Human Toxicity Caused by Indole and Indazole Carboxylate Synthetic Cannabinoid Receptor Agonists: From Horizon Scanning to Notification.

BACKGROUND: The emergence of novel psychoactive substances (NPS), particularly synthetic cannabinoid receptor agonists (SCRA), has involved hundreds of potentially harmful chemicals in a highly dynamic international market challenging users', clinicians', and regulators' understanding of what circulating substances are causing harm. We describe a toxicovigilance system for NPS that predicted the UK emergence and identified the clinical toxicity caused by novel indole and indazole carboxylate SCRA. METHODS: To assist early accurate identification, we synthesized 5 examples of commercially unavailable indole and indazole carboxylate SCRA (FUB-NPB-22, 5F-NPB-22, 5F-SDB-005, FUB-PB-22, NM-2201). We analyzed plasma and urine samples from 160 patients presenting to emergency departments with severe toxicity after suspected NPS use during 2015 to 2016 for these and other NPS using data-independent LC-MS/MS. RESULTS: We successfully synthesized 5 carboxylate SCRAs using established synthetic and analytical chemistry methodologies. We identified at least 1 SCRA in samples from 49 patients, including an indole or indazole carboxylate SCRA in 17 (35%), specifically 5F-PB-22 (14%), FUB PB-22 (6%), BB-22 (2%), 5F NPB-22 (20%), FUB NPB-22 (2%), and 5F-SDB-005 (4%). In these 17 patients, there was analytical evidence of other substances in 16. Clinical features included agitation and aggression (82%), reduced consciousness (76%), acidosis (47%), hallucinations and paranoid features (41%), tachycardia (35%), hypertension (29%), raised creatine kinase (24%), and seizures (12%). CONCLUSIONS: This toxicovigilance system predicted the emergence of misuse of indole and indazole carboxylate SCRA, documented associated clinical harms, and notified relevant agencies. Toxicity appears consistent with other SCRA, including mental state disturbances and reduced consciousness.

Identification of a novel ligand for the ATAD2 bromodomain with selectivity over BRD4 through a fragment growing approach.

ATAD2 is an ATPase that is overexpressed in a variety of cancers and associated with a poor patient prognosis. This protein has been suggested to function as a cofactor for a range of transcription factors, including the proto-oncogene MYC and the androgen receptor. ATAD2 comprises an ATPase domain, implicated in chromatin remodelling, and a bromodomain which allows it to interact with acetylated histone tails. Dissection of the functional roles of these two domains would benefit from the availability of selective, cell-permeable pharmacological probes. An in silico evaluation of the 3D structures of various bromodomains suggested that developing small molecule ligands for the bromodomain of ATAD2 is likely to be challenging, although recent reports have shown that ATAD2 bromodomain ligands can be identified. We report a structure-guided fragment-based approach to identify lead compounds for ATAD2 bromodomain inhibitor development. Our findings indicate that the ATAD2 bromodomain can accommodate fragment hits (Mr < 200) that yield productive structure-activity relationships, and structure-guided design enabled the introduction of selectivity over BRD4.

Combined PI3K and CDK2 inhibition induces cell death and enhances in vivo antitumour activity in colorectal cancer.

BACKGROUND: The phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway is commonly deregulated in human cancer, hence many PI3K and mTOR inhibitors have been developed and have now reached clinical trials. Similarly, CDKs have been investigated as cancer drug targets. METHODS: We have synthesised and characterised a series of 6-aminopyrimidines identified from a kinase screen that inhibit PI3K and/or mTOR and/or CDK2. Kinase inhibition, tumour cell growth, cell cycle distribution, cytotoxicity and signalling experiments were undertaken in HCT116 and HT29 colorectal cancer cell lines, and in vivo HT29 efficacy studies. RESULTS: 2,6-Diaminopyrimidines with an O(4)-cyclohexylmethyl substituent and a C-5-nitroso or cyano group (1,2,5) induced cell cycle phase alterations and were growth inhibitory (GI50<20 µM). Compound 1, but not 2 or 5, potently inhibits CDK2 (IC50=0.1 nM) as well as PI3K, and was cytotoxic at growth inhibitory concentrations. Consistent with kinase inhibition data, compound 1 reduced phospho-Rb and phospho-rS6 at GI50 concentrations. Combination of NU6102 (CDK2 inhibitor) and pictilisib (GDC-0941; pan-PI3K inhibitor) resulted in synergistic growth inhibition, and enhanced cytotoxicity in HT29 cells in vitro and HT29 tumour growth inhibition in vivo. CONCLUSIONS: These studies identified a novel series of mixed CDK2/PI3K inhibitors and demonstrate that dual targeting of CDK2 and PI3K can result in enhanced antitumour activity.

