A Novel Piperazine-Based Drug Lead for Cryptosporidiosis from the Medicines for Malaria Venture Open-Access Malaria Box.

Cryptosporidiosis causes life-threatening diarrhea in children under the age of 5 years and prolonged diarrhea in immunodeficient people, especially AIDS patients. The standard of care, nitazoxanide, is modestly effective in children and ineffective in immunocompromised individuals. In addition to the need for new drugs, better knowledge of drug properties that drive in vivo efficacy is needed to facilitate drug development. We report the identification of a piperazine-based lead compound for Cryptosporidium drug development, MMV665917, and a new pharmacodynamic method used for its characterization. The identification of MMV665917 from the Medicines for Malaria Venture Malaria Box was followed by dose-response studies, in vitro toxicity studies, and structure-activity relationship studies using commercial analogues. The potency of this compound against Cryptosporidium parvum Iowa and field isolates was comparable to that against Cryptosporidium hominis Furthermore, unlike nitazoxanide, clofazimine, and paromomycin, MMV665917 appeared to be curative in a NOD SCID gamma mouse model of chronic cryptosporidiosis. MMV665917 was also efficacious in a gamma interferon knockout mouse model of acute cryptosporidiosis. To determine if efficacy in this mouse model of chronic infection might relate to whether compounds are parasiticidal or parasitistatic for C. parvum, we developed a novel in vitro parasite persistence assay. This assay suggested that MMV665917 was parasiticidal, unlike nitazoxanide, clofazimine, and paromomycin. The assay also enabled determination of the concentration of the compound required to maximize the rate of parasite elimination. This time-kill assay can be used to prioritize early-stage Cryptosporidium drug leads and may aid in planning in vivo efficacy experiments. Collectively, these results identify MMV665917 as a promising lead and establish a new method for characterizing potential anticryptosporidial agents.

Estrogen receptor-beta potency-selective ligands: structure-activity relationship studies of diarylpropionitriles and their acetylene and polar analogues.

Through an effort to develop novel ligands that have subtype selectivity for the estrogen receptors alpha (ERalpha) and beta (ERbeta), we have found that 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) acts as an agonist on both ER subtypes, but has a 70-fold higher relative binding affinity and 170-fold higher relative potency in transcription assays with ERbeta than with ERalpha. To investigate the ERbeta affinity- and potency-selective character of this DPN further, we prepared a series of DPN analogues in which both the ligand core and the aromatic rings were modified by the repositioning of phenolic hydroxy groups and by the addition of alkyl substituents and nitrile groups. We also prepared other series of DPN analogues in which the nitrile functionality was replaced with acetylene groups or polar functions, to mimic the linear geometry or polarity of the nitrile, respectively. To varying degrees, all of the analogues show preferential binding affinity for ERbeta (i.e., they are ERbeta affinity-selective), and many, but not all of them, are also more potent in activating transcription through ERbeta than through ERalpha (i.e., they are ERbeta potency-selective). meso-2,3-Bis(4-hydroxyphenyl)succinonitrile and dl-2,3-bis(4-hydroxyphenyl)succinonitrile are among the highest ERbeta affinity-selective ligands, and they have an ERbeta potency selectivity that is equivalent to that of DPN. The acetylene analogues have higher binding affinities but somewhat lower selectivities than their nitrile counterparts. The polar analogues have lower affinities, and only the fluorinated polar analogues have substantial affinity selectivities. This study suggests that, in this series of ligands, the nitrile functionality is critical to ERbeta selectivity because it provides the optimal combination of linear geometry and polarity. Furthermore, the addition of a second nitrile group beta to the nitrile in DPN or the addition of a methyl substitutent at an ortho position on the beta-aromatic ring increases the affinity and selectivity of these compounds for ERbeta. These ERbeta-selective compounds may prove to be valuable tools in understanding the differences in structure and biological function of ERalpha and ERbeta.

Estrogen receptor subtype-selective ligands: asymmetric synthesis and biological evaluation of cis- and trans-5,11-dialkyl- 5,6,11, 12-tetrahydrochrysenes.

We have recently reported that racemic 5,11-cis-diethyl-5,6,11, 12-tetrahydrochrysene-2,8-diol (THC, rac-2b) acts as an agonist on estrogen receptor alpha (ERalpha) and as a complete antagonist on estrogen receptor beta (ERbeta) (Sun et al. Endocrinology 1999, 140, 800-804). To further investigate this novel ER subtype-selective estrogenic activity, we have synthesized a series of cis- and trans-dialkyl THCs. cis-Dimethyl, -diethyl, and -dipropyl THCs 2a-c were prepared in a highly enantio- and diastereoselective manner by the acyloin condensation of enantiomerically pure alpha-alkyl-beta-arylpropionic esters, followed by a Lewis acid-mediated double cyclization under conditions of minimal epimerization. ERalpha and ERbeta binding affinity of both cis and trans isomers of dimethyl, diethyl, and dipropyl THCs was determined in competitive binding assays, and their transcriptional activity was determined in reporter gene assays in mammalian cells. Nearly all THCs examined were found to be affinity-selective for ERbeta. All these THCs are agonists on ERalpha, and THCs with small substituents are agonists on both ERalpha and ERbeta. As substituent size was increased, ERbeta-selective antagonism developed first in the (R,R)-cis enantiomer series and finally in the trans diastereomer and (S,S)-cis enantiomer series. The most potent and selective ligand was identified as (R,R)-cis-diethyl THC 2b, which mimicked the ERbeta-selective antagonist character of racemic cis-diethyl THC 2b. This study illustrates that the antagonist character in THC ligands for ERbeta depends in a progressive way on the size and geometric disposition of substituent groups and suggests that the induction of an antagonist conformation in ERbeta can be achieved with these ligands with less steric perturbation than in ERalpha. Furthermore, antagonists that are selectively effective on ERbeta can have structures that are very different from the typical antiestrogens tamoxifen and raloxifene, which are antagonists on both ERalpha and ERbeta.

Novel ligands that function as selective estrogens or antiestrogens for estrogen receptor-alpha or estrogen receptor-beta.

We report on the identification of novel, nonsteroidal ligands that show pronounced subtype-selective differences in ligand binding and transcriptional potency or efficacy for the two estrogen receptor (ER) subtypes, ER alpha and ER beta. An aryl-substituted pyrazole is an ER alpha potency-selective agonist, showing higher binding affinity for ER alpha and 120-fold higher potency in stimulation of ER alpha vs. ER beta in transactivation assays in cells. A tetrahydrochrysene (THC) has a 4-fold preferential binding affinity for ER beta; it is an agonist on ER alpha, but a complete antagonist on ER beta. Intriguingly, the antagonist activity of THC is associated with the R,R-enantiomer (R,R-THC). The S,S-enantiomer (S,S-THC) is an agonist on both ER alpha and ER beta but has a 20-fold lower affinity for ER beta than R,R-THC. This difference in binding affinity accounts for the full ER beta antagonist activity of the THC racemate (a 1:1 mixture of R,R-THC and S,S-THC). These compounds should be useful in probing the conformational changes in these two ERs that are evoked by agonists and antagonists, and in evaluating the distinct roles that ER beta and ER alpha may play in the diverse target tissues in which estrogens act.

Facile synthesis of high affinity styrylpyridine systems as inherently fluorescent ligands for the estrogen receptor.

A series of styrylpyridine derivatives containing two phenols was prepared via an efficient two-step synthesis. These compounds were designed to maximize the estrogen receptor binding affinity of a known series of inherently fluorescent styrylpyridines. While significant improvements were achieved in receptor affinity, the fluorescence intensity of this series of compounds is poor.