Discovery of nonpeptidic small-molecule AP-1 inhibitors: Lead hopping based on a three-dimensional pharmacophore model

Article abstract of DOI:10.1021/jm050550d

We designed and synthesized small-molecule activator protein-1 (AP-1) inhibitors based on a three-dimensional (3D) pharmacophore model that we had previously derived from a cyclic decapeptide exhibiting AP-1 inhibitory activity. New AP-1 inhibitors with a

Full text of DOI:10.1021/jm050550d

80
J. Med. Chem. 2006, 49, 80-91
Discovery of Nonpeptidic Small-Molecule AP-1 Inhibitors: Lead Hopping Based on a
Three-Dimensional Pharmacophore Model
Keiichi Tsuchida,*,† Hisaaki Chaki,‡ Tadakazu Takakura,† Hironori Kotsubo,‡ Tadashi Tanaka,† Yukihiko Aikawa,‡
Shunichi Shiozawa,#, and Shuichi Hirono§
DiscoVery Laboratories and Research Laboratories, Toyama Chemical Co., Ltd., 4-1 Shimookui 2-chome, Toyama 930-8508, Japan,
Department of Rheumatology, Faculty of Health Science, Kobe UniVersity School of Medicine, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142,
Japan, Rheumatic Disease DiVision, Kobe UniVersity Hospital, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan, and School of
Pharmaceutical Sciences, Kitasato UniVersity, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
ReceiVed June 13, 2005
We designed and synthesized small-molecule activator protein-1 (AP-1) inhibitors based on a three-
dimensional (3D) pharmacophore model that we had previously derived from a cyclic decapeptide exhibiting
AP-1 inhibitory activity. New AP-1 inhibitors with a 1-thia-4-azaspiro[4.5]decane or a benzophenone scaffold,
which inhibit the DNA-binding and transactivation activities of AP-1, were discovered using a “lead hopping”
procedure. An additional investigation of the benzophenone analogues confirmed the reliability of the
pharmacophore model, its utility to discover AP-1 inhibitors, and the potency of the benzophenone derivatives
as a lead series.
Introduction
Activator protein-1 (AP-1) is a transcription factor that has a
crucial role in cellular signal transduction, as it is responsible
for the induction of a number of genes that are involved in cell
proliferation, differentiation, and immune and inflammatory
responses.1,2 It has been implicated in various diseases, such as
rheumatoid arthritis.3,4
AP-1 contains members of the Fos and Jun families, which
form either Jun-Jun homodimers or Fos-Jun heterodimers and
bind to the consensus DNA sequence 5′-TGAGTCA-3′, which
is known as the AP-1 binding site.1 An investigation of the X-ray
crystal structure of the basic region-leucine zipper (bZIP)
domains of c-Fos and c-Jun bound to a DNA fragment
containing the AP-1 binding site revealed that both domains
form continuous R-helices, and the heterodimer grips the major
groove of the DNA, similar to a pair of forceps.5
Natural products such as curcumin,6 dihydroguaiaretic acid,7
Figure 1. Binding model of peptide 1 (yellow) resulting from MD
simulation. Ribbon representation of the basic domains (c-Fos, cyan;
and an anthraquinone derivative8 were reported to inhibit the
c-Jun, magenta). The residues of c-Fos and c-Jun that are involved in
binding of AP-1 to the AP-1 binding site. However, three-
interactions are labeled with one-letter codes, and the residues of peptide
1 that are involved in interactions are labeled with three-letter codes.
dimensional (3D) structural information about the AP-1 binding
of these inhibitors that is necessary for structure-based drug
design is not well-known. In a previous report,9 we discovered
a new cyclic disulfide decapeptide Ac-cyclo[Cys-Gly-Gln-Leu-
Asp-Leu-Ala-Asp-Gly-Cys]-NH2 (peptide 1) that exhibits AP-1
inhibitory activity, using a de novo approach that exploited
molecular modeling methods, such as molecular dynamics (MD)
simulations, and docking studies based on the 3D structure of
the bZIP domains derived from the X-ray structure.5 Further-
more, we built a 3D pharmacophore model based on the
chemical and structural features of peptide 1. These data were
obtained from an alanine scan and structural studies involving
a combination of MD simulation of the bZIP-peptide 1 complex
Red broken lines indicate putative hydrogen bonds and green broken
lines indicate putative hydrophobic interactions. Hydrogen atoms are
not shown for clarity.
with explicit water molecules (Figure 1) and NMR measure-
ments of the peptide in water.9
Peptides generally have unfavorable properties for therapeutic
drugs, such as poor bioavailability.10 Several approaches have
been reported to convert bioactive peptides into nonpeptidic drug
candidates known as peptidomimetics.10-12 In many cases, these
peptidomimetics are peptide-like molecules, such as peptoids,
and so further modifications are essential.
To avoid these problems, we designed nonpeptidic small
molecules using a molecular modeling method based on a 3D
pharmacophore model. This procedure could be described as
“lead hopping” and has been employed as part of several recent
in silico approaches.13 There are two general computational
approaches, de novo design and 3D database searching, to
identify new lead candidates with desired biological activity
using a pharmacophore model.14 Although several successful
* To whom correspondence should be addressed. Phone: +81-76-431-
8218.
Fax:
+81-76-431-8208.
E-mail:
keiichi_tuchida@
toyama-chemical.co.jp.
† Discovery Laboratories, Toyama Chemical Co., Ltd.
‡ Research Laboratories, Toyama Chemical Co., Ltd.
# Kobe University School of Medicine.
Kobe University Hospital.
§ Kitasato University.
10.1021/jm050550d CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/01/2005

Nonpeptidic Small-Molecule AP-1 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 81
Figure 3. Fitting images of two scaffolds, 1-thia-4-azaspiro[4.5]decane
(a) and benzophenone (b) to the trapezoid shown in Figure 2.
Figure 2. Pharmacophore model derived from peptide 1. The phar-
macophoric residues and the supplementary parts are colored yellow
and gray, respectively. The pharmacophoric elements on peptide 1 are
shown as circles and an ellipse in magenta. The main chain forms a
trapezoid shape, the dimensions of which are shown by the arrows.
An anthracene molecule of the same scale is depicted in green for a
comparison of the sizes. Hydrogen atoms are not shown for clarity.
examples of these have been reported, the problems inherent
in them have also been pointed out.15,16 In de novo design,
output structures are particularly apt to be difficult to synthe-
size.15,16 In 3D database searching, novel structures cannot be
obtained.15 Thus, we chose to search the compound library in
our company to identify synthetically accessible scaffolds before
general 3D database searching. We succeeded in finding several
good scaffolds in the library. These scaffolds are synthetically
accessible and easy to modify by our organic chemists. Through
design efforts based on these scaffolds, we discovered new AP-1
inhibitors: 1-thia-4-azaspiro[4.5]decane and benzophenone
derivatives.
Figure 4. Illustration of the lead hopping of benzophenone. The
superposition of the pharmacophoric residues of peptide 1 and the
benzophenone scaffold is shown. The pharmacophoric elements on
peptide 1 are shown as magenta-colored circles and an ellipse. The
connections of the substituents from benzophenone are denoted by
curved arrows.
as an anthracene molecule, which was therefore assumed to be
the maximum size for a scaffold.
Although the main chain of the five pharmacophoric residues
of peptide 1 had a planar trapezoid shape, the pharmacophoric
elements were not located on the same plane. Thus, to maintain
the pharmacophoric arrangement, it was desirable that the
scaffold was not planar, but rather was twisted and compact,
within limits.
In the current paper, we describe the design by a lead-hopping
strategy and synthesis of nonpeptidic AP-1 inhibitors and their
inhibitory activities as evaluated by AP-1 binding and cell-based
reporter gene assays.
On the basis of these considerations, by searching our
compound library, we selected the two scaffolds that are
depicted in Figure 3: 1-thia-4-azaspiro[4.5]decane (a), and
benzophenone (b) scaffolds.
The Design of Nonpeptidic AP-1 Inhibitors. Lead-Hopping
Strategy. Our pharmacophore model of AP-1 binding com-
pounds is illustrated in Figure 2, which shows the 3D arrange-
ment of the side-chain functional groups of the Gln-Leu-Asp-
Leu-Ala residues in peptide 1.9 A visual inspection revealed
that the main chain of the five residues of peptide 1 has a â-turn-
like conformation and a trapezoid shape (Figure 2). Its dimen-
sions, which were measured as the distances between the
R-carbons of Gln3-Leu4, Leu4-Asp5, Asp5-Ala7, and Gln3-
Ala7, are 3.89, 3.89, 5.99, and 8.40 Å, respectively. Thus, we
decided to design candidate small molecules for synthesis and
evaluation by combining a suitably sized scaffold placed onto
the trapezoid structure with the corresponding pharmacophoric
elements, which constitute pivotal functional groups within the
side chains of the five pharmacophoric residues of peptide 1,
to retain the 3D arrangement in the pharmacophore model.
Scaffold Design. In general, it is assumed that a scaffold
should have a certain level of rigidity to maintain the correct
spatial arrangement of each of the pharmacophoric elements.
