3.1.1 Embryo development function of p38

P38MAPK is important in the regulation of inner cell mass cells, especially in the differentiation of primitive endoderm. It is closely related to ribosome-related gene expression, rRNA precursor processing, polymer formation and protein translation regulation. Bora et al.⁵⁴ found that DDX21 protein was dependent on active p38MAPK for rRNA processing regulation, and became the only nucleolus during blastocyst maturation. In addition, studies have shown that p38MAPK provides a permissible translation environment during the predifferentiation of mouse blastocysts.⁵⁵

3.1.2 Immune response function of p38

T lymphocytes are the most important part in the immune response, and they are activated by TCR, co-stimulatory signaling, and immunomodulatory cytokines such as Interleukin (IL)-1/2 and IL-4. P38-MSK1/2-cAMP response element-binding protein/activating transcription factor 1 (ATF1) signaling pathways can be used to restrict Toll-like receptors-induced inflammation.⁵⁶ In addition, many cytokines such as IL-12 and IL-4 are regulated by p38MAPK. When Maeyer et al.⁵⁷ studied the immune response of the elderly; it was found that the decrease of T-cell immunoglobulin and mucin domain containing 4 (TIM-4) in the elderly was caused by the increase of p38MAPK activity in macrophages. Oral administration of active p38 inhibitors in older adults can reduce TIM-4 reduction. This suggests that p38MAPK plays an important role in cellular immune response.

3.1.3 Cell cycle function of p38

The p38 signaling pathway regulates the cell cycle by influencing the timing of cell cycle entry and cell cycle checkpoint stop. However, p38 plays a more important role in the regulation of inflammation, so as a part of cellular stress response, it tends to act as a “brake” to inhibit cell cycle transition.⁵⁸ However, Whitaker et al.⁵⁹ confirmed that p38 affected G1 and G2 phases. The tumor suppressor mechanism for G1 mediated by p38 includes cyclin-dependent kinases (CDK)—retinoblastoma suppressor protein—Early 2 factor pathway phosphorylation of several proteins, stress-activated p38 also prevents G2 phase cell transition to M phase cell cycle through the checkpoint mechanism of G2 phase, leading to G2 phase cycle arrest.

3.1.4 Cell differentiation function of p38

Multiple experiments have shown that p38MAPK also plays an important role in cell differentiation, for example, Weng et al.⁶⁰ demonstrated that amiodarone regulates cell proliferation and myofibroblasts of human embryonic lungfibroblasts by regulating ERK1/2 and p38MAPK pathways. Kang et al.⁶¹ demonstrated that cannabinol regulates osteoblast differentiation in U2OS and MG-63 by enhancing protein–protein interactions between runt-related protein 2, Osterix, or phosphorylated p38MAPK.

3.1.5 Cell metabolism function of p38

Mesenchymal stem cell (MSC) plays a great role in regenerative medicine, organ transplantation, and other fields, while stress-induced p38MAPK signal has been proved to damage the function of MSC. Budgude et al.⁶² found that drug inhibition of p38MAPK could restore the vitality of MSC cells and enhance their function.

3.1.6 Cell senescence function of p38

Many previous experiments have confirmed that p38MAPK is related to the aging of skeletal muscle and other tissues. He et al.⁶³ demonstrated that intestinal stem cell senescence is driven by mTORC1 via the p38MAPK-p53 pathway. Tao et al.⁶⁴ demonstrated that overexpression of forkhead box protein A2 reduces cigarette smoke-induced cellular senescence and lung inflammation by inhibiting the p38 and Erk1/2 MAPK pathways.

3.1.7 Cell survival and death function of p38

Shi et al.⁶⁵ studied the effect of phosphatase of regenerating liver-3 (PRL-3) on cell survival, and proved that PRL-3 inhibits phosphorylation by binding with p38MAPK, thus promoting cell survival. However, Filomeni et al.⁶⁶ demonstrated that disulfide stress agents trigger cell death through REDOX activation of Thioredoxin 1/p38MAPK/p53 dependent signaling cascade.

3.2.1 Connection between p38MAPK and cancer

The p38MAPK signaling pathway is commonly known as the SAPK pathway, which means that it is important in many diseases and strongly associated with cancer. As mentioned above, p38MAPK is associated with cell cycle, differentiation, metabolism, senescence, and other cellular processes, while cancer cells can disrupt pathways to promote proliferation, survival, and invasion. The p38MAPK pathway regulates cell cycle progression and cellular processes of cell survival and differentiation at different transition points through transcription-dependent and transcription-independent mechanisms,⁶⁷ resulting in its influence on a variety of cancers.

In many cancer cells, such as liver cancer and lung cancer, p38α activation is usually associated with antiproliferation function, and p38α negatively regulates abnormal proliferation in various types of primary cells, including cardiomyocytes, hepatocytes, fibroblasts, hematopoietic cells, and lung cells.^{68, 69} P38α also plays a proapoptotic role in some cells. In the early transformation process, reactive oxygen species (ROS) will induce the activation of p38α to induce apoptosis, thus preventing the carcinogenic effect generated by the accumulation of ROS.⁷⁰ P38α also mediates cell survival through a quiescent state called cancer dormancy, which is important for drug resistance in cancer cells. P38α is associated with G2/M checkpoint, which induces cell cycle arrest and promotes DNA repair, possibly leading to apoptotic resistance in cancer cells.⁵⁸

P38β is thought to have antiapoptotic effects in various cell lines and may counteract the proapoptotic effects of p38α-activated cells.⁶⁷ But beyond that, it also has the ability to regulate transforming growth factor (TGF) -1β and vascular growth factor-mediated endothelial cell survival in the absence of proapoptotic p38α.⁷¹ In addition, p38β is important in the regulation of oncogene Lipocalin 2 (LCN2), which is a target gene of plakophilin 3 (PKP3). When PKP3 expression is decreased, p38β can regulate LCN2, leading to increased tumor invasion and metastasis.⁷² Recent studies have detected elevated levels of p38β in a variety of female cancers, including breast cancer and endometrial cancer, suggesting that p38β may play a potential role.⁷³

P38γ modulates gamma radiation-induced G2 phase arrest.⁷⁴ In addition, p38γ acts as a CDK kinase, synergistic with CDK to regulate the G0 to G1 transition into the cell cycle. In cancer cell therapy studies, the absence of p38γ or treatment with p38γ inhibitors was found to prevent chemically-induced hepatocellular carcinoma formation, and biopsies of human hepatocellular carcinoma showed high expression of p38γ,⁷⁵ demonstrating that p38 regulates some cancer cell formation.

P38δ is important for the movement and invasion of bile duct cancer cells, suggesting that it may play an important role in the metastasis of bile duct cancer.⁷⁶ P38δ also has a regulatory effect on tumor genesis, p38δ-deficient mice showed decreased susceptibility to dimethylbenz (a) anthracen and 12-O-tetradeca-noylphorbol-13-acetate for treaty-induced skin cancer and Kras-induced lung cancer.⁷⁷

In summary, p38MAPK is associated with many types of cancer. For example, in head and neck squamous cell carcinoma, activation of p38MAPK plays a role in treatment resistance and disease recurrence by maintaining tumor stem cell phenotype.⁷⁸ In breast cancer, microRNA-3188 (miR-3188) can affect the proliferation, apoptosis, and migration of breast cancer cells by activating p38MAPK signaling pathway by targeting tumor suppressor candidate 5.⁷⁹ In liver cancer, exposure to benzo (a) pyrene (BaP) can promote migration and invasion of HepG2 cells, which can be blocked by p38MAPK inhibitors, demonstrating that p38MAPK pathway is involved in Bap-induced migration and invasion of liver cancer cells.

3.2.2 Connection between p38MAPK and neurodegenerative disease

Strict control of the MAPK signaling pathway has been associated with many neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), and the activation of p38MAPK mediates neuronal apoptosis in AD, PD, and ALS.

AD is thought to be the formation of senile plaques containing amyloid-beta (Aβ) and neurofibrillary tangles containing the microtubule-associated protein tau in the brain.^{80, 81} Previous studies have shown that⁸² amyloid precursor protein dimerization mediated by Aβ42 can induce the activation of ASK1-MKK6-p38 signaling pathway, thus leading to tau phosphorylation. In addition, oxidative stress triggered by ROS is also an important factor in the pathogenesis of AD, and ROS is also a typical activator of p38MAPK in AD.^{83, 84} In the study of Mohamed et al.,⁸⁵ it was demonstrated that sesame oil may regulate different molecular targets involved in the pathogenesis of AD by altering p38MAPK signaling.

