- Email: [email protected]
Discovery of an irreversible HCV NS5B polymerase inhibitor
Bioorganic & Medicinal Chemistry Letters 23 (2013) 6585–6587
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Discovery of an irreversible HCV NS5B polymerase inhibitor Qingbei Zeng ⇑, Anilkumar G. Nair ⇑, Stuart B. Rosenblum, Hsueh-Cheng Huang, Charles A. Lesburg, Yueheng Jiang, Oleg Selyutin, Tin-Yau Chan, Frank Bennett, Kevin X. Chen, Srikanth Venkatraman, Mousumi Sannigrahi, Francisco Velazquez, Jose S. Duca, Stephen Gavalas, Yuhua Huang, Haiyan Pu, Li Wang, Patrick Pinto, Bancha Vibulbhan, Sony Agrawal, Eric Ferrari, Chuan-kui Jiang, Cheng Li, David Hesk, Jennifer Gesell, Steve Sorota, Neng-Yang Shih, F. George Njoroge, Joseph A. Kozlowski Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e
i n f o
Article history: Received 3 October 2013 Revised 25 October 2013 Accepted 28 October 2013 Available online 6 November 2013
a b s t r a c t The discovery of lead compound 2e was described. Its covalent binding to HCV NS5B polymerase enzyme was investigated by X-ray analysis. The results of distribution, metabolism and pharmacokinetics were reported. Compound 2e was demonstrated to be potent (replicon GT-1b EC50 = 0.003 lM), highly selective, and safe in in vitro and in vivo assays. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: HCV NS5B polymerase
Chronic infection with the hepatitis C virus (HCV) is a life threatening global public health issue. An estimated 170 million people worldwide are infected with hepatitis C virus according to the World Health Organization.1 If left untreated, hepatitis C can result in liver damage, which can lead to serious conditions such as cirrhosis or liver cancer. Until recently, the standard treatment is a combination of pegylated interferon-alpha-2a or pegylated interferon-alpha-2b with ribavirin for a period of 24 or 48 weeks, depending on hepatitis C virus genotype.2 However, responses to these treatments are low, especially in genotype 1 patients. More recently, FDA approved two HCV protease inhibitors, Victrelis™ (boceprevir) and INCIVEK™ (telaprevir), which show significantly improved response when added to the standard of care, but the side-effects and potential drug resistance remain.3 Therefore, our continued efforts are to look for alternative therapies. HCV NS5B polymerase is an RNA dependent RNA polymerase that is essential for the viral replication.4 As a therapeutic target, it has been extensively investigated and clinically validated.5 Lead compound 1 is a novel non-nucleoside HCV NS5B inhibitor which was synthesized and evaluated in our laboratories.6 Our investigations demonstrated that it is an irreversible HCV NS5B polymerase inhibitor. This compound showed very good activity (EC50 = 0.001 lM) and excellent oral exposure in rats, dogs, and monkeys. However, the NO2 group in the molecule could be considered a
⇑ Corresponding authors. E-mail address: [email protected] (A.G. Nair). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.060
potential toxic liability. In this Letter, we report the discovery of compound 2e, another irreversible HCV NS5B inhibitor with an improved toxicity acceptability profile. HN O
HN
O
O O
OH
N
F
O 2N F 1
N
OH
R 2
Our strategy to replace the NO2 group was to maintain a displaceable halogen such as chloro which is activated by a non-nitro structural element. Since substitutions at the indole C-2, C-3, C-4, C-5, and C-6 positions were optimized in our early SAR explorations, we maintained the optimal region as shown in structure 2 and focused our efforts to modifications of indole N-1 area (R in structure 2). Scheme 1 illustrates the general synthesis of 2. Compound 3, prepared by the established method,7 was subject to boronic acid and palladium complexation. Suzuki coupling affords 4 in good yield. After the indole alkylation, the resulting compound 5 was demethylated using a solution of HCl in dioxane in a pressure vessel at 90 °C. The last hydrolysis step was facile in refluxing aqueous lithium hydroxide solution.
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Q. Zeng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6585–6587
N H O
F
N
N
I i
O
ii
O O N H
F
3
N
F
O
NH
NH iv
O O N
F
R
O
R 5
4
iii
O O
O OH F
O
N R
6
O 2
Scheme 1. Reagents and conditions: (i) 2-methoxypyridine-3-boronic acid, [1,10 bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane, sodium carbonate, DME, reflux, 4 h; (ii) RCH2Cl, cesium carbonate, DMF, rt; (iii) HCl in dioxane (4 M), 90 °C, 24 h; (iv) LiOH (aq), reflux, 3 h.
