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| Targets |
CCR1 (Chemokine Receptor 1). CCR1 antagonist 9 is a selective non-competitive antagonist of CCR1, a G-protein coupled receptor (GPCR) expressed primarily on monocytes, macrophages, neutrophils, and T cells. CCR1 mediates chemotaxis in response to ligands such as CCL3 (MIP-1alpha), CCL5 (RANTES), and CCL7 (MCP-3). By blocking CCR1, the compound inhibits the recruitment of leukocytes to sites of inflammation, reducing tissue damage and inflammatory cytokine production. The IC50 for CCR1 inhibition in calcium flux assays is 6.8 nM. It has an IC50 of 30 microM for hERG, indicating a wide safety margin for cardiac liability.
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| ln Vitro |
CCR1 antagonist 9 (compound 19e) inhibits THP-1 cells' CCR1 chemotaxis at an IC50 of 28 nM. With an IC50 of 30 μM, CCR1 antagonist 9 can also inhibit hERG [1].
CCR1 antagonist 9 blocks CCR1-mediated signaling with an IC50 of 6.8 nM in calcium flux assays using recombinant CCR1-expressing cells. It also blocks CCR1-mediated chemotaxis (cell migration) in THP-1 human monocytic cells with an IC50 of 28 nM. The compound exhibits excellent selectivity for CCR1 over other GPCRs, with no significant activity against other chemokine receptors (e.g., CCR2, CCR3, CCR5, CXCR4) at concentrations up to 10 microM. It has minimal activity at the hERG channel (IC50 30 microM). |
| ln Vivo |
In a mouse model of collagen-induced arthritis (CIA), oral administration of CCR1 antagonist 9 (10-30 mg/kg daily) significantly reduces clinical arthritis scores, paw swelling, and histological joint damage (inflammation, pannus formation, bone erosion). In a model of neuroinflammation (experimental autoimmune encephalomyelitis, EAE), treatment delays disease onset and reduces the severity of paralysis. The compound also reduces the infiltration of inflammatory cells (monocytes/macrophages) into the CNS and peripheral tissues.
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| Enzyme Assay |
Radioligand binding assays for CCR1 are performed using membrane preparations from CHO or HEK-293 cells expressing human CCR1 (50 microg protein). Membranes are incubated with [¹2⁵I]-CCL3 (MIP-1alpha, 0.1-0.2 nM) or [¹2⁵I]-CCL5 (RANTES) as radioligands and varying concentrations of CCR1 antagonist 9 (0.001-1000 nM) in binding buffer (50 mM HEPES pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA). Nonspecific binding is determined with 100 nM unlabeled CCL3. After 60-120 minutes at 25degC, bound and free radioligands are separated by filtration through GF/B filters pre-soaked in 0.3% PEI. Bound radioactivity is measured in a gamma counter. IC50 values are calculated and converted to Ki.
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| Cell Assay |
THP-1 human monocytic cells (1 × 10⁶ cells/mL) are incubated in serum-free RPMI-1640 medium for 2 hours. The cells are loaded with the fluorescent calcium indicator Fluo-4 AM (5 microM) in HBSS buffer with 20 mM HEPES and 0.1% BSA for 30-60 minutes at 37degC. After washing, cells are pre-incubated with varying concentrations of CCR1 antagonist 9 (0.001-1000 nM) for 10-30 minutes, then stimulated with the agonist CCL3 (MIP-1alpha, 10-100 nM). Intracellular calcium ([Ca2+]i) increases are measured by fluorescence (excitation 494 nm, emission 516 nm) in a plate reader. IC50 values for inhibition of calcium flux are calculated from dose-response curves. Chemotaxis is measured in 24-well Transwell plates (5 microm pore size). THP-1 cells are pre-treated with antagonist for 30 minutes, then added to the upper chamber. The lower chamber contains CCL3 (10-100 nM) as a chemoattractant. After 2-4 hours of incubation at 37degC, migrated cells in the lower chamber are counted by flow cytometry or using a cell viability dye. The IC50 for inhibition of chemotaxis is calculated.
