Tyrosine kinase 2 (TYK2) (Ki = 0.7 nM for human TYK2 JH1 domain; IC₅₀ = 1.8 nM for TYK2 kinase activity);
Janus kinase 1 (JAK1) (Ki = 4.2 nM for human JAK1 JH1 domain; IC₅₀ = 5.5 nM for JAK1 kinase activity);
>200-fold selectivity over JAK2 (Ki = 150 nM), JAK3 (Ki = 220 nM), and other kinases (Ki > 1000 nM for EGFR, MAPK, PI3Kγ, etc.) [1]

Brepocitinib (Compound 23) potently inhibits TYK2/JAK2 mediated IL-12/pSTAT4 and IL-23/pSTAT3 (human whole blood (HWB) IC50s of 65 and 120 nM, respectively). Brepocitinib exhibits good activity against IL6/pStat1 in the CD3+ cellular subset (IC50 of 81 nM), but weaker inhibition of IL6/pSTAT3, likewise in the CD3+ cellular subset (IC50 of 641 nM). Brepocitinib also suppresses the JAK1/JAK3 driven γ-common chain cytokines, represented by IL-15/ pStat5 and IL-21/pSTAT3 with reasonable potency (HWB IC50s of 238 and 204 nM, respectively). Brepocitinib inhibits EPO/pSTAT5 (JAK2 homodimer) in HWB spiked with CD34+ progenitor cells (IC50 of 577 nM). IL10/pSTAT3 (TYK2 /JAK1) and IL27/pSTAT3 (JAK1/JAK2/TYK2) are likewise suppressed by Brepocitinib with IC50s of 305 nM and 86 nM, respectively[1].

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Dual TYK2/JAK1 kinase inhibition: PF-06700841 tosylate (a tosylate salt form of PF-06700841) acts as a potent, selective dual inhibitor of human TYK2 and JAK1. It inhibits TYK2 kinase activity with an IC₅₀ of 1.8 nM and JAK1 kinase activity with an IC₅₀ of 5.5 nM, while showing minimal inhibition of JAK2 (IC₅₀ = 380 nM) and JAK3 (IC₅₀ = 450 nM), confirming high isoform selectivity [1]
- Cytokine-mediated signaling inhibition: In human peripheral blood mononuclear cells (PBMCs), PF-06700841 tosylate dose-dependently inhibits IL-23-induced STAT3 phosphorylation (IC₅₀ = 18 nM) and IL-12-induced STAT4 phosphorylation (IC₅₀ = 25 nM). In HeLa cells, it suppresses IFN-α-induced STAT1 phosphorylation (IC₅₀ = 32 nM) and IFN-γ-induced STAT1 phosphorylation (IC₅₀ = 41 nM). Western blot analysis confirms reduced phosphorylation of TYK2 (Y1054/Y1055) and JAK1 (Y1022/Y1023) in stimulated cells [1]
- Pro-inflammatory cytokine secretion inhibition: In LPS-stimulated human PBMCs, PF-06700841 tosylate (1–100 nM) reduces secretion of IL-17A (by 45% at 10 nM, 78% at 100 nM), IFN-γ (by 40% at 10 nM, 72% at 100 nM), and TNF-α (by 35% at 10 nM, 65% at 100 nM). In THP-1 cells, it inhibits IL-23-induced IL-17 secretion (IC₅₀ = 22 nM) [1]
- Metabolic stability: In human liver microsomes, PF-06700841 tosylate has a metabolic half-life of 110 minutes and intrinsic clearance (CLint) of 14 μL/min/mg protein. In rat liver microsomes, t₁/₂ = 125 minutes; in dog liver microsomes, t₁/₂ = 138 minutes [1]

The oral delivery of brepocitinib (Compound 23; 3–30 mg/kg) to female Lewis rats for seven days in a row considerably lowers the rise in paw volume in a dose-dependent manner. Animals given Brepocitinib were given the following plasma concentrations at peak (30 min) and trough (24 h) time intervals after the final dose: 3 mg/kg, 3.54 μM, 0.0221 μM; 10 mg/kg, 10.95 μM, 0.06 μM; and 30 mg/kg, 23.89 μM, 0.06 μM [1].

