- Bruton’s tyrosine kinase (BTK) - Biochemical IC₅₀: 5.9 nM (recombinant BTK assay) [1]
- Cellular IC₅₀: 6.4 nM (B cell receptor (BCR)-stimulated Ramos cells)
- In vivo IC₅₀: 1.1 μM (mouse blood, BTK phosphorylation inhibition) / 5.6 μM (rat blood)

With an IC50 value of 5.9±1.1 nM and a Hill slope value of -0.84±0.07 (mean±SE), GDC-0834 inhibits BTK kinase activity [1]. With IC50 values ranging from 0.86 to 1.87 μM, GDC-0834 was demonstrated to be a strong and reversible inhibitor of six recognized aldehyde oxidase (AO) substrates[2].

---

1. AOX-Mediated Amide Hydrolysis Activity: When GDC-0834 was incubated with recombinant human AOX, it underwent dose-dependent and time-dependent amide hydrolysis, producing a carboxylic acid metabolite (designated as M1). Enzyme kinetic analysis revealed that the Michaelis constant (Km) for this reaction was approximately 11.2 μM, and the maximum reaction rate (Vmax) was approximately 1.8 nmol/min/mg protein [2]
2. Species-Specific Metabolic Difference: In liver microsomes from different species, the hydrolysis activity of GDC-0834 showed significant species differences. Human liver microsomes exhibited the highest hydrolysis efficiency, while liver microsomes from monkey, dog, rat, and mouse had minimal hydrolysis activity (the hydrolysis rate was less than 10% of that in human liver microsomes) [2]
3. Enzyme Specificity Verification: When GDC-0834 was co-incubated with human liver microsomes and AOX-specific inhibitors (e.g., raloxifene), the formation of metabolite M1 was reduced by more than 80%, confirming that AOX was the key enzyme mediating the amide hydrolysis of GDC-0834. In contrast, co-incubation with cytochrome P450 (CYP) enzyme inhibitors had no effect on M1 formation, indicating that CYP enzymes were not involved in this metabolic process [2]

---

1. BTK Kinase Inhibition - Potently suppresses BTK autophosphorylation (Tyr223) in biochemical assays (IC₅₀ = 5.9 nM) and BCR-stimulated Ramos cells (IC₅₀ = 6.4 nM). Inhibition is reversible and dose-dependent, with a Hill slope of -0.84 ± 0.07
2. Selectivity - >100-fold selectivity over other kinases (e.g., JAK2, SYK, EGFR) at 1 μM concentration
3. Metabolic Activity - Undergoes human aldehyde oxidase (AOX)-mediated amide hydrolysis in vitro, generating a carboxylic acid metabolite (M1). Km = 11.2 μM, Vmax = 1.8 nmol/min/mg (recombinant human AOX) [2]

GDC-0834 treatment of BALB/c mice led to pBTK-Tyr223 inhibition in a dose-dependent manner. The blood levels of pBTK-Tyr223 were completely inhibited in animals given 150 or 100 mg/kg GDC-0834 for two hours, with average inhibition rates of 97% and 96%, respectively. GDC-0834 suppressed pBTK-Tyr223 in rat blood in a dose-dependent manner in a research on CIA rats. Rats' estimated IC50 for pBTK-Tyr223 inhibition is 5.6±1.6 μM, with a mean±standard error of 0.51±0.087 [1].

---

1. Antiinflammatory Efficacy (Rat CIA Model) - Oral administration (30–100 mg/kg) dose-dependently reduces ankle swelling (ED₅₀ = 45 mg/kg) and histological arthritis severity (e.g., synovial hyperplasia, neutrophil infiltration) in collagen-induced arthritis (CIA) rats. Maximal inhibition (70% reduction in swelling) at 100 mg/kg
- Correlates with BTK phosphorylation inhibition in peripheral blood mononuclear cells (PBMCs): ≥90% inhibition at 100 mg/kg (rat) [1]
2. Pharmacokinetic Species Differences - In humanized PXB chimeric mice, low clearance (CL < 10 mL/min/kg) and poor oral bioavailability (<5%) were observed. Plasma concentrations in humans (35–105 mg oral dose) were mostly undetectable (<1 ng/mL), likely due to rapid AOX-mediated hydrolysis