In addition, the substitution position to which a pharmacophoric
element might be connected via a suitable linker should be
flexible to ensure synthetic feasibility. In a different perspective,
at least three substituents for each pharmacophoric element could
be introduced that would allow sufficient pharmacophoric
interactions. As depicted in Figure 2, the trapezoid was as large
Definition of Pharmacophoric Elements. Four pharma-
cophoric elements were defined from the five residues of the
pharmacophore model: the carboxamide group of Gln3, the
isobutyl group of Leu4, the carboxyl group of Asp5, and an
isobutyl group that was adopted as a proxy for Leu6 and Ala7.
The reason for using a proxy was that the two residues in
question were adjacent to one another and could be regarded
as one large hydrophobic element. Among these, the carboxyl
group of Asp5 was regarded as a key pharmacophoric element,
because substitution of the Asp5 had a significant effect in a
previous alanine scan experiment;9 we therefore assumed that
an acidic group at this position was essential for interactions
with the basic regions of the bZIP domains. In addition, this
carboxyl group appeared to be the only charged group and might
have conferred desirable features in terms of drug design. We
therefore produced various combinations of three or four
pharmacophoric elements, while ensuring that a carboxyl group
was incorporated into the newly designed molecules.
Scaffold Placement and Substitution Points. Only those
atoms on a scaffold for which the introduction of a suitable
substituent was synthetically feasible were viewed as potential

82 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Tsuchida et al.
Table 1. Chemical Structure of Compounds 2-5 and Their Inhibitory Activities against AP-1
a Inhibition of the binding of the AP-1 bZIP peptide to synthetic oligonucleotides containing the AP-1 binding site. b Inhibition of the expression of
AP-1-luciferase by TPA-stimulated NIH3T3 cells. c NT: not tested.
substitution points. Each scaffold was manually placed onto the
trapezoid depicted in Figure 2 and interactively checked using
a visual display. The position and orientation of the scaffolds
were designed so that as many substitution points as possible
were pointed in a favorable direction toward the corresponding
pharmacophoric elements.
Combination of Scaffold and Pharmacophore Elements.
The candidate molecules for the synthesis were constructed by
connecting the relevant pharmacophoric elements to suitable
substitution points on each scaffold via linkers.
An example of substituent introduction featuring benzophe-
none is shown in Figure 4. The connections between the
pharmacophoric elements at suitable substitution points are
indicated by curved arrows. In this case, the pharmacophoric
elements corresponding to Gln3, Leu4, and Asp5 could be
connected by any atoms on the left-hand ring of the scaffold
via linkers. Likewise, the centroid of Leu6 and Ala7 could be
connected from any atom on the right-hand ring. The lengths
and types of linkers used were carefully selected, taking
synthetic feasibility into consideration.
Computational Evaluation of Designed Molecules. We
examined the various possible combinations of substitution
points, and the lengths and types of linkers, so that each
pharmacophoric element was well suited to the pharmacophore
model. Each of the designed molecules was computationally
evaluated to verify the fact that the 3D pharmacophore require-
ments were met by a low-energy conformer. Conformational
analyses were carried out to obtain low-energy conformers. All
unique conformers that were within 6 kcal mol-1 of the lowest
energy were collected for each of the designed molecules. The
selected low-energy conformers were then systematically su-
perimposed onto peptide 1 using the least-squares fit17 of three
or four of the pharmacophoric atoms: the δ-carbon atom of
Gln3, the γ-carbon atoms of Leu4 and Asp5, and the centroid
of the γ- and δ-carbon atoms of Leu6 and the â-carbon atom
of Ala7. In addition, the overlapping volume between each
conformer and the corresponding pharmacophoric residues in
peptide 1, and its ratio to the volume of the corresponding
residues in peptide 1, were calculated. Those compounds in
which at least one conformer had a root-mean-square deviation

Nonpeptidic Small-Molecule AP-1 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 83
Table 2. Benzophenone Derivatives 4, 6-13 and Their Inhibitory Activities in Binding Assay
% inhibition
at 500 µM
compd
R1
R2
R3
at 1 mM
IC50 (µM)a
4
6
7
8
OiBu
H
OiBu
OiBu
H
CH2CH2CHO2H
CH2CH2CHO2H
CH2CH2CHO2H
H
CH2CH2CHO2H
CH2CH2CHO2H
CH2CH2CHO2H
CH2CH2CHO2H
CH2CH2CONH2
28
10
7
91
14
9
610
>2000
>2000
NDc
910
OiBu
OiBu
OnPr
OiBu
OCH2Ph
OiBu
OiBu
OiBu
OiBu
OnPr
OiBu
OCH2Ph
OiBu
3
-
b
9
26
25
51
45
5
55
42
97
10
11
12
13
930
420
b
-
-
NDc
NDc
b
a 50% inhibition concentration was determined from the concentration-response curve (125-2000 µM). b % inhibition was not determined at this
concentration due to precipitation. c ND: not determined.
Scheme 1a
Scheme 2a
a Reagents: (a) (EtO)2P(O)CH2CO2Et, NaH, DMF; (b) H2, Pd-C, EtOH;
(c) oxalyl chloride, DMF, CH2Cl2; (d) isobutoxybenzene, AlCl3, CH2Cl2;
(e) NaOH-H2O, EtOH.
Syntheses of mono- and di-isobutoxy derivatives of 3-(3-
benzoylphenyl)propionic acid (4-7) were carried out as il-
lustrated in Schemes 2-4. 3-[3-(4-Isobutoxybenzoyl)phenyl]-
propionic acid (6) was prepared via the Horner-Emmons
reaction of benzyl 3-formylbenzoate (19) with ethyl dieth-
ylphosphonoacetate and NaH, which was followed by Pd-C-
catalyzed hydrogenation to yield 3-(ethoxycarbonylethyl)benzoic
acid (20). The corresponding acid chloride generated by
treatment with oxalyl chloride was allowed to react with
isobutoxybenzene19 in the presence of AlCl3 in CH2Cl2 to give
an ethyl ester, which on alkaline ester hydrolysis provided
compound 6 (Scheme 2). Compound 7 (Scheme 3), which is a
regioisomer of the isobutoxy group of 6, was prepared via
isobutyl 3-(2-isobutyloxyphenyl)propionate (22), which was
subjected to a regioselective Friedel-Crafts benzoylation.
Compound 22 was also employed for the synthesis of 3-[2-
isobutoxy-5-(4-isobutoxybenzoyl)phenyl]propionic acid (4). The
regioselective formylation reaction20 of 22 followed by Na-
ClO2-H2O2 oxidation21 afforded the monocarboxylic acid 23,
and coupling its acyl chloride with isobutoxybenzene afforded
compound 4. The regioisomer 5, 3-[2-isobutoxy-5-(3-isobu-
toxybenzoyl)phenyl]propionic acid, was prepared via coupling
of 22 with the acyl chloride of 3-isobutoxybenzoic acid (25) in
the presence of AlCl3 (Scheme 4).
Exchange of either one of the two isobutyloxy groups in 4
with a propyloxy or benzyloxy ether moiety was also considered
as outlined in Scheme 5. The methyl ester of 4 (compound 26)
was subjected to AlCl3-catalyzed lactonization accompanied by
cleavage of the isobutoxy linkage to produce 6-(4-hydroxyben-
zoyl)-3,4-dihydrocoumarin (27). The liberated phenolic hydroxyl
group was then subjected to the Mitsunobu reaction with R2-
OH (2-methylpropanol, propanol, or benzyl alcohol) in the
presence of diisopropyl azodicarboxylate (DIAD) and triph-
enylphosphine in tetrahydrofuran (THF) followed by MeONa-
a Reagents: (a) Me2CHCH2CH2CH2PPh3Br (for 15) or Me2CHCH2CH2-
PPh3I (for 17), n-butyllithium, THF; (b) 6 M HCl, 1,4-dioxane; (c)
L-cysteine, aq EtOH; (d) 4-methylpentanoic acid (for 2) or 5-methylhexanoic
acid (for 3), oxalyl chloride, Et3N, CH2Cl2.
(RMSD) value e 1.0 Å under a three- or four-point superposi-
tion with the corresponding residues of peptide 1, and an
overlapping volume ratio g 0.5, were selected as small-molecule
inhibitor candidates for synthesis and biological evaluation.
Biological Evaluation of the Compounds Synthesized. The
AP-1 inhibitory activities of the compounds synthesized were
evaluated using enzyme-linked immunosorbent assay (ELISA)-
based AP-1 DNA-binding and cell-based reporter assays (Table
1; compounds 2-5).
Furthermore, the synthesis of benzophenone derivatives and
the evaluation of their inhibitory activities were performed to
verify our pharmacophore model (Table 2; compounds 6-13).
Chemistry
Synthesis of the (R)-4-(4-methylpentanoyl)-1-thia-4-azaspiro-
[4.5]decane compound 2 was carried out as outlined in Scheme
1. The starting material, 1,4-cyclohexanedione monoethylene
ketal (14), was subjected to the Wittig reaction with 4-meth-
ylpentyltriphenylphosphonium bromide and n-butyllithium to
afford 4-(4-methylpentylidene)cyclohexanone (15) after an acid-
catalyzed deprotection procedure. Treatment of 15 with L-
cysteine in aqueous EtOH produced the spiro[cyclohexane-1,2′-
thiazolidine] compound 16, which was treated with 4-methyl-
pentanoyl chloride in the presence of Et3N to provide the target
molecule 2.18 The structurally analogous spiro compound 3,
1-thia-4-azaspiro[4.5]decane-3-carboxylic acid, 8-(3-methylbu-
tylidene)-4-(5-methylhexanoyl), was prepared by the same
procedure.

