Expert Opinion on Therapeutic Patents
ISSN: 1354-3776 (Print) 1744-7674 (Online) Journal homepage: http://www.tandfonline.com/loi/ietp20
Inhibitors of interleukin-1 receptor-associated kinase 4 (IRAK4): a patent review (2012-2015)
William Michael Seganish
To cite this article: William Michael Seganish (2016): Inhibitors of interleukin-1 receptor- associated kinase 4 (IRAK4): a patent review (2012-2015), Expert Opinion on Therapeutic Patents, DOI: 10.1080/13543776.2016.1202926
To link to this article: http://dx.doi.org/10.1080/13543776.2016.1202926Accepted author version posted online: 16 Jun 2016.
Published online: 16 Jun 2016.
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Inhibitors of interleukin-1 receptor-associated kinase 4 (IRAK4): a patent review (2012- 2015)
William Michael Seganish1*
1 Merck Sharp and Dohme Corp., 2015 Galloping Hill Rd, Kenilworth, NJ 07033 USA
Abstract
Introduction: IRAK4 plays a key role in both inflammation and oncology-related disorders. IRAK4 is located proximal to TLR/IL-1 receptors, and in preclinical studies, inhibits downstream signaling from these receptors. The development of novel small molecule inhibitors of this kinase has the potential to lead to new therapeutics to treat diseases such as rheumatoid arthritis, lupus, and lymphomas.
Areas Covered: The aim of this review is to summarize the recent patent literature (2012-2015) surrounding small molecule inhibitors of IRAK4. Both IRAK4 selective and dual IRAK4/IRAK1 inhibitors will be included. Specific examples of the chemical matter from each patent will be discussed, including any data that are presented for the examples highlighted. If data on compounds exemplified in the patents are available from other sources (peer reviewed literature, conference abstracts) it will be included as appropriate.
Expert Opinion: There are currently many examples of highly potent and kinase selective IRAK4 inhibitors and some have been tested in various in vivo disease models, demonstrating robust pre-clinical efficacy. Several compounds appear to have the “drug-like” properties to advance to the clinic, with Pfizer having already initiated several phase I studies. It seems likely that within the next several years, the efficacy of IRAK4 inhibition in a human disease state will be tested.
Keywords: IRAK4, inflammation, TLR signaling, IL-R signaling, serine-threonine kinase inhibitor, PF-06650833
Article highlights
IRAK4 is involved in MyD88 signaling cascades and is hypothesized to play a role in inflammation related disorders as well as in oncology.
Many IRAK4 inhibitors appear to have dual IRAK4/IRAK1 inhibition due to a high sequence homology between the two kinases, however it is not clear as to the biological relevance of selective IRAK4 versus dual inhibitors.
The development of IRAK4 kinase inhibitors has accelerated over the past few years with multiple companies now reporting very potent inhibitors that are active in cellular, whole blood and animal models of disease.
Although several IRAK4 inhibitors appear poised to enter human trials, only one is currently in the clinic: PF-06650833 which has multiple ongoing phase I trials.
1. Introduction
Interleukin-1 receptor associated kinase-4 (IRAK4) is a serine-threonine kinase that is known to have biologically important kinase activity 1. IRAK4, and all members of the family (including IRAK1, IRAK2 and IRAKM) facilitate key protein scaffolding and regulatory functions in various signaling pathways 2, 3. IRAK4 in particular, participates as a key mediator in the interleukin-1/Toll-like receptor (IL-1/TLR) signaling cascades 4, 5. The IL-1/TLR receptors play a vital role in the innate immune response to pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) 6. PAMPs include components of viral and bacterial particles that, when detected, can trigger an immune response. DAMPs include host components such as cytokines and cellular debris, but also can include external stimuli.
Signaling through IL-1R/TLR results in the activation of adaptor protein myeloid differentiation primary response gene 88 (MyD88) which recruits IRAK4 and IRAK1 to form a signaling complex. This complex then interacts with a series of kinases, adaptor proteins and ligases eventually resulting in the activation of the transcription factors kappa light chain enhancer of activated B cells (NF-B) and activator protein-1 (AP-1), ultimately inducing the generation of inflammatory cytokines 6-8. In normal homeostasis, the inflammatory response elicited by these molecular patterns is transient and fades after the stimuli are removed. However, several inflammatory diseases involve chronic stimulation of the inflammatory response which leads to significant damage to the surrounding tissue and resulting disease pathology. Indeed, defects in IL-1R and TLR signaling have been associated with various diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease and psoriasis 9-15. While biologic-based therapeutics have been validated for cytokine mediated inflammatory disorders 16, 17, and have identified the key role modulating IL-1/TLR pathways can have on
disease, opportunities exist for the development of novel, safe and orally bioavailable small molecule therapeutics that can effect these pathways.
