Improvement of pharmacological properties of irreversible thyroid receptor coactivator binding inhibitors

Article abstract of DOI:10.1021/jm9002704

We have previously reported the discovery and preliminary structure activity relationships of a series of β-aminoketones that disrupt the binding of coactivators to TR. However, the most active compounds had moderate inhibitory potency and relatively high cytotoxicity, resulting in narrow therapeutic index. Additionally, preliminary evaluation of in vivo toxicology revealed a significant dose related cardiotoxicity. Here we describe the improvement of pharmacological properties of thyroid hormone receptor coactivator binding inhibitors. A comprehensive survey of the effects of substitutents in key areas of the molecule was carried out based on mechanistic insight from the earlier report. This study revealed that both electron withdrawing and hydrophobic substituents on the aromatic ring led to higher potency. On the other hand, moving from an alkyl to a sulfonyl alkyl side chain led to reduced cytotoxicity. Finally, utilization of amine moieties having low pK_a's resulted in lowered ion channel activity without any loss of pharmacological activity.

Full text of DOI:10.1021/jm9002704

3892
J. Med. Chem. 2009, 52, 3892–3901
Improvement of Pharmacological Properties of Irreversible Thyroid Receptor Coactivator
Binding Inhibitors
Jong Yeon Hwang,† Leggy A. Arnold,† Fangyi Zhu,† Aaron Kosinski,† Thomas J. Mangano,‡ Vincent Setola,‡ Bryan L. Roth,‡
and R. Kiplin Guy†,*
St. Jude Children’s Hospital, Department of Chemical Biology and Therapeutics, 262 Danny Thomas Place, Memphis, Tennessee 38105-3678
ReceiVed March 3, 2009
We have previously reported the discovery and preliminary structure activity relationships of a series of
ꢀ-aminoketones that disrupt the binding of coactivators to TR. However, the most active compounds had
moderate inhibitory potency and relatively high cytotoxicity, resulting in narrow therapeutic index.
Additionally, preliminary evaluation of in vivo toxicology revealed a significant dose related cardiotoxicity.
Here we describe the improvement of pharmacological properties of thyroid hormone receptor coactivator
binding inhibitors. A comprehensive survey of the effects of substitutents in key areas of the molecule was
carried out based on mechanistic insight from the earlier report. This study revealed that both electron
withdrawing and hydrophobic substituents on the aromatic ring led to higher potency. On the other hand,
moving from an alkyl to a sulfonyl alkyl side chain led to reduced cytotoxicity. Finally, utilization of amine
moieties having low pKa’s resulted in lowered ion channel activity without any loss of pharmacological
activity.
Introduction
T3 have been reported, no antagonist drugs are yet approved to
treat thyroid receptor malfunctions.9-12
The thyroid hormone receptors (TRsa), belonging to the
superfamily of nuclear receptors (NRs), regulate development,
growth, and metabolism.1-3 The thyroid hormone (T3) induces
the majority of transcriptional responses mediated by TR’s in
vivo.4 The TR’s have two isoforms, TRR and TRꢀ, encoded
by two genes, with each isoform having two distinct subtypes
due to alternative splicing.5 Although these isoforms are widely
expressed, there are distinct patterns of expression that vary with
tissue and developmental stage. TRR or TRꢀ knockout mice
display unique phenotypes, suggesting that the different TR
isoforms have unique regulatory roles.4,6
We have previously reported the discovery and preliminary
structure-activity relationships of a series of ꢀ-aminoketones
that disrupt the binding of coactivators to TR without affecting
T3 binding.13-15 These time-dependent irreversible inhibitors
were proposed to work by in situ generation of an enone
followed by a reaction between the electrophilic enone and a
nucleophilic cysteine in the coactivator binding pocket. The
recently reported X-ray structure of TRꢀ bound to an enone
derived by elimination from one of these aminoketones supports
this hypothesis.16
TRꢀ is unique among the nuclear receptors in having three
cysteine residues (C294, C298, and C309) located in or near
the coactivator binding site. Active site mutagenesis and mass
spectroscopy revealed that the enones derived from this series
of ꢀ-aminoketones selectively attack C298, even in the presence
of 10 mM ꢀ-mercaptoethanol. A preliminary SAR study of
ꢀ-aminoketones and the various electrophilic compounds con-
firmed important features of these selective small molecule
inhibitors.15 However, the most active compounds emerging for
this study had apparent IC50’s in the low micromolar range with
relatively high cytotoxicity. Additionally, preliminary evaluation
of in vivo toxicology revealed a significant dose-related car-
diotoxicity, which is consistent with the original use of these
ꢀ-aminoketones as sodium channel targeted local anesthetics.17,18
Reduction of cytotoxicity, lowering of ion channel activity, and
potentially improvement of potency, as long as selectivity could
be maintained, would increase the therapeutic window and
enable us to utilize these compounds in vivo to study the role
of coactivator recruitment in thyroid hormone endocrine action.
TR has three functional domains: a N-terminal ligand
independent transcription activation domain (AF-1), central
DNA binding domain (DBD), and a carboxy terminal ligand
binding domain (LBD) containing a thyroid hormone inducible
coactivator binding domain (AF-2).7 Upon binding of thyroid
hormone, TR undergoes a conformational change, releasing
corepressor proteins and recruiting coactivator proteins, such
as steroid receptor coactivators (SRC’s), to regulate gene
transcription.8 Although two structurally distinct antagonists of
* To whom correspondence should be addressed. Phone: (901) 595-5714.
Fax: (901) 595-5715. E-mail: kip.guy@stjude.org.
†
Department of Chemical Biology and Therapeutics, St. Jude Children’s
Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105.
