Synthesis and biological activity of 2-cyanoacrylamide derivatives tethered to imidazopyridine as TAK1 inhibitors

Abstract The importance of transforming growth factor beta-activated kinase 1 (TAK1) to cell survival has been demonstrated in many studies. TAK1 regulates signalling cascades, the NF-κB pathway and the mitogen-activated protein kinase (MAPK) pathway. TAK1 inhibitors can induce the apoptosis of cancerous cells, and irreversible inhibitors such as (5Z)-7-oxozeaenol are highly potent. However, they can react non-specifically with cysteine residues in proteins, which may have serious adverse effects. Reversible covalent inhibitors have been suggested as alternatives. We synthesised imidazopyridine derivatives as novel TAK1 inhibitors, which have 2-cyanoacrylamide moiety that can form reversible covalent bonding. A derivative with 2-cyano-3-(6-methylpyridin-2-yl)acrylamide (13h) exhibited potent TAK1 inhibitory activity with an IC50 of 27 nM. It showed a reversible reaction with β-mercaptoethanol, which supports its potential as a reversible covalent inhibitor.


Introduction
Transforming growth factor beta-activated kinase 1 (TAK1), also known as mitogen-activated protein kinase kinase kinase 7 (MAP3K7) or MEK kinase 7 (MEKK7), is a serine/threonine kinase encoded by MAP3K7 gene. Since it was first found to be activated by transforming growth factor beta (TGFb) and bone morphologic protein (BMP) 1 , TAK1 has been reported to mediate signal transduction for the regulation of cell proliferation and apoptosis pathways 2 . TAK1 is activated by various exogenous stimuli, including interleukin-1 (IL-1), lipopolysaccharide (LPS), tumour necrosis factor alpha (TNFa), and TGFb 3,4 . TNFa has critical roles in signalling pathways for both cell survival and death [5][6][7] .
Because TAK1 is a key signalling element that is required for cell survival and death in TNF a signalling, it has emerged as a potential therapeutic target for cancer and inflammatory disease [8][9][10] . In TNFa stimulated breast cancer cells, inhibition of TAK1 causes apoptosis by switching from NFjB pro-survival signalling to induction of effector caspases 11 . In-vivo studies have provided evidence of a strong relationship between TAK1 and various malignancies, including pancreatic cancer 12 , colon cancer 13 , and breast cancer 14 . A number of small molecules have been reported to inhibit TAK1 ( Figure 1). (5Z)-7-Oxozeaenol (5Z7O, 1) 15 and epoxyquinol B (EPQB, 2) 16 are compounds derived from fungi which possess a resorcylic lactone and an epoxide, respectively. Imidazo [1,2-b]pyridazine (3) was developed as a reversible type I inhibitor. It fits into an active DFG-in conformation with TAK1 17 . Pyrazole urea (4) 18 and 1H-pyrrolo [2,3-b]pyridine (5) 19 have been reported as Type II inhibitors which bind to TAK1 in the inactive DFG-out confirmation.
5Z7O is a potent irreversible TAK1 inhibitor 15 , although it is also a promiscuous kinase inhibitor. The cis-enone of 5Z7O has off-target effects because it forms covalent bonds with reactive cysteine residues 20 . Acrylamide Michael acceptors irreversibly bind to nucleophiles such as cysteine under physiological conditions 21 . Michael acceptors that undergo dual activation by electron-withdrawing groups form reversible covalent bonds by increasing the a-carbon acidity of covalent adducts 22 . Converting an irreversible warhead to a reversible warhead can limit off-target binding and increase the probability of binding the target site 23 .
In this study, we designed imidazopyridines with 2-cyanoacrylamide moiety for reversible covalent TAK1 inhibition. The screening of our in-house chemical library led to identification of a pyrimidine compound (6) with an IC 50 of 413 nM against TAK1. To identify novel scaffold as TAK1 inhibitors, imidazopyridine scaffold was designed based on a bioisosteric replacement strategy. The imidazopyridine 14 showed retained activity although its IC 50 was approximately twice that of the pyrimidine (6). Target molecules were designed for reversible covalent chemistry by replacing the acrylamide moiety with various 2-cyanoacrylamide moieties ( Figure 2).