Trifluoroacetic acid in 2,2,2-trifluoroethanol facilitates S(N)Ar reactions of heterocycles with arylamines.

Small-molecule drug discovery requires reliable synthetic methods for attaching amino compounds to heterocyclic scaffolds. Trifluoroacetic acid-2,2,2-trifluoroethanol (TFA-TFE) is as an effective combination for achieving SN Ar reactions between anilines and heterocycles (e.g., purines and pyrimidines) substituted with a leaving group (fluoro-, chloro-, bromo- or alkylsulfonyl). This method provides a variety of compounds containing a "kinase-privileged fragment" associated with potent inhibition of kinases. TFE is an advantageous solvent because of its low nucleophilicity, ease of removal and ability to solubilise polar substrates. Furthermore, TFE may assist the breakdown of the Meisenheimer-Jackson intermediate by solvating the leaving group. TFA is a necessary and effective acidic catalyst, which activates the heterocycle by N-protonation without deactivating the aniline by conversion into an anilinium species. The TFA-TFE methodology is compatible with a variety of functional groups and complements organometallic alternatives, which are often disadvantageous because of the expense of reagents, the frequent need to explore diverse sets of reaction conditions, and problems with product purification. In contrast, product isolation from TFA-TFE reactions is straightforward: evaporation of the reaction mixture, basification and chromatography affords analytically pure material. A total of 45 examples are described with seven discrete heterocyclic scaffolds and 2-, 3- and 4-substituted anilines giving product yields that are normally in the range 50-90 %. Reactions can be performed with either conventional heating or microwave irradiation, with the latter often giving improved yields.

Model system for irreversible inhibition of Nek2: thiol addition to ethynylpurines and related substituted heterocycles.

Recent studies have shown that irreversible inhibition of Nek2 kinase [(Never in mitosis gene a)-related kinase 2], overexpression of which is observed in several cancers, can be achieved using Michael acceptors containing an ethynyl group, which target the enzyme's cysteine 22 residue lying near the catalytic site. The model studies described herein demonstrate an analogous capture of the ethynyl moiety in a series of ethynyl-heterocycles (e.g. 6-ethynyl-N-phenyl-9H-purin-2-amine) by N-acetylcysteine methyl ester in the presence of 1,4-diazabicyclo[2.2.2]octane in either dimethyl sulfoxide or N,N-dimethylformamide. Kinetic studies showed a 50-fold range in reactivity with 7-ethynyl-N-phenyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine being the most reactive compound, whereas 4-ethynyl-N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine was the least reactive. Studies of the isomeric compounds, 2-(3-((6-ethynyl-7-methyl-7H-purin-2-yl)amino)phenyl)acetamide and 2-(3-((6-ethynyl-9-methyl-9H-purin-2-yl)amino)phenyl)acetamide, revealed the N(7)-methyl isomer to be 5-fold more reactive than the 9-methyl isomer, which is ascribed to a buttressing effect in the N(7)-methyl compound. Comparison of the crystal structures of these isomers showed that the ethynyl group is significantly displaced away from the methyl group exclusively in the N(7)-methyl isomer with an sp(2) bond angle of 124°, whereas the corresponding angle in the N(9)-methyl isomer was the expected 120°. The results of this study indicate heterocyclic scaffolds that are likely to be more promising for inhibition of Nek2 and other kinases containing a reactive cysteine.

Trifluoroethanol solvent facilitates selective N-7 methylation of purines.

Purines protected at N-9 by p-methoxybenzyl are methylated or ethylated in 2,2,2-trifluoroethanol at N-7 by trimethyl- or triethyl-oxonium borofluorate, respectively. Subjecting the resulting cationic species to microwave irradiation releases an N(7)-methyl- or ethyl-purine. This one-pot procedure is an efficient regiospecific method applicable to diverse substrates.

Potent enantioselective inhibition of DNA-dependent protein kinase (DNA-PK) by atropisomeric chromenone derivatives.

Substitution at the 7-position of the chromen-4-one pharmacophore of 8-(dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one NU7441, a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, with allyl, n-propyl or methyl enabled the resolution by chiral HPLC of atropisomers. Biological evaluation against DNA-PK of each pair of atropisomers showed a marked difference in potency, with biological activity residing exclusively in the laevorotatory enantiomer.

Mapping Ligand Interactions of Bromodomains BRD4 and ATAD2 with FragLites and PepLites─Halogenated Probes of Druglike and Peptide-like Molecular Interactions.