‡
Department of Pharmacology, University of North Carolina, 8032
Burnett-Womack Building, Chapel Hill, NC 27514.
a Abbreviations: AF-1, activation domain; ARO, human anaplastic
thyroid cell line; ATCC, American Type Culture Collection; ATP, adenosine
triphosphate; CoA, coactivator; CoR, coregulator; DBD, DNA-binding
domain; DMSO, dimethyl sulfoxide; LBD, ligand-binding domain; LCMS,
liquid chromatography-mass spectrometry; MOE, molecular operating
environment software; NCoR, nuclear receptor corepressor; NID, nuclear
receptor interacting domain; NR, nuclear hormone receptor; PAMPA,
parallel artificial membrane permeation assay; PBS, phosphate buffered
saline;PDA,photodiodearray;RXR,retinoidXreceptor;SAR,structure-activity
relationship; SMRT, silencing mediator of retinoic acid; SRC, steroid
receptor coactivator; SRC2-2, steroid receptor coactivator 2 peptide
representing the second NID; TR, thyroid receptor; U2OS, human osteosa-
rcoma epithelial cell line.
Herein, we present the synthesis and characterization of
ꢀ-aminoketones with improved properties with an emphasis on
(i) the orientation of carbonyl group, (ii) substitution of phenyl
core structure, (iii) substitution on R-position of the aminoke-
tone, (iv) alternate ꢀ-amino moieties, and (v) functionality of
alkyl side chain (Figure 1). These chemical features were
optimized in order to provide the best balance between maximal
10.1021/jm9002704 CCC: $40.75  2009 American Chemical Society
Published on Web 05/26/2009

IrreVersible Thyroid Receptor CoA Binding Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3893
reflecting the conditions required for activity in cell-based
assays. Finally, as a marker for cardiac ion channel activity,
each compound was evaluated its effects on the hERG channel
using a potassium-induced cellular depolarization assay.
The Effects of Substitution Patterns of the Aromatic
Ring on Activity. Our original studies showed that the hexyl
side chain could be successfully replaced by substitutents that
were roughly the same size and hydrophobic character. To
simplify exploring the effects of ring substitutions, we replaced
the original hexyl substituent with a hexyloxy group. This
substitution enhanced solubility and suppressed potential meta-
bolicoxidationbycytochrome450satthebenzylicmethylene.26-28
The prior SAR study showed that a para relationship between
the hydrophobic substituent and the aminoketone appeared to
be an essential feature of potent ꢀ-aminophenylketones.15
However, one ortho-substituted compound, hexyl 2-(acryloy-
loxy)benzoate, showed high affinity for TR. To investigate this
SAR relationship more carefully, ꢀ-aminophenylketones with
hexyloxy substituents in ortho, meta, and para positions were
synthesized and characterized as described above. The results
are summarized in Table 1.
Figure 1. Structure of ꢀ-aminoketone 1 and five parts of SAR
modification.
potency, efficacy, and selectivity, minimal cellular toxicity,
favorable physiochemical properties, and minimal activity at
cardiac ion channels.
Results and Discussion
Chemistry. The synthesis of the ꢀ-aminoketone compounds
was accomplished by two different routes (Scheme 1): (a)
Friedel-Crafts acylation or (b) Mannich reaction. Activated
aromatic compounds like 2 were acylated under Friedel-Crafts
conditions resulting in the formation of ortho- and para-
substituted ketones. These reactions proceeded in high yield and
with generally high selectivities depending on the substituent.
In general, analogues of 2 were alkylated and subsequently
reacted with 3-chloropropionyl chloride in the presence of AlCl3
at 0 °C generating ortho- and para-acylated compounds 3 and
4. Subsequent treatment with dimethylamine (2 M in THF)
resulted in the formation of the desired ꢀ-aminoketones 5 and
6 in high yield.
Derivatives of 2 with strong electron withdrawing substituents
(e.g., R ) NO2, CF3, and SO2Me) could not be made using this
general route. Additionally, steric hindrance slowed reaction
rates for the 3-MeO, 3-MeS, and most disbustituted rings
resulting in low yield of acylated products. In these cases, the
desired ꢀ-aminoketones were obtained using a Mannich reac-
tion.19 In general, this three-component reaction utilizes harsh
reaction conditions and suffers from low yields. The application
of microwave assisted heating increased the yields and enabled
us to synthesize ꢀ-aminoketones that were inaccessible using
the Friedel-Crafts acylation route.20,21 Most of the hydroxyl
acetophenones starting materials were commercially available
or were synthesized from the corresponding phenol.22 Heating
these compounds in the microwave at 120 °C in water/
acetonitrile (1:9) in the presence of paraformaldehyde and
dimethylamine hydrochloride salt resulted in the formation of
desired ꢀ-aminoketones in good yields.
Evaluation of ꢀ-Aminoketones. Preliminary evaluation of
all compounds included assessment of biochemical activity and
cytotoxicity. Additionally, the solubility and passive perme-
ability of the small molecules were investigated to support
biochemical and cell-based assay results and to guide the earliest
stages of probe discovery toward tractable compounds.23-25The
ability of the synthesized ꢀ-aminoketones to bind to both TR
isoforms and inhibit the interaction with coregulator peptide
SRC2-2 (second NID box of SRC2) was evaluated using a
previously reported competitive fluorescence polarization (FP)
assay.13 The cytotoxicity of ꢀ-aminoketones was evaluated in
human liver cancer cells (HepG2) using a commercially
available luminescence cell viability assay (CellTiter-Glo,
Promega Corp.). The solubility of each ꢀ-aminoketone was
determined in PBS buffer containing 5% DMSO, reflecting the
conditions of the fluorescence polarization assay. The perme-
ability of each compound across a membrane bilayer was
measured using a parallel artificial membrane permeation assay
(PAMPA). The assay was carried out at neutral pH (pH ) 7.4),
The meta-substituted ꢀ-aminophenylketone 10 showed the
highest potency (IC50 ) 7.1 µM) in the SRC binding assay with
TRꢀ, while the para-substituted 9 is less potent, with an IC50
)
16.5 µM. ortho-Substituted compound 11 showed no measurable
activity. We examined other ortho-substituted compounds, with
ethyl, methoxy, chloro, bromo, and iodo substituents, but none
had measurable activity in either biochemical or cytotoxicity
assays (data not shown). Although the meta-substituted ꢀ-ami-
nophenylketone was more potent, careful exploration of its SAR
was hindered by synthetic difficulties in producing a diverse
set of analogues. Therefore, we performed the SAR analysis of
the ring substitutions using para-substituted compounds.