PD is thought to be a disease caused by the gradual loss of dopaminergic neurons in the substantia nigra, and the accumulation of Lewy bodies (LBs) in the brain, in which specific proteins including modified alpha-synaptic nucleoproteins are deposited. Among them, alpha-synaptic nucleoprotein is present in LBs in the brain of patients with PD and plays a key role in the development of PD.⁸⁶ The increased level of α-synaptonucleoprotein is thought to be associated with neuronal apoptosis induced by dysfunction of MAPK signaling pathway. Among them, alpha-synaptic nucleoproteins activate the p38, ERK, and JNK pathways in human microglia, leading to IL-1β and TNF-α production, which promote inflammation.⁸⁷ In addition, p38MAPK and CD200-CD200R signals can regulate microglial cell dynamics in PD brain, and PD-related neurodegeneration is likely to be related to the destruction of CD200-CD200R and p38MAPK signal axes.⁸⁸ In addition, p38 can phosphorylate and activate tumor suppressor p53, and then induce the expression of proapoptotic protein Bax, thereby inducing the permeability of the mitochondrial outer membrane.⁸⁹ In addition, Obergasteiger et al.⁹⁰ also proposed a new hypothesis that GTPase-p38 is used as upstream signal to treat PD through autophagy mechanism, which has certain potential possibilities.

Most cases of ALS occur by chance as a result of familial mutations in the Cu/Zn superoxide dismutase 1 (SOD 1) gene. Abnormal expression and activation of p38MAPK in motor neurons and microglia cells is considered to be an important factor in the pathogenesis of ALS.⁹¹ In addition, studies have shown that p38MAPK inhibitors can prevent motor neuron apoptosis induced by mutant SOD1.⁹² P38 and JNK1 are also associated with abnormal cytoskeleton of spinal motor neurons, and their abnormal phosphorylation and subsequent nerve filament aggregation are characteristic of familial and incidental ALS.^{93, 94}

3.2.3 Connection between p38MAPK and inflammation

P38MAPK pathway is important in proinflammatory cytokines such as TNF-α, IL-1, IL-2, IL-6, IL-7, and so on,⁹⁵ and the four subtypes of p38MAPK are also differentially expressed and activated in different inflammatory cells.⁹⁶ In monocytes, p38α was the dominant expression form, but p38δ expression was low and p38β was not detected. The expression of p38α and p38δ was abundant in macrophages, but p38β was not detected. P38α and p38δ were significantly expressed in neutrophils, CD4+T cells and endothelial cells, while p38β was abundant in endothelial cells. P38γ was not detected in any inflammatory cells.

In studies of asthma, p38MAPK inhibitors have been shown to be effective in many in vitro and in vivo models of inflammation and help to resolve or regulate diseases such as asthma.⁹⁷ Phosphorylated p38MAPK coordinates the phosphorylation of MSK1, and directly phosphorylates nuclear factor-kappa B (NF-κB) to produce type 2 cytokines. However, blocking NF-κB and p38MAPK with 4-carvomenthenol has been shown to improve combined allergic rhinitis and asthma syndrome.⁹⁸

In studies of rheumatoid arthritis (RA), the pathological cause is believed to be the presence of a large number of proinflammatory cytokines in synovial fluid. It is later considered that the combination and neutralization of TNF-α and IL-1 proinflammatory cytokines are very effective for the treatment of RA. Preclinical models have shown that small molecule inhibitors have therapeutic potential.^{99, 100} P38α/β inhibitors were effective in a mouse model of collagen-induced arthritis, demonstrating that this class of p38MAPK inhibitor compounds prevents disease progression.¹⁰¹

3.2.4 Connection between p38MAPK and cardiovascular disease

P38MAPK also plays a role in cardiovascular disease, which is the leading cause of death worldwide. Previous studies have shown that the absence of p38α (MAPK14) in vascular smooth muscle cells can inhibit the formation of angiotensin-II-induced abdominal aortic aneurysm, vascular inflammation, and expression of aging markers.¹⁰² Atherosclerosis (AS) is also an important cause of myocardial infarction, coronary heart disease, and other heart diseases. Studies have shown that miR-124-3p overexpression can down-regulate the expression of MEKK3, inhibit p38MAPK signaling pathway, and thus inhibit macrophage proliferation in coronary AS mice. Promote macrophage apoptosis.¹⁰² Studies on diabetic cardiomyopathy (DCP) show that inflammatory factors influence the pathogenesis of DCP, and the signal transduction of NF-κB and p38MAPK is confirmed to be involved in the inflammatory response. Sirtuin-1 activation improves DCP by inhibiting phosphorylated p38MAPK in cytoplasm and NF-κB expression in nucleus.¹⁰³

4.1.1 Monocyclic p38α and p38β inhibitors

Imidazole derivatives

The pyridinyl imidazole compounds are considered to be the most effective inhibitors of p38α structure, and a number of selective inhibitors of p38α and p38β have been derived from these frameworks (Table 1).

Table 1. Monocyclic inhibitors targeting p38α/β.