The initial SAR study was to use a Cl group in the similar position of F group shown in 1 and activate the nucleophilic substitution by both para F and pyridine nitrogen (2a). This compound, however, showed reduced activity compared to 1. In order to test if an ‘activated’ chloro in a different orientation could help activity, compound 2b was prepared. Interestingly, this compound showed comparable intrinsic activity (IC50) but weaker potency in the cellbased replicon assay (EC50). An alternative functional group to activate chloride was quinoline. The first quinoline analog was 2c, which showed 0.06 lM of binding activity and >1 lM of cellular base replicon potency. When 20 Cl was removed, the resulting compound 2d displayed significantly improved activity in both enzyme and cellular assays. This suggested that di-substitution at 20 and 40 positions of quinoline was not tolerated. Indeed, when monochloro was introduced to quinoline 20 position, the compound (2e) showed further improved replicon activity (0.003 lM). To evaluate the affect of a substituent on the quinoline, compounds 2f–2i were prepared. These inhibitors showed slightly diminished replicon activities. These data are summarized in Table 1. In order to better understand the binding mode of the quinoline-containing compounds, compound 2e was soaked into a preformed crystal of HCV NS5B. The crystal diffracted to 1.63 Å and
Table 1 NS5B inhibitors Entry
R
NS5B (D21) IC50 (lM)8
Human replicon EC50 (lM)8
0.012
1
0.009
>1
0.06
>1
0.006
0.02
0.013
0.003
0.02
0.025
0.006
0.01
0.02
0.02
F
2a 2b
N
Cl
Cl
N Cl
2c N
Cl
Cl
2d N
2e
N
2f
N
2g 2h
Cl
O
F
Cl
N
Cl
N
Cl
N
Cl
F
2i
F
0.01
0.03
Figure 1. X-ray structure of compound 2e/NS5B adduct. After soaking preformed crystals of NS5B with compound 2e, high quality X-ray diffraction data to 1.63 Å were collected and the crystal structure solved. The location and conformation of the compound are identical to those observed in previous reports of analogous compounds [Refs. 6,7b]. Briefly, the compound forms dual hydrogen bonds from the C3 pyridone group to the backbone of residues Ile-447 and Tyr-448. The indole core protrudes into a lipophilic cavity bounded in part by Met-414. The C2 carboxylic acid group interacts indirectly with the backbone of Gly-449 via a water-mediated hydrogen bonding network. Specific to this structure, however, is a covalent adduct observed at Cys-366 resulting from nucleophilic substitution of the chloro group by the sidechain thiol. Two conformations of the protein side chain were observed, one of which was consistent with adduct formation. As there was only one conformation of the compound observed, the other sidechain conformation was assigned to be that of the unreacted free thiol. Only the conformation corresponding to the adduct is shown in the figure, which was prepared using PYMOL.10 The crystal structure has been deposited at the PDB with code 4MZ4.
provided very high quality diffraction data with an Rmerge of 5.3%. Analysis of the resulting data shows that compound 2e binds to the palm site of NS5B and forms an irreversible covalent adduct with the enzyme at a conserved residue, Cys-366 by displacing the quinoline chlorine (Fig. 1). Since compound 2a and compound 2e showed comparable intrinsic activity (IC50) and significantly different potency in the cell-based replicon assay (EC50), 2a was also examined crystallographically. In this case, analysis showed that the chloro group on the pyridine moiety was not substituted by Cys-366 (data not shown). These facts suggest that the formation of covalent bond dramatically improves the cellular activity. The chemical reactivity and selectivity were evaluated both biochemically and in the cellular replicon assay. As mentioned above, the 2-chloroquinoline moiety is stable to aqueous lithium hydroxide under refluxing conditions. This moiety is also stable to ammonia in dioxane (0.5 M) at 100 °C for one day. When compound 2e was incubated with HCV NS5B enzyme, mass spectroscopy analysis identified a single adduct which formed only within the tryptic peptide comprising residues 346–379. This showed that residue 366 is the only cysteine to be alkylated of the 21 cysteines present in the enzyme, 14 of which are at least somewhat solvent-exposed. In addition, adduct formation was observed with native NS5B but