We explored the effects of other substitutents on the central
aromatic ring using a modified Topliss scheme in which all
substitutents were simultaneously explored and the substituent
set expanded according to Hansch to give an understanding of
the interlocking effects of sterics, hydrophobics, and electrostatics.29,30
The substituent set used is shown in Figure 2. Suitable starting
materials were converted into the corresponding ꢀ-aminophe-
nylketone using a Friedel-Crafts acylation (A) or Mannich
reaction (B). Details of the synthetic procedures and supporting
data are provided in the Supporting Information. All compounds
were evaluated in the assays discussed above using independent,
replicate dose-response methods to generate IC50 or EC50
constants that are summarized as a heat map in Figure 3
(tabulated data with errors can be found in the Supporting
Information).
Most of the substituted ꢀ-aminophenylketones investigated
were more potent than unsubstituted compound 9 except
compounds 12{4}, 12{7}, and 12{28}. The 3-methoxy substi-
tuted compound 12{4} showed reduced potency, while both
3-dimethylamino substituted 12{7} and 2-benzyl substituted
12{28} failed to inhibit the TRꢀ-coactivator interaction.
ꢀ-Aminophenylketones bearing strong electron withdrawing
substituents including monohalogens 12{9}-12{15}, 3-sulfone
12{17}, nitro 12{20}, and trifluoromethyls 12{18} and 12{19}
showed affinities in the low-micromolar range (1-5 µM).
However, the 2-methylsulfone 12{16} gave low inhibitory
potency (8 µM) even though it has strong electron withdrawing
group. This implies that a hydrophobic electron withdrawing
group gives better potency. More hydrophobic substitutents such
as alkyl groups 12{1}-12{3}, 12{8}, and thioether 12 {5-6}
gave slightly improved potency (4-10 µM) compared to

3894 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Scheme 1. General Synthetic Routes for ꢀ-Aminoketonesa
Hwang et al.
a Reagents and conditions: (a) hexyl bromide, K2CO3, DMF, 80-90 °C, 12 h; (b) 3-chloropropionyl chloride, AlCl3, DCM, 0 °C; (c) 10 eq dimethylamine
(2 M in THF), THF, rt, 30 min; (f) paraformaldehyde, Me2NH·HCl, H2O/MeCN (1/9, v/v), MW, 120 °C, 2 h.
Table 1. ꢀ-Aminophenylketone Regioisomers
ꢀ-aminophenylketones exhibited good permeability at pH ) 7.4
(Supporting Information, Table 1).
Replacement of the Phenyl Ring. A general trend correlating
increased core hydrophobicity with increased potency suggested
exploring alternate core scaffolds. From the initial screening
data, we observed an increase of efficacy inhibition of SRC2-2
recruitment by TRꢀ from 12% to 33% at a fixed 10 µM dose
with exchange of the phenyl core structure by a naphthalene
structure.15 On the basis of this observation, several alternate
cored ꢀ-aminoketones were synthesized and subjected to the
same array of assays discussed earlier. The results are sum-
marized in Table 2.
ꢀ-Aminonaphthylketone 13{1} showed an improved inhibi-
tory potency in the SRC binding competition for TRꢀ (IC50
)
4 µM) but was more toxic in comparison with ꢀ-aminophe-
nylketone 9. In addition, compound 13{1} was less soluble than
compound 9, although it retained high permeability (Supporting
Information, Table 2). Biphenyl ꢀ-aminoketone 13{2} and 1,6-
substituted ꢀ-aminonaphthylketone 13{3} were both failed to
inhibit the interaction between TRꢀ and SRC2-2 under the assay
conditions. These studies indicated that no significant advantage
would arise from using a larger, polyaromatic, and core
structure.
a Values are the mean of three independent experiments in triplicate.
b Values are means of two independent experiments in triplicate. The general
error limits are (5%.
compound 9. This implies that electron donation generally is
more driving than hydrophobicity. For disubstituted compounds,
compounds 12{22}-12{26} showed potent inhibition (0.8-1.8
µM) in comparison with their monosubstituted counterparts
regardless of their electronic properties. Cell viability in the
presence of ꢀ-aminoketones was also investigated. Most of
compounds exhibited weak inhibition of cell proliferation (EC50
> 20 µM). Notable exceptions were the 3,5-dimethyl 12{22},
3-trifluoromethyl 12{19}, 2-tert-butyl 12{8}, and 2-phenyl
12{27} compounds. The halogen substituted compounds were
Substitution of r-Carbon. Initial evaluation of lead com-
pounds had indicated that they were ion channel active
cardiotoxins that exhibited QT prolongation. For this reason, it
was desired to lower interactions with the hERG and other ion
channels. Mitigation of ion channel effects of substituted amines
generally follows two strategies: increased steric bulk around
the amine and/or modulation of the basicity of the amine. To
examine the effect of R-carbon substituents of ꢀ-aminophe-
nylketones, we synthesized the compounds depicted in Table
3. All compounds were analyzed by the same array of assays
discussed earlier.
all weakly growth inhibitory among active compounds (IC50
>
10 µM) with the exception of 2,6-dichloro compound 12{26}.
The therapeutic index shows that 2,3- and 2,5-dichloro com-
pounds 12{24} and 12{25} were not only the most potent
compound but also the least toxic compounds. Compounds
12{24} and 12{25} also had acceptable solubility and perme-
ability (Supporting Information, Table 1, 10 µM and 600 ×
10-6 cm/s for 12{24} and 20 µM and 1040 × 10-6 cm/s for
12{25}). Generally solubility of this series of compounds was
good (between 2-185 µM, Supporting Information, Table 1).
