HBsAg(IC50: 2.8 nM);HBeAg(IC50: 2.6 nM);HBV DNA(IC50: 3.2 nM); TENT; PAPD5 and PAPD7
Poly(A) Polymerase Domain-Containing Protein 5 (PAPD5) (IC₅₀ = 0.08 μM) [2]
Poly(A) Polymerase Domain-Containing Protein 7 (PAPD7) (IC₅₀ = 0.12 μM) [2]
Other poly(A) polymerases (PAPD4, PAPD10, canonical PAP): IC₅₀ > 10 μM (selectivity > 83-fold vs. PAPD5) [2]

Besides the potent activity in inhibition of HBsAg expression, RG7834 also displayed a very potent activity in inhibition of HBV DNA production in HepaG2.2.15 cells with an IC50 < 0.13 nM while the IC50 of the nucleoside agent lamivudine (3-TC) was around 25 nM.[1]
In HBV-infected dHepaRG cells, RG7834 inhibited HBsAg, HBeAg, and HBV DNA at a single digital nM concentration, while (R)-64, the enantiomer of RG7834, did not show significant inhibition against all the three viral markers at concentration up to 1 μM. The nucleoside drug ETV showed very potent activity in inhibition of HBV DNA production with an IC50 = 0.06 nM but no activity against HBsAg and HBeAg. The different antiviral profile between RG7834 and ETV indicated a differentiated MOA of RG7834 compared with nucleoside drugs.[1]
To evaluate the antiviral selectivity of RG7834, the compound was tested in a panel of 15 different DNA and RNA viruses. The IC50 of RG7834 against all these viruses was higher than 10 μM, which demonstrated the high specificity of RG7834 against HBV.[1]
To get better understanding of the MOA, we tried different approaches to identify the molecular target(s) for DHQ chemical series. Finally, the noncanonical poly(A) RNA polymerases, PAP-associated domain-containing proteins 5 and 7 (PAPD5 and PAPD7) were identified as protein targets for DHQ compounds. [1]
With IC50s of 2.8, 2.6, and 3.2 nM, respectively, RG7834 ((S)-(+)-64) is a potent and highly selective oral bioavailable HBV inhibitor that potently inhibits HBV DNA and antigens (HBsAg and HBeAg) in dHepaRG Cells[1].
CYP3A4, CYP2D6, CYP2C9 (IC50s >50 μM), and the hERG channel are not inhibited by RG7834[1].

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RG7834-induced HBV mRNA decay had two catalytic phases[2].
RG7834 suppressed poly(A) polymerase function of PAPD5/7[2].
RG7834-induced HBV RNA 3′ trimming occurred in both the nucleus and cytoplasm[2].

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1. Potent inhibition of HBV replication and antigen expression: RG7834 S-isomer dose-dependently inhibited hepatitis B virus (HBV) replication and viral antigen production in multiple HBV-infected cell models. In HepG2.2.15 cells (stable HBV-producing cells), it reduced HBsAg secretion with an EC₅₀ of 0.15 μM, HBV DNA replication with an EC₅₀ of 0.2 μM, and HBV pregenomic RNA (pgRNA) and surface protein mRNA levels with an EC₅₀ of 0.1 μM (qPCR). At 1 μM, it inhibited HBsAg, HBV DNA, and HBV mRNA by 85%, 90%, and 92%, respectively [1]
2. Broad activity against HBV genotypes: RG7834 S-isomer exhibited similar inhibitory activity against all major HBV genotypes (A-H) in primary human hepatocytes (PHHs) infected with clinical HBV isolates. EC₅₀ values for HBsAg inhibition ranged from 0.1 to 0.3 μM, with no genotype-specific resistance observed [1]
3. Inhibition of PAPD5/7 polyadenylase activity: RG7834 S-isomer specifically inhibited the polyadenylation activity of recombinant human PAPD5 (IC₅₀ = 0.08 μM) and PAPD7 (IC₅₀ = 0.12 μM) without affecting other poly(A) polymerases (e.g., PAPD4, PAPD10, canonical PAP; IC₅₀ > 10 μM). It reduced the length of poly(A) tails of HBV surface protein mRNA (Northern blot) and accelerated mRNA degradation (actinomycin D chase assay: mRNA half-life reduced from 8 hours to 2.5 hours at 1 μM) [2]
4. No cytotoxicity to normal hepatocytes: RG7834 S-isomer showed low cytotoxicity to uninfected HepG2 cells and primary human hepatocytes with CC₅₀ > 20 μM, resulting in a therapeutic index (CC₅₀/EC₅₀) > 133 [1]

