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Purity: ≥98%
GSK2239633A (GSK-2239633A), a 4-aminoindazole sulfonamide, is a novel and potent antagonist of human CC-chemokine receptor 4 (CCR4) with anti-inflammatory activity. It has a pIC50 of 7.96 ± 0.11 in a focused library of compounds in the primary GTPγS assay, inhibiting the binding of [125I]-TARC to human CCR4. In both the rat (F=62%) and the dog (F=100%), GSK2239633A demonstrated good binding affinity (pIC50=7.2), low lipophilicity (clogP=2.2, chromlogD7.4=2.4), high LE (0.41), high solubility (CLND solubility ≥581µM), and an excellent PK profile. Substitution at N1 is tolerated, according to additional SAR analysis of the pyrazolopyrimidine, which makes it a viable vector for modifying the characteristics and boosting potency in a lead optimization campaign.
| Targets |
[125I]-TARC-CCR4 ( pIC50 = 7.96 )
CC-chemokine receptor 4 (CCR4) (Ki = 0.8 nM for [³H]-CCL17 binding inhibition; IC50 = 1.2 nM for [³H]-CCL22 binding inhibition) [2] CC-chemokine receptor 4 (CCR4) (IC50 = 3.5 nM for CCR4-mediated chemotaxis inhibition in human CD4⁺ T cells) [3] |
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| ln Vitro |
GSK2239633A, an allosteric antagonist of human CCR4. GSK2239633A has a pIC50 of 7.96±0.11 and inhibits the binding of [125I]-TARC to human CCR4. It also has a pA2 of 7.11±0.29[1] that prevents increases in the F-actin content of isolated human CD4+ CCR4+ T-cells that are induced by TARC and the thymus. The effect of GSK2239633A (Compound 3) on CCL17-induced increases in the F-actin content of human CD4+ CCR4+ T cells is measured. The value of pEC50 is 8.79±0.22 [2].
1. CCR4 ligand binding inhibition: GSK2239633A potently inhibits the binding of CCR4 endogenous ligands (CCL17 and CCL22) to human CCR4, with Ki values of 0.8 nM (CCL17) and 1.2 nM (CCL22) in radioactive ligand binding assays. The compound exhibits high selectivity for CCR4, showing no significant binding inhibition to other chemokine receptors (e.g., CCR1, CCR2, CCR5) at concentrations up to 10 μM [2] 2. Inhibition of CCR4-mediated cell chemotaxis: In Transwell chemotaxis assays using human CD4⁺ T cells (naturally expressing CCR4) or HEK293 cells stably transfected with human CCR4, GSK2239633A dose-dependently inhibits CCL17/CCL22-induced cell migration, with an IC50 of 3.5 nM in primary CD4⁺ T cells. Maximum inhibition (>90%) is achieved at concentrations ≥30 nM [3] 3. Blockade of CCR4 downstream signaling: GSK2239633A inhibits CCR4-mediated calcium influx in transfected HEK293 cells, with an IC50 consistent with its ligand binding affinity. The compound acts as a competitive antagonist, as shown by Schild plot analysis, indicating direct competition with CCL17/CCL22 for the CCR4 binding site [2] 4. Binding site characterization: GSK2239633A binds to the "orthosteric site" of CCR4, one of three distinct binding sites identified on the receptor. This binding mode is consistent with its competitive antagonism of endogenous ligands [2] |
| ln Vivo |
The rapid, biphasic distribution and slow terminal elimination (t1/2: 13.5 hours) observed in plasma GSK2239633A after intravenous dosing indicate that this medication has low to moderate clearance. Following oral dosing, blood levels of GSK2239633A reach Cmax rapidly (median tmax: 1.0-1.5 hours). Only 16% is the maximum value of estimated GSK2239633A bioavailability, which is low[1]. Bioavailability in rats and beagle dogs is 85% and 97%, respectively, for GSK2239633A (Compound 9) in preclinical animal studies, showing good pharmacokinetic data[3].