The development of ligands for biological targets is critically dependent on the identification of sites on proteins that bind molecules with high affinity. A set of compounds, called FragLites, can identify such sites, along with the interactions required to gain affinity, by X-ray crystallography. We demonstrate the utility of FragLites in mapping the binding sites of bromodomain proteins BRD4 and ATAD2 and demonstrate that FragLite mapping is comparable to a full fragment screen in identifying ligand binding sites and key interactions. We extend the FragLite set with analogous compounds derived from amino acids (termed PepLites) that mimic the interactions of peptides. The output of the FragLite maps is shown to enable the development of ligands with leadlike potency. This work establishes the use of FragLite and PepLite screening at an early stage in ligand discovery allowing the rapid assessment of tractability of protein targets and informing downstream hit-finding.

DNA-dependent protein kinase (DNA-PK) inhibitors: structure-activity relationships for O-alkoxyphenylchromen-4-one probes of the ATP-binding domain.

Introduction of an O-alkoxyphenyl substituent at the 8-position of the 2-morpholino-4H-chromen-4-one pharmacophore enabled regions of the ATP-binding site of DNA-dependent protein kinase (DNA-PK) to be probed further. Structure-activity relationships have been elucidated for inhibition of DNA-PK and PI3K (p110α), with N-(2-(cyclopropylmethoxy)-4-(2-morpholino-4-oxo-4H-chromen-8-yl)phenyl)-2-morpholinoacetamide 11a being identified as a potent and selective DNA-PK inhibitor (IC(50)=8 nM).

MDM2-p53 protein-protein interaction inhibitors: a-ring substituted isoindolinones.

Structure-activity relationships for the MDM2-p53 inhibitory activity of a series of A-ring substituted 2-N-benzyl-3-(4-chlorophenyl)-3-(1-(hydroxymethyl)cyclopropyl)methoxy)isoindolinones have been investigated, giving rise to compounds with improved potency over their unsubstituted counterparts. Isoindolinone A-ring substitution with a 4-chloro group for the 4-nitrobenzyl, 4-bromobenzyl and 4-cyanobenzyl derivatives (10a-c) and substitution with a 6-tert-butyl group for the 4-nitrobenzyl derivative (10j) were found to confer additional potency. Resolution of the enantiomers of 10a showed that potent MDM2-p53 activity resided in the (-)-enantiomer ((-)-10a; IC(50)=44 ± 6 nM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compounds 10a and (-)-10a increase p53 protein levels, activate p53-dependent MDM2 and p21 transcription in MDM2 amplified cells, and show improved selectivity for growth inhibition in wild type p53 cell lines over the parent compound.

Versatile synthesis of functionalised dibenzothiophenes via Suzuki coupling and microwave-assisted ring closure.

Amino-substituted biphenyls were obtained by Suzuki cross-coupling of 2,6-dibromoaniline with a phenylboronic acid (substituted with Me, NO(2), OH, OMe or Cl) preferably assisted by microwave irradiation. Conversion of the amino group into a thiol preceded a base-induced intramolecular substitution, also facilitated by microwave heating, to generate the second C-S bond of the target dibenzothiophene. The 1-, 2-, 3- or 4-substituted 6-halodibenzothiophenes obtained were subjected to a palladium-mediated coupling with 2-morpholin-4-yl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4H-chromen-4-one to give the respective 6-, 7-, 8- or 9-substituted dibenzothiophen-4-ylchromenones. These compounds were evaluated as inhibitors of DNA-dependent protein kinase (DNA-PK) and compared to the parent 8-(dibenzo[b,d]thiophen-4-yl)-2-morpholin-4-yl-4H-chromen-4-one. Notably, derivatives bearing hydroxy or methoxy substituents at C-8 or C-9 retained activity, whereas substitution at C-7 lowered activity. Substitution with chloro at C-6 was not detrimental to activity, but a chloro group at C-7 or C-8 reduced potency. The data indicate permissive elaboration of hydroxyl at C-8 or C-9, enabling the possibility of improved pharmaceutical properties, whilst retaining potency against DNA-PK.

An Alkynylpyrimidine-Based Covalent Inhibitor That Targets a Unique Cysteine in NF-κB-Inducing Kinase.

NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome. Analysis of existing inhibitor scaffolds and early structure-activity relationships (SARs) led to the design of C444-targeting covalent inhibitors based on alkynyl heterocycle warheads. Mass spectrometry provided proof of the covalent mechanism, and the SAR was rationalized by computational modeling. Profiling of more potent analogues in tumor cell lines with constitutively activated NIK signaling induced a weak antiproliferative effect, suggesting that kinase inhibition may have limited impact on cancer cell growth. This study shows that alkynyl heterocycles are potential cysteine traps, which may be employed where common Michael acceptors, such as acrylamides, are not tolerated.

Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein-Protein Interaction.

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

Mapping the ATP-binding domain of DNA-dependent protein kinase (DNA-PK) with coumarin- and isocoumarin-derived inhibitors.

Replacement of the core heterocycle of a defined series of chromen-4-one DNA-PK inhibitors by the isomeric chromen-2-one (coumarin) and isochromen-1-one (isocoumarin) scaffolds was investigated. Structure-activity relationships for DNA-PK inhibition were broadly consistent, albeit with a reduction of potency compared with the parent chromenone.

Synthesis of sulfonamide-based kinase inhibitors from sulfonates by exploiting the abrogated SN2 reactivity of 2,2,2-trifluoroethoxysulfonates.

The attenuated S(N)2 reactivity of the 2,2,2-trifluoroethyl group has been exploited for the synthesis of a series of 6-cyclohexylmethoxy-2-arylaminopurines in which a sulfonamide moiety was attached to the aryl ring via a methylene group. These were required as potential inhibitors of serine-threonine kinases of interest for the treatment of cancer. 3-Nitrophenylmethanesulfonyl chloride was converted into the corresponding 2,2,2-trifluoroethoxysulfonyl ester by reaction with 2,2,2-trifluoroethanol in the presence of triethylamine/4-dimethylaminopyridine. Catalytic hydrogenation of the nitro group employing 2,2,2-trifluoroethanol as solvent gave 2,2,2-trifluoroethyl 3-aminophenylmethanesulfonate, which was reacted with 6-cyclohexylmethoxy-2-fluoropurine in 2,2,2-trifluoroethanol/trifluoroacetic acid to afford 2,2,2-trifluoroethyl 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonate. 3-(6-Cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamides were synthesised by microwave heating of the trifluoroethoxysulfonate with an amine and 1,8-diazabicycloundec-7-ene in tetrahydrofuran. The mechanism of this process was shown to involve an intermediate sulfene by a deuterium-labelling experiment. 3-(6-Cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide derivatives were assayed as inhibitors of human cyclin-dependent kinase 2. Previous structure-activity studies demonstrated that relocating the sulfonamide group of O(6)-cyclohexylmethoxy-2-(4'-sulfamoylanilino)purine from the 4- to the 3-position on the 2-arylamino ring resulted in a 40-fold reduction in potency against CDK2. In the present study, no further loss of activity was observed on introducing a methylene group between the sulfonamide and the aryl ring, 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide proving equipotent with O(6)-cyclohexylmethoxy-2-(3'-sulfamoylanilino)purine (IC(50) = 0.21 microM). N-Alkylation of the sulfonamide reduced CDK-2 inhibitory activity, while a substituted benzyl or 3-phenylpropyl group on the sulfonamide resulted in a loss of potency compared with 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide. The dimethylaminopropyl derivative, 1-[3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenyl]-N-(3-dimethylaminopropyl)methanesulfonamide was only 2-fold less potent than 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide, suggesting an interaction between the basic dimethylamino group and the kinase. The presence of alicyclic groups on the pendant sulfonamide showed IC(50) values in the 0.5-1.5 microM range. N-(4-tert-Butylphenyl)-1-[3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenyl]methanesulfonamide was markedly less active (IC(50) = 34 microM), suggesting a steric effect within the ATP-binding domain.

Atropisomeric 8-arylchromen-4-ones exhibit enantioselective inhibition of the DNA-dependent protein kinase (DNA-PK).

Substitution at the 3-position of the dibenzothiophen-4-yl ring of 8-(dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one NU7441, a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, with propyl, allyl or methyl enabled the separation by chiral HPLC of atropisomers. This is a consequence of restricted rotation about the dibenzothiophene-chromenone bond. Biological evaluation against DNA-PK of the pairs of atropisomers showed a marked difference in potency, with only one enantiomer being biologically active.

Synthesis and biological evaluation of 5-substituted O4-alkylpyrimidines as CDK2 inhibitors.

