Discovery of potent anti-inflammatory 4-(4,5,6,7-tetrahydrofuro[3,2-c]pyridin-2-yl) pyrimidin-2-amines for use as Janus kinase inhibitors

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Abstract

The Janus kinase (JAK) family of tyrosine kinases has been proven to provide targeted immune modulation. Orally available JAK inhibitors have been used for the treatment of immune-mediated inflammatory diseases, such as rheumatoid arthritis (RA). Here, we report the design, synthesis and biological evaluation of 4-(4,5,6,7-tetrahydrofuro[3,2-c]pyridin-2-yl) pyrimidin-2-amino derivatives as JAK inhibitors. Systematic structure–activity relationship studies led to the discovery of compound 7j, which strongly inhibited the four isoforms of JAK kinases. Molecular modeling rationalized the importance of cyanoacetyl and phenylmorpholine moieties. The in vivo investigation indicated that compound 7j possessed favorable pharmacokinetic properties and displayed slightly better anti-inflammatory efficacy than tofacitinib at the same dosage. Accordingly, compound 7j was advanced into preclinical development.

Introduction

Janus kinases (JAKs) are members of a family of intracellular tyrosine kinases. JAKs are important in cytokine receptor-mediated signal transduction. JAK is considered a desirable target, due to the versatile JAK-STAT-PI3K-MAPK signal network.1, 2 JAK kinase family consists of four isoforms. JAK3 is confined to hematopoietic, myeloid, and lymphoid cells, while JAK1, JAK2 and TYK2 are ubiquitously expressed.3

Many JAK-dependent cytokines have been shown to contribute to inflammatory diseases, especially most efficacious in treating rheumatoid arthritis (RA).4, 5, 6, 7, 8 As a consequence, JAKs have received significant interest from academic and industrial researchers in the past decades. Dozens of small molecule JAK inhibitors have been approved or entered into clinical trials for treatment of inflammatory and autoimmune diseases (Fig. 1). These approved and late-stage clinical JAK inhibitors are divided into three classes and exemplified as follows: pan-JAK inhibitors (tofacitinib, 19 and peficitinib, 210), JAK1/2 inhibitors (baricitinib, 311 and filgotinib, 412) and selective JAK1 inhibitor (upadacitinib, 513). Overall, JAK1, JAK3, and Tyk2 have been indicated as efficacious targets for treating inflammatory diseases.14 JAK2 was thought to mediate side effects such as anemia and leukopenia.15 However, for tofacitinib-treatment groups, only small decreases in hemoglobin (1–3 g/dL) occurred in about 8% of patients, with no statistical difference versus placebo.5 In patients treated with baricitinib, mild thrombocytosis was instead observed despite the inhibition of erythropoietin receptor signaling via JAK2 inhibition.7 Isoform selectivity of some compounds in the kinase assay did not provide a significant cellular selectivity and a sufficient block downstream signaling in vivo.16, 17 Large gaps existed between exclusive enzymatic inhibition of some isoform and cellular potency.18 Importantly, clinical resistance to JAK inhibitors has been reported as point mutations or abnormal heterodimerization between JAK isoforms.19 Therefore, there is still great need to discover new classes of JAK inhibitors, with strengthened efficacy and improved safety. Herein, we report our preliminary results on this subject.

Recently, we have disclosed the identification of 4-(2-furanyl) pyrimidin-2-amines as JAK2 inhibitors (Fig. 2).20 A series containing a 4-(4,5,6,7-tetrahydrofuro[3,2-c]pyridin-2-yl) pyrimidin-2-amine scaffold were found to exhibit high potency. Simply switching the hydrogen atom on the 5-position of pyrimidine (6a) to a methyl group (6b) led to a significant increase in the JAK2 activity. We hypothesized that this methyl group occupied the hydrophobic pocket of the active site, which contributed to the enhanced binding. Therefore, to establish a comprehensive structure–activity relationship (SAR), we performed an extensive structural exploration based on this scaffold and pharmacophore. On the other hand, the present design of these new JAK inhibitors follows the principles of bioisosterism and cyclization, as Momelotinib (CYT387)21 as a start point. We now report the synthesis and pharmacological evaluation of these new 4-(4,5,6,7-tetrahydrofuro[3,2-c]pyridin-2-yl) pyrimidin-2-amine analogues as JAK inhibitors, of which the pharmacokinetic properties and in vivo anti-inflammatory efficacy of compound 7j was further evaluated.

Section snippets

Chemistry

The synthetic route of compounds 7 is outlined in Scheme 1, which is similar to that of 6b. The key intermediate 5-benzyl-4,5,6,7-tetrahydrofuro[3,2-c]pyridine (8) was readily prepared at a hectogram scale from feedstock furfural. Lithiation and trap with chlorotributyltin of 8 gave organostannane 9, which underwent a Pd-catalyzed Stille coupling with 2,4-dichloro-5-methyl pyrimidine to afford 10. Nucleophilic aromatic substitution (SNAr), with varied aniline concentration, under microwave

Conclusions

We have designed a series of novel 4-(4,5,6,7-tetrahydrofuro[3,2-c]pyridin-2-yl) pyrimidin-2-amine derivatives. In this study, a comprehensive SAR was conducted, including the JAK inhibitory activities and cellular anti-proliferative activities. The binding mode of the optimal compound 7j was rationalized by molecular docking. Both the cyanoacetyl and phenylmorpholine moieties were of critical importance for inhibitory activity. Furthermore, compound 7j exhibited favorable PK properties. The in

Experimental

All of the compounds were synthesized as shown in Scheme 1. Detailed procedures, characterization data, 1H NMR and 13C NMR spectra are presented in the Supplementary Material. JAK enzymatic inhibition assays20 and Pharmacokinetic evaluation in SD rats28 were performed as our previous procedures. Molecular modeling was performed using Glide 5.9 in Schrödinger 2013 suite and the highest scoring pose was visualized by PyMol.29

Conflict of interest

The authors have no conflicts of interest to declare.

Acknowledgments

This work was supported by the National Major Science and Technology Project of China (Innovation and Development of New Drugs, No. 2011ZX09401-008-07), the National Natural Science Foundation of China (No. 21402089, 201502063), and the self-determined research funds of CCNU from the colleges' basic research and operation of MOE (No. CCNU18TS009).

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    Present address: Nanjing Sanhome Pharmaceutical Co. Ltd.

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