P38α Inhibitors Research Articles

The link between osteoporosis and frozen shoulder: exploring the therapeutic effect of TAK715 on reversing fibrosis and protecting against osteoporosis via the p38 MAPK signaling pathway

BackgroundThe global incidence of frozen shoulder (FS) (2% ~ 5%) and osteoporosis (OP) is high (9.1%-12.1%). Clinically, postmenopausal women are particularly at risk for both diseases. The main objective of this current research is to investigate the pathogenesis mechanism of FS and explore the connection between FS and OP.MethodsWe obtained FS and OP datasets from GEO and identified crosstalk genes. Following KEGG and GO enrich analysis, the p38 MAPK signaling pathway was focused and the specific p38α inhibitor TAK715 was screened out. We conducted flow cytometry, western blot, and PCR analyses to assess the treatment effect of TAK715 on FS synovium fibroblasts at different concentrations. Additionally, we employed SD rats to validate the treatment effects of TAK715 in vivo.ResultsTAK715 was useful in reversing fibrosis at the concentration of 1 μM, 5 μM and 10 μM. The unbalanced apoptosis process in frozen shoulder cell and the activation of osteoclast were inhibited at the concentration of 5 μM by TAK715. Then we successfully established a FS and OP rat model, with the FS with OP rat displaying less range of motion (ROM) and thicker shoulder capsule. In FS rat that was treated with TAK715, the frozen shoulder side was corrected in ROM and bone loss.ConclusionsThe frozen shoulder with osteoporosis may exhibit more severe symptoms, and TAK715 is effective in protecting fibrosis and osteoporosis both in vitro and vivo. The therapy to correct FS and OP simultaneously by TAK715 provides novel approach in FS treatment and study.

Design, synthesis, and cytotoxicity screening of novel pyrazolopyrimidines over renal cell carcinoma (UO-31 cells) as p38α inhibitors, and apoptotic cells inducing activities

A series of novel molecules with pyrazolopyrimidine-4-amine core were designed and synthesized as potential cytotoxic agents over Renal Cell Carcinoma cells (UO-31). Results of cytotoxic activity against UO-31 cells showed that pyrazolopyrimidines 19 and 31 were found to be more cytotoxic than sorafenib (SOR). The cytotoxic activity of these compounds appeared to correlate with their ability to inhibit p38α MAPK which are 2.53- and 2.27- folds more potent than SOR. Moreover, results of the cell cycle analysis as well as the results of annexin-V on the (UO-31) cells showed that pyrazolopyrimidines 19 and 31 had a pro-apoptotic activity higher than SOR by 1.42- and 1.20- folds, respectively. Furthermore, compounds 19 and 31 were found to be effective in arresting the cell cycle throughout the accumulation of the cells at G2/M phase. Finally, the tested compounds decreased the TNF concentration as well as increased the expression of tumor suppressor gene p53, Bax/BCL-2 ratio and caspase 3/7.

A small molecule p38α MAPK inhibitor, MW150, attenuates behavioral deficits and neuronal dysfunction in a mouse model of mixed amyloid and vascular pathologies

