Aminothiazoles: Hit to lead development to identify antileishmanial agents.

As part of Drugs for Neglected Diseases initiative's lead optimization program for the development of new chemical entities to treat visceral leishmaniasis (VL), a series of aminothiazoles were synthesized and screened for in vitro efficacy, solubility and microsomal stability. The primary aim of identifying a lead structure with sub-micromolar activity was achieved. Out of 43 compounds synthesized, 16 compounds showed in vitro activity at less than 1 µM against VL. Compound 32 showed excellent antileishmanial potency (IC50 = 3 nM) and had all the acceptable properties except for metabolic instability. Blocking the metabolic soft spots in compound 32, where the 4-methoxy pyridine substituent was replaced by 5-ethoxy group, led to compound 36 (IC50 = 280 nM) with improved stability. To understand the disposition of 36, in vivo pharmacokinetic study was conducted in a mouse model. Compound 36 showed high clearance (91 mL/min/kg); short half-life (0.48 h) after intravenous administration (1 mg/kg) and exposure (AUC0-24) following oral administration was 362 ng h/mL with absolute bioavailability of 8%. To summarize, 43 analogs were synthesized out of which 15 compounds showed very potent sub-nanomolar efficacy in in vitro systems but the liability of metabolic instability seemed to be the major challenge for this chemical class and remains to be addressed.

Design, synthesis, ADME characterization and antileishmanial evaluation of novel substituted quinoline analogs.

In vitro ADME characterization of the lead compound 1 identified for visceral leishmaniasis was undertaken and further structural analogs were synthesized for antileishmanial screening. Compound 1 was highly permeable in intestinal PAMPA model (31 × 10(-6)cm/s) and was moderately bound to mouse and human plasma proteins (% bound 85-95%), its blood to plasma concentration ratio was less than 1, but the compound was unstable in blood. Compound 1 was found to have no CYP450 liability with CYP2C9, 2C19, 2D6 and 3A4. It showed inhibition with CYP1A2 with an IC50 value of 0.50 µM. Analogs of 1 were synthesized and subsequently characterized for in vitro activity against the intracellular form of Leishmania donovani. Resulting quinolines were found to have similar efficacy as 1 against the parasite. Compounds 8b and 8f were found to be the most active with IC50 values of 0.84 µM and 0.17 µM, respectively compared to 0.22 µM for compound 1. Of all the analogs tested, 8d was stable in hamster, mouse and human liver microsomes but lost the efficacy with an IC50 of 6.42 µM. Based on the in vitro efficacy and DMPK profile, compounds 8b and 8f seem the best candidates to be screened in further assays.

Design, synthesis and biological evaluation of 2-substituted quinolines as potential antileishmanial agents.

An analogous library of 2-substituted quinoline compounds was synthesized with the aim to identify a potential drug candidate to treat visceral leishmaniasis. These molecules were tested for their in vitro and in vivo biological activity against Leishmania donovani. Metabolic stability of these compounds was also improved through the introduction of halogen substituents. Compound (26g), found to be the most active; exhibited an IC50 value of 0.2 µM and >180 fold selectivity. The hydrochloride salt of (26g) showed 84.26 ± 4.44 percent inhibition at 50 mg/kg × 5 days (twice daily, oral route) dose in L. donovani/hamster model. The efficacy was well correlated with the PK data observed which indicating that the compound is well distributed.

Acridones circumvent P-glycoprotein-associated multidrug resistance (MDR) in cancer cells.

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (P-gp) is one of the best characterized transporter-mediated barriers to successful chemotherapy in cancer patients. Chemosensitizers are the agents that increase the sensitivity of multidrug-resistant cells to the toxic influence of previously less effective drugs. In an attempt to find such vital chemosensitizers, a series of N(10)-substituted-2-chloroacridone analogous (1-17) have been synthesized. Compound 1 was prepared by the Ullmann condensation of o-chlorobenzoic acid and p-chloroaniline followed by cyclization. The N-(omega-chloroalkyl) analogues were found to undergo iodide catalyzed nucleophilic substitution reaction with secondary amines and the resultant products were characterized by spectral methods. The lipophilicity expressed in log(10)P and pK(a) of compounds has been determined. All compounds were examined for their ability to increase the uptake of vinblastine (VLB) in MDR KBCh(R)-8-5 cells and the results showed that the compounds 6, 8, 11-14, 16, and 17 at their respective IC(50) concentrations caused a 1.0- to 1.7-fold greater accumulation of VLB than did a similar concentration of the standard modulator, verapamil (VRP). Results of the efflux experiment showed that VRP and each of the modulators significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp. All modulators effectively competing with [(3)H]azidopine for binding to P-gp pointed out this transport membrane protein as their likely site of action. Compounds at IC(10) were evaluated for their efficacy to modulate the cytotoxicity of VLB and the results showed that modulators 11, 13, 14, 16, and 17 were able to completely reverse the 25-fold resistance of KBCh(R)-8-5 cells to VLB. Examination of the relationship between lipophilicity and antagonism of MDR showed a reasonable correlation suggesting that hydrophobicity is one of the determinants of potency for anti-MDR activity of 2-chloroacridones. The results allowed us to draw preliminary conclusions about structural features of 2-chloroacridones important for MDR modulation.

Nanomolar affinity small molecule correctors of defective Delta F508-CFTR chloride channel gating.

Deletion of Phe-508 (Delta F508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. Delta F508-CFTR has defects in both channel gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective Delta F508-CFTR Cl- channel gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing Delta F508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which Delta F508-CFTR was targeted to the plasma membrane by culture at 27 degrees C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated DeltaF508-CFTR Cl- conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after Delta F508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective Delta F508-CFTR gating was also demonstrated in human bronchial epithelial cells from a Delta F508 cystic fibrosis subject after 27 degrees C temperature rescue. In conjunction with correctors of defective Delta F508-CFTR processing, small molecule potentiators of defective Delta F508-CFTR gating may be useful for therapy of cystic fibrosis caused by the Delta F508 mutation.