1. Recombinant Human AOX Catalysis Assay: GDC-0834 was prepared into solutions with concentrations ranging from 1 μM to 50 μM. Each concentration of GDC-0834 was mixed with recombinant human AOX in a buffer system containing nicotinamide adenine dinucleotide phosphate (NADPH) (to maintain the catalytic activity of AOX), and the mixture was incubated at 37°C. Samples were collected at 0, 15, 30, 45, and 60 minutes respectively. After collection, acetonitrile containing an internal standard was added to each sample to quench the reaction, followed by centrifugation to remove precipitated proteins. The supernatant was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the content of metabolite M1. Finally, the kinetic parameters (Km and Vmax) of the hydrolysis reaction were calculated by fitting the experimental data to the Michaelis-Menten equation [2]
2. Liver Microsome Metabolic Assay: Liver microsomes from human, monkey, dog, rat, and mouse were separately suspended in an appropriate buffer to prepare a microsome suspension. GDC-0834 was added to the microsome suspension to a final concentration of 10 μM, and the mixture was incubated at 37°C for 30 minutes. For the enzyme inhibition verification experiment, AOX inhibitors or CYP inhibitors were pre-incubated with the microsome suspension for 10 minutes before adding GDC-0834. After the incubation, the reaction was terminated by adding acetonitrile, and the sample was centrifuged. The supernatant was detected by LC-MS/MS to determine the amount of M1 produced, so as to evaluate the hydrolysis activity of GDC-0834 in different species' liver microsomes and the effect of inhibitors on the reaction [2]

---

- Biochemical Assay: Recombinant human BTK (10 nM) was incubated with ATP (10 μM) and GDC-0834 (0.01–1000 nM) in kinase buffer. Phosphorylation of a peptide substrate (KKLPQpYASL) was quantified via ELISA. IC₅₀ = 5.9 nM was calculated from triplicate dose-response curves [1]
- Cellular Assay: Ramos B cells (1 × 10⁶ cells/mL) were stimulated with anti-IgM (10 μg/mL) and GDC-0834 (0.1–100 nM) for 15 min. Phospho-BTK (Tyr223) levels were measured by flow cytometry, yielding IC₅₀ = 6.4 nM [1]

- Model: Male Lewis rats (180–200 g) induced with bovine type II collagen (CIA). Treated orally with GDC-0834 (30, 60, 100 mg/kg) or vehicle (0.5% methylcellulose) daily from day 21 post-induction for 7 days [1]
- Dosing: Formulated as a suspension in 0.5% methylcellulose. Ankle circumference measured daily; joints harvested for histopathology (H&E staining) [1]
- PK/PD Modeling: Plasma GDC-0834 concentrations (LC-MS/MS) correlated with BTK phosphorylation inhibition in PBMCs (r² = 0.89), establishing an EC₅₀ of 5.6 μM for in vivo efficacy [1]

1. In vitro metabolic pathway: The main in vitro metabolic pathway of GDC-0834 in humans was identified as AOX-mediated amide hydrolysis to produce carboxylic acid metabolite M1. This metabolic pathway is considered a novel function of AOX, as AOX is traditionally known to catalyze the oxidation of heterocyclic compounds rather than the hydrolysis of amide bonds [2]. 2. Species-dependent metabolism: GDC-0834 exhibits significant species-dependent metabolism in vitro. Only human liver microsomes showed significant hydrolytic activity for GDC-0834, while liver microsomes from common preclinical experimental animals (monkeys, dogs, rats, mice) showed almost no hydrolytic activity. This suggests that these non-human species cannot be used as animal models to predict the metabolism of GDC-0834 in humans [2]. Metabolism: Mainly metabolized to M1 via amide hydrolysis by human AOX (rather than CYP enzymes). Hydrolysis in non-human species (monkeys, dogs, rats) is negligible and therefore not applicable for predicting human pharmacokinetics [2]
- Clearance: The clearance rate in rats was 25 mL/min/kg; the clearance rate in mice was 18 mL/min/kg. Given the strong antioxidant enzyme activity in humans, the clearance rate in humans is expected to be higher [2,3]
- Oral bioavailability: Less than 5% in humans (possibly due to first-pass metabolism), compared to 25% in rats [2]

Safety: No significant toxicity was observed in rats at doses up to 200 mg/kg (10 times the therapeutic dose). Mild thrombocytopenia (platelet count ↓15%) was observed at a dose of 100 mg/kg, which was reversible upon discontinuation of the drug [1]. - hERG inhibition: No significant inhibition (>30%) was observed at a concentration of 10 μM, indicating a low risk of QT interval prolongation.

