Pharmacophore-based design of sphingosine 1-phosphate-3 receptor antagonists that include a 3,4-dialkoxybenzophenone scaffold

Article abstract of DOI:10.1021/jm060834d

Sphingosine 1-phosphate (S1P) receptors are G-protein-coupled receptors. Among the five identified subtypes S1P₁-5, the SIP₃ receptor expressed on vascular endothelial cells has been shown to play an important role in cell proliferation, migration, and inflammation. A pharmacophore-based database search was used to identify a potent scaffold for an S1P₃ receptor antagonist by common feature-based alignment and further validated using the Güner-Henry (GH) scoring method. Assumed excluded volumes were merged into this model to evaluate the steric effect with the S1P₃ receptor. Three commercially available compounds were identified as S1P₃ receptor antagonists, with IC₅₀ values <5 μM. The synthesis of further derivatives revealed that the 3,4-dialkoxybenzophenone scaffold is a potent component of an S1P₃ receptor antagonist. Our results indicate that pharmacophore-based design of S1P₃ receptor antagonists can be used to expand the possibility of structural modification through scaffold-hopping based on a database search.

Full text of DOI:10.1021/jm060834d

442
J. Med. Chem. 2007, 50, 442-454
Pharmacophore-Based Design of Sphingosine 1-phosphate-3 Receptor Antagonists That Include
a 3,4-Dialkoxybenzophenone Scaffold
Yuuki Koide,*,†,§ Kazuhiro Uemoto,† Takeshi Hasegawa,† Tomoyuki Sada,† Akira Murakami,† Hiroshi Takasugi,†,‡
Atsuko Sakurai,‡ Naoki Mochizuki,‡ Atsuo Takahashi,† and Atsushi Nishida§
Drug Research Department, Tokyo Research Laboratories, TOA EIYO Ltd., 2-293-3 Amanuma, Oomiya, Saitama 330-0834, Japan, Department
of Structural Analysis, National CardioVascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan, and Graduate
School of Pharmaceutical Sciences, Chiba UniVersity, 1-33 Yayoi, Inage, Chiba 263-8522, Japan
ReceiVed July 16, 2006
Sphingosine 1-phosphate (S1P) receptors are G-protein-coupled receptors. Among the five identified subtypes
S1P1-5, the S1P3 receptor expressed on vascular endothelial cells has been shown to play an important role
in cell proliferation, migration, and inflammation. A pharmacophore-based database search was used to
identify a potent scaffold for an S1P3 receptor antagonist by common feature-based alignment and further
validated using the Gu¨ner-Henry (GH) scoring method. Assumed excluded volumes were merged into this
model to evaluate the steric effect with the S1P3 receptor. Three commercially available compounds were
identified as S1P3 receptor antagonists, with IC50 values <5 µM. The synthesis of further derivatives revealed
that the 3,4-dialkoxybenzophenone scaffold is a potent component of an S1P3 receptor antagonist. Our results
indicate that pharmacophore-based design of S1P3 receptor antagonists can be used to expand the possibility
of structural modification through scaffold-hopping based on a database search.
Introduction
Sphingosine 1-phosphate (S1Pa) is a potent lipid mediator
produced from the metabolism of sphingomyelin (Figure 1).
S1P acts on a family of G-protein-coupled receptors (GPCRs)
and transduces intracellular signals involved in numerous
cellular processes.1,2 The S1P1 receptor, which was the first S1P
receptor to be identified, was initially named endothelium
differentiation gene-1 (Edg-1) receptor.3 The five subtypes of
S1P-induced Edg receptors were renamed S1P receptors,
consistent with the official IUPHAR nomenclature for rational
classification; S1P1 (formerly Edg-1), S1P2 (formerly Edg-5),
S1P3 (formerly Edg-3), S1P4 (formerly Edg-6), and S1P5
(formerly Edg-8).4
S1P3 receptors expressed on vascular endothelial cells play
several important roles, such as in cell proliferation based on
extracellular signal-regulated kinase (ERK) activation in a
pertussis toxin (PTX)-sensitive manner,5 developmental and
pathological angiogenesis,6 endothelial cell migration,7 and the
activation of eNOS.8 The role of vascular S1P3 receptors has
been reviewed in detail.9-12
The advent of the novel immunosuppressant 2 has inspired
considerable interest in agonists and antagonists of S1P receptors
(Figure 1).13 It has been demonstrated that 2 is metabolized
across various species to a monophosphate ester 3, which is a
high-affinity ligand for S1P1, S1P3, S1P4, and S1P5, but not
S1P2.14,15 Administration of 2 resulted in the sequestration of
Figure 1. S1P, reported S1P1 receptor agonists and S1P3 receptor
circulating lymphocytes within secondary lymphoid tissues. Its
antagonists (3, S1P1, 3-5 receptor agonist; 2, immunosuppressant which
is the precursor for 3; 4 and 5, S1P1/S1P3 receptor antagonists; 6, S1P3
receptor antagonist.).
egress-blocking effect occurs through the inactivation of the
S1P1 receptor.16 Therefore, an S1P1 receptor-selective agonist
or antagonist has been expected to be a more effective
Clinical studies with 2 have identified dose-dependent
immunosuppressant than 2.
transient symptomatic bradycardia in stable renal transplant
patients.17 Bradycardia is consistent with the S1P-induced
* To whom correspondence should be addressed. Tel: +81(48)6477971.
activation of muscarinic receptor-activated inwardly rectifying
K+ current (IK.Ach).18-20 Recent studies in S1P3 null mice have
shown that the induction of bradycardia is mediated through
the activation of S1P3 receptors.21,22 Meanwhile, S1P2 and S1P3,
which are abundantly expressed in airway smooth muscle
Fax: +81(48)6480078. E-mail: koide.yuuki@toaeiyo.co.jp.
† TOA EIYO Ltd.
‡ National Cardiovascular Center Research Institute.
§ Chiba University.
a Abbreviations: S1P, sphingosine 1-phosphate; EDG, endothelium
differentiation gene; GH score, Gu¨ner-Henry score.
10.1021/jm060834d CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/11/2007

Sphingosine 1-phosphate-3 Receptor Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3 443
Chart 1. Chart for Deriving a Pharmacophore Model of S1P3
Receptor Antagonists
(ASM), mobilize calcium and mediate airway hypercontractil-
ity.23 Administration of 2 induces bronchoconstriction and
elevates airway constriction, while administration of an S1P1
receptor-selective agonist has no effect on bradycardia or airway
resistance.24,25
Taken together, these results suggest that an S1P3 receptor
antagonist may be useful for preventing vascular proliferation,
bradycardia, and bronchoconstriction. Furthermore, elucidation
of the structural requirements for S1P3 receptor antagonist may
enable the development of a more selective S1P1 receptor
antagonist. While several S1P1 receptor-selective agonists have
been reported, there have been few reports on S1P3 antagonists.26
Although suramin has been reported to be an S1P3 receptor-
selective antagonist, it also acts as a reversible P2-purinoceptor
antagonist,27,28 an inhibitor of DNA topoisomerase II,29 and a
potent inhibitor of the reverse transcriptase of RNA tumor
viruses.30 Suramin has also been reported to act as a direct
antagonist of heterotrimeric G proteins.31 Recently, practical
S1P1/S1P3 receptor antagonists 4 and 5, which have meta-
alkylated phenyl structures, were reported from the University
of Virginia (Figure 1).32,33 However, the activities of these
antagonists toward S1P3 receptors are at least one-fifth less than
those toward S1P1 receptors.
To identify structurally novel agonists or antagonists ef-
ficiently, a computational approach for scaffold-hopping has
been extensively applied.34 Structure-based virtual screening,
in which the 3D structure of a target protein (usually derived
from X-ray crystallography) is used in protein-ligand docking
calculations, has been used extensively to explore potent
compounds. However, this approach to scaffold-hopping in
GPCRs is limited due to issues involving purification and
crystallization. Meanwhile, a pharmacophore is a generic
description of the key features of an active molecule and is
usually built by overlaying different conformations of active
compounds. Pharmacophore models derived from active ligands
have been widely used to explore GPCR ligands.35
We developed 2-alkyl or aryl thiazolidine-4-carboxylic acid
derivatives as potent S1P3 antagonists at 10 µM by a pharma-
cophore-based database search which included information on
the conformations of S1P.36 However, very recently, one of these
compounds, (4R)-4-undecylthiazolidine-2-carboxylic acid 6
(Figure 1), has been reported to be inappropriate for identifying
selective antagonism at the S1P3 receptor due to its low potency
and reduced selectivity.37 More potent and practical S1P3
receptor antagonists are still needed.
We have continued to study more potent S1P3 receptor
antagonists, which may be useful for influencing vascular
proliferation and inflammation, or at least for elucidating the
detailed mechanism of S1P3 receptor antagonism. In this report,
we describe (i) the generation and assessment of a pharma-
cophore model for S1P3 receptor antagonists, (ii) the identifica-
tion of novel potent S1P3 antagonists retrieved from among
commercially available compounds by a pharmacophore-based
database search, (iii) the structure-activity relationship of
synthesized derivatives as S1P3 receptor antagonists, and (iv)
the structural requirements for S1P3 receptor antagonists.
were validated to identify preferable models that could be used
to efficiently exclude inactive compounds. Third, information
regarding steric properties was added to the pharmacophore
models, since these models could not exclude inactive com-
pounds which probably had decreased potency due to extreme
molecular volumes. Finally, these modified pharmacophore
models were validated if they made it possible to efficiently
exclude inactive compounds and to identify active compounds
that were not used for pharmacophore generation (test set). The
most reliable pharmacophore model was then identified. The
details of each step are described below.
(i) Generation of the Pharmacophore Model. A feature-
based pharmacophore model derived from the active compound
set with diverse conformations without quantitative activity data
was applied.38 It describes possible pharmacophoric space as
an arrangement of features. For example, a feature, such as
hydrogen-bond acceptor, is described which defines the kind
of interaction with the target protein.
In this paper, we used three common features: hydrogen-
bond acceptor, hydrophobic, and negatiVe ionizable. The
hydrophobic feature matches the following types of groups of
atoms: a contiguous set of atoms that are not adjacent to any
concentrations of charge (charged atoms or electronegative
atoms) in a conformer such that the atoms have surface
accessibility, including phenyl, cycloalkyl, isopropyl, and methyl
groups. The hydrogen-bond acceptor feature matches the
following types of atoms or groups of atoms with surface
accessibility: sp or sp2 nitrogens that have a lone pair and charge
less than or equal to zero, sp3 oxygens or sulfurs that have a
lone pair and charge less than or equal to zero, or nonbasic
amines that have a lone pair. The negatiVe ionizable feature
matches atoms or groups of atoms that are likely to be
deprotonated at physiological pH, such as trifluoromethyl
sulfonamide hydrogens, sulfonic acids, phosphonic acids,
sulfinic, carboxylic, or phosphinic acids, tetrazoles, or negative
charges not adjacent to a positive charge.
Methods
(ii) Validation of the Quality of Generated Pharmacophore
Models. The Gu¨ner-Henry (GH) scoring method39-41 was used
to assess the quality of pharmacophore models. The GH score
is a metric for quantifying the precision of hits and the recall
of actives mined from a database consisting of known actives
and inactives (Chart 2). It is considered a relevant metric, since
it takes into account both the percent yield of actives in a
1. Generation and Validation of Pharmacophore Models.
The scheme for generating reliable pharmacophore models for
S1P3 receptor antagonists is outlined in Chart 1. First, pharma-
cophore models were constructed by aligning active compounds
as S1P3 receptor antagonists with multiple conformations
(training set). Second, the generated pharmacophore models