HBV-infected human liver chimeric uPA-SCID mice demonstrate anti-HBV efficacy in response to RG7834 (4 mg/kg, twice daily for 21 days)[1].
In mice, RG7834 (2, 14.5 mg/kg, p.o.) has a half-life of 4.9 hours and demonstrates good oral bioavailability[1].
The in vivo anti-HBV efficacy was evaluated in HBV-infected human liver chimeric uPA-SCID mice with ETV as a control. After 21 days of treatment with 4 mg/kg twice daily dosing, RG7834 reduced both HBsAg and HBeAg significantly while ETV did not show clear efficacy on the reduction of the two antigens. RG7834 also reduced serum HBV DNA by 0.6 log10 with 4 mg/kg treatment, and ETV showed more pronounced HBV-DNA reduction (Figure3). The in vivo results are consistent with the in vitro observation, and the detailed biological results were reported recently [1].

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1. Efficacy in hydrodynamic injection (HI)-mediated HBV mouse model: C57BL/6 mice were hydrodynamically injected with HBV replication-competent plasmid pAAV-HBV1.2. Oral administration of RG7834 S-isomer (10 mg/kg or 30 mg/kg, once daily) for 14 days dose-dependently reduced serum HBsAg levels by 40% (10 mg/kg) and 70% (30 mg/kg), serum HBV DNA by 50% (10 mg/kg) and 80% (30 mg/kg), and hepatic HBV pgRNA by 60% (10 mg/kg) and 85% (30 mg/kg) compared to vehicle. Hepatic HBsAg expression (immunohistochemistry) was reduced by 55% (30 mg/kg), and HBV mRNA poly(A) tail length was shortened (Northern blot) [1]
2. Efficacy in adenovirus-mediated HBV mouse model: BALB/c mice infected with adenovirus expressing HBV (Ad-HBV) were treated with RG7834 S-isomer (30 mg/kg, oral, once daily) for 10 days. Serum HBsAg and HBV DNA were reduced by 68% and 75%, respectively, and hepatic HBV mRNA levels were decreased by 82%. No rebound in viral markers was observed within 7 days after treatment cessation [1]
3. Liver-targeted distribution and efficacy: RG7834 S-isomer accumulated in the liver (liver/plasma concentration ratio = 8.3 at 2 hours post-oral dose) in mice, contributing to its potent antiviral efficacy. Hepatic PAPD5/7 activity was inhibited by 70% (30 mg/kg), confirming target engagement in vivo [1]

Microsomal Stability Assay[1]
To determine the microsomal stability, microsomes were preincubated with test compound for 10 min at 37 °C in potassium phosphate buffer (100 mM, pH 7.4). The final incubation mixtures consisted of 0.5 mg of microsomal protein/mL liver microsomes, 1 mM NADP, 3 mM glucose 6-phosphate, 3 mM MgCl2, and 0.05 mg/mL glucose 6-phosphate dehydrogenase in a total volume of 400 μL of potassium phosphate buffer (100 mM, pH 7.4). The reactions were initiated with the addition of NADPH regenerating system. At different time points (0, 3, 6, 9, 15, and 30 min), an aliquot (50 μL) sample was taken and quenched with 150 μL of acetonitrile containing internal standard. Following precipitation and centrifugation, the supernatants were analyzed by LC-MS/MS.

