High-Throughput Screening and Hit Validation of Extracellular-Related Kinase 5 (ERK5) Inhibitors

Article abstract of DOI:10.1021/acscombsci.5b00155

The extracellular-related kinase 5 (ERK5) is a promising target for cancer therapy. A high-throughput screen was developed for ERK5, based on the IMAP FP progressive binding system, and used to identify hits from a library of 57-617 compounds. Four distinct chemical series were evident within the screening hits. Resynthesis and reassay of the hits demonstrated that one series did not return active compounds, whereas three series returned active hits. Structure-activity studies demonstrated that the 4-benzoylpyrrole-2-carboxamide pharmacophore had excellent potential for further development. The minimum kinase binding pharmacophore was identified, and key examples demonstrated good selectivity for ERK5 over p38α kinase.

Full text of DOI:10.1021/acscombsci.5b00155

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Article
High-throughput screening and hit validation of
extracellular-related kinase 5 (ERK5) inhibitors.
Stephanie May Myers, Ruth Helen Bawn, Louise C Bisset, Timothy J. Blackburn,
Betty Cottyn, Lauren Molyneux, Ai-Ching Wong, Celine Cano, William Clegg, Ross W.
Harrington, Hing Leung, Laurent Rigoreau, Sandrine Vidot, Bernard Thomas Golding,
Roger John Griffin, Tim Hammonds, David R. Newell, and Ian Robert Hardcastle
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.5b00155 • Publication Date (Web): 11 Jul 2016
Downloaded from http://pubs.acs.org on July 12, 2016
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High-throughput screening and hit validation of extracellular-
related kinase 5 (ERK5) inhibitors.
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Stephanie M. Myers1, Ruth H. Bawn1, Louise C. Bisset3, Timothy J. Blackburn1, Betty Cottyn1, Lauren
Molyneux1, Ai-Ching Wong4, Celine Cano1, William Clegg2, Ross. W. Harrington2, Hing Leung5, Laurent
Rigoreau4, Sandrine Vidot,1 Bernard T. Golding1, Roger J. Griffin1, Tim Hammonds4, David R. Newell3, Ian R.
Hardcastle1*
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1 Newcastle Cancer Centre, Northern Institute for Cancer Research and School of Chemistry, Bedson
Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK
2 School of Chemistry, Bedson Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK
3 Newcastle Cancer Centre, Northern Institute for Cancer Research, Paul O’Gorman Building, Medical
School, Framlington Place, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
4 Cancer Research Technology Ltd, Discovery Laboratories, Wolfson Institute for Biomedical Research, The
Cruciform Building , Gower Street, London, WC1E 6BT, UK
5 The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61
1BD, UK
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Abstract
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The extracellular-related kinase 5 (ERK5) is a promising target for cancer therapy. A high-throughput screen was
developed for ERK5, based on the IMAP FP Progressive Binding System, and used to identify hits from a library of
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57,617 compounds. Four distinct chemical series were evident within the screening hits. Resynthesis and re-assay of
the hits demonstrated that one series did not return active compounds, whereas three series returned active hits.
Structure-activity studies demonstrated that the 4-benzoylpyrrole-2-carboxamide pharmacophore had excellent
potential for further development. The minimum kinase binding pharmacophore was identified, and key examples
demonstrated good selectivity for ERK5 over p38α kinase.
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INTRODUCTION
The extracellular-related kinase 5 (ERK5), also known as Big Map Kinase (BMK1), is a 816 amino acid protein kinase
that forms part of a non-canonical MAP kinase pathway in cells.1 Extracellular stimulation, in the form of growth
factors, such as epidermal growth factor (EGF), nerve growth factor (NGF), vascular endothelium growth factor
(VEGF), and fibroblast growth factor-2 (FGF-2), initiates a signalling cascade from the cell surface to nuclear
transcription factors via ERK5.2 Unlike the linear canonical Ras/Raf/MEK/ERK pathway, the ERK5 signalling cascade
occurs independently of Raf.3 ERK5 is activated specifically by MEK5, which is in turn activated by MEKK2/3.1a, 4
ERK5 is structurally different from the other members of the ERK sub-families. A unique loop-12 structure and
extended C-terminal domain (~400 amino acids) gives ERK5 its characteristic structure.2, 4 The C-terminal extension
harbours nuclear localisation and export sequences, two proline-rich domains, a transcriptional activating domain,
and MEF2 interacting region. The large C-terminal unique to ERK5 is auto-phosphorylated at multiple sites, resulting
in an increase in transcriptional activity.5
Phosphorylation of ERK5 results in activation of a number of transcription factors including MEF2, c-Myc, c-Jun, c-
Fos, Fra-1, and NFκB.6 ERK5 also phosphorylates and activates p90 RSK, also involved in signal transduction.7 An
increasing body of mechanistic data indicates that ERK5 plays a key role in tumor biology, i.e. cell proliferation and
survival, invasion and metastasis, and angiogenesis.8
Expression of ERK5 is significantly up-regulated in advanced prostate cancer and has been identified as an
independent prognostic biomarker for aggressive disease.9 ERK5 is over-expressed in 20% of breast cancer patients
and its expression is an independent prognostic biomarker for reduced disease-free survival.10 In hepatocellular
carcinoma (HCC), ERK5 overexpression has been reported, associated with gene amplification at the 17p11
chromosome fragment, harboring the MAPK7 gene.11 High levels of ERK5 were found to correlate with more
