p38 MAPK (IC50 = 110 nM)
p38 kinase-alpha (p38α) (IC₅₀ < 200 nmol/L for inhibiting tumor necrosis factor-alpha (TNFα) release in vitro and in vivo); modest selectivity over p38β, and selective over p38γ, p38delta and 50 other kinases screened [1]
- p38 MAP kinase (p38α) [2]

SD0006 is selective for p38α kinase over 50 other kinases screened (including p38γ and p38δ with modest selectivity over p38β). In cellular studies, it reduces the release of inflammatory mediators. Multiple proinflammatory proteins are expressed less frequently when SD0006 is present, both at the transcriptional and translational levels. While SD0006 has no direct COX-1 or COX-2 inhibitory activity in RASFs, it was able to completely inhibit the release of IL-1β-stimulated PGE2 with an IC50 of 96.2 nmol/l[1].

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1. SD-0006 (SD-06), a diarylpyrazole, is a potent and selective inhibitor of p38α kinase in vitro, with selectivity over 50 other kinases (including p38γ, p38delta, and modest selectivity over p38β). Crystal structure analysis showed that SD-0006 binds to the ATP site of p38α and forms additional residue interactions outside the ATP pocket unique to p38α, which confers advantages over other ATP-competitive inhibitors. In cellular models (U937 cells, human primary monocytes, human whole blood (HWB) cells), SD-0006 preincubated for 60 min inhibited lipopolysaccharide (LPS)-stimulated TNFα release in a concentration-dependent manner (IC₅₀ < 200 nmol/L for TNFα release inhibition), with ex vivo equivalence of inhibition observed across different cell types. In human rheumatoid arthritis synovial fibroblast (RASF) cell lines stimulated with IL-1β (1 ng/ml), SD-0006 added 1 h before stimulation inhibited PGE₂ release in a concentration-dependent manner (IC₅₀ determined from two experiments) and selectively suppressed COX-2 protein expression (but not COX-1) after 4 h of stimulation. In RASF cells preincubated with SD-0006 for 60 min and then challenged with IL-1β (1 ng/ml, 30 min), SD-0006 inhibited p38 kinase activation and activity (as shown by reduced phosphorylation of Hsp27 detected via Western blotting and densitometry, and downregulated p38 activation measured by electrochemiluminescence-based ELISAs), but had no effect on ERK, JNK activation or phosphorylation of cJun (JNK substrate) [1]
2. SD-0006 is a C(5)-substituted diaryl pyrazole derivative developed from an initial HTS screening lead, with the switch from pyridyl to pyrimidine at the C(4)-position contributing to its potent inhibition of p38α kinase. It showed potent activity in human whole blood-based cell assays [2]

In animal models of chronic inflammation and disease, SD0006 is successful in vivo. It has significant protective effects on bone density and paw joint integrity in the rat streptococcal-cell-wall induced arthritis model, demonstrating its efficacy. Additionally, SD0006 exhibits good oral anti-inflammatory efficacy and excellent interspecies agreement in rats, cynomolgus monkeys, and humans. In a rodent model of acute inflammation, it reduces pain and swelling[1].

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1. In vivo, SD-0006 inhibited LPS-stimulated TNFα release with an IC₅₀ < 200 nmol/L, and this inhibitory effect was confirmed in a phase 1 clinical trial. In the rat carrageenan paw model: (1) Prophylactically, SD-0006 administered 2 h before carrageenan injection reduced pain (change in paw withdrawal latency time) and edema (change in paw volume) in a dose-dependent manner, with 10 mg/kg and 30 mg/kg doses showing statistically significant effects compared to vehicle control (p < 0.05); (2) Therapeutically, SD-0006 (30 mg/kg) administered orally 3 h after carrageenan injection reduced pain with efficacy equivalent to dexamethasone (3 mg/kg), and was significantly different from dexamethasone at 2 h post-treatment (p < 0.05). In the rat streptococcal-cell-wall (SCW)-induced arthritis model: (1) SD-0006 administered b.i.d. at 15 mg/kg from days 18 to 21 inhibited the transcription of multiple proinflammatory cytokines (TNFα, IL-1β, IL-6, MMP-3 etc.) with efficacy comparable to anti-rat TNFα biologic (statistically significant vs vehicle control for all except MMP-3, p < 0.05); (2) SD-0006 administered from days 10 to 21 inhibited paw swelling in a dose-dependent manner (ED₅₀ and EC₅₀ determined via 4-parameter logistical model); (3) SD-0006 (15 mg/kg b.i.d.) showed dramatic protective effects on paw joint integrity and bone density (assessed by computed tomography and bone density analysis), with 7.9 mg/kg and 25 mg/kg b.i.d. doses resulting in bone density statistically indistinguishable from normal control (p ≥ 0.994). In the murine collagen-induced arthritis (CIA) model, SD-0006 administered from day 21 to 56 reduced the incidence of arthritis in a dose-dependent manner, with efficacy equivalent to anti-TNFα antibody treatment. In cynomolgus monkeys challenged with LPS, SD-0006 inhibited TNFα production in a time-dependent manner, and cross-species correlation (rat, cynomolgus monkey, human) was demonstrated for the inhibition of LPS-stimulated TNFα production by SD-0006 [1]
2. SD-0006 showed efficacy in multiple animal models for rheumatoid arthritis [2]

