Elsevier

Bioorganic & Medicinal Chemistry

Volume 26, Issue 22, 1 December 2018, Pages 5852-5869
Bioorganic & Medicinal Chemistry

Synthesis and in vitro evaluation of diverse heterocyclic diphenolic compounds as inhibitors of DYRK1A

https://doi.org/10.1016/j.bmc.2018.10.034Get rights and content

Abstract

Dual-specificity tyrosine phosphorylation-related kinase 1A (DYRK1A) is a dual-specificity protein kinase that catalyses phosphorylation and autophosphorylation. Higher DYRK1A expression correlates with cancer, in particular glioblastoma present within the brain. We report here the synthesis and biological evaluation of new heterocyclic diphenolic derivatives designed as novel DYRK1A inhibitors. The generation of these heterocycles such as benzimidazole, imidazole, naphthyridine, pyrazole-pyridines, bipyridine, and triazolopyrazines was made based on the structural modification of the lead DANDY and tested for their ability to inhibit DYRK1A. None of these derivatives showed significant DYRK1A inhibition but provide valuable knowledge around the importance of the 7-azaindole moiety. These data will be of use for developing further structure-activity relationship studies to improve the selective inhibition of DYRK1A.

Introduction

Protein kinases catalyse protein phosphorylation and are therefore linked to the occurrence of multiple diseases including neurodegenerative disorders and cancer.1, 2 Inhibitors targeting protein kinases are regarded as potential new therapeutic agents for researchers.3, 4 DYRK1A (dual-specificity tyrosine phosphorylation-related kinase 1A) is a dual-specificity protein kinase that catalyses not only autophosphorylation on its own tyrosine residue, but the phosphorylation of serine and threonine residues of its substrates.5 The DYRK1A gene is the only DYRK member (others including DYRK1B, DYRK2, DYRK3 and DYRK4) located on 21q22.2 of human chromosome 21,6 which encodes more than 300 annotated genes, one third of which are overexpressed in Down syndrome (DS).7 Recent studies indicated that the DYRK1A expression level is correlated with cancer. For example, an upregulation of over 30% of DYRK1A levels has been found in glioblastoma cells, a cancer that represents approximately 15% of brain tumours.8 It has been suggested that DYRK1A inhibition by DYRK1A inhibitors leads to cancer cell apoptosis.9 The development of novel DYRK1A inhibitors has consequently attracted considerable interest as new chemotherapeutic agents.

There are several compounds with inhibitory activity for DYRK1A. Some have been isolated from natural sources, with examples including harmine,10 variolins,11 meriolins12 and staurosporine.13, 14 All of these natural leads possess an indolocarbazole ring system. Purely synthetic inhibitors also exist, though they possess very similar structural features namely with the heterocyclic cores, which include imidazopyridine,15 imidazopyridazine,16 pyridoquinazoline,17 thiazoloquinazoline,18 pyrrolopyrimidines.9, 19 Recently discovered lead compounds DANDY (1) represent one chemotype of the most potent DYRK1A inhibitors.19 That study showed that variable hydroxy substitution gave potent compounds, including mono substitution (1a, IC50 = 23.1 nM) and dihydroxylation (1b, IC50 = 3.0 nM) among others. In our previous studies,9 we were able to improve potency without increasing phenyl substitution and thus generate a molecule that was more likely to cross the blood-brain barrier. Our previous studies also suggested that the Nsingle bondCsingle bondN moiety of DANDY plays an important role in maintaining DYRK1A inhibition.9, 20 It is still unclear if other heterocyclic cores with a similar arrangement are tolerated, or potentially provide a more potent scaffold. Herein, we present the synthesis of a new and diverse group of heterocyclic compounds. They correspond to the 7-azaindole DANDY by maintaining the characteristic diphenol moieties and dinitrogen functionality (shown in pink in Fig. 1). Different spatial arrangements of two nitrogen atoms were studied by designing heterocycles such as benzimidazole-2-amine 2, 2-acetamide 3, imidazole 4, naphthyridine 5, pyrazole-pyridines 6 and 7, bipyridine 8, and triazolopyrazines 9 and 10. Additionally, the 7-membered analogue 11 with an intrinsic diphenol has also been investigated. We decided to maintain single hydroxy substitution on the phenyl groups to improve drug-like properties and reduce toxicity associated with oxidation to the quinone. With these novel analogues in hand, we then explored their inhibitory activity against DYRK1A.

