JP-2022516075-A5 -

Dates

Publication Date

20221227

Application Date

20191226

Description

Example 39 Galectin-3 Activity-ELISA Assay The ability of galectin-3 antagonists to inhibit the binding of galectin-3 to the Galβ1-3GlcNAc carbohydrate structure was evaluated. The detailed protocol was as follows: A 1 ug/mL suspension of Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-PAA-biotin polymer (Glycotech, catalog no. 01-096) was prepared. 100 uL aliquots of this polymer were added to the wells of a 96-well streptavidin-coated plate (R&D Systems, catalog no. CP004). 100 uL aliquots of 1X Tris-buffered saline (TBS, Sigma, catalog no. T5912-10X) were added to the control wells. The polymer was bound to streptavidin-coated wells at room temperature for 1.5 hours. The contents of these wells were discarded, and 200 μL of 1X TBS containing 1% bovine serum albumin (BSA) was added to each well as a blocking agent, and the plate was maintained at room temperature for 30 minutes. These wells were washed three times with 1X TBS containing 0.1% BSA. Serial dilutions of the test compound were prepared in a separate V-bottom plate (Corning, catalog no. 3897). A 75 μL aliquot of the highest concentration of the compound to be tested was added to the first well of the column in the V-bottom plate, and then 15 μL was sequentially transferred to 60 μL of 1X TBS across the remaining wells in the column to produce serial dilutions 1 to 5. 60 μL aliquots of 2 μg/mL galectin-3 IBL (catalog no. IBATGP0414) were added to each well of this V-bottom plate. 100 μL aliquots of the galectin-3/test compound mixture were transferred from this V-bottom plate to an assay plate containing Galβ1-3GlcNAc polymer. Four sets of control wells from this assay plate were prepared in double-row configurations, containing: 1) both Galβ1-3GlcNAc polymer and galectin-3, 2) neither polymer nor galectin-3, 3) galectin-3 alone without polymer, or 4) polymer alone without galectin-3. The plates were gently shaken at room temperature for 1.5 hours. These wells were washed four times with TBS/0.1% BSA. 100 μL aliquots of horseradish peroxidase-conjugated anti-galectin-3 antibody (from R&D Systems, DGAL30 kit) were added to each well, and the plate was maintained at room temperature for 1 hour. These wells were washed four times with TBS/0.1% BSA. 100 μL aliquots of TMB substrate solution were added to each well. This TMB substrate solution was prepared by creating a 1:1 mixture of TMB peroxidase substrate (KPL, catalog no. 5120-0048) and peroxidase substrate solution B (KPL, catalog no. 5120-0037). The plate was maintained at room temperature for 10–20 minutes. Color development was stopped by adding 100 μL of 10% phosphoric acid (RICCA Chemical Co., catalog no. 5850-16). The absorbance at 450 nm ( A450 ) was measured using a FlexStation 3 plate reader (Molecular Devices). The A450 vs. test compound concentration plot and IC50 determination were performed using GraphPad Prism. This was done using option 6. In one embodiment, for example, the following items are provided. (Item 1) Compound of formula (I): A compound selected from a prodrug of the compound of formula (I), and a pharmaceutically acceptable salt of any of the above, During the ceremony, R1 is selected from -CN, -CH2CN, and -C(=O)Q groups, where Q is selected from -OZ1 , -NHOH , -NHOCH3 , -NHCN , and -NZ1Z2 groups , where Z1 and Z2 may be the same or different, and are independently selected from H, C1-8 alkyl , C2-12 heterocyclyl , C6-18 aryl , and C1-13 heteroaryl groups, where the C2-12 heterocyclyl , C6-18 aryl , and C1-13 heteroaryl groups are halo, C1-8 alkyl, C1-8 hydroxyalkyl, C1-8 haloalkyl, C6-18 aryl , -OT1 , -C(=O)OT1 , -C(=O) NT1T2 , -CN , and -ST 1 , S(O)T1 , and -SO2 are optionally substituted with one or more groups independently selected from the T1 group , where T1 and T2 may be the same or different, independently selected from H, C1-8 alkyl, and C1-8 haloalkyl groups, or T1 and T2 together with the nitrogen atom to which they are bonded to form a ring, or Z1 and Z2 together with the nitrogen atom to which they are bonded to form a ring; R2 is selected from H, C1-8 alkyl, C2-8 alkenyl, C2-8 alkynyl , C1-8 haloalkyl, C2-8 haloalkenyl , C2-8 haloalkynyl , C4-16 cycloalkylalkyl , C6-18 aryl , C1-13 heteroaryl, C7-19 arylalkyl, and C2-14 heteroarylalkyl, where the C1-8 alkyl, C2-8 alkenyl , C2-8 alkynyl, C1-8 haloalkyl , C2-8 haloalkenyl , C2-8 haloalkynyl , C4-16 cycloalkylalkyl , C6-18 aryl, C1-13 heteroaryl, C7-19 arylalkyl, and C2-14 heteroarylalkyl are halo, C1-8 alkyl , C1-8 hydroxyalkyl , C The groups are optionally substituted with one or more groups independently selected from 1-8 haloalkyl, C6-18 aryl, -OZ3 , -C(=O)OZ3 , -C(=O)NZ3Z4 , and -SO2Z3 groups , where Z3 and Z4 may be the same or different, independently selected from H, C1-8 alkyl , and C1-8 haloalkyl, or Z3 and Z4 together with the nitrogen atom to which they are bonded to form a ring; R3 is selected from C6-18 aryl and C1-13 heteroaryl groups, where the C6-18 aryl and C1-13 heteroaryl groups are optionally subst