444 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3
Koide et al.
Chart 2. Metrics for Analyzing Hit Lists by a Pharmacophore
models for S1P3 receptor antagonists were generated using five
of seven active compounds: 6, 8, 10, 11 and 12, which were
previously identified (Figure 2).36 Compounds 7 and 9 were
used as the test set in validation. Twelve patterns of pharma-
cophore generation were applied, based on three major param-
eters (Table 1): (i) The number of maximum features applied
in a pharmacophore model was varied from three to five. (ii)
NegatiVe ionizable and hydrophobic features were applied for
the generation of all models. Meanwhile, models were separately
generated with or without the hydrogen-bond acceptor feature,
since it was not clear whether this feature was important. (iii)
2-Alkyl or aryl thiazolidine-4-carboxylic acid derivatives 6, 8,
10 and 11 in the training set are diastereomers at C2 ((2R)- and
(2S)-isomer). This complication may hamper conformational
analysis and subsequent pharmacophore generation. Therefore,
two training sets that included (2R)-thiazolidine-4-carboxylic
acid derivatives 6, 8, 10 and 11 or their (2S)-derivatives were
used. Compound 12 was used in both training sets. As a result,
these three conditions gave 307 pharmacophore models.
Model-Based Database Searcha
a Ha, number of actives in the hit list (true positives); Ht, number of
hits retrieved; A, number of active compounds in the database; D, number
of compounds in the database; % A, ratio of actives retrieved in the hit list
(precision); % Y, yield of actives (recall); E, enrichment of the concentration
of actives by the model relative to random screening without any
pharmacophoric approach; GH, Gu¨ner-Henry score.
database (% Y, recall) and the percent ratio of actives in the
hit list (% A, precision). The GH score ranges from 0,
which indicates the null model, to 1, which indicates the
ideal model. On the basis of S1P-induced cytosolic Ca2+
mobilization in CHO cells expressing the S1P3 receptor, a
compound with more than 30% inhibitory activity at 10 µM
was defined as an “active” and a compound with less than 20%
inhibitory activity at 10 µM was defined as an “inactive”. For
GH score assessment, all compounds with inhibitory activities
of 20 to 30% at 10 µM were excluded to simplify the
classification.
(iii) Addition of Excluded Volumes to Pharmacophore
Models. Steric interaction can be approximated by the strategic
placement of excluded volumes (spheres that cannot be pen-
etrated by the ligand) in the pharmacophore model. The
inclusion of excluded volumes in pharmacophore models has
been reported to improve the pruning of database hit lists by
30-75%.42 To obtain a more desirable pharmacophore model,
appropriate excluded volumes were added by the GH scoring
method.
(ii) Validation of Generated Pharmacophore Models. The
quality of pharmacophore models was assessed using the GH
score as a metric to search a database that consisted of 100
inactive compounds and seven active compounds 6-12 (all of
their structures are shown in the Supporting Information). Since
the results of validation were strongly influenced by the quality
of the database, the composition of the active and inactive
compounds in the database is the predominant factor in GH
score assessment. Thus, the inclusion of inactive compounds
was limited according to the following criteria. (A) All inactive
compounds should match at least three types of features
(negatiVe ionizable, hydrophobic and hydrogen-bond acceptor
features), the same as active compounds, since an inactive
compound without one of these features in a pharmacophore
model is excluded easily and thus it cannot reflect its ability.
(B) Eighteen inactive compounds with thiazolidine-4-carboxylic
acid scaffolds were included to avoid obtaining a too-strict and
rigid pharmacophore model that could identify only the thia-
zolidine scaffold as an S1P3 receptor antagonist.
(iv) Determination of the Most Relevant Pharmacophore
Model. Pharmacophore models with excluded volumes were
validated to identify the most relevant model by the same GH
scoring method as described in section ii. In addition, pharma-
cophore models that could not identify any compound in the
test set were excluded.
The following criteria were applied to identify desirable
pharmacophore models: A GH score of more than 0.4 was used
to limit the number of models by less than 50%. A recall value
of more than 0.5 was used for desirable pharmacophore models.
Three hundred seven pharmacophore models were analyzed in
a scatter diagram between the GH score and the recall of active
compounds (Figure 3). Plots were divided into four blocks by
the above criteria. Most of the plots were in the lower squares,
which indicated a GH score of less than 0.4 and that these were
nonselective pharmacophore models. The upper-left square
showed too-exclusive pharmacophore models that could only
identify a few active compounds. Meanwhile, in the upper-right
square (GH score of more than 0.4 and recall of 0.5), the
pharmacophore models can selectively retrieve most active
compounds. The seven pharmacophore models in this upper-
right square, which fulfilled the criteria, were selected from
among the 307 models (Figure 4).
2. Database Searches. Composite databases, which contained
a total of approximately three million commercially available
compounds, were searched using the pharmacophore model. Hit
compounds (hit list) can be ranked according to the fit value,
which is the degree of consistency with the pharmacophore
model. In this paper, the fit value is defined as the relative
percentage compared to the maximum fit value. To decrease
the number of hits, a minimum fit value, which is the lowest
limit to qualify as a hit compound, was applied. In this paper,
we used 90% of the minimum fit value to prune the hit list.
3. Biological Assay. The IC50 values for S1P1, S1P2, and
S1P3 receptors were quantitatively determined in terms of the
Fura-2 fluorescent intensity. Dye was loaded into CHO cells
that stably expressed S1P1, S1P2 or S1P3 receptors. After
addition of an antagonist, the inhibition of the transient increase
in the intracellular calcium ion concentration ([Ca2+]i) induced
by S1P was measured.
Pharmacophore models R1, R2, R3, and R4 were generated
by the training set that included (2R)-thiazolidine-4-carboxylic
acid derivatives. Meanwhile, pharmacophore models S1, S2 and
S3 were generated by the model that consisted of (2S)-
derivatives. All seven models included four features: one
negatiVe ionizable, one hydrogen-bond acceptor, and two
hydrophobic features.
Results and Discussion
1. Generation and Validation of Pharmacophore Models.
(iii) Addition of Excluded Volumes to Pharmacophore
(i) Generation of Pharmacophore Models. Pharmacophore
Models by Considering the GH Score. Our previous study

