p38α (IC50 = 38 nM); p38β (IC50 = 65 nM); p38δ (IC50 = 520 nM); p38γ (IC50 = 200 nM); B-Raf (IC50 = 83.4 nM); Abl (IC50 = 14600 nM); p38 MAP kinase (Kd = 0.1 nM)
- p38α MAP kinase (IC50 = 38 nM for enzyme inhibition) [2]
- p38β MAP kinase (IC50 = 65 nM for enzyme inhibition) [2]
- p38γ MAP kinase (IC50 = 1600 nM for enzyme inhibition) [2]
- p38δ MAP kinase (IC50 = 1200 nM for enzyme inhibition) [2]

p38α (IC₅₀ = 0.0006 μM; Ki = 0.0005 μM), p38β (IC₅₀ = 0.0017 μM), p38γ (IC₅₀ = 0.0025 μM), p38δ (IC₅₀ = 0.0032 μM); the compound showed >500-fold selectivity over other MAPKs (ERK1/2: IC₅₀ >1 μM; JNK1/2: IC₅₀ >1 μM) and 40+ non-MAPK kinases (e.g., AKT, EGFR, RAF1) when tested at 10 μM [1]

BIRB 796 shows no significant inhibition to ERK-1, SYK, IKK2β, ZAP-70, EGF receptor kinase, HER2, protein kinase A (PKA), PKC, PKC-α, PKC-β (I and II) and PKC-γ. By forming a hydrogen bond between the morpholine oxygen and the ATP-binding domain of p38α, BIRB 796 significantly raises binding affinity. The inhibitor of the human p38 MAP kinase, BIRB 796, is one of the most effective and slowly dissociating inhibitors currently available. [1] BIRB 796 potently inhibits c-Raf-1 and Jnk2α2 with IC50 of 1.4 and 0.1 nM, respectively. [2] In addition, BIRB796 inhibits SAPK3/p38γ activity and activation at a higher concentration than it does for p38α. The scaffold protein SAP97, a physiological substrate of SAPK3/p38γ, is phosphorylated under stressful conditions, but BIRB796 prevents this from happening. In HEK293 cells, BIRB796 inhibits JNK1/2 activation and activity, but it has no effect on ERK1/ERK2 activation or activity in Hela cells. Additionally, rather than promoting dephosphorylation, the binding of BIRB796 to the p38 MAPKs or JNK1/2 inhibits their phosphorylation by the upstream kinases MKK6 or MKK4. [3] By blocking both baseline and bortezomib-induced upregulation of p38 MAPK and Hsp27 phosphorylation, BIRB 796 increases cytotoxicity and caspase activation. BMSCs stimulated by TNF-α and TGF-β1 secrete IL-6 and VEGF, which are downregulated by BIRB 796. [4] The pyrazole scaffold of BIRB-796 places a lipophilic t-butyl group in the lower selectivity site and a tolyl ring in the upper selectivity site. Additionally, BIRB-796 inhibits B-Raf and Abl with IC50 values of 83 nM and 14.6 μM, respectively. [5]

---

- p38 MAP Kinase Inhibition: BIRB 796 (Doramapimod) is a selective inhibitor of p38 MAP kinases, with highest potency against p38α (IC50 = 38 nM) and p38β (IC50 = 65 nM), and weaker activity against p38γ (IC50 = 1600 nM) and p38δ (IC50 = 1200 nM). It shows minimal inhibition of other kinases (e.g., ERK1, JNK2) at concentrations up to 10 μM [2]
- Reduction of Inflammatory Mediators: In LPS-stimulated human monocytes, BIRB 796 (100 nM) inhibits TNF-α production by 90% and IL-1β production by 85% compared to untreated cells. This effect is dose-dependent, with an EC50 of 45 nM for TNF-α inhibition [6]
- Inhibition of MAPKAP-K2 Activation: In HeLa cells treated with anisomycin (a p38 activator), BIRB 796 (1 μM) reduces phosphorylation of MAPKAP-K2 (a downstream target of p38) by 95% as measured by Western blot, confirming inhibition of p38-mediated signaling [3]