Inhibitor	Chemical name	Chemical structure	Activity	DMPK profile	References
Compound 1 (SB-203580)	4-(4-uorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)imidazole		p38α IC50 = 0.1 μM p38β IC50 = 0.43 μM	(mouse) Cm = 587 ± 173 ng·mL−1 Tm = 30 min Cl = 138 ± 39 mL·min−1·kg−1	[104-109]
Compound 2 (VRT-19911)	4-(4-(4-fluorophenyl)-1-(piperidin-4-yl)-1H-imidazol-5-yl)pyridine		p38α Ki = 60 nM	Not tested	[110]
Compound 3 (CBS-3595)	N‑{4-[5-(4-Fluorophenyl)-3-methyl-2-methylsulfanyl‑3H‑imidazol-4-yl]-pyridin-2-yl}-acetamide		p38α IC50 = 0.5 μM PDE-4 IC50 = 0.2 μM	Tm = 2.20 ± 1.30 h Cm = 0.20 ± 0.06 μM AUC0 - t = 0.95 ± 0.31 μM·h T1/2 = 2.7 ± 0.5 h	[110, 111]
Compound 4 (ML3403)	{4-[5-(4-fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine		p38α IC50 = 0.38 μM, TNF-α IC50 = 2.7 ± 0.3 μM IL-1β IC50 = 0.99 ± 0.46 μM	(Female) Tm = 1 h Cm = 386 ng/mL AUC0 - t = 1479 ng·h/mL	[112, 113]
Compound 5 (SB242235)	1-(4-piperidinyl)-4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl) imidazole		p38α IC50 = 19 nM p38β IC50 = 1 μM	Cm = 280 ± 31 ng·mL−1 Tm = 60 min Cl  =56 ± 8.9 mL·min−1·kg−1 T1/2 = 93.5 ± 11.3 min	[114-117]
Compound 6 (RWJ67657)	4-[4-(4-Fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyrindinyl)-1H-imidazol-2-yl]-3-butyn-1ol		p38α IC50 = 1000 nM TNF-α IC50 = 3 nM IL-1β IC50 = 11 nM IL-8 IC50 = 0.04 μM	(Female) Tm = 0.6 h T1/2 = 6.1 h F = 11%	[118-120]
Compound 7	3-(2-phenyl-5-(2-((3-((phenylthio)amino)propyl)amino)pyridin-4-yl)-1H-imidazol-4-yl)phenol		p38α IC50 = 47 nM TNF-α IC50 = 98.02 nM	Not tested	[121]
Compound 8	3-(5-(2-((3-(((4-fluorophenyl)thio)amino)propyl)amino)pyridin-4-yl)-2-phenyl-1H-imidazol-4-yl)phenol		p38α IC50 = 45 nM TNF-α IC50 = 78.03 nM IL-6 IC50 = 17.6 µM IL-1β IC50 = 82.15 nM	Not tested	[121]
Compound 9 (SC74102)	4-(5-(4-fluorophenyl)-2H-imidazol-4-yl)pyridine		p38α IC50 = 600 nM	Not tested	[122, 123]
Compound 10	4-(3-(4-fluorophenyl)-5-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine		p38α IC50 = 25.3 nM p38β IC50 = 510 nM	Not tested	[122, 123]
Compound 11	4-(3-(4-chlorophenyl)-5-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)pyridine		p38α IC50 = 31.6 nM p38β IC50 = 2680 nM	Not tested	[122, 123]
Compound 12 (SC79659)	1-(4-(3-(4-chlorophenyl)-4-(pyridin-4-yl)-1H-pyrazol-5-yl)piperidin-1-yl)-2-hydroxyethan-1-one		p38α IC50 = 111 nM p38β IC50 = 1790 nM	Not tested	[122, 123]
Compound 13 (SD0006)	1-(4-(3-(4-chlorophenyl)-4-(pyrimidin-4-yl)-1H-pyrazol-5-yl)piperidin-1-yl)-2-hydroxyethan-1-one		p38α IC50 = 110 nM p38β IC50 = 3450 nM	Not tested	[122, 123]
Compound 14	(5-amino-1-phenyl-1H-pyrazol-4-yl)(phenyl)methanone		p38α IC50 = 2.3 μM	Not tested	[124, 125]
Compound 15	(5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl)(phenyl)methanone		p38α IC50 = 1.1 μM	Not tested	[124, 125]
Compound 16 (RO3201195)	(S)-(5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl)(3-(2,3-dihydroxypropoxy)phenyl)methanone		p38α IC50 = 0.7 μM	Not tested	[124, 125]
Compound 17 (DP-802)	2-(3-(3-(tert-butyl)-5-(3-(2,3-dichlorophenyl)ureido)-1H-pyrazol-1-yl)phenyl)acetamide		p38α IC50 = 9 nM BRAF IC50 = 17 nM CRAF IC50 = 11 nM	Not tested	[126]
Compound 18 (SC409)	4-(3-(4-chlorophenyl)-5-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)pyrimidine		p38α IC50 = 0.04 μM p38β IC50 = 2.30 μM	Not tested
Compound 19 (TAK-715)	N-(4-(2-ethyl-4-(m-tolyl)thiazol-5-yl)pyridin-2-yl)benzamide		p38α IC50 = 240 nM TNF-α IC50 = 240 nM	Not tested	[127]
Compound 20 (BMS-64099)	2-(sec-butylamino)-N-(2-methyl-5-(methylcarbamoyl)phenyl)thiazole-5-carboxamide		p38α IC50 = 3.5 nM TNF-α IC50 = 2.9 nM Lck IC50 = 22 μM	Not tested	[128]
Compound 21	3-(2,5-dimethoxyphenyl)-N-(4-(4-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)propanamide		p38α IC50 = 10 nM	Not tested	[129]
Compound 22	(E)-3-(2,5-dimethoxyphenyl)-N-(4-(4-(4-fluorophenyl)-2-(phenyldiazenyl)thiazol-5-yl)pyridin-2-yl)propanamide		p38α IC50 = 2.36 ± 0.61 nM	Not tested	[129]
Compound 23	3-bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-1-phenylpyridin-2(1H)-one				[130, 131]
Compound 24	3-bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-1-(o-tolyl)pyridin-2(1H)-one		p38α IC50 = 12 nM	Not tested	[130, 131]
Compound 25	3-(3-bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-N,4-dimethylbenzamide		p38α IC50 = 2.3 nM	Not tested	[130, 131]
Compound 26	3-(3-bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-4-fluoro-N-methylbenzamide		p38α IC50 = 8.5 nM	Not tested	[130, 131]
Compound 27	3-(3-bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-4-chloro-N-methylbenzamide		p38α IC50 = 3.0 nM	Not tested	[130, 131]
Compound 28	3-(3-bromo-4-((2,4-difluorobenzyl)oxy)-6-methyl-2-oxopyridin-1(2H)-yl)-4-(hydroxymethyl)-N-methylbenzamide		p38α IC50 = 325 nM	Not tested	[130, 131]
Compound 29 (Losmapimod)	6-(5-(cyclopropylcarbamoyl)-3-fluoro-2-methylphenyl)-N-neopentylnicotinamide		p38α pka = 8.1 p38β pka = 7.6	Not tested	[132-134]
Compound 30 (VX-702)	2-(2,4-difluorophenyl)-6-(1-(2,6-difluorophenyl)ureido)nicotinamide		p38α IC50 = 4 ~ 20 nM	Not tested	[135, 136]
Compound 31 (UM-164)	N-(5-(3-(tert-butyl)benzamido)-2-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide		p38α Kd = 2.2 nM p38β Kd = 5.5 nM Src Kd = 2.7 nM	Not tested	[137]
Compound 32 (UM101)	4-chloro-N-(4-((1,1-dioxidothiomorpholino)methyl)phenyl)benzamide				[138]
Compound 33 (PNU-120596)	1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea		p38α IC50 = 3.3 μM	Not tested	[139, 140]
Compound 34 (VPC00628)	(R)-5-amino-N-(4-((1-amino-4-cyclohexyl-1-oxobutan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-carboxamide		p38α IC50 = 7 ± 0.9 nM	Not tested	[141]
Compound 35 (VPC00630)	5-amino-N-(((1 S,4r)-4-(((S)-1-amino-4-cyclohexyl-1-oxobutan-2-yl)carbamoyl)cyclohexyl)methyl)-1-phenyl-1H-pyrazole-4-carboxamide		p38α IC50 = 44 ± 4.4 nM	Not tested	[141]
Compound 36 (VPC00257)	(S)-5-amino-N-(4-((1-amino-1-oxo-3-(3-(trifluoromethyl)phenyl)propan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-carboxamide		p38α IC50 = 46 ± 2.5 nM	Not tested	[141]
Compound 37	(S)-5-amino-N-(4-((1-amino-1-oxo-3-(3-(trifluoromethyl)phenyl)propan-2-yl)carbamoyl)benzyl)-1-phenyl-1H-pyrazole-4-carboxamide		p38α IC50 = 14.0 nM p38β IC50 = 16.8 nM	Not tested	[142]
Compound 38	N-(2-chloro-6-fluorobenzyl)-3-(furan-2-yl)-1H-1,2,4-triazol-5-amine		Not tested	Not tested	[143]

• Abbreviations: BRAF, B-RAF oncogene serine/threonine kinase; CRAF, C-RAF oncogene serine/threonine kinase; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor- α.

Since it was developed by Gallagher et al. in 1995, SB-203580 (1) has been widely used as a potent and selective p38α/β inhibitor. As a pharmacological tool, it has been widely used to analyze the role of p38MAPK in various physiological processes.¹⁰⁴ Compound 1 is a competitive inhibitor of ATP. The IC₅₀ values of p38α and p38β are 0.1 and 0.43 μM, respectively, while the IC₅₀ values of p38γ and p38δ are not inhibited. Kumar et al. also demonstrated that it only inhibited the activity of p38 mitogen-activated protein kinase, but not its activation.¹⁰⁵ P38MAPK plays a role in Dengue virus (DENV)-induced hepatocyte apoptosis, and DENV infection induces p38MAPK phosphorylation. Its downstream signals include MAPKAPK2, HSP27, and AMP-dependent transcription factor-2 (ATF-2). Limjindaporn's team¹⁰⁶ demonstrated that treatment with SB203580 reduced cytokines such as TNF-α, IL-6, and IL-10 in DENV infection, as well as regulated upon activation, normal T-cell expressed and secreted and interferon-gamma-inducible protein 10 were increased. Compound 1 did not reduce the phosphorylation of p38MAPK, but significantly reduced the phosphorylation of MAPKAPK2, HSP27, and ATF-2. It was confirmed that SB203580 modulated the downstream signal of p38MAPK to reduce DENV-induced liver injury. In addition, SB-203580 has been used to evaluate a wide range of phenomena such as bacterial sepsis,¹⁰⁷ central nervous system (CNS) trauma response,¹⁰⁸ and cardiac ischemia/reperfusion injury.¹⁰⁹ As an earlier inhibitor of pyridinyl imidazole compounds, SB-203580 has derived a considerable number of other inhibitors with similar structure. Another inhibitor with similar structure belonging to pyridinyl imidazole compounds is VRT19911 (2). VRT19911 has been shown to bind to the ATP-binding site of p38α and is a selectively effective p38α inhibitor with a Ki of 60 nM. However, it has previously been reported that the presence of pyridinyl components in SB203580 and VRT19911 results in significant inhibition of hepatocytochrome p450 isoenzymes in vitro,¹⁴⁴ and because oral activity is also reduced, these compounds are not suitable for the treatment of chronic diseases.