The genetic evidence in support of a key role of IRAK4 in inflammation responses has been demonstrated with studies of IRAK4 kinase dead, knock-in mice. Mice possessing this genotype are resistant to joint inflammation in several rodent arthritis models 18. Additionally, a small IRAK4 deficient human population has been identified 19. These patients suffer from an increased susceptibility to bacterial infections at a young age. This susceptibility to infections is also observed in patients with autosomal recessive MyD88 mutations 20, making the clinical phenotype indistinguishable between the two conditions 21. It was originally thought that, with age, the susceptibility to infections decreased. However, a recent report indicates that IRAK4 deficient patients may still suffer from a susceptibility to infections, including staphylococcal skin infections, long past childhood 22. When peripheral blood monocyte cells (PBMCs) from IRAK4 deficient patients are stimulated with IL-1R/TLR agonists in vitro, the cells are found to have impaired responses to this stimulation 19. The genetic data in rodents and humans, as well as the proximal location of the kinase to the IL-1R/TLR receptor implies that inhibiting IRAK4 could be a viable strategy to modulate the production of inflammatory cytokines and cytokine induced pathologies.
Recent data suggest an additional potential indication for IRAK4 inhibitors is cancer. IRAK4 is proposed to play a role in lymphomas that contain a mutation in the MYD88 adaptor protein (L265P) 23. This mutation leads to a constitutively active signaling cascade that enhances the survival of the tumor cells. The role of IRAK1 and 4 in activated B cell-like subtype of diffuse large B cell lymphoma (ABC DLBCL) has been studied 23. Through IRAK1 kinase dead mutations, it was found that IRAK1 kinase activity was not required, although the kinase appears to play a structural role. A dual IRAK4/IRAK1 kinase inhibitor previously disclosed by Amgen 24, killed ABC DLBCL cell lines that possess the MYD88 mutation, but it did not affect cells without this mutation 23. A subsequent study using a highly potent and selective IRAK4
inhibitor showed a similar effect on the growth of ABC DLBCL cell lines in vitro as well as in tumor xenographs, and this effect may be synergistic with other B cell receptor signaling blockers (BTK, SYK, PI3Kδ inhibitors) 25. Taken together, this data indicate a promising therapeutic role for inhibition of IRAK4 in treating B cell malignancies.
2. Patent Application Evaluations
This review is organized alphabetically by assignee company name. In addition to evaluation of patent application publications, data from relevant selected peer reviewed literature and other sources are discussed as it pertains to the chemical matter presented in the patent literature. When kinase selectivity is discussed, “selectivity” specifically refers to biochemical kinase selectivity and may not necessarily represent functional (cellular, in vivo) selectivity. Finally, while the focus of this review is patent applications published between 2012 and 2015, selected patents from pre-2012 and 2016 will be covered when pertinent. For a more comprehensive review of IRAK4 chemical matter, the reader is directed to several review articles 26-28, as well as a recent report of IRAK4 inhibitors not explicitly covered in detail below
29.
2.1 Ares Trading/Merck KGaA
A series of pyrazole-pyrimidine-phenyl compounds (1) have been disclosed as IRAK inhibitors useful for the treatment of cancer and other diseases related to IRAK overexpression (Figure 1) 30. The majority of compounds presented possess an N-methyl pyrazole in the R2 position, although several other heterocycles (5- and 6-membered) appear to be tolerated.
Many of these compounds are reported to have IRAK4 IC50 values less than 100 nM in a radiochemical enzymatic assay. A range of functional groups appeared to be tolerated at the R1 position (compounds 2 and 3), enabling changes in size and polarity while retaining potent IRAK4 enzyme inhibition activity.
2.2 Aurigene Discovery Technologies
The first disclosure of IRAK4 chemical matter from Aurigene occurred in 2013 31. This publication describes a series of fused benzoheterocycle amides (4) (Figure 2). The fused heterocycle examples are exemplified by benzoxazole and benzothiazole, as in compound 5. Multiple compounds are described as having IRAK4 enzyme inhibition activity less than 50 nM. The vast majority of compounds possess a morpholino ring at the 2-position of the benzoheterocycle core. The biaryl ring system appeared tolerant to changes, although the 2- pyridyl pendant to the amide appeared to be preferred. No data on cellular activity or kinase selectivity are presented. However, compound 5 was evaluated in a lipopolysaccharide (LPS, a TLR4 agonist) rat model of inflammation. Compound 5 was dosed orally (30 mpk) and demonstrated a 53% reduction in tumor necrosis factor- (TNF-) levels compared to vehicle control, lower doses of compound 5 were not effective in lowering TNF- levels.
Several recent patent publications have emerged from Aurigene investigating aza- benzoheterocyle amides 32 and indazole amides (Figure 3) 33, 34 as inhibitors for IRAK kinases, in particular IRAK4. The azabenzoheterocycle examples include both benzoxazole (6) and benzothiazole (7) cores, similar to the previous disclosure 31. In this instance, additional substituents are explored at the 5-position of the benzoheterocycle ring, and many of the examples include an oxazole ring in place of the 2-pyridyl that was employed previously (cf. 5 and 7). Again, like the previous disclosure 31, the distal pyridine ring could accommodate changes including being replaced with non-aromatic heterocyclic rings.
The reported indazole amides 8-12 adopt the similar oxazole-pyridine amide fragment as the benzoheterocycle disclosures. Additionally, these compounds also share the disubstitution at the 5 and 6 positions of the heterocyclic ring system. Interestingly, two indazole analogs are reported, compounds 8 and 10 that have corresponding pro-drugs (9 and 11, respectively) exemplified in the patent application publication. Many of the compounds reported have activities less than 50 nM, although no additional data are presented. Recently, it has been
announced that Curis Inc. has entered into a collaboration, licensing and option agreement with Aurigene to develop and commercialize their IRAK4 inhibitors 35.
