A novel chemical series of 5-LO activating protein (FLAP) inhibitors for

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A novel chemical series of 5-LO activating protein (FLAP) inhibitors for treatment of Coronary Artery Disease Malin Lemurell, Johan Ulander, Hans Emtenäs, Susanne Winiwarter, Johan Broddefalk, Marianne Swanson, Martin A Hayes, Luna Prieto Garcia, Annika Westin Eriksson, Johan Meuller, Johan Cassel, Gabrielle Saarinen, Zhong-Qing Yuan, Christian Löfberg, Staffan Karlsson, Monica Sundqvist, and Carl Whatling J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b02012 • Publication Date (Web): 30 Mar 2019 Downloaded from http://pubs.acs.org on March 31, 2019

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Journal of Medicinal Chemistry

A novel chemical series of 5-LO activating protein (FLAP) inhibitors for treatment of Coronary Artery Disease Malin Lemurell*†, Johan Ulander†, Hans Emtenäs‡, Susanne Winiwarter†, Johan Broddefalk†, Marianne Swanson†, Martin A. Hayes║, Luna Prieto Garcia†, Annika Westin Eriksson§, Johan Meuller║, Johan Cassel†¥, Gabrielle Saarinen†¥, Zhong-Qing Yuan†, Christian Löfberg†¥, Staffan Karlsson‡, Monica Sundqvist†¥, Carl Whatling†

†

Cardiovascular, Renal and Metabolism IMED Biotech Unit; ‡Pharmaceutical Science IMED

Biotech Unit; ║Discovery Science IMED Biotech Unit; §Drug Safety & Metabolism IMED Biotech Unit; AstraZeneca Gothenburg, Pepparedsleden 1, Mölndal, 43183, Sweden. ¥

See present addresses.

KEYWORDS: FLAP, 5-Lipoxygenase Activating Protein inhibitor, 5-LO pathway, Leukotriene B4 (LTB4), inflammatory disease, Coronary Artery Disease (CAD)

ABSTRACT: 5-lipoxygenase activating protein (FLAP) inhibitors have proven to attenuate 5-LO pathway activity and leukotriene production in human clinical trials. However, previous clinical

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candidates have been discontinued and the link between FLAP inhibition and outcome in inflammatory diseases remains to be established. We here describe a novel series of FLAP inhibitors identified from a screen of 10k compounds and the medicinal chemistry strategies undertaken to progress this series. Compound 4i showed good overall properties and a pIC50 hWBfree of 8.1 and an LLE of 5.2. Target engagement for 4i was established in dog using ex vivo measurement of LTB4 levels in blood with good correlation to in vitro potency. A predicted human dose of 280 mg BID suggests a wide margin to any identified in vitro off-target effects and sufficient exposure to achieve an 80% reduction of LTB4 levels in humans. Compound 4i is progressed to pre-clinical in vivo safety studies.

INTRODUCTION 5-lipoxygenase activating protein (FLAP) is critical for the cellular production of leukotrienes, the lipid mediators derived from arachidonic acid that have inflammatory and vasoactive actions and play a role in innate immunity.1 In leukotriene producing cells such as neutrophils, macrophages and mast cells, FLAP is primarily located within the nuclear membrane. Here it may act as a docking site for 5-lipoxygenase (5-LO) following cell activation and facilitates the transfer of arachidonic acid released from membrane phospholipids to the active site of 5-LO which initiates the production of leukotrienes (Figure 1).


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Figure 1: FLAP facilitates the transfer of arachidonic acid from membrane phospholipids to the active site of 5-LO. Inhibition of FLAP attenuates the production of the leukotriene precursor LTA4 and the formation of the biologically active leukotrienes LTB4, LTC4, LTD4 and LTE4 In addition to a role in innate immunity, leukotriene production has been associated with several chronic diseases that have an inflammatory component in their pathophysiology including cardiovascular diseases such as atherosclerotic coronary artery disease (CAD).2 It is possible to block 5-LO pathway activity completely by targeting 5-LO or FLAP, preventing formation of the precursor leukotriene A4 (LTA4) that is required for production of the proinflammatory and vasoactive leukotrienes LTB4, LTC4, LTD4 and LTE4 (Figure 1). Moreover, it has been suggested that FLAP inhibition might deliver a more robust and sustained suppression of the 5-LO pathway activity in vivo than can be achieved by 5-LO inhibition.3 Both FLAP and 5-LO inhibitors have been evaluated in human clinical trials and have shown robust target engagement and sustained suppression of the 5-LO pathway as indicated by reduction


3


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in ex vivo LTB4 production in blood and LTE4 levels in urine.4-5 In a Phase II study in coronary artery disease (CAD) patients that had experienced a recent (within 1 month) acute coronary syndrome (ACS) event, the 5-LO inhibitor VIA-2291 delivered a reduction in non-calcified atherosclerotic plaque volume and a reduction in the incidence of new coronary artery lesion formation after 6 months dosing during which LTB4 production was suppressed by over 80%.6 Despite these promising results VIA-2291 has not been progressed into Phase III clinical studies. Different chemical series of FLAP inhibitors have been described,7-10 some representative compounds are exemplified in Figure 2.

DG-031

AM803

AZD6642

BI665915

Figure 2: Examples of the most advanced FLAP inhibitors from previously described chemical series, which have later been discontinued during pre-clinical toxicology studies or in development.

None of these chemical series have delivered a clinical candidate that has progressed beyond Phase II despite being very potent inhibitors (~0.5-10 nM). To the best of our knowledge the reasons for discontinuation were related to either efficacy (DG-031 and AM803) or a variety of safety


4

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liabilities, which for the class of compounds leading up to AM803 potentially could be attributed to their highly lipophilic nature (ClogP>5) and promiscuous in vitro secondary pharmacology profiles.7-8 We have previously demonstrated that it is possible to generate a FLAP inhibitor with lower lipophilicity, high lipophilic ligand efficiency (LLE) and good in vitro safety profile in AZD6642.11 This compound was however stopped due to toxicity in pre-clinical animal models (unpublished results). Another later generation FLAP inhibitor, BI665915,9 showed testicular toxicological effects.12 In light of these challenges, we saw the need to identify and optimize a new, structurally distinct, chemical series of FLAP inhibitors for selection of a candidate drug with the aim to evaluate the benefits of inhibiting the 5-LO pathway in patients with CAD.

RESULTS Hit Finding A new hit finding campaign was initiated to identify novel FLAP inhibitors with the aim to open up chemical space with the possibility to identify compounds with improved LLE and safety profiles. A competitive binding assay with 3H-MK-591 as the ligand was used as primary screening13 and it was assumed that the assay would identify inhibitors which bound to the same site in the transmembrane region of FLAP as MK-591 and other reported FLAP inhibitors.3, 14 The low throughput of the FLAP-binding assay precluded a full-scale HTS of the AZ compound collection and a virtual screen was instead performed from which a subset of 10k compounds were selected. The focus was on ligand-based techniques since the large cavity and low resolution of existing X-ray structures 2Q7M, 2Q7R (4.0, 4.2 Å)14 did limit the use of docking approaches.


5


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Some of the features of the putative binding pocket were however incorporated into pharmacophore searches. MOE calculation was used for a pharmacophore/excluded volume search.15 To avoid entropic penalties and to focus attention on bioactive conformations a Boltzmann-weighted scoring of the conformational ensembles was used for the 3D-scoring metrics (shape / pharmacophoric /Tversky/ electrostatic fit tanimotos). Approximately 20% of the selected compounds originated from multi-fingerprint search techniques (Trust, alfi, foyfi, ecfi)15-17 and 80 % from 3-D techniques such as shape or pharmacophore searches.18-19 More potent compounds were then evaluated in a secondary assay measuring the effect on LTB4 production in human whole blood (hWB) after calcium ionophore stimulation. In parallel, representative compounds from each chemical cluster identified were subject to in vitro ADME and safety evaluation, e.g. solubility, metabolic stability, CYP inhibition, hERG inhibition, and reactive metabolite formation. A series of highly interesting neutral compounds with a trans-benzoyl cyclohexane carboxamide core was identified, generic structure shown in the heading of Table 1.

Chemistry The strategy during the lead generation phase was to design routes which allowed late stage diversification. As a result, early exploration of this series was ideally set up for starting from trans-2-(4-bromobenzoyl)cyclohexane-1-carboxylic acid (1) followed by routes for the variation of the amines or the Suzuki reagents (Scheme 1). Trans-2-(4-bromobenzoyl)cyclohexane-1carboxylic acid (1) was either purchased as racemic material or obtained as the pure enantiomer starting from (1R,2R)-2-(methoxycarbonyl) cyclohexane carboxylic acid.20 The final compounds


6

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4a-u were made from 1 either by conversion to the amides 2a-k followed by Suzuki reaction or compound 1 was first used in a Suzuki coupling to construct intermediates (3a-c) followed by amide coupling. The affinity for FLAP was found to reside in the (R,R)-enantiomer of these compounds and from here on any compound number will refer to the (R,R)-enantiomer unless denoted (rac) or (S,S). Scheme 1. Late stage variation of the amines and Suzuki reactants to provide compounds 4a-u either as racemates or pure enantiomers. See Table 1 for chemical structure and stereochemistrya,b


7


a

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Commonly used reagents and conditions: (a) R1-NH2, TBTU, DIPEA, DMF or R1-NH2, T3P®,

Et3N, 50 °C; (b) R2-B(OH)2/R2-Bpin, Pd(dppf)Cl2, K3PO4, DMF-H2O, 100 °C or R2-B(OH)2/R2Bpin, Pd(dtbpf)Cl2, K2CO3, dioxane-water, 80 °C. b

See experimental section for synthesis details. No synthesis details given for 4a (rac) and 4b (rac)

as they were identified as hits during the HTS from the AstraZeneca compound collection.

To introduce variation in the aryl moiety we instead used racemic (3a,7a-trans)-hexahydro-2benzofuran-1,3-dione as starting material. As a result, this was reacted with the organometallic regents made from either 2,5-dibromo pyridine or 2-iodo-5-bromo toluene to furnish compounds 5 (rac) and 6 (rac) respectively. These could then be modified by the chemistry depicted in Scheme 2 to form compounds 7 (rac) and 8 (rac).

