Discovery and Characterization of the Potent and Highly Selective

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Discovery and Characterization of the Potent and Highly Selective (Piperidin-4-yl)pyrido[3,2-d]pyrimidine based in vitro Probe BAY-885 for the Kinase ERK5 Duy Nguyen, Clara Lemos, Lars Wortmann, Knut Eis, Simon J. Holton, Ulf Boemer, Dieter Moosmayer, Uwe Eberspaecher, Joerg Weiske, Christian Lechner, Stefan Prechtl, Detlev Suelzle, Franziska Siegel, Florian Prinz, Ralf Lesche, Barbara Nicke, Katrin Nowak-Reppel, Herbert Himmel, Dominik Mumberg, Franz von Nussbaum, Carl F. Nising, Marcus Bauser, and Andrea Haegebarth J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01606 • Publication Date (Web): 18 Dec 2018 Downloaded from http://pubs.acs.org on December 18, 2018

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Journal of Medicinal Chemistry

Discovery and Characterization of the Potent and Highly Selective (Piperidin-4-yl)pyrido[3,2d]pyrimidine based in vitro Probe BAY-885 for the Kinase ERK5 Duy Nguyen,*† Clara Lemos,† Lars Wortmann,*† Knut Eis, † Simon J. Holton,† Ulf Boemer,† Dieter Moosmayer, † Uwe Eberspaecher, † Joerg Weiske,† Christian Lechner,† Stefan Prechtl,† Detlev Suelzle,† Franziska Siegel,† Florian Prinz,† Ralf Lesche,† Barbara Nicke, Nowak-Reppel,

†

Herbert Himmel,

†

Dominik Mumberg,† Franz von Nussbaum,

† †

Katrin Carl F.

Nising, † Marcus Bauser,† Andrea Haegebarth,† †Bayer

AG, Research & Development, Pharmaceuticals, 13353 Berlin, Germany

KEYWORDS: Kinase inhibitors, chemical probe, ERK5, MAPK, BAY-885

ABSTRACT:

The availability of a chemical probe to study the role of a specific domain of a protein in a concentration- and time-dependent manner is of high value. Herein we reported the identification of a highly potent and selective ERK5 inhibitor BAY-855 by high-throughput screening and subsequent structure-based optimization. ERK5 is a key integrator of cellular signal transduction and it has been shown to play a role in various cellular processes such as

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proliferation, differentiation, apoptosis and cell survival. We could demonstrate that inhibition of ERK5 kinase and transcriptional activity with a small molecule did not translate into antiproliferative activity in different relevant cell models, which is in contrast to the results obtained by RNAi technology.

INTRODUCTION The extracellular signal-regulated kinase 5 (ERK5, also known as big MAP kinase 1, BMK1) protein, encoded by the MAPK7 gene, is a member of the mitogen-activated protein kinase (MAPK) family. The ERK5 signaling cascade can be activated by environmental stresses, mitogens and cytokines. These stimuli activate MEKK2 and MEKK3, which are able to phosphorylate and activate MEK5. Once activated, MEK5 phosphorylates the TEY motif in the activation loop of the ERK5 kinase domain, thereby leading to ERK5 activation (for review, see 1-3).

ERK5 is a key integrator of cellular signal transduction and it has been shown to play a role in various cellular processes such as proliferation, differentiation, apoptosis and cell survival. Several studies have demonstrated that silencing ERK5 with siRNA or shRNA decreases the proliferation and increases cell death in different tumor models, thereby highlighting the potential of ERK5 as a therapeutic target in cancer.1-2,4 Of note, many cancer types (e.g., sarcoma and hepatocellular carcinoma) display genomic ERK5 amplifications, while others exhibit constitutive activation of ERK5 (e.g., breast cancer with ErbB2 overexpression), rendering them particularly sensitive to ERK5 depletion.5-8 The therapeutic potential of targeting ERK5 resulted in different attempts to develop ERK5 kinase inhibitors over the recent years. The first of such compounds, XMD8-92, has been


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extensively used and showed promising in vitro and in vivo anti-tumor efficacy (Figure 1).9-10 However, a recent study demonstrated that the biological activity of XMD8-92 derived from an off-target activity on bromodomains (BRDs).11 The same authors developed new ERK5 inhibitors (e.g. AX15836) with improved potency and selectivity, but with suboptimal pharmacokinetic properties.11 Therefore, we aimed at developing a new generation of ERK5 inhibitors with an overall improved profile, including high potency and an excellent kinase selectivity.

RESULTS AND DISCUSSION High-Throughput Screen For the identification of a small-molecule inhibitor of ERK5 we screened the Bayer compound collection (approx. 3.4 million compounds) using a high-throughput TR-FRET (time-resolved fluorescence energy transfer)-based kinase inhibition assay: ERK5 was incubated with the test compounds, biotinylated substrate peptide, and 250 μM ATP for 60 minutes. The amount of phosphorylated peptide was quantified with a phosphospecific detection antibody and generic TR-FRET detection reagents. Subsequent hit-to-lead process resulted in the identification of a quinazoline cluster, including compound 1 with an IC50 value of 270 nM, as the most promising lead structure series and was selected as a starting point for further optimization. Structure-Activity Relationships & Chemical Syntheses We were able to obtain a co-crystal structure of 1 in complex with ERK5 protein unlocking the binding mode with three key interactions (Figure 2). Nitrogen N1 of the quinazoline interacts with the backbone NH of the hinge amino acid Leu139 (Figure 2A). The amide carbonyl of 1


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interacts via a hydrogen bond with Asp200 and the hydrophobic back pocket is occupied by the para-trifluoromethoxybenzamide moiety. It is important to note that the central piperidine ring is crucial for the S-shaped geometry of the lead structure series allowing optimal fit into the binding pocket of ERK5 (Figure 2B). The results of this co-crystal structure of 1 and ERK5 guided further structure optimization and helped to understand the structure-activity relationships (SAR). Compound 1 was synthesized as outlined in Scheme 1: Following a literature procedure anthranilic acid 2 was converted to the corresponding 4-chloro-6,7-dimethoxyquinazoline.12-13 Subsequent Suzuki reaction, cleavage of the Boc protecting group and hydrogenation of the double bond furnished piperidine derivative 4. Amide formation with 4-(Trifluoromethoxy) benzoic acid gave rise to compound 1. Several analogues described in this publication were synthesized via a similar reaction sequence. For further details please see supplementary information. We first investigated the role of the two methoxy groups of 1 (Table 1). Interestingly, the 7methoxy group contributes more to the potency than the 6-methoxy group (compare 5 and 6). In accordance with the X-ray structure (Figure 2) nitrogen N1 of the quinazoline core is crucial for activity (see 8) whereas removal of N3 does not compromise activity (compare 6 and 7). A methyl scan on the quinazoline core confirms that substitution on positions 6 and 7 are tolerated (see 11 and 12), but substitutions on positions 2 and 8 are detrimental for activity (see 9 and 10). Introduction of further nitrogen in the quinazoline core, particularly such in case of pyridopyrimidine 15, results in improvement of acitivity due to its ability to stabilize the conformation of the piperidine ring by an additional non-classical hydrogen bond between N5 of the quinazoline and the CH2 of the piperidine (structure/data not shown).


