Identification of a Potent, Selective, and Efficacious

Article pubs.acs.org/jmc

Identification of a Potent, Selective, and Efficacious Phosphatidylinositol 3‑Kinase δ (PI3Kδ) Inhibitor for the Treatment of Immunological Disorders Qingjie Liu,† Qing Shi,† David Marcoux,*,† Douglas G. Batt,† Lyndon Cornelius,† Lan-Ying Qin,† Zheming Ruan,† James Neels,† Myra Beaudoin-Bertrand,† Anurag S. Srivastava,† Ling Li,† Robert J. Cherney,† Hua Gong,† Scott H. Watterson,† Carolyn Weigelt,† Kathleen M. Gillooly,† Kim W. McIntyre,† Jenny H. Xie,† Mary T. Obermeier,† Aberra Fura,† Bogdan Sleczka,† Kevin Stefanski,† R. M. Fancher,† Shweta Padmanabhan,‡ Thatipamula RP,‡ Ipsit Kundu,‡ Kallem Rajareddy,§ Rodney Smith,† James K. Hennan,† Dezhi Xing,† Jingsong Fan,† Paul C. Levesque,† Qian Ruan,† Sidney Pitt,† Rosemary Zhang,† Donna Pedicord,† Jie Pan,† Melissa Yarde,† Hao Lu,† Jonathan Lippy,† Christine Goldstine,† Stacey Skala,† Richard A. Rampulla,† Arvind Mathur,† Anuradha Gupta,‡ Pirama Nayagam Arunachalam,‡ John S. Sack,† Jodi K. Muckelbauer,† Mary Ellen Cvijic,† Luisa M. Salter-Cid,† Rajeev S. Bhide,‡ Michael A. Poss,† John Hynes,† Percy H. Carter,† John E. Macor,∥ Stefan Ruepp,† Gary L. Schieven,† and Joseph A. Tino† †

Research & Development, Bristol-Myers Squibb Company, Route 206 and Province Line Road, Princeton, New Jersey 08543, United States ‡ Department of Discovery Synthesis, Biocon Bristol-Myers Squibb Research Centre, Biocon Park, Bommasandra IV Phase, Jigani Link Road, Bengaluru 560099, India S Supporting Information *

ABSTRACT: PI3Kδ plays an important role controlling immune cell function and has therefore been identified as a potential target for the treatment of immunological disorders. This article highlights our work toward the identification of a potent, selective, and efficacious PI3Kδ inhibitor. Through careful SAR, the successful replacement of a polar pyrazole group by a simple chloro or trifluoromethyl group led to improved Caco-2 permeability, reduced Caco-2 efflux, reduced hERG PC activity, and increased selectivity profile while maintaining potency in the CD69 hWB assay. The optimization of the aryl substitution then identified a 4′-CN group that improved the human/rodent correlation in microsomal metabolic stability. Our lead molecule is very potent in PK/PD assays and highly efficacious in a mouse collagen-induced arthritis model.

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INTRODUCTION Over the last few decades, phosphatidylinositol 3-kinases (PI3Ks) have been identified as important targets for the potential treatment of oncologic and immunologic disorders.1 Class I PI3Ks, comprising PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ, have been targeted the most. The primary cellular function of class I PI3Ks is to phosphorylate phosphatidylinositol bisphosphate (PIP2), converting it to phosphatidylinositol triphosphate (PIP3).2 PIP3 then interacts with a variety of protein kinases that possess a pleckstrin homology (PH) domain, such as AKT, PDK1, and BTK. For example, the activation of AKT contributes to the control of growth, survival, and proliferation of hematopoietic cells through the stimulation of the mTOR pathway.3 PI3Kγ and PI3Kδ have been shown to be more suitable targets for the treatment of immunological disorders because © 2017 American Chemical Society

they are predominantly expressed in hematopoietic cells. PI3Kα and -β, on the other hand, are ubiquitously expressed and are reported to play a role in sugar metabolism.4 PI3Kα is also reported to affect platelet function.5 Selectivity over PI3Kγ may also be desirable considering its expression level in cardiomyocytes6 and the reduced ability of PI3Kγ KO mice to cope with cardiac distress.7 In the past decade, PI3Kδ has been revealed to play an important role controlling immune cell function.8 Guided by these reports and as part of an effort to identify new potential treatments for immunological disorders, this article will discuss work toward the identification of a potent, selective, and efficacious PI3Kδ inhibitor.9,10 Received: April 27, 2017 Published: May 25, 2017 5193

DOI: 10.1021/acs.jmedchem.7b00618 J. Med. Chem. 2017, 60, 5193−5208

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human whole blood (hWB) assay, this molecule lacked selectivity over PI3Kα and PI3Kγ. Furthermore, its high polar surface area (PSA = 124 Å2) resulted in poor in vitro ADME properties. The first area of the molecule that was explored was the heterocyclic C-5 moiety. In order to do so, the synthetic plan described in Schemes 1 and 2 was followed. The piperazinone ring was prepared by reacting 3-bromoaniline (3) with bromoacetyl bromide 4 yielding amide 5 in high yield. Silver mediated displacement of the tertiary bromide 5 with ethanol amine (6) afforded alcohol 7. Mitsunobu reaction on alcohol 7 followed by acetylation afforded piperazinone 9. Pd-catalyzed cross coupling of aryl bromide 9 with bispinacol diboronate gave rise to the desired boronic ester 10. With respect to the synthesis of the core, two procedures were utilized depending on the heterocyclic moiety (Scheme 2). 1,5-Pyrazoles were prepared from the previously disclosed pyrrolotriazine 11.13 Hydrolysis of ester 11 followed by Weinreb’s amide formation afforded amide 12. The addition of an alkynyl lithium reagent to amide 12 followed by the addition of dimethylamine afforded ketone 13. Condensation with various hydrazines 14 gave rise to 1,5-pyrazoles 15 as the major regioisomer. Finally, Suzuki cross-coupling with boronic ester 10 led to the desired analogues. Alternatively, previously disclosed bromo compound 17 was allowed to react with boronate ester 10 under Suzuki cross-coupling reaction condition. The resulting pyrrolotriazine 18 was then iodinated at the C-5 position using NIS which facilitated SAR studies using palladium-catalyzed cross coupling reactions.

Several PI3Kδ inhibitors with varying degrees of selectivity over PI3Kγ have been reported.11 Two of them, highlighted in Figure 1, have been studied in late stage clinical trials for

Figure 1. Clinical PI3Kδ inhibitors.

oncologic disorders. Idelalisib (1a) was approved by the FDA for the treatment of blood cancer while duvelisib (1b) was recently terminated in a phase III clinical trial. Both PI3Kδ inhibitors showed an approximately 20- to 30-fold selectivity over PI3Kγ. In this article, PI3Kδ inhibitors with high selectivity over PI3Kα, -β, and -γ as well as the efficacy of a lead molecule in a mouse preclinical model of arthritis are disclosed.

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CHEMISTRY Pyrrolotriazine 2 was recently reported as a very potent PI3Kδ inhibitor (Figure 2).12 Despite excellent potency in a CD69

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RESULTS AND DISCUSSION Table 1 summarizes different C-5 heterocyclic moieties that were initially studied. Reduction of PSA using hydrophobic side chains (see 21 and 22) provided improved Caco-2 (A−B) permeability. 2,2,2-Trifluoroethyl derivative 22 showed a similar selectivity profile to 2, although it is about 8-fold less potent in the CD69 hWB assay. 1,4-Pyrazole analogues 23 and 24, on the other hand, exhibited improved PI3Kδ selectivity while being about 2-fold more potent in the hWB assay compared to 22. However, their high efflux ratios resulted in low mouse in vivo exposures (exposure for 24 in BALB/c mice at 10 mg/kg po: 470 ± 120 nM Cmax, 1100 ± 320 nM·h AUC24h). A variety of heteroaromatic moieties were then studied (25−30). Although the Caco-2 efflux ratios and

Figure 2. Previously reported PI3Kδ inhibitor 2.

Scheme 1. Synthesis of Piperazinone Intermediate 10a

Reagents and conditions: (a) N-ethyl-N-isopropylpropan-2-amine, DCM, 0 °C, 98%; (b) silver oxide, MeCN, 23 °C, 75%; (c) PPh3, DIAD, THF, 23 °C, 90%; (d) AcCl, Et3N, DCM, 0 °C, 93%; (e) bispinacol diboronate, PdCl2(dppf), KOAc, dioxane, 90 °C, 68%. a

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Scheme 2. Synthetic Schemes for C-5 Heterocyclic SARa

a Reagents and conditions: (a) NaOH, water/MeOH 40 °C; (b) Weinreb’s salt, T3P, N-ethyl-N-isopropylpropan-2-amine, THF, 23 °C, 81% (2 steps); (c) ethynyltrimethylsilane, BuLi, THF, −78 °C; (d) Me2NH, EtOH/water, 90 °C, 89% (2 steps); (e) TEA, EtOH, 80 °C, 85−94%; (f) 10, PdCl2(dppf), K3PO4, dioxane, 100 °C, 55−86%; (g) NIS, THF, 23 °C, 88%.

h AUC24h). The in vitro cardiac channel safety profile (hERG patch clamp (PC) IC50 ≈ 17 μM)15 and high free fraction of 22 suggested a potentially narrow safety window with respect to cardiovascular liabilities. This was confirmed upon evaluation of 22 in a rabbit electrophysiology (EP) study where QTc prolongation exceeded 10 ms at free concentration levels that were deemed unacceptable for progression. Additional undesirable effects included concentration-dependent increases in heart rate and decreases in blood pressure. In order to reduce cardiovascular liabilities, SAR efforts were focused on reducing hERG PC activity (QTc prolongation), increasing PI3Kγ selectivity (potential hemodynamic effect),16 reducing the free Cmax by improving hWB potency, increasing protein binding, and improving ADME properties to achieve a lower peak to trough ratio. In order to accomplish this, replacement of the relatively large and polar C-5 heterocyclic moiety was pursued. Smaller groups at C-5 were investigated, and the results are summarized in Table 3. As seen with analogue 18, which showed low affinity for PI3Kδ, a steep SAR was observed when the C-5 position was unsubstituted. However, simple methyl or nitrile substitution improved hWB potencies comparable to 22 while also improving PI3Kγ selectivity and permeability (see 31 and 32). Unfortunately, the hERG PC activity of nitrile 32 was comparable to that of pyrazole 22.17 The hERG activity could be lowered by introducing a trifluoromethyl group or a halogen at C-5. Indeed, the hERG inhibition at 3 μM was reduced to 12−14% with analogues 33, 35, and 36. More importantly, their improved protein binding profiles (7−12% free in human serum), their increased PI3Kγ selectivities (>100×), and their reduced Caco-2 efflux ratios (1−2) indicated that these molecules were worthy of further investigation. It is believed that the reduction of PSA (97 Å2 for 33 and 34) is responsible for these improvements. Although iodo analogue 19 and bromo analogue 36 are more potent in the hWB assay, these C-5 substituents were deprioritized due to their light sensitivity which was identified in a reactive oxygen generation assay (ROS, data not shown). This effective replacement of the relatively large pyrazole at C-5 in 22 by a simple chloro or trifluoromethyl group was surprising. To understand this further, a computational model of analog 35 based on the X-ray cocrystal structure of pyrazole

Table 1. SAR of the C-5 Heterocyclic Group

PI3Kδ IC50 (nM)a 21 22 23 24 25 26 27 28 29 30 a

2.0 3±1 4.1 1.9 ± 0.5 3.3 4.2 3.3 5.5 1.4 ± 0.6 1.4 ± 0.5

PI3Kγ PI3Kα selectivitya selectivitya 11× 37× 210× 360× 43× 79× 60× 210× 280× 140×

ADP Glo functional assay. parentheses.

