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.

Physicochemical Properties

Molecular Formula	C23H21CLN2O3
Molecular Weight	408.88
Exact Mass	408.124
Elemental Analysis	C, 67.56; H, 5.18; Cl, 8.67; N, 6.85; O, 11.74
CAS #	1443437-74-8
Related CAS #	1443437-74-8
PubChem CID	71681561
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Boiling Point	656.0±55.0 °C at 760 mmHg
Flash Point	350.5±31.5 °C
Vapour Pressure	0.0±2.0 mmHg at 25°C
Index of Refraction	1.633
LogP	3.76
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	4
Heavy Atom Count	29
Complexity	579
Defined Atom Stereocenter Count	0
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
InChi Key	HERLZBNILRVHQN-UHFFFAOYSA-N
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)
Chemical Name	N-(4-chlorophenyl)-1-[3-(furan-2-yl)benzoyl]piperidine-3-carboxamide
Synonyms	CCG203971; CCG 203971; CCG-203971
HS Tariff Code	2934.99.9001
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)

Biological Activity

Targets	Rho; SRE.L ( IC50 = 0.64 μM )
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].
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].
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.
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.
References	[1]. Bioorg Med Chem Lett . 2017 Apr 15;27(8):1744-1749. [2]. J Pharmacol Exp Ther . 2014 Jun;349(3):480-6.

Solubility Data

Solubility (In Vitro)

DMSO: 81~250 mg/mL (198.1~611.4 mM) Water: < 1mg/mL Ethanol: ~4 mg/mL (~9.8 mM)

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. 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. 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. Solubility in Formulation 4: 10% DMSO: 40% PEG300: 5% Tween-80: 45% saline: ≥ 2.5 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

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.