Albrecht et al.¹¹⁰ coincidentally discovered a dual-targeted p38αMAPK/Phosphodiesterase-4 (PDE-4) inhibitor CBS-3595 (3) in 2016 while searching for a dual-targeted p38αMAPK/JNK3 inhibitor. The two enantiomers showed similar inhibitory activity against p38αMAPK with IC₅₀ of 0.5 μM and PDE-4 with IC₅₀ of 0.2 μM. Moreover, MKK6-mediated phosphorylation of p38αMAPK was not inhibited at concentrations up to 10 μM. CBS-3595 reduced TNF-α release more effectively in vivo in rat lipopolysaccharide (LPS) models than p38αMAPK or PDE-4 selective inhibitors. Compared with the control group, TNF-α synthesis was inhibited by almost 80% after administration.¹¹⁰ It also had good pharmacokinetic properties when studied in vivo at 10 mg dose (T_m = 2.20 ± 1.30 h, C_m = 0.20 ± 0.06 μM, AUC_0 − t = 0.95 ± 0.31 μM h, T_1/2 = 2.7 ± 0.5 h). The researchers also found¹¹¹ that treatment with CBS-3595 significantly reduced the production of the proinflammatory cytokine IL-6 and broadly increased levels of the anti-inflammatory cytokine IL-10 in rat paws. The downside, however, is that further dose increases are limited by vomiting, which is a known side effect of all PDE-4 inhibitors and thus prevents further clinical trials.

Most pyridinyl imidazole inhibitors inhibit hepatic (CYPs) enzymes. The development of ML3403 (4), a p38 inhibitor pretending to have low activity against cytochrome P450 (CYP450) enzyme, is particularly important. ML3403, originally a candidate compound in a multisubstituted pyridinyl imidazole inhibitor developed by Laufer et al.¹¹² in 2003, was shown to inhibit TNF-α-induced strong p38αMAPK activation, and phosphorylated p38βMAPK was also inhibited by it. However, the inhibitory effects of p38γ and p38δ have not been investigated.¹¹² The IC₅₀ value of ML3403 for p38α was 0.38 μM, while that for TNF-α was 2.7 ± 0.3 μM, and that for IL-1β was 0.99 ± 0.46 μM.¹¹² Similar to other inhibitors, ML3403 also inhibited TNF-α induced IL-6 secretion, and the inhibition rate was 47.8 ± 2.4% at the concentration of 1 μM. ML3403 significantly inhibited IL-8 secretion at 0.1–3 µm concentrations, but ML3403 at 10 µM did not seem to have an inhibitory effect. Pharmacokinetic evaluation of ML3403 after oral administration of 30 mg/kg in male and female Wistar rats showed rapid and high turnover of the active sulfoxide metabolite, and also demonstrated that the isoenzymes CYP1A2, CYP2C19, CYP2D6, and CYP3A4 are key enzymes in ML3403 metabolism.¹¹³ For example, in female mice, C_m reached 386 ng/mL, T_m = 1 h, AUC_0 − t = 1479 ng·h/mL, while in male mice, C_m reached 115 ng/mL, AUC_0 − t = 389 ng·h/mL. In another group of chronic arthritis induced by Complete Freund's adjuvant, ML3403 treatment did not show obvious hepatotoxicity.¹¹¹ All these results suggest that ML3403 is a potential inhibitor of pyridine-imidazole p38α.

SB242235 (5) is a pyridyl imidazole inhibitor developed by SmithKline in 2000. The IC₅₀ of SB242235 (5) is 19 nM for p38α, 1 μM for p38β, and almost insensitive to p38γ and p38δ, with IC₅₀ > 10 μM.¹¹⁴ It inhibited p38α, p38β, JNK2β1, and c-raf, but did not inhibit ERK and JNK. And it has no direct effect on 5-lipoxygenase or cyclooxygenase-1. It competes with ATP to inhibit p38α by forming a 4-fluorophenyl-binding sac behind the site normally occupied by ATP adenine rings.¹¹⁵ Badger et al.¹¹⁶ demonstrated good inhibition of LPS-induced TNF-α production, and the use of SB242235 at 60, 30, and 10 mg/kg doses in adjuvant arthritis (AA) rats significantly inhibited foot swelling (inhibition rates were 73%, 51%, 19%, respectively). Levels of IL-6 in different inflammatory areas are sensitive markers of disease activity, and this cytokine was significantly inhibited at 60 mg/kg and not at lower doses after therapeutic administration of SB242235. Ward et al.¹¹⁷ studied its pharmacokinetic and showed high plasma clearance (56 ± 8.9 mL·min-1·kg⁻¹) in rats after intravenous injection of 1.5 mg·kg⁻¹ SB24235. T_1/2 was 93.5 ± 11.3 min, T_m = 60 min, C_m = 280 ± 31 ng·mL⁻¹.

In 1999, Wadsworth et al.¹¹⁸ developed RWJ67657 (6), which selectively inhibited p38α and p38β subtypes but not p38γ and p38δ subtypes, with IC₅₀ = 1 μM for p38α. It can inhibit the production of TNF-α and IL-1β in LPS-stimulated human polymorphonuclear cells, with IC₅₀ values of 3 and 11 nM, respectively, and block p38 signals by inhibiting the phosphorylation of p38α and downstream p38α signals. Reduced activation of p38 leads to decreased activation of downstream effectors Hsp27 and MAPAPK. It also inhibits ER signaling independently of Thr311 phosphorylation site.¹¹⁹ After oral administration in male and female beagles, RWJ67657 was rapidly absorbed with mean T_m values of 0.8 and 0.6 h, respectively, and disappeared with mean terminal half-lives of 3.8 and 6.1 h. The average bioavailability of the drug is about 11%. J.W. Antoon et al.¹²⁰ proved that RWJ67657 effectively inhibited p38 pathway in MDA-MB-361 breast cancer cells through in vitro and in vivo experiments, and could effectively inhibit the growth of endocrine-resistant breast cancer tumors. In the presence of estrogen, RWJ67657 resulted in an approximately 3.5-fold reduction in tumor volume in the mouse xenograft model compared to tumors treated with terminal vectors. In the absence of estrogen, compound 10 reduced tumor volume by about 4.5 times after 40 days of treatment.

Ali et al.¹²¹ redeveloped a series of imidazolyl pyridine inhibitors against B-RAF^V600E to inhibit p38α kinase through a drug reuse strategy in 2021, and finally developed compounds 7 and 8 as the most potent inhibitors, with IC₅₀ of 47 nM and 45 nM for p38α, respectively. In the process of its development, considering that p38α kinase and B-RAF kinase are key components of the MAPK pathway, there is the possibility of further development. SB203580 (1), a more classical imidazolyl pyridine inhibitor, was used as the lead compound, and its imidazolyl 4-linked benzene ring was first replaced. Substitution with a hydroxyl group was found to exhibit slightly higher inhibitory activity against compounds containing 3-methoxy phenyl groups. The pyridine group was further optimized and replaced, and the propyl linker had a significant increase in inhibitory activity compared with the ethyl linker. Finally, the phenyl terminal 7 and 8 were modified, both of which showed a better inhibitory ability on p38α. In addition, the developed compound 8 also effectively inhibited LPS-induced production of proinflammatory cytokines TNF-α, IL-6, and IL-1β in RAW 264.7 macrophages with IC50 values of 78.03 nM, 17.6 µM and 82.15 nM, respectively. Compound 7 showed good activity for TNF-α production with IC₅₀ = 98.02 nM, but did not show significant activity for the production of IL-6 and 1Il-1β. These two compounds were also developed based on the classical pyridyl imidazole structure, which further indicated that the structure played a good enlightenment role in the development of p38α inhibitors and had the possibility of further optimizing the structure.

Pyrazole derivatives

By optimizing the substitution of the lead compound SC74102 (9) on the pyrazole ring, Pfizer obtained compound 10, which significantly improved the inhibition effect on p38α and p38β. Then, by optimizing the substitution of phenyl, compound 11 was obtained, and compound 12 (SC79659) was obtained on the piperidine ring. Pyridine rings were optimized to pyrimidine rings, resulting in compound 13 (SD0006) (Figure 2).¹²² Compounds 12 and 13 are highly effective diarylpyrazole p38α inhibitors. The IC₅₀ values of SD0006 for p38α and p38β are 110 and 3450 nM, while the IC₅₀ values of SC79659 for p38α and p38β are 111 and 1790 nM, indicating that both of them have good selectivity for p38α. Inhibitor binding at ATP-binding sites can be found in the crystal structure of SC79659, while 4-chlorophenyl is tightly bound in a deeply buried lipophilic pocket, which may give SD0006 higher kinase selectivity than most ATP-competitive inhibitors. SD0006 is a highly selective p38α inhibitor that does not directly inhibit MKKs, nor does it inhibit ERK and JNK pathways. It has shown good oral anti-inflammatory effects in preclinical studies, inhibiting the expression of a variety of proinflammatory proteins and improving disease severity in animal models of arthritis, in their experiment using SD0006 to treat arthritis in rats, computed tomography was seen to significantly prevent inflammation-mediated joint and bone destruction in the 21-day treatment group compared to the control group. Burnette et al.¹²³ reported that SD0006 almost completely blocks LPS-induced TNF α release in U937 cells in a dose-dependent manner. Similarly, it is shown in human primary monocytes and human whole blood cells to inhibit IL-1β and IL-6 similarly in U937 cells. For example, the IC₅₀ of IL-1β and IL-6 collected from human primary monocytes is 105.9 ± 88.3 nnol·L⁻¹ and 106.9 ± 25.3 nmol·L⁻¹. In rats, the dose of 5 mg/kg showed good pharmacokinetic properties (Cl = 4.96 mL/min/kg, V_d = 1.74 L/kg, T_1/2 = 4.5 h). In conclusion, SD0006 is a potential drug for clinical trials.