Scheme 2. Variation of the aryl ring introduced by ring opening of (3a,7a-trans)-hexahydro-2benzofuran-1,3-dionea


8

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a

Reagents and conditions: (a) (3a,7a-trans)-hexahydro-2-benzofuran-1,3-dione, n-BuLi, Et2O

and/or THF, -78 oC; 20-32% (b) 1H-pyrazol-3-ylboronic acid, Pd(dppf), K3PO4 or K2CO3, DME/EtOH/H2O or Dioxane/H2O, heated in a microwave reactor at 130 oC or reflux, 30-42%; (c) 1,3-dimethyl-1H-pyrazol-4-amine hydrogen chloride, TBTU, NMM or DIPEA, DMF or CH2Cl2, rt, 6-77%; (d) NH4CO2H, Pd(OH)2, CH3OH, heated in a microwave reactor at 100 oC, quant; (e) K2CO3, PhN(Tf)2, DMF, rt, 69%; (f) chiral separation.


9


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Introduction of an ortho-fluoro substituent in the aryl ring turned out to be problematic using this approach due to instability of the organometallic reagents. Following a similar procedure as previously described16 compound 10 (rac) could be obtained in moderate yield by reacting the benzyl protected phenol21 9 with the (3a,7a-trans)-hexahydro-2-benzofuran-1,3-dione under lithiation conditions. After amide coupling using standard conditions and de-benzylation, the obtained phenol 11 (rac) was used in the following Suzuki coupling by first converting the phenol 11 (rac) to the corresponding triflate followed by direct coupling with heteroaryl boronic acid. The racemic mixture of 12 (rac) was separated by chiral chromatography to give both individual enantiomers (12 and 12 (S,S)). A limiting factor using the anhydride route, was the lack of appropriate commercially available (hetero) aryl halides as starting material. More importantly, the use of anhydrides was considered a safety risk due to the intrinsic allergenic properties of these compounds. Our attention instead turned to develop a Diels-Alder based route (Scheme 3). β-Acyl acrylic acids of type 13 are known to perform well in Diels-Alder reactions with dienes.22 Thus, compound 13 was reacted with transbutadiene to give the cyclohexene 14 (rac) in high yield.

Scheme 3. Synthesis of ortho-fluoro compounds using a Diels-Alder approacha


10

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a

Reagents and conditions: (a) Oxoacetic acid, CH3CO2H, CH3SO3H, 130 oC, 73%; (b) buta-1,3-

diene, toluene, 60 oC, 90%; (c) chiral separation; (d) 1,3-dimethyl-1H-pyrazol-4-amine hydrogen chloride, T3P®, Et3N, EtOAc, 80 oC, 70%; (e) 1H-Pyrazol-3-ylboronic acid or 1-(tetrahydro-2Hpyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole,20 Pd(dtbpf)Cl2, K2CO3, dioxane-H2O, 85 oC, 52-58%; (f) Pd/C, CH3OH, H2 (g, 1atm), rt, 56%; (g) 5% Rh/C, THF, H2 (g, 2bar), rt, 91%; (h) 4-Amino-1-methyl-1H-pyrazole-3-carboxamide, HATU, DIPEA, DMF, rt; (i) 1.25 M HCl in CH3OH, 16 oC, 69%.


11


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To avoid chiral separation of all final compounds in this series, we choose to separate the cyclohexene derivative 14 (rac). The (+) isomer of 14 was found to give 12 (S,S) (the least active enantiomer) by applying the chemistry described in Scheme 1 followed by reducing the double bond by hydrogenation, thus concluding that the (-)-isomer of 14 had the desired (R,R)stereochemistry. Moreover, a shorter route to the final compounds was desired and our strategy aimed to reduce the double bond at an earlier stage. Initial attempts to reduce the double bond of 14 by hydrogenation with Pd/C turned out to be difficult and gave rise to by-products such as reduced ketone as well as debromination. After a thorough screen of catalyst and reaction conditions, we found that Rh/C gave a good result. Thus, the cyclohexene 14 was converted to 16 by hydrogenation. The final compound 17 where made by applying similar chemistry as in Scheme 1 followed by acidic hydrolysis (Scheme 3). Moreover, to introduce variation in the cyclohexyl ring system, we first choose to react the anhydride with the amine forming the imide 18. After hydrogenation of the double bond, the imide 19 could be reacted with organometallic reagent forming compound 20 (rac). When applying standard Suzuki conditions, a mixture of our desired screening compound 21 (rac) together with the cyclized hemi-amidal 22 was isolated (Scheme 4).

Scheme 4. Introduction of bridged cyclohexyl ring systema


12

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a

Reagents and conditions: (a) 1,3-dimethyl-1H-pyrazol-4-amine hydrogen chloride, CDI, DIPEA,

CH2Cl2, rt, 91%; (b) 5% Pd/C, THF, H2, rt, 99%; (c) 2-fluoro-5-bromo-bromobenzene, n-BuLi, THF, -78 oC, 11%; (d) 1H-pyrazol-5-ylboronic acid, Pd(dppf)2CH2Cl2, K2CO3, dioxane-H2O, 14%.

Our attention then turned into functionalize the double bond further and we focused on the installation of a cyclopropyl group. In this case we performed the Diels-Alder reaction with the ester 24 to give the cyclohexene 25 (rac). Cyclopropanation using the Furukawa modification of the Simmons–Smith reaction gave compound 26 (rac) as a mixture of stereoisomers. Finally, the desired compound 27 could be synthesized by hydrolysis of the ester, followed by standard amine coupling and Suzuki coupling and then by separation of the four stereoisomers by chiral chromatography. The stereochemical arrangement of the cyclopropyl group was assigned by NMR (NOE experiments).


13


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Scheme 5. Introduction of cyclopropyl fused cyclohexyl ring systema

a

Reagents and conditions: (a) buta-1,3-diene, hydroquinone, toluene, 160C, 95%; (b) Et2Zn,

ClCH2I, DCE/heptane, 0 °C→rt; (c) LiOH, CH3OH-THF-H2O, 60 °C; (d) 1-methyl-3(trifluoromethyl)-1H-pyrazol-4-amine, T3P®, Et3N, 50 °C, 64% (from 25 (rac)); (e) 1H-pyrazol3-ylboronic acid, Pd(dtbpf)Cl2, K2CO3, dioxane-water, 80 °C, 80%. (f) chiral separation; (g) 1methyl-5-(trifluoromethyl)-1H-pyrazol-4-amine, T3P®, Et3N, 80 °C, 47% (from 25); (h) 1Hpyrazol-3-ylboronic acid, Pd(dtbpf)Cl2, K2CO3, dioxane-water, 80 °C, 63%.

To minimize the cumbersome separation of all isomers in the end, we decided to first separate the enantiomers of the cyclohexyl 25 (rac). The (-)-isomer of the cyclohexyl 25 was deduced from 1


14

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and was further converted to 26 as mixture of two diastereomers by cyclopropanation. Analogous to 27, the desired screening compound 28 was obtained (Scheme 5). We also wanted to evaluate modifications of the aryl ketone. Initial attempts to introduce the 1,2benzisoxazole group via a late stage modification of ortho-fluoro compounds, e.g. 12, using hydroxylamine proved to be very inefficient and provided poor yield. We therefore decided to introduce the 1,2-benzisoxazole group earlier in the synthetic sequence. In a stepwise procedure oxime 29 could be prepared from 4-bromo-2-fluoro cyclohexylarylketone 16, subsequent deprotonation by NaH to facilitate the intramolecular SNAr reaction gave the 1,2-benzisoxazole 30. The desired compound 32 was then prepared by Suzuki coupling to give acid 31 followed by amide bond formation.

Scheme 6. Synthesis of a 1,2-benzisoxazole derivativesa


15


a

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Reagents and conditions: (a) HONH2·HCl, pyridine, 100 C; (b) NaH, DMF, rt, 59% (over 2

steps); (c) 3-methyl-1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)-1H-pyrazole, PdCl2(PPh3)2, K2CO3, dioxane-H2O, 90 C, 96%; (d) 4-amino-1-methyl-1Hpyrazole-3-carboxamide, HATU, DIPEA, DMF, rt; (e) HCl(aq), dioxane-H2O, rt, 74% (over 2 steps); (f) H2SO4, EtOH, ; (g) Et2Zn, ClCH2I, DCE/heptane, 0 °C→rt; (h) LiOH, CH3OH-THFH2O, 50 °C; (i) separation of diastereomers.

Finally, 35, an analogue of 1,2-benzisoxazole compound 32 also containing the bicyclo[4.1.0]heptane ring system was prepared. Esterification of cyclohexen derivate 14 followed by cyclopropanation and ester hydrolysis gave 33. The 1,2-benzioxazole moiety of 34 was installed


16

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in a manner analogous to compound 32. Suzuki coupling, amide bond formation and subsequent separation of diastereomers gave the analogue 35.

Structure Activity Relationships and In Vitro Data Early hits from this series demonstrated good affinity to FLAP with binding pIC50 higher than 6 and hWB pIC50 close to 6, here exemplified by 4a (rac) and 4b (rac), Table 1. The structure activity relationship (SAR) of the series was developed using the competitive binding assay and the hWB assay. In the hWB assay the standard compound incubation time was 30 min, but as described by Stock et.al., a prolonged incubation time (4 h) was required for some compounds to appreciate the full response on LTB4 reduction as indicated in Table 1 and Table 2.23 The permeability of the compounds was found to be a key parameter influencing the onset to full effect, Figure 3. Further, the IC50 hWB contains large effects from non-specific protein binding. The free concentration is what drives the biological response and the relevant design parameter is therefore the free potency, IC50u .24 To account for the effect of non-specific binding, the free potency IC50 hWBfree was estimated using the measured compound fraction unbound (fu) in human plasma. The binding assay contained lipid membranes with some effects from non-specific partitioning. The correlation between the uncorrected IC50’s from the binding assay and IC50 hWBfree was however sufficient (R2=0.8) to guide prioritization of compounds for further study. LLE25 was a key optimization parameter to identify compounds with highly specific interactions with FLAP and here derived from pIC50 hWBfree-logD.


