 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C10H14N5O4P |
| Molecular Weight | 299.22 |
| Exact Mass | 299.078 |
| CAS # | 441785-25-7 |
| Related CAS # | Besifovir Dipivoxil maleate;441785-26-8; 441785-25-7; 1039623-01-2 |
| PubChem CID | 5270766 |
| Appearance | Light yellow to yellow solid powder |
| Density | 1.9±0.1 g/cm3 |
| Boiling Point | 668.4±65.0 °C at 760 mmHg |
| Flash Point | 358.0±34.3 °C |
| Vapour Pressure | 0.0±2.1 mmHg at 25°C |
| Index of Refraction | 1.826 |
| LogP | -2.56 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 8 |
| Rotatable Bond Count | 5 |
| Heavy Atom Count | 20 |
| Complexity | 415 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | C1CC1(CN2C=NC3=CN=C(N=C32)N)OCP(=O)(O)O |
| InChi Key | KDNSSKPZBDNJDF-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C10H14N5O4P/c11-9-12-3-7-8(14-9)15(5-13-7)4-10(1-2-10)19-6-20(16,17)18/h3,5H,1-2,4,6H2,(H2,11,12,14)(H2,16,17,18) |
| Chemical Name | [1-[(2-aminopurin-9-yl)methyl]cyclopropyl]oxymethylphosphonic acid |
| Synonyms | Besifovir; 441785-25-7; Besifovir [INN]; ((1-((2-Amino-9H-purin-9-yl)methyl)cyclopropoxy)methyl)phosphonic acid; UNII-4PLG22CQUU; 4PLG22CQUU; PHOSPHONIC ACID, [[[1-[(2-AMINO-9H-PURIN-9-YL)METHYL]CYCLOPROPYL]OXY]METHYL]- (9CI); [1-[(2-aminopurin-9-yl)methyl]cyclopropyl]oxymethylphosphonic acid; |
| 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/hepatitis B virus |
| ln Vitro | In this article, we describe a novel phosphonate nucleoside, 9-[1-(phosphonomethoxycyclopropyl)methyl]guanine (PMCG, 1), which exhibits a highly potent and selective anti-HBV activity (EC50 = 0.5 μM). Its orally available drug candidate, 9-[1-(phosphonomethoxycyclopropyl)methyl]-6-deoxyguanine Dipivoxil (Besifovir Dipivoxil (PMCDG Dipivoxil), 2) (Figure 1) is also described. Evaluation of anti-HBV activity of these synthetic compounds was conducted with HepG2 2.2.15 cells transfected with the HBV genome. As shown in Table 1, 1 and 16 are the most potent (EC50 = 0.5 μM). The other modified guanine base analogues also exhibit moderate to high anti-HBV activity (EC50 = 1.5−8.0 μM), while the thymine base analogue 11 and 6-ethylaminoguanine analogue 14 do not show anti-HBV activity at 30 μM. Among the modified guanine base analogues, the anti-HBV activity of 8, 15, and 16 are comparable to that of 1. Compounds 8, 15, and 16 would most likely be converted to 1 by oxidation or hydrolysis inside the cell. All novel phosphonate nucleoside analogues show an excellent cellular toxicity profile with CC50 > 1.0 mM and do not inhibit replication of HIV-1 and HSV-1 at 30 μM [6]. |
| ln Vivo |
In conclusion, the introduction of a cyclopropyl moiety at the 2‘-position to restrict conformational mobility of an acyclic phosphonate nucleoside was shown to result in highly potent, specific, and selective anti-HBV activity. The orally available drug candidate Besifovir Dipivoxil (PMCDG Dipivoxil)(2) is a double prodrug of PMCG (1), which drastically reduced DNA titers of woodchuck hepatitis B virus (WHBV) in an in vivo study with woodchucks at 5 mg/kg per day.19 The drug candidate 2 has successfully completed phase I and is currently undergoing phase II clinical study for evaluation of efficacy in human.[6] Orally administered prodrug LB80380 (30, 60, 120, and 240 mg) is quickly absorbed and transforms into Besifovir (LB80331), the parent medication (LB80380 was not found in plasma). For LB80331, the range of the median Tmax was 1.0–2.0 hours after dosage. After that, the concentration of LB80331 in plasma dropped monoexponentially, with an average t1/2 of 2.5–3.3 hours. Background: LB80380 is potent antiviral agent against hepatitis B virus (HBV) in vitro and in the woodchuck model. It has an excellent preclinical safety profile including lower potential for renal toxicity than adefovir. It is effective against both wild-type and YMDD mutant HBV. LB80380 is converted to