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Purity: ≥98%
CCG-203971 is a novel, 2nd generation small-molecule inhibitor of the Rho/MRTF/SRF [RhoA/myocardin-related transcription factor A (MRTF-A)] pathway with an IC50 value of 0.64 μM for SRE. CCG-203971 suppresses the expression of collagen 1 (COL1A2), α-smooth muscle actin (α-SMA), and connective tissue growth factor (CTGF) in SSc fibroblasts as well as fibroblasts stimulated by transforming growth factor β (TGFβ) and lysophosphatidic acid (LPA). At a concentration of 25 μM, CCG-203971 significantly suppresses MKL1 expression induced by TGF-β.
| Targets |
Rho; SRE.L ( IC50 = 0.64 μM )
Rho/MRTF/SRF transcriptional pathway [1] |
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| ln Vitro |
In vitro activity: CCG-203971 suppresses the expression of collagen 1 (COL1A2), α-smooth muscle actin (α-SMA), and connective tissue growth factor (CTGF) in SSc fibroblasts as well as fibroblasts stimulated by transforming growth factor β (TGFβ) and lysophosphatidic acid (LPA)[2]. 1. CCG-203971 inhibited the expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA, ACTA2), and collagen 1 (COL1A2) in dermal fibroblasts from patients with diffuse cutaneous systemic sclerosis (SSc) in a concentration-dependent manner; it also suppressed the upregulation of these fibrotic genes in NIH-3T3 fibroblasts stimulated by 10 μM lysophosphatidic acid (LPA) (assessed by qPCR, with COL1A2 primers targeting newly synthesized heterogeneous nuclear RNA, expression quantified relative to GAPDH). [2] 2. CCG-203971 (30 μM) reduced the proliferation rate of SSc dermal fibroblasts (assessed by WST-1 assay for viable cell density after 3 days of culture), which was faster than normal dermal fibroblasts; 10 μM CCG-203971 inhibited the TGFβ-induced (10 ng/ml, 3 days) myofibroblast transition of normal human dermal fibroblasts (assessed by immunocytochemistry for α-SMA with nuclear DAPI staining), and also decreased the fraction of α-SMA-positive cells in SSc dermal fibroblasts in a concentration-dependent manner (scored by blinded observer). [2] |
| ln Vivo |
CCG-203971 shows encouraging anti-fibrotic activity in a number of animal disease models, including intestinal, pulmonary, and dermal fibrosis, both in vitro and in vivo. CCG-203971 has a half-life of only 1.6 minutes in mouse liver microsomes (MLM), which suggests that it is highly susceptible to oxidative metabolism[1]. Moreover, bleomycin-induced skin thickening and collagen deposition are prevented by CCG-203971 in vivo treatment[2].
1. CCG-203971 (100 mg/kg, intraperitoneal injection, twice daily) prevented bleomycin-induced (0.1 mg intradermal injection for 2 weeks) skin thickening and collagen deposition in C57BL/6J mice (n=7 per group); skin thickness (triplicate measures per mouse) and hydroxyproline content (collagen marker) were significantly reduced compared with bleomycin + vehicle group (assessed by Masson’s trichrome staining and hydroxyproline assay, one-way ANOVA with Bonferroni post-test: P < 0.05 for hydroxyproline, P < 0.001 for skin thickness). [2] 2. CCG-203971 showed modest in vivo potency and poor pharmacokinetic properties, making it unsuitable for long-term efficacy studies; its analog CCG-232601 (8f) exhibited oral efficacy (50 mg/kg daily oral gavage for 14 days) in inhibiting bleomycin-induced dermal fibrosis in mice (n=8 per group), comparable to CCG-203971 at a 4-fold higher intraperitoneal dose. [1] |
| Cell Assay |
Plated into a 96-well plate, human dermal fibroblasts (2.0 × 104) are cultured in DMEM containing 10% FBS for an entire night. After removing the media, they are replaced with DMEM that contains 0.1% DMSO control or 30 μM CCG-203971 along with 2% FBS. WST-1 dye is added to each well after 72 hours, and the absorbance at 490 nm is measured after 60 minutes.
