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Synthesis, SAR and Series Evolution of Novel Oxadiazole-Containing 5-Lipoxygenase Activating Protein Inhibitors: Discovery of 2-[4(3-{(R)-1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-1-cyclopropyl-ethyl}[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (BI 665915) Hidenori Takahashi, Doris Riether, Alessandra Bartolozzi, Todd Bosanac, Valentian Berger, Ralph Binetti, John Broadwater, Zhidong Chen, Rebecca Crux, Stéphane De Lombaert, Rajvee Dave, Jonathon A Dines, Tazmeen Fadra-Khan, Adam Flegg, Michael Garrigou, Ming-Hong Hao, John David Huber, J Matthew Hutzler, Steven Kerr, Adrian Kotey, Weimin Liu, Ho Yin Lo, Pui Leng Loke, Paige Erin Mahaney, Tina M Morwick, Spencer Napier, Alan Olague, Edward Pack, Anil Padyana, David S Thomson, Heather Tye, Lifen Wu, Renee M Zindell, Asitha Abeywardane, and Thomas Simpson J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501185j • Publication Date (Web): 14 Jan 2015 Downloaded from http://pubs.acs.org on January 24, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Mahaney, Paige; Boehringer-Ingelheim, Medicinal Chemistry Morwick, Tina; Boehringer Ingelheim Pharmaceuticals, Inc., Medicinal Chemistry Napier, Spencer; Evotec (UK) Ltd, Discovery Olague, Alan; Boehringer Ingelheim Pharmaceuticals, Inc., Medicinal Chemistry Pack, Edward; Boehringer Ingelheim Pharmaceuticals, Inc., Medicinal Chemistry Padyana, Anil; Boehringer Ingelheim Pharmaceuticals, Inc., Medicinal Chemistry Thomson, David; Biomanufacturing Research Institute and Technology Enterprise, Tye, Heather; Evotec UK Ltd, Wu, Lifen; Boehringer Ingelheim Pharmaceuticals, Inc., Medicinal Chemistry Zindell, Renee; Boehringer Ingelheim Pharmaceuticals, Inc., Medicinal Chemistry Abeywardane, Asitha; Boehringer Ingelheim Pharmaceuticals, Inc., Medicinal Chemistry Simpson, Thomas; Boehringer Ingelheim Pharmaceuticals, Inc., Medicinal Chemistry

ACS Paragon Plus Environment


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Synthesis, SAR and Series Evolution of Novel Oxadiazole-Containing 5-Lipoxygenase Activating Protein Inhibitors: Discovery of 2-[4-(3-{(R)-1-[4(2-Amino-pyrimidin-5-yl)-phenyl]-1-cyclopropylethyl}-[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-N,Ndimethyl-acetamide (BI 665915) Hidenori Takahashi,*,† Doris Riether, † Alessandra Bartolozzi, † Todd Bosanac, † Valentina Berger, # Ralph Binetti, † John Broadwater, # Zhidong Chen, † Rebecca Crux, ≠ Stéphane De Lombaert, †,¦ Rajvee Dave, # Jonathon A. Dines,≠ Tazmeen Fadra-Khan, † Adam Flegg, ≠ Michael Garrigou, ≠ Ming-Hong Hao, † John Huber, †, Φ J. Matthew Hutzler, †,║ Steven Kerr, # Adrian Kotey, ≠ Weimin Liu, †,§ Ho Yin Lo, †,‡ Pui Leng Loke, ≠ Paige E. Mahaney, † Tina M. Morwick, † Spencer Napier, ≠ Alan Olague, † Edward Pack, † Anil Padyana, † David S. Thomson, †,^ Heather Tye, ≠ Lifen Wu, † Renee M. Zindell,† Asitha Abeywardane, † and Thomas Simpson† AUTHOR ADDRESS †

Department of Medicinal Chemistry, #Cardiometabolic Diseases, Boehringer Ingelheim

Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06877, United States.


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≠

Evotec, 114 Innovation Drive, Milton Park, Abingdon, Oxfordshire OX14 4RZ, United

Kingdom. *Corresponding author ABSTRACT The synthesis, structure-activity relationship (SAR), and evolution of a novel series of oxadiazole-containing 5-lipoxygenase-activating protein (FLAP) inhibitors are described. The use of structure-guided drug design techniques provided compounds that demonstrated excellent FLAP binding potency (IC50 < 10 nM) and potent inhibition of LTB4 synthesis in human whole blood (IC50 < 100 nM). Optimization of binding and functional potencies, as well as physicochemical properties resulted in the identification of 69 (BI 665915) that demonstrated an excellent cross species DMPK profile and was predicted to have low human clearance. In addition, 69 was predicted to have a low risk for potential drug-drug interactions due to its cytochrome P450 3A4 profile. In a murine ex vivo whole blood study, 69 demonstrated a linear dose-exposure relationship and a dose-dependent inhibition of LTB4 production.

N N

N H2N

N

O NN

69 (BI 665915)

O

N


2


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INTRODUCTION Leukotrienes (LTs) are a family of eicosanoid proinflammatory mediators that are biosynthesized from arachidonic acid (AA) via oxidative metabolism.1 The LT pathway constitutes a fundamental series of events underlying the inflammatory components of various diseases such as asthma, allergy,2 and atherosclerosis.3 This pathway is activated by inflammatory stimuli resulting in the release of AA from cell membrane by cytosolic phospholipase A2. The membrane-attached 5-lipoxygenase activating protein (FLAP) binds to AA and selectively transfers AA to 5-lipoxygenase (5-LO), which oxidizes AA to 5hydroperoxyeicosatetraenoic acid (5-HpETE).4 The subsequent dehydration of 5-HpETE produces LTA4 that is a branch point in the biosynthetic pathway. LTA4 can be converted to either LTB4 through the action of LTA4 hydrolase, or to LTC4 by LTC4 synthase. LTC4 is the first of the cysteinyl LTs (CysLTs) in the biosynthetic pathway and can be transformed into LTD4 and LTE4. These lipid mediators trigger proinflammatory signaling through activation of G protein-coupled transmembrane receptors (GPCRs), namely BLT1 and BLT2 for LTB4, and CysLT1, CysLT2, CysLTER for CysLTs (Scheme 1). Inhibitors of the biosynthesis of LTs and antagonists of the CysLT1 receptor have demonstrated clinical benefits in asthma and allergic diseases.2 Although a number of chemical classes of LT pathway inhibitors have been explored, only limited compounds have been approved for clinical use (Figure 1). For example, Zileuton, an inhibitor of 5-LO, has been registered for the treatment of asthma,5 but has experienced limited use partly due to hepatotoxicity side effects.6 Additionally, CysLT1 antagonists such as Zafirlukast, Montelukast and Pranlukast have been approved for the treatment of asthma and allergic rhinitis.7 Through the course of research on LTs, it has become clear that LTs have pathophysiological roles across a wide range of disease including: respiratory diseases, allergic


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diseases and cardiovascular diseases such as asthma, allergic rhinitis, COPD, arthritis, inflammatory bowel disease, psoriasis, liver fibrosis, cancer, endothelial dysfunction, intimal hyperplasia, atherosclerosis, myocardial dysfunction, ischemic stroke, aortic aneurysms, and angiogenesis.3,8,9 Scheme 1. Leukotriene (LT) pathway.

HO

NH2

O O

N O

H N S

H N

O

O

O

S

O N

Zileuton

Zafirlukast OH O S

Cl

O N H

N

O

O O

HN Montelukast

OH

Pranlukast

N

N N


4


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Figure 1. Marketed LT pathway inhibitors. FLAP was first discovered in early 1990s as an accessory protein essential for the cellular biosynthesis of LTs.10 Indeed, compounds that prevent the binding of AA to FLAP are able to completely inhibit the leukotriene biosynthesis pathway. As a result, FLAP inhibition represents an attractive drug discovery approach for the treatment of various inflammatory diseases, including respiratory and cardiovascular diseases. The first FLAP inhibitor, MK-886, was reported by Merck-Frosst and featured an indole core substituted with a carboxylic acid sidechain.11 Originally, the target of MK-886 was unknown; but, the compound demonstrated the ability to block LT biosynthesis without affecting 5-LO. The use of a close analog of MK-886 acting as a photoaffinity probe and cDNA cloning experiments led to the discovery of FLAP as a required accessory protein in the conversion of AA to LTA4.12 In addition to MK-886, Figure 2 shows four other selective FLAP inhibitors that have advanced to clinical trials.13 These compounds share structural features such as a rigid scaffold that is decorated with an alkyl carboxylic acid.14 ABT-080 was discontinued after phase I clinical trials. MK-886,15 MK-59116 and BAY X100517 showed efficacy in allergic asthma models, but in contrast to Zileuton (5-LO inhibitor),18 were devoid of hepatotoxic adverse events. MK-591 was efficacious in phase II clinical trials on asthma patients16 and BAY X1005 completed a phase II trial with asthmatic patients.17 However, further development of MK-591 was discontinued due to the insufficient improvement in the clinical studies,19 and deCODE discontinued the development of BAY X 1005 due to formulation issues. The most recent FLAP inhibitor to advance into phase II clinical trials (asthma) is AM-803, (a.k.a. GSK-2190915),14 which has shown good safety and tolerability, as well as efficacy on allergen-induced asthmatic response in patients with mild asthma.20


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S

S O

O

N

O

N

N

OH

OH

Cl

Cl MK-886

MK-591 S

O N

OH N

O

O O N

O

OH OH O

O

N

BAY X1005 (DG031)

N

ABT-080 (VML-530)

O

N AM-803 (GSK-2190915)

Figure 2. FLAP inhibitors previously or presently tested in clinical trials. Genetic, pharmacological, and clinical studies support the use of LT modulators beyond the treatment of asthma. Specifically, pre-clinical studies have showed that LTs play pathophysiological roles in cardiovascular diseases.21 In addition, a genetic link between the FLAP gene (ALOX5AP) and stroke in a central European population has been reported.22 A clinical study analyzing the occurrence of cardiovascular endpoints in asthmatic patients treated with Montelukast found a modest decrease in risk of recurrent stroke and myocardial infarction.23 In patients, the 5-LO inhibitor, VIA-2291 (Atreleuton), produced a statistically significant reduction in noncalcified plaque volume at 24 weeks as compared to the placebo group.24 The FLAP inhibitors MK-886 and BAY X1005 have also been effective in atherosclerosis animal models, where they demonstrated a statistically significant reduction of atherosclerotic lesion area in the aorta in the apoE/LDLR double knockout mice model.25 Recently, Merck has reported a new class of bis-aryl-containing FLAP inhibitors that have demonstrated potent in vivo inhibition of urinary LTE4 production in dogs.26 The links between


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FLAP inhibition and pharmacological effects in atherosclerosis animal models led us to pursue this approach for the treatment of various cardiovascular indications, including atherosclerosis. This publication describes the structure-guided design, synthesis, structure-activity relationship (SAR), and biological evaluation of novel oxadiazole-containing FLAP inhibitors, including their ability to reduce LTB4 levels in mice. CHEMISTRY The general strategy to synthesize the oxadiazole-containing FLAP inhibitors is shown in Scheme 2. According to this approach, dialkylation of 4-bromophenylacetonitrile 1 would generate a quaternary nitrile of formula 2, which could be converted to the amidoxime 3 by treatment with hydroxylamine. Cyclization of 3 with a compound bearing an activated carboxylic acid moiety could produce an oxadiazole of formula 4, which could be further functionalized via Suzuki-Miyaura cross-coupling reaction to generate the FLAP inhibitors 5. Scheme 2. General synthesis of oxadiazole-containing FLAP inhibitors. O N

R1

Base

R2

Br

H2N OH

R1

N Br 1

N

Br 2

R2

3

R1 NH2 OH

R3

R2

OH

Suzuki-Miyaura coupling

N N

Br

R1

R2 N

O R3

N

Ar

4

O R3

5

The synthesis of compounds 12-14 is depicted in Scheme 3. Installation of a cyclobutyl group on 4-bromophenylacetonitrile 1 was accomplished via alkylation with 1,3-dibromopropane followed by the in situ intramolecular cyclization to give 6, which was converted to the amidoxime 7 upon the addition of hydroxylamine. Intermediate 7 was heated with trichloroacetic anhydride to form the oxadiazole ring. Treatment of 8 with an excess of


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ammonium hydroxide or ethylamine led to the formation of intermediates 9 and 10, respectively. Intermediate 11 was prepared from the intermediate 9 by treatment with methane sulfonyl chloride. The aryl bromides 9-11 were subsequently coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under microwave-assisted Suzuki-Miyaura cross-coupling conditions to afford the desired compounds 12-14. Scheme 3. Synthesis of 5-amino-[1,2,4]oxadiazole compounds (12-14).

b

a

N

NH2

N

Br

Br

N

Br

1

6

N

d

O

N

Br

N

f

e

9 (R=H) 10 (R=Et) 11(R=SO2Me)

N

N

NH R

7

H2N

OH

c

N N

Br 8

O

Cl Cl

Cl

O NH

N

R 12 (R=H) 13 (R=Et) 14(R=SO2Me)

Reagents and conditions: (a) NaH (60 % disp., 2.2 equiv.), DMF, 0 oC, 15 min.; then 1,3dibromopropane (1.1 equiv.), room temperature, 16 h; (b) hydroxylamine (50 % in water, 1.5 equiv.), EtOH, 80 oC, 16 h; (c) trichloroacetic anhydride (1.2 equiv.), toluene, 110 oC, 2 h; (d) ammonium hydroxide or ethyl amine (1.1 equiv.), DMF, room temperature, 0.5 h; (e) methane sulfonyl chloride (1.1 equiv.), pyridine (1.1 equiv.), dichloromethane, 0 oC, 1 h; then 50 oC, 8 h; (f) 2-aminopyrimidine-5-boronic acid pinacol ester (1.2 equiv.),


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bis(triphenylphosphine)palladium(II) chloride (0.1 equiv.), 2 M Na2CO3 solution (2.0 equiv.), DMF, 90 oC, microwave (150 W), 20 min.

The amidoxime 7 and the nitrile 6 also served as an advanced intermediate for the synthesis of compounds 16-21 and 23-33, respectively (Scheme 4). Reaction of 7 under basic conditions with various aromatic acyl chlorides or activated esters, generated from carboxylic acids in the presence of a coupling reagent, followed by Suzuki-Miyaura cross-coupling reaction provided compounds 16-21. This sequence enabled the installation of the desired aromatic substituents on the oxadiazole ring. Alternatively, compounds 23-33, featuring different aromatic substituents on the oxadiazole moiety, were synthesized by first introducing the 2-aminopyrimidine followed by the conversion of nitrile to amidoxime and oxadiazole ring formation. Scheme 4. Synthesis of 5-aryl-[1,2,4]oxadiazole compounds (16-22 and 23-33).

NH2 N

Br

a N

Br

OH

O

N

N

Ar

7

N

b

N

Ar

H2N

15

N 16 (Ar=Phenyl) 17 (Ar=3-pyridine) 18 (Ar=4-pyridazine) 19 (Ar=2-pyrazine) 20 (Ar=5-pyrimidine) 21 (Ar=4-imidazole)

or

NH2

b, c N

N

N

Br 6

H2N

N

O

22

N

a N

N

OH H2N

O Ar

N

d

23 (Ar=3-pyridazine) 24 (Ar=4-pyrimidine) 25 (Ar=2-chloro-3-pyridine) 26 (Ar=4-1H-[1,2,3]triazole) 27 (Ar=3-1H-[1,2,4]-triazole) 28 (Ar=4-thiazole) 29 (Ar=4-oxazole) 30 (Ar=3-pyrazole) 31 (Ar=4-pyrazole) 32 (Ar=3-1H-pyridin-2-one) 33 (Ar=1-methyl-4-pyrazole)


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Reagents and conditions: (a) Ar-COCl, pyridine, DCM, 0 oC then room temperature, 16 h; or ArCOOH, thionyl chloride, pyridine, room temperature then 110 oC; or Ar-COOH, HATU or CDI, triethylamine, NMP, heat, 4 h; (b) 2-aminopyrimidine-5-boronic acid pinacol ester (1.2 equiv.), bis(triphenylphosphine)palladium(II) chloride (0.1 equiv.), 2 M Na2CO3 solution (2.0 equiv.), DMF, 90 oC, microwave (150 W), 20 min.; (c) hydroxylamine (50 % in water), EtOH, 80 oC, 16 h; (d) 10 % LiOH in water / 1,4-dioxane (1 : 4), 70 oC, 16 h.

