HBV(IC50= 12 nM )
Hepatitis B Virus (HBV) replication (IC₅₀ = 12 nmol/L for both wild-type and adefovir-resistant HBV) [1]

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].

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GLS4 is a potent inhibitor of Hepatitis B Virus (HBV) replication, targeting HBV capsid formation. It inhibits the replication of both wild-type and adefovir-resistant HBV with an IC₅₀ of 12 nmol/L. [1]
Incubation of radiolabeled GLS4 with dog and human liver microsomes and recombinant human CYP3A4 showed extensive metabolism within 15 minutes, with less than 20% of the parent compound remaining. The major metabolic pathways were morpholine N-dealkylation and morpholine N,N-di-dealkylation. These metabolic pathways were almost completely inhibited by the CYP3A-specific inhibitor ketoconazole, indicating that GLS4 is a sensitive substrate of CYP3A. [1]

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].

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In a nude mouse model subcutaneously inoculated with HBV-replicating HepAD38 cells (which form tumors and lead to viremia), oral administration of GLS4 for 14 days resulted in a dose-dependent reduction of serum HBV DNA levels during the treatment period. Doses of 15, 30, and 60 mg/kg/day showed highly significant suppression compared to carrier-treated mice. The suppression persisted for up to 2 weeks after treatment ended (rebound period) at doses ≥ 15 mg/kg/day. The antiviral effect during and after treatment was comparable to that of BAY 41-4109 (60 mg/kg/day) and superior to lamivudine (100 mg/kg/day), which showed significant viral rebound after treatment cessation. [2]
Tumors extracted from mice treated with GLS4 (60 mg/kg/day) at the end of treatment (week 3) showed at least a 4-fold lower level of HBc polypeptide by Western blot compared to tumors from mice at the end of the rebound period (week 5) (P < 0.005). Immunohistochemistry confirmed weaker and scattered HBc staining in tumors at the end of treatment compared to strong staining at the end of the rebound period. [2]

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].

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The in vitro metabolism of GLS4 was assessed using incubation assays. Radiolabeled [¹⁴C]GLS4 (10 μmol/L) was incubated at 37°C for 15 minutes with either dog liver microsomes (1.0 mg protein/mL), human liver microsomes (1.0 mg protein/mL), or recombinant human CYP3A4 (50 pmol/mL) in phosphate buffer (pH 7.4). Reactions were initiated by adding NADPH (1 mmol/L) and terminated with ice-cold acetonitrile. For inhibition studies, ketoconazole (1 μmol/L) was pre-incubated with the microsomes and substrate before initiating the reaction with NADPH. Metabolites were separated and detected using a gradient HPLC system coupled with a dynamic online radio flow detector. [1]

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].

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Cytotoxicity Assay (MTT): Primary human hepatocytes or HepAD38 cells were treated with increasing concentrations of GLS4, BAY 41-4109, or no drug. After 48 hours of treatment, cell viability was measured using the MTT assay. Absorbance was read to determine the percentage of viable cells relative to untreated controls. [2]
Antiviral Activity Assay (qPCR): HepAD38 cells were seeded and treated with different concentrations of GLS4, BAY 41-4109, or lamivudine for 7 days, with daily refreshment of medium and drug. Cell culture supernatants were collected, and HBV DNA levels were quantified using real-time PCR. [2]
Intracellular HBV DNA Analysis (Southern Blot): HepAD38 cells were treated with drugs for 7 days. Total intracellular DNA was extracted and subjected to Southern blot hybridization using an HBV-specific probe to detect replicative intermediate DNA forms (relaxed circular, double-stranded, single-stranded) and cccDNA. Signals were quantified using a phosphorimager. [2]
Western Blot for HBc Antigen: HepAD38 cells were treated with drugs for a specified period. Cell lysates were prepared, proteins were separated by SDS-PAGE, transferred to a membrane, and probed with an antibody against Hepatitis B core antigen (HBc). Signals were normalized to a loading control (e.g., β-actin) and quantified by gel scanning. [2]

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].