84 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Tsuchida et al.
Scheme 3a
a Reagents: (a) isobutyl bromide, K2CO3, DMF; (b) benzoyl chloride, AlCl3, CH2Cl2; (c) NaOH-H2O, acetone (for 4) or EtOH (for 7); (d) Cl2CHOMe,
AlCl3, CH2Cl2, then MeOH, ∆; (e) NaClO2, H2O2, NaH2PO4, aq. MeCN; (f) oxalyl chloride, DMF, CH2Cl2; (g) isobutoxybenzene, AlCl3, CH2Cl2.
Scheme 4a
a Reagents: (a) isobutyl bromide, K2CO3, DMF; (b) NaOH-H2O, acetone; (c) oxalyl chloride, DMF, CH2Cl2; (d) compound 22, AlCl3, CH2Cl2.
Scheme 5a
a Reagents: (a) AlCl3, 1,2-dichloroethane; (b) R2-OH, DIAD, Ph3P, THF; (c) MeONa-MeOH, THF; (d) R1-Br, K2CO3, DMF; (e) NaOH-H2O, acetone;
(f) isobutyl bromide, K2CO3, DMF.
catalyzed methanolysis to yield the corresponding alkyl ethers
28-30. Compound 28 was alkylated with R1-Br (propyl bromide
or benzyl bromide), followed by hydrolysis with alkali to yield
9 and 11 as depicted in Scheme 5. The regioisomeric analogues
10 and 12 were prepared similarly via 29 (R2 ) propyl) and 30
(R2 ) benzyl), respectively.
Scheme 6a
Synthesis of carboxamide of 4 (compound 13) was carried
out as outlined in Scheme 6. Compound 4 was treated with
oxalyl chloride and catalytic DMF leading to the acid chloride,
a Reagents: (a) oxalyl chloride, DMF, THF, then NH4OH.

Nonpeptidic Small-Molecule AP-1 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 85
Figure 5. Stereoview of the superposition of Leu-Asp-Leu-Ala of peptide 1 (yellow) with compound 2 (white). The fitted points are shown as
magenta-colored circles and an ellipse. Hydrogen atoms are not shown for clarity.
Figure 6. Stereoviews of the superposition of Leu-Asp-Leu-Ala of peptide 1 (yellow) with (a) compound 4 (white) and (b) compound 11 (white).
The fitted points are shown as magenta-colored circles and an ellipse. Hydrogen atoms are not shown for clarity.
which was converted to propionamide 13 with ammonium
hydroxide. Bis(4-isobutoxyphenyl)methanone (8) was prepared
from commercially purchased bis(4-hydroxyphenyl)methanone
by O-isobutylation with isobutyl bromide and K2CO3 in DMF.
points within compound 2 were the carbon atom of the carboxyl
group plus the carbon atoms at the branched positions of the
4-methylpentanoyl and 4-methylpentylidene groups. The RMSD
value resulting from the superposition of the three pharmacoph-
oric points was 0.89 Å, and the energy of this best-fitted
Results and Discussion
conformer relative to the lowest energy was 2.01 kcal mol-1
.
The three overlapped points within compound 4 were the carbon
atom of the carboxyl group plus the carbon atoms at the
branched positions of the isobutyl groups. The RMSD value
resulting from the superposition of the three pharmacophoric
points was 0.92 Å, and the energy of this best-fitted conformer
relative to the lowest energy was 2.46 kcal mol-1. The structural
isomers 2 and 3, in which the lengths of methylene linkers
between the 1-thia-4-azaspiro[4.5]decane scaffold and each
isobutyl pharmacophore element were different, showed similar
activity. The RMSD value obtained by the best-fit superposition
of compound 3 and peptide 1 was 0.64 Å, and the energy of
the best-fitted conformer relative to the lowest energy was 1.73
kcal mol-1; thus the 3D pharmacophore requirement was met
by compound 3, as well as compound 2. Because compounds
Evaluation of Designed Compounds. The designed com-
pounds were synthesized and evaluated using an ELISA-based
AP-1 DNA-binding assay. Among these, compounds 2 and 3
bearing 1-thia-4-azaspiro[4.5]decane as a scaffold, and com-
pounds 4 and 5, bearing benzophenone as a scaffold, inhibited
the binding of AP-1 bZIP and oligonucleotides containing the
AP-1 binding site (Table 1; IC50 ) 460-650 µM). The three
pharmacophoric elements of these compounds corresponded to
Leu4, Asp5, and the centroid of Leu6 and Ala7 of peptide 1.
As examples, the best-fit superpositions of compounds 2 and 4
onto peptide 1 based on the pharmacophore model are shown
in Figures 5 and 6a, respectively. The three pairs of pharma-
cophoric points that were used for overlapping are shown as
circles and an ellipse in these figures. The three overlapped

86 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Tsuchida et al.
4 and 5, in which only the substitution position of an isobutoxy
moiety corresponding to the centroid of Leu6 and Ala7 differed,
showed similar activity, there might be some allowance in the
hydrophobic region of the pharmacophore. This was consistent
with our assumption that Leu6 and Ala7 comprised one large
hydrophobic element. These small-molecule compounds showed
lower inhibitory activity than peptide 1 (IC50 ) 64 µM). The
results would be acceptable, given that these compounds have
only three pharmacophore elements compared to the cyclic
decapeptide.
Subsequently, we evaluated the effects of these compounds
on the transactivation activity of AP-1 using a cell-based AP-1
reporter gene assay (Table 1). Compounds 2-5 showed dose-
dependent inhibitory activities in this assay, similar to AP-1
DNA-binding inhibition. Thus, taking into account both the
inhibitory activities and synthetic feasibility, we carried out
further structural development of the benzophenone derivatives.
Conversion of Substituents on Benzophenone Derivatives.
To verify the pharmacophoric elements, we designed and
synthesized several derivatives based on compound 4, and
evaluated their inhibitory activities on AP-1 DNA-binding
(Table 2).
derivatives, were discovered using this combination of compu-
tational methods and medicinal chemistry intuition. Our study
demonstrated the reliability of the 3D pharmacophore model
and its utility in the discovery of AP-1 inhibitors. Additionally,
the possibility of further improvement of the benzophenone
derivatives was suggested by the results of the conversion of
the substituents. The benzophenone derivatives were considered
to be an attractive lead series, because they exhibited AP-1
inhibitory activities not only in the binding assay but also in
the cell-based assay. The continuous optimization of this series
might produce drugs that can be used against AP-1 for the
treatment of various diseases.
Experimental Section
Molecular Modeling. All molecular modeling was performed
using the SYBYL software package version 6.522 running on a
Silicon Graphics Power Indigo2 workstation. All of the compounds
designed for use in this study were built using the SKETCH module.
The carboxyl group of the designed compounds was deprotonated
to imitate physiological conditions. The designed compounds were
minimized using the Powell method in the MAXIMIN2 module of
SYBYL to reach a final convergence of 0.01 kcal mol-1 Å-1; the
MMFF94 force field and the MMFF94 charges23 were employed
for the energy minimizations. Electrostatic interactions were taken
into consideration using a distance-based dielectric constant of 78.3
to simulate an aqueous environment.
Conformational analysis of each of the designed compounds was
carried out using the Random Search module implemented in
SYBYL. An absolute energy cutoff of 300 kcal mol-1 was used to
remove high energy conformations. The energy values during the
optimization were computed with the MMFF94 force field and the
MMFF94 charges. The number of maximum cycles was set to 1000.
Within each cycle, the bonds that were defined as rotatable were
set to random torsion angles and the compounds were then
minimized. The number of maximum hits was set to 6. Unique
conformers were distinguished by setting a RMS threshold of 0.2
Å, with chirality checking if necessary. The systematical superposi-
tion of collected low-energy conformers was carried out using the
FIT routine in SYBYL.
The overlapping volume and the volume of the corresponding
residues in peptide 1 as a template were calculated using the
MVOLUME and VOLUME routines in SYBYL, respectively.
Chemistry. Melting points were determined using a B-545
melting point apparatus (Bu¨chi Lab., Flawil, Switzerland) and were
uncorrected. Proton NMR (1H NMR) spectra were recorded on a
JEOL JNM-AL 400 spectrometer (JEOL Ltd., Tokyo, Japan) using
tetramethylsilane as the internal standard. Chemical shifts are
reported in ppm (δ units), and the signal patterns are designated as
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br
(broad). Column chromatography was carried out using PSQ100B
silica gel of 75-200 µm (Fuji Silysia Chemical, Aichi, Japan). Low-
resolution mass spectra (MS) were obtained on a Hitachi M-8000
mass spectrometer system equipped with an electrospray ionization
interface (Hitachi Ltd., Tokyo, Japan). Elemental analysis (C, H,
and N) was performed at Toyama Industrial Technology Center
(Toyama, Japan), and all results were within (0.4% of the
theoretical values. Unless otherwise noted, all solvents, chemicals,
and reagents were obtained commercially and used without
purification. Peptide 1 has been previously described.9
4-(4-Methylpentylidene)cyclohexanone (15). To a stirred and
cooled (-45 °C) suspension of 4-methylpentyltriphenylphospho-
nium bromide (13.1 g, 30.7 mmol) in THF (40 mL) was added
dropwise n-butyllithium (1.6 M in hexane, 19.2 mL, 30.7 mmol).
