High-Throughput ¹H NMR and HPLC Characterization of a 96-Member Substituted Methylene Malonamic Acid Library

Abstract

A solid phase organic synthesis method has been developed for the preparation of substituted methylene malonamic acids and malonic ester mono acids 5. Two substituents are introduced into the core molecule 5 by preparation of unsymmetrical malonic acid derivatives 2, followed by Knoevenagel condensation with aromatic or aliphatic aldehydes, giving resin-bound 4. Evaluation of the scope of these reactions led to the preparation of a 96-member library from a set of eight amines/alcohols (seven amines and one alcohol) and 11 aldehydes leading to 88 substituted methylene malonamic/malonate mono acids 5 and eight unsymmetrical malonamic/malonate mono acids 3. Structural validation and quantitation for every member of the library was obtained by evaluation of 1H NMR and HPLC, respectively. The 1H NMR data were obtained using automated delivery of DMSO solutions of every library member from a 96-deep well microtiter plate to a flow probe-equipped NMR spectrometer. HPLC data were used for determination of the extent of conversion of malonamic/malonate esters 2 to the products 5 by an external standard method. Summary information from the 1H NMR and HPLC data is viewed as plate diagrams for analysis of the final library.

Synthesis of highly substituted 5-(trifluoromethyl)ketoimidazoles using a mixed-solid/solution phase motif

“…2,3 The yields indicate the ratio of the observed loading to the theoretical loading for each resin.…”

“…Analysis of the polymer loading was determined by direct cleavage 1 H NMR using hexamethyldisiloxane (HMDS) as an internal standard. 2,3 An aliquot of resin (50-100 mg) was weighed into a fritted syringe barrel and cleaved with 10 mM HMDS in TFA/CDCl 3 (1:1) for 30 min. The resin was washed 3 times with CDCl 3 , and the combined filtrates used for analysis.…”

“…As our understanding of the available methods or "toolkit" has grown, we have gained the ability to apply these techniques to specific targeted compounds for lead discovery and structure-activity relationship studies. [2][3][4] Both solidphase and solution-phase methods have been described which are amenable to efficient, parallel synthesis. Not surprisingly, specific targeted compounds can provide unique challenges for parallel-synthesis methods that are not directly amenable to either a purely solid-phase or solution-phase route.…”

Automated structure verification based on 1H NMR prediction

“…4 The main bottleneck in the application of proton NMR spectroscopy to combinatorial chemistry and parallel synthesis is the analysis of the huge amounts of data generated. In a previous demonstration by Hamper et al, a qualitative manual inspection of a set of NMR spectra was performed using stacked plots for each plate row (A-H) in a 96-well plate, 5 paying attention to the presence of peaks expected in the desired product(s). Although the results were shown to Ł Correspondence to: Brent A. Lefebvre, Advanced Chemistry Development Inc. (ACD/Labs), 110 Yonge Street, 14th floor, Toronto, ON M5C 1T4, Canada.…”

A program for the visualisation, extraction, and reporting of NMR data from plate chemistry samples

“…New challenges have appeared for the NMR analysis and evaluation of compounds derived in such a high-throughput manner, but a robust and general solution to the problem has been elusive. 10,11 Spurred by recent efforts of chemists to generate compound libraries with high purity, 12,13 we chose to write our own software to overcome this challenging analysis. The program is called Spadez, and runs on standard PCs fitted with Microsoft Windows 2000 or higher.…”