2.3 Bayer Pharma
A series of indazole amide IRAK4 inhibitors has been disclosed by Bayer Pharma for the treatment of proliferative and inflammatory diseases (Figure 4) 36. This patent application publication provides examples of benzo-fused heterocycles that possess structure similarity to the Aurigene compounds (Figures 2 and 3). Both the Aurigene and Bayer compounds possess a conserved amide; with the 2-pyridyl ring being exemplary in both series of compounds (cf. 5 and 13). While the Bayer patent also describes alternatives to this pyridyl ring, it is highly conserved; in particular the 6-trifluoromethyl substituted pyridine is present in many of the examples, including some of the most highly characterized analogs (compounds 13 and 14).
The vast majority of the compounds presented have IRAK4 IC50 values between 100 and 1000 nM. Most of the compounds were also evaluated in a cellular assay. In this assay, LPS was used to trigger TNF- release in THP-1 cells. Compounds were evaluated for their ability to inhibit this release. Potencies ranged from triple digit nanomolar to micromolar with most of the compounds falling into the micromolar range. The IC50 of compound 13 was 640 nM and compound 14 was 730 nM.
These two compounds were profiled in additional IRAK4 signaling dependent assays.
Compound 13 was evaluated in human PBMCs that were stimulated with LPS and it was found that TNF- production was inhibited at a concentration of 1 μM. NF-B signaling was also interrogated in hDLBCL cells using a lentiviral NF-B reporter construct. Inhibition of NF-B activity was measured for compound 14 and an IC50 of 1-10 μM was reported. Additionally these cell lines were used to evaluate the effects of IRAK4 inhibitors on IL-6 and 10 secretion. Data for compound 14 were presented that showed an IC50 of 1-10 μM for both IL-6 and IL-10.
Compound 13 was also evaluated in several in vivo models. In one, mice were dosed with compound 13 (p.o.) followed by LPS administration and the levels of several inflammatory cytokines were measured. In particular, TNF- levels were shown to be reduced in a dose dependent manner (reduction at 40 and 80 mpk). The effects of compound 13 on IL-1 mediated cytokine release were also evaluated. Mice were treated with compound 13 followed by IL-1 and, like the LPS stimulated animals, a dose dependent decrease in TNF- levels was observed. While cellular and in vivo work appears to have been conducted on this series of compounds, no data on kinase selectivity were presented.
2.4 Biogen IDEC
A report describing macrocyclic IRAK4 inhibitors for inflammation related disorders was published in 2014 (Figure 5) 37. These inhibitors possess a macrolactam embedding three aromatic rings. Aromatic rings 2 and 3, as shown in macrocyles 15 and 16 were held constant in the majority of the compounds with ring 2 being either an oxazole or thiazole ring and ring 3 being a pyridine. Biochemical potency (fluorescent based assay) was generally good with many inhibitors exhibiting IC50 values of less than 100 nM. Cellular data were also generated using A549 cells that were stimulated with IL-1 after incubation with an IRAK4 inhibitor. Levels of IL-6 were then quantified via ELISA to generate EC50 values. Macrocycles 15 and 16 both produced EC50 values in this assay of <1000 nM.
From the examples presented in the patent, it appears that substitution of ring 1 was well tolerated, and this ring could be optionally exchanged for various heterocycles. Additionally, as demonstrated with macrocycle 17, ring 1 could be removed completely, leaving only the two carbons that are part of the macrocycle. This change still resulted in a compound with <100 nM potency. However, the size of the macrocycle appears important as evidenced by macrocycle
18. This compound has one carbon excised from the ring relative to macrocycle 15 resulting in a
complete loss of potency. Although no data on kinase selectivity were presented, it would be interesting to compare the selectivity of these macrocyles to non-macrocyclic IRAK4 inhibitors.
A recent publication claims a series of indazole analogs as IRAK4 inhibitors (Figure 6) 38.
Attached to the indazole core are two additional heteroaromatic rings, with 2-pyridine and pyrimidine appearing in the most number of examples, such as compounds 19 and 20. A key structural feature appears to be a hydroxyl methyl group on the pyridine attached to the indazole N-1 position. This motif is present in almost all of the compounds. Additionally, the presence of a basic nitrogen on the left hand aromatic ring appears to be important as it is also present in all the examples. Using the same biochemical and cellular assays described previously in the macrocycle patent 37, many potent compounds (IC50 <100 nM) are reported.
Compounds were also profiled in several fibrosis assays 38. In mouse kidney fibroblasts, an “IRAK4 inhibitor” (not identified) demonstrated lowering of IL-6 levels in response to IL-1 or LPS. In human kidney fibroblasts, an “IRAK4 inhibitor” lowered levels of fibrotic and inflammatory gene transcripts in response to DAMPs, transforming growth factor beta (TGF-), or activated histone stimulation. Finally, several “IRAK4 inhibitors” were evaluated in mice subjected to ischemic kidney injury (IKI, a model of kidney fibrosis) 38. The inhibitors reduced the levels of profibrotic and inflammatory gene transcripts compared to vehicle control.
