HCV NS3 serine protease (overall inhibition constant Ki = 7 ± 1 nM for genotype 1b; Ki = 0.7 nM for genotype 1a; Ki = 3 nM for genotype 2a; Ki = 7 nM for genotype 3a) [2]
HCV NS3 serine protease (Ki = 6 nM, HNE/HCV = 600) [1]

In Vitro: Narlaprevir (SCH 900518) inhibited replicon RNA at EC50 of 20 ± 6 nM and EC90 of 40 ± 10 nM in a 72-hour assay using genotype 1b HCV replicon system. No cytotoxicity was observed in replicon cells by MTS assay (50% cytotoxic concentration > 25 μM). [2]
Prolonged exposure to 1.2 × EC90 of Narlaprevir (SCH 900518) resulted in a 2-log-unit decrease in RNA levels by day 14. Dosing with 5 × EC90 resulted in a 3-log-unit decrease in RNA levels by day 14. [2]
When interferon alfa-2b was coadministered with Narlaprevir (SCH 900518), there was a concentration-dependent, enhanced inhibition of replicon RNA compared to treatment with the compound alone. Combination indices (CI) at 50%, 75%, and 90% inhibition levels were calculated to be 0.3 ± 0.1, 0.6 ± 0.2, and 0.6 ± 0.1, respectively, indicating at least additive activity. [2]
Culturing of replicon-bearing cells in the presence of 3 × EC90 of Narlaprevir (SCH 900518) for 3 weeks resulted in emergence of resistant colonies at approximately 0.13%. Increases in concentration to 6 × EC90 and 15 × EC90 reduced resistant colonies by about 14- and 65-fold, respectively. The emergence of resistant colonies was reduced about 10-fold when 3 × EC90 of the compound was used with interferon alfa-2b (30 IU/ml) compared to the compound alone. [2]
Selection of replicon cells with Narlaprevir (SCH 900518) resulted in outgrowth of resistant mutants with T54A/S mutations (approximately 10-fold increase in EC90) and A156S/T/V mutations (higher level resistance). A double mutation (T54A and A156T) was observed at higher selecting concentration, conferring resistance even at 40 μM. [2]
Narlaprevir (SCH 900518) was cross-resistant to mutations raised against boceprevir. Fold increases in EC90 values: V36M (8-fold), T54A (11-fold), R155K (15-fold), A156S (70-fold), A156T (>1,000-fold), D168V (1-fold), V170A (10-fold), V36M+R155K (130-fold). [2]
Preclinical resistance studies indicated that Narlaprevir (SCH 900518) was cross-resistant to mutations raised against boceprevir in both enzymatic and replicon assays, but retained more activity against many mutants due to its higher intrinsic potency compared to boceprevir. [1]
Narlaprevir (SCH 900518) displayed no significant issues in in vitro CYP or hERG assay. [1]
Unlike an early compound containing P' allyl moiety, no glutathione or acyl glucuronide conjugates were observed with Narlaprevir (SCH 900518) in rats, dogs and monkeys, indicating no reactive metabolite issue. [1]

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Ketoamides are efficiently inhibited by nallaprevir (SCH 900518), with a Ki value of 7 nM [2]. Replicon RNA is efficiently inhibited by nalaprevir (SCH 900518), with an EC90 value of 40 nM [2].

In Vivo: In a rat in vivo study, Narlaprevir (SCH 900518) displayed very good AUC of 6.5 μM·h, and 46% bioavailability when dosed orally. Rat liver concentration at 8 h post dosing (C8h) was 750 ng/g. [1]
Dog oral exposure for Narlaprevir (SCH 900518) was moderate (AUC = 0.9 μM·h, F = 29%). [1]
In monkeys, Narlaprevir (SCH 900518) displayed an AUC of 1.1 μM·h, with 46% oral bioavailability. [1]

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Narlaprevir (SCH 900518), when administered orally (rat 10 mg/kg, dog 3 mg/kg, monkey 3 mg/kg), demonstrates a moderate oral bioavailability (rat 46%, dog 29%) [1]. After intravenous dosing (four mg/kg in rats and one mg/kg in dogs), nallaprevir (SCH 900518) has an intermediate half-life (4.8 hours in rats and 2 hours in dogs) [1].