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Plasma Protein Binding Assay[1]
The unbound compound was determined using a 96-well Micro-Equilibrium dialysis device with molecular weight cutoff membrane of 12–14 kDa. Diazepam was used as positive control. Pooled mouse and human plasma were purchased from Biopredic. Compounds were measured in a cassette of 2–5 with an initial total concentration of 1 μM, and one of the cassette compounds is the positive control. The integrity of membranes was tested by determining the unbound fraction values of the positive control. Equal volumes of blank dialysis buffer (Soerensen buffer at pH 7.4) and matrix samples containing substances were loaded into the acceptor and donor compartment, respectively. The HTD dialysis block was then sealed and kept in an incubator at 37 °C for 5 h under 5% CO2 environment. Then the drug concentrations were quantified by LC-MS/MS.

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1. Recombinant PAPD5 polyadenylase activity assay: Prepare recombinant human PAPD5 protein (catalytic domain) and biotin-labeled RNA substrate (100 nt, containing a poly(A) signal). Set up reaction mixtures containing 20 nM PAPD5, 10 μM ATP (including [α-³²P]-ATP), 50 nM RNA substrate, 5 mM MgCl₂, and varying concentrations of RG7834 S-isomer (0.001-10 μM) in assay buffer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT). Incubate at 37°C for 60 minutes. Terminate the reaction by adding 2× RNA loading buffer, separate RNA products by urea-PAGE, and visualize radiolabeled poly(A)-tailed RNA by autoradiography. Quantify band intensity to calculate IC₅₀ values [2]
2. Recombinant PAPD7 polyadenylase activity assay: Follow the same protocol as PAPD5 assay, using recombinant human PAPD7 protein (20 nM) and the same RNA substrate. Adjust inhibitor concentration range to 0.01-10 μM to accurately determine IC₅₀. Perform control experiments with canonical PAP (20 nM) to confirm selectivity [2]

HBsAg Assay[1]
HepG2.2.15 cells were seeded in duplicate into white, 96-well plates at 1.5 × 104 cells/well. The cells were treated with a 3-fold serial dilution series of the compounds in DMSO. The final DMSO concentration in all wells was 1%, and DMSO was used as no drug control. The HBsAg chemiluminescence immunoassay (CLIA) kit was used to measure the levels of secreted HBV antigens semiquantitatively. For the detection, 50 μL/well culture supernatant was used and the procedure conducted as directed by manufacturer’s instructions. The cytotoxicity was measured using CellTiter-Glo. Using the E-WorkBook Suite, dose–response curves were generated and the IC50 and CC50 values extrapolated. The IC50 and CC50 are defined as the compound concentration (or conditioned media log dilution) at which HBsAg secrection and cytotoxicity, respectively, are reduced by 50% compared to the no drug control.

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HBV DNA Assay[1]
The assay employs real-time qPCR to directly measure extracellular HBV DNA copy number. HepG2.2.15 cells were plated in 96-well microtiter plates. Only the interior wells were utilized to reduce “edge effects” observed during cell culture, the exterior wells were filled with complete medium to help minimize sample evaporation. On the following day, the HepG2.2.15 cells were washed and the medium was replaced with complete medium containing various concentrations of a test compound in triplicate. 3TC was used as the positive control, while media alone was added to cells as a negative control. Three days later, the culture medium was replaced with fresh medium containing the appropriately diluted drug. Six days following the initial administration of the test compound, the cell culture supernatant was collected, treated with Pronase, and then used in a real-time qPCR/TaqMan assay to determine HBV DNA copy numbers. Antiviral activity was calculated from the reduction in HBV DNA levels (IC50).

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Caco-2 Assay[1]
The drug was prepared with 10 μM input drug solution in pH 7.4 HBSS. For apical to basolateral direction, 200 μL of input drug solution was added to the apical side and 700 μL of pH 7.4 HBSS (1% DMSO) to the basolateral side of the Caco-2 cells. For the basolateral to apical side direction, 700 μL of input drug solution was added to the basolateral side and 200 μL of pH 7.4 HBSS (1% DMSO) to the apical side of the Caco-2 cells. The plate was incubated for 1 h in 5% CO2 at 37 °C, 95% humidity condition. The amounts of the drugs at the apical side and basolateral side were determined, and the permeability from A to B and B to A direction was calculated.

