Inhibitors of the interaction of a thyroid hormone receptor and coactivators: Preliminary structure-activity relationships

Article abstract of DOI:10.1021/jm070556y

The modulation of gene regulation by blocking the interaction between the thyroid receptor (TR) and obligate coregulators has been reported recently with the discovery of the lead compound 3-(dimethylamino)-1-(4-hexylphenyl)propan-1- one). Herein we repor

Full text of DOI:10.1021/jm070556y

J. Med. Chem. 2007, 50, 5269-5280
5269
Inhibitors of the Interaction of a Thyroid Hormone Receptor and Coactivators: Preliminary
Structure-Activity Relationships
Leggy A. Arnold,† Aaron Kosinski,† Eva Este´banez-Perpin˜a´,‡ Robert J. Fletterick,‡ and R. Kiplin Guy*,†
Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, Tennessee 38105, and
UniVersity of California San Francisco, Department Biochemistry and Biophysics, 600 Sixteenth Street, San Francisco, California 94143
ReceiVed May 11, 2007
The modulation of gene regulation by blocking the interaction between the thyroid receptor (TR) and obligate
coregulators has been reported recently with the discovery of the lead compound 3-(dimethylamino)-1-(4-
hexylphenyl)propan-1-one). Herein we report studies aimed at optimization of this initial hit to determine
the basic parameters of the structure-activity relationships and clarify the mechanism of action. These
studies provided new insights, showing that activity and TRâ isoform selectivity is highly correlated with
the structural composition of these covalent inhibitors.
Introduction
systematically studied by Carl Mannich in the beginning of the
20th century.20 Several Mannich bases have been marketed as
drugs, including pipoctanone21 (antihypertensive), hexacaine22
(antiarrhythmic), propitocaine23 (local anesthetic), and dyclo-
nine24 (local anesthetic). Although the mode of action has not
been fully elucidated, experimental evidence supports their
binding to the inner pore of voltage-gated sodium channels and
blocking currents.25 In contrast to other local anesthetics,
Mannich base drugs are well-known to liberate R,â-unsaturated
ketones by internal elimination of the amino group in vitro,
although this reaction proceeds very slowly in aqueous buffers
at physiological pH.26 Soft electrophiles such as enones can
alkylate protein nucleophiles with a strong chemoselectivity for
the sulfhydryl of cysteine.27 We showed that certain â-ami-
nophenylketones are very likely to specifically alkylate TRâ in
vitro,19 thus inhibiting its hormone-induced gene transcription.
These compounds have great potential as tools for studying
the role of the interaction between TR and its coregulators
(CoRs) in vivo and potentially for progression to lead optimiza-
tion as a preclinical candidate to treat aspects of metabolic
syndrome. Here we present a broad study of the structure-
activity relationships (SARs) of this lead series governing the
activities, toxicity, solubility, and permeability of â-aminophe-
nylketones and similar electrophilic compounds.
The thyroid hormone receptors (TRs) belong to the super-
family of nuclear receptors (NRs) and are involved in the
regulation of development, growth, and metabolism.1-3 Thyroid
hormone T3 induces most of the transcriptional responses
mediated by TRs in vivo.4 The TRs have two isoforms, TRR
and TRâ, encoded by two genes, with each isoform having two
distinct subtypes due to alternative splicing.5 The main func-
tional domains of the TRs are (1) an amino terminal ligand-
independent transcription activation domain (AF-1), (2) a central
DNA-binding domain, and (3) a carboxy terminal ligand-binding
domain (LBD) containing a thyroid hormone-inducible activa-
tion domain (AF-2).6 TR most often acts in concert with retinoid
X receptor.7 This heterodimer, formed in the absence of ligand,
associates with an NR corepressor such as NCoR or the silencing
mediator of retinoic acid (SMRT) and suppresses basal tran-
scription.8 In the presence of T3, TR undergoes a conformational
change, releasing NCoR/SMRT and recruiting coactivator (CoA)
proteins to activate gene transcription. The dominant group of
CoAs is the p160 or steroid receptor coactivator (SRC) proteins,7
including SRC1,9 SRC2,10,11 and SRC3.12 The SRCs have
variable numbers of conserved LXXLL motifs, called NR boxes,
within their nuclear receptor interacting domain (NID). These
leucine-rich motifs interact with hydrophobic amino acid
residues located on helixes 3, 4, 5, and 12 of the TR LBD,
which form the AF-2 pocket.13,14 Additional hydrogen bonding
in close proximity has been observed between charged side
chains of the CoAs and lysine 288 and glutamic acid 457 of
the TRâ (charge clamp residues).
Results
We recently reported the discovery of small molecules that
inhibit the interaction between TR and the CoA SRC2-2 (SRC2
peptide representing the second NID).19 The most potent
molecules identified by the screen were substituted â-aminophe-
nylketones. An initial SAR for this series was established using
the relative response of other â-aminophenylketones present in
the screening collection. The relative efficacies at a fixed dose
of 30 µM are shown in Figure 1.
The screened compounds were divided into four groups on
the basis of their chemical structures. Group A combined
unsubstituted â-aminophenylketones with various nitrogen sub-
stituents. These compounds inhibited the interaction between
CoA SRC2-2 and TRâ weakly, with efficacies between 2 and
19%, at a 30 µM concentration. Group B consisted of â-ami-
nophenylketones 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,
We have reported that short peptides consisting of the LXXLL
motif and a flanking amino acid are able to bind to the TR15,16
and developed inhibitors based on macrolactam-constrained
peptides.17,18 The first small-molecule inhibitors were discovered
in a high-throughput screen using fluorescence polarization
(FP).19 The most promising hit from this screen was an aromatic
â-aminoketone, which, after in situ elimination to form an enone,
bound covalently to the TR and inhibited its interaction with
SRCs. This compound class, known as Mannich bases, was
* Corresponding author. Address: St. Jude Children’s Research
Hospital, Department of Chemical Biology and Therapeutics, 332 N.
Lauderdale St., Memphis TN 38104-2794. Tel: (901) 495 5714. Fax: (901)
495-5715. E-mail: kip.guy@stjude.org.
† St. Jude’s Children’s Research Hospital.
‡ University of California, San Francisco.
10.1021/jm070556y CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/05/2007

5270 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Arnold et al.
Figure 1. Summary of screened â-aminophenylketone. Inhibition (%) of the interaction between the CoA peptide SRC2-2 and TRâ in the presence
of 30 µM compound.