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Figure 3: Compounds with reduced permeability required prolonged incubation time (4 h vs 30 min) in the hWB assay to attenuate the full response on LTB4 reduction. Dashed line gives the linear fit (residual standard error = 0.32 log units which corresponds to a factor 2), R2=0.44; dotted lines correspond to a ratio 4x difference from fitted line.

The

series

was

expanded

using

the

commercially

available

racemic

trans-2-(4-

bromobenzoyl)cyclohexane-1-carboxylic acid 1 suitable for library synthesis by amide formation (R1) and cross coupling reactions (R2). From compound 4c (rac) and 4d (rac) it was evident that potency could be improved in this series with a reduction in lipophilicity compared to 4a (rac) and 4b (rac). Chiral separation of the more active compounds, e.g. 4d (rac), showed that FLAP inhibition primarily resided in the (R,R)-isomer as shown for the corresponding two enantiomers 4d and 4d (S,S) (Table 1). A set of C-C and C-N cross coupling reactions of the aryl bromide to introduce various aliphatic and aromatic substituents as R2 showed a relatively narrow SAR (Table 1). The R2 pyrazole derivative 4d (rac) appeared to offer beneficial interactions in this region of the binding site and matched pair analysis confirmed this group as a preferred aromatic R2 motif. The R2 2-pyridine


18

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and 2-pyrrole motifs of 4e (rac) and 4f (rac) respectively were both tolerated but nonetheless exhibited reduced binding affinity compared to 4d (rac). Methylation of the 2-N of the pyrazole (4g (rac)) had a significant negative impact on binding affinity. This suggests the R2 pyrazole nitrogens form one or two hydrogen bond interactions at the binding site. Introduction of a methyl substituent in the 4-position of the pyrazole (4h (rac)) resulted in an affinity drop of an order of magnitude compared to 4d (rac). More successful was the introduction of a methyl substituent in the 5-position of the pyrazole (4i), while larger groups such as ethyl, iso-propyl or trifluoro methyl (data not shown) were not well tolerated. The relatively high binding affinity of 4i translated into good potency in the hWB assay. The free hWB pIC50 was as high as 8.1 resulting in an LLE of 5.2. A broad set of amides with various substitutions were synthesized as analogues of 4d to explore the SAR of the R1 region (Table 1). This dataset suggested a rather high degree of tolerance for structural and electronic variations in this region and several compounds were identified with sub 100 nM binding affinity. However, to move away from aromatic amides and a potential genotoxic metabolite liability, aliphatic amines were introduced with generally poor success. Two of the more promising compounds with good affinity and high hWB activity were 4j and 4k. A smaller drop in affinity was observed for the tertiary amide 4l in comparison with 4d. From this example it was not apparent that the hydrogen bond donor of the amide bond is important for the compounds’ interaction to the binding site even though many of the tertiary amides showed reduced affinity to a varying degree. Reversing the amide bond or replacing it with a sulfonamide was detrimental to binding affinity (data not shown). Exploration of a wide range of 5-membered and 6-membered aromatic amines resulted in highly potent compounds, in particular 4m, which exhibited a free hWB pIC50 of 9.3. It was also found


19


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that large substituents elongating out from the vector of the amide in R1 as shown with the parasubstituent in 4n (rac) were tolerated. The 5-membered amino pyrazole introduced in 4d was found to be of particular interest and used as basis for further exploration. Substitutions in the position next to the amide bond as exemplified by biaryl 4o and by 4p, 4q, 4r, 4s, 4t and 4u resulted in novel FLAP inhibitors which engaged the protein with high LLE between 4.8 and 5.5. It is known that certain amino pyrazoles are genotoxic26-28 and hence interesting amino pyrazoles were tested in a 2-strain Ames assay (with and without metabolic activation using rat S9 fraction) throughout the optimization of this series. Compounds containing a masked Ames positive aromatic amine were not progressed. Among the potent compounds highlighted above, only the amine in 4s was measured Ames positive. The amino pyrazole in 4r was not chemically stable and therefore could not be reliably tested in the Ames assay. Interestingly it was observed that the substitution pattern of the amino pyrazole was important and while the amino pyrazole in 4t did not show any Ames liability, its analogue with the methyl moved to the adjacent pyrazole nitrogen was measured Ames positive.

Table 1. Structure Activity Relationship for the described series of FLAP inhibitors. Data is given for the (R,R) enantiomer unless denoted (rac or (S,S)).a


20

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-NHR1

-R2

Compd

cLogP

LogD7.4

pIC50 bind.b

pIC50 hWBc

pIC50 hWBfreed

LLE hWBfreee

4a (rac)

3.5

3.9

5.6

ND

ND

ND

4b (rac)

3.5

3.6

6.5

5.7

7.4

3.8

4c (rac)

2.5

3.1

6.7

6.3

ND

ND

4d (rac) 4d 4d (S,S)

2.6

2.6

7.1 7.3 5.7

6.3 6.7 5.1

7.1 7.4 5.8

4.5 4.8 3.2

4e (rac)

3.0

2.8

6.3

5.7

6.8

4.0

4f (rac)

3.1

3.1

6.0

ND

ND

ND

4g (rac)

2.5

2.5

4.6

4.5

5.1

2.5

4h (rac)

2.6

3.1

6.0

ND

ND

ND

4i

2.9

3.0

7.2

7.2

8.1

5.2

4j

3.1

3.0

6.6

5.6

6.7

3.7

4k

2.6

2.1

7.2

6.9f

7.4

5.3


21


a

Page 22 of 94

4l

2.6

3.0

6.7

6.6

7.5

4.5

4m

4.1

3.9

7.8

7.8

9.3

5.4

4n (rac)

3.3

3.2

7.8

7.0f

8.3

5.2

4o

3.4

2.9

7.5

7.4

8.4

5.5

4p

2.6

3.4

7.6

7.3

8.5

5.1

4q

2.6

3.6

7.4

7.3

8.4

4.8

4r

2.8

3.0

7.5

7.0

8.5

5.5

4s

2.8

3.5

8.0

7.2

8.5

5.0

4t

2.4

3.5

7.7

7.0

8.5

5.0

4u

2.2

3.1

7.4

6.7

7.9

4.8

Defined in experimental section, bpIC50 in the FLAP binding assay, cpIC50 in the human whole

blood assay measuring the inhibition of the downstream LTB4 production with 30 min incubation time of the compound, dpIC50 in the hWB assay taking into account the plasma protein binding, e

LLE calculated from pIC50 hWBfree minus LogD7.4, f pIC50 in the hWB assay after 4 h incubation

time of the compound, ND = not determined.


22

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We then turned to explore the core phenyl ring. Introduction of heteroatoms or small substitutions resulted in reduced affinity, as exemplified by 7 (rac) and 8 (rac) (Table 2) compared to 4d (rac), except for the introduction of halogens ortho to the ketone. The high hWB potency and LLE was maintained upon introduction of an ortho-fluoro substituent, e.g. 12 and 17 compared to 4d and 4u, respectively. The major metabolic in vitro clearance pathway for compounds in this series was CYP mediated oxidation in the aliphatic cyclohexyl ring. Significant effort was put into blocking metabolism or reducing lipophilicity in this part of the molecule, though with limited success with respect to simultaneously maintaining binding affinity. Replacement of the cyclohexyl ring with alternative aliphatic 3-, 4-, 5- or 7-membered rings were unsuccessful. Complete removal of the ring and replacement with an ethylene or ethyl chain was also not tolerated, nor was the introduction of a methylene or ether bridge between the 3- and 6-carbon. The analogue 21 (rac) with an ethylene bridge showed reasonable affinity but with the penalty of increased lipophilicity and increased metabolic clearance. Overall, small modifications that affected the conformation of the cyclohexyl and the 1, 2- substituents led to detrimental effects on affinity, which we hypothesized to be due to distortion of the vectors positioning the 1, 2 - substituents into a preferred binding mode in the FLAP binding site. Introduction of fluorine in the cyclohexyl ring to block oxidation sites caused issues with chemical or metabolic instability due to HF elimination. However, addition of a cyclopropyl ring in 27 and 28 which altered the sp3 carbons most labile to CYP mediated oxidation into ones with more sp2 like character resulted in highly potent compounds with free hWB pIC50 of 9.0 and 8.5, respectively.


23


Page 24 of 94

Considering the generally reactive and electrophilic nature of a ketone group, we were initially keen to understand if it could be replaced. However, it turned out that the ketone was difficult to replace. Replacing the ketone with the corresponding alcohol, replacement by methylene, ether, thioether, sulfoxide or sulfone were examples of modifications that resulted in no or poor binding affinity to FLAP. The benzisoxazole in 32 and 35 was identified as a tolerated ketone bioisostere which retained potency in the binding and hWB assays, e.g. 32 versus 4u (Table 2).


24

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Table 2. Structure Activity Relationship for alternative central core structures of the described series of FLAP inhibitors. Data is given for the (R,R) enantiomer unless denoted (rac).a Structure

Compd

cLogP

pIC50 bind.b

pIC50 hWBc

A=N R=H

7 (rac)

1.8

1.8

6.0

5.4

5.8

4.1

A=CH R=Me

8 (rac)

3.1

3.0

6.0

6.1

6.9

3.9

A=CH R=F

12

2.3

2.9

6.9

7.2

8.0

5.1

NA

17

1.9

3.5

7.0

6.8

8.3

4.8

NA

21 (rac)

3.1

4.0

7.2

6.7

7.1

3.1

LogD7.4

pIC50 hWBfreed

LLE hWBfreee


25


Page 26 of 94

R1 = 27

2.4

3.7

7.9

7.6

9.0

5.3

28

2.4

3.7

7.9

7.3

8.5

4.8

n=0

32

1.8

3.4

7.1

7.2f

8.5

5.1

n=1

35

1.5

3.5

7.4

6.9f

8.5

5.0

R1=

a

Defined in experimental section, bpIC50 in the FLAP binding assay, cpIC50 in the human whole

blood assay measuring the inhibition of the downstream LTB4 production, dpIC50 in the hWB assay taking into account the plasma protein binding, eLLE calculated from pIC50 hWBfree minus LogD7.4, f pIC50 in the hWB assay after for 4h incubation time of the compound.