Sphingosine 1-phosphate-3 Receptor Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3 445
Figure 2. Previously identified S1P3 receptor antagonists. Commercially available compounds are shown by the catalog number with the supplier’s
name in parentheses. The IC50 value was measured by the inhibition of S1P-induced cytosolic Ca2+ mobilization in CHO cells expressing S1P3
receptor in terms of Fura-2 fluorescence (see Experimental Section). For compounds 9 and 11, while IC50 values were over 20 µM, dose-dependent
inhibitions were observed. The asterisk (*) represents the training set for S1P3 receptor antagonists that was used to generate pharmacophore
models.
Table 1. Conditions for Pharmacophore Generation along with the
Number of Features, with or without the Hydrogen-Bond Acceptor
Feature and the Chirality at C2 of the (2R)- or (2S)-Thiazolidine
Scaffold in the Training Set
number of featuresa
HBA
number of modelsb
NegI
HYD
2R
2S
1
1
1
1
1
1
1
0
1
0
1
0
1
2
2
3
3
4
18
15
60
17
52
0
17
12
49
15
47
5
a Number of maximum features used in pharmacophore generation. NegI,
negative ionizable feature; HBA, hydrogen-bond acceptor feature; HYD,
hydrophobic feature. b Number of models generated by using a training set
that included (2R)- or (2S)-thiazolidine-4-carboxylic acid derivatives.
Figure 3. Scatter-diagram analysis of pharmacophore model validations
based on the GH score and recall of active compounds. A GH score of
1 means the best (ideal) model and 0 means the worst (null) model.
Recall means the yield of known active compounds registered in a
database (also called yield of actives (% Y)). For example, if a
pharmacophore model identifies all seven known actives 6-12 as hits
among 107 compounds, the recall value is 1 (100%).
showed that the S1P3 antagonist activity of thiazolidine-4-
carboxylic acid was increased by lengthening the side alkyl chain
at the 2-position up to 11 methylenic groups and then decreased
as the number of methylenic groups increased further.36 One
possible explanation for this observation is based on the steric
effects of the methyl group C13 in 2-tridecyl thiazolidine-4-
carboxylic acid 13 (Figure 5). All seven models in Figure 4
identified 13 as an active compound due to the lack of
information about steric properties. Therefore, these models
should be improved to take into account steric properties and
then further validated.
Overlap between the pharmacophore model and 13 could
reveal the 3D-coordinates of C13 positions which are expected
as excluded volumes. For that purpose, the following methods
were used: (A) All of the generated conformations of 13 with
the (2R)- or (2S)-isomer were superimposed on each pharma-
cophore model derived from the training set consisting of (2R)-
or (2S)-derivatives, respectively. All of the 3D-coordinates of
C13 of 13 were transferred to the center coordinates of excluded
volumes (spheres) with radius 1.5 Å and merged with the
pharmacophore model. (B) All active compounds 6-12 were
aligned with each pharmacophore model with the excluded
volumes. If an excluded volume sphere partially overlapped any
sphere of an atom in active compounds with radius 1.5 Å, this
excluded volume was discarded. Consequently, excluded vol-
umes outside the active space were retained. For example, in
model R3, 95 excluded volumes surrounding the active space
were retained (Figure 6a). (C) Each excluded volume was
assessed by the GH score using the above-mentioned database.
Only excluded volumes that improved the GH score were
retained, since they could filter out inactive compounds,
probably based on steric effects. For example, in model R3,
three of the 95 excluded volumes were assigned to be associated
with steric interaction based on improvement of the GH score
(Figure 6b,c).
(iv) Determination of the Most Relevant Pharmacophore
Model. As an S1P3 antagonist pharmacophore model, model
R3 with three excluded volumes (R3 Ex) was chosen among
seven models with excluded volumes based on the GH score
(Table 2).
The GH score of model R3 without an excluded volume was
0.59, which was the best among the seven models, and with
three excluded volumes this value increased to 0.93, which was
also the best among the seven models with excluded volumes.
In addition, model R3 could identify both 7 and 9 in the test
set that were not used for pharmacophore generation.

446 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3
Koide et al.
Figure 4. Seven pharmacophore models that consisted of one negatiVe ionizable, one hydrogen-bond acceptor, and two hydrophobic features
fulfilled the criteria. The letters R and S with each name indicate the pharmacophore model generated by a training set consisting of (2R)- and
(2S)-thiazolidine derivatives, respectively. Four features: NegIonizable (negatiVe ionizable), HYDROPHOBIC (hydrophobic) 2.11 and 3.11, and
HBA (hydrogen-bond acceptor) 4.11 features are represented as spheres with radius 1.72 Å. The HBA (hydrogen-bond acceptor) 4.21 feature is
the projected point of HBA 4.11 and is represented as a sphere with radius 2.32 Å.
14 as an S1P3 receptor antagonist, derivatives of 14 were
synthesized (Scheme 1). We replaced the piperidine group with
a phenyl group to simplify synthesis, since we assumed that it
acted as a hydrophobic feature.
Compound 19 or 20 was coupled with the benzophenone
derivative to elucidate whether two carboxylic acids were
essential, as the model predicted. Five benzophenone building
blocks 21-24 were synthesized to clarify the substituent effects
of 4-cetyloxy and 3-methoxy groups on the phenyl ring. Both
geometric isomers (E and Z isomers) with regard to the
hydrazone moiety were obtained by a coupling reaction. They
Figure 5. Assumed steric repulsion between S1P3 receptor and 13.
As a possible explanation for the significant decrease in the potency
of 13 compared to 6, steric repulsion with the S1P3 receptor can be
approximated at C13 of 13. In a pharmacophore model, a kind of steric
repulsion can be represented by the strategic placement of excluded
volume (a sphere that cannot be penetrated by the ligand).
were purified separately as single isomers by column chroma-
tography. The geometry of the hydrazone bond was assigned
by comparison of the 13C NMR chemical shift of the hydrazone
carbon of the E and Z isomers. A steric compression shift that
results in an upfield shift is observed in sterically hindered Z
isomer of oxime or hydrazone by 13C NMR spectroscopy43-46
andhasbeenusedinthestudiesofdiverseorganiccompounds.47-50
For example, the 13C NMR chemical shift of the hydrazone
carbon of E isomer 25E was 150.04 ppm and that of Z isomer
25Z was 149.26 ppm. Thus, a steric compression shift of 0.78
ppm was observed for the Z isomer. Compounds 25-29 were
purified separately as E or Z isomers. We obtained the
corresponding carboxylic acids 30-34 by hydrolysis.
2. Database Searches To Identify S1P3 Receptor Antago-
nists, Biological Assay, and Results. Model R3 Ex was used
to search databases consisting of commercially available
compounds. Among the hit list, 36 compounds were selected,
purchased, and assayed to identify S1P3 antagonist activity (the
structures of all assayed compounds are shown in the Supporting
Information).
Among the screened compounds, isophthalic acid derivatives
14, 15, and 16 were identified as antagonists for S1P3 receptor
(Figure 7). While they showed similar potencies, we chose
isophthalic acid derivative 14 for further studies to clarify the
structural requirements because it contained fewer carboxylic
acids than 15 and 16. Isophthalic acid 14 was aligned to model
R3 Ex (Figure 8a). One of the carboxylic acids on the phenyl
ring was recognized as a negatiVe ionizable feature. The other
carboxylic acid was assumed to act as the hydrogen-bond
acceptor feature. The 3-methoxyphenyl group, but not the
4-cetyloxy group, was mapped as a hydrophobic feature. The
direction of the 4-cetyloxy group was different from that of the
m-substituted alkyl chain in 8 (Figure 8a,b). The piperidine
group, which was recognized as a hydrophobic feature, was also
considered to play an important role in regulating the active
conformation.
S1P3 receptor antagonist activities of these synthesized
derivatives 30-34 were evaluated (Table 3). Isophthalic acid
14 retrieved from the database showed an IC50 value of 3.44
µM, and this also showed moderate antagonist activity for the
S1P1 receptor. Synthesized 30E showed an IC50 value of 2.96
µM. We also synthesized a derivative without this phenyl group,
corresponding to compound 30E; however, S1P3 antagonist
activity was not observed at all (data not shown). Therefore,
this phenyl group was implicated in the interaction with the
S1P3 receptor. None of the synthesized sulfohydrazone deriva-
tives showed more than 50% inhibitory activity for the S1P2
receptor at 10 µM.
In the assay of 32E, 31E, and 30E with 4, 8, and 16 alkyl
chain carbons, respectively, the IC50 values for the S1P3 receptor
and the S1P1 receptor were found to decrease with a decrease
in the length of the alkyl chain. Therefore, the length of the
alkyl chain at the 4-position of the phenyl ring was the dominant
factor for not only the S1P3 receptor but also S1P1 receptor
3. Synthesis of Derivatives and Identification of the
Structure-Activity Relationship in an S1P3 Receptor An-
tagonist. To clarify the minimum structural requirements for

Sphingosine 1-phosphate-3 Receptor Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3 447
Figure 6. Addition of all excluded volumes to model R3 to identify 13 as inactive. Overlap between the optimized model (R3 Ex) and 8. (a)
Ninety-five 3D coordinates of C13 in compound 13 aligned with model R3 were transferred to the corresponding excluded volumes with radius 1.5
Å and merged into that model. (b and c) Viewpoints of model R3 Ex aligned with compound 8. Three excluded volumes 1-3 are abbreviated as
Excl-1, -2, and -3 and indicated with black mesh.
Table 2. Comparison of Database Searches Using Models R3 and R3 Exa
model
all
actives
hits
tp
fp
tn
fn
%A
%Y
E
GH
R3
R3 Ex
107
107
7
7
11
5
6
5
5
0
95
100
1
2
54.5
100
85.7
71.4
8.34
15.3
0.59
0.93
a All, number of compounds in the database; actives, number of actives in the database; hits, number of hits retrieved by the model (minimum fit value
of 90%); tp, number of true positives in the hit list; fp, number of false positives in the hit list; tn, number of true negatives in the hit list; fn, number of false
negatives in the hit list.
Figure 8. Overlap between model R3 Ex and 14 and 8. (a) 14 with
model R3 Ex. (b) 8 with model R3 Ex.
Figure 7. Newly identified S1P3 receptor antagonists obtained via a
database search. The supplier’s name is given in parentheses. The IC50
value was measured by the inhibition of S1P-induced [Ca2+]i mobiliza-
tion in CHO cells expressing the S1P3 receptor in terms of Fura-2
fluorescence.
30E. This assumption regarding the pharmacophore model is
consistent with the results of the biological assay. The impor-
tance of the 3-methoxy group as the hydrophobic feature was
supported by the finding that compound 33E, which does not
contain a 3-methoxy group, was less potent than compound 30E.
As mentioned earlier, the 4-cetyloxy group did not correspond
to the meta-substituted alkyl chain of compound 8 (Figure 8a,b).
However, the 4-cetyloxy group in isophthalic acid derivatives
was significantly associated with antagonist activities for S1P1
and S1P3 receptors. Taken together, these results suggest that
the 4-cetyloxy group plays a role corresponding to the alkyl
chain of S1P. Initially, we hypothesized that the hydrophobic
space represented by hydrophobic 2.11 and 3.11 corresponded
to the alkyl chain of S1P. However, we now believe that it works
differently than S1P and is significantly associated with
antagonist activity for the S1P3 receptor. Thus, a 3,4-dialkoxy-
benzophenone scaffold which fulfilled this hydrophobic space
could be a potent component of an S1P3 receptor antagonist.
Meanwhile, since a 4-cetyloxy group may play a role similar
to that of the alkyl chain of S1P, it is assumed to contribute to
antagonist activity. This tendency was also observed for 31Z
and 30Z.
As expected based on the pharmacophore model, the car-
boxylic acid of isophthalic acid, which was identified as a
hydrogen-bond acceptor feature, was also essential for S1P3
antagonist activity, since benzoic acid derivatives 34E and 34Z
did not show clear potencies. Compound 30Z exhibited a similar
potency (IC50 3.57 µM) to 30E. Both of the active conformations
30E and 30Z were compared along with model R3 Ex (Figure
9a,b). 3,4-Dialkoxybenzophenone acted as hydrophobic 2.11 and
3.11 in both 30E and 30Z. The 3-methoxy group of 30E was
superimposed on hydrophobic 3.11, as in compound 14.
Meanwhile, the 3-methoxy group of 30Z could also be
superimposed on hydrophobic 3.11, since the symmetry of
isophthalic acid allowed each carboxylic acid to act as either a
negatiVe ionizable or hydrogen-bond acceptor feature, with the
result that 30Z could be superimposed on the model similar to