2.5 Bristol-Meyers Squibb Company
BMS has published a series of patents on a suite of closely related diaminopyridine analogs (Figure 7) 39-41. Many examples of potent (single digit nM IC50 value) compounds are provided. A benzothiazole ring was common at the 2-amino position and an isopropyl group appeared to be favored at the 4-amino position. Small aromatic heterocycles were tolerated at the 5-position, and substituents on these rings varied in molecular complexity, as in compounds 21 and 22, while still preserving potency.
A series of three subsequent publications disclosed a series of potent diaminopyrimidine amide inhibitors 42-44. Most of the compounds presented in these publications have IRAK4 IC50 values in the single digit nanomolar range. In contrast to the previous publications, the heterocycles at the 5-position of the pyridine ring have been replaced with an amide (Figure 8). A highly optimized amide with a tertiary alcohol and a chiral fluorine, as in aminopyrimidine 23 appear in many of the examples. However, as shown by compound 24, a simple ethyl amide was tolerated. Compound 24 also possesses a highly functionalized substitution on the 4-amino substituent of the pyridine ring. Fused heterocycles at the 2-amino positions are explored in one publication (compounds 23 and 24) 42, while another report describes 6-membered heteroaromatic rings (compounds 25, 26 and 27) 43. In both publications, single digit nanomolar IRAK4 inhibitors were reported. In compound 26, the tertiary hydroxyl group has been methylated with no loss of potency. Compound 27 is presented as an active compound, and there is an example of a phosphate prodrug version of this compound (attached via the tertiary hydroxyl group), although no data are reported. In addition to biochemical enzyme inhibition data, cellular data were also provided. Human PBMCs were incubated with IRAK4 inhibitors and stimulated with lipoteichoic acid (TLR2 agonist) and levels of IL-6 were measured to generate IC50 or EC50 values. Potency values < 100 nM could be achieved with modest cell shifts for the majority of the compounds tested.
A recent disclosure by BMS in the same series of diaminopyridine amides presents compounds that have 5-6-fused heterocycles directly attached to the 2-position of the pyridine core (Figure 9) 44. In particular, the examples cover indazole (28) and indole (29) rings connected via N-1 of the respective ring systems. This change could have implications for the kinase selectivity of these compounds. Like in the previous publications, compounds are uniformly potent in the enzyme inhibition assay (single digit nanomolar) as well as in a cell based assay (PBMCs), including several examples of compounds with minimal or no cellular potency shift (compounds 28 and 29).
No in vivo or kinase selectivity data are provided in any of the BMS patents.
2.6 Hoffmann-La Roche
Pyrazolopyrimidine and thienopyrimidine amides have been published as compounds that can modulate IRAK4 and/or IRAK1 (Figure 10) 45. Additional data are presented for several compounds indicating that they are also spleen tyrosine kinase (SYK) inhibitors.
2.7 Ligand Pharmaceuticals
A series of substituted 2-aminopyridine compounds has been published as IRAK4 inhibitors by Ligand Pharmaceuticals (Figure 11) 46. The majority of compounds have a benzothiazole ring at the 2-amino position of the pyridine core as in compounds 32 and 33. A range of substituents appears to be tolerated at the 4-pyridine position. These substituents include, for example, a methylene hydroxyl group (compound 32) and a much larger substituted piperizine ring (compound 33). An assay is described that details experimental conditions for performing both an IRAK4 and an IRAK1 enzyme inhibition assay. Additionally, a table of data for selected compounds is provided. However, it is not clear as to whether this data are IRAK4 or IRAK1 inhibition values. Compounds are described as having EC50 values of “A” or “B,” but “A” and “B” are not defined. It is unclear as to whether the compounds presented are selective IRAK1 or IRAK4 inhibitors or possibly dual inhibitors.
Ligand Pharmaceuticals has partnered with TG Therapeutics to develop their IRAK4 inhibitor compounds, with the current status indicating pre-clinical development 47. A joint poster was presented at the annual meeting of the American Association for Cancer Research (AACR)
48. Two compounds were described: LG0224912 (IRAK4 IC50 = 0.7 nM), and LG0250276 (IRAK4 IC50 = 2.7 nM), with LG0224912 having been previously presented at a 2011 meeting of the American College of Rheumatology 49. No structures have been disclosed for either compound, accordingly it is not clear if the compounds are related to those presented in the Ligand patent application publication 46. The recent AACR poster indicates that both compounds also inhibit IRAK1 and have selectivity against a “broad range of kinases.” The compounds had
anti-proliferative activity against ABC DLBCL lymphomas and could be combined with ibrutinib (BTK inhibitor) or TGR-1201 (PI3Kδ inhibitor) resulting in synergistic anti-proliferative effects. The poster also indicates that IND-enabling nonclinical toxicity studies are underway.
2.8 Merck KGaA
Merck KGaA has published several applications describing IRAK4 inhibitors. The first application contains a series of indazolyl triazoles (Figure 12) 50. The compounds all possess an indazole-triazole-phenyl core, with few examples of modifications to this core. The majority of the modifications are made on the phenyl ring, with amides being common (as in compounds 34 and 35), but aromatic and saturated ring systems in place of amides are disclosed. The compounds are reported to have dual IRAK4 and IRAK1 activity, with many compounds having a dual potency of <100 nM in the enzyme inhibition assays. Selected compounds were also profiled in a THP-1 cell assay. Cells were incubated with compound and stimulated with IL-1.