However, ꢀ-aminophenylketones with 2-tert-butyl 12{8}, 2-tri-
fluoromethyl 12{18}, 2-phenyl 12{27}, and 2-benzyl 12{28}
substituents were poorly soluble (solubility <10 µM). Most of
R-Substituted compounds 14{1}-14{3} (tested as racemates)
were not able to inhibit the interaction between TR and SRC2-
2. In contrast, cyclic compounds 14{4}-14{6} were highly
potent, exhibiting IC50 values of 1-2 µM. However, they carried
the liability of an increased growth inhibitory potency for the
HepG2 cell line, giving poor therapeutic index in comparison
with compound 9. These studies indicated that increased steric
bulk and/or conformational restriction of the ꢀ-aminoketone
moiety were both deleterious with respect to functional inhibition

IrreVersible Thyroid Receptor CoA Binding Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3895
Figure 2. Diversity of aromatic substitutents.
Figure 3. Activity summary for ring-substituted ꢀ-aminophenylketones: Heat map shows IC50 values for the inhibition of coregulatory peptide
binding (SRC2-2) by TRꢀ, EC50 values for cellular proliferation inhibition (HepG2), and calculated therapeutic index (TI, ratio of two prior values).
Compound order is sorted by therapeutic index. (a) Values are the mean of three independent experiments in triplicate. (b) Values are means of two
independent experiments in triplicate. (c) Values are EC50 over IC50.
of thyroid signaling due to loss of efficacy or increased growth
inhibition, respectively.
The Role of Amine Substitutions. The majority of ion-
Table 2. Summary of ꢀ-Aminoarylketones with Alternate Aromatic
Rings
channel modulators bear amine functionalities with pKa values
between 8.0-9.0, ensuring high permeability of the uncharged
amine and binding of the protonated amine at the receptor site.
A strategy to develop compounds that are active toward their
primary target but not binding hERG has been to reduce the
pKa value of the amine. The identification of ꢀ-aminoketones
with low amine pKa’s that still inhibited cofactor binding to
TR in the initial hit exploration indicated that this strategy might
afford compounds with better in vivo toxicology profiles. Prior
to careful exploration of amine substitutions with respect to ion
channel effects, a more broad structure-activity relationship
needed to be established. TR inhibition by ꢀ-aminoketones is
dependent on elimination rate of the pro-drug into the active
enone. In Mannich bases, this elimination rate is pKa dependent.
To establish the range of this component of the SAR,
ꢀ-aminophenylketones with pKa values between 3 to 10 were
synthesized and tested. (Figure 4) The compounds were
synthesized using a Friedel-Crafts reaction to afford the
haloketone, followed by treatment with different amines in the
a Values are the mean of three independent experiments in triplicate.
b Values are means of two independent experiments in triplicate. The general
error limits are ( 5%.

3896 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Table 3. Summary of R-Substituted ꢀ-Aminophenylketones
Hwang et al.
Table 4. Summary of ꢀ-Aminophenylketones Bearing Different
Nitrogen Substituents
SRC binding
inhibition,
TRꢀ (IC50, µM)a (EC50, µM)
cell viability hERG inhibition
compd
no.
HepG 2
(% terfenadine calculated
b
effect)c
pKa
9
16.5 ( 1.5
65 ( 19
61 ( 9
9.12
15{1}
15{2}
15{3}
15{4}
15{5}
15{6}
15{7}
15{8}
15{9}
15{10}
15{11}
15{12}
2.7 ( 0.4
9.1 ( 1.2
18.9 ( 6.8
22.0 ( 2.4
5.6 ( 0.9
3.8 ( 0.3
4.1 ( 0.5
>50
48 ( 20
66 ( 34
59 ( 17
67 ( 22
54 ( 13
81 ( 32
56 ( 19
>130
48 ( 15
75 ( 2
69 ( 9
35 ( 4
60 ( 7
29 ( 12
32 ( 13
8 ( 11
22 ( 1
18 ( 3
32 ( 3
-11 ( 6
9.95
9.61
9.28
6.45
8.1
6.13
6.5
5.26
4.38
3.67
d
>50
>130
>50
>130
54 ( 31
>130
>50
>50
d
a Values are the mean of three independent experiments in triplicate.
b Values are means of two independent experiments in triplicate. The general
error limits are (5%. c Values are the mean of two independent experiments
in quadruplicate. Depolarization inhibition data in HEK293-hERG cells are
expressed as % inhibition of KCl-stimulated depolarization as measured
using a Vm-sensitive dye (Molecular Devices), scaled as follows: %
inhibition for compound ) 100(value for test compound - value for
negative control)/(value for 10 uM terfenadine - value for negative control).
d Not available.
a Values are the mean of three independent experiments in triplicate.
b Values are means of two independent experiments in triplicate. The general
error limits are ( 5%.
readily generate the active enone form of the compounds. The
piperazine and morpholine analogues (15{5}-15{7}), with
intermediate pKa values (8.1, 6.13, and 6.45, respectively),
showed relatively potent inhibition (3.8-5.6 µM). We hypoth-
esize that this is due to a ꢀ-atom effect raising the pKa. The
ꢀ-aminophenylketones 15{11} and 15{12}, bearing imidazole
and succinimide substituents, respectively, were inactive. Unlike
activity against the target, the cytotoxicity of this series of
compounds showed little change (48-81 µM) regardless of
amino group. This series of compounds was tested to see if
KCl-stimulated depolarization of HEK293-hERG cells was
affected by the amino group. Most of the compounds active
against TR also inhibited KCl-stimulated depolarization of
HEK293-hERG cells in a manner similar to that of a prototypical
hERG blocker (terfenadine). Among the active compounds,
aziridine 15{4}, phenylpiperazine 15{6}, and morpholine
compound 15{7} showed the lowest inhibition of KCl-
stimulated depolarization of HEK293-hERG cells (35%, 29%,
and 32% of the inhibitory effect of terfenadine, respectively).
In contrast, all of the inactive compounds showed no or poor
blockade of KCl-stimulated depolarization of HEK293-hERG
depolarization. Examining the relationship between pKa and
antagonism of KCl-stimulated depolarization of HEK293-hERG
cells (Figure 5) revealed that increasing basicity correlates fairly
strongly with inhibition of depolarization responses in HEK293-
hERG cells (R2 ) 0.8, P ) 0.0002). The two most notable
outliers were 15{1}and 15{8}, which carry significantly more
bulky substitutents around the amine.