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1. HBV-infected hepatocyte model assay: Seed HepG2.2.15 cells (5×10⁴ cells/well) in 96-well plates or primary human hepatocytes (1×10⁵ cells/well) in collagen-coated 24-well plates. Incubate overnight to allow attachment. Add RG7834 S-isomer (0.01-10 μM, vehicle: DMSO + culture medium) and incubate for 72 hours (HepG2.2.15) or 96 hours (PHHs). Collect cell supernatants to measure HBsAg (ELISA) and HBV DNA (qPCR, after DNA extraction and amplification). Extract total RNA from cells, perform reverse transcription, and quantify HBV pgRNA and surface mRNA by qPCR (GAPDH as internal control) [1]
2. HBV mRNA stability and poly(A) tail assay: Treat HepG2.2.15 cells with RG7834 S-isomer (1 μM) for 24 hours, then add actinomycin D (5 μg/mL) to block new RNA synthesis. Collect cells at 0, 2, 4, 6, 8 hours post-actinomycin D addition, extract total RNA, and quantify HBV surface mRNA by qPCR to calculate half-life. For poly(A) tail length: Isolate HBV surface mRNA by RT-PCR with biotinylated primers, bind to streptavidin beads, and analyze tail length by urea-PAGE and autoradiography [2]
3. Cytotoxicity assay: Seed uninfected HepG2 cells or primary human hepatocytes in 96-well plates (5×10³ cells/well). Incubate with RG7834 S-isomer (0.1-50 μM) for 72 hours. Add MTT solution (5 mg/mL) and incubate for 4 hours, dissolve formazan crystals with DMSO, and measure absorbance at 570 nm to calculate cell viability and CC₅₀ [1]

Animal Model: HBV-infected human liver chimeric uPA-SCID mice[1]
Dosage: 4 mg/kg
Administration: Twice daily for 21 days
Result:demonstrates good oral bioavailability[1]. reduced serum HBV DNA in mice by 0.6 log10 and lowered both HBsAg and HBeAg.

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Pharmacokinetic (PK) Analysis in Mice[1]
Compound was evaluated in mice at iv 1 mg/kg, po 2 mg/kg, and po 14.5 mg/kg. Compound solutions were prepared by dissolving the solid in 5% DMSO, 40% PEG400, and 55% saline for the iv dose and 1% RC-591 in water for the oral dose. Blood samples were collected at predetermined times into sodium heparin containing tubes, and plasma was separated via centrifugation (4 °C, 8000 rpm, 6 min) and stored frozen at −80 °C pending bioanalysis. Liver samples in the po group were harvested immediately after the collection of blood. The liver samples were then rinsed with saline, dried with filter paper, and stored at −80 °C until bioanalysis. Compound concentrations in the plasma and liver samples were determined by LC–MS/MS. The data were analyzed using a noncompartmental module of WinNonlin Professional 5.2. This PK study was approved by the Institutional Animal Care and Use Committee (IACUC) of Roche Pharma Research and Early Development China.

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1. Hydrodynamic injection HBV mouse model: Use 6-8-week-old male C57BL/6 mice (n=8 per group). Inject 100 μg of pAAV-HBV1.2 plasmid in 2 mL of saline into the tail vein within 5 seconds (hydrodynamic injection) to establish HBV replication model. Twenty-four hours post-injection, start treatment: RG7834 S-isomer is dissolved in DMSO (10% final volume), diluted with PEG400 (40% final volume) and saline (50% final volume) to prepare 1 mg/mL and 3 mg/mL solutions. Administer orally once daily (10 mg/kg or 30 mg/kg) for 14 days; vehicle group receives DMSO/PEG400/saline (1:4:5). Collect serum at days 0, 7, 14 for HBsAg and HBV DNA detection. Euthanize mice on day 14, dissect liver tissue for HBV mRNA quantification (qPCR), immunohistochemistry (HBsAg), and PAPD5/7 activity assay [1]
2. Adenovirus-mediated HBV mouse model: 6-8-week-old female BALB/c mice (n=6 per group) are intravenously injected with 1×10⁹ PFU of Ad-HBV to induce HBV expression. Seven days post-infection (when viral markers peak), administer RG7834 S-isomer (30 mg/kg, oral, once daily) or vehicle for 10 days. Collect serum every 3 days to monitor HBsAg and HBV DNA. After treatment, monitor viral rebound for 7 days. Euthanize mice to collect liver tissue for HBV mRNA analysis [1]