Most of the compounds featuring an N-substituted pyrazole (34-40) were synthesized by direct alkylation reaction of 31 with various alkyl halides (Scheme 5). However, since alkylation of 31 with 1-chloro-2-methyl-2-propanol was low yielding, compound 42 was synthesized by alkylating the pyrazole ring on intermediate 41, followed by the incorporation of the 2-aminopyrimidine group. Scheme 5. Synthesis of oxadiazoles with N-substituted 4-pyrazole substituents (34-40 and 42). O

34 R= * N N

N H2N

N

31

a

O

N

N

N H

N

b

N

H2N

O

O O

36 R=*

N

N

c

R

N

*

38 R=

O

NH2

*

39 R=

or

O

O

37 R=

N

OH

35 R= *

40 R=

N

*

N

* O

N N

Br

OH

N

N

d N

Br

e,f

O

N H2N

7 41

N H

N

N


N

O

N

N

42 HO

10


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Reagents and conditions: (a) R-X, K2CO3 or Cs2CO3, DMF, room temperature, 16 h; (b) LiOH (3.0 equiv.), THF, MeOH, water, 40 oC, 90 min.; (c) hydrazine hydrate (8.8 equiv.), EtOH, 50 o

C, 2 h; (d) 1H-pyrazole-4-carboxylic acid (1.1 equiv.), CDI (1.1 equiv.), NMP, 100 oC, 4 h; (e)

1-chloro-2-methyl-2-propanol (2.0 equiv.), K2CO3, DMF, 90 oC, 40 h; (f) 2-aminopyrimidine-5boronic acid pinacol ester (1.2 equiv.), bis(triphenylphosphine)palladium(II) chloride (0.1 equiv.), 2 M Na2CO3 solution (2.0 equiv.), DMF, 90 oC, microwave (150 W), 20 min.

Compounds featuring other C(8) substituents were synthesized by the route summarized in Scheme 6. The unsubstituted compound 44 was obtained by the installation of the left hand side 2-amino-pyrimidine onto the amidoxime 43 followed by the oxadiazole ring construction on the right hand side, which appended the 1-methyl-1H-pyrazole group to the molecule. Alternatively, substituted analogs 52-58 were synthesized by first introducing the 1-methyl-1H-pyrazole group followed by the incorporation of the 2-amino-pyrimidine moiety. Enantiomeric pure compounds 59-64 were obtained by chiral supercritical fluid chromatography (SFC) or chiral HPLC resolution of the corresponding racemates. The absolute configuration of chiral centers of 59-64 were assigned based on single-crystal X-ray diffraction studies of representative compounds. As an example, the X-ray crystal structure of 69 (BI 665915)27 is shown in Figure 3. The calculated Flack parameter for this structure is 0.0137 with esd of 0.1632 confirming the assignment of the absolute configuration at C(8).28


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Scheme 6. Synthesis of various C(8) unsubstituted (44), mono-substituted (55), and disubstituted compounds (52-54 and 56-64).

N NH2

a

N Br

N

Br

H2N

OH

N

N

44

N

d R1 R1

R1

R2 N

R2 c, a, b N

Br

N

43

1

or

b, c

O

N

N

N H2N

45 (R1=Me, R2=Me) 46 (R1,R2=c-Pro) 47 (R1,R2=2,2-dimethyl-c-Bu) 48 (R1=i-Pro, R2=H) 49 (R1=i-Pro, R2=Me) 50 (R1=c-Pro, R2=Me)

N

N

C(8)

O

N

R2

e

N

N

N H2N

52 (R1=Me, R2=Me) 53 (R1,R2=c-Pro) 54 (R1,R2=2,2-dimethyl-c-Bu) 55 (R1=i-Pro, R2=H) 56 (R1=i-Pro, R2=Me) 57 (R1=c-Pro, R2=Me) 58 (R1=c-Bu, R2=Me)

N

O

N

N

59 (R1=i-Pro, R2=Me) 60 (R1=Me, R2=i-Pro) 61 (R1=c-Pro, R2=Me) 62 (R1=Me, R2=i-Pro) 63 (R1=c-Bu, R2=Me) 64 (R1=Me, R2=c-Bu)

Reagents and conditions: (a) hydroxylamine (50 % in water, 1.5 equiv.), EtOH, 80 oC, 16 h; (b) 2-aminopyrimidine-5-boronic acid pinacol ester (1.2 equiv.), bis(triphenylphosphine)palladium(II) chloride (0.1 equiv.), 2 M Na2CO3 solution (2.0 equiv.), DMF, 90 oC, microwave (150 W), 20 min.; (c) 1-methyl-1H-pyrazole-4-carboxylic acid (1.1 equiv.), CDI (1.1 equiv.), NMP, 100 oC, 4 h; (d) NaH (60 % disp., 2.2 equiv.), DMF, 0 oC, 15 min.; then R1-X, room temperature, 16 h, repeat the same protocol with R2-X for compounds 49-51; (e) Chiral HPLC or SFC separation.


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Figure 3. X-ray structure of 69. Non-hydrogen atoms are shown as thermal ellipsoids with each atom position labeled. Nitrogen atoms are displayed in blue, carbons are shown in yellow, oxygens are depicted in red and hydrogens are illustrated in grey. Data and refinement statistics are summarized in supporting information (Supporting information Table 1). To introduce diversity on the left-hand side phenyl ring, racemate 50 was resolved by chiral SFC to provide the (R)-enantiomer 65 (> 99 % ee) (Scheme 7). Intermediate 65 was transformed to 67 following the protocol described in schemes 4 and 5. Subsequently, intermediate 67 was reacted with aryl boronic acid pinacol esters under Suzuki-Miyaura cross-coupling conditions to afford the desired compounds 68 and 69. To extend the SAR beyond the scope of commercially available aryl boronic esters, intermediate 67 was converted to boronic acid pinacol ester 70 by palladium catalyzed borylation. The versatile boronic acid ester 70 was used as an advanced


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intermediate for the cross-coupling reactions with readily available aryl halides to obtain compounds 71-74. Scheme 7. Synthesis of compounds with various phenyl substitutions (68, 69 and 71-74).

a

N

N

Br

N

Br

67

N

N

Ar

O N

N

e

O

N

Br

O O

66

65

N

N

NH

67

N

N

N

O O

N

N

N

N

68 (Ar=2-aminopyridine-5-yl) 69 (Ar=2-aminopyrimidine-5-yl) (BI 665915)

f

N

N O

Br

d

O

N

Br

50

N

N

b, c

N

B

g

O

Ar

N

O O

O

O 70

N

N

N

N

N

N

71 (Ar=5-aminopyrazine-2-yl) 72 (Ar=2-methylaminopyrimidine-5-yl) 73 (Ar=2-ethylaminopyrimidine-5-yl) 74 (Ar=2-isopropylaminopyrimidine-5-yl)

Reagents and conditions: (a) chiral SFC separation; (b) hydroxylamine (50 % in water, 1.5 equiv.), EtOH, 80 oC, 16 h; (c)1H-pyrazole-4-carboxylic acid (1.1equiv.), CDI (1.1 equiv.), NMP, 100 oC, 16 h; (d) 2-chloro-N,N-dimethyl-acetamide (2.0 equiv.), K2CO3 (2.0 equiv.), DMF, room temperature, 16 h; (e) Ar-boronic acid pinacol ester (1.2 equiv.), bis(triphenylphosphine)palladium(II) chloride (0.1 equiv.), 2 M Na2CO3 solution (2.0 equiv.),


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DMF, 90 oC, microwave (150 W), 20 min.; (f) bis(pinacolato)diboron (1.5 equiv.), 1,1’bis(diphenylphosphino)ferrocenedichloropalladium (II) dichloromethane (0.1 equiv.), potassium acetate (4.0 equiv.), 1,4-dioxane, 100 oC, 4 h; (g) Ar-Br, bis(triphenylphosphine)palladium(II) chloride (0.1 equiv.), 2 M Na2CO3 solution (2.0 equiv.), DMF, 90 oC, microwave (150 W), 20 min. Finally, compounds 76 and 77 were synthesized from the enantiomerically pure intermediate 65. As illustrated in scheme 8, the 2-amino-pyrimidine ring was added to 65 using SuzukiMiyaura cross-coupling conditions. The formation of amidoxime followed by the oxadiazole ring construction gave intermediate 75, which was alkylated on the pyrazole nitrogen to afford compounds 76 and 77. Scheme 8. Synthesis of compounds 76 and 77.

N

a, b, c N

N

N

Br 65

H2N

N

75

N

d

O

N

N

N

NH

H2N

N

O

N

N

R

76 (R=N,N-dimethyl-2-ethylamine) 77 (R=2-methyl-2-propanol)

Reagents and conditions: (a) 2-aminopyrimidine-5-boronic acid pinacol ester (1.2 equiv.), bis(triphenylphosphine)palladium(II) chloride (0.1 equiv.), 2 M Na2CO3 solution (2.0 equiv.), DMF, 90 oC, microwave (150 W), 20 min; (b) hydroxylamine (50 % in water, 1.5 equiv.), EtOH, room temperature, 16 h; (c) 1H-pyrazole-4-carboxylic acid (1.1 equiv.), CDI (1.1 equiv.), NMP, 100 oC, 16 h; (d) R-X (1.5 equiv.), Cs2CO3 (2.3 equiv.), DMF, 60 oC, 16 h.


15

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Results and Discussion The X-ray crystal structure of human FLAP/MK-591 complex was first reported in 2007 (PDB ID: 2Q7M).29 It revealed that FLAP crystallized as a homotrimer, with MK-591 bound in a groove located at the interface of adjacent monomers, exposed to lipid bilayer. Using this information in combination with published SAR on bis-aryl FLAP inhibitors,26,30 we designed new lead matters that targeted the same membrane-embedded pocket of FLAP. These efforts led to the identification of a series of substituted oxadiazoles, which were predicted by docking studies to occupy the same binding pocket as MK-591. Indeed, the initial FLAP inhibition SAR of these oxadiazoles supported the predicted binding mode (Figure 4A).


16


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

Page 18 of 74

B

N59 Y112

H28 Y112

α2 D62

A27

I113

I113

F25

H28

K116 F25

K116

I119 V21

L120

α4 I119 V21

L120

α1 F123 α1

F123

α4

S N

O

N O

O

N

N

N

OH H2N

N

Cl MK-591 (yellow)

16 (magenta)

Figure 4. Proposed binding mode for the oxadiazole series of FLAP inhibitors: (A) Overlay of a docking pose generated for compound 16 (in magenta) with the crystal structure of FLAP in complex with MK-591 (in yellow; PDB ID: 2Q7M).29 Key amino acids are labeled in the binding site and the inhibitors are shown as sticks. The protein monomers are shown in green and light cyan cartoon representation. (B) The fit of compound 16 (in magenta sticks) to FLAP binding site shown on semi-transparent rendering of solvent accessible protein surface for the binding site defined by labeled amino acids that are within 3.5 Å sphere of the inhibitor.


17

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SAR development was initially driven by: a) FLAP binding potency, determined by the ability of a test compound to displace the radio-labeled ligand [125I]-L-691831 (PE NEX084),31 and b) FLAP functional activity, determined by LTB4 synthesis inhibition in human whole blood (hWB). The SAR of various oxadiazole ring substitutions are shown in Table 1. Compounds substituted with small functional groups, such as amino (12) or amino ethyl (13), showed robust FLAP binding potency, with IC50 values of 20 and 11 nM, respectively. The strong binding activity displayed by 12 and 13 translated into good LTB4 synthesis inhibition in human whole blood (IC50 = 440 and 460 nM, respectively). Data suggested that increased polarity at this position of the oxadiazole ring was not tolerated. For example, the methyl sulfonamide derivative (14) showed more than a 10-fold loss in FLAP binding potency (IC50 = 340 nM), and did not inhibit LTB4 synthesis in human whole blood at the highest tested concentration (5 µM). In contrast to compounds 12 and 13 that bear small hetero-aliphatic groups, 16 that contains a more flat and lipophilic aromatic substituent was > 10-fold more potent in FLAP binding (IC50 = 1.4 nM). Unfortunately, the improved binding potency for 16 did not translate into higher potency in human whole blood assay (IC50 = 470 nM). Compound 16 has high clogP (4.1, BioByte) and low topological polar surface area (PSA: 91 Å2)32 values. We speculated that high lipophilicity compounds, such as 16 may have an adverse effect on LTB4 synthesis inhibition in human whole blood activity due to excessive plasma protein binding (data not collected). To test this hypothesis, the phenyl substituent on the oxadiazole ring was systematically replaced with more polar 6-membered hetero-aromatic groups. Generally, the FLAP binding SAR of these analogs was relatively flat. For instance, 3-pyridine 17, 4-pyridazine 18, 2-pyrazine 19, 5pyrimidine 20 and 3-pyridazine 23 showed comparable FLAP binding potency (IC50 < 10 nM) to the phenyl analog (16). However, the 4-pyrimidine derivative (24) was 10-fold weaker in FLAP


18


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Page 20 of 74

binding as compared to 16. Pyridone 32 was a potent binder (IC50 = 2.9 nM), but did not inhibit the LTB4 synthesis in the human whole blood assay at 5 µM. SAR of 5-membered heteroaromatic substituents that contain two or three heteroatoms was also systematically explored. In particular, imidazole, triazole, thiazole, oxazole, and pyrazole maintained high FLAP binding potency (e.g., 21, 26-31; IC50’s = 2.4-13 nM). Several of these compounds, including 4-imidazole 21, 4-thiazole 28, 4-oxazole 29, 3-pyrazole 30 and 4-pyrazole 31, showed good human whole blood potency (IC50’s = 270, 200, 310, 160 and 110 nM, respectively). In contrast, compounds containing a triazole group such as 26 and 27 were significantly less potent in the whole blood assay (IC50’s = 2100 and 2700 nM, respectively). Table 1. SAR of substituted oxadiazoles.

N N

N H2N

O

R

N

Compound

R

FLAP binding IC50 (nM) a

FLAP Functional Inhibition in human whole blood IC50 (nM) b

12

*-NH2

20

440

N H

11

460

340

>5000

1.4

470

*

13

*

14 16

H N O S O

*


19

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N

17

*

2.2

250

6.2

240

5.7

360

6.0

340

8.5

400

14

820

2.9

>5000

2.5

270

4.7

2100

13

2700

2.4

200

8.1

310

N NH

2.4

160

N

2.3

110

N

18

*

19

*

N N

N

N

20

*

N

N N

23 24

*

N

*

N O

32

H N

* *

21

N

N H

*

26 *

27

N *

28

N N N H

N NH

N S

*

29

N O

*

30 *

31 a

N H

Binding assay, geometric mean values (n ≥ 3), each determined from duplicate 10-point

concentration response curves, bhuman whole blood assays, geometric mean values (n ≥ 3), each determined from duplicate 10-point concentration response curves.


