New AZT conjugates as potent anti-HIV agents.

In an attempt to discover anti-HIV agents with much reduced cytotoxicity from the currently available HIV-reverse transcriptase inhibitors, AZT conjugates of cholanic acids, 2-imidazolidone-4-carboxylic acid and its derivatives, and N,N'-disubstituted 5-hydroxy-tetrahydropyrimidin-2-ones have been synthesized and their anti-HIV profiles determined with CEM-SS cell line. The AZT conjugates with 2-imidazolidone-4-carboxylic acid and 2-pyrrolidone-5-carboxylic acid through an ester linkage, and with N,N'-diphenyl-5-hydroxy-tetrahydropyrimidin-2-one through a succinate tether showed significantly higher therapeutic indexes than AZT while they also retained or enhanced AZT's anti-HIV activity. Thus, structural features that favor the desired therapeutic profile of the conjugates appear to include a five-membered ring cyclic urea or lactam, and six-membered ring cyclic urea with N,N'-diphenyl substitution.

Synthesis and pharmacological evaluations of new steroidal anti-inflammatory antedrugs: 9alpha-Fluoro-11beta,17alpha,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16alpha-carboxylate (FP16CM) and its derivatives.

In continuing efforts to develop potent anti-inflammatory steroids without systemic adverse effects, methyl 9alpha-fluoro-11beta,17alpha,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16alpha-carboxylate (FP16CM) and its 16-alkoxycarbonyl derivatives (FP16CE, FP16CP and FP16CB) were synthesized based on the antedrug concept. The steroids were evaluated for their pharmacological activities and adverse systemic effects. All steroidal antedrugs showed both binding affinity to the glucocorticoid receptor in liver cytosol and inhibitory effect on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cell. These compounds also inhibited croton-oil-induced ear edema and showed no systemic effects such as thymus atrophy and suppression of corticosterone level after 5-day treatment. Among those compounds tested, FP16CM showed the highest activities in receptor binding, NO inhibition and ear edema, these activities were comparable to those of prednisolone. Hydrolysis study in plasma showed that FP16CB was hydrolyzed rapidly, with the half-live (T1/2) of 3.2 min and the half-lives of other compounds were between 16.9 and 29.4 min. These results support the antedrug concept, of which the decrease in systemic adverse effects is attributed to fast hydrolysis to inactive metabolite in the systemic circulation.

Chirality effect of C-20 on metabolism of methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oate derivatives.

Epimers at C-20 of methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oates, their 9alpha-fluoro analogs, their carbonate derivatives, and their acetonide derivatives were subjected to metabolism study in rat plasma and rat liver homogenate. These steroids were synthesized based on the antedrug concept. In rat plasma, the carboxy ester bonds of 20beta-triols and their acetonides were hydrolyzed with half-lives (T(1/2)) of between 5.7 and 7.7 min, while their corresponding alpha-epimers had longer half-lives of more than 2.5 h. A more profound difference was observed between the alpha- and beta-epimers of the carbonates, with the latter showing a T(1/2) less than 1 min (0.3 and 0.43 min for P20beta- and PF20beta-carbonate, respectively), while that of the former about 3 h (165 min for P20alpha-carbonate and 191 min for PF20alpha-carbonate). In rat liver homogenate, the triol and acetonide derivatives showed greater stability than they did in rat plasma, with T(1/2) for the beta-group in the range of 54-108 min, and T(1/2) for the alpha-group over 7 h. A significant difference in hydrolysis of the carbonate derivatives was also observed in rat liver homogenate. The half-lives of P20beta- and PF20beta-carbonate were 0.67 and 0.66 min, respectively, and the alpha-isomers showed the similar metabolic rate with other alpha-isomers. An esterase inhibitor effectively blocked the hydrolysis of the ester bond, indicating that this metabolism is an enzymatic reaction. Molecular modeling studies show that steric hindrance around the ester group of the alpha-epimers is much greater than that of their beta-counterparts, affording one explanation for the large difference in the metabolic hydrolysis rate; i.e. the carboxy ester bond of beta-isomer which is less hindered sterically than their counter alpha-isomers is hydrolyzed faster than that of alpha-isomers. In conclusion, this study confirms that chirality at C-20 had profound effects on metabolism and pharmacological profile of the steroid acid ester derivatives.

Synthesis, anti-HIV, and cytotoxic activity of new AZT conjugates of steroid acids.