25× 110× 110× 170× 33× 110× 100× 96× 160× 260× b

hWB CD69 IC50 (nM)

Caco-2 A−B (nm/s)b

38 58 ± 39 24 ± 7 31 ± 11 100 ± 20 410 160 500 ± 280 140 ± 110 280 ± 130

36 (15) 54 (6) 35 (18) 23 (28) 13) 240 (1) 170 (2) 120 (4) 120 (4) ND

Permeability. Efflux ratio is in

selectivity profiles were much improved in most cases, the low hWB potency precluded further investigation. Considering its combination of good potency and lower Caco-2 efflux, trifluoroethylpyrazole 22 was selected for further profiling. Figure 3 represents an X-ray cocrystal structure of pyrazole 22 in PI3Kδ. Key interactions between 22 and the hinge included V828 and E826 and a close interaction between the carbonyl of the acetamide group and T750. The pyrazole group of 22 filled a hydrophobic pocket formed by I825 and participated in an edge-to-face interaction with Y813. Pyrazole 22 was further profiled, and the results are provided in Table 2. This molecule was potent in an IFNγ hWB assay, was moderately stable in microsomal stability assays, and displayed a 30% free fraction in human serum. Despite an efflux ratio of 6,14 moderate exposures were observed in BALB/c mice at a dose of 10 mg/kg po (1800 ± 300 nM Cmax, 4100 ± 1200 nM· 5195

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Figure 3. X-ray cocrystal structure of pyrazole 22 bound to PI3Kδ (PBD code 5EM).

which from the modeling did not appear to fill the pocket as well (see 34, Table 3). Unfortunately, trifluoromethyl analogue 33 and chloro analogue 35 suffered from a human/mouse disconnect in an in vitro metabolic stability assay (Figure 5). In vitro liver microsome metabolite identification revealed that both 33 and 35 underwent extensive oxidation on the piperazinone ring especially in rodents (Figure 5). This posed a problem since the in vivo models for program progression were in rodents making it difficult to assess proof of concept in an efficacy study. In an effort to saturate clearance mechanisms, high dose mouse PK studies with chloro compound 35 were conducted. Unfortunately, even at doses of 30 mg/kg and 100 mg/kg, the exposures were low with AUC24h of 2.9 ± 0.3 μM·h and 17 ± 6 μM·h, respectively. These exposures were much lower than those observed with pyrazole 22 at a 10 mg/kg dose (vide supra). As part of the effort to improve the microsomal stability of these molecules, new piperazinone motifs and phenyl substitutions were pursued to sterically block cytochrome P450s from accessing the metabolic soft spots. Of the two approaches, modification of the phenyl substitution was found

Table 2. Profiling of pyrazole 22 parameter hWB CD69 IC50 (nM) hWB IFNγ IC50 (nM) CYP inhibition Met Stab (h, r, m; % rem) t1/2 (h, r, m; min) Prot binding (h, r, m, rb; % free) Caco-2 (nm/s) hERG PC (% inh at 3, 10, 30 μM) in vivo PK (BALB/c, 10 mg/kg) Cmax (nM) AUC (nM·h)

result 58 ± 39 71 ± 40 >20 μM (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4) 80, 70, 62 57, 31, 26 30, 31, 32, 26 54 (A−B) (efflux ratio = 6) 23 ± 2, 42 ± 6, 59 ± 3 1800 ± 300 4100 ± 1200

22 in PI3Kδ, showed that the fairly large chloro group filled the hydrophobic pocket nicely as well as interacted with Y813 in a similar fashion to pyrazole 22, making it an adequate replacement (Figure 4). In contrast, reduced affinity was observed when a smaller fluoro group was introduced at C-5 Table 3. Replacement of C-5 Heterocyclic Moiety

compd

R

PI3Kδ IC50 (nM)

PI3Kγ selectivity

18 31 32 33 34 35 36 19

H Me CN CF3 F Cl Br I

>150 9 5±2 2.4 ± 0.8 20 3±1 2.1 ± 0.9 2.0 ± 0.9

ND 240× 110× 270× 120× 180× 75× 97×

a

hWB CD69 IC50 (nM) ND 74 ± 36 ± 72 ± 570 95 ± 39 ± 35

34 17 46 21 14

Caco-2 A−B (nm/s)a

Prot bind (h, % free)

ND 110 (5) 84 (7) 290 (1) 190 (2) 200 (2) 290 (1) 250 (1)

ND 17 30 12 23 9 7 5

hERG PC (% inh at 3 μM) ND ND 28 ± 12 ± ND 12 ± 14 ± ND

4 1 2 2

Permeability. Efflux ratio is found in parentheses. 5196

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Figure 4. Computational model of chloro compound 35, based on the X-ray cocrystal structure of pyrazole 22 bound to the kinase domain of PI3Kδ (PDB code 5EM).18

On the other hand, a much better correlation between human and mouse microsomal stability was observed when a methyl group was introduced at the C-4 position (see 40, Table 4). Importantly, hWB potency was also improved by about 2-fold. The improvement in potency was surprising since this substitution was originally predicted to extend toward a solvent exposed area of the PI3Kδ binding pocket. It was hypothesized that C-4 substitution locks rotation of the piperazine ring in its preferred bioactive conformation. Although substitution at C-5 significantly improved rat microsomal stability, hWB potency was greatly diminished (see 41, Table 4). C-4 substitution, therefore, appeared to be a viable strategy for improving in vitro mouse metabolic stability. Considering poor microsomal stability can result from aromatic methyl substitution, a variety of groups were investigated at the C-4 position (Table 5). Overall, several groups were tolerated. In all cases, metabolic stability was improved compared to 35. However, when the C-5 position of the pyrrolotriazine ring was substituted by a chloro group (see 42−45, Table 5), potencies in both the IFNγ and CD69 hWB assays were reduced. The racemic secondary alcohol 46 showed good potency in the hWB CD69 assay but had low Caco-2 A-B permeability and a high efflux ratio. Nonetheless, this compound provided a potential prodrug handle if needed. Nitrile 47 showed good potency in the hWB IFNγ but lesser potency in the hWB CD69. Interestingly, when the C-5 position of the pyrrolotriazine ring was substituted with a trifluoromethyl group (see 48−52, Table 5), hWB potencies were generally improved. This was especially true for racemic secondary alcohol 51 and nitrile 52 which exhibited hWB potencies of 48× 97× 90×

95 ± 21 ND 38 ± 28 550 (2)

100, 57, 17 84, 19, 1 82, 60, 66 92, 100, 45 5197

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Table 5. Optimization of the C-4 Phenyl Substitution

compd

R1

R2

42 43 44 45 46 47 48 49 50 51 52

Cl Cl Cl Cl Cl Cl CF3 CF3 CF3 CF3 CF3

CF3 OMe Cl SO2Me CHOHME CN CF3 OMe SO2Me CHOHMe CN

a

PI3Kδ IC50 (nM) 1.6 2.2 1.5 2.3 4.8 1.0 1.9 2.7 1.9 3.5 1.7

± 0.8 ± 0.9 ± 0.7 ± 1.2 ± 0.3 ± 1.3 ± 1.1 ± 0.9

PI3Kγ selectivity

PI3Kα selectivity

PI3Kδ INFγ (nM)

PI3Kδ WB CD69 (nM)

Met Stab (h, r, m; % rem)

Caco-2 A−B (nm/s)a

Prot bind (h, % free)

31× 96× 59× 34× 43× 59× 22× 140× 53× 41× l00×

850× 230× 340× 390× 300× 440× 910× 290× 960× 650× 700×

98 ± 67 120 ± 80 300 ± 310 560 120 ± 60 28 ± 16 82 ± 47 56 ± 34 140 ± 110 36 ± 7 35 ± 15

220 ± 120 230 ± 280 370 ± 300 150 31 ± 7 90 ± 61 230 ± 130 92 ± 70 79 ± 34 16 ± 2 35 ± 20

95, 87, 88 94, 91, 94 85, 52, 67 100, 100, 84 100, 100, 100 90, 89, 89 70, 77, 99 95, 100, 100 96, 96, 97 100, 100, 100 94, 91, 81

150 (1) 170 (3) ND ND 31 (18) 200 (2) ND 190 (2) 95 (4) 56 (5) 110 (4)

ND 17 ND ND 34 17 ND 13 ND 34 16

Permeability. Efflux ratio is in parentheses.

Table 6. Study of the Addition of Methyl Groups to the Piperazinone Ring

Table 7. Profiling of Trifluoromethyl 52 parameter ADP-Glo PI3Kδ IC50 (nM) HTRF PI3Kδ IC50 (nM) ADP-Glo PI3Kγ IC50 (nM) ADP-Glo PI3Kα IC50 (nM) ADP-Glo PI3Kβ IC50 (nM) hWB CD69 IC50 (nM) hWB IFNγ IC50 (nM) CYP inhibition

compd

R1

PI3Kδ IC50 (nM)

PI3Kγ selectivity

hWB CD69 IC50 (nM)

53a 53b 54a 54b

Cl Cl CF3 CF3

1.3 2.4 1.7 1.1

± ± ± ±

40× 23× 67× 52×

66 ± 44 42 ± 28 105 ± 25 18 ± 9

0.3 1.1 1.0 0.5

Met Stab (h, r, m; % rem) 82, 99, 90, 89,

59, 94, 43, 91,

Met Stab (h, r, m; % rem) in vitro t1/2 (h, r, m; min) Prot binding (h, r, m, rb; % free) PAMPA, pH 7.4 (nm/s) Caco-2 (nm/s) hERG PC % inh at 3, 10 μM kinase selectivity (HTRF assay) IC50 (nM) PI3Kγ MNK1 PI3Kα MLCK CLK4 others

22 85 23 100

hWB assay. Interestingly, its enantiomer 54a is less potent by about 5-fold in the same assay. Although trifluoromethyl analogue 54b may offer improved potency over trifluoromethyl analogue 52, the latter was selected for additional studies due to its improved PI3Kγ selectivity profile. Trifluoromethyl analogue 52 was profiled further in vitro (Table 7). This molecule showed >100-fold selectivity over the other PI3K isoforms. Kinome selectivity was also excellent with >660-fold selectivity over MNK1 and others in HTRF assays (235 kinases tested including phosphatidylinositol 3-kinase related kinases). Importantly, 52 had an improved human/ rodent in vitro stability correlation and a good permeability profile. This translated to much improved mouse PK (BALB/c, 10 mg/kg, po) compared to pyrazole 22, with a Cmax of 8.2 ± 1.2 μM and an AUC24h of 17 ± 3 μM·h. PK studies in higher species were also performed revealing excellent oral bioavailability across species (Table 8). Clearance was found to be higher in mouse but low in dog resulting in a longer iv t1/2 in dog of 29 ± 8 h. However, the incorporation of the nitrile group reintroduced hERG PC activity. Although this level of activity was similar to pyrazole 22, it was hoped that the improved protein binding profile, PK, potency, and PI3Kγ

result 1.7 ± 0.9 5000 35 ± 20 35 ± 15 >20 μM (1A2, 2B6, 2C8, 2C6, 2C19, 2D6, 3A4) 94, 91, 81 >120, 67, 72 16, 11, 14, 9 590 110 (A−B) (efflux ratio = 4) 25 ± 5, 40 ± 10 41 ± 11 66 ± 24 370 ± 65 510 ± 110 700 ± 500 >2000

selectivity of 52 would help mitigate cardiovascular risk. Compound 52 was therefore evaluated in a rabbit EP study (Table 9). Some degree of QTc prolongation was observed at doses of 10 and 20 mg/kg, while the 3 mg/kg dose did not produce significant changes. Although hemodynamic effects were profound at the 20 mg/kg dose, it is important to note that observed concentrations at 3 mg/kg represent approximately a 10-fold safety margin over the predicted free Cmax (vide infra). Trifluoromethyl analogue 52 was further evaluated in a basophil ex vivo pharmacodynamic study. This assay consisted of stimulating blood taken from mice 1 h after oral administration of 52 and anti-mIgE. The levels of CD63+ 5198

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Table 8. Pharmacokinetic Profile of Trifluoromethyl 52 in Preclinical Species species

dose route

dose (mg/kg)

mouse

iv po iv po iv po iv

3 6 2 5 1 2 1

rat dog monkey

Cmax (μM) 5.9 ± 0.8 14 ± 2

CL (mL min−1 kg−1)

AUC (μM h)

Vss (L/kg)

t1/2 (h)

17 ± 4

2.7 ± 0.3

4.9 ± 1.2

2.3 ± 0.3

0.5 ± 0.1

3.5 ± 0.2

0.24 ± 0.04

0.61 ± 0.06

29 ± 8

4.6 ± 1.3

3.6 ± 0.8

10 ± 2

12 ± 6

94 ± 21

54 ± 25

6.3 ± 3.4

F (%)

71 ± 34

90 ± 37

75 ± 28

Table 9. In Vivo Rabbit EP Study with Trifluoromethyl 52 dose (mg/kg)

compd concn (μM)

free compd concn (μM)

QTc (ms)

heart rate (%)

blood pressure (%)

3 10 20

10 ± 2 27 ± 3 54 ± 9

0.9 ± 0.2 2.4 ± 0.5 4.8 ± 1.0

+2.2 ± 0.5 +13 ± 2 +25 ± 4

+3.3 ± 0.5 +27 ± 3 +100 ± 10

−4.1 ± 1.0 −22 ± 4 −58 ± 10

due to the fact that IgG levels were recorded at trough. Overall, based on CD69 inhibition, the results from the study suggested three different doses (0.5, 2, and 5 mg/kg b.i.d.) for an in vivo efficacy study. Arthritis was induced in mice by injecting collagen in the tail of arthritis prone DBA-1 mice. Analogue 52 was dosed in a preventive mode orally b.i.d. for 42 days, starting on day 1 (Figure 7). Methotrexate was used as a positive control.

cells were determined, and the results are shown in Figure 6. Analogue 52 demonstrated a dose dependent inhibition of

Figure 6. Anti-mIgE basophile mouse ex vivo assay with trifluoromethyl 52.

CD63+ cells that was maximal with a dose of 10 mg/kg. These data indicated a mouse ex vivo EC50 of 2.0 ± 0.8 nM and an EC90 of 9 ± 5 nM. These values appear to be consistent with the mWB assay (IC50 = 5.1 ± 1.8 nM). Analogue 52 was further evaluated in a keyhole limpet hemocyanin (KLH) mouse humoral model. The compound was administered orally b.i.d. at five different doses to mice that had been immunized intraperitoneally with KLH. On day 13, blood levels of CD69 and IgG titers at trough (24 h) were recorded following ex vivo KLH treatment (Table 10). Importantly, the IgG response was suppressed in a doseproportional fashion and this correlated with the inhibition of CD69 measured ex vivo. The data indicated that greater inhibition of CD69 was required (based on calculated % inhibition) for robust inhibition of IgG, although this could be

Figure 7. Mouse CIA efficacy model with trifluoromethyl compound 52.

Although lower than expected exposures were observed for 52 compared to healthy DBA-1 mice (data not shown), a dosedependent reduction of the clinical score was observed. Doses of 2 and 5 mg/kg showed greater than 50% suppression of paw swelling. Taking the exposure into consideration, EC50 of 10 nM at 24 h (ED50 of ∼1.25 mg/kg) was derived. This level of efficacy was similar to other compounds reported in the literature20 and corresponded to about a 2-fold multiple of the IC50 at trough.