1. LPA-stimulated fibrotic gene expression assay: NIH-3T3 cells were treated with various concentrations of CCG-203971 or DMSO for 23 hours; 1 hour before RNA isolation, cells were stimulated with 10 μM LPA; total RNA was extracted, and qPCR was performed to detect mRNA expression of CTGF, ACTA2, and COL1A2 (COL1A2 primers targeting hnRNA), with expression levels normalized to GAPDH; experiments were repeated independently twice, data presented as mean ± S.D. [2] 2. SSc fibroblast gene expression assay: Primary human dermal fibroblasts from normal donors or SSc patients (≤5 passages) were cultured; for SSc fibroblasts, cells were treated with indicated concentrations of CCG-203971 or 300 μM pirfenidone (PFD) for 24 hours before RNA isolation; qPCR was used to quantify CTGF, ACTA2, and COL1A2 mRNA expression, data presented as mean ± S.E.M. (n≥4 individuals for SSc, n=3 for normal). [2] 3. Fibroblast proliferation assay: Normal/SSc dermal fibroblasts were plated in 96-well plates and cultured for 3 days with 30 μM CCG-203971 or DMSO; viable cell density was measured by WST-1 assay (enzymatic reduction of water-soluble tetrazolium dye), data presented as mean ± S.E.M. (n=3 individuals). [2] 4. Myofibroblast transition assay: Normal human dermal fibroblasts were plated on coverslips, treated with 10 ng/ml TGFβ for 3 days (with/without 10 μM CCG-203971 or DMSO); SSc dermal fibroblasts were plated on coverslips and treated with indicated concentrations of CCG-203971; cells were fixed, immunocytochemistry performed for α-SMA (nuclear DAPI staining), and α-SMA-positive cell fraction scored by blinded observer (data mean ± S.E.M., n≥4 individuals). [2] |
| Animal Protocol |
Mice: In C57BL/6 mice (female, 8 weeks old), a local intracutaneous injection of 100 μL of Bleomycin (1 mg/mL) in phosphate-buffered saline (PBS) is administered daily for two weeks in a predetermined area (~1 cm2) on the upper back to induce skin fibrosis. A control is a 100 μL intracutaneous injection of PBS. A total of 21 mice are used, divided into three groups. Two groups are given bleomycin challenges while the first group is given PBS injections. During the first two weeks of the Bleomycin challenge, 100 mg/kg of CCG-203971 administered intraperitoneally twice daily in 50 μL of DMSO is started. The vehicle control system is DMSO. The animal groups are as follows: (1) Bleomycin/DMSO; (2) PBS/DMSO; and (3) Bleomycin/CCG-203971. Following therapy, cervical dislocation is used to humanely kill the animals, and tissue is gathered.
1. Bleomycin-induced skin fibrosis model in mice: Three groups of C57BL/6J mice (n=7 per group) received intradermal injections of bleomycin (0.1 mg) or PBS (control) for 2 weeks; mice in the treatment group received twice daily intraperitoneal injections of CCG-203971 (100 mg/kg) or vehicle (50 μl DMSO); at the end of treatment, skin samples were collected for Masson’s trichrome staining (skin thickness measurement) and hydroxyproline content analysis. [2] 2. Pharmacokinetic optimization study in mice: CCG-203971 was evaluated for metabolic stability and solubility; analogs (including CCG-232601/8f) were tested for plasma exposure in mice (over 10-fold increase vs CCG-203971); for the efficacy study of CCG-232601, three groups of mice (n=8 per group) were treated for 14 days: Control (intracutaneous PBS), Bleo (intracutaneous bleomycin + oral vehicle), Bleo + 8f (intracutaneous bleomycin + 50 mg/kg 8f daily oral gavage). [1] |
| ADME/Pharmacokinetics |
1. CCG-203971 has poor pharmacokinetic (PK) characteristics, including low metabolic stability and low solubility; its analogues (e.g., CCG-232601/8f) showed more than 10-fold increased plasma exposure in mice after optimization. [1]
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| References | |
| Additional Infomation |
1. CCG-203971 is a novel small molecule inhibitor that can inhibit the Rho/MRTF/SRF transcriptional pathway, which is a key regulator of myofibroblast activation; it can block the nuclear localization of MRTF, thereby inhibiting the transcriptional program of profibrotic genes activated by multiple stimulating factors (LPA, TGFβ) during fibrosis. [2] 2. CCG-203971 is a potential antifibrotic therapy that can be used to treat systemic scleroderma (SSc) and other fibrotic diseases; current SSC therapies mainly focus on anti-inflammatory drugs or single receptor targets, while CCG-203971 targets the common downstream pathway of multiple profibrotic factors (Rho/MRTF/SRF), and therefore may have a wider range of therapeutic effects. [2]