CDK2 inhibitory structure-activity relationships have been explored for a range of 5-substituted O(4)-alkylpyrimidines. Variation of the 5-substituent in the 2,6-diaminopyrimidine series confirmed the 5-nitroso substituent as optimal, and showed that 5-formyl and 5-acetyl substituents were also tolerated at this position. A series of O(4)-alkyl-N(2)-aryl-5-substituted-6-aminopyrimidines revealed interesting structure-activity relationships. In the 5-nitroso series, the optimum O(4)-alkyl substituents were cyclohexylmethyl or sec-butyl, combined with a 2-sulfanilyl group. By contrast, in the N(2)-arylsulfonamido-5-formyl series, the cyclohexylmethyl compound showed relatively poor activity compared with the sec-butyl derivative (22j, (R)-4-(4-amino-6-sec-butoxy-5-formylpyrimidin-2-ylamino)benzenesulfonamide; CDK2 IC(50) = 0.8 nM). Similarly, in the N(2)-arylsulfonamido-5-(hydroxyiminomethyl) series the O(4)-sec-butyl substituent conferred greater potency than the cyclohexylmethyl (23c, (rac)-4-(4-amino-6-sec-butoxy-5-(hydroxyiminomethyl)pyrimidin-2-ylamino)benzenesulfonamide; CDK2 IC(50) = 7.4 nM). The 5-formyl derivatives show selectivity for CDK2 over other CDK family members, and are growth inhibitory in tumour cells (e.g. 22j, GI(50) = 0.57 microM).

2-Arylamino-6-ethynylpurines are cysteine-targeting irreversible inhibitors of Nek2 kinase.

Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers. We now report the design, synthesis and biological evaluation of 6-ethynylpurines that act as covalent inhibitors of Nek2 by capturing a cysteine residue (Cys22) close to the catalytic domain of this protein kinase. Examination of the crystal structure of the non-covalent inhibitor 3-((6-cyclohexylmethoxy-7H-purin-2-yl)amino)benzamide in complex with Nek2 indicated that replacing the alkoxy with an ethynyl group places the terminus of the alkyne close to Cys22 and in a position compatible with the stereoelectronic requirements of a Michael addition. A series of 6-ethynylpurines was prepared and a structure activity relationship (SAR) established for inhibition of Nek2. 6-Ethynyl-N-phenyl-7H-purin-2-amine [IC50 0.15 µM (Nek2)] and 4-((6-ethynyl-7H-purin-2-yl)amino)benzenesulfonamide (IC50 0.14 µM) were selected for determination of the mode of inhibition of Nek2, which was shown to be time-dependent, not reversed by addition of ATP and negated by site directed mutagenesis of Cys22 to alanine. Replacement of the ethynyl group by ethyl or cyano abrogated activity. Variation of substituents on the N-phenyl moiety for 6-ethynylpurines gave further SAR data for Nek2 inhibition. The data showed little correlation of activity with the nature of the substituent, indicating that after sufficient initial competitive binding to Nek2 subsequent covalent modification of Cys22 occurs in all cases. A typical activity profile was that for 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide [IC50 0.06 µM (Nek2); GI50 (SKBR3) 2.2 µM] which exhibited >5-10-fold selectivity for Nek2 over other kinases; it also showed > 50% growth inhibition at 10 µM concentration against selected breast and leukaemia cell lines. X-ray crystallographic analysis confirmed that binding of the compound to the Nek2 ATP-binding site resulted in covalent modification of Cys22. Further studies confirmed that 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide has the attributes of a drug-like compound with good aqueous solubility, no inhibition of hERG at 25 µM and a good stability profile in human liver microsomes. It is concluded that 6-ethynylpurines are promising agents for cancer treatment by virtue of their selective inhibition of Nek2.

FragLites-Minimal, Halogenated Fragments Displaying Pharmacophore Doublets. An Efficient Approach to Druggability Assessment and Hit Generation.

Identifying ligand binding sites on proteins is a critical step in target-based drug discovery. Current approaches to this require resource-intensive screening of large libraries of lead-like or fragment molecules. Here, we describe an efficient and effective experimental approach to mapping interaction sites using a set of halogenated compounds expressing paired hydrogen-bonding motifs, termed FragLites. The FragLites identify productive drug-like interactions, which are identified sensitively and unambiguously by X-ray crystallography, exploiting the anomalous scattering of the halogen substituent. This mapping of protein interaction surfaces provides an assessment of druggability and can identify efficient start points for the de novo design of hit molecules incorporating the interacting motifs. The approach is illustrated by mapping cyclin-dependent kinase 2, which successfully identifies orthosteric and allosteric sites. The hits were rapidly elaborated to develop efficient lead-like molecules. Hence, the approach provides a new method of identifying ligand sites, assessing tractability and discovering new leads.

Identification of a novel orally bioavailable ERK5 inhibitor with selectivity over p38α and BRD4.

Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82 µM for ERK5; IC50 > 120 µM for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.