Synthesis and In vitro Efficacy of Tetracyclic Benzothiazepines Against Blood-Stage Plasmodium falciparum and Liver-Stage P. berghei.

OBJECTIVE: A series of novel, substituted tetracyclic benzothiazepines were designed and prepared in an effort to optimize the potency of this chemical class against drug-resistant strains of the malaria parasite. METHODS: Tetracyclic benzothiazepines bearing structural modification at seven distinct positions within the structure were synthesized in Knoevenagel condensation reactions followed by sequential intermolecular thio-Michael and then intramolecular imine formation reactions. Following purification and chemical characterization, the novel compounds were tested for in vitro efficacy against blood-stage P. falciparum and liver-stage P. berghei and also for in vivo efficacy against P. berghei. RESULTS: Benzothiazepines bearing structural modification at the sulfur atom and at the three carbocycles within the molecule were successfully synthesized. The majority of analogs inhibited bloodstage P. falciparum with submicromolar IC50 values. The potency of an 8-methoxy-substituted analog 12 exceeded that of chloroquine in all three P. falciparum strains tested. The parent benzothiazepine 1 possessed liver-stage activity, inhibiting P. berghei sporozoites infecting HepG2 cells with an IC50 of 106.4 nM and an IC90 of 408.9 nM, but failed to enhance the longevity of P. berghei infected mice compared to the controls. Compounds displayed modest toxicity toward HepG2 cells and were tolerated by mice at the highest dose tested, 640 mg/kg/dose once daily for three days. CONCLUSION: The tetracyclic benzothiazepine described, which inhibits P. berghei infected hepatic cells with an IC50 of 106.4 nM, would appear to warrant further investigation. Optimization of ADME properties may be required since the most active analogs are probably excessively lipophilic.

Correlation between Cyclin Dependent Kinases and Artemisinin-Induced Dormancy in Plasmodium falciparum In Vitro.

BACKGROUND: Artemisinin-induced dormancy provides a plausible explanation for recrudescence following artemisinin monotherapy. This phenomenon shares similarities with cell cycle arrest where cyclin dependent kinases (CDKs) and cyclins play an important role. METHODS: Transcription profiles of Plasmodium falciparum CDKs and cyclins before and after dihydroartemisinin (DHA) treatment in three parasite lines, and the effect of CDK inhibitors on parasite recovery from DHA-induced dormancy were investigated. RESULTS: After DHA treatment, parasites enter a dormancy phase followed by a recovery phase. During the dormancy phase parasites up-regulate pfcrk1, pfcrk4, pfcyc2 and pfcyc4, and down-regulate pfmrk, pfpk5, pfpk6, pfcrk3, pfcyc1 and pfcyc3. When entering the recovery phase parasites immediately up-regulate all CDK and cyclin genes. Three CDK inhibitors, olomoucine, WR636638 and roscovitine, produced distinct effects on different phases of DHA-induced dormancy, blocking parasites recovery. CONCLUSIONS: The up-regulation of PfCRK1 and PfCRK4, and down regulation of other CDKs and cyclins correlate with parasite survival in the dormant state. Changes in CDK expression are likely to negatively regulate parasite progression from G1 to S phase. These findings provide new insights into the mechanism of artemisinin-induced dormancy and cell cycle regulation of P. falciparum, opening new opportunities for preventing recrudescence following artemisinin treatment.

In vitro efficacy of 2,N-bisarylated 2-ethoxyacetamides against Plasmodium falciparum.

Investigation of a series of 2,N-bisarylated 2-ethoxyacetamides resulted in the identification of four inhibitors 5, 20, 24, 29 with single-digit micromolar in vitro efficacy against two drug-resistant Plasmodium falciparum strains. These compounds are analogs of structurally-related 1,3-bisaryl-2-propen-1-ones (chalcones), the latter showing efficacy in vitro but not in a malaria-infected mouse. The 2,N-bisarylated 2-ethoxyacetamides (e.g., 2, 5, 20) were shown to possess significantly greater stability in the presence of metabolizing enzymes than the corresponding 1,3-bisaryl-2-propen-1-ones (e.g., 1, 3, 18).