Table 10. Results of the Mouse KLH Study for Compound 52 dose of 52, po (mg/kg b.i.d.) 0.05 0.15 0.45 1.5 4.5

% inh of CD69 63 88 92 96 97

± ± ± ± ±

4 4 2 3 2

% inh of IgG

fold of in vitro hWB IC90 at trough

± ± ± ± ±

0.l× 0.5× 0.7× 1.9× 4.5×

32 40 67 71 93

3 3 3 2 2

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CONCLUSION Several highly potent and selective PI3Kδ inhibitors were identified. The initial analogue, pyrazole 2, suffered from poor ADME properties and kinase selectivity. Two significant improvements to 2 were made. First, replacement of the 5199

DOI: 10.1021/acs.jmedchem.7b00618 J. Med. Chem. 2017, 60, 5193−5208

Journal of Medicinal Chemistry

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pyrazol-5-yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (22 mg, 65% yield). HPLC tR = 2.90 min (method C). LCMS (ESI) m/z calcd for C28H32N8O3 [M + H]+ 529.3, found 529.3. 1 H NMR (500 MHz, DMSO-d6) δ 8.11−8.04 (m, 2H), 8.04 (s, 1H), 7.68 (s, 1H), 7.54 (t, J = 7.9 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.27 (s, 1H), 6.47 (s, 1H), 4.35 (m, 1H), 3.92−3.85 (m, 2H), 3.83 (m, 2H), 3.77 (m, 2H), 3.43−3.40 (m, 2H), 2.20−2.00 (m, 2H), 2.10 (s, 3H), 1.78 (m, 2H), 1.70 (s, 6H). 2-Bromo-N-(3-bromophenyl)-2-methylpropanamide (5). A mixture of 3-bromoaniline (3) (2.0 g, 12 mmol) and N-ethyl-Nisopropylpropan-2-amine (4.0 mL, 23 mmol) in DCM (100 mL) was cooled to 0 °C, and 2-bromo-2-methylpropanoyl bromide (4) (2.2 mL, 17 mmol) was added dropwise. The mixture was allowed to warm up to room temperature over 2 h and was diluted with DCM (100 mL). The organic phase was washed with water, 1 N HCl, saturated NaHCO3, and brine. The resulting solution was dried over Na2SO4 and concentrated to give the titled compound in 98% yield which was used without further purification. HPLC tR = 1.01 min (method A). LCMS (ESI) m/z calcd for C10H11Br2NO [M + H]+ 321.6, found 321.7. N-(3-Bromophenyl)-2-((2-hydroxyethyl)amino)-2-methylpropanamide (7). Crude bromide 5 and 2-aminoethanol (6) (1.4 g, 23 mmol) were dissolved in acetonitrile (95 mL) followed by the addition of silver oxide (5.4 g, 23 mmol). The mixture was stirred at room temperature for 1 h and then filtered through Celite. The filtrate was diluted with ethyl acetate (250 mL) and washed with saturated NaHCO3 and brine. The resulting solution was dried over Na2SO4 and concentrated to give crude solid which was used without further purification (75% yield). HPLC tR = 1.95 min (method C). LCMS (ESI) m/z calcd for C12H17BrN2O2 [M + H]+ 302.6, found 302.7. 1H NMR (400 MHz, chloroform-d) δ 9.79 (br s, 1H), 7.90 (t, J = 2.0 Hz, 1H), 7.54 (dt, J = 7.8, 1.7 Hz, 1H), 7.27−7.11 (m, 2H), 3.86−3.75 (m, 2H), 2.83−2.69 (m, 2H), 1.44 (s, 6H). 1-(3-Bromophenyl)-3,3-dimethylpiperazin-2-one (8). To a solution of diisopropyl azocarboxylate (2.4 g, 12 mmol) in THF (50 mL) at room temperature was added triphenylphosphine (3.1 g, 12 mmol). After 10 min, a yellow solid formed. A solution of N-(3bromophenyl)-2-((2-hydroxyethyl)amino)-2-methylpropanamide (7) (1.2 g, 4 mmol) in THF (50 mL) was added. After stirring overnight at room temperature, the mixture was diluted with 100 mL of ethyl acetate and was washed with saturated NaHCO3 and brine. The resulting solution was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by acid/base extraction to give 1-(3-bromophenyl)-3,3-dimethylpiperazin-2-one in 90% yield. HPLC tR = 1.71 min (method C). LCMS (ESI) m/z calcd for C12H15BrN2O [M + H]+ 284.8, found 284.8. 1H NMR (400 MHz, chloroform-d) δ 7.50−7.45 (m, 1H), 7.44−7.38 (m, 1H), 7.29−7.22 (m, 2H), 3.75−3.67 (m, 2H), 3.31−3.22 (m, 2H), 1.50 (s, 6H). 4-Acetyl-1-(3-bromophenyl)-3,3-dimethylpiperazin-2-one (9). To a solution of 1-(3-bromophenyl)-3,3-dimethylpiperazin-2-one (8) (0.82 g, 2.7 mmol) in DCM (10 mL) were added triethylamine (1 mL) and acetic anhydride (0.33 g, 3.2 mmol), and the resulting mixture was stirred for 2 h at room temperature. The reaction was diluted with DCM (20 mL), washed with water, saturated NaHCO3, and brine. The resulting solution was dried over Na2SO4 and concentrated under reduced pressure. The crude mixture was purified by silica gel chromatography, eluting with a gradient of 0−100% ethyl acetate in hexanes to give 4-acetyl-1-(3-bromophenyl)-3,3-dimethylpiperazin-2-one (9) (0.9 g, 93%). HPLC tR = 0.79 min (method A). LCMS (ESI) m/z calcd for C14H17BrN2O2 [M + H]+ 326.7, found 326.8. 1H NMR (400 MHz, chloroform-d) δ 7.51 (dt, J = 1.9, 1.0 Hz, 1H), 7.44 (dt, J = 7.3, 1.9 Hz, 1H), 7.33−7.22 (m, 2H), 3.83−3.73 (m, 4H), 2.20 (s, 3H), 1.86 (s, 6H). 4-Acetyl-3,3-dimethyl-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazin-2-one (10). A mixture of 4-acetyl1-(3-bromophenyl)-3,3-dimethylpiperazin-2-one (9) (1.95 g, 6.00 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (1.7 g, 6.60 mmol), PdCl2(dppf)−DCM adduct (0.150 g, 0.180 mmol), and potassium acetate (1.2 g, 12 mmol) in dioxane (15 mL) in a capped pressure reaction vial was degassed by vacuum, then filled with

polar pyrazole group by a simple chloro or trifluoromethyl group led to improved Caco-2 permeability, reduced Caco-2 efflux, and an improved selectivity profile while maintaining potency in a CD69 hWB assay. Furthermore, analogues 33 and 35 demonstrated reduced activity in a hERG PC assay and a better protein binding profile which together may mitigate cardiovascular risk. However, these molecules suffered from an undesired human/rodent disconnect in metabolic stability. Second, the identification of nitrile 52 improved mouse metabolic stability while maintaining an excellent profile. This molecule was shown to be very potent ex vivo in a mouse basophil assay as well as in vivo in a mouse KLH study. Furthermore, 52 was shown to be efficacious in a mouse arthritis model. Finally, from this effort, trifluoromethyl analogue 52 was selected for additional preclinical toxicity studies, including evaluation for cardiovascular risk in higher species.

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EXPERIMENTAL SECTION

All commercially available chemicals and solvents were used without further purification. Reactions were performed under an atmosphere of nitrogen. All new compounds gave satisfactory 1H NMR and/or LC/ MS results. 1H NMR spectra were obtained on a Bruker 400 MHz or a JEOL 500 MHz NMR spectrometer using TMS as internal reference. Electrospray ionization (ESI) mass spectra were obtained on a Waters ZQ single quadrupole mass spectrometer. We do not believe that this series of compounds has a PAINS liability. All compounds were tested in four different PI3K isoforms ADP-Glo assays as well as in >200 kinases HTRF assays. Clear isoform selectivity and general kinase selectivity for the compounds discussed in the paper support that this is not an issue. Additionally, in vivo efficacy studies have confirmed activity for compound 52. The purity of tested compounds determined by analytical HPLC was >95%. HPLC methods are listed below. Method A. Waters Acquity UPLC, BEH C18 2.1 mm × 50 mm, 1.7 μm particles. Mobile phase A: 98:2 water/acetonitrile with 0.05% TFA. Mobile phase B: acetonitrile with 0.05% TFA. Temperature 50 °C. Gradient 2−98% B over 1 min, then 0.5 min hold at 100% B. Flow: 0.8 mL/min. Detection: UV at 200 nm. Method B. Waters Acquity UPLC, BEH C18 2.1 mm × 50 mm, 1.7 μm particles. Mobile phase A: 5:95 acetonitrile/water with 10 mM ammonium acetate. Mobile phase B: 95:5 acetonitrile/water with 10 mM ammonium acetate. Temperature: 50 °C. Gradient: 0−100% B over 2 min, then a 0.5 min hold at 100% B. Flow: 1.0 mL/min. Detection: UV at 220 nm. Method C. Sunfire C18 3.0 mm × 150 mm, 3.5 μm particles. Mobile phase A: 5:95 acetonitrile/water with 0.05% TFA. Mobile phase B: 95:5 acetonitrile/water with 0.05 TFA. Temperature: 25 °C. Gradient: 0−100% B over 3 min, then a 1 min hold at 100% B. Flow: 1.0 mL/min. Detection: UV at 254 nm. Method D (Preparative). Luna C18 30 mm × 100 mm, 5 μm particles; 2 mL injection. Mobile phase: 0.1% TFA in water. Mobile phase B: 0.1% TFA in acetonitrile. Temperature: 50 °C. Gradient: 20− 100% B over 5 min, then a 10 min hold at 100% B. Flow: 30 mL/min. Detection: UV at 220 nm. 4-Acetyl-1-(3-(4-amino-5-(1-(tetrahydro-2H-pyran-4-yl)-1Hpyrazol-5-yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (2). A pressure vial was charged with boronic ester 10 (52 mg, 0.18 mmol), water (0.3 mL), 7-bromo-5-(1(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-5-yl)pyrrolo[2,1-f ][1,2,4]triazin-4-amine (15a) (33 mg, 0.15 mmol), Na2CO3 (49 mg, 0.46 mmol), and PdCl2(dppf)−DCM adduct (25 mg, 0.03 mmol) in dioxane (1.2 mL). The mixture was purged with nitrogen, and then the vessel was sealed and the mixture was stirred at 110 °C for 5 h. The reaction mixture was cooled, diluted with 15 mL of water, and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by preparative HPLC (method D) to obtain 4-acetyl-1-(3-(4-amino-5-(1-(tetrahydro-2H-pyran-4-yl)-1H5200

DOI: 10.1021/acs.jmedchem.7b00618 J. Med. Chem. 2017, 60, 5193−5208

Journal of Medicinal Chemistry

Article

7.27 (m, 1H), 6.92 (d, J = 4.6 Hz, 1H), 6.73 (d, J = 4.6 Hz, 1H), 3.92− 3.87 (m, 2H), 3.85−3.79 (m, 2H), 2.21 (s, 3H), 1.90 (s, 6H). 4-Acetyl-1-(3-(4-amino-5-iodopyrrolo[1,2-f ][1,2,4]triazin-7yl)phenyl)-3,3-dimethylpiperazin-2-one (19). A solution of 4acetyl-1-(3-(4-aminopyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3dimethylpiperazin-2-one (18) (300 mg, 0.8 mmol) and Niodosuccinimide (180 mg, 0.8 mmol) in DMF (10 mL) was stirred under nitrogen at room temperature for 15 h. More NIS (45 mg, 0.2 mmol) and TFA (1 drop) were added to the reaction mixture, and stirring was continued for 2 h allowing full conversion. The reaction mixture was diluted with saturated NaHCO3 (50 mL) and stirred vigorously for 15 min. The precipitated solid was collected by filtration, washed with water and then diethyl ether. The solid was dried under vacuum affording 4-acetyl-1-(3-(4-amino-5-iodopyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (350 mg, 0.7 mmol, 88% yield) as a light brown solid. HPLC tR = 0.7 min (method A). LCMS (ESI) m/z calcd for C20H21I1N6O2 [M + H]+ 505.1, found 505.0. 1H NMR (500 MHz, DMSO-d6) δ 8.01−7.96 (m, 2H), 7.93 (s, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.37−7.31 (m, 2H), 3.84− 3.73 (m, 4H), 2.10 (s, 3H), 1.70 (s, 6H). 4-Acetyl-1-(3-(4-amino-5-(1-cyclopropyl-1H-pyrazol-5-yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (21). Compound was synthesized according to the procedure outlined for the synthesis of pyrazole 2. HPLC tR = 2.99 min (method C). LCMS (ESI) m/z calcd for C26H28N8O2 [M + H]+ 485.2, found 485.4. 1H NMR (500 MHz, DMSO-d6) δ 8.12 (s, 1H), 8.07−7.99 (m, 2H), 7.60−7.50 (m, 2H), 7.45−7.31 (m, 2H), 6.45 (s, 1H), 3.86−3.73 (m, 4H), 2.08 (s, 3H), 1.79−1.64 (m, 7H), 1.09−1.00 (m, 2H), 0.92−0.80 (m, 2H). 4-Acetyl-1-(3-(4-amino-5-(1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (22). Compound was synthesized according to the procedure outlined for the synthesis of pyrazole 2. HPLC tR = 2.61 min (method C). LCMS (ESI) m/z calcd for C25H25F3N8O2 [M + H]+ 527.2, found 527.4. 1H NMR (400 MHz, methanol-d4) δ 8.92− 8.80 (m, 3H), 8.59 (s, 1H), 8.40−8.30 (m, 1H), 8.20−8.13 (m, 1H), 8.05 (s, 1H), 7.43 (s, 1H), 5.91−5.78 (m, 2H), 4.70−4.52 (m, 4H), 2.91 (s, 3H), 1.63 (s, 6H). 4-Acetyl-1-(3-(4-amino-5-(1-methyl-1H-pyrazol-4-yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (23). A mixture of 4-acetyl-1-(3-(4-amino-5-iodopyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (19) (100 mg, 0.2 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1Hpyrazole (50 mg, 0.24 mmol), PdCl2(dppf)−DCM adduct (8 mg, 10 μmol), and K3PO4 (2 N, 0.2 mL, 0.4 mmol) in dioxane (3 mL) in a capped pressure reaction vial was degassed by vacuum and backfilled with nitrogen twice. The vial was placed in a 90 °C heating block and stirred for 3 h. The crude material was purified via preparative HPLC (method D) yielding 40 mg of the desired product. HPLC tR = 0.61 min. LCMS (ESI) m/z calcd for C24H26N8O2 [M + H]+ 459.2, found 459.2. 1H NMR (500 MHz, DMSO-d6) δ 8.08−8.01 (m, 1H), 8.01− 7.88 (m, 3H), 7.69−7.59 (m, 1H), 7.57−7.45 (m, 1H), 7.36−7.24 (m, 1H), 7.06 (s, 1H), 3.91 (s, 3H), 3.85−3.68 (m, 4H), 2.09 (s, 3H), 1.69 (s, 6H). 4-Acetyl-1-(3-(4-amino-5-(1-cyclopropyl-1H-pyrazol-4-yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (24). Compound was synthesized according to the procedure outlined for the synthesis of pyrazole 23. HPLC tR = 1.90 min (method C). LCMS (ESI) m/z calcd for C26H28N8O2 [M + H]+ 485.2, found 485.4. 1H NMR (500 MHz, DMSO-d6) δ 8.08−8.01 (m, 1H), 8.01−7.88 (m, 3H), 7.69−7.59 (m, 1H), 7.57−7.45 (m, 1H), 7.36−7.24 (m, 1H), 7.06 (s, 1H), 3.85−3.68 (m, 5H), 2.09 (s, 3H), 1.69 (s, 6H), 1.20−1.08 (m, 2H), 1.08−0.90 (m, 2H). 4-Acetyl-1-(3-(4-amino-5-(2-chloro-1-methyl-1H-imidazol-5yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (25). Compound was synthesized according to the procedure outlined for the synthesis of pyrazole 23. HPLC tR = 2.64 min (method C). LCMS (ESI) m/z calcd for C24H25Cl1N8O2 [M + H]+ 493.2, found 493.4. 1H NMR (500 MHz, DMSO-d6) δ 8.13−8.06 (m, 1H), 8.06−7.93 (m, 2H), 7.57−7.47 (m, 1H), 7.37−7.27 (m, 1H),