aggressive and metastatic stages in fresh samples from human clear cell renal cell carcinoma.12
To date, three small-molecule inhibitors of the MEK5/ERK5 pathway have been described. The indolinone based
inhibitors, BIX02188 (1a) and BIX02189 (1b), are dual inhibitors of the MEK5/ERK5 cascade. Both compounds inhibit
MEK5 with nanomolar potency, whereas modest activity was reported for ERK5.13 Benzo[e]pyrimido-[5,4-
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b]diazepine-6(11H)-one (XMD8-92, 2) is a potent ERK5 inhibitor which exhibits anti-proliferative and anti-angiogenic
effects on HeLa cells in mouse xenograft models.14 An X-ray crystal structure of 2 bound to ERK5 shows the inhibitor
bound to the Met140 in the kinase hinge via a pair of hydrogen bonds from the aniline and pyrimidine nitrogens, and
an additional water-bridged hydrogen bond from the diazepinone carbonyl to Asp200 and Glu102 in the DFG loop.15
However, some of the in vivo activity of 2 has been attributed to off-target activities e.g. inhibition of doublecortin-
like kinase 1 (DCLK-1) in pancreatic cancer.16 Inhibition of ERK5 or siRNA knockdown has been shown to inhibit the
growth of HCC cell lines, in vitro and in vivo.17 ERK5 signalling has been shown to be essential for chemically induced
carcinogenesis in skin by erk5 gene deletion or ERK5 inhibition (with 2).18 The combination of doxorubicin and either
erk5 gene deletion or ERK5 inhibition (with 2) was found to be additive in inflammation-driven tumour models.
Thus, pharmacological inhibition of ERK5 may provide an opportunity for the treatment of inflammation-driven,
invasive or metastatic cancers where ERK5 is deregulated. Given the strong link between ERK5 mediated signalling
and malignancy, there remains a strong need to develop selective tool molecules to fully elucidate the effect of ERK5
inhibition in vivo. For this reason, we sought to discover novel ERK5 inhibitory chemotypes through high-throughput
screening as chemical tools and for development as therapeutic agents.
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In this paper, we describe the development of a high-throughput screening assay for ERK5 inhibition based on the
IMAP FP format that was used to identify four discrete series of hit molecules. Validation of each series was
attempted by resynthesis and retesting of selected members from each series. Preliminary structure activity studies
were obtained resulting in the identification of one series for further optimisation.
DEVELOPMENT AND EXECUTION OF ERK5 IMAP SCREEN
Assay development
The discovery of kinase inhibitors using high-thoughput screening is well established.19 In our case, expression of
active ERK5 protein required co-expression of MEK5. The screen was developed using the IMAP format (Molecular
Devices) that relies on the high specificity interaction of phospho groups on a fluorescently tagged peptide with M3+
containing nanoparticles. The IMAP format was chosen as a robust and efficient method of determining kinase
activity using a ‘mix-and-measure’ format with a non-radioactive, fluorescence output.
Preliminary experiments to determine a suitable peptide substrate for the IMAP FP assay used a commercial source
of the enzyme and were based on sequence of the natural substrate of ERK5, and were augmented with a substrate
finder kit. Five peptide sequences were designed based around the reported site of phosphorylation of MEF2C by
ERK5, with the expected serine phosphorylation site highlighted in red (Table 1).1 The peptides were tested using the
IMAP FP Progressive Binding System (Molecular Devices) in the absence and presence of ERK5 (Carna Biosciences).
The IMAP FP substrate finder kit for serine/threonine kinases plate 2 (Molecular Devices) covering CMGC, CK1, STE,
and TKL portions of the kinome was used to identify potential peptide substrates phosphorylated by ERK5. The FAM-
EGFR-derived peptide (LVEPLTPSGEAPNQK-5FAM-COOH) proved optimal.
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Kinetic studies determined the ERK5 KMapp to be 300 µM. Ideally, kinase screens are run with the ATP concentration
equal to the KM, however, in this case the IMAP format did not return acceptable results at a high ATP concentration
(300 µM), presumably due to interference of ATP with the interaction of the phospho peptide with the metal
nanoparticles. For this reason, the HTS assay was run at the maximum acceptable ATP concentration (100 µM) to
allow ‘mix-and-measure’ determinations.
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High-throughput Screening for ERK5 inhibitors
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To identify inhibitors of ERK5 the IMAP HTS assay was set up and a library of 57,617 small molecules was screened
(final DMSO concentration of 4% and a total reaction volume of 40 µL). The library was composed of a 48,479
member diverse library and a 9136 member kinase focussed library, both libraries were sourced from commercial
vendors. Z’ factors for each plate were calculated using Equation 2, and were typically 0.6-0.8. Plates with Z’ factors
below 0.4 were re-screened (Supporting Information).
HTS Results
The HTS assay returned 245 active compounds (0.5 % hit rate), i.e. >50% inh at 30 ꢀM, from the 57,617 member
library. Active compounds (245) showing >50% inhibition in the screen were resupplied from stock or commercial
vendors and retested at 30, 10 and 3.3 ꢀM. 71 active compounds, giving >30% mean inhibition, were treated as
confirmed hits (0.10% overall hit-rate) and assayed over a full IC50 range. IC50 determinations required a two stage
process whereby the reaction occurred initially in the absence of the IMAP reagent, followed by subsequent addition
of the IMAP reagent, to allow the KM concentration of ATP to be used.