1. For the kinase selectivity assay of SD-0006: p38α, p38β, p38γ, p38delta and 50 other kinases were screened in vitro. The kinase activity was measured by incubating the kinase with its specific substrate and ATP in the presence of different concentrations of SD-0006. The amount of phosphorylated substrate was detected to determine the inhibitory effect of SD-0006 on each kinase, and the selectivity of SD-0006 for p38α over other kinases was evaluated based on the IC₅₀ values. For the crystal structure analysis of p38α/SD-0006 complex: SC79659 (differing from SD-0006 only by pyridine ring substituted with pyrimidine, same binding conformation) was used to form complex with p38α. Crystals of p38α/inhibitor complex were obtained, diffraction data were measured, and the binding mode of the inhibitor at the ATP binding site of p38α (hinge region between two lobes of the enzyme) was analyzed, including interactions with protein residues and ordered water molecules, and potential hydrogen bonds [1]

1. Cytokine release assay (U937 cells, human primary monocytes, HWB cells): U937 cells, human primary monocytes (3 donors) and HWB cells (6 donors) were preincubated with SD-0006 for 60 min, then challenged with LPS. Medium or plasma was collected at specific time points (4 h for TNFα and IL-6, 16 h for IL-1β; 16 h for IL-6 in U937 cells) to determine cytokine release. Curve fitting, IC₅₀ determinations and standard errors were calculated using Grafit 5(2) software, with HWB values corrected for plasma free fraction (FF). RASF cell PGE₂ release and COX-2 expression assay: Human RASF cell lines were preincubated with SD-0006 for 1 h, then stimulated with 1 ng/ml IL-1β. Supernatants were collected after 20 h to assay PGE₂ levels (IC₅₀ determined from two experiments), and cells were collected after 4 h of stimulation for Western blotting to detect COX-1 and COX-2 protein expression. RASF cell p38/ERK/JNK activation assay: RASF cells were preincubated with SD-0006 for 60 min, then challenged with 1 ng/ml IL-1β for 30 min. Cell lysates were prepared: (1) Phosphorylation of Hsp27 was detected by Western blotting with phospho-specific antibodies and quantified via densitometry (standard deviation from 3 assays); (2) Activation states of p38, ERK, JNK and phosphorylation of cJun were determined by electrochemiluminescence-based ELISAs (average values from 2 assays) [1]
2. Human whole blood-based cell assay: Human whole blood cells were treated with different concentrations of SD-0006, and the inhibitory effect on p38α kinase activity was evaluated [2]

8- to 12-week-old DBA/1 mice
3.75, 7.5 and 15 mg/kg.
Orally twice daily.