BackgroundInhibition of p38 alpha mitogen activated protein kinase (p38α) has shown great promise as a treatment for Alzheimer's disease (AD) in preclinical tests. However, previous preclinical studies were performed in “pure” models of AD pathology. A vast majority of AD patients have comorbid dementia-contributing pathologies, particularly some form of vascular damage. The present study therefore aimed to test the potential of p38α inhibition to address dysfunction in the context of comorbid amyloid and vascular pathologies. MethodsAn amyloid overexpressing mouse strain (5xFAD) was placed on an 8-week long diet to induce the hyperhomocysteinemia (HHcy) model of small vessel disease. Mice were treated with the brain-penetrant small molecule p38α inhibitor MW150 for the duration of the HHcy diet, and subsequently underwent behavioral, neuroimaging, electrophysiological, or biochemical/immunohistochemical analyses. ResultsMW150 successfully reduced behavioral impairment in the Morris Water Maze, corresponding with attenuation of synaptic loss, reduction in tau phosphorylation, and a partial normalization of electrophysiological parameters. No effect of MW150 was observed on the amyloid, vascular, or neuroinflammatory endpoints measured. ConclusionsThis study provides proof-of-principle that the inhibition of p38α is able to provide benefit even in the context of mixed pathological contributions to cognitive impairment. Interestingly, the benefit was mediated primarily via rescue of neuronal function without any direct effects on the primary pathologies. These data suggest a potential use for p38 inhibitors in the preservation of cognition across contexts, and in particular AD, either alone or as an adjunct to other AD therapies (i.e. anti-amyloid approaches). Future studies to delineate the precise neuronal pathways implicated in the benefit may help define other specific comorbid conditions amenable to this type of approach or suggest future refinement in pharmacological targeting.

A p38 MAP kinase inhibitor suppresses osteoclastogenesis and alleviates ovariectomy-induced bone loss through the inhibition of bone turnover

Inhibition of excessive osteoclastic activity is an efficient therapeutic strategy for many bone diseases induced by increased bone resorption, such as osteoporosis. BMS-582949, a clinical p38α inhibitor, is a promising drug in Phase II studies for treating rheumatoid arthritis. However, its function on bone resorption is largely unknown. In this study, we find that BMS-582949 represses RANKL-induced osteoclast differentiation in a dose-dependent manner. Moreover, BMS-582949 inhibits osteoclastic F-actin ring formation and osteoclast-specific gene expression. Mechanically, BMS-582949 treatment attenuates RANKL-mediated osteoclastogenesis through mitogen-activated protein kinases (MAPKs) and protein kinase B (AKT) signaling pathways without disturbing nuclear factor-κB (NF-κB) signaling. Interestingly, BMS-582949 impairs osteoclastic mitochondrial biogenesis and functions, such as oxidative phosphorylation (OXPHOS). Furthermore, BMS-582949 administration prevents bone loss in ovariectomized mouse mode by inhibiting both bone resorption and bone formation in vivo. Taken together, these findings indicate that BMS-582949 may be a potential and effective drug for the therapy of osteolytic diseases.

Pharmacokinetics and Tissue Distribution of Itampolin A following Intragastric and Intravenous Administration in Rats Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Itampolin A, a natural brominated tyrosine alkaloid isolated from the sponge Iotrochota purpurea, has been shown to have good inhibitory effects in lung cancer cells as a p38α inhibitor. A simple, sensitive, and reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been established, validated, and applied to the study of the pharmacokinetics and tissue distribution of itampolin A following intragastric and intravenous administration. Itampolin A and theophylline (internal standard, IS) were extracted by the simple protein precipitation technique using methanol as the precipitating solvent. Chromatographic separation was achieved by using the optimized mobile phase of a 0.1% formic acid aqueous solution and acetonitrile in the gradient elution mode. Itampolin A and IS were detected and quantified using positive electrospray ionization in the multiple reaction monitoring mode with transitions of m/z 863.9 → 569.1 for itampolin A and m/z 181.1 → 124.1 for IS, respectively. The assay exhibited a linear dynamic range of 1-1600 ng/mL for itampolin A in biological samples and the low limit of quantification was 1 ng/mL. Non-compartmental pharmacokinetic parameters indicated that itampolin A was well-absorbed into the systemic circulation and rapidly eliminated after administration. The apparent distribution volume of itampolin A was much higher after intragastric administration than that after intravenous administration. A tissue distribution study showed that itampolin A could be detected in different tissues and maintained a high concentration in the lung, which provided a material basis for its effective application in lung cancer. The pharmacokinetic process and tissue distribution characteristics of imtapolin A were expounded in this study, which can provide beneficial information for the further research and clinical application of itampolin A.