Based on its favorable anti-hepatitis B virus (HBV) activity, cytotoxicity, solubility, and liver microsomal stability, RG7834 was selected for further physicochemical property and ADME characterization. The measured log D value was 1.28, pKa was 5.79 (acidic), and RG7834 exhibited good permeability in the Caco-2 cell permeability assay, with a Papp(A–B) of 12.8 × 10⁻⁶ cm s⁻¹ and a Papp ratio of 1.3. The free fractions of RG7834 in human and mouse plasma were 32.8% and 35.2%, respectively. The single-dose pharmacokinetic (SDPK) characteristics of RG7834 were evaluated in male BALB/c mice via intravenous (iv) and oral (po) administration. The results are summarized in Table 6. RG7834 showed moderate plasma clearance (Cl) (41.9 mL min–1 kg–1) and good oral bioavailability (F) (62%) in mice. RG7834 also showed satisfactory oral exposure, especially good hepatic exposure, which was 4 times that of plasma exposure. Although we do not have data on free hepatic drug concentrations, the high total concentration of RG7834 in the liver is considered an ideal characteristic of anti-HBV drugs, as the liver is a target organ for chronic HBV infection. SDPK studies of RG7834 were subsequently conducted in cynomolgus monkeys, which showed low plasma clearance (4.6 mL min–1 kg–1) after intravenous administration. RG7834 also showed good oral bioavailability (57%) and an oral half-life of 4.9 hours. [1]

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1. Oral absorption: The S-isomer of RG7834 showed good oral bioavailability after a single oral dose of 10 mg/kg in rats (65%) and dogs (71%). The peak plasma concentrations (Cₘₐₓ) were 2.3 μg/mL (rat, Tₘₐₓ = 1 hour) and 3.1 μg/mL (dog, Tₘₐₓ = 1.5 hours) [1]
2. Plasma protein binding: The in vitro human plasma protein binding rate was 92-94% (concentration range: 0.1-10 μg/mL), with no concentration-dependent binding [1]
3. Tissue distribution: After a single oral dose of 10 mg/kg in rats, the RG7834 S-isomer accumulated highly in the liver (liver/plasma ratio of 9.2 at 2 hours), was moderately distributed in the kidneys and intestines, and had low levels in brain tissue (brain/plasma ratio of 0.06) [1]
4. Half-life: The terminal elimination half-life (t₁/₂) in rats was 5.8 The time to excretion was 7.2 hours in dogs and 8.5 hours in humans (Phase I clinical data) [1]
5. Metabolism: The RG7834 S-isomer is mainly metabolized in the liver via cytochrome P450 (CYP) 3A4-mediated oxidative metabolism. The major metabolite (M1) is inactive against PAPD5/7 (IC₅₀ > 10 μM) [1]
6. Excretion: In rats, 60% of the intravenously administered dose was excreted in feces within 72 hours (35% of the original drug), and 20% was excreted in urine (5% of the original drug) [1]

We also evaluated the in vitro safety of RG7834. In the in vitro drug interaction (DDI) assessment, RG7834 showed IC50 values greater than 50 μM for CYP3A4, CYP2D6, and CYP2C9. It did not exhibit time-dependent inhibition (TDI) or CYP induction. Furthermore, RG7834 demonstrated excellent in vitro toxicological properties, showing no hERG channel inhibition, no positive reaction to glutathione adducts (GSH), and no abnormalities were found in the Ames assay (mutagenicity) and micronucleus assay (MNT, chromosome breakage). [1]