Initial in vitro ADME and safety evaluation of the early compounds in this series, such as 4c (rac) and 4d (rac), showed good solubility and medium metabolic stability (Table 3). Throughout the advancement of the series, selected compounds were profiled more broadly, and the series was optimized on parameters such as solubility, permeability and metabolic stability as the combination of these properties along with the compounds’ potency influences the predicted human dose. Additionally, properties that indicated potential risks for adverse effects, e.g. CYP inhibition, reactive metabolite formation, and hERG inhibition were monitored.


26

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The more lipophilic compounds in the series, such as 4m and 27 showed low solubility whereas 28, an isostere of 27 with similar lipophilicity, demonstrated moderate solubility. The ketone replacement benzisoxazole introduced in 32 and 35 had a negative impact on solubility which turned out to be a general disadvantage for this ketone replacement. Some of the modifications in the amino pyrazole such as the introduction of a nitrile group or a primary amide in 4t or 4u reduced the solubility compared to 4i. Despite that the solubility of 4u was still moderate, the primary amide likely contributed to the poor solubility of 17, 32 and 35 as well. The compounds displayed in general good permeability in Caco-2 cells, often with no or low levels of efflux (data not shown). Introduction of a second amide bond hampered permeability significantly in 4n (rac). Also, the Papp of 32 with the additional amide substituent on the amino pyrazole was relatively low whereas higher permeability was measured for the analogues 4u, 17 and 35. Only low levels of cytochrome P450 enzyme inhibition were observed across the series from screening a panel of the most common CYP isoforms and in general no or only minor levels of reactive metabolite (RM) formation were seen using glutathione (GSH) as a trapping agent in human liver microsome incubations. The higher levels of RM observed could be assigned to oxidation followed by elimination in the R1 dimethyl oxazole in 4c (rac) and in the aromatic biaryl ring of 4o. Ketone reactivity was assessed on a subset of compounds using methoxylamine as trapping agent.27 No or only minimal imine formation was detected for the compounds tested (data not shown).


27


Page 28 of 94

Table 3. In vitro data for selected compounds (solubility, CYP inhibition, metabolic stability in human hepatocytes, Caco-2 permeability, reactive metabolite (RM) trapping, contribution of CYP3A4 to metabolism and hERG inhibition

aq. Sol.a (M)

max pIC50 CYP inhb (isoform)c

HH Clintd (l/min/ 106 cells)

Caco-2 Pappe (10-6cm/s)

trapping

(GSH)f

Contr 3A4g/ fm, CYP3A4h (%)

hERGi pIC50/ inh. (%)

4c (rac)

92

5.4 (3A4)

6

44

yes

ND/ND

99.9% ee): [α]20D –80.3 (c 1.0, CH3CN); HRMS (ESI) m/z calcd for C22H24FN5O2 [M + H]+ 410.1992, found 410.1977. The second eluted compound was collected


69


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and evaporated to give 12 (0.561 g, 49%, 97.9% ee). [α]20D +72.0 (c 1.0, CH3CN); 1H NMR (400 MHz, CDCl3) δ 7.86 (t, J = 7.9 Hz, 1H), 7.72 (s, 1H), 7.51 – 7.63 (m, 3H), 7.16 (s, 1H), 6.66 (d, J = 2.4 Hz, 1H), 3.72 (s, 3H), 3.63 – 3.71 (m, 1H), 2.79 – 2.89 (m, 1H), 2.13 – 2.22 (m, 4H), 2.00 – 2.08 (m, 1H), 1.89 (t, J = 12.4 Hz, 2H), 1.68 – 1.81 (m, 1H), 1.30 – 1.56 (m, 3H), 1.24 (qd, J = 12.8, 3.2 Hz, 1H); HRMS (ESI) m/z calcd for C22H24FN5O2 [M + H]+ 410.1992, found 410.1991.

(1S,2S)-N-(1,3-Dimethyl-1H-pyrazol-4-yl)-2-[2-fluoro-4-(1H-pyrazol-3yl)benzoyl]cyclohexane-1-carboxamide (12 (S,S)) T3P® (50% in EtOAc, 0.55 mL, 0.92 mmol) was added to a solution of (1S,6S)-6(4-bromo-2-fluorobenzoyl)cyclohex-3-ene-1-carboxylic acid (14 (S,S), 0.20 g, 0.61 mmol), 1,3dimethyl-1H-pyrazol-4-amine hydrogen chloride (0.18 g, 1.22 mmol) and Et3N (0.51 mL, 3.67 mmol) in EtOAc (3.0 mL) and the reaction mixture was stirred at rt for 3 days and then heated in a microwave reactor at 80 oC for 1 h. The reaction mixture was partitioned between EtOAc and saturated NaHCO3(aq). The organic phase was dried (phase-separator) and concentrated in vacuo. The crude product was purified by preparative HPLC (C18, 15→65% CH3CN in H2O/CH3CN/NH3 95/5/0.2) to give (1S,6S)-6-(4-bromo-2-fluorobenzoyl)-N-(1,3-dimethyl-1Hpyrazol-4-yl)cyclohex-3-ene-1-carboxamide (0.18 g, 70 %): MS m/z 422.1 [M + H]+. 1H-Pyrazol-3-ylboronic acid (0.064 g, 0.57 mmol) and a degassed solution of K2CO3 (0.21 g, 1.52 mmol) in water (2 mL) was added to a solution of (1S,6S)-2-(4-bromo-2fluorobenzoyl)-N-(1,3-dimethyl-1H-pyrazol-4-yl)cyclohexane-1-carboxamide

(0.16

g,

0.38

mmol) in dioxane (2 mL) and the reaction mixture was heated in a microwave reactor at 85 oC for 1 h. The reaction mixture was diluted with EtOAc and the organic phase was washed with saturated


70

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NaCl(aq). The aqueous phase was extracted twice with EtOAc and the combined organic phase was dried (phase-separator) and concentrated in vacuo. The crude product was purified by preparative HPLC (C18, 15→65% CH3CN in H2O/CH3CN/NH3 95/5/0.2) to give (1S,6S)-N-(1,3dimethyl-1H-pyrazol-4-yl)-6-[2-fluoro-4-(1H-pyrazol-3-yl)benzoyl]cyclohex-3-ene-1carboxamide (15 (S,S), 0.080 g, 52%): MS m/z 408.3 [M + H]+. Palladium catalyst (Pd/C, 10%, 0.050 g, 0.050 mmol) was added to a solution of compound 15 (S,S) (0.080 g, 0.20 mmol) in CH3OH (5 mL) and the reaction mixture was stirred at rt under hydrogen (1 atm) for 3 days. The reaction mixture was filtrated and concentrated in vacuo and the crude compound was purified by preparative HPLC (C18, 5→70% CH3CN in H2O/CH3CN/HCO2H 95/5/0.2) to give 12 (S,S) (0.045 g, 56 %): 1H NMR (500 MHz, DMSO-d6) δ 13.16 (s, 1H), 9.36 (s, 1H), 7.66 – 7.89 (m, 5H), 6.90 (d, J = 1.7 Hz, 1H), 3.62 (s, 3H), 3.45 – 3.55 (m, 1H), 2.86 (t, J = 11.1 Hz, 1H), 2.00 (m, 5H), 1.80 (d, J = 10.4 Hz, 2H), 1.23 – 1.45 (m, 3H), 1.15 (q, J = 12.6 Hz, 1H); 19F NMR (470 MHz, DMSO) δ -111.86 (s); HRMS (ESI) m/z calcd for C22H24FN5O2 [M + H]+ 410.1992, found 410.1977; [α]20D –76.3 (c 1.0, CH3CN).

(2E)-4-(4-Bromo-2-fluorophenyl)-4-oxobut-2-enoic acid (13) A mixture of 1-(4-bromo-2-fluorophenyl)ethan-1-one (100 g, 460.8 mmol), oxoacetic acid (55.1 g, 599 mmol) and methane sulfonic acid (2.99 mL, 46.1 mmol) in CH3CO2H (500 mL) under N2 was heated at 130 oC for 20 h. The reaction mixture was allowed to cool to 80 o

C and water (750 mL) was slowly added. The reaction mixture was cooled to 20 oC during 2 h,

and then stirred at rt for 1 h. The suspension was filtered and the solid cake was washed with 25% CH3CO2H (aq) (200 mL) and then water (3 x 150 mL). The solids were dried under reduced pressure at 45 oC for 3 days to give 13 (92 g, 73 %). 1H NMR (400 MHz, CDCl3) δ 10.26 (bs, 1H),


71


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7.53 – 7.73 (m, 2H), 7.17 – 7.37 (m, 2H), 6.70 (dd, J = 15.5, 1.4 Hz, 1H); MS m/z 270.8 [M – H]. trans-6-(4-Bromo-2-fluorobenzoyl)cyclohex-3-ene-1-carboxylic acid (14 (rac)) A suspension of buta-1,3-diene (20 % in toluene, 227 mL, 671.7 mmol) was added to a mixture of 13 (91.7 g, 335.8 mmol) in toluene (500 mL) in a steel cylinder, and the reaction mixture was heated at 60 oC and 1 bar overnight. EtOAc (2 x 200 mL) was added to the reaction mixture and the mixture was concentrated in vacuo. EtOAc (100 mL) was added to the crude product, followed by heptane (300 mL) and the suspension was heated to 80o C. The suspension was slowly allowed to attain rt and then stirred at rt for 1 h. The solids were collected by filtration and washed with EtOAc (20 % in heptane, 50 mL) and then with heptane (150 mL) to give 14 (rac) (98.9 g, 90 %): 1H NMR (500 MHz, CDCl3) δ 7.59 (t, J = 8.1 Hz, 1H), 7.18 – 7.34 (m, 2H), 5.54 – 5.69 (m, 2H), 3.52 (td, J = 10.9, 5.3 Hz, 1H), 2.95 (dddd, J = 11.0, 7.4, 5.6, 2.7 Hz, 1H), 2.27 – 2.5 (m, 2H), 2.06 – 2.21 (m, 1H), 1.88 (ddd, J = 16.9, 14.2, 2.8 Hz, 1H); ); MS m/z 326 [M – H]-.