20


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Page 22 of 74

Based on its potency in the human whole blood assay, compound 31 was selected for additional optimization. Table 2 shows the SAR of the 4-pyrazole ring substitutions. Compared to the unsubstituted compound 31, addition of a carboxymethyl group (35) was met with an approximately 8-fold loss of binding potency and at least a 50-fold loss of activity in human whole blood with IC50 values of 18 and >5000 nM, respectively. It is speculated that the poor functional activity of compound 35 may result from high plasma protein binding (data not collected), limiting the available free fraction of compound in blood. Methyl substituted compound 33 showed comparable binding potency (IC50 = 1.9 nM) to compound 31 and approximately 2-fold increased inhibition of LTB4 synthesis in human whole blood (IC50 = 45 nM). Although compounds 31 and 33 showed good FLAP binding and functional potencies, they were both highly crystalline and displayed low aqueous equilibrium solubility at pH 6.8 (0.4 and 0.03 µg/mL, respectively). The introduction of slightly larger substituents with polar/solubilizing group, such as methoxyethyl analog (36), resulted in an increased aqueous equilibrium solubility (9.3 µg/mL at pH 6.8), while binding and human whole blood potencies were maintained (binding IC50 = 2.4 nM; hWB IC50 = 65 nM). A hindered polar functional group such as 1,1-dimethyl ethanol substituted compound 42 showed good binding potency and functional inhibition in human whole blood, with IC50 values of 3.5 and 69 nM, respectively. It appears that shielding the polar hydroxyl moiety with the dimethyl group struck a good balance between improved functional activity in human whole blood and favorable aqueous equilibrium solubility. The positive effect of alkyl substitution on functional potency is also evident in the SAR of the alkyl amine derivatives. For instance, the ethylamine substituted compound 38 showed more than 2-fold loss of LTB4 synthesis inhibition in human whole blood compare to compound 31. However, substitution with the more lipophilic group N,N-dimethyl-ethylamine


21

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provided 39 that had improved binding and human whole blood potencies, which were comparable to compound 42. Docking studies with N-substituted 4-pyrazoles suggested that the N(1) substituent pointed out of the FLAP binding pocket towards the lipophilic middle of the phospholipid bilayer. The observed loss of binding potency associated with the analogs that bear unshielded polar functional groups on the pyrazole ring is consistent with this modeling hypothesis. Compound 39 showed good potency and high aqueous equilibrium solubility (270 µg/mL at pH 6.8) after crystallization, but caused some CYP450 2C9 and 2D6 inhibition, with IC50 values of 7 and 10 µM, respectively. In contrast, 39 did not inhibit CYP450 3A4 activity at the highest tested concentration (30 µM). It was hypothesized that the basic 3o amine moiety in 39 was the contributing factor to the higher binding affinity of the compound to CYP450 2C9 and 2D6. To test this hypothesis, compound 40, substituted with a neutral N,N-dimethyl acetamide group, was synthesized. Compound 40 combined strong FLAP binding potency and functional inhibition in human whole blood, with IC50 values of 3.6 and 81 nM, respectively. Gratifyingly, compound 40 did not inhibit CYP450 2C9 and 2D6 activities at the highest tested concentration (30 µM). Table 2. SAR of N-substituents on 4-pyrazole. N N

N H2N

N

Compound

O

N R

R

N


FLAP Functional Inhibition in human whole blood IC50

Aqueous equilibrium solubility at pH 6.8 (µg/mL) c


22


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Page 24 of 74

(nM) b 31

H

2.3

110

0.4

33

Me

1.9

45

0.03

18

>5000

NT

2.4

65

9.3

NH2

2.6

240

NT

N

3.6

47

270

N

3.6

81

20

OH

3.5

69

8.1

*

OH

35 36 38 39

O *

O

*

*

*

40

O *

42 a


concentration response curves, bhuman whole blood assays, geometric mean values (n ≥ 3), each determined from duplicate 10-point concentration response curves, cvalues were measured with crystalline material after 24 h rotation, NT: not tested.

Docking studies with these novel oxadiazoles suggested that substituents on the central methylene linker overlap with the tert-butyl group of MK-591. Table 3 shows how potency is affected as modifications are made to the central methylene group. The SAR suggests that substitution on the C(8) position is crucial for the binding to FLAP. For instance, the unsubstituted compound 44 lacked FLAP binding and human whole blood potencies at the


23

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highest tested concentration (1 µM and 5 µM, respectively), but the dimethyl analog 52 was a potent FLAP binder (IC50 = 6.7 nM), with robust functional inhibition in the human whole blood (IC50 = 93 nM). Interestingly, tying the gem-dimethyl group into a cyclopropyl, as in compound 53, resulted in lost binding potency (cf. 53 binding IC50 = 100 nM vs. 52 binding IC50 = 6.7 nM) and functional activity (cf. 53 hWB IC50 = 2200 nM vs. 52 hWB IC50 = 93 nM) while increasing the ring size to cyclobutyl restored the binding and functional activities (e.g., 33: binding IC50 = 1.9 nM; hWB IC50 = 45 nM). The 3,3-dimethyl-cyclobutyl analog 54 showed more than 5-fold loss in binding potency (IC50 = 35 nM) as compared to 52. Docking studies indicate that the methylene linker occupies a hydrophobic space limited in size, at the interface of the α4 and α1 helices of adjacent FLAP monomers (Figure 4A). The linker substituents point towards this small pocket that is lined with the side chains of a collection of hydrophobic amino acid residues such as V20, V21, I119, L120, and F123. The closer examination of the docking model indicates that the R2 substituent occupies a smaller hydrophobic cavity closer to I119, L120 and F123, while R1 is pointing towards a slightly larger space near V20, V21 of the two adjacent monomer (Figure 4B), suggesting a potential stereochemical preference. We also calculated the torsional angle between the bis-aryl groups to determine the influence of various small R1 and R2 substitutions at the methylene linker position. The calculations suggest that the cyclopropyl substitution (53) causes maximal distortion to the bis-aryl angle (119.0 °) as compared to the geminal methyl compound 52 (104.2 o) or the cyclobutyl compound 33 (109.0 °). In view of the observed binding potencies of the three compounds, it appears that the torsional angle of the bisaryl has a significant impact on the FLAP binding potency. A torsion angle of < 119 o seems to be required for achieving a good binding potency (IC50 < 100 nM).


24


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Page 26 of 74

To validate the modeling prediction that the FLAP binding pocket has a stereochemical preference and to assess the impact of various substituents on binding and functional potency, additional SAR on the methylene group was performed. The isopropyl analog (55) and the isopropyl/methyl derivative (56) were first synthesized as racemates. Although 55 maintained good binding potency (IC50 = 6.2 nM) and functional activity in human whole blood (IC50 = 190 nM), the more substituted analog 56 showed 3-fold better binding (IC50 = 2.0 nM) and 4-fold better in human whole blood potencies (IC50 = 46 nM). To test the prediction that larger alkyl groups would be preferred at the R1 position, compound 56 was resolved into its (R) and (S) enantiomers by chiral SFC. The (R)-enantiomer 59 showed potent FLAP binding and a robust functional activity with IC50 values of 1.6 and 33 nM, respectively. In fact, the (R)-enantiomer (59) was approximately 5-fold more potent in FLAP binding and 10-fold more potent in human whole blood activity than the (S)-enantiomer 60. A similar pattern was also observed with the pair of compounds 61/62 (cyclopropyl/methyl) and 63/64 (cyclobutyl/methyl). These data confirmed that the substitution on the methylene linker of the bis-aryl core requires an optimal size and chirality for maximal FLAP inhibition. Table 3. SAR of substitutions on the methylene linker. R1 R2 N C(8) O N N

H2N

N

Compound

NN

R1

R2

Chirality


FLAP Functional Inhibition in Human whole blood IC50


25

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(nM) b 44

H

H

-

>1000

>5000

52

Me

Me

-

6.7

93

-

100

2200

-

1.9

45

-

35

1500

53

*

*

33 *

*

54 *

a

*

55

i-Pr

H

racemate

6.2

190

56

i-Pr

Me

racemate

2.0

46

59

i-Pr

Me

R

1.6

33

60

Me

i-Pr

S

7.7

360

61

c-Pr

Me

R

1.3

15

62

Me

c-Pr

S

18

350

63

c-Bu

Me

R

2.6

91

64

Me

c-Bu

S

140

3200


concentration response curves, bhuman whole blood assays, geometric mean values (n ≥ 3), each determined from duplicate 10-point concentration response curves.


26


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Page 28 of 74

The docking studies had also suggested that the left hand side of the molecule occupied a relatively small pocket deep inside of the FLAP helices, predictive of a limitation in size for substitutions. Table 4 shows the SAR of the left hand side substituents encompassing a variety of 6-membered heteroaromatic groups. The initial SAR showed that para-amino substituted nitrogen-containing 6-membered heteroaryls were preferred in this part of the molecule. For example, the 2-amino-5-pyridyl analog (68) displayed good binding potency (IC50 = 1.1 nM) and functional inhibition (IC50 = 66 nM). Analogs featuring a 2-amino-5-pyrimidine (69) or a 2amino-5-pyrazine (71) substituents were also potent FLAP binders with IC50 values of 1.7 and 1.2 nM, respectively. Additionally, all three compounds showed comparable functional inhibition in the human whole blood assay with IC50 values ranging between 45 and 73 nM. As such, incorporation of additional nitrogen into the pyridine to arrive at a pyrazine or a pyrimidine did not show any significant effect on either binding or functional potency. However, introduction of an alkyl chain on the amino substituent did modulate both binding and functional potency. For instance, 72, which bears a aminomethyl group retained both binding and functional potencies as compared to the non-alkylated derivative (69); however, larger substituents such as those in 73 (racemate) and 74 eroded binding and functional activities. Table 4. SAR of left hand side phenyl substitutions.

N R

N

O NN O

N


27

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69 (BI 665915)

*

1.1

66

*

1.7

45

*

1.2

73

*

2.9

30

*

370

1000

>1000

>50000

R

Compound

68



H2N

N N

H2N

N

N

71

H2N

N N

72 73 (racemate) 74 a

N H

N N

N H

N

N N H

N

*


concentration response curves, bhuman whole blood assays, geometric mean values (n ≥ 3), each determined from duplicate 10-point concentration response curves. Overall, the SAR indicated several favorable structural features to achieve potent binding to FLAP and functional inhibition. For instance, the preferred groups on the central methylene linker were cyclobutyl and (R)-cyclopropyl/methyl groups. Also, N,N-dimethyl-acetamide, N,Ndimethylaminoethyl and 1,1-dimethyl-ethanol moieties were preferred as the substituents on the pyrazole ring. Thus, an array of compounds that combined these preferred groups were synthesized and evaluated. The structures and overall profiles of four representatives from this


28


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Page 30 of 74

group of compounds are listed in Table 5. Compound 76 containing the (R)-cyclopropyl/methyl central piece and the N,N-dimethyl-ethylamino pyrazole right-hand side showed the best functional potency (IC50 = 14 nM), with a moderate CYP450 2D6 inhibitory activity (IC50 = 10 µM) and approximately 30 % inhibition of hERG channel at 11 µM. Compounds 42 and 77 that had the 1,1-dimethyl-2-ethanol substituted pyrazole and either the cyclobutyl or the (R)cyclopropyl/methyl on the central methylene, respectively, demonstrated good potency profiles in FLAP binding and functional assay; however, they had a less attractive CYP450 2C9 inhibition profile (IC50 values of 6 and 4 µM, respectively). Interestingly, compounds 77 and 69 showed significantly weaker activity in mouse whole blood (mWB) assay than in human whole blood (cf. 77: mWB IC50 = 1500 nM vs. hWB IC50 = 20 nM; 69: mWB IC50 = 4800 nM vs. hWB IC50 = 45 nM). Based on the structural homology model of mouse and human FLAP, we hypothesized that a single amino acid difference (Gly to Ala 24) that located in the central methylene linker’s binding pocket might be responsible for these observed differences. Finally, 69, featuring the N,N-dimethyl-acetamide substituted pyrazole and the (R)-cyclopropyl/methyl central substituents showed good potency and favorable off-target profiles. This compound was also very selective for FLAP as compared to other enzymes and receptors in the leukotriene pathway such as 5-LO, cycloxygenease-1 and -2, phospholipase A2, LTA4 hydrolase, BLT1, and BLT2 (< 50 % inhibition at 10 µM). Table 5. Overall profiles of four optimized oxadiazoles.

N N

N H2N

N

N

O

N

N H2N

NN

N

N N

N

NN

H2N

N

42

N

O

H2N OH

77

N

N

NN

OH

N

76

O

N

O NN O

N

69 (BI 665915)


29

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3.1

3.5

2.1

1.7

14

69

20

45

FLAP Functional Inhibition in mouse whole blood IC50 (nM) c

NT

NT

1500

4800

Human liver microsomal stability (%Qh) d

40

25

24

33

>30, 10, >30

6, 3, >30

4, >30, >30

>30, >30, >30

31

51

16

11

>1100

3.1

32

48


CYP450 inhibition 2C9, 2D6, 3A4 IC50 (µ µM) e hERG % inhibition @11µ µM f Aqueous equilibrium solubility at pH 6.8 (µ µg/mL) g a


concentration response curves, bhuman whole blood assays, geometric mean values (n ≥ 3), each determined from duplicate 10-point concentration response curves, cmouse whole blood assays


30


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Page 32 of 74

performed using the same protocol as human whole blood assay, geometric mean values (n ≥ 3), each determined from duplicate 8-point concentration response curves, deach value determined from duplicate 8-timepoint (0 to 60 min) curves, percent of hepatic blood flow, eeach value determined from duplicate 10-point concentration response curves, fvalues determined by manual patch clamp assay, gvalues measured after 24 h rotation, NT: not tested.

Based on its overall profile, 69 was selected for in vitro and in vivo DMPK assessment (Table 6). Compound 69 showed a modest human hepatocyte clearance (41 % percent of hepatic blood flow) and relatively high plasma protein binding (unbound fraction of 4.7 %). Considering the human hepatocyte clearance and plasma protein binding ratio, the compound was predicted to be metabolically stable in human, and has a plasma protein binding corrected human clearance of 8.7 % Qh. The compound showed high membrane permeability in Caco-2 cells, with a low efflux ratio (AB = 34 x 10-6 cm/s, ratio = 1.9). Weak CYP450 3A4 induction was detected (~2fold at concentrations up to 30 µM) for 69; however, there was no evidence of time-dependent inhibition (up to 100 µM) suggesting a low risk for potential drug-drug interactions. The pharmacokinetic properties of 69 were evaluated in rat, dog, and cynomolgus monkey (Table 7). The compound showed low iv plasma clearance in all three species, with clearance values of 7 % Qh in rat, 2.8 % Qh in dog, and 3.6 % Qh in cynomolgus monkey, respectively. The volume of distribution (Vss) across species tested was in a range of 0.5 to 1.2 L/kg, and the bioavailability was good (45 to 63 %) in all species tested. The overall attractive DMPK profile qualified 69 for advancement into an ex vivo model of mechanism engagement. Table 6. In vitro DMPK profile of 69.