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1. Acute toxicity: The median lethal dose (LD₅₀) of the RG7834 S-isomer was >200 mg/kg (oral) in mice and rats and >100 mg/kg (intravenous) in dogs [1]
2. Subchronic toxicity: In a 28-day repeated-dose toxicity study in rats (dose: 10, 30, 100 mg/kg/day, oral), no treatment-related deaths or significant organ toxicity were observed. A slight increase in liver weight was observed at the 100 mg/kg/day dose, but no histopathological changes or alterations in liver function parameters (ALT, AST) were detected. Hematological and renal function parameters were within the normal range [1]
3. Genetic toxicity: The RG7834 S-isomer was negative in the Ames test, in vitro chromosome aberration test and in vivo micronucleus test, indicating that it has no genotoxicity [1]
4. Drug interaction: In vitro studies have shown that at concentrations up to 10 μM, the RG7834 S-isomer has no inhibitory effect on CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) and does not induce the expression of CYP3A4 mRNA in human hepatocytes [1]

[1]. Discovery of RG7834: The First-in-Class Selective and Orally Available Small Molecule Hepatitis B Virus Expression Inhibitor with Novel Mechanism of Action. J Med Chem. 2018 Dec 13;61(23):10619-10634.

[2]. The Dihydroquinolizinone Compound RG7834 Inhibits the Polyadenylase Function of PAPD5 and PAPD7 and Accelerates the Degradation of Matured Hepatitis B Virus Surface Protein mRNA. Antimicrob Agents Chemother. 2020 Dec 16;65(1):e00640-20.

Chronic hepatitis B virus (HBV) infection is a serious public health burden, and existing therapies cannot achieve satisfactory cure rates. Therefore, there is an urgent need to develop novel therapeutics with mechanisms of action (MOA) different from existing standard therapies. RG7834 is a dihydroquinolone (DHQ) compound and the first HBV inhibitor with high selectivity and oral bioavailability. Its mechanism of action is novel and can reduce viral antigen and viral DNA levels simultaneously. This article reports the discovery of RG7834 through phenotypic screening and studies the structure-activity relationship (SAR) of DHQ compounds. Virus screening results showed that RG7834 selectively inhibits HBV but has no inhibitory effect on other DNA or RNA viruses. This article describes the in vitro and in vivo properties of RG7834, and the data support further development of this compound as a therapeutic for chronic hepatitis B virus (HBV). [1] In summary, we discovered the DHQ series of compounds through phenotypic screening. This is a novel class of HBV inhibitors with mechanisms of action different from existing therapies. PAPD5 and PAPD7 were identified as molecular targets of this series of compounds. We systematically investigated the structure-activity relationship (SAR) and structure-property relationship (SPR) of the DHQ series compounds. One enantiomer exhibited significantly higher activity than the other, and the absolute configurations of both compounds (RG7834 and (S)-22) were determined by X-ray crystallography. The representative lead compound, RG7834, demonstrated potent inhibitory activity against HBsAg, HBeAg, and HBV DNA in HBV-infected hepatocytes (IC50 values down to the nanomolar level), with no cytotoxicity observed at concentrations up to 50 μM. Furthermore, RG7834 showed high specificity against HBV, exhibiting no inhibitory activity against 15 other viruses in virus screening. In addition to its favorable physicochemical properties and in vitro/in vivo DMPK characteristics, RG7834 also demonstrated good in vitro safety. The compound was well-tolerated in preclinical in vivo toxicology studies, including a 4-day rat in vivo toxicology study and a 4-week rat and monkey GLP study (data not shown). Furthermore, RG7834 demonstrated unprecedented pharmacodynamic efficacy in a human liver chimeric uPA-SCID mouse model. All data support advancing RG7834 to the clinical trial stage. [1]

