Design, synthesis, and biological evaluation of 3-(1-aryl-1 h -indol-5-yl)propanoic acids as new indole-based cytosolic phospholipase a2α inhibitors

Article abstract of DOI:10.1021/jm500494y

This article describes the design, synthesis, and biological evaluation of new indole-based cytosolic phospholipase A₂α (cPLA₂α, a group IVA phospholipase A₂) inhibitors. A screening-hit compound from our library, (E)-3-{4-[(4-chlorophenyl)thio]-3-nitrophenyl}acrylic acid (5), was used to design a class of 3-(1-aryl-1H-indol-5-yl)propanoic acids as new small molecule inhibitors. The resultant structure-activity relationships studied using the isolated enzyme and by cell-based assays revealed that the 1-(p-O-substituted)phenyl, 3-phenylethyl, and 5-propanoic acid groups on the indole core are essential for good inhibitory activity against cPLA₂α. Optimization of the p-substituents on the N1 phenyl group led to the discovery of 56n (ASB14780), which was shown to be a potent inhibitor of cPLA₂α via enzyme assay, cell-based assay, and guinea pig and human whole-blood assays. It displayed oral efficacy toward mice tetradecanoyl phorbol acetate-induced ear edema and guinea pig ovalbumin-induced asthma models.

Full text of DOI:10.1021/jm500494y

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Article
Design, Synthesis, and Biological Evaluation of 3-(1-
Aryl-1H-indol-5-yl)propanoic Acids as New Indole-
2
based Cytosolic Phospholipase A# Inhibitors
Toshiyuki Tomoo, Takashi Nakatsuka, Toyoko Katayama, Yasuhiro
Hayashi, Yusuke Fujieda, Maki Terakawa, and Kazuhiro Nagahira
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500494y • Publication Date (Web): 07 Aug 2014
Downloaded from http://pubs.acs.org on August 16, 2014
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Design, Synthesis, and Biological Evaluation of 3ꢀ
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(
1ꢀArylꢀ1Hꢀindolꢀ5ꢀyl)propanoic Acids as New
Indoleꢀbased Cytosolic Phospholipase A α
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Inhibitors
,
†
†
‡
§
Toshiyuki Tomoo,* Takashi Nakatsuka, Toyoko Katayama, Yasuhiro Hayashi, Yusuke
||
#
#
Fujieda, Maki Terakawa, and Kazuhiro Nagahira
†
‡
§
||
Faculty of Pharmaceutical Chemistry, R&D administration, Exploratory Technology, Drug
#
Discovery Technology, Pharmacology I, Asubio Pharma Co., Ltd, 6ꢀ4ꢀ3 Minatojimaꢀ
Minamimachi, Chuoꢀku, Kobe, Hyogo 650ꢀ0047, Japan
ABSTRACT
This paper describes the design, synthesis, and biological evaluation of new indoleꢀbased
cytosolic phospholipase A α (cPLA α, a group IVA phospholipase A ) inhibitors. A screeningꢀ
2
2
2
hit compound from our library, (E)ꢀ3ꢀ{4ꢀ[(4ꢀchlorophenyl)thio]ꢀ3ꢀnitrophenyl}acrylic acid (5),
was used to design a class of 3ꢀ(1ꢀarylꢀ1Hꢀindolꢀ5ꢀyl)propanoic acids as new small molecule
inhibitors. The resultant structure–activity relationships studied using the isolated enzyme and by
cellꢀbased assays revealed that the 1ꢀ(pꢀOꢀsubstituted)phenyl, 3ꢀphenylethyl, and 5ꢀpropanoic
acid groups on the indole core are essential for good inhibitory activity against cPLA α.
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Optimization of the pꢀsubstituents on the N1 phenyl group led to the discovery of 56n
(ASB14780), which was shown to be a potent inhibitor of cPLA α via enzyme assay, cellꢀbased
2
assay, and guinea pig and human wholeꢀblood assays. It displayed oral efficacy toward mice
tetradecanoyl phorbol acetateꢀinduced ear edema and guinea pig ovalbuminꢀinduced asthma
models.
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INTRODUCTION
Phospholipase A (PLA ) is an enzyme that specifically hydrolyzes the ester bond at the snꢀ2
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position of phospholipids to yield fatty acids and lysophospholipids. The PLA superfamily
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comprises at least six types of PLA s: (i) secreted PLA (sPLA ), (ii) cytosolic PLA (cPLA ),
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+
(iii) Ca ꢀindependent PLA (iPLA ), (iv) lysosomal PLA (LPLA ), (v) adiposeꢀspecific PLA
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(AdPLA), and (vi) plateletꢀactivating factor acetyl hydrolase (PAFꢀAH), which were identified
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and classified in mammals based on factors such as their locality, Ca ꢀdependency for activity,
1
and substrate specificity. cPLA α (a group IVA phospholipase A ), which is an isoform of
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cPLA , is the first cloned cPLA with a molecular weight of 85 kDa. Extensive studies have
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revealed that cPLA α is widely expressed in mammalian cells and plays a central role in the
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stimuliꢀinduced production of lipid mediators because of the following factors: it selectively
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a, 3
releases arachidonic acid (AA) from glycerophospholipids in the cytoplasm;
it is activated by
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+
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submicromolar Ca concentrations;
and its activity is regulated through phosphorylation by
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mitogenꢀactivated protein kinase (MAPK) or by direct binding with other lipid second
6
messengers.
AA released by cPLA α is further converted to inflammatory lipid mediators such as
2
prostaglandins (PGs) and thromboxanes (TXs) through the cyclooxygenase pathway and
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leukotrienes (LTs) through the lipoxygenase pathway. Lysophospholipids, which are another
product of the hydrolysis, can also be converted to plateletꢀactivating factor (PAF) via biological
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acetylation. These lipid mediators are involved in the initiation or exacerbation of various
