Grazoprevir hydrate (previously known as MK 5172; MK-5172; MK5172; Zepatier), the mono-hydrated form of grazoprevir, is a second generation inhibitor of HCV (Hepatitis C Virus) NS3/4A Protease and a marketed broad spectrum anti-HCV drug with activity across genotypes and resistant variants. It inhibits HCV genotype 1a, 1B, and 4 with IC50 values of 7pM, 4pM, and 62pM, respectively. In 2016, Grazoprevir was approved by FDA for the treatment of hepatitis C along with the NS5A replication complex inhibitor elbasvir, either with or without ribavirin.
Physicochemical Properties
| Molecular Formula | C38H52N6O10S |
| Molecular Weight | 784.93 |
| Exact Mass | 784.347 |
| Elemental Analysis | C, 58.15; H, 6.68; N, 10.71; O, 20.38; S, 4.08 |
| CAS # | 1350462-55-3 |
| Related CAS # | Grazoprevir;1350514-68-9;Grazoprevir potassium salt;1206524-86-8;Grazoprevir sodium salt;1425038-27-2 |
| PubChem CID | 71576667 |
| Appearance | White to off-white solid powder. |
| LogP | 5.576 |
| Hydrogen Bond Donor Count | 4 |
| Hydrogen Bond Acceptor Count | 12 |
| Rotatable Bond Count | 8 |
| Heavy Atom Count | 55 |
| Complexity | 1580 |
| Defined Atom Stereocenter Count | 7 |
| SMILES | S(C1([H])C([H])([H])C1([H])[H])(N([H])C([C@]1(C([H])([H])[C@@]1([H])C([H])=C([H])[H])N([H])C([C@]1([H])C([H])([H])[C@]2([H])C([H])([H])N1C([C@]([H])(C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])N([H])C(=O)O[C@]1([H])C([H])([H])[C@@]1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1C(=NC3C([H])=C(C([H])=C([H])C=3N=1)OC([H])([H])[H])O2)=O)=O)=O)(=O)=O.O([H])[H] |
| InChi Key | RXSARIJMSJWJLZ-CIAYNJNFSA-N |
| InChi Code | InChI=1S/C38H50N6O9S.H2O/c1-6-22-19-38(22,35(47)43-54(49,50)25-13-14-25)42-32(45)29-18-24-20-44(29)34(46)31(37(2,3)4)41-36(48)53-30-16-21(30)10-8-7-9-11-27-33(52-24)40-28-17-23(51-5)12-15-26(28)39-27;/h6,12,15,17,21-22,24-25,29-31H,1,7-11,13-14,16,18-20H2,2-5H3,(H,41,48)(H,42,45)(H,43,47);1H2/t21-,22-,24-,29+,30-,31-,38-;/m1./s1 |
| Chemical Name | (33R,35S,91R,92R,5S)-5-(tert-butyl)-N-((1R,2S)-1-((cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)-17-methoxy-4,7-dioxo-2,8-dioxa-6-aza-1(2,3)-quinoxalina-3(3,1)-pyrrolidina-9(1,2)-cyclopropanacyclotetradecaphane-35-carboxamide hydrate |
| Synonyms | MK5172 hydrate; MK 5172; MK-5172 hydrate; 1350462-55-3; MK-5172 (hydrate); MK-5172 hydrate; Grazoprevir [USAN]; Grazoprevir monohydrate; 4O2AB118LA; Grazoprevir (USAN); Trade name: Zepatier. |
| 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 | gt1b(Ki=0.01±<0.01 nM);gt1a(Ki=0.01±0.01 nM);gt2a(Ki=0.08±0.02 nM);gt2b(Ki=0.15±0.06 nM);gt3a(Ki=0.90±0.2 nM) | ||
| ln Vitro | MK-5172 (Grazoprevir) is effective in biochemical assays against major genotypes and variants engineered with common resistant mutations, with Ki of 0.01±<0.01 nM (gt1b), 0.01±0.01 nM (gt1a), 0.08±0.02 nM (gt2a), 0.15±0.06 nM (gt2b), 0.90±0.2 nM (gt3a), 0.07±0.01 nM (gt1bR155K), 0.14±0.03 nM (gt1bD168V), 0.30±0.04 nM (gt1bD168Y), 5.3±0.9 nM (gt1bA156T), and 12±2 nM (gt1bA156V), respectively. In the replicon assay, MK-5172 demonstrates subnanomolar to low-nanomolar EC50s against genotypes 1a, 1b, and 2a, with EC50s of 0.5±0.1 nM, 2±1 nM, and 2±1 nM for gt1bcon1, gt1a, and gt2a, respectively. MK-5172 is potent against a panel of HCV replication mutants NS5A (Y93H) (EC50=0.7±0.3 nM), NS5B nucleosides (S282T) (EC50=0.3±0.1 nM), and NS5B (C316Y) (EC50=0.4±0.2). MK-5172 maintains the excellent potency against the gt 3a enzyme as well as a broad panel of mutant enzymes, has excellent potency in the replicon system [gt1b IC50(50% NHS)=7.4 nM; gt1a IC50(40% NHS)=7 nM], and shows excellent rat liver exposure. | ||
| ln Vivo |
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| Enzyme Assay | recombinant HCV NS3/4A enzymes are expressed and purified from E. Coli. Enzyme sequences are derived from genotype 1a (gt1a) H77, gt1b con1, gt2a JFH1, gt2b HCJ8, and gt3a NZL1. Inhibition of HCV NS3/4A protease activity in reaction mixtures containing MK-5172 (Grazoprevir), Vaniprevir, or the reference compounds Danoprevir and TMC435 is determined in a time-resolved fluorescence assay. Cell-based HCV replicon assays are conducted in genotype 1b (con1) stable cell line HB1 or a gt2a cell line (JFH) in the presence of either 10% fetal bovine serum (FBS) or 40% normal human serum (NHS). Determinations of 50% effective concentrations (EC50s) against the panel of genotype or mutant replicon cell lines are conducted using a TaqMan-based assay. The 50% cytotoxic concentration (CC50) is determined in the HCV replicon cell line with the use of an MTS assay. Potency determinations against clinical genotype 1 NS3/4A sequences are made using a transient cell-based phenotype assay. The NS3/4A patient isolates are cloned from human plasma infected with HCV. Broad counterscreening, in which MK-5172 is evaluated for its inhibitory potency at a concentration of 10 μM, is conducted at MDS Pharma Services. | ||