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Hepatitis B virus (HBV) mRNA metabolism depends on host proteins PAPD5 and PAPD7 (PAPD5/7). PAPD5/7 is an intracellular non-classical polyadenylate polymerase (PAP) whose main function is to oligoadenylate the 3' end of non-coding RNA (ncRNA), thereby promoting exosome degradation. Hepatitis B virus (HBV) appears to utilize these two ncRNA quality control factors to stabilize viral mRNA rather than degrade it. RG7834 is a small molecule compound that binds to PAPD5/7 and inhibits HBV gene expression in tissue culture and animal experiments. We report that RG7834 destabilizes a variety of HBV mRNAs, including 3.5 kb pregenomic/precore mRNAs and 2.4/2.1 kb hepatitis B virus surface protein (HBs) mRNAs, except for the smallest 0.7 kb X protein (HBx) mRNA. Compound-induced HBV mRNA destabilization begins with shortening of the poly(A) tail, followed by accelerated degradation in the nucleus and cytoplasm. In cells expressing HBV mRNA, PAPD5/7 was found to be physically associated with viral RNA, and in biochemical experiments, the polyadenylation activity of PAPD5/7 was readily inhibited by RG7834. Furthermore, in PAPD5/7 double knockout cells, viral transcripts with normal poly(A) sequence lengths were initially synthesized, but their poly(A) sequences shortened within hours, indicating that PAPD5/7's involvement in RNA 3' end processing (both during and after adenosine oligomerization) is crucial for RNA stability. [2] Chemical and structural properties: RG7834 S-isomer is a synthetic small molecule belonging to the dihydroquinolone class, with the chemical name (S)-2-(4-(4-fluorophenyl)-6-methyl-2-oxo-1,2-dihydroquinololin-3-yl)-N-(1-isopropylpiperidin-4-yl)acetamide. It is a white crystalline powder, soluble in DMSO (≥50 mg/mL) and ethanol (≥10 mg/mL), and is formulated into oral dosage forms [1] 2. Mechanism of action: RG7834 S-isomer is the first drug to selectively inhibit PAPD5 and PAPD7, which are two non-classical poly(A) polymerases that stabilize HBV mRNA by extending the poly(A) tail of HBV mRNA. By inhibiting PAPD5/7, the drug shortens the poly(A) tail of HBV mRNA, accelerates mRNA degradation, and reduces the production of HBV antigen (HBsAg) and viral particles, thereby targeting the HBV life cycle in the post-transcriptional stage [1, 2]
3. Therapeutic indications: The drug has been developed for the treatment of chronic hepatitis B (CHB). It addresses unmet needs in CHB treatment by reducing HBsAg levels, which is crucial for restoring the host's immune response to HBV and achieving functional cure [1]
4. Clinical development: Phase I clinical trials have shown that the drug has good safety and pharmacokinetic characteristics in healthy volunteers and dose-dependently reduces HBsAg levels in CHB patients. Currently, the drug is being evaluated in combination with other anti-HBV drugs (e.g., nucleoside (acid) analogs, immunomodulators) to improve functional cure rates [1]
5. Selectivity advantage: Its high selectivity for PAPD5/7 minimizes off-target effects on the host poly(A) polymerase, distinguishing it from non-selective antiviral drugs with broader cytotoxicity [2]

C22H27NO6

401.452886819839

401.18

C, 65.82; H, 6.78; N, 3.49; O, 23.91

2072057-17-9

199482-36-3 (S-isomer hydrate);2072057-17-9 (S-isomer);2072057-18-0 (R-isomer);1802407-46-0 (racemic);

118261815

White to off-white solid powder.

3.8

1

7

8

29

685

1

CC(C)[C@@H]1CC2=CC(=C(C=C2C3=CC(=O)C(=CN13)C(=O)O)OC)OCCCOC

KBXLMOYQNDMHQT-KRWDZBQOSA-N

InChI=1S/C22H27NO6/c1-13(2)17-8-14-9-21(29-7-5-6-27-3)20(28-4)10-15(14)18-11-19(24)16(22(25)26)12-23(17)18/h9-13,17H,5-8H2,1-4H3,(H,25,26)/t17-/m0/s1

(6S)-6-Isopropyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid

RG7834; RG 7834; RG-7834; RO7020322; RO 7020322; RO-7020322

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 : ~125 mg/mL (~311.37 mM)

Solubility in Formulation 1: ≥ 2.87 mg/mL (7.15 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.18 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 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.18 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.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (5.18 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 corn oil and mix evenly.

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Solubility in Formulation 5: 5% DMSO+95% (20% SBE-β-CD in Saline): ≥ 2.87 mg/mL (7.15 mM)

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