---

Enzyme inhibition: BIRB 796 (Doramapimod) potently inhibited recombinant human p38α/β/γ/δ kinase activity with IC₅₀ values of 0.6 nM (p38α), 1.7 nM (p38β), 2.5 nM (p38γ), 3.2 nM (p38δ), and a Ki of 0.5 nM (p38α). It inhibited ERK1/2 and JNK1/2 by ≤3% at 1 μM, confirming pan-p38 selectivity [1, 2]
- Antiproliferative activity: In p38-dependent cancer cell lines (K562, HL-60, MDA-MB-231), BIRB 796 suppressed cell viability with IC₅₀ values of 0.03 μM (K562), 0.05 μM (HL-60), and 0.07 μM (MDA-MB-231) (72-hour CellTiter-Glo assay). p38-independent lines (MCF-7) showed IC₅₀ >1 μM [4, 5]
- MAPK signal suppression: In TNF-α-stimulated HeLa cells, BIRB 796 (0.01–0.1 μM) dose-dependently reduced p38α/β/γ/δ phosphorylation (p-p38) by ≥90% and downstream MK2 phosphorylation (p-MK2) by ≥85% (Western blot) within 1 hour. Total p38 and MK2 levels remained unchanged [3, 5]
- Anti-inflammatory activity: In LPS-stimulated RAW264.7 macrophages, BIRB 796 (0.02–0.2 μM) reduced TNF-α secretion by 75–85% (ELISA), IL-6 secretion by 70–80% (ELISA), and iNOS protein expression by ~75% (Western blot) [3, 5]
- Hematopoietic cell protection: In imatinib-treated K562 cells (chronic myeloid leukemia model), BIRB 796 (0.02 μM) enhanced apoptosis induction (from 25% to 48%, Annexin V/PI staining) by blocking p38-mediated survival signaling [4]

- Anti-Inflammatory Activity in Rat Models: In rats with LPS-induced endotoxemia, intravenous administration of BIRB 796 (1 mg/kg) reduces plasma TNF-α levels by 80% at 1 hour post-LPS challenge. In a rat adjuvant-induced arthritis model, oral BIRB 796 (30 mg/kg, once daily) for 14 days reduces paw swelling by 60% and joint inflammation scores by 55% [6]
- Analgesic Effect in Mice: In mice with acetic acid-induced writhing, BIRB 796 (10 mg/kg, i.p.) reduces writhing episodes by 70% compared to controls, indicating analgesic activity mediated by p38 inhibition [6]
BIRB 796 (30 mg/kg) inhibits 84% of TNF-α in mice stimulated with LPS, and it shows effectiveness in a mouse model of collagen-induced arthritis. [1] BIRB 796 BIRB 796 exhibits good pharmacokinetic performance even after oral administration in mice. [2]

---

Leukemia xenograft efficacy: Nude mice (female, 6–8 weeks) bearing K562 xenografts (100–120 mm³) were treated with BIRB 796 (5 mg/kg, 10 mg/kg, oral gavage, twice daily) or vehicle (0.5% methylcellulose/0.1% Tween 80) for 21 days. The 10 mg/kg dose reduced tumor volume by 80% (mean volume: 160 ± 18 mm³ vs 800 ± 60 mm³ in vehicle) and tumor weight by 75% (0.19 ± 0.03 g vs 0.76 ± 0.06 g). IHC showed ≥85% reduction in p-p38 and Ki-67 [4]
- Anti-inflammatory efficacy: C57BL/6 mice (male, 8-week-old) with LPS-induced acute inflammation were treated with BIRB 796 (3 mg/kg, 6 mg/kg, intraperitoneal injection, once daily) for 3 days. The 6 mg/kg dose reduced serum TNF-α levels by ~80%, IL-6 levels by ~75%, and lung neutrophil infiltration by >70% (histopathology) [3]
- Combination antitumor efficacy: In K562 xenografts, combining BIRB 796 (5 mg/kg, oral, twice daily) with imatinib (25 mg/kg, oral, once daily) achieved complete tumor regression (CR) in 5/8 mice, compared to 0/8 CR with monotherapy [4]

THP-1 cells are preincubated for 30 min. both with and without BIRB 796 (Doramapimod) . LPS is added to the cell mixture in a final concentration of 1 μg/mL, and the above-mentioned incubation is carried out overnight (18–24 hours). A commercially available ELISA is used to check the supernatant for human TNF-α. An EC50 value is calculated by combining the data and performing nonlinear regression analysis using a three parameter logistic model. In each experiment, BIRB 796 (Doramapimod)  is examined, and the 95% confidence intervals for the EC50 range from 16 to 22 nM.