nitrogen. This process was repeated twice. The reaction mixture was placed in a 90 °C heating block for 2.5 h. The reaction mixture was filtered through Celite and the filtrate was purified by silica gel chromatography, eluting with a gradient of 0−100% ethyl acetate in hexanes affording 4-acetyl-3,3-dimethyl-1-(3-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)phenyl)piperazin-2-one (1.75 g, 4.7 mmol, 68% yield) as a white solid. HPLC tR = 0.87 min (method A). LCMS (ESI) m/z calcd for C19H27BN2O4 [M + H]+ 359.1, found 359.0. 1H NMR (400 MHz, chloroform-d) δ 7.77−7.73 (m, 1H), 7.68 (d, J = 1.5 Hz, 1H), 7.45−7.42 (m, 2H), 3.84−3.74 (m, 4H), 2.20 (s, 3H), 1.86 (s, 6H), 1.37 (s, 12H). (E)-1-(4-Amino-7-bromopyrrolo[2,1-f ][1,2,4]triazin-5-yl)-3(dimethylamino)prop-2-en-1-one (13). To a solution of ethynyltrimethylsilane (1.7 mL, 12 mmol) in THF (30 mL) at −78 °C was added n-butyllithium (4.6 mL, 11 mmol) dropwise. After stirring for 5 min at −78 °C, 4-amino-7-bromo-N-methoxy-Nmethylpyrrolo[2,1-f ][1,2,4]triazine-5-carboxamide (12)21 (1.2 g, 4 mmol) was added as a solution in THF (6 mL). After 30 min, acetic acid (0.6 mL) was added to give a bright yellow solution. The mixture was partitioned between ethyl acetate (40 mL) and water (20 mL). The organic layer was washed with brine, dried over Na2SO4, and concentrated to afford alkyne 13 (1.3 g) as a yellow solid used without further purification. The solid (1.1 g, 3.3 mmol) was suspended in ethanol (16 mL), and dimethylamine (0.620 mL, 4.9 mmol, 40% solution in water) was added. The mixture was heated at 80 °C for 0.5 h and then cooled to room temperature. The solids were removed through filtration and were washed with ethanol, ethyl acetate, and hexanes, and volatiles were removed under vacuum providing (E)-1(4-amino-7-bromopyrrolo[2,1-f ][1,2,4]triazin-5-yl)-3(dimethylamino)prop-2-en-1-one (904 mg, 2.91 mmol, 89% yield) as a yellow/green solid. HPLC tR = 0.86 min (method C). LCMS (ESI) m/z calcd for C11H12BrN5O [M + H]+ 310.1, found 310.2. 1H NMR (400 MHz, DMSO-d6) δ 10.86 (br s, 1H), 8.17 (br s, 1H), 8.03−7.93 (m, 1H), 7.78 (d, J = 12.1 Hz, 1H), 7.64−7.56 (m, 1H), 5.92 (d, J = 12.1 Hz, 1H), 3.17 (s, 3H), 2.97 (s, 3H). 7-Bromo-5-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-5-yl)pyrrolo[2,1-f ][1,2,4]triazin-4-amine (15a). (E)-1-(4-Amino-7bromopyrrolo[2,1-f ][1,2,4]triazin-5-yl)-3-(dimethylamino)prop-2-en1-one (13) (1.0 g, 3.2 mmol) was suspended in ethanol (40 mL), and (tetrahydro-2H-pyran-4-yl)hydrazine, HCl salt (0.92 g, 6.3 mmol) was added. The suspension was stirred vigorously for 2 min, and triethylamine (2.2 mL, 16 mmol) was added. The mixture was refluxed for 18 h, then cooled to room temperature followed by the addition of water (50 mL). The mixture was concentrated to 15 mL. The solid formed was filtered and dried under vacuum providing 7bromo-5-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-5-yl)pyrrolo[2,1f ][1,2,4]triazin-4-amine (3.24 g, 94% yield) as a white solid. HPLC tR = 0.69 min (method A). LCMS (ESI) m/z calcd for C14H15BrN6O [M + H]+ 363.1, found 363.2. 1H NMR (400 MHz, DMSO-d6) δ 8.06 (s, 1H), 7.65 (d, J = 1.5 Hz, 1H), 7.04 (s, 1H), 6.42 (d, J = 1.8 Hz, 1H), 4.33−4.23 (m, 1H), 3.87 (dd, J = 11.2, 3.5 Hz, 2H), 3.37−3.31 (m, 2H), 2.06 (qd, J = 12.2, 4.7 Hz, 2H), 1.84−1.68 (m, 2H). 4-Acetyl-1-(3-(4-aminopyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (18). A pressure vial was charged with boronic ester 10 (500 mg, 1.5 mmol), water (3 mL), 7bromopyrrolo[2,1-f ][1,2,4]triazin-4-amine (17) (330 mg, 1.5 mmol), Na2CO3 (489 mg, 4.61), and PdCl2(dppf)−DCM adduct (251 mg, 0.308 mmol) in dioxane (12 mL). The mixture was purged with nitrogen, the vessel was sealed, and the mixture was stirred at 100 °C for 5 h. The reaction mixture was cooled, diluted with 15 mL of water, and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel chromatography, eluting with a gradient of 0−100% ethyl acetate in hexanes to obtain 4-acetyl-1-(3-(4-aminopyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (320 mg, 60% yield). HPLC tR = 1.90 min (method C). LCMS (ESI) m/z calcd for C20H22N6O2 [M + H]+ 379.3, found 379.4. 1H NMR (400 MHz, chloroform-d) δ 8.02−7.99 (m, 2H), 7.94−7.88 (m, 1H), 7.54−7.48 (m, 1H), 7.31− 5201

DOI: 10.1021/acs.jmedchem.7b00618 J. Med. Chem. 2017, 60, 5193−5208

Journal of Medicinal Chemistry

Article

1H), 8.03 (d, J = 7.7 Hz, 1H), 7.96 (br s, 1H), 7.64 (s, 1H), 7.55 (t, J = 7.9 Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H), 6.97 (s, 1H), 3.97−3.77 (m, 4H), 3.14−3.03 (m, 1H), 2.11 (s, 3H), 1.72 (s, 6H), 1.31 (d, J = 7.1 Hz, 6H). 4-Acetyl-1-(3-(4-amino-5-methylpyrrolo[2,1-f ][1,2,4]triazin7-yl)phenyl)-3,3-dimethylpiperazin-2-one (31). A mixture of 7iodo-5-methylpyrrolo[2,1-f ][1,2,4]triazin-4-amine (23 mg, 0.084 mmol),23 4-acetyl-3,3-dimethyl-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazin-2-one (10) (38 mg, 0.1 mmol), PdCl2(dppf)−DCM adduct (3 mg, 4 μmol), and K3PO4 (2 N, 0.084 mL, 0.17 mmol) in DMF (1 mL) in a capped reaction vial was degassed by vacuum, then backfilled with nitrogen through the septum. This process was repeated twice. The vial was placed in a 90 °C heating block and stirred for 2 h. The reaction mixture was filtered and the crude material was purified via preparative HPLC (method D) yielding 21 mg of the desired compound. HPLC tR = 2.28 min (method C). LCMS (ESI) m/z calcd for C21H24N6O2 [M + H]+ 393.3, found 393.4. 1H NMR (500 MHz, DMSO-d6) δ 8.05−7.99 (m, 1H), 7.99−7.87 (m, 2H), 7.58−7.47 (m, 1H), 7.39−7.31 (m, 1H), 7.08− 6.98 (m, 1H), 3.88−3.70 (m, 4H), 2.61 (s, 3H), 2.09 (s, 3H), 1.65 (s, 6H). 7-(3-(4-Acetyl-3,3-dimethyl-2-oxopiperazin-1-yl)phenyl)-4aminopyrrolo[2,1-f ][1,2,4]triazine-5-carbonitrile (32). A mixture of 4-acetyl-1-(3-(4-amino-5-iodopyrrolo[2,1-f ][1,2,4]triazin-7-yl) phenyl)-3,3-dimethylpiperazin-2-one (19) (30 mg, 0.06 mmol), copper(I) cyanide (53 mg, 0.60 mmol), and anhydrous pyridine (0.6 mL) was placed in a 5 mL conical pressure vial. The vial was flushed with nitrogen, capped and the mixture stirred at 120 °C in an oil bath. After 3 h, the reaction mixture was concentrated under reduced pressure, and the residue was partitioned between DCM (10 mL), DMF (3 mL), and 1 M HCl (10 mL). The organic phase was separated and concentrated under reduced pressure. The crude material was purified via preparative HPLC (method D) yielding 7-(3-(4-acetyl-3,3dimethyl-2-oxopiperazin-1-yl)phenyl)-4-aminopyrrolo[2,1-f ][1,2,4]triazine-5-carbonitrile (8 mg, 33%). HPLC tR = 0.71 min (method A). LCMS (ESI) m/z calcd for C21H21N7O2 [M + H]+ 404.2, found 404.3. 1 H NMR (500 MHz, DMSO-d6) δ 8.17 (s, 1H), 7.99−7.87 (m, 2H), 7.66 (s, 1H), 7.53 (t, J = 7.9 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 3.80 (br s, 2H), 3.74 (br s, 2H), 3.49 (d, J = 5.4 Hz, 2H), 2.08 (s, 3H), 1.69 (s, 6H). 4-Acetyl-1-(3-(4-amino-5-(trifluoromethyl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (33). A mixture of 4-acetyl-1-(3-(4-amino-5-iodopyrrolo[2,1-f ][1,2,4]triazin7-yl) phenyl)-3,3-dimethylpiperazin-2-one (19) (60 mg, 0.12 mmol), di-tert-butyl dicarbonate (0.03 mL, 0.13 mmol), N-ethyl-N-isopropylpropan-2-amine (0.03 mL, 0.18 mmol), DMAP (3 mg, 0.02 mmol), and THF (1 mL) was stirred at room temperature for 16 h. More ditert-butyl dicarbonate (0.03 mL, 0.13 mmol) and N-ethyl-Nisopropylpropan-2-amine (0.03 mL, 0.18 mmol) were added. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with DCM, washed with 1.5 M K2HPO4, water, and brine. The organic mixture was dried over Na2SO4 and concentrated under reduced pressure to give 100 mg of a yellow solid as bis-boc protected product. A mixture of CuI (27 mg, 0.14 mmol) and di-tert-butyl (7-(3-(4acetyl-3,3-dimethyl-2-oxopiperazin-1-yl)phenyl)-5-iodopyrrolo[2,1-f ][1,2,4]triazin-4-yl) biscarbamate (100 mg, 0.14 mmol) in a capped pressure reaction vial was placed under vacuum, then backfilled with nitrogen twice. DMF (1 mL) and HMPA (0.1 mL) were added, and the degassing procedure was repeated again 3 times. Methyl 2,2difluoro-2-(fluorosulfonyl)acetate (14 mg, 0.71 mmol) was added, and the reaction mixture was heated at 80 °C for 2 days. The reaction mixture was diluted with ethyl acetate, washed with saturated NH4Cl, 1.5 M Na2HPO4, and water. The organic layer was then dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified via preparative HPLC (method D) yielding 4-acetyl-1-(3(4-amino-5-(trifluoromethyl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)3,3-dimethylpiperazin-2-one (7.7 mg, 12% yield). HPLC tR = 0.79 min (method A). LCMS (ESI) m/z calcd for C21H21F3N6O2 [M + H]+ 447.2, found 447.1. 1H NMR (500 MHz, DMSO-d6) δ 8.16 (s, 1H),

