Synthesis and evaluation of pyrazolo[3,4-b]pyridines and its structural analogues as TNF-α and IL-6 inhibitors

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Abstract

In the present article, we have synthesized three different series of pyrazolo[3,4-b]pyridines and their structural analogues using novel synthetic strategy involving one-pot condensation of 5,6-dihydro-4H-pyran-3-carbaldehyde/2-formyl-3,4,6-tri-O-methyl-d-glucal/chromone-3-carbaldehyde with heteroaromatic amines. All synthesized compounds were evaluated for their anti-inflammatory activity against TNF-α and IL-6. Out of 28 compounds screened, 40, 51, 52 and 56 exhibited promising activity against IL-6 with 60–65% inhibition at 10 μM concentration. Amongst these, 51, 52 and 56 showed potent IL-6 inhibitory activity with IC50’s of 0.2, 0.3 and 0.16 μM, respectively. Compound 56 was not cytotoxic in CCK-8 cells up to the concentration of >100 μM.

Graphical abstract

Three different series of pyrazolo[3,4-b]pyridines were synthesized and evaluated for their anti-inflammatory activity against TNF-α and IL-6. Several compounds showed promising IL-6 inhibitory activity, amongst which most potent analogue has IC50 0.16 μM and is not cytotoxic (IC50 >100 μM).

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Introduction

Proinflammatory cytokines are involved in the pathogenesis of a variety of autoimmune and inflammatory diseases. Interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) are two multifunctional proinflammatory cytokines involved in the pathogenesis of cardiovascular, neurodegenerational diseases and cancer through a series of cytokine signalling pathways.1, 2 Inhibition of release of cytokines has become a major focus of current drug discovery and development, and an important method for evaluating the bioactivity of drugs. The inhibition of cytokines, in particular TNF-α, has been successful in several clinical trials for the treatment of rheumatoid arthritis.3

The inflammatory process is characterized by the production of leukotrienes, histamine, bradykinin, and a variety of cytokines and chemokines by tissues and migrating cells. Amongst many cytokines responsible for regulating cellular physiology, TNF-α has been proven to play a dominant role both in normal immune function and in disturbances leading to autoimmune disease. Overexpression of TNF-α can lead to a variety of pathological conditions including rheumatoid arthritis (RA), multiple sclerosis, cachexia, sepsis, ulcerative colitis, congestive heart failure and Crohn’s disease. To date, much research has been directed towards the inhibition of TNF-α production, the antagonism of TNF-α and the shedding of TNF-α from the cell surface. Thus, TNF-α has received a considerable amount of attention as a molecular target for the treatment of diseases mentioned above.4

Amongst proinflammatory cytokines, interleukin-6 (IL-6) is considered to contribute to the initiation and extension of the inflammatory process. Interleukin-6 is a multifunctional cytokine produced by wide range of cells, usually at sites of tissue inflammation, and it regulates hepatic acute-phase response, the immune response, inflammation and haematopoiesis. It appears to be the central mediator in a range of inflammatory diseases, including end-stage renal disease and rheumatoid arthritis.5 Inhibition of IL-6 has not received desired attention in drug discovery.

Pyrazolo[3,4-b]pyridine class of compounds are reported to possess diverse range of biological activities. Misra et al. identified 4-substituted 1H-pyrazolo[3,4-b]pyridine derivatives (1, SQ-67563: IC50 0.11 μM) as a new class of cdk2 inhibitors.6 Lin et al. identified substituted pyrazolo[3,4-b]pyridine analogue 2 as a potent and selective cyclin-dependent kinase and cellular anti-proliferative inhibitor (IC50 0.7 nM) against CDK1/cyclin B.7 Witherington et al. discovered 6-heteroaryl-pyrazolo[3,4-b]pyridine 3 as a glycogen synthase kinase-3 (GSK-3) inhibitor (IC50 0.8 nM).8 Pyrazolo[3,4-b]pyridines are also reported to possess anti-microbial (4, Escherichia coli: IC50 22 μM; Candida albicans: IC50 18 μM),9, 10 anti-chagasic (5, Trypanosoma cruzi: IC50 1.9 μg/mL)11 and anti-leishmanial (6, Leishmania amazonensis: IC50 0.12 μM)12 activities. It has also been reported that pyrazolo[3,4-b]pyridine analogue, Y-25510 (7), stimulates production of IL-1α and IL-6 at the level of messenger RNA expression in cultured human monocytes as well as enhances in vivo production of IL-lα and IL-6 in mice.13, 14 Compound 8 showed potent p38α MAP kinase inhibitory activity (IC50 0.7 nM) and potent in vivo TNF-α inhibition (97% inhibition of LPS-induced TNF-α release in mice at 20 mg/kg po.).15