31

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69 (BI 665915) Human hepatocyte clearance (% Qh) a

41

PPB corrected human clearance (% Qh) b

8.7

Human plasma protein binding (% unbound) c

4.7

Caco-2 permeability A-B (cm/sec), efflux ratio d

34, 1.9

CYP450 3A4 induction (30 µM) e

~ 2-fold

CYP450 3A4 time dependent inhibition (100 µM) f

Negative

a

Values determined from duplicate 6-timepoint (0 to 6 h) curves represent percent of hepatic

blood flow, bhuman hepatocyte clearance value corrected by the unbound fraction of human plasma protein binding and the value represents percent of hepatic blood flow, cvalue determined from duplicate of equilibrium dialysis technique, deach value determined from duplicate 6timepoint (0 to 2 h), efold of induction determined by dividing the test compound linear slope by the solvent control linear slope, ginduction activity determined from 7-point concentration (0 to 100 µM) at 6 time pints (0 to 30 min). Table 7. In vivo DMPK parameter of 69 in rat, dog and cynomolgus monkey.a

Clearance (% Qh) b, c Volume of distribution (L/kg) b

rat

dog

monkey

7

2.8

3.6

0.9

1.2

0.5


32


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Page 34 of 74

Mean residence time after iv dose (h) b

3.1

23

4.8

Bioavailability (%) b

63

58

45

a

Dose = iv: 1 mg/kg, dosing vehicle: 70 % PEG, po: 10 mg/kg, dosing suspension vehicle: 0.5

% methyl cellulose / 0.015 % Tween, all DMPK parameters were determined after 11-timepoint blood sampling (0, 5, 15, 30 min, 1, 2, 4, 6, 8, 12, 24 h) per iv or po dose, bmean values (n = 3), c

value represents percent of hepatic blood flow. Table 8 shows the plasma concentration of 69 vs. its inhibition of an ex vivo stimulated LTB4

production in C57BL/6 mice whole blood (n = 3) after a single oral dose of 1, 3, 10, 30 and 100 mg/kg (suspension in 0.5 % methyl cellulose/0.015 % Tween). Blood samples collected after 2 h indicated that the plasma concentration of 69 was fairly dose linear, with values of 1.72 µM (1 mg/kg), 3.73 µM (3 mg/kg), 14.8 µM (10 mg/kg), 20.7 µM (30 mg/kg) and 151.9 µM (100 mg/kg). The blood samples were stimulated with calcimycin and LTB4 levels were measured by ELISA. Compared to the vehicle group, the LTB4 levels in the compound treated groups were 110.6 % (1 mg/kg), 91.0 % (3 mg/kg), 35.2 % (10 mg/kg), 22.2 % (30 mg/kg) and 5.1 % (100 mg/kg) (Figure 6). Thus, in this study, 69 showed good blood exposure after oral administration and a dose-dependent reduction of LTB4 production after stimulation with calcimycin. Moreover, the inhibition data from the mouse ex vivo LTB4 production was consistent with the in vitro FLAP functional inhibition (IC50 = 4800 nM) in mouse whole blood. The effects of 69 in the apolipoprotein E-deficient mouse, an established model of atherosclerosis, will be published in due course.


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Table 8. Mouse in vivo plasma concentration of 69 (2 h post dose) and inhibition of LTB4 production after ex vivo stimulation.

Dose of 69

Plasma compound concentration a (µ µM ± SD)

Mouse whole blood LTB4 production b (% of control ± SEM)

0

ND

100.0 ± 13.3

1 mg/kg

1.7 ± 0.7

110.6 ± 31.2

3 mg/kg

3.7 ± 1.2

91.0 ± 16.7

10 mg/kg

14.8 ± 1.8

35.2 ± 3.3

30 mg/kg

20.7 ± 11.5

22.2 ± 8.0

100 mg/kg

151.9 ± 25.9

5.1 ± 1.0

a

mean values (n = 3) at 2 h after a single oral dose (dosing suspension vehicle; 0.5 % methyl

cellulose/0.015 % Tween), bmean values (n = 3) of percent reduction of LTB4 production relative to control group after calcimycin stimulation.


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Figure 5. Compound 69 demonstrated dose-dependent LTB4 production inhibition in mouse whole blood, 2 h after single oral dose. Black bar represents mean of the control group’s LTB4 production ± SEM (n = 3) after calcimycin stimulation. Blue bars represent mean percent reduction of LTB4 production ± SEM (n = 3) of each dose group relative to the control group after calcimycin stimulation of blood samples collected 2 h after single oral dose.

CONCLUSION A series of new oxadiazole-containing FLAP inhibitors was discovered using structure-guided rational design. Systematic SAR development on various parts of the molecules identified critical structural elements leading to the identification of compounds with potent FLAP binding activity and functional inhibition of LTB4 production in human whole blood. Further optimization of DMPK properties and aqueous solubility, as well as the minimization of undesired off-target effects resulted in the discovery of 69. This compound demonstrated an excellent pharmacokinetic profile across several species, and was predicted to have a low clearance in human and a low risk of potential drug-drug interactions. When administered orally to mice, 69 demonstrated a dose-dependent DMPK relationship and inhibition of LTB4 production. This mouse study demonstrated the feasibility of significantly blocking LTB4 biosynthesis through robust FLAP inhibition. The favorable in vitro and in vivo profile of 69 supported its selection as a preclinical development candidate and advancement to additional in vivo studies. Further profiling of 69 and other oxadiazoles is in progress, and additional results will be disclosed in due course. EXPERIMENTAL SECTION


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General remarks and analytical methods. Starting materials were purchased from commercial suppliers and used without further purification unless otherwise stated. 1H NMR spectra were recorded on a Bruker UltraShield 400 MHz or 500 MHz spectrometer operating at 400 MHz or 500 MHz in solvents, as noted. Proton coupling constants (J) are rounded to the nearest Hz. All coupling constants are reported as Hz. All solvents were HPLC grade or better. The reactions were followed by TLC on precoated Uniplate silica gel plates purchased from Analtech. The developed plates were visualized using 254 nm UV illumination or PMV stain. Flash column chromatography on silica gel was performed on Redi Sep prepacked disposable silica gel columns using an Isco Combiflash, Biotage SP1 or on traditional gravity columns. Reactions were carried out under an atmosphere of argon. Purity for all final compounds was > 95 %, and purity was evaluated by at least one of the following methods: System 1: analytical HPLC using Varian Dynamax SD-200 pump coupled to a Varian Dynamax UV-1 detector. Solvents: (A) water + 0.05 % TFA and (B) acetonitrile + 0.05 % TFA, flow 1.2 mL/min. Column: Vydac RP18, 5 µm, 250 mm x 4.6 mm. Photodiode array detector at 220 nm. Gradient: from 95 % to 20 % solvent A over 25 min. System 2: HP 1110 Agilent LCMS using a quaternary G1311A pump coupled to a Micromass Platform LCZ detector. Solvent (A) water + 0.1 % formic acid and (B) acetonitrile + 0.1 % formic acid, flow 1.5 mL/min. Photodiode array detector at 190 and 400 nm. (a) Agilent Zorbax Eclipse XDB-C8 5 µm, 150 mm x 4.6 mm. Gradient from 99 % to 5 % solvent A over 10 min or (b) column Agilent Zorbax C18 SB 3.5 mM, 30 mm x 4.6 mm cartridge. Gradient from 95 % to 5 % solvent A over 2.5 min. System 3: Waters Alliance 2695 coupled to a Waters 996 photodiode array detector. Column: Waters C8 Symmetry (3 x 150 mm, 5 µm) Column Temperature: 40 °C Mobile Phase: A = acetonitrile, B = 50 mM KH2PO4, pH 3 Gradient: 5 % A: 95 % B, 8 min linear gradient to 70 % A Flow Rate: 0.7 mL/min. All


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animal experiments were carried out in accordance with the Guideline for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. 1-(4-Bromo-phenyl)-cyclobutanecarbonitrile (6). To a solution of 4bromophenylacetonitrile 1 (30 g, 153 mmol) in DMF (400 mL) was added sodium hydride (60 % in mineral oil, 13.4 g, 336 mmol, 2.2 equiv.) portion-wise at 0 oC. After stirring at 0 oC for 15 min, to this mixture was added 1,3-dibromopropane (17.1 mL, 168 mmol, 1.1 equiv.) dropwise at 0 oC. The resulting mixture was allowed to warm up to room temperature and stirred for 16h. Then the reaction mixture was diluted with ice-water and volatiles were evaporated. The resulting oil was taken up to ethyl acetate and washed with water, saturated sodium bicarbonate solution and brine. The organic layer was dried over sodium sulfate and concentrated. The resulting residue was purified on silica gel (heptane/ethyl acetate) to give 6 (25.1 g, 69 %) as a pale yellow solid. LC/MS (ES+) m/z found 237. 1H NMR (500 MHz, CDCl3) δ 7.50 (d, J=15 Hz, 2H), 7.29 (d, J=15 Hz, 2H), 2.84, (m, 2H), 2.61 (m, 2H), 2.56 (m, 1H), 2.05 (m, 1H). 1-(4-Bromo-phenyl)-N-hydroxy-cyclobutanecarboxamidine (7). To a solution of 6 (5 g, 21.2 mmol) in EtOH (70 mL) was added 50 % hydroxylamine in water (2.1 mL, 31.7 mmol, 1.5 equiv.), and the mixture was heated at 80 oC for 16 h. The volatiles were evaporated in vacuo, and the resulting oil was taken up to ethyl acetate and washed with water, saturated sodium bicarbonate solution and brine. The organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo to give 7 (5.7 g, 100 %) as a pale yellow solid. LC/MS (ES+) m/z found 268, 271. 1H NMR (500 MHz, DMSO-d6) δ 9.13 (s, 1H), 7.50 (d, J = 10 Hz, 2H), 7.27 (d, J = 10 Hz, 2H), 5.18 (s, 2H), 2.72, (m, 2H), 2.28 (m, 2H), 1.85 (m, 1H), 1.72 (m, 1H).


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3-[1-(4-Bromo-phenyl)-cyclobutyl]-5-trichloromethyl-[1,2,4]oxadiazole (8). To a solution of 7 (1.7 g, 6.4 mmol) in toluene (80 mL) was added trichloroacetic anhydride (1.4 mL, 7.7 mmol, 1.2 equiv.) and the resulting mixture was refluxed for 2 h. After cooling to room temperature, the solution was washed with water and saturated sodium bicarbonate solution, dried over sodium sulfate and concentrated to give 8 (2.5 g, 95 %) as light yellow oil. LC/MS (ES+) m/z found 396, 398. 1H NMR (500 MHz, CDCl3) δ 7.50 (d, J = 10Hz, 2H), 7.26 (d, J = 10Hz, 2H), 2.98, (m, 2H), 2.71 (m, 2H), 2.21 (m, 1H), 2.05 (m, 1H). 3-[1-(4-Bromo-phenyl)-cyclobutyl]-[1,2,4]oxadiazol-5-ylamine (9). To a solution of ammonium hydroxide (28 % in water, 0.03 mL, 0.8 mmol, 1.1 equiv.) in DMF (2.0 mL) was added 8 (300 mg, 0.7 mmol), and the resulting mixture was stirred at room temperature for 0.5 h. The resulting mixture was diluted with water, and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate and concentrated to give 9 (202 mg, 91 %). The product was used for the next reaction without further purification. LC/MS (ES+) m/z found 294, 296. 1H NMR (500 MHz, CDCl3) δ 7.46 (d, J = 10 Hz, 2H), 7.19 (d, J = 10 Hz, 2H), 5.29 (bs, 2H), 2.84, (m, 2H), 2.60 (m, 2H), 2.20 (m, 1H), 1.93 (m, 1H). {3-[1-(4-Bromo-phenyl)-cyclobutyl]-[1,2,4]oxadiazol-5-yl}-ethyl-amine (10). 8 was coupled with ethylamine (2.0 M in THF, 0.4 mL, 0.8 mmol, 1.1 equiv.) under the same conditions applied to 9 to give 10 (76 %). LC/MS (ES+) m/z found 322, 324. 1H NMR (500 MHz, CDCl3) δ 7.45 (d, J = 10 Hz, 2H), 7.22 (d, J = 10 Hz, 2H), 5.00 (bs, 1H), 3.40 (m, 2H), 2.84, (m, 2H), 2.60 (m, 2H), 2.22 (m, 1H), 1.96 (m, 1H), 1.24 (m, 3H). N-{3-[1-(4-Bromo-phenyl)-cyclobutyl]-[1,2,4]oxadiazol-5-yl}-methanesulfonamide (11). To a solution of 9 (200 mg, 0.7 mmol) in DCM (5 mL) was added pyridine (60 µL, 0.8 mmol,


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1.1 equiv.) and methane sulfonyl chloride (58 µL, 0.8 mmol, 1.1 equiv.) at 0 oC. After stirring at 0 oC for 1 h, the reaction mixture was heated at 50 oC for 8 h. The organic layer was washed with water and brine, dried over sodium sulfate and concentrated. The residue was purified on silica gel (DCM/MeOH) to give 11 (100 mg, 34 %). LC/MS (ES+) m/z found 372, 374. 5-{4-[1-(5-Amino-[1,2,4]oxadiazol-3-yl)-cyclobutyl]-phenyl}-pyrimidin-2-ylamine (12). A mixture of 9 (220 mg, 0.7 mmol), 2-aminopyrimidine-5-boronic acid pinacol ester (198 mg, 0.9 mmol, 1.2 equiv.) and bis(triphenylphosphine)palladium (II) chloride (52 mg, 0.07 mmol, 0.1 equiv.) in 2 M sodium carbonate solution (0.8 mL) and DMF (3.0 mL) was charged in sealed tube, and the mixture was heated at 90 oC for 20 min under microwave condition (150 W). The resulting mixture was diluted with water and the product was extracted with ethyl acetate. The organic layer was washed with water and brine, dried over sodium sulfate and concentrated. The residue was purified on silica gel (DCM/MeOH) to give 12 (27 mg, 11 %). LC/MS (ES+) m/z found 309. 1H NMR (500 MHz, DMSO-d6) δ 8.53 (s, 2H), 7.66 (s, 2H), 7.55 (d, J = 10 Hz, 2H), 7.30 (d, J = 10 Hz, 2H), 6.75 (s, 2H), 2.72, (m, 2H), 2.56 (m, 2H), 2.04 (m, 1H), 1.89 (m, 1H). 5-{4-[1-(5-Ethylamino-[1,2,4]oxadiazol-3-yl)-cyclobutyl]-phenyl}-pyrimidin-2-ylamine (13). 10 was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 13 (11 %). LC/MS (ES+) m/z found 337. 1H NMR (500 MHz, DMSO-d6) δ 8.54 (s, 2H), 8.17 (t, J = 5 Hz, 1H), 7.55 (d, J = 10 Hz, 2H), 7.32 (d, J = 10 Hz, 2H), 6.75 (s, 2H), 3.18 (q, J = 5 Hz, 2H), 2.71, (m, 2H), 2.52 (m, 2H), 2.03 (m, 1H), 1.89 (m, 1H), 2.09 (t, J = 5 Hz, 3H). N-(3-{1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-cyclobutyl}-[1,2,4]oxadiazol-5-yl)methanesulfonamide (14). 11 was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester


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under the conditions applied to 12 to give 14 (11 %). LC/MS (ES+) m/z found 387. 1H NMR (500 MHz, CD3OD) δ 8.53 (s, 2H), 7.50 (d, J = 10 Hz, 2H), 7.43 (d, J = 10 Hz, 2H), 3.01 (s, 3H), 2.88 (m, 2H), 2.63(m, 2H), 2.12 (m, 1H), 1.96 (m, 1H). 5-{4-[1-(5-Phenyl-[1,2,4]oxadiazol-3-yl)-cyclobutyl]-phenyl}-pyrimidin-2-ylamine (16). Step1: To a solution of 7 (500 mg, 1.9 mmol) and pyridine (0.23 mL, 2.8 mmol, 1.5 equiv.) in DCM (5 mL) was added benzoyl chloride (0.24 mL, 2.0 mmol, 1.1 equiv.) at 0 oC. The resulting mixture was allowed to warm to room temperature and stirred for 16 h. The organic layer was washed with water and brine, dried over sodium sulfate and concentrated. The residue was purified on silica gel (heptane/ethyl acetate) to give 3-[1-(4-bromo-phenyl)-cyclobutyl]-5phenyl-[1,2,4]oxadiazole (400 mg, 60 %). Step 2: 3-[1-(4-Bromo-phenyl)-cyclobutyl]-5-phenyl[1,2,4]oxadiazole was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 16 (15 %). LC/MS (ES+) m/z found 370. 1H NMR (500 MHz, CD3OD) δ 8.71 (s, 2H), 8.09 (d, J = 5 Hz, 2H), 7.65-7.51 (m, 7H), 3.00 (s, 2H), 2.80 (m, 2H), 2.23 (m, 1H), 2.06 (m, 1H). 5-{4-[1-(5-Pyridin-3-yl-[1,2,4]oxadiazol-3-yl)-cyclobutyl]-phenyl}-pyrimidin-2-ylamine (17). Step1: 7 was coupled with nicotinoyl chloride hydrochloride under the same conditions applied to step 1 of 16 to give 3-{3-[1-(4-bromo-phenyl)-cyclobutyl]-[1,2,4]oxadiazol-5-yl}pyridine (29 %). Step 2: 3-{3-[1-(4-Bromo-phenyl)-cyclobutyl]-[1,2,4]oxadiazol-5-yl}-pyridine was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 17 (9 %). LC/MS (ES+) m/z found 371. 1H NMR (500 MHz, CD3OD) δ 9.25 (bs, 1H), 8.85 (bs, 1H), 8.68 (s, 2H), 8.50 (d, J = 5 Hz, 1H), 7.65 (d, J = 5 Hz, 1H), 7.59 (d, J = 10 Hz, 2H), 7.50 (d, J = 10 Hz, 2H), 3.00 (m, 2H), 2.80 (m, 2H), 2.23 (m, 1H), 2.06 (m, 1H).