Enzyme Assay: Recombinant single-chain HCV NS3 and NS4A proteases from genotypes 1 to 3 were prepared. HCV protease assays were performed using a 200-μl reaction mixture. Typically, 100 μl protease was added to 100 μl of assay buffer containing the chromogenic substrate acetyl-DTEDVVPV(norvaline)-O-4-phenylazophenol. The change in absorbance was monitored over 60 minutes with a microtiter plate reader. [2]
Progress curve analysis: Curves of time-dependent progress of product formation in the presence of various concentrations of inhibitor were first fit to the modified Morrison equation to obtain initial velocity (vi), steady-state velocity (vs), and the constant for apparent rate of reaching steady state (kobs). No-inhibitor controls yielded uninhibited initial velocity (vo). A plot of vi/vo vs inhibitor concentration was fit to equation 2 to derive apparent inhibition constant Kiapp for the initial, fast binding step. A plot of vs/vo vs inhibitor concentration was fit to equation 3 to derive overall apparent inhibition constant Kiapp for the subsequent slow isomerization step. A plot of kobs vs inhibitor concentration was fit to equation 4 to derive isomerization rate constants k3 (from EI to EI) and k4 (from EI to EI) while fixing or floating Kiapp. Kiapp and Kiapp were converted into Ki and Ki using equations 5a and 5b with input of substrate concentration and predetermined Michaelis-Menten constant (Km). The inhibition constant Ki was used as a measure of inhibitor potency. To calculate koff (overall rate constant of dissociation of enzyme-inhibitor complex), k2 (rate of dissociation of EI to E and I) was estimated from Ki and reasonable ranges of k1 (10^5 to 10^8 M^-1 s^-1) using equation 6. koff was calculated with equation 7. The lower limit of the half-life of dissociation (t1/2) was calculated using equation 8. [2]
The two-step, postbinding slow isomerization model is E + I ↔ EI ↔ EI. [2]
The overall inhibition constant (Ki) of Narlaprevir (SCH 900518) for genotype 1b NS3 protease was estimated to be 7 ± 1 nM (n = 12 experiments). The compound was estimated to dissociate from the enzyme-inhibitor complex with a lower limit of half-life of 1 to 2 h. [2]

Cell Assay: Replicon (genotype 1b) construction was performed. To measure antireplicon activity, replicon cells were seeded in 96-well collagen I-coated plates. Narlaprevir (SCH 900518) was added at 24 h post-seeding, and plates were incubated for 3 days, at which point cells were lysed and replicon RNA level was measured by real-time PCR. EC50 was the drug concentration necessary to achieve an increase in cycle threshold (CT) of 1 over the projected baseline CT. EC90 was the drug concentration necessary to achieve an increase in CT of 3.2 over the projected baseline CT. [2]
For combination treatment with alpha interferon, replicon cells were treated for 3 days with Narlaprevir (SCH 900518), serially diluted 1:2.5 for a 5-point titration. At each concentration of the compound, alpha interferon was titrated in. Replicon RNA level was measured by real-time PCR (TaqMan assay) with GAPDH RNA as an endogenous control. Relative replicon RNA inhibition (dCT) was calculated as: dCT = NS5B CT - GAPDH CT. Combination index (CI) values were calculated for alpha interferon in combination with multiple fixed concentrations of the compound using the nonexclusive formula. [2]
To select replicon cells resistant to Narlaprevir (SCH 900518), subconfluent monolayers of genotype 1b replicon cells and parental line Huh-7 were cultured with various concentrations of the compound (EC90 = 65 nM in the resistance study) with or without alpha interferon. All cells were passed at 1:10 ratio when they reached 95% confluence. Colonies that survived selection were pooled and expanded for further analysis. To identify mutations in NS3 protease domain conferring resistance, total cellular RNA was isolated from pooled colonies and amplified by RT-PCR. RT-PCR products were purified and sequenced, or cloned into TOPO TA vector and plasmid DNA from 5-6 bacterial colonies was sequenced. [2]

