, 7.5 Hz, 1H), 6.57 (s, 2H), 6.39−5.86 (m, 2H), 4.49 (td, J = 7.5, 3.9 Hz, 1H), 1.98−1.43 (m, 2H), 0.66 (q, J = 6.9 Hz, 3H). ES/MS m/z = 448.1 (M + H+). (S)-2,4-Diamino-6-((5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)(cyclopropyl)methylamino)pyrimidine-5carbonitrile (19). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.65 (d, J = 2.5 Hz, 1H), 8.58−8.42 (m, 1H), 7.95 (ddt, J = 8.2, 2.7, 1.3 Hz, 1H), 7.87−7.72 (m, 1H), 7.66 (ddt, J = 8.2, 4.2, 1.1 Hz, 1H), 7.63−7.44 (m, 1H), 7.38 (dd, J = 8.1, 4.8 Hz, 1H), 6.72−6.43 (m, 3H), 6.18 (s, 2H),

5.72 (d, J = 1.0 Hz, 1H), 4.53−4.27 (m, 1H), 1.46−0.95 (m, 2H), 0.07 (dd, J = 5.7, 2.8 Hz, 2H). ES/MS m/z = 460.5 (M + H+). (S)-2-Chloro-6-(2-((2,6-diamino-5-cyanopyrimidin-4-yl)amino)propanamido)-N-(pyridin-3-yl)benzamide (22). (S)-2,4Diamino-6-((1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile (11) (75 mg) was dissolved in acetonitrile (2 mL) and 1 N hydrochloric acid (2 mL) and heated at 40 °C for 12 h. LCMS indicated completion. Solvents were removed in vacuo and crude was dry loaded onto SiO2 and purified via column chromatography, eluting with 0−100% ethyl acetate in hexanes and then 0−20% methanol in ethyl acetate. Appropriate fractions were pooled and concentrated to afford the title compound as a white amorphous solid (35 mg). 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 9.57 (s, 1H), 8.87−8.82 (m, 1H), 8.36 (dd, J = 4.9, 1.4 Hz, 1H), 8.16 (ddd, J = 8.4, 2.5, 1.4 Hz, 1H), 7.78 (s, 2H), 7.72 (dd, J = 8.2, 1.1 Hz, 1H), 7.69 (s, 1H), 7.53−7.43 (m, 2H), 7.39 (dd, J = 8.1, 1.1 Hz, 1H), 4.66 (q, J = 7.0 Hz, 1H), 1.31 (d, J = 7.1 Hz, 3H). ES/MS m/z = 452.1 (M + H+). 9237



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Figure 9. Histological evaluation revealed that 35 exhibited robust disease modifying activity in rats with established CIA. Ankle tissue was microscopically examined and graded on a five-point scale for each disease parameter (see Supporting Information SI16 for scoring guidelines).

Figure 10. Progression path to the discovery of 35. (S)-2,4-Diamino-6-(1-(5-chloro-3-(2-methylpyridin-3-yl)-4oxo-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (23). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.31 (dd, J = 5.2, 1.5 Hz, 1H), 7.94(dd, J = 7.8, 1.3 Hz, 1H), 7.82 (t, 8.11 Hz, 1H), 7.72 (dd, J = 8.11, 1.16 Hz, 1H), 7.61 (dd, J = 7.94, 1.04 Hz, 1H), 7.34 (dd, J = 7.85, 4.85 Hz, 1H), 5.18 (m, 1H), 2.23 (s, 3H), 1.38 (d, J = 6.7 Hz, 3H). ES/MS 448.8 m/z = (M + H+). (S)-2,4-Diamino-6-(1-(5-chloro-3-(2-methylpyridin-3-yl)-4oxo-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (24). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.4 (dd, J = 4.7, 1.6 Hz, 1H), 7.81 (dd, J = 8.0, 1.6 Hz, 1H), 7.80 (t, 8.03 Hz, 1H), 7.66 (dd, J = 8.21, 1.17 Hz, 1H), 7.59 (dd, J = 7.8, 1.17 Hz, 1H), 7.27 (dd, J = 7.9, 4.8 Hz, 1H), 4.68 (m, 1H), 2.25 (s, 3H), 1.26 (d, J = 6.67 Hz, 3H). ES/MS 448.8 m/z = (M + H+). (S)-2,4-Diamino-6-(1-(5-chloro-3-(4-methylpyridin-3-yl)-4oxo-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-car-