The inhibition of IL-8 secretion was quantified, with potencies in a similar range to the biochemical enzyme inhibition data. Additionally, several compounds were evaluated in human PBMCs that were stimulated with IL-1, monitoring IL-6 secretion. Again, multiple compounds appeared to have sub-micromolar activity in this assay. Compound 34 was further profiled in a murine model of inflammation. Mice were stimulated with LPS and the effect of inhibitor 34 on TNF- and IL-6 cytokine levels was measured. Data presented showed that inhibitor 34 demonstrated a dose dependent reduction in the levels of these cytokines (doses used were 10, 30 and 60 mpk). No data on kinase selectivity (other than IRAK1) were disclosed.
Merck KGaA has also reported a series of pyridazinone amides 51, and pyridazinone macrocyclic lactams (Figure 13) 52. Both structural classes are reported to be dual IRAK4/IRAK1 inhibitors. The pyridazinone amide publication has 17 examples, with only 4 compounds
reported to have dual IRAK4/IRAK1 activities <100 nM, with compound 36 being an example. Examples of substitutions are limited to the pyridine and benzimidazole ring.
Pyridazinone macrocyles are claimed that have dual IRAK4/IRAK1 potency 52. The macrocyles (e. g. 37 and 38) are structurally similar to the amides, with modifications again being confined to the benzimidazole and aryl/pyridyl ring. However, the size of the macrocycle could be increased by one atom (c. f. 37 and 38), with no apparent loss in dual potency. Several macrocyles (including 37 and 38) were evaluated in a PBMC assay where the cells were stimulated with a TLR7 agonist (not defined), and levels of IL-6 cytokine production were measured. Multiple compounds were reported to be active in this assay although precise IC50 values were not provided.
2.9 Merck Sharp & Dohme Corp.
Multiple patent application publications from Merck Sharp and Dohme Corp. describe an amidopyrazole scaffold (Figure 14) 53-55. In the initial publication 53, pyrazole N-1 aryls and heteroaryls appear preferred, and the amidopyrazole moiety is relatively unchanged. A significant number of examples display diversity at the 3-position of the pyrazole. A recent publication has identified compound 39 as an analog from the patent application that has potent IRAK4 enzyme inhibition activity and cellular activity (THP-1 cells stimulated with LPS) 56. When the compound was administered to mice that had been given the TLR2 agonist PAM2CSK4 (i. p., 100 mpk), it reduced the levels of several cytokines including TNF-, IL-1, IL-6 and IL-8.
Additionally, compound 39 was evaluated in a mouse antibody induced arthritis model and was found to statistically significantly reduce paw swelling. The compound was also reported to be selective against a panel of 108 kinases (only IRAK4 >80% inhibition @ 1 μM) 56.
Regioisomeric amidopyrazole 40 displays modest IRAK4 potency 54, while amidopyrazole 41 is one of 3 examples from a publication where structural changes are described that lead to an improvement of in vivo Cmax for amidopyrazole 41 over 39 (1 μM to 8.6
μM respectively). It is interesting to note that amidopyrazole 40 is similar to a series of potent IRAK4 inhibitors published by Astellas in 2011 (Figure 15) 57.
A series of diaminopyrimide58 and diaminopyrimidinone59 cores have also been published by Merck Sharp and Dohme Corp. (Figure 16). Many of the analogs described in these two publications have a unique cyclopentane amine moiety. IRAK4 enzyme inhibition values for many compounds are in the low nanomolar range. A subsequent journal article identifed diaminopyrimidine 43 from the above mentioned patent application publication as a compound of interest 60. This series was identifed as originating from a high throughput screening (HTS) hit. While compound 43 was potent against IRAK4, a kinase selectivity screen against 308 kinases showed the compound to be only modestly selective (57/308 kinases had
>80% inhibition @ 1 μM). An additional publication showed diaminopyrimidine 44 to have improved kinase selectivity (96% of 112 kinases tested had >100 fold selectivity for IRAK4) 61. Cellular data (THP-1 cells stimulated with LPS) were also presented 61. Additional compounds within the patent that have been functionalized on the aminocylopentane ring, such as compound 45, appear potent, but no additional data are available 58.
Compounds from the diaminopyrimidinone publication 59 share many of the same structural features as the diaminopyrimidines such as the benzothiazole and aminocyclopentane rings. However, compound 47 demonstrated that the aminocyclopentane could be modified and still preserve IRAK4 potency. Additional data on compounds 46 and 47 were disclosed in a separate publication 62. Cellular potencies (THP-1 cells stimulated with LPS and PBMCs stimulated with IL-1) are provided as well as kinase selectivity data. The selectivity data indicate that both compounds are selective against 96% of a panel of 111 kinases (>100 fold) with a key off target being FLT3. More potent compounds such as pyrimidinone 48 are described in the patent publication, but no additional data are provided.