The mixture was then warmed to 0 °C over 20 min. A solution of
1,4-cyclohexanedione monoethylene ketal (14) (4.00 g, 25.6 mmol)
in THF (20 mL) was added dropwise to the resulting phosphorane
solution at 0-5 °C. Subsequently, the reaction mixture was allowed
to warm to room temperature and was stirred for 1 h. The mixture
was quenched with water (100 mL) and extracted with EtOAc (1
The conversion of each substituent of compound 4 to
hydrogen (compounds 6-8) resulted in a considerable loss of
inhibitory activity in the binding assay; thus, the importance of
the three pharmacophore elements was confirmed.
In addition, the conversion of each isobutoxy group to an
n-propoxy group (compounds 9 and 10) resulted in a moderate
loss of inhibitory activity at 1 mM. This finding may suggest a
requirement for bulkiness of the branched chain alkyl groups.
Furthermore, compounds 11 and 12, in which each isobutoxy
group was substituted with a benzyloxy group, showed similar
inhibitory activities to compound 4, which implied that both
aliphatic and aromatic substituents were acceptable hydrophobic
elements at this position. The best-fit superposition of compound
11 and peptide 1 is shown in Figure 6b. The three overlapped
points within compound 11 are the carbon atom of the carboxyl
group, the carbon atom at the branched position of the isobutyl
group, and the carbon atom at the root of the benzene ring in
the benzyl group. The RMSD value obtained by the superposi-
tion of the three pharmacophoric points was 0.93 Å, and the
energy of this best-fitted conformer relative to the lowest energy
was 4.58 kcal mol-1. The conformation and orientation of
compound 11 were similar to those of compound 4 (Figure 6).
Finally, we converted the R3 substituent to investigate the
importance of the carboxyl group. The conversion into car-
boxamide (compound 13) resulted in a considerable loss of
inhibitory activity. This result clearly demonstrates the signifi-
cance of the ionic interactions. This appears to support our initial
hypothesis that the basic lysine and/or arginine in the basic
region of bZIP domains might take part in the interactions with
peptide 1.
The above-mentioned results demonstrate that the optimal
arrangement of at least three pharmacophoric elements is
fundamental. In addition, we suggest that it might be possible
to enhance the inhibitory activity by the further introduction of
a fourth pharmacophoric element corresponding to Gln3 of
peptide 1 that showed more potent inhibitory activity.
Conclusions
We converted cyclic peptide 1 to small-molecule AP-1
inhibitors using a lead-hopping approach based on a 3D
pharmacophore model that was derived from the peptide. New
AP-1 inhibitors, 1-thia-4-azaspiro[4.5]decane and benzophenone

Nonpeptidic Small-Molecule AP-1 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 87
× 100 mL). The aqueous layer was extracted with EtOAc (1 × 50
mL). The combined organic extracts were washed with water (1 ×
100 mL) and brine (1 × 100 mL), dried over MgSO4, and
concentrated under reduced pressure. The residue was purified by
chromatography (silica gel 150 g, 5:1 hexane/EtOAc) to give 8-(4-
methylpentylidene)-1,4-dioxaspiro[4.5]decane (5.40 g, 94%) as a
colorless oil: 1H NMR (400 MHz, CDCl3) δ 0.87 (6H, d, J ) 6.6
Hz), 1.21 (2H, q, J ) 7.5 Hz), 1.46-1.62 (1H, m), 1.62-1.72 (4H,
m), 1.99 (2H, q, J ) 7.5 Hz), 2.21 (2H, t, J ) 6.3 Hz), 2.27 (2H,
t, J ) 6.5 Hz), 3.97 (4H, s), 5.13 (1H, t, J ) 7.5 Hz).
A mixture of the ketal (1.00 g, 4.46 mmol) obtained above and
6 M HCl (5 mL) in 1,4-dioxane (10 mL) was stirred at room
temperature for 9 h before dilution with CHCl3 (50 mL) and water
(30 mL). The aqueous layer separated was extracted with CHCl3
(1 × 30 mL), and the combined organic extracts were washed with
water (1 × 30 mL) and brine (1 × 30 mL), dried over MgSO4, and
concentrated under reduced pressure. The residue was purified by
chromatography (silica gel 100 g, 10:1 hexane/EtOAc) to give 15
(0.77 g, 96%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ
0.90 (6H, d, J ) 6.6 Hz), 1.16-1.32 (2H, m), 1.48-1.66 (1H, m),
2.04 (2H, q, J ) 7.6 Hz), 2.36-2.56 (8H, m), 5.30-5.38 (1H, m).
4-(3-Methylbutylidene)cyclohexanone (17). Compound 14 was
treated by the procedure described for 15 except using 3-methyl-
butyltriphenylphosphonium iodide to afford 17 (65%) as a colorless
oil: 1H NMR (400 MHz, CDCl3) δ 0.91 (6H, d, J ) 6.6 Hz), 1.56-
1.72 (1H, m), 1.90-1.97 (2H, m), 2.36-2.54 (8H, m), 5.37 (1H,
t, J ) 7.3 Hz); MS m/z 167 (M + H)+.
(R)-8-(4-Methylpentylidene)-1-thia-4-azaspiro[4.5]decane-3-
carboxylic Acid (16). A mixture of 15 (1.20 g, 6.66 mmol) and
L-cysteine (1.21 g, 9.99 mmol) in a mixture of 7:3 EtOH/water (36
mL) was stirred at room temperature for 3 h and at 40 °C for 5 h.
The reaction mixture was evaporated under reduced pressure before
dilution with water (100 mL). The product precipitated was
collected by filtration and washed with water (3 × 30 mL) and
hexane (3 × 30 mL) to afford 16 (1.18 g, 63%) as a light brown
solid: 1H NMR (400 MHz, CDCl3) δ 0.88 (6H, d, J ) 6.6 Hz),
1.20 (2H, q, J ) 7.3 Hz), 1.46-1.60 (1H, m), 1.84-2.64 (10H,
m), 3.22-3.34 (1H, m), 3.36-3.50 (1H, m), 4.33 (1H, t, J ) 7.2
Hz), 5.10-5.22 (1H, m), 5.30-5.70 (2H, br); MS m/z 284 (M +
H)+, 282 (M - H)-.
(R)-8-(3-Methylbutylidene)-4-(5-methylhexanoyl)-1-thia-4-
azaspiro[4.5]decane-3-carboxylic Acid (3). Compound 18 (500
mg, 1.86 mmol) was treated by the procedure described for 2 except
using 5-methylhexanoic acid to afford 3 (305 mg, 43%) as a white
solid, which was crystallized from CH2Cl2/hexane to give colorless
crystals, mp 135-138 °C: 1H NMR (400 MHz, CDCl3) δ 0.87
(12H, d, J ) 6.8 Hz), 1.10-1.24 (2H, m), 1.48-1.74 (5H, m),
1.76-2.50 (7H, m), 2.56-2.72 (1H, m), 2.84-3.34 (5H, m), 4.90-
5.00 (1H, m), 5.10-5.20 (1H, m); MS m/z 382 (M + H)+, 380 (M
- H)-. Anal. (C21H35NO3S) C, H, N.
3-(2-Ethoxycarbonylethyl)benzoic Acid (20). To a stirred
suspension of NaH (60% dispersion in mineral oil, 1.37 g, 34.3
mmol) in DMF (75 mL) at 10-15 °C was added dropwise ethyl
diethylphosphonoacetate (7.69 g, 34.3 mmol). The mixture was then
stirred at room temperature for 30 min. To the stirred and ice-
cooled mixture was added dropwise benzyl 3-formylbenzoate (19)
(7.50 g, 31.2 mmol) before being stirred at room temperature for
30 min. The reaction mixture was poured into a mixture of EtOAc
(100 mL) and 1 M HCl (50 mL), and the aqueous layer separated
was extracted with EtOAc (1 × 50 mL). The organic extracts were
washed with water (1 × 100 mL) and brine (1 × 100 mL), dried
over MgSO4, and concentrated under reduced pressure. The residue
was purified by chromatography (silica gel 100 g, 3:1 hexane/
EtOAc) to give benzyl 3-((E)-2-ethoxycarbonylvinyl)benzoate (9.12
g, 94%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 1.34
(3H, t, J ) 7.1 Hz), 4.27 (2H, q, J ) 7.1 Hz), 5.38 (2H, s), 6.50
(1H, d, J ) 16.0 Hz), 7.31-7.56 (6H, m), 7.70 (1H, d, J ) 8.1
Hz), 7.70 (1H, d, J ) 16.0 Hz), 8.08 (1H, d, J ) 7.8 Hz), 8.22
(1H, s); MS m/z 311 (M + H)+.
