Phenamil methanesulfonate is an analogue of Amiloride, a more potent and irreversible epithelial sodium channel (ENaC) blocker with IC50 of 400 nM. Phenamil methanesulfonate is also a competitive inhibitor of TRPP3. In Ca2+ uptake experiments, the IC50 for inhibiting TRPP3-mediated Ca2+ transport was 140 nM. Phenamil methanesulfonate is a small molecule that promotes bone repair by strongly activating the BMP signaling pathway. Phenamil methanesulfonate is being studied in cystic fibrosis lung disease.

Physicochemical Properties

Molecular Formula	C13H16CLN7O4S
Molecular Weight	401.83
Exact Mass	401.067
CAS #	1161-94-0
PubChem CID	135419184
Appearance	Light yellow to yellow solid powder
Boiling Point	621.7ºC at 760 mmHg
Flash Point	329.8ºC
Vapour Pressure	2.22E-15mmHg at 25°C
LogP	3.381
Hydrogen Bond Donor Count	5
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	3
Heavy Atom Count	26
Complexity	492
Defined Atom Stereocenter Count	0
InChi Key	MHPIZTURFVSLTJ-UHFFFAOYSA-N
InChi Code	InChI=1S/C12H12ClN7O.CH4O3S/c13-8-10(15)19-9(14)7(18-8)11(21)20-12(16)17-6-4-2-1-3-5-6;1-5(2,3)4/h1-5H,(H4,14,15,19)(H3,16,17,20,21);1H3,(H,2,3,4)
Chemical Name	3,5-diamino-6-chloro-N-(N'-phenylcarbamimidoyl)pyrazine-2-carboxamide;methanesulfonic acid
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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.
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	TRPC3 140 nM (IC50)
ln Vitro	TRPP3, a cation channel that belongs to the transient receptor potential (TRP) superfamily, is triggered by Ca2+ and permeable to Ca2+, Na+, and K+. In spinal cord neurons, TRPP3 is involved in the control of pH-sensitive action potentials. In radiotracer uptake experiment, phenamil methanesulfonate (1 μM) reduces 45Ca2+ absorption. With an IC50 value of 0.28 μM in oocytes expressing TRPP3 or oocytes injected with H2O, it inhibits TRPP3-mediated Ca2+ transport[1]. The epithelial sodium channel (ENaC) is inhibited by phenamil methanesulfonate, a more powerful ENaC blocker than amiloride, with an IC50 of 400 nM (vs 776 nM for amiloride)[2]. In both human and ovine bronchial epithelia cells, phenolmethyl methanesulfonate suppresses baseline short-circuit currents with IC50 values of 75 and 116 nM, respectively[3]. In C3H10T1/2 cells, phenamil methanesulfonate (0–20 μM; 14 days) controls adipogenesis and increases the expression of PPARγ, Fabp4, lipoprotein lipase, and adipogenic genes in a concentration-dependent manner[4]. Methanesulfonate (0–20 μM; 7 or 14 days) boosts concentration-dependently the activity of alkaline phosphatase (ALP) in MC3T3–E1 cells and modifies their osteoblastic differentiation[4].
ln Vivo	PAH caused by chronic hypoxia is lessened by phenamil methanesulfonate (subcutaneous injection; 15 or 30 mg/kg; 21 days; infusion rate of 1 ml/h). Furthermore, Phenamil reduces the mRNA levels of SMA, SM22, Id3, and Trb3 in the lung sample in rats under hypoxia or normoxia. Phenamil, however, has minimal effects on the pulmonary vasculature in a physiological setting[5].
Cell Assay	RT-PCR[4] Cell Types: C3H10T1/2 cells Tested Concentrations: 0 μM and 20 μM Incubation Duration: 14 days Experimental Results: Increased PPARγ, Fabp4, and lipoprotein lipase (LPL) mRNA expression.
Animal Protocol	Animal/Disease Models: Male SD (Sprague-Dawley) rats[5] Doses: 15 or 30 mg /kg Route of Administration: subcutaneous (sc) injection; 15 or 30 mg/kg; 21 days; infusion rate of 1 ml/h Experimental Results: decreased hypoxia-induced pulmonary hypertension and vascular remodeling.
References	[1]. Inhibition of TRPP3 channel by amiloride and analogs. Mol Pharmacol. 2007 Dec;72(6):1576-85. [2]. Design, synthesis, and structure-activity relationships of novel 2-substituted pyrazinoylguanidine epithelial sodium channel blockers: drugs for cystic fibrosis and chronic bronchitis. J Med Chem. 2006 Jul 13;49(14):4098-115. [3]. Evaluation of second generation amiloride analogs as therapy for cystic fibrosis lung disease.J Pharmacol Exp Ther. 2004 Dec;311(3):929-38. [4]. The amiloride derivative phenamil attenuates pulmonary vascular remodeling by activating NFAT and the bone morphogenetic protein signaling pathway. Mol Cell Biol.

Solubility Data

Solubility (In Vitro)

DMSO: 25 mg/mL (62.22 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.22 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 25.0 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.4886 mL	12.4431 mL	24.8861 mL
	5 mM	0.4977 mL	2.4886 mL	4.9772 mL
	10 mM	0.2489 mL	1.2443 mL	2.4886 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.