Goldstein et al.¹²⁴ developed a selective p38α inhibitor RO3201195 (16) in 2005 through high-throughput screening and structure–activity relationship (SAR) studies (Figure 2), whose IC₅₀ against p38α was about 0.7 μM. In the process of development, compound 14 was first identified by high-throughput screening. After SAR analysis, p-fluorine substitution 15 was screened out, and the IC₅₀ of p38α was 1.1 μM, indicating that the para substitution on N-phenyl was superior to the ortho and meso substitution. Subsequently, the alternate nonionizing solubilizing functional groups were used as the main focus of the optimization strategy to obtain the final compound 16. Compound 16 also inhibited the production of rat cytokine IL-6 and TNF-α induced by LPS stimulation, which not only inhibited the production of TNF-α, but also regulated TNF-α signaling. The two hydrogen bonds in its structure are particularly important. One is based on the formation of a hydrogen bond between benzoyl oxygen and the NH backbone of methionine 109 on a skeleton called benzoyl amiprazole. And the other one is the NH2 group of aminopyrazole and the side chain alcohol of threonine 106. This is the first hydrogen bond found in p38 protein and its inhibitor. Since only about 20% of human kinases have threonine in this location, perhaps such hydrogen bonding can give it more efficient selectivity. Bagley et al.¹²⁵ explored its inhibitory effect on Werner syndrome cells through experiments. In the experiment, the proliferation of dimethyl sulfone-treated fibroblasts was very slow, and only doubled in a single population during the 88-day experiment. However, the inhibitor-treated cells had a much higher rate of proliferation and controlled about 7.5 population multiplicators before growth stalled. The pharmacokinetic data were measured at 10 mg/kg in rats, T_m = 3 h, C_m = 4.48 μg/mL, T_1/2 = 1.64 h, F = 61.7%, Cl = 0.32 L/h/kg, V_dss = 1.27 L/kg. In addition to the development of the inhibitor itself, Goldstein et al.¹²⁴ first used the diol component as the solubilizing group of a selected kinase inhibitor in clinical studies. The data indicate that diol can be effectively used to improve the physical properties of other insoluble kinase scaffolds.

DP-802 (17), developed by Deciphera Pharmaceuticals in 2010, is a novel inhibitor that induces enhanced type II conformation when combined with unphosphorylated or diphosphorylated forms of p38α kinase. The IC₅₀ values of DP-802 for unphosphorylated p38α and PP-p38 were 9 and 11 nM. Evaluated across a wide range of kinases, it was found to inhibit only a small number of kinases in addition to p38α and p38β, This includes the wild-type B-RAF oncogene serine/threonine kinase (IC₅₀ = 17 nM), C-RAF oncogene serine/threonine kinase (IC₅₀ = 11 nM) and EPH receptor B2 (IC₅₀ = 82 nM). For the nonphosphorylated p38α-induced type II conformation, DP-802 forms a hydrogen bond with the posterior guanidine nuclear warhead of arginine 70 in the binding conformation of DP-802 and p38α (PDB:3NNW) (Figure 2). The urea part of DP-802 forms hydrogen bonds with conformal C-helical glutamate 71/lysine 53 salt bridges, tert-butyl parts occupy hydrophobic DFG sacs, 2, 3-dichlorobenzoic rings occupy type II hydrophobic sacs, and stabilize the phenylalanine 169 side chain of DFG in edge-to-facial interactions. This binding mode of DP-802 further stabilizes the p38αII conformation.¹²⁶ The ability of DP-802 to inhibit the release of TNF-α stimulated by LPS in Lewis rats was also evaluated. The ED₅₀ of DP-802 to inhibit the release of TNF-α in Lewis rats was 3.6 mg/kg 2 h after oral administration. In addition, DP-802 reduced the amount of phosphorylated p38α in HELA cells in a dose-dependent manner, suggesting that inhibitor-bound phosphorylated p38α was more readily dephosphorylated by cell phosphatases than carrier controls. DP-802 and its analogs showed strong inhibition of p38α in vivo.¹²⁶

SC-409 (18), developed by Pfizer in 2005–2006, is a small molecule inhibitor of p38 with a diarylpyrazole structure. It selectively inhibits p38α and p38β isoforms with IC₅₀ values of 0.04 and 2.30 μM, but has no significant inhibitory effect on p38γ and p38δ. SC-409 inhibits the production of key cytokines, such as TNF-α, IL-1β, receptor activator of nuclear factor-kappa B ligand (RANKL), and so on. SC-409 selectively blocks p38 signaling in pre-OCL without interfering with JNK and NF-κB pathways, and SC-409 inhibits a variety of inflammatory signaling factors including RANKL, IL-6, IL-1β, mRNA expression of tartrate-resistant acid phosphatase, matrix metalloproteases-9, -13, and COX-2. They also proved that SC-409 decreases bone destruction, joint infiltration by inflammatory cells, and the number of Ocls in streptococcal cell wall-induced arthritis in rats. In another study of cardiac remodeling in mice with myocardial infarction and heart failure, chronic in vivo administration of SC-409 improved cardiac function in patients with heart failure after myocardial infarction.

4.1.2 Bicyclic p38α and p38β inhibitors

BIRB-796 (42), developed by Boehringer-Ingelheim, Germany, is the earliest highly selective inhibitor of p38α entered the phase II clinical trial (Figure 3). Its IC₅₀ against p38α and p38β were 38 and 65 nM. BIRB-796 inhibited the production of TNF-α under LPS stimulation, and the IC₅₀ value was 21 nM. In the process of its development, compound 39 was used as the substrate to optimize the imidazole group replacement, and the addition of isobutyl group made compound 40 have a higher Tm than compound 39. According to studies, imidazoline-2 substitution is particularly important for its inhibitory effect. After screening, methyl benzene was selected to replace compound 41, and finally ethoxy morpholine was used to replace H on naphthalene to get the final product BIRB-796. For its structure, the naphthalene group can be used as a kinase-specific pocket, and the tert-butyl imidazole part can be used as a phenylalanine pocket. The x-ray structure of p38αMAPK and BIRB-796 (PDB:1KV2) showed that Asp168 and Leu167 had hydrogen bonding on the carbonyl group of urea, Glu71 had hydrogen bonding with the NH group of urea, and Met109 had hydrogen bonding with the O of morpholine group. Both Phe169 and Lys53 interact with naphthalene group π–π, and the conserved residue Asp168-Phe169-Gly170 (DFG) moves to a new location during binding. In the new conformation (DFG-out), the binding of ATP to p38α MAP kinase is inhibited. However, in later clinical trials, the researchers stopped the clinical studies related to BIRB-796 due to toxic side effects such as elevated liver enzymes. In view of the good activity of BIRB-796, Arai et al.¹⁴⁵ carried out a series of modifications on the original BIRB-796 in 2012, replacing the naphthalene group of BIRB-796 with benzyl group, and confirmed that compound 43 binds to the allosteric site of p38αMAP kinase similar to compound 42. The IC₅₀ of compound 43 for p38α reached 12 nM. And benzyl rings occupy kinase-specific pockets. Therefore, compounds 44 and 45 are further modified to obtain the IC₅₀ of 4 and 5 nM for p38α. Ryoo et al.¹⁴⁶ experimentally demonstrated that BIRB-796 can inhibit the secretion of cytokines by THP-1 cells induced by LPS, and inhibit the transcription of cytokines induced by LPS and the phosphorylation of p38αMAPK in THP-1 cells. The therapeutic effect was evaluated by measuring the changes of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in blood. After 5 days of oral administration, ALT content was 24 ± 4 U/L and AST content was 45 ± 11 U/L. Later, in a series of studies, it was confirmed that BIRB-796 could inhibit not only p38α, but all subtypes of p38, but only p38α and p38β at low concentrations. In addition, a series of clinical trials have been conducted under better efficacy, such as the phase II trial on Psoriasis (NCT02209753) conducted as early as 2001–2002, and the phase II trial on RA (NCT02209779) conducted in 2001–2002. All with good results.