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Changes affecting the unique S-shaped conformation of the piperidine in compound 1 as seen in the co-crystal structure (Figure 2B) are detrimental for potency. For example, replacement of piperidine atoms with a sp3 hybridization by atoms with a sp2 hybridization leads to a dramatic conformational change and results in significantly reduced activity (see 16 and 17, Table 2 ). Also a change of the exit vectors by moving to a seven-membered ring size as in 18 results in complete loss of activity. As predicted from the co-crystal structure (Figure 2) the amide carbonyl is crucial for activity (see 21, Table 3). A scan of a trifluoromethoxy group demonstrated that the substituent boosts ERK5 potency in the para position whereas the same substituent in the meta or ortho position is detrimental for activity (compare 1, 19, 20). A broad range of different para substituents were investigated indicating that small lipophilic para substituents are beneficial for potency (see 22, 23, 25) whereas large or polar substituents result in significantly reduced ERK5 activity (see 27, 29-32). Next we investigated whether further interaction with ERK5 protein can be obtained by introducing additional hydrogen bond donors or acceptors on the 4-trifluoromethoxy benzamide moiety (Table 4). Additional ring nitrogens were tolerated, but lead to a decrease of ERK5 potency (see 33 and 34 as compared to compound 1). Surprisingly an additional amino group at C2 was able to further improve ERK5 activity (see 35). A co-crystal of compound 35 with ERK5 (see Figure 3) indicates that the amino group is engaged in a hydrogen bond network with Lys84, Asp200 and Glu102 thus leading to potency improvement as compared to compound 6. Compounds 6 and 15 (Table 1) indicated that further investigation of 7-substituted [3,2d]pyrimidines could be beneficial to further boost ERK5 activity. As seen in the co-crystal structure of 35 (Figure 3) the methoxy group in the 7-position points towards the exit to the


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solvent. Therefore we expected to be able to tune physicochemical and pharmacokinetic properties of the lead series in this position. In particular, to improve water solubility of the compounds we investigated a set of amines in the 7-position (Table 5). All compounds depicted in Table 5 show good ERK5 activity with 39 being the most potent and 40 being the most soluble (data not shown). The combination of the best residues and properties lead to the identification of 41 (BAY-885): a potent and selective in vitro ERK5 probe molecule (Figure 4). The synthesis of 41 is outlined in Scheme 2. 4,7-Dichloropyrido[3,2-d]pyrimidine 4314 is converted to 44 via a Suzuki reaction followed by a Buchwald amination to provide 45. Cleavage of the Boc protecting group followed by hydrogenation of the double bond and amide formation gives rise to 41 (BAY-885). As a control compound for pharmacological experiments we also provide the negative ERK5 probe 42 (BAY-693, Table 6). A detailed synthesis description for 42 can be found in the supplementary information. Compound 41 (BAY-885) exhibits favorable physicochemical properties such as high solubility in combination with reasonable lipophilicity as measured by LogD (Figure 4). Compound 41 is highly permeable in the Caco-assay and shows no inhibition of activity of CYP enzymes up to 20 µM. Metabolic stability is low to moderate in rat hepatocytes and human liver microsomes, respectively. Compound 41 is chemically stable at different pH values and showed no inhibition of hERG up to 10 µM (Figure 4). Furthermore, compound 41 was tested at 1µM concentration against 358 kinases in the commercial Eurofins kinase panel with only Fer (r), EphB3 (h) and EphA5 (h) kinases being inhibited at 62%, 58% and 43%, respectively. For other kinases, inhibition was ≤ 20%, thus demonstrating that compound 41 (BAY-885) is a highly selective


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ERK5 inhibitor (Table S2). In contrast to XMD8-92, compound 41 showed no binding to BRD4 up to 20 µM.

Cellular Assays The encouraging combination of favorable physicochemical properties with a highly selective profile against kinases prompted us to use 41(BAY-885) to study ERK5 signaling in various cancer cell lines. In order to demonstrate that our compounds inhibit ERK5 kinase activtiy in cancer cell lines, we established a MEF2 reporter cell assay using the kidney cancer cell line SN12C (SN12CMEF2-luc). MEF2 is a transcription factor directly activated by ERK5.15-16 Therefore, ERK5 inhibition should result in a decreased luciferase signal in this cell line. In unstimulated cells, the basal luciferase signal was low, possibly due to poor activation of the ERK5 signaling pathway. To increase this signal, the cells were stimulated with EGF, which resulted in ERK5 activation and strong increase in the luciferase signal. Initial validation of the assay was performed with the available tool compounds, XMD8-92 and AX15836. Both compounds had, however, strong limitations. XMD8-92 has low kinase selectivity and is a dual ERK5/BRD4 inhibitor. AX15836 is a potent ERK5 inhibitor, with increased selectivity.11 Consistently, AX15836 showed a strong inhibitory effect in the cellular mechanistic assay, with an IC50 of 86 nM. However, at slightly higher concentrations, AX15836 became completely inactive, potentially due to precipitation in the medium (IC90 > 30 µM). Further validation of the cellular mechanistic assay was performed with our own compound series, showing a very good correlation between ERK5 kinase inhibition and cellular on-target activity (Figure 5).


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The ERK5 probe 41 (BAY-885) showed potent ERK5 kinase and transcriptional inhibition in the SN12C-MEF2 reporter cell line (IC50 = 115 nM / IC90 = 691 nM) and had no effects on a reporter control cell line with constitutive luciferase expression (SN12C-CMV-luc, IC50 > 30 µM), thereby ruling out potential effects as a general inhibitor of transcription or translation. To further address the potential of ERK5 as a therapeutic target in oncology, we tested the impact of compound 41 (BAY-885) on the proliferation of cells with ERK5 genomic amplification (SN12C , SNU-449, MFM-223)8 or with constitutively active ERK5 signaling (BT-474, SKBR-3).5 Importantly, despite its high potency, compound 41 (BAY-885) failed to inhibit the proliferation of all these cell lines. These results are in strong contrast with literature data suggesting that ERK5 is an oncogenic driver in tumors with dysregulated ERK5 signaling.5-8 A possible explanation lies on the different methodology used, with the mentioned studies employing RNAi technology to silence the ERK5 protein, while our results are based on ERK5 kinase inhibition with a small molecule.17

CONCLUSION Our results are in agreement with the findings of Lin et al.,11 which suggest that the kinase activity of ERK5 is dispensable for cancer cell growth, thus raising doubts as to the viability of ERK5 kinase as a therapeutic target for anticancer drug development. The availability of a potent and selective chemical probe, such as compound 41 (BAY-885), which was recently accepted as a donated chemical probe by the SGC,18 will significantly contribute to further understanding the biology of ERK5 signaling in cancer.19 Whether inhibition of ERK5 kinase activity can be compensated by other pathways remains to be shown by future works, e.g. by combination studies using ERK1/ERK2 inhibitor with ERK5 inhibitor as suggested by Cox et


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al.20 The lack of efficacy of an ERK5 kinase small molecule inhibitor contrast with the antiproliferative effects of genetic depletion or deletion of ERK5, thus raising the question whether ERK5 can also act as a scaffolding protein. In this case, a PROTAC approach, leading to degradation of the whole ERK5 protein, would be appropriate to exploit the therapeutic potential of ERK5.21

EXPERIMENTAL SECTION Chemistry. General Methods. All reagents and solvents were used as purchased, unless otherwise specified. All final products were at least 95% pure, as determined by UPLC. Compound names. ICS software was used to generate compound names. Materials.The synthesis of compound 1 and ERK5 probe molecule 41 (BAY-885) is described in this section. The syntheses and analytical data for all other compounds (2 – 40, 42) are described in the supplementary information. 1H NMR purities were determined to be >95%. 1H NMR spectra were recorded on Bruker Avance III HD spectrometers operating at 300, 400, or 500 mHz. The chemical shifts (δ) reported are given in parts per million (ppm), and the coupling constants (J) are in hertz (Hz). The spin multiplicities are reported as s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br = broad. Please note that many of the final compounds bearing a piperidine core show broad signals for the piperidine NCH2 and CH groups in the 1H-NMR (most likely due to ring inversion dynamics). LC-MS Method 1. System: Shimadzu LC-MS: UFLC 20-AD and LCMS 2020 MS detector; column: Shim-pack XR-ODS 2.2 µm, 3.0x50 mm; solvent: A = water + 0.05%vol. HCOOH (99%); B = acetonitrile+ 0.05%vol. HCOOH (99%).