Table 2 Pharmacokinetics data of compound 2e Species
PO AUC
F (%)
T1/2 (h)
Clearance (ml/min/kg)
Rat Monkey Dog
35 lM h @ 10 mpk 11.5 lM h @ 3 mpk 3.2 lM h @ 3 mpk
14 38 38
7 5 2
1.5 3.5 13
Q. Zeng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6585–6587 Table 3 Pharmacokinetics data of single rising dose of compound 2e Dose (mg/kg)
PO AUC (lM h)
Oral Cmax (lM)
Rat
30 100 250 500
176 375 1090 1040
21 32 66 65
Monkey
10 30 100
9 15 17
0.9 1.3 2
not the denatured protein. Furthermore, the NMR-based ALARM assay8 indicated that no reaction occurs between compound 2e and active thiols from the human La antigen.9 Thus, all above facts led to the conclusion that the covalent bond formation was highly selective and largely affinity-driven. It may involve two steps: the first was highly selective non-covalent binding to enzyme palm site and second step was the substitution of chloro at quinoline moiety by the Cys-366 sidechain thiol. In the second step, the proximity between the ‘activated’ chloro group and Cys-366 with suitable alignment of the chloroquinoline plane are required for formation of covalent bond. Based on its high activity in the cellular assay and its unique mode of action, compound 2e was further profiled in vitro and in vivo. The in vivo distribution, metabolism and pharmacokinetics (DMPK) were evaluated in Sprague Dawley rats, Cynomolgus monkeys, and Beagle dogs (Table 2). A crystalline sodium salt was used in these studies. The oral (PO) AUC level of rats and monkeys was high (35 lM h @ 10 mpk and 11.5 lM h @ 3 mpk respectively) and that in dogs was moderate (3.2 lM h @ 3 mpk). The oral bioavailability in rats, monkeys and dogs was 14%, 38%, and 38%, respectively. In rats and monkeys, the IV half life was 7 and 5 h respectively. In dogs the half-life was 2 h. Compound 2e also shows low to moderate tissue distribution in rats, dogs and monkeys with steady-state volumes of distribution of 0.3, 1.7, and 1.2 L/kg, respectively. The liver and brain uptake of 2e was evaluated following oral administration (10 mg/kg) to fasted male rats. The mean liver/plasma ratio is 0.4 and brain/plasma ratio is less than 0.01. The extent of plasma protein binding of 2e is high with 99.8%, 99.6%, 99.5%, and 99.7% in rat, dog, monkey, and human plasma, respectively. In order to assess the dose exposure of compound 2e, it was orally administrated to male Sprague Dawley rats (fed), and male Cynomolgus monkeys (fed). The pharmacokinetics data is summarized in Table 3. Both rat AUC and Cmax values increase less than proportional with increasing dose from 30 mg/kg to 500 mg/kg and reach a plateau at about 250 mg/kg. As shown in the table, the monkey AUC values also increase less than proportional with increasing dose of 10–100 mg/kg and plateau at about 30 mg/kg. In order to study the metabolic pathway, radio-labeled [3H]-2e was administrated to monkey. The primary routes of clearance for the monkey are both biliary and renal excretion of unchanged drug (81% of excreted material in bile is parent compound) after a single oral administration of [3H]-2e. The major metabolic pathway for [3H]-2e in monkey was observed to be oxidation to either the indole or pyridine region of the molecule. No glutathione or sulfate conjugates were observed. The properties of compound 2e to inhibit CYPs 3A4, 1A2, 2C8, 2C9, 2C19, 2D4, and 2D6 were evaluated in human liver microsomes. The concentrations (concurrent/pre-incubation) that
6587