A suspension of the R,â-unsaturated ester (8.50 g, 274 mmol)
obtained above in EtOH (85 mL) was stirred at 40 °C under the
atmospheric pressure of H2 in the presence of 10% Pd-C (1.70
g). The mixture was filtered through Celite after 3 h, and the residue
was washed with EtOH (3 × 20 mL). The filtrate was concentrated
under reduced pressure to afford the semisolid residue, which was
triturated with hexane (100 mL) to give 20 (5.87 g, 96%) as an
off-white solid: 1H NMR (400 MHz, CDCl3) δ 1.24 (3H, t, J )
7.2 Hz), 2.67 (2H, t, J ) 7.7 Hz), 3.03 (2H, t, J ) 7.7 Hz), 4.14
(2H, q, J ) 7.2 Hz), 7.41 (1H, t, J ) 7.7 Hz), 7.47 (1H, d, J ) 7.7
Hz), 7.92-8.03 (2H, m); MS m/z 221 (M - H)-.
(R)-8-(3-Methylbutylidene)-1-thia-4-azaspiro[4.5]decane-3-
carboxylic Acid (18). Compound 17 (5.00 g, 30.1 mmol) was
treated by the procedure described for 16 to afford 18 (4.40 g, 54%)
as a white solid: 1H NMR (400 MHz, CDCl3) δ 0.87 (6H, d, J )
6.3 Hz), 1.50-1.64 (1H, m), 1.82-2.14 (6H, m), 2.16-2.64 (4H,
m), 3.22-3.32 (1H, m), 3.42 (1H, dd, J ) 11.3, 7.4 Hz), 3.78 (2H,
br s), 4.32 (1H, t, J ) 7.4 Hz), 5.19 (1H, t, J ) 7.3 Hz); MS m/z
268 (M - H)-.
Isobutyl 3-(2-Isobutoxyphenyl)propionate (22). A mixture of
3-(2-hydroxyphenyl)propionic acid (21) (10.0 g, 60.2 mmol), K2-
CO3 (41.6 g, 301 mmol), and isobutyl bromide (33.0 g, 241 mmol)
in DMF (100 mL) was heated with vigorous stirring at 140 °C for
1 h. After cooling to room temperature, the reaction mixture was
treated with EtOAc (150 mL) and water (300 mL) followed by
acidification to pH 4 with 6 M HCl. The organic extract was washed
with water (1 × 50 mL) and brine (1 × 50 mL), dried over MgSO4,
and concentrated under reduced pressure. The residue was purified
by chromatography (silica gel 300 g, 5:1 hexane/EtOAc) to give
22 (15.4 g, 92%) as a colorless oil: 1H NMR (400 MHz, CDCl3)
δ 0.91 (6H, d, J ) 7.1 Hz), 1.05 (6H, d, J ) 7.1 Hz), 1.84-1.96
(1H, m), 2.06-2.18 (1H, m), 2.63 (2H, t, J ) 7.9 Hz), 2.97 (2H,
t, J ) 7.9 Hz), 3.74 (2H, d, J ) 6.4 Hz), 3.85 (2H, d, J ) 6.6 Hz),
6.78-6.88 (2H, m), 7.12-7.20 (2H, m); MS m/z 279 (M + H)+.
4-Isobutoxy-3-(2-methoxycarbonylethyl)benzoic Acid (23). To
a stirred solution of 22 (10.0 g, 35.9 mmol) in CH2Cl2 (50 mL) at
-30 °C were successively added AlCl3 (9.58 g, 71.8 mmol) and
dichloromethyl methyl ether (4.96 g, 43.1 mmol), and the mixture
was stirred at -10 °C for 1 h. After addition of MeOH (50 mL),
the resulting mixture was stirred under reflux for 2 h before cooling
to room temperature. The reaction mixture was poured into ice-
water (200 mL), and the organic layer was sequentially washed
with 1 M HCl (1 × 50 mL) and water (1 × 50 mL), and
concentrated under reduced pressure to give aldehyde as a brown
oil, which was used without further purification. The aldehyde thus
obtained was dissolved in CH3CN (20 mL). To the stirred solution
were added a solution of NaH2PO4‚2H2O (15.1 g, 96.8 mmol) in
water (20 mL) and 30% aqueous H2O2 (6.11 g, 53.9 mmol) in
sequence. A solution of NaClO2 (80% purity, 6.50 g, 57.5 mmol)
in water (10 mL) was added dropwise over 10 min to the ice-cooled
(R)-4-(4-Methylpentanoyl)-8-(4-methylpentylidene)-1-thia-4-
azaspiro[4.5]decane-3-carboxylic Acid (2). To a stirred solution
of 4-methylpentanoic acid (225 mg, 1.94 mmol) in CH2Cl2 (5 mL)
at room temperature was added dropwise oxalyl chloride (246 mg,
1.94 mmol) followed by the addition of DMF (50 µL), and the
mixture was stirred at the same temperature for 2 h. The mixture
was cooled in an ice bath before the sequential addition of 16 (500
mg, 1.76 mmol) and Et3N (535 mg, 5.29 mmol), and then the
mixture was stirred at room temperature for 2 h. The resulting
mixture was treated with CHCl3 (30 mL) and water (30 mL),
followed by acidification to pH 2 with 6 M HCl, and the layers
were separated. The aqueous layer was extracted with CHCl3 (30
mL), and the combined organic extracts were washed with water
(1 × 30 mL) and brine (1 × 30 mL), dried over MgSO4, and
concentrated under reduced pressure. The residue was purified by
chromatography (silica gel 50 g, 10:1 CHCl3/MeOH) to give 2 (380
mg, 56%) as a white solid, which was crystallized from CH2Cl2/
hexane to give colorless crystals, mp 144-145 °C: 1H NMR (400
MHz, CDCl3) δ 0.87 (6H, d, J ) 6.6 Hz), 0.88 (6H, d, J ) 5.6
Hz), 1.20 (2H, q, J ) 7.0 Hz), 1.34-1.78 (5H, m), 1.86-2.72 (9H,
m), 2.84-3.38 (4H, m), 4.90-5.02 (1H, m), 5.06-5.16 (1H, m),
5.25-5.45 (1H, br); MS m/z 381 (M - H)-. Anal. (C21H35NO3S)
C, H, N.

88 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Tsuchida et al.
mixture with stirring before being stirred at room temperature for
1 h. The solid material that had precipitated during the reaction
was filtered and washed with water (2 × 20 mL) to give 23 (7.32
g, 73%) as a pale yellow solid: 1H NMR (400 MHz, CDCl3) δ
1.06 (6H, d, J ) 6.6 Hz), 2.08-2.22 (1H, m), 2.64 (2H, t, J ) 7.8
Hz), 3.00 (2H, t, J ) 7.8 Hz), 3.69 (3H, s), 3.82 (2H, d, J ) 6.6
Hz), 6.86 (1H, d, J ) 8.7 Hz), 7.91 (1H, d, J ) 2.2 Hz), 7.98 (1H,
dd, J ) 8.7, 2.2 Hz); MS m/z 279 (M - H)-.
7.22-7.32 (2H, m), 7.35 (1H, t, J ) 7.9 Hz), 7.68-7.78 (2H, m);
MS m/z 397 (M - H)-. Anal. (C24H30O5) C, H.
3-[3-(4-Isobutoxybenzoyl)phenyl]propionic Acid (6). Com-
pound 20 and isobutoxybenzene were treated by the procedure
described for 4 to afford 6 (77%) as a white solid, which was
crystallized from CH2Cl2/hexane to give colorless crystals, mp 98-
99 °C: 1H NMR (400 MHz, CDCl3) δ 1.05 (6H, d, J ) 6.6 Hz),
2.06-2.18 (1H, m), 2.73 (2H, t, J ) 7.7 Hz), 3.03 (2H, t, J ) 7.7
Hz), 3.80 (2H, d, J ) 6.6 Hz), 6.95 (2H, d, J ) 8.8 Hz), 6.91-
6.98 (3H, m), 7.36-7.46 (2H, m), 7.54-7.64 (2H, m), 7.80 (2H,
d, J ) 8.8 Hz); MS m/z 327 (M + H)+, 325 (M - H)-. Anal.
(C20H22O4) C, H.
3-(5-Benzoyl-2-isobutoxyphenyl)propionic Acid (7). Com-
pound 22 and benzoyl chloride were treated by the procedure
described for 4 to afford 7 (40%) as a white solid, which was
crystallized from CH2Cl2/hexane to give colorless crystals, mp 95-
97 °C: 1H NMR (400 MHz, CDCl3) δ 1.07 (6H, d, J ) 6.6 Hz),
2.08-2.24 (1H, m), 2.71 (2H, t, J ) 7.7 Hz), 3.00 (2H, t, J ) 7.7
Hz), 3.83 (2H, d, J ) 6.3 Hz), 6.88 (1H, d, J ) 8.3 Hz), 7.44-
7.52 (2H, m), 7.52-7.60 (1H, m), 7.68-7.80 (4H, m); MS m/z
327 (M + H)+, 325 (M - H)-. Anal. (C20H22O4) C, H.