Figure 4. Amine substituents examined.
presence of DBU. The pKa values of each compound were
calculated using the imbedded calculator in Pipeline Pilot
(Accelrys Software Inc.). The compounds were evaluated with
the same array of assays described earlier. In addition, the
compounds’ abilities to block KCl-stimulated depolarization of
HEK293-hERG cells (at 10 µM concentration) was assessed
using a Vm-sensitive fluorescent dye-based assay (Molecular
Devices). A comprehensive description of the assay (performed
by the National Institute of Mental Health Psychoactive Drug
Screening Program), including a detailed protocol, sample data,
and analysis procedures, is available online (http://pubchem.ncbi.
nlm.nih.gov/assay/assay.cgi?aid)376&loc)ea_ras or http://pdsp.
med.unc.edu/UNC-CH%20Protocol%20Book.pdf). The results
are summarized in Table 4.
All ꢀ-aminophenylketones bearing alkyl amines that were
tested inhibited the interaction between TR and SCR2-2 (Table
4, 15{1}-15{7}). In contrast, ꢀ-aminophenylketones bearing
anilines were inactive (Table 4, 15{8}-15{10}). These com-
pounds possess amines with pKa values lower than 5.3 that are
unlikely to be protonated at pH 7.4 and thus probably do not
Functionality of Hydrophobic Side Chain. In our prelimi-
nary SAR studies of ꢀ-aminophenylketones, we established that
para-alkyl substitutents between 6 and 8 carbon atoms were
essential for highly active inhibitors of the TR-coactivator
interaction.15 Such freely rotable, highly hydrophobic side chains
carried a correlated liability of poor solubility. To balance these
issues, the incorporation of more polar groups in the side chain,
incorporated relatively closely to the ring, was explored. All

IrreVersible Thyroid Receptor CoA Binding Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3897
16{4} showed moderate toxicity (40.7 uM). The amide 16{6}
and reverse amide 16{7} also showed reasonable inhibitory
potency for TRꢀ (8.1 and 12.4 µM, respectively) with low
toxicity. However, sulfonamide 16{8} and the urea 16{9} were
inactive in the SRC2-2 binding assay. Aside from placement
of polar groups being highly important, no clear structure-activity
relationships were evident for this part of the molecule. This
compound series was also tested for inhibition of KCl-stimulated
depolarization of HEK293-hERG cells. Most of compounds
showed moderate inhibitory activity (3-68%), consistent with
the other substitution patterns of the aryl ring and aminoketone.
Among the active compounds, sulfone 16{5} and amides 16{6}
and 16{7} showed fairly weak inhibition of KCl-stimulated
depolarization of HEK293-hERG. All of active compounds had
good solubility (58-224.5 µM) and permeability (565-1926
× 10-6 cm/s). This study suggested that utilization of a
heteroatom attachment for this side chain might prove fruitful
as a strategy to circumvent both toxicity and inhibition of KCl-
stimulated depolarization of HEK293-hERG cells without
substantial loss of activity.
Figure 5. Relationship between the pKa of the amino group of
ꢀ-aminophenylketones and inhibition of KCl-stimulated depolarization
of HEK293-hERG cells.
Table 5. Summary the Activities of Hydrophobic Side Chain Modified
ꢀ-Aminophenylketones
Selectivity between the Isoforms TRr and TRꢀ. In addition
to testing all compounds against the TRꢀ-LBD, all compounds
were also tested against TRR to determine if this series could
afford selective antagonists. Most compounds exhibited similar
potency of antagonism toward both TRR and TRꢀ.
Modeling of Physiochemical Behaviors. Having prepared
a range of derivatives that systematically varied steric and
electronic factors, quantitative structure-activity and structure-
property relationships were explored. The importance of genera-
tion of an active conjugated enone in the mechanism of action
implied that Hammett analysis might help in explaining the SAR
of substituted ꢀ-aminoketones. Meanwhile, the importance of
both solubility and permeability in activity indicated that Hansch
constants (π) might be critical in predicting both biochemical
and cellular activity. Compounds outlined in Figure 3 and Table
5 were used for this analysis and the results are presented in
Figure 6.
The IC50 values were plotted against literature σ- and
π-constants of substituents. In general, we observed that electron
withdrawing substituents (positive σ-values) improved the ability
of ꢀ-aminophenylketones to inhibit the interaction between
SRC2-2 and TR, regardless of substitution location, whereas
ꢀ-aminophenylketones with electronic donating groups (positive
σ-values) exhibit weaker activities (Figure 6A). While this
correlation was significant, the trend was not particularly strong
(slope ) 9.6, p ) 0.003). The substituent effect of ꢀ-aminophe-
nylketones with regard to their hydrophobicity was also analyzed
(Figure 6B,C). While no effect was evident with meta substi-
tutents, a relatively strong substituents effect could be found
with ortho-substituted compounds (Figure 6C, slope ) 19.7, p
) 0.002). These studies indicated that the major effector of
potency is the hydrophobicity of the ortho-substituent, relative
to the ketone. Overall, this analysis served to focus future work
on incorporation of ortho hydrophobic, electron withdrawing
(halogens in particular) on the ring.
We hypothesized that this arises from a steric effect that alters
electrophilicity of the eneone. An examination of minimized
structures of unsubstituted compound 9 and 3,5-dimethyl
substituted compound 12{22} by MOE (Forecefield-MMFF94)
showed that compound 9 has small predicted dihedral angle
(0°) and low energy barrier for rotation of the ketone (6 kcal/
mol), while 3,5-disubstituted compound 12{22} has large
predicted dihedral angle (∼90°) and high energy barrier for
rotation (>50 kcal/mol). This distorted dihedral angle disturbs
a Values are the mean of three independent experiments in triplicate.
b Values are means of two independent experiments in triplicate. The general
error limits are (5%. c Values are the mean of two independent experiments
in quadruplicate.
compounds were subjected to the same array of assays discussed
earlier, and the results are summarized in Table 5.