inhibited 50% of the enzyme activity were all >50 lM except for CYP 2C8 which was 21/30 lM. Compound 2e was also evaluated for the potential induction of human CYPs 1A2 and 3A4 in vitro using human hepatocytes at concentrations up to 40 lM. No induction was observed. Compound 2e is selective for the HCV NS5B polymerase. It shows no inhibition of other tested HCV viral enzymes (NS3 protease and helicase) as well as other viral polymerases (poliovirus polymerase and HIV polymerase). No major alerts were noted in a broad counterscreen panel (CEREP). Compound 2e inhibited hERG current by 6% in voltage clamp assay at 2 lM. The potential toxicity evaluations began with mini-Ames assay and showed that compound 2e is not mutagenic to the tested bacterial strains (TA 97a, TA 100 and TA 102). The single dose toxicity assessment with rats at 250, 500, 1000, and 2000 mg/kg showed that clinical signs of toxicity were limited to stool (soft, mucoid) in 1000 and 2000 mg/kg groups within one day. There was no mortality. The further two-week toxicity studies in rats at dose of 10, 30, 150, or 500 mg/kg also demonstrated the compound is safe. In summary, compound 2e is a novel irreversible HCV NS5B polymerase inhibitor. Biological evaluations demonstrated 2e is very active and selective. In vitro and in vivo assays showed 2e exhibited good oral exposure and safety profile. Therefore, compound 2e is a promising candidate for development to combat HCV infection. References and notes 1. World Health Organization Wkly Epidemiol. Rec. 1999, 74, 421. 2. (a) Neumann, A. U.; Lam, N. P.; Dahari, H.; Gretch, D. R.; Wiley, T. E.; Layden, T. J.; Perelson, A. S. Science 1998, 282, 103; (b) Rosen, H. R.; Gretch, D. R. Mol. Med. Today 1999, 5, 393; (c) Di Bisceglie, A. M.; McHutchison, J.; Rice, C. M. Hepatology 2002, 35, 224; (d) Zeuzem, S.; Berg, T.; Moeller, B.; Hinrichsen, H.; Mauss, S.; Wedemeyer, H.; Sarrazin, C.; Hueppe, D.; Zehnter, E.; Manns, M. P. J. Viral Hepat. 2009, 16, 75. 3. Hofmann, W. P.; Zeuzem, S. Nat. Rev. Gastroenterol. Hepatol. 2011, 8, 257. 4. Moradpour, D.; Penin, F.; Rice, C. M. Nat. Rev. Microbilol. 2007, 5, 453. 5. (a) Birerdinc, A.; Younossi, Z. M. Expert Opin. Emerging Drugs 2010, 15, 535; (b) Legrand-Abravanel, F.; Nicot, F.; Izopet, J. Expert Opin. Investig. Drugs 2010, 19, 863. 6. Chen, K. X.; Lesburg, C. A.; Vibulbhan, V.; Yang, W.; Chan, T.-Y.; Venkatraman, S.; Velazquez, F.; Zeng, Q.; Bennet, F.; Anilkumar, G. N.; Duca, J.; Jiang, J.; Pinto, P.; Wang, L.; Huang, Y.; Selyutin, O.; Gavalas, S.; Pu, H.; Agrawal, S.; Feld, B.; Huang, H.-C.; Li, C.; Cheng, K.-C.; Shih, N.-Y.; Kozlowski, J. A.; Rosenblum, S. B.; Njoroge, G. F. J. Med. Chem. 2012, 55, 2089. 7. (a) Robertson, W. M.; Kastrinsky, D. B.; Hwang, I.; Boger, D. L. Bioorg. Med. Chem. Lett. 2010, 20, 2722; (b) Anilkumar, G. N.; Lesburg, C. A.; Selyutin, O.; Rosenblum, S. B.; Zeng, Q.; Jiang, Y.; Chan, T.-Y.; Pu, H.; Vaccaro, H.; Wang, L.; Bennett, F.; Chen, K. X.; Duca, J.; Gavalas, S.; Huang, Y.; Pinto, P.; Sannigrahi, M.; Velazquez, F.; Venkatraman, S.; Vibulbhan, B.; Agrawal, S.; Butkiewicz, N.; Feld, B.; Ferrari, E.; He, Z.; Jiang, C.-K.; Palermo, R. E.; McMonagle, P.; Huang, H.C.; Shih, N.-Y.; Njoroge, G.; Kozlowski, J. A. Bioorg. Med. Chem. Lett. 2011, 21, 5336. 8. HCV NS5B polymerase activity was measured in a radiolabeled nucleotide incorporation assay as described in reference (a) Cheng, C. C.; Shipps, G. W., Jr.; Yang, Z.; Kawahata, N.; Lesburg, C. A.; Duca, J. S.; Bandouveres, J.; Bracken, J. D.; Jiang, C. K.; Agrawal, S.; Ferrari, E.; Huang, H. C. Bioorg. Med. Chem. Lett. 2010, 20, 2119; (b) To measure cell-based anti-HCV activity, replicon cells (1b-Con1) were seeded at 5000 cells/well in 96-well plates one day prior to inhibitor treatment. Various concentrations of an inhibitor in DMSO were added to the replicon cells, with the final concentration of DMSO at 0.5% and fetal bovine serum at 5% in the assay media. Cells were harvested 3 days post dosing. The replicon RNA level was measured using real-time RT-PCR (Taqman assay) with GAPDH RNA as endogenous control. EC50 values were calculated from experiments with 10 serial twofold dilutions of the inhibitor in duplicate. 9. Huth, J. R.; Song, D.; Mendoza, R. R.; Black-Schaefer, C. L.; Mack, J. C.; Dorwin, S. A.; Ladror, U. S.; Severin, J. M.; Walter, K. A.; Bartley, D. M.; Hajduk, P. J. Chem. Res. Toxicol. 2007, 20, 1752. 10. The PYMOL Molecular Graphics System, Version 1.3, Schrödinger LLC.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Discovery of an irreversible HCV NS5B polymerase inhibitor Qingbei Zeng ⇑, Anilkumar G. Nair ⇑, Stuart B. Rosenblum, Hsueh-Cheng Huang, Charles A. Lesburg, Yueheng Jiang, Oleg Selyutin, Tin-Yau Chan, Frank Bennett, Kevin X. Chen, Srikanth Venkatraman, Mousumi Sannigrahi, Francisco Velazquez, Jose S. Duca, Stephen Gavalas, Yuhua Huang, Haiyan Pu, Li Wang, Patrick Pinto, Bancha Vibulbhan, Sony Agrawal, Eric Ferrari, Chuan-kui Jiang, Cheng Li, David Hesk, Jennifer Gesell, Steve Sorota, Neng-Yang Shih, F. George Njoroge, Joseph A. Kozlowski Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e