bonitrile (25). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H), 8.37 (d, J = 4.9 Hz, 1H), 8.31 (bs, 1H), 7.90 (bs, 1H), 7.83 (t, 8.09 Hz, 1H), 7.74 (dd, J = 8.09, 1.22 Hz, 1H), 7.62 (dd, J = 7.75, 1.22 Hz, 1H), 7.30 (d, J = 5.25 Hz, 1H), 5.25 (m, 1H), 2.13 (s, 3H), 1.40 (d, J = 6.4 Hz, 3H). ES/MS m/z = 448.8 (M + H+). (S)-2,4-Diamino-6-(1-(5-chloro-3-(4-methylpyridin-3-yl)-4oxo-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (26). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.4 (s, 1H), 8.38 (d, J = 5.0 Hz, 1H), 8.11 (bs, 1H), 7.90 (bs, 1H), 7.83 (t, 7.86 Hz, 1H), 7.71 (dd, J = 7.9, 1.25 Hz, 1H), 7.63 (dd, J = 7.9, 1.25 Hz, 1H), 7.44 (d, J = 5.0 Hz, 1H), 4.75 (m, 1H), 2.12 (s, 3H), 1.34 (d, J = 6.6 Hz, 3H). ES/MS m/z = 448.8 (M + H+). (S)-2,4-Diamino-6-(1-(5-chloro-3-(5-fluoro-4-methylpyridin3-yl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (27). The title compound was synthesized according the general procedure described in Scheme 1 (see 9238



