We recently reported the discovery of small molecules that inhibit the interaction between TR and the CoA SRC2-2.19
The most potent molecules identified by the screen were substituted β-aminophenylketones. An initial SAR for this series was established using the relative response of other β-aminophenylketones present in the screening collection. The relative efficacies at a fixed dose of 30 μM are shown in .
Summary of screened β-aminophenylketone. Inhibition (%) of the interaction between coactivator peptide SRC2-2 and TRβ in the presence of 30 μM compound is depicted in bold.
The screened compounds were divided into four groups on the basis of their chemical structures. Group A combined unsubstituted β-aminophenylketones with various nitrogen substituents. These compounds inhibited the interaction between CoA SRC2-2 and TRβ weakly, with efficacies between 2% and 19%, at a 30 μM concentration. Group B consisted of β-aminophenylketones with small alkoxy/hydroxy and/or halogen substituents in a para position to the ketone functionality. The highest efficacy in this group was 9% at a 30 μM concentration. Little SAR was established in these two groups. In contrast, group C combining β-aminophenylketones with larger hydrophobic substituents in a para position to the ketone showed dramatic SAR. The compounds with 4-ethyl cyclohexyl and n-hexyl substituents exhibited inhibition values of 87% and 97%, respectively, whereas only 9% efficacy was found for the p-ethyl β-aminophenylketone. β-Aminonaphthylketones in group D were more active (26–30% inhibition) at 30 μM than were the β-aminophenylketones in group A (12%). This analysis showed that a hydrophobic moiety at the para position of β-aminophenylketone is an important feature of potent inhibitors of the TR-CoA interaction.
To address the importance of nitrogen substituents, analogues of group C, β-aminophenylketones bearing n
-hexyl substituents at the para
position of the aromatic ring, were synthesized (). 1-Phenylhexane reacted with acryloyl chloride in the presence of aluminum chloride, giving compounds 1
in a ratio of 8:2 after acidic work-up.28
Treatment of this mixture with secondary amines afforded a focus library of β-aminophenylketones in 85–97% yield ().
Synthesis of β-aminophenylketones 3a–k.a
Table 1 Summary of β-aminophenylketones 3a–k data: Yield, inhibition (IC50) of the interaction between coregulatory peptide SRC2-2 and TRα/TRβ, viability (LD50) of U2OS and ARO cells in the presence of compound, solubility in PBS (more ...)
Four major factors come into play in the optimization of compounds for use in biochemical and cellular assays: potency and efficacy against the target, cytotoxicity and other off-target effects, solubility, and cellular permeability. Proper optimization requires seeking the best balance between these factors. The potency and efficacy of the β-aminophenylketones synthesized were investigated using a competitive fluorescence polarization assay. Labeled SRC2-2 peptide, TR binding domain (TR-LBD) protein, and thyroid hormone T3 were incubated with varying concentrations of the β-aminophenylketones 3a–k
for 3 h.29
The resulting measured fluorescence polarization reflected the ratio of bound to unbound CoA. Two TR isoforms were investigated: TRα and TRβ. Additionally, these compounds were incubated in various concentrations with cultured human osteosarcoma epithelial cells (U2OS) and human anaplastic thyroid cancer cells (ARO) to determine cell viability after 48 h of exposure. Bone cancer cell lines have a minimal expression of TR, whereas expression of TRβ has been reported for the thyroid cancer cell line ARO.30, 31
A commercially available luminescence cell viability assay, CellTiter-Glo (Promega Corp.) was used to quantify ATP in viable cells. The solubility of each β-aminophenylketone in PBS buffer containing 5% DMSO was determined by measuring the absorption at 280 nm after filtration of the saturated solution. The condition used (PBS containing 5% DMSO) reflected the liquid conditions of the fluorescence polarization assay. Finally, we measured the permeability using a parallel artificial membrane permeation assay (PAMPA). The partition of the β-aminophenylketones between a donor well and acceptor well separated by a lipid layer was measured by UV absorption. The assay was carried out at pH 7.4, imitating absorbance in a cellular system. The results are summarized in .
All β-aminophenylketones 3a–k were able to inhibit the interaction between the CoA and TR in a low micromolar potency range. Between the TR isoforms, there was a roughly twofold selectivity toward TRβ. In a cell-based assay, these compounds were generally more toxic to the hormone-responsive thyroid cancer cell line ARO than to the T3-insensitive U2OS cells. LD50 ranges were 2.5–25.6 μM for ARO and 13.2–38.4 μM for U2OS, except for compound 3k, which was significantly less toxic (, entry 11). The solubility of these compounds depended on the nitrogen substituents and varied between 6 μM and 442 μM in PBS containing 5% DMSO. β-Aminophenylketones bearing small nitrogen alkyl substituents, such as 3d, 3h, and 3k, were the most soluble compounds (, entries 4, 8, and 11). Although solubility is a basic requirement for permeability, we observed different Pe values among the very soluble compounds 3d, 3h, and 3k. In contrast to 3d and 3h, which showed high diffusion rates, 3k poorly penetrated the lipid layer. β-Aminophenylketones 3b, 3e, and 3g, which showed moderate solubilities (97–152 μM in PBS with DMSO), had relatively high permeabilities (965–1957 × 10−6 cm/s) (, entries 2, 5, and 7).