In vitro biotransformation, in vivo efficacy and pharmacokinetics of antimalarial chalcones.

4'-n-Butoxy-2,4-dimethoxy-chalcone (MBC) has been described as protecting mice from an otherwise lethal infection with Plasmodium yoelii when dosed orally at 50 mg/kg/dose, daily for 5 days. In contrast, we found that oral dosing of MBC at 640 mg/kg/dose, daily for 5 days, failed to extend the survivability of P. berghei-infected mice. The timing of compound administration and metabolic activation likely contribute to the outcome of efficacy testing in vivo. Microsomal digest of MBC yielded 4'-n-butoxy-4-hydroxy-2-methoxy-chalcone and 4'-(1-hydroxy-n-butoxy)-2,4-dimethoxy-chalcone. We propose that the latter will hydrolyze in vivo to 4'-hydroxy-2,4-dimethoxy-chalcone, which has greater efficacy than MBC in our P. berghei-infected mouse model and was detected in plasma following oral dosing of mice with MBC. Pharmacokinetic parameters suggest that poor absorption, distribution, metabolism and excretion properties contribute to the limited in vivo efficacy observed for MBC and its analogs.

In vitro efficacy of 7-benzylamino-1-isoquinolinamines against Plasmodium falciparum related to the efficacy of chalcones.

A series of 1,7-diaminoisoquinolinamines, that are expected to mediate antimalarial activity by the same mechanism employed by the chalcones, were produced. Six 7-benzylamino-1-isoquinolinamines were found to be submicromolar inhibitors in vitro of drug-resistant Plasmodium falciparum, with the best possessing activity comparable to chloroquine. Despite being developed from a lead that is a DHFR inhibitor, these compounds do not mediate their antimalarial effects by inhibition of DHFR.

Selective inhibition of Pfmrk, a Plasmodium falciparum CDK, by antimalarial 1,3-diaryl-2-propenones.

The cyclin dependent protein kinases, Pfmrk and PfPK5, most likely play an essential role in cell cycle control and differentiation in Plasmodium falciparum and are thus an attractive target for antimalarial drug development. Various 1,3-diaryl-2-propenones (chalcone derivatives) which selectivity inhibit Pfmrk in the low micromolar range (over PfPK5) are identified. Molecular modeling shows a pair of amino acid residues within the Pfmrk active site which appear to confer this selectivity. Predicted interactions between the chalcones and Pfmrk correlate well with observed potency. Pfmrk inhibition and activity against the parasite in vitro correlate weakly. Several mechanisms of action have been suggested for chalcone derivatives and our study suggests that kinase inhibition may be an additional mechanism of antimalarial activity for this class of compounds.

In vitro and in vivo efficacy and in vitro metabolism of 1-phenyl-3-aryl-2-propen-1-ones against Plasmodium falciparum.

Investigation of a series of 1-phenyl-3-aryl-2-propen-1-ones resulted in the identification of nine inhibitors with submicromolar efficacy against at least one Plasmodium falciparum strain in vitro. These inhibitors were inactive when given orally in a Plasmodium berghei infected mouse model. Significant compound degradation occurred upon their exposure to a liver microsome preparation, suggesting metabolic instability may be responsible for the lack of activity in vivo.

N-(3-phenylsulfonyl-3-piperidinoyl)-phenylalanine derivatives as potent, selective VLA-4 antagonists.

The SAR of 1-sulfonyl-cyclopentyl carboxylic acid amides, ligands for the VLA-4 integrin, was investigated. This effort resulted in the identification of N-(3-phenylsulfonyl-3-piperidinoyl)-(L)-4-(2',6'-dimethoxyphenyl)phenylalanine 52 as a potent, selective VLA-4 antagonist (IC(50)=90 pM). Expansion of the SAR demonstrated that this structural unit can be used to identify a diverse series of sub-nanomolar antagonists.

The Total Synthesis of Eleutherobin: A Surprise Ending.

An "sp2 -sp3 Stille coupling" of the vinyl triflate 1 and the stannyl compound 2 is a key step toward the completion of the total synthesis of eleutherobin, a natural product exhibiting taxol-like cytotoxic activity.