The hit compounds produced inhibition curves with IC50 values ranging from 0.6 to 76 µM (1 compound >120 µM).
Confirmed hits were clustered according to common structures, revealing four promising chemical series. SAR
around the hits was expanded by assaying related in-house compounds and close analogues from commercial
suppliers. From these results, four compound series were selected for validation by resynthesis prior to progressing
to hit-to-lead studies: 2-amino-N,N-alkylbenzo[d]thiazole-6-sulfonamides (Table 2, 3a-c); 4-substituted-2-
(substitutedthio)-6-phenylnicotinonitriles (Table 3, 4a,b); 4-amino-2-(arylamino)pyrimidine-5-carbonitriles (Table 5,
5a-c); and 4-aroyl-N-alkyl-1H-pyrrole-2-carboxamides (Table 6, 6a-e).
SYNTHESIS
Benzothiazole Series 3
Numerous methods have been described for the synthesis of benzothiazoles.20 For the synthesis of compounds 3a,b
and analogues, we used the reported reaction of anilines with potassium thiocyanate-copper(II) sulfate (Scheme 1).21
The required 4-aminophenylsulfonamides (7a-c) were prepared by reduction of the corresponding nitro compounds
(8a-c). The nitro precursors were obtained by coupling the relevant amine with 4-nitrobenzenesulfonyl chloride. The
5-sulfonamide isomer 9a, was prepared via the same method (Scheme 2). Thus, 3-nitrobenzenesulfonyl chloride was
reacted with pyrrolidine or N-methylethylamine, and the resulting sulfonamides 10a,b were reduced to the
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respective anilines 11a,b (Scheme 2). Reactions of 11a,b with with potassium thiocyanate-copper(II) sulfate gave in
each case, besides the desired 5-substituted benzothiazole 9a,b, a significant quantity of a thiocyanatobenzene
(12a,b).
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Nicotinonitrile Series 4
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The first synthetic approach considered for the synthesis of 3-cyanopyridines was based on the route reported by
Shestopalov et al.22 For example, 4-fluorochalcone 13a, prepared via Claisen-Schmidt condensation of acetophenone
with 4-fluorobenzaldehyde, was treated with elemental sulfur and morpholine in ethanol at reflux, followed by
malononitrile, to give pyridinethione 14a in moderate yield (Method A, Scheme 3). Isolation of the intermediate
pyridinethiones 14 required extensive purification, attributed to the propensity of this intermediate to tautomerise,
and its readiness to oxidise under atmospheric conditions.
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Consideration of the likely mechanism of the one-pot sequence of the cyclisation reaction prompted the
replacement of the sulfur and malononitrile with 2-cyanothioacetamide for the Michael addition in an alternative
route (Method B, Scheme 3). Reactions were performed under nitrogen to avoid oxidative side-reactions. The crude
intermediate 14 was used directly in the alkylation step, to avoid a lengthy purification, giving cyanopyridines 15a-k.
Method B allowed isolation of 15a in an improved 54% yield over 2 steps. Deprotection with TFA gave acids 4a-k in
near quantitative yield (99%). The carboxamide 17 was prepared from 4a by a HBTU-mediated coupling with p-
methoxybenzylamine giving amide 16, which was deprotected with TFA.
Further variations to the thioether group were introduced via alkylation of 14a (Schemes 4-7). The acetamide
derivatives 31 and 32 were prepared by the alkylation of 14a and 14g, respectively, with bromoacetamide 30 which
was obtained by reaction of aminoacetone hydrochloride 29 with bromoacetylchloride (Scheme 8).23
Cyanopyrimidine Series 5
A small series of 4-amino-2-anilinopyrimidine-5-carbonitriles (5a-c) were prepared by the reaction of the appropriate
aniline with chloropyrimidine (33) at 100 oC in DMF (Scheme 9).24
4-Benzoylpyrrole-2-carboxamide Series 6
A selection of 4-benzoylpyrrole-2-carboxamides were prepared by Friedel-Crafts acylation of methyl 1H-pyrrole-2-
carboxylate (34) with a substituted benzoyl chloride giving pyrrole (35). Hydrolysis of the methyl ester with lithium
hydroxide gave carboxylic acid 36 that was coupled with the appropriate amine using CDI to give the desired
carboxamides (6a-r)(Scheme 10). The N-methyl derivative 6m was prepared by methylation of ester 35a, followed by
hydrolysis and coupling with 3-pyridylmethylamine (Scheme 11).
2-Substituted-4-benzoylpyrrole derivatives
The alkene derivative 40 was prepared by aldol condensation of ketone 39 and isonicotinaldehyde (Scheme 12).
Selective reduction was achieved by refluxing alkene 40 in aqueous ethanol with indium metal and ammonium
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chloride giving alkane 41 in moderate yield. 25 The cyclopropyl analogue 42 was prepared by a Corey Chaykovsky
reaction.26 Thus, alkene 40 was reacted with trimethylsulfoxonium iodide and potassium tert-butoxide giving 42 in
12% yield.27 Diketone 44 was prepared via a Claisen condensation between 1-(1H-pyrrol-2-yl)ethanone and methyl
isonicotinate diketone 43 (Scheme 13). Friedel-Crafts acylation with 2,3-dichlorobenzoyl chloride gave 44.