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1. Rat carrageenan paw model: (1) Prophylactic study: SD0006 was administered orally to rats (5 per group) at doses of 0, 10, 30 mg/kg 2 h before carrageenan injection. Pain (paw withdrawal latency time) and edema (paw volume) were measured 3 h after carrageenan injection. One-way ANOVA and Dunnett‘s t test were used for statistical analysis. (2) Therapeutic study: Vehicle, SD-0006 (30 mg/kg) or dexamethasone (3 mg/kg) were administered orally to rats 3 h after carrageenan injection. Pain was measured 1, 2, and 3 h post-treatment, and repeated ANOVA was used for longitudinal data analysis. Murine CIA model: DBA mice were treated with anti-murine TNFα antibody (n = 70) or SD-0006 at different doses (n = 19 per dose) from day 21 to 56 post-immunization, and the incidence of arthritis was monitored. Rat SCW-induced arthritis model: (1) For cytokine transcription analysis: Rats (8 per group) were inoculated with SCW, and SD-0006 (15 mg/kg) was administered b.i.d. from days 18 to 21. On day 21, total RNA was isolated from paw tissues and subjected to TaqMan analysis to detect proinflammatory cytokine mRNA levels (one-way ANOVA for statistical analysis). (2) For paw swelling analysis: SD-0006 was administered from days 10 to 21, plasma was collected at various times on day 21 to determine compound levels (Cₘₐₓ and AUC from 3 animals per time point), and paw volume was measured (two observations per animal, 4-8 animals per group). Inhibition of edema was analyzed by 4-parameter logistical model to determine ED₅₀ and EC₅₀. (3) For joint/bone protection analysis: Rats inoculated with SCW were orally given SD-0006 b.i.d. 10 days later (4 per group). On day 21, computed tomography was used to assess paw joint integrity, and bone density of hind paws was analyzed (Dunnett‘s t test for statistical analysis). Cynomolgus monkey LPS challenge model: Cynomolgus monkeys were treated with SD-0006, then challenged with LPS. Blood samples were collected at different time points to measure TNFα levels (mean of 3 samples per group, standard errors < 10% of the mean) [1]

1. SD-0006 is orally administered and has good oral anti-inflammatory effects, and exhibits excellent cross-species relevance among rats, cynomolgus monkeys, and humans [1]

[1]. SD0006: a potent, selective and orally available inhibitor of p38 kinase. Pharmacology. 2009;84(1):42-60.

[2]. Identification of SD-0006, a potent diaryl pyrazole inhibitor of p38 MAP kinase. Bioorg Med Chem Lett. 2010 Apr 15;20(8):2634-8.

SD-06 belongs to the pyrazole class of compounds, with the structure 1H-pyrazole, where the hydrogen atoms at positions 3, 4, and 5 are substituted with N-(hydroxyacetyl)piperidin-4-yl, pyrimidin-4-yl, and p-chlorophenyl, respectively. It belongs to the pyrazole, pyrimidine, N-acylpiperididine, monochlorobenzene, and primary alcohol classes. 1. SD-0006 is a diarylpyrazole derivative; its binding to p38α involves interactions outside the ATP-binding pocket specific to p38α, making it superior to other ATP-competitive inhibitors. In cell models, in vivo experiments, and Phase I clinical trials, a direct correlation between p38α activity inhibition and LPS-stimulated TNFα release has been demonstrated. SD-0006 inhibits the expression of multiple pro-inflammatory proteins at both the transcriptional and translational levels, suggesting that its therapeutic efficacy is broader than that of monotherapy targeting cytokines [1]. 2. SD-0006 originated from the initial high-throughput screening lead compound, and its key structural modification was the replacement of the pyridinyl group at the C(4) position of the diarylpyrazole skeleton with a pyrimidinyl group. This improved its efficacy in human whole blood-based cell assays and animal models of rheumatoid arthritis, and it was identified as a clinical candidate drug [2].

C20H20CLN5O2

397.86

397.13

C, 60.38; H, 5.07; Cl, 8.91; N, 17.60; O, 8.04

271576-80-8

271576-80-8;

9865587

White to off-white solid powder

1.4±0.1 g/cm3

652.6±55.0 °C at 760 mmHg

348.5±31.5 °C

0.0±2.1 mmHg at 25°C

1.638

1.5

2

5

4

28

523

0

OCC(N1CCC(CC1)C2=NNC(C3=CC=C(C=C3)Cl)=C2C4=NC=NC=C4)=O

CATQHDWESBRRQA-UHFFFAOYSA-N

InChI=1S/C20H20ClN5O2/c21-15-3-1-13(2-4-15)19-18(16-5-8-22-12-23-16)20(25-24-19)14-6-9-26(10-7-14)17(28)11-27/h1-5,8,12,14,27H,6-7,9-11H2,(H,24,25)

1-[4-[3-(4-chlorophenyl)-4-pyrimidin-4-yl-1H-pyrazol-5-yl]piperidin-1-yl]-2-hydroxyethanone

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)

DMSO: ~80 mg/mL ( ~201.1 mM)
Water: <2 mg/mL
Ethanol: Insoluble

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.28 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 25.0 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 2: ≥ 2.5 mg/mL (6.28 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 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 3: ≥ 2.5 mg/mL (6.28 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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 (Please use freshly prepared in vivo formulations for optimal results.)