3. CCG-203971 was initially developed as a Rho-mediated gene transcription inhibitor. It was effective in acute fibrosis animal models (including scleroderma) after intraperitoneal injection, but its pharmacokinetic properties were poor, and medicinal chemistry optimization was needed to improve its metabolic stability and solubility. [1] |
| Molecular Formula |
C23H21CLN2O3
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|---|---|---|
| Molecular Weight |
408.88
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| Exact Mass |
408.124
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| Elemental Analysis |
C, 67.56; H, 5.18; Cl, 8.67; N, 6.85; O, 11.74
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| CAS # |
1443437-74-8
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| Related CAS # |
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| PubChem CID |
71681561
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
656.0±55.0 °C at 760 mmHg
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| Flash Point |
350.5±31.5 °C
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| Vapour Pressure |
0.0±2.0 mmHg at 25°C
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| Index of Refraction |
1.633
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| LogP |
3.76
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
29
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| Complexity |
579
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C([H])=C([H])C(=C([H])C=1[H])N([H])C(C1([H])C([H])([H])N(C(C2=C([H])C([H])=C([H])C(C3=C([H])C([H])=C([H])O3)=C2[H])=O)C([H])([H])C([H])([H])C1([H])[H])=O
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| InChi Key |
HERLZBNILRVHQN-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H21ClN2O3/c24-19-8-10-20(11-9-19)25-22(27)18-6-2-12-26(15-18)23(28)17-5-1-4-16(14-17)21-7-3-13-29-21/h1,3-5,7-11,13-14,18H,2,6,12,15H2,(H,25,27)
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| Chemical Name |
N-(4-chlorophenyl)-1-[3-(furan-2-yl)benzoyl]piperidine-3-carboxamide
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (6.11 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.09 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (5.09 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 10% DMSO: 40% PEG300: 5% Tween-80: 45% saline: ≥ 2.5 mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4457 mL | 12.2285 mL | 24.4571 mL | |
| 5 mM | 0.4891 mL | 2.4457 mL | 4.8914 mL | |
| 10 mM | 0.2446 mL | 1.2229 mL | 2.4457 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.
LPA activates fibrotic gene expression in 3T3 fibroblasts in a Rho/MRTF-dependent manner. NIH-3T3 cells were treated with the indicated concentration of CCG-203971 or DMSO for 23 hours. One hour prior to RNA isolation, cells were stimulated with 10μM LPA.J Pharmacol Exp Ther.2014 Jun;349(3):480-6. |
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SSc-patient dermal fibroblasts show increased expression of fibrosis markers/MRTF target genes, which are inhibited by CCG-203971.J Pharmacol Exp Ther.2014 Jun;349(3):480-6. |
 Scleroderma dermal fibroblasts proliferate faster than normal cells, and this is inhibited by CCG-203971.J Pharmacol Exp Ther.2014 Jun;349(3):480-6. |
 CCG-203971 modulates myofibroblast transition of dermal fibroblasts.J Pharmacol Exp Ther.2014 Jun;349(3):480-6. |
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CCG-203971 prevents bleomycin-induced fibrosis in vivo.J Pharmacol Exp Ther.2014 Jun;349(3):480-6. |
8f (CCG-203971)inhibits bleomycin-induced skin fibrosis in mice. Three groups of mice (n=8) were treated for 14 days with one of the following daily protocols: Control: intracutaneous injections of PBS; Bleo: intracutaneous injections of bleomycin with concurrent oral gavage of vehicle; or Bleo + 8f: intracutaneous injections of bleomycin with concurrent oral gavage of 50 mg/kg8f.Bioorg Med Chem Lett.2017 Apr 15;27(8):1744-1749. |
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