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inflammatory diseases, food allergies (e.g., allergic colitis), allergic rhinitis, allergic airway
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inflammation (e.g., asthma), and other respiratory diseases (e.g., acute lung injury), multiple
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sclerosis, osteoporosis, and so on. Therefore, inhibition of the cPLA α activity to mitigate the
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release of AA and lysophospholipids should suppress the production of lipid mediators
downstream of the metabolic cascade providing effective treatment or prevention of the diseases
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described above. In cPLA αꢀdeficient mice, alleviation of asthma, rheumatoid arthritis, acute
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lung injury, pulmonary fibrosis, inflammatory bone resorption, multiple sclerosis, cerebral
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ischemia/reperfusion injury, atherosclerosis, hepatic fibrosis, and other diseases is
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observed. For these diseases, it is believed that cPLA α is involved in the onset or aggravation
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of the disease; therefore, these diseases could be remedied and healed by inhibiting this enzyme.
cPLA α inhibitors have been described in reviews, and some of the chemical inhibitors have
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1b, 25
been reported to be effective for some animal disease models.
According to literature,
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tricarbonyls,
methyl arachidonyl fluorophosphonates,
trifluoromethyl ketones,
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pyrrolidines, pyrroles, indoles, 2ꢀoxoamides, and 1,3ꢀdisubstituted propanꢀ2ꢀones are
effective cPLA α inhibitors in in vitro or in vivo experiments. However, none of the inhibitors
2
described above has been commercialized as a pharmaceutical yet; most of these chemical
inhibitors have long alkyl side chains, which is reflective of natural substrates, or large
hydrophobic group substituents such as biphenyl, benzhydryl, and trityl in the molecule and thus
have high molecular weights.
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The bestꢀdeveloped inhibitors are Wyeth’s indole compounds (1, 2), which display oral efficacy
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in in vivo models and have advanced to clinical trials.
Using a substrate mimetic approach,
their exploration began with the application of the indole template to zafirlukast, which is the
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leukotriene D receptor antagonist. The Lehr’s group also developed a series of indole
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derivatives as part of 1,3ꢀdisubstituted propanꢀ2ꢀone inhibitors (3, 4), most of which bear
3
3b, 36
hydrophobic long alkyl chains in the molecule and have low bioavailability;
their
application to the indole template proceeded from their previously developed pyrrole derivatives
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0a, 31a
(Figure 1).
We searched for new cPLA α inhibitors and identified a class of 3ꢀ(1ꢀarylꢀ1Hꢀindolꢀ5ꢀ
2
yl)propanoic acid derivatives as new indoleꢀbased inhibitors. These novel indolic inhibitors were
originally designed based on our screeningꢀhit compound (5) and were developed as relatively
small molecules without hydrophobic long alkyl chains; therefore, they have desirable
bioavailability and oral efficacy.
RESULTS AND DISCUSSION
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Design and Synthesis. We searched for nonpeptide and low molecularꢀweight inhibitors of
cPLA α and identified (E)ꢀ3ꢀ[4ꢀ(4ꢀchlorophenylthio)ꢀ3ꢀnitrophenyl]acrylic acid (5) as a hit from
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our compound library. Compound 5 showed cPLA α inhibitory activity with an IC value of 1
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ꢀM in the enzyme assay. However, because the SAR studies of compound 5 did not reveal
suitable information for increasing its potency, we analyzed its Xꢀray crystal structure (Figure 2).
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The intramolecular nonbonded S–O interaction between the sulfur atom of the sulfide moiety
and an oxygen atom of the nitro group results in coplanarity of the nitroꢀsulfide loop with the
bridging benzene ring and controls the conformation of the chlorophenyl residue; this inspired us
to design a bicyclic indole scaffold. Replacing the pseudoꢀ5ꢀmembered ring system comprising
the N–O moiety of the nitro group, sulfur atom of the sulfide group, and two carbon atoms of the
benzene ring with a connected 5ꢀmembered ring system resulted in a more rigid conformation of
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the protrudent aryl residue in the new 3ꢀ(1ꢀarylꢀ1Hꢀindolꢀ5ꢀyl)propenoic acid (6) scaffold for
cPLA α inhibitors.
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Compared to that of compound 5, compound 6 had a lower potency with an IC value of 10
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ꢀM for the enzyme assay. However, pilot syntheses revealed that the introduction of a
hydrophobic moiety at the C3 position of 6 increased its cPLA α inhibitory potency above that
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of compound 5. Furthermore, conversion of the twoꢀcarbon linker at the C5 position of the
indole ring from a double to single bond further increased the inhibitory potency. These findings
are consistent with our speculation that more hydrophobic and flexible character, similar to that
of a natural substrate, is necessary for increased potency.
Simple synthetic strategies that allowed functionalization