All compounds bearing heteroatoms within the hydrophobic
chain in any position other than immediately adjacent to the
phenyl ring were inactive in TR receptor binding assay (Table
5, 16{1-3}). This is not unexpected given that our cocrystal
structure revealed that this group threads through a narrow
hydrophobic cleft on the protein surface. All of these compounds
were less toxic in comparison with compound 1, although this
might be due to the reduced permeability exhibited by most
(Supporting Information, Table 5). The thioether 16{4} and
sulfone 16{5} compounds showed reasonable potency (5.9 and
4.9 µM, respectively for TRꢀ). Interestingly, sulfone 16{5} was
nontoxic to HepG2 cells at high concentrations, while thioether

3898 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Hwang et al.
Figure 6. Hammett and Hansch analysis of ꢀ-aminoketones in dependency of inhibition of TRꢀ. (A) electronic effect (meta and para substituents);
(B) hydrophobicity (meta substituents); (C) hydrophobicity (ortho substituents).
All of the second generation compounds were evaluated to
determine potency for antagonism of TRꢀ binding to SRC2-2.
The majority possessed inhibitory potency in a narrow range
(0.4-5 µM). The exception was observed in 3,5-dimethyl
substituted oxopiperazine compounds 17{1,5,4} and 17{2,5,4}
(Supporting Information, Table 6, 18.9 and 11.2 µM, respec-
tively), which were significantly weaker. This trend indicates
that the initial SAR models have guided the evolution of these
compounds into a potency maxima that is reasonable for the
targeted interaction. In contrast, the differences in growth
inhibition of HepG2 were considerably more pronounced. The
sulfone side chain compounds 17{3,y,z} showed generally
significantly less growth inhibition than ethers 17{1,y,z} or
thioethers 17{2,y,z}. Thus, lowering of cellular toxicity was the
dominant driver for good therapeutic index. With respect to ring
substitutions, the 3,5-dimethyl compounds were generally the
most toxic (28.1-50.4 µM in sulfone series), while many of
the other compounds showed reasonable potency and therapeutic
index.
We also explored solubility and permeability of the three
series and found significant evident differences. The sulfone
series generally possessed moderate, but reasonable, solubility
(1-30 µM), whereas both the ether and thioether series showed
poor solubility (<10 and <5 µM, respectively). As the effective
available intracellular concentration is a product of the combina-
tion of solubility and permeability, we prefer to analyze this
data by graphical comparison of these two sets of data (Figure
9). This allows one to focus attention on those compounds with
the best mix of properties. Taking these considerations in mind,
together with therapeutic index, allow one to understand that
high therapeutic index is not due to poor solubility or perme-
ability. Indeed aggregate analysis indicates that the compounds
that have therapeutic index more than 100 are those most likely
to achieve high cellular concentrations.
Figure 7. Array of compounds targeted for second generation studies.
the π-π overlap between the carbonyl group and the aromatic
ring, thus decreasing the resonance stability of the elimination
intermediate and giving both an increased rate of elimination
and increased reactivity of the resulting enone for Michael
addition.
Second Generation Compounds. On the basis of the
evaluation of the initial suite of ꢀ-aminophenylketones, we
designed and synthesized a set of second generation compounds
to test hypotheses about SAR. These focused on a substitution
pattern containing: (i) para-orientation between hydrophobic
chain and carbonyl group, (ii) 3-positioned electron withdrawing
substituents, or disubstituted rings, (iii) an unsubstituted R-car-
bon, (iv) an amino ketone with an amine having a pKa between
6 and 8, and (v) an ether, thioether, or sulfonyl attached side
chain. We targeted three chemsets for the second generation as
shown Figure 7. A secondary focus was to emphasize groups
that gave low hERG activity in the previous work. To fully
explore potential synergy between substituents, the second
generation compounds were produced as a fully combinatorial
array (17{X, R1, R2R3NH}, 3 × 5 × 6 ) 90 compounds).
Details of the synthetic procedures and supporting data are
provided in the Supporting Information. This set of compounds
was evaluated using the suite of assays discussed above, and
the results are summarized as a hit map in Figure 8.
Next we explored the ability of these compounds to antago-
nize KCl-stimulated depolarization of HEK293-hERG cells as
above, focusing predominantly on sulfonyl compounds
17{3,x,y}. The ether 17{1,y,z} and thioether 17{2,y,z} series
mostly showed low inhibitiory effect on KCl-treated HEK293-
hERG cells, but this is most likely due to their poor solubility
(data not shown). Most of sulfonyl compounds have no effect
on KCl responses from HEK293-hERG cells (<20%) or weak
responses (20-40%). The exception was the 3-chloro substituted
compounds 17{3,1,3}-17{3,1,6}, which have good therapeutic
index but moderately active blockers of KCl-stimulated depo-
larization of HEK293-hERG cells (45-61%) at 10 µM. Among
the compounds that have therapeutic index more than 100, the

IrreVersible Thyroid Receptor CoA Binding Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3899
Figure 8. Activity summary of the second generation compounds sorted by therapeutic index (TI): heat map shows inhibitory constants for inhibition
of interaction of coregulatory peptide SRC2-2 and TRꢀ (IC50), HepG2 proliferation inhibition (EC50), and calculated therapeutic index (ratio of two
prior values). (a) Values are the mean of three independent experiments in triplicate. (b) Values are means of two independent experiments in
triplicate. (c) Values are EC50 over IC50. (d) Values are the mean of two independent experiments in quadruplicate.
Figure 9. Solubility and permeability of the second generation
chemsets.
oxypiperazines (17{3,3,4} and 17{3,2,4}) and acylpiperazine
(17{3,3,5}) showed no effect on KCl responses from HEK293-
hERG cells.
Conclusion
Figure 10. Best four compounds based on TI and hERG results.