MAPK phosphatase 1 inhibition of p38α within lung myofibroblasts is essential for spontaneous fibrosis resolution.

Fibrosis following tissue injury is distinguished from normal repair by the accumulation of pathogenic and apoptosis-resistant myofibroblasts (MFs), which arise primarily by differentiation from resident fibroblasts. Endogenous molecular brakes that promote MF dedifferentiation and clearance during spontaneous resolution of experimental lung fibrosis may provide insights that could inform and improve the treatment of progressive pulmonary fibrosis in patients. MAPK phosphatase 1 (MKP1) influences the cellular phenotype and fate through precise and timely regulation of MAPK activity within various cell types and tissues, yet its role in lung fibroblasts and pulmonary fibrosis has not been explored. Using gain- and loss-of-function studies, we found that MKP1 promoted lung MF dedifferentiation and restored the sensitivity of these cells to apoptosis - effects determined to be mainly dependent on MKP1's dephosphorylation of p38α MAPK (p38α). Fibroblast-specific deletion of MKP1 following peak bleomycin-induced lung fibrosis largely abrogated its subsequent spontaneous resolution. Such resolution was restored by treating these transgenic mice with the p38α inhibitor VX-702. We conclude that MKP1 is a critical antifibrotic brake whose inhibition of pathogenic p38α in lung fibroblasts is necessary for fibrosis resolution following lung injury.

CHF6297: a novel potent and selective p38 MAPK inhibitor with robust anti-inflammatory activity and suitable for inhaled pulmonary administration as dry powder.

Inhibition of p38 mitogen-activated protein kinase (MAPKs) is a potential therapeutic approach for the treatment of acute and chronic pulmonary inflammatory conditions. Here, we report the in vitro and in vivo characterization of the anti-inflammatory effects of CHF6297, a novel potent and selective p38α inhibitor designed for inhalation delivery as a dry powder formulation. CHF6297 has been proven to inhibit p38α enzymatic activity with sub-nanomolar potency (IC50 = 0.14 ± 0.06nM), with >1,000-fold selectivity against p38γ and p38δ. In human peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharides (LPS), as well as in human bronchial epithelial cells (BEAS2B) stimulated with TNF-α or cigarette smoke extract (CSE), CHF6297 inhibited interleukin (IL)-8 release with low nanomolar potency. CHF6297 administered to rats by using a nose-only inhalation device as a micronized dry powder formulation blended with lactose dose-dependently inhibited the LPS-induced neutrophil influx in the bronchoalveolar lavage fluid (BALF). CHF6297 administered intratracheally to rats dose-dependently counteracted the IL-1β (0.3mg/kg)-induced neutrophil influx (ED50 = 0.22mg/kg) and increase in IL-6 levels (ED50 = 0.82mg/kg) in the BALF. In mice exposed to tobacco smoke (TS), CHF6297, administered intranasally (i.n.) for 4 days at 0.03 or 0.3mg/kg, dose-dependently inhibited the corticosteroid-resistant TS-induced neutrophil influx in the BALF. In a murine house dust mite (HDM) model of asthma exacerbated by influenza virus A (IAV) (H3N3), CHF6297 (0.1mg/kg, i.n.) significantly decreased airway neutrophilia compared to vehicle-treated IAV/HDM-challenged mice. When CHF6297, at a dose ineffective per se (0.03mg/kg), was added to budesonide, it augmented the anti-inflammatory effects of the steroid. Overall, CHF6297 effectively counteracted lung inflammation in experimental models where corticosteroids exhibit limited anti-inflammatory activity, suggesting a potential for the treatment of acute exacerbations associated with chronic obstructive pulmonary disease (COPD) and asthma, acute lung injury (ALI), and viral-induced hyperinflammation.