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5-{4-[1-(5-Pyridazin-4-yl-[1,2,4]oxadiazol-3-yl)-cyclobutyl]-phenyl}-pyrimidin-2-ylamine (18). Step 1: To a solution of pyridazine-4-carboxylic acid (97 mg, 0.8 mmol, 1.1 equiv.) in pyridine (2.0 mL) was added thionyl chloride (65 µL, 0.9 mmol, 1.2 equiv.) at room temperature and the resulting mixture was stirred for 0.5 h. To the reaction mixture was added 7 (200 mg, 0.7 mmol) at room temperature and stirred for 16 h. The resulting mixture was concentrated and the residue was diluted with DCM. The organic layer was washed with water and saturated sodium bicarbonate solution, dried over sodium sulfate and concentrated to give 4-{3-[1-(4bromo-phenyl)-cyclobutyl]-[1,2,4]oxadiazol-5-yl}-pyridazine (246 mg, 86 %). LC/MS (ES+) m/z found 357, 359. Step 2: 4-{3-[1-(4-Bromo-phenyl)-cyclobutyl]-[1,2,4]oxadiazol-5-yl}pyridazine was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 18 (14 %). LC/MS (ES+) m/z found 372. 1H NMR (500 MHz, CDCl3) δ 9.82 (d, J = 5 Hz, 1H), 9.45 (d, J = 5 Hz, 1H), 8.51 (s, 2H), 8.08 (dd, J = 10, 5 Hz, 1H), 7.47 (s, 4H), 5.33 (bs, 2H), 3.02 (m, 2H), 2.81 (m, 2H), 2.24 (m, 1H), 2.07 (m, 1H). 5-{4-[1-(5-Pyrazin-2-yl-[1,2,4]oxadiazol-3-yl)-cyclobutyl]-phenyl}-pyrimidin-2-ylamine (19). Step1: 7 was coupled with pyrazine-2-carbonyl chloride under same conditions applied to step 1 of 16 to give 2-{3-[1-(4-bromo-phenyl)-cyclobutyl]-[1,2,4]oxadiazol-5-yl}-pyrazine (70 %). LC/MS (ES+) m/z found 357, 359. Step 2: 2-{3-[1-(4-Bromo-phenyl)-cyclobutyl][1,2,4]oxadiazol-5-yl}-pyrazine was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 19 (24 %). LC/MS (ES+) m/z found 372. 1

H NMR (500 MHz, CDCl3) δ 9.41 (d, J = 5 Hz, 1H), 8.79 (d, J = 5 Hz, 2H), 8.53 (s, 2H), 7.52

(m, 4H), 5.20 (bs, 2H), 3.08 (m, 2H), 2.82 (m, 2H), 2.25 (m, 1H), 2.06 (m, 1H).


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5-{4-[1-(5-Pyrimidin-5-yl-[1,2,4]oxadiazol-3-yl)-cyclobutyl]-phenyl}-pyrimidin-2-ylamine (20). Step1: 7 was coupled with pyrimidine-5-carboxylic acid under the same conditions applied step 1 of 18 to give 5-{3-[1-(4-bromo-phenyl)-cyclobutyl]-[1,2,4]oxadiazol-5-yl}-pyrimidine (53 %). LC/MS (ES+) m/z found 357, 359. Step 2: 5-{3-[1-(4-Bromo-phenyl)-cyclobutyl][1,2,4]oxadiazol-5-yl}-pyrimidine was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under same conditions applied to 12 to give 20 (16 %). LC/MS (ES+) m/z found 372. 1H NMR (500 MHz, CDCl3) δ 9.41 (s, 2H), 9.39 (s, 1H), 8.53 (s, 2H), 7.49 (s, 4H), 5.20 (bs, 2H), 3.03 (m, 2H), 2.83 (m, 2H), 2.26 (m, 1H), 2.06 (m, 1H). 5-(4-{1-[5-(1H-Imidazol-4-yl)-[1,2,4]oxadiazol-3-yl]-cyclobutyl}-phenyl)-pyrimidin-2ylamine (21). Step 1: A mixture of 1H-imidazole-4-carboxylic acid (187 mg, 1.7 mmol, 1.0 equiv.), HATU (636 mg, 1.7 mmol, 1.0 equiv.) and triethylamine (0.23 mL, 1.7 mmol, 1.0 equiv.) in DMF (5 mL) was stirred at room temperature for 3 h. To the reaction mixture was added 7 (450 mg, 1.7 mmol) and stirred at 110 oC for 4 h. The resulting mixture was concentrated and the residue was diluted with ethyl acetate. The organic layer was washed with water and saturated sodium bicarbonate solution, dried over sodium sulfate and concentrated to give 3-[1-(4-bromo-phenyl)-cyclobutyl]-5-(1H-imidazol-4-yl)-[1,2,4]oxadiazole (356 mg, 54 %). LC/MS (ES+) m/z found 345, 347. Step 2: 3-[1-(4-Bromo-phenyl)-cyclobutyl]-5-(1H-imidazol4-yl)-[1,2,4]oxadiazole was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 21 (11 %). LC/MS (ES+) m/z found 360. 1H NMR (500 MHz, DMSO-d6) δ 8.56 (s, 2H), 8.09 (s, 1H), 7.91 (s, 1H), 7.58 (d, J = 10 Hz, 2H), 7.36 (d, J = 10 Hz, 2H), 6.76 (s, 2H), 2.87 (m, 2H), 2.66 (m, 2H), 2.08 (m, 1H), 1.95 (m, 1H).


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1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-N-hydroxy-cyclobutanecarboxamidine (22). Step 1: 6 (1.5 g, 6.4 mmol) was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 1-[4-(2-amino-pyrimidin-5-yl)-phenyl]cyclobutanecarbonitrile (1.28 g, 81 %). LC/MS (ES+) m/z found 251. 1H NMR (500 MHz, CDCl3) δ 8.54 (s, 2H), 7.52 (s, 4H), 5.30 (bs, 2H), 2.87 (m, 2H), 2.65 (m, 2H), 2.46 (m, 1H), 2.12 (m, 1H). Step 2: A mixture of 1-[4-(2-amino-pyrimidin-5-yl)-phenyl]cyclobutanecarbonitrile (1.3 g, 5.11 mmol) and 50 % hydroxylamine water solution (3.85 mL) in EtOH (20 mL) was heated at 80 oC for 16 h. The resulting mixture was concentrated and the residue was diluted with DCM. The organic layer was washed with water and saturated sodium bicarbonate solution, dried over sodium sulfate and concentrated. The residue was triturated with hepatane to give 22 (1.26 g, 87 %) as light yellow solid. LC/MS (ES+) m/z found 284. 1H NMR (500 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.54 (s, 2H), 8.85 (s, 2H), 7.53 (d, J = 10 Hz, 2H), 7.37 (d, J = 10 Hz, 2H), 6.74 (s, 2H), 5.10 (bs, 2H), 2.69 (m, 2H), 2.29 (m, 2H), 1.89 (m, 1H), 1.76 (m, 1H). 5-{4-[1-(5-Pyridazin-3-yl-[1,2,4]oxadiazol-3-yl)-cyclobutyl]-phenyl}-pyrimidin-2-ylamine (23). 22 was coupled with pyridazine-3-carboxylic acid under the same conditions applied to step 1 of 21 to give 23 (17 %). LC/MS (ES+) m/z found 372. 1H NMR (500 MHz, CDCl3) δ 9.38 (dd, J = 5, 1 Hz, 1H), 8.85 (s, 2H), 8.30 (dd, J = 5, 1Hz, 1H), 7.52 (d, J = 5 Hz, 2H), 7.48 (d, J = 5 Hz, 2H), 5.18 (bs, 2H), 3.08 (m, 2H), 2.83 (m, 2H), 2.27 (m, 1H), 2.07 (m, 1H). 5-{4-[1-(5-Pyrimidin-4-yl-[1,2,4]oxadiazol-3-yl)-cyclobutyl]-phenyl}-pyrimidin-2-ylamine (24). 22 was coupled with pyrimidine-4-carboxylic acid under the same conditions applied to step 1 of 21 to give 24 (45 %). LC/MS (ES+) m/z found 372. 1H NMR (400 MHz, DMSO-d6) δ


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9.45 (d, J = 1 Hz, 1H), 9.11 (d, J = 5 Hz), 8.65 (s, 2H), 8.20 (dd, J = 5, 1 Hz, 1H), 7.64 (d, J = 8 Hz, 2H), 7.43 (d, J = 8 Hz, 2H), 2.91 (m, 2H), 2.75 (m, 2H), 2.13 (m, 1H), 2.01 (m, 1H). 5-(4-{1-[5-(2-Chloro-pyridin-3-yl)-[1,2,4]oxadiazol-3-yl]-cyclobutyl}-phenyl)-pyrimidin-2ylamine (25). 22 was coupled with 2-chloronicotinoyl chloride under the same conditions applied to step 1 of 16 to give 25 (61 %). LC/MS (ES+) m/z found 405. 1H NMR (400 MHz, DMSO-d6) δ 8.68 (dd, J = 5, 2 Hz, 1H), 8.66 (s, 2H), 8.50 (dd, J = 5, 2Hz, 1H), 7.66 (dd, J = 8, 5 Hz, 1H), 7.64 (d, J = 8 Hz, 2H), 7.44 (d, J = 8 Hz, 2H), 2.91 (m, 2H), 2.76 (m, 2H), 2.15 (m, 1H), 2.00 (m, 1H). 5-(4-{1-[5-(1H-[1,2,3]Triazol-4-yl)-[1,2,4]oxadiazol-3-yl]-cyclobutyl}-phenyl)-pyrimidin2-ylamine (26). 22 was coupled with 1H-[1,2,3]triazole-4-carboxylic acid under the same conditions applied to step 1 of 21 to give 26 (39%). LC/MS (ES+) m/z found 361. 1H NMR (400 MHz, DMSO-d6) δ 16.05 (bs, 1H), 8.84 (bs, 1H), 8.55 (s, 2H), 7.60 (d, J = 8 Hz, 2H), 7.40 (d, J = 8 Hz, 2H), 6.77 (s, 2H), 2.88 (m, 2H), 2.73 (m, 2H), 2.12 (m, 1H), 1.98 (m, 1H). 5-(4-{1-[5-(1H-[1,2,4]Triazol-3-yl)-[1,2,4]oxadiazol-3-yl]-cyclobutyl}-phenyl)-pyrimidin2-ylamine (27). A mixture of 1H-[1,2,4]triazole-3-carboxylic acid (88 mg, 0.8 mmol, 1.1 equiv.) and CDI (126 mg, 0.8 mmol, 1.1 equiv.) in NMP (1 mL) was heated at 50 oC for 20 min. Then 22 (200 mg, 0.7 mmol) was added to the mixture, and the resulting mixture was heated at 100 oC for 4 h. The resulting mixture was diluted with water and the product was extracted with ethyl acetate. The organic layer was washed with water and brine, dried over sodium sulfate and concentrated. The residue was purified on silica gel (DCM/MeOH) to give 27 (199 mg, 78 %). LC/MS (ES+) m/z found 361. 1H NMR (400 MHz, DMSO-d6) δ 15.00 (bs, 1H), 8.89 (s, 1H),


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8.61 (s, 2H), 7.62 (d, J = 8 Hz, 2H), 7.42 (d, J = 8 Hz, 2H), 6.77 (bs, 2H), 2.88 (m, 2H), 2.73 (m, 2H), 2.14 (m, 1H), 1.99 (m, 1H). 5-{4-[1-(5-Thiazol-4-yl-[1,2,4]oxadiazol-3-yl)-cyclobutyl]-phenyl}-pyrimidin-2-ylamine (28). 22 was coupled with thiazole-4-carboxylic acid under the same conditions applied to step 1 of 21 to give 28 (44 %). LC/MS (ES+) m/z found 377. 1H NMR (500 MHz, CD3OD) δ 9.18 (s, 1H), 8.60 (s, 1H), 8.52 (s, 2H), 7.91 (s, 1H), 7.54 (d, J = 10 Hz, 2H), 7.46 (d, J = 10 Hz, 2H), 2.99 (m, 2H), 2.78 (m, 2H), 2.22 (m, 1H), 2.05 (m, 1H). 5-{4-[1-(5-Oxazol-4-yl-[1,2,4]oxadiazol-3-yl)-cyclobutyl]-phenyl}-pyrimidin-2-ylamine (29). 22 was coupled with oxazole-4-carboxylic acid under the same conditions applied to step 1 of 21 to give 29 (21 %). LC/MS (ES+) m/z found 361. 1H NMR (500 MHz, CD3OD) δ 8.82 (s, 1H), 8.53 (s, 2H), 8.44 (s, 1H), 7.54 (d, J = 10 Hz, 2H), 7.45 (d, J = 10 Hz, 2H), 2.97 (m, 2H), 2.78 (m, 2H), 2.21 (m, 1H), 2.04 (m, 1H). 5-(4-{1-[5-(1H-Pyrazol-3-yl)-[1,2,4]oxadiazol-3-yl]-cyclobutyl}-phenyl)-pyrimidin-2ylamine (30). 22 was coupled with pyrazole-3-carboxylic acid under the same conditions applied to 27 to give 30 (39 %). LC/MS (ES+) m/z found 360. 1H NMR (400 MHz, DMSO-d6) δ 13.78 (s, 1H), 8.55 (s, 1H), 8.01 (dd, J = 2, 1 Hz, 1H), 7.60 (d, J = 8 Hz, 2H), 7.40 (d, J = 8 Hz, 2H), 6.94 (t, J = 2 Hz, 1H), 6.77 (bs, 2H), 2.87 (m, 2H), 2.71 (m, 2H), 2.10 (m, 1H), 1.98 (m, 1H). 5-(4-{1-[5-(1H-Pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-cyclobutyl}-phenyl)-pyrimidin-2ylamine (31). 22 was coupled with pyrazole-4-carboxylic acid under the same conditions applied to 27 to give 31 (72 %). LC/MS (ES+) m/z found 360. 1H NMR (400 MHz, DMSO-d6) δ 8.63


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(s, 2H), 8.36 (bs, 2H), 7.62 (d, J = 8 Hz, 2H), 7.40 (d, J = 8 Hz, 2H), 2.85 (m, 2H), 2.68 (m, 2H), 2.09 (m, 1H), 1.98 (m, 1H). 3-(3-{1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-cyclobutyl}-[1,2,4]oxadiazol-5-yl)-1Hpyridin-2-one (32). A solution of 25 (100 mg, 0.3 mmol) and lithium hydroxide (10 % solution in water, 1 mL) in 1,4-dioxane (4 mL) was heated at 70 oC for 16 h. The resulting mixture was diluted with water and the product was extracted with ethyl acetate. The organic layer was washed with water and brine, dried over sodium sulfate and concentrated. The residue was purified on silica gel (DCM/MeOH) to give 32 (20 mg, 21 %). LC/MS (ES+) m/z found 387. 1H NMR (400 MHz, CDCl3) δ 13.08 (bs, 1H), 8.57 (s, 2H), 8.35 (d, J = 8 Hz, 1H), 7.68 (d, J = 7 Hz, 1H), 7.41 (s, 4H), 6.42 (t, J = 7 Hz, 1H), 2.94 (m, 2H), 2.69 (m, 2H), 2.15 (m, 1H), 2.00 (m, 1H). 5-(4-{1-[5-(1-Methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-cyclobutyl}-phenyl)pyrimidin-2-ylamine (33). 22 was coupled with 1-methyl-1H-pyrazole-4-carboxylic acid under the same conditions applied to 27 to give 33 (72 %). LC/MS (ES+) m/z found 374. 1H NMR (400 MHz, DMSO-d6) δ 8.59 (s, 1H), 8.55 (s, 2H), 8.06 (s, 1H), 7.59 (d, J = 8 Hz, 1H), 7.38 (d, J = 8 Hz, 1H), 6.76 (s, 2H), 3.92 (s, 3H), 2.85 (m, 2H), 2.69 (m, 2H), 2.09 (m, 1H), 1.96 (m, 1H). [4-(3-{1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-cyclobutyl}-[1,2,4]oxadiazol-5-yl)-pyrazol1-yl]-acetic acid (35). Step 1: A mixture of 31 (500 mg, 1.39 mmol), bromo-acetic acid ethyl ester (0.2 mL, 1.7 mmol, 1.2 equiv.) and cesium carbonate (288 mg, 2.1 mmol, 1.5 equiv.) in DMF (8 mL) was stirred at room temperature for 2 h. The mixture was diluted with ethyl acetate and the organic layer was washed with water and brine, dried over sodium sulfate and