its parent drug, Besifovir (LB80331), after oral absorption, and further metabolized to its active form, LB80317. Aims/methods: This randomized placebo-controlled Phase I/II clinical study of LB80380 was conducted to assess the safety, antiviral activity and pharmacokinetics of its parent drug Besifovir (LB80331) and its active form LB80317 in 29 Asian adults with chronic hepatitis B positive for hepatitis B e antigen in four escalating dose groups (30, 60, 120 and 240 mg once per day) for 4 weeks with a 12-week follow-up period. Results: The mean maximum HBV DNA reduction was 3.05, 4.20, 3.67 and 3.68 log10 copies/ml for 30, 60, 120 and 240 mg per day, respectively. Viral dynamic analysis suggested a high degree of inhibition of HBV replication at doses of 60 mg or higher per day. LB80380 was well tolerated at all dose groups, and no dose-related clinical or laboratory adverse event was reported. Conclusion: LB80380 is shown to be a potent and safe antiviral agent for HBV. Marked HBV DNA suppression was observed in all dose groups. The HBV DNA suppression was approximately constant at doses of 60 mg and higher over the 28-day dosing period. The dose response of LB80380 will be evaluated further in large clinical studies.[2] |
| Enzyme Assay |
Procedure To Evaluate Anti-HBV Activity and Cytotoxicity.Anti-HBV Activity. [6] The compounds listed above were evaluated for anti-HBV activity, referring to the article.13 The HepG2 2.2.15 cells, hepatitis B virus (HBV) producing cell line, were cultured at 37 °C in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 1% Antibiotic-Antimycotics, and 400 μg/mL Geneticin in the presence of 5% CO2. The HepG2 2.2.15 cells were plated at a density of 1.5 × 104 cells/well on 96-well plates and incubated for 2 days. When confluence was about 80−90%, the media were replaced with fresh DMEM containing 2% FBS, 1% ABAM, and 400 μg/mL G 418. The synthetic compounds with concentrations of 100, 20, 4, 0.8, 0.16, and 0 μM were treated every other day for 9 days. On day 10 the culture media were collected, boiled, and diluted serially. The samples were analyzed by real-time PCR using a Rotor-Gene 2000. Amplification primers were HBV2005F (5‘-TCA GCT CTG TAT CGG GAA GCC TTA G-3‘) and HBV2122R (5‘-CAC CCA CCC AGG TAG CTA GAG TCA-3‘), and TaqMan probe was 5‘-6-FAM-CCT CAC CAT ACT GCA CTC AGG CAA-BHQ-1−3‘. PCR samples were denatured for 10 min at 94 °C, followed by 40 cycles of 94° C for 30 s, 55 °C for 30 s, and 86 °C for 30 s. Emitting fluorescence was detected at 86 °C and the data analyzed statistically using PRISM. |
| Cell Assay |
Cytotoxicity. [6] The 50% cytotoxic concentration of each compound was determined in HepG2 2.2.15 cell line. After compounds treatments for the evaluation of anti-HBV activity, each well of the 96-well plate was treated with 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazdium bromide (MTT) and incubated for 2 h at 37 °C. At the end of the incubation period, the medium was removed, and the converted dye was solubilized with acidic isopropyl alcohol (0.05% HCl in 95% isopropyl alcohol). The metabolized formazan reduction product was colorimetrically measured at 490 nm. |
| Animal Protocol |
Methods: An open-label, single- dose, randomized-sequence, 2-treatment crossover study was conducted in 32 Korean male volunteers. Subjects received either a combination of 60 and 90 mg tablets of the free base LB80380 formulation or a 183 mg (150 mg as a free base) tablet of the maleate LB80380 formulation. Then, after a 14- day washout period, each subject received the other formulation. Plasma and urine concentrations of LB80331 and LB80317 (active metabolites of LB80380) were measured by validated liquid chromatographytandem mass spectrometry assays. A safety assessment, which included vital signs, adverse events, electrocardiograms and clinical laboratory tests, was performed for each subject.