i n f o
Article history: Received 3 October 2013 Revised 25 October 2013 Accepted 28 October 2013 Available online 6 November 2013
a b s t r a c t The discovery of lead compound 2e was described. Its covalent binding to HCV NS5B polymerase enzyme was investigated by X-ray analysis. The results of distribution, metabolism and pharmacokinetics were reported. Compound 2e was demonstrated to be potent (replicon GT-1b EC50 = 0.003 lM), highly selective, and safe in in vitro and in vivo assays. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: HCV NS5B polymerase
Chronic infection with the hepatitis C virus (HCV) is a life threatening global public health issue. An estimated 170 million people worldwide are infected with hepatitis C virus according to the World Health Organization.1 If left untreated, hepatitis C can result in liver damage, which can lead to serious conditions such as cirrhosis or liver cancer. Until recently, the standard treatment is a combination of pegylated interferon-alpha-2a or pegylated interferon-alpha-2b with ribavirin for a period of 24 or 48 weeks, depending on hepatitis C virus genotype.2 However, responses to these treatments are low, especially in genotype 1 patients. More recently, FDA approved two HCV protease inhibitors, Victrelis™ (boceprevir) and INCIVEK™ (telaprevir), which show significantly improved response when added to the standard of care, but the side-effects and potential drug resistance remain.3 Therefore, our continued efforts are to look for alternative therapies. HCV NS5B polymerase is an RNA dependent RNA polymerase that is essential for the viral replication.4 As a therapeutic target, it has been extensively investigated and clinically validated.5 Lead compound 1 is a novel non-nucleoside HCV NS5B inhibitor which was synthesized and evaluated in our laboratories.6 Our investigations demonstrated that it is an irreversible HCV NS5B polymerase inhibitor. This compound showed very good activity (EC50 = 0.001 lM) and excellent oral exposure in rats, dogs, and monkeys. However, the NO2 group in the molecule could be considered a
⇑ Corresponding authors. E-mail address: [email protected] (A.G. Nair). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.10.060
potential toxic liability. In this Letter, we report the discovery of compound 2e, another irreversible HCV NS5B inhibitor with an improved toxicity acceptability profile. HN O
HN
O
O O
OH
N
F
O 2N F 1
N
OH
R 2
Our strategy to replace the NO2 group was to maintain a displaceable halogen such as chloro which is activated by a non-nitro structural element. Since substitutions at the indole C-2, C-3, C-4, C-5, and C-6 positions were optimized in our early SAR explorations, we maintained the optimal region as shown in structure 2 and focused our efforts to modifications of indole N-1 area (R in structure 2). Scheme 1 illustrates the general synthesis of 2. Compound 3, prepared by the established method,7 was subject to boronic acid and palladium complexation. Suzuki coupling affords 4 in good yield. After the indole alkylation, the resulting compound 5 was demethylated using a solution of HCl in dioxane in a pressure vessel at 90 °C. The last hydrolysis step was facile in refluxing aqueous lithium hydroxide solution.
6586
Q. Zeng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6585–6587
N H O
F
N
N
I i
O
ii
O O N H
F
3
N
F
O
NH
NH iv
O O N
F
R
O
R 5
4
iii
O O
O OH F
O
N R
6
O 2
Scheme 1. Reagents and conditions: (i) 2-methoxypyridine-3-boronic acid, [1,10 bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane, sodium carbonate, DME, reflux, 4 h; (ii) RCH2Cl, cesium carbonate, DMF, rt; (iii) HCl in dioxane (4 M), 90 °C, 24 h; (iv) LiOH (aq), reflux, 3 h.