Chemistry
Unless otherwise noted, all reagents and solvents were purchased from a commercial vendor and used without further purification.
Reactions were monitored via thin-layer chromatography (TLC) using Merck TLC silica gel 60 F 254 250 mm plates. Flash column chromatography was performed using ZEOprep 60 silica gel (Zeochem, 40-63 mm) and a CombiFlash system (Teledyne ISCO) loaded with pre-packed silica gel flash column cartridges (Welux TM ). 1 H and 13 C NMR spectra were obtained using a Resonance ECZ 600R NMR spectrometer (JEOL). 1 H NMR spectra were collected at 600 MHz, and 13 C spectra were collected at 150 MHz using tetramethylsilane (TMS) as an internal standard. Chemical shifts are reported in parts per million (ppm, d) downfield of TMS, and the coupling constant (J) is reported in hertz (Hz). Splitting patterns are reported with the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartette; p, pentet; dd, doublet of doublets; dt, doublet of triplets; td, triplet of doublets; m, multiplet; br, broad signal. High-resolution mass spectrometry (HRMS) was performed using a Q-Exactive MS (ThermoScientific) coupled with an Ultimate 3000 LC system (Dionex). A ThermoScientific Hypersil GOLD C18 column (2.1 mm Â 50 mm, 1.9 mm) was used for separation.

HPLC analysis for reversible addition of BME to 13h
Phosphate-buffered saline (PBS) was prepared by mixing a solution of 91.2 mg monobasic potassium phosphate (KH 2 PO 4 ) in 10 ml H 2 O and a solution of 116.7 mg dibasic potassium phosphate (K 2 HPO 4 ) in 10 ml H 2 O. The 0.067 M phosphate solutions were mixed to obtain a pH 7.4 phosphate buffer. A solution of 48 mM b-mercaptoethanol (BME) in PBS (0.25 ml) was added to a solution of 13h (1 mg, $2 mmol) in dimethylsulphoxide (DMSO, 0.75 ml). Analysis of the reaction mixture after 30 min showed full conversion to the BME adduct. To determine whether the reaction between 13h and BME was reversible, the mixture was diluted 1:10 with PBS in DMSO and analysed via HPLC and mass spectra. The analysis was performed using a Waters HPLC system equipped with a 1525 pump, a PDA2998 detector, and a SunFire C18 column (4.6 Â 150 mm, 5 lm). The eluent system consisted of 0.05% TFA in 8:2 to 1:9 water/acetonitrile. Inhibitory activity of compounds for TAK1 was evaluated using LanthaScreen V R Eu Kinase Binding Assay (Invitrogen, Waltham, MA). The kinase profile of compound 13h was determined by the kinase HotSpot Profiling service of Reaction Biology Corporation. All assays were performed at Km for ATP.

Cell culture
MDA-MB-231 cells were obtained from the Korean Cell Line Bank. Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% foetal bovine serum and 1% penicillin/ streptomycin at 37 C with 5% CO 2 under humidified atmosphere.

Caspase-3/7 assay
The assay was performed using Apo-ONE V R Homogeneous Caspase-3/7 Assay kits (Catalogue No. G7790, Promega, Madison, WI). MDA-MB-231 cells in a concentration of 5000 cells/100 ml were seeded in each well of a black 96-well plate and starved after adhering to the plate. The cells were incubated for 24 h, and the serum-starved cells were treated with either Takinib or 13h in the presence or absence of TNFa (10 ng/ml). All samples and the control contained DMSO in a final concentration of 0.5%. After another 24 h of incubation, 100 mL of Apo-ONE V R caspase-3/7 reagent was added to each well, and the cells were incubated at room temperature in darkness for 2 h. Fluorescence was then measured at 530 nm with excitation at 485 nm using a FlexStation 3 Multi-Mode microplate reader (Molecular Devices). Statistical analyses of the data included a one-way ANOVA, followed by Tukey's multiple comparison test.