448 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3
Koide et al.
Scheme 1. Synthesis of 5-sulfohydrazono-1,3-isophthalic Acid Derivatives and 3-Sulfohydrazonobenzoic Acid Derivativesa
a
Reagents and conditions: (a) NH2NH2‚H2O, THF, 0 °C; (b) 19 or 20, MeOH, reflux; (c) 1N NaOH, THF-MeOH.
Table 3. S1P3 Receptor Antagonist Activities of Synthesized
Isophthalic Acid Derivativesa
IC50 (µM)
compd
S1P3
S1P1
S1P2
14
3.44
2.96
3.57
11.39
13.23
(6.7%)
9.74
(34.4%)
(8.3%)
6.18
6.64
4.28
(12.7%)
(32.9%)
(24.2%)
(17.2%)
(34.8%)
(1.0%)
(35.9%)
(40.9%)
(9.5%)
(-4.6%)
(14.0%)
(6.2%)
(20.5%)
(-0.1%)
(-8.7%)
30E
30Z
31E
31Z
32E
33E
34E
34Z
a The IC50 value was measured by the inhibition of S1P-induced [Ca2+
]
i
mobilization in CHO cells expressing S1P1, S1P2, or S1P3 receptor in terms
of Fura-2 fluorescence. For a compound with lower potency, for which it
was difficult to measure an accurate IC50 value, the percent inhibition at
10 µM is given in parentheses.
all S1P receptors and to lack selectivity for S1P3 receptor
antagonism. Thus, 31E with a moderate alkyl chain may be
useful not only for exploring a more selective pharmacophore
model for S1P3 receptor antagonists but also as a better starting
point to obtain a more potent and selective S1P3 receptor
antagonist than 14 and 30E.
4. Assessment of the Model Using Different S1P3 Receptor
Antagonists. Although the pharmacophore model identified a
potent scaffold, it was based on a training set that consisted of
compounds with limited diversity and activities. To generate a
feature-based pharmacophore model, structural diversity is
Figure 9. Overlap analysis of E and Z isomers. (a) 30E (E isomer)
with model R3 Ex. (b) 30Z (Z isomer) with model R3 Ex.
important for exploring the key structural features. In addition,
there were too few compounds in the test set. Thus, the
pharmacophore model was further evaluated using different S1P3

Sphingosine 1-phosphate-3 Receptor Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3 449
Figure 10. S1P1/S1P3 receptor agonists and antagonists reported by
the University of Virginia. The three asterisks (*) represent the reported
S1P1/S1P3 receptor antagonists, and their antagonist activities for the
S1P3 receptor were determined by a [32P]-S1P binding assay (4; Ki )
1175 nM, 5; Ki ) 98 nM, 50; Ki ) 1586 nM).32 Compounds 35-42
and 47-49 were reported to be S1P1/S1P3 receptor agonists with EC50
values for the S1P3 receptor that were greater than 1000 nM based on
a [35S]- γ-GTP binding assay.51,52 Compounds 43 and 46 were reported
to be both S1P1 receptor agonists and S1P3 receptor partial agonist
and weak partial agonist, respectively.32 Compound 44 was reported
to be an S1P1 receptor antagonist and 45 was reported as an S1P1/
S1P4 receptor agonist.32
Figure 11. Overlap between model R3 Ex and 5 with a C7 alkyl chain
and 44 with a C6 alkyl chain. (a) 5 with model R3 Ex. (b) 44 with
model R3 Ex. Since 5 with model R3 Ex shows a better fit than 44,
the terminal alkyl group of the C7 alkyl chain in 5 is assumed to be
important for S1P3 receptor antagonism. This perspective is consistent
with their reported Ki values.
alkyl chains, respectively.32 As mentioned, the pharmacophore
model provided one possible explanation for the sensitivity of
these antagonist activities for the S1P3 receptor. This perspective
is consistent with our results. In addition, the antagonist activities
of these compounds for the S1P1 receptor were reported to not
be influenced by the length of their alkyl chains.32 If we consider
all of these results, while the pharmacophore model has not
clarified the role of hydrogen-bond acceptor feature, at least
the hydrophobic space including hydrophobic 2.11 and 3.11
features surrounded by excluded volumes is assumed to play
the key important role for S1P3 receptor antagonism.
receptor antagonists to see whether the model could identify
the same key features for S1P3 receptor antagonism in our result.
Recently, researchers at the University of Virginia reported
that phosphates 4, 5, and 50 were S1P1/S1P3 receptor antagonists
based on the result of [35S]-γ-GTP binding and [32P]-S1P
binding assays (Figure 10).32 Meanwhile, most of the derivatives
in Figure 10 were reported to act as S1P3 receptor agonists.32,51,52
They were useful for assessing the potency of the pharmacoph-
ore model for S1P3 receptor antagonists, since they included
agonists (35-43 and 46-49), antagonists (4, 5, and 50) and
inactive compounds (44 and 45) for the S1P3 receptor. Model
R3 Ex was applied to elucidate if it could efficiently identify
the antagonists among them.
Conclusion
Our results demonstrate that a pharmacophore model con-
structed by common feature-based alignment can be used for
scaffold-hopping to efficiently identify diverse active compounds
prior to biological testing. For that purpose, the GH score based
on well-selected structures was useful as a metric for determin-
ing relevant excluded volumes and obtaining a reliable phar-
macophore model. Isophthalic acid derivatives were identified
as novel and potent S1P3 receptor antagonists by a pharma-
cophore-based database search. On the basis of a consideration
of structure-activity relationships, the 3,4-dialkoxybenzophe-
none scaffold was found to be a potent component of an S1P3
receptor antagonist. The key feature for S1P3 receptor antago-
nism which was described by hydrophobic features with
excluded volumes was assumed to be different from the space
occupied by the alkyl chain of S1P. Further studies are underway
to identify selective S1P3 receptor antagonists by pharmacoph-
ore-based design.
As a result, model R3 Ex identified only 5 as an active
compound among the 18 derivatives in Figure 10. Although 4
and 50 were reported to be S1P3 receptor antagonists, their Ki
values were less than one-tenth that of 5. Thus, the results based
on the pharmacophore model are consistent with the reported
S1P3 receptor antagonist actives. The terminal alkyl group of
the C7 alkyl chain in 5 was superimposed on hydrophobic 3.11,
as in 8 (Figure 11a). Meanwhile, 44, with a C6 alkyl chain,
which was reported to not act as an S1P3 receptor agonist or
antagonist, had an alkyl chain that was too short to reach
hydrophobic 3.11 (Figure 11b) and thus showed a lower fit value
than 5. In addition, since 4 with a C8 alkyl chain, which is one
carbon longer than that in 5, also shows a lower fit value than
5, it may have reduced the potency due to repulsion between
excluded volumes.
The potency for S1P3 receptor antagonism was reported to
differ significantly among 44, 5, and 4 with C6, C7, and C8

