Pradefovir (formerly known as Remofovir, ICN-2001-3, MB-06866, MB-6866; ICN-20013) is an RTI (reverse transcriptase) inhibitor that has the potential for the treatment of chronic HBV infection. Pradefovir is prodrug of adefovir that is designed to target liver. Pradefovir can be activated to PMEA (9-(2-phosphonylmethoxyethyl)adenine ) in human liver microsomes with with a Km of 60 μM, a maximum rate of metabolism of 228 pmol/min/mg protein, and an intrinsic clearance of about 359 ml/min.

Physicochemical Properties

Molecular Formula	C17H19CLN5O4P
Molecular Weight	423.79
Exact Mass	423.086
CAS #	625095-60-5
Related CAS #	625095-60-5 freebase
PubChem CID	9604654
Appearance	Typically exists as solid at room temperature
LogP	3.988
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	6
Heavy Atom Count	28
Complexity	576
Defined Atom Stereocenter Count	2
SMILES	ClC1=CC=CC(=C1)[C@@H]1CCOP(COCCN2C=NC3C(N)=NC=NC2=3)(=O)O1
InChi Key	GWNHAOBXDGOXRR-HJFSHJIFSA-N
InChi Code	InChI=1S/C17H19ClN5O4P/c18-13-3-1-2-12(8-13)14-4-6-26-28(24,27-14)11-25-7-5-23-10-22-15-16(19)20-9-21-17(15)23/h1-3,8-10,14H,4-7,11H2,(H2,19,20,21)/t14-,28+/m0/s1
Chemical Name	(2R,4S)-2-((2-(6-amino-9H-purin-9-yl)ethoxy)methyl)-4-(3-chlorophenyl)-1,3,2-dioxaphosphinane 2-oxide
Synonyms	Remofovir; ICN-2001-3; MB-06866; MB 06866; MB06866; MB6866; MB-6866; MB 6866; ICN-20013; ICN20013; ICN2001-3;
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	Pradefovir was converted to PMEA in human liver microsomes with a K(m) of 60 microM, a maximum rate of metabolism of 228 pmol/min/mg protein, and an intrinsic clearance of about 359 ml/min. Addition of ketoconazole and monoclonal antibody 3A4 significantly inhibits the conversion of pradefovir to PMEA in human liver microsomes, suggesting the predominant role of CYP3A4 in the metabolic activation of pradefovir. Pradefovir at 0.2, 2, and 20 microM was neither a direct inhibitor nor a mechanism-based inhibitor of CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 in human liver microsomes.[1]
ln Vivo	In rats, the liver was the site of metabolic activation of pradefovir, whereas the small intestine did not play a significant role in the metabolic conversion of pradefovir to PMEA. Daily oral dosing (300 mg/kg of body weight) to rats for 8 days showed that pradefovir was not an inducer of P450 enzymes in rats. Furthermore, pradefovir at 10 microg/ml was not an inducer of either CYP1A2 or CYP3A4/5 in primary cultures of human hepatocytes.[1]
Cell Assay	Pradefovir (1.63, 4.08, 16.3, or 40.8 μM) or testosterone (50 μM) was also incubated with pooled human liver microsomes (0.4 mg/ml), with or without ketoconazole (various concentrations), or CYP3A4-inhibitory monoclonal antibody. All incubations were performed in 1.5-ml Eppendorf tubes containing potassium phosphate buffer (50 mM; pH 7.4). The mixtures were preheated at 37°C for 3 min.[1] The preparations of hepatocytes cultured from three separate human livers were treated with dimethyl sulfoxide (0.1 vol/vol), pradefovir (0.1, 1, or 10 μg/ml), PMEA (0.01, 0.1, or 1 μg/ml), and two known human P450 inducers (β-naphthoflavone [33 μM] or rifampin [20 μM]) once daily for three consecutive days. The cultured hepatocytes were assessed daily by light microscopy to assess them for morphological normalcy, with confluence adequate for treatments.[1]
Animal Protocol	Rats were fasted overnight and given 30 mg/kg of [14C]pradefovir mesylate by oral gavage. Blood samples were simultaneously collected from the portal vein and systemic vein cannulas at 2, 5, 10, 20, 40, and 60 min after dosing and placed in heparinized tubes; and whole blood was centrifuged to harvest the plasma. The plasma concentrations of pradefovir and PMEA were determined by a validated LC-MS/MS method.[1] Five rats received pradefovir (300 mg/kg/day) orally for 8 days. At 24 h after administration of the last dose, the rats were killed and liver samples were collected. Body weight, liver weight, liver protein content, and liver microsomal P450 contents were determined. The apoprotein levels for CYP1A1, CYP2B1/2B2, CYP3A1/3A2, and CYP4A1/4A3 and the enzyme activities for CYP1A, CYP2B, and CYP3A were also determined by Western blot and LC analysis, respectively.[1]
References	Antimicrob Agents Chemother.2006 Sep;50(9):2926-31.
Additional Infomation	Pradefovir is a prodrug of [adefovir]. Pradefovir is a cyclodiester antiviral prodrug with specific activity against hepatitis B virus (HBV). Pradefovir is specifically metabolized in the liver by hepatic enzymes, mainly by CYP4503A4, to adefovir. In turn, adefovir is phosphorylated by cellular kinases to its activated form adevofir diphosphate. By competing with the natural substrate dATP, the diphosphate form is incorporated into viral DNA and inhibits RNA-dependent DNA polymerase. This causes DNA chain termination and eventually results in an inhibition of HBV replication.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 50 mg/mL Water:N/A Ethanol:N/A

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.3597 mL	11.7983 mL	23.5966 mL
	5 mM	0.4719 mL	2.3597 mL	4.7193 mL
	10 mM	0.2360 mL	1.1798 mL	2.3597 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.