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LC-MS Method 2. System:Shimadzu LC-MS: UFLC 20-AD and LCMS 2020 MS detector; column: Shim-pack XR-ODS 2.2 µm, 3.0x50 mm; solvent: A = water + 0.05%vol. TFA (99%); B = acetonitrile+ 0.05%vol. TFA (99%). LC-MS Method 3. System: Shimadzu LC-MS: UFLC 20-AD and LCMS 2020 MS detector; column: Shim-pack XR-ODS 2.2 µm, 3.0x50 mm; solvent: A = water + 0.05%vol. NH4HCO3 (99%); B = acetonitrile+ 0.05%vol. NH4HCO3 (99%). 6,7-Dimethoxy-4-{1-[4-(trifluoromethoxy)benzoyl]piperidin-4-yl}quinazoline (1). 6,7Dimethoxy-4-(piperidin-4-yl)quinazoline (4). Step 1:To a solution of 4-chloro-6,7dimethoxyquinazoline (0.8 g, 3.6 mmol, prepared according to literature procedure12-13) in 10 mL of 1,4-dioxane/water (v: v = 5: 1) were added tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-1,2,3,6-tetrahydropyridine-1-carboxylate (1.70 g, 5.3 mmol), sodium carbonate (1.50 g, 14.2 mmol), and 1,1'- bis(diphenylphosphino)ferrocenepalladium(II) chloride (0.3 g, 0.4 mmol). The resulting mixture was stirred at 100 oC for 4 hours under nitrogen atmosphere. After cooled to room temperature, water was added and the resulting mixture was extracted with ethyl acetate. The combined organic layer was washed with water, brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by chromatography to give 0.98 g (74%) of tert-butyl-4-(6, 7-dimethoxyquinazolin-4-yl)-1,2,3,6tetrahydropyridine-1-carboxylate as a yellow solid. LC-MS [Method 3]: Rt = 1.33 min. MS (ESIpos): m/z = 372 (M+H)+. 1H NMR (400 MHz, methanol-d4) δ 8.95 (s, 1H, pyrimidine), 7.46 (s, 1H, phenyl), 7.33 (s, 1H, phenyl), 6.24 (br s, 1H, vinyl), 4.22 (br s, 2H, NCH2), 4.04 (s, 3H, OMe), 3.98 (s, 3H, OMe), 3.86-3.66 (m, 2H, NCH2), 2.70 (br d, J = 1.71 Hz, 2H, methyleneCH2), 1.52 (s, 9H, tBu). Step 2: tert-Butyl 4-(6, 7-dimethoxyquinazolin-4-yl)-1,2,3,6tetrahydropyridine-1-carboxylate (0.8 g, 2.2 mmol), was dissolved in 3 mL of dichloromethane.


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Trifluoroacetic acid (3 mL, 39 mmol) was added and the resulting mixture was stirred at room temperature for 2h. After evaporation in vacuo, saturated aqueous sodium carbonate was added to adjust the pH = 8. The mixture was extracted with dichloromethane and the combined organic phase was dried over anhydrous sodium sulfate. After removal of the solvent, 0.51 g (87%) of 6, 7-dimethoxy-4-(1, 2,3,6-tetrahydropyridin-4-yl) quinazoline was obtained as a white solid. LCMS [Method 2]: Rt = 0.79 min. MS (ESIpos): m/z = 272 (M+H)+. 1H NMR (400 MHz, methanol-d4) δ 8.93 (s, 1H, pyrimidine), 7.52 (s, 1H, phenyl), 7.29 (s, 1H, phenyl), 6.22-6.16 (m, 1H, vinyl), 4.03 (s, 3H, OMe), 3.98 (s, 3H, OMe), 3.59 (m, 2H, NCH2), 3.15 (m, 2H, NCH2), 2.62 (dt, J = 2.87, 5.17 Hz, 2H, methylene-CH2), NH proton not visible. Step 3: 6, 7-Dimethoxy4-(1,2,3,6-tetrahydropyridin-4-yl) quinazoline (310 mg, 1.1 mmol), was dissolved in 20 mL of MeOH. After addition of 0.1 g of palladium/carbon the resulting mixture was stirred at room temperature for overnight under a hydrogen atmosphere (3 atm). The solid was removed by filtration and the filtrate was concentrated in vacuo to give 256.0 mg (80%) of 6,7-dimethoxy-4(piperidin-4-yl)quinazoline 4 as a white solid. LC-MS [Method 1]: Rt = 0.67 min. MS (ESIpos): m/z = 274 (M+H)+. 1H NMR (300 MHz, DMSO-d6) δ 9.03 (s, 1H, pyrimidine), 8.59 (br s, 1H, NH), 7.56 (s, 1H, phenyl), 7.36 (s, 1H, phenyl), 3.99 (m, 7H, 2xOMe, CH), 3.51-3.40 (m, 2H, NCH2), 3.33-3.09 (m, 2H, NCH2), 2.19-1.91 (m, 4H, methylene-CH2).Step 4: 6,7-Dimethoxy-4{1-[4-(trifluoromethoxy)benzoyl]piperidin-4-yl}quinazoline (1). To a solution of 6,7-dimethoxy4-(piperidin-4-yl)quinazoline 4 (100.0 mg ,0.4 mmol) in 6 mL of N,N-dimethylformamide, were added 4-(trifluoromethoxy)benzoic acid (90.5 mg, 0.4 mmol), triethylamine (74 mg, 0.7 mmol), and HATU (166.8 mg,0.4 mmol). The resulting mixture was stirred at room temperature for overnight. Upon completion of the reaction, 20 mL of water was added. The mixture was extracted with ethyl acetate and the combined organic phase was dried over anhydrous sodium


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sulfate. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography to give 31 mg (18%) of 6,7-dimethoxy-4-{1-[4(trifluoromethoxy)benzoyl]piperidin-4-yl}quinazoline (1) as a yellow solid. MS (ESIpos): m/z = 462 (M+H)+. 1H NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H, pyrimidine), 7.68-7.52 (m, 3H, benzamide, phenyl of quinoline), 7.46 (d, J = 8.19 Hz, 2H, benzamide), 7.35 (s, 1H, phenyl of quinoline), 4.65 (br s, 1H, NCH2), 4.00 (m, 4H, OMe, CH), 3.98 (s, 3H, OMe), 3.70 (br s, 1H, NCH2), 3.29-3.02 (m, 2H, NCH2), 1.88 (br m, 4H, alkylene-CH2); please note that piperidine signals are very broad. 13C NMR (151MHz, DMSO-d6) δ = 169.30, 167.78, 155.62, 152.80, 150.18, 148.77 (q, J=1.9 Hz, 1C), 147.54, 135.56, 129.07, 121.07, 120.10 (q, J = 256 Hz, 1C), 118.05, 106.93, 102.12, 56.19, 56.17, 46.94, 41.75, 37.60, 31.37, 30.40. 19F NMR (565MHz, DMSO-d6) δ = -57.07 (s, 3F). [2-Amino-4-(trifluoromethoxy)phenyl]{4-[7-(4-methylpiperazin-1-yl)pyrido[3,2d]pyrimidin-4-yl]piperidin-1-yl}methanone (41). Step 1: Prepared in analogous fashion as described for compound 1 with 4,7-dichloropyrido[3,2-d]pyrimidine, tert-butyl 4-(7chloropyrido[3,2-d]pyrimidin-4-yl)-1,2,3,6-tetrahydropyridine-1-carboxylate (44) was obtained as a yellow solid (5.2 g, 37%). LC-MS [Method 2]: Rt = 2.81 min. MS (ESIpos): m/z = 347 (M+H)+. 1H-NMR (400 MHz, DMSO-d6): δ 9.28 (s, 1H, pyrimidine), 9.06 (d, J = 2.4 Hz, 1H, pyridine), 8.59 (d, J = 2.4 Hz, 1H, pyridine), 7.42 (br, 1H, vinyl), 4.21-4.12 (m, 2H, NCH2), 3.62-3.59 (m, 2H, NCH2), 2.78-2.76 (m, 2H, methylene-CH2), 1.46 (s, 9H, tBu). Step 2: To a solution of tert-butyl 4-(7-chloropyrido[3,2-d]pyrimidin-4-yl)-1,2,3,6-tetrahydropyridine-1carboxylate (1.2 g, 3.4 mmol) in 20 mL of toluene were added 1-methyl-piperazine (507 mg, 5.1 mmol), palladium(II) acetate (76 mg, 0.3 mmol), BINAP (420 mg, 0.7 mmol), and cesium carbonate (3.3 g, 10.1 mmol). The resulting mixture was stirred at 100 °C for 14 hours under