2.10 Nimbus Therapeutics
Nimbus has been very active in publishing patent applications on a series of thienopyrimidines. Both the initial patent application 63, and a subsequent application cover a very similar set of compounds (Figure 17) 64. A later journal article states that the original hit for this series originated from a virtual screen of 1.3 million compounds from a commercially available library 25. This virtual screen was conducted using the published X-ray structure of IRAK4 65. All of the compounds presented in the above patent application have few modifications to the thienopyrimidine core and most changes occur on the periphery of the compounds. IRAK4 enzyme inhibition data are provided, however the lower range for the potency (<5000 nM) is too high to adequately judge the structure activity relationships (SAR). Selected compounds were also profiled in a THP-1 cellular assay, whereby the cells were stimulated with LPS and inhibition of TNF and IL-8 was monitored. Compounds 49 and 51 both are claimed to have activity <500 nM against the secretion of both cytokines. The 2013 application 64, as well as a journal article published in 2015 25 provide a significant amount of additional profiling data including more precise values for assay data as well as mouse pharmacokinetics (PK) on compounds 50 (also called ND-2110) and 51 (also called ND-2158)
25. This data set is summarized in Table 1.
In vitro kinase profiling data were published showing that both compounds 50 and 51 had selectivity against a broad range of kinases with key off targets being CLK1 and 2 and CDK8. Additional in vivo profiling of compound 51 demonstrated efficacy in a mouse collagen- induced arthritis model and a mouse inflammatory gout model 25. Compound 51 was also evaluated for its anti-proliferative activity. In ABC DLBCL cells, 51 was found to reduce the levels of inflammatory cytokines (IL-6 and 10) in vitro, and also reduced the growth of ABC DLBCL tumors in vivo 25, 64. Further studies showed that the antiproliferative activity of 51 was
synergistic with a BTK inhibitor (ibrutinib), SYK inhibitor (PRT062607), and BCL-2 inhibitor (ABT-199) 25.
More recent patent application publications from Nimbus Therapeutics have described similar structures with additional modifications to the core (Figure 18). Benzothienylpyrimidine compounds, such as compound 52 66, and thienylpyridine compounds (compound 53) have been reported 67. Benzothienylpyrimidine 52 was reported to be active in an enzyme inhibition assay, as well as a THP-1 cell assay (stimulation with LPS, monitoring secretion of TNF- and IL-8). Additional procedures are provided for testing compounds in whole blood and in a rat in vivo assay whereby LPS induces TNF- production, but no data for any compounds are provided. Thienylpyridine 53 is reported to have an IRAK4 enzyme inhibition potency of < 5000 nM (the lowest value reported in the publication). Out of 208 examples, enzyme inhibition data are only provided for 8 compounds. Combined with the relatively broad definition of potency (<5000 nM), it is difficult to assess the impact of this core modification on the overall activity of the compounds. Like the previous publication 66, assays are described (THP-1 cell data, whole blood, and in vivo cytokine measurements) but no data for any compounds are provided.
Another core that has been disclosed is exemplified by pyrrolopyrimidine 54 68. Selected compounds have IRAK4 enzyme inhibition activity presented, with the most potent being in the 100-1000 nM range (like compound 54). Although a potency range of <100 nM is defined, no compounds are identified as having this value. Similar to previous publications, additional assay protocols (THP-1, PMBCs, whole blood, and rat in vivo cytokine measurements) are provided with no data on compounds tested.
Two additional cores were published in 2015 by Nimbus Therapeutics: a thiazolopyrimidine (compound 55) 69 and a quinazoline (compound 56) 70. The thiazolopyrimidine compounds share many of the same structural features present in the more active compounds that Nimbus has disclosed (Figure 17 and Table 1). However, one of the
compounds profiled in greater detail in this publication is thiazolopyrimidine 55 that possesses an elaborated aminopyrazole ring. This compound is reported to be potent in the enzyme assay with good activity in the THP-1 (LPS induced TNF and IL-8), as well as whole blood activity (LPS induced TNF-). An additional statement is provided indicating that compounds (not defined) were assayed in a panel of 334 kinases and found to be “highly selective” for IRAK4 69. Thiazolopyrimidine 55 was also evaluated in rats stimulated with LPS and it was found that it inhibited TNF- production by 50% at 10 mpk (p.o., 1.05 μM plasma drug concentration).
A quinazoline core has been disclosed (compound 56) 70 and, like many of the other cores published by Nimbus Therapeutics, shares many peripheral structural features. The quinazoline 56 is reported to have IRAK4 enzyme inhibition activity of <5000 nM (lowest range disclosed in the patent). However the THP-1 cellular potency (LPS stimulated TNF- and IL-8) is reported to be <500 nM and the potency in human whole blood (LPS induced TNF) is reported to be less than 250 nM, although the actual potency shift in whole blood is unknown due to the lack of definition of the biochemical potency. Nevertheless, quinazoline 56 was also evaluated in rats stimulated with LPS and it was found that it inhibited TNF production with a “minimally efficacious dose” of 5 mpk (p.o.), although the definition of “minimally efficacious dose” is not provided. Assuming that this minimal efficacy is 50% inhibition of TNF- production (from the thiazolopyrimidine patent) 69, this represents a lower efficacious dose than observed for the thiazolopyrimidines (compound 55).