Pyrazolo[3,4-b]pyridines, 9 [IC50 15.7 nM (TNF-α), >1 μM (PDE-IV)] and 10 [IC50 27 nM (TNF-α), >1 μM (PDE-IV)], are reported to exhibit anti-inflammatory activity by inhibition of TNF-α and PDE-IV.16 Similarly biologically active pyrazolo[1,5-a]pyrimidines, 11 (non-steroidal anti-inflammatory drug)17 and 12 (COX-2 inhibitor)18, and isoxazolo[5,4-b]pyridine 13 (TACE inhibitor)19 are also reported (Fig. 1).

There are several reports on synthesis of pyrazolo-pyridines and pyrazolo-pyrimidines using different synthetic routes. Lavecchia et al. synthesized pyrazolo[3,4-b]pyridines via indirect iodination of 2-chloro-nicotinonitrile to yield 2-chloro-5-iodonicotinonitrile, which was cyclized with methyl hydrazine leading to 3-amino-5-iodopyrazolo[3,4-b]pyridine.20 Chebanov et al. synthesized pyrazolo[3,4-b]pyridines via refluxing 5-amino-3-methyl-1-phenylpyrazole with arylidenepyruvic acids.21 Jachak et al. synthesized pyrazolo[3,4-b]pyridines and pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidines via Friedlander condensation of 5-amino-pyrazole-4-carbaldehyde with various active methylene compounds.22, 23 Goda et al. in 2004 devised a novel route for synthesis of pyrazolo-pyridines starting from 1,3-diaryl chalcones and ketones to get substituted pyrazolo-pyridines.9 Zheng et al. reported one-pot conversion of 5-azidopyrazole-4-carboxaldehyde to pyrazolo[3,4-b]pyridines via diazo-transfer and subsequent Friedlander reaction.24 Apart from these reports, several other researchers published synthesis of substituted 1H-pyrazolo[3,4-b] pyridines and related skeletons.25, 26, 27, 28

Based on the interesting biological activity profile of 1H-pyrazolo[3,4-b]pyridines and as a part of our continuing efforts towards discovery of new class of compounds against different therapeutic areas, we have designed and synthesized different series of pyrazolo-pyridines and their structural analogues for evaluation of anti-inflammatory activity against TNF-α and IL-6.

Our dual interest in this work was to synthesize new series of hybrid structures containing heterocycle and a carbohydrate (d-glucal) moiety as recognition element, and to evaluate these against different therapeutic targets. Herein, we report synthesis of three different series of pyrazolo[3,4-b]pyridines and other hybrid heterocycles via novel short and efficient synthetic route.

Section snippets

Chemistry

Our initial aim in this work was to devise a short and efficient synthetic strategy for synthesis of hybrid structures containing heterocycle and a carbohydrate component. Based on literature reports on construction of heterocyclic skeletons using 3-alkoxyacroleins,29, 30 we designed synthetic strategy involving condensation of 3-alkoxyacrolein analogues with heterocyclic amines to get pyrazolo[3,4-b]pyridines via imine intermediate as shown in Fig. 2.

Based on this strategy, we visualized

Conclusion

In conclusion, three different series of pyrazolo[3,4-b]pyridines and other related structural analogues were synthesized using new, short and efficient synthetic strategy involving condensation of 3-alkoxyacroleins with different heterocyclic amines. From the activity results, we could conclude that only three analogues, 51, 52 and 56 (IC50 0.2, 0.3 and 0.16 μM, respectively), are the most promising compounds. Moreover, since compound 56 did not show cytotoxicity even up to 100 μM concentration,

General

Melting points were recorded on Labindia visual melting point apparatus. 1H NMR spectra were recorded on 300 MHz Bruker FT-NMR (Avance DPX300) spectrometer using tetramethylsilane as internal standard, and chemical shifts are reported in δ units. Mass spectra were recorded on either GCMS (Focus GC with TSQ II mass analyzer and thermoelectron) with auto sampler/direct injection (EI/CI) or LCMS (APCI/ ESI; Bruker Daltonics MicroTOF Q). HPLC purity was checked using Waters Alliance or Dionex Ultima

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