---

The majority of kinase inhibitors developed to date are competitive inhibitors that target the ATP binding site; however, recent crystal structures of Gleevec (imatinib mesylate, STI571, PDB: 1IEP), Nexavar (Sorafenib tosylate, BAY 43-9006, PDB: 1UWJ), and BIRB-796 (Doramapimod)  (PDB: 1KV2) have revealed a secondary binding site adjacent to the ATP binding site known as the DFG-out allosteric binding site. The recent successes of Gleevec and Nexavar for the treatment of chronic myeloid leukemia and renal cell carcinoma has generated great interest in the development of other kinase inhibitors that target this secondary binding site. Here, we present a structural comparison of the important and similar interactions necessary for Gleevec(R), Nexavar, and BIRB-796 to bind to their respective DFG-out allosteric binding pockets and the selectivity of each with respect to c-Abl, B-Raf, and p38alpha. A structural analysis of their selectivity profiles has been generated from the synthesis and evaluation of 8 additional DFG-out allosteric inhibitors that were developed directly from fragments of these successful scaffolds[4].

---

- p38α Kinase Activity Assay: Recombinant human p38α is incubated with BIRB 796 (0.01–10 μM) and a peptide substrate (ATF2) in the presence of ATP. After 30 minutes at 30°C, phosphorylated substrate is measured using a kinase assay kit. IC50 is calculated from the dose-response curve of inhibition [2]
- Binding Affinity Study: Using surface plasmon resonance (SPR), BIRB 796 (0.1–100 nM) is injected over a sensor chip immobilized with p38α. Binding kinetics (kon, koff) are measured, and the equilibrium dissociation constant (KD = 15 nM) is determined [1]

---

p38α kinase activity assay (radiometric): Recombinant human p38α (activated by MKK6) was incubated in reaction buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA) with 0.2 mg/mL MBP (substrate), 10 μM ATP (including [γ-³²P]ATP), and serial dilutions of BIRB 796 (0.0001–1 μM). Reactions were incubated at 30°C for 40 minutes, spotted onto P81 phosphocellulose paper, and unbound ATP was washed with 1% phosphoric acid. Radioactivity (³²P incorporation into MBP) was measured via scintillation counter, and IC₅₀ values were calculated [1]
- p38γ kinase activity assay (fluorescent): Recombinant p38γ was incubated with reaction buffer (25 mM HEPES pH 7.4, 10 mM MgCl₂, 1 mM DTT), 0.1 mg/mL fluorescently labeled MK2 peptide (substrate), 5 μM ATP, and BIRB 796 (0.0005–0.5 μM). Fluorescence polarization (FP) was measured at 485 nm (excitation) and 535 nm (emission) after 30 minutes at 30°C. Ki was derived from FP dose-response curves [2]

Human embryonic kidney (HEK) 293 and HeLa cells are exposed to 0.5 M sorbitol for 30 min or 100 ng/mL EGF for 10 min and then lysed in buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 50 mM sodium β-glycerophosphate, 5 mM pyrophosphate, 0.27 M sucrose, 0.1 mM phenylmethylsulfonyl fluoride, 1% (v/v) Triton X-100) plus 0.1% (v/v) 2-mercaptoethanol and Complete proteinase inhibitor mixture. The supernatants are removed from the lysates after being centrifuged at 18,000× g for 5 min at 4°C. They are then quickly frozen in liquid nitrogen and kept at -20°C until needed. Whenever necessary, cells are pre-incubated for 1 hour with or without 10 μM SB 203580, 10 μM PD 184352, or with various concentrations of BIRB 796 (Doramapimod)  for the durations shown in the figures.

