Readily available N-acyl-5-vinyl-2,3-dihydro-4-pyridones undergo Diels-Alder cyclization with various dienophiles to afford novel octahydroquinolines containing synthetically useful functionality. With dihydropyridone 5 and cis-disubstituted dienophiles, the resulting cycloadducts were obtained as single diastereomers in good to excellent yield. The corresponding reaction of 5 with methyl acrylate, acrylonitrile, and phenyl vinyl sulfone showed modest preference for the endo adducts. The effect of the dihydropyridone C-2 and C-4 substituents on the degree of diastereofacial control was examined. By using this methodology, the core decahydroquinoline skeleton of gephyrotoxin was prepared in a stereocontrolled fashion. Interesting reactivity was observed with certain dienophiles leading to ring-opening of the initially formed cycloadducts. This tandem reaction provides a route to uniquely substituted beta-aminoketones, alcohols, and unnatural amino acids.

[reaction: see text] Readily available 5-vinyldihydropyridones 2 undergo Diels-Alder cyclization with various dienophiles to afford novel octahdroquinolines. The process is highly stereoselective and provides heterocyclic products containing synthetically useful functionality.

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An asymmetric synthesis of (2S,4R)-4-hydroxypipecolic acid was accomplished in eight steps and 31% overall yield.

In this paper a new graphical method, derived from the Steric Mass Action model of non-linear ion-exchange chromatography, is presented for the determination of solute affinity in ion exchange displacement systems. The affinity of solutes in these systems is described by a dynamic affinity parameter which is a function of the linear steric mass action parameters of the solutes and the characteristic velocity of the displacer front. This method can be employed to determine the elution order of the feed solutes in the isotachic displacement train as well as the ability of a given molecule to act as a displacer for a given protein mixture. The ideal model of ion-exchange displacement chromatography and a transient model are employed to study selectivity reversals and resolving power in ion-exchange displacement systems. The work presented here provides a theoretical framework for studying dynamic affinity, resolving power, and displacer design for ion-exchange displacement chromatography.

One of the major advantages of displacement chromatography is the simultaneous concentration and purification that can be effected during the process. The steric mass-action model of non-linear ion-exchange chromatography is employed to investigate the affects of mobile phase salt concentration and displacer equilibrium properties on the concentration of proteins in isotachic displacement zones. The results indicate that the salt microenvironment in displacement zones plays a major role in determining the concentration of the displaced proteins. The parameters which affect the salt microenvironment are both the initial salt concentration in the carrier as well as the induced gradient produced during the displacement process. For a given breakthrough volume, the induced gradient is determined by the ratio of the steric factor to the characteristic charge of the displacer. These results indicate that the use of relatively low salt concentrations in the carrier along with displacers with relatively high steric factor to characteristic charge ratios will produce significant concentration of the proteins during the purification process.

In this chapter we present a steric mass-action (SMA) ion-exchange equilibrium formalism which explicitly accounts for the steric hindrance of salt counter-ions upon protein binding in multicomponent equilibria. A simple method is presented for the rapid calculation of effluent profiles of displaced proteins and induced salt gradients under ideal chromatographic conditions. Theoretical predictions are compared to experimental results in both cation and anion exchange systems. Both the induced salt gradients and the displacement profiles match well with the theoretical predictions. These displacements differ from the traditional vision of displacement in several important ways: the isotherm of the displacer does not lie above the feed component isotherms; the concentrations of displaced proteins exceed the concentration of the displacer; and the induced salt gradient produces different salt microenvironments for each displaced protein. These results demonstrate the ability of the SMA formalism to predict complex behavior present in ion-exchange protein displacement systems. Furthermore, the development of a rapid method for obtaining ideal isotachic displacement profiles with this formalism facilitates methods development and optimization of ion-exchange protein displacement separations.