This paper describes the improvement of pharmacological
index as well as no observable inhibition of KCl-stimulated
depolarization of HEK293-hERG cells. These compounds are
currently undergoing evaluation in in vivo models.
properties of thyroid hormone receptor coactivator binding
inhibitors. A major focus of optimization effort was to improve
therapeutic index and to reduce cardiac toxicity. On the basis
of the evaluation of the initial SAR study of ꢀ-aminophenylke-
tones, we designed and synthesized a set of second generation
compounds to improve pharmacological properties. Electron
withdrawing substituents led to high efficacy and the sulfonyl
attached side chain led to reduced cytotoxicity. The combination
of these elements elevated the small therapeutic index of early
hit compounds. In addition, utilization of amine moiety having
low pKa’s resulted in lowering ion channel activity without any
loss of target activity. The best compounds, based on therapeutic
index and ion channel activity, are shown in Figure 10. These
four compounds, 17{3,2,4}-17{3,2,5} and 17 {3,3,4}-
17{3,3,5}, showed both outstanding thyroid hormone receptor
coactivator interaction inhibitory potency and good therapeutic
Experimental Section
Chemistry. All materials were obtained from commercial
suppliers and used without further purification. All solvents used
were dried using an aluminum oxide column. Thin-layer chroma-
tography was performed on precoated silica gel 60 F254 plates.
Purification of compounds was carried out by normal phase column
chromatography (SP1 [Biotage], Silica gel 230-400 mesh) followed
by evaporation (HT-4X evaporator (Genevac)). Initiator (Biotage)
was used for microwave reaction. Purity determination were
performed using UPLC-MS (BEH C18 1.7 µ, 2.1 mm × 50 mm
column, Waters Corp.). Data were acquired using Masslynx v.4.1
and analyzed using the Openlynx software suite. The flow was then
split to an evaporative light scattering detector (ELSD) and SQ

3900 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Hwang et al.
mass spectrometer. The total flow rate was 1.0 mL/min and gradient
program started at 90% A (0.1% formic acid in H2O), changed to
95% B (0.1% formic acid in ACN), and then to 90% A. The mass
spectrometer was operated in positive-ion mode with electrospray
ionization. All compounds presented were confirmed at 95% purity
or better using the method (either ELSD or UV chromatogram)
that reported the lowest purity. NMR spectra are recorded on a
Bruker 400 MHz and referenced internally to the residual resonance
in CDCl3 (δ ) 7.26 ppm) for hydrogen and (δ ) 77 ppm) for
carbon atoms. NMR peaks were assigned by MestRec (4.9.9.6) and
MestReNova (5.2.2).
General Procedure for Friedel-Crafts Acylation. To a
solution phenol 2 (5.3 mmol, 1 equiv) in DMF (15 mL) was added
K2CO2 (1.47 g, 10.6 mmol, 2 equiv) and n-hexylbromide (1.12 mL,
8.0 mmol, 1.5 equiv). The reaction mixture was stirred at 85 °C
for overnight, poured into water (30 mL), and extracted with Et2O
(2 × 30 mL). The combined organic layer were washed with brine,
dried over MgSO4, and concentrated in vacuo to yield crude
hexylphenyl ether intermediate. This crude product was used for
the next step without purification. To a solution of hexylphenyl
ether (2.81 mmol, 1 equiv) in DCM (10 mL) was added 3-chlo-
ropropanoic chloride (0.35 mL, 3.65 mmol, 1.3 equiv) and AlCl3
(0.49 g, 3.65 mmol, 1.3 equiv) at 0 °C and stirred for 1 h. The
reaction mixture was poured into ice-water (50 mL) and extracted
with DCM (2 × 30 mL). The combined organic layer was washed
with brine, dried over MgSO4, and concentrated in vacuo. The crude
product was purified by flash column chromatography (SP1, 25 M
(SiO2), flow rate: 25 mL/min; gradient: 1-30% ethyl acetate in
hexanes over 20 CV).
General Procedure for 5 and 6. To a solution compound 3 or
4 (1 equiv) in THF was added dimethylamine (2 M in THF, 10
equiv) and stirred at rt for 1 h. The reaction mixture was filtered
and washed with ether (×2). The combined organic layer was
concentrated in vacuo to yield crude aminoketone 5 or 6 and used
without further purification.
General Procedure for Mannich Reaction. To a solution
phenol 7 (5.3 mmol, 1 equiv) in DMF (15 mL) was added K2CO2
(1.47 g, 10.6 mmol, 2 equiv) and n-hexylbromide (1.12 mL, 8.0
mmol, 1.5 equiv). The reaction mixture was stirred at 85 °C for
overnight, poured into water (30 mL), and extracted with Et2O (2×,
30 mL). The combined organic layer were washed with brine and
dried over MgSO4 and concentrated in vacuo to yield crude
hexyloxy acetopheon intermediate. This crude product was used
for the next step without purification. A solution of hexyloxy
acetophenon (1 equiv), dimethylamine hydrochloride (2 equiv),
paraformaldehyde (2 equiv), c-HCl (cat.), and H2O/actonitrile (1/
9) was heated at 120 °C in MW for 2 h. The reaction mixture was
concentrated in vacuo and purified by flash column chromatography
(SP1, gradient: 1-10% MeOH in hexanes over 15 CV).
µL was transferred into the 384-cell culture plates (Corning 3917),
yielding a final compound concentration range of 200-0.01 µM.
After 48 h incubation at 37 °C 25 µL of CellTiter-Glo (Promega)
was added and mixed by aspiration and dispense and luminescence
was detected (EnVision, PerkinElmer) after 10 min.
Solubility Study. The assay was carried out using the Multi-
Screen Solubility assay developed by Millipore. Briefly, 16 test
compounds were dispensed into each 96-well polypropylene plate
(Costar 3365) as serial diluted concentration ranges from from
10000 µM to 625 µM in DMSO starting from columns 1-5 and
7-11. Columns 6 and 12 were filled with DMSO. Then 5 µL of
each well was transferred into the 96-well disposable UV-Star
(Greiner Bio-One). Acetonitrile (97.5 µL) and PBS buffer (97.5
µL) was added to each well, and the plate was agitated for 30 min
(IKA microtiterplate shaker). The UV spectra from 200-500 nm
was measured for all wells and subtracted from the background
(DMSO). The correlations between concentrations and absorbance
at 260, 280, and 300 nm were determined as slopes. Then, 5 µL of
each well of the polypropylene plate was added to a MultiScreen
solubility filter plate (Millipore) and diluted with 195 µL of PBS.