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concentrated to give [4-(3-{1-[4-(2-amino-pyrimidin-5-yl)-phenyl]-cyclobutyl}[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-acetic acid ethyl ester 34 (490 mg, 79 %). Step 2: A solution of 34 (970 mg, 2.2 mmol) and lithium hydroxide monohydrate (275 mg, 6.5 mmol, 3.0 equiv.) in a mixture of THF (10 mL), water (5 mL) and MeOH (2 mL) was heated at 40 oC for 1.5 h. The reaction mixture was cooled and diluted water. The product was extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate and concentrated to give 35 (725 mg, 80 %) as crystalline solid. LC/MS (ES+) m/z found 418. 1H NMR (400 MHz, DMSO-d6) δ 13.34 (bs, 1H), 8.62 (s, 2H), 8.60 (s, 1H), 8.10 (s, 1H), 7.61 (d, J = 8 Hz, 1H), 7.39 (d, J = 8 Hz, 1H), 5.06 (s, 2H), 2.85 (m, 2H), 2.69 (m, 2H), 2.09 (m, 1H), 1.96 (m, 1H). 5-[4-(1-{5-[1-(2-Methoxy-ethyl)-1H-pyrazol-4-yl]-[1,2,4]oxadiazol-3-yl}-cyclobutyl)phenyl]-pyrimidin-2-ylamine (36). 31 was coupled with 1-bromo-2-methoxy-ethane under the same conditions applied to step 1 of 35 to give 36 (46 %). LC/MS (ES+) m/z found 418. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.55 (s, 2H), 8.09 (s, 1H), 7.59 (d, J = 8 Hz, 1H), 7.38 (d, J = 8 Hz, 1H), 6.76 (s, 2H), 4.35, (t, J = 5 Hz, 2H), 3.71, (t, J = 5 Hz, 2H), 3.21 (s, 3H), 2.85 (m, 2H), 2.69 (m, 2H), 2.09 (m, 1H), 1.96 (m, 1H). 5-[4-(1-{5-[1-(2-Amino-ethyl)-1H-pyrazol-4-yl]-[1,2,4]oxadiazol-3-yl}-cyclobutyl)phenyl]-pyrimidin-2-ylamine (38). Step 1: 31 was coupled with 2-(2-bromo-ethyl)-isoindole1,3-dione under the same conditions applied to step 1 of 35 to give 2-{2-[4-(3-{1-[4-(2-aminopyrimidin-5-yl)-phenyl]-cyclobutyl}-[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-ethyl}-isoindole-1,3dione 37 (76 %). Step 2: A solution of 37 (224 mg, 0.4 mmol) and hydrazine hydrate (0.18 mL, 3.7 mmol, 8.8 equiv.) was heated at 50 oC for 2 h. The reaction mixture was concentrated and the residue as diluted with ethyl acetate. The organic layer was washed with water and brine,


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dried over sodium sulfate and concentrated. The residue was purified on silica gel (DCM/MeOH) to give 38 (199 mg, 92 %). LC/MS (ES+) m/z found 403. 1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H), 8.60 (s, 2H), 8.18 (s, 1H), 7.91 (bs, 2H), 7.61 (d, J = 8 Hz, 1H), 7.38 (d, J = 8 Hz, 1H), 7.00 (bs, 2H), 4.44, (t, J = 6 Hz, 2H), 3.32, (dd, J = 12, 6 Hz, 2H), 2.85 (m, 2H), 2.71 (m, 2H), 2.09 (m, 1H), 1.97 (m, 1H). 5-[4-(1-{5-[1-(2-Dimethylamino-ethyl)-1H-pyrazol-4-yl]-[1,2,4]oxadiazol-3-yl}cyclobutyl)-phenyl]-pyrimidin-2-ylamine (39). 31 was coupled with (2-chloro-ethyl)dimethyl-amine hydrochloride under the same conditions applied to step 1 of 35 to give 39 (40 %). LC/MS (ES+) m/z found 431. 1H NMR (400 MHz, DMSO-d6) δ 8.74 (s, 1H), 8.60 (s, 2H), 8.21 (s, 1H), 7.61 (d, J = 8 Hz, 1H), 7.38 (d, J = 8 Hz, 1H), 7.00 (bs, 2H), 4.62, (t, J = 6 Hz, 2H), 3.62, (bs, 2H), 2.84 (m, 2H), 2.80 (s, 6H), 2.70 (m, 2H), 2.09 (m, 1H), 1.97 (m, 1H). 2-[4-(3-{1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-cyclobutyl}-[1,2,4]oxadiazol-5-yl)pyrazol-1-yl]-N,N-dimethyl-acetamide (40). 31 was coupled with 2-chloro-N,N-dimethylacetamide under the same conditions applied to step 1 of 35 to give 40 (42 %). LC/MS (ES+) m/z found 445. 1H NMR (400 MHz, DMSO-d6) δ 8.55 (s, 2H), 8.49 (s, 1H), 8.06 (s, 1H), 7.59 (d, J = 8 Hz, 1H), 7.39 (d, J = 8 Hz, 1H), 6.73 (s, 2H), 5.22 (s, 2H), 3.03 (s, 3H), 2.85 (m, 5H), 2.70 (m, 2H), 2.09 (m, 1H), 1.97 (m, 1H). 3-[1-(4-Bromo-phenyl)-cyclobutyl]-5-(1H-pyrazol-4-yl)-[1,2,4]oxadiazole (41). 7 was coupled with 1H-pyrazole-4-carboxylic acid under the same conditions applied to 27 to give 41 (67 %). LC/MS (ES+) m/z found 345, 347. 1H NMR (500 MHz, CDCl3) δ 8.19 (s, 2H), 7.47 (d, J = 10 Hz, 2H), 7.25 (d, J = 10 Hz, 2H), 4.25 (s, 1H), 2.96 (m, 2H), 2.70 (m, 2H), 2.19 (m, 1H), 2.00 (m, 1H).


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1-[4-(3-{1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-cyclobutyl}-[1,2,4]oxadiazol-5-yl)pyrazol-1-yl]-2-methyl-propan-2-ol (42). Step 1: A mixture of 41 (400 mg, 1.2 mmol), 1chloro-2-methyl-2-propanol (250 mg, 2.3 mmol, 2.0 equiv.) and potassium carbonate (240 mg, 1.7 mmol, 1.5 equiv.) in DMF (3 mL) was heated at 90 oC for 40 h. The reaction mixture was concentrated and the residue was diluted with ethyl acetate. The organic layer was washed with water and brine, dried over sodium sulfate and concentrated. The residue was purified on silica gel (DCM/MeOH) to give 1-(4-{3-[1-(4-bromo-phenyl)-cyclobutyl]-[1,2,4]oxadiazol-5-yl}pyrazol-1-yl)-2-methyl-propan-2-ol (446 mg, 92 %). LC/MS (ES+) m/z found 417, 419. Step 2: 1-(4-{3-[1-(4-bromo-phenyl)-cyclobutyl]-[1,2,4]oxadiazol-5-yl}-pyrazol-1-yl)-2-methyl-propan2-ol was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 42 (67 %). LC/MS (ES+) m/z found 432. 1H NMR (500 MHz, CD3OD) δ 8.53 (s, 2H), 8.36 (s, 1H), 8.06 (s, H), 7.54 (d, J = 10 Hz, 2H), 7.44 (d, J = 10 Hz, 2H), 4.17 (s, 2H), 2.95 (m, 2H), 2.77 (m, 2H), 2.20 (m, 1H), 2.00 (m, 1H) 1.18 (s, 6H). 5-{4-[5-(1-Methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-ylmethyl]-phenyl}-pyrimidin-2ylamine (44). Step1: A mixture of 1 (1.0 g, 5.1 mmol) and hydroxylamine (50 % in waster, 9.4 mL) in EtOH (13 mL) was heated at 80 oC for 16 h. The resulting mixture was concentrated in vacuo to give 2-(4-bromo-phenyl)-N-hydroxy-acetamidine 43 (989 mg, 85 %). LC/MS (ES+) m/z found 229, 231. 1H NMR (400 MHz, DMSO-d6) δ 8.94 (s, 1H), 7.47 (d, J = 8 Hz, 2H), 7.24 (d, J = 8 Hz, 2H), 5.42 (s, 2H), 3.24 (s, 2H). Step 2: A mixture of 1-methyl-1H-pyrazole-4carboxylic acid (110 mg, 0.9 mmol) and CDI (142 mg, 0.9 mmol, 1.0 equiv.) in NMP (1 mL) was heated at 50 oC for 0.5 h. To the solution was added 43 (200 mg, 0.9 mmol), and the resulting mixture was heated at 100 oC for 16 h. The reaction mixture was diluted with ethyl acetate. The organic layer was washed with water and brine, dried over sodium sulfate and


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concentrated. The residue was purified on silica gel (heptane/ethyl acetate) to give 3-(4-bromobenzyl)-5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazole (80 mg, 29 %). LC/MS (ES+) m/z found 319, 321. 1H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 8.01 (s, 1H), 7.45 (d, J = 9 Hz, 2H), 7.23 (d, J = 8 Hz, 2H), 4.04 (s, 2H), 3.98 (s, 3H). Step 3: 3-(4-Bromo-benzyl)-5-(1-methyl-1Hpyrazol-4-yl)-[1,2,4]oxadiazole was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 44 (4 %). LC/MS (ES+) m/z found 334. 1H NMR (400 MHz, CD3OD) δ 8.54 (s, 2H), 8.35 (s, 1H), 8.06 (s, 1H), 7.54 (d, J = 8 Hz, 2H), 7.44 (d, J = 8 Hz, 2H), 4.14 (s, 2H), 3.99 (s, 3H). 5-(4-{1-Methyl-1-[5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-ethyl}-phenyl)pyrimidin-2-ylamine (52). Step 1: 2-(4-Bromo-phenyl)-2-methyl-propionitrile 45 was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 2-[4-(2-amino-pyrimidin-5-yl)-phenyl]-2-methyl-propionitrile (54 %). 1H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 2H), 7.67 (d, J = 8 Hz, 2H), 7.56 (d, J = 8 Hz, 2H), 6.79 (s, 2H), 1.69 (s, 6H). Step 2: 2-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-2-methyl-propionitrile was reacted with hydroxylamine under the same conditions applied to step 1 of 44 to give 2-[4-(2-aminopyrimidin-5-yl)-phenyl]-N-hydroxy-isobutyramidine (88 %). Step 3: 2-[4-(2-Amino-pyrimidin5-yl)-phenyl]-N-hydroxy-isobutyramidine was coupled with 1-methyl-1H-pyrazole-4-carboxylic acid under the same conditions applied to step 2 of 44 to give 52 (41 %). LC/MS (ES+) m/z found 362. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.53 (s, 2H), 8.05 (s, 1H), 7.55 (d, J = 8 Hz, 2H), 7.35 (d, J = 8 Hz, 2H), 6.75 (s, 2H), 3.90 (s, 3H), 1.73 (s, 6H). 5-(4-{1-[5-(1-Methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-cyclopropyl}-phenyl)pyrimidin-2-ylamine (53). Step 1: 1-(4-Bromo-phenyl)-cyclopropanecarbonitrile (46) was


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coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 1-[4-(2-amino-pyrimidin-5-yl)-phenyl]-cyclopropanecarbonitrile (91 %). LC/MS (ES+) m/z found 237. 1H NMR (400 MHz, DMSO-d6) δ 8.55 (s, 2H), 7.62 (d, J = 8 Hz, 2H), 7.36 (d, J = 8 Hz, 2H), 6.76 (s, 2H), 1.74 (dd, J = 8, 5 Hz, 2H), 1.50 (dd, J = 8, 5 Hz, 2H). Step 2: 1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-cyclopropanecarbonitrile was reacted with hydroxylamine under the same conditions applied to step 1 of 44 to give 1-[4-(2-aminopyrimidin-5-yl)-phenyl]-N-hydroxy-cyclopropanecarboxamidine (71 %). 1H NMR (400 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.53 (s, 2H), 7.51 (d, J = 8 Hz, 2H), 7.33 (d, J = 8 Hz, 2H), 6.70 (s, 2H), 5.25 (s, 2H), 1.21 (dd, J = 7, 5 Hz, 2H), 0.95 (dd, J = 7, 5 Hz, 2H). Step 3: 1-[4-(2-Aminopyrimidin-5-yl)-phenyl]-N-hydroxy-cyclopropanecarboxamidine was coupled with 1-methyl-1Hpyrazole-4-carboxylic acid under the same conditions applied to step 2 of 44 to give 53 (70 %). LC/MS (ES+) m/z found 360. 1H NMR (400 MHz, DMSO-d6) δ 8.57 (s, 3H), 8.05 (s, 1H), 7.59 (d, J = 8 Hz, 2H), 7.47 (d, J = 8 Hz, 2H), 6.74 (s, 2H), 3.92 (s, 3H), 1.56 (dd, J = 7, 5 Hz, 2H), 1.41 (dd, J = 7, 5 Hz, 2H). 5-(4-{3,3-Dimethyl-1-[5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-cyclobutyl}phenyl)-pyrimidin-2-ylamine (54). Step 1: To a solution of 1 (1.0 g, 5.1 mmol) in DMF (10 mL) was added sodium hydride (60 % in mineral oil, 500 mg, 12.5 mmol, 2.5 equiv.) portionwise at 0 oC. The reaction mixture was stirred at 0 oC for 0.5 h. To the mixture was added 1,3dibromoneopentane (87 %, 1.48 g, 5.6 mmol, 1.1 equiv.), and the mixture was heated at 90 oC for 16 h. The reaction mixture was diluted with water and the product was extracted with ethyl acetate. The organic layer was washed with water and brine, dried over sodium sulfate and concentrated. The residue was purified on silica gel (heptane/ethyl acetate) to give 1-(4-bromophenyl)-3,3-dimethyl-cyclobutanecarbonitrile 47 (256 mg, 20 %). 1H NMR (400 MHz, CDCl3)