Animal Protocol: For rat in vivo study, Narlaprevir (SCH 900518) was dosed orally (po) at 10 mpk or intravenously (iv) at 4 mpk. Vehicle: po, 0.4% HPMC or 20% HPBCD; iv, 40% HPBCD. [1]
For dog in vivo study, Narlaprevir (SCH 900518) was dosed orally (po) at 3 mpk or intravenously (iv) at 1 mpk. Vehicle: po, 0.4% HPMC or 20% HPBCD; iv, 40% HPBCD. [1]
For monkey in vivo study, Narlaprevir (SCH 900518) was dosed orally (po) at 3 mpk. Vehicle: po, 0.4% HPMC or 20% HPBCD. [1]
Replicon cells were treated with various concentrations of Narlaprevir (SCH 900518). Replicon cells were refreshed with the compound every 2 to 3 days, and cells were split when they became confluent. [2]

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Animal/Disease Models: Rats, dogs, monkeys[1]
Doses: rats PO/IV 10/4 mg/kg; dogs PO/IV 3/1 mg/kg; monkeys PO 3 mg/kg
Route of Administration: intravenous (iv) (iv)(iv ) or po (oral gavage)
Experimental Results: T1/2 in rats and dogs were 4.8 and 2 hrs (hrs (hours)) respectively.

ADME/Pharmacokinetics: For Narlaprevir (SCH 900518) in rats (po/iv, 10/4 mpk): AUC = 6.5 μM·h; F = 46%; Cmax = 1.3 μM; t1/2 = 4.8 hr; Conc (6h)/EC90 = 11; Rat liver conc (C8h) = 750 ng/g. [1]
For Narlaprevir (SCH 900518) in dogs (po/iv, 3/1 mpk): AUC = 0.9 μM·h; F = 29%; Cmax = 0.8 μM; t1/2 = 2 hr. [1]
For Narlaprevir (SCH 900518) in monkeys (po, 3 mpk): AUC = 1.1 μM·h; F = 46%. [1]
Narlaprevir (SCH 900518) displayed very good rat exposure (AUC = 6.5 μM·h), and significantly improved monkey exposure (AUC = 1.1 μM·h) in comparison with boceprevir. [1]
Plasma protein binding data (in vitro, human) were essentially similar for both boceprevir and Narlaprevir (SCH 900518). [1]

Toxicity/Toxicokinetics: No cytotoxicity was observed with Narlaprevir (SCH 900518) in replicon cells by MTS assay (50% cytotoxic concentration > 25 μM). [2]
Narlaprevir (SCH 900518) displayed no significant issues in the in vitro CYP or hERG assay. [1]
Unlike an early compound containing P' allyl moiety, no glutathione or acyl glucuronide conjugates were observed with Narlaprevir (SCH 900518) in rats, dogs and monkeys, thus indicating no reactive metabolite issue. [1]
Plasma protein binding data (in vitro, human) were essentially similar for both boceprevir and Narlaprevir (SCH 900518). [1]

[1]. Discovery of Narlaprevir (SCH 900518): A Potent, Second Generation HCV NS3 Serine Protease Inhibitor. ACS Med Chem Lett. 2010 Feb 15;1(2):64-9.

[2]. Preclinical characterization of the antiviral activity of SCH 900518 (narlaprevir), a novel mechanism-based inhibitor of hepatitis C virus NS3 protease. Antimicrob Agents Chemother. 2010 Jun;54(6):2365-70.

