Novel inhibitors of hepatitis C virus replication
An alkyl and compound technology, applied in the field of compounds for the treatment of hepatitis C virus infection, can solve the problems of treatment failure and patients without treatment alternatives, etc.
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Examples
example A
[0552] Example A : NS3-NS4 protease assay
[0553] NS3 complex formation with NS4A-2
[0554] Recombinant E. coli or baculovirus full-length NS3 was diluted to 3.33 μΜ with assay buffer and the material was transferred to an eppendorf tube and placed in a water bath in a 4°C refrigerator. Add an appropriate amount of NS4A-2 up to 8.3 mM in assay buffer to equal the volume of NS3 in step 2.1.1 (conversion factor - 3.8 mg / 272 μL assay buffer). The material was transferred to an Eppendorf tube and placed in a water bath in a 4°C refrigerator.
[0555] After equilibrating at 4°C, equal volumes of NS3 and NS4A-2 solutions were combined in Eppendorf tubes, mixed gently with a manual pipette, and the mixture was incubated in a 4°C water bath for 15 minutes. The final concentrations in the mixture were 1.67 [mu]M NS3, 4.15 mM NS4A-2 (2485-fold molar excess of NS4A-2).
[0556] After 15 minutes at 4°C, the NS3 / NS4A-2 Eppendorf tubes were removed and placed in a room temperature wa...
example B
[0557] Example B : NS3 Inhibition Assay
[0558] Sample compounds were dissolved in DMSO up to 10 mM and then diluted to 2.5 mM in DMSO (1:4). Compounds are typically added to assay plates at a concentration of 2.5 mM and diluted to give a starting concentration of 50 [mu]M in the assay inhibition curve. Compounds were serially diluted in assay buffer to provide lower concentrations of test solutions.
[0559] E. coli NS3 / NS4A-2 was diluted to 4 nM NS3 (1:417.5 of 1.67 μM stock - 18 μL of 1.67 μM stock + 7497 μL assay buffer). BV NS3 / NS4A-2 was diluted to 6 nM NS3 (1:278.3 of 1.67 μM stock - 24 μL of 1.67 μM stock + 6655 μL assay buffer). Using a manual multichannel pipette, being careful not to introduce air bubbles into the plate, 50 μL of assay buffer was added to wells A01-H01 of a black Costar 96-well polypropylene storage plate.
[0560] Using a manual multichannel pipette, being careful not to introduce air bubbles into the plate, add 50 µL of the diluted NS3 / NS4A-...
example
[0574] table 3
[0575] compound
NS3-NS4 IC 50
90
D
91
C
101
A
102
B
103
B
104
A
105
A
106
A
107
A
108
B
109
A
110
B
111
A
112
B
[0576] where A indicates an IC between 10 and 50 μM 50 , B indicates IC between 1 and 10 μM 50 , C represents an IC between 0.1 and 1 μM 50 , and D represents an IC of less than 0.1 μM 50 .
[0577] in conclusion
[0578] Potent small molecule inhibitors of the HCV NS3 protease have been developed.
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