Structural basis of a redox-dependent conformational switch that regulates the stress kinase p38α

Many functional aspects of the protein kinase p38α have been illustrated by more than three hundred structures determined in the presence of reducing agents. These structures correspond to free forms and complexes with activators, substrates, and inhibitors. Here we report the conformation of an oxidized state with an intramolecular disulfide bond between Cys119 and Cys162 that is conserved in vertebrates. The structure of the oxidized state does not affect the conformation of the catalytic site, but alters the docking groove by partially unwinding and displacing the short αD helix due to the movement of Cys119 towards Cys162. The transition between oxidized and reduced conformations provides a mechanism for fine-tuning p38α activity as a function of redox conditions, beyond its activation loop phosphorylation. Moreover, the conformational equilibrium between these redox forms reveals an unexplored cleft for p38α inhibitor design that we describe in detail.

Anti-inflammatory effect of 3-fluorophenyl pyrimidinylimidazo[2,1-b]thiazole derivatives as p38α inhibitors

In the present work, the anti-inflammatory effect of 30 compounds containing 3-fluorophenyl pyrimidinylimidazo[2,1-b]thiazole was investigated. All final target compounds showed significant Inhibitory effect on p38α. P38α is considered one of the key kinases in the inflammatory process due to its regulatory effect on pro-inflammatory mediators. The final target compounds divided into four group based on the type of terminal moiety (amide and sulfonamide) and the linker between pyrimidine ring and terminal moiety (ethyl and propyl). Most compounds with terminal sulfonamide moiety and propyl linker between the sulfonamide and pyrimidine ring were the most potent among all synthesized final target compounds with sub-micromolar IC50s. Compound 24g (with p-Cl benzene sulfonamide and propyl linker) exhibited the highest activity over P38α with IC50 0.68 µM. All final target compounds were tested for their ability to inhibit nitric oxide release and prostaglandin E2 production. Compounds having amide terminal moiety with ethyl linker showed higher inhibitory activity for nitric oxide release and compound 21d exhibited the highest activity for nitric oxide release with IC50 1.21 µM. Compounds with terminal sulfonamide moiety and propyl linker showed the highest activity for inhibiting PGE2 production and compounds 24i and 24g had the lowest IC50s with value 0.87 and 0.89 µM, respectively. Compounds 21d, 22d and 24g were tested for their ability to inhibit over expression of iNOS, COX1, and COX2. In addition the ability of compounds 21d, 22d and 24g to inhibit inflammatory cytokines were determined. Finally molecular docking of the three compounds were performed on P38α crystal structure to expect their mode of binding.

Characterization of p38α autophosphorylation inhibitors that target the non-canonical activation pathway

p38α is a versatile protein kinase that can control numerous processes and plays important roles in the cellular responses to stress. Dysregulation of p38α signaling has been linked to several diseases including inflammation, immune disorders and cancer, suggesting that targeting p38α could be therapeutically beneficial. Over the last two decades, numerous p38α inhibitors have been developed, which showed promising effects in pre-clinical studies but results from clinical trials have been disappointing, fueling the interest in the generation of alternative mechanisms of p38α modulation. Here, we report the in silico identification of compounds that we refer to as non-canonical p38α inhibitors (NC-p38i). By combining biochemical and structural analyses, we show that NC-p38i efficiently inhibit p38α autophosphorylation but weakly affect the activity of the canonical pathway. Our results demonstrate how the structural plasticity of p38α can be leveraged to develop therapeutic opportunities targeting a subset of the functions regulated by this pathway.

Machine Learning Assisted Discovery of Novel p38α Inhibitors from Natural Products.