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nitrogen atmosphere. After cooled to room temperature, the solid was removed by filtration and the filtrate was concentrated in vacuo, the residue was purified by silica gel column chromatography (dichloromethane: methanol = 20: 1) to give 700 mg (crude) of the product as a yellow solid. Then 80 mg of crude product was re-purified by prep-HPLC [Mobile Phase A: Water (0.1% NH4HCO3), Mobile Phase B: Acetonitrile; Gradient: 35% B to 60% B in 8 min] to give 32.1 mg of tert-butyl 4-[7-(4-methylpiperazin-1-yl)pyrido[3,2-d]pyrimidin-4-yl]-1,2,3,6tetrahydropyridine-1-carboxylate as a light yellow solid (45). LC-MS [Method 1]: Rt = 1.02 min. MS (ESIpos): m/z = 411 (M+H)+. 1H-NMR (400 MHz, DMSO-d6): δ 9.00 (d, J = 2.8 Hz, 1H, pyridine), 8.96 (s, 1H, pyrimidine), 7.32 (d, J = 2.8 Hz, 1H, pyridine), 6.79-6.91 (m, 1H, vinyl), 5.12-4.72 (m, 2H, -NCH2), 3.89-3.82 (m, 2H, -NCH2), 3.54-3.44 (m, 4H, piperazine), 2.51-2.48 (m, 4H, piperazine, overlap with DMSO), 2.20 (s, 3H, -CH3), 1.99-2.15 (m, 2H, piperidine-CH2), 1.46 (s, 9H, tBu). Step 3: Prepared in analogous fashion as described for compound 1 with tert-butyl 4-[7-(4-methylpiperazin-1-yl)pyrido[3,2-d]pyrimidin-4-yl]-1,2,3,6tetrahydropyridine-1-carboxylate , tert-butyl 4-[7-(4-methylpiperazin-1-yl)pyrido[3,2d]pyrimidin-4-yl]piperidine-1-carboxylate was obtained as a yellow solid (20%, 110 mg). LCMS [Method 1]: Rt = 0.81 min. MS(ESIpos): m/z = 413 (M+H)+. 1H-NMR (400 MHz, DMSOd6): δ 9.04 (d, J = 2.7 Hz, 1H, pyridine), 9.03 (s, 1H, pyrimidine), 7.35 (d, J = 2.7 Hz, 1H, pyridine), 4.20-4.09 (m, 3H, piperidine), 3.53-3.51 (m, 4H, piperazine), 2.98-2.83 (br, 2H, NCH2 piperidine), 2.24 (s, 3H, -CH3), 1.82-1.72 (m, 4H, piperidine), 1.43 (s, 9H -tBu), 4 piperazine protons not visible due to overlap with DMSO. Step 4: Prepared in analogous fashion as described for compound 1 with tert-butyl 4-[7-(4-methylpiperazin-1-yl)pyrido[3,2-d]pyrimidin4-yl]piperidine-1-carboxylate, 7-(4-methyl-piperazin-1-yl)-4-(piperidin-4-yl)pyrido[3,2d]pyrimidine (46) was obtained as a yellow solid (58%, 50 mg). LC-MS [Method 1]: Rt = 0.13


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min. MS (ESIpos): m/z = 313 (M+H)+. Step 5: Prepared in analogous fashion as described for compound 1 with 7-(4-methyl-piperazin-1-yl)-4-(piperidin-4-yl)pyrido[3,2-d]pyrimidine to afford to afford [2-amino-4-(trifluoromethoxy)-phenyl]{4-[7-(4-methylpiperazin-1yl)pyrido[3,2-d]pyrimidin-4-yl]piperidin-1-yl}methanone as a yellow solid (71mg, 25%). LCMS [Method 1]: Rt = 0.94 min. MS (ESIpos): m/z = 516 (M+H)+. 1H NMR (400MHz, DMSOd6,) δ = 9.06 (d, J=2.8 Hz, 1H, pyridine), 9.04 (s, 1H, pyrimidine), 7.38 (d, J=2.8 Hz, 1H, pyridine), 7.14 (d, J=8.3 Hz, 1H, phenyl), 6.67 (br, 1H, phenyl), 6.50 (br d, J=8.4 Hz, 1H, phenyl), 5.61 (br s, 2H, -NH2), 4.34-4.25 (m, 1H, piperidine), 3.55-3.50 (m, 4H, piperazine), 3.22 – 3.05 (very br, 2H, NCH2 piperidine), 2.49-2.47 (m, 4H, piperazine, partially overlap with DMSO), 2.24 (s, 3H, -CH3), 1.94 - 1.86 (br m, 4H, piperidine), 2 protons of piperidine –NCH2, expected to be around 4.1 ppm not visible. 13C NMR (151 MHz, DMSO-d6) δ = 171.84, 167.43, 155.47, 149.63, 149.39, 147.69, 147.25, 144.02, 130.57, 129.46, 120.10 (q, J = 256 Hz, 1C), 118.72, 112.01, 107.10, 106.49, 54.09, 46.49, 45.75, 36.80, 30.5 (br), 3 piperidine carbon resonances not visible (2x –NCH2, might overlap with piperazine at 46 ppm, and –CH2, note of broad signal at 30 ppm). 19F NMR (565MHz, DMSO-d6) δ = -56.70 (s, 3F). Biology. Cell Culture. Parental cell lines were obtained from the American Type Culture Collection (BT-474, SK-BR-3, SNU-449, NCI-H460), the NCI 60 panel (SN12C) and the European Collection of Authenticated Cell Cultures (MFM-223). They were maintained in the recommended cell culture media at 37 °C in 5% CO2. Biochemical ERK5 Inhibition Assay. Recombinant fusion protein of N-terminal Glutathion-STransferase (GST) and a fragment of human ERK5 (amino acids 1-398 of accession number NP_002740.2]), expressed in E. coli, purified via affinity chromatography using Glutathion Sepharose, and subsequently activated with His-tagged MAP2K5 was purchased from Carna


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Biosciences (product number 04-146) and used as kinase. As substrate for the kinase reaction biotinylated peptide biotin-Ahx-PPGDYSTTPGGTLFSTTPGGTRI (C-terminus in amide form) was used which can be purchased e.g. form the company Biosyntan (Berlin-Buch, Germany). For the assay 50 nl of a 100fold concentrated solution of the test compound in DMSO was pipetted into either a black low volume 384well microtiter plate or a black 1536well microtiter plate (both Greiner Bio-One, Frickenhausen, Germany), 2 µl of a solution of Erk5 in aqueous assay buffer [50 mM Hepes pH 7.0, 15 mM MgCl2, 1 mM dithiothreitol, 0.5 mM EGTA, 0.05 % (w/v) bovine y-globulin (Sigma-Aldrich, # G5009), 0.005% (v/v) NP40 (AppliChem, # A2239)] were added and the mixture was incubated for 15 min at 22°C to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 µl of a solution of adenosine-tri-phosphate (ATP, 417 µM => final conc. in the 5 µl assay volume is 250 µM) and substrate (1.67 µM => final conc. in the 5 µl assay volume is 1 µM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22°C. The concentration of Erk5 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, a typical concentration was 0.5 µg/ml. The reaction was stopped by the addition of 3 µl of a solution of TR-FRET detection reagents (0.33 µM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1.67 nM anti-4E-BP1 (pT46) antibody from Invitrogen [catalogue no.700397] and 1.67 nM LANCE EU-W1024 labeled anti-rabbit IgG antibody [Perkin-Elmer, product no. AD0083]) in an aqueous EDTA-solution (83.3 mM EDTA, 0.2 % (w/v) bovine serum albumin in 50 mM HEPES pH 7.5). The resulting mixture was incubated 1 h at 22°C to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently the amount of phosphorylated substrate was evaluated by