Methyl 3-[2-Hydroxy-5-(4-isobutoxybenzoyl)phenyl]propi-
onate (28). A mixture of 26 (1.00 g, 2.42 mmol) and AlCl3 (1.94
g, 14.5 mmol) in 1,2-dichloroethane (10 mL) was stirred under
reflux for 8 h before cooling to room temperature. The mixture
was diluted with 2-butanone (50 mL) and then poured into a mixture
of ice (15 g) and 12 M HCl (15 mL). The organic layer separated
was sequentially washed with 1 M HCl (2 × 10 mL), water (1 ×
10 mL), and brine (1 × 10 mL), dried over MgSO4, and
concentrated under reduced pressure. The residual solid was
triturated with hexane (10 mL) to give 27 (0.55 g) as a pale red
solid: 1H NMR (400 MHz, DMSO-d6) δ 2.85 (2H, t, J ) 7.3 Hz),
3.08 (2H, t, J ) 7.3 Hz), 6.90 (2H, d, J ) 8.7 Hz), 7.19 (1H, d, J
) 8.3 Hz), 7.59 (1H, dd, J ) 8.3, 2.2 Hz), 7.65-7.67 (1H, m),
7.66 (2H, d, J ) 8.7 Hz), 10.43 (1H, s); MS m/z 269 (M + H)+,
267 (M - H)-. To a suspension of 27 (0.55 g), isobutyl alcohol
(0.18 g, 2.43 mmol), and triphenylphosphine (0.65 g, 2.48 mmol)
in THF (6 mL) at temperatures below 40 °C was added dropwise
DIAD (0.50 g, 2.47 mmol). After being stirred for 30 min, NaOMe
(28 wt % in MeOH, 1.98 g, 10.3 mmol) was added dropwise to
the ice-cooled mixture with stirring, and then the resulting mixture
was stirred at room temperature for 30 min. The reaction mixture
was treated with toluene (10 mL) and 1 M HCl (20 mL). The
organic layer was sequentially washed with water (1 × 10 mL)
and brine (1 × 10 mL), dried over MgSO4, and concentrated under
reduced pressure. The residue was purified by chromatography
(silica gel 100 g, 4:1 hexane/EtOAc) to give 28 (0.60 g, 69%) as
a white solid: 1H NMR (400 MHz, CDCl3) δ 1.05 (6H, d, J ) 6.8
Hz), 2.06-2.18 (1H, m), 2.76 (2H, t, J ) 6.3 Hz), 2.95 (2H, t, J
) 6.3 Hz), 3.72 (3H, s), 3.80 (2H, d, J ) 6.6 Hz), 6.94 (1H, d, J
) 8.3 Hz), 6.95 (2H, d, J ) 8.7 Hz), 7.58 (1H, dd, J ) 8.3, 2.2
Hz), 7.63 (1H, d, J ) 2.2 Hz), 7.76 (2H, d, J ) 8.7 Hz), 7.95 (1H,
br s); MS m/z 357 (M + H)+, 355 (M - H)-.
3-Isobutoxybenzoic Acid (25). A mixture of 3-hydroxybenzoic
acid (24) (10.0 g, 72.4 mmol), K2CO3 (25.0 g, 181 mmol), and
isobutyl bromide (29.8 g, 217 mmol) in DMF (100 mL) was heated
with stirring at 120 °C for 2 h. The reaction mixture was then cooled
to room temperature, treated with water (300 mL) followed by
acidification to pH 4 with 6 M HCl, and extracted with EtOAc
(100 mL). The organic layer was washed with water (1 × 100 mL)
and brine (1 × 100 mL), dried over MgSO4, and evaporated under
reduced pressure to afford di-O-isobutylated product as a pale
yellow oil. The di-O-isobutylated product thus obtained was
dissolved in acetone (100 mL), and then 20 wt % aqueous NaOH
(50.0 g) was added to the mixture at room temperature. The reaction
mixture was allowed to cool to room temperature after being stirred
at 50 °C for 2 h, and then water (300 mL) was added. The mixture
was acidified to pH 1 with 12 M HCl and stirred at room
temperature for 30 min, and the resulting precipitate was collected
by filtration and washed with water (2 × 50 mL) to give 25 (9.85
g, 70%) as a white solid: 1H NMR (400 MHz, CDCl3) δ 1.05 (6H,
d, J ) 6.4 Hz), 2.04-2.18 (1H, m), 3.78 (2H, d, J ) 6.6 Hz), 7.16
(1H, ddd, J ) 7.9, 2.6, 0.5 Hz), 7.37 (1H, t, J ) 7.9 Hz), 7.62 (1H,
dd, J ) 2.6, 1.6 Hz), 7.70 (1H, d, J ) 7.9 Hz); MS m/z 193 (M -
H)-.
3-[2-Isobutoxy-5-(4-isobutoxybenzoyl)phenyl]propionic Acid
(4). To a stirred solution of 23 (2.00 g, 7.13 mmol) and DMF (0.1
mL) in CH2Cl2 (20 mL) at room temperature was added oxalyl
chloride (1.09 g, 8.59 mmol). After continuous stirring for 1 h, the
mixture was cooled with ice-water before the sequential addition
of AlCl3 (2.09 g, 15.7 mmol) and isobutoxybenzene (1.29 g, 8.59
mmol). After being stirred at 5-10 °C for 30 min, the reaction
mixture was poured into a mixture of ice (20 g) and 6 M HCl (30
mL). The layers were separated, and the organic layer was washed
with water (1 × 20 mL) and brine (1 × 20 mL), dried over MgSO4,
and concentrated under reduced pressure. The residue was purified
by chromatography (silica gel 50 g, 5:1 hexane/EtOAc) to give
methyl 3-[2-isobutoxy-5-(4-isobutoxybenzoyl)phenyl]propionate
(26) (2.36 g, 80%) as a white crystalline solid, mp 78-79 °C: 1H
NMR (400 MHz, CDCl3) δ 1.05 (6H, d, J ) 6.8 Hz), 1.07 (6H, d,
J ) 6.8 Hz), 2.06-2.22 (2H, m), 2.64 (2H, t, J ) 7.8 Hz), 3.00
(2H, t, J ) 7.8 Hz), 3.67 (3H, s), 3.80 (2H, d, J ) 6.6 Hz), 3.82
(2H, d, J ) 6.3 Hz), 6.87 (1H, d, J ) 8.3 Hz), 6.95 (2H, d, J ) 8.7
Hz), 7.64 (1H, d, J ) 2.0 Hz), 7.67 (1H, dd, J ) 8.3, 2.0 Hz), 7.76
(2H, d, J ) 8.7 Hz); MS m/z 413 (M + H)+.
A mixture of 26 (10.4 g, 25.2 mmol) and 20 wt % aqueous NaOH
(10.4 g) in acetone (30 mL) was stirred at 50 °C for 2 h before
dilution with water (100 mL). The product that was precipitated
by acidification to pH 1 with 12 M HCl was collected by filtration
and washed with water (2 × 50 mL) to afford 4 (9.44 g, 94%) as
a white solid, which was crystallized from CH2Cl2/hexane to give
colorless crystals, mp 102-103 °C: 1H NMR (400 MHz, CDCl3)
δ 1.04 (6H, d, J ) 6.6 Hz), 1.07 (6H, d, J ) 6.8 Hz), 2.06-2.22
(2H, m), 2.71 (2H, t, J ) 7.7 Hz), 3.00 (2H, t, J ) 7.7 Hz), 3.80
(2H, d, J ) 6.4 Hz), 3.82 (2H, d, J ) 6.6 Hz), 6.87 (1H, d, J ) 8.5
Hz), 6.95 (2H, d, J ) 8.8 Hz), 7.66 (1H, d, J ) 2.2 Hz), 7.69 (1H,
dd, J ) 8.5, 2.2 Hz), 7.76 (2H, d, J ) 8.8 Hz); MS m/z 397 (M -
H)-. Anal. (C24H30O5) C, H.
