Discovery and structural characterization of peficitinib (ASP015K) as a novel and potent JAK inhibitor

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Abstract

Janus kinases (JAKs) are considered promising targets for the treatment of autoimmune diseases including rheumatoid arthritis (RA) due to their important role in multiple cytokine receptor signaling pathways. Recently, several JAK inhibitors have been developed for the treatment of RA. Here, we describe the identification of the novel orally bioavailable JAK inhibitor 18, peficitinib (also known as ASP015K), which showed moderate selectivity for JAK3 over JAK1, JAK2, and TYK2 in enzyme assays. Chemical modification at the C4-position of lead compound 5 led to a large increase in JAK inhibitory activity and metabolic stability in liver microsomes. Furthermore, we determined the crystal structures of JAK1, JAK2, JAK3, and TYK2 in a complex with peficitinib, and revealed that the 1H-pyrrolo[2,3–b]pyridine-5-carboxamide scaffold of peficitinib forms triple hydrogen bonds with the hinge region. Interestingly, the binding modes of peficitinib in the ATP-binding pockets differed among JAK1, JAK2, JAK3, and TYK2. WaterMap analysis of the crystal structures suggests that unfavorable water molecules are the likely reason for the difference in orientation of the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide scaffold to the hinge region among JAKs.

Introduction

Rheumatoid arthritis (RA) is one of the most common chronic inflammatory autoimmune diseases; it targets synovial tissues and causes progressive joint disability.1 Over the past decades, disease-modifying anti-rheumatic drugs (DMARDs) like methotrexate (MTX), which is the anchor drug in RA therapy, have formed the basis of RA treatments. Several approved biological agents, including tumor necrosis factor antagonists, T-cell co-stimulation blockade and interleukin (IL)-6 receptor antagonists, have been shown to be effective in patients who had poor response to conventional DMARDs.1., 2., 3. However, the disadvantages of these current drugs, such as inconvenient routes of administration, side effects such as serious infection, and lack or loss of efficacy due to anti-drug antibody production, warrant additional treatment options for RA.1., 4. Therefore, there is still a significant unmet need for orally available small molecules with improved efficacy.5., 6.

JAKs are a family of cytoplasmic protein tyrosine kinases with four known members (JAK1, JAK2, JAK3, and TYK2) that are associated with various cytokine-mediated signal transduction pathways.7., 8., 9. Binding of the ligands to their corresponding receptors induces JAK activation and subsequent phosphorylation of the receptors. Subsequently, the activated JAKs phosphorylate signal transducers and activators of transcription (STAT) proteins, which form dimers, translocate to the nucleus, and promote cytokine-responsive gene expression.10., 11., 12.

In the JAK family, JAK3 is selectively expressed in hematopoietic cells, while the other three members are ubiquitously expressed.13., 14., 15. JAK3 is specifically associated with common γ-chain (γc) cytokines such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, which play key roles in T-cell differentiation, proliferation, and survival.7., 8., 9., 10., 11., 12. In addition, JAK3 knockout mice exhibit severe combined immunodeficiency but no other remarkable phenotypes.16., 17. JAK1 is also linked to the IL-2 receptor and regulates T cell proliferation in concert with JAK3. Additionally, JAK1 is associated with the signaling pathways of IL-6 and interferon (IFN)-γ for inflammatory responses in psoriasis and RA.7., 8., 9., 10., 11., 12. In contrast, JAK2 has the most characteristic profile of the four JAK kinases. JAK2 mediates signaling of hematopoietic growth factors such as erythropoietin (EPO) and thrombopoietin, and JAK2-knockout mice are embryonic lethal because of defective erythropoiesis.7., 11. Therefore, JAK2 inhibition may affect some essential functions regulated by EPO.18 TYK2 has been shown to regulate the cytokine-mediated signals of IFN-α, IL-12, IL-23, which are related to psoriasis and inflammatory bowel disease.19., 20.