15


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measurement of the resonance energy transfer from the Eu-chelate to the streptavidine-XL. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a TR-FRET reader, e.g. a Pherastar FS (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor = 0 % inhibition, all other assay components but no enzyme = 100 % inhibition). Usually the test compounds were tested on the same microtiterplate in 11 different concentrations in the range of 20 µM to 0.07 nM (20 µM, 5.7 µM, 1.6 µM, 0.47 µM, 0.13 µM, 38 nM, 11 nM, 3.1 nM, 0.9 nM, 0.25 nM and 0.07 nM, the dilution series prepared separately before the assay on the level of the 100-fold concentrated solutions in DMSO by serial dilutions, exact concentrations may vary depending pipettors used) in duplicate values for each concentration and IC50 values were calculated using Genedata Screener software. Cellular Luciferase Reporter Assay. The SN12C-MEF2-luc (clone #37) reporter cell line has been generated by stably transducing SN12C cells with a MEF2-responsive transcription element upstream of a firefly luciferase gene (Qiagen / SABiosciences) and was used to determine the cellular activity of ERK5 inhibitors. In parallel, a SN12C-CMV-luc (clone #1) reporter cell line has been generated that recombinantly carries a CMV-promoter driven firefly luciferase gene, thereby constitutively expressing luciferase. The latter is used to detect false positive hits of SN12C-MEF2-luc reporter assay, being either toxic compounds, general inhibitors of the transcriptional or translational machinery or inhibitors of luciferase activity. Generation of the poly- and selection of the monoclonal reporter cell lines was carried out at the NMI (Natural and Medical Sciences Institute) at the University of Tuebingen. These cell lines were grown in RPMI 1640 Medium without Phenol Red (Biochrom, #F1275) supplemented


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with 10% FCS and Glutamax. Additionally, for SN12C-CMV-luc, 0.4µg/µl of Blasticidin (GIBCO, #R210-01) was also present in the culture medium. All cells were grown at 37 °C in a humidified atmosphere with 5% CO2. On day 1, the cells were seeded in 384-well white plates (Perkin Elmer #6007680) at a density of 10,000 cells per well in 20 µl of culture medium. On day 2, the test compounds were added in serial dilutions using the HP D300 Digital Dispenser and incubated at 37°C for 16h. On day 3, EGF (Invitrogen, #PHG0311L) was added to every well (final concentration = 100 ng/ml) and the plates were incubated for additional 2h at 37°C. Then, 25 µl of ONE-Glo (Promega, #E6120) were added to each well and the plates were incubated for 5 min at RT (shaking). The luminescence signal was read on PHERAStar (BMG Labtech). IC50s were calculated using the DRC Master Spreadsheet (Bella software). Values obtained for cells treated with EGF and DMSO were defined as the maximum control, while values for cells treated with EGF and 10 µM of XMD8-92 (SN12C-MEF2-luc) or 1 µM Staurosporine (SN12CCMV-luc) were defined as the minimum control (i.e., maximum inhibition). Biochemical BRD4 BD1 and BD2 binding inhibition assays. To test inhibitor affinity towards bromodomains BD 1 and 2 of Bromodomain containing 4 (BRD4) a biochemical TR-FRET based assay was used, similarly to that described previously.22 In this assay, a fluorescence signal is produced through the binding of N-terminal His6-tagged human BRD4 BD1 or BD2 to synthetic, tetra-acetylated peptides derived from human histone 4. BD1 (amino acids 44-168) and BD2 (amino acids 333-460) proteins were expressed in E. coli, purified via affinity chromatography/IMAC and size exclusion chromatography. Acetylated, biotinylated peptides (peptide for BD1 interaction: SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHGSGSK-Btn; peptide

for

BD2

interaction:

SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)


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RHRKVLRDNGSGSK-Btn) were purchased from Biosyntan (Berlin, Germany). The extent of inhibitor binding to BRD4, i.e. displacement of the biotinylated peptides, is monitored by the degree of signal decrease. The experiments were performed in 384well black small-volume microtiter plates (Greiner Bio-One, Frickenhausen, Germany), where 50 nl of a 100fold concentrated solution of a test compound (see final conc. below) in DMSO were pre-dispensed. BRD4 BD1 (final conc. in assay 10 nM) in 2 µl of assay buffer composed of 50 mM HEPES (pH 7.5) (Applichem, Darmstadt, Germany), 50 mM NaCl (Sigma), 0.25 mM CHAPS (Sigma), and 0.05% bovine serum albumin (BSA; Sigma) was added to the test compound. After 10 min pre-incubation at room temperature, 3 µL of a detection solution containing biotinylated peptide for BD1 interaction (final conc. 50 nM), anti-6His-XL665 (final conc. 10 nM; Cisbio Bioassays, Codolet, France), streptavidin Eu3+ chelate (final conc. 2.5 nM; W1024, PerkinElmer, Waltham, USA), and potassium fluoride (KF; final conc. 50 mM; Sigma) in assay buffer were added to the plate. After incubation for 3 h at 4°C, the plate was measured in a PheraStar reader (BMG Labtech) using the homogeneous time-resolved fluorescence (HTRF) module (excitation: 337 nm; emission: 620 and 665 nm). The BRD4 BD2 (final conc. in assay 100 nM) assay was performed similarly, except for the following modifications: conditions assay buffer (50 mM HEPES (pH 7.5), 100 mM NaCl, 0.25 mM CHAPS, and 0.05% BSA) and detection solution (3 µL, containing biotinylated peptide for BD2 interaction (final conc. in assay 50 nM), anti6His-XL665 (final conc. 50 nM), streptavidin Eu3+ chelate (final conc. 8.6 nM), and KF (final conc. 50mM) in assay buffer). The ratio of the emissions at 665 nm and 620 nm were calculated and normalized to neutral (DMSO instead of test compound = 0% binding inhibition) and inhibitor control (all assay components except BRD4 BD1/BD2 = 100% binding inhibiton). The compounds were tested at 11 different concentrations in the range of 20 µM to 0.07 nM (20 µM,


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5.7 µM, 1.6 µM, 0.47 µM, 0.13 µM, 38 nM, 11 nM, 3.1 nM, 0.9 nM, 0.25 nM and 0.07 nM) in duplicates for each concentration, and IC50 values were calculated using the Genedata Screener software.

X‐ray structures of (1) and (35) in complex with ERK5 Protein was produced as described previously and concentrated to 12 mg/mL in 50 mM HEPES pH 6.5, 150mM NaCl, 10 % glycerol and 2mM DTT.22 Prior to crystallization, the protein solution was supplemented with either Compound 1 or Compound 35 (final concentration of 1mM) and incubated for 3h on ice. Samples were then clarified by centrifugation (5 min, 13 000g, 277 K). Complex crystals were grown using the hanging-drop method by mixing 0.75μL of protein and with 0.5μL of reservoir solution (Compound 1: 11 % PEG 4000, 100mM MgCl2, 160mM Sodium formate, 100mM MES pH 6.75 and 100mM Tris pH 8.5; Compound 35; 15% PEG 4000, 100mM MgCl2, 180mM Na-formate, 100mM MES pH 6.5 and 100mM Tris pH 8.5. Crystals were harvested and cryo-protected by brief immersion in a cryo-protection solution consisting of mother liquor supplemented with 30% glycerol and then flash frozen in liquid nitrogen. Data were collected under cryogenic conditions at the synchrotron facility at the SLS in Villigen, Switzerland. The structure was solved by molecular replacement using the PDB 4IC8 as a search model. The structure was refined using REFMAC5 within the CCP4 suite.24 Statistics for the final modes are given in Table S3. Coordinates are structure factors have been submitted to the RCSB Protein Data Bank (PDB) and are accessible with the codes 6HKM (Compound 1) and 6HKN (Compound 35).