Previously, we proposed that the β-aminophenylketones function as prodrugs for the true active species, the unsaturated ketones.19
This is supported by the fact that the ability to bind to TR is very similar for all described β-aminophenylketones because all these compounds afford the same elimination product, 1-(4-hexylphenyl)prop-2-en-1-one, regardless of their amino moieties (, entry 1). To better understand the importance of the electrophilic character of the inhibitors, we investigated various aromatic unsaturated ketones. 1-Phenylhexane was reacted with acid chlorides or anhydrides under Friedel-Crafts reaction conditions to obtain the corresponding ketones ().
Table 2 Summary of enones 1 and 4a–e and allylic alcohols 5b–e: Yield, IC50 values of the inhibition between coregulatory peptide SRC2-2 and TRα/TRβ, viability (LD50) of U2OS and ARO cells in the presence of compound, solubility (more ...)
Synthesis of ketones 1, 4a, and 4g.a
Compounds 1, 4a
, and 4g
were obtained in moderate yields (45–85%) (, entries 1, 2, and 8). The saturated ketone 4a
was synthesized as a nonelectrophilic control compound for the TR-CoA competition assay. The use of substituted acryloyl chloride derivatives under Friedel-Crafts reaction conditions resulted in complex reaction mixtures. An alternative two-step synthesis involving a halogen-exchange reaction of 4-bromo-heptylbenzene with n
-butyllithium at −78°C followed by addition of substituted acroleins was used to obtain the corresponding allylic alcohols 5b–f.
The oxidation to obtain the corresponding unsaturated ketones 4b–f
was carried out using 4-methylmorpholine N-oxide in the presence tetrapropylammonium perruthenate.32
The resulting compounds were studied using the methods outlined above, and the results are summarized in .
The inhibition of the interaction between TR and CoA SRC2-2 depends strongly on the substitution pattern of the unsaturated ketones. Unsubstituted ketone 1 was identified as the most potent compound with the greatest ability to select between TRα and TRα. The IC50 values were 1.5 μM for TRβ and 28.1 μM for TRα (, entry 1). Derivatives of 1 bearing a methyl substituent in the α or β position (4b and 4c) or a carboxy substituent in the β position (4a) were less active (, entries 2–4). The dimethyl- and phenyl-substituted unsaturated ketones 4d, 4e, and 4f could not inhibit the interaction between TR and the CoA peptide (, entries 5–7). The saturated non-electrophilic ketone 4g showed no activity (, entry 8). A correlation between in vitro activity and toxicity was observed in ARO and U2OS cells. Active compounds 1 and 4a–c were considerably more cytotoxic than the inactive analogues 4d–g. LD50 values below 11 μM were found for compounds 1 and 4a–c in ARO cells. U2OS cells were less sensitive, especially in the case of 4a–b (, entries 2–3). The solubility of the ketones was limited in PBS containing 5% DMSO except 4a, bearing an acid functionality. All the ketones tested exhibited poor permeability.
We reported previously that aromatic acrylates inhibited the interaction between TR and SRC2-2.19
For this reason, acrylates and acrylamides were explored as a potential scaffolds. Variation of the aromatic ring was the focus of the acrylate library. Thirteen acrylates were synthesized using various phenols, hydroxylcyclohexane, and hydroxyltetrahydronaphthalene. These starting materials were converted into the corresponding acrylates by using acryloyl chloride in presence of triethylamine ().
Synthesis of substituted acrylates 6a–l.a
The unsaturated esters were obtained in 71–94% yield (). Their ability to inhibit the interaction between TR and CoA was measured using an FP assay with TRα and TRβ. The cytotoxicity was determined in U2OS and ARO cells, and the results are summarized in .
Summary of IC50 values of 6a–m: Yields; inhibition of coregulatory peptide SRC2-2 and TRα/TRβ binding; viability (LD50 ) of U2OS and ARO cell in the presence of compounds.
