Structure-based design and discovery of protein tyrosine phosphatase inhibitors incorporating novel isothiazolidinone heterocyclic phosphotyrosine mimetics.

Structure-based design led to the discovery of novel (S)-isothiazolidinone ((S)-IZD) heterocyclic phosphotyrosine (pTyr) mimetics that when incorporated into dipeptides are exceptionally potent, competitive, and reversible inhibitors of protein tyrosine phosphatase 1B (PTP1B). The crystal structure of PTP1B in complex with our most potent inhibitor 12 revealed that the (S)-IZD heterocycle interacts extensively with the phosphate binding loop precisely as designed in silico. Our data provide strong evidence that the (S)-IZD is the most potent pTyr mimetic reported to date.

The isolation and identification of a toxic impurity in XP315 drug substance.

In early safety assessment studies with the experimental anti-neoplastic drug XP315, a toxic reaction was observed in dogs immediately after intravenous (iv) infusion. The reaction was characterized by severe erythema around the ears, eyes, face and body; ocular hyperemia; head shaking; swelling around the eyes, face, paws, head, neck and legs; scratching; and reddened gums, which lasted several hours after dosing. By fractionating the drug substance using preparative HPLC and then infusing the residues into dogs by iv, this reaction was traced to an impurity in the drug substance. Following the preparative isolation of the toxic impurity, characterization was performed using a combination of NMR and mass spectral methods. The proposed impurity was found to be structurally related and nearly twice the molecular weight of XP315, resulting from a dimerization by ring fusion of two 3-aminonaphthalene fragments during the synthetic process. This paper details the steps taken to isolate the toxic impurity and characterize its structure using off-line methods.

Two-pump at-column-dilution configuration for preparative liquid chromatography-mass spectrometry.

Preparative liquid chromatography-mass spectrometry (LC-MS) is widely used in parallel synthesis schemes to expedite purification. Recently, an alternative sample loading scheme, at column dilution, has been shown to dramatically increase the mass loading capacity of LC-MS purification methods. The prototype system utilized separate sample loading and binary gradient pumps. We report here a configuration for effecting at-column dilution using only the two pumps that provide the binary gradient flow. The advantages of a two-pump configuration are reduced cost, reduced space requirements, simplified control, and reduced service and maintenance issues. The two-pump at-column-dilution configuration is demonstrated for large- and small-scale LC-MS purifications. Purification on scales appropriate for high-throughput parallel synthesis can be achieved with small-scale chromatography using at-column dilution; purification of 20 mg of material is demonstrated using a 4.6 mm x 150 mm column and a flow rate of 3 mL/m. Reducing the scale of chromatography required for LC-MS purification has significant benefits, including the following. It requires less expensive columns, consumes less solvent, generates smaller-volume fractions (shorter dry-down time and the ability to collect into small-volume collector formats, such as 96-well plates), and has the potential for faster separations.

Preparative LC-MS purification: improved compound-specific method optimization.

One of the remaining challenges in providing effective preparative LC-MS purification is balancing throughput and compound purity. We describe here an approach to optimizing preparative LC-MS methods that provides significantly better chromatographic resolution and, hence, better compound purity than generic preparative LC methods consuming the same amount of time. This approach is easier to implement, is more rugged, and permits significantly greater flexibility than previously reported approaches. The instrument configurations and protocols presented here are specifically tailored for open access support, but the basic approach is equally suitable and effective in high-throughput situations.

Kinetics and mechanism of hydrolysis of efavirenz.

PURPOSE: To determine the kinetics and mechanism of hydrolysis of efavirenz [(S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one] in aqueous solutions. METHODS: The solution stability was examined at 60 degrees C and an ionic strength of 0.3 M over the pH range of 0.6 to 12.8. The loss of efavirenz and the appearance of degradants were followed with a reverse-phase high-performance liquid chromatography assay. Characterization of the degradants was accomplished with liquid chromatography-mass spectrometry. RESULTS: The degradation of efavirenz followed apparent first-order kinetics over the pH range of 0.6 to 12.8 at 60 degrees C. The catalytic effect of phosphate and borate buffers was negligible while acetate and citrate demonstrated buffer catalysis. The overall rate constant indicated a pH minimum (the pH of maximum stability) of approximately 4. Mass spectra data identified a degradant with a molecular weight consistent with hydrolysis of the cyclic carbamate to the corresponding amino alcohol. The degradation route was confirmed with spiking experiments with an authentic sample of the amino alcohol indicating that the carbamate hydrolysis pathway was the predominant reaction throughout the pH range studied. Subsequent degradation of the amino alcohol proceeded at the extremes of the pH range studied via rearrangement to the quinoline. CONCLUSIONS: The pH-rate profile was consistent with a combination of a V-shaped profile in the pH range of 0-9 and a sigmoid-shaped profile in the pH range of 4-13. The plateau that began at pH 10-11 is a result of the ionization of the amine of the carbamate inhibiting the base-catalyzed hydrolysis of efavirenz, given that the ionized form of the carbamate is resonance-stabilized toward hydroxide-catalyzed degradation. Thus, increasing the pH resulted in a parallel decrease in the unionized fraction and increase in hydroxide ion concentration resulting in a constant k(obs) value.