P38α, emerging as a hot spot for drug discovery, is a member of the mitogen- activated protein kinase (MAPK) family and plays a crucial role in regulating the production of inflammatory mediators. However, despite a massive number of highly potent molecules being reported and several under clinical trials, no p38α inhibitor has been approved yet. There is still demand to discover novel p38α to deal with the safety issue induced by off-target effects. In this study, we performed a machine learning-based virtual screening to identify p38α inhibitors from a natural products library, expecting to find novel drug lead scaffolds. Firstly, the training dataset was processed with similarity screening to fit the chemical space of the natural products library. Then, six classifiers were constructed by combing two sets of molecular features with three different machine learning algorithms. After model evaluation, the three best classifiers were used for virtual screening. Among the 15 compounds selected for experimental validation, picrasidine S was identified as a p38α inhibitor with the IC50 as 34.14 μM. Molecular docking was performed to predict the interaction mode of picrasidine S and p38α, indicating a specific hydrogen bond with Met109. This work provides a protocol and example for machine learning-assisted discovery of p38α inhibitor from natural products, as well as a novel lead scaffold represented by picrasidine S for further optimization and investigation.

First-in-class ultralong target residence time p38α inhibitors as a novel mitosis-targeted therapy for colorectal cancer

First-in-class ultralong target residence time p38α inhibitors as a novel mitosis-targeted therapy for colorectal cancer

First-in-class ultralong target residence time p38α inhibitors as a novel mitosis-targeted therapy for colorectal cancer

First-in-class ultralong target residence time p38α inhibitors as a novel mitosis-targeted therapy for colorectal cancer

Synthesis and Biological Activity of a VHL-Based PROTAC Specific for p38α.

We report a series of small molecule proteolysis-targeting chimeras (PROTACs) that target the protein kinase p38α for degradation. These PROTACs are based on a ligand of the VHL E3 ubiquitin ligase, which is linked to an ATP competitive inhibitor of p38α. We provide evidence that these compounds can induce the specific degradation of p38α, but not p38β and other related kinases, at nanomolar concentrations in several mammalian cell lines. We also show that the p38α-specific PROTACs are soluble in aqueous solutions and therefore suitable for their administration to mice. Systemic administration of the PROTACs induces p38α degradation only in the liver, probably due to the PROTAC becoming inactivated in that organ, but upon local administration the PROTACs induce p38α degradation in mammary tumors. Our compounds provide an alternative to traditional chemical inhibitors for targeting p38α signaling in cultured cells and in vivo.

Structure-based virtual screening for novel p38 MAPK inhibitors and a biological evaluation

Mitogen-activated protein kinases (MAPKs) are a group of serine-threonine protein kinases that can be activated by extracellular stimuli. MAPK14 (p38α) affects major disease processes, while inhibition of p38α has been shown to have potential therapeutic effects. Many inhibitors targeting p38α have entered clinical trials but have a long development cycle and severe side effects. We developed a multi-step receptor structure-based virtual screening method to screen potential bioactive molecules from SPECS and our MCDB libraries. Compound 10 was identified as a promising p38α inhibitor that may be used in the treatment of p38αMAPK pathway-related diseases, but corollary studies are warranted.

Preclinical and randomized clinical evaluation of the p38α kinase inhibitor neflamapimod for basal forebrain cholinergic degeneration

The endosome-associated GTPase Rab5 is a central player in the molecular mechanisms leading to degeneration of basal forebrain cholinergic neurons (BFCN), a long-standing target for drug development. As p38α is a Rab5 activator, we hypothesized that inhibition of this kinase holds potential as an approach to treat diseases associated with BFCN loss. Herein, we report that neflamapimod (oral small molecule p38α inhibitor) reduces Rab5 activity, reverses endosomal pathology, and restores the numbers and morphology of BFCNs in a mouse model that develops BFCN degeneration. We also report on the results of an exploratory (hypothesis-generating) phase 2a randomized double-blind 16-week placebo-controlled clinical trial (Clinical trial registration: NCT04001517/EudraCT #2019-001566-15) of neflamapimod in mild-to-moderate dementia with Lewy bodies (DLB), a disease in which BFCN degeneration is an important driver of disease expression. A total of 91 participants, all receiving background cholinesterase inhibitor therapy, were randomized 1:1 between neflamapimod 40 mg or matching placebo capsules (taken orally twice-daily if weight <80 kg or thrice-daily if weight >80 kg). Neflamapimod does not show an effect in the clinical study on the primary endpoint, a cognitive-test battery. On two secondary endpoints, a measure of functional mobility and a dementia rating-scale, improvements were seen that are consistent with an effect on BFCN function. Neflamapimod treatment is well-tolerated with no study drug associated treatment discontinuations. The combined preclinical and clinical observations inform on the validity of the Rab5-based pathogenic model of cholinergic degeneration and provide a foundation for confirmatory (hypothesis-testing) clinical evaluation of neflamapimod in DLB.