450 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3
Koide et al.
chromatography was carried out on E. Merck Kieselgel 60 (230-
400 mesh) silica gel.
Experimental Section
Pharmacophore Modeling Studies. Pharmacophore models
were generated with the HipHop module of Catalyst 4.938,53 on an
Intel-based PC running the Red Hat Enterprise Linux WS 3.0
operating system. Conformational models of all compounds were
calculated using an energy cutoff of 15 kcal in Best mode. The
number of conformers generated for each molecule was limited to
a maximum of 255. Pharmacophore generation was conducted by
the feature-based alignment of five active compounds: 6, 8, 10,
11, and 12. Compound 8 was used as the central compound to
generate pharmacophore models. The database used for GH score-
based validation was constructed using Catalyst/catDB, and the
maximum number of conformers per compound was set at 255 in
Best mode.
Conformers of (2R)- and (2S)-13 were generated with the above-
mentioned conditions and 182 and 204 conformers were obtained,
respectively. All conformations in (2R)- or (2S)-13 were aligned
with the pharmacophore model derived from the training set
consisting of (2R)- or (2S)-derivatives, respectively. All superim-
posed 3D-coordinates of C13 of 13 in the pharmacophore models
were exported using the Catalyst/citest command and converted
into the excluded volumes (spheres) with radius 1.5 Å. The seven
active compounds of the training set were also aligned with the
pharmacophore models, and the 3D-coordinates of all atoms were
exported using the Catalyst/citest command and converted into atom
spheres with radius 1.5 Å. Excluded volumes that overlapped any
atom sphere with radius 1.5 Å were omitted because of inappropri-
ate 3D-coordinates for an excluded volume. Appropriate excluded
volumes that improved the GH score were identified and incorpo-
rated into the pharmacophore model. The Minimum Fit for Search
option of the pharmacophore model was set at 3.6, which was 90%
of the maximum fit value. A database search was conducted with
a fast flexible search.
Stable Expression of S1P Receptors in CHO-K1 Cells. CHO-
K1 cells (Dainippon Sumitomo Pharma) were transfected using
Lipofectamine 2000 (Invitrogen) with pcDNA3.1 (+) plasmid
(Sigma-Aldrich) encoding the human S1P1 or S1P3 receptor and
neomycin (G418) resistance or pFLAG-CMV9 plasmid (Sigma-
Aldrich) encoding the human S1P2 receptor and G418 resistance.
Cells were grown in Ham’s F12K medium supplemented with G418
(200 µg/mL, Sigma-Aldrich).
Measurement of Intracellular Calcium Mobilization. A Flex-
Station II fluorimeter (Molecular Devices) was used to measure
the intracellular calcium concentration ([Ca2+]i) in CHO-K1 cells
that had been stably transfected with S1P1, S1P2, or S1P3 receptors.
Suspensions of cells in a nutrient mixture of Ham’s medium
containing 1% (v/v) fetal bovine serum (FBS) were loaded (20 000
cells/well) in 96-well, clear-bottom black microplates (Greiner Bio-
One) and left for 24 h at 37 °C. The cells were washed and then
incubated in Ham’s medium for 24 h at 37 °C.
5-Chlorosulfonyl Isophthalic Acid Dimethyl Ester (17). To a
suspension of dimethyl 5-sulfoisophthalate sodium salt (5.70 g,
19.24 mmol) in CCl4 (250 mL) were added PCl5 (20.23 g, 96.21
mmol) and diisopropylamine (2.5 mL, 17.66 mmol). The mixture
was refluxed for 6 h, diluted with H2O at 0 °C, extracted with
CHCl3, washed sequentially with H2O and brine, dried over Na2-
SO4, filtered, and concentrated. Recrystallization from diethylether/
hexane gave 17 as a colorless powder (3.16 g, 56%). mp 91-92
°C; 1H NMR (300 MHz, CDCl3) δ 9.00 (s, 1H), 8.85 (s, 2H), 4.03
(s, 6H); 13C NMR (75 MHz, CDCl3) δ 163.88, 145.13, 136.48,
132.66, 131.57, 53.19; MS (FAB) m/z 292 (M+); Anal. (C10H9-
ClO6S) calcd, C 41.04; H 3.10; found, C 41.35, H 3.10.
5-Hydrazosulfonyl Benzene-1,3-Carboxylic Acid Dimethyl
Ester (19). A solution of hydrazine hydrate (3.28 g, 65.60 mmol)
in EtOH (50 mL) was added to a stirred 17 (3.84 g, 13.12 mmol)
in CHCl3 (100 mL). The mixture was stirred for 6 h, diluted with
CHCl3, washed sequentially with H2O and brine, dried over Na2-
SO4, filtered, and concentrated. Recrystallization from diethylether/
hexane gave 19 as colorless needles (2.11 g, 56%). mp 142-143
°C; 1H NMR (300 MHz, CDCl3) δ 8.91 (s, 1H), 8.73 (d, J ) 1.65
Hz, 2H), 4.00 (s, 6H); 13C NMR (75 MHz, CDCl3) δ 164.56,
139.10, 135.04, 132.95, 132.09, 52.98; MS (FAB) m/z 288 (M+);
Anal. (C10H12N2O6S) calcd, C 41.66; H 4.20; N, 9.72; found, C,
41.72; H, 4.09; N, 9.55.
3-Methoxycarbonylbenzenesufohydrazide (20). A suspension
of 5-chlorosulfonylbenzoic acid (1.10 g, 5.00 mmol) in thionyl
chloride (5 mL) was refluxed for 2 h. The mixture was concentrated,
dissolved in methanol (10 mL), stirred for 15 min, and concentrated.
The residue of 18 was dissolved in THF (10 mL). To the solution
was added hydrazine hydrate (0.36 mL, 7.50 mmol). The mixture
was stirred for 30 min at 0 °C, diluted with CHCl3, washed
sequentially with H2O and brine, dried over Na2SO4, filtered, and
concentrated. Recrystallization from chloroform/diethylether gave
20 as a colorless powder (367 mg, 32%). mp 87-89 °C; 1H NMR
(300 MHz, CDCl3) δ 8.55 (brs, 1H), 8.33 (s, 1H), 8.19 (d, J )
7.71 Hz, 1H), 8.04 (d, J ) 7.94 Hz, 1H), 7.76 (dd, J ) 7.94, 7.71
Hz, 1H), 4.21(brs, 2H), 3.90 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ 165.36, 137.19, 134.30, 132.23, 131.41, 129.57, 129.21, 52.70;
MS (FAB) m/z 230 (M+); Anal. (C8H10N2O4S) calcd, C, 41.73; H,
4.38; N, 12.17; found, C, 42.08; H, 4.26; N, 11.95.
4-Cetyloxy-3-methoxybenzophenone (21). To a solution of
4-hydroxy-3-methoxybenzophenone55 (342 mg, 1.50 mmol) in DMF
(10 mL) were added sodium carbonate (249 mg, 1.80 mmol) and
cetyl bromide (0.69 mL, 2.25 mmol). The mixture was stirred for
2 h at 80 °C, diluted with ethyl acetate, washed sequentially with
H2O and brine, dried over Na2SO4, filtered, and concentrated.
Column chromatography (10:1 hexane:ethyl acetate) provided 21
1
as a colorless powder (605 mg, 89%). mp 57-58 °C; H NMR
(300 MHz, CDCl3) δ 7.75 (dd, J ) 8.63, 1.65 Hz, 2H), 7.59-7.54
(m, 1H), 7.50-7.45 (m, 3H), 7.36 (dd, J ) 8.42, 2.01 Hz, 1H),
6.88 (d, J ) 8.42 Hz, 1H), 4.09 (t, J ) 7.03 Hz, 2H), 3.93 (s, 3H),
1.89 (dt, J ) 7.03, 6.89 Hz, 2H), 1.49-1.42 (m, 2H), 1.38-1.22
(m, 24H), 0.88 (t, J ) 6.61 Hz, 3H); 13C NMR (75 MHz, CDCl3)
δ 195.64, 152.70, 149.21, 138.37, 131.81, 129.87, 129.72, 128.15,
125.53, 112.40, 110.76, 69.09, 56.11, 31.93, 29.69, 29.66, 29.60,
29.55, 29.37, 28.96, 25.91, 22.70, 14.13; MS (FAB) m/z 452 (M+);
Anal. (C30H44O3) calcd, C, 79.60; H, 9.80; found, C, 79.35; H, 10.0.
4-Octyloxy-3-methoxybenzophenone (22). Compound 22 was
prepared according to the procedure described for 21 but with octyl
bromide instead of cetyl bromide. Compound 22 was obtained as
a colorless powder (604 mg, quant). mp 34-35 °C; 1H NMR (300
MHz, CDCl3) δ 7.76 (dd, J ) 8.44, 1.47 Hz, 2H), 7.59-7.54 (m,
1H), 7.50-7.45 (m, 3H), 7.36 (dd, J ) 8.44, 2.02 Hz, 1H), 6.88
(d, J ) 8.44 Hz, 1H), 4.09 (t, J ) 6.97 Hz, 2H), 3.93 (s, 3H), 1.87
(dt, J ) 6.97, 6.79 Hz, 2H), 1.49-1.46 (m, 2H), 1.33-1.23 (m,
8H), 0.89 (t, J ) 6.97 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ
195.38, 152.61, 149.12, 138.24, 131.65, 129.76, 129.56, 128.00,
125.37, 112.32, 110.70, 68.94, 55.94, 31.66, 29.20, 29.07, 28.86,
Cells were washed and dye-loaded with Fura2-AM ester (5 µM,
Molecular Probes) in Hanks balanced salt solution (HBSS) contain-
ing sulfinpyrazone (0.25 mmol/l, Sigma-Aldrich) for 60 min at 37
°C. After cell monolayers were washed with HBSS containing
sulfinpyrazone (0.25 mmol/L), loading buffer was added, and cells
were exposed to the test compound for 5 min. After S1P was added
(10 nmol, Sigma-Aldrich), fluorescence (excitation at 335 and 362
nm; emission at 505 and 512 nm) was immediately measured for
45 s at intervals of 3 s with a FlexStation II. A curve-fitting
algorithm (Model 08: Sigmoidal Inhibition Curve) implemented
in MDL Assay Explorer54 was used to calculate the IC50 value.
Chemistry. General Information. Melting points were deter-
mined with a Yanako MP-500V micro melting point apparatus
1
(uncorrected), and H and 13C NMR spectra were recorded on a
JEOL JNM-AL-300, using CDCl3 or DMSO-d6 as solvents, with
Me4Si as an internal standard. Mass spectra were recorded on either
a JEOL HX-110A (FAB) or Finnigan LCQ (ESI). Elemental
analysis (C, H, N) was performed on a Carloerba EA-1108 at Tokyo
Chemical Industry Co., Ltd. Reactions were monitored by TLC
analysis using E. Merck silica gel 60F254 thin layer plates. Flash