Polar and primary metabolites of cyclosporin A (CsA) have successfully been isolated by a novel separation protocol. An efficient, easy-to-scale-up chromatographic adsorption/desorption operation recovers polar and primary CsA metabolite pools from large volumes of urine; purified CsA metabolites are subsequently obtained by high-resolution preparative elution chromatography of the semipurified metabolite pools. Separations performed on a semipreparative scale [with a 250 x 9.4 mm (i.d.) reversed-phase HPLC column] yielded microgram quantities of CsA metabolites at > 97% purity, as determined by fast atom bombardment mass spectrometry. These separations also yielded two previously unreported CsA metabolites, similar to AM1A but with an additional hydroxylation. The yield of metabolites was increased to several milligrams by performing the separations with a preparative-scale [250 x 21.2 mm (i.d.)] reversed-phase column. The production rate of purified primary CsA metabolites was greatly increased by performing the separation with the preparative-scale column under conditions of severe mass overloading. In a single chromatographic run, we successfully isolated 11.0 and 5.0 mg of AM1 and AM1c, respectively, at a purity of > 97%. As expected, this increase in the yield of purified metabolites was accompanied by a decrease in the overall recovery. This separation scheme enables the rapid processing of large volumes of urine for isolation of the milligram quantities of CsA metabolites necessary to assess their biological activity. The procedure is applicable to small- or large-scale metabolite isolation and provides a ready source of purified metabolites for in vitro and whole-animal studies.

AM1 (M17) is the major metabolite of cyclosporine found in the blood of human transplant recipients, and trough levels of this derivative exceed those of the parent compound approximately two-fold. Studies performed in vitro indicate that AM1 retains only 10-20% of the biological activity of the parent compound, but very little is known about its in vivo immunosuppressive effects. We therefore developed a rapid and sensitive method, based on the rejection of allogeneic L1210 (H-2d) leukemia cells by C57BL/6 (H-2b) mice, to assess the immunosuppressive activity of AM1 in vivo. Rejection of the leukemia allograft was determined by analyzing the spleens from mice injected intravenously with 10(5) L1210 cells for the presence of H-2K(d)-positive cells by flow cytometry using an FITC-conjugated monoclonal anti-H-2K(d) antibody. Nonimmunosuppressed mice rejected the allogeneic cells and survived indefinitely. Spleens from these mice were virtually free of H-2K(d)-positive cells (0.51 +/- 0.21%) by day 7. In contrast, C57BL/6 mice treated with 10 mg/kg/day s.c. of CsA all died from the L1210 challenge (mean survival time of 9 +/- 1 days). Spleens from mice treated in this manner contained 11.02 +/- 3.31% H-2K(d)-positive cells on day 7. There was a direct correlation between the dose of CsA administered (7.5-50 mg/kg/day) and the percentage of H-2K(d)-positive cells in the spleen. We then compared the immunosuppressive activity of AM1 and CsA in this model. AM1 was purified from the urine of CsA-treated renal allograft recipients by a combination of preparative adsorption-desorption chromatography and preparative elution high-performance liquid chromatography. AM1 at a dose of 10 mg/kg/day exhibited no demonstrable immunosuppressive effect, and trough levels of AM1 on day 7 were only 36 +/- 4 ng/ml. Increasing the dose of AM1 to 50 mg/kg/day resulted in only 1.05 +/- 0.16% H-2K(d)-positive cells in the spleens (P = NS) and a mean trough level of 221 +/- 27 ng/ml. In contrast, mice treated with 50 mg/kg/day of CsA exhibited 17.7 +/- 2.9% H-2K(d)-positive cells in their spleens and a mean trough CsA level of 3036 +/- 277 ng/ml. The half-life of a single subcutaneous dose of 10 mg/kg of AM1 (4.6 hr) was significantly shorter than that of CsA (9.7 hr) in mice. Compared with CsA, the lack of immunosuppressive effect of AM1 in vivo therefore appears to be due to a combination of decreased immunosuppressive activity and increased rate of clearance in mice.

The study of nonlinear competitive equilibrium is of fundamental importance in understanding the behavior of proteins in preparative ion-exchange chromatographic separations. In this work we present a steric mass-action (SMA) ion-exchange equilibrium formalism, which explicitly accounts for the steric hindrance of salt counterions upon protein binding in multicomponent equilibria. An analytical solution has been derived for the calculation of isotachic effluent profiles of displaced proteins and induced salt gradients under ideal chromatographic conditions. A stability analysis has been employed to establish the order of the feed components in the displacement train. Theoretical predictions are compared to experimental results for the separation of proteins by cation-exchange displacement chromatography. These results demonstrate the efficacy of the SMA formalism in predicting complex behavior present in ion-exchange displacement systems. Furthermore, the analytical solution of ideal isotachic displacement profiles with the SMA formalism enables rapid methods development and optimization of ion-exchange displacement separations.