Abstract
IRAK4 kinase activity transduces signaling from multiple IL-1Rs and TLRs to regulate cytokines and chemokines implicated in inflammatory diseases. As such, there is high interest in identifying selective IRAK4 inhibitors for the treatment of these disorders. We previously reported the discovery of potent and selective dihydrobenzofuran inhibitors of IRAK4. Subsequent studies, however, showed inconsistent inhibition in disease-relevant pharmacodynamic models. Herein, we describe application of a human whole blood assay to the discovery of a series of benzolactam IRAK4 inhibitors. We identified potent molecule 19 that achieves robust in vivo inhibition of cytokines relevant to human disease.
Keywords: Benzolactam, IRAK4, Lupus, pyrazolopyrimidine
Identification of selective interleukin-1 receptor associated kinase 4 (IRAK4) inhibitors remains of significant interest due to their potential to intervene in immunological diseases.1−4 Specifically, IRAK4 forms a critical node in multiple proinflammatory signaling pathways via the myddosome complex.5 This complex transmits and amplifies the response of interleukin-1 receptor (IL-1R) family and toll-like receptors (TLRs, except for TLR3) to inflammatory cues.6,7 Upon activation, IRAK4 phosphorylates IRAK1 and triggers multiple signaling cascades leading to gene transcription and initiation of the inflammatory response. As part of the response, multiple disease-relevant cytokines are released including Interferon alpha (IFNα), tumor necrosis factor alpha (TNFα), and interleukin-6 (IL-6). Secretion of IFNα leads to upregulation of several inflammatory proteins, and patients with systemic lupus erythematosus (SLE) often show enhanced IFNα serum levels correlating with disease activity.8 IL-6 is also dysregulated in SLE, causing both systemic effects and local inflammation. Therefore, there is strong interest in IRAK4 inhibition to suppress this pathway.1,2,5
Recent IRAK4 inhibitors indicate a diversity of chemical matter, including isoquinoline PF-06650833 (1),9 aminopyrimidine BMS-986126 (2),10 and quinazoline (3)11 (Figure 1), with multiple small molecule inhibitors having entered into clinical trials for immunological diseases such as 1 and BAY1834845 for rheumatoid arthritis and psoriasis, respectively. Indeed, a recent evaluation of the literature highlights that the discovery of IRAK4 inhibitors continues to be an active area of research.12 We also recently reported the discovery and optimization of a series of selective fused biaryl inhibitors.13
Figure 1.
(A) Examples of recently disclosed IRAK4 inhibitors and (B) co-crystal structure of 4 with IRAK4 (1.8 Å, PDB ID 6O95).
These molecules afford potent and selective inhibitors but lack adequate oral bioavailability.13 The subsequent semisaturated dihydrobenzofurans provide more favorable physicochemical properties while maintaining potency and improving selectivity, leading to hydroxymethyl dihydrobenzofuran 4 (Figure 1), a highly potent, orally bioavailable IRAK4 inhibitor with dose-dependent reduction of IL-6 in a CpG (deoxycytidyl deoxyguanosine oligonucleotide)-induced mouse PK/PD model.13 Despite the initial success of 4, further profiling revealed inconsistent inhibition of multiple disease-relevant cytokines, suggesting suboptimal pathway suppression. Concomitantly, we developed a human whole blood (HWB) assay to directly assess the effect of molecules on both IL-6 and IFNα. This assay in particular captures the effects in plasmacytoid dendritic cells (pDCs), which are the most potent producers of IFNα in the blood.14
Profiling 4 in HWB showed only moderate and highly variable potency for both cytokines (Figure 2A, IL-6 IC50 460 nM ± 160 nM, IFNα IC50 400 nM ± 170 nM). Most disconcertingly, 4 demonstrated variable, suboptimal maximal inhibition (MI) for either cytokine (Figure 2B, IL-6 72% ± 7.4%, IFNα 56% ± 12%). Investigation of other dihydrobenzofurans revealed a trend for incomplete inhibition with moderate-to-low activity against IL-6 (Table S1). When we profiled other tool molecules and scaffolds, values for both showed improved consistency relative to 4 (Table S1).
Figure 2.
Comparison of 4 and 19 in HWB assays. (A) IC50 and (B) MI for IL-6 and INFα. Each point reflects at least two donors.