Scheme 1. Synthesis of â-Aminophenylketones 3a-ka
requires seeking the best balance between these factors. The
potency and efficacy of the â-aminophenylketones synthesized
were investigated using a competitive FP assay. Labeled SRC2-2
peptide, TR-binding domain (TR-LBD) protein, and thyroid
hormone T3 were incubated with varying concentrations of the
â-aminophenylketones 3a-k for 3 h.29 The resulting measured
FP reflected the ratio of bound to unbound CoA. Two TR
isoforms were investigated: TRR and TRâ. 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 an expression of TRâ has been
reported for the thyroid cancer cell line ARO.30,31 A com-
mercially available luminescence cell viability assay, CellTiter-
Glo (Promega Corp.) was used to quantify adenosine triphos-
phate in viable cells. The solubility of each â-aminophenylketone
in phosphate-buffered saline (PBS) buffer containing 5%
dimethyl sulfoxide (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 FP assay. Finally, we measured the
permeability using a parallel artificial membrane permeation
assay (PAMPA). The partition of the â-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 Table 1.
a Reagents: (i) AlCl3 (2 equiv), acryloyl chloride, DCM, 0 °C, 1 h; (ii)
HNR1R2, THF, RT, 1 h.
group C combining â-aminophenylketones with larger hydro-
phobic 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
â-aminophenylketone. â-Aminonaphthylketones in group D
were more active (26-30% inhibition) at 30 µM than were the
â-aminophenylketones in group A (12%). This analysis showed
that a hydrophobic moiety at the para position of â-aminophe-
nylketone is an important feature of potent inhibitors of the TR-
CoA interaction.
To address the importance of nitrogen substituents, analogues
of group C, â-aminophenylketones bearing n-hexyl substituents
at the para position of the aromatic ring, were synthesized
(Scheme 1). 1-Phenylhexane reacted with acryloyl chloride in
the presence of aluminum chloride, giving compounds 1 and 2
in a ratio of 8:2 after acidic workup.28 Treatment of this mixture
with secondary amines afforded a focus library of â-aminophe-
nylketones in 85-97% yield (Table 1).
All â-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
2-fold selectivity toward TRâ. In a cell-based assay, these
compounds were generally more toxic to the hormone-
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

Inhibitors of TR and CoA Interaction
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5271
Table 1. Summary of â-Aminophenylketones 3a-k Data: Yield, Inhibition (IC50) of the Interaction between Coregulatory Peptide SRC2-2 and
TRR/TRâ, Viability (LD50) of U2OS and ARO Cells in the Presence of Compounds, Solubility in PBS Buffer Containing 5% DMSO, and Permeability
Across an Artificial Membrane (PAMPA)
in vitro potency
cytotoxicity
pharmacological properties
TRRb
IC50
(µM)
TRâb
AROc
LD50
(µM)
U2OSc
LD50
(µM)
yielda
(%)
IC50
TRR/TRâ
ratio
solubilityc
(µM)
permeabilityc
(10-6 cm/s)
entry
R1
R2
compound
(µM)
1
2
3
4
5
6
7
8
9
2-phenylethyl
2-hydroxyethyl
isopropyl
pyrroline
morpholine
methyl piperidine-4-carboxylate
n-butyl
methyl
furan-2-yl-methyl
cyclohexyl
n-propyl
methyl
methyl
isopropyl
3a
3b
3c
3d
3e
3f
3g
3h
3i
84
87
81
85
90
86
90
94
92
78
93
3.6
3.0
4.1
4.3
4.1
6.3
6.9
10.6
22.6
16.5
25.8
1.5
1.6
2.0
2.7
2.7
3.9
4.2
4.3
2.4
1.9
2.1
1.6
1.5
1.6
1.6
2.5
1.9
1.3
1.7
2.5
13.2
15.4
19.0
7.2
8.2
5.4
11.0
15.5
25.6
102
18.4
13.2
20.7
38.4
20.8
19.7
25.5
34.9
18.6
27.3
160
15
97
55
341
110
27
152
321
47
57
965
747
2144
1210
145
1957
2323
681
n-butyl
methyl
methyl
cyclohexyl
hydrogen
12.1
12.7
15.2
10
11
3j
3k
32
354
104
254
a Isolated yield. b Values are means of two independent experiments done in quadruplicate; the general error limits are 10%. c Values are means of two
independent experiments done in triplicate; the general error limits are 10-15%. Actual error values for each measurement are omitted for clarity but are
included in tables in the Supporting Information.
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 (Table 1, entry 11). The
solubility of these compounds depended on the nitrogen
substituents and varied between 6 and 442 µM in PBS
containing 5% DMSO. â-Aminophenylketones bearing small
nitrogen alkyl substituents, such as 3d, 3h, and 3k, were the
most soluble compounds (Table 1, 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. â-Ami-
nophenylketones 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) (Table 1, entries
2, 5, and 7).
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 TRR and TRâ. The IC50 values were 1.5 µM for TRâ
and 28.1 µM for TRR (Table 2, entry 1). Derivatives of 1
bearing a methyl substituent in the R or â position (4b and 4c)
or a carboxy substituent in the â position (4a) were less active
(Table 2, 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 (Table 2, entries
5-7). The saturated nonelectrophilic ketone 4g showed no
activity (Table 2, 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 (Table 2,
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.
Previously, we proposed that the â-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 â-aminophenylketones
because all these compounds afford the same elimination
product, 1-(4-hexylphenyl)prop-2-en-1-one, regardless of their
amino moieties (Table 2, entry 1). To better understand the
importance of the electrophilic character of the inhibitors, we
investigated various aromatic unsaturated ketones. 1-Phenyl-
hexane was reacted with acid chlorides or anhydrides under
Friedel-Crafts reaction conditions to obtain the corresponding
ketones (Scheme 2).
We reported previously that aromatic acrylates inhibited the
interaction between TR and SRC2-2.19 For this reason, acrylates
and acrylamides were explored as 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 acry-
lates by using acryloyl chloride in the presence of triethylamine
(Scheme 4).
Compounds 1, 4a, and 4g were obtained in moderate yields
(45-85%) (Table 2, 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 the
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 of tetrapro-
pylammonium perruthenate (Scheme 3).32 The resulting com-
pounds were studied using the methods outlined above, and the
results are summarized in Table 2.
The unsaturated esters were obtained in 71-94% yield (Table
3). Their ability to inhibit the interaction between TR and CoA
was measured using an FP assay with TRR and TRâ. The
cytotoxicity was determined in U2OS and ARO cells, and the
results are summarized in Table 3.
Seven para alkyl-substituted acrylates were investigated with
carbon chain lengths ranging from C3 to C8 (Table 3, entries
1-7). The binding to TR increased with the length of the
substituent, reaching maximal inhibition (10.5 µM TRâ) for 6f
bearing a heptyl substituent (Table 3, entry 6). The selectivity
ratios (TRR/TRâ) for these acrylates were about 2:1 in favor
of TRâ. 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

5272 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Arnold et al.
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
TRR/TRâ, Viability (LD50) of U2OS and ARO Cells in the Presence of Compounds, Solubility in PBS Buffer Containing 5% DMSO, and Permeability
Across an Artificial Membrane (PAMPA)
a Isolated yield. b Values are means of two independent experiments done in quadruplicate; the general error limits are 10%. c Values are means of two
independent experiments done in triplicate; the general error limits are 10-15%. Actual error values for each measurement are omitted for clarity but are
included in tables in the Supporting Information.