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DISCUSSION
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Selected examples of the HTS hits in the benzothiazole series (3a, 3b, 3i) were synthesised and re-assayed. The ERK5
inhibitory activity for the resynthesized benzothiazoles were 1000-fold lower than for the library material (Table 2).
Comparision of the 1H-NMR and LCMS spectra of the resynthesized and screened samples of 3a suggested that the
library material was the 5-sulfonamide 9a, so authentic samples of isomers 9a and 9b were prepared. In order to
eliminate the possibility of mis-identification of the compounds by spectroscopic methods, the identity of
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isothiocyanate 12a and benzothiazoles 3a and 3i were elucidated by small-molecule X-ray crystallography (Figures 1-
3).
The assay results for these isomers also failed to replicate the initial IC50 values from the screening samples.
Interestingly, the isothiocyanate side-product 12a showed 10-fold greater potency than the benzothiazole, although
this result was not replicated for the analogue 12b. Time-dependent enzyme inactivation by isothiocyanates, via
their reaction with lysine residues, has been reported.28 Further investigations into the mechanism of action of 12a
were not conducted. Some aminothiazoles have recently been identified as frequent hitters from a fragment
screening set and dubbed promiscuous 2-aminothiazoles (PrATs).29 The reason for the discrepancy between the
activity of the HTS sample and the resynthesized material is not clear. Numerous mechanisms for false positives in
HTS are possible, including the presence of trace impurities or protein aggregation, and further effort was not
expended eliminating these possibilities.30
Three HTS hits in the nicotinonitrile series (4a, 19, and 31) were synthesised and reassayed (Tables 3 and 4). The
results for the glycine derivative 4a and proline methyl ester derivative 19 were in good agreement with the HTS IC50
values. In contrast, the propan-2-one derivative 31 was 50-fold less active than the HTS result. On this basis, a
limited series of compounds was prepared to establish preliminary SARs and to determine the minimum inhibitory
pharmacophore. The SARs for the 4- and 6-substitutents were delineated keeping the 2-thio substituent as the
glycine amide (Table 3). The 4,6-diphenyl, 4-phenyl-6-methyl and 4,6-dimethyl compounds (4d, 4j and 4k,
respectively) were each devoid of activity. The 4-(2-fluorophenyl) derivative 4c was 7-fold less active than the 4-(4-
fluorophenyl) derivative 4a, whereas the 4-(3-fluorophenyl) derivative 4b lacked measurable potency. The
combination of 2-fluoro and 4-fluoro substituents (4f) was not additive and resulted in a 15-fold loss of potency
compared with 4a. The 4-(4-pyridyl) derivative 4g exhibited a 13-fold loss in potency compared to 4a, despite the
similar electronic properties of the rings. Substitution of the 4-phenyl group with 4-trifluoromethyl 4e, or 4-methoxy
4h resulted in loss of activity.
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The SARs for the thioether side-chain were investigated (Table 4). The pyridine thiol 14a lacking the amide side-chain
showed a 20-fold loss in potency compared to 4a. The glycine ethyl ester 15l showed similar activity to the proline
methyl ester derivative 19, and was 4-fold less potent than the corresponding glycine derivative 4a. In contrast, the
glycine amide 17 lacked measurable activity. The shorter, unsubstituted amide 20 showed weak activity, whereas
the corresponding ester 21 was inactive. Two thioalkyl carboxylic acids 23a and 23b were inactive, as was the
corresponding amine 28, demonstrating the requirement for the amide group in the sidechain for potency.
Comparison of the 4-fluorophenyl propan-2-one derivative 31 with the 4-pyridyl derivative 32 showed a 5-fold loss in
potency consistent with the results in the glycine amide series (4a and 4g).
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Overall, each of the changes made to the hit compounds in the nicotinonitrile series (4a, 19, and 31) resulted in loss
of potency. Modifications to the aromatic and side-chain substituents revealed a highly constrained pharmacophore
and limited SAR. As a result, no further optimisation of this series was attempted. Interestingly, 3-cyano-4,6-
diphenyl-pyridines have been identified recently as inhibitors of the PA-PB1 protein-protein interaction for
influenza.31
The cyanopyrimidines 5a-c showed reasonable activity against ERK5, with IC50 values in the 12-88 ꢀM range (Table
5), and generally consistent with the HTS values. The activity against ERK5 in this series was promising, but the series
had also been selected for development against another target internally. For this reason, no further analogues were
prepared. Kinase inhibitors incorporating a 5-cyanopyrimidine core have been reported, e.g. Wee1 inhibitors32, and
CDK2 inhibitors33.
Five 4-benzoylpyrrole-2-carboxamides (6a-e) gave good potency in the HTS. Upon resynthesis and retesting, the 2,3-
dichlorobenzoyl-N-(4-fluorobenzyl) substitued analogue 6a maintained significant activity (IC50 = 3.7 μM) despite a 5-
fold loss in potency compared to the HTS result (Table 6). Similarly, the 2-trifluoromethylbenzoyl-N-methyl
substituted analogue 6d gave a two-fold drop in activity (IC50 = 9.6 μM) compared to the HTS result, and the benzoyl-
N-methyl-3-pyridyl derivative 6e gave a 3-fold drop in activity (IC50 = 26 μM). In contrast, the resynthesized 2,4-
dichlorobenzoyl analogues 6b and 6c bearing either the N,N-dimethylamide or N-phenethylamide substituents,
respectively, showed no activity.