(-)-trans-6-(4-Bromo-2-fluorobenzoyl)cyclohex-3-ene-1-carboxylic acid (14) and (+)-trans-6(4-bromo-2-fluorobenzoyl)cyclohex-3-ene-1-carboxylic acid (14 (S,S)) The enantiomers of 14 (rac) were separated by chiral SFC chromatography on a Lux C2 column (5μm, 250x30mm). 800 mg (200 mg/mL in CH3OH) was injected and eluted with 20% CH3OH in CO2 (g) (175 bar) at a flow rate of 130 mL/min and detected at 280 nm. The first eluted compound was collected and evaporated, dissolved in EtOAc (30 mL) and then treated with heptane (250 mL). The suspension formed was cooled on an ice-bath and filtered to give 14 (42.3


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g, 43%, 99.9% ee): 1H NMR (500 MHz, CDCl3) δ 7.59 (t, J = 8.1 Hz, 1H), 7.16 – 7.30 (m, 2H), 5.60 (qdt, J = 7.0, 4.8, 2.3 Hz, 2H), 3.51 (td, J = 10.9, 5.3 Hz, 1H), 2.94 (tdd, J = 10.9, 5.6, 2.0 Hz, 1H), 2.24 – 2.49 (m, 2H), 2.12 (ddtd, J = 16.0, 11.1, 4.4, 2.4 Hz, 1H), 1.88 (dddt, J = 17.3, 11.1, 4.2, 2.3 Hz, 1H); [α]20D -16.8 (c 1.0, CH3CN). The second eluted compound was collected and evaporated, dissolved in EtOAc (30 mL) and then treated with heptane (250 mL). The suspension formed was cooled on an ice-bath and filtered to give 14 (S,S) (45 g, 45%, 99.6% ee): 19F NMR (470 MHz, CDCl3) δ -108.82 (s); [α]20D 18.6 (c 1.0, CH3CN).

(1R,2R)-2-(4-Bromo-2-fluorobenzoyl)cyclohexane-carboxylic acid (16) Rhodium catalyst (5% Rh/C, 0.63 g, 0.31 mmol) was added to a solution of 14 in THF (100 mL). The reaction mixture was stirred at rt under hydrogen (2 bar) for 16 h. The crude product was filtered through celite, the filtrate was evaporated and the crude product was recrystallized from methyl tert-butylether and heptane. The solid formed was dried in vacuo to give 16 (9.16 g, 91%): 1H NMR (500 MHz, CDCl3) δ 7.60 (t, J = 8.1 Hz, 1H), 7.18 – 7.29 (m, 2H), 3.19 – 3.30 (m, 1H), 2.73 (t, J = 11.3 Hz, 1H), 2.03 – 2.13 (m, 1H), 1.94 (d, J = 13.3 Hz, 1H), 1.66 – 1.79 (m, 2H), 1.15 – 1.4 (m, 3H), 0.97 – 1.07 (m, 1H); MS m/z 308.8 [M – H]-.

4-({(1R,2R)-2-[2-Fluoro-4-(3-methyl-1H-pyrazol-5-yl)benzoyl]cyclohexane-1carbonyl}amino)-1-methyl-1H-pyrazole-3-carboxamide (17) 4-Amino-1-methyl-1H-pyrazole-3-carboxamide (0.33 g, 2.38 mmol) was added to a solution of 16 (0.52 g, 1.59 mmol), HATU (0.60 g, 1.59 mmol) and DIPEA (0.55 mL, 3.18 mmol)


73


Page 74 of 94

in DMF (3 mL), and the reaction mixture was stirred at rt for 3 h. The reaction mixture was diluted with EtOAc and the organic phase was washed twice with saturated NaHCO3(aq). The water phase was extracted once more with EtOAc and the combined organic phase was dried (phase-separator), evaporated and the crude product was dried in vacuo overnight to give 4-({[(1R,2R)-2-(4-bromo2-fluorobenzoyl)cyclohexyl]-carbonyl}amino)-1-methyl-1H-pyrazole-3-carboxamide (1.29 g). MS m/z 453.1 [M + H]+. K2CO3 (0.88 g, 6.36 mmol) and Pd(dtbpf)Cl2 (0.092 g, 0.14 mmol) were added to a solution of 4-({[(1R,2R)-2-(4-bromo-2-fluorobenzoyl)cyclohexyl]carbonyl}-amino)-1-methyl1H-pyrazole-3-carboxamide (1.29 g) and 1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)-1H-pyrazole20 (0.70 g, 2.39 mmol) in dioxane (6 mL) and water (6 mL). The reaction mixture was evacuated and purged with N2 three times, and then heated in a preheated oil-bath at 80 °C for 1 h. The reaction mixture was diluted with EtOAc and the organic phase was washed with saturated NaCl(aq). The aqueous phase was extracted with EtOAc and the combined organic phase was dried (phase-separator) and evaporated in vacuo. The crude product was purified by silica gel flash chromatography (75% →100% EtOAc in heptane) to give 4{[(1R,2R)-2-{2-fluoro-4-[3-methyl-1-(oxan-4-yl)-1H-pyrazol-5-yl]benzoyl}cyclohexane-1carbonyl]amino}-1-methyl-1H-pyrazole-3-carboxamide (0.49 g, 58 %): MS m/z 521.4 [M – H]-. HCl (1.25 M in CH3OH, 0.5 mL, 0.63 mmol) was added to a solution of 4({[(1R,2R)-2-{2-fluoro-4-[3-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5yl]benzoyl}cyclohexyl]carbonyl}amino)-1-methyl-1H-pyrazole-3-carboxamide (0.22 g, 0.40 mmol) in CH3OH (10 mL) and the reaction mixture was concentrated in vacuo at 16 °C. The residue was dissolved in CH3OH (10 mL) and concentrated in vacuo at 16 °C. The crude product was purified by preparative HPLC (C18, 5→80% CH3CN in H2O/CH3CN/NH3 95/5/0.2) to give


74

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17 (0.13 g, 69%): 1H NMR (500 MHz, CD3OD) δ 7.99 (s, 1H), 7.83 (t, J = 8.0 Hz, 1H), 7.54 – 7.65 (m, 2H), 6.53 (s, 1H), 3.84 (s, 3H), 3.56 – 3.64 (m, 1H), 2.86 (t, J = 10.6 Hz, 1H), 2.35 (s, 3H), 2.12 (dd, J = 18.0, 14.0 Hz, 2H), 1.90 (d, J = 10.4 Hz, 2H), 1.59 (q, J = 12.4 Hz, 1H), 1.41 – 1.52 (m, 2H), 1.33 – 1.22 (m, 1H); 19F NMR (470 MHz, CD3OD) δ -112.78 (s); HRMS (ESI) m/z calcd for C23H25FN6O3 [M + H]+ 905.4023, found 905.4113.

(rac)-N-(1,3-Dimethyl-1H-pyrazol-4-yl)-3-(2-fluoro-4-(1H-pyrazol-3yl)benzoyl)bicyclo[2.2.2]octane-2-carboxamide (21 (rac)) DIPEA (1.960 mL, 11.22 mmol) was added to a suspension of bicyclo[2.2.2]oct-5-ene-2,3dicarboxylic

anhydride

(1.00

g,

5.61

mmol)

and

1,3-dimethyl-1H-pyrazol-4-amine

dihydrochloride (1.033 g, 5.61 mmol) in CH2Cl2 (30 mL). The mixture was stirred at rt for 30 min, at which time 1,1'-carbonyl-diimidazole (1.876 g, 11.22 mmol) was added and the mixture was stirred at rt for 60 min. The mixture was diluted with CH2Cl2 and washed with saturated NH4Cl(aq) (x2), saturated NaHCO3(aq) and saturated NaCl(aq). The organic phase was dried, and the solvent was removed under reduced pressure to give 2-(1,3-dimethyl-1H-pyrazol-4-yl)-3a,4,7,7atetrahydro-1H-4,7-ethanoisoindole-1,3(2H)-dione 18 (1.382 g, 91 %) as a solid which was used in the next step without further purification. Pd/C (0.524 g, 0.25 mmol) was added to a solution of 18 (1.336 g, 4.92 mmol) in THF (70 mL). The mixture was hydrogenated at RT, at 1 atm for 1.5 h. The reaction mixture was filtered and the solvent was removed under reduced pressure to give 2-(1,3-dimethyl-1H-pyrazol-4yl)hexahydro-1H-4,7-ethanoisoindole-1,3(2H)-dione 19 (1.331 g, 99 %) as a solid which was used in the next step without purification.