7.20 (s, 1H), 6.85−6.72 (m, 2H), 6.68−6.55 (m, 1H), 3.87−3.67 (m, 4H), 2.74 (s, 1H), 2.10 (s, 3H), 1.71 (s, 6H). 4-Acetyl-1-(3-(4-amino-5-(oxazol-2-yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (26). A mixture of 4-acetyl-1-(3-(4-amino-5-bromopyrrolo[2,1-f ][1,2,4]triazin-7yl)phenyl)-3,3-dimethylpiperazin-2-one (37) (30 mg, 0.07 mmol), 1tributyltinoxazole (27 mg, 0.07 mmol), Pd2(dba)3 (6 mg, 7 μmol), and tri(2-furyl)phosphine (3 mg, 0.01 mmol) in DMF (1 mL) was heated at 120 °C for 1 h. The crude material was purified via preparative HPLC (method D) yielding 7 mg of the desired product. HPLC tR = 0.69 min (method A). LCMS (ESI) m/z calcd for C23H23N7O3 [M + H]+ 446.2, found 446.1. 1H NMR (500 MHz, DMSO-d6) δ 10.31 (s, 1H), 8.41 (s, 1H), 8.26 (s, 1H), 8.14−7.93 (m, 3H), 7.62 (s, 1H), 7.58−7.49 (m, 1H), 7.49−7.41 (m, 1H), 7.41−7.31 (m, 1H), 3.89− 3.66 (m, 4H), 2.11 (s, 3H), 1.71 (s, 6H). 4-Acetyl-1-(3-(4-amino-5-(5-methyl-1,3,4-thiadiazol-2-yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (27). Compound was synthesized according to to the procedure outlined for the synthesis of pyrazole 23. HPLC tR = 0.67 min (method A). LCMS (ESI) m/z calcd for C23H24N8O2S1 [M + H]+ 477.2, found 477.1. 1H NMR (500 MHz, DMSO-d6) δ 10.09 (br s, 1H), 8.41 (br s, 1H), 8.11−7.88 (m, 3H), 7.61−7.46 (m, 2H), 7.43−7.32 (m, 1H), 3.88−3.67 (m, 4H), 2.78 (s, 3H), 2.11 (s, 3H), 1.71 (s, 6H). 4-Acetyl-1-(3-(4-amino-5-(thiazol-4-yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (28). Compound was synthesized according to the procedure outlined for the synthesis of oxazole 26. HPLC tR = 0.69 min (method A). LCMS (ESI) m/z calcd for C23H23N7O2S1 [M + H]+ 462.2, found 462.1. 1H NMR (500 MHz, DMSO-d6) δ 10.37 (br s, 1H), 9.36 (br s, 1H), 8.28−8.19 (m, 1H), 8.13 (s, 1H), 8.07−8.01 (m, 1H), 7.99 (s, 1H), 7.93 (s, 1H), 7.70 (s, 1H), 7.60−7.47 (m, 1H), 7.40−7.29 (m, 1H), 3.88−3.66 (m, 4H), 2.11 (s, 3H), 1.72 (s, 6H). 4-Acetyl-1-(3-(4-amino-5-(5-isopropyl-1,3,4-oxadiazol-2-yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (29). Compound was synthesized according to the procedure outlined for the synthesis of pyrazole 23. HPLC tR = 0.79 min (method A). LCMS (ESI) m/z calcd for C25H28N8O3 [M + H]+ 489.3, found 489.1. 1H NMR (400 MHz, chloroform-d) δ 10.25 (br s, 1H), 8.06 (s, 1H), 7.97 (s, 1H), 7.95−7.89 (m, 1H), 7.59−7.51 (m, 1H), 7.40−7.33 (m, 2H), 5.87 (br s, 1H), 3.97−3.77 (m, 4H), 3.39− 3.21 (m, 1H), 2.22 (s, 3H), 1.90 (s, 6H), 1.51 (d, 6H). 4-Acetyl-1-(3-(4-amino-5-(3-isopropylisoxazol-5-yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (30). To a heterogeneous mixture of 7-bromo-5-ethynylpyrrolo[2,1f ][1,2,4]triazin-4-amine (100 mg, 0.4 mmol)22 and N-hydroxyisobutyrimidoyl chloride (103 mg, 0.8 mmol) in 1,2-dichloroethane (3 mL) immersed in an oil bath at 65 °C was added triethylamine (0.29 mL, 2.1 mmol) dropwise. The reaction mixture was stirred at 65 °C for 3 h, during which time the mixture became nearly homogeneous. The mixture was diluted with DCM, washed with water, and dried over Na2SO4. Concentration under reduced pressure followed by purification by silica gel chromatography using a gradient of 20−35% ethyl acetate in hexanes afforded 7-bromo-5-(3-isopropylisoxazol-5-yl)pyrrolo[2,1-f ][1,2,4]triazin-4-amine (80 mg, 0.246 mmol, 58.3% yield) as a white solid. HPLC tR = 0.77 min (method A). LCMS (ESI) m/z calcd for C12H12BrN5O [M + H]+ 323.8, found 323.9. A 1 dram pressure vial was flushed with nitrogen, to which were added dioxane (2 mL), 7-bromo-5-(3-isopropylisoxazol-5-yl)pyrrolo[2,1-f ][1,2,4]triazin-4-amine (20 mg, 0.06 mmol), and 4-acetyl-3,3dimethyl-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazin-2-one (28 mg, 0.07 mmol). K3PO4 (0.093 mL, 0.19 mmol) and PdCl2(dppf)−DCM adduct (5 mg, 6 μmol) were added, and the reaction mixture was purged with nitrogen for 5 min. The reaction vessel was sealed and heated to 110 °C for 2 h. The residue was purified by preparative HPLC (method D) to obtain 4-acetyl-1-(3-(4amino-5-(3-isopropylisoxazol-5-yl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin-2-one (11 mg, 35% yield). HPLC tR = 0.77 min (method A). LCMS (ESI) m/z calcd for C26H29N7O3 [M + H]+ 488.3, found 488.4. 1H NMR (500 MHz, DMSO-d6) δ 8.12 (s, 5202

DOI: 10.1021/acs.jmedchem.7b00618 J. Med. Chem. 2017, 60, 5193−5208

Journal of Medicinal Chemistry

Article

8.02 (d, J = 8.1 Hz, 1H), 7.96 (s, 1H), 7.58−7.50 (m, 2H), 7.37 (d, J = 7.7 Hz, 1H), 3.85−3.73 (m, 4H), 2.10 (s, 3H), 1.70 (s, 6H). 4-Acetyl-1-(3-(4-amino-5-fluoropyrrolo[2,1-f ][1,2,4]triazin7-yl)phenyl)-3,3-dimethylpiperazin-2-one (34). To a solution of 7-bromopyrrolo[2,1-f ][1,2,4]triazin-4-amine (12) (50 mg, 0.2 mmol) was added Selectfluor (170 mg, 0.5 mmol). The reaction was stirred for 1 h when LCMS showed full consumption of starting material. The reaction was diluted with ethyl acetate and washed with water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure yielding 7-bromopyrrolo[2,1-f ][1,2,4]triazin-4-amine and 7bromo-5-fluoropyrrolo[2,1-f ][1,2,4]triazin-4-amine. To the resulting solid and 4-acetyl-3,3-dimethyl-1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazin-2-one (10) (160 mg, 0.43 mmol) in DMF (1.1 mL) were added PdCl2(dppf)−DCM adduct (9 mg, 11 μmol) and K3PO4 (2 N, 330 μL, 0.6 mmol). The reaction vial was capped and degassed by vacuum, then filled with nitrogen. This process was repeated twice. The vial was placed in a 90 °C heating block and stirred for 3 h. The crude material was purified via preparative HPLC (method D) to yield 5 mg of the desired product. HPLC tR = 0.61 min (method A). LCMS (ESI) m/z calcd for C20H21FN6O2 [M + H]+ 397.2, found 397.1. 1H NMR (500 MHz, DMSO-d6) δ 8.05−7.99 (m, 1H), 7.96 (s, 1H), 7.85 (s, 1H), 7.54− 7.45 (m, 1H), 7.36−7.28 (m, 1H), 7.01 (s, 1H), 3.85−3.69 (m, 4H), 2.11 (s, 3H), 1.70 (s, 6H). 4-Acetyl-1-(3-(4-amino-5-chloropyrrolo[1,2-f ][1,2,4]triazin7-yl)phenyl)-3,3-dimethylpiperazin-2-one (35). To a solution of 4-acetyl-1-(3-(4-aminopyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3dimethylpiperazin-2-one (18) (100 mg, 0.264 mmol) in DMF (2.64 mL) was added NCS (42.3 mg, 0.317 mmol), and the reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with DMF and purified by preparative HPLC (method D) affording 4-acetyl-1-(3-(4-amino-5-chloropyrrolo[2,1-f ][1,2,4]triazin7-yl)phenyl)-3,3-dimethylpiperazin-2-one (63 mg, 0.15 mmol, 56% yield). HPLC tR = 0.66 min (method A). LCMS (ESI) m/z calcd for C20H21Cl1N6O2 [M + H]+ 413.2, found 413.2. 1H NMR (500 MHz, DMSO-d6) δ 7.99 (d, J = 7.7 Hz, 1H), 7.94 (s, 2H), 7.51 (t, J = 8.1 Hz, 1H), 7.33 (d, J = 7.7 Hz, 1H), 7.22 (s, 1H), 3.86−3.71 (m, 4H), 2.10 (s, 3H), 1.70 (s, 6H). 4-Acetyl-1-(3-(4-amino-5-bromopyrrolo[1,2-f ][1,2,4]triazin7-yl)phenyl)-3,3-dimethylpiperazin-2-one (36). A solution of 4acetyl-1-(3-(4-aminopyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3dimethylpiperazin-2-one (18) (1 g, 2.6 mmol) and NBS (0.400 g, 2.2 mmol) in THF (10 mL) was stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate, washed with 1.5 M K2HPO4 and water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure to give 4-acetyl-1-(3-(4-amino-5bromopyrrolo[2,1-f ][1,2,4]triazin-7-yl)phenyl)-3,3-dimethylpiperazin2-one (1.03 g, 2.3 mmol, 85% yield) as a gray solid. HPLC tR = 2.47 min (method C). LCMS (ESI) m/z calcd for C20H21Br1N6O2 [M + H]+ 457.2, found 457.2. 1H NMR (500 MHz, DMSO-d6) δ 8.03−7.91 (m, 3H), 7.53−7.46 (m, 1H), 7.35−7.29 (m, 1H), 7.29 (s, 1H), 3.85− 3.71 (m, 4H), 2.10 (s, 3H), 1.70 (s, 6H). 7-Bromo-5-chloropyrrolo[1,2-f ][1,2,4]triazin-4-amine (37). To a solution of 7-bromopyrrolo[2,1-f ][1,2,4]triazin-4-amine (18) (0.48 g, 2.3 mmol) in DMF (11 mL) was added NCS (0.36 g, 2.7 mmol), and the reaction mixture was stirred at room temperature overnight. The reaction mixture was partitioned between 1.5 M K2HPO4 and ethyl acetate. The organic phase was washed with 10% LiCl and brine. The organic layer was then dried over Na2SO4 and concentrated under reduced pressure to afford a white solid as the crude product. The crude product was then purified by silica gel chromatography, eluting with a gradient of 30−75% ethyl acetate in hexanes. The desired product was obtained as a white solid (0.48 g) in 86% yield. HPLC tR = 0.67 min (method A). LCMS (ESI) m/z calcd for C6H4BrClN4 [M + H]+ 248.7, found 248.8. 1H NMR (400 MHz, DMSO-d6) δ 8.17 (d, J = 8.4 Hz, 1H), 7.93 (s, 1H), 7.21−7.00 (m, 1H), 6.98 (s, 1H). 7-Bromo-5-(trifluoromethyl)pyrrolo[1,2-f ][1,2,4]triazin-4amine (38). A solution of 7-bromopyrrolo[2,1-f ][1,2,4]triazin-4amine (17) (3 g, 14 mmol) in DMF (30 mL) was treated with 4-