Encouraged by these results, a small series of aroylpyrroles was prepared. Compounds were designed to establish
the minimum kinase binding pharmacophore, and to explore possibilities to gain potency and selectivity by variation
of the amide substituent. The benzoyl substituent was fixed as most potent 2,3-dichlorophenyl for all these
examples.
Series (6f-n) was prepared to explore simple variations to the amide moiety (Table 7). Monomethyl amide 6f was
equipotent with the parent 4-fluorobenzyl amide 6a, whereas the dimethyl amide 6g was 7-fold less potent.
Introduction of the 3-pyridylmethyl amide from 6e or the 4-pyridylmethyl amide 6h, retained potency, whereas the
benzyl derivative 6k and 2-pyridyl derivative 6j were less potent, and phenylethyl amide 6l was inactive. Similar to
6g, N-methyl-(3-pyridylmethyl) amide derivative 6n was 7-fold less potent than primary amide 6i. Importantly,
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methylation of the pyrrole NH (6m) completely abolished ERK5 activity, indicating an essential interaction with the
kinase at this position. In contrast, the relatively small drop in activity for the secondary amides 6g and 6n suggested
the amide NH was not forming a critical interaction, and that the drop in potency could be related to the
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conformational preference of the amide group. With this in mind, a limited number of conformationally restricted, 5-
and 6-membered cyclic secondary amides were investigated (Table 8). The 3,4-dihydro-2,6-naphthyridinyl and
isoindolinyl derivatives (6o and 6q) were inactive. In contrast, 3,4-dihydroisoquinolinyl 6p was 5-fold less potent than
6h, a comparable to the loss in potency seen for the N-methyl analogues, whereas the pyrrolidinopyridinyl 6r was
equipotent with 6h. Selected examples in this series were assayed in an orthogonal LANCE™ assay format (see
supporting information), based on time-resolved fluorescence resonance energy transfer (FRET), to eliminate the
possibility of false positives. In all cases, the LANCE results were comparable with those obtained using the IMAP
assay.
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In order to establish the minimum kinase binding pharmacophore, systematic isosteric replacements to the amide
group were made. The acetyl derivative 39 and the 1,3-diketone 44 were inactive (Table 9). In contrast, the
unsaturated ketone 40 and the cyclopropyl ketone 42 retained similar activity to the parent 6a, whereas the
saturated ketone 41 was 10-fold less active. These results confirm that the amide NH is not required for activity, and
that conformational rigidity at this position is favourable. The loss of activity for diketone 44 was explained by the
preferred enol tautomer lacking an essential H-bond to the kinase via the ketone adjacent to the pyrrole.
The most potent pyrrole inhibitor 6h was submitted for a kinase selectivity screen and gave a promising selectivity
profile. Of the 20 kinases, screened only one kinase (SAPK2a or p38α MAP kinase) was inhibited at >50% inhibition
(10 µM). Subsequent to our identification of pyrrole-2-carboxamides as ERK5 inhibitors, similar compounds, e.g. 45,
have been independently identified as p38α MAP kinase inhibitors with micromolar activity.34 The X-ray structure of
45 shows it bound to the hinge of the kinase via hydrogen bonds from the pyrrole NH and the carboxamide carbonyl,
with the aryl portion occupying the lipophilic region close to the gatekeeper, and the furan binding in the outer
lipophilic region. ERK5 shares 48% sequence homology with p38α MAP kinase, and 58% homology in the kinase
domain. In addition, the gatekeeper residues of the kinases are similar, with leucine in ERK5 and threonine in p38α
MAP kinase. The ERK5 SAR for our series is consistent with a similar binding mode to ERK5 as seen in the p38 X-ray
structure, in particular the donor/acceptor doublet of H-bonds from the pyrrole NH and amide carbonyl to the
kinase.
At this point, given the similarity between the published p38α MAP kinase inhibitors and our hit series, we needed
to establish selectivity vs p38α MAP kinase to provide useful ERK5 tool compounds or therapeutic agents.34-35 Thus,
selected compounds were counterscreened against p38α MAP kinase using a LANCE assay. As anticipated, the 2-
pyridyl derivatives 6j and 3,4-dihydroisoquinolinyl derivative 6p were equipotent for both p38α MAP kinase and
ERK5. Importantly, the pyrrolidinopyridinyl derivative 6r was inactive in the p38α assay. The ability to eliminate p38α
MAP kinase activity whilst maintaining ERK5 activity by variation of the amide side chain was not readily predicted
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from the published p38α X-ray structure and points to differing structural requirements around the amide side-chain
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CONCLUSIONS
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The IMAP FP high-throughput screen for ERK5 returned four distinct chemical series as hits. Synthesis of the hits and
selected close analogues demonstrated that the HTS activity of the benzothiazoles 3 was not reproducable, activity
for the cyanopyridine hits (4a, 19) was reproducable, but the limited scope to develop the SAR ruled this series out,
and two series with confirmed active hits. The lack of activity of these hits was disappointing but not atypical in
screening campaigns. The cyanopyrimidine hits 5a-c were not pursued for reasons of competition. The remaining
series, the pyrrole carboxamides 6a-e, demonstrated consistent ERK5 activity, with SARs consistent with a kinase
hinge binder. Selectivity against the close homologue p38α MAP kinase was achieved without loss of ERK5 activity
through minor structural modification, and a representative example 6h showed an acceptable kinase selectivity
profile in a panel. At this stage the pyrrole carboxamides demonstrated tractable synthesis, intelligible preliminary
SARs, and promising selectivity. The relatively modest kinase inhibitory activity achieved at this stage did not give
any concern as the pharmacophore established presented opportunities to optimise potency at both the benzoyl
and amide portions, indepenently. Having demonstrated the necessary requirements to progress to the hit-to-lead
optimisation stage, further SAR studies were undertaken, with an initial focus on improving potency, which will be
reported seperately.36
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EXPERIMENTAL SECTION
IMAP Substrate mapping
Non-phosphorylated and phosphorylated versions of each ERK5 sequence (Table 1) were obtained from the CRUK
Peptide Synthesis Research Services group. The substrate finder kit was used according to the manufacturer’s
instructions. Reaction buffer (10 µL) containing ATP (100 µM) was added to wells of the plate to reconstitute 5-FAM
labeled substrates. Reaction buffer (10 µL) with or without ERK5 (6.4 ng/µL) was added to appropriate wells of the
plate, to generate background controls and positive controls. The reaction was incubated for 1 hour at ambient
temperature after which IMAP Binding Solution (60 µL) was added. After a further 1 hour of incubation the
fluorescence polarisation was measured. The results were analysed using the IMAP Substrate Mapper provided with
the kit.
