Synthesis and Evaluation of Sulfonylnitrophenylthiazoles (SNPTs) as Thyroid Hormone Receptor–Coactivator Interaction Inhibitors

We previously identified a series of methylsulfonylnitrobenzoates (MSNBs) that block the interaction of the thyroid hormone receptor with its coactivators. MSNBs inhibit coactivator binding through irreversible modification of cysteine 298 of the thyroid hormone receptor (TR). Although MSNBs have better pharmacological features than our first generation inhibitors (β-aminoketones), they contain a potentially unstable ester linkage. Here we report the bioisosteric replacement of the ester linkage with a thiazole moiety, yielding sulfonylnitrophenylthiazoles (SNPTs). An array of SNPTs representing optimal side chains from the MSNB series was constructed using parallel chemistry and evaluated to test their antagonism of the TR-coactivator interaction. Selected active compounds were evaluated in secondary confirmatory assays including regulation of thyroid response element driven transcription in reporter constructs and native genes. In addition the selected SNPTs were shown to be selective for TR relative to other nuclear hormone receptors (NRs)

Piperidinyl Ureas Chemically Control Defective in Cullin Neddylation 1 (DCN1)-Mediated Cullin Neddylation

We previously discovered and validated a class of piperidinyl ureas that regulate defective in cullin neddylation 1 (DCN1)-dependent neddylation of cullins. Here, we report preliminary structure–activity relationship studies aimed at advancing our high-throughput screen hit into a tractable tool compound for dissecting the effects of acute DCN1–UBE2M inhibition on the NEDD8/cullin pathway. Structure-enabled optimization led to a 100-fold increase in biochemical potency and modestly increased solubility and permeability as compared to our initial hit. The optimized compounds inhibit the DCN1–UBE2M protein–protein interaction in our TR-FRET binding assay and inhibit cullin neddylation in our pulse-chase NEDD8 transfer assay. The optimized compounds bind to DCN1 and selectively reduce steady-state levels of neddylated CUL1 and CUL3 in a squamous cell carcinoma cell line. Ultimately, we anticipate that these studies will identify early lead compounds for clinical development for the treatment of lung squamous cell carcinomas and other cancers

Piperidinyl Ureas Chemically Control Defective in Cullin Neddylation 1 (DCN1)-Mediated Cullin Neddylation

We previously discovered and validated a class of piperidinyl ureas that regulate defective in cullin neddylation 1 (DCN1)-dependent neddylation of cullins. Here, we report preliminary structure–activity relationship studies aimed at advancing our high-throughput screen hit into a tractable tool compound for dissecting the effects of acute DCN1–UBE2M inhibition on the NEDD8/cullin pathway. Structure-enabled optimization led to a 100-fold increase in biochemical potency and modestly increased solubility and permeability as compared to our initial hit. The optimized compounds inhibit the DCN1–UBE2M protein–protein interaction in our TR-FRET binding assay and inhibit cullin neddylation in our pulse-chase NEDD8 transfer assay. The optimized compounds bind to DCN1 and selectively reduce steady-state levels of neddylated CUL1 and CUL3 in a squamous cell carcinoma cell line. Ultimately, we anticipate that these studies will identify early lead compounds for clinical development for the treatment of lung squamous cell carcinomas and other cancers

Optimization of a Novel Series of Ataxia-Telangiectasia Mutated Kinase Inhibitors as Potential Radiosensitizing Agents

We previously reported a novel inhibitor of the ataxia-telangiectasia mutated (ATM) kinase, which is a target for novel radiosensitizing drugs. While our initial lead, compound <b>4</b>, was relatively potent and nontoxic, it exhibited poor stability to oxidative metabolism and relatively poor selectivity against other kinases. The current study focused on balancing potency and selectivity with metabolic stability through structural modification to the metabolized site on the quinazoline core. We performed extensive structure–activity and structure–property relationship studies on this quinazoline ATM kinase inhibitor in order to identify structural variants with enhanced selectivity and metabolic stability. We show that, while the C-7-methoxy group is essential for potency, replacing the C-6-methoxy group considerably improves metabolic stability without affecting potency. Promising analogues <b>20</b>, <b>27g</b>, and <b>27n</b> were selected based on in vitro pharmacology and evaluated in murine pharmacokinetic and tolerability studies. Compound <b>27g</b> possessed significantly improve pharmacokinetics relative to that of <b>4</b>. Compound <b>27g</b> was also significantly more selective against other kinases than <b>4</b>. Therefore, <b>27g</b> is a good candidate for further development as a potential radiosensitizer

Optimization of a Novel Series of Ataxia-Telangiectasia Mutated Kinase Inhibitors as Potential Radiosensitizing Agents

We previously reported a novel inhibitor of the ataxia-telangiectasia mutated (ATM) kinase, which is a target for novel radiosensitizing drugs. While our initial lead, compound <b>4</b>, was relatively potent and nontoxic, it exhibited poor stability to oxidative metabolism and relatively poor selectivity against other kinases. The current study focused on balancing potency and selectivity with metabolic stability through structural modification to the metabolized site on the quinazoline core. We performed extensive structure–activity and structure–property relationship studies on this quinazoline ATM kinase inhibitor in order to identify structural variants with enhanced selectivity and metabolic stability. We show that, while the C-7-methoxy group is essential for potency, replacing the C-6-methoxy group considerably improves metabolic stability without affecting potency. Promising analogues <b>20</b>, <b>27g</b>, and <b>27n</b> were selected based on in vitro pharmacology and evaluated in murine pharmacokinetic and tolerability studies. Compound <b>27g</b> possessed significantly improve pharmacokinetics relative to that of <b>4</b>. Compound <b>27g</b> was also significantly more selective against other kinases than <b>4</b>. Therefore, <b>27g</b> is a good candidate for further development as a potential radiosensitizer