The plate was agitated for 2 h and filtered into a 96-well disposable
UV-Star plate, and the UV absorbance at 260, 280, and 300 nm
was measured. The aqueous solubility (aqueous solubility ) Amax
filtrate/slope) was determined for all three wavelengths and given
as the average value.
PAMPA Procedure. The procedure was conducted using a
developed procedure by pION. Briefly, all liquid-handling steps
for the PAMPA assay are performed on a Biomek FX laboratory
automation workstation (Beckman-Coulter) and analyzed by pION’s
(London, UK) PAMPA Evolution 96 Command software. The
PAMPA Evolution 96 permeability assay kit includes the acceptor
sink buffer (ASB), double-sink lipid solution, and a PAMPA
sandwich plate, preloaded with magnetic disks. Then 3 µL of lipid
were transferred onto the support membrane in the acceptor well,
followed by addition of 200 µL of ASB (pH 7.4). Then, 180 µL of
diluted test compound (50 µM in system buffer at pH 7.4 starting
from a 10 mM DMSO solution) was added to the donor wells.
The PAMPA sandwich plate was assembled and placed on the Gut-
Box and stirred for 30 min. The distribution of the compounds in
the donor and acceptor buffers (100 µL aliquot) was determined
by measurement of the UV spectra from 200 to 500 nm using the
SpectraMax reader (Molecular Devices). The permeability coef-
ficient is determined using the maximum absorbance from 200 to
500 nm using the following formula:
Pe ) 2.3VD/[A(t - tLAG)]log10{1/(1 - R)CD(t)/CD(0)}
Fluorescence Polarization Assay (FPA). This assay has been
described in detail previously.13,14 The concentration of each
compound required to inhibit 50% of the binding between TRꢀ-
LBD and SRC2-2 peptide is presented in Tables 1-5. hTRꢀ1 LBD
(His6 T209-D461) was expressed in BL21 (DE3) (invitrogen), and
the peptide SRC2-2 (CLKEKHKILHRLLQDSSSPV) was labeled
with 5-iodoacetamidofluorescien (Molecular Probes), as described.
For each reported value, two independent experiments were carried
out in quadruplicate on two separate days using alternate batches
of reagents. The competition binding experiments were evaluated
using PrismPad, and the IC50 values were obtained by fitting data
to equation: Sigmoidal dose-response (variable slope) or four
parameter logistic equation; Y ) bottom + (top - bottom)/(1 +
VD is the donor well volume (cm3), A is the filter area (cm2),
CD(0) is the sample concentration in the donor well at time 0 (mol/
cm3), CD(t) is the sample concentration in the donor well at time t
(mol/cm3), t is the interval of time (s), tLAG is the lag time needed
to reach steady state conditions (s), and R is the membrane retention
(related to the membrane/water partition coefficient). Standards used
were verapamil (Pe ) 1505 × 10-6 cm/s) as a high permeability
standard, carbamazepine (Pe ) 150 × 10-6 cm/s) as medium
permeability standard, and ranitidine (Pe ) 2.3 × 10-6 cm/s) as
low permeability standard. The compounds were measured in
triplets and reported as average values.
hERG Binding Assay. This screen was performed by the
National Institute of Mental Health Psychoactive Drug Screening
Program (PDSP), directed by Dr. Bryan L. Roth, at University of
North Carolina Chapel Hill (http://pdsp.med.unc.edu/indexR.html).
Detailed protocols and data analysis procedures are available online
(http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid)376&loc)
ea_ras or http://pdsp.med.unc.edu/UNC-CH%20Protocol%20Book.
pdf). Samples and internal controls (known hERG blockers) were
submitted in a blind fashion to the PDSP. The assays reliably and
reproducibly identified the blinded internal controls as “hits”.
10((LogEC -X)HillSlope)); X is the logarithm of concentration, Y is the
50
response. Tabulated data are reported as mean values across all
experiments and replicates with associated 95% confidence limits.
Cytotoxicity Study. HepG2 cells were at 37 °C in minimum
essential medium (MEM), 10% fetal bovine serum, 50 mL of
penicillin/streptomycin. Cells were grown to 80% confluence,
collected, and dispensed in 384 well plates at 50 µL per well (6000
cells). The small molecules were serially diluted from 50000 µM
to 2.5 µM in DMSO into a 386-well plate (Costar 3365) and 0.2

IrreVersible Thyroid Receptor CoA Binding Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3901
(15) Arnold, L. A.; Kosinski, A.; Estebanez-Perpina, E. R. J. F.; Guy, R. K.
Inhibitors of the Interaction of a Thyroid Hormone Receptor and
Coactivators: Preliminary Structure-Activity Relationships. J. Med.
Chem. 2007, 50, 5269–5280.
Acknowledgment. This work was supported by the NIH
(DK58080), the American Lebanese Syrian Associated Charities
(ALSAC), and St. Jude Children’s Research Hospital.
(16) Estebanez-Perpina, E.; Arnold, L. A.; Jouravel, N.; Togashi, M.;
Blethrow, J.; Mar, E.; Nguyen, P.; Phillips, K. J.; Baxter, J. D.; Webb,
P.; Guy, R. K.; Fletterick, R. J. Structural Insight into the Mode of
Action of a Direct Inhibitor of Coregulator Binding to the Thyroid
Hormone Receptor. Mol. Endocrinol. 2007, 21, 2919–2928.
(17) Bruhova, I.; Tikhonov, D. B.; Zhorov, B. S. Access and Binding of
Local Anesthetics in the Closed Sodium Channel. Mol. Pharmacol.
2008, 74, 1033–1045.
Supporting Information Available: Experimental procedures,
tabulated activity data, and characterization data for all final
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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