Sphingosine 1-phosphate-3 Receptor Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3 451
25.79, 22.52, 13.97; MS (FAB) m/z 340 (M+); Anal. (C22H28O3)
calcd, C, 77.61; H, 8.29; found, C, 77.39; H, 8.41.
(dt, J ) 7.89, 6.97 Hz, 2H), 1.51-1.47 (m, 2H), 1.35-1.30 (m,
8H), 0.89 (t, J ) 6.79 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ
164.68, 155.37, 150.29, 150,09, 139.85, 136.32, 134.81, 132.81,
131.87, 130.14, 128.25, 127.89, 122.34, 121.13, 113.07, 111.27,
69.13, 56.17, 52.89, 31.82, 29.34, 29.23, 29.04, 25.98, 22.66, 14.12;
MS (ESI) m/z 610 (M+); Anal. (C32H38N2O8S) calcd, C, 62.93; H,
6.27; N, 4.59; found, C, 62.93; H, 6.26; N, 4.61. Compound 26Z:
mp 110-111 °C; 1H NMR (300 MHz, CDCl3) δ 8.88 (d, J ) 1.65
Hz, 1H), 8.84-8.82 (m, 2H), 7.54-7.52 (m, 3H), 7.33 (d, J )
1.65 Hz, 1H), 7.15-7.12 (m, 2H), 6.69-6.63 (m, 2H), 4.00-3.95
(m, 8H), 3.87 (s, 3H), 1.85-1.79 (m, 2H), 1.49-1.45 (m, 2H),
1.39-1.24 (m, 8H), 0.87 (t, J ) 6.97 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 164.62, 155.31, 150.71, 149.25, 139.90, 134.74, 132.84,
131.83, 130.89, 130.20, 129.75, 128.58, 128.22, 122.30, 111.22,
109.17, 68.91, 55.91, 52.85, 31.75, 29.29, 29.16, 28.93, 25.86,
22.61, 14.08; MS (ESI) m/z 610 (M+); Anal. (C32H38N2O8S) calcd,
C, 62.93; H, 6.27; N, 4.59; found, C, 62.64; H, 6.10; N, 4.40.
4-Butylloxy-3-methoxybenzophenone (E)-3,5-Dimethoxycar-
bonylphenylsulfohydrazone (27E). Compound 27E was prepared
according to the procedure described for 25E and 25Z but with 23
instead of 21. Compound 27E was obtained as a colorless powder
4-Butyloxy-3-methoxybenzophenone (23). Compound 23 was
prepared according to the procedure described for 21 but with butyl
bromide instead of cetyl bromide. Compound 23 was obtained as
1
a colorless powder (435 mg, 87%). mp 75-76 °C; H NMR (300
MHz, CDCl3) δ 7.77 (dd, J ) 8.26, 1.29 Hz, 2H), 7.59-7.54 (m,
1H), 7.50-7.45 (m, 3H), 7.36 (dd, J ) 8.26, 2.02 Hz, 1H), 6.88
(d, J ) 8.26 Hz, 1H), 4.10 (t, J ) 6.79 Hz, 2H), 3.92 (s, 3H), 1.86
(dt, J ) 7.52, 6.79 Hz, 2H), 1.56-1.48 (m, 2H), 0.99 (t, J ) 7.34
Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 195.52, 152.69, 149.19,
138.31, 131.73, 129.85, 129.64, 128.08, 125.44, 112.42, 110.77,
68.70, 56.05, 30.96, 19.11, 13.78; MS (FAB) m/z 284 (M+); Anal.
(C18H20O3) calcd, C, 76.03; H, 7.09; found, C, 75.97; H, 7.18.
4-Cetyloxybenzophenone (24). Compound 24 was prepared
according to the procedure described for 21 but with 4-hydroxy-
benzophenone instead of 4-hydroxy-3-methoxybenzophenone. Com-
pound 24 was obtained as a colorless powder (1.14 g, 90%). mp
1
66-67 °C; H NMR (300 MHz, CDCl3) δ 7.82 (d, J ) 8.84 Hz,
2H), 7.75 (dd, J ) 8.44, 1.46 Hz, 2H), 7.59-7.53 (m, 1H), 7.49-
7.44 (m, 2H), 6.95 (d, J ) 8.84 Hz, 2H), 4.03 (t, J ) 6.61 Hz,
2H), 1.82 (dt, J ) 6.90, 6.61 Hz, 2H), 1.52-1.22 (m, 26H), 0.88
(t, J ) 6.61 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 195.57, 162.84,
138.29, 132.55, 131.81, 129.80, 129.70, 128.13, 113.94, 68.24,
31.90, 29.68, 29.64, 29.58, 29.54, 29.35, 29.08, 25.96, 22.68, 14.12;
MS (FAB) m/z 422 (M+); Anal. (C29H42O2) calcd, C, 82.41; H,
10.02; found, C, 82.55; H, 10.08.
1
(760 mg, 91%). mp 179-181 °C; H NMR (300 MHz, CDCl3) δ
8.89 (t, J ) 1.47 Hz, 1H), 8.84 (d, J ) 1.47 Hz, 2H), 7.78 (s, 1H),
7.48 (dd, J ) 8.44, 1.65 Hz, 2H), 7.36-7.29 (m, 3H), 6.98 (d, J )
8.26 Hz, 1H), 6.71 (dd, J ) 8.26, 2.02 Hz, 1H), 6.61 (d, J ) 2.02
Hz, 1H), 4.09 (t, J ) 6.79 Hz, 2H), 4.00 (s, 6H), 3.80 (s, 3H), 1.88
(dt, J ) 7.16, 6.79 Hz, 2H), 1.58-1.50 (m, 2H), 1.01 (t, J ) 7.34
Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 164.68, 155.36, 150.25,
139.81, 136.29, 134.82, 132.81, 131.85, 130.15, 128.25, 127.88,
122.29, 121.11, 114.00, 113.00, 111.22, 68.75, 56.15, 52.90, 31.05,
19.21, 13.86; MS (ESI) m/z 554 (M+); Anal. (C28H30N2O8S) calcd,
C, 60.64; H, 5.45; N, 5.05; found, C, 60.74; H, 5.34; N, 5.27.
4-Cetyloxybenzophenone (E)-3,5-dimethoxycarbonylphenyl-
sulfohydrazone (28E). Compound 28E was prepared according
to the procedure described for 25E and 25Z but with 24 instead of
21. Compound 28E was obtained as a colorless powder (84 mg,
4-Cetyloxy-3-methoxybenzophenone (E)-3,5-dimethoxycar-
bonylphenylsulfohydrazone (25E) and 4-Cetyloxy-3-methoxy-
benzophenone (Z)-3,5-dimethoxycarbonylphenylsulfohydrazone
(25Z). To a solution of 19 (562 mg, 1.95 mmol) in methanol (13
mL) was added 21 (588 mg, 1.30 mmol). The mixture was refluxed
for 18 h, diluted with CHCl3, washed with 1 N hydrochloric acid
and brine, dried over Na2SO4, filtered, and concentrated. Column