methoxybenzyl chloride (4.9 g, 31 mmol) and Cs2CO3 (8.4 g, 25 mmol). The mixture was stirred at room temperature for 22 h. The mixture was diluted with water and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure to provide a yellow solid. The material was purified by silica gel chromatography, eluting with a gradient of 5−30% ethyl acetate in hexanes affording 7-bromoN,N-bis(4-methoxybenzyl)pyrrolo[2,1-f ][1,2,4]triazin-4-amine (4.3 g, 68% yield). HPLC tR = 1.12 min (method A). LCMS (ESI) m/z calcd for C22H21BrN4O2 [M + H]+ 453.0, found 453.0. 1H NMR (400 MHz, chloroform-d) δ 8.14 (s, 1H), 7.23 (d, J = 8.6 Hz, 4H), 6.93−6.88 (m, 4H), 6.61 (d, J = 1.3 Hz, 2H), 4.94 (s, 4H), 3.83 (s, 6H). A solution of 7-bromo-N,N-bis(4-methoxybenzyl)pyrrolo[2,1-f ][1,2,4]triazin-4-amine (4.3 g, 9.5 mmol), NIS (2.1 g, 9.5 mmol) in DMF (20 mL), and 10 drops of TFA was stirred at room temperature for 16 h. Additional NIS (130 mg, 0.05 equiv) was added and the mixture was stirred at room temperature for an additional 1 h. The reaction mixture was poured into an ice−water and 1.5 M K2HPO4 (∼1:1) mixture to afford a yellow precipitate. The mixture was filtered, and the filter cake was washed twice with water. The filter cake was triturated with ethyl acetate to give 2.98 g of product as a white solid. The mother liquor was concentrated and triturated with MeOH to give another 1.43 g of crystalline product as a white solid. Total yield of 7-bromo-5-iodo-N,N-bis(4-methoxybenzyl)pyrrolo[2,1-f ][1,2,4]triazin-4-amine: 4.4 g, 7.6 mmol, 80% yield. HPLC tR = 1.26 min (method A). LCMS (ESI) m/z calcd for C22H20BrIN4O2 [M + H]+ 578.9, found 578.9. 1H NMR (400 MHz, chloroform-d) δ 8.13 (s, 1H), 7.29 (s, 1H), 7.07−7.03 (m, 4H), 6.89−6.85 (m, 4H), 4.64 (s, 4H), 3.83 (s, 6H). A mixture of 7-bromo-5-iodo-N,N-bis(4-methoxybenzyl)pyrrolo[2,1-f ][1,2,4]triazin-4-amine (3 g, 5.2 mmol), CuI (0.99 g, 5.2 mmol), and KF (0.9 g, 16 mmol) in a capped pressure reaction vial was placed under vacuum and backfilled with nitrogen twice. To the above solid mixture was added DMF (8 mL). The resulting suspension was degassed three times. To the above suspension was added methyl 2,2difluoro-2-(fluorosulfonyl)acetate (5 g, 26 mmol). The reaction vial was placed in an 80 °C heating block and stirred for 3 h. The reaction mixture was cooled to room temperature, then filtered through Celite. The filter cake was washed with ethyl acetate three times. The combined filtrate was washed twice with 5% ammonia, once with 10% LiCl, and once with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure to give 7-bromo-N,N-bis(4methoxybenzyl)-5-(trifluoromethyl)pyrrolo[2,1-f ][1,2,4]triazin-4amine (2.5 g, 93% yield). HPLC tR = 1.31 min (method A). LCMS (ESI) m/z calcd for C23H20BrF3N4O2 [M + H]+ 521.0, found 521.0. 1 H NMR (400 MHz, chloroform-d) δ 8.21 (s, 1H), 7.10 (s, 1H), 6.96−6.93 (m, 4H), 6.84−6.82 (m, 4H), 4.58 (s, 4H), 3.81−3.79 (m, 6H). 7-Bromo-N,N-bis(4-methoxybenzyl)-5-(trifluoromethyl)pyrrolo[2,1-f ][1,2,4]triazin-4-amine (1.2 g, 2.3 mmol) in TFA (10 mL) in a pressure reaction vial was placed in a 110 °C heating block and stirred for 4 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in DCM and purified by silica gel chromatography, eluting with a gradient of 0−100% ethyl acetate in hexanes affording 7-bromo-5-(trifluoromethyl)pyrrolo[2,1-f ][1,2,4]triazin-4-amine (500 mg, 78% yield). HPLC tR = 0.83 min (method A). LCMS (ESI) m/z calcd for C7H4BrF3N4 [M + H]+ 281.1, found 281.0. 1H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.38 (s, 1H). Synthesis of Boronic Ester Coupling Partner. The sequence utilized for the synthesis of boronic ester 10 was used to access these boronic esters: 4-Acetyl-3,3-dimethyl-1-(2-methyl-3-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)phenyl)piperazin-2-one. HPLC tR = 0.89 min (method A). LCMS (ESI) m/z calcd for C21H31B1N2O4 [M + H]+ 387.3, found 387.2. 4-Acetyl-3,3-dimethyl-1-(2-methyl-5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)phenyl)piperazin-2-one. HPLC tR = 0.89 min (method A). LCMS (ESI) m/z calcd for C21H31B1N2O4 [M + H]+ 387.3, found 387.2. 5203

DOI: 10.1021/acs.jmedchem.7b00618 J. Med. Chem. 2017, 60, 5193−5208

Journal of Medicinal Chemistry

Article

C21H20ClF3N6O2 [M + H]+ 480.9, found 481.0. 1H NMR (500 MHz, DMSO-d6) δ 8.46 (d, J = 8.1 Hz, 1H), 8.22 (br s, 1H), 8.11 (s, 1H), 8.00 (s, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.43 (s, 1H), 7.12 (br s, 1H), 3.61−3.86 (m, 4H), 2.10 (s, 3H), 1.68 (s, 3H), 1.65 (s, 3H). 4-Acetyl-1-(5-(4-amino-5-chloropyrrolo[2,1-f ][1,2,4]triazin7-yl)-2-methoxyphenyl)-3,3-dimethylpiperazin-2-one (43). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.70 min (method A). LCMS (ESI) m/z calcd for C21H23ClN6O3 [M + H]+ 442.9, found 443.0. 1H NMR (500 MHz, DMSO-d6) δ 8.04 (dd, J = 8.6, 1.9 Hz, 1H), 7.91 (s, 1H), 7.83 (d, J = 2.0 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 7.12 (s, 1H), 3.81 (s, 3H), 3.73 (br s, 2H), 3.59 (d, J = 4.4 Hz, 2H), 2.08 (s, 3H), 1.67 (s, 6H). 4-Acetyl-1-(5-(4-amino-5-chloropyrrolo[2,1-f ][1,2,4]triazin7-yl)-2-chlorophenyl)-3,3-dimethylpiperazin-2-one (44). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.73 min (method A). LCMS (ESI) m/z calcd for C20H20Cl2N6O2 [M + H]+ 447.2, found 447.0. 1H NMR (500 MHz, DMSO-d6) δ 8.19 (d, J = 8.4 Hz, 1H), 8.07 (s, 1H), 7.97 (s, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 3.57−3.88 (m, 4H), 2.10 (s, 3H), 1.63−1.79 (m, 6H). 4-Acetyl-1-(5-(4-amino-5-chloropyrrolo[2,1-f ][1,2,4]triazin7-yl)-2-(methylsulfonyl)phenyl)-3,3-dimethylpiperazin-2-one (45). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.61 min (method A). LCMS (ESI) m/z calcd for C21H23ClN6O4S [M + H]+ 491.2, found 491.1. 1H NMR (500 MHz, DMSO-d6) δ 8.51−8.56 (m, 1H), 8.15 (s, 1H), 8.07 (d, J = 8.41 Hz, 1H), 8.03 (s, 1H), 7.51 (s, 1H), 3.62−3.89 (m, 4H), 3.18 (s, 3H), 2.11 (s, 3H), 1.73 (s, 3H), 1.71 (s, 3H) rac-4-Acetyl-1-(5-(4-amino-5-chloropyrrolo[2,1-f ][1,2,4]triazin-7-yl)-2-(1-hydroxyethyl)phenyl)-3,3-dimethylpiperazin2-one (46). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.63 min (method A). LCMS (ESI) m/z calcd for C22H25ClN6O3 [M + H]+ 457.3, found 457.2. 1H NMR (500 MHz, DMSO-d6) δ 8.38 (br s, 1H), 8.08 (d, J = 8.2 Hz, 1H), 7.96 (s, 1H), 7.72−7.78 (m, 1H), 7.60−7.68 (m, 1H), 7.36 (s, 1H), 7.22−7.26 (m, 1H), 4.65−4.77 (m, 1H), 3.76 (d, J = 10.7 Hz, 4H), 2.06−2.12 (m, 3H), 1.68−1.75 (m, 3H), 1.65 (s, 3H), 1.21−1.32 (m, 3H). 2-(4-Acetyl-3,3-dimethyl-2-oxopiperazin-1-yl)-4-(4-amino-5chloropyrrolo[1,2-f ][1,2,4]triazin-7-yl)benzonitrile (47). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.71 min (method A). LCMS (ESI) m/z calcd for C21H20ClN7O2 [M + H]+ 438.1, found 438.0. 1H NMR (500 MHz, DMSO-d6) δ 8.39 (d, J = 8.4 Hz, 1H), 8.35−8.11 (m, 2H), 8.03−7.96 (m, 2H), 7.46 (s, 1H), 7.13 (br s, 1H), 3.81 (br s, 4H), 2.11 (s, 3H), 1.72 (s, 6H). 4-Acetyl-1-(5-(4-amino-5-(trifluoromethyl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)-2-(trifluoromethyl)phenyl)-3,3-dimethylpiperazin-2-one (48). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.81 min (method A). LCMS (ESI) m/z calcd for C22H20F6N6O2 [M + H]+ 515.3, found 515.0. 1H NMR (500 MHz, DMSO-d6) δ 8.51 (d, J = 8.4 Hz, 1H), 8.22 (s, 1H), 8.17 (s, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.78 (s, 1H), 3.62−3.86 (m, 4H), 2.10 (s, 3H), 1.67 (d, J = 14.5 Hz, 6H). 4-Acetyl-1-(5-(4-amino-5-(trifluoromethyl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)-2-methoxyphenyl)-3,3-dimethylpiperazin2-one (49). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.75 min (method A). LCMS (ESI) m/z calcd for C22H23F3N6O3 [M + H]+ 477.4, found 477.0. 1H NMR (500 MHz, DMSO-d6) δ 8.14 (s, 1H), 8.09 (dd, J = 8.8, 1.7 Hz, 1H), 7.86 (d, J = 1.7 Hz, 1H), 7.46 (s, 1H), 7.22 (d, J = 8.8 Hz, 1H), 3.82 (s, 3H), 3.73 (br s, 2H), 3.61 (m, 2H), 2.08 (s, 3H), 1.67 (s, 6H). 4-Acetyl-1-(5-(4-amino-5-(trifluoromethyl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)-2-(methylsulfonyl)phenyl)-3,3-dimethylpiperazin-2-one (50). Compound was synthesized according to the

4-Acetyl-3,3-dimethyl-1-(3-methyl-5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)phenyl)piperazin-2-one. HPLC tR = 0.89 min (method A). LCMS (ESI) m/z calcd for C21H31B1N2O4 [M + H]+ 387.3, found 387.2. ( 3-(4 -Acetyl -3, 3-di met hyl- 2- oxop ip erazin -1- yl) -4(trifluoromethyl)phenyl)boronic Acid. HPLC tR = 0.75 min (method A). LCMS (ESI) m/z calcd for C15H18BF3N2O4 [M + H]+ 359.1, found 359.0. 1H NMR (400 MHz, DMSO-d6) δ 7.84 (q, J = 7.7 Hz, 2H), 7.74 (s, 1H), 3.78 (br s, 2H), 3.59−3.66 (m, 2H), 2.08 (s, 3H), 1.67 (s, 3H), 1.64 (s, 3H), 1.32 (s, 12H). 4-Acetyl-1-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,3-dimethylpiperazin-2-one. HPLC tR = 0.85 min (method A). LCMS (ESI) m/z calcd for C21H31BN2O5 [M + H]+ 403.3, found 403.1. 4-Acetyl-1-(2-chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,3-dimethylpiperazin-2-one. HPLC tR = 0.88 min (method A). LCMS (ESI) m/z calcd for C20H28BClN2O4 [M + H]+ 407.3, found 407.0. ( 3-(4 -Acetyl -3, 3-di met hyl- 2- oxop ip erazin -1- yl) -4(methylsulfonyl)phenyl)boronic Acid. HPLC tR = 0.70 min (method A). LCMS (ESI) m/z calcd for C15H21BN2O6S [M + H]+ 369.2, found 369.1. rac-4-Acetyl-1-(2-(1-hydroxyethyl)-5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)phenyl)-3,3-dimethylpiperazin-2-one. HPLC tR = 0.71 min (method A). LCMS (ESI) m/z calcd for C22H33BN2O5 [M + H]+ 417.3, found 417.2. ( 3-(4 -Acetyl -3, 3-di met hyl- 2- oxop ip erazin -1- yl) -4cyanophenyl)boronic Acid. HPLC tR = 0.60 min (method A). LCMS (ESI) m/z calcd for C15H18BN3O4 [M + H]+ 316.2, found 316.0. 1H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J = 7.3 Hz, 1H), 7.79 (s, 1H), 7.77 (dd, J = 7.6, 1.0 Hz, 1H), 3.71−3.84 (m, 4H), 2.10 (s, 3H), 1.70 (s, 6H). (R)-(3-(4-Acetyl-3,3,6-trimethyl-2-oxopiperazin-1-yl)-4cyanophenyl)boronic Acid. HPLC tR = 0.61 min (method A). LCMS (ESI) m/z calcd for C16H20BN3O4 [M + H]+ 330.2, found 330.1. (S)-(3-(4-Acetyl-3,3,6-trimethyl-2-oxopiperazin-1-yl)-4cyanophenyl)boronic Acid. HPLC tR = 0.61 min (method A). LCMS (ESI) m/z calcd for C16H20BN3O4 [M + H]+ 330.2, found 330.1. 4-Acetyl-1-(3-(4-amino-5-chloropyrrolo[2,1-f ][1,2,4]triazin7-yl)-2-methylphenyl)-3,3-dimethylpiperazin-2-one (39). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.71 min (method A). LCMS (ESI) m/z calcd for C21H23ClN6O2 [M + H]+ 426.9, found 427.0. 1H NMR (500 MHz, DMSO-d6) δ 7.87 (s, 1H), 7.30−7.39 (m, 3H), 6.89 (s, 1H), 3.48− 3.84 (m, 4H), 2.08 (s, 3H), 1.92 (s, 3H), 1.68 (d, J = 10.8 Hz, 6H). 4-Acetyl-1-(5-(4-amino-5-chloropyrrolo[2,1-f ][1,2,4]triazin7-yl)-2-methylphenyl)-3,3-dimethylpiperazin-2-one (40). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.71 min (method A). LCMS (ESI) m/z calcd for C21H23ClN6O2 [M + H]+ 426.9, found 427.0. 1H NMR (500 MHz, DMSO-d6) δ 7.94−8.04 (m, 2H), 7.83 (s, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.25 (s, 1H), 3.48−3.91 (m, 4H), 2.15 (s, 3H), 2.09 (s, 3H), 1.73 (s, 3H), 1.67 (s, 3H). 4-Acetyl-1-(3-(4-amino-5-chloropyrrolo[2,1-f ][1,2,4]triazin7-yl)-5-methylphenyl)-3,3-dimethylpiperazin-2-one (41). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.71 min (method A). LCMS (ESI) m/z calcd for C21H23ClN6O2 [M + H]+ 426.9, found 427.0. 1H NMR (500 MHz, DMSO-d6) δ 7.99 (s, 1H), 7.79 (s, 1H), 7.74 (s, 1H), 7.23 (s, 1H), 7.16 (s, 1H), 3.69−3.81 (m, 4H), 2.37 (s, 3H), 2.08 (s, 3H), 1.68 (s, 6H). 4-Acetyl-1-(5-(4-amino-5-chloropyrrolo[2,1-f ][1,2,4]triazin7-yl)-2-(trifluoromethyl)phenyl)-3,3-dimethylpiperazin-2-one (42). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.76 min (method A). LCMS (ESI) m/z calcd for 5204