In an attempt to discover potent anti-HIV agents devoid of the serious toxicity of the current HIV-reverse transcriptase inhibitors, three steroid prodrugs of AZT have been synthesized and their anti-HIV profiles determined with CEM-SS cell line. Two of the prodrugs were active against HIV, though weaker than AZT. The third agent was totally inactive against HIV. However, it demonstrated remarkably high anti-growth activities. Further experiments established that growth inhibition of the third agent was caused by induction of apoptosis rather than general necrosis.

Metabolism of steroidal anti-inflammatory antedrugs in vitro: methyl 3,20-dioxo-11beta,17alpha,21-trihydroxy-1,4-pregnadiene-16alpha-carboxylate; its 9alpha-fluorinated, and their 21-O-acyl derivatives.

The in vitro hydrolysis rates of steroidal anti-inflammatory antedrugs, methyl 3,20-dioxo-11beta,17alpha,21-trihydroxy-1,4-pregnadiene-16alpha-carboxylate (P16CM), its 9alpha-fluorinated analogue (FP16CM), and their 21-O-acyl derivatives (P16CM-acetyl, FP16CM-acetyl, FP16CM-propionyl, FP16CM-valeryl, and FP16CM-pivalyl) were investigated in rat plasma. These steroids were synthesized based on the antedrug concept. P16CM and FP16CM were hydrolyzed to inactive steroid-16-carboxylate, with half-lives of 90.0 and 99.4 min, respectively. The metabolite was positively identified by NMR and elemental analysis. To determine the relative hydrolysis rate of the C21-O-acyl versus the C16-methoxycarbonyl group, P16CM- and FP16CM-21-O-acyl derivatives were also studied. The hydrolysis rates of all 21-O-acyl groups were much faster than that of the 16-methoxycarbonyl group. The half-lives of P16CM-acetyl, FP16CM-acetyl, FP16CM-valeryl, and FP16CM-propionyl were 6.3, 16.8, 23.2, and 18.4 min, respectively. On the other hand, FP16CM-pivalyl showed relatively slow hydrolysis rate (T(1/2): 59.7 min). These results clearly indicate that 21-O-acyl group is metabolized first to active compound, P16CM or FP16CM, followed by the hydrolysis of 16-methoxycarbonyl to corresponding inactive steroid-16-carboxylates as the major metabolites. Collectively, the results of the present study support the previous reports where decrease in adverse systemic effects without losing local anti-inflammatory activity was attributed to the hydrolysis of the active agents to inactive acidic metabolites in the systemic circulation. This study thus shows that the incorporation of a 16-methoxycarbonyl coupled with a 21-O-acyl moiety may be a fundamentally sound synthetic strategy in the development of locally active anti-inflammatory steroids having reduced systemic adverse activities.

Prodrug and antedrug: two diametrical approaches in designing safer drugs.

The prodrug and antedrug concepts, which were developed to overcome the physical and pharmacological shortcomings of various therapeutic classes of agents, employ diametrically different metabolic transformations. The prodrug undergoes a predictable metabolic activation prior to exhibiting its pharmacological effects in a target tissue while the antedrug undergoes metabolic deactivation in the systemic circulation upon leaving a target tissue. An increased therapeutic index is the aspiration for both approaches in designing as well as evaluation criteria. The recent research endeavors of prodrugs include the gene-directed and antibody-directed enzymatic activation of a molecule in a targeted tissue, organ specific delivery, improved bioavailabilities of nucleosides and cellular penetration of nucleotides. As for antedrugs, emphasis in research has been based upon the design and synthesis of systemically inactive molecule by incorporating a metabolically labile functional group into an active molecule.

Effect of chirality at C-20 of methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oate derivatives on antiinflammatory activity.

In an effort to determine the C-20 chirality effect on the antiinflammatory activity of 17beta-glycolate esters, methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oate and its 9alpha-fluoro analog, their acetonide and their carbonate derivatives were synthesized and evaluated. The agents were tested for their binding potency to the macrophage glucocorticoid receptor, and their effect on LPS-induced nitric oxide generation in RAW 264.7 cells. The acetonide derivatives showed the highest binding affinity while the triols and carbonates bound rather poorly to the receptors. With the exception of the triols, the alpha-isomer in each pair of the agents exhibited higher binding affinity to the receptor than its corresponding beta-isomer, clearly indicating that C-20 chirality has a significant effect on antiinflammatory activity. In addition, the alpha-isomers of the acetonides showed substantially higher binding affinity than the parent compound, prednisolone. In contrast to the high binding activity exhibited by some of the acetonides, all of the agents showed weak inhibitory effect on NO generation. Metabolic inactivation during assessment of NO inhibition may play a role in the divergence noted between receptor affinity and the measured biologic activity resulting from the binding.