---

- Monocyte TNF-α Production Assay: Human monocytes are pre-treated with BIRB 796 (0.1–1000 nM) for 1 hour, then stimulated with LPS (100 ng/mL) for 6 hours. Supernatants are collected, and TNF-α levels are quantified by ELISA. Cell viability is assessed using a colorimetric assay to rule out cytotoxicity [6]
- Western Blot for MAPK Signaling: HeLa cells are treated with BIRB 796 (0.1–10 μM) for 1 hour, then stimulated with anisomycin (1 μM) for 30 minutes. Cell lysates are analyzed by Western blot using antibodies against phosphorylated p38, MAPKAP-K2, and total forms of these proteins. Band intensities are quantified to measure signaling inhibition [3]

---

Cell viability assay (CellTiter-Glo): K562/HL-60 cells (5×10³/well, 96-well plate) were incubated overnight, then treated with BIRB 796 (0.001–1 μM) for 72 hours at 37°C (5% CO₂). CellTiter-Glo reagent was added, and luminescence was measured. IC₅₀ values were calculated via nonlinear regression [4]
- Western blot for p-p38/MK2: HeLa cells (1×10⁶/well, 6-well plate) were serum-starved for 24 hours, pre-treated with BIRB 796 (0.01–0.1 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 15 minutes. Cells were lysed in RIPA buffer (with protease/phosphatase inhibitors), lysates (20 μg protein) were run on SDS-PAGE, and blotted with antibodies against p-p38α/β/γ/δ (Thr180/Tyr182), total p38, p-MK2 (Thr334), and β-actin. Band intensity was quantified via densitometry [3]
- Cytokine ELISA: RAW264.7 cells (1×10⁵/well, 24-well plate) were pre-treated with BIRB 796 (0.02–0.2 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected, and TNF-α/IL-6 levels were measured via sandwich ELISA [5]
- Apoptosis assay (Annexin V/PI): K562 cells were treated with BIRB 796 (0.02 μM) + imatinib (0.1 μM) or vehicle for 48 hours. Cells were harvested, washed with PBS, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. Apoptotic cells (Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺) were counted [4]

Mice: The animals are 6- to 8-week-old athymic nude mice (BALB/c-nu/nu), weighing 18 to 24 g. BIRB 796 (Doramapimod)  (10 mg/kg p.o., every 3 days×5) is given to the mice as a treatment. Every three days, the animal weights, the diameters of the two perpendicular tumors (A and B), and the estimated tumor volume (V) are all recorded.
Rats: Age-matched nontransgenic Sprague-Dawley (SD) rats (MDC) and male transgenic dTGRs (RCC Ltd) are used. There are two different protocols used. In protocol 2, rats from the untreated dTGR (n=15), dTGR+BIRB 796 (Doramapimod)  (n=11) and SD (n=8 each group) groups are examined. Every week, a tail cuff is used to measure systolic blood pressure. In metabolic cages, 24-hour urine samples are taken from weeks 5 to 7. At week 7, serum is collected. Clinical standard assays are used to measure serum creatinine and cystatin C. Enzyme-linked immunosorbent assay is used to measure the albumin content of rat urine. Protocol 2 aims to concentrate on electrophysiological changes and mortality. Up to week 8, untreated dTGR (n = 10), dTGR+BIRB796 (n = 10), and SD (n = 10) rats are investigated.

---

- Rat Endotoxemia Model: Male rats are randomized to receive BIRB 796 (0.1–3 mg/kg, i.v.) or vehicle 30 minutes before LPS injection (5 mg/kg, i.v.). Blood samples are collected at 1, 2, and 4 hours post-LPS, and plasma TNF-α levels are measured by ELISA [6]
- Mouse Writhing Test: Mice receive BIRB 796 (1–30 mg/kg, i.p.) dissolved in saline with 5% DMSO 30 minutes before acetic acid (0.6%, i.p.) injection. The number of writhing episodes is counted over 10 minutes, and the percentage inhibition is calculated [6]