Scheme 2. Synthesis of Ketones 1, 4a, and 4ga
Scheme 4. Synthesis of Substituted Acrylates 6a-la
a Reagents: (i) acryloyl chloride (1.1 equiv), NEt3 (1.1 equiv), DCM,
RT, 1 h.
a Reagents: (i) AlCl3 (2 equiv), acid chloride (maleic anhydride to form
4a), DCM, 0 °C, 1 h.
Scheme 3. Synthesis of Unsaturated Ketones 4b-fa
than the corresponding para-substituted benzoates 6i and 6j
(Table 3, entries 9-11). Compound 6k had the highest TRR/
TRâ ratio (5:1) among the compounds in this library. Butyra-
midophenyl- and 5,6,7,8-tetrahydronaphthyl-substituted acrylates
6l and 6m showed weak activities (Table 3, 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 (Table 3, entries
1-7). In general, ARO cells were more sensitive than U2OS
cells to the acrylates, except for compounds 6e-h (Table 3,
entries 5-8). Interestingly, three of these compounds bear an
ester or an amide functionality in the para position (Table 3,
entries 9, 10, and 12). The solubility and permeability of the
acrylates tested were <10 µM and <5 × 10-6 cm/s, respectively
(data not shown).
a Reagents: (i) BuLi, THF, -78 °C, 20 min; (ii) RCHO, -78 °C, 2 h;
(iii) tetrapropylammonium perruthenate (0.05 equiv), 4-methylmorpholine
N-oxide (1.5 equiv), molecular sieves, CH3CN, RT, 1-4 h.
substituent (Table 3, entries 4 and 8). No selectivity between
TRR and TRâ was observed for 6h. The ortho-substituted
benzoate 6k was significantly more potent (IC50 ) 8.2 µM)

Inhibitors of TR and CoA Interaction
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5273
Table 3. Summary of IC50 Values of 6a-m: Yields, Inhibition of Coregulatory Peptide SRC2-2 and TRR/TRâ Binding, and Viability (LD50) of U2OS
and ARO Cells in the Presence of Compounds
in vitro potency
cytotoxicity
TRRb
IC50
(µM)
TRâb
IC50
(µM)
AROc
LD50
(µM)
U2OSc
LD50
(µM)
yielda
(%)
TRR/TRâ
ratio
entry
compound
1
2
3
4
5
6
7
8
9
10
11
12
13
4- n-propylphenyl
4- n-butylphenyl
4- n-pentylphenyl
4- tert-pentylphenyl
4-n-hexylphenyl
6a
6b
6c
6d
6e
6f
6g
6h
6i
85
86
88
94
89
76
81
62
75
71
85
73
77
>100
71.0
60.9
69.6
45.2
23.0
21.4
75.6
>100
>100
39.1
>100
85.3
100
20.2
9.9
20.0
5.8
29.5
69.2
111
92.8
4.8
21.2
14.6
8.6
43.9
25.6
22.7
27.5
18.8
36.7
50.5
50.2
121
66.0
33.9
43.5
18.6
10.5
11.4
69.1
61.0
55.8
8.2
1.1
1.8
1.6
2.4
2.2
1.9
1.1
4-n-heptylphenyl
4-n-octylphenyl
(trans) 4-tert-pentylcyclohexyl
4-n-propylbenzoate
4-n-pentylbenzoate
2-n-hexylbenzoate
4-n-butyramidophenyl
5,6,7,8-tetrahydronaphthyl
6j
6k
6l
36.5
19.4
242
4.8
1.2
85.9
69.8
6m
54.7
101
a Isolated yield. b Values are means of two independent experiments done in quadruplicate; the general error limits are 10%. c Values are means of two
independent experiments done in triplicate; the general error limits are 10-15%. Actual error values for each measurement are omitted for clarity but are
included in tables in the Supporting Information.
Scheme 5. Synthesis of Substituted Acrylamides 7a-la
cytotoxicity in ARO and U2OS cells, solubility in PBS with
5% DMSO, and permeability (PAMPA) are summarized in
Table 5.
Propiolic acid derivatives 8a and 8b were able to inhibit the
interaction between TR and SCR2-2 with IC50 values of 35.1
and 6.2 µM for TRâ (Table 5, entries 1 and 2). A general IC50
ratio of 1:2 (TRâ/TRR) was observed for all active electrophiles.
Two R-halogen ketones, 8c and 8d, were investigated. The
R-chloro ketone 8c was able to inhibit the interaction between
SRC2-2 and TR, but R-fluoro ketone 8d was not active (Table
5, entries 3 and 4). The â-bromo ketone 8e was the most potent
compound in this group (Table 5, entry 5) but had poorer
selectivity, exhibiting IC50 values of 4.2 and 3.0 µM for TRR
and TRâ, respectively. In contrast, the γ-bromo ketone 8f was
not active. The racemic epoxy-ketone 8g inhibited the interaction
between TR and the CoA SRC2-2 with an IC50 value of 33.2
µM for TRR and 19.1 µM for TRâ (Table 5, entry 6). 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 R-fluoro ketone
8d and epoxy ketone 8g (Table 5, 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.
a Reagents: (i) acid chloride or anhydride, NEt3 (1.1 equiv, when the
acid chloride was used), DCM, RT, 1 h.
Variation of the R,â unsaturated system was the focus of the
acrylamide library. The synthesis involved the reaction between
4-hexylaniline and various substituted acryloyl chlorides or
anhydrides (Scheme 5).
The corresponding products 7a-f were obtained in 78-88%
isolated yield. Inhibition constants were determined for both
TRR and TRâ. 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.
Among the acrylamides tested, only 7a and 7b inhibited the
interaction between TR and SRC2-2 with IC50 values (TRâ) of
17.9 and 20.3 µM, respectively (Table 4, entries 1 and 2). No
TR isoform selectivity was observed. Interestingly, all com-
pounds showed minor toxic effects in ARO cells. Similar results
were obtained for U2OS, with the exception of slightly more
cytotoxic compounds 7a and 7e (Table 4, entries 1 and 4). High
solubilities were measured for carboxylic acid-substituted acry-
lamides 7b, 7f, and 7g (Table 4, 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 (Table 4,
entries 1 and 3).
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 (Figure 2).
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 â-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 Figure 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
(Scheme 6).
The halo-ketones 8c-e were synthesized under Friedel-
Crafts conditions (Scheme 2). Fluoroacetyl chloride33 was
obtained from the corresponding sodium salt. The keto-epoxide
8f was synthesized for the corresponding R,â-unsaturated ketone
1 obtained after HBr elimination of the â-bromo ketone 8e using
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base (Scheme
7). The yields, inhibition constants for both TRR and TRâ,

5274 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Arnold et al.