DOI: 10.1021/acs.jmedchem.7b00618 J. Med. Chem. 2017, 60, 5193−5208

Journal of Medicinal Chemistry

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procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.75 min (method A). LCMS (ESI) m/z calcd for C22H23F3N6O4S [M + H]+ 525.3, found 525.1. 1H NMR (500 MHz, DMSO-d6) δ 8.54−8.59 (m, 1H), 8.24 (s, 1H), 8.19 (s, 1H), 8.08−8.13 (m, 1H), 7.81−7.85 (m, 1H), 3.64−3.91 (m, 4H), 3.19 (s, 3H), 2.11 (s, 3H), 1.73 (s, 3H), 1.71 (s, 3H). rac-4-Acetyl-1-(5-(4-amino-5-(trifluoromethyl)pyrrolo[2,1f ][1,2,4]triazin-7-yl)-2-(1-hydroxyethyl)phenyl)-3,3-dimethylpiperazin-2-one (51). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.75 min (method A). LCMS (ESI) m/z calcd for C23H25F3N6O3 [M-H20+H]+ 473.3, found 473.2. 1H NMR (500 MHz, DMSO-d6) δ 8.13−8.18 (m, 2H), 7.78− 7.83 (m, 1H), 7.64−7.69 (m, 1H), 7.54−7.57 (m, 1H), 5.25−5.19 (m, 1H), 4.68−4.78 (m, 1H), 3.61−3.85 (m, 4H), 2.08−3.12 (m, 3H), 1.70−1.75 (m, 3H), 1.66 (s, 3H), 1.32−1.24 (m, 3H). 2-(4-Acetyl-3,3-dimethyl-2-oxopiperazin-1-yl)-4-(4-amino-5(trifluoromethyl)pyrrolo[1,2-f ][1,2,4]triazin-7-yl)benzonitrile (52). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.81 min (method A). LCMS (ESI) m/z calcd for C22H20F3N7O2 [M + H]+ 472.3, found 472.0. 1H NMR (500 MHz, DMSO-d6) δ 8.43 (d, J = 8.4 Hz, 1H), 8.25 (s, 1H), 8.22 (s, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.79 (s, 1H), 3.82 (d, J = 3.0 Hz, 4H), 2.11 (s, 3H), 1.72 (s, 6H). (R)-2-(4-Acetyl-3,3,6-trimethyl-2-oxopiperazin-1-yl)-4-(4amino-5-chloropyrrolo[2,1-f ][1,2,4]triazin-7-yl)benzonitrile (53a). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.73 min (method A). LCMS (ESI) m/z calcd for C22H22ClN7O2 [M + H]+ 452.2, found 452.1. 1H NMR (400 MHz, DMSO-d6) δ 8.48−8.37 (m, 1H), 8.23 (s, 1H), 8.07−7.94 (m, 2H), 7.50 (s, 1H), 4.26−3.96 (m, 1H), 3.94−3.67 (m, 2H), 2.12 (s, 3H), 1.88 (s, 3H), 1.60 (s, 3H), 1.32−1.20 (m, 3H). (S)-2-(4-Acetyl-3,3,6-trimethyl-2-oxopiperazin-1-yl)-4-(4amino-5-chloropyrrolo[2,1-f ][1,2,4]triazin-7-yl)benzonitrile (53b). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.73 min (method A). LCMS (ESI) m/z calcd for C22H22Cl1N7O2 [M + H]+ 452.2, found 452.1. 1H NMR (400 MHz, DMSO-d6) δ 8.48−8.37 (m, 1H), 8.23 (s, 1H), 8.07−7.94 (m, 2H), 7.50 (s, 1H), 4.26−3.96 (m, 1H), 3.94−3.67 (m, 2H), 2.12 (s, 3H), 1.88 (s, 3H), 1.60 (s, 3H), 1.32−1.20 (m, 3H). (R)-2-(4-Acetyl-3,3,6-trimethyl-2-oxopiperazin-1-yl)-4-(4amino-5-(trifluoromethyl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)benzonitrile (54a). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.83 min (method A). LCMS (ESI) m/z calcd for C23H22F3N7O2 [M + H]+ 486.3, found 486.2. 1H NMR (400 MHz, DMSO-d6) δ 8.54−8.43 (m, 1H), 8.34− 8.20 (m, 2H), 8.11−8.00 (m, 1H), 7.82 (s, 1H), 4.26−3.99 (m, 1H), 3.93−3.69 (m, 2H), 2.09 (s, 3H), 1.88 (s, 3H), 1.59 (s, 3H), 1.33− 1.22 (m, 3H). (S)-2-(4-Acetyl-3,3,6-trimethyl-2-oxopiperazin-1-yl)-4-(4amino-5-(trifluoromethyl)pyrrolo[2,1-f ][1,2,4]triazin-7-yl)benzonitrile (54b). Compound was synthesized according to the procedure outlined for the synthesis of analogue 18 using the appropriate coupling partner. HPLC tR = 0.83 min (method A). LCMS (ESI) m/z calcd for C23H22F3N7O2 [M + H]+ 486.3, found 486.2. 1H NMR (400 MHz, DMSO-d6) δ 8.54−8.43 (m, 1H), 8.34− 8.20 (m, 2H), 8.11−8.00 (m, 1H), 7.82 (s, 1H), 4.26−3.99 (m, 1H), 3.93−3.69 (m, 2H), 2.09 (s, 3H), 1.88 (s, 3H), 1.59 (s, 3H), 1.33− 1.22 (m, 3H). General Methods for Biology Experiments. All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee and conformed to the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH Publication No. 85-23, revised 2011). ADP-Glo Format PI3K Assays. The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (PerkinElmer no. 6008280). The final assay volume was 2 μL prepared from 1 μL

additions of enzyme/PIP2:PS lipid (Invitrogen no. PV5100) mixture and 1 μL of ATP (provided in kit, Promega no. V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 min (PI3Kα, -β, -γ) or 3 h for PI3Kδ. ADP-Glo (2 μL), followed by kinase detection reagent (4 μL), was added to the reaction following the initial incubation and allowed to incubate for 40 min at room temperature. The reaction mixture was analyzed on the TOPCOUNT (PerkinElmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3Kα [0.5 nM], PI3Kβ [2 nM], PI3Kγ [20 nM], PI3Kδ [0.5 nM]. ATP final concentrations are as follows: for α [10 μM], for β [12.5 μM], for γ [6.5 μM], for δ [100 μM]. Lipid final concentration was the same for all enzymes [25 μM]. Dose−response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in DMSO and evaluated at 11 concentrations. The IC50 values were derived by nonlinear regression analysis. HTRF Format PI3K Assays. IC50 values of compounds were determined in a HTRF binding competition assay. The final reaction mixture contained 1 nM kinase, 0.2 nM terbium-labeled anti-His tag antibody, ATP competitive fluorescein-labeled kinase tracer at the respective Kd for each kinase and test compounds in assay buffer consisting of 20 mM HEPES, pH 7.5, 10 mM MgCl2, 0.015% (w/v) Brij-35, 2 mM DTT, and 50 mg/mL BSA. Compounds were dissolved in DMSO and evaluated at 11 concentrations with 3-fold serial dilution. The reactions were incubated at room temperature for 60 min, and then fluorescence intensities at emission wavelengths for fluorescein acceptor (520 nm) and terbium donor (495 nm) were measured on an Envision plate reader (PerkinElmer). Inhibition data were calculated from the 520/495 ratio from no protein control reactions for 100% inhibition and DMSO-only reactions for 0% inhibition. PI3K CD69 Whole Blood Assay. ACD-treated human whole blood patient samples are requested as needed. On the day of the assay, test compounds were 3-fold, 11-point serially diluted in DMSO in a REMP assay plate (Matrix catalog no. 4307) using Tecan, and 5 μL of compound solution was transferred to an ECHO 384-well microplate (Labcyte no. LP-0200). Compounds were dotted 10 nL Matrix microplates, 384-well, polypropylene (Matrix catalog no. 4312) using a Labcyte Echo liquid handling system quickly followed by the addition of 9 μL of whole blood using the CybiWell. Plates were incubated for 1 h in an incubator at 37 °C. Subsequently, dextran conjugated anti-human IgD (Fina Biosolution LLC, catalog no. 0002, rat IgG) was diluted in assay medium consisting of RPMI 1640 with 10% FCS and Pen-strep, to a final concentration of 100 ng/mL. 1 μL of IgD was added to the wells and incubated overnight at 37 °C. The following day, 1 μL/well each of anti-hCD19-APC (BD no. 555415) and anti-hCD69-FITC (BD no. 555530) was added to each well and mixed. Plates were subsequently incubated for 1 h in the dark with gentle shaking at 4 °C. Finally, an amount of 90 μL of warm Lyse/Fix buffer (BD no. 558049) was added to each well, mixed, and incubated for an additional 45 min at 37 °C. Plates were then centrifuged at 2000 rpm for 5 min, and the supernatant was removed using a plate washer. Plates were washed a total of 3 times using dPBS/BSA:1% BSA (Sigma A8327). Samples were then analyzed using the IntelliCyt. Test compounds ranged from final concentration of 10 nM to 170 pM. Inhibition data for the test compound over a range of concentrations were plotted as percentage activation of the test compound. After correcting for background [(sample mean of low control)/(mean of high control − mean of low control)] (low control is DMSO without any compound), IC50 values were determined. PI3K IFNγ AlphaLisa Whole Blood Assay. Human heparinized whole blood (unknown donor) was collected, and 22 μL was added to a 384-well matrix plate (Matrix Microplates, 384-well, polypropylene, 4312). 10 nL of a compound solution (1 mM), serial diluted 3-fold, was then added to the blood and shaken for 30 s. The plates were then 5205