A recent press release has announced an exclusive worldwide licensing agreement with Genentech to discover and develop IRAK4 inhibitors 71. Financial terms have not been disclosed, but Genentech will be responsible for all preclinical and clinical development, manufacturing and commercialization.
2.11 Pfizer, Inc.
Pfizer has previously published a series of indoloquinoline IRAK4 inhibitors (such as 57, Figure 19) the core of which was discovered as an impurity in an HTS screen for novel IRAK4 inhibitors 72. In summary, while these compounds possessed potent IRAK4 enzyme inhibition and facilitated the development of an in vivo tool compound, a significant cell and whole blood shift, coupled to modest PK and off target liabilities limited the advancement of these compounds 72, 73.
A recent patent application publication has revealed a new class of quinoline and isoquinoline compounds (Figure 20) 74, and it was recently disclosed that these compounds originated from a fragment screen 75. Initial compounds presented in the patent application, such as quinoline 58, possess an oxygen linked aliphatic group at the 4-position of the quinoline ring, an isopropyl ether at the 6-position and a carboxamide at the 7-position. As the IRAK4 potency improved, both the ether and the carboxamide are retained. The ether side chain was also retained and optimized, to an un-substituted pyrrolidinone ring (compound 59), then to highly functionalized pyrrolidinone 60. Compound 60 has sub-nanomolar IRAK4 enzyme inhibition potency. Importantly, activity in the human PBMC (R848 induced TNF secretion) assay demonstrated single digit nanomolar potency with a cell shift of only 12 fold, compared to greater than 900 fold for compound 57. Another interesting point of SAR for this series is the change from quinoline (compound 58) to isoquinoline (compounds 59 and 60).
Isoquinoline 59 was evaluated in a mouse model of inflammation. Mice were stimulated with LPS and effects of compounds on the reduction of TNF- secretion were quantified.
Isoquinoline 59 elicited a dose dependent (10, 30, 100 mpk) decrease in TNF secretion. This compound was also evaluated in a rat collagen-induced arthritis model. At 10, 30 and 100 mpk
p.o. BID dosing (for 8 days); isoquinoline 59 demonstrated statistically significant dose dependent reductions in hind paw swelling at the conclusion of this study. An additional mouse in vivo inflammation study was conducted with isoquinoline 60. In this study, inflammation was
induced on the ear of the mice using imiquimod 76, and the effects of IRAK4 inhibitors were evaluated for their ability to reduce this swelling. After five days of treatment (100 mpk, p.o., BID) ear swelling had reduced by 51% in the IRAK4 inhibitor treated group. No additional data, pharmacokinetics, nor kinase selectivity is presented in the publication.
Pfizer recently presented data on PF-06650833 (compound 60) which has entered clinical trials 75. Optimization of this series led to a highly ligand efficient IRAK4 inhibitor with broad kinase selectivity. This compound possesses properties amenable to clinical development and as a result, is the only IRAK4 inhibitor currently in clinical trials.77 Two trials have been completed: a study of modified release formulations (NCT02609139) and a single ascending dose safety study (NCT02224651). One study is currently enrolling: a safety, tolerability and pharmacokinetic study of oral PF-06650833 in healthy subjects (NCT02485769). The current targeted indication is systemic lupus erythematosus (SLE) 78.
2.13 Takeda Pharmaceutical Company
Takeda has recently published a series of IRAK4 inhibitors (Figure 21) 79. These compounds bear structural similarity to those amidopyrazoles published by Astellas 57 and Merck Sharpe and Dohme 54. IRAK4 enzyme inhibition data are provided for selected compounds; however, data are presented as a percent inhibition at 1 μM, and most compounds have close to 100% inhibition at that concentration. It does appear that less elaborated compounds such as amidopyrazole 61 have weaker potency than more functionalized analogs 62 and 63. Despite the lack of clarity on the biochemical potency, amidopyrazoles 62 and 63 were both tested in vivo in a rat R848 induced cytokine production model. Compounds were administered (p.o.) 1 h before R848 injection and the resulting levels of TNF- were measured. Both compounds produced a dose dependent decrease in TNF- levels. Maximum effect was achieved with amidopyrazole 62 at 10 mpk which produced a 79% lowering of TNF- levels
compared to vehicle (1 and 3 mpk also produced TNF- lowering effects, but 0.3 mpk did not). Amidopyrazole 62 was also evaluated in a NCI-induced nephritis rat model (model of kidney inflammation). It was found that after 35 days of treatment (3 mpk, p.o., BID) the cohort treated with the IRAK4 inhibitor elicited a drop in proteinuria, possibly indicating less inflammation related kidney damage in the treatment group compared to vehicle control.
3. Conclusion
Over the past several years there have been a significant number of patent application publications reflecting a high interest in pursuing small molecule IRAK inhibitors. The potential therapeutic applications of these inhibitors can be hinted at by the data published in the respective patents, as well as in the peer reviewed literature. These therapeutic applications can range from inflammation related disorders in multiple organ types, to anti-fibrosis (these effects can be related to inflammation effects) and cancer. A substantial amount of pre-clinical data has been accumulated with multiple chemotypes in a variety of animal models. The next steps will be the demonstration of human clinical efficacy with a suitable IRAK4 inhibitor. At the time of this writing, Pfizer has already completed two phase I clinical trials with PF-06650833 and other companies may be preparing to enter the clinic.
