Design and synthesis of 5-[(2-chloro-6-fluorophenyl)acetylamino]-3-(4-fluorophenyl)-4-(4-pyrimidinyl)isoxazole (AKP-001), a novel inhibitor of p38 MAP kinase with reduced side effects based on the antedrug concept

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Abstract

Inhibitors of p38 mitogen-activated protein (MAP) kinase, which are closely involved in the production of inflammatory cytokines, are considered promising curative drugs for chronic inflammatory disorders. However, there is also a growing concern regarding its systemic side effects. To reduce the occurrence of side effects, we have identified a novel p38 MAP kinase inhibitor that shows properties of an antedrug, which imparts its effect solely on the inflammatory site and is metabolically inactivated right after. We have designed isoxazole derivatives through the addition of a fresh interacting fourth site to the structure of the prototypical p38 MAP kinase inhibitor that harbors three point interactive sites. The derivative 26d (AKP-001) shows excellent p38 MAP kinase inhibitory activity and a high selectivity for various kinases. Its rapid metabolism has been confirmed in rats. Moreover, 26d has been shown to be effective in animal models of inflammatory bowel disease.

Introduction

Inflammatory bowel disease (IBD) is a collective name for intestinal disorders that include Crohn’s disease (CD) and ulcerative colitis (UC). These intestinal inflammatory and intractable diseases of unknown etiology are characterized by repetitive periods of activity and remission. The incidence of IBD has steadily increased in recent years, predominantly affecting young adults, thus resulting in the deterioration of their quality of life (QOL) during the acute period and forcing diet regulations even during remission. Treatment regimens for IBD include 5-aminosalicylic acid (5-ASA) formulations such as mesalazine,1 which is used mainly during the remission period, and steroids such as prednisolone, which is generally administered during the active period. In case of the former, however, a high dose is required to observe an effect, whereas the latter is associated with various side effects or may result in drug dependency. Tumor necrosis factor-α (TNF-α) inhibitors such as infliximab and adalimumab have been clinically used and have shown excellent results,2, 3 although these are relatively costly and their route of administration has been described as not very ideal. Based on these conditions, there is a need to develop a small molecule drug of novel function that shows excellent efficacy and reduced side effects.

Mitogen-activated protein kinases (MAPKs) represent a class of serine/threonine kinases that are widely distributed across different cell types and play an important role in various cell functions. Of these, p38 MAP kinase is involved in the signaling pathway that regulates cellular responses to cytokines or stress. Its activation is responsible for the formation of inflammatory cytokines such as TNF-α or interleukin 1β (IL-1β). Accordingly, the p38 MAPK inhibitor acts as a therapeutic drug for the treatment of chronic inflammatory diseases such as rheumatic arthritis (RA) by regulating the secretion of cytokines. Many inhibitors have been reported in literature. Based on its structure and interaction with p38 MAP kinase, these inhibitors can be divided into 3 types (Fig. 1).4 Compounds that competitively interact with the ATP-binding site are classified as prototypes (teardrop binders) such as SB203580,5 and linear binders such as VX-745,6, 7 and PH-797804.8, 9, 10 Teardrop binders carry a 5-membered heterocyclic ring as the nucleus and characteristically forms hydrogen bonds with Lys53. Extended binders, represented by BIRB796,11 undergo allosteric binding with a p38 MAP kinase that results in an inactive DFG-out conformation. p38 MAP kinase is involved not only in the production of inflammatory cytokines, but also in cell differentiation and proliferation, as well as cell-cycle regulation or apoptosis. It is ubiquitously expressed in various systems of the body and thus, long-term exposure to inhibitors may result in toxicity.12, 13 Although not all cases are class effects, adverse events such as hepatotoxicity, cardiac toxicity, and disorders involving the central nervous system have been reported, prompting authorities to terminate its development.14 Leftheris et al. have previously reported that p38 MAP kinase inhibitors contain a triazine structure that has a short duration of action, which suggests that its adverse effects can possibly be reduced.15 Recently, Pfizer released the p38 MAPKs inhalant drug, PF-03715455, for the treatment of respiratory diseases.16 Although the site of drug application was different, we agree with the study that minimizing the exposure of the rest of the body by using a drug with high clearance and low absorption is extremely imperative. Most anti-inflammatory drugs that have been designed with reduced side effects by limiting its site or period of action are steroids that are commonly used in the area of dermatology. After imparting its effect on the disease site, steroids are rapidly inactivated during metabolism. This reaction has served as the foundation of the concept of an antedrug.17 We believe that anti-inflammatory drugs showing the properties of an antedrug may be applicable to not only the skin or specific respiratory regions, but also to inflammatory bowel disease. Therefore, we have attempted to design p38 MAP kinase inhibitors that exclusively follows a topical action.

Section snippets

Design concept

The X-ray crystal structure of SB203580, which is a representative of the prototypical p38 MAP kinase inhibitor, has been previously reported (Fig. 2).18 SB203580 interacts with the ATP-binding site of p38 MAP kinase, and a hydrogen bond is formed between the nitrogen of the imidazole 3-position and Lys53. Its p-fluorophenyl group interacts with a lipophilic pocket (hydrophobic region I) consisting of Thr106 (gatekeeper residual group), Leu75, Leu86, and Leu104 through the formation of van der

Chemistry

Melting points were determined on a micro hot plate melting point apparatus of Mettler-Toledo (Central Processor: Mettler FP80HT, Hot Stage: Mettler FP82HT) and were uncorrected. Proton NMR (1H NMR) spectra were recorded on a JEOL JNM-ECP 400. Chemical shifts (δ) are expressed in parts per million using tetramethylsilane as the internal standard. Mass spectra (MS) were obtained on a Shimadzu GC/MS QP-5000 mass spectrometer system, with electrospray ionization methodology. Infrared spectra were

Chemistry

Although not fulfilling the conditions for the antedrug concept (Fig. 3), first, 5-phenylpropylpyrazole 6 was synthesized by using the procedure presented below to confirm the effect of the new fourth interaction site (Scheme 1). By the reaction of enamine 1 and hydrazine monohydrate, the pyrazole framework was constructed to generate 2,25 and then 3 was obtained through the reaction of pyrrolidine and formalin. After deprotonation of the 5-position using n-butyl lithium and introduction of the

Conclusions

In the present study, we have discovered a novel compound 26d using isoxazole as the nucleus and a p38 MAP kinase inhibitor of prototypical structure. The fourth interaction site and 5-position substituent, which effectively interacts with p38α MAP kinase, was discovered and an antedrug was designed with reduced side effects due to its rapid inactivation during metabolism. Compound 26d showed a high clearance in metabolic stability tests using human liver S9 cells. Furthermore, 26d underwent

Acknowledgments

We thank Katsuya Ueno (Cosmo Bio Co., Ltd) as a mediator of SB Drug Discovery in the biological testing. We are also grateful to Hiroshi Uchida and Makoto Okada for the assistance in the docking studies, Katsuyuki Keino and Akira Asagarasu for a comprehensive support of writing this paper (each of ASKA Pharmaceutical Co., Ltd).

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