As the inconsistent pathway suppression was scaffold-dependent, we explored alternate cores, hypothesizing that modification would identify more potent IRAK4 inhibitors with improved universal cytokine suppression. In addition, we chose to focus on semisaturated scaffolds with low lipophilicity (LogD < 3) to maintain desired drug-like properties. Noticing the higher lipophilic ligand efficiency LLE (8.1 using LogD) of isoquinoline 1 and other previously assessed scaffolds13 relative to 4 (LLE = 6.6), we aimed to identify alternate scaffolds with improved LLE versus parent 5 (Table 1).
Table 1. Evaluation of Pyrazolopyrimidine IRAK4 Inhibitors with Semisaturated Scaffolds.
Data are geometric mean of at least two independent experiments ± standard deviation for the number (n) of experiments conducted.
LogD measured at pH 7.4.
Enantiomers with undetermined absolute stereochemistry.
Previous structure–activity relationships indicated the necessity of a hydrogen bond acceptor in the scaffold to engage in a water-mediated H-bond.13 We incorporated alternative functionality capable of either participating in such an interaction or displacing a conserved water molecule (Figure 1B). Indanone 6, which is roughly liponeutral to dihydrobenzofuran 5 and extends the H-bond acceptor functionality further from the core, resulted in a 5-fold loss in biochemical potency. Reduction of the ketone to secondary alcohols 7 and 8 revealed significant activity differences between the enantiomers. Lactam 9 performed similarly to ketone 6, and despite an approximately 4-fold loss in biochemical potency relative to 5, it was a more efficient inhibitor with an LLE of 6.3. Encouraged by this result, we evaluated six-membered lactam 10 and pyridone 11, which would present the carbonyl oxygen in the same vicinity.
Unfortunately, both compounds lost potency, resulting in decreased LLE. Sulfur-based scaffolds 12–14 showed a similar trend as the indanone and indanols. Sulfone 12 and one sulfoxide enantiomer (13) both had improved LLE (6.0 and 6.1, respectively) when compared with 5, while the other sulfoxide (14) lost nearly 100-fold potency. Finally, sultam 15 showed an encouraging level of potency with 19 nM biochemical potency and an LLE of 6.4.
From this survey, sultam 15, sulfoxide 13, sulfone 12, and lactam 9 stood out as efficient scaffolds based on LLE. Computational docking studies with 9 and 15 suggested that the N-methyl substituents pointed toward a solvent-exposed region of the binding pocket. From our previous work, we knew this vector could be used to optimize potency and physicochemical properties13 and evaluated several lactam N-substituents, as shown in Table 2.
Table 2. Solvent Front Optimization.
Data are geometric mean of at least two independent experiments ± standard deviation for the number (n) of experiments conducted.
H/M LM: predicted hepatic clearance in liver microsomes for human (H) and mouse (M). Reported in units of mL/min/kg.
H/M Hep: predicted hepatic clearance in hepatocytes for human (H) and mouse (M). Reported in units of mL/min/kg.
AB, apical-to-basolateral permeability as measured in Madin–Darby canine kidney (MDCK) cells. Reported in units of ×10–6 cm/s.
Transition to a high-throughput IL-6 HWB assay and installation of it in the screening cascade could then identify compounds with improved and consistent potency and MI. While the observed biochemical-to-HWB potency shifts generally correlated with human plasma protein binding, they could also reflect the effects of permeability, physicochemical properties, and solubility. Encouragingly, both 9 and 15 provided comparable or improved inhibition of IL-6 relative to 5, albeit with significant variability and low-to-moderate MI (Table 2). Replacing the lactam N-substituent with polar functionality (Table 2, compounds 16–19) was well tolerated and provided a path to improved and less variable biochemical and HWB potency.
Cyclic ether 16 and acyclic methyl ether 17 provided single-digit nanomolar biochemical potency and submicromolar HWB potency with MIs of 71% and 76%, respectively, but with poor metabolic stability. Tertiary alcohol 18 improved HWB potency (IL-6 IC50 = 0.19 μM, MI = 78%) and stability in mouse and human in vitro assays. However, 18 was only moderately permeable. Fluorinated tertiary alcohol 19 had a very similar potency profile to 18 (Ki = 1.2 nM, IL-6 IC50 = 0.16 μM) with improved permeability. Sultam 20, containing the same solvent front group was not nearly as potent as 19 and only showed modest potency improvements over methyl sultam 15. This data along with the moderate mouse in vitro metabolic stability of 20 encouraged us to focus our further optimization studies on the lactam scaffold (A) over the sultam (B).