[1] Results: A total of 32 healthy subjects was enrolled, and 26 subjects completed the study. Single oral administrations of LB80380 maleate tablets did not result in clinically significant differences in the safety profile compared to the LB80380 free base tablets. The 90% confidence intervals (CIs) for the geometric mean ratios of Cmax and AUClast for LB80331 of the two treatments (maleate versus free base formulation) were 0.986 - 1.1240 and 0.9848 - 1.0533, respectively. The 90% CIs for the geometric mean ratios of Cmax and AUClast for LB80317 were 0.8379 - 0.9696 and 0.7224 - 0.9196. [1] Conclusions: In healthy male subjects, the 183 mg LB80380 maleate tablet was pharmacokinetically equivalent to the 60 and 90 mg LB80380 free base tablets. |
| ADME/Pharmacokinetics | Besifovir (LB80380) is a relatively new oral acyclic nucleotide phosphonate. We reviewed the pharmacokinetic characteristics of LB80380 and discussed its role in the treatment of chronic hepatitis B infection. Areas covered: LB80380 is a prodrug of LB80331 and LB80317. It is rapidly absorbed when taken orally. Escalating doses of besifovir produce linear increase of the plasma concentration. Doses above 60mg are effective for inhibiting HBV in human. Using 60mg as an example, the maximal concentration of LB80331 in plasma is 397 ng/mL. The time required to reach maximal concentration in plasma and elimination half-life are 2.0 and 3.0 h, respectively. Besifovir and its metabolites are mainly excreted via the kidneys. Its antiviral efficacy is non-inferior to ETV 0.5mg daily. It is generally safe in terms of renal and bone toxicity. The most common adverse event is carnitine depletion which affects almost all patients on besifovir requiring carnitine supplementation. Expert opinion: Besifovir demonstrated predictable pharmacokinetic characteristics in human subjects. Few clinical studies on besifovir have been conducted. More data are expected particularly for special populations. The adverse events upon long term exposure should be monitored. Large scale head-to-head trials comparing besifovir with existing NA, especially tenofovir alafenamide, should be conducted. [3] |
| Toxicity/Toxicokinetics | There was no significant difference in the rates of overall adverse events between the 2 groups throughout the study period (Table 3). Consistently, between weeks 48 and 96, there was no significant difference in the adverse events that were newly reported in 61 BSV-BSV group patients (53.5%) and 57 TDF-BSV group patients (50.0%). Adverse events with an incidence ≥5% included nasopharyngitis, dyspepsia, nausea, back pain, headache, dizziness, fatigue, and ALT elevation in the BSV-BSV group and nasopharyngitis, dyspepsia, gastritis, diarrhea, pruritus, and urticaria in the TDF-BSV group. Whereas no dyspepsia was reported in the BSV group, 8 dyspepsia events occurred in 7 patients in the TDF group (8.33%) during the first 48-week treatment period. Although there was no serious adverse event that caused death during the study period, 1 BSV group patient with HCC and 2 TDF group patients with either HCC or creatine phosphokinase elevation discontinued study drug treatment during the first 48-week treatment period. In addition, 1 BSV-BSV group patient developed tuberculous colitis, and an additional TDF-BSV group patient developed essential thrombocythemia, both of whom discontinued treatment with the study drugs during the extended period. https://pubmed.ncbi.nlm.nih.gov/30448598/ |
| References |
[1]. Pharmacokinetic comparison of the maleate and free base formulations of LB80380, a novel nucleotide analog, in healthy male volunteers. Int J Clin Pharmacol Ther. 2012 Sep;50(9):657-64. [2]. A randomized placebo-controlled, dose-finding study of oral LB80380 in HBeAg-positive patients with chronic hepatitis B. Antivir Ther. 2006;11(8):977-83. [3]. Pharmacokinetic evaluation of besifovir for the treatment of HBV infection. Expert Opin Drug Metab Toxicol. 2018 Jan;14(1):101-106. [4]. Efficacy and Safety of Besifovir Dipivoxil Maleate Compared With Tenofovir Disoproxil Fumarate in Treatment of Chronic Hepatitis B Virus Infection. Clin Gastroenterol Hepatol.2018 Nov 15. pii: S1542-3565(18)31244-8;[5]. Hepatitis B virus: new therapeutic perspectives. Liver Int.2016 Jan;36 Suppl 1:85-92. [6]. A novel class of phosphonate nucleosides. 9-[(1-phosphonomethoxycyclopropyl)methyl]guanine as a potent and selective anti-HBV agent. J Med Chem. 2004 May 20;47(11):2864-9. |