Section snippets

Results and discussion

We synthesised benzimidazole-2-amine 2 and benzimidazole-2-acetamide 3 starting from commercially available 2-fluoroaniline (12) (Scheme 1). The selectively brominated intermediate 13 was achieved by treatment with N-bromosuccinimide (NBS) in chloroform in 90% yield. The aniline 13 was then oxidised to nitro compound 14 in the presence of hydrogen peroxide in triflouroacetic acid (TFA) at 75 °C for 2 h. It was planned that the subsequent nucleophilic substitution could be achieved by treatment

Conclusions

We have presented a series of synthetic methods in this paper that have generated 12 novel compounds containing a variety of heterocyclic cores. All these novel compounds were assessed in a DYRK1A inhibition assay. However, none of these derivatives showed significant DYRK1A inhibition. Regardless, biological studies have provided us with the valuable knowledge that the 7-azaindole heterocyclic core shows more potent activity against DYRK1A than other heterocycles, even when maintaining the

General chemical synthesis details

Unless noted otherwise, commercially obtained reagents were used as purchased without further purification. Solvents for flash chromatography were distilled prior to use, or used as purchased for HPLC grade, with the eluent mixture reported as the volume/volume ratio (v/v). Unless otherwise stated, reactions were performed under an atmosphere of nitrogen. Flash chromatography was performed using Merck Kieselgel 60 (230–400 mesh) silica gel. Analytical thin-layer chromatography (TLC) was

General procedure A for the TBS protection

The compounds could be prepared according to the literature.40 To a solution of hydroxy substrate (1.0 equiv.) in anhydrous DMF (0.05 M) was added imidazole (2.5 or 5.0 equiv.) at 0 °C, followed by the addition of TBSCl (1.5 or 3.5 equiv.). The reaction mixture was allowed to warm to RT for 12 h. After completion monitored by TLC, the mixture was extracted with ethyl acetate (3 × 30 mL) and H2O (50 mL), the organic layers were dried over MgSO4 and concentrated in vacuo.

General procedure B for Suzuki coupling reaction

To a solution of aryl-halide (1.0 equiv.) in 1,4-dixoane (0.02 M) was added arylboronic acid (1.2 equiv.), aq. K2CO3 (2 M, 2.0 equiv.), and Pd(PPh3)4 (2 mol%), and the reaction was heated to 100–110 °C for 4 h under argon. After completion monitored by TLC, the reaction mixture was cooled to RT, solvent was removed under reduce pressure, and then partitioned between H2O and ethyl acetate. The aqueous layer was extracted with ethyl acetate, and the combined organic layers were dried over MgSO4

General procedure C for condensation reactions

The compounds ware prepared following a literature procedure.45, 46 To a solution of halogenated pyridine substrate (1.0 equiv.) in anhydrous toluene (0.05 M) was added n-BuLi (1.1 equiv., 2.5 M in hexane) dropwise at −78 °C, and the resulting mixture was stirred for 30 min. N, N-dimethylacetamide or DMF (1.5 equiv.) was added dropwise and the mixture was stirred for another 30 min. After completion, the reaction was quenched with sat. NH4Cl (aq.) and extracted with ethyl acetate (3 × 15 mL).

General procedure D for benzyl protection

The compounds were prepared following a literature procedure.48 To a solution of hydroxyaryl substrate (1.0 equiv., 1 mmol) in DMF (0.05 M) was added K2CO3 (1.5 equiv., 1.5 mmol), followed by the dropwise addition of benzyl bromide (1.2 equiv., 1.2 mmol) at RT, and the mixture was stirred at RT for 12 h. After completion monitored by TLC, the solvent was removed under reduced pressure, and the residue was washed with H2O (30 mL) and extracted with ethyl acetate (3 × 20 mL), the organic layers

General procedure E for aldol condensation

LDA formation: To a solution of N,N-diisopropylethylamine (1.3 equiv.) in anhydrous THF was added n-BuLi (1.2 equiv.) dropwise at −78 °C. After addition, the resulting mixture was stirred at −78 °C for 30 min before using.

To a solution of LDA (1.3 equiv.) in solvent (10 mL) was added ketone (1.1 equiv.) at −78 °C, and the resulting mixture was stirred for 30 min. To the mixture above was added a solution of aldehyde (1.0 equiv.) in the corresponding solvent (0.1 M) dropwise, after addition, the

General procedure F for the oxidation of alcohols

To a solution of alcohol substrate (1.0 equiv.) in CH2Cl2 (0.05 M) was added Dess-Martin periodinane (DMP) (1.5 equiv.), and the mixture was stirred at RT for 30 min. After completion monitored by TLC, the mixture was quenched with sat. NaHCO3 (aq.) (5 mL) and the resulted mixture was extracted with CH2Cl2 (3 × 10 mL), the organic layers were dried over MgSO4 and concentrated in vacuo.