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2H), 8.10−7.80 (m, 1H), 7.71 (dd, J = 9.7, 8.7 Hz, 1H), 7.64−7.38 (m, 2H), 6.76 (dd, J = 20.9, 7.5 Hz, 1H), 6.58 (s, 2H), 6.19 (d, J = 38.7 Hz, 2H), 4.54 (tt, J = 7.8, 5.3 Hz, 1H), 1.91−1.59 (m, 2H), 0.69 (td, J = 7.2, 4.4 Hz, 3H). ES/MS m/z = 466.1 (M + H+). (S)-2,4-Diamino-6-((5-chloro-8-fluoro-4-oxo-3-(pyridin-3-yl)3,4-dihydroquinazolin-2-yl)(cyclopropyl)methylamino)pyrimidine-5-carbonitrile (35). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid in 100% ee. 1H NMR (400 MHz, DMSO-d6) δ 8.76−8.39 (m, 2H), 8.00−7.64 (m, 2H), 7.68−7.22 (m, 2H), 6.74−6.41 (m, 3H), 6.15 (d, J = 24.2 Hz, 2H), 4.48 (td, J = 7.8, 1.5 Hz, 1H), 1.54−1.24 (m, 1H), 0.53−0.21 (m, 3H), 0.07 (m, 1H). ES/MS m/z = 478.1 (M + H+). PI3K Biochemical Assay. PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ biochemical assays were performed as previously reported.9 PI3Kδ EC50, Human Whole Blood Basophil. HWB basophil EC50 assay was performed as described previously.9 EC50, Whole Blood Basophil-PI3Kγ. For the analysis of p110γ signaling, basophil activation was measured in 25% serum, and using isolated WBCs in low serum using the Flow2 CAST kit (Buhlmann Laboratories AG, Baselstrasse, Switzerland, distributed by ALPCO Diagnostics), p110γ was activated with fMLP in the absence or presence of increasing concentrations of compound. To monitor the basophil cell population and cellular activation, anti-CD63-FITC and anti-CCR3-PE antibodies were added to each sample. Flow cytometric analysis of the basophil activation was performed on a FC500MPL flow cytometer (Beckman Coulter Inc., Fullerton, CA). CCR3-staining and side scatter were applied to gate at least 150 basophils for 25% serum and at least 75 basophils for low-serum that expressed a high density of CCR3. The percentage of CD63 positive cells within the gated basophil population were determined in treatment groups and normalized to the vehicle control (0.3% DMSO). The final compound concentration was adjusted to correct for the dilution effect of added reagents. EC50, MEF-PI3Kα. For the analysis of p110α signaling, MEFs were seeded in a 12-well plate at 2 × 105 in DMEM containing 15% FBS and incubated overnight at 37 °C/5% CO2. The cells were removed from FBS and starved for 2 h in DMEM containing 0.1% FBS, then treated for 2 h with 35 at various concentrations followed by stimulation with PDGF (10 ng/mL; Cell Signaling, Danvers, MA) for 10 min at 37 °C. After washing once in cold PBS, the cell pellet was resuspended in 1× lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM Na pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, supplemented with 1× cOmplete Mini protease inhibitor (Roche Diagnostics Corp, Indianapolis, IN), and 1× phosphatase inhibitor cocktail set I, II (EMD Millipore, Billerica, MA) for 10 min on ice. Whole-cell lysates were obtained by centrifugation at 14 000g for 10 min at 4 °C, and the soluble protein was denatured by boiling for 2 min in 1× NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA), and run on a Peggy instrument as directed by the manufacturer (Proteinsimple, Santa Clara, CA) and probed with the antibodies for total Akt and phosphor-Akt S473 (Cell Signaling, Danvers, MA), at a 1:50 dilution. EC50, PC3-PI3Kβ. For the analysis of p110β signaling, the PC3 cells culture medium was replaced with low serum DMEM (0.1% FBS/1% PSG) and incubated for 2 h at 37 °C/5% CO2 to quiesce the cells. Cells were harvested and plated into 384-well plates at 25 000 cells/ well that have been prespotted with 35 at various concentrations. The assay plate was incubated at 37 °C/5% CO2 for 2 h, and the resulting cells were lysed in order to detect total and Akt and P-Akt S473. P-Akt S473 was determined using the CISBio Phosphor-AKT (Ser473) timeresolved fluorescence resonance energy transfer (TR-FRET) kit (catalog no. 64AKSPEI). This assay detection method utilizes two different specific antibodies: an Akt antibody labeled with Eu-cryptate (donor label) and phosphor-AKT (Ser473) antibody labeled with d2 (acceptor label). Binding of both antibodies to phosphorylated Ser473 Akt in lysed cells triggers TR-FRET signal that is detected on an Envision plate reader (excitation, 320 nm; emission, 615/665 nm; 100

Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 8.45 (s, 1H), 8.33 (bs, 1H), 7.87 (t, J = 8.0 Hz, 1H), 7.78 (dd, J = 8.2, 1.2 Hz, 1H), 7.67 (dd, J = 7.8, 1.2 Hz, 1H), 7.31 (bs, 1H), 5.30 (m, 1H), 2.08 (s, 3H), 1.44 (d, J = 6.5 Hz, 3H). ES/MS m/z = 466.8 (M + H+). (S)-2,4-Diamino-6-(1-(5-chloro-3-(5-fluoro-4-methylpyridin3-yl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (28). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H), 8.32 (s, 1H), 7.87 (t, J = 8.0 Hz, 2H), 7.76 (dd, J = 8.2, 1.2 Hz, 1H), 7.67 (dd, J = 7.9, 1.25 Hz, 1H), 4.86 (m, 1H), 2.14 (s, 3H), 1.39 (d, J = 6.67 Hz, 3H). ES/MS m/z = 466.8 (M + H+). (S)-2,4-Diamino-6-(1-(8-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (29). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.75 (d, J = 2.5 Hz, 1H), 8.65−8.42 (m, 2H), 8.16−7.94 (m, 3H), 7.92−7.58 (m, 1H), 7.65−7.47 (m, 2H), 7.51−7.38 (m, 1H), 7.31 (s, 2H), 4.94 (h, J = 6.7 Hz, 1H), 1.39 (dd, J = 6.6, 2.6 Hz, 3H). ES/MS m/z = 434.9 (M + H+). (S)-2,4-Diamino-6-(1-(5,8-dichloro-4-oxo-3-(pyridin-3-yl)3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (30). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.87−8.38 (m, 4H), 8.02 (tdd, J = 7.4, 6.6, 5.6 Hz, 2H), 7.89−7.65 (m, 2H), 7.65− 7.32 (m, 3H), 5.03−4.68 (m, 1H), 1.57−1.15 (m, 3H). ES/MS m/z = 469.2 (M + H+). (S)-2,4-Diamino-6-(1-(5,8-difluoro-4-oxo-3-(pyridin-3-yl)-3,4dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (31). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.73 (d, J = 2.5 Hz, 1H), 8.65−8.36 (m, 1H), 8.02 (dt, J = 8.2, 2.0 Hz, 1H), 8.02−7.70 (m, 6H), 7.55 (dd, J = 8.1, 4.8 Hz, 1H), 7.47 (dd, J = 8.1, 4.8 Hz, 1H), 7.47−7.25 (m, 1H), 4.83 (q, J = 6.7 Hz, 1H), 1.48−1.26 (m, 3H). ES/ MS m/z = 436.4 (M + H+). (S)-2,4-Diamino-6-(1-(5-chloro-8-fluoro-4-oxo-3-(pyridin-3yl)-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine-5-carbonitrile (32). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.80−8.42 (m, 2H), 8.12−7.81 (m, 1H), 7.80−7.68 (m, 1H), 7.62−7.51 (m, 1H), 7.46 (dt, J = 10.6, 5.3 Hz, 1H), 6.87 (dd, J = 16.8, 7.0 Hz, 1H), 6.51 (d, J = 5.7 Hz, 2H), 6.21 (d, J = 21.8 Hz, 2H), 4.76−4.60 (m, 1H), 1.30 (dd, J = 6.6, 1.5 Hz, 3H). ES/MS m/z = 452.1 (M + H+). (S)-2,4-Diamino-6-(1-(5-chloro-8-fluoro-3-(5-fluoropyridin-3yl)-4-oxo-3,4-dihydroquinazolin-2-yl)ethylamino)pyrimidine5-carbonitrile (33). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.83− 8.25 (m, 2H), 8.23−7.66 (m, 2H), 7.59 (dd, J = 8.8, 4.5 Hz, 1H), 6.90 (dd, J = 23.2, 7.2 Hz, 1H), 6.52 (d, J = 9.2 Hz, 2H), 6.24 (s, 2H), 4.97−4.46 (m, 1H), 1.33 (dd, J = 6.6, 1.6 Hz, 3H). ES/MS m/z = 470.1 (M + H+). (S)-2,4-Diamino-6-(1-(5-chloro-8-fluoro-4-oxo-3-(pyridin-3yl)-3,4-dihydroquinazolin-2-yl)propylamino)pyrimidine-5-carbonitrile (34). The title compound was synthesized according the general procedure described in Scheme 1 (see Supporting Information or WO2014201409A1 for experimental details). Obtained as a white amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ 8.81−8.46 (m, 9239



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μs delay; and 200 μs read window). Data were normalized based on a positive (1 μM wortmannin) and negative (DMSO) controls, and EC50 values were calculated from the fit of the dose−response curves to a four-parameter equation. EC50, B-Cell Activation Assay (Human and Rat). B-cell activation assays were performed as previously reported.9 Hydrolytic Degradation of Pyridine Compounds in pH 2 and pH 7 Aqueous Solutions. The chemical stability of pyridine compounds was studied in pH 2 and pH 7 solutions at 40 °C using 50 mM phosphate buffer (pH 2 or pH 7) adjusted to a constant ionic strength of 0.15 M using NaCl. Concentrated stocks of pyridine compounds were prepared in acetonitrile or DMSO. Reaction solutions were prepared by diluting concentrated stocks of pyridine compound to a final concentration of 100 μg/mL in phosphate buffer. Reaction solutions were placed in a UPLC autosampler chamber equilibrated to 40 °C. Samples were injected at predetermined time points and analyzed using the UPLC−UV method described herein. The pseudo-first-order kinetic rate constants of the degradation of the pyridine compounds were calculated using a linear least-squares regression analysis of the data according to eq 1: ln ct = − kobst + ln c0

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01169. Absolute configuration, screen data, selectivity scores, scoring criteria, synthesis procedures, and arthritis model (PDF) Molecular formula strings and some data (CSV) Accession Codes

PDB code for compound 27 is 5T7F. PDB code for compound 35 is 5T8I. Authors will release the atomic coordinates and experimental data upon article publication.