Seven para alkyl substituted acrylates were investigated with carbon chain lengths ranging from C3 to C8 (, entries 1–7). The binding to TR increased with the length of the substituent reaching maximal inhibition (10.5 μM TRβ) for 6f bearing a heptyl substituent (, entry 6). The selectivity ratios (TRα:TRβ) for these acrylates were about 2:1 in favor of TRβ. Compound 6h, bearing a cyclohexyl ring structure element and a tert-pentyl substituent, was less active than 6d, which had a phenyl ring structure element and the same substituent (, entries 4 and 8). No selectivity between TRα and TRβ was observed for 6h. The ortho-substituted benzoate 6k was significantly more potent (IC50 = 8.2 μM) than the corresponding para-substituted benzoates 6i and 6j (, entries 9–11). Compound 6k had the highest TRα:TRβ ratio (5:1) among the compounds in this library. Butyramidophenyl- and 5,6,7,8-tetrahydronaphthyl-substituted acrylates 6l and 6m showed weak activities (, entries 11 and 12). The measurement of the viability of ARO and U2OS cells in the presence of acrylates revealed that the cytotoxicity of the compounds gradually decreased with increasing length of the n-alkyl para substituent, except in the case of 6a (, entries 1–7). In general, ARO cells were more sensitive than U2OS cells to the acrylates, except for compounds 6e–h (, entries 5–8). Interestingly, three of these compounds bear an ester or an amide functionality in the para position (, entries 9, 10, and 12). The solubility and permeability of the acrylates tested was <10 μM and <5 × 10−6 cm/s, respectively (data not shown).
Variation of the α,β unsaturated system was the focus of the acrylamide library. The synthesis involved the reaction between 4-hexylaniline and various substituted acryloyl chlorides or anhydrides ().
Synthesis of substituted acrylamides 7a–l.a
The corresponding products 7a–f were obtained in 78–88% isolated yield. Inhibition constants were determined for both TRα and TRβ. Additionally, we determined toxicity in ARO and U2OS cells, solubility in PBS buffer containing 5% DMSO, and artificial membrane permeability. All these results are summarized in .
Table 4 Summary of acrylamides 7a–g: Yield, IC50 values of the inhibition between coregulatory peptide SRC2-2 and TRα/TRβ, viability (LD50) of U2OS and ARO cells in the presence of compound, solubility in PBS buffer containing 5% DMSO, (more ...)
Among the acrylamides tested, only 7a and 7b inhibited the interaction between TR and SRC2-2 with IC50 values (TRβ) of 17.9 μM and 20.3 μM, respectively (, entries 1 and 2). No TR isoform selectivity was observed. Interestingly, all compounds showed minor toxic effects in ARO cells. Similar results were obtained for U2OS, with the exception of slightly more cytotoxic compounds 7a and 7e (, entries 1 and 4). High solubilities were measured for carboxylic acid-substituted acrylamides 7b, 7f, and 7g (, entries 2, 6, and 7). These compounds exhibit limited permeabilities in the PAMPA assay (Pe < 60 × 10−6 cm/s. In contrast, good diffusion rates were observed for the less soluble compounds 7a and 7c (, entries 1 and 3).
Finally, several other potential electrophilic moieties were explored. First, propiolic acid derivatives 8a and 8b were obtained using diisopropylcarbodiimide as a coupling reagent, starting from the corresponding phenol or aniline ().
The halo-ketones 8c–e
were synthesized under Friedel-Crafts conditions (). Fluoroacetyl chloride33
was obtained from the corresponding sodium salt. The keto-epoxide 8f
was synthesized for the corresponding α,β-unsaturated ketone 1
obtained after HBr elimination of the β-bromo ketone 8e
using DBU as a base (). The yields, inhibition constants for both TRα and TRβ, cytotoxicity in ARO and U2OS cells, solubility in PBS with 5% DMSO, and permeability (PAMPA) are summarized in .
Table 5 Summary of electrophilic compounds 8a–f: Yield, IC50 values of the inhibition between coregulatory peptide SRC2-2 and TRα/TRβ, viability (LD50) of U2OS and ARO cells in the presence of compound, solubility in PBS buffer containing (more ...)
Propiolic acid derivatives 8a and 8b were able to inhibit the interaction between TR and SCR2-2 with IC50 values of 35.1 μM and 6.2 μM for TRβ (, entries 1 and 2). A general IC50 ratio of 1:2 (TRβ:TRβ) was observed for all active electrophiles. Two α-halogen ketones, 8c and 8d, were investigated. The α-chloro ketone 8c was able to inhibit the interaction between SRC2-2 and TR, but α-fluoro ketone 8d was not active (, entries 3 and 4). The β-bromo ketone 8e was the most potent compound in this group (, entry 5) but had poorer selectivity, exhibiting IC50 values of 4.2 μM and 3.0 μM for TRα and TRβ, respectively. In contrast, the γ-bromo ketone 8f was not active. The racemic epoxy-ketone 8f inhibited the interaction between TR and the CoA SRC2-2 with an IC50 value of 47.7 μM for TRα and 23.1 μM for TRβ (Table 6, entry 7). The viability of ARO and U2OS cells in the presence of these electrophiles was limited. Major differences in sensitivities between the two cell lines were measured for α-fluoro ketone 8d and epoxy ketone 8g (, entries 4 and 7). Although most of the electrophiles were not very soluble in PBS buffer containing 5% DMSO, we observed high permeabilities for 8c–e and 8g.