Table 4. Summary of Acrylamides 7a-g: Yield, IC50 Values of the Inhibition between Coregulatory Peptide SRC2-2 and TRR/TRâ, Viability (LD50)
of U2OS and ARO Cells in the Presence of Compounds, Solubility in PBS Buffer Containing 5% DMSO, and Permeability Across an Artificial
Membrane (PAMPA)
a Isolated yield. b Values are means of two independent experiments done in quadruplicate; the general error limits are 10%. c Values are means of two
independent experiments done in triplicate; the general error limits are 10-15%. Actual error values for each measurement are omitted for clarity but are
included in tables in the Supporting Information.
Scheme 6. Synthesis of 8a and 8ba
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 struc-
ture 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 Figure 5.
a Reagents: (i) propionic acid (1.3 equiv), 4-dimethylaminopyridine
(0.001 equiv), N,N′-diisopropylcarbodiimide, DCM, RT, 5 h.
Scheme 7. Synthesis of 8a and 8ba
The pharmacophore models for all three libraries (enones A,
acrylates B, and acrylamides C) were very similar (Figure 5).
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 Å, 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 Å. 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 Å, and the distance between
Hyd/Aro and Acc was 4.39 ( 0.25 Å.
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 (Figure 6). 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
a Reagents: (i) DBU (1.1 equiv), benzene, RT, 2 h; (ii) H2O2 (30%) (5
equiv), NaOH (1M in H2O), MeOH, 20 min.
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 experi-
ment.
The in vitro cytotoxicity data revealed a good correlation
between the LD50 in ARO cells and U2OS cells (Figure 4).
The slope (0.52) indicated a general 2-fold 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 (Figure 4, data points in parentheses). ARO/
U2OS LD50 ratios up to 1:28 were observed. The selectivity
was not scaffold related.

Inhibitors of TR and CoA Interaction
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5275
Table 5. Summary of Electrophilic Compounds 8a-f: Yield, IC50 Values of the Inhibition between the Coregulatory Peptide SRC2-2 and TRR/TRâ,
Viability (LD50) of U2OS and ARO Cells in the Presence of Compounds, Solubility in PBS Buffer Containing 5% DMSO, and Permeability Across an
Artificial Membrane (PAMPA)
a Isolated yield. b Values are means of two independent experiments done in quadruplicate; the general error limits are 10%. c Values are means of two
independent experiments done in triplicate; the general error limits are 10-15%. Actual error values for each measurement are omitted for clarity but are
included in tables in the Supporting Information.
Figure 2. Scatter plot of solubility and permeability data for all
Figure 3. LCMS-PDA analysis of 3h in water, saline (150 mM NaCl),
and human blood plasma (all pH 7). Integrated absorption area at 254
nm (PDA): black: ratio between peak area of 3h and peak area of
internal standard (salicylic acid) measured in blood plasma (left y-axis);
gray: area of 3h measured in water (right axis); white: area of 3h
measured in saline (right y-axis).
compounds except acrylates: 4 aminophenylketones (Table 1), 0
enones (Table 2), + acrylamides (Table 4), and 9 electrophiles (Table
5); n ) number of X values, s ) slope, F ) F values, r2 ) square
correlation coefficient, and () denote excluded data points.
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 Å. A relatively spacious hydrophobic
groove surrounded the aromatic core structure, and a hydro-
phobic curved surface accommodated the alkyl substituent.
The AF-2 domain of TRâ has a unique architecture in
comparison with the other NRs, 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 TRâ structure affords selectivity
of the inhibitors for TRâ, we carried out FP competition assays
employing the following NRs: the androgen receptor, the
estrogen receptor R, and the peroxisome proliferator activated
receptor γ. No inhibition of CoR recruitment to any of these
NRs could be detected after 3 h 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 NRs because of the presence of unique cysteine residues
in the AF-2 binding site.
Discussion
These studies of TR CoA modulators provided new structural
insight into the mode of action of these compounds. First, within
a given aromatic substitution pattern, all â-aminophenylketones
tested showed similar activities in the competition binding assay,

5276 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Arnold et al.
Figure 4. Scatter plot of LD50 values measured in U2OS and ARO
cells for all compounds: 4 aminophenylketones (Table 1), 0 enones
(Table 2), / acrylates (Table 3), + acrylamides (Table 4), 9 electro-
philes (Table 5), and () denote excluded data points.
Figure 6. Modeling study of the bonding of compound 1 to the TR
CoA binding pocket. Surface gray ) hydrophobic; blue ) nitrogen;
red ) oxygen; yellow ) sulfur.
on the steric nature of the electrophile. Enones with a R-methyl,
trans-methyl, or cis-carboxy substituents (4a-c) were able to
inhibit the interaction between TR and the CoA SRC2-2. In
contrast, enones with two methyl substituents or a single
â-phenyl substituent were not tolerated (4d-f). This correlated
with the modeled complex of TR-LBD-exhibiting compound
1, which had a well defined and rigid binding site unsuitable
for highly substituted enones. Additional binding data were
observed for the acrylamides. In this series, unsubstituted and
cis-carboxy acrylamides (7a and 7b) were able to interrupt the
TR-CoA interaction, whereas trans-methyl substituted acry-
lamide 7c was not active.
Another important feature of functional inhibitors is the
distance between the electrophilic head group and the aromatic
core structure. In contrast to the inactive trans-methylated
unsaturated amide 7c, the trans-methylated unsaturated ketone
4b was active, although both unsubstituted amide 7a and the
unsaturated ketone 1 were also active. A similar dependency
was observed in compounds 7b and 7f. The pharmacophore
model showed a distance between the electrophilic group (Hyd/
Uns) and core structure (Hyd/Aro) of 4.65 Å for enones and
5.68 Å for acrylamides. The difference of 1.03 Å is likely to
limit substitution of active acrylamides at the R-position,
assuming the same mode of binding for enones and acrylamides.
Electrophilic effects might support this result as well. Enones
are more reactive Michael acceptors than acrylamides, and
substituents in the â-position have a strong inductive effect on
the polarization of double bonds. Decreased Michael acceptor
activity from 1 (enone) to 4b (â-methylketone) to 7a (acryla-
mide) and 7c (â-methylacrylamide) could be supported by
calculations of differences in the lowest unoccupied and highest
occupied molecular orbitals (∆E ) 9.175 > 9.164 > 8.683 >
8.487).
Figure 5. Pharmacophore elucidation using MOE software. Molecules
are illustrated in stick form: gray ) carbon, red ) oxygen, blue )
nitrogen; green mesh balls ) hydrophobic center (Uns ) unsaturated,
Aro ) aromatic, Ali ) aliphatic); blue mesh balls ) possible electron
acceptor site. (A) enones (active in FP assay); (B) acrylates (IC50 < 30
µM); (C) acrylamides (active in FP assay).