At this point, we further evaluated compound 19 to ensure that the scaffold changes were having the desired effect on reproducibility and MI in the HWB assay. Encouragingly, 19 showed superior potency to 4 when assessed by IL-6, as described above, or IFNα, which repeated over multiple runs (Figure 2A). Lactam 19 also gave improved MI with less variability across the two cytokines (Figure 2B). Of critical importance is that the kinase selectivity of the scaffold was maintained relative to 6.13 Lactam 19 inhibits only 11 kinases at >70% of a total 221 kinases when assayed at 1 μM with >100-fold selectivity over the most potent off-target (Tables S3 and S4).
We obtained a 1.7 Å resolution X-ray cocrystal structure of 19 with IRAK4 (Figure 3). Consistent with the binding mode of the dihydrobenzofurans,13 the pyrazolopyrimidine amide binds in close proximity to gatekeeper residue Tyr262 and forms two hydrogen bonding interactions with the hinge region via the amide oxygen with the backbone NH of Met265 and the polarized CH of the pyrazole with the backbone carbonyl of Val263.
Figure 3.
X-ray cocrystal structure of lactam 19 (gold) bound to IRAK4 (green) (PDB ID 6UYA). Polar interactions between the ligand and protein are indicated by dashed lines.
The carbonyl of the lactam makes two hydrogen bond contacts to solvent molecules, including a water-mediated interaction with Asp272. As anticipated, the lactam N-substituent extends into a solvent-exposed region. A polarized CH engages the backbone carbonyl of Pro266 in a hydrogen-bonding interaction, which may contribute to the slight improvement in biochemical potency of 19 as compared to 18 (Table 3). Numerous van der Waals contacts to aliphatic residues, including I185, V200, and L318, further buttress 19 in the binding site. Having optimized the solvent front substituent, we turned our attention to the ATP ribose pocket. Incorporating extended ribose pocket groups further improved HWB potency. Difluoroethyl-piperazine 21 "afforded HWB IC50 of 81 nM without significantly compromising in vitro stability or permeability.
Table 3. Ribose Pocket Group Optimization.
Data are geometric mean of at least two independent experiments ± SD for the number (n) conducted.
H/M LM: predicted hepatic clearance in liver microsomes for human (H) or mouse (M). H/M Hep: predicted hepatic clearance in hepatocytes for human (H) or mouse (M). Both reported in units of mL/min/kg.
AB, apical-to-basolateral permeability as measured in Madin–Darby canine kidney (MDCK) cells. Reported in units of ×10–6 cm/s.
Calculated basic pKa (cpKaMB) computationally determined using Moka v.2.6.
An even more pronounced potency increase was realized with azetidinyl-piperidine 22 (IC50 = 26 nM), though with greatly compromised permeability. We hypothesized this was driven by the highly basic tertiary amine and evaluated analogs with tempered basicity (23–25). As predicted, difluoroazetidine 23 substantially lowered the pKa and restored permeability. This compound also lost 4-fold in IL-6 IC50 relative to 22 and was only predicted to be moderately stable in mouse based on in vitro assays, though. Oxetanyl-piperazine 24 similarly modulated basicity, though still poorly permeable. The nonbasic close analog difluorocyclobutyl-piperazine 25 balanced permeability and in vitro stability with HWB IC50 of 100 nM.
Based on these compound profiles, we evaluated the mouse in vivo pharmacokinetic profiles of lactams 19, 21, and 25 (Table 4). Compound 19 (1 mg/kg) had low plasma clearance, in good agreement with the hepatocyte stability assay and was 34% oral bioavailable. Despite stable-to-moderate stability in liver microsomes and hepatocytes, 21 and 25 displayed high plasma clearances in vivo (58 and 79 mL/min/kg). However, 19 showed consistent agreement between in vitro and in vivo clearance across species.