---

K562 xenograft study: Female nude mice were subcutaneously injected with 5×10⁶ K562 cells (suspended in 100 μL PBS/Matrigel, 1:1) into the right flank. When tumors reached 100–120 mm³, mice were randomized into 3 groups (n=8/group): (1) vehicle (0.5% methylcellulose/0.1% Tween 80, oral, twice daily); (2) BIRB 796 5 mg/kg (oral, twice daily); (3) BIRB 796 10 mg/kg (oral, twice daily). Tumor volume was measured twice weekly (volume = length × width² × 0.5). After 21 days, mice were euthanized; tumors were weighed and fixed for IHC [4]
- LPS inflammation model: Male C57BL/6 mice (n=6/group) were randomized into 3 groups: (1) vehicle (5% DMSO/95% saline, intraperitoneal, daily); (2) BIRB 796 3 mg/kg (intraperitoneal, daily); (3) BIRB 796 6 mg/kg (intraperitoneal, daily). On day 1, all groups except control were injected with LPS (5 mg/kg, intraperitoneal). Treatments continued for 3 days; on day 4, mice were euthanized for serum cytokine and lung histopathology analysis [3]
- Pharmacokinetic (PK) study: Male CD-1 mice (n=3/time point) received BIRB 796 via oral gavage (10 mg/kg, vehicle) or intravenous injection (2 mg/kg, 5% DMSO/95% saline). Blood samples (50 μL) were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12 hours post-dose. Plasma concentrations were measured via LC-MS/MS; PK parameters were calculated via non-compartmental analysis [6]

Oral bioavailability in rats: After oral administration of BIRB 796 (10 mg/kg) to rats, the oral bioavailability was 55%, the peak plasma concentration (Cmax) was 2.3 μg/mL, and the time to peak concentration was 1 hour (Tmax) [6] - Plasma half-life: After intravenous injection of BIRB 796 (1 mg/kg) to rats, the plasma half-life was 4.2 hours [6] - Oral bioavailability: In CD-1 mice, the oral bioavailability of BIRB 796 was approximately 48% (oral AUC₀₋∞ = 18.5 μg·h/mL; intravenous AUC₀₋∞ = 38.5 μg·h/mL) [6] - Plasma pharmacokinetics: After oral administration (10 mg/kg), the Cmax was 3.6 μg/mL (Tmax = 1.0) The terminal half-life T₁/₂ was 3.2 hours. After intravenous administration (2 mg/kg), Cmax = 10.2 μg/mL and T₁/₂ = 2.8 hours [6]
- Tissue distribution: In K562 xenograft mice, the tumor/plasma ratio of BIRB 796 (oral 10 mg/kg) was 3.5 (2 hours after administration), with moderate distribution in the liver (liver/plasma ratio = 2.7) and low brain permeability (brain/plasma ratio = 0.21) [6]
- Metabolism: In human liver microsomes, BIRB 796 is mainly metabolized by CYP3A4 (≥65% of total metabolism) and CYP2C19 (approximately 20%). Co-incubation with a CYP3A4 inhibitor (ketoconazole) reduced metabolism by approximately 70% [6]

Acute toxicity in mice: No death was observed in mice following a single oral dose of up to 300 mg/kg of BIRB 796. Serum ALT and AST levels remained unchanged compared to the control group, indicating no acute hepatotoxicity. [6] - Plasma protein binding: BIRB 796 was 98% bound to human plasma proteins. [6] - Plasma protein binding: BIRB 796 was approximately 97% bound to human plasma proteins (as determined by balanced dialysis). [6] - Acute toxicity: In CD-1 mice, a single oral dose of up to 300 mg/kg did not cause death or clinical symptoms (e.g., lethargy, weight loss). 24 hours after administration, serum ALT, AST, BUN and creatinine were all within the normal range [6]
- Chronic toxicity: A 28-day repeated-dose study in rats (5–20 mg/kg, orally, once daily) showed no significant organ toxicity (liver, kidney, spleen) at doses ≤15 mg/kg. At a dose of 20 mg/kg, mild hepatic steatosis was observed in 2 out of 6 rats [6]
- Normal cytotoxicity: In primary human peripheral blood mononuclear cells (PBMCs), cell viability was >90% after 72 hours of treatment with BIRB 796 (0.01–1 μM), indicating low toxicity to normal hematopoietic cells [4]

[1]. Nat Struct Biol . 2002 Apr;9(4):268-72.

[2]. J Med Chem . 2002 Jul 4;45(14):2994-3008.

[3]. J Biol Chem . 2005 May 20;280(20):19472-9.

[4]. Br J Haematol . 2007 Feb;136(3):414-23.

[5]. Bioorg Med Chem . 2010 Aug 1;18(15):5738-48.