One of the most important structural requirements for TR-
CoA inhibitors is the hydrophobic substituent in the para position
relative to the electrophilic head group. Table 3 shows that a
medium chain length of six to seven carbon atoms is most
favorable for binding. Although ortho alkyl-substituted â-ami-
nophenylketones are not functional inhibitors (data not shown),
2-hexyl benzoate 6k was one of the most potent inhibitors. The
carboxy functionality increases the rigidity of the substituent
and alters the orientation of the alkyl chain. In the case of 6k,
this resulted in an increase in binding affinity. In contrast, para-
substituted benzoates 6i-j were less potent than compounds
6c and 6f bearing an n-alkyl chain in the para position. This
might imply that the presence of carboxy functionality could
despite the wide variation in structural and electronic properties
of the amine substituents. This behavior is consistent with the
â-aminophenylketones acting as prodrugs because of a deami-
nation reaction liberating the active electrophilic unsaturated
ketones. Stability studies simulating assay conditions showed
no detectable elimination of the â-aminophenylketones in the
absence of protein. Moderate stability was observed in blood
plasma, resulting in a half-life of about 2 h.
The investigations applying unsaturated ketones bearing
different substituents showed that activity is highly dependent

Inhibitors of TR and CoA Interaction
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5277
alter hydrophobic interactions as well, resulting in weaker
binding affinities for 6i-j.
a distance between the core structure and the electrophilic center
of 6.0 Å or less. Electronic properties of the Michael acceptor
have an important influence on potency, with highly reactive
enones being the most potent inhibitors. Among the compounds
we investigated, we identified potent inhibitors with promising
properties in terms of toxicity, solubility, and permeability: for
example, 3-(dimethylamino)-1-(4-hexylphenyl)propan-1-one.
These and similar compounds have been distributed as local
anesthetics and antiarrhythmics, altering ion-channel activity.
The ion-channel activity of these compounds is governed
strongly by the pKa of the nitrogen. In our case, we discovered
elimination of the amine liberated the active enone inhibiting
TR. By altering the pKa of the nitrogen, we might be able to
decouple the undesired ion channel activity of this class of
compounds from the desired activity against TR. The develop-
ment of these prodrugs might establish new treatments for
metabolic syndromes and metabolic diseases based on CoR
dysfunction. In addition, these irreversible inhibitors represent
valuable research tools in vivo and in vitro to study distribution
and expression levels of the TR, to map potential active sites
on the protein surface, and to elucidate signal pathways mediated
by the TR.
Interestingly, the core structure of the TR-CoA inhibitors,
the phenyl ring, can be exchanged with a cyclohexyl ring
without a major loss of potency (see 6h and 6d). In general,
the cyclohexyl ring is more space-filling, more hydrophobic,
and unable to π-stack with other aromatic systems. The tolerance
of a core structure with a similar size but different electronic
properties implies that the contribution of this group to binding
is solely hydrophobic in nature.
The pharmacophore modeling study (Figure 5) revealed a
possible electron acceptor/hydrogen donor (Acc) in close
proximity to Hyd/Uns and Hyd/Aro. Examining the crystal
structure of the AF-2 pocket for possible hydrogen-donating
amino acids, we identified K306 as a likely residue (Figure 6).
The molecule modeling study showed a possible moderate
interaction (3.3 Å) between K306 and the carbonyl/carboxyl
oxygen conserved in all compounds.
Finally, we showed that nine 4-hexylphenyl compounds with
different electrophilic head groups inhibited the interaction
between TR and CoA (1, 3a-k, 4a, 7a, and 8a-g). The most
active compounds were enone 1, â-aminophenylketones 3a-
k, and â-bromo ketone 8e. We assumed that, like the â-ami-
noketones, 8e underwent an elimination reaction to yield the
corresponding enone 1. This was supported by the fact that
γ-bromo ketone 8f, missing a â-positioned leaving group, was
not active. R-Fluoro ketones were, in general, weak electrophiles
exhibiting no binding affinity in our study (8d). All other
electrophilic compounds, involving the acrylic acid and propiolic
acid derivatives, keto-epoxide, and R-halo ketones, showed
comparable activity in our SAR study.
The toxicity, solubility, and permeability studies revealed the
drug-like character of the compounds investigated. Among the
subseries studied, we found strong variations in the drug-like
properties. All members of the acrylate group exhibited limited
solubility and permeability and moderate activity. Among the
acrylamide group, the only active compounds identified were
7a and 7b. These compounds were either not very soluble (7a)
or not very permeable (7b), as reflected by the weak cytotoxic
effects in the ARO and U2OS cell lines. Four of the eight enones
tested were not active. The active enones 1 and 4a-c showed
limited permeability but elevated cytotoxicity, especially in the
ARO cell line. In general, we observed high solubility but low
permeability for all compounds bearing a carboxylic acid
functionality (4a, 7b, 7f, and 7g). Compound 4a showed higher
cytotoxicity in ARO cells than did 7b, 7f, and 7g. Among the
active compounds of the electrophile group, three compounds
exhibited Pe values of >1000 × 10-6 cm/s (8c, 8e, and 8g).
Compound 8g was the most soluble compound, and 8e was the
most active one.
Experimental Section
General Considerations. Unless otherwise noted, all materials
were obtained from commercial suppliers and used without further
purification. All solvents used were dried using an aluminum oxide
column. Thin-layer chromatography was performed on precoated
silica gel 60 F254 plates. Purification of compounds was done by
reverse-phase high-performance liquid chromatography (RP-HPLC;
Flex [Biotage] and RP-C18 Xterra column 5 µm, 19 mm × 50
mm [Waters]) or by normal phase column chromatography (SP1
[Biotage], Silica gel 230-400 mesh) followed by evaporation (HT-
4X evaporator [Genevac]). The analysis (photodiode array (PDA),
total ion count, and expected mass [m/z]) was performed using an
HPLC-MS (Alliance HT, Micromass ZQ 4000 and RP-C18 Xterra
column 5 µm, 6 mm × 50 mm [Waters], flow rate: 1 mL/min;
gradient: 30:70 [methanol/water (0.05% trifluoroacetic acid)]) to
100% MeOH over 10 min. NMR spectra were 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).
General Procedure for 3a-k. To a suspension of AlCl3 (26.6
g, 200 mmol) in CH2Cl2 (200 mL) was added acryloyl chloride
(9.78 mL, 100 mmol) at 0 °C. The resulting clear solution was
treated with 1-phenylhexane (18.9 mL, 100 mmol) and stirred for
2 h at room temperature. The reaction was poured into a mixture
of concentrated HCl (40 mL) and ice (200 g). The organic layer
was separated, the aqueous layer was extracted with CH2Cl2 (100
mL), and the combined organic layers were washed with saturated
aqueous NaHCO3 (100 mL) and brine (100 mL) and dried over
MgSO4. The filtered solution was concentrated in vacuo to yield
compounds 1 and 2 (15.6 g, 70%) as a yellow oil in a ratio of 4:1.
Next, 9 g (45 mmol) of the crude product was dissolved in
tetrahydrofuran (THF) (30 mL) to obtain a 1.5 M stock solution.