Kinetic characterisation of ERK5
Reactions were carried out with varying concentrations of ATP at constant substrate and enzyme concentrations.
Due to limitations of the IMAP FP assay with respect to ATP concentrations, we utilised a transfer method to
increase the maximum concentration of ATP that can be used. Reactions were conducted as normal in 10 µL reaction
volume. After either 1, 2, 3 or 4 hour incubation period at 37°C, 4 µL of the reaction was transferred to 196 µL of
reaction buffer followed by a subsequent transfer of 10 µL of this solution to 30 µL of IMAP Binding Solution. Rates
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of reaction at 1 hour reaction time at the range of substrate concentrations were determined, and kinetic
parameters were determined by non-linear regression fitting of the data to the Michaelis-Menten equation
(Equation 1) ; curve fitting was performed using GraphPad Prism software.37
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ERK5 High-throughput Screen
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Compounds were assayed in a 10 µL reaction mixture per well containing: 1 in 700 dilution of ERK5 stock from CRT,
100 nM peptide R7129 and 100 µM of ATP. The reactions were performed with 10 mM Tris-HCl (pH 7.2), 10 mM
MgCl2, 0.05% NaN3 and 0.01% Tween-20. Reactions were incubated for 3 hours at 37°C, followed by addition of 30
µL of IMAP binding solution (1 in 600 dilution of IMAP binding reagent in 60% Binding Buffer A and 40% Binding
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Buffer B) and a further incubation for 2 hours at ambient temperature. Plates were read on an Analyst HT microplate
reader and the data analysed using ActivityBase.
ERK5 IC50 Determination (IMAP)
The enzyme reaction was run as described for the HTS but using 300 µM ATP, 250 nM peptide and a reduced
incubation time of 2 hours at 37°C. 1 µL of this reaction was then transferred to a new assay plate and 9 µL of
reaction buffer was added followed by 30 µL of IMAP binding solution.
X-ray crystallography
Data were collected on an Oxford Diffraction Gemini A Ultra diffractometer for 3i, using MoKα radiation (λ = 0.71073
Å) at 150K, and on a Bruker Apex2 diffractometer for 3a and 12a, using synchrotron radiation (λ = 0.6946 Å; SRS
station 9.8, Daresbury Laboratory) at 120 K because of the very small size of crystals available. Corrections were
made for synchrotron beam decay and for absorption and other systematic effects on the basis of repeated and
equivalent data. The structures were solved by direct methods and refined on all unique F2 values with anisotropic
non-hydrogen atoms, with freely refined isotropic H atoms bonded to N, and with a riding model for H atoms
bonded to C. All four structures are fully ordered; 3i have two independent molecules in the asymmetric unit, and
the non-centrosymmetric but achiral crystal structure of 3i displays inversion twinning with essentially equal
components. Full crystallographic details are given in the Supporting information. Programs were standard Oxford
Diffraction CrysAlisPro38 and Bruker Apex239 for data collection and processing, and SHELXTL40 and SHELXL-
2014_ENREF_52_ENREF_5341 for structure solution and refinement. CCDC references: 1410001, 141003, and
1410004.
ASSOCIATED CONTENT
Supporting Information
Additional screening and synthesis information, X-ray crystal structure data for compounds 12a, 3a, and 3i, synthetic
procedures, ERK5 and p38α LANCE assay protocols, kinase selectivity data for 6h.
AUTHOR INFORMATION
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Corresponding Author
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*E-mail: Ian.Hardcastle@ncl.ac.uk
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Funding
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This work was supported by Cancer Research UK (C240/A7409, C1296/A6963 and C2115/A21421), EPSRC equipment
grant (EP/F03637X/1), and the synchrotron component of the UK National Crystallography Service 2001–2010
(EP/D07746X/1).
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Notes
The authors declare no competing financial interests.
ACKNOWLEDGEMENTS
We thank CCLRC for access to synchrotron diffraction facilities at SRS. The use of the EPSRC National Mass
Spectroscopy Facility, Swansea University is gratefully acknowledged.