General procedure G for the formation of pyrazoles

The compounds were prepared following a literature procedure.27 To a solution of 1,3-dione (1.0 equiv.) in MeOH (0.05 M) was added N2H4·H2O (64%, 2.0 equiv.), and the resulting mixture was stirred at RT for 48 h under nitrogen. After completion monitored by TLC, the solvent was removed under reduced pressure, and the residue was treated with H2O (10 mL) and extracted with ethyl acetate (3 × 10 mL). The combined organic layers were dried over MgSO4 and concentrated in vacuo.

General procedure H for the synthesis of hydrazones

The compounds were prepared following a literature procedure.31 To a solution of chloro-pyrazine substrate (1.0 equiv.) in ethanol (0.05 M) was added hydrazine monohydrate (1.1 equiv.). The resulting mixture was stirred at 80 °C for 8 h. After completion monitored by TLC, the reaction mixture was cooled to RT, and the precipitate obtained filtered, washed by ethanol and dried to get a pale yellow solid. To a solution of the crude intermediate (1.0 equiv.) in ethanol (0.05 M) was added

General procedure I for the oxidative heterocyclisation

The compounds were prepared following a literature procedure.32 To a solution of hydrazone (1.0 equiv.) in CH2Cl2 (0.05 M) was added PhI(OAc)2 (1.0 equiv.). The resulting mixture was stirred at RT for 12 h. After completion monitored by TLC, the solvent was removed under reduced pressure.

General procedure J for the synthesis of ethers

To a solution of 49 (1.0 equiv.) in toluene (0.05 M) was added KOH (3.0 equiv.) and 18-crown-6 (0.07 equiv.), followed by the addition of 50a-b (1.1 equiv.). The resulting mixture was stirred at 40 °C for 4 h. After completion monitored by TLC, the reaction mixture was washed with H2O and extracted with ethyl acetate (3 × 10 mL). The combined organic layers were dried over MgSO4 and concentrated in vacuo.

General procedure K for the benzyl hydrogenolysis

To a solution of benzyl protected substrates (1.0 equiv.) in MeOH (0.05 M) was added Pd/C (10 wt%) under nitrogen atmosphere, and the mixture was stirred at RT for 2 h under 1 atm of hydrogen. After completion monitored by TLC, the Pd-C was filtered through Celite®, and the solvent was removed under reduced pressure.

General procedure L for amide coupling reactions

The compounds were prepared following a literature procedure.57 To a solution of carboxylic acid (1.0 equiv.) in anhydrous CH2Cl2 (0.1 M) was added oxalyl chloride (1.5 equiv.) and DMF (0.1 equiv.) at 0 °C, and the resulting mixture was allowed to warm to RT for 2 h. Then the volatiles were concentrated under reduced pressure, and the residue was dissolved in anhydrous CH2Cl2 (0.1 M), followed by the addition of aniline (1.0 equiv.) and triethylamine (1.1 equiv.), and the resulting mixture was

General procedure M for the deprotection of TBS ethers

The compounds were prepared following a literature procedure.35 To a solution of TBS-protected substrate (1.0 equiv.) in THF (0.05 M) was added TBAF (1 M, 2.0 equiv.). The resulting mixture was stirred at RT for 30 min. After completion monitored by TLC, the solvent was removed in vacuo.

Purification of His-DYRK1a

Human Dyrk1A Kinase domain (126-490aa) with an N-terminal Histidine tag was expressed in E. coli BL21 (DE3) cells. 1 mL of a 10 mL O/N culture containing 50ug/ml kanamycin and 34 ug/mL chloramphenicol, was used to inoculate a 1 L of Luria-Bertani (LB) media supplemented with the same antibiotics. The culture was grown at 37 °C until an OD600 of 0.5 was reached, the temperature was then reduced to 18 °C. Expression was induced with the addition of 1 mM IPTG (isopropyl B-d-thiogalactoside) and

Kinase inhibition assay

Active DYRK1A was assayed in Tris buffer (50 mM Tris-HCl, pH 7.5) containing 0.1 mM EGTA, 15 mM DTT, MgAc/ATP cocktail (0.5 mM HEPES pH 7.4; 10 mM Mg(CH3COO)2; 0.1 mM ATP), [γ-32P]-ATP 100–300 cpm/pmol and test compounds diluted in deionised water. As substrate, Woodtide (50 µM, Genscript) was used in DYRK1A assay. The reaction was initiated with 1 ng/µL DYRK1A. The reaction mixture was incubated at 30 °C for 10 min. Reaction was stopped by pipetting 10 µL of the reaction mixture onto P81 paper

Acknowledgements

This work was supported by the National Health & Medical Research Council of Australia (APP1106145).

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