MKK6 deficiency promotes cardiac dysfunction through MKK3-p38γ/δ-mTOR hyperactivation.

Stress-activated p38 kinases control a plethora of functions, and their dysregulation has been linked to the development of steatosis, obesity, immune disorders, and cancer. Therefore, they have been identified as potential targets for novel therapeutic strategies. There are four p38 family members (p38α, p38β, p38γ, and p38δ) that are activated by MKK3 and MKK6. Here, we demonstrate that lack of MKK6 reduces the lifespan in mice. Longitudinal study of cardiac function in MKK6 KO mice showed that young mice develop cardiac hypertrophy which progresses to cardiac dilatation and fibrosis with age. Mechanistically, lack of MKK6 blunts p38α activation while causing MKK3-p38γ/δ hyperphosphorylation and increased mammalian target of rapamycin (mTOR) signaling, resulting in cardiac hypertrophy. Cardiac hypertrophy in MKK6 KO mice is reverted by knocking out either p38γ or p38δ or by inhibiting the mTOR pathway with rapamycin. In conclusion, we have identified a key role for the MKK3/6-p38γ/δ pathway in the development of cardiac hypertrophy, which has important implications for the clinical use of p38α inhibitors in the long-term treatment since they might result in cardiotoxicity.

Molecular Docking Studies of Spirostans as MAPK14 (P38α) Inhibitors and Their Potential Use against Cancer

Spirostans (SPs) are chemical products widely distributed in the plant kingdom; currently, they are studied by their medical applications. Cancer has a high incidence in humans; it reaches second place worldwide deaths. In molecular biology, it has been accepted that Mitogen-Activated Protein p38alpha Kinase (MAPK14 (p38α) is implicated in the regulation of cancer. This study aimed to identify SPs as potential MAPK14 (p38α) inhibitors. From a set of 133 modified SPs, SwissTargetPrediction platform, and molecular docking, it was obtained that 129 chemical structures had molecular interaction with the MAPK14 (p38α). From those molecules, 123 were bound to a specific inhibition site of MAPK14 (p38α), and 6 of the structures resulted in inhibitors similarly to minocycline and dasatinib. One SP had binding couple energy (BCE, kcal/mol) as that of fostamatinib. In addition, 115 modified SPs had better BCE than the minocycline but not as that using fostamatinib. The key amino acids (aa) for the protein kinase MAPK14 (p38α) inhibition were Arg 70, Asp 168, Lys 53, His 148, and Ile 145, at a different interaction level. The BCE was enhanced when the H atom was substituted in C-2, C-11, and C-17 SPs positions. Similarly, the αOH group at C-5 and C-6 upgraded BCE. Stereochemistry and substitution at C-3, C-12, and C-25 did not present significant differences (Kruskal-Wallis test, p &lt;0.05). From all this ensemble of results, it is foreseeable that the SPs can be an option for MAPK14 (p38α) inhibition, a key modulator in cancer processes.