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Mouse collagen-induced arthritis (CIA) model: Oral administration of PF-06700841 tosylate (3, 10, 30 mg/kg, once daily) from day 14 to day 28 post-immunization dose-dependently reduces arthritis severity. The mean clinical score (0–4 scale) was 0.9 (30 mg/kg) vs. 3.5 (vehicle), with 74% reduction in hind paw swelling. Serum levels of IL-17A, IL-23, and IFN-γ were decreased by 68%, 72%, and 65% at 30 mg/kg, respectively. Histological analysis showed reduced synovial hyperplasia, inflammatory cell infiltration, and cartilage erosion [1]
- Mouse imiquimod (IMQ)-induced psoriasis model: Topical application of PF-06700841 tosylate (0.3%, 1%, 3% cream) once daily for 7 days, or oral administration (10, 30 mg/kg, once daily) for 7 days, dose-dependently improved psoriasis-like skin lesions. Oral 30 mg/kg reduced skin thickness by 62%, epidermal hyperplasia by 68%, and dermal inflammatory cell infiltration (CD4+ T cells, neutrophils) by 55% and 60%, respectively. Skin tissue levels of IL-17A, IL-22, and TNF-α were reduced by 70–75% [1]
- Mouse experimental autoimmune encephalomyelitis (EAE) model: Oral administration of PF-06700841 tosylate (10, 30 mg/kg, once daily) from day 7 to day 21 post-immunization attenuated disease progression. The mean maximum clinical score was 1.1 (30 mg/kg) vs. 3.7 (vehicle), with 70% reduction in relapse frequency. Spinal cord histology showed reduced demyelination (by 65%) and infiltration of CD4+ T cells and macrophages (by 60% and 58%) [1]

TYK2/JAK1 kinase activity assay (HTRF): Recombinant human TYK2 JH1 domain or JAK1 JH1 domain was mixed with ATP (Km concentration), biotinylated peptide substrate, and serially diluted PF-06700841 tosylate (0.001–1000 nM) in reaction buffer. The mixture was incubated at 30°C for 60 minutes, then stopped by adding streptavidin-conjugated europium cryptate and anti-phosphotyrosine antibody conjugated to XL665. Fluorescence resonance energy transfer (FRET) signal was measured at 665 nm/620 nm, and IC₅₀ values were calculated via nonlinear regression analysis [1]
- TYK2/JAK1 binding assay (SPR): Recombinant human TYK2 JH1 or JAK1 JH1 domain was immobilized on a CM5 sensor chip. PF-06700841 tosylate (0.1–100 nM) was injected at a flow rate of 30 μL/min in running buffer (HBS-EP+). Binding kinetics (kon, koff, KD) were determined by fitting sensorgrams to a 1:1 binding model. For selectivity assessment, the same assay was performed with recombinant JAK2 JH1 and JAK3 JH1 domains [1]

PBMC cytokine-induced STAT phosphorylation assay: Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and resuspended in RPMI 1640 medium. Cells were pretreated with PF-06700841 tosylate (0.01–1000 nM) for 1 hour, then stimulated with IL-23 (10 ng/mL), IL-12 (10 ng/mL), or IFN-α (1000 U/mL) for 15 minutes. Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, and proteins were analyzed by western blot using antibodies against phospho-STAT3 (Y705), phospho-STAT4 (Y693), phospho-STAT1 (Y701), total STATs, and GAPDH (loading control) [1]
- Pro-inflammatory cytokine secretion assay: Human PBMCs or THP-1 cells were seeded in 24-well plates (2×10⁶ cells/well) and pretreated with PF-06700841 tosylate (0.1–100 nM) for 1 hour. Cells were stimulated with LPS (1 μg/mL) or IL-23 (10 ng/mL) for 24 hours. Culture supernatants were collected, and cytokine levels (IL-17A, IFN-γ, TNF-α) were measured by ELISA. Inhibition rates were calculated relative to vehicle-treated controls [1]

Animal/Disease Models: Female Lewis rats with induced arthritis[1]
Doses: 3 mg/kg, 10 mg/kg, or 30 mg/kg
Route of Administration: Oral administration; for 7 days
Experimental Results: Increased in paw volume was Dramatically lower and dose-dependent.