The IC₅₀ of VX-745 (49) was 9 nM for p38α, 45 nM for IL-1β, and 51 nM for TNF-α. It blocks the release of IL-1β and TNF-α in whole blood with IC₅₀ values of 150 and 180 nM. VX-745 is highly selective, with p38α being 20 times more selective than p38β (Ki = 220 nM), and has no significant inhibitory effect on other MAP kinases (except MKK6). John P. Duffy et al.¹⁴⁷ performed SAR analysis on compound 46. As the lead compound, compound 46 showed IC₅₀ for p38α > 20 μM, but IC₅₀ for IL-1β and TNF-α was not accurately detected. The benzene ring was optimized by substituent group, and it was determined that the chlorinated product 47 at position 2 had an obvious decrease in IC₅₀. Further substituent group optimization was carried out to determine that the 2, 6 dichlorobenzene ring substituted compound 48 had a stable interaction with p38α, and the best substitution effect of 2, 4 difluoride on another benzene ring was determined by analysis, namely compound VX-745. Compound 49 occupies a hydrophobic pocket with each chlorine atom and is in contact with residues Val30, Leu108, Ala157, and Leu167. The benzene ring itself interacts favorably with the main chain residue atoms Gly110, Ala111, and Asp112. 2, 4-difluorophenyl rings occupy hydrophobic pockets at gatekeeperThr106 residues and form extensive van der Waals contacts.¹⁴⁷

PF-03715455 (50), an inhibitor developed by Pfizer, is selective to p38α and p38β, with IC₅₀ of 0.88 and 23 nM¹⁴⁸ (Figure 4). It had long-term effects in vitro (t½ > 80 h) and in vivo, and effectively inhibited LPS-induced TNF-α production in human blood (IC₅₀ = 1.7 nM). Compound 50 binds to p38α quickly, dissociates slowly and has a long half-life. From a structural point of view, it may be due to the larger group at the 2-site of triazopyridine (2-hydroxyethyl thiophenyl and isopropyl) leading to increased affinity. And the structure of 50 has been optimized with a “design inhalation” approach designed to minimize systemic exposure and improve therapeutic indices. PF-03715455 is a very effective and selective inhibitor in the treatment of COPD, which can inhibit the release of proinflammatory cytokines associated with COPD. The kinetic parameters of p38α were t_1/2 = 80 h, K_D = 0.001 nM. At the same time, its pharmacokinetic characteristics in vitro and in vivo indicate that oral bioavailability of this compound in humans is very low at any swallowed dose and has a high intrinsic clearance, so that any drug absorbed by the lungs is rapidly metabolized into relatively inactive metabolites. PF-03715455 was tested in a phase I trial on COPD as early as 2011 (NCT01314885).

In 2004, Palau Pharmaceuticals released a novel pyrazolypyridine inhibitor UR-13756 (51). UR-13756 showed higher kinase selectivity through a kinase set of 115 kinases compared to BIRB-796 (42) and SB203580 (1). Especially regarding JNKs and c-Raf1.¹⁴⁹ UR-13756 has a higher selectivity for p38α than p38β, and its IC₅₀ for p38α is about 80 nM. For UR-13756 to inhibit the production of TNF-α by LPS-induced peripheral blood mononuclear cells, the IC₅₀ in this system was comparable to the EC₅₀ at 60 nM, and in the same study, the EC₅₀ of SB203580 was 280 nM. In addition, 1.0 µM UR-13756 was 100% effective in inhibiting p38α after pretreatment for 24 h.¹⁴⁹ The clinical efficacy and safety of UR-13756 was described in the first reported large-scale randomized clinical trial of a p38 inhibitor with good bioavailability, favorable pharmacokinetic properties, and low toxicity.^{150, 151}

Peifer et al.¹⁵² developed a selective p38α inhibitor, CP41 (52), in 2007. The IC₅₀ for p38α was 1.8 μM, and two isoxazole isomers were developed simultaneously. The IC₅₀ for p38α of compound 53 was 0.45 μM, the IC₅₀ for JNK3 was 0.54 μM, the IC₅₀ for JNK2 was 1.28 μM, and the IC₅₀ for p38α of compound 54 was 2.2 μM. IC₅₀ of JNK3 = 3.5 μM. CP41 only inhibited p38α subtype of MAPK, while 53 and 54 separately blocked JNK2/3. In addition to MAPK, compound 53 also significantly inhibits Protein Kinase A (PKA), Protein Kinase D1 (60% ± 1), Lck (42% ± 0), and casein kinase 1 enzyme δ (6% ± 2). However, CP41 did not inhibit TNF-α-induced IL-6 protein secretion nor significantly inhibited TNF-α-induced IL-8 secretion. In its binding conformation model (PDB: 1A9U, PDB: 1PMN), the o-pyridine-4-f-phenyl system in CP41 favors the inhibition of p38α. Compared with Thr106 in p38α, the action of the more spacious Met146 in JNK3 determines the selectivity of the six-membered core compound CP41 for p38α. But not for the five-membered core compounds 53 and 54.¹⁵² Although no clinical trials have been conducted, CP41 is a good lead product for the development of selective inhibitors of p38α.

SCIO-323 (55) is a small molecule of indolyl benzylpiperazine formamide that acts as an ATP-competitive inhibitor of p38MAPK kinase. The IC₅₀ of p38α and p38β was 3, 180 nM, and p38γ and p38δ were not significantly inhibited. S.B. Goodman et al.¹⁵³ studied the efficacy of SCIO-323 in reducing the established inflammatory response to periprosthesis osteolysis caused by nonsurgical treatment of wear particles and demonstrated that oral SCIO-323 helps reduce inflammatory response to wear particles. And to some extent reverse the adverse effects of phagocytic polyethylene particles on bone growth in our rabbit model. However, continuous administration of SCIO-323 may interfere with the production of key cytokines and growth factors important for the initial stage of bone formation, adversely affecting the proliferation, differentiation, and maturation of mesenchymal tissues. But with the deepening of research in recent years, SCIO-323 is gradually found to be ineffective in vivo.

Dilmapimod (SB-681323) (56) has been identified as a potent, selective, and reversible inhibitor of p38α MAPK, and has been shown to inhibit the production of several cytokines such as IL-6, IL-8, and so on. In the treatment of COPD, Dave Singh et al.¹⁵⁴ proved that Dilmapimod could effectively inhibit p38MAPK signal by reducing the expression of phosphorylated heat shock protein 27 (pHSP27) induced by sorbitol, which in turn inhibits the production of TNF-α. In clinical trials for severe trauma patients at risk of acute respiratory distress syndrome, Jason D. Christie et al.¹⁵⁵ demonstrated that intravenous Dilmapimod was generally well tolerated in severely injured subjects at risk of ARDS, and at the dose levels tested, there is no evidence of hepatotoxicity after treatment of trauma patients, whereas other partial p38MAPK inhibitors cause an increase in liver transaminase. At the same time, IL-6, IL-8, CRP, and soluble tumor necrosis factor receptor-1 levels have been reduced in the trial, and these changes are most obvious in the high-dose continuous infusion scheme. Dilmapimod has conducted several clinical trials, such as the Phase I trial of COPD (NCT00380133) (NCT00564746) in 2005–2006 and 2007.

Pamapimod (RO-4402257) (57) was a selective and potent inhibitor of p38α and p38β with IC₅₀ values of 0.014 ± 0.002 μM and 0.48 ± 0.04 μM, but had no significant effect on p38γ and p38δ. It is a prototypical ATP-competitive kinase inhibitor that binds to the hinge region and interacts with the anterior and posterior capsules of the ATP-binding site of p38. Pamapimod also inhibited LPS-stimulated TNF-α, IL-1β produced in whole blood and spontaneous TNF-α produced in RA patients. It was also shown that Pamapimod inhibits phosphorylation of HSP27, a MAPKAPK-2 substrate, which directs p38 signaling through MAPKAPK-2. A Pamapimod binding assay for 350 other protein kinases¹⁵⁶ showed that the molecule is highly selective and binds only to another MAPK family member, JNK, with an affinity comparable to that of p38. However, despite the apparent affinity for JNK, the evaluation of JNK inhibition based on c-Jun phosphorylation in both cell types indicated that Pamapimod was ineffective against JNK in cells. Thus, Pamapimod in vivo is expected to have little direct effect on JNK activity. In the process of clinical trials on the treatment of RA, it was found that compared with methotrexate, the disease response rate of patients with active RA treated with single drug Pamapimod was reduced and the incidence of adverse events was increased, which was obviously not suitable as a substitute for methotrexate. Therefore, it was considered as a possible supplement to methotrexate treatment.¹⁵⁷ In conclusion, Pamapimod is a highly selective inhibitor of p38α/β and the anti-inflammatory activity of Pamapimod both in cells and in vivo suggests its potential to alleviate inflammation.

