Bis(2-haloacetamido)-compounds for use as linking agents and resultant products which comprise antibodies, half-antibodies and antibody fragments
A linker compound, compound technology, applied in the preparation of organic compounds, the preparation of carboxylic acid amides, chemical instruments and methods, etc., can solve problems such as low yield
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Embodiment 11
[0211] Example 11 provides a good illustration of this regioselectivity. Fully reduced Tmab (4 disulfides were reduced) was treated with only two equivalents of linker compound. The 3,4-compound (X=I) showed a high preference for LC-HC with little intra-HC bridging. 3,5-Compounds (X=I) show a high preference for intra-HC, almost all HCs have one linker, but also form HC-LC conjugates with two linkers (meaning that the initial The HC internal product can then be further reacted with another linker to attach the LC). HC-LC products containing only one linker molecule were not observed.
[0212] In the case of the example related to rituximab described herein, the inventors believe that these preferences extend to other mAbs.
[0213] It will thus be recognized that site selection of a mAb (e.g. trastuzumab) can be achieved by treating a fully reduced mAb with a limited amount (less than 4 equivalents) of any of the linker compounds of the invention by taking advantage of the ...
Embodiment 1
[0248] General Synthesis of Methyl 3,5-Bis(2-Haloacetamido)benzoate
[0249] The methyl 3,5-bis(2-haloacetamido)benzoate linked compounds of the invention can be prepared according to the following general synthesis, wherein X is suitably Br or Cl.
[0250]
[0251] Compound 1
[0252] 2-Chloroacetyl chloride (1.35 g, 12.04 mmol, 2 equiv) was added dropwise to a cooled solution of methyl 3,5-diaminobenzoate (1 g, 6.02 mmol) in DCM. The mixture was allowed to warm and stirred at room temperature for 2 hours. The resulting solution was washed with water, saturated ammonium chloride solution, washed with MgSO 4 It was dried, and the solvent was evaporated under reduced pressure to give acetamide (1) (1.7 g, 89%) as a pale yellow solid.
[0253] 1 H NMR (CDCl 3 , 400MHz): δ=8.31(s, 2H, NH), 8.20(s, 1H, Ar), 7.93(s, 2H, Ar), 4.16(s, 2H, 2x CH 2 ), 3.88(s, 3H, Me). 13 C NMR (CDCl 3 , 100MHz): δ165.82, 164.10, 137.58, 131.88, 117.45, 115.56, 52.45, 42.71
[0254] ESI-HR...
Embodiment 2
[0390] Example 2 - Water Stability
[0391] The stability of ester and amide functionalized linker compounds was determined and compared.
[0392] In the first experiment, the aqueous stability of linker compounds 1 to 6 was observed over 4 days. use 1 H NMR solvent inhibition method Stability was determined in phosphate buffer (100 mM, pH 7.5) in the presence of 10% DMF-d7. The data indicated that ortho-substituted (1,2-)dihaloacetamides were less aqueous stable than meta-substituted (1,3-). See figure 2 .
[0393] The aqueous stability of arylbishaloacetamide linkers with ester and amide functionalization was then compared by determining the residual percentages of the bishaloacetamide derivatives (2, 3, 5, 6, and 14 to 16) over 4 days. sex. Stability was reassessed using the solvent inhibition method in phosphate buffer (100 mM, pH 7.5) at a final concentration of 10% DMF-d7. see image 3 .
[0394] Surprisingly, the inventors observed that replacing the ester f...
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