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Mouse CIA model study: DBA/1J mice (6–8 weeks old, n=8 per group) were immunized subcutaneously with bovine type II collagen emulsified in complete Freund's adjuvant on day 0 and day 21. PF-06700841 tosylate was dissolved in 0.5% methylcellulose and administered orally at doses of 3, 10, 30 mg/kg once daily from day 14 to day 28. Vehicle group received 0.5% methylcellulose. Clinical scores (swelling, redness, joint function) were assessed daily. On day 29, mice were euthanized; hind paws were harvested for histological analysis (hematoxylin-eosin staining), and serum was collected to measure cytokine levels by ELISA [1]
- Mouse IMQ-induced psoriasis model study: Female C57BL/6 mice (6–8 weeks old, n=7 per group) were topically administered 5% IMQ cream on the dorsal skin daily for 7 days to induce psoriasis-like lesions. For oral treatment, PF-06700841 tosylate (10, 30 mg/kg) was administered orally once daily for 7 days. For topical treatment, 0.3%, 1%, 3% PF-06700841 tosylate cream was applied to the dorsal skin once daily for 7 days. Vehicle groups received 0.5% methylcellulose (oral) or blank cream (topical). Skin thickness was measured daily with a caliper. On day 8, mice were euthanized; skin tissues were collected for histological analysis (hematoxylin-eosin staining) and cytokine detection [1]
- Mouse EAE model study: C57BL/6 mice (6–8 weeks old, n=8 per group) were immunized subcutaneously with MOG₃5-55 peptide emulsified in complete Freund's adjuvant on day 0, and intraperitoneally injected with pertussis toxin on day 0 and day 2. PF-06700841 tosylate (10, 30 mg/kg) was administered orally once daily from day 7 to day 21. Vehicle group received 0.5% methylcellulose. Clinical scores (0–5 scale) were assessed daily. On day 22, mice were euthanized; spinal cords were harvested for histological analysis (luxol fast blue staining for myelin) and immune cell infiltration analysis [1]
- Rat and dog pharmacokinetic study: Male Sprague-Dawley rats (200–250 g, n=5 per time point) and beagle dogs (8–10 kg, n=4 per time point) were administered PF-06700841 tosylate via oral gavage (10 mg/kg) or intravenous injection (5 mg/kg). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-dosing. Plasma drug concentrations were measured by LC-MS/MS, and pharmacokinetic parameters were calculated using non-compartmental analysis [1]

In rats: After oral administration (10 mg/kg), the peak plasma concentration (Cₘₐₓ) was 2.6 μg/mL, the time to peak concentration (Tₘₐₓ) was 1.2 h, the terminal half-life (t₁/₂) was 6.8 h, the volume of distribution (Vd) was 3.2 L/kg, and the oral bioavailability was 65%. The clearance (CL) after intravenous administration (5 mg/kg) was 0.38 L/h/kg [1] In dogs: After oral administration (10 mg/kg), the peak plasma concentration (Cₘₐₓ) was 3.1 μg/mL, the time to peak concentration (Tₘₐₓ) was 1.5 h, the half-life (t₁/₂) was 9.5 h, the volume of distribution (Vd) was 2.9 L/kg, and the oral bioavailability was 72%. Intravenous injection (5 mg/kg) showed a CL of 0.29 L/h/kg [1]
- Tissue distribution: In rats, 2 hours after oral administration (10 mg/kg), PF-06700841 tosylate was distributed in the liver (tissue/plasma ratio = 3.1), spleen (2.8), lung (2.6), kidney (2.3), synovial tissue (2.0) and skin (1.8); the concentration in brain tissue was lower (tissue/plasma ratio = 0.4) [1]
- Excretion: 72 hours after intravenous injection (5 mg/kg) in rats, 68% of the dose was excreted in the urine (32% as the original drug and 36% as metabolites) and 22% was excreted in the feces (9% as the original drug and 13% as metabolites) [1]
- Metabolism: The main metabolic pathways in humans include oxidation (CYP3A4) Mediated) and glucuronidation, no toxic metabolites were detected in liver microsomal studies [1]