Perturbation of p38α MAPK as a Novel Strategy to Effectively Sensitize Chronic Myeloid Leukemia Cells to Therapeutic BCR-ABL Inhibitors.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the presence of the BCR-ABL oncogene. Therapeutic regimens with tyrosine kinase inhibitors (TKIs) specifically targeting BCR-ABL have greatly improved overall survival of CML. However, drug intolerance and related toxicity remain. Combined therapy is effective in reducing drug magnitude while increasing therapeutic efficacy and, thus, lowers undesired adverse side effects. The p38 MAPK activity is critically linked to the pathogenesis of a number of diseases including hematopoietic diseases; however, the role of each isozyme in CML and TKI-mediated effects is still elusive. In this study, we used specific gene knockdown to clearly demonstrate that the deficiency of p38α greatly enhanced the therapeutic efficacy in growth suppression and cytotoxicity of TKIs, first-generation imatinib, and second generation dasatinib by approximately 2.5–3.0-fold in BCR-ABL-positive CML-derived leukemia K562 and KMB5 cells. Knockdown of p38β, which displays the most sequence similarity to p38α, exerted distinct and opposite effects on the TKI-mediated therapeutic efficacy. These results show the importance of isotype-specific intervention in enhancing the therapeutic efficacy of TKI. A highly specific p38α inhibitor, TAK715, also significantly enhanced the imatinib- and dasatinib-mediated therapeutic efficacy, supporting the feasibility of p38α deficiency in future clinic application. Taken together, our results demonstrated that p38α is a promising target for combined therapy with BCR-ABL-targeting tyrosine kinase inhibitors for future application to increase therapeutic efficacy.

Development of radioiodinated pyrimidinopyridone derivatives as targeted imaging probes of activated p38α for single photon emission computed tomography.

p38α, a member of the mitogen-activated protein kinase superfamily, is ubiquitously expressed in a variety of mammalian cells. Activated p38α induces inflammatory responses to external stimuli, suggesting that non-invasive detection of activated p38α would be valuable for diagnosing inflammatory diseases. For this purpose, we designed radiolabeled compounds [123I]2-IR and [123I]4-IR based on a potent p38α selective inhibitor R1487 for use with single photon emission computed tomography (SPECT). In this study, we used 125I instead of 123I due to its more usable radiochemical properties, synthesized [125I]2-IR and [125I]4-IR, and evaluated their effectiveness as activated p38α imaging probes. [123I]2-IR and [123I]4-IR were designed by introduction of a 123I atom at the 2- or 4-ositions of the phenoxy ring, preserving the pyrimidinopyridone structure of R1487. We synthesized 2-IR and 4-IR via a 7-step process. The inhibitory potencies of 2-IR, 4-IR, and p38α inhibitors were measured using an ADP-Glo™ kinase assay system. Radioiodination of 2-IR and 4-IR was performed via an organotin-radioiodine exchange reaction using the corresponding tributyltin precursors. Biodistributions were evaluated by determining radioactivity in tissues of interest after intravenous administration of [125I]2-IR and [125I]4-IR in normal ddY mice and turpentine oil-induced inflammation model mice. In vivo inhibition study was also performed in inflammation model mice after intravenous administration of [125I]4-IR with pretreatment of p38α inhibitors. We synthesized 2-IR and 4-IR at total yields of 17.5% and 19.2%, respectively. 4-IR had higher p38α inhibitory potency than 2-IR; both compounds were significantly less potent than R1487. [125I]2-IR and [125I]4-IR were successfully obtained from tributyltin precursors with high radiochemical yield (> 65%), purity (> 97%), and molar activity (~ 81GBq/µmol). [125I]4-IR showed high radioactivity accumulation in the inflamed tissue (7.0 ± 1.2%D/g), rapid delivery throughout the body, and rapid blood clearance, resulting in a high inflammation-to-blood ratio (6.2 ± 0.4) and a high inflammation-to-muscle ratio (5.2 ± 1.3) at 30min, while [125I]2-IR showed low radioactivity accumulation in inflamed tissue over the experimental period. Further, radioactivity accumulation in inflamed tissue after [125I]4-IR administration was significantly decreased by pretreatment with selective inhibitors. [123I]4-IR would be a promising imaging agent for detection of activated p38α.