3-[2-Isobutoxy-5-(3-isobutoxybenzoyl)phenyl]propionic Acid
(5). Compounds 22 and 25 were treated by the procedure described
for 4 to afford 5 (85%) as a white solid, which was crystallized
from CH2Cl2/hexane to give colorless crystals, mp 77-78 °C: 1H
NMR (400 MHz, CDCl3) δ 1.03 (6H, d, J ) 6.8 Hz), 1.07 (6H, d,
J ) 6.8 Hz), 2.04-2.22 (2H, m), 2.70 (2H, t, J ) 7.7 Hz), 3.00
(2H, t, J ) 7.7 Hz), 3.77 (2H, d, J ) 6.4 Hz), 3.83 (2H, d, J ) 6.3
Hz), 6.87 (1H, d, J ) 9.3 Hz), 7.10 (1H, dd, J ) 7.9, 2.6 Hz),
Methyl 3-[2-Hydroxy-5-(4-propoxybenzoyl)phenyl]propionate
(29). Compound 26 (1.00 g, 2.42 mmol) was treated by the
procedure described for 28 except using n-propyl alcohol to afford
29 (0.56 g, 67%) as a white solid: 1H NMR (400 MHz, CDCl3) δ
1.06 (3H, t, J ) 7.3 Hz), 1.80-1.90 (2H, m), 2.76 (2H, t, J ) 6.2
Hz), 2.95 (2H, t, J ) 6.2 Hz), 3.72 (3H, s), 4.00 (2H, t, J ) 6.6
Hz), 6.94 (1H, d, J ) 8.4 Hz), 6.95 (2H, d, J ) 8.8 Hz), 7.57 (1H,
dd, J ) 8.4, 2.1 Hz), 7.63 (1H, d, J ) 2.1 Hz), 7.76 (2H, d, J )
8.8 Hz), 7.92 (1H, s); MS m/z 341 (M - H)-.
Methyl 3-[5-(4-Benzyloxybenzoyl)-2-hydroxyphenyl]propi-
onate (30). Compound 26 (1.00 g, 2.42 mmol) was treated by the
procedure described for 28 except using benzyl alcohol to afford
30 (0.66 g, 70%) as a white solid: 1H NMR (400 MHz, CDCl3) δ
2.76 (2H, t, J ) 6.2 Hz), 2.95 (2H, t, J ) 6.2 Hz), 3.72 (3H, s),
5.15 (2H, s), 6.94 (1H, d, J ) 8.4 Hz), 7.03 (2H, d, J ) 8.9 Hz),
7.32-7.48 (5H, m), 7.58 (1H, dd, J ) 8.4, 2.2 Hz), 7.64 (1H, d, J
) 2.2 Hz), 7.77 (2H, d, J ) 8.9 Hz), 7.94 (1H, s); MS m/z 391 (M
+ H)+, 389 (M - H)-.

Nonpeptidic Small-Molecule AP-1 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 89
3-[5-(4-Isobutoxybenzoyl)-2-propoxyphenyl]propionic Acid
(9). To a stirred suspension of 28 (3.00 g, 8.42 mmol) and K2CO3
(1.16 g, 8.39 mmol) in DMF (15 mL) at room temperature was
added n-propyl bromide (1.55 g, 12.6 mmol), and the mixture was
stirred at 90-100 °C for 1 h. After cooling to room temperature,
the reaction mixture was treated with toluene (15 mL) and water
(60 mL) followed by acidification to pH 4 with 6 M HCl. The
organic layer was washed with water (1 × 20 mL) and brine (1 ×
20 mL), dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by chromatography (silica gel
75 g, 2:1 hexane/EtOAc) to give methyl 3-[5-(4-isobutoxybenzoyl)-
2-propoxyphenyl]propionate (2.73 g, 81%) as a white solid: 1H
NMR (400 MHz, CDCl3) δ 1.02-1.12 (9H, m), 1.81-1.93 (2H,
m), 2.00-2.20 (1H, m), 2.64 (2H, t, J ) 7.8 Hz), 2.99 (2H, t, J )
7.8 Hz), 3.67 (3H, s), 3.80 (2H, d, J ) 6.3 Hz), 4.02 (2H, t, J )
6.3 Hz), 6.87 (1H, d, J ) 8.4 Hz), 6.95 (2H, d, J ) 8.7 Hz), 7.64
(1H, d, J ) 2.2 Hz), 7.67 (1H, dd, J ) 8.4, 2.2 Hz), 7.77 (2H, d,
J ) 8.7 Hz); MS m/z 400 (M + H)+.
A mixture of the ester (1.00 g, 2.51 mmol) obtained above and
20 wt % aqueous NaOH (1.50 g) in acetone (4 mL) was stirred at
50 °C for 1 h before dilution with water (12 mL). The product that
was precipitated by acidification to pH 1 with 6 M HCl was
collected by filtration and washed with water (2 × 5 mL) to afford
9 (0.91 g, 94%) as a white crystalline solid, mp 107-108 °C: 1H
NMR (400 MHz, CDCl3) δ 1.04 (6H, d, J ) 6.6 Hz), 1.07 (3H, t,
J ) 7.4 Hz), 1.80-1.94 (2H, m), 2.04-2.20 (1H, m), 2.71, 2.99
(each 2H, t, J ) 7.6 Hz), 3.79 (2H, d, J ) 6.6 Hz), 4.01 (2H, t, J
) 6.5 Hz), 6.88 (1H, d, J ) 8.3 Hz), 6.95 (2H, d, J ) 8.7 Hz),
7.66 (1H, d, J ) 2.1 Hz), 7.69 (1H, dd, J ) 8.3, 2.1 Hz), 7.76 (2H,
d, J ) 8.7 Hz); MS m/z 383 (M - H)-. Anal. (C23H28O5) C, H.
3-[2-Benzyloxy-5-(4-isobutoxybenzoyl)phenyl]propionic Acid
(11). Compound 28 was treated by the procedure described for 9
except using benzyl bromide to afford 11 (67%) as a white solid,
which was crystallized from hexane/diisopropyl ether to give
colorless crystals, mp 100 °C: 1H NMR (400 MHz, CDCl3) δ 1.04
(6H, d, J ) 6.6 Hz), 2.02-2.18 (1H, m), 2.72 (2H, t, J ) 7.6 Hz),
3.04 (2H, t, J ) 7.6 Hz), 3.79 (2H, d, J ) 6.6 Hz), 5.17 (2H, s),
6.91-6.98 (3H, m), 7.30-7.48 (5H, m), 7.64-7.70 (2H, m), 7.76
(2H, d, J ) 8.8 Hz); MS m/z 431 (M - H)-. Anal. (C27H28O5) C,
H.
3-[2-Isobutoxy-5-(4-propoxybenzoyl)phenyl]propionic Acid
(10). Compound 29 was treated by the procedure described for 9
except using isobutyl bromide to afford 10 (85%) as a white
crystalline solid, mp 128-129 °C: 1H NMR (400 MHz, CDCl3) δ
1.00-1.12 (9H, m), 1.76-1.92 (2H, m), 2.08-2.24 (1H, m), 2.71
(2H, t, J ) 7.6 Hz), 3.00 (2H, t, J ) 7.6 Hz), 3.82 (2H, d, J ) 6.4
Hz), 4.00 (2H, t, J ) 6.6 Hz), 6.87 (1H, d, J ) 8.4 Hz), 6.95 (2H,
d, J ) 8.7 Hz), 7.66 (1H, d, J ) 2.1 Hz), 7.69 (1H, dd, J ) 8.4,
2.1 Hz), 7.76 (2H, d, J ) 8.7 Hz); MS m/z 383 (M - H)-. Anal.
(C23H28O5) C, H.
3-[5-(4-Benzyloxybenzoyl)-2-isobutoxyphenyl]propionic Acid
(12). Compound 30 was treated by the procedure described for 9
except using isobutyl bromide to afford 12 (79%) as a white solid,
which was crystallized from CH2Cl2/hexane to give colorless
crystals, mp 150-151 °C: 1H NMR (400 MHz, CDCl3) δ 1.06
(6H, d, J ) 6.6 Hz), 2.08-2.20 (1H, m), 2.71 (2H, t, J ) 7.7 Hz),
3.00 (2H, t, J ) 7.7 Hz), 3.82 (2H, d, J ) 6.3 Hz), 5.15 (2H, s),
6.87 (1H, d, J ) 8.3 Hz), 7.03 (2H, d, J ) 8.7 Hz), 7.30-7.48
(5H, m), 7.60-7.72 (2H, m), 7.77 (2H, d, J ) 8.7 Hz); MS m/z
432 (M - H)-. Anal. (C27H28O5) C, H.
extracted with EtOAc (1 × 30 mL). The combined organic extracts
were washed with water (1 × 30 mL) and brine (1 × 30 mL),
followed by the addition of CHCl3 (30 mL). The solution was dried
over MgSO4 and concentrated under reduced pressure. The residue
was triturated with hexane (20 mL) to afford 8 (2.88 g, 94%) as a
white solid, which was crystallized from CH2Cl2/hexane to give
colorless crystals, mp 118-119 °C: 1H NMR (400 MHz, CDCl3)
δ 1.05 (12H, d, J ) 6.8 Hz), 2.04-2.20 (2H, m), 3.80 (4H, d, J )
6.6 Hz), 6.95 (4H, d, J ) 8.5 Hz), 7.77 (4H, d, J ) 8.5 Hz); MS
m/z 327 (M + H)+. Anal. (C21H26O3) C, H.
3-[2-Isobutoxy-5-(4-isobutoxybenzoyl)phenyl]propionamide
(13). To a stirred solution of 4 (5.00 g, 12.5 mmol) in THF (50
mL) at room temperature was sequentially added oxalyl chloride
(1.91 g, 15.0 mmol) and DMF (50 µL), and the mixture was stirred
for 3 h. The resulting mixture was slowly poured into ice-cooled
25 wt % NH4OH (50 mL) with vigorous stirring. After being stirred
at 5 °C for 30 min, the mixture was adjusted to pH 6 with 6 M
HCl and extracted with EtOAc (1 × 100 mL). The aqueous layer
separated was extracted with EtOAc (1 × 50 mL), and the organic
extracts were washed with water (1 × 50 mL) and brine (1 × 50
mL), dried over MgSO4, and concentrated under reduced pressure.