75


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Compound 19 (0.600 g, 2.20 mmol) and 1,4-dibromo-2-fluorobenzene (0.557 g, 2.20 mmol) were dissolved in THF (20 mL) and cooled to -78 °C and n-butyllithium (1.32 mL, 3.29 mmol) was added. The mixture was stirred at -78 °C for 60 min and was then allowed to reach rt before being quenched with saturated NH4Cl(aq). The solvent was removed under reduced pressure and the residue was partitioned between EtOAc and saturated NaHCO3(aq). The organic phase was washed with saturated NH4Cl(aq) and saturated NaCl(aq) and then dried, before the solvent was removed under reduced pressure. The residue was purified by flash column chromatography (50 %→100 % of CH3OH/EtOAc 1:9 in heptane) and preparative HPLC (Xbridge, 40→80 % CH3CN in H2O/CH3CN/NH3 95/5/0.2 buffer). Organic solvent from the combined fractions were removed under reduced pressure and the aqueous residue was extracted with EtOAc and CH2Cl2. The organic phases were dried (phase separator) and the solvent was removed under reduced pressure. To facilitate precipitation the residue was dissolved in CH2Cl2/pentane and then the solvent was removed under reduced pressure. (rac)-3-(4-bromo-2fluorobenzoyl)-N-(1,3-dimethyl-1H-pyrazol-4-yl)bicyclo[2.2.2]octane-2-carboxamide 20 (rac) (0.106 g, 10.77 %) was collected as a solid. MS m/z 448.1 [M+H]+ K2CO3 (98 mg, 0.71 mmol) and Pd(dppf)Cl2·CH2Cl2 (19 mg, 0.02 mmol) were added to a solution of 20 (rac) (106 mg, 0.24 mmol) and 1H-pyrazol-3-ylboronic acid (40 mg, 0.35 mmol) in dioxane (2 mL) and water (2 mL). The mixture was degassed and backfilled with N2 and then heated at reflux for 60 min. More 1H-pyrazol-3-ylboronic acid (13 mg, 0.12 mmol) was added together with Pd(dppf)Cl2·CH2Cl2 (19 mg, 0.02 mmol) and the mixture was heated at reflux for another 30 min. The mixture was allowed to reach rt and was then diluted with EtOAc and water. The aqueous phase was extracted once with EtOAc. The combined organic phase was washed with saturated NaHCO3(aq), saturated NaCl(aq), saturated NH4Cl(aq) and saturated NaCl(aq), before


76

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the organic phase was dried (phase separator) and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (50 % →100 % CH3OH/EtOAc 1:9 in heptane) to give (rac)-N-(1,3-dimethyl-1H-pyrazol-4-yl)-3-(2-fluoro-4-(1H-pyrazol-3yl)benzoyl)bicyclo[2.2.2]octane-2-carboxamide (21 (rac), 14 mg, 14 %).

1

H NMR (600 MHz,

DMSO-d6) δ 13.17 (s, 1H), 9.41 (s, 1H), 7.85 – 7.88 (m, 1H), 7.78 – 7.84 (m, 3H), 7.75 (d, J = 12.9 Hz, 1H), 6.79 – 7.00 (m, 1H), 4.09 (d, J = 6.5 Hz, 1H), 3.69 (s, 3H), 3.44 (d, J = 6.9 Hz, 1H), 2.09 (s, 3H), 1.98 (s, 1H), 1.94 (s, 1H), 1.72 – 1.81 (m, 1H), 1.63 – 1.71 (m, 1H), 1.46 – 1.61 (m, 3H), 1.32 – 1.43 (m, 2H), 1.22 – 1.3 (m, 1H); HRMS (ESI) m/z calcd for C24H27FN5O2 [M + H]+ 436.2143, found 436.2158.

(1R,6R)-6-(4-bromobenzoyl)cyclohex-3-ene-1-carboxylate (25) A suspension of (E)-ethyl 4-(4-bromophenyl)-4-oxobut-2-enoate (24, 5.3 g, 18.7 mmol) in toluene (16 mL) was transferred to a steel bomb with a teflon insert. Hydroquinone (0.021 g, 0.19 mmol) and excess of cooled -78 °C buta-1,3-diene (7.9 mL, 94 mmol) were added and the reaction mixture was stirred at rt for 1 h then at 160 °C overnight and 200 °C for 2 h before being cooled to RT. The reaction mixture was then concentrated in vacuo and the residue was purified by flash column chromatography (5→10% EtOAc in heptane) to give ethyl trans-6-(4bromobenzoyl)cyclohex-3-ene-1-carboxylate (25 (rac), 6.0 g, 95%); 1H NMR (500 MHz, CDCl3) δ 7.85 – 7.90 (m, 2H), 7.60 – 7.66 (m, 2H), 5.68 – 5.83 (m, 2H), 3.98 – 4.15 (m, 2H), 3.73 – 3.85 (m, 1H), 3.05 – 3.17 (m, 1H), 2.51 – 2.63 (m, 1H), 2.32 – 2.43 (m, 1H), 2.18 – 2.32 (m, 1H), 2.02 – 2.16 (m, 1H), 1.17 (t, J = 7.1 Hz, 3H).


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The isomers of 25 (rac) (4.6 g, 13.6 mmol) were separated by chiral SFC chromatography on a Lux C2 250x30 mm, 5 μm column. 575 mg (115 mg/ml in CH3OH) was injected and eluted at 40 °C with 10% CH3OH in CO2, 120 bar at a flow rate of 130 mL/min and detected at 254 nm. The first eluted compound was collected and evaporated to yield 25 (2.23 g, 99.9% ee); [α]D25 – 31.4 (c 1.0, CH3CN).

(1S,3R,4R,6R)-N-[1-Methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]-4-[4-(1H-pyrazol-3yl)benzoyl]bicyclo[4.1.0]heptane-3-carboxamide (27) trans-Ethyl 6-(4-bromobenzoyl)cyclohex-3-enecarboxylate (25 (rac), 510 mg, 1.51 mmol) was dissolved in DCE (3.8 mL) and cooled to 0 °C, before the mixture was degassed and backfilled with N2 (x2). Diethylzinc (1M in heptane, 3.02 mL, 3.02 mmol) was added to the solution followed by dropwise addition of chloroiodomethane (0.441 mL, 6.05 mmol). The mixture was stirred at 0 °C for 30 min and was then stirred at rt overnight. The reaction mixture was diluted with CH2Cl2 and saturated Na2EDTA(aq) and the mixture was stirred for a couple of min, then a small portion of saturated NaHCO3(aq) was added and the mixture was stirred until two clear phases were formed. The mixture was washed with saturated NaHCO3(aq). The aqueous phase was extracted once with CH2Cl2 and the organic phases were combined and dried (phase separator), before the solvent was removed under reduced pressure:

1

H NMR showed

product:starting material ~ 5:1. The crude mixture was dissolved in DCE (3.8 mL) and cooled to 0 °C, before the mixture was degassed and backfilled with N2 (x2). Diethylzinc (1M in heptane, 3.02 mL, 3.02


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mmol) was added to the solution followed by dropwise addition of chloroiodomethane (0.441 mL, 6.05 mmol). The mixture was stirred at 0 °C for 30 min and was then stirred at rt overnight. The reaction mixture was diluted with CH2Cl2 and saturated Na2EDTA(aq) and the mixture was stirred for a couple of min, then a small portion of saturated NaHCO3(aq) was added and the mixture was stirred until two clear phases were formed. The mixture was washed with saturated NaHCO3(aq). The aqueous phase was extracted once with CH2Cl2 and the organic phases were combined and dried (phase separator), before the solvent was removed under reduced pressure. Ethyl (trans-4(4-bromobenzoyl)bicyclo[4.1.0]heptane-3-carboxylate (26 (rac), 524 mg, 99 %) was collected as a colorless oil and used without further purification; MS m/z 351.0 [M + H]+. Lithium hydroxide (0.5 M in water) (11 mL, 5.5 mmol) was added to a solution of 26 (rac) (521 mg, 1.48 mmol) in THF (2 mL) and CH3OH (2 mL). The mixture was heated at 60 °C for 6 h and then stirred at rt overnight. The mixture was concentrated under reduced pressure. The residue was dissolved in water and 3.8M HCl(aq) was added until a white precipitate formed and pH was ~2. The mixture was extracted with EtOAc (x3). The organic phases were combined, dried (phase separator), and the solvent was removed under reduced pressure. trans-4-(4Bromobenzoyl)bicyclo[4.1.0]heptane-3-carboxylic acid (478 mg, 100 %) was collected as a white foam; MS m/z 321.1 [M – H]-. Et3N 0.603 mL, 4.33 mmol) and then a solution of T3P® (50% in EtOAc, 1.03 mL, 1.73 mmol) were added to a mixture of trans-4-(4-bromobenzoyl)bicyclo[4.1.0]heptane-3carboxylic acid (466 mg, 1.44 mmol) and 1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-amine (313 mg, 1.90 mmol) in EtOAc (13 mL). The reaction mixture was stirred at rt overnight. More 1methyl-3-(trifluoromethyl)-1H-pyrazol-4-amine (119 mg, 0.72 mmol), Et3N (0.300 mL, 2.16 mmol), and a solution of T3P® (0.515 mL, 0.87 mmol) were added and the mixture was stirred at


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rt overnight. The mixture was diluted with EtOAc and washed with saturated NaHCO3(aq) (x2), saturated NH4Cl(aq) and saturated NaCl(aq). The organic phase was dried (phase separator) and the solvent was removed under reduced pressure. The residue was purified by preparative HPLC (35→80% CH3CN in H2O/CH3CN/NH3 95/5/0.2 buffer). Product containing fractions were partially concentrated and extracted with CH2Cl2 (x2). The organic phase was dried (phase separator) and concentrated in vacuo to give trans-4-(4-bromobenzoyl)-N-(1-methyl-3(trifluoromethyl)-1H-pyrazol-4-yl)bicyclo[4.1.0]heptane-3-carboxamide (431 mg, 64 %) as an off-white solid; MS m/z 470.0 [M + H]+. K2CO3 (505 mg, 3.66 mmol) and Pd(dtbpf)Cl2 (59 mg, 0.09 mmol) were added to a solution

of

trans-4-(4-bromobenzoyl)-N-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-

yl)bicyclo[4.1.0]heptane-3-carboxamide (430 mg, 0.91 mmol) and 1H-pyrazol-3-ylboronic acid (153 mg, 1.37 mmol) in dioxane (4.5 mL) and water (4.5 mL). The mixture was degassed and backfilled with N2 (x3) and then heated at 80 °C for 45 min in a microwave reactor. The mixture was diluted with EtOAc and washed with H2O. The aqueous phase was extracted once with EtOAc. The combined organic phases were washed with saturated NH4Cl(aq) and saturated NaCl(aq). The EtOAc layer was dried (phase separator) and the solvent was removed under reduced pressure. The residue was dissolved in EtOAc and filtered (SiO2). The compound was eluted with EtOAc and the solvent was then removed under reduced pressure. The crude compound was purified by preparative HPLC (15→75% CH3CN in H2O/CH3CN/NH3 95/5/0.2 buffer). Product containing fractions were partially concentrated and extracted with CH2Cl2 (x3). The organic phase was dried (phase separator) and concentrated in vacuo to give trans-4-(4-(1H-pyrazol-3-yl)benzoyl)-N-(1methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)bicyclo[4.1.0]heptane-3-carboxamide (335 mg, 80 %) as a white solid; MS m/z 458.1 [M + H]+.