■

(2)

t 90 =

− ln(0.9) kobs

(3)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (206) 832-2058. Address: Gilead Sciences, Inc., 199 E Blaine Street, Seattle, Washington 98102, United States.

where kobs is the first-order rate constant, t is time, c0 is the initial concentration of compound in the reaction solution, and ct is the concentration of compound in the reaction solution at time t. The time required for compound concentration in the reaction solution to decrease by 50% (t50) and 10% (t90) was calculated using eq 2 and eq 3, respectively: ln(2) kobs

ASSOCIATED CONTENT

S Supporting Information *

(1)

t50 =

Article

Present Addresses

† J.E.: Acerta Pharma, 15400 SE 30th Place, Suite 206, Bellevue, WA 98007, U.S. ‡ K.D.P.: Celgene Corporation, 10300 Campus Point Drive, Suite 100, San Diego, CA 92121, U.S.

Notes

The authors declare the following competing financial interest(s): The authors are employees of Gilead Sciences except for J.E and K.D.P who were employed at Gilead Sciences during this research. All authors are shareholders of Gilead Sciences.

UPLC−UV Method. Pyridine compound aqueous reaction solutions were analyzed on a Waters Acquity UPLC H-class system consisting of an H-class quaternary pump, an autosampler, a column manager, a photodiode array (PDA) detector, and Empower 2 software. The reverse-phase UPLC method used a Waters HSS T3, 1.8 μm particle size, 2.1 mm × 100 mm colum (Waters Corporation, Milford, MA, part no. 186003539). The method parameters and gradient are summarized below. Detection: UV at 220 nm/PDA collection between 210−400 nm Flow rate: 0.6 mL/min Run time: 10 min Injection volume: 1.0 μL Column temperature: 40 °C Sample temperature: 40 °C Mobile phase A: 0.1% trifluoroacetic acid in water Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile X-ray Crystallography. Methods used to crystallize and determine the structure of PI3Kδ bound with 27 and with 35 have been described previously.7a DiscoveRx Screen. The KINOMEscan (DiscoveRx, San Diego, CA) platform was used to assess the kinase selectivity profile of compound 35 at a concentration of 10 μM. Results for the primary screen binding interactions are reported as “percent control”, where lower numbers indicate stronger hits. See Supporting Information for assay results. Pharmacokinetics. Pharmacokinetic studies for 35 were performed in a similar fashion to that described previously.9 The intravenous (1 mg/kg) and oral (5 mg/kg) doses were formulated in a vehicle of 5% ethanol, 55% PEG 400, and 40% water for rats and dogs; 5% ethanol, 22% PEG 400, and 73% water for dosing in cynomolgus monkeys; and 5% ethanol, 25% PEG 400, and 70% water for dosing in rhesus monkeys. Rat Collagen-Induced Arthritis (CIA) Model. Performed at Bolder BioPATH, Inc.16a,b (see Supporting Information SI21 for experimental details).

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ACKNOWLEDGMENTS The authors thank Julian Codelli and Jennifer Loyer-Drew for helpful discussions during the preparation of this manuscript. The authors also thank Gayatri Balan for help with assigning the absolute axial conformation of 27.

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ABBREVIATIONS USED PI3K, phosphoinositide 3-kinase; Akt, protein kinase B; BCR, B-cell receptor; RA, rheumatoid arthritis; HWB, human whole blood; WBC, white blood cell; CLpr, predicted clearance; hHeps, human hepatocytes; MMFF, Merck molecular force field; EWG, electron withdrawing group; CIA, collagen-induced arthritis; IgD, immunoglobulin D

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