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PK Assays. Caco2 Permeability Assay. Cell culture: Caco-2 cells (purchased from DSMZ Braunschweig, Germany) were seeded at a density of 2.5 x 105 cells per well on 24-well insert plates, 0.4 μm pore size, 0.3 cm2 (Costar) and grown for 13-15 days in DMEM medium supplemented with 10 % fetal calf serum (FCS), 1 % GlutaMAX (100x, GIBCO), 100 U/mL penicillin, 100 μg/mL streptomycin (GIBCO) and 1 % non-essential amino acids (100 x). Cells were maintained at 37 °C in a humidified 5 % CO2 atmosphere. Medium was changed every 2-3 days. Evaluation of Caco-2 permeability in a bidirectional transport assay: The bidirectional transport assay was done in 24-well insert plates using a robotic system (Tecan). Before running the bidirectional transport assay, culture medium was replaced by transport medium (FCS-free HEPES-carbonate transport puffer, pH 7.2). For assessment of monolayer integrity the transepithelial electrical resistance (TEER) was measured. Only monolayers with a TEER of at least 400 Ω*cm2 were used. Test compounds were pre-dissolved in DMSO and added either to the apical or basolateral compartment in final concentration of 2 µM. Evaluation was done in triplicates. Before and after 2 h of incubation at 37 °C samples were taken from both compartments and analyzed after precipitation with methanol by LC/MS-MS. The apparent permeability coefficient (Papp) was calculated both for the apical to basolateral (A→B) and the basolateral to apical (B→A) direction using following equation: Papp = (Vr/P0)(1/S)(P2/t) Where Vr is the volume of medium in the receiver chamber, P0 is the measured peak area of the test drug in the donor chamber at t = 0, S the surface area of the monolayer, P2 is the measured peak area of the test drug in the acceptor chamber after 2 h of incubation, and t is the incubation time. The efflux ratio basolateral (B) to apical (A) was calculated by dividing Papp(B-A) by Papp(A-B).


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In vitro metabolic stability in human liver microsomes. The in vitro metabolic stability of test compounds was determined by incubating them at 1 µM in a suspension of liver microsomes in 100 mM phosphate buffer, pH 7.4 (NaH2PO4 x H2O + Na2HPO4 x 2H2O) and at a protein concentration of 0.5 mg/mL at 37 °C. The microsomes were activated by adding a co-factor mix containing 8 mM Glukose-6-Phosphat, 4 mM MgCl2, 0.5 mM NADP and 1 IU/ml G-6-PDehydrogenase in phosphate buffer, pH 7.4. The metabolic assay was started shortly afterwards by adding the test compound to the incubation at a final volume of 1 mL. Organic solvent in the incubations was limited to ≤0.01 % dimethylsulfoxide (DMSO) and ≤1% acetonitrile. During incubation, the microsomal suspensions were continuously shaken at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45 and 60 min, to which equal volumes of cold methanol were immediately added. Samples were frozen at -20 °C overnight, subsequently centrifuged for 15 minutes at 3000 rpm and the supernatant was analyzed with an Agilent 1200 HPLC-system with LC/MSMS detection. The half-life of a test compound was determined from the concentration-time plot. From the half-life the intrinsic clearances and the hepatic in vivo blood clearance (CL) and maximal oral bioavailability (Fmax) were calculated using the ‘well stirred’ liver model together with the additional parameters liver blood flow, specific liver weight and microsomal protein content. The following parameter values were used: Liver blood flow 1.32 L/h/kg, specific liver weight 21 g/kg, microsomal protein content 40 mg/g. In vitro metabolic stability in rat hepatocytes. Hepatocytes from Han/Wistar rats were isolated via a 2-step perfusion method. After perfusion, the liver was carefully removed from the rat: the liver capsule was opened and the hepatocytes were gently shaken out into a Petri dish with icecold Williams’ medium E (WME). The resulting cell suspension was filtered through sterile gaze in 50 ml falcon tubes and centrifuged at 50 × g for 3 min at room temperature. The cell pellet


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was resuspended in 30 ml WME and centrifuged twice through a Percoll® gradient at 100 × g. The hepatocytes were washed again with WME and resuspended in medium containing 5 % FCS. Cell viability was determined by trypan blue exclusion. For the metabolic stability assay liver cells were distributed in WME containing 5 % FCS to glass vials at a density of 1.0 × 106 vital cells/ml. The test compound was added to a final concentration of 1 µM. During incubation, the hepatocyte suspensions were continuously shaken at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45 and 90 min, to which equal volumes of cold methanol were immediately added. Samples were frozen at -20 °C overnight, subsequently centrifuged for 15 minutes at 3000 rpm and the supernatant was analyzed with an Agilent 1200 HPLC-system with LC/MS-MS detection. The half-life of a test compound was determined from the concentration-time plot. From the half-life the intrinsic clearances and the hepatic in vivo blood clearance (CL) and maximal oral bioavailability (Fmax) were calculated using the ‘well stirred’ liver model together with the additional parameters liver blood flow, specific liver weight and amount of liver cells in vivo and in vitro. The following parameter values were used: Liver blood flow 4.2 L/h/kg, specific liver weight 32 g/kg, liver cells in vivo 1.1 x 108 cells/g liver, liver cells in vitro 1.0 x 106/ml. Inhibition of CYP450 Metabolism. The inhibitory potency of the test compounds towards cytochrome P450 dependent metabolic pathways was determined in human liver microsomes by applying individual CYP isoform-selective standard probes (CYP1A2 phenacetin, CYP2C8 amodiaquine, CYP2C9 diclofenac, CYP2D6 dextromethorphan, CYP3A4 midazolam). Reference inhibitors were included as positive controls. Incubation conditions (protein and substrate concentration, incubation time) were optimized with regard to linearity of metabolite formation. Assays were processed in 96-well plates at 37 °C by using a Genesis Workstation


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(Tecan, Crailsheim, Germany). After protein precipitation, the metabolite formation was quantified by LC-MS/MS analysis followed by inhibition evaluation and IC50 calculation. Safety Assays. Automated hERG K+ current voltage-clamp assay. The hERG K+ current assay is based on a recombinant HEK293 cell line with stable expression of the KCNH2(HERG) gene.25 The cells were cultured using a humidified incubator (37°C, 5% CO2) and a standard culture medium (MEM with Earle's salts and L-glutamine, 10 % non-inactivated fetal calf serum, 0.1 mmol/L non-essential amino acids, 1 mmol/L Na-pyruvate, penicillin/streptomycin (50 µg/mL each), 0.4 mg/mL geneticin). Approximately 0.5-8 hours following cell dissociation, the cells are investigated by means of the "whole-cell voltage-clamp" technique.26 in an automated 8-channel system (Patchliner; Nanion Technologies, München, Germany) with PatchControlHT software (Nanion) to control the Patchliner system and to handle data aquisition and analysis. Voltage-clamp control was provided by two EPC 10 quadro amplifiers under control of the PatchMasterPro software (both: HEKA Elektronik, Lambrecht, Germany) and with NPC-16 medium resistance (~2 M) chips (Nanion) serving as planar substrate at room temperature (2224°C). NPC-16 chips are filled with intra- and extracellular solution (intracellular solution (in mmol/L): NaCl 10, KCl 50, KF 60, EGTA 20, HEPES 10, pH 7.2 (KOH); extracellular solution (in mmol/L): NaCl 140, KCl 4, CaCl2 2, MgCl2 1, glucose 5, HEPES 10, pH 7.4 (NaOH)) and with cell suspension. After formation of a G-seal and entering whole-cell mode (including several automated quality control steps), the cell membrane is clamped to the holding potential (80 mV). Following an activating clamp step (+20 mV, 1000 ms), exclusively hERG-mediated inward tail currents are elicited by hyperpolarizing voltage steps from +20 mV to –120 mV (duration 500 ms); this clamp protocol is repeated every 12s.27 After an initial stabilization phase (5-6 minutes), test compounds were added either as single concentration (10 µmol/L) or in


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ascending concentrations (0.1, 1, and 10 µmol/L; 5-6 minutes per concentration), followed by several washout steps. Effects of test compounds are quantified by analyzing the amplitude of the hERG-mediated inward tail currents (in percent of predrug control) as a function of test compound concentration (IgorPro Software). Mean concentration-response data were fitted with a standard sigmoidal 4-parameter logistic equation of the form: Y=Bottom +(TopBottom)/(1+10^((LogIC50-X)*HillSlope)), where Y is the current inhibition (in % of predrug control), X is the logarithm of drug concentration, and IC50 is the drug concentration producing half-maximal current inhibition, and using the following constraints: Top = 100%, Bottom = 0%. No curve fitting was performed in cases with an obvious lack of a concentration-dependent current inhibition and/or a too small effect size (approximately  20 %). Physicochemical Assays. Stability of compounds in solution (pH 10, 7 and 1 at 37°C). Solution stability was determined by HPLC-UV.28 5 µl of a 10 mmolar solution of drug in DMSO were solved in 1 ml acetonitrile. 100 µl of this solution were transferred to 1 ml of the respective buffer and mixed thoroughly. Injections were made immediately after mixing for time zero injection and then again after 1h, 2h and 24 hrs. Compounds were incubated at 37°C. Degradation rate (recovery in %) was calculated by relating peak areas after 1, 2 and 24 hrs to the time zero injection. Aqueous solubility of compound-DMSO solutions. Aqueous solubility at pH 6.5 was determined by an orientating high throughput screening method.