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The

isomers

of

trans-4-(4-(1H-pyrazol-3-yl)benzoyl)-N-(1-methyl-3-

(trifluoromethyl)-1H-pyrazol-4-yl)bicyclo[4.1.0]heptane-3-carboxamide (317 mg, 0.69 mmol) were separated by chiral SFC chromatography on a ReproSil 250x30 mm, 8 μm column. 20 mg (65 mg/ml in EtOH) was injected and eluted at 40 °C with 20% EtOH/Et3N 100/0.5 in CO2, 120 bar at a flow rate of 70 mL/min and detected at 260 nm. The third eluted compound was collected and evaporated to yield 27 (48 mg, 98% ee); [α]D25 + 86.4 (c 0.50, CH3CN); 1H NMR (500 MHz, CDCl3) δ 8.02 (s, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H), 7.66 (br s, 1H), 7.43 (br s, 1H), 6.72 (br s, 1H), 3.82 (s, 3H), 3.47 – 3.58 (m, 1H), 2.68 – 2.79 (m, 1H), 2.39 – 2.53 (m, 1H), 2.31 (dd, J = 13.6, 4.2 Hz, 1H), 1.77 – 1.88 (m, 2H), 1.04 – 1.17 (m, 2H), 0.75 – 0.86 (m, 1H), 0.31 (q, J = 5.1 Hz, 1H); HRMS (ESI) m/z calcd for C23H23F3N5O2 [M + H]+ 458.1798; found 458.1840.

(1S,3R,4R,6R)-N-[1-Methyl-5-(trifluoromethyl)-1H-pyrazol-4-yl]-4-[4-(1H-pyrazol-3yl)benzoyl]bicyclo[4.1.0]heptane-3-carboxamide (28) Compound 28 was prepared from 25 in a manner analogous to compound 27 described above. Use of 1-methyl-5-(trifluoromethyl)-1H-pyrazol-4-amine gave (3R,4R)-4-(4bromobenzoyl)-N-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-4-yl]bicyclo[4.1.0]heptane-3carboxamide (274 mg, 47%); MS m/z 470.0 [M + H]+. Subsequent use of 1H-pyrazol-3-ylboronic acid provided (3R,4R)-N-[1-methyl-5(trifluoromethyl)-1H-pyrazol-4-yl]-4-[4-(1H-pyrazol-3-yl)benzoyl]bicyclo[4.1.0]heptane-3carboxamide (122 mg, 63%); MS m/z 458.1 [M + H]+.


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The isomers of (3R,4R)-N-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-4-yl]-4-[4(1H-pyrazol-3-yl)benzoyl]bicyclo[4.1.0]heptane-3-carboxamide 120 mg, 0.26 mmol) were separated by chiral SFC chromatography on a ReproSil 250x30 mm, 8 μm column. 80 mg (40 mg/ml in EtOH) was injected and eluted at 40 °C with 30% EtOH in CO2, 150 bar at a flow rate of 80 mL/min and detected at 270 nm. The first eluted compound was collected and evaporated to yield 28 (43 mg, 99.6% de); [α]D25 + 90.7 (c 1.0, CH3CN); 1H NMR (500 MHz, CDCl3) δ 8.01 (s, 1H), 7.98 (d, J = 5.4 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 2.3 Hz, 1H), 7.38 (s, 1H), 6.72 (d, J = 2.1 Hz, 1H), 3.92 (s, 3H), 3.48 – 3.60 (m, 1H), 2.66 – 2.78 (m, 1H), 2.40 – 2.52 (m, 1H), 2.30 (dd, J = 13.7, 4.4 Hz, 1H), 1.76 – 1.90 (m, 2H), 1.02 – 1.16 (m, 2H), 0.74 – 0.88 (m, 1H), 0.31 (q, J = 5.2 Hz, 1H); HRMS (ESI) m/z calcd for C23H23F3N5O2 [M + H]+ 458.1798; found 458.1814.

1-Methyl-4-({(1R,2R)-2-[6-(5-methyl-1H-pyrazol-3-yl)-1,2-benzoxazol-3-yl]cyclohexane-1carbonyl}amino)-1H-pyrazole-3-carboxamide (32) Hydroxylamine hydrochloride (0.633 g, 9.11 mmol) was added to a solution of 16 (1.00 g, 3.04 mmol) in pyridine (10 mL). The reaction mixture was stirred at 100 °C overnight (16 h). The reaction mixture was then concentrated in vacuo and the residue was partitioned between CH2Cl2 and 1 M KHSO4(aq). The aqueous phase was extracted with CH2Cl2 and the combined organic phases were dried (phase separator) and concentrated in vacuo to give crude (1R,6R)-6[(4-bromo-2-fluorophenyl)(hydroxyimino)methyl]cyclohex-3-ene-1-carboxylic acid (29). The crude material was taken to the next step without further purifications. MS m/z 342.2 [M – H]-.


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Sodium hydride (0.608 g, 15.2 mmol) (60% in mineral oil) was added to a solution of crude 29 (1.046 g, 3.04 mmol) in DMF (15 mL). The reaction mixture was stirred at rt overnight (15 h). The mixture was then partitioned between EtOAc and 1M KHSO4(aq), the aqueous phase was extracted with EtOAc and the combined organic phases were dried (Na2SO4) and concentrated in vacuo. The residue was purified by reversed phase HPLC (30→95% CH3CN in 0.2% HO2H (aq)) to give (1R,6R)-6-(6-bromo-1,2-benzoxazol-3-yl)cyclohex-3-ene-1-carboxylic acid (30, 0.581 g, 59 %): 1H NMR (500 MHz, CDCl3) δ 7.73 – 7.75 (m, 1H), 7.54 – 7.58 (m, 1H), 7.42 (dd, J = 8.4, 1.5 Hz, 1H), 3.28 – 3.38 (m, 1H), 2.97 – 3.07 (m, 1H), 2.19 – 2.28 (m, 1H), 2.09 – 2.17 (m, 1H), 1.86 – 1.96 (m, 2H), 1.55 – 1.66 (m, 2H), 1.4 – 1.54 (m, 2H); MS m/z 322.1 [M – H]-. A solution of K2CO3 (0.989 g, 7.16 mmol) in degassed water (10 mL) was added to mixture of 30 (0.580 g, 1.79 mmol), crude 3-methyl-1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.202 g, 4.12 mmol) and PdCl2(PPh3)2 (0.188 g, 0.27 mmol) in degassed dioxane (20 mL) and the reaction mixture was heated at 90 °C for 15 min. The reaction mixture was cooled to rt and then partitioned between CH2Cl2 and saturated NH4Cl(aq). The aqueous phase was extracted with CH2Cl2 and the combined organic phases were dried (phase separator) and concentrated in vacuo. The residue was purified by reversed phase HPLC (C8, 30→100% CH3CN in 0.2% CH3CO2H (aq)) to give (1R,6R)-6-{6-[3-methyl-1-(oxan2-yl)-1H-pyrazol-5-yl]-1,2-benzoxazol-3-yl}cyclohex-3-ene-1-carboxylic acid (31, 0.704 g, 96 %): 1H NMR (500 MHz, CDCl3) δ 7.77 (dd, J = 8.1, 2.5 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.42 – 7.47 (m, 1H), 6.20 (s, 1H), 5.15 (d, J = 9.7 Hz, 1H), 4.16 (d, J = 11.7 Hz, 1H), 3.54 – 3.67 (m, 1H), 3.35 – 3.47 (m, 1H), 3.05 – 3.18 (m, 1H), 2.53 – 2.65 (m, 1H), 2.35 (s, 3H), 2.17 – 2.31 (m, 2H), 2.01 – 2.06 (m, 1H), 1.90 – 1.98 (m, 2H), 1.72 – 1.86 (m, 2H), 1.60 – 1.70 (m, 2H), 1.46 – 1.58 (m, 4H); MS m/z 408.4 [M – H]-.


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DIPEA (0.698 mL, 4.01 mmol) and HATU (0.559 g, 1.47 mmol) were added to a solution of 31 (0.547 g, 1.34 mmol) and 4-amino-1-methyl-1H-pyrazole-3-carboxamide (0.281 g, 2.00 mmol) in DMF (8 mL) and the reaction mixture was stirred at rt overnight (18 h). The reaction mixture was then partitioned between EtOAc and saturated NaHCO3(aq). The aqueous phase was extracted with EtOAc and the combined organic phases were dried (Na2SO4) and concentrated in vacuo. The crude product, 1-methyl-4-{[(1R,2R)-2-{6-[3-methyl-1-(oxan-2-yl)-1H-pyrazol-5-yl]1,2-benzoxazol-3-yl}cyclohexane-1-carbonyl]amino}-1H-pyrazole-3-carboxamide, was taken to the next step without further purifications. MS m/z 532.3 [M + H]+. 3.8 M HCl(aq) (0.388 mL, 1.47 mmol) was added to a solution of crude 1-methyl-4{[(1R,2R)-2-{6-[3-methyl-1-(oxan-2-yl)-1H-pyrazol-5-yl]-1,2-benzoxazol-3-yl}cyclohexane-1carbonyl]amino}-1H-pyrazole-3-carboxamide (0.712 g, 1.34 mmol) in dioxane (40 mL) and water (10 mL) and the reaction mixture was stirred at rt for 4.5 h. The reaction mixture was then quenched by addition of saturated NaHCO3(aq). The mixture was transferred to a separatory funnel with the aid of EtOAc and was washed with saturated NaHCO3(aq). The aqueous phase was extracted with EtOAc (x2) and the combined organic phases were dried (Na2SO4) and concentrated in vacuo. The residue was purified by reversed phase HPLC (XBridge C18, 15→70% CH 3CN in 0.2% NH3(aq)) to give 32 (0.444 g, 74 %). 1H NMR (500 MHz, CD3OD) δ 7.93 (s, 1H), 7.84 (br s, 2H), 7.74 (br s, 1H), 6.52 (s, 1H), 3.79 (s, 3H), 3.43 – 3.52 (m, 1H), 2.97 – 3.07 (m, 1H), 2.34 (s, 3H), 2.10 – 2.24 (m, 2H), 1.91 – 2.00 (m, 2H), 1.69 – 1.80 (m, 2H), 1.51 – 1.66 (m, 2H); HRMS (ESI) m/z calcd for C23H26N7O3 [M + H]+ 448.2092, found 448.2105.