The residual solid was triturated with hexane (100 mL) to afford
13 (4.67 g, 94%) as a white solid, which was crystallized from
CH2Cl2/hexane to give colorless crystals, mp 123 °C: 1H NMR
(400 MHz, CDCl3) δ 1.05 (6H, d, J ) 6.8 Hz), 1.08 (6H, d, J )
6.6 Hz), 2.06-2.22 (2H, m), 2.56 (2H, t, J ) 7.7 Hz), 3.02 (2H, t,
J ) 7.7 Hz), 3.80 (2H, d, J ) 6.6 Hz), 3.83 (2H, d, J ) 6.3 Hz),
5.29 (1H, br s), 5.38 (1H, br s), 6.88 (1H, d, J ) 8.1 Hz), 6.95
(2H, d, J ) 8.5 Hz), 7.62-7.70 (2H, m), 7.76 (2H, d, J ) 8.5 Hz);
MS m/z 399 (M + H)+. Anal. (C24H31NO4) C, H, N.
ELISA-Based AP-1 Binding Assay: Preparation of c-Fos and
c-Jun bZIP Peptides. The resin and all of the protected amino
acids and coupling reagents were purchased from Watanabe
Chemical Industries (Hiroshima, Japan). All of the reagents and
solvents were reagent grade or better and were used without further
purification. High-performance liquid chromatography (HPLC) was
performed using a Hitachi L-7100 apparatus equipped with an
L-7400 UV detector (peak detection at 230 nm) and a PROTEINE-
RP column (YMC-Pack, YMC Co., Kyoto, Japan) of 250 × 20
mm for preparative HPLC or 150 × 4.6 mm for analytical HPLC.
c-Jun bZIP peptide [c-Jun (263-324) Cys278Ser] and biotinylated
c-Fos bZIP peptide [N-biotinyl-(Gly)4-c-Fos (139-200) Cys154Ser]5
were synthesized manually using standard solid-phase peptide
chemistry with tert-butoxycarbonyl (Boc)-protected amino acids
on Boc-His(benzyloxymethyl)-PAM resin24 at a 0.30 mmol scale.
The Boc protecting group was removed by treatment with trifluo-
roacetic acid (10 mL) at room temperature for 3 min. Coupling
with Boc-protected amino acids (4 equiv) was then performed in
the presence of N,N-diisopropylethylamine (6 equiv) and 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(3.8 equiv) in N-methyl-2-pyrrolidone (8 mL) at room temperature
for 30 min.25 Subsequently, the peptide resin was treated with
decanoic anhydride (0.3 mol/L in 1:1 DMF/CH2Cl2, 10 mL) at room
temperature for 30 min. The protected c-Jun bZIP peptide resin
(0.50 g) was treated with a reagent containing anhydrous HF (1.5
mL) and dimethyl sulfide (4.5 mL) with stirring at 0 °C for 1.5 h
to remove the remaining O- and N-protecting groups.26 After the
evaporation of HF, ether was added to the resin, which was then
filtered, washed in CH2Cl2 (5 × 10 mL), followed by dried in vacuo.
The resin was treated with a reagent containing anhydrous HF (4.5
mL) and p-cresol (1.5 mL) with stirring at 0 °C for 1.5 h to induce
cleavage of the peptide from the resin. The precipitate was collected
by centrifugation and dissolved in water. The exhausted resin was
filtered, and the filtrate was purified by reverse phase HPLC eluted
with a linear gradient of acetonitrile in water containing 0.1%
trifluoroacetic acid at a flow rate of 7.0 mL/min before lyophiliza-
tion. Biotinylated c-Fos bZIP peptide was synthesized according
to the procedure that was used to prepare the c-Jun bZIP peptide.
The peptides were separately dissolved in 20 mM Tris-HCl buffer
(pH 7.5) containing 50 mM KCl, 1 mM ethylenediaminetetraacetic
acid (EDTA), 10 mM MgCl2, 1 mM dithiothreitol (DTT), 0.5 M
Bis(4-isobutoxyphenyl)methanone (8). To a stirred suspension
of bis(4-hydroxyphenyl)methanone (2.00 g, 9.34 mmol) and K2-
CO3 (3.87 g, 28.0 mmol) in DMF (20 mL) at room temperature
was added isobutyl bromide (3.84 g, 28.0 mmol), and then the
mixture was heated with stirring at 100 °C for 1 h. Additional K2-
CO3 (1.29 g, 9.33 mmol) and isobutyl bromide (1.28 g, 9.34 mmol)
were then added, and the heating was continued for a further 1.5
h. The reaction mixture was cooled to room temperature before
filtration, and the residue was rinsed with EtOAc (3 × 30 mL).
The filtrate was treated with water (50 mL) followed by acidification
to pH 2 with 6 M HCl, and the aqueous layer separated was

90 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Tsuchida et al.
guanidine HCl, and 30% glycerol. Equimolar quantities of both of
the solutions were mixed together and the resulting mixture was
used as the AP-1 bZIP peptide.
Tokyo, Japan) according to the manufacturer’s protocol. The firefly
luciferase activity was normalized for transfection efficiency based
on the Renilla luciferase activity. Taking the activity in the absence
of sample as 100%, the inhibition rate of each sample was calculated
from the absorbance in the presence of the sample. The IC50 values
were calculated based on a logistic concentration-response curve
using the SAS system version 8.2.
Binding Assay of AP-1 bZIP Peptide and AP-1 Double-
Stranded Oligonucleotides. The inhibitory activities of the syn-
thetic compounds on the DNA-binding activity of AP-1 were
evaluated using an ELISA-based AP-1 binding assay with synthetic
double-stranded oligonucleotides that contained the AP-1 binding
site (shown in bold) and the AP-1 bZIP peptide. The AP-1 bZIP
peptide (10 pmol/well) was added to an avidin-coated 96-well
ELISA plate, washed, and then blocked with bovine serum albumin.
Digoxigenin-labeled double-stranded oligonucleotides (22-mer)
containing an AP-1 binding sequence (5′-CTAGTGATGAGT-
CAGCCGGATC-3′ and 5′-GATCCGGCTGACTCATCACTAG-
3′) (Nisshinbo Industries, Inc., Tokyo, Japan) were reacted in the
presence or absence of each sample at room temperature for 1 h in
a binding reaction solution [25 mM Tris-HCl (pH 7.9) containing
0.5 mM EDTA, 0.05% Nonidet P-40 and 10% glycerol]. Subse-
quently, the unbound labeled oligonucleotide was washed out with
HEPES-KOH (pH 7.9) buffer containing 0.5 mM EDTA, 50 mM
KCl, 10% glycerol, 0.1% BSA, and 0.05% Tween-20. The
peroxidase-conjugated anti-digoxigenin antibody (Roche Diagnos-
tics, Indianapolis, IN) was then added and reacted with the labeled
oligonucleotide bound to AP-1. After washing out the excess
antibody with HEPES-KOH buffer containing 0.05% Tween-20,
the residue was reacted for a predetermined period of time in 100
mM citrate buffer (pH 5.0) containing 0.03% H2O2 using o-
phenylenediamine as a substrate. After adding sulfuric acid solution
to each well, the absorbance at 492 nm was measured with a
Microplate spectrophotometer (Bio-Rad Laboratories, Hercules,
CA). Taking the absorbance in the absence of sample as 100%,
the inhibition rate of each sample was calculated from the
absorbance in the presence of the sample. The IC50 values were
calculated based on a logistic concentration-response curve using
the SAS system version 8.2 (SAS Institute, Cary, NC).
Cell-Based Assay: Plasmids. Reporter plasmids containing the
firefly luciferase reporter gene driven by a basic promoter element
(TATA box) plus a defined inducible cis-enhancer element were
utilized (PathDetect in Vivo Signal Transduction Pathway cis-
Reporting Systems, Stratagene, La Jolla, CA). The firefly luciferase
gene in the reporter plasmid (pAP-1-Luc) was controlled by
synthetic enhancer sequences comprising seven repeats of the
binding sites for AP-1; TGACTAA. A control plasmid expressing
Renilla reniformis luciferase driven by the herpes simplex virus
thymidine kinase promoter, phRL-TK Vector (Promega, Madison,
WI), was used to correct the efficiency of transfection.
Cell Culture. The murine fibroblast cell line NIH3T3 was
obtained from the American Type Culture Collection (ATCC No.
CRL-1658; Rockville, MD) and grown in Dulbecco’s modified
Eagle’s medium (DMEM; Nissui Pharmaceutical, Tokyo, Japan)
supplemented with 4 mM l-glutamine (Wako Pure Chemical, Osaka,
Japan), 1.5 g/L glucose (Wako Pure Chemical), 10% heat-
inactivated fetal calf serum (FCS; JRH Biosciences, Lenexa, KS),
and 60 µg/mL kanamycin sulfate (Sigma-Aldrich, St. Luis, MO)
at 37 °C and 5% CO2.
Acknowledgment. This study was partially supported by
the Japan Science and Technology Agency.
Supporting Information Available: Characterization data for
esters of compounds 5-7 and 10-12 plus elemental analysis data
for compounds 2-13. This material is available free of charge via
the Internet at http://pubs.acs.org.
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JM050550D