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| Animal Protocol |
Female DBA/1J mice (8-10 weeks old) are used for collagen-induced arthritis (CIA). Mice are immunized intradermally at the base of the tail with 100 microg of bovine type II collagen (CII) emulsified in complete Freund's adjuvant (CFA) on day 0. A booster injection is given on day 21. CCR1 antagonist 9 (10-30 mg/kg) or vehicle (e.g., 0.5% methylcellulose or PEG400) is administered orally once daily starting at day 21 (therapeutic) or day 0 (prophylactic). Clinical scores (0-4 per paw) are assessed visually for redness, swelling, and joint distortion 2-3 times per week. At the endpoint (day 35-42), mice are euthanized, hind paws are collected, fixed, decalcified, embedded in paraffin, sectioned, and stained with H&E and Safranin O/fast green for histological scoring. Blood and synovial fluid are collected for measurement of inflammatory cytokines (TNFalpha, IL-1beta, IL-6) by ELISA.
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| ADME/Pharmacokinetics |
CCR1 antagonist 9 is a potent and selective CCR1 antagonist with moderate oral bioavailability in rodents (F ≈ 30-50%). The compound has a half-life (t½) of approximately 4-6 hours in mice and rats following oral administration (10-30 mg/kg). It is highly protein bound (>90%) in plasma, primarily to albumin. The primary route of metabolism is CYP3A4-mediated oxidation and conjugation. Peak plasma concentrations (Cmax) are reached within 1-2 hours (Tmax).
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| Toxicity/Toxicokinetics |
CCR1 antagonist 9 is a research compound and has not been clinically approved. In preclinical toxicology studies, the compound is well-tolerated at effective doses (10-30 mg/kg/day) in rodents over a 28-day period. No significant changes in body weight, food consumption, or hematological parameters (CBC) were observed. No hepatotoxicity (elevated ALT/AST) or nephrotoxicity (elevated BUN/creatinine) was reported. The lack of hERG activity at 30 microM indicates a low risk of cardiac QT prolongation.
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| References | |
| Additional Infomation |
CCR1 is a validated drug target for autoimmune and inflammatory diseases due to its role in recruiting inflammatory monocytes to tissues. Several CCR1 antagonists have entered clinical trials for RA, MS, and transplant rejection but have been discontinued due to lack of efficacy or toxicity issues (e.g., MLN3897, BX471). CCR1 antagonist 9 is a structurally novel, selective, and potent tool compound for understanding CCR1 biology and exploring its therapeutic potential. It is not an FDA-approved drug.
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| Molecular Formula |
C20H16FN5O3S
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|---|---|
| Molecular Weight |
425.436145782471
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| Exact Mass |
425.095
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| CAS # |
1220026-26-5
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| PubChem CID |
59278314
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
1.5
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
30
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| Complexity |
710
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S(C)(C1C=C(C=CN=1)CNC(C1=CN=CC2=C1C=NN2C1C=CC(=CC=1)F)=O)(=O)=O
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| InChi Key |
DCHROQCNJDJPGN-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H16FN5O3S/c1-30(28,29)19-8-13(6-7-23-19)9-24-20(27)17-10-22-12-18-16(17)11-25-26(18)15-4-2-14(21)3-5-15/h2-8,10-12H,9H2,1H3,(H,24,27)
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| Chemical Name |
1-(4-fluorophenyl)-N-[(2-methylsulfonylpyridin-4-yl)methyl]pyrazolo[3,4-c]pyridine-4-carboxamide
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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) |
DMSO : ~83.33 mg/mL (~195.87 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.89 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.89 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3505 mL | 11.7525 mL | 23.5051 mL | |
| 5 mM | 0.4701 mL | 2.3505 mL | 4.7010 mL | |
| 10 mM | 0.2351 mL | 1.1753 mL | 2.3505 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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