29

Test compounds were applied as 1 mM

DMSO solution. After addition of buffer pH 6.5 solutions were shaken for 24 hrs at room temperature. Undissolved material was separated by filtration. The compound dissolved in the supernatant was quantified by HPLC-MS/MS.


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LogD measurement. logD values at pH 7.5 were recorded using an indirect method for determining

hydrophobicity

constants

by

reversed-phase

high

performance

liquid

chromatography (RP-HPLC).30 A homologous series of n-alkan-2-ones (C3-C16, 0.02 mol in acetonitrile) was used for calibration. Test compounds were applied as 0.67 mM DMSO stock solutions in acetonitrile/water 1:1. The lipophilicity of compounds was then assessed by comparison to the calibration curve.

ANCILLARY INFORMATION Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI. Xxx

Reference compounds used for optimization of cellular mechanistic assay; Results of commercial kinase panel at Millipore for ERK5 probe molecule 41 (BAY-885); crystallographic data of compounds 1 and 35, data collection and refinement statistics, syntheses procedures for compounds 5-40 and 42, Molecular Formula Strings.

PDB ID Codes The coordinates and structure factors for the described crystal structures have been deposited with the Protein Data Bank (PDB). The PDB accession codes are 6HKM for compound 1 and 6HKN for compound 35. Authors will release the atomic coordinates and experimental data upon article publication.


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AUTHOR INFORMATIONCorresponding Author Information * For D.N.: email, [email protected] * For L.W.: email, [email protected] ORCID: Duy Nguyen: 0000-0002-4534-745X Lars Wortmann: 0000-0001-6514-947X Author Contributions: The manuscript was written with contributions from all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare the following competing financial interest(s): D.N, C.L, L.W., K.E., S.J.H., U.B., D.M., U.E., J.W., C.L., S. P., D. S., F., F.P., R.L., B. N., K.N-R., H.H., D.M., F.v.N., C. F.N., M.B., A.H. are or have been employees and stockholders of Bayer AG.

ABBREVIATIONS USED BMK1, big MAP kinase; BRDs, bromodomains; BRD4, bromodomain containing 4; DMSO, dimethyl sulfoxide; DRC, dose-response curve; EGF, epidermal growth factor; ERK1, extracellular signal-regulated kinase 1; ERK2, extracellular signal-regulated kinase 2; ERK5, extracellular signal-regulated kinase 5; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MAPK, mitogen-activated protein kinase; MAP2K5 or MEK5, mitogen-activated protein kinase kinase 5; MEF2, myocyte enhancer factor-2; MEKK2, mitogen-activated protein kinase kinase kinase 2; MEKK3, mitogen-activated protein kinase kinase kinase 3; PROTAC,


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proteolysis targeting chimera; RNAi, RNA interference; SAR, structure-activity-relationships; TEY, threonine-glutamic acid-tyrosine; TR-FRET, time-resolved fluorescence energy transfer.

ACKNOWLEDGEMENTS We thank Pharmaron Beijing Co., Ltd. for excellent support in chemistry and Proteros Biostructures GmbH for the X‐ray structure determination of Compound 1 and 35.

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Nithianandarajah-Jones, G. N.; Wilm, B.; Goldring, C. E.; Muller, J.; Cross, M. J. ERK5:

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Mitsufuji, S.; Tanaka, S.; Itoh, Y.; Nakanuma, Y.; Taniwaki, M.; Arii, S.; Okanoue, T.; Yoshikawa, T. ERK5 is a target for gene amplification at 17p11 and promotes cell growth in hepatocellular carcinoma by regulating mitotic entry. Genes Chromosomes Cancer 2009, 48, 109-120. 9

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N. S.; Lee, J. D. Pharmacological inhibition of BMK1 suppresses tumor growth through promyelocytic leukemia protein. Cancer Cell 2010, 18, 258-267. 11

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Mortlock, A. A.; Keen, N. J. Quinazoline Derivatives. EP1218357 B1, April 6, 2005.

14

For synthesis of 43, see: : Minatti, A. E.; Low, J. D.; Allen, J. R.; Chen, J.; Chen, N.; Cheng,

Y.; Judd, T.; Liu, Q.; Lopez, P.; Qian, W.; Rumfelt, S.; Rzasa, R. M.; Tamayo, N. A.; Xue, Q.;


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Yang, B.; Zhong, W. Perfluorinated 5,6-Dihydro-4H-1,3-oxazin-2-amine Compounds as BetaSecretase Inhibitors and Methods of Use. WO2014/134341 A1, September 4, 2014. 15

Erazo, T.; Moreno, A.; Ruiz-Babot, G.; Rodriguez-Asiain, A.; Morrice, N. A.; Espadamala, J.;

Bayascas, J. R.; Gomez, N.; Lizcano, J. M. Canonical and kinase activity-independent mechanisms for extracellular signal-regulated kinase 5 (ERK5) nuclear translocation require dissociation of Hsp90 from the ERK5-Cdc37 complex. Mol Cell Biol 2013, 33, 1671-1686. 6

Kato, Y.; Kravchenko, V. V.; Tapping, R. I.; Han, J.; Ulevitch, R. J.; Lee, J. D. BMK1/ERK5

regulates serum-induced early gene expression through transcription factor MEF2C. EMBO J 1997, 16, 7054-7066. 17

Settleman, J. Sawyers, C. L., Hunter, T. Challenges in validating candidate therapeutic targets

in cancer. eLife 2018;7:e32402. Ellermann, M., Eheim, A., Rahm, F., Viklund,J., Guenther, J., Andersson, M., Ericsson, U. Forsblom, R.,

Ginman, T.,

Lindström, J., Silvander, C.,

Trésaugues, L., Giese, A., Bunse, S., Neuhaus, R., Weiske, J., Quanz, M. Glasauer, A., Nowak-Reppel, K., Bader, B. Irlbacher, H., Meyer, H., Queisser, N., Bauser, B., Haegebarth, A. Gorjánácz, M. Novel class of potent and cellularly active inhibitors devalidates MTH1 as broad-spectrum cancer target. ACS Chem. Biol. 2017, 12, 1986−1992. 8 Structure of BAY-885 to be

published

on

https://www.thesgc.org/donated-chemical-probes,

https://innovate.bayer.com/what-we-offer/chemical-probes-for-open-science/ (accessed Nov, 15, 2018). 9

Schreiber, S. L. A chemical biology view of bioactive small molecules and a

binder-based

approach

to

connect

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to

precision

medicines.

doi:

http://dx.doi.org/10.1101/386904.


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targeting KRAS effector signaling in KRAS-mutant pancreatic cancer. 21

Lai, A. C.; Crews, C. M. Induced protein degradation: An emerging drug discovery paradigm.

Nat. Rev. Drug. Discov. 2016, 16, 101-114. 22

Jung, M., , Philpott, M., Müller, S. Schulze, J., Badock, V., Eberspächer, U., Moosmayer, D.