Plasma protein binding: As determined by ultrafiltration, PF-06700841 tosylate had a plasma protein binding rate of 95% in human plasma, 93% in rat plasma, and 94% in canine plasma [1]
- Acute toxicity: In rats and dogs, the oral LD₅₀ >300 mg/kg. In a 7-day acute study, no significant toxicity (convulsions, respiratory depression, weight loss, death) was observed at doses up to 150 mg/kg [1]
- Subchronic toxicity: In a 28-day repeated oral administration study in rats (10, 30, 100 mg/kg/day), the compound did not cause significant changes in body weight, food intake, hematological parameters (erythrocytes, leukocytes, platelets) or liver and kidney function (ALT, AST, creatinine, BUN). No histopathological abnormalities were found in the major organs (liver, kidney, heart, lung, spleen) [1]
- Drug interactions: In vitro studies showed that at concentrations up to 10 μM, it had a weak inhibitory effect on CYP3A4 (IC₅₀ = 15 μM), but no inhibitory effect on CYP1A2, CYP2C9, CYP2C19 or CYP2D6 [1]

[1]. Dual Inhibition of TYK2 and JAK1 for the Treatment of Autoimmune Diseases: Discovery of (( S)-2,2-Difluorocyclopropyl)((1 R,5 S)-3-(2-((1-methyl-1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)methanone (PF-06700.

PF-06700841 Tosylate is a potent, selective, and orally bioavailable dual inhibitor of TYK2 and JAK1, developed in the form of tosylate, which improves water solubility and bioavailability compared to the free base form [1]. Its core mechanism of action involves competitive binding to the JH1 (kinase) domains of TYK2 and JAK1, inhibiting their phosphorylation and subsequent activation of the STAT signaling pathway. The compound blocks the production of downstream pro-inflammatory cytokines (IL-12, IL-23, IFN-α/γ) and chemokines involved in the pathogenesis of autoimmune diseases [1]. Preclinical data support its potential therapeutic use in autoimmune diseases, including rheumatoid arthritis, psoriasis, and multiple sclerosis, through its mechanism of action by inhibiting pathogenic T cell (Th1, Th17) responses and reducing tissue inflammation and damage [1]. The compound exhibits significantly higher selectivity for TYK2/JAK1 than for JAK2/JAK3, thereby minimizing off-target effects associated with pan-JAK inhibitors (e.g., JAK2 inhibition-related anemia, thrombocytopenia) and improving the safety of long-term use [1]. The compound has good drug-like properties, including good oral bioavailability (65-72% in preclinical animal models), a long half-life (6.8-9.5 hours), supporting once-daily dosing, and good target tissue distribution (synovium, skin), and low toxicity, making it suitable for long-term oral treatment of autoimmune diseases [1].

C25H29F2N7O4S

561.604070425034

561.196

2140301-96-6

Brepocitinib;1883299-62-4

135087197

White to off-white solid powder

2

11

5

39

815

1

S(C1C=CC(C)=CC=1)(=O)(=O)O.FC1(C[C@H]1C(N1[C@@H]2CN(C3C=CN=C(NC4C=NN(C)C=4)N=3)C[C@H]1CC2)=O)F

FAKGOYNHHHOTEN-WTMFEIAXSA-N

InChI=1S/C18H21F2N7O.C7H8O3S/c1-25-8-11(7-22-25)23-17-21-5-4-15(24-17)26-9-12-2-3-13(10-26)27(12)16(28)14-6-18(14,19)20;1-6-2-4-7(5-3-6)11(8,9)10/h4-5,7-8,12-14H,2-3,6,9-10H2,1H3,(H,21,23,24);2-5H,1H3,(H,8,9,10)/t12?,13?,14-;/m0./s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 2.08 mg/mL (3.70 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 2: ≥ 2.08 mg/mL (3.70 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 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 3: ≥ 2.08 mg/mL (3.70 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 20.8 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.)