4. Expert Opinion
Protein kinases have been investigated as therapeutic targets for decades, but the first approval of a drug did not occur until imatinib gained FDA approval for chronic myologenous leukemia in 2001. Since then the field has maintained a significant investment in novel kinase targets and inhibitors as therapeutics 80-82. The scope of diseases has expanded significantly to include non-oncology indications including inflammation, hypertension and Parkinson’s disease
81.
There has been significant advancement in the development of novel IRAK4 inhibitors. Initially reported compounds displayed modest potency and selectivity. The available chemical
matter has evolved to generate multiple diverse chemotypes of potent inhibitors (sub-nanomolar IC50 values) that have excellent kinase selectivities. The sources of these compounds have been equally diverse from range from traditional high throughput screening hits 60, to structure based virtual screening 25, to fragment based drug design 75. Additionally these compounds have evolved to have low nanomolar IC50 values in cellular and whole blood assays, and many have now been published that have efficacy in inflammation, oncology and/or anti-fibrotic models. The pharmacokinetic properties of several series of compounds have matured to the standpoint where several companies including Ligand Pharmaceuticals/TG Therapeutics and Nimbus Therapeutics/Genentech are claiming to be preparing to enter clinical trials. Pfizer has already begun clinical trials with their selective IRAK4 inhibitor PF-06650833. These accomplishments point toward the most important data set that has yet to be attained – clinical validation of a selective IRAK4 inhibitor in a human patient population.
Two areas of research that require future exploration are: (1) the value of selective IRAK4 inhibitors versus dual IRAK4/1 inhibitors and, (2) potential species differences in IRAK4 signaling. Multiple patent applications claim compounds to be dual inhibitors, with others claiming selective IRAK4 inhibitors. The advanced compounds disclosed by Ligand/TG appear to be dual inhibitors (<10 fold selective), while the Nimbus Therapeutics compounds may be 300-1000 fold selective for IRAK4 over IRAK1. While some studies have shown that IRAK1 kinase activity is not necessary for signaling in certain indications 23, IRAK1 kinase activity does appear important in other signaling pathways 83. Additionally, it has been reported by Chiang et al. 84 that there are species and cell type differences in the requirements for IRAK4 and IRAK1 scaffolding versus kinase activity, with some cell types (B-cell, T-cell, plasmacytoid dendritic and myeloid cells) in rodents having a requirement for both scaffolding and kinase activity.
Conversely, signaling in some human cells may not be dependent on IRAK1 nor IRAK4 kinase activity (B-cell, T-cell, and myeloid cells), but sometimes dependent on both (plasmacytoid dendritic cells).84 Cushing et al. have shown IRAK4 kinase activity to be important in thegeneration of IL-6 and TNF in human monocytes, but not dermal fibroblasts, highlighting a difference in signaling between human cell types 85. However, the effects on monocytes are in contrast to those published by Chiang et al.84 where inhibition of IRAK4 in human monocytes did not impact cytokine production. This difference in signaling effects may be due to the difference in potency of the IRAK4 inhibitors used in the two studies 85.
A recent report studied the differences between human and mouse TLR signaling in macrophages using siRNA 86. Knockdown of mouse IRAK4 showed robust impairment of TLR signaling, whereas in human macrophages knockdown of IRAK4 to similar levels as in the mouse (<20%), only weakly affected TLR signaling. Additionally, knockdown of IRAK1 had little effect on TLR signaling in murine macrophages, whereas in human macrophages, significant impairment of TLR signaling occurred 86. Based on the data presented in these studies, the true test of efficacy of a selective IRAK4 inhibitor will need to be demonstrated in human patients in an appropriate disease setting.
Pfizer has indicated its clinical trials are aimed at SLE, the most common form of lupus. In order to treat this chronic inflammatory condition, it would be expected that an IRAK4 inhibitor would have uniformly high kinase selectivity, which was one of the greatest obstacles to early IRAK4 kinase inhibitors 26-28. Indeed the first kinase inhibitor approved for a non-oncology indication (RA) 87 was developed by Pfizer (tofacitinib, JAK3 inhibitor), and is highly selective against a panel of kinases 88. Although the exact kinase selectivity of PF-06650833 has not been disclosed, it would be anticipated to be at least as good as tofacitinib. However, the pursuit of an oncology indication (lymphoma) with an IRAK4 inhibitor could lower the threshold for selectivity. This could be the strategy of Ligand/TG as presentations on their IRAK4 inhibitor compounds indicated that kinase selectivity could be an issue. Although Nimbus Therapeutics has published data indicating that their compounds are efficacious in treating B cell malignancies, disclosed kinase selectivity data seem to indicate that the compounds ND-2110 and ND-2158 have good selectivity against a panel of 334 kinases 25. This would imply thatthese compounds could have potential in the treatment of chronic inflammatory conditions. Regardless, it appears that several companies may be poised to test the value of IRAK4 inhibition in patients.
The clinical data generated over the next several years will be critical for determining the future of IRAK4 inhibitors, but it seems likely that compounds have now been identified that can properly evaluate the value of targeting IRAK4 for treating human disease.
Declaration of interest
WM Seganish is an employee of Merck Sharp and Dohme Corp. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
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