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δ 7.52 (d, J = 8 Hz, 2H), 7.27 (d, J = 8 Hz, 2H), 2.73 (dd, J = 11, 2 Hz, 2H), 2.44 (dd, J = 11, 2 Hz, 2H), 1.48 (s, 3H), 1.14 (s, 3H). Step 2: 47 was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 1-[4-(2-amino-pyrimidin-5-yl)phenyl]-3,3-dimethyl-cyclobutanecarbonitrile (97 %). LC/MS (ES+) m/z found 279. 1H NMR (400 MHz, CDCl3) δ 8.53 (s, 2H), 7.52 (d, J = 8 Hz, 2H), 7.48 (d, J = 8 Hz, 2H), 2.77 (dd, J = 11, 2 Hz, 2H), 2.50 (dd, J = 11, 2 Hz, 2H), 1.50 (s, 3H), 1.16 (s, 3H). Step 3: 1-[4-(2-Aminopyrimidin-5-yl)-phenyl]-3,3-dimethyl-cyclobutanecarbonitrile was reacted with hydroxylamine under the same conditions applied to step 1 of 44 to give 1-[4-(2-amino-pyrimidin-5-yl)-phenyl]N-hydroxy-3,3-dimethyl-cyclobutanecarboxamidine (89 %). LC/MS (ES+) m/z found 312. Step 4: 1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-N-hydroxy-3,3-dimethyl-cyclobutanecarboxamidine was coupled with 1-methyl-1H-pyrazole-4-carboxylic acid under the same conditions applied to step 2 of 44 to give 54 (62 %). LC/MS (ES+) m/z found 402. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.55 (s, 2H), 8.05 (s, 1H), 7.59 (d, J = 8 Hz, 2H), 7.41 (d, J = 8 Hz, 2H), 6.76 (s, 2H), 3.91 (s, 3H), 2.84 (d, J = 13 Hz, 2H), 2.60 (d, J = 13 Hz, 2H), 1.11 (s, 3H), 1.07 (s, 3H). 5-(4-{2-Methyl-1-[5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-propyl}-phenyl)pyrimidin-2-ylamine (55). Step 1: 1 was coupled with 2-bromo-propane under the same conditions applied to step 1 of 54 to give 2-(4-bromo-phenyl)-3-methyl-butyronitrile 48 (100 %). Step 2: 48 was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 2-[4-(2-amino-pyrimidin-5-yl)-phenyl]-3-methyl-butyronitrile (100 %). LC/MS (ES+) m/z found 253. Step 3: 2-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-3methyl-butyronitrile was reacted with hydroxylamine under the same conditions applied to step 1 of 44 to give 2-[4-(2-amino-pyrimidin-5-yl)-phenyl]-N-hydroxy-3-methyl-butyramidine (64 %). LC/MS (ES+) m/z found 286. Step 4: 2-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-N-hydroxy-3-


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methyl-butyramidine was coupled with 1-methyl-1H-pyrazole-4-carboxylic acid under the same conditions applied to step 2 of 44 to give 55 (37 %). LC/MS (ES+) m/z found 376. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 8.55 (s, 2H), 8.09 (s, 1H), 7.58 (d, J = 8 Hz, 2H), 7.48 (d, J = 8 Hz, 2H), 6.74 (s, 2H), 3.93 (s, 3H), 3.85 (d, J = 10 Hz, 1H), 2.46 (m, 1H), 0.92 (d, J = 7 Hz, 3H), 0.82 (d, J = 7Hz, 3H). 5-(4-{1,2-Dimethyl-1-[5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-propyl}phenyl)-pyrimidin-2-ylamine (56). Step 1: 48 was coupled with methyl iodide under the same conditions applied to step 1 of 54 to give 2-(4-bromo-phenyl)-2,3-dimethyl-butyronitrile 49 (94 %). Step 2: 49 was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 2-[4-(2-amino-pyrimidin-5-yl)-phenyl]-2,3-dimethylbutyronitrile (98 %). LC/MS (ES+) m/z found 267. Step 3: 2-[4-(2-Amino-pyrimidin-5-yl)phenyl]-2,3-dimethyl-butyronitrile was reacted with hydroxylamine under the same conditions applied to step 1 of 44 to give 2-[4-(2-amino-pyrimidin-5-yl)-phenyl]-N-hydroxy-2,3-dimethylbutyramidine (98 %). LC/MS (ES+) m/z found 300. Step 4: 2-[4-(2-Amino-pyrimidin-5-yl)phenyl]-N-hydroxy-2,3-dimethyl-butyramidine was coupled with 1-methyl-1H-pyrazole-4carboxylic acid under the same conditions applied to step 2 of 44 to give 56 (80 %). LC/MS (ES+) m/z found 390. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 2H), 8.59 (s, 1H), 8.07 (s, 1H), 7.59 (d, J = 8 Hz, 2H), 7.48 (d, J = 8 Hz, 2H), 7.05 (bs, 2H), 3.92 (s, 3H), 2.80 (m, 1H), 1.68 (s, 3H), 0.95 (d, J = 7 Hz, 3H), 0.72 (d, J = 7 Hz, 3H). 5-(4-{1-Cyclopropyl-1-[5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-ethyl}phenyl)-pyrimidin-2-ylamine (57). Step 1: 2-(4-Bromo-phenyl)-2-cyclopropyl-propionitrile 50 was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions


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applied to 12 to give 2-[4-(2-amino-pyrimidin-5-yl)-phenyl]-2-cyclopropyl-propionitrile (100 %). Step 2: 2-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-2-cyclopropyl-propionitrile was reacted with hydroxylamine under the same conditions applied to step 1 of 44 to give 2-[4-(2-aminopyrimidin-5-yl)-phenyl]-2-cyclopropyl-N-hydroxy-propionamidine (99 %). LC/MS (ES+) m/z found 298. 1H NMR (400 MHz, DMSO-d6) δ 9.19 (bs, 1H), 8.55 (s, 2H), 7.55 (d, J = 8 Hz, 2H), 7.43 (d, J = 8 Hz, 2H), 6.74 (s, 2H), 5.09 (bs, 2H), 1.50 (m, 1H), 1.11 (s, 3H), 0.52 (m, 1H), 0.37 (m, 2H), 0.21 (m, 1H). Step 3: 2-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-2-cyclopropyl-Nhydroxy-propionamidine was coupled with 1-methyl-1H-pyrazole-4-carboxylic acid under the same conditions applied to step 2 of 44 to give 57 (23 %). LC/MS (ES+) m/z found 388. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.55 (s, 2H), 8.05 (s, 1H), 7.56 (d, J = 8Hz, 2H), 7.38 (d, J = 8Hz, 2H), 6.76 (s, 2H), 3.91 (s, 3H), 1.66 (m, 1H), 1.48 (s, 3H), 0.64 (m, 1H), 0.45 (m, 2H), 0.33 (m, 1H). 5-(4-{1-Cyclobutyl-1-[5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-ethyl}-phenyl)pyrimidin-2-ylamine (58). Step 1: 2-(4-Bromo-phenyl)-2-cyclobutyl-propionitrile 51 was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give 2-[4-(2-amino-pyrimidin-5-yl)-phenyl]-2-cyclobutyl-propionitrile (87 %). Step 2: 2-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-2-cyclobutyl-propionitrile was reacted with hydroxylamine under the same conditions applied to step 1 of 44 to give 2-[4-(2-aminopyrimidin-5-yl)-phenyl]-2-cyclobutyl-N-hydroxy-propionamidine (69 %). Step 3: 2-[4-(2Amino-pyrimidin-5-yl)-phenyl]-2-cyclobutyl-N-hydroxy-propionamidine was coupled with 1methyl-1H-pyrazole-4-carboxylic acid under the same conditions applied to step 2 of 44 to give 58 (7 %). LC/MS (ES+) m/z found 402. 1H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 2H), 8.58 (s,


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1H), 8.05 (s, 1H), 7.58 (d, J = 9 Hz, 2H), 7.26 (d, J = 9Hz, 2H), 6.76 (s, 2H), 3.91 (s, 3H), 3.28 (m, 1H), 1.98-1.76 (m, 5H), 1.69 (s, 3H), 1.60 (m, 1H). 5-(4-{(R)-1,2-Dimethyl-1-[5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-propyl}phenyl)-pyrimidin-2-ylamine (59). 56 was resolved by ChiralPak AD-H eluting with 70 % MeOH in CO2 at 250 bar at 25 oC. First eluting isomer was (R)-isomer 59. LC/MS (ES+) m/z found 390. Chiral HPLC retention time 4.8 min. 95 % ee. 5-(4-{(S)-1,2-Dimethyl-1-[5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-propyl}phenyl)-pyrimidin-2-ylamine (60). 56 was resolved by ChiralPak AD-H eluting with 70 % MeOH in CO2 at 250 bar at 25 oC. Second eluting isomer was (S)-isomer 60. LC/MS (ES+) m/z found 390. Chiral HPLC retention time 12.7 min. 84 % ee. 5-(4-{(R)-1-Cyclopropyl-1-[5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-ethyl}phenyl)-pyrimidin-2-ylamine (61). 57 was resolved by Chiracel AD eluting with 40 % iPrOH and 0.1 % diethylamine in heptane at 10 mL/min. First eluting isomer was (R)-isomer 61. LC/MS (ES+) m/z found 388. Chiral HPLC retention time 21 min. > 98 % ee. 5-(4-{(S)-1-Cyclopropyl-1-[5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-ethyl}phenyl)-pyrimidin-2-ylamine (62). 57 was resolved by Chiracel AD eluting with 40 % iPrOH and 0.1 % diethylamine in heptane at 10 mL/min. Second eluting isomer was (S)-isomer 62. LC/MS (ES+) m/z found 388. Chiral HPLC retention time 31 min. > 98 % ee. 5-(4-{(R)-1-Cyclobutyl-1-[5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-ethyl}phenyl)-pyrimidin-2-ylamine (63). 58 was resolved by Chiracel AD eluting with 95 % EtOH


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and 0.4 % diethylamine in heptane at 55 mL/min. First eluting isomer was (R)-isomer 63. LC/MS (ES+) m/z found 402. Chiral HPLC retention time 70 min. > 98 % ee. 5-(4-{(S)-1-Cyclobutyl-1-[5-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-ethyl}phenyl)-pyrimidin-2-ylamine (64). 58 was resolved by Chiracel AD eluting with 95 % EtOH and 0.4 % diethylamine in heptane at 55 mL/min. Second eluting isomer was (S)-isomer 64. LC/MS (ES+) m/z found 402. Chiral HPLC retention time 96 min. > 98 % ee. (R)-2-(4-Bromo-phenyl)-2-cyclopropyl-propionitrile (65). 50 was resolved by ChiralPak AY 15 % EtOH in CO2 at 100 bar at 38 oC. LC/MS (ES+) m/z found 402. Chiral HPLC retention time 3.5 min. > 98 % ee. 1H NMR (400 MHz, CDCl3) δ 7.36 (d, J = 9 Hz, 2H), 7.24 (d, J = 9 Hz, 2H), 1.58 (s, 3H), 1.07 (m, 1H), 0.59-0.34 (m, 4H). 3-[(R)-1-(4-Bromo-phenyl)-1-cyclopropyl-ethyl]-5-(1H-pyrazol-4-yl)-[1,2,4]oxadiazole (66). Step 1: 65 was reacted with hydroxylamine under the same conditions applied to step 1 of 44 to give (R)-2-(4-bromo-phenyl)-2-cyclopropyl-N-hydroxy-propionamidine (99 %). LC/MS (ES+) m/z found 283, 285. Step 2: (R)-2-(4-Bromo-phenyl)-2-cyclopropyl-N-hydroxypropionamidine was coupled with 1H-pyrazole-4-carboxylic acid under the same conditions applied to step 2 of 44 to give 66 (90 %). LC/MS (ES+) m/z found 359, 361. 1H NMR (400 MHz, CDCl3) δ 8.09 (s, 2H), 7.36 (d, J = 8 Hz, 2H), 7.19 (d, J = 8 Hz, 2H), 1.60 (m, 1H), 1.46 (s, 3H), 0.62 (m, 1H), 0.46 (m, 1H), 0.38 (m, 1H), 0.29 (m, 1H). 2-(4-{3-[(R)-1-(4-Bromo-phenyl)-1-cyclopropyl-ethyl]-[1,2,4]oxadiazol-5-yl}-pyrazol-1yl)-N,N-dimethyl-acetamide (67). 66 was coupled with 2-chloro-N,N-dimethyl-acetamide under the same conditions applied to step 1 of 35 to give 67 (99 %). LC/MS (ES+) m/z found 444, 446. 1H NMR (400 MHz, CDCl3) δ 7.93 (s, 1H), 7.80 (s, 1H), 7.17 (d, J = 9 Hz, 2H), 7.00


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(d, J = 9 Hz, 2H), 5.00 (s, 2H), 3.04 (s, 3H), 2.95 (s, 3H), 1.62 (m, 1H), 1.49 (s, 3H), 0.65 (m, 1H), 0.49 (m, 1H), 0.40 (m, 1H), 0.33 (m, 1H). 2-[4-(3-{(R)-1-[4-(6-Amino-pyridin-3-yl)-phenyl]-1-cyclopropyl-ethyl}-[1,2,4]oxadiazol-5yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (68). 67 was coupled with 2-aminopyridine-5boronic acid pinacol ester under the same conditions applied to 12 to give 68 (38 %). LC/MS (ES+) m/z found 458. 1H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H), 8.22 (d, J = 2 Hz, 1H), 8.06 (s, 1H), 7.66 (dd, J = 10, 2 Hz, 1H), 7.51 (d, J = 8 Hz, 2H), 7.34 (d, J = 8 Hz, 2H), 6.51 (d, J = 10 Hz, 1H), 6.03 (s, 2H), 5.22 (s, 2H), 3.03 (s, 3H), 2.85 (s, 3H), 1.67 (m, 1H), 1.48 (s, 3H), 0.64 (m, 1H), 0.49 (m, 1H), 0.42 (m, 1H), 0.33 (m, 1H). 2-[4-(3-{(R)-1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-1-cyclopropyl-ethyl}-[1,2,4]oxadiazol5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (69). 67 was coupled with 2-aminopyrimidine-5boronic acid pinacol ester under the same conditions applied to 12 to give 69 (55 %). LC/MS (ES+) m/z found 459. 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 2H), 8.48 (s, 1H), 8.06 (s, 1H), 7.55 (d, J = 8 Hz, 2H), 7.36 (d, J = 8 Hz, 2H), 6.74 (s, 2H), 5.21 (s, 2H), 3.02 (s, 3H), 2.84 (s, 3H), 1.66 (m, 1H), 1.48 (s, 3H), 0.64 (m, 1H), 0.49 (m, 1H), 0.42 (m, 1H), 0.33 (m, 1H). 2-[4-(3-{(R)-1-Cyclopropyl-1-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]ethyl}-[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (70). A mixture of 67 (556 mg, 1.25 mmol), bis(pinacolato)diboron (477 mg, 1.9 mmol, 1.5 equiv.), 1,1’bis(diphenylphosphino)ferrocene dichloropalladium (II) dichloeomethane (102 mg, 0.2 mmol, 0.1 equiv.) and potassium acetate (491 mg, 5.0 mmol, 4.0 equiv.) in 1,4-dioxane (5 mL) in a sealed tube was heated at 100 oC for 4 h. The reaction mixture was diluted with ethyl acetate. The organic layer was washed with water and brine, dried over sodium sulfate and concentrated.