DOI: 10.1021/acs.jmedchem.7b00618 J. Med. Chem. 2017, 60, 5193−5208

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covered and incubated at 37 °C for 60 min. Anti-hCD3 ((OKT3), BMS internal product, 1.65 mg/mL, mouse IgG2a, lot no. CR26sun, 2012) and anti-hCD28 (BD Bioscience no. 340975, mouse IgG1, 1 mg/mL, low azide) antibodies were prepared at 10 μg/mL each and 40 μg/mL of X-link (goat anti-mouse IgG, Pierce no. 31164, 1.8 mg/ mL) antibody in DMEM. The plate was incubated at 37 °C overnight. The following day, 5 μL of warm DMEM was added to each well and the plate was spun at 2500 rpm for 5 min. The AlphaLisa assay (PerkinElmer kit product no. AL217F 5000) was then performed base on the manufacturer’s protocol. 2 μL of supernatant was added to a 384-well Proxiplate (PerkinElmer 6008280). 4 μL AlphaLisa beads/ biotin was then added, and the mixture was incubated at room temperature for 60 min. Subsequently, 4 μL SA donor beads were added to each well and incubated at room temperature for 30 min. The plate was then read on the PerkinElmer Envision. Data were plotted using the Toolset/CurveMaster (BMS proprietary data analysis tools) suite for IC50 determination. Metabolic Stability Assay. Compounds (0.5 μM initial concentration) are incubated with liver microsomes (concentration of microsomal protein of 1 mg/mL) at 37 °C for 6 min. The reaction is initiated by the addition of NADPH (1 mM in a solution of 100 mM Na3PO4, pH 7.4, and 5 mM MgCl2) at 37 °C. Aliquots at times 0 and 10 min are quenched and precipitated at 2700 rpm for 15 min. The supernatant is collected and analyzed by LC−MS/MS. The % remaining is calculated as the peak area at 10 min divided by the peak area at 0 min. Mouse Basophil ex Vivo Assay. Heparin-treated whole blood was obtained from compound dosed in BALB/c mice 1 h after oral administration. Mouse heparinized blood was diluted 1:2 with 1.5% BSA/RPMI1640/p/s. It was then stimulated with 40 ng/mL anti-m IgE for 30 min at 37 °C. After letting sit for 10 min, the blood was stained with anti-IgE-FITC and anti-CD63-PE for 30 min on ice. 7.5 vol of Fix/lyse buffer was added to lyse the red blood cells and fix the other cells. Cells were washed with 2-fold of FACS buffer and resuspended with FACS buffer. Samples were run on BD FACS Canto, and the data were analyzed with Flowjo software. Analysis of each sample for its % CD63+ cells in IgE population was done, and comparison of samples treated with compound to those treated with vehicle was reported. Electrophysiological Effects in Anesthetized Rabbits. Experiments were performed in six adults, male New Zealand white rabbits (3−3.5 kg, fed with normal diet). Rabbits were given compound (n = 3/group) in an iv cumulative dosage of 3, 10, and 20 mg/kg or the same amount of vehicle (n = 3). Vehicle and dosing concentration: PEG400, with dosing solution concentration of 20 mg/mL. Rabbits were preanesthetized with ketamine (25 mg/kg, IM) and xylazine (15 mg/kg, IM), and anesthesia was maintained with a mixture of propofol (Diprivan, AstraZeneca Pharmaceuticals LP, 0.6 mg kg−1 min−1, iv) and fentanyl citrate (0.5 μg kg−1 min−1, iv). Rabbits were intubated and ventilated with 100% oxygen by a mechanical ventilator (Harvard Apparatus, model 665) at a volume of 15 mL/kg and a rate of 8−12 strokes/min. In control experiments, this method yielded an end-tidal CO2 (ETCO2) of approximately 40 mmHg as sampled by an infrared ETCO2 monitor (Datex Normocap 200) positioned in line with the respiratory circuit. The right and left jugular veins were cannulated for intravenous infusion of compound and for anesthetics, respectively. When not infusing anesthetic, normal saline (0.9%) was administered at a rate of 0.3 mL/min via the left jugular vein. The left carotid artery was cannulated and a 4F multielectrode catheter was inserted and advanced to the aortic root for His-bundle electrocardiogram recording. The femoral artery was cannulated for arterial blood pressure monitoring. A recovery period of at least 30 min was used to allow for stabilization of heart rate, blood pressure, and ECG prior to infusion of test compounds. The blood pressure transducer and ECG lead II were connected to a Ponemah system (DATA Sciences International, St. Paul, MN) for data acquisition and analysis. Body surface ECG and His-bundle electrocardiogram were continuously monitored and recorded during the study using Prucka EP system (GE, Milwaukee, WI). Compound was given intravenously over time via an infusion pump at incrementing doses of 3, 10, and 20 mg/kg.

Each dose involved a 10 min infusion of test compound followed by a 2 min monitoring period. The interval between the start of each dose was 12 min. Electrophysiological (EP) parameters measured include QTc interval, intra- and interventricular conduction and atrial to ventricular conduction (QRS duration and PR interval), atrial to ventricular conduction (AH and HV intervals from His-bundle electrogram), as well as heart rate (HR) and arterial blood pressure (BP). Blood (∼0.5 mL/sample) was sampled at baseline and immediately at the end of each infusion. After the last infusion, additional blood samples were taken at 10, 20, and 30 min. Blood samples were collected in tubes of appropriate size containing K2EDTA, inverted several times to ensure mixing, and centrifuged immediately for collecting plasma. All plasma samples were stored frozen at or below −20 °C. The total blood volume taken was ∼5.0 mL/rabbit, which was below the maximum blood volume (14 mL based on 7% of 200 mL blood) that can be removed without significant disturbance to rabbit normal physiology.24 Study parameters were averaged from 1 min ECG recording at baseline and immediately after each infusion, as well as at time points of blood sampling. QTc interval was corrected for heart rate effects (Fridericia or Van De Water correction). Effects on AH and HV intervals from His-bundle electrogram were assessed by manual measurements from ≥3 cardiac cycles. KLH-Induced Serum IgG Production in Mice. Female BALB/c mice (20 g, Envigo) were immunized intraperitoneally with 250 μg/ mouse of keyhole limpet hemocyanin (KLH, Sigma-Aldrich, St. Louis, MO) diluted in phosphate buffered saline. Test compound (dissolved in 85% PEG300/5% Pluronic L44/10% 400 mM citric acid) was administered by oral gavage twice daily beginning on the day of immunization. Blood samples were collected on day 13 under isoflurane anesthesia. 4-Fold serial dilutions of serum that had been separated from clotted samples by centrifugation (5 min, 5000g, room temperature) were added in duplicate to flat-bottomed 96-well ELISA plates precoated with KLH (10 μg/mL) overnight at 4 °C and blocked with milk diluent. After a 2 h incubation at room temperature, the plates were washed, and horseradish peroxidase-conjugated goat antimouse anti-IgG detection antibody (Southern Biotechnology, Birmingham, AL) was added. Following a final washing step, the plates were developed with tetramethylbenzidine substrate (Kirkegard and Perry, Gaithersburg, MD) and read on a spectrophotometer at 450 nm. Anti-KLH titers were expressed as the reciprocal of the serum dilution that yielded an optical density of 1.0 (within the linear portion of the dilution curve). Collagen-Induced Arthritis in Mice. Male DBA/1 mice (20−25 g, Harlan) were immunized with 200 μg/mouse of bovine type II collagen (Elastin Products Company, Owensville, MO) in Sigma Adjuvant System (Sigma-Aldrich, St. Louis, MO) in 0.1 mL at the base of the tail on day 0 and on day 21. Vehicle or test compound in vehicle was administered by oral gavage twice daily beginning on day 1. Mice were monitored after the second immunization for the development of paw inflammation. Each paw was individually scored as follows: 0 = normal; 1 = one or more swollen digits; 2 = mild paw swelling; 3 = moderate paw swelling; 4 = fusion of joints/ankylosis. The scores for all four paws are summed for each mouse, and results are shown as the mean of n = 12 mice per treatment group. Statistical significance among the groups was determined using the nonparametric Mann− Whitney U test.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00618. Computational model of chloro compound 35 (CC( O)N1CCN(C2CC(CCC2)C2CC(Cl) C3N2NCNC3N)C(O)C1(C)C) based on the X-ray cocrystal structure of pyrazole 22 bound to the kinase domain of PI3Kδ (PDB code 5EM) (PDB) 5206

DOI: 10.1021/acs.jmedchem.7b00618 J. Med. Chem. 2017, 60, 5193−5208

Journal of Medicinal Chemistry

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Accession Codes

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X-ray cocrystal structure of pyrazole 22 (CC(O)N1CCN(C2CCCC(C2)C2CC(C3CCNN3CC(F)(F)F)C3N2NCNC3N)C(O)C1(C)C) in PI3Kδ (PDB code 5EM). Authors will release the atomic coordinates and experimental data upon article publication.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (609) 252-3980. ORCID

David Marcoux: 0000-0002-0295-7717 Present Addresses §

K.J.: School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas 79106. ∥ J.E.M.: Integrated Drug Discovery, Sanofi, 153 2nd Avenue, Waltham, MA 02451. Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Proteros Biostructures GmbH for the X-ray cocrystal structure of compound 22 in PI3Kδ. We are also grateful to Claude Quesnelle, Maude Poirier, Lalgudi Harikrishnan, John Duncia, Samuel Gerritz, and Ruth Wexler for carefully reviewing this manuscript and for helpful discussions.

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ABBREVIATIONS USED PI3K, phosphoinositide 3-kinase; KLH, keyhole limpet hemocyanin; hERG, human ether-a-go-go-related gene; CIA, collagen induced arthritis; PIP2, phosphatidylinositol 4,5bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PH, pleckstrin homology; AKT, protein kinase B; PDK1, phosphoinositide-dependent kinase 1; BTK, Burton’s tyrosine kinase; mTOR, mechanistic target of rapamycin; KO, knockout; FDA, Food and Drug Administration; PSA, polar surface area; CD, cluster differentiation; ADME, absorption, distribution, metabolism, and excretion; hWB, human whole blood; ADP, adenosine diphosphate; Met Stab, metabolic stability; Prot, protein; BMS, Bristol-Myers Squibb; % rem, % remaining; PAMPA, parallel artificial membrane permeability assay; SAR, structure−activity relationship; po, per os, oral; AUC, area under the curve; EP, electrophysiology; IC, inhibitory concentration; EC, effective concentration; Cmax, maximal concentration; ROS, reactive oxygen generation; PK, pharmacokinetic; MNK1, MAP kinase-interacting serine/threonineprotein kinase 1; MLCK, myosin light-chain kinase; HTRF, homogeneous time-resolved fluorescence; iv, intravenous; CYP, cytochrome P450; CLK4, CDC-like kinase 4; mIgE, mouse immunoglobulin E; b.i.d., twice a day

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REFERENCES

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O’Brien, S. M. Idelalisib and rituximab in relapsed chronic lymphocytic leukemia. N. Engl. J. Med. 2014, 370, 997−1007. (b) Winkler, D. G.; 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. PI3Kδ and PI3K-γ inhibition by IPI-145 abrogates immune responses and suppresses activity in autoimmune and inflammatory disease models. Chem. Biol. 2013, 20, 1364−1374. (12) Qin, L.-Y.; Ruan, Z.; Cherney, R. J.; Dhar, T. G.; Neels, J.; Weigelt, C. A.; Sack, J. S.; Srivastava, A. S.; Cornelius, L. A.; Tino, J. A.; Stefanski, K.; Gu, X.; Xie, J.; Susulic, V.; Yang, X.; Yarde-Chinn, M.; Skala, S.; Bosnius, R.; Goldstein, C.; Davies, P.; Ruepp, S.; Salter-Cid, L.; Bhide, R. S.; Poss, M. A. Discovery of 7-(3-(piperazin-1yl)phenyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine derivatives as highly potent and selective PI3Kδ inhibitors. Bioorg. Med. Chem. Lett. 2017, 27, 855−861. (13) Bhide, R. S.; Neels, J.; Qin, L.-Y.; Ruan, Z.; Stachura, S.; Weigelt, C.; Sack, J. S.; Stefanski, K.; Gu, X.; Xie, J. H.; Goldstine, C. B.; Skala, S.; Pedicord, D. L.; Ruepp, S.; Dhar, T. G.; Carter, P. H.; Salter-Cid, L. M.; Poss, M. A.; Davies, P. Discovery and SAR of pyrrolo[2,1f][1,2,4]triazin-4-amines as potent and selective PI3Kδ inhibitors. Bioorg. Med. Chem. Lett. 2016, 26, 4256−4260. (14) A clear correlation between efflux and exposure has been observed with this chemotype. In general, exposure is affected with efflux of >6 while efflux of >10 always lead to low exposure. (15) This molecule has otherwise a clean Na and Ca channels profile. Na channel % inhibition at 10 μM is 0.1% (1 Hz) and 1% (4 Hz). Ca L-type channel % inhibition at 10 μM is 5%. (16) In vivo analysis also showed that the inhibition of PI3Kγ reduces blood pressure supposedly due to L-type Ca channel (LTCC) subunit trafficking: Carnevale, D.; Vecchione, C.; Mascio, G.; Esposito, G.; Cifelli, G.; Martinello, K.; Landolfi, A.; Selvetella, G.; Grieco, P.; Damato, A.; Franco, E.; Haase, H.; Maffei, A.; Ciraolo, E.; Fucile, S.; Frati, S.; Mazzoni, O.; Hirsch, E.; Lembo, G. PI3Kγ inhibition reduces blood pressure by a vasorelaxant Akt/L-type calcium channel mechanism. Cardiovasc. Res. 2012, 93, 200−209. (17) Analogue 32 was not tested in a hERG PC assay due to low in vitro metabolic stability. (18) See supporting PDB file. (19) Marcoux, D.; Qin, L.-Y.; Ruan, Z.; Shi, Q.; Ruan, Q.; Weigelt, C.; Qiu, H.; Schieven, G.; Hynes, J.; Bhide, R.; Poss, M.; Tino, J. Identification of highly potent and selective PI3Kδ inhibitors. Bioorg. Med. Chem. Lett. 2017, 27, 2849−2853. (20) (a) Aragoneses-Fenoll, L.; Montes-Casado, M.; Ojeda, G.; Acosta, Y. Y.; Herranz, J.; Martinez, S.; Blanco-Aparicio, C.; Criado, G.; Pastor, J.; Dianzani, U.; Portolés, P.; Rojo, J. M. ETP-46321, a dual p110α/δ class IA phosphoinositide 3-kinase inhibitor modulates T lymphocyte activation and collagen-induced arthritis. Biochem. Pharmacol. 2016, 106, 56−69. (b) See ref 11b. (21) Markwalder, J. A.; Fink, B. E.; Gavai, A. V.; He, L.; Kim, S.-H.; Seitz, S. P. Substituted pyrrolotriazines as protein kinase inhibitors. WO 2011/123493, 2011. (22) Bhide, R. S.; Ruan, Z.; Cherney, R. J.; Cornelius, L. A. M.; Dhar, T. G. M.; Gong, H.; Marcoux, D.; Poss, M. A.; Qin, L.-Y.; Shi, Q.; Tino, J. A. Heteroaryl substituted pyrrolotriazine amine compounds as PI3K inhibitors. WO 2016064958 A1, 2016. (23) Bhide, R. S.; Batt, D. G.; Cherney, R. J.; Cornelius, L. A. M.; Liu, Q.; Marcoux, D.; Neels, J.; Poss, M. A.; Qin, L.-Y.; Ruan, Z.; Shi, Q.; Srivastava, A. S.; Tino, J. A.; Watterson, S. H. Bicyclic heteroaryl amine compounds as PI3K inhibitors. WO 2016064957 A1, 2016. (24) Veterinary Sciences SOPHopewell, Animal Care and Use Subcommittee SOP.

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