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EQUATIONS
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Equation 1
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ꢀ
ꢂꢃꢄꢅ[ꢆ]
[ ]
(ꢇꢈ + ꢆ )
=
[ꢁ]
8
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Equation 2
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3ꢋꢃꢌ + 3ꢋꢃꢍ
ꢉꢊ = 1 −
|
|
μꢃꢌ − μꢃꢍ
Where σ and µ represent the standard deviation and mean of the positive (c+) and negative (c-) plate controls,
respectively.
TABLES
Table 1
Number
166
Sequence
5-FAM-AGRSPVD
168
5-FAM-EAGRSPVDS
5-FAM-HEAGRSPVDSL
5-FAM-RHEAGRSPVDSLS
5-FAM-TRHEAGRSPVDSLSS
170
172
174
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Table 2
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
ERK5 IC50 (μM)
Resynthesizeda
Compound Structure
R1
R2
HTS
0.057
0.087
0.087
0.11
0.13
0.13
0.46
0.60
0.89
0.93
5.13
-
3a
3b
3c
A
A
A
A
A
A
A
A
A
A
A
B
B
C
C
-(CH2)4-
51 ± 5.0
Et
Me
Et
Me
H
85 ± 5.0
-
3d
3e
3f
H
-
CH2=CHCH2-
H
-
s-Bu
H
-
3g
3h
3i
-(CH2)5-
-
-
i-Pr
n-Pr
Et
H
H
>120b
3j
Et
-
3k
9a
9b
12a
12b
-(CH2)2O(CH2)2-
-
-(CH2)4-
29 ± 1.3
21 ± 1.5
2.3 ± 1.5
>120b
Et
Et
Me
-
-(CH2)4-
-
Me
-
a) Values are the mean of at least 3 determinations ± SD; b) n = 2
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ACS Combinatorial Science
Table 3: ERK5 inhibitory activity of nicotinonitrile series 4a-k.
1
2
3
4
5
6
7
8
9
ERK5 IC50 (µM)
HTS Resynthesizeda
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
Compd
4a
R1
4-F-Ph
R2
Ph
1.6
4.9 ± 0.3
>120b
4b
4c
4d
4e
4f
3-F-Ph
2-F-Ph
Ph
Ph
Ph
-
-
-
-
-
-
-
-
-
-
34.3 ± 6.4
>120b
>120b
Ph
4-(CF3)-Ph
2,4-di-F-Ph
4-Py
Ph
Ph
72.9 ± 26.1
65.1 ± 12.4
>120b
4g
4h
4i
Ph
4-MeOPh
Ph
Ph
4-MeOPh
CH3
CH3
>120b
4j
Ph
>120b
4k
CH3
>120b
a) Values are the mean of at least 3 determinations ± SD; b) n = 2
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Table 4: ERK5 inhibitory activity of nicotinonitrile series 14a,15l, 17,19-21, 23, 28 and 31-32.
1
2
3
4
5
6
7
8
9
ERK5 IC50 (µM)
Compound
14a
R
R1
R2
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
HTS
Resynthesizeda
H
Ph
4-F-Ph
-
111 ± 6.5
15l
17
4-F-Ph
4-F-Ph
-
-
20.9 ± 1.6c
>120
Ph
Ph
19
4-F-Ph
31
29.4 ± 3.7c
Ph
20
21
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
-
-
-
-
-
117 ± 18
>120
Ph
Ph
Ph
Ph
Ph
23a
23b
28
>120d
>120d
>120
31
32
4-F-Ph
4-Py
0.4
-
20.5 ± 1.3
Ph
Ph
104.9 ± 6.5
a) Values are the mean of at least 3 determinations ± SD b) n = 1; c) n = 2; d)precipitation observed at 1.2 mM in
40% DMSO;
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Table 5: ERK5 inhibitory activity of pyrimidine series 5a-c.
1
2
3
4
5
6
7
8
9
ERK5 IC50 (µM)
Compound
R
HTS Resynthesizeda
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
5a
5b
5c
2-CH3
3-OCH3 11
4-F 6.5
26
88 ± 3
23 ± 7
12 ± 3
a) Values are the mean of at least 3 determinations ± SD
Table 6 : ERK5 inhibitory activity of pyrrole carboxamides (6a-e)
ERK5 IC50 (µM)
Resynthesizeda
Compound
6a
Ar
R1
R2
HTS
H
CH3
H
0.66
3.7
6b
6c
CH3
1.89
3.50
>120b
>120b
6d
6e
CH3
H
H
4.32
8.0
9.6 ± 3.9
26.0 ± 1.2
a) Values are the mean of at least 3 determinations ± SD; b) n = 2
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Table 7. ERK5 SAR for pyrrole carboxamides (6f-n)
Page 20 of 31
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
ERK5 IC50 (µM)
Compound
R1
H
R2
H
R3
IMAPa
LANCEa
6f
CH3
CH3
3.3 ± 1.0b
3.6 ± 1.0
6g
H
CH3
24 ± 27c
44 ± 24
-
6h
6i
H
H
H
H
2.0 ± 2.0d
3.8 ± 3.7c
1.1 ± 0.4c
3.9 ± 0.8
6j
H
H
H
H
7.2 ± 0.02
21e
6k
-
-
6l
6m
6n
H
CH3
H
H
H
>120
>120
-
CH3
25 ± 1.3
13 ± 1.9
a) determinations ± standard deviation (mean of n = 2 unless otherwise stated); b) IC50 mean of n = 4; c) IC50 mean of n = 6; d)
IC50 mean of n = 10; e) IC50 n = 1.
