H. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4864–4868
4867
Table 3
Cytochrome P450 inhibition data for compounds 5b, 5d and BMS-790052a
Compd
CYP1A2 (lM)
CYP3A4 (lM)
CYP2D6 (lM)
CYP2C9 (lM)
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
5b
5d
>100
>100
>100
>100
>100
>100
13.8 11.2
3.1 2.3
7.2 1.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
81.6 26.1
>100
59.5 13.6
>100
>100
>100
BMS-790052
a
Mean SD of at least two independent assays; IC50 = 50% inhibitory concentration. The following positive controls were used (IC50
,
l
M):
a
-naphthoflavone for 1A2 (>93%
at 3
l
M); ketoconazole for 3A4 (>93% at 10 M); PH-053 (proprietary) for 2D6 (>70% at 100 M); sulfaphenazole for 2C9 (>90% at 10
l
l
lM).
(4-R)-azido compound 10 with an EC50 = 9.5 pM appeared as po-
tent as reference compound 1 without displaying any signs of tox-
ited at therapeutically relevant concentrations. In addition, these
two halogenated compounds displayed improved stability toward
human liver microsomes versus BMS-790052 while maintaining
comparable anti-HCV replication activity. Finally, further mutation
selection studies are currently being investigated in our laboratory
and will be the subject of future publications.
icity up to 100
lM in PBM, CEM and Vero cells. Interestingly, (4-S)-
azido compound 9 appeared slightly less active (EC50 = 23 pM) and
showed toxicities in PBM and CEM cells (IC50 = 20.3 and 18.4
respectively).
lM,
Based on these results, a combination of our best modifications
in both zone II and III was also studied. Thus, (4-S)- and (4-R)-azido
compounds 9 and 10 were halogenated at the 4-position of the
imidazoles rings by using either NBS or NCS in CH2Cl2 (Scheme
4) and all the synthesized compounds displayed an EC50 610 pM.
Interestingly, compound 17 showed an EC50 of 2.6 pM with no tox-
Acknowledgments
This work was supported in part by NIH Grant 5P30-AI-50409
(CFAR), 5R01-AI-071846 and by the Department of Veterans Af-
fairs. Dr. Schinazi is the founder and a major shareholder of RFS
Pharma, LLC. Emory received no funding from RFS Pharma, LLC to
perform this work and vice versa.
icity toward Vero cells up to 100 lM (see Table 1).
Among all the molecules synthesized, the most promising com-
pounds 5a–d were further investigated. First of all, their stability at
pH 3 and 7 in phosphate buffer was evaluated. After 48 h at 20 °C,
LC/MS analysis at time 0, 1, 8, 24 and 48 h revealed no evidence of
degradation of these compounds. Next stability in human liver
References and notes
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microsomes was established (Table 2). Compounds 5a–d (1 lM)
were incubated with human liver microsomes in potassium phos-
phate buffer and 1 mg/mL microsomal protein. Samples were col-
lected at 5, 15, 30 and 60 min, analyzed by LC–MS/MS and half-
life (t1/2) and intrinsic clearance (CLint) for each compound were
calculated based on the elimination rate constant (k). Interestingly,
compounds 5b and 5d show higher metabolic stability and there-
fore lower clearance (CLint <8.6) compared with BMS-790052.
Drug–drug interactions have become an important issue in
modern health care. It is now apparent that many drug–drug inter-
actions can be explained by alterations in the metabolic enzymes
that are present in the liver and other extra-hepatic tissues. Many
of the major pharmacokinetic interactions between drugs are due
to hepatic cytochrome P450 (CYP450) enzymes being affected by
one or more of the drugs present in a given individual. Further-
more, drug interactions can be a result of inhibition or induction
of CYP450 enzymes. The potential drug–drug interaction liabilities
of these compounds were investigated by a CYP450 reversible inhi-
bition assay. Compounds 5b and 5d did not appear to inhibit the
major CYP450 enzymes at the IC90 level and had a favorable CYP
profile, which is suggestive of no potential drug–drug interactions
(Table 3). Due to the large therapeutic window, the inhibition ob-
served for 3A4 (in the micromolar range) is expected not to be clin-
ically relevant at therapeutic doses.
Initial resistant virus selection studies using 5b and BMS-
790052 were performed and mutations identified by population
sequencing. Mutations observed for BMS-790052 included L31 V,
which was previously reported,13 in addition to several unreported
mutations, which we will disclose in full detail in subsequent pub-
lications. Compound 5b selected for several different mutations
including Y93H, which was previously reported as an NS5A resis-
tance mutation.13
In conclusion, extremely potent new NS5A inhibitors (picomo-
lar activity) with a wide therapeutic window (>104) are disclosed.
Among all the compounds prepared 5b and 5d exhibited improved
liver microsome stability compared to reference compound BMS-
790052 and no inhibition of major human CYP enzymes was exhib-
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