HBV-IN-4, a phthalazinone derivative, is a novel and selective HBV DNA replication inhibitor with an IC50 of 14 nM. HBV-IN-4 has effective anti activity and induces the formation of genome-free capsids.

Physicochemical Properties

Molecular Formula	C24H19CLFN5O3
Molecular Weight	479.8908
Exact Mass	479.12
CAS #	2305897-84-9
PubChem CID	146653485
Appearance	White to off-white solid powder
LogP	3.6
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	7
Heavy Atom Count	34
Complexity	799
Defined Atom Stereocenter Count	0
SMILES	ClC1=C(N=C(C(=C1[H])C1C2=C([H])C([H])=C([H])C([H])=C2C(N(C([H])([H])C2C([H])=C([H])C(C#N)=C([H])C=2[H])N=1)=O)F)N([H])C([H])([H])C([H])(C([H])([H])O[H])O[H]
InChi Key	WTCNPITUIDRZKR-UHFFFAOYSA-N
InChi Code	InChI=1S/C24H19ClFN5O3/c25-20-9-19(22(26)29-23(20)28-11-16(33)13-32)21-17-3-1-2-4-18(17)24(34)31(30-21)12-15-7-5-14(10-27)6-8-15/h1-9,16,32-33H,11-13H2,(H,28,29)
Chemical Name	4-[[4-[5-chloro-6-(2,3-dihydroxypropylamino)-2-fluoropyridin-3-yl]-1-oxophthalazin-2-yl]methyl]benzonitrile
Synonyms	HBV-IN-4
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	HBV DNA replication ( IC50 = 14 nM )
ln Vitro	In HepG2.2.15 cells, HBV-IN-4 (compound 19f; 0-1 μM; 8 days) treatment suppresses the different types (relaxed circular [rc] and single-stranded [ss] HBV DNA) in a dose-dependent manner. Treatment with HBV-IN-4 may also decrease capsid-associated DNAs in a dose-dependent manner. A faster-migrating phenotype of genome-free capsids may be induced by HBV-IN-4[1].
ln Vivo	In AAV-HBV/mouse model, HBV-IN-4 (Compound 19f; 50-150 mg/kg; oral administration; twice a day; for 4 weeks; Balb/c male mice) treatment results in 2.67 log viral load reduction[1]. When given orally at a dose of 20 mg/kg to mice, HBV-IN-4 (compound 19f) demonstrates good drug exposure (AUC0-t=49 744 h·ng/L), low plasma clearance (CL=4.1 mL/min/kg), T1/2 (2.15), and oral bioavailability (F=60.4%). In hepatic exposure, HBV-IN-4 likewise exhibits an excellent distribution[1].
Animal Protocol	Animal/Disease Models: Balb/c male mice (8weeks old) injected with a recombinant adenoassociated virus (AAV[1] Doses: 50 mg/kg, 150 mg/kg Route of Administration: Oral administration; twice a day ; for 4 weeks Experimental Results: Resulted in a 2.67 log reduction of the HBV DNA viral load during a 4-week treatment.
References	[1]. Discovery of Phthalazinone Derivatives as Novel Hepatitis B Virus Capsid Inhibitors. J Med Chem. 2020 Jul 21.

Solubility Data

Solubility (In Vitro)

DMSO : ~100 mg/mL (~208.4 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.21 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 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. Solubility in Formulation 2: 2.5 mg/mL (5.21 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 ultrasonication. 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 3: ≥ 2.5 mg/mL (5.21 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 25.0 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	2.0838 mL	10.4191 mL	20.8381 mL
	5 mM	0.4168 mL	2.0838 mL	4.1676 mL
	10 mM	0.2084 mL	1.0419 mL	2.0838 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.