chromatography (5:1 hexane:ethyl acetate) provided 25E as a
colorless powder (656 mg, 70%) and 25Z as a colorless powder
(64 mg, 7%). Compound 25E: mp 124-126 °C; 1H NMR (CDCl3)
δ 8.89 (t, J ) 1.55 Hz, 1H), 8.84(d, J ) 1.65 Hz, 2H), 7.77 (brs,
1H), 7.49 (dd, J ) 8.08, 1.65 Hz, 2H), 7.38-7.29 (m, 3H), 6.97
(d, J ) 8.11 Hz, 1H), 6.71 (dd, J ) 8.11, 1.80 Hz, 1H), 6.60 (d, J
) 1.80 Hz, 1H), 4.08 (t, J ) 7.02 Hz, 2H), 4.00 (s, 6H), 3.80 (s,
3H), 1.89 (dt, J ) 7.02, 6.89 Hz, 2H), 1.52-1.22 (m, 26H), 0.88
(t, J ) 6.21 Hz, 3H); 13C NMR (CDCl3) δ 164.63, 155.34, 150.17,
150.04, 139.78, 136. 27, 134.77, 132.78, 131.80, 130.11, 128.21,
127.84, 122.27, 121.09, 112.93, 111.16, 69.06, 56.10, 52.87, 31.89,
29.67, 29.59, 29.55, 29.37, 29.34, 29.00, 25.93, 22.66, 14.12; MS
(ESI) m/z 722 (M+); Anal. (C40H54N2O8S) calcd, C, 66.46; H, 7.53;
N, 3.87; found, C, 66.45; H, 7.53; N, 3.89. Compound 25Z: mp
73-74 °C; 1H NMR (CDCl3) δ 8.88 (t, J ) 1.65 Hz, 1H), 8.82 (d,
J ) 1.65 Hz, 2H), 8.54 (brs, 1H), 7.54-7.49 (m, 3H), 7.33 (d, J )
1.71 Hz, 1H), 7.14-7.11 (m, 2H), 6.67 (d, J ) 8.43 Hz, 1H), 6.61
(dd, J ) 8.43, 1.71 Hz, 1H), 3.99 (t, J ) 6.80 Hz, 2H), 3.98 (s,
6H), 3.87 (s, 3H), 1.81 (dt, J ) 7.21, 6.80 Hz, 2H), 1.44-1.22 (m,
26H), 0.88 (t, J ) 6.43 Hz, 3H); 13C NMR (CDCl3) δ 164.64,
155.33, 150.76, 149.26, 139.93, 134.76, 132.86, 131.86, 130.92,
130.23, 129.77, 128.61, 128.25, 122.31, 111.25, 109.22, 68.94,
55.94, 52.87, 31.93, 29.69, 29.65, 29.59, 29.53, 29.36, 28.98, 25.90,
22.70, 14.13; MS (ESI) m/z 722 (M+); Anal. (C40H54N2O8S) calcd,
C, 66.46; H, 7.53; N, 3.87; found, C, 66.34; H, 7.56; N, 3.90.
4-Octyloxy-3-methoxybenzophenone (E)-3,5-Dimethoxycar-
bonylphenylsulfohydrazone (26E) and 4-Octyloxy-3-methoxy-
benzophenone (Z)-3,5-dimethoxycarbonylphenylsulfohydrazone
(26Z). Compounds 26E and 26Z were prepared according to the
procedure described for 25E and 25Z but with 22 instead of 21.
Compound 26E was obtained as a colorless powder (500 mg, 72%),
and 26Z was obtained as a colorless powder (187 mg, 27%).
Compound 26E: mp 136-137 °C; 1H NMR (300 MHz, CDCl3) δ
8.89 (t, J ) 1.47 Hz, 1H), 8.84 (d, J ) 1.47 Hz, 2H), 7.76 (brs,
1H), 7.48 (d, J ) 8.44 Hz, 2H), 7.36-7.29 (m, 3H), 6.98 (d, J )
8.26 Hz, 1H), 6.71 (dd, J ) 8.26, 2.02 Hz, 1H), 6.61 (d, J ) 2.02
Hz, 1H), 4.08 (t, J ) 6.97 Hz, 2H), 4.00 (s, 6H), 3.80 (s, 3H), 1.88
1
61%). mp 141-143 °C; H NMR (300 MHz, CDCl3) δ 8.89 (t,
J ) 1.48 Hz, 1H), 8.83 (d, J ) 1.48 Hz, 2H), 7.45 (d, J ) 7.17
Hz, 2H), 7.36-7.28 (m, 4H), 7.10 (d, J ) 8.62 Hz. 2H), 7.01 (d,
J ) 8.62 Hz, 2H), 4.02 (t, J ) 6.41 Hz, 2H), 4.00 (s, 6H), 1.83 (dt,
J ) 7.01, 6.41 Hz, 2H), 1.24-1.51 (m, 26H), 0.88 (t, J ) 6.63 Hz,
3H); 13C NMR (75 MHz, CDCl3) δ 164.68 160.56, 139.82, 136.51,
134.82, 132.83, 131.86, 130.11, 130.00, 128.22, 127.93, 122.13,
115.61, 68.27, 52.88, 31.93, 29.70, 29.60, 29.36, 29.15, 26.04,
22.70, 14.13; MS (ESI) m/z 692 (M+); Anal. (C39H52N2O7S) calcd,
C, 67.60; H, 7.56; N, 4.04; found, C, 67.39; H, 7.62; N, 4.02.
4-Cetyloxy-3-methoxybenzophenone (E)-3-Methoxycarbon-
ylphenylsulfohydrazone (29E) and 4-Cetyloxy-3-methoxyben-
zophenone (Z)-3-Methoxycarbonylphenylsulfohydrazone (29Z).
Compounds 29E and 29Z were prepared according to the procedure
described for 25E and 25Z, but with 20 instead of 19. Compound
29E was obtained as a colorless powder (84 mg, 32%) and 29Z
was obtained as a colorless powder (132 mg, 50%). Compound
29E: mp 85-86 °C; 1H NMR (300 MHz, CDCl3) δ 8.66 (dd, J )
1.83, 1.38 Hz, 1H), 8.27(ddd, J ) 7.91, 2.93, 1.38 Hz, 1H), 8.19
(ddd, J ) 7.83, 2.93, 1.83 Hz, 1H), 7.75 (brs, 1H), 7.65 (dd, J )
7.91, 7.83 Hz, 1H), 7.47 (dd, J ) 7.89, 1.55 Hz, 2H), 7.38-7.27
(m, 3H), 6.97 (d, J ) 8.30 Hz, 1H), 6.69 (dd, J ) 8.30, 1.83 Hz,
1H), 6.59 (d, J ) 1.83 Hz, 1H), 4.07 (t, J ) 6.81 Hz, 2H), 3.97 (s,
3H), 3.79 (s, 3H), 1.89 (dt, J ) 7.02, 6.81 Hz, 2H), 1.54-1.44 (m,
2H), 1.40-1.23 (m, 24H), 0.88 (t, J ) 6.40 Hz, 3H); 13C NMR
(75 MHz, CDCl3) δ 165.43, 154.98, 150.25, 149.99, 139.14, 136.45,
134.07, 131.96, 131.30, 130.02, 129.28, 129.08, 128.22, 127.80,
122.48, 121.08, 113.03, 111.23, 69.11, 56.13, 52.63, 31.93, 29.70,
29.62, 29.59, 29.41, 29.36, 29.05, 25.98, 22.70, 14.13; MS (ESI)
m/z 664 (M+); Anal. (C38H52N2O6S) calcd, C, 68.64; H, 7.88; N,
4.21; found, C, 68.66; H, 7.93; N, 4.22. Compound 29Z: mp 75-
77 °C; 1H NMR (300 MHz, CDCl3) δ 8.66 (dd, J ) 1.61, 1.54 Hz,
1H), 8.27 (ddd, J ) 7.70, 2.94, 1.61 Hz, 1H), 8.16 (ddd, J ) 7.90,
2.94, 1.54 Hz, 1H), 7.63 (dd, J ) 7.90, 7.70 Hz, 1H), 7.54-7.49
(m, 3H), 7.47 (brs, 1H), 7.29 (d, J ) 1.80 Hz, 1H), 7.13-7.10 (m,