[3]. Bardoxolone and bardoxolone methyl, two Nrf2 activators in clinical trials, inhibit SARS-CoV-2 replication and its 3C-like protease. Signal Transduct Target Ther. 2021 May 29;6(1):212.

Narlaprevir (SCH 900518) is a ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine (Ser139) of NS3 protease. The covalent enzyme-inhibitor complex dissociates with a half-life of 1 to 2 h. [2]
Binding of Narlaprevir (SCH 900518) to the active site was confirmed by X-ray crystal structure of the protease bound complex (PDB code 3LON). The P4 cyclohexyl moiety was positioned for additional hydrophobic contact with the enzyme S4 pocket, and one of the P4 sulfone oxygen atoms was in close proximity for hydrogen bonding interaction with Cys-159. [1]
Narlaprevir (SCH 900518) is a single isomer (unlike boceprevir which was a mixture of diastereomers at P1), and was obtained in crystalline form. The entire synthesis sequence required no silica gel purification. [1]
Preclinical resistance studies indicated that Narlaprevir (SCH 900518) was cross-resistant to mutations raised against boceprevir in both enzymatic and replicon assays. However, it retained more activity against many of the mutants due to its higher intrinsic potency compared to boceprevir. [1]
Narlaprevir (SCH 900518) is currently undergoing phase II clinical studies in humans. [1]

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Narlaprevir is an azabicyclohexane compound with the structure (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane, substituted at the 2S and 3 positions with [(3S)-1-(cyclopropylamino)-1,2-dioxohepane-3-yl]aminoacyl and N-({1-[(tert-butylsulfonyl)methyl]cyclohexyl}carbamoyl)-3-methyl-L-valine groups, respectively. It is a hepatitis C virus (HCV) NS3/4A serine protease inhibitor (Ki = 6 nM) used to treat chronic hepatitis C. It possesses various activities, including as a hepatitis C protease inhibitor, an antiviral drug, an EC 3.4.22.69 (SARS coronavirus main protease) inhibitor, and an anticoronavirus drug. It is a sulfone, a urea compound, a tertiary carboxamide, a aziridine bicyclohexane, a pyrrolidine carboxamide, a secondary carboxamide, and also a cyclopropane compound.

C36H61N5O7S

707.96384

707.429

C, 61.08; H, 8.69; N, 9.89; O, 15.82; S, 4.53

865466-24-6

11857239

White to off-white solid powder

1.2±0.1 g/cm3

1.558

3.61

4

7

15

49

1400

5

CCCC[C@@H](C(=O)C(=O)NC1CC1)NC(=O)[C@@H]2[C@@H]3[C@@H](C3(C)C)CN2C(=O)[C@H](C(C)(C)C)NC(=O)NC4(CCCCC4)CS(=O)(=O)C(C)(C)C

RICZEKWVNZFTNZ-LFGITCQGSA-N

InChI=1S/C36H61N5O7S/c1-10-11-15-24(27(42)30(44)37-22-16-17-22)38-29(43)26-25-23(35(25,8)9)20-41(26)31(45)28(33(2,3)4)39-32(46)40-36(18-13-12-14-19-36)21-49(47,48)34(5,6)7/h22-26,28H,10-21H2,1-9H3,(H,37,44)(H,38,43)(H2,39,40,46)/t23-,24-,25-,26-,28+/m0/s1

(1R,2S,5S)-3-(N-(((1-((tert-butylsulfonyl)methyl)cyclohexyl)amino)carbonyl)-3-methyl-l- valyl)-N-((1s)-1-((cyclopropylamino)(oxo)acetyl)pentyl)-6,6-dimethyl-3- azabicyclo(3.1.0)hexane-2-carboxamide

SCH-900518; SCH 900518; Narlaprevir; 865466-24-6; SCH 900,518; SCH-900,518; SCH900,518; SCH900518

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 : ≥ 50 mg/mL (~70.63 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.53 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.53 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.

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