[1]. Antiarthritis effect of a novel Bruton's tyrosine kinase (BTK) inhibitor in rat collagen-induced arthritis and mechanism-based pharmacokinetic/pharmacodynamic modeling: relationships between inhibition of BTK phosphorylation and efficacy. J Pharmacol Exp Ther. 2011 Jul;338(1):154-63.

[2]. A novel reaction mediated by human aldehyde oxidase: amide hydrolysis of GDC-0834. Drug Metab Dispos. 2015 Jun;43(6):908-15.

1. New understanding of AOX function: GDC-0834 can undergo AOX-mediated amide hydrolysis, which expands the known substrate range of AOX. Previously, AOX was mainly thought to play a role in oxidizing heterocyclic structures in drugs; however, this study confirms that AOX can also catalyze the hydrolysis of amide bonds, providing a new understanding of the catalytic diversity of AOX [2]
2. Implications for drug development: The species-specific metabolism of GDC-0834 poses a challenge to the selection of preclinical animal models in its development process. Since non-human animal models cannot simulate the metabolic process of GDC-0834 in humans, other methods (e.g., in vitro human systems) must be used to evaluate its metabolic characteristics. In addition, this study shows that individual differences in AOX activity (e.g., AOX gene polymorphism) may lead to differences in the in vivo exposure and metabolic pathways of GDC-0834 in different populations, which needs to be addressed in subsequent clinical studies [2]

---

- Mechanism: Competitive BTK inhibitors bind to ATP binding pockets (crystal structure: PDB 5V9P). Blocking BCR signaling and reducing the secretion of pro-inflammatory cytokines (TNF-α, IL-6) [1,5] - Clinical significance: Despite poor pharmacokinetic data in humans, the drug has rapidly entered Phase I clinical trials due to unmet medical needs in the field of autoimmune diseases. Limited systemic exposure in humans suggests that local or topical administration may be necessary [2,3] - Patent status: It is protected by patent WO2010037798A1 (2010), which relates to BTK inhibitors for the treatment of arthritis [3]

C33H36N6O3S

596.742345809937

596.256

C, 66.42; H, 6.08; N, 14.08; O, 8.04; S, 5.37

1133432-49-1

GDC-0834 Racemate;1133432-46-8;GDC-0834 S-enantiomer;1133432-50-4; 1133432-49-1 (R-isomer); 1133432-47-9 (racemate TFA)

25234918

Light yellow to yellow solid powder

1.4±0.1 g/cm3

1.697

3.68

2

6

6

43

1140

1

CC1=C(C=CC=C1NC(=O)C2=CC3=C(S2)CCCC3)C4=CN(C(=O)C(=N4)NC5=CC=C(C=C5)[C@@H]6C(=O)N(CCN6C)C)C

CDOOFZZILLRUQH-GDLZYMKVSA-N

InChI=1S/C33H36N6O3S/c1-20-24(9-7-10-25(20)36-31(40)28-18-22-8-5-6-11-27(22)43-28)26-19-39(4)33(42)30(35-26)34-23-14-12-21(13-15-23)29-32(41)38(3)17-16-37(29)2/h7,9-10,12-15,18-19,29H,5-6,8,11,16-17H2,1-4H3,(H,34,35)(H,36,40)/t29-/m1/s1

(R)-N-(3-(6-((4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenyl)amino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide

GDC0834; GDC-0834; 1133432-49-1; FM7JG3L4SR; Benzo(b)thiophene-2-carboxamide, N-(3-(6-((4-((2R)-1,4-dimethyl-3-oxo-2-piperazinyl)phenyl)amino)-4,5-dihydro-4-methyl-5-oxo-2-pyrazinyl)-2-methylphenyl)-4,5,6,7-tetrahydro-; GDC 0834; RefChem:142767; UNII-FM7JG3L4SR; GDC 0834.

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 : ≥ 32 mg/mL (~53.62 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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