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]

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]

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- 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]

- 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]

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.

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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].

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- 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]

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.

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- 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]

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.

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- 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]

- Oral Bioavailability in Rats: After oral administration of BIRB 796 (10 mg/kg) to rats, oral bioavailability is 55%, with a peak plasma concentration (Cmax) of 2.3 μg/mL at 1 hour (Tmax) [6]
- Plasma Half-Life: In rats, the plasma half-life of BIRB 796 is 4.2 hours after i.v. administration (1 mg/kg) [6]

- Acute Toxicity in Mice: No mortality is observed in mice after single oral doses of BIRB 796 up to 300 mg/kg. Serum ALT and AST levels remain unchanged compared to controls, indicating no acute hepatotoxicity [6]
- Plasma Protein Binding: BIRB 796 binds to human plasma proteins at 98% [6]

[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 member of the class of pyrazoles that is an immunomodulator used for treatment of rheumatoid arthritis, Crohn's disease and psoriasis. It has a role as an immunomodulator and an EC 2.7.11.24 (mitogen-activated protein kinase) inhibitor. It is a member of morpholines, a member of pyrazoles, a member of naphthalenes, a member of ureas and an aromatic ether.
Doramapimod is a P38 MAP kinase inhibitor.

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The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics, consistent with the requirement for conformational change. Improving interactions in this allosteric pocket, as well as establishing binding interactions in the ATP pocket, enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series, BIRB 796, has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture.[1]

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We report on a series of N-pyrazole, N'-aryl ureas and their mode of binding to p38 mitogen activated protein kinase. Importantly, a key binding domain that is distinct from the adenosine 5'-triphoshate (ATP) binding site is exposed when the conserved activation loop, consisting in part of Asp168-Phe169-Gly170, adopts a conformation permitting lipophilic and hydrogen bonding interactions between this class of inhibitors and the protein. We describe the correlation of the structure-activity relationships and crystallographic structures of these inhibitors with p38. In addition, we incorporated another binding pharmacophore that forms a hydrogen bond at the ATP binding site. This modification affords significant improvements in binding, cellular, and in vivo potencies resulting in the selection of 45 (BIRB 796) as a clinical candidate for the treatment of inflammatory diseases.[2]

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The compound BIRB796 inhibits the stress-activated protein kinases p38alpha and p38beta and is undergoing clinical trials for the treatment of inflammatory diseases. Here we report that BIRB796 also inhibits the activity and the activation of SAPK3/p38gamma. This occurs at higher concentrations of BIRB796 than those that inhibit p38alpha and p38beta and at lower concentrations than those that inhibit the activation of JNK isoforms. We also show that at these concentrations, BIRB796 blocks the stress-induced phosphorylation of the scaffold protein SAP97, further establishing that this is a physiological substrate of SAPK3/p38gamma. Our results demonstrate that BIRB796, in combination with SB203580, a compound that inhibits p38alpha and p38beta, but not the other p38 isoforms, can be used to identify physiological substrates of SAPK3/p38gamma as well as those of p38alpha and p38beta.[3]

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We have previously shown 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 examined anti-MM activity of a novel p38 MAPK inhibitor, BIRB 796, alone and in combination with conventional and novel therapeutic agents. BIRB 796 blocked baseline and bortezomib-triggered upregulation of p38 MAPK and Hsp27 phosphorylation, thereby enhancing cytotoxicity and caspase activation. The Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin (17-AAG) upregulated protein expression and phosphorylation of Hsp27; conversely, BIRB 796 inhibited this phosphorylation and enhanced 17-AAG-induced cytotoxicity. Importantly, BIRB 796 inhibited Hsp27 phosphorylation induced by 17-AAG plus bortezomib, thereby enhancing cytotoxicity. In bone marrow stromal cells (BMSC), BIRB 796 inhibited phosphorylation of p38 MAPK and secretion of interleukin-6 (IL-6) and vascular endothelial growth factor triggered by either tumour necrosis factor-alpha or tumour growth factor-beta1. BIRB 796 also inhibited IL-6 secretion induced in BMSCs by adherence to MM cells, thereby inhibiting tumour cell proliferation. These studies therefore suggest that BIRB 796 overcomes drug-resistance in the BM microenvironment, providing the framework for clinical trials of a p38 MAPK inhibitor, alone and in combination with bortezomib, Hsp90 inhibitor, or Dex, to improve patient outcome in MM.[4]

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We report on the structure-activity relationships (SAR) of 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl]urea (BIRB 796), an inhibitor of p38alpha MAP kinase which has advanced into human clinical trials for the treatment of autoimmune diseases. Thermal denaturation was used to establish molecular binding affinities for this class of p38alpha inhibitors. The tert-butyl group remains a critical binding element by occupying a lipophilic domain in the kinase which is exposed upon rearrangement of the activation loop. An aromatic ring attached to N-2 of the pyrazole nucleus provides important pi-CH(2) interactions with the kinase. The role of groups attached through an ethoxy group to the 4-position of the naphthalene and directed into the ATP-binding domain is elucidated. Pharmacophores with good hydrogen bonding potential, such as morpholine, pyridine, and imidazole, shift the melting temperature of p38alpha by 16-17 degrees C translating into K(d) values of 50-100 pM. Finally, we describe several compounds that potently inhibit TNF-alpha production when dosed orally in mice.[5]

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

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.

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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.

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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.

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

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