Site-Specific Tandem Knoevenagel Condensation–Michael Addition To Generate Antibody–Drug Conjugates

Expanded ligation techniques are sorely needed to generate unique linkages for the growing field of functionally enhanced proteins. To address this need, we present a unique chemical ligation that involves the double addition of a pyrazolone moiety with an aldehyde-labeled protein. This ligation occurs via a tandem Knoevenagel condensation–Michael addition. A pyrazolone reacts with an aldehyde to generate an enone, which undergoes subsequent attack by a second pyrazolone to generate a bis-pyrazolone species. This rapid and facile ligation technique is performed under mild conditions in the absence of catalyst to generate new architectures that were previously inaccessible via conventional ligation reactions. Using this unique ligation, we generated three site-specifically labeled antibody–drug conjugates (ADCs) with an average of four drugs to one antibody. The in vitro and in vivo efficacies along with pharmacokinetic data of the site-specific ADCs are reported

Aldehyde Tag Coupled with HIPS Chemistry Enables the Production of ADCs Conjugated Site-Specifically to Different Antibody Regions with Distinct in Vivo Efficacy and PK Outcomes

It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody–drug conjugates (ADCs). Site-specific payload placement allows for control over both the drug-to-antibody ratio (DAR) and the conjugation site, both of which play an important role in governing the pharmacokinetics (PK), disposition, and efficacy of the ADC. In addition to the DAR and site of conjugation, linker composition also plays an important role in the properties of an ADC. We have previously reported a novel site-specific conjugation platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone. This chemistry results in a stable C–C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs. The flexibility and versatility of the aldehyde tag conjugation platform has enabled us to undertake a systematic evaluation of the impact of conjugation site and linker composition on ADC properties. Here, we describe the production and characterization of a panel of ADCs bearing the aldehyde tag at different locations on an IgG1 backbone conjugated using Hydrazino-<i>iso</i>-Pictet-Spengler (HIPS) chemistry. We demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate

Discovery of (<i>R</i>)‑4-Cyclopropyl-7,8-difluoro-5-(4-(trifluoromethyl)phenylsulfonyl)-4,5-dihydro‑1<i>H</i>‑pyrazolo[4,3‑<i>c</i>]quinoline (ELND006) and (<i>R</i>)‑4-Cyclopropyl-8-fluoro-5-(6-(trifluoromethyl)pyridin-3-ylsulfonyl)-4,5-dihydro‑2<i>H</i>‑pyrazolo[4,3‑<i>c</i>]quinoline (ELND007): Metabolically Stable γ‑Secretase Inhibitors that Selectively Inhibit the Production of Amyloid‑β over Notch

Herein, we describe our strategy to design metabolically stable γ-secretase inhibitors which are selective for inhibition of Aβ generation over Notch. We highlight our synthetic strategy to incorporate diversity and chirality. Compounds <b>30</b> (ELND006) and <b>34</b> (ELND007) both entered human clinical trials. The in vitro and in vivo characteristics for these two compounds are described. A comparison of inhibition of Aβ generation in vivo between <b>30</b>, <b>34</b>, Semagacestat <b>41</b>, Begacestat <b>42</b>, and Avagacestat <b>43</b> in mice is made. <b>30</b> lowered Aβ in the CSF of healthy human volunteers