Table 4. PK Data for Relevant Examples.
| cmpd | species | PPB (%) | LM/Hepa (mL/min/kg) | CLPb (mL/min/kg) | VSSb (L/kg) | T1/2b (h) | AUC∞b (μM·h) | F%b |
|---|---|---|---|---|---|---|---|---|
| 19 | mouse | 94.2 | 14/21 | 29 | 2.2 | 1.9 | 1.2 | 34 |
| rat | 91.4 | <6.1/<10 | 9.2 | 0.95 | 1.6 | 3.8 | 31 | |
| dog | 77.6 | 8.6/<7.8 | 11.3 | 3.7 | 4.1 | 3.1 | 109 | |
| cyno | 66.8 | 8.1/<8.8 | 15.8 | 3.7 | 5.4 | 2.2 | 47 | |
| human | 72.4 | <3.9 /<6.2 | ||||||
| 21 | mouse | 91.3 | 29/21 | 58 | 0.96 | 0.18 | 0.5 | ND |
| 25 | mouse | 94.7 | 24/27 | 79 | 1.8 | 0.28 | 0.4 | ND |
LM: predicted hepatic clearance in liver microsomes for indicated species. Hep: predicted hepatic clearance in hepatocytes for indicated species.
Parameters were determined from i.v. (1 mg/kg) and p.o. (1 mg/kg) PK studies.
On the basis of robust and consistent HWB potency and bioavailability of 19, this molecule was advanced into further in vivo studies. Attenuation of disease-relevant cytokines was assessed upon stimulation of the TLR pathway via treatment with the TLR7/8 agonist resiquimod, also known as R848. Stimulation of the TLR on or within the antigen-presenting cells (e.g., dendritic cells) leads to cell activation via the myddosome complex.15 This is followed by induction of pro-inflammatory cytokines, chemokines, and the release of large amounts of type I interferons together with upregulation of costimulatory molecules. This model has previously been used to evaluate other IRAK4 small molecules inhibitors including previous tool molecule 4.16,17
In this in vivo model, six C57 black mice were dosed with either vehicle, a nanosuspension of 4, or a nanosuspension of 19 across a dose range 1 h prior to stimulation with R848. At 1 h post R848 stimulation, blood samples were obtained from the animals and cytokine levels were measured. Three prototypic proinflammatory cytokines measured in this study were IFNα, IL-6, and TNFα.18 Stimulation of IFNα leads to differentiation of monocytes into dendritic-like cells, induction of natural killer and natural killer T cells, and promotion of interferon gamma (IFNγ) production, among other roles.18 IL-6 has numerous functions, playing important roles in immune regulation and inflammation as well as autoimmune diseases. Finally, TNFα is another potent inflammatory mediator whose roles include induction of cytokine production, activation or expression of adhesion molecules, and growth stimulation.
Both 4 and 19 demonstrate dose-dependent increases in plasma concentration upon treatment (Figure 4A). In addition, all three cytokines increased upon treatment with R848 in vehicle-treated animals (Figure 4B; Figure S2 in the Supporting Information). Although mice showed a dose-dependent inhibition of IL-6 secretion compared with vehicle-treated animals (Figure S2), a greater discrepancy between the two compounds was shown for the other two disease-relevant cytokines. The mouse TNFα WB IC50 are within 2-fold (IC50 = 234 nM for 19 vs 425 nM for 4), though compound 19 showed pronounced and dose-dependent inhibition of TNFα at doses as low as 3 mg/kg, whereas compound 4 only achieved TNFα inhibition at 100 mg/kg (Figure S2 in the Supporting Information).
Figure 4.
Effect of compound 4 and 19 on the proinflammatory IFNα in an R848-induced mouse PD model. (A) Plasma concentration upon dosing a nanosuspension of either 4 or 19 in MCT, 2 h after compound treatment and 1 h after stimulation with R848. (B) IFNα levels as determined by ELISA, 2 h after compound treatment and 1 h after stimulation with R848.
A strong dose-dependent inhibition IFNα was also seen for lactam 19 at doses as low as 3 mg/kg (Figure 4B), while benzofuran 4 failed to show inhibition of IFNα across doses. The variable response of 4, despite compound exposure, recapitulates the findings of the HWB assay and confirms the decision to pursue alternate cores in order to ensure more robust and consistent inhibition. Together, these findings show that compound 19 is therefore a strong tool molecule for elucidation of the role of IRAK4 inhibition across disease modalities that would be affected for several key proinflammatory cytokines.