The initial SAR study was to use a Cl group in the similar position of F group shown in 1 and activate the nucleophilic substitution by both para F and pyridine nitrogen (2a). This compound, however, showed reduced activity compared to 1. In order to test if an ‘activated’ chloro in a different orientation could help activity, compound 2b was prepared. Interestingly, this compound showed comparable intrinsic activity (IC50) but weaker potency in the cellbased replicon assay (EC50). An alternative functional group to activate chloride was quinoline. The first quinoline analog was 2c, which showed 0.06 lM of binding activity and >1 lM of cellular base replicon potency. When 20 Cl was removed, the resulting compound 2d displayed significantly improved activity in both enzyme and cellular assays. This suggested that di-substitution at 20 and 40 positions of quinoline was not tolerated. Indeed, when monochloro was introduced to quinoline 20 position, the compound (2e) showed further improved replicon activity (0.003 lM). To evaluate the affect of a substituent on the quinoline, compounds 2f–2i were prepared. These inhibitors showed slightly diminished replicon activities. These data are summarized in Table 1. In order to better understand the binding mode of the quinoline-containing compounds, compound 2e was soaked into a preformed crystal of HCV NS5B. The crystal diffracted to 1.63 Å and
Table 1 NS5B inhibitors Entry
R
NS5B (D21) IC50 (lM)8
Human replicon EC50 (lM)8
0.012
1
0.009
>1
0.06
>1
0.006
0.02
0.013
0.003
0.02
0.025
0.006
0.01
0.02
0.02
F
2a 2b
N
Cl
Cl
N Cl
2c N
Cl
Cl
2d N
2e
N
2f
N
2g 2h
Cl
O
F
Cl
N
Cl
N
Cl
N
Cl
F
2i
F
0.01
0.03
Figure 1. X-ray structure of compound 2e/NS5B adduct. After soaking preformed crystals of NS5B with compound 2e, high quality X-ray diffraction data to 1.63 Å were collected and the crystal structure solved. The location and conformation of the compound are identical to those observed in previous reports of analogous compounds [Refs. 6,7b]. Briefly, the compound forms dual hydrogen bonds from the C3 pyridone group to the backbone of residues Ile-447 and Tyr-448. The indole core protrudes into a lipophilic cavity bounded in part by Met-414. The C2 carboxylic acid group interacts indirectly with the backbone of Gly-449 via a water-mediated hydrogen bonding network. Specific to this structure, however, is a covalent adduct observed at Cys-366 resulting from nucleophilic substitution of the chloro group by the sidechain thiol. Two conformations of the protein side chain were observed, one of which was consistent with adduct formation. As there was only one conformation of the compound observed, the other sidechain conformation was assigned to be that of the unreacted free thiol. Only the conformation corresponding to the adduct is shown in the figure, which was prepared using PYMOL.10 The crystal structure has been deposited at the PDB with code 4MZ4.
provided very high quality diffraction data with an Rmerge of 5.3%. Analysis of the resulting data shows that compound 2e binds to the palm site of NS5B and forms an irreversible covalent adduct with the enzyme at a conserved residue, Cys-366 by displacing the quinoline chlorine (Fig. 1). Since compound 2a and compound 2e showed comparable intrinsic activity (IC50) and significantly different potency in the cell-based replicon assay (EC50), 2a was also examined crystallographically. In this case, analysis showed that the chloro group on the pyridine moiety was not substituted by Cys-366 (data not shown). These facts suggest that the formation of covalent bond dramatically improves the cellular activity. The chemical reactivity and selectivity were evaluated both biochemically and in the cellular replicon assay. As mentioned above, the 2-chloroquinoline moiety is stable to aqueous lithium hydroxide under refluxing conditions. This moiety is also stable to ammonia in dioxane (0.5 M) at 100 °C for one day. When compound 2e was incubated with HCV NS5B enzyme, mass spectroscopy analysis identified a single adduct which formed only within the tryptic peptide comprising residues 346–379. This showed that residue 366 is the only cysteine to be alkylated of the 21 cysteines present in the enzyme, 14 of which are at least somewhat solvent-exposed. In addition, adduct formation was observed with native NS5B but