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A pharmacokinetic and drug-drug interaction study was conducted in beagle dogs. Eight healthy male beagle dogs (10-15 kg) were used and divided into two groups (A and B). GLS4 was dissolved in 0.5% carboxymethylcellulose sodium for oral gavage administration. On the first day (pre-treatment phase), all dogs received a single oral dose of GLS4 (15 mg/kg). Blood samples were collected from the jugular vein before dosing and at 0.5, 1, 1.5, 2, 3, 5, 7, 9, 12, and 24 hours post-dosing.
After a three-day wash-out period, Group A received an oral dose of ketoconazole (100 mg, capsule) once daily for 8 consecutive days. Group B received an oral dose of rifampicin (100 mg, capsule) once daily for 8 consecutive days. On the 12th day (post-treatment phase), immediately after the final dose of ketoconazole or rifampicin, all dogs received another single oral dose of GLS4 (15 mg/kg). Blood sampling was repeated as in the pre-treatment phase. Plasma was separated and stored at -70°C until analysis by HPLC-MS/MS. [1]

GLS4 is metabolized extensively, primarily through CYP3A-mediated N-dealkylation and N,N-didealkylation of morpholine in canine and human liver microsomes. In beagle dogs, the pharmacokinetic parameters (mean ± standard error) of GLS4 after a single oral dose of 15 mg/kg were: Cmax = 0.445 ± 0.098 μg/mL, Tmax = 0.875 ± 0.250 h, AUC₀–∞ = 1.81 ± 0.38 μg·h·mL⁻¹, T₁/₂ = 12.1 ± 2.7 h, CL/F = 8.58 ± 1.80 L·h⁻¹·kg⁻¹. Pretreatment with the CYP3A inhibitor ketoconazole significantly increased GLS4 exposure: Cmax increased 3.3-fold. The Cmax of GLS4 was 1.38 ± 0.34 μg/mL, and the AUC₀–∞ increased by 4.4-fold to 7.21 ± 1.72 μg·h·mL⁻¹. The clearance rate (CL/F) decreased by 75.8%. Conversely, pretreatment with the CYP3A inducer rifampin significantly reduced GLS4 exposure: Cmax decreased by 83.2% to 0.0810 ± 0.0127 μg/mL, and AUC₀–∞ decreased by 88.5% to 0.204 ± 0.012 μg·h·mL⁻¹. The clearance rate (CL/F) increased by 9.2-fold. These results indicate that GLS4 is a sensitive substrate of CYP3A, and its first-pass metabolism plays an important role in its elimination. [1]

The study highlighted the potential risks of drug interactions: co-administration with potent CYP3A inhibitors (such as ketoconazole) could significantly increase plasma exposure to GLS4, potentially leading to safety issues if the therapeutic window is narrow. Conversely, co-administration with potent CYP3A inducers (such as rifampin) could significantly reduce plasma exposure to GLS4, potentially affecting its efficacy. [1]

[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.

GLS4 is a novel heteroaryl dihydropyrimidine compound developed from Bay41-4109. It represents a new class of anti-hepatitis B virus drugs that inhibit viral replication by targeting and disrupting the hepatitis B virus capsid assembly. This mechanism is considered less likely to induce drug resistance compared to nucleoside analogs. Currently, GLS4 is undergoing Phase I clinical trials in China, and preliminary studies have shown that it has good pharmacokinetic properties and tolerability. This study in beagle dogs showed that GLS4 is a sensitive substrate for CYP3A. The significant interactions observed with ketoconazole and rifampin suggest that caution should be exercised in clinical practice when GLS4 is used in combination with other drugs that modulate CYP3A activity, as this may lead to clinically significant changes in GLS4 exposure, thereby affecting its safety and efficacy. [1]

C21H22BRFN4O3S

509.39178609848

508.06

C, 49.52; H, 4.35; Br, 15.69; F, 3.73; N, 11.00; O, 9.42; S, 6.29

1092970-12-1

1793065-08-3 (R-isomer);2093044-32-5 (S-isomer);1092970-12-1 (racemic);1646361-04-7 (mesylate);

25144422

Light yellow to yellow solid powder

2.5

1

8

7

31

719

0

SQGRDKSRFFUBBU-UHFFFAOYSA-N

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)

ethyl 4-[2-bromo-4-fluorophenyl]-6-[morpholino-methyl]-2-[2-thiazolyl]-1,4-dihydro-pyrimidine-5-carboxylate

Morphothiadine; GLS-4; GLS 4.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)