To date, a number of JAK inhibitors have been developed for the treatment of RA.21., 22. Tofacitinib (compound 1, Fig. 1) was the first developed small molecule JAK inhibitor, and was approved in 2012 as a pan JAK inhibitor for the treatment of RA.23., 24. Most recently, the JAK1 and JAK2 inhibitor baricitinib (compound 2, Fig. 1) was approved in the EU as a monotherapy or in combination with MTX for patients with RA not responding adequately to one or more DMARDs.25 Further, specific JAK1 inhibitors filgotinib and upadacitinib (compound 3 and compound 4, respectively, Fig. 1) are currently being evaluated in clinical trials for RA.26., 27.

In our previous research, 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivative 5 was identified as a lead compound and a potent and moderately selective JAK3 inhibitor (Fig. 2).28 However, lead compound 5 showed poor metabolic stability in liver microsomes and in vivo Pharmacokinetic (PK) profiles due to high molecular lipophilicity. Additionally, docking calculations to JAK3 indicated that the pyrrolopyridine scaffold of 5 interacted with the hinge region of the ATP-binding site and that the C4-substituent of the pyrrolopyridine ring occupied the hydrophobic cavity.28

Here, we report the design, synthesis, and structural characterization of a novel JAK inhibitor with moderate selectivity for JAK3 inhibition, peficitinib (ASP015K), as alternative option for the treatment of RA.

Section snippets

Chemistry

1H-Pyrrolo[2,3-b]pyridine-5-carboxamide derivatives were synthesized as shown in Scheme 1. Compound 6 was prepared as previously described.28 Nucleophilic substitution of the chloride at the C4-position with several amines under microwave irradiation yielded the desired compounds 7a–c.

Aminoadamantane derivatives were synthesized as summarized in Scheme 2. 4-Aminoadamantan-1-ol 829 was protected with benzyl chloroformate (CbzCl). The diastereomers were chromatographically separated to obtain

In vitro structure-activity relationships (SARs)

As JAK3 and JAK1 collaboratively regulate the IL-2 signaling pathway, we evaluated our newly synthesized compounds for human JAK3 and JAK1 inhibitory activity. We also investigated JAK2 inhibitory activity because it is potentially associated with hematopoietic side effects such as anemia.31 Additionally, we also evaluated the compounds’ metabolic stability in rat liver microsomes.

As shown in Table 1, lead compound 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivative 5 showed potent JAK3

Conclusion

We investigated the effect of converting the C4-cyclohexane ring of lead compound 5 to a bridged ring, and found that the 2-adamantyl moiety played an important role in increasing JAK3 inhibitory activity. In addition, we examined the location at which a polar group is introduced to the adamantyl moiety using docking calculations for 7b for human JAK3. We found that introducing a hydroxyl group to the C5-position of the 2-adamantyl moiety led to improved metabolic stability, and 18

Chemistry

1H NMR spectra were measured using a Brucker DPX200, Avance 400, AV400M, Brucker Avance III HD500, or Varian VNS-400 spectrometer. Chemical shifts are expressed in δ units (ppm) using tetramethylsilane as an internal standard. Abbreviations of 1H NMR signal patterns are as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Mass spectra (MS) were recorded on Agilent 1100 LC/MSD, Thermo Fisher Scientific LCQ Advantage, or Waters UPLC/SQD. Electrospray ionization

Acknowledgements

The authors thank Dr. Kazuo Oda for evaluating the compounds, and Dr. Yukihiro Tateishi and Mr. Yusuke Tomimoto for providing the recombinant proteins. The authors also thank Mr. Hiroyuki Kaizawa, Dr. Hiroaki Yamagishi, and Dr. Tatsuya Niimi for their support in the preparation of this manuscript. In addition, the authors are grateful to the staff of the Division of Analytical Science Laboratories for conducting elemental analysis and spectral measurements.

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    Present address: Maruho Co., Ltd, Bldg. 5, Kyoto Research Park, 93 Awata-cho, Chudoji, Shimogyo-ku, Kyoto 600-8815, Japan.

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