Synthesis and Evaluation of 7-Substituted 4-Aminoquinoline Analogues for Antimalarial Activity

We previously reported that substituted 4-aminoquinolines with a phenyl ether substituent at the 7-position of the quinoline ring and the capability of intramolecular hydrogen bonding between the protonated amine on the side chain and a hydrogen bond acceptor on the amine’s alkyl substituents exhibited potent antimalarial activity against the multidrug resistant strain <i>P</i>. <i>falciparum</i> W2. We employed a parallel synthetic method to generate diaryl ether, biaryl, and alkylaryl 4-aminoquinoline analogues in the background of a limited number of side chain variations that had previously afforded potent 4-aminoquinolines. All subsets were evaluated for their antimalarial activity against the chloroquine-sensitive strain 3D7 and the chloroquine-resistant K1 strain as well as for cytotoxicity against mammalian cell lines. While all three arrays showed good antimalarial activity, only the biaryl-containing subset showed consistently good potency against the drug-resistant K1 strain and good selectivity with regard to mammalian cytotoxicity. Overall, our data indicate that the biaryl-containing series contains promising candidates for further study

Top hits for inhibition of growth of <i>L</i>. <i>mexicana</i> amastigotes.

<p>EC₅₀ values represent the mean ± standard deviation for n = 2 and were calculated from dose-response curves against intracellular amastigotes of <i>L</i>. <i>mexicana</i> and <i>L</i>. <i>donovani</i>, the bloodstream form of <i>T</i>. <i>brucei</i>, and the host macrophage J774A.1. TI was calculated as EC₅₀ J774A.1/EC₅₀ <i>L</i>. <i>mexicana</i> amastigotes. None of the nine compounds inhibited proliferation of normal fibroblasts (BJ cells) at 20 μM. *Exact compound has been previously reported as exhibiting antileishmanial activity. For J774.A5 macrophages, compounds were tested up to 10 μM concentration, and those that showed no inhibition of growth were reported to have an EC₅₀ value of >10μM.</p

Plasma protein binding for compounds 4, 5, and the control drug propranolol.

<p>Plasma protein binding for compounds 4, 5, and the control drug propranolol.</p

Discovery of novel, orally bioavailable, antileishmanial compounds using phenotypic screening

<div><p>Leishmaniasis is a parasitic infection that afflicts approximately 12 million people worldwide. There are several limitations to the approved drug therapies for leishmaniasis, including moderate to severe toxicity, growing drug resistance, and the need for extended dosing. Moreover, miltefosine is currently the only orally available drug therapy for this infection. We addressed the pressing need for new therapies by pursuing a two-step phenotypic screen to discover novel, potent, and orally bioavailable antileishmanials. First, we conducted a high-throughput screen (HTS) of roughly 600,000 small molecules for growth inhibition against the promastigote form of the parasite life cycle using the nucleic acid binding dye SYBR Green I. This screen identified approximately 2,700 compounds that inhibited growth by over 65% at a single point concentration of 10 μM. We next used this 2700 compound focused library to identify compounds that were highly potent against the disease-causing intra-macrophage amastigote form and exhibited limited toxicity toward the host macrophages. This two-step screening strategy uncovered nine unique chemical scaffolds within our collection, including two previously described antileishmanials. We further profiled two of the novel compounds for <i>in vitro</i> absorption, distribution, metabolism, excretion, and <i>in vivo</i> pharmacokinetics. Both compounds proved orally bioavailable, affording plasma exposures above the half-maximal effective concentration (EC₅₀) concentration for at least 12 hours. Both compounds were efficacious when administered orally in a murine model of cutaneous leishmaniasis. One of the two compounds exerted potent activity against trypanosomes, which are kinetoplastid parasites related to <i>Leishmania</i> species. Therefore, this compound could help control multiple parasitic diseases. The promising pharmacokinetic profile and significant <i>in vivo</i> efficacy observed from our HTS hits highlight the utility of our two-step phenotypic screening strategy and strongly suggest that medicinal chemistry optimization of these newly identified scaffolds will lead to promising candidates for an orally available anti-parasitic drug.</p></div

Screening flow chart and high throughput primary screen of promastigotes.

<p><b>a.</b> Schematic of the high-throughput screening workflow. SP refers to single point, or single concentration, and DR represents dose-response. The cutoff value of 65% inhibition in the first step was chosen based in ROC analysis, the cutoff of 2 μM and TI > 5 in step 2 was somewhat arbitrary but produced a reasonable number of hits for subsequent analysis, and the cutoff of 1 μM and TI > 10 for the final step is consistent with recommendations for lead identification for leishmaniasis [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006157#pntd.0006157.ref012" target="_blank">12</a>]. <b>b.</b> Scatter plot of primary screen data shown as normalized percent growth inhibition. Each dot represents the activity of one compound. Negative controls (DMSO treated) are in red, positive controls (pentamidine treated) are in green, test compounds are in blue (hits) or black (non-hits). The orange and purple horizontal lines indicate the 95% and 99% quantiles of activity respectively. <b>c.</b> Primary screen quality control: Z-prime value per assay plate screened, lower outlier bound (in purple), yellow lines separate screen runs. <b>d.</b> Receiver operating characteristic (ROC) curve (red); the combined ROC set AUC is 0.893. The blue line represents an AUC of 0.5 that is indicative of an assay with random results. TP is true positive, FP is false positive, FN is false negative, and TN is true negative.</p