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1-Methyl-4-({(1S,3R,4R,6R)-4-[6-(5-methyl-1H-pyrazol-3-yl)-1,2-benzoxazol-3yl]bicyclo[4.1.0]heptane-3-carbonyl}amino)-1H-pyrazole-3-carboxamide (35) Sulfuric acid (0.034 mL, 0.61 mmol) was added to a solution of 14 (2.00 g, 6.11 mmol) in EtOH (15 mL) and the mixture was heated at reflux for 2 days. The mixture was allowed to cool to rt and was concentrated under reduced pressure. The residue was partitioned between saturated NaHCO3(aq) and EtOAc. The aqueous phase was extracted once with EtOAc and the combined organic phases were dried (phase separator) and the solvent removed under reduced pressure. Ethyl (1R,6R)-6-(4-bromo-2-fluorobenzoyl)cyclohex-3-ene-1-carboxylate (2.171 g, 100 %) was collected as a pale yellow oil; 1H NMR (500 MHz, CDCl3) δ 7.73 (t, J = 8.1 Hz, 1H), 7.40 (dd, J = 8.4, 1.7 Hz, 1H), 7.36 (dd, J = 10.4, 1.7 Hz, 1H), 5.67 – 5.82 (m, 2H), 4.04 – 4.14 (m, 2H), 3.63 – 3.74 (m, 1H), 3.01 – 3.13 (m, 1H), 2.41 – 2.59 (m, 2H), 2.14 – 2.30 (m, 1H), 1.98 – 2.11 (m, 1H), 1.20 (t, J = 7.1, 7.1 Hz, 3H). Cyclopropanation of ethyl (1R,6R)-6-(4-bromo-2-fluorobenzoyl)cyclohex-3-ene-1carboxylate was performed in a manner analogous to compound 26 described above and ethyl (3R,4R)-4-(4-bromo-2-fluorobenzoyl)bicyclo[4.1.0]heptane-3-carboxylate was taken to the next step without further purifications. MS m/z 369.1 [M + H]+. Lithium hydroxide (0.275 g, 11.48 mmol) was dissolved in water (10 mL) and added to a solution of crude ethyl (3R,4R)-4-(4-bromo-2-fluorobenzoyl)bicyclo[4.1.0]heptane-3carboxylate (1.06 g, 2.87 mmol) in THF (10 mL) and CH3OH (10 mL). The reaction mixture was heated at 50 °C for 80 min, was allowed to cool to RT, and was then concentrated under reduced pressure. The residue was partitioned between water and EtOAc. 3.8M HCl(aq) was added until pH was ~3. The phases were separated and the aqueous phase was extracted with EtOAc. The


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combined organic phases were dried (phase separator) and the solvent was removed under reduced pressure. (3R,4R)-4-(4-bromo-2-fluorobenzoyl)bicyclo[4.1.0]heptane-3-carboxylic acid (33, 0.996 g, quatitative) was collected as an off-white foam and used without purifications. Compound

34

was

prepared

from

(3R,4R)-4-(4-bromo-2-

fluorobenzoyl)bicyclo[4.1.0]heptane-3-carboxylic acid (33) in a manner analogous to compound 30 described above (0.353 g, 37%); MS m/z 334.1 [M + H]+. 1-Methyl-4-({(3R,4R)-4-[6-(5-methyl-1H-pyrazol-3-yl)-1,2-benzoxazol-3yl]bicyclo[4.1.0]heptane-3-carbonyl}amino)-1H-pyrazole-3-carboxamide was prepared from 34 in a manner analogous to compound 32 described above (0.255 mg, 57% overall yield); MS m/z 460.3 [M + H]+. The

isomers

of

1-methyl-4-({(3R,4R)-4-[6-(5-methyl-1H-pyrazol-3-yl)-1,2-

benzoxazol-3-yl]bicyclo[4.1.0]heptane-3-carbonyl}amino)-1H-pyrazole-3-carboxamide (237 mg, 0.52 mmol) were separated by chiral SFC chromatography on a Whelk-O1 250x30 mm, 5 μm column. The sample (20 mg/ml in EtOH) was injected and eluted at 40 °C with 35% CH3OH in CO2, 150 bar at a flow rate of 120 mL/min and detected at 250 nm. The first eluted compound was collected and evaporated to yield 35 (91 mg, 98.9% de); 1

H NMR (500 MHz, DMSO-d6) δ 12.72 (s, 1H), 9.70 (s, 1H), 7.93 – 7.98 (m, 2H),

7.91 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.57 (s, 1H), 7.41 (s, 1H), 6.60 (s, 1H), 3.32 (s, 3H), 3.16 – 3.25 (m, 1H), 2.91 – 3.02 (m, 1H), 2.43 – 2.49 (m, 1H), 2.28 (s, 3H), 2.19 – 2.26 (m, 1H), 2.02 – 2.13 (m, 1H), 1.61 – 1.74 (m, 1H), 1.03 – 1.17 (m, 2H), 0.65 – 0.76 (m, 1H), 0.41 (q, J = 4.9 Hz, 1H); HRMS (ESI) m/z calcd for C24H26N7O3 [M + H]+ 460.2092, found 460.2136.


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ASSOCIATED CONTENT Supporting Information General experimental set-up for analysis and characterization, FLAP binding assay, whole blood LTB4 assay, assays related to ADME and safety profiling (logD, solubility, CYP inhibition, hepatocyte CLint (human, rat, dog), Caco-2 Papp assay, RM trapping (GSH and CH3ONH2), CYP reaction profiling (CYP3A4 contribution and fraction metabolised), plasma protein binding (human, rat, dog), Fraction unbound in the incubation, Blood-plasma ratio, hERG inhibition, inhibition of other cardiac ion channels, liver panel, Phospholipidosis, hAhR Activation, Ames test, in vitro Micronucleus assay, whole blood COX-1 and COX-2 assays), MetID experimental details, in vivo PK and PK/PD experimental details and dose prediction equations are supplied as supporting information. This material is available free of charge via the Internet at http://pubs.acs.org at the DOI: Molecular formula string (CSV)

AUTHOR INFORMATION Corresponding Author *Email: [email protected] Present Addresses ¥

Contribution to this work was performed as employee of the Cardiovascular, Renal and

Metabolism IMED Biotech Unit, AstraZeneca Gothenburg, Pepparedsleden 1, Mölndal, 43183,


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Sweden. Current affiliation for Monica Sundqvist is Department of DMPK, Research, LEO Pharma A/S

Author Contributions The manuscript was written through contributions from Lemurell, Ulander, Emtenäs, Winiwarter, Broddefalk, Swanson, Hayes, Garcia, Westin Eriksson and Whatling. All authors have given approval to the final version of the manuscript. Funding Sources The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors would like to thank Magnus Johansson, Jan Lindberg for synthetic chemistry support; the Separation Science Laboratory and Structure Analysis Support at AstraZeneca Gothenburg for help with compound purification and analysis; For development of biological assays and generation of screening results the authors acknowledge Johan Meuller, Anita Dellsén, Anna Rönnborg and Angela Menschik-Lunden; Ulf Bredberg for input on ADME and PK; Anna Carlsson for biotransformation and MetID determinations; Patrik Johansson for help with graphical abstract FLAP; Margareta Herslöf for project lead during early hit finding and lead generation; Jörgen Jensen for project lead during lead optimisation.

ABBREVIATIONS


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ACS, acute coronary syndrome; Bpin, 4,4,5,5-tetramethyl-1,3,2-dioxaborolane; CAD, coronary artery disease; Cav3.2, human cardiac ion channel ICaT; CDI, carbonyldiimadazole; COX, Cyclooxygenas;

DIPEA,

N-ethyl-N-(propan-2-yl)propan-2-amine;

Dppf,

1,1′-

bis(diphenylphosphino)ferrocene; Dtbpf, 1, 1′-bis(di-t-butylphosphino(ferrocene); eD2M, early dose to man; EDCI, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; FLAP, 5-Lipoxygenase activating Protein; GSH, glutathione; hAhR, human aromatic hydrocarbon receptor; HATU, N[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide; hBSEP, human Bile Salt Export Pump (ABCB11); HCN4, human cardiac ion channel IF; hMrp2, human Multi-Drug Resistance Protein 2; HOBt, Hydroxybenzotriazole; hWB, human whole blood; IKs, human cardiac ion channel hKv7.1/hKCNE1; Ito, human cardiac ion channel hKv4.3/hKChIP2.2; Kv1.5, human cardiac ion channel IKUR; LLE, lipophilic ligand efficiency; LTA4, Leukotriene A4; LTB4, Leukotriene B4; LTC4, Leukotriene C4; LTD4, Leukotriene D4; LTE4, Leukotriene E4; 5-LO, 5-Lipoxygenase; LVEF; left ventricular ejection fraction; MEC, minimum effective concentration; MOE, Molecular Operating Environment; Nav1.5, human cardiac channel INa; NMM, N-Methyl morpholine; PGE2, Prostaglandin E2; PhN(Tf)2, 1,1,1-trifluoro-N-phenyl-N-(trifluoromethanesulfonyl)methanesulfonamide; QTOF, Quadrupole Time of Flight; Rac, Racemic; RM, reactive metabolites; T3P®, n-propanephosphonic acid anhydride; TBTU, O-(Benzotriazol-1-yl)N,N,N',N'-tetramethyluronium tetrafluoroborate; Thromboxane B2 (TXB2). REFERENCES 1.

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A novel chemical series of 5-LO activating protein (FLAP) inhibitors for

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