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Table 8. SAR for cyclic pyrrole carboxamides (6j, 6o-r) against ERK5 and p38α.
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
ERK5 IC50 (µM)a
p38α LANCE
IC50 (µM)a
Compound ID
6j
R
IMAP
LANCE
7.2 ± 0.03
4.3 ± 1.2b
6o
6p
>120
-
11 ± 2.3b
25 ± 1.8
28 ± 20
6q
6r
>120
-
-
2.7 ± 0.4
> 120
a) determinations ± standard deviation (mean of n = 2 unless otherwise stated); b) IC50 mean of n = 4.
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Table 9. ERK5 SAR for pyrroles (39-42, 44).
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
ERK5 IC50 (µM)a
Compound ID
39
R
IMAP
LANCE
CH3
>120
-
40
41
3.1 ± 0.1b
23 ± 4.4
-
N
24 ± 2.2
42
44
6.8 ± 2.0
16 ± 5.7c
>120
-
a) determinations ± standard deviation (mean of n = 2 unless otherwise stated); b) mean of n = 4; c) mean of n = 6
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FIGURES
ACS Combinatorial Science
1
2
Figure 1: Crystal structure of 3-(pyrrolidin-1-ylsulfonyl)-4-thiocyanatobenzenamine 12a.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
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21
22
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34
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2: Crystal structure of 6-(pyrrolidine-1-sulfonyl)-benzothiazol-2-ylamine 3a.
Figure 3: Crystal structure of 2-amino-N-propylbenzo[d]thiazole-6-sulfonamide 3i.
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STRUCTURES
1
2
3
4
O
N
N
5
6
7
8
HN
N
N
N
EtO
9
10
11
12
13
14
15
16
17
18
19
20
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22
23
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44
45
46
47
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49
50
51
52
53
54
55
56
57
58
59
60
OH
1a R = H, 1b R = Me
2
45
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SCHEMES
Scheme 1.
ACS Combinatorial Science
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59
60
Reagents and conditions: a) pyrrolidine or N-methylethylamine or propylamine, Et3N, DCM; b) Pd/C, H2, EtOAc; c)
KSCN, Cu(II)SO4, MeOH.
Scheme 2.
Reagents and conditions: a) pyrrolidine or N-methylethylamine, Et3N, DCM; b) Pd/C, H2, EtOAc; c) KSCN, Cu(II)SO4,
MeOH.
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Scheme 3
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2
3
4
5
6
7
8
9
10
11
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59
60
Reagents and Conditions: a) KOH, EtOH, RT; b) Method A, S8, morpholine, EtOH, 80 oC 30 min then malononitrile; or
Method B, 2-cyanothioacetamide, 1.6 M NaOMe in MeOH, 80 oC; c) tert-butyl or ethyl 2-(2-
bromoacetamido)acetate, K2CO3 or KOH, DMF, 100 oC; d) TFA, RT; e) p-methoxybenzylamine, HBTU, DIPEA, DMF, 60
oC; f) TFA, 70 oC. NB: No base was required in step c after step b (Method B), as an excess of NaOMe was used in step
b
Scheme 4
Reagents and Conditions: a) bromoacetyl chloride, CaCO3, CHCl3, H2O, 0 oC; b) 14a, KOH, DMF, reflux.
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Scheme 5
ACS Combinatorial Science
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5
6
7
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9
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51
52
53
54
55
56
57
58
59
60
Reagents and Conditions: a) methyl bromoacetate, KOH, DMF, reflux, or chloroacetamide, NaOAc.3H2O, ethanol,
reflux.
Scheme 6
Reagents and Conditions: a) RBr, K2CO3, THF, 100 oC; b) TFA, RT.
Scheme 7
Reagents and Conditions: a) ethanolamine, RT; b) Boc2O, Et3N, DCM, 0 oC-RT; c) MsCl, Et3N, DCM, 0 oC-RT; d) 14a,
DMF, 100 oC; e) TFA, RT.
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Scheme 8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
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23
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37
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Reagents and Conditions: a)i) Ac2O, pyridine, reflux; ii) HCl, H2O, reflux; b) bromoacetyl chloride, CaCO3, DCM, reflux;
c) 14a or 14g, K2CO3, DMF, 100 oC
Scheme 9
Reagents and Conditions: a) DMF, 100 oC.
Scheme 10
Reagents and Conditions: a) ArCOCl, AlCl3, DCM, 0 oC-RT; b) LiOH, THF, H2O, 60 oC; c) i) CDI, THF, 70 oC; ii) R1R2NH, 50
oC - RT.
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Scheme 11
ACS Combinatorial Science
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2
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4
5
6
7
8
9
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Reagents and Conditions: a) NaH, DMF, MeI; b) LiOH, THF, H2O, 60 oC; c) i) CDI, THF, 70 oC; ii) 3-pyridylmethylamine,
50 oC - RT.
Scheme 12
Reagents and Conditions: a) AlCl3, 2,3-dichlorobenzoyl chloride, DCM, 0 oC-RT, 18 h.; b) Isonicotinaldehyde, KOH,
EtOH, H2O, 0 oC–RT, 18 h.; c) Indium powder, NH4Cl, EtOH, H2O, reflux, 8 h; d) (CH3)2SO+I-, KOtBu, DMSO, RT, 24 h.
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