Results and discussion
Transforming the irreversible terminal acrylamide to 2-cyanoacrylamide was key for the synthesis of reversible derivatives. The synthetic route for the imidazopyridine derivatives is outlined in Scheme 1. Synthesis of target molecules commenced with nucleophilic addition of aminopyrrolidine to 7, which gave 8 in a moderate yield. The nitro compound (8) was reduced in the presence of Fe and acetic acid at 40 C to yield 9, which was followed by ring closure with 4-(4-methylpiperazin-1-yl)benzaldehyde to give imidazopyridine 10 24 . The Boc group was removed under acidic conditions, and a subsequent reaction with cyanoacetic acid provided the key intermediate (12). Target compounds 13a-s were obtained from 12 via Knoevenagel condensation with various aldehydes. Irreversible derivative 14 was synthesised by reacting 11 with acryloyl chloride at 0 C.
A structure-activity relationship (SAR) study was performed to optimise the R group on the 2-cyanoacrylamide moiety (Table 1). Phenyl derivative 13a had an IC 50 of 385 nM, which was $2.5-fold lower than the IC 50 of 14. Among the compounds with five-membered heterocycles (13b-d), the 4-methylthiazolyl derivative (13b) exhibited the highest potency. Conversion of the phenyl group (13a) to pyridine without a substituent afforded 13e, 13m, and 13q, which had activities that were 8-fold to 14-fold higher. Further SAR analysis was performed for substituted pyridine derivatives. The IC 50 values of the 2-pyridinyl derivatives increased with additional bulky substituents on the aromatic ring (e.g. 13g, 13i, and 13k) whereas small substituents (e.g. 13f and 13h) maintained or improved potency. Interestingly, the potency of the derivatives with methyl substituents (13h, 13j, and 13l) was excellent regardless of the methyl position. Unlike the 2-pyridinyl derivatives, the position of the methyl substituent affected the activity of 3-pyridinyl derivatives (13n-p). Introducing a substituent to the 4-pyridinyl group resulted in lower activity (13q-s). Covalent docking studies of the 13h and 14 with C174 of the TAK1 kinase domain were conducted. The free energies of binding were estimated to be À9.65 kcal/mol and À7.05 kcal/mol for 13h and 14, respectively. Imidazopyridine of both compounds formed two hydrogen bonds with A107 in the hinge region. The pyridinyl group of 13h occupied the back pocket of TAK1 and formed a hydrogen bonding with D175. These results correspond to the TAK1 inhibitory activity of 13h and 14 (Figure 3). A representative imidazopyridine derivative (13h) had an IC 50 of 27 nM for TAK1, but inhibition of other MAP kinases by 13h (1 mM) was as low as 10-15% (Table 2). Although limited, the kinase profile indicated that 13h was selective for TAK1 over other MAP kinases and BRAF.
To evaluate the reversible covalent properties of the described compounds, we reacted 13h with b-mercaptoethanol (BME) and  25 . Imidazopyridine core interacts with hinge region of TAK1 and pyridinyl nitrogen forms a hydrogen bond with D175. The estimated free energy of binding was found to be À9.65 kcal/mol and À7.05 kcal/mol for 13h and 14, respectively. Covalent docking study was performed using Autodock via flexible side chain method 26 . The figure was visualised using Discovery Studio 2020 Visualiser. 13k 132 a In-vitro enzymatic assay data obtained from Invitrogen TM . determined the reversibility of covalent adduct formation using a previously reported method (Scheme 2) 22 .
The reaction between 13h and BME generated a covalent adduct, which was identified via high-resolution mass spectrometry (HRMS, Figure S1). The BME adduct mixture was diluted 10fold to confirm that adduct formation was reversible. After dilution, the BME adduct gradually reverted to 13h ( Figure S1). TAK1 inhibition has been shown to induce the apoptosis of TNFa-stimulated breast cancer cells 11 . To assess its activity in a cell-based model, the caspase-3/7 activity of 13h was measured in MDA-MB-231 cells. Takinib, a potent TAK1 inhibitor 11 , was used as a positive control. Like Takinib, 13h (0.5 mM) induced significant caspase activation in the presence of TNFa, indicating that 13h strongly inhibited TAK1 in the cells (Figure 4).

Conclusions
We discovered potent imidazopyridine TAK1 inhibitors derived from 2-cyanoacrylamide-bearing pyrimidine derivatives. The introduction of the phenyl group into 2-cyanoacrylamide moiety led to increased activity. Among substituents of 2-cyanoacrylamide, pyridines exhibited better activity than the phenyl group or 5-membered heterocycles. These data indicated that aryl group of 2cyanoacrylamide should provide a contribution to the interaction with TAK1. We postulate that they will act as reversible covalent TAK1 inhibitors based on the reversible reaction between 13h and BME. Our results may contribute to the identification of novel kinase inhibitors or reversible covalent inhibitors.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This research was financially supported by the Advanced Research Center Program [2015R1A5 A1008958] of the Korean Government.