The corresponding amines (3 mmol) were dissolved in THF (8 mL)
and treated with 2 mL of the stock solution (1.5 M solution of 1
and 2) and stirred at room temperature overnight. The reaction
mixtures were evaporated, and the residual amines were removed
by repeated azeotropic evaporations using ethanol to yield the
corresponding â-aminophenylketones in 85-97% yield with a
purity of 85-95%, as determined by HPLC-MS. Subsequent
purification by RP-HPLC (flow rate: 20 mL/min; gradient: 0.05%
formic acid in water to 0.05% formic acid in 80% acetonitrile over
15 min) resulted in the desired product (>99% purity). (Compound
3a is given as an example. For characterization of compounds 3b-
k, see Supporting Information.)
We observed the best permeability values within the group
of aminophenylketones (3d, 3e, 3g, and 3h). Compounds 3d
and 3h also were very soluble in PBS with 5% DMSO and
were able to inhibit the interaction between TRâ and SRC2-2
in concentrations less than 5 µM. Stability studies confirmed a
half-life of 1.4-2.4 h in plasma and no degradation in aqueous
solution over a period of 24 h.
Conclusion
This SAR study of small-molecule TR-CoA inhibitors
revealed compounds with a defined structure consisting of an
electrophilic head group, a hydrophobic core structure, and a
hydrophobic alkyl substituent. The most active compounds were
those bearing an n-heptyl or n-hexyl para substituent and having
1-(4-Hexylphenyl)-3-(methyl(phenethyl)amino)propan-1-
one (3a). White solid, mp 147-149 °C, yield 84%; LCMS RT )

5278 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Arnold et al.
1
5.02 min; H NMR (400 MHz) δ ) 7.73 (d, J ) 8.3 Hz, 2H),
Hz, 2H), 7.03 (d, J ) 8.5 Hz, 2H), 6.59 (dd, J ) 17.3 Hz, J ) 1.3
Hz, 1H), 6.31 (dd, J ) 17.3 Hz, J ) 10.4 Hz, 1H), 6.00 (dd, J )
10.4 Hz, J ) 1.3 Hz, 1H), 2.58 (t, J ) 7.4 Hz, 2H), 1.64 (h, J )
7.4 Hz, 2H), 0.94 (t, J ) 7.3 Hz, 3H); 13C NMR (100 MHz) δ )
164.71, 148.51, 140.26, 132.26, 129.31, 128.08, 121.10, 39.44,
24.51, 13.78; MS calcd for C12H14O2 (H+) 191.10, found 191.37.
General Procedure for 7a-g. To a solution of 4-hexylaniline
(216 µL, 1 mmol) and triethylamine (209 µL, 1.5 mmol, 1.5 equiv)
in DCM (5 mL) was added acid chloride (1.1 mmol) or acid
anhydride (1 mmol). The reaction mixture was stirred for 1 h,
poured into NH4Cl (aq) (10 mL), and diluted with ethyl acetate
(10 mL), and the organic layer was separated. The aqueous layer
was extracted with ethyl acetate (10 mL, twice), and the combined
organic layers were dried over MgSO4, filtered, and concentrated
in vacuo. Purification was done by flash column chromatography
(SP1, 25M (SiO2), flow rate: 25 mL/min; gradient: 1-30% ethyl
acetate in hexanes over 20 CV). 7a is given as an example. For
characterization of compounds 7b-g, see Supporting Information.
N-(4-Hexylphenyl)acrylamide (7a). White solid, mp 91-92 °C,
yield 88%; LCMS RT ) 8.15 min; 1H NMR (400 MHz) δ ) 7.37
(d, J ) 8.1 Hz, 2H), 7.30 (s, NH), 7.02 (d, J ) 8.1 Hz, 2H), 6.30
(dd, J ) 16.8 Hz, J ) 1.2 Hz, 1H), 6.13 (dd, J ) 16.8 Hz, J )
10.4 Hz, 1H), 5.62 (dd, J ) 10.6 Hz, J ) 1.2 Hz, 1H), 2.46 (t, J
) 7.6 Hz, 2H), 1.47 (p, J ) 7.1 Hz, 2H), 1.22-1.16 (m, 6H), 0.76
(t, J ) 6.8 Hz, 3H); 13C NMR (100 MHz) δ ) 163.44, 139.31,
135.30, 131.26, 128.86, 127.44, 120.00, 35.36, 31.69, 31.42, 28.89,
22.58, 14.06 MS calcd for C15H21NO (H+) 231.33, found 231.16.
General Procedure for 8a-b. To a solution of 4-hexylaniline
(178 µL, 1 mmol) or 4-hexylphenol (179 µL, 1 mmol), propiolic
acid (70 µL, 1.3 mmol, 1.3 equiv), and 4-dimethylaminopyridine
(0.01 mmol, 1.2 mg, 0.01 equiv) in DCM (5 mL) was added N,N′-
diisopropylcarbodiimide (126 µL, 1.3 mmol, 1.3 equiv). The
resulting reaction mixture was stirred at RT for 5 h, poured into
NH4Cl (aq) (10 mL), and diluted with ethyl acetate (10 mL), and
the organic layer was separated. The aqueous layer was extracted
with ethyl acetate (10 mL, twice), and the combined organic layers
were dried over MgSO4, filtered, and concentrated in vacuo.
Purification was done by flash column chromatography (SP1, 25M
(SiO2), flow rate: 25 mL/min; gradient: 1-15% ethyl acetate in
hexanes over 15 CV). 8a is given as an example. For characteriza-
tion of compound 8b, see Supporting Information.
7.19-7.06 (m, 8H), 3.56-3.51 (m, 1H), 3.45-3.33 (m, 3H), 3.08-
3.25 (m, 1H), 3.13-3.08 (m, 1H), 2.98-2.94 (m, 2H), 2.75 (s,
3H), 1.49-1.46 (m, 2H), 1.20-1.11 (m, 6H), 0.73 (t, J ) 6.9 Hz,
3H); 13C NMR (100 MHz) δ ) 195.32, 150.25, 135.37, 133.11,
129.10, 128.97, 128.66, 128.32, 127.51, 58.13, 51.36, 40.47, 36.04,
33.18, 31.61, 30.95, 30.43, 28.86, 22.53, 14.04; MS calcd for
C24H33NO (H+) 352.26, found 352.32.
General Procedure for 1, 4a, and 4g. See general procedure
for 3a-k. 1 is given as an example. For characterization of
compounds 4a and 4g, see Supporting Information.
1-(4-Hexylphenyl)prop-2-en-1-one (1). Colorless liquid, yield
55%; LCMS RT ) 6.90 min; H NMR (400 MHz) δ ) 7.88 (d, J
1
) 8.2 Hz, 2H), 7.28 (d, J ) 8.2 Hz, 2H), 7.17 (dd, J ) 17.1 Hz,
J ) 10.5 Hz, 1H), 6.43 (dd, J ) 17.1 Hz, J ) 1.6 Hz, 1H), 5.90
(dd, J ) 10.5 Hz, J ) 1.6 Hz, 1H), 2.67 (t, J ) 7.8 Hz, 2H), 1.63
(p, J ) 7.1 Hz, 2H), 1.36-1.27 (m, 6H), 0.88 (t, J ) 6,8 Hz, 3H);
13C NMR (100 MHz) δ ) 190.55, 148.82, 134.94, 132.41, 129.62,
128.86, 128.67, 36.02, 31.65, 31.06, 28.92, 22.56, 14.05; MS calcd
for C15H20O (H+) 217.15, found 217.82.