The relationships between biochemical activities, cellular activities, and pharmacological properties are important in the identification of potential drug candidates. To assess such relationships, scatter plots based on measured data were constructed. First, we visualized the relationship between their physical properties, solubility and permeability ().
Scatter plot of solubility and permeability data for all compounds except acrylates.a
The scatter plot revealed a significant relationship between both properties. The linear regression resulted in a positive slope of 0.11, indicating a positive correlation between permeability and solubility. In contrast to the general trend, we observed low permeability for highly soluble carboxylic acids (4a, 7b, 7f, and 7g) and the secondary amine 3k. The compounds with the highest solubility and permeability were β-aminophenylketones 3d and 3h. Compound 3h was selected for further stability studies. Over a period of 24 h, we monitored the stability of an aqueous and saline solution (150 mM NaCl) of 3h at pH 7 using liquid chromatography-mass spectrometry (LCMS). The results are given in .
LCMS-PDA analysis of 3h in water, saline (150 mM NaCl), and human blood plasma (all pH 7).a
No degradation of 3h was detected in water or saline at pH 7 during this time course. Additionally, we performed a stability study in human blood plasma using 5 μM 3h. Degradation with a half-life of 1.4–2.4 h was determined during a 24-h experiment.
The in vitro cytotoxicity data revealed a good correlation between the LD50 in ARO cells and U2OS cells ().
Scatter plot of LD50 values measured in U2OS and ARO cells for all compounds.a
The slope (0.52) indicated a general twofold higher cytotoxicity in thyroid cancer cells (ARO) than in bone cancer cells (U2OS). Compounds 4a, 6i, 6l, and 8d were highly selective between the two cell lines (, data point in parentheses). ARO:U2OS LD50 ratios up to 1:28 were observed. The selectivity was not scaffold related.
Given the general utility of the wide range of the electrophiles and the variance in the specific nature of inhibition, we wished to understand the relationship between three-dimensional structure and activity. Pharmacophore modeling is an important tool for elucidating a general molecular structure underlying the active compounds in a bioactive screen. The study was carried out using Molecular Operating Environment (MOE) software.34
The different compound libraries we analyzed separately anticipating the same binding mode for all compounds. The results are illustrated in .
Pharmacophore elucidation using MOE software.a
The pharmacophore models for all three libraries (enones A, acrylates B, and acrylamides C) were very similar (). All had an electrophilic head group (Hyd/Uns), an aromatic core structure (Hyd/Aro), and a hydrophobic center (Hyd/Ali) represented by green mesh balls. The distance between Hyd/Uns and Hyd/Aro differed for A, B, and C between 4.65 and 5.69
, depending on the linkage C(O), OC(O), or NHC(O). The distance between Hyd/Ali and Hyd/Aro varied between 3.89 and 5.32
. The analysis of a possible electron acceptor interaction revealed an electron acceptor site (Acc) for all pharmacophores. The average distance between Hyd/Uns and Acc for all models was 4.31 ± 0.37
, and the distance between Hyd/Aro and Acc was 4.39 ± 0.25
To elucidate the mode of binding of these molecules to the TR-CoA binding site, we modeled compound 1
with a covalent bond to C309 (). Point mutation analysis showed significantly less binding affinity for the TR C309A mutant than for the wild type, as determined by two independent assays.19
We observed a ridged binding site for the electrophilic head group and a possible activation of a carbonyl functionality by K306. The distance between the carbonyl oxygen and the K306 amide hydrogen was 3.3
. A relatively spacious hydrophobic groove surrounded the aromatic core structure, and a hydrophobic curved surface accommodated the alkyl substituent.
Modeling study of the bonding of compound 1 to the TR coactivator binding pocket.a
The AF-2 domain of TRβ has a unique architecture in comparison the other NR -- containing three cysteine residues (Cys309, Cys298, and Cys294) not present in any other NR AF-2. Two of the sulfhydryl functionalities of these unique cysteines (Cys309 and Cys298) are exposed to the solvent. To confirm that this aspect of the TRβ structure affords selectivity of the inhibitors for TRβ, we carried out fluorescence polarization competition assays employing the following nuclear receptors: the androgen receptor, the estrogen receptor α, and the peroxisome proliferator activated receptor γ. No inhibition of coregulator recruitment to any of these NR could be detected after 3h of exposure to 3h at concentrations up to 100 μM (data not shown). Thus, this class of inhibitors appears to be intrinsically selective for TR relative to other NR due to the presence of unique cysteine residues in the AF-2 binding site.