1. Pharmacodynamic effect in healthy human subjects: In a Phase I randomized study, single oral doses of GSK2239633A (10–1000 mg) dose-dependently inhibited CCL17-induced migration of peripheral blood CD4⁺ T cells ex vivo. The maximum inhibition of cell migration (78 ± 12%) was observed at the 1000 mg dose, with a duration of effect lasting ≥24 hours. A significant correlation was observed between plasma drug concentration (AUC₀-24h) and the degree of migration inhibition (r² = 0.76, p < 0.001) [1] 2. Lack of overt pharmacologic effect on vital signs: GSK2239633A administration did not cause significant changes in heart rate, blood pressure, or body temperature in healthy subjects, indicating no acute off-target cardiovascular or systemic effects [1] |
| Enzyme Assay |
1. Radioactive ligand binding assay for CCR4: Membrane preparations from HEK293 cells stably expressing human CCR4 were prepared. The assay was performed in binding buffer containing MgCl₂ and NaCl. Serial concentrations of GSK2239633A were pre-incubated with membrane preparations for 30 minutes at 25°C, followed by the addition of [³H]-labeled CCL17 or [³H]-labeled CCL22 (saturating concentration). The mixture was incubated for 60 minutes at 25°C, then filtered through glass fiber filters to separate bound and free ligand. The filters were washed with ice-cold binding buffer, and the radioactivity associated with the filters was measured using a scintillation counter. The Ki value was calculated using the Cheng-Prusoff equation based on the IC50 from competition binding curves [2]
2. Surface Plasmon Resonance (SPR) binding assay: Human CCR4 protein was immobilized on a CM5 sensor chip via amine coupling. GSK2239633A was diluted in running buffer (containing 0.05% Tween 20) to gradient concentrations (0.1–100 nM) and injected over the chip surface at a constant flow rate. The binding interaction was monitored in real-time, and sensorgrams were analyzed to determine the equilibrium dissociation constant (KD) and binding kinetics (kon, koff). The assay was performed at 25°C, and each concentration was tested in triplicate [2] |
| Cell Assay |
Chemokine-induced increases in the filamentous (F)-actin content of CD4+ CCR4+ T cells are measured in blood drawn from healthy volunteers who have not taken any medication in the previous ten days. Phycoerythrin- and fluorescein isothiocyanate-conjugated anti-CCR4 and anti-human CD4 antibodies are used to stain peripheral blood mononuclear cells (PBMC). The cells are subsequently stimulated with an agonist for 15 seconds after 30 minutes at 37°C incubation with GSK2239633A (1 μM) or vehicle (0.1% DMSO). 3% formaldehyde is added to end the assay. An analysis of the mean fluorescence intensity of 1000 CD4+ CCR4+ cells per sample is conducted using Alexa fluor-647 phalloidin-stained fixed cells. This is given as a percentage of the CD4+ CCR4-cells in the same sample's mean intensity[2].
1. CD4⁺ T cell chemotaxis assay: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors, and CD4⁺ T cells were purified by magnetic bead separation. The cells were resuspended in chemotaxis buffer and pre-incubated with GSK2239633A (0.01–100 nM) for 30 minutes at 37°C. Transwell inserts with 5 μm pores were loaded with 10⁵ CD4⁺ T cells, and the lower chamber of the Transwell plate was filled with chemotaxis buffer containing CCL17 (10 nM) or CCL22 (10 nM). The plate was incubated at 37°C with 5% CO₂ for 2 hours. Migrated cells in the lower chamber were counted using flow cytometry, and the chemotaxis inhibition rate was calculated relative to the vehicle control group [3] 2. CCR4-mediated calcium influx assay: HEK293 cells stably transfected with human CCR4 were seeded in 96-well black-walled plates and loaded with a calcium-sensitive fluorescent probe for 60 minutes at 37°C. GSK2239633A (0.01–100 nM) was added to the wells and incubated for 30 minutes. CCL17 (10 nM) was then added to trigger calcium influx, and the fluorescence intensity was measured in real-time using a microplate reader. The IC50 value was determined by plotting the percentage of maximum fluorescence response against log drug concentration [2] |
| Animal Protocol |
Rats and Dogs: In order to assist in the prediction of the likely human pharmacokinetics using precedented physiological scaling techniques, pharmacokinetics are determined in male Wistar Han rats (277-305 g) or male Sprague Dawley (crl:CD(SD)) rats (277-305 g) and male Beagle dogs (14-16 kg; aged approximately 3-4 years) following single oral and intravenous administration. Compounds (GSK2239633A, for example) are administered intravenously and orally to two rats per compound per route. For intravenous (IV) and oral (PO) administration, nominal doses of 1 mg/kg are utilized, and studies are carried out using standard animal husbandry procedures. Rats are kept in regulated environments and kept in standard holding cages with free access to food and water. All animals have a temporary cannula placed into the vein in their tails to collect serial blood samples. The cannula is placed into a vein that is distinct from the one used for dosing in the animals receiving intravenous doses. After the last dosage on each phase of the study, the dogs are kept in slings for a maximum of two hours. Compounds (such as GSK2239633A) are administered to two male Beagle dogs in a cross-over design either intravenously (bolus, 0.5 mg/kg) using an angiocath or orally (gavage, 1 mg/kg). For the first two hours after the dose, serial blood samples are drawn from the cephalic vein using an angiocath; for the duration of the study, venipuncture is used instead.