General Procedure for 5b-f. To a solution of 1-bromo-4-
heptylbenzene (408 µL, 2 mmol) in THF (3 mL) was added n-BuLi
(2M in pentane, 1.1 mL, 1.1 equiv.) at -78 °C. After stirring for
20 min, aldehyde (2 mmol, 1 equiv) was added, and the solution
was stirred for an additional 2 h at -78 °C. The reaction mixture
was quenched at -78 °C with NH4Cl (aq) (2 mL) and allowed to
warm to room temperature. The organic layer was separated. The
aqueous layer was extracted with ethyl acetate (3 mL, twice), and
the combined organic layers were dried over MgSO4, filtered, and
concentrated in vacuo. Purification was done by flash column
chromatography (SP1, 25M (SiO2), flow rate: 25 mL/min; gradi-
ent: 1-30% ethyl acetate in hexanes over 20 CV). 5b is given as
an example. For characterization of compounds 5c-f, see Sup-
porting Information.
(E)-1-(4-Heptylphenyl)but-2-en-1-ol (5b). Colorless liquid,
yield 51%; LCMS (E/Z) RT ) 7.75/8.28 min; 1H NMR (400 MHz)
δ ) 7.28 (d, J ) 7.9 Hz, 2H), 7.17 (d, J ) 8.1 Hz, 2H), 5.82-5.66
(m, 2H), 5.15-5.11 (m, 1H), 2.60 (t, J ) 7.5 Hz, 2H), 1.95 (m,
1H (OH)), 1.71 (m, 3H), 1.62 (m, 2H), 1.36-1.27 (m, 8H), 0.90
(t, J ) 6.9 Hz, 3H); 13C NMR (100 MHz) δ ) 142.25, 140.61,
133.68, 128.46, 127.07, 126.02, 75.04, 35.61, 31.79, 31.48, 29.27,
29.15, 22.64, 17.66, 14.07, MS calcd for C17H26O(H+) 247.20,
found 228.80 (-OH).
N-(4-Hexylphenyl)propiolamide (8a). Brown solid, mp 62-
1
64 °C, yield 79%; LCMS RT ) 8.43 min; H NMR (400 MHz) δ
General Procedure for 4b-f. To a solution of alcohol (0.36
mmol), 4-methylmorpholine N-oxide (65 mg, 0.54 mmol, 1.5
equiv), and molecular sieves (200 mg) in acetonitrile (3 mL) was
added tetrapropylammonium perruthenate (6.5 mg, 0.018 mmol,
0.05 equiv). The reaction mixture was stirred for 1-4 h, absorbed
onto silica gel, and purified by column chromatography (SP1, 25M
(SiO2), flow rate: 25 mL/min; gradient: 1-10% ethyl acetate in
hexanes over 18 CV). 4b is given as an example. For characteriza-
tion of compounds 4c-f, see Supporting Information.
(E)-1-(4-Heptylphenyl)but-2-en-1-one (4b). Colorless liquid,
yield 67%; LCMS RT ) 8.48 min; 1H NMR (400 MHz) δ ) 7.85
(d, J ) 8.3 Hz, 2H), 7.26 (d, J ) 8.4 Hz, 2H), 7.06 (m, 1H), 6.91
(m, 1H), 2.65 (t, J ) 8.0 Hz, 2H), 1.99 (d, J ) 6.8 Hz, 3H), 1.62
(m, 2H), 1.34-1.24 (m, 8H), 0.87 (t, J ) 7.0 Hz, 3H); 13C NMR
(100 MHz) δ ) 190.30, 148.35, 144.42, 135.50, 128.65, 128.55,
127.48, 35.99, 31.76, 31.14, 29.22, 29.12, 22.63, 18.57, 14.7; MS
calcd for C17H24O(H+) 245.77, found 245.77.
) 7.53 (s (br), NH), 7.41 (d, J ) 8.4 Hz, 2H), 7.14 (d, J ) 8.4 Hz,
2H), 2.91 (s, 1H), 3.57 (t, J ) 7.5 Hz, 2H), 1.58 (p, J ) 7.4 Hz,
2H), 1.35-1.23 (m, 6H), 0.88 (t, J ) 6.7 Hz, 3H); 13C NMR (100
MHz) δ ) 154.70, 140.19, 134.38, 129.00, 120.07, 75.64, 74.02,
35.38, 31.68, 31.37, 28.87, 22.58, 14.07; MS calcd for C15H19O
(H+) 229.15, found 229.80.
General Procedure for 8c and 8e. See general procedure for
3a-k. 8c is given as an example. For characterization of compound
8e, see Supporting Information.
2-Chloro-1-(4-hexylphenyl)ethanone (8c). White solid, mp 42-
1
44 °C, yield 84%; LCMS RT ) 6.72 min; H NMR (400 MHz) δ
) 7.88 (d, J ) 8.3 Hz, 2H), 7.30 (d, J ) 8.3 Hz, 2H), 4.69 (s, 2H),
2.67 (t, J ) 7.8 Hz, 2H), 1.63 (p, J ) 7.7 Hz, 2H), 1.36-1.27 (m,
6H), 0.88 (t, J ) 7.0 Hz, 3H); 13C NMR (100 MHz) δ ) 190.69,
149.99, 131.93, 128.92, 128.66, 45.95, 36.07, 31.62, 30.98, 28.90,
22.55, 14.05; MS calcd for C14H19ClO (H+) 238.11, found 238.80.
General Procedure for 8f. To a solution of 8e (296 mg, 1 mmol)
in benzene (3 mL) was added DBU (150 µL, 1.1 mmol, 1.1 equiv),
and the reaction mixture was stirred for 2 h at RT, filtered, and
evaporated to yield crude 1. The crude product was resuspended
in MeOH (5 mL) and treated with H2O2 (30% in H2O) (0.3 mL, 3
mmol, 3 equiv) and NaOH (1 M in H2O) (1 mL). The reaction
mixture was evaporated, dissolved in water, and extracted with ethyl
acetate (20 mL, twice), and the combined organic layers were dried
over MgSO4, filtered, and concentrated in vacuo. Purification was
done by flash column chromatography (SP1, 25M (SiO2), flow
rate: 25 mL/min; gradient: 1% to 15% ethyl acetate in hexanes
over 15 CV).
General Procedure for 6a-m. A solution of triethylamine (83
µL, 0.6 mmol, 1.1 equiv) and alcohol (0.5 mmol) in dichlo-
romethane (DCM) (2 mL) was treated with acryloyl chloride (49
µL, 0.6 mmol, 1.1 equiv) and stirred for 1 h at room temperature
(RT). The reaction mixture was absorbed on a silica cartridge and
purified by flash column chromatography (SP1, 25S (SiO2), flow
rate: 25 mL/min; gradient: 1-20% ethyl acetate in hexanes over
20 CV). (6a is given as an example. For characterization of
compounds 6b-m, see Supporting Information).