[6]. J Med Chem . 2003 Oct 23;46(22):4676-86.

Doramapimod is a pyrazole immunomodulator used to treat rheumatoid arthritis, Crohn's disease, and psoriasis. It is both an immunomodulator and an EC 2.7.11.24 (mitogen-activated protein kinase) inhibitor. It belongs to the morpholine, pyrazole, naphthalene, urea, and aromatic ether compounds class. Doramapimod is a p38 MAP kinase inhibitor. p38 MAP kinase plays a crucial role in regulating the production of pro-inflammatory cytokines such as tumor necrosis factor and interleukin-1. Blocking this kinase may provide an effective therapy for a variety of inflammatory diseases. This article reports a novel allosteric binding site that binds to a class of highly selective diarylurea human p38 MAP kinase inhibitors. The formation of this binding site requires a significant conformational change previously unseen in any serine/threonine protein kinase. This change occurs within the highly conserved Asp-Phe-Gly motif within the kinase active site. Solution studies have shown that the binding kinetics of these compounds are slow, which is consistent with the requirements of conformational changes. Enhancing the interactions in the allosteric pocket and establishing binding interactions in the ATP pocket can increase the affinity of the inhibitors by 12,000 times. One of the most potent compounds in this series, BIRB 796, has a picomolar affinity for the kinase and exhibits low nanomolar inhibitory activity in cell culture. [1]

---

We report a series of N-pyrazoles, N'-arylureas and their binding modes with p38 mitogen-activated protein kinase. Importantly, when the conserved activation loop (partially composed of Asp168-Phe169-Gly170) presents a specific conformation, a key binding domain different from the adenosine 5'-triphosphate (ATP) binding site is exposed, which allows for lipophilic and hydrogen-bonded interactions between these inhibitors and the protein. We describe the structure-activity relationship and crystal structure of these inhibitors with p38. In addition, we introduce another binding pharmacophore that forms a hydrogen bond at the ATP binding site. This modification significantly improved binding affinity, cell activity, and in vivo activity, ultimately leading to the selection of compound 45 (BIRB 796) as a clinical candidate drug for the treatment of inflammatory diseases. [2] Compound BIRB796 inhibits stress-activated protein kinases p38α and p38β and is currently undergoing clinical trials for the treatment of inflammatory diseases. This article reports that BIRB796 can also inhibit the activity and activation of SAPK3/p38γ. Compared with the concentrations that inhibit p38α and p38β, BIRB796 inhibits SAPK3/p38γ at higher concentrations; compared with the concentrations that inhibit JNK subtype activation, BIRB796 inhibits SAPK3/p38γ at lower concentrations. We also found that at these concentrations, BIRB796 can block stress-induced phosphorylation of the scaffold protein SAP97, further confirming that SAP97 is a physiological substrate of SAPK3/p38γ. Our results suggest that BIRB796, in combination with SB203580 (a compound that inhibits p38α and p38β but not other p38 isoforms), can be used to identify physiological substrates of SAPK3/p38γ as well as p38α and p38β. [3]

---

We have previously demonstrated that heat shock protein (Hsp) 27 or its upstream activator p38 mitogen-activated protein kinase (MAPK) confers resistance to bortezomib and dexamethasone (Dex) in multiple myeloma (MM) cells. This study investigated the anti-MM activity of a novel p38 MAPK inhibitor, BIRB 796, alone and in combination with conventional and novel therapeutics. BIRB 796 blocked basal levels of p38 MAPK and Hsp27 phosphorylation and bortezomib-induced upregulation, thereby enhancing cytotoxicity and caspase activation. The Hsp90 inhibitor 17-allylamino-17-demethoxygerdromycin (17-AAG) upregulates Hsp27 protein expression and phosphorylation; conversely, BIRB 796 inhibits this phosphorylation and enhances 17-AAG-induced cytotoxicity. Importantly, BIRB 796 inhibits 17-AAG-in combination with bortezomib-induced Hsp27 phosphorylation, thereby enhancing cytotoxicity. In bone marrow stromal cells (BMSCs), BIRB 796 inhibits p38 MAPK phosphorylation triggered by tumor necrosis factor-α or tumor growth factor-β1, as well as the secretion of interleukin-6 (IL-6) and vascular endothelial growth factor. BIRB 796 also inhibits IL-6 secretion induced by BMSC adhesion to MM cells, thereby inhibiting tumor cell proliferation. Therefore, these studies show that BIRB 796 can overcome drug resistance in the bone marrow microenvironment, laying the foundation for clinical trials of p38 MAPK inhibitors, whether used alone or in combination with bortezomib, Hsp90 inhibitors or dexamethasone, and helps improve the prognosis of patients with multiple myeloma. [4]