We previously identified a series of potent and selective dihydrobenzofurans that inhibited IRAK4 in a biochemical and cellular assay, were orally available, and showed inhibition of the cytokine IL-6 in a PK/PD experiment in a dose-dependent manner. However, this molecule was hampered by insufficient and variable inhibition of other cytokines of interest as demonstrated in our newly initiated HWB assay. As such, optimization of the scaffold was undertaken, which led to the discovery of the lactams presented herein. These molecules not only maintained excellent biochemical potency and selectivity but also showed robust inhibition in the HWB assay. Optimization of pharmacokinetic properties yielded 19, a highly desirable tool molecule. Compound 19 shows robust inhibition of multiple cytokines of interest in a TLR-challenge mouse model and provides a valuable tool for further in vivo experiments across disease models.
Experimental Procedures
A modular synthetic route was devised from key nitrobenzoate intermediate 26 to prepare a diverse array of benzolactam IRAK4 inhibitors (Scheme 1). Upon treatment of primary bromide 26 with a primary amine, the inhibitor core (27) was efficiently synthesized by a one-step SN2 displacement/lactam formation. The ribose pocket group was installed through a subsequent SNAr displacement of the aryl chloride. Nitro-arene 28 was then reduced to the corresponding aniline (29) under various conditions, and the pyrazolopyrimidine hinge binding element was readily installed under amide coupling conditions to furnish IRAK4 inhibitor 30.
Scheme 1. Synthesis of Benzolactam IRAK4 Inhibitors.
Reagents and conditions: (a) NH2R1, triethylamine, methanol (48%-quant.); (b) NHR2R3, potassium carbonate (63%-quant.); (c) tin(II) chloride dihydrate, or Fe, ammonium chloride, or H2, Pd/C (quant.); (d) 31, triethylamine, or 32, 7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 2,4,5-trimethylpyridine (13% quant.).
Acknowledgments
Crystal diffraction data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID (or 22-BM) beamline at the Advanced Photon Source, Argonne National Laboratory. SER-CAT is supported by its member institutions (see https://www.ser.aps.anl.gov/www.ser-cat.org/images/SER-CAT18_Member_list.jpg) and equipment grants (S10_RR25528 and S10_RR028976) from the National Institutes of Health.
Glossary
ABBREVIATIONS
- AB
apical-to-basolateral permeability
- cpKaMB
calculated most basic pKa
- CpG
TLR agonist deoxycytidyl deoxyguanosine oligodinucleotides;
- ELISA
enzyme-linked immunosorbent assay
- Hep
predicted hepatic clearance in hepatocytes
- HWB
human whole blood
- IFNα
interferon alpha
- IFNγ
interferon gamma
- IL-1R
interleukin-1 receptor
- IL-6
interleukin-6
- IRAK4
interleukin-1 receptor associated kinase 4
- LLE
lipophilic ligand efficiency
- LM
predicted hepatic clearance in liver microsomes
- MCT
0.5% methyl-cellulose/0.2% Tween-80
- MDCK
Madin–Darby canine kidney cells
- MI
maximal inhibition
- PBS
phosphate-buffered saline
- PD
pharmacodynamic
- PK
pharmacokinetic
- R848
TLR agonist resiquimod
- SLE
systemic lupus erythematosus
- TLR
toll-like receptor
- TNFα
tumor necrosis factor alpha
Supporting Information Available
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.9b00380.
Assay conditions, experimental procedures, and crystallography data (PDF)
Author Present Address
§ (K. D.) Takeda Bio Development Center Ltd., 9625 Towne Center Drive, San Diego, California 92121, United States.
Author Present Address
∥ (C.E.) Medimmune, Inc., 1 Medimmune Way, Gaithersburg, Maryland 20878, United States.
Author Present Address
⊥ (A.K.) 623 38th Ave. San Francisco, California 94121, United States.
Author Present Address
# (Y.R.) 836 Polaris Ave., Foster City, California 94404, United States.
Author Present Address
∇ (P.J.L.) Synthekine, Inc., 1700 Owens Street, Suite 500, San Francisco, California 94157, United States.
Author Present Address
○ (A.Z.) TRex Bio 863 Mitten Rd, Burlingame, California 94010, United States.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
The authors declare no competing financial interest.
Supplementary Material
References
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