| Additional Infomation |
Besifovir dipivoxil is a small-molecule orally available inhibitor of the HBV polymerase. The HBV polymerase is the enzyme that catalyzes the production of new RNA from the existing strand of RNA. Besifovir dipivoxil is believed to inhibit viral proliferation by interrupting the replicating machinery of the virus. See also: Besifovir (annotation moved to). Drug Indication Investigated for use/treatment in hepatitis (viral, B). Mechanism of Action Besifovir dipivoxil is an inhibitor of the HBV polymerase. It is believed to inhibit viral proliferation by interrupting the replicating machinery of the virus. Current antiviral therapies have dramatically improved the long-term outcomes of patients with chronic hepatitis B virus (HBV) infection. Both interferon (IFN) and nucleos(t)ide analogue (NA) treatments have been shown to reduce the progression of liver disease in chronic hepatitis B (CHB) patients. However, persistent covalently closed circular DNA (cccDNA) can result in a viral relapse after discontinuation of antiviral treatment. On the basis of extensive research on the HBV lifecycle and virus-host interactions, several new agents focusing on viral and host targets are under development to cure HBV. New polymerase inhibitors, tenofovir alafenamide and besifovir provide effective and safer treatment for CHB patients. Agents targeting cccDNA, such as engineered site-specific nucleases and RNA interference therapeutics could eliminate cccDNA or silence cccDNA transcription. Inhibitors of HBV nucleocapsid assembly suppress capsid formation and prevent synthesis of HBV DNA. The HBV entry inhibitor, Myrcludex-B, has been shown to effectively inhibit amplification of cccDNA as well as the spread of intrahepatic infection. Agents targeting host factors that enhance innate and adaptive immune responses, including the lymphotoxin-β receptor agonist, toll-like receptor agonist, immune checkpoint inhibitors and adenovirus-based therapeutic vaccine, could play a critical role in the elimination of HBV-infected cells. With all of these promising approaches, we hope to reach the ultimate goal of a cure to HBV in the near future. https://pubmed.ncbi.nlm.nih.gov/26725903/ 9-[1-(Phosphonomethoxycyclopropyl)methyl]guanine (PMCG, 1), representative of a novel class of phosphonate nucleosides, blocks HBV replication with excellent potency (EC(50) = 0.5 microM) in a primary culture of HepG2 2.2.15 cells. It exhibits no significant cytotoxicity in several human cell lines up to 1.0 mM. It does not inhibit replication of human immunodeficiency virus (HIV-1) or herpes simplex virus (HSV-1) at 30 microM. Many purine base analogues of 1 also exhibit inhibitory activity against HBV, but at 30 microM, pyrimidine analogues do not. 1 is 4 times more potent than 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), which was used as a positive control (EC(50) = 2.0 microM). The characteristic cyclopropyl moiety at the 2'-position of 1 was prepared by titanium-mediated Kulinkovich cyclopropanation. 1 was modified to give the orally available drug candidate, Besifovir Dipivoxil (PMCDG Dipivoxil)(2). Compound 2 exhibited excellent efficacy when administered at 5 mg per kg per day in a study with woodchucks infected with woodchuck hepatitis B virus (WHBV). Drug candidate 2 has successfully completed phase I clinical trials and is currently undergoing phase II clinical studies for evaluation of efficacy. |
Solubility Data
| Solubility (In Vitro) |
DMSO: 250 mg/mL (835.51 mM) H2O: ≥ 100 mg/mL (334.20 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (6.95 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (6.95 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (6.95 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 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 | 3.3420 mL | 16.7101 mL | 33.4202 mL | |
| 5 mM | 0.6684 mL | 3.3420 mL | 6.6840 mL | |
| 10 mM | 0.3342 mL | 1.6710 mL | 3.3420 mL |