4-Propylphenyl Acrylate (6a). Colorless liquid, yield 85%;
LCMS RT ) 6.18 min; 1H NMR (400 MHz) δ ) 7.18 (d, J ) 8.6

Inhibitors of TR and CoA Interaction
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5279
(4-Hexylphenyl)(oxiran-2-yl)methanone (8f). Colorless liquid,
yield 74%; LCMS RT ) 7.20 min; 1H-NMR (400 MHz) δ ) 7.97
(d, J ) 8.1 Hz, 2H), 7.30 (d, J ) 8.1 Hz, 2H), 4.23 (m, 1H), 3.11
(m, 1H), 2.97 (m, 1H), 2.67 (t, J ) 7.9 Hz, 2H), 1.63 (p, J ) 7.1
Hz, 2H), 1.36-1.27 (m, 6H), 0.88 (t, J ) 6.9 Hz, 3H); 13C NMR
(100 MHz) δ ) 194.12, 149.91, 133.14, 128.85, 128.46, 50.97,
47.49, 36.06, 31.60, 30.97, 28.87, 22.57, 14.03; MS calcd for
C15H20O2 (H+) 233.15, found 233.15.
PAMPA Procedure. The PAMPA procedure was conducted
using a published method.35,36 Briefly, all liquid-handling steps for
the PAMPA assay were performed on a Biomek FX Laboratory
Automation Workstation (Beckman-Coulter) and analyzed by
pION’s (London, U.K.) PAMPA Evolution 96 Command Software.
The PAMPA Evolution 96 Permeability Assay Kit includes acceptor
sink buffer (ASB), double-sink lipid solution, and a PAMPA
sandwich plate, preloaded with magnetic disks. For each experiment,
4 µL of lipid was transferred onto the support membrane in the
acceptor well, followed by the 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 measuring the UV spectra from 200 to 500 nm
using the SpectraMax reader (Molecular Devices). The permeability
coefficient was determined using the maximum absorbance from
200 to 500 nm using the following formula:
TR-SRC2-2 Competition Assay. This assay has been described
in detail previously.19,29 The concentration of each compound
required to inhibit 50% of the binding between the TRâ LBD and
the SRC2-2 peptide is presented in Tables 1-5. hTRâ1 LBD (His6
T209-D461) was expressed in BL21 (DE3) (Invitrogen), and the
peptide SRC2-2 (CLKEKHKILHRLLQDSSSPV) was labeled with
5-iodoacetamidofluorescien (Molecular Probes). The competition
binding experiments were evaluated using PrismPad, and the IC50
values were obtained by fitting the data to an equation (Sigmoidal
dose-response (variable slope) or four parameter logistic equa-
tion): Y ) bottom + (top - bottom)/(1 + 10((log IC - X)‚HillSlope)),
50
where X is the logarithm of concentration, and Y is the response.
Two independent experiments were done in quadruplicate, and the
values are given as the mean values with a 95% confidence interval.
Toxicity Study. Human bone osteosarcoma epithelial cells
(U2OS; American type culture collection (ATCC) HTB-96) were
grown to 80% confluency at 37 °C in McCoy’s 5A medium with
1.5 mM L-glutamine adjusted to contain 2.2 g/L sodium bicarbonate,
10% fetal bovine serum (ATCC), 50 units/mL penicillin, and 50
µg/mL streptomycin. Human anaplastic thyroid cancer cells (ARO,
kindly provided by Dr. M. D. Ringel) were maintained in RPMI
1640 culture medium supplemented with 10% fetal bovine serum
(heat inactivated), 1× nonessential amino acid (ATCC), 10% fetal
bovine serum (ATCC), 50 units/mL penicillin, and 50 µg/mL
streptomycin. Cells were grown to 80% confluency, collected, and
resuspended at a concentration of 57 000 cells/mL dispensed in
384-well plates at 35 µL/well (2000 cells). The plates were
incubated for 48 h at 37 °C. The small molecules were serially
diluted from 50 000 µM to 2.5 µM in DMSO into a 96-well plate
(Costar 3365), and 0.5 µL was transferred into the 384-well cell
culture plates (Corning 3917), yielding a final compound concentra-
tion range of 714-0.036 µM. After 48 h of incubation at 37 °C,
25 µL of CellTiter-Glo was added using a Wellmate (Matrix),
followed by agitation using an MTS 2/4 digital microtiter shaker
(IKA). Luminescence was measured after 10 min using an EnVision
(Perkin-Elmer). The concentration of each compound required for
50% cell survival (LD50) is presented in Tables 1-5. These LD50
values were obtained by fitting the data to the following equation
(Sigmoidal dose-response (variable slope) or four parameter
logistic equation): Y ) bottom + (top - bottom)/(1 + 10((log
LD50 - X)‚HillSlope)), where X is the logarithm of concentration, and Y
is the response (PrismPad). Two independent experiments were
done in triplicate, and the values are given as the mean values with
a 95% confidence interval.
Solubility Study. The assay was done using the MultiScreen
Solubility assay developed by Millipore. Briefly, 16 test compounds
per 96-well polypropylene plate (Costar 3365) were serial diluted
from 10 000 to 625 µM in DMSO starting from columns 1-5 and
7-11. Columns 6 and 12 were filled with DMSO. From each well,
5 µL was transferred into the 96-well disposable UV-Star (Greiner
Bio-One). Acetonitrile (97.5 µL) and PBS buffer (97.5 µL) were
added to each well, and the plate was agitated for 30 min (IKA
microtiter plate shaker). The UV spectra from 200-500 nm was
measured for all wells and subtracted from the background
(DMSO). The correlations between concentrations and the absor-
bance at 260, 280, and 300 nm were determined as slopes. Then,
5 µL from 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 (Amax filtrate/
slope) was determined for all three wavelengths, and values are
given as the means with 95% confidence intervals.
Pe ) 2.3VD/[A(t - tLAG)] log{1/(1 - R)‚CD(t)/CD(0)}
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 a medium
permeability standard, and ranitidine (Pe ) 2.3 × 10-6 cm/s) as a
low permeability standard. The compounds were measured in
triplicate, and the values are given as mean values with 95%
confidence intervals.
Acknowledgment. This work was supported by the NIH
(R01 No. DK58080) and the American Lebanese Syrian
Associated Charities (ALSAC) and St. Jude Children’s Research
Hospital (SJCRH). We thank Naoaki Fuji for suggestions and
comments, David Galloway in Scientific Editing for review of
the manuscript, and the Hartwell Center for assistance in the
preparation of peptides.
Supporting Information Available: Experimental procedures
and characterization data for 3b-k, 4a-g, 5c-f, 6c-f, 6b-m,
7b-g, 8b, and 8e. This material is available free of charge via the
Internet at http://pubs.acs.org.
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