Table 2 Pharmacokinetics data of compound 2e Species
PO AUC
F (%)
T1/2 (h)
Clearance (ml/min/kg)
Rat Monkey Dog
35 lM h @ 10 mpk 11.5 lM h @ 3 mpk 3.2 lM h @ 3 mpk
14 38 38
7 5 2
1.5 3.5 13
Q. Zeng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6585–6587 Table 3 Pharmacokinetics data of single rising dose of compound 2e Dose (mg/kg)
PO AUC (lM h)
Oral Cmax (lM)
Rat
30 100 250 500
176 375 1090 1040
21 32 66 65
Monkey
10 30 100
9 15 17
0.9 1.3 2
not the denatured protein. Furthermore, the NMR-based ALARM assay8 indicated that no reaction occurs between compound 2e and active thiols from the human La antigen.9 Thus, all above facts led to the conclusion that the covalent bond formation was highly selective and largely affinity-driven. It may involve two steps: the first was highly selective non-covalent binding to enzyme palm site and second step was the substitution of chloro at quinoline moiety by the Cys-366 sidechain thiol. In the second step, the proximity between the ‘activated’ chloro group and Cys-366 with suitable alignment of the chloroquinoline plane are required for formation of covalent bond. Based on its high activity in the cellular assay and its unique mode of action, compound 2e was further profiled in vitro and in vivo. The in vivo distribution, metabolism and pharmacokinetics (DMPK) were evaluated in Sprague Dawley rats, Cynomolgus monkeys, and Beagle dogs (Table 2). A crystalline sodium salt was used in these studies. The oral (PO) AUC level of rats and monkeys was high (35 lM h @ 10 mpk and 11.5 lM h @ 3 mpk respectively) and that in dogs was moderate (3.2 lM h @ 3 mpk). The oral bioavailability in rats, monkeys and dogs was 14%, 38%, and 38%, respectively. In rats and monkeys, the IV half life was 7 and 5 h respectively. In dogs the half-life was 2 h. Compound 2e also shows low to moderate tissue distribution in rats, dogs and monkeys with steady-state volumes of distribution of 0.3, 1.7, and 1.2 L/kg, respectively. The liver and brain uptake of 2e was evaluated following oral administration (10 mg/kg) to fasted male rats. The mean liver/plasma ratio is 0.4 and brain/plasma ratio is less than 0.01. The extent of plasma protein binding of 2e is high with 99.8%, 99.6%, 99.5%, and 99.7% in rat, dog, monkey, and human plasma, respectively. In order to assess the dose exposure of compound 2e, it was orally administrated to male Sprague Dawley rats (fed), and male Cynomolgus monkeys (fed). The pharmacokinetics data is summarized in Table 3. Both rat AUC and Cmax values increase less than proportional with increasing dose from 30 mg/kg to 500 mg/kg and reach a plateau at about 250 mg/kg. As shown in the table, the monkey AUC values also increase less than proportional with increasing dose of 10–100 mg/kg and plateau at about 30 mg/kg. In order to study the metabolic pathway, radio-labeled [3H]-2e was administrated to monkey. The primary routes of clearance for the monkey are both biliary and renal excretion of unchanged drug (81% of excreted material in bile is parent compound) after a single oral administration of [3H]-2e. The major metabolic pathway for [3H]-2e in monkey was observed to be oxidation to either the indole or pyridine region of the molecule. No glutathione or sulfate conjugates were observed. The properties of compound 2e to inhibit CYPs 3A4, 1A2, 2C8, 2C9, 2C19, 2D4, and 2D6 were evaluated in human liver microsomes. The concentrations (concurrent/pre-incubation) that