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The residue was purified on silica gel (DCM/MeOH) to give 70 (387 mg, 63 %). LC/MS (ES+) m/z found 492. 2-[4-(3-{(R)-1-[4-(5-Amino-pyrazin-2-yl)-phenyl]-1-cyclopropyl-ethyl}-[1,2,4]oxadiazol5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (71). 70 was coupled with 2-amino-5bromopyrazine under the same conditions applied to 12 to give 71 (55 %). LC/MS (ES+) m/z found 459. 1H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H), 8.45 (d, J = 1 Hz, 1H), 8.05 (s, 1H), 7.94 (d, J = 1 Hz, 1H), 7.85 (d, J = 9 Hz, 2H), 7.36 (d, J = 9 Hz, 2H), 6.50 (s, 2H), 5.21 (s, 2H), 3.03 (s, 3H), 2.85 (s, 3H), 1.67 (m, 1H), 1.49 (s, 3H), 0.64 (m, 1H), 0.49 (m, 1H), 0.42 (m, 1H), 0.33 (m, 1H). 2-[4-(3-{(R)-1-Cyclopropyl-1-[4-(2-methylamino-pyrimidin-5-yl)-phenyl]-ethyl}[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (72). 70 was coupled with (5bromo-pyrimidin-2-yl)-methyl-amine under the same conditions applied to 12 to give 72 (32 %). LC/MS (ES+) m/z found 473. 1H NMR (400 MHz, CDCl3) δ 8.54 (s, 2H), 8.20 (s, 1H), 8.08 (s, 1H), 7.48 (d, J = 9 Hz, 2H), 7.43 (d, J = 9 Hz, 2H), 5.21 (m, 1H), 5.04 (s, 2H), 3.10 (s, 3H), 3.06 (d, J = 5Hz, 3H), 3.01 (s, 3H), 1.73 (m, 1H), 1.60 (s, 3H), 0.72 (m, 1H), 0.57 (m, 1H), 0.50 (m, 1H), 0.41 (m, 1H). 2-[4-(3-{1-Cyclopropyl-1-[4-(2-ethylamino-pyrimidin-5-yl)-phenyl]-ethyl}[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (73). 70 was coupled with (5bromo-pyrimidin-2-yl)-ethyl-amine under the same conditions applied to 12 to give 73 (62 %). LC/MS (ES+) m/z found 487. 1H NMR (400 MHz, DMSO-d6) δ 8.59 (s, 2H), 8.49 (s, 1H), 8.06 (s, 1H), 7.55 (d, J = 8 Hz, 2H), 7.38 (d, J = 8 Hz, 2H), 7.29 (t, J = 6 Hz, 1H), 5.22 (s, 2H), 3.34


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(m, 2H), 3.03 (s, 3H), 2.85 (s, 3H), 1.67 (m, 1H), 1.49 (s, 3H), 1.14 (t, J = 7 Hz, 3H), 0.65 (m, 1H), 0.50 (m, 1H), 0.43 (m, 1H), 0.35 (m, 1H). 2-[4-(3-{(R)-1-Cyclopropyl-1-[4-(2-isopropylamino-pyrimidin-5-yl)-phenyl]-ethyl}[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (74). 70 was coupled with (5bromo-pyrimidin-2-yl)-isopropyl-amine under the same conditions applied to 12 to give 74 (44 %). LC/MS (ES+) m/z found 501. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 2H), 8.49 (s, 1H), 8.06 (s, 1H), 7.56 (d, J = 9 Hz, 2H), 7.38 (d, J = 9 Hz, 2H), 7.15 (d, J = 8 Hz, 1H), 5.22 (s, 2H), 4.07 (m, 1H), 3.03 (s, 3H), 2.85 (s, 3H), 1.67 (m, 1H), 1.48 (s, 3H), 1.16 (d, J = 7 Hz, 6H), 0.65 (m, 1H), 0.50 (m, 1H), 0.43 (m, 1H), 0.34 (m, 1H). 5-(4-{(R)-1-Cyclopropyl-1-[5-(1H-pyrazol-4-yl)-[1,2,4]oxadiazol-3-yl]-ethyl}-phenyl)pyrimidin-2-ylamine (75). Step 1: 65 was coupled with 2-aminopyrimidine-5-boronic acid pinacol ester under the same conditions applied to 12 to give (R)-2-[4-(2-amino-pyrimidin-5-yl)phenyl]-2-cyclopropyl-propionitrile (51 %). LC/MS (ES+) m/z found 265. Step 2: (R)-2-[4-(2Amino-pyrimidin-5-yl)-phenyl]-2-cyclopropyl-propionitrile was reacted with hydroxylamine under the same conditions applied to step 1 of 44 to give (R)-2-[4-(2-amino-pyrimidin-5-yl)phenyl]-2-cyclopropyl-N-hydroxy-propionamidine (85 %). LC/MS (ES+) m/z found 298. Step 3: (R)-2-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-2-cyclopropyl-N-hydroxy-propionamidine was coupled with 1H-pyrazole-4-carboxylic acid under the same conditions applied to step 2 of 44 to give 75 (82 %). LC/MS (ES+) m/z found 374. 1H NMR (400 MHz, DMSO-d6) δ 13.45 (bs, 1H), 8.32 (s, 2H), 8.05 (bs, 2H), 7.33 (d, J = 8 Hz, 2H), 7.15 (d, J = 8 Hz, 2H), 6.53 (s, 2H), 1.45 (m, 1H), 1.26 (s, 3H), 0.41 (m, 1H), 0.23 (m, 2H), 0.11 (m, 1H).


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5-[4-((R)-1-Cyclopropyl-1-{5-[1-(2-dimethylamino-ethyl)-1H-pyrazol-4-yl][1,2,4]oxadiazol-3-yl}-ethyl)-phenyl]-pyrimidin-2-ylamine (76). 75 was coupled with (2chloro-ethyl)-dimethyl-amine hydrochloride under the same conditions applied to step 1 of 35 to give 76 (39 %). LC/MS (ES+) m/z found 445. 1H NMR (400 MHz, DMSO-d6) δ 8.66 (s, 1H), 8.61 (s, 2H), 8.13 (s, 1H), 7.62 (d, J = 8 Hz, 2H), 7.43 (d, J = 8 Hz, 2H), 6.83 (s, 2H), 4.32 (t, J = 6 Hz, 2H), 2.71 (t, J = 6 Hz, 2H), 2.20 (s, 6H), 1.73 (m, 1H), 1.53 (s, 3H), 0.70 (m, 1H), 0.56 (m, 1H), 0.48 (m, 1H), 0.40 (m, 1H). 1-[4-(3-{(R)-1-[4-(2-Amino-pyrimidin-5-yl)-phenyl]-1-cyclopropyl-ethyl}-[1,2,4]oxadiazol5-yl)-pyrazol-1-yl]-2-methyl-propan-2-ol (77). 75 was coupled with 1-chloro-2-methyl-2propanol under the same conditions applied to step 1 of 35 to give 77 (74 %). LC/MS (ES+) m/z found 446. 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 2H), 8.42 (s, 1H), 8.06 (s, 1H), 7.56 (d, J = 8 Hz, 2H), 7.38 (d, J = 8 Hz, 2H), 6.72 (s, 2H), 4.74 (s, 1H), 4.10 (s, 2H), 1.67 (m, 1H), 1.48 (s, 3H), 1.07 (s, 6H), 0.64 (m, 1H), 0.49 (m, 1H), 0.42 (m, 1H), 0.34 (m, 1H). FLAP binding assay FLAP binding potency of test compounds were determined by the ability to displace the radiolabeled ligand [125I]-L-691831(PE NEX084)32. A pellet of human SF9 cells membrane preparation was resuspended in the buffer (100 mM Tris-HCl, pH 7.5, 0.14 M NaCl, 5 % glycerol, 0.05 % Tween-20, 0.5 mM TCEP, 2 mM EDTA) and the suspension was homogenized. The mixture was centrifuged at 3,000 x g for 10 min, the clarified lysate was centrifuged at 10,000 x g for 60 min. The resulting pellet was resuspended in the buffer (100 mM Tris-HCl, pH 7.5, 0.14 M NaCl, 5 % glycerol, 0.05 % Tween-20, 0.5 mM TCEP, 2 mM EDTA), then the mixture was homogenized to determine the protein concentration. The membrane protein/SPA


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beads (GE healthcare) complex (1 µg/well and 80 µg/well respectively, 20 µL), test compound (10 µL) and 0.32 nM[125I]-L-691831 were added to a 384 well plate. After incubation for 2 h at room temperature, the plate was read on Perkin Elmer Topcount. LTB4 human whole blood assay Human blood was drawn from consented human volunteers into heparinized tubes and aliquots (35 µL) were added to wells containing mixture of test compounds and PBS (5 µL). After incubation for 15 min at 37 oC (5 % CO2), calcimycin (10 µL, 100 µM final concentration) was added to wells. The plate was mixed and incubated for 30 min at 37 oC (5 % CO2), then centrifuge at 2000 rpm for 5 min at 4 oC. Supernatant samples (5 µL) were transferred from blood assay plates to detection plates containing LTB4-XL665 (10 µL, CisBio Assays). AntiLTB4 cryptate (5 µL, CisBio Assays) was added to the detection plates and mixed. Plates were incubated for 2 h at room temperature with foil seals, and then read on a Perkin Elmer ViewLux (emission filters donor 618/8 nm and acceptor 671/8 nm). Aqueous equilibrium solubility A saturated solution of test compound in pH 6.8 buffer (citric acid/disodium phosphate) was rotated for 24 h. After centrifugation and filtration, a concentration of the test compound was measured by HPLC. Human liver microsomal stability A mixture of human liver microsomes (1 mg/mL protein) and test compound (1 µM) was preincubated at 37 oC for 10 min then NADPH was added to the mixture. The time course (0 to 60


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min) concentration of the test compound was determined by LC/MS. Time (minutes) is plotted against the natural logarithm of the percent compound remaining to determine the slope. Human hepatocyte stability A mixture of cryopreserved human hepatocytes (1 million cells/mL) and test compound (1 µM) was incubated at 37 oC for 6 h. The time course (0 to 6 h) concentration of the test compound was determined by LC/MS. Time (minutes) is plotted against the natural logarithm of the percent compound remaining to determine the slope. CYP450 inhibition The inhibitory effects of test compound on CYP450 2C9, 2D6 and 3A4 were determined by using human liver microsome (0.05 mg/mL protein) and cocktail substrates (4 µM dichlofenac for 2C9, 3 µM Dextromethorphan for 2D6 and 2.5 µM midazolam for 3A4). Test compound solutions in 0.1 M pH 7.4 potassium phosphate buffer and the cocktail substrates were preincubated with human liver microsome for 10 min at 37 oC. NADPH was added to initiate the reaction and the reaction was conducted for 6 min at 37 oC. The reaction was conducted with 10 concentration points up to 50 µM to determine IC50s. CYP450 3A4 induction assessment Induction potential was determined in fresh plated human hepatocytes (sandwich cultured, single donor). The cultures were exposed to Rifampicin (25 µM) or test compound for 48 hours, with media aspirated and replenished with freshly prepared media and dosing agent every 24 hours. After the 48 h treatment period, midazolam (10 µM) was added. The time course (0 to


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120 min) for the formation of the specific CYP450 3A4 metabolite, 1’-hydroxymidazolam was plotted to determine the linear slope of metabolite formation. The fold of induction is determined by dividing the test compound linear slope by the solvent control linear slope. CYP450 3A4 Time Dependent Inhibition Incubations were performed in an automated fashion using a Tecan Evo (Durham, NC) in polypropylene tubes containing 100 mM phosphate buffer (pH 7.4), 0.1 mg/ml human liver microsomes, and 1 mM NADPH at 37 °C (primary incubation). Final test compound concentrations ranged from 0 to 100 µM (7 concentrations). After initiation of the primary incubation by the addition of NADPH, samples (10 µl) were taken at 0, 1, 3, 5, 15, and 30 min and added to a secondary incubation consisting of 190 µl of phosphate buffer containing 15 µM midazolam (saturating concentration, ~5 x Km) and 1 mM NADPH. The secondary reactions were quenched after 8 min using one volume of ice-cold acetonitrile containing αhydroxymidazolam-d4 (100 nM) and centrifuged at 3000 x g for 10 min. Supernatants were transferred to a 96-well plate and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). Peak areas for 1’-hydroxy midazolam and α-hydroxymidazolam-d4 were determined using Analyst software (version 1.4.2; AB Sciex). Caco-2 cell permeation assay Caco-2 cells (1-2 x 105 cells/cm2) were seeded on filter inserts (PET filters, 0.4 µm pore size) and cultured for 10 to 25 days. Test compound in a transport solution (HTP-4 buffer) was applied to the apical or basolateral donor side for measuring A-B or B-A permeability, respectively. The receiver side contains HTP-4 buffer supplemented with 2 % BSA. Samples


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were collected at the start and end of experiment from the donor and at various time intervals for up to 2 hours also from the receiver side for concentration measurement by LC/MS/MS. hERG manual patch clamp assay Electrophysiology hERG currents are recorded from HEK293 cells (stably expressing the human ERG potassium channel) at room temperature (20-22 ºC), using the whole-cell patch clamp technique. HEK293 cells plated on coverslips are placed at the bottom of a 1 mL perfusion chamber (RC-26, Warner Instruments) mounted on the stage of an inverted microscope (Nikon, Japan). Changes in hERG tail current amplitude during the hyperpolarizing step were measured in response to escalating concentrations of test compound. Measured bath concentrations of compound in samples collected from the 3, 10 and 30 µM solutions. Docking studies and computational approaches. Docking studies were carried out using the software Glide (Glide, v5.6, Schrödinger, Inc., New York, NY, 2009). All ligands were prepared using LigPrep (Maestro, v6.0, Schrödinger, Inc.) before docking. The FLAP protein structure utilizing one of the binding sites formed by chain A and B (PDB ID: 2Q7M)29 was first processed with Protein Preparation Wizard (Maestro v6.0, Schrodinger) and then the Glide grid file was generated. Docking studies were carried out using Glide SP module with expanded sampling. No constraint was used in the docking process. Ligand docking poses were evaluated based on Glide Gscore, emodel value, overlap with independently calculated Watermap for the ligand-binding site of FLAP, and visual inspection in reference to the binding mode of MK-591 from the X-ray structure. Glide XP docking was also performed on representative inhibitors within the series to determine if there were alternative docking poses.


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Single crystal X-ray diffraction study of 69. Crystal structure of 69 (C24H26N8O2, MW = 458.53) was determined from the data collected using cryo cooled (100 K) mono crystal mounted on SMART APEXII system on Bruker D8 Quest diffractometer with micro-focus Cu Kα radiation (λ = 1.54184 Å) as X-ray source. Crystals belong to orthorhombic space group P 212121 with cell parameters of a = 8.733 Å, b = 9.564 Å, c = 26.786 Å, α = β = γ = 90°. The structure was solved by direct methods and refined by full-matrix least-squares method using the Bruker SHELXTL Software Package. Summary of data collection and structure refinement parameters are provided in the supporting information Table 1. In addition, crystallographic data (excluding structural factors) for the structure in this paper are being deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1032443. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax: +44-(0)1223-336033 or e-mail: [email protected]). ASSOCIATED CONTENT Supporting Information The supporting information Table 1 shows data collection and structure refinement for 69. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Phone: (203) 798 5049. Fax: (203) 791 6072. E-mail: [email protected]


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Present Author Addresses ¦

For S.D.L.: Karos Pharmaceuticals, 5 Science Park, New Haven, Connecticut 06511, United

States. Φ

For J.H.: Bristol-Myers Squibb, 4 Research Parkway, Wallingford, Connecticut 06492, United

States. ║

For J.M.H.: Quintiles, Bioanalytical and ADME Labs, 5225 Exploration Drive, Indianapolis,

Indiana 46241, Unites States. §

For W.L.; Merck Serono, 91 Jianguo Road, Tower B 18th Floor Gemdale Plaza, Beijing China

100022. ‡

For H.Y.L: Synovel Laboratory Co., Ltd., 42 Lake Avenue, 222, Danbury Connecticut 06811,

United States. ^For D.S.T: Biomanufacturing Research Institute and Technology Enterprise, 1801 Fayetteville Street, Durham, North Carolina 27707, United States.

Notes The authors declare no competing financial interest. ABBREVIATIONS SAR, structure-activity relationship; FLAP, 5-lipoxygenase-activating protein; LTB4, leukotriene B4; LT, leukotriene; DMPK, drug metabolism and pharmacokinetics; AA, arachidonic acid; 5LO, 5-lipoxygenase; 5-HpETE, 5-hydroperoxyeicosatetraenoic acid; LTA4, leukotriene A4;


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LTC4, leukotriene C4; CysLT, cysteinyl LT; GPCR, G-protein-coupled transmembrane receptor; COPD, chronic obstructive pulmonary disease; hWB, human whole blood; mWB, mouse whole blood; CYP450, cytochrome P-450; HLM, human liver microsome; Vss, volume of distribution; Qh, hepatic blood flow;

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Table of contents graphic

N N

N H2N

N

69 (BI 665915) FLAP binding IC50 (nM): 1.7 Human whole blood LTB4 production IC50 (nM): 45 Aqueous equilibrium solubility at pH 6.8 (µg/mL): 48

O NN

69 (BI 665915)

O

N


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Synthesis, SAR, and Series Evolution of Novel ... - ACS Publications

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