| Cell Assay | HB1 cells (30,000 per well) are seeded of a 6-well tissue culture plate per drug concentration. The next day (day 0), the medium is replenished with fresh medium and MK-5172 at the appropriate drug concentration. Cells from a single well per drug concentration are harvested on days 0, 1, and 2, washed, and stored frozen until evaluation. The fourth well is similarly harvested on day 3.5 except that 30,000 cells are reseeded with fresh medium and MK-5172 at the appropriate drug concentration. For additional time points, cells are passaged and harvested every one-half week for 2 weeks. For the third week, cells are similarly treated except that cells received replenishing medium which contained 0.5 mg/ml G418 without protease inhibitor. | ||
| Animal Protocol |
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| Toxicity/Toxicokinetics |
Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation Grazoprevir has not been studied in nursing mothers being treated for hepatitis C infection. Because it is greater than 98.9% bound to maternal plasma proteins, amounts in breastmilk are likely to be very low. Some sources recommend against breastfeeding when grazoprevir is used with ribavirin. Hepatitis C is not transmitted through breastmilk and breastmilk has been shown to inactivate hepatitis C virus (HCV). However, the Centers for Disease Control recommends that mothers with HCV infection should consider abstaining from breastfeeding if their nipples are cracked or bleeding. It is not clear if this warning would apply to mothers who are being treated for hepatitis C. Infants born to mothers with HCV infection should be tested for HCV infection; because maternal antibody is present for the first 18 months of life and before the infant mounts an immunologic response, nucleic acid testing is recommended. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. |
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| References |
[1]. Antimicrob Agents Chemother.2012 Aug;56(8):4161-7. [2]. ACS Med Chem Lett.2012 Mar 2;3(4):332-6. [3]. Signal Transduct Target Ther. 2021 May 29;6(1):212. |
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| Additional Infomation |
◉ Summary of Use during Lactation
Grazoprevir has not been studied in nursing mothers being treated for hepatitis C infection. Because it is greater than 98.9% bound to maternal plasma proteins, amounts in breastmilk are likely to be very low. Some sources recommend against breastfeeding when grazoprevir is used with ribavirin. Hepatitis C is not transmitted through breastmilk and breastmilk has been shown to inactivate hepatitis C virus. However, the Centers for Disease Control recommends that mothers with HCV infection should consider abstaining from breastfeeding if their nipples are cracked or bleeding. It is not clear if this warning would apply to mothers who are being treated for hepatitis C. Infants born to mothers with HCV infection should be tested for HCV infection; because maternal antibody is present for the first 18 months of life and before the infant mounts an immunologic response, nucleic acid testing is recommended. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.[1] A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.[2] |
Solubility Data
| Solubility (In Vitro) | DMSO : ~50 mg/mL (~63.70 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.19 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 (3.19 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. Solubility in Formulation 3: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (3.19 mM) (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.2740 mL | 6.3700 mL | 12.7400 mL | |
| 5 mM | 0.2548 mL | 1.2740 mL | 2.5480 mL | |
| 10 mM | 0.1274 mL | 0.6370 mL | 1.2740 mL |