Morphothiadin (formerly GLS-4; GLS4) is a novel, potent and selective inhibitor of HBV replication, effective against both wild-type and adefovir-resistant HBV with an IC50 of 12 nM. Morphothiadin demonstrated potent inhibitory activities in HBV HepG2.2.15 cell assay with an EC50 value of 1nM, and it also exhibited high potency against various drug-resistant HBV viral strains with EC50 values in the range of 10-20nM, more potent than the typical HBV polymerase inhibitors such as lamivudine, telbivudine, and entecavir. Pharmacokinetic profiles of GLS4 were favorable and safety evaluation including acute toxicity and repeated toxicity study indicated that GLS4 was safe enough to support clinical experiments in human.

Physicochemical Properties

Molecular Formula	C21H22BRFN4O3S
Molecular Weight	509.39178609848
Exact Mass	508.06
Elemental Analysis	C, 49.52; H, 4.35; Br, 15.69; F, 3.73; N, 11.00; O, 9.42; S, 6.29
CAS #	1092970-12-1
Related CAS #	1793065-08-3 (R-isomer);2093044-32-5 (S-isomer);1092970-12-1 (racemic);1646361-04-7 (mesylate);
PubChem CID	25144422
Appearance	Light yellow to yellow solid powder
LogP	2.5
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	7
Heavy Atom Count	31
Complexity	719
Defined Atom Stereocenter Count	0
InChi Key	SQGRDKSRFFUBBU-UHFFFAOYSA-N
InChi Code	InChI=1S/C21H22BrFN4O3S/c1-2-30-21(28)17-16(12-27-6-8-29-9-7-27)25-19(20-24-5-10-31-20)26-18(17)14-4-3-13(23)11-15(14)22/h3-5,10-11,18H,2,6-9,12H2,1H3,(H,25,26)
Chemical Name	ethyl 4-[2-bromo-4-fluorophenyl]-6-[morpholino-methyl]-2-[2-thiazolyl]-1,4-dihydro-pyrimidine-5-carboxylate
Synonyms	Morphothiadine; GLS-4; GLS 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(IC50= 12 nM )
ln Vitro	With an IC50 of 12 nM, morphothiadin is a strong inhibitor of both adefovir-resistant and wild-type HBV replication. Up to 25 μM, morphothiadin (GLS4) exhibits no toxicity. For primary hepatocytes, the cytotoxic dose (CC50) at which 50% of cells die is 115 μM for morphothiadin (P<0.001). In HepAD38 cells, the CC90 for morphothiadin is 190 μM (P<0.01). At 25 nM to 100 nM, phothiadin significantly reduces the amount of virus that accumulates in the supernatant of HepAD38 cells (P<0.02). The core protein in cells treated with morphothiadin decreases in a concentration-dependent manner, according to the results[2].
ln Vivo	Morphothiadin (GLS4) has an area under the concentration-time curve (AUC0-24) of 556 h•ng/mL from 0 to 24 hours. Following the intravenous injection of 10 mg/kg of morphothiadin, the apparent volume distribution and total plasma clearance are 7.38 liters/kg and 4.2 liters/h/kg, respectively. Morphothiadin has a bioavailability of 25.5%. It is discovered that the mice treated with 3.75 mg/kg of morphothiadin per day had an increase in virus titers of 83.5 times, the mice treated with 7.5 mg/kg per day had an increase of 28.3 times, and the mice treated with the higher doses of morphothiadin had an increase of only 3 to 6 times.The amount of morphothiadin and the virus titer are generally inversely correlated; mice treated with 3.75 mg/kg of morphothiadin per day showed the largest rebound (540-fold), while mice treated with 60 mg/kg per day showed the smallest rebound (23-fold) (P<0.001). During the course of treatment, phothiadin doses exceeding 7.5 mg/kg per day significantly suppress the virus replication cycle. Moreover, phothiadin doses exceeding 15 mg/kg per day continue to suppress the virus for up to two weeks following the end of treatment[2].
Enzyme Assay	Dog and human liver microsomes and CYP3A4 were incubated with [(14)C]GLS4 for 15 min and then analyzed using a HPLC-dynamic online radio flow detection method. Two groups of beagle dogs were used for in vivo studies. Group A were orally administered a single dose of GLS4 (15 mg/kg) with or without ketoconazole pretreatment (100 mg/d for 8 consecutive days). Group B were orally administered a single dose of GLS4 (15 mg/kg) with or without rifampicin pretreatment (100 mg/d for 8 consecutive days). Plasma was sampled after GLS4 dosing. GLS4 concentrations were determined by HPLC-tandem mass spectrometry [1].
Cell Assay	Tetracycline (0.3 μg/mL) is used to grow HepAD38 cells to approximately 80% confluence. Following TET removal, the cells are either left untreated or given varying dosages of morphothiadin (GLS4). The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay is used to measure cell viability[2].
Animal Protocol	The pharmacokinetic (PK) characteristics of morphothiadin (GLS4) are assessed in ICR mice. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) is used to measure the amount of morphothiadin in plasma after oral administration of 10 mg/kg (of body weight) to male mice. ICR mice are given morphothiadin by gavage over a 4-week period for toxicity studies, after which they are kept off medication for an additional 2-week period. Twenty male and twenty female mice per group are given a vehicle (1% methyl-2-hydroxyethyl cellulose) at doses of 35.7, 118.9, or 356.6 mg/kg daily in a volume equivalent to 20 mL/kg. Two weeks after the end of the drug treatment, ten mice per dose group are put to death. Serum albumin levels, body weight, food intake, and side effects are calculated[2].
References	[1]. Effects of ketoconazole and rifampicin on the pharmacokinetics of GLS4, a novel anti-hepatitis B virus compound, in dogs. Acta Pharmacol Sin. 2013 Nov;34(11):1420-6. [2]. Preclinical characterization of GLS4, an inhibitor of hepatitis B virus core particle assembly. Antimicrob Agents Chemother. 2013 Nov;57(11):5344-54.

Solubility Data

Solubility (In Vitro)

DMSO : ~62.5 mg/mL (~122.70 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 3 mg/mL (5.89 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 30.0 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: ≥ 3 mg/mL (5.89 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 30.0 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	1.9631 mL	9.8157 mL	19.6313 mL
	5 mM	0.3926 mL	1.9631 mL	3.9263 mL
	10 mM	0.1963 mL	0.9816 mL	1.9631 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.