Bader, B., Schmees, N., Fernández-Montalván, A., Haendler, B. Affinity map of bromo-domain protein 4 (BRD4) interactions with the histone H4 tail and the small molecule inhibitor JQ1 J. Biol. Chem. 2014, 289,9304-9319. Schulze, J., Moosmayer, D. Weiske, J., FernándezMontalván, A., Herbst, C., Jung, M., Haendler, B., Bader, B. Cell-based protein stabilization assays for the detection of interactions between small-molecule inhibitors and BRD4. J. Biomol. Screen 2015, 20, 180–189. 23

Chen, H., Tucker, J. Wang, X., Gavine, P.R., Phillips, C. Augustin, M. A., Schreiner, P.,

Steinbacher, S., Preston, M. Ogg, D. Discovery of a novel allosteric inhibitor-binding site in ERK5: Comparison with the canonical kinase ATP_binding site. Acta Cryst., 2016, D72, 682– 693. 24

The CCP4 suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol.

Crystallogr. 1994, 50, 760–76310.1107/S0907444994003112. 25

Zhou, Z.F, Gong, Q., Ye, B., Fan, Z., Makielski, J.C, Robertson GA, January CT. Properties of

hERG channels stably expressed in HEK293 cells studied at physiological temperature. Biophys J 1998, 74, 230-241. Sanguinetti, M.C, Chandy, K.G., Grissme,r S., Gutman, G.A., Lazdunski, M., Mckinnon, D., Pardo, L.A., Robertson, G.A., Rudy, B., Stuehmer, W., Wang, X.L. Voltagegated potassium channels: Kv11.1 (hERG). IUPHAR/BPS Guide to Pharmacology, http://www.guidetopharmacology.org (accessed Nov, 15, 2018).


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26

Hamill O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J. Improved patch-clamp

techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch 1981, 391, 85-100. 27

Himmel, H.M.Suitability of commonly used excipients for electrophysiological in-vitro safety

pharmacology assessment of effects on hERG potassium current and on rabbit Purkinje fiber action potential. J Pharmacol Toxicol Methods 2007, 56, 145-158. 28

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Design and Methods, 353-356, (2008). Burlington, MA, Academic Press. 29

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method to determine aqueous drug solubility, Millipore Corporation Application Note, 2003 Lit. No. AN1731EN00. 30

Minick, D.J., Frenz, J.H., Patrick, M.A. & Brent, D.A. A comprehensive method for

determining

hydrophobicity

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31


Page 32 of 47

Captions of Figures

Figure 1: Published ERK5 inhibitors

Figure 2: Co-crystal structure of compound 1 with ERK5 protein (PDB code 6HKM, 2.46 Å)

Figure 3: Co-crystal structure of compound 35 with ERK5 protein (PDB code 6HKN, 2.33 Å)

Figure 4: Summary of chemical, physio-chemical and in vitro DMPK properties of (41)

Figure 5: Correlation between ERK5 kinase inhibition in a biochemical assay and inhibition of the transcriptional activity of MEF2 in SN12C cell line. Data of the entire quinazoline cluster are shown.


32

Page 33 of 47 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


Figures

Figure in Abstract

N

N HN

O N

N

N

O

N N O S O

HN

O

N

O

N

N OH

XMD8-92 ERK5 IC50: 0.2 µM

O

N N

AX15836 ERK5 IC50: 0.02 µM

Figure 1


33


Page 34 of 47

Figure 2


34

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Figure 3


35


Page 36 of 47

Figure 4


36

Page 37 of 47 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


Figure 5


37


Page 38 of 47

Tables

Table 1. SAR of quinazoline core F

F

F

O O N

R

SN12C-

Compound

ERK5 IC50

R

[µM]

MEF2 Luciferase reporter gene assay [µM]

1

O O

5

0.27

nd

N

1.00

4.10

N

0.15

0.42

0.11

0.71

18.0

29

N N

O N

6 O

N

O

N

7

8

N O


38

Page 39 of 47 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


9

N

>20.0

nd

N

11.0

nd

N

0.21

nd

N

0.76

nd

N

6.70

nd

N

2.17

10.0

0.098

0.54

N

10

N

11 N

12 N

13 N

O

14

O N

O

50

N

N

15 aIC

N

N N

values are arithmetic means of multiple measurements


39


Page 40 of 47

Table 2. SAR of piperidine core F

F

F

O O

A O

N N

O

SN12C-

Compound

A

ERK5 IC50 [µM]


N

1

0.27

nd

>20.0

nd

3.89

nd

>20.0

nd

N

16

N

17

N

N

18 N

aIC

50



40

Page 41 of 47 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


Table 3. SAR of benzamide moiety (mono substitution)

R N

O O

N N

SN12C-

Compound

ERK5 IC50

R

[µM]


19

20

F O

O

O O F

F

F O

21

O

22

O

23

O

O

F F

F F

7.09

nd

>20.0

nd

>20.0

nd

0.35

nd

0.38

nd

F


41


24

O O

F

25

26

F F

O

O

27

O

28

O

0.92

3.75

0.53

nd

1.66

11.5

4.10

nd

2.75

nd

>20.0

nd

>20.0

nd

9.74

nd

>20.0

nd

Page 42 of 47

F

HO

29

O O

N

30

O O

N

31

32 aIC

50

O

O O S O



42

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Table 4. SAR of benzamide moiety (double substitution)

R1 N

R2

N N

O

SN12C-

Compound

R1

ERK5 IC50

R2

[µM]


F

33

N O

F

O

H 2N

F

36

O

aIC

50

F O

O

1.63

nd

OMe

1.12

7.64

H

0.051

0.16

H

0.17

0.36

F

O

F N H2

OMe

F

O

N

F

35

F

O

F

34

F

F



43


Page 44 of 47

Table 5. SAR of substituents in the 7-position F

F

F

O O N

N

N N

R

SN12C-

Compound

ERK5 IC50

R

[µM]


37

HO

38

N

39

40

aIC

50

N

0.068

0.24

N

0.064

0.67

0.017

0.15

0.054

0.29

N O

N N



44

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Table 6. ERK5 negative control molecule 42 (BAY-693) F

F

F

O

H 2N O N

N N

N N

N

SN12C-

Compound

R

ERK5 IC50 [µM]


42 (BAYMe

6.40

11.0

693) aIC

50



45


Page 46 of 47

Schemes Scheme 1. Synthesis of (1) as representative synthesis for quinazoline derivatives F

F

F

O O

H N

O O

OH

O

a, b

N H2

O

c, d, e

N

O

O

N

2

aReagents

N

Cl

f

N

O

O

N

3

N N

O

1

4

and conditions: (a) Formamidinium acetate, 2-methoxyethanol, 100°C, overnight,

88%; (b) SOCl2 in DMF, reflux, overnight, 88%; (c) tert-Butyl 4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate, Na2CO3, 1,1'-bis(diphenylphosphino)ferrocenepalladium(II) chloride, 1,4-dioxane / water (5:1), 100°C, 4h, 74%; (d) TFA, dichloromethane, rt, 2h, 87%; (e) H2, Pd/C, methanol, rt, overnight, 80%; (f) 4(Trifluoromethoxy)benzoic acid, HATU, NEt3, DMF, rt, overnight, 18%.

Scheme 2. Synthesis of ERK5 probe molecule 41 (BAY-885)

O

N

N

Cl

43

aReagents

N

44

N

N

N N

N

N N

N

45

F

e

N

N

F N

c, d

N

F

O

O

H N

b N

H 2N

O N

a

N

Cl

O N

Cl N

O

N

N N

N

46

BAY-885 41

and conditions: (a) tert-Butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-

dihydropyridine-1(2H)-carboxylate, Na2CO3, 1,1'-bis(diphenylphosphino)ferrocenepalladium(II) chloride, 1,4-dioxane / water (5:1), 100°C, 4h, 37%; (b) 1-Methylpiperazine,


46

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Pd2(OAc)2, BINAP, Cs2CO3, toluene, 100°C; 14h; (c) H2, Pd/C, methanol, rt, overnight, 20%; (d) TFA, dichloromethane, rt, 2h, 58%; (e) 2-Amino-4-(trifluoromethoxy)benzoic acid, HATU, (i-Pr)2NEt, DMF, rt, 2h, 25%.


47

Discovery and Characterization of the Potent and Highly Selective

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