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inhibited 50% of the enzyme activity were all >50 lM except for CYP 2C8 which was 21/30 lM. Compound 2e was also evaluated for the potential induction of human CYPs 1A2 and 3A4 in vitro using human hepatocytes at concentrations up to 40 lM. No induction was observed. Compound 2e is selective for the HCV NS5B polymerase. It shows no inhibition of other tested HCV viral enzymes (NS3 protease and helicase) as well as other viral polymerases (poliovirus polymerase and HIV polymerase). No major alerts were noted in a broad counterscreen panel (CEREP). Compound 2e inhibited hERG current by 6% in voltage clamp assay at 2 lM. The potential toxicity evaluations began with mini-Ames assay and showed that compound 2e is not mutagenic to the tested bacterial strains (TA 97a, TA 100 and TA 102). The single dose toxicity assessment with rats at 250, 500, 1000, and 2000 mg/kg showed that clinical signs of toxicity were limited to stool (soft, mucoid) in 1000 and 2000 mg/kg groups within one day. There was no mortality. The further two-week toxicity studies in rats at dose of 10, 30, 150, or 500 mg/kg also demonstrated the compound is safe. In summary, compound 2e is a novel irreversible HCV NS5B polymerase inhibitor. Biological evaluations demonstrated 2e is very active and selective. In vitro and in vivo assays showed 2e exhibited good oral exposure and safety profile. Therefore, compound 2e is a promising candidate for development to combat HCV infection. References and notes 1. World Health Organization Wkly Epidemiol. Rec. 1999, 74, 421. 2. (a) Neumann, A. U.; Lam, N. P.; Dahari, H.; Gretch, D. R.; Wiley, T. E.; Layden, T. J.; Perelson, A. S. Science 1998, 282, 103; (b) Rosen, H. R.; Gretch, D. R. Mol. Med. Today 1999, 5, 393; (c) Di Bisceglie, A. M.; McHutchison, J.; Rice, C. M. Hepatology 2002, 35, 224; (d) Zeuzem, S.; Berg, T.; Moeller, B.; Hinrichsen, H.; Mauss, S.; Wedemeyer, H.; Sarrazin, C.; Hueppe, D.; Zehnter, E.; Manns, M. P. J. Viral Hepat. 2009, 16, 75. 3. Hofmann, W. P.; Zeuzem, S. Nat. Rev. Gastroenterol. Hepatol. 2011, 8, 257. 4. Moradpour, D.; Penin, F.; Rice, C. M. Nat. Rev. Microbilol. 2007, 5, 453. 5. (a) Birerdinc, A.; Younossi, Z. M. Expert Opin. Emerging Drugs 2010, 15, 535; (b) Legrand-Abravanel, F.; Nicot, F.; Izopet, J. Expert Opin. Investig. Drugs 2010, 19, 863. 6. Chen, K. X.; Lesburg, C. A.; Vibulbhan, V.; Yang, W.; Chan, T.-Y.; Venkatraman, S.; Velazquez, F.; Zeng, Q.; Bennet, F.; Anilkumar, G. N.; Duca, J.; Jiang, J.; Pinto, P.; Wang, L.; Huang, Y.; Selyutin, O.; Gavalas, S.; Pu, H.; Agrawal, S.; Feld, B.; Huang, H.-C.; Li, C.; Cheng, K.-C.; Shih, N.-Y.; Kozlowski, J. A.; Rosenblum, S. B.; Njoroge, G. F. J. Med. Chem. 2012, 55, 2089. 7. (a) Robertson, W. M.; Kastrinsky, D. B.; Hwang, I.; Boger, D. L. Bioorg. Med. Chem. Lett. 2010, 20, 2722; (b) Anilkumar, G. N.; Lesburg, C. A.; Selyutin, O.; Rosenblum, S. B.; Zeng, Q.; Jiang, Y.; Chan, T.-Y.; Pu, H.; Vaccaro, H.; Wang, L.; Bennett, F.; Chen, K. X.; Duca, J.; Gavalas, S.; Huang, Y.; Pinto, P.; Sannigrahi, M.; Velazquez, F.; Venkatraman, S.; Vibulbhan, B.; Agrawal, S.; Butkiewicz, N.; Feld, B.; Ferrari, E.; He, Z.; Jiang, C.-K.; Palermo, R. E.; McMonagle, P.; Huang, H.C.; Shih, N.-Y.; Njoroge, G.; Kozlowski, J. A. Bioorg. Med. Chem. Lett. 2011, 21, 5336. 8. HCV NS5B polymerase activity was measured in a radiolabeled nucleotide incorporation assay as described in reference (a) Cheng, C. C.; Shipps, G. W., Jr.; Yang, Z.; Kawahata, N.; Lesburg, C. A.; Duca, J. S.; Bandouveres, J.; Bracken, J. D.; Jiang, C. K.; Agrawal, S.; Ferrari, E.; Huang, H. C. Bioorg. Med. Chem. Lett. 2010, 20, 2119; (b) To measure cell-based anti-HCV activity, replicon cells (1b-Con1) were seeded at 5000 cells/well in 96-well plates one day prior to inhibitor treatment. Various concentrations of an inhibitor in DMSO were added to the replicon cells, with the final concentration of DMSO at 0.5% and fetal bovine serum at 5% in the assay media. Cells were harvested 3 days post dosing. The replicon RNA level was measured using real-time RT-PCR (Taqman assay) with GAPDH RNA as endogenous control. EC50 values were calculated from experiments with 10 serial twofold dilutions of the inhibitor in duplicate. 9. Huth, J. R.; Song, D.; Mendoza, R. R.; Black-Schaefer, C. L.; Mack, J. C.; Dorwin, S. A.; Ladror, U. S.; Severin, J. M.; Walter, K. A.; Bartley, D. M.; Hajduk, P. J. Chem. Res. Toxicol. 2007, 20, 1752. 10. The PYMOL Molecular Graphics System, Version 1.3, Schrödinger LLC.