452 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 3
Koide et al.
2H), 6.68 (d, J ) 8.39 Hz, 1H), 6.63 (dd, J ) 8.39, 1.80 Hz, 1H),
3.97 (t, J ) 6.99 Hz, 2H), 3.95 (s, 3H), 3.86 (s, 3H), 1.81 (dt, J )
7.70, 6.58 Hz, 2H), 1.46-1.44 (m, 2H), 1.40-1.21 (m, 24H), 0.88
(t, J ) 6.37 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 165.38, 155.01,
150.65, 149.20, 146.37, 139.14, 134.07, 131.90, 131.29, 131.05,
130.16, 129.73, 129.23, 128.82, 128.27, 122.17, 111.30, 109.43,
68.94, 55.93, 52.61, 31.93, 29.69, 29.59, 29.54, 29.37, 28.99, 25.90,
22.70, 14.13; MS (ESI) m/z 664 (M+); Anal. (C38H52N2O6S) calcd,
C, 68.64; H, 7.88; N, 4.21; found, C, 68.73; H, 7.99; N, 4.05.
(E)-5-[1-(4-Cetyloxy-3-methoxyphenyl)-1-phenylmethylene]-
sulfohydrazono isophthalic Acid (30E). To a solution of 25E (108
mg, 0.15 mmol) in THF (3 mL) and methanol (1 mL) was added
1 N NaOH (1 mL). The mixture was stirred for 19 h, diluted with
CHCl3, washed with 1 N hydrochloric acid and brine, dried over
Na2SO4, filtered, and concentrated. Recrystallization from isopropyl
alcohol gave 30E as a pale yellow powder (97 mg, 93%). mp 161-
(E)-5-[1-(4-Butyloxy-3-methoxyphenyl)-1-phenylmethylene]-
sulfohydrazono isophthalic Acid (32E). Compound 32E was
prepared according to the procedure described for 30E but with
27E instead of 25E. Compound 32E was obtained as a colorless
1
powder (632 mg, 92%). mp 196-199 °C; H NMR (300 MHz,
DMSO-d6) δ 10.65 (brs, 1H), 8.66 (s, 3H), 7.39-7.32 (m, 5H),
7.08 (d, J ) 8.81 Hz, 1H), 6.77-6.74 (m, 2H), 4.02 (t, J ) 6.42
Hz, 2H), 3.71 (s, 3H), 1.73 (dt, J ) 6.79, 6.42 Hz, 2H), 1.49-1.42
(m, 2H), 0.95 (t, J ) 7.34 Hz, 3H); 13C NMR (75 MHz, DMSO-
d6) δ 165.41, 155.68, 149.16, 148.71, 139.77, 137.28, 133.62,
132.26, 132.14, 129.92, 128.27, 127.70, 124.17, 121.98, 112.55,
112.41, 67.77, 55.62, 30.76, 18.79, 13.69; MS (ESI) m/z 525
(MH-); Anal. (C26H26N2O8S) calcd, C, 59.31; H, 4.98; N, 5.32;
found, C, 58.99; H, 4.93; N, 5.16.
(E)-5-[1-(4-Cetyloxyphenyl)-1-phenylmethylene]sulfohydra-
zono isophthalic Acid (33E). Compound 33E was prepared
according to the procedure described for 30E but with 28E instead
of 25E. Compound 33E was obtained as a pale yellow powder (13
1
163 °C; H NMR (300 MHz, DMSO-d6) δ 13.82 (brs, 2H), 10.65
(brs, 1H), 8.65 (s, 3H), 7.43-7.30 (m, 5H), 7.06 (d, J ) 8.80 Hz,
1H), 6.77-6.73 (m, 2H), 3.99 (t, J ) 6.54 Hz, 2H), 3.69 (s, 3H),
1.73 (dt, J ) 7.09, 6.54 Hz, 2H), 1.42-1.19 (m, 26H), 0.83 (t, J )
7.09 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 165.42, 155.66,
149.16, 148.72, 139.78, 137.29, 133.63, 132.28, 132.14, 129.91,
128.26, 127.70, 124.18, 121.98, 112.57, 112.43, 68.11, 55.62, 31.32,
29.07, 28.77, 28.74, 25.58, 22.12, 13.98; MS (ESI) m/z 693 (MH-);
Anal. (C38H50N2O8S) calcd, C, 65.68; H, 7.25; N, 4.03; found, C,
65.90; H, 7.36; N, 3.98.
1
mg, 26%). mp 175-176 °C; H NMR (300 MHz, DMSO-d6) δ
13.82 (brs, 2H), 10.64 (brs, 1H), 8.66 (brs, 3H), 7.42-7.26 (m,
5H), 7.17 (d, J ) 7.71 Hz, 2H), 7.03 (d, J ) 7.71 Hz, 2H), 4.01 (t,
J ) 6.40 Hz, 2H), 1.73-1.69 (m, 2H), 1.46-1.14 (m, 26H), 0.84
(t, J ) 6.71 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 165.38,
159.52, 155.92, 139.62, 137.45, 132.20, 130.65, 128.23, 127.72,
124.16, 114.46, 67.56, 31.26, 29.01, 28.98, 28.71, 28.67, 28.58,
25.48, 22.06, 13.93; MS (ESI) m/z 663 (MH-); Anal. (C37H48N2O7S)
calcd, C, 66.84; H, 7.28; N, 4.21; found, C, 66.73; H, 7.50;
N, 4.29.
(Z)-5-[1-(4-Cetyloxy-3-methoxyphenyl)-1-phenylmethylene]-
sulfohydrazono isophthalic Acid (30Z). Compound 30Z was
prepared according to the procedure described for 30E but with
25Z instead of 25E. Compound 30Z was obtained as a colorless
(E)-3-[1-(4-Cetyloxy-3-methoxyphenyl)-1-phenylmethylene]-
sulfohydrazono benzoic Acid (34E). Compound 34E was prepared
according to the procedure described for 30E, but with 29E instead
of 25E. Compound 34E was obtained as a colorless powder (54
1
powder (53 mg, 85%). mp 179-181 °C; H NMR (300 MHz,
DMSO-d6) δ 13.81 (brs, 2H), 10.44 (brs, 1H), 8.65 (s, 3H), 7.51-
7.49 (m, 2H), 7.23-7.19 (m, 2H), 7.10 (d, J ) 1.71 Hz, 1H), 6.82
(d, J ) 8.32 Hz, 1H), 6.45 (dd, J ) 8.32, 1.71 Hz, 2H), 3.90 (t, J
) 6.51 Hz, 2H), 3.68 (s, 3H), 1.66 (dt, J ) 6.59, 6.41 Hz, 2H),
1.38-1.16 (m, 26H), 0.83 (t, J ) 7.09 Hz, 3H); 13C NMR (75
MHz, DMSO-d6) δ 165.43, 156.57, 150.05, 148.65, 139.74, 133.64,
132.53, 132.35, 132.23, 129.42, 129.14, 128.91, 128.56, 122.05,
111.65, 108.62, 68.07, 55.04, 31.29, 29.02, 28.95, 28.70, 28.57,
25.43, 22.09, 13.96; MS (ESI) m/z 693 (MH-); Anal. (C38H50N2O8S)
calcd, C, 65.68; H, 7.25; N, 4.03; found, C, 65.89; H, 7.32; N,
3.97.
1
mg, 83%). mp 134-135 °C; H NMR (300 MHz, DMSO-d6) δ
13.50 (brs, 1H), 10.56 (brs, 1H), 8.50 (s, 1H), 8.20 (d, J ) 7.83
Hz, 1H), 8.13 (d, J ) 7.72 Hz, 1H), 7.77 (dd, J ) 7.83, 7.72 Hz,
1H), 7.39-7.28 (m, 5H), 7.06 (d, J ) 7.94 Hz, 1H), 6.74-6.72
(m, 2H), 4.00 (t, J ) 6.30 Hz, 2H), 3.70 (s, 3H), 1.72 (dt, J )
6.80, 6.32 Hz, 2H), 1.45-1.19 (m, 26H), 0.84 (t, J ) 6.80 Hz,
3H); 13C NMR (75 MHz, DMSO-d6) δ 166.11, 154.98, 149.06,
148.76, 139.35, 137.32, 133.45, 131.64, 131.53, 129.75, 129.66,
128.64, 128.26, 127.55, 124.28, 121.85, 112.63, 112.37, 68.09,
55.60, 31.30, 29.06, 28.71, 25.57, 22.10, 13.97; MS (ESI) m/z 649
(MH-); Anal. (C37H50N2O6S) calcd, C, 68.28; H, 7.74; N, 4.30;
found, C, 68.23; H, 7.80; N, 4.25.
(E)-5-[1-(4-Octyloxy-3-methoxyphenyl)-1-phenylmethylene]-
sulfohydrazono isophthalic Acid (31E). Compound 31E was
prepared according to the procedure described for 30E, but with
26E instead of 25E. Compound 31E was obtained as a pale yellow
(Z)-3-[1-(4-Cetyloxy-3-methoxyphenyl)-1-phenylmethylene]-
sulfohydrazono benzoic Acid (34Z). Compound 34Z was prepared
according to the procedure described for 30E, but with 29Z instead
of 25E. Compound 34Z was obtained as a colorless powder (45
mg, 69%). mp 72-74 °C; 1H NMR (300 MHz, DMSO-d6) δ 13.49
(brs, 1H), 10.37 (brs, 1H), 8.49 (s, 1H), 8.20 (d, J ) 7.80 Hz, 1H),
8.14 (d, J ) 7.69 Hz, 1H), 7.76 (dd, J ) 7.80, 7.69 Hz, 1H), 7.51-
7.49 (m, 3H), 7.22-7.19 (m, 2H), 7.04 (d, J ) 1.78 Hz, 1H), 6.82
(d, J ) 8.37 Hz, 1H), 6.47 (dd, J ) 8.37, 1.78 Hz, 1H), 3.90 (t,
J ) 6.42 Hz, 2H), 3.67 (s, 3H), 1.67 (dt, J ) 6.57, 6.42 Hz, 2H),
1.38-1.17 (m, 26H), 0.84 (t, J ) 6.82 Hz, 3H); 13C NMR (75
MHz, DMSO-d6) δ 166.14, 149.93, 148.59, 139.20, 137.09, 133.48,
132.61, 131.98, 131.48, 129.52, 129.37, 128.95, 128.81, 128.57,
122.31, 121.79, 111.71, 108.99, 68.08, 55.13, 31.29, 29.02, 28.96,
28.70, 28.57, 25.44, 22.10, 13.97; MS (ESI) m/z 649 (MH-); Anal.
(C37H50N2O6S) calcd, C, 68.28; H, 7.74; N, 4.30; found, C, 68.30;
H, 7.80; N, 4.28.
1
powder (231 mg, 82%). mp 190-192 °C; H NMR (300 MHz,
DMSO-d6) δ 10.65 (brs, 1H), 8.66 (s, 3H), 7.41-7.32 (m, 5H),
7.07 (d, J ) 8.81 Hz, 1H), 6.77-6.75 (m, 2H), 4.01 (t, J ) 6.42
Hz, 2H), 3.70 (s, 3H), 1.75 (dt, J ) 6.42, 6.06 Hz, 2H), 1.44-1.42
(m, 2H), 1.40-1.24 (m, 8H), 0.87 (t, J ) 6.97 Hz, 3H); 13C NMR
(75 MHz, DMSO-d6) δ 165.47, 155.68, 149.17, 148.71, 139.77,
137.31, 133.69, 132.33,132.17,129.95, 128.30, 127.74, 124.19,
122.02, 112.55, 112.40, 68.12, 55.64, 31.32, 28.75, 25.62, 22.15,
14.04; MS (ESI) m/z 581 (MH-); Anal. (C30H34N2O8S) calcd, C,
61.84; H, 5.88; N, 4.81; found, C, 62.00; H, 5.88; N, 4.68.
(Z)-5-[1-(4-Octyloxy-3-methoxyphenyl)-1-phenylmethylene]-
sulfohydrazono isophthalic Acid (31Z). Compound 31Z was
prepared according to the procedure described for 30E but with
26Z instead of 25E. Compound 31Z was obtained as a colorless
1
powder (421 mg, 91%). mp 209-210 °C; H NMR (300 MHz,
DMSO-d6) δ 10.44 (brs, 1H), 8.66 (s, 3H), 7.52-7.50 (m, 3H),
7.24-7.21 (m, 2H), 7.11 (d, J ) 2.02 Hz, 1H), 6.84 (d, J ) 8.44
Hz, 1H), 6.47 (dd, J ) 8.44, 2.02 Hz, 1H), 3.92 (t, J ) 6.61 Hz,
2H), 3.69 (s, 3H), 1.68 (dt, J ) 7.34, 6.61 Hz, 2H), 1.40-1.22 (m,
10H), 0.85 (m, 3H); 13C NMR (75 MHz, DMSO-d6) δ 165.46,
156.61, 150.06, 148.67, 139.74, 133.68, 132.55, 132.37, 132.25,
129.44, 129.14, 128.94, 128.58, 122.08, 111.66, 108.61, 68.09,
55.05, 31.25, 28.71, 28.65, 28.59, 25.48, 22.09, 13.99; MS (ESI)
m/z 581 (MH-); Anal. (C30H34N2O8S) calcd, C, 61.84; H, 5.88; N,
4.81; found, C, 61.65; H, 5.82; N, 4.71.
Acknowledgment. We thank Ms. Y. Kato, Ms. A. Fu-
rukawa, and Ms. Y. Hironaka at TOA EIYO Ltd. for their
dedicated technical assistance and Accelrys K.K., Japan, for
providing excellent technical support. This study was supported
by a research grant for ‘Research on Health Sciences Focusing
on Drug Innovation’ (KH21004) from the Japan Health Sciences
Foundation and by a Grant-in-Aid for Scientific Research on
Priority Areas (A) “Exploitation of Multi-Element Cyclic

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Molecules” from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
Supporting Information Available: One hundred seven struc-
tures in the database for assessing the GH score and the structures
of 36 assayed compounds. All elemental analyses (C, H, N) for
synthesized compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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