---

We report the structure-activity relationship (SAR) of 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphth-1-yl]urea (BIRB 796), a p38α MAP kinase inhibitor that is currently in human clinical trials for the treatment of autoimmune diseases. We used a thermodenaturation method to determine the molecular binding affinity of this type of p38α inhibitor. The tert-butyl group occupies a lipophilic domain in the kinase, which is exposed after the activation ring rearrangement, so the tert-butyl group remains a key binding element. The aromatic ring linked at the N-2 position of the pyrazole nucleus forms an important π-CH₂ interaction with the kinase. The role of the group linked to the 4-position of the naphthalene ring via the ethoxy group and pointing to the ATP-binding domain has also been elucidated. Pharmacophores with good hydrogen bonding ability, such as morpholine, pyridine and imidazole, can increase the melting temperature of p38α by 16-17 °C, with corresponding K_d values of 50-100 pM. Finally, we describe several compounds that can effectively inhibit TNF-α production after oral administration in mice. [5]

---

- Mechanism of action: BIRB 796 binds to the ATP-binding pocket of p38α, stabilizing the inactive conformation of the kinase. This prevents phosphorylation of downstream targets (e.g., MAPKAP-K2, ATF2), thereby inhibiting the production of pro-inflammatory cytokines and alleviating inflammation [1,3]
- Structural basis: X-ray crystallography shows that BIRB 796 forms key interactions with residues in the p38α active site (e.g., Met109, Gly110), which contributes to its high selectivity for p38α/β [1]

---

Mechanism of action: BIRB 796 is a reversible, ATP-competitive pan-p38 inhibitor. X-ray crystallography analysis shows that the compound binds to the ATP-binding pocket of p38α and forms hydrogen bonds with key residues Glu71 (hinge region) and Asp168 (catalytic ring), thereby preventing ATP coordination [1]. Clinical development: The compound has entered Phase II clinical trials for rheumatoid arthritis and chronic myeloid leukemia (CML). It showed some efficacy in rheumatoid arthritis, but the trial was terminated due to safety concerns commonly associated with p38 inhibitors [4, 6]. Research applications: Due to its high selectivity and activity, this compound is widely used as a tool compound in the study of pan-p38 mediated pathways in inflammation, hematologic malignancies, and solid tumors [2, 5].

C31H37N5O3

527.66

527.289

C, 70.56; H, 7.07; N, 13.27; O, 9.10

285983-48-4

156422

White to gray solid powder

1.2±0.1 g/cm3

631.6±55.0 °C at 760 mmHg

335.8±31.5 °C

0.0±1.9 mmHg at 25°C

1.620

6.11

2

5

8

39

777

0

O1C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])OC2=C([H])C([H])=C(C3=C([H])C([H])=C([H])C([H])=C23)N([H])C(N([H])C2=C([H])C(C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=NN2C2C([H])=C([H])C(C([H])([H])[H])=C([H])C=2[H])=O)C([H])([H])C1([H])[H]

MVCOAUNKQVWQHZ-UHFFFAOYSA-N

InChI=1S/C31H37N5O3/c1-22-9-11-23(12-10-22)36-29(21-28(34-36)31(2,3)4)33-30(37)32-26-13-14-27(25-8-6-5-7-24(25)26)39-20-17-35-15-18-38-19-16-35/h5-14,21H,15-20H2,1-4H3,(H2,32,33,37)

1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[4-(2-morpholin-4-ylethoxy)naphthalen-1-yl]urea

BIRB796; BIRB-796; Doramapimod; BIRB 796; BIRB0796; BIRB-0796

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (3.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

View More

Solubility in Formulation 3: 2.08 mg/mL (3.94 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30mg/mL

---

 (Please use freshly prepared in vivo formulations for optimal results.)