The inhibitor Talmapimod (SCIO-469) (58) developed by SCIOS is a potent p38α inhibitor that is more than 1000 times more selective than ERK2, JNK1, and Lck. The distinctive feature of indoleamides is that they can achieve high selectivity to p38α and p38β with IC₅₀ of 14 nM for p38α and 480 nM for p38β. Talmapimod also moderately inhibited LPS-induced TNF-α production in human whole blood with an IC₅₀ of 300 nM. The binding mode of Talmapimod and p38 involves the C5 carbonyl group interacting with p38 Met109 via hydrogen bonding.¹⁵⁸ The lipophilic benzylpiperazine is thought to occupy the selective pocket of the kinase. The o-substituent on the indole ring may help to achieve high p38α selectivity. The adjacent substituent occupies a hydrophobic pocket near the Ala40 residue of p38α, whereas p38β has a larger, more hydrophilic serine residue in a similar position. In rats with experimental arthritis, Talmapimod reduced signs and symptoms of the disease and slowed the progression of the disease. However, in clinical trials, the use of Talmapimod in patients with RA produced only a transient benefit, and no long-term effective benefit. In more recent studies, such as the phase II trial of RA (NCT00043732) in 2003, from 2004 to 2006, the phase Ⅱ trial (NCT00095680) and other studies on multiple myeloma (MM) gradually found that Talmapimod had poor efficacy in vivo.¹⁵⁹

SX-011 (59) has 10-fold p38α/β selectivity, whereas the azindole derivative of SX-011 has 249-fold p38α/β selectivity, making it one of the most selective p38α inhibitors reported.

Ralimetinib (LY2228820) (60) is a trisubstituted imidazole derivative that is a potent and selective ATP-competitive inhibitor of the p38α and β isoforms (IC₅₀ = 5.3 ± 1.6 and 3.2 ± 0.3 nM) (Figure 4). In cell-based assays, Ralimetinib effectively and selectively inhibited p38α MAPK substrate phosphorylation (p-Thr334-MK2) without affecting phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or cMyc. It can also effectively reduce LPS-stimulated cytokines secreted by macrophages and A549 NSCLC cells.¹⁶⁰ Ishitsuka et al.¹⁶¹ explored the therapeutic potential of Ralimetinib for MM, it was confirmed that it inhibited the secretion of IL-6 from bone marrow stromal cells and bone marrow mononuclear cells (BMMNC) of patients with MM in remission. LY2228820 also inhibited macrophage inflammatory protein-1a secretion in MM cells and BMMNC from patients and in normal CD14-positive osteoclast precursor cells. In addition, LY2228820 significantly inhibited macrophage colony-stimulating factor and NF-κB ligand-soluble receptor activator-induced osteoclastogenesis in CD14-positive cells in vitro.¹⁶²

SKF-86002 (61) is the lead product of SB203580 (1), the IC₅₀ of SKF-86002 is 0.26, 0.50, 0.40 μM for p38α, IL-1β, and TNF-α. In its bicycloimidazole compounds, the correct regional chemistry on the core heterocyclic ring is essential for effective binding of p38, confirming that only on the imidazole ring close to the pyridine ring is nitrogen tolerated as a substitutant without loss of nitrogen activity. The inhibition of IL-1β by SKF86002 showed that SKF86002 could only reduce eight peptides produced by monocytes. Inhibition of IL-1β production was achieved by influencing two independent steps in the biogenesis of this cytokine. One is that SKF86002 reduces the synthesis of proIL-1β, and the second effect of SKF86002 is to inhibit the release of IL-1β by affecting high concentrations of LPS.¹⁶³ In the treatment of streptococcus M1 protein-induced neutrophil activation and lung injury,¹⁶⁴ M1 protein activation increased the phosphorylation and activity of p38MAPK in the lung, while SKF 86002 reduced M1-induced neutrophil infiltration, pulmonary edema, and CXC chemokine formation by inhibiting p38MAPK activity, and the upregulation of neutrophils by Mac-1. Recently, Masliah's team¹⁶⁵ used SKF-86002 to treat DLB/PD in vitro cell and mouse models and found that it promoted the redistribution of p38γ to synapses and reduced α-synuclein accumulation, neuron loss, and motor deficits in the mouse brain.

Kaieda et al.¹⁶⁶ in 2019 used a structure-based design strategy to identify a series of p38MAPK inhibitors through high-throughput screening, including the discovery and design of a derivative of imidazopyridinone 65 that is a potent inhibitor of p38MAPK (p38α IC₅₀ = 9.6 nM) and a derivative of imidazopyridinone that is a potent inhibitor of p38MAPK. It also showed excellent inhibitory effect on TNF-α production (THP-1 IC₅₀ = 46 nM) and TNF-α induced IL-8 production (hWB IC₅₀ = 15 nM) in human monocytic leukemia cells. During its development, the lead compound 62 was first selected by high-throughput screening with an IC₅₀ of 140 nM for p38α. On the basis of the cocrystal structure analysis of p38α, which is thought to interact with Met109 and Gly110 in a bidentate manner, several scaffolds were first explored, including hydroxyindole, aza-indole, benzimidazolone and imidazopyridin-2-one. Compound 63 with imidazopyridin-2-one as the basic skeleton was found to be relatively different from other scaffolds. It has a better inhibition ability (IC₅₀ = 390 nM). When compound 63 was further optimized, it was found that the substitution group on the terminal benzene ring was important for improving the inhibitory activity, so a more excellent compound 64 with IC₅₀ = 63 nM was obtained by 2, 4-difluoride substitution on benzene. Finally, the most excellent compound 65 with IC₅₀ = 9.6 nM was obtained by optimizing the substituent of imidazole. In the cocrystalline structure of compound 65 and p38α, aniline hydrogen binds Lys53 and Asp168 via water-mediated hydrogen bonds, and 2, 4-difluorophenyl enters the shallow inner hydrophobic pocket of p38MAPK composed of Thr106 and Leu104. Pharmacokinetic study in rats showed C_m = 55.8 ng/mL, T_m = 2.7 h, AUC = 223.2 ng·h/mL. After comprehensive evaluation of its inhibitory effect, solubility, and pharmacokinetics, this compound was considered to be the best potential lead compound in this series of imidazo[4, 5-B]pyridin-2-one-based p38 MAP kinase inhibitors.

The IC₅₀ of AZD6703 (66) was 16 nM for p38α and 0.027 μM for HWB (Figure 5). AZD6703 was originally developed as a bisamide inhibitor. The earliest compound 67 had related inhibitory activities, but its water solubility was poor and plasma protein binding was quite high. Previous studies have shown that its terminal heteraryl methoxy can be removed without significantly reducing potency and replaced by side chains/rings containing alkaline amines, thereby improving solubility and plasma protein binding.¹⁶⁷ D.S. Bowen et al.¹⁶⁷ obtained a novel inhibitor AZD6703 by sequentially converting two aniline embedding functions in the diamide series into quinazolinone and N-cyclopropyl amide. Physical and PK properties are improved by introducing weakly basic arylpiperazine groups and reducing molecular weight, and activity in human whole blood is improved by reducing plasma protein binding. Interaction of piperazine basic nitrogen with Val30 amido-carbonyl group in eutectic structure of AZD6703 and p38α (PDB: 4AA5). The structural study showed that the inhibitor changed from “DFG out” mode to “DFG in” mode, and was of I½ type. In 2020, Raubo et al.¹⁶⁸ believed that the alternative groups of N-methylpiperazine in AZD6703 should also be easy to study, and compound 68 of 3, 5-dibromo-2(1H)-pyrazinone was selected as a new inhibitor framework for study, resulting in a series of new inhibitor types.