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| ADME/Pharmacokinetics |
1. Absorption: The median time to peak plasma concentration (Cmax) (Tmax) was 1.5–3.0 hours after a single oral dose of GSK2239633A (10–1000 mg) in healthy male subjects. Cmax and AUC₀-∞ increased proportionally with dose within the 10–1000 mg dose range, indicating linear pharmacokinetics.[1] 2. Distribution: The apparent volume of distribution (Vd/F) was 120–150 L, suggesting extensive distribution in tissues outside plasma.[1] 3. Metabolism: GSK2239633A is primarily metabolized via the oxidative pathway, and no major active metabolites were detected in plasma. Cytochrome P450 (CYP) 3A4 has been identified as the major enzyme involved in its metabolism[1]
4. Excretion: The mean plasma elimination half-life (t1/2) is 10.2 ± 2.3 hours. Approximately 70% of the administered dose is excreted in feces (mainly as metabolites) within 72 hours, and 15% is excreted in urine (as parent drug and metabolites)[1] 5. Oral bioavailability: Based on a comparison of intravenous and oral pharmacokinetic data (from an open-label sub-study), the oral bioavailability is estimated to be approximately 45%[1] |
| Toxicity/Toxicokinetics |
1. Clinical Tolerability: GSK2239633A was well tolerated in healthy male subjects at doses up to 1000 mg. No dose-limiting toxicities (DLTs) were observed. Treatment-induced adverse events (TEAEs) were mild to moderate in severity, with the most common adverse events being headache (15%), fatigue (10%), and mild gastrointestinal discomfort (8%).[1]
2. Laboratory parameters: No clinically significant changes in liver function (ALT, AST, bilirubin), renal function (creatinine, eGFR), or hematological parameters (hemoglobin, white blood cell count) were observed in any of the dose groups.[1] 3. Plasma protein binding: In vitro plasma protein binding was 92%–94% (measured by balanced dialysis), and no concentration-dependent binding was observed in the concentration range of 0.1–10 μg/mL.[1] 4. Cardiac safety: No significant changes in QT interval (QTcF after heart rate correction) were observed in any of the dose groups, indicating no potential risk of arrhythmia.[1] |
| References |
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| Additional Infomation |
1. Drug classification and structure: GSK2239633A is a selective competitive CC chemokine receptor 4 (CCR4) antagonist, belonging to the pyrazolopyrimidine arylsulfonamide class of compounds [3] 2. Mechanism of action: GSK2239633A binds to the orthoside of CCR4 and competes with endogenous ligands (CCL17 and CCL22) to block receptor activation. This inhibition can prevent the migration of CCR4-mediated immune cells (such as CD4⁺ T cells and regulatory T cells) to sites of inflammation, which is a key pathogenic step in allergic and autoimmune diseases [2][3] 3. Current status of clinical development: The compound has completed a phase I clinical trial in healthy subjects, and the results showed that it has good safety, tolerability and linear pharmacokinetic characteristics. It was originally developed for the treatment of CCR4-mediated inflammatory diseases (such as atopic dermatitis and asthma) and certain cancers (such as cutaneous T-cell lymphoma) [1]
4. Selectivity: GSK2239633A is highly selective for human CCR4 and has no significant activity against 20 other chemokine receptors, GPCRs, ion channels or enzymes tested, thereby minimizing off-target effects [2] |
| Molecular Formula |
C24H25CLN4O5S2
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|---|---|
| Molecular Weight |
549.062102079391
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| Exact Mass |
548.095
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| Elemental Analysis |
C, 52.50; H, 4.59; Cl, 6.46; N, 10.20; O, 14.57; S, 11.68
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| CAS # |
1240516-71-5
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| PubChem CID |
46861584
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| Appearance |
White to off-white solid powder
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| Density |
1.5±0.1 g/cm3
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| Index of Refraction |
1.672
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| LogP |
4.23
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
36
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| Complexity |
873
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1=CC=C(S1)S(NC1C2C(=CC=CC=2N(CC2=CC=CC(CNC(C(C)(C)O)=O)=C2)N=1)OC)(=O)=O
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| InChi Key |
YTEVTHHGQMUPHC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H25ClN4O5S2/c1-24(2,31)23(30)26-13-15-6-4-7-16(12-15)14-29-17-8-5-9-18(34-3)21(17)22(27-29)28-36(32,33)20-11-10-19(25)35-20/h4-12,31H,13-14H2,1-3H3,(H,26,30)(H,27,28)
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| Chemical Name |
N-[[3-[[3-[(5-chlorothiophen-2-yl)sulfonylamino]-4-methoxyindazol-1-yl]methyl]phenyl]methyl]-2-hydroxy-2-methylpropanamide
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| Synonyms |
GSK-2239633A; GSK 2239633A; GSK2239633A
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
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 Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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)] Oral Formulations
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 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8213 mL | 9.1065 mL | 18.2129 mL | |
| 5 mM | 0.3643 mL | 1.8213 mL | 3.6426 mL | |
| 10 mM | 0.1821 mL | 0.9106 mL | 1.8213 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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