5-(N,N)-Dimethylamiloride Hydrochloride (Hexamethylene amiloride), an amiloride derivative, is a Na(+)-H+ exchange inhibitor that decreases the intracellular pH (pHi) and induces apoptosis in leukemic cells. 5-(N,N-Hexamethylene)-amiloride (Hexamethylene amiloride) is also an inhibitor of the HIV-1 Vpu virus ion channel and inhibits mouse hepatitis virus (MHV) replication and human coronavirus 229E (HCoV229E) replication in cultured L929 cells with EC50s of 3.91 μM and 1.34 μM, respectively.

Physicochemical Properties

Molecular Formula	C₁₂H₁₈CLN₇O
Molecular Weight	311.77
Exact Mass	311.126
CAS #	1428-95-1
PubChem CID	1794
Appearance	Light yellow to yellow solid powder
Density	1.63g/cm3
Boiling Point	638.2ºC at 760mmHg
Flash Point	339.8ºC
Vapour Pressure	3.47E-16mmHg at 25°C
Index of Refraction	1.742
LogP	2.553
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	2
Heavy Atom Count	21
Complexity	393
Defined Atom Stereocenter Count	0
InChi Key	RQQJJXVETXFINY-UHFFFAOYSA-N
InChi Code	InChI=1S/C12H18ClN7O/c13-8-10(20-5-3-1-2-4-6-20)18-9(14)7(17-8)11(21)19-12(15)16/h1-6H2,(H2,14,18)(H4,15,16,19,21)
Chemical Name	3-amino-5-(azepan-1-yl)-6-chloro-N-(diaminomethylidene)pyrazine-2-carboxamide
Synonyms	Hexamethylene amiloride HMA
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

ln Vitro	5-(N,N-hexamethylene)-amiloride inhibits human cardiac ion channels hERG (in CHO cells), Nav1.5, and Cav1.2 (in EHK293 cells) in electrophysiological assays. The concentrations of the inhibitors are 3.3 μM, 30 μM, and 8.3 μM, respectively [3]. At 1 μM, it demonstrated microsomal stability [3]. At 1 μg/mL, it demonstrated mouse plasma stability and plasma protein binding [4–6 hours at 37 °C]. At 20 μM, it demonstrated Caco-2 cell permeability and cardiac ion channel activity [4–6 hours at 37 °C].
ln Vivo	5-(N,N-hexamethylene)-amiloride (2.5 mg/kg; intravenous injection; single dose) has a limited oral bioavailability of 4.5% and a short half-life [3]. Rat or mouse in vivo pharmacokinetic models [3]. The dosage is 2.5 mg/kg. Single-dose intravenous injection administered 10 and 60 minutes following treatment. t1/2 (hour) (mL/min/kg) Plasma CLint Vss of Plasma (L/kg) (h·μM) Plasma AUC0-inf B/P percentage CL of blood (mL/min/kg) Blood Volume (L/kg) ) Mouse, female Balb/c 0.62-86 2.0 1.559 1.4 1.5 The Dawley rat Sprague urine excreted from an IV dose (0–24 hours): 3.2 83.5 5.3 1.6 1.8 46.2 2.9% (mL/min/kg) for renal blood CL CL of non-renal blood (mL/min/kg) Rat Spraggle Dawley 0.5 0.2 46.0 Note: Female Balb/c mouse (17–27 g, non-fasting); male Sprague Dawley Rats (238–325 g, overnight fasting); B/P is the distribution ratio of blood to plasma.
Cell Assay	Cell Viability Assay[3] Cell Types: In vitro pharmacokinetic properties Tested Concentrations: 1 μM Incubation Duration: 0-60 minutes Experimental Results: t1/2 (min) CLint (μL/min/mg protein) CLint (μL/min/mg Protein) Human liver microsomes in vitro 73 24 74 Mouse liver microsomes in vitro 2.4 726 2243
References	[1]. Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na(+)/H(+) exchanger. Blood. 2000 Feb 15;95(4):1427-34. [2]. Hexamethylene amiloride blocks E protein ion channels and inhibits coronavirus replication. Virology. 2006 Sep 30;353(2):294-306. Epub 2006 Jul 3. [3]. Systematic evaluation of structure-property relationships and pharmacokinetics in 6-(hetero)aryl-substituted matched pair analogs of amiloride and 5-(N,N-hexamethylene)amiloride. Bioorg Med Chem. 2021 May 1;37:116116.
Additional Infomation	5-(N,N-hexamethylene)amiloride is a member of the class of pyrazines that is amiloride in which the two amino hydrogens at position N-5 are replaced by a hexamethylene moiety, resulting in the formation of an azepane ring. It has a role as a sodium channel blocker, an apoptosis inducer, an antineoplastic agent and an odorant receptor antagonist. It is a member of pyrazines, an organochlorine compound, a member of azepanes, a member of guanidines, an aromatic amine and a monocarboxylic acid amide. It is functionally related to an amiloride.

Solubility Data

Solubility (In Vitro)

DMSO : ~100 mg/mL (~320.75 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 2.08 mg/mL (6.67 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication. 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 (6.67 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 3: ≥ 2.08 mg/mL (6.67 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	3.2075 mL	16.0375 mL	32.0749 mL
	5 mM	0.6415 mL	3.2075 mL	6.4150 mL
	10 mM	0.3207 mL	1.6037 mL	3.2075 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.