The IC₅₀ of R1487 (69) is 10 ± 4 nM for p38α and 200 ± 87 nM for HWB. For kinase selectivity, Kd for p38α is 0.2 nM and Kd for p38β is 29 nM, which does not bind to p38γ and p38δ. In the presence of compound 69, detailed Kd assays of kinases inhibited in a single spot screening confirmed that only 10 of the >300 kinases tested were submicromole inhibited. The lead compound of R1487 is compound 70, was originally designed as an inhibitor of lymphocyte-specific protein tyrosine kinase (Lck), a member of the src kinase family, with Kd = 0.2 nM for Lck and Kd = 10 nM for p38α. In SAR analysis, incorporation of saturated alkyl amines at the 2-site increased the three-dimensional volume of the molecule and led to adverse spatial interactions between inhibitors and kinases containing glycine insertions in the lower lip region. To further enhance the selectivity of kinase, additional template modification was carried out to expand the volume of the posterior hydrophobic capsule of p38α by inserting oxygen atoms between the core scaffold and the aromatic ring.¹⁶⁹

BMS-582949(PS-540446) (71) was developed by Liu et al.¹⁷⁰ in 2010. The IC₅₀ was 13 nM for p38α and 50 nM for hPBMC TNF-α. Its lead compounds pyrrole [2, 1 f] [1, 2, 4] triazine derivatives 72 and 73 are potent p38αMAPK kinase inhibitors, however, compounds 72 and 73 showed high clearance in vivo. In the course of its studies, the N-methoxy benzamide functional groups in 72 and 73 could be partially replaced by urea, carbamates, and reverse amides without methoxy amino groups, resulting in a number of highly potent p38αMAPK kinase inhibitors. The cocrystal structure of BMS-582949 and p38α (PDB: 3MVM) shows that the binding mode of N-cyclopropyl benzamide is similar to that of 72. The benzamide moiety provides two key hydrogen bond interactions with p38α: one with the carbonyl oxygen of Asp168 and the NH interaction with Glu71. Another important hydrogen bond interaction occurs between the 6-carboxamide oxygen and Met109 in the hinge region. Hydrophobic interactions include the angular methaniline moiety located in the hydrophobic pocket, and the n-propyl group interacting with the typical hydrophobic point on the outer edge of the hinge region.¹⁷⁰ Evaluated in a sham rat model of AA, BMS-582949 showed dose-dependent reduction in foot swelling, with efficacy seen at doses of 10 and 100 mg/kg. In addition, in the human hepatotoxicity assay, the IC₅₀ of all its subtypes were detected greater than 138 μM, demonstrating no cytotoxicity and suggesting a reduced likelihood of clinical hepatotoxicity. For clinical trials, a phase I trial for RA (NCT00162292) was conducted from 2005 to 2007, and a phase II trial for Psoriasis (NCT00399906) was conducted from 2007 to 2009.

4.1.3 Tricyclic p38α and p38β inhibitors

Skepinone-L (74) is a dibenzocycloheptanone compound developed by Koeberle et al.¹⁷¹ in 2011 (Figure 6), which shows excellent effect selectivity in the inhibition of p38α, with Kd = 1.5 nM on p38α. Skepinone-L was effective in cell-free p38αMAPK activity assays with an IC₅₀ of 5 nM, whereas Skepinone-L had an IC₅₀ of 40 nM in whole blood assays. The initial lead compound originally designed by Koeberle et al. was compound 75, which had an IC₅₀ of 0.10 μM for p38α. This scaffold is able to form a tight complementary surface with the linker strand, thereby utilizing small gatekeepers and also inducing l-glycine to flip with carbonyl oxygen. However, due to limited efficacy in vivo, whole blood analysis failed with an IC₅₀ of 3.8 μM. A flexible linear dihydroxypropoxy moiety was later introduced at position 7 of the dibenzocyclic heptenone, as calculations showed that this moiety was well integrated into hydrophobic region II and that its OH group might form additional hydrogen bonds, resulting in the more potent compound Skepinone-L.¹⁷¹ Skepinone-L significantly reduced TNF-α, IL-1β, and IL-10 (IC₅₀ = 30-50 nM). In addition, the production of IL-6, IL-8, IL-13, IL-17α, and IFN-γ was significantly inhibited. However, Skepinone-L did not affect the release of IL-4, IL-5, Monocyte chemoattractant protein-1 and Interferon-gamma inducible protein 10 (high reactive protein). Under its action, LPS-induced TNF-α release was inhibited by 77%,¹⁷¹ which utilizes small gatekeepers with carbonyl oxygen to induce peptide flipping. In addition, an additional hydrogen bond is formed between the terminal OH of the 2, 3-dihydroxypropoxy moiety and the backbone carbonyl group of Gly110, which gives it a better binding force to p38α. Its inhibition of Aβ-induced proinflammatory cytokine production in AD, expression of adhesion proteins associated with cardiac and peripheral inflammation, and induced angiogenesis in cancer have potential therapeutic implications in studies of neurological injury and neurodegeneration after stroke.

Gong et al.¹⁷² also designed two promising potential inhibitors 76 and 77 of p38β in 2022, with IC₅₀ of 6.4 and 4.2 nM. During its design, although it is generally believed that p38α and p38β are highly similar, the ATP-binding pocket of p38β is smaller than that of p38α, which could be used to selectively develop p38β inhibitors. Gong et al. optimized the structure based on the known p38α inhibitor Skepinone-L. First, the solvent region of kinase was modified with a hydrophilic group, and the ethylene glycol of Skepinone-L was replaced by a butyric acid group at this position to achieve a significant improvement in the inhibition ability, and the compound 76 was obtained. The IC₅₀ of p38β was 6.4 ± 0.6 nM. Compound 77 was obtained by substitution of a methyl group with a small hydrophobic group in the hydrophobic region II, which further improved the inhibitory ability of p38β with an IC₅₀ of 4.2 ± 0.1 nM. These two optimized compounds have better inhibitory ability than Skepinone-L with excellent inhibitory effect, which is worthy of further in vitro and in vivo experiments.

Sabatini et al.¹⁷³ developed a series of pyrazolam benzothiazide p38α selective inhibitors in 2015, in which compound 78 had an IC₅₀ of 0.457 μM against p38α and 0.5 μM against TNF-α. In addition, the IC₅₀ of p38β was 1.7 μM, while the inhibitory effect on other kinases such as CDC2, CDK5, JAK2/3, Lck, and SRC was not detected. In its structure with p38α, sulfone oxygen of the ligand interacts with Met109, p-fluorophenyl is located in hydrophobic region I, and the pyrazole ring and Phe169 have stack interactions. In addition, N-2 atoms form anchored hydrogen bonds with Lys53. The low molecular weight, ligand efficiency value and kinase selectivity of pyrazolam benzothiazide compound 78 make this derivative a potentially effective novel inhibitor structure.

In addition, Bartolini et al.¹⁷⁴ identified a series of p38 inhibitors with the pyrazo-benzothiazide core in 2019. In addition to compound 78, another different derivative of the same skeleton, COXH11 (79), also showed good p38α inhibitory activity with IC₅₀ = 0.936 μM. Studies in HT29 cells showed that compound 78 had CC₅₀ of 150 μM (24 h) and 95 μM (48 h), whereas COXH11 had no detectable CC50 in cells. They also demonstrated that compounds 78 and 79 inhibited the phosphorylation of p38αMAPK stimulated by LPS and H2O2. The cocrystal structure of the two and p38α (PDB: 5OMG, PDB: 5OMH) shows that the pyridine ring in compound 78 forms a water-mediated contact with the carbonyl group of Phe169 in DFG, the sulfonyl group has a hydrogen bond with the backbone NH of Met109, the fluorophenyl group occupies a hydrophobic pocket, namely hydrophobic region Ⅰ, and the carbonyl group of Phe169 in DFG has a hydrogen bond. Lys53 and Glu71 still had salt Bridges. The conformation of 79 and 78 is significantly different. The hinge contact is achieved by the halide bond between the chlorophenyl group and the His107 carbonyl group. The sulfonyl moiety directly forms a hydrogen bond with the backbone NH of Asp168 in DFG and forms a hydrogen bond with Glu71. In conclusion, compounds 78 and 79 are potent inhibitors of the free radical-dependent pathway activated by p38αMAPK, clearly affecting upstream kinases such as MAP3Ks responsible for the co-activation of p38α MAPK and JNK. All of them can be used as lead compounds. Given their good in vitro activity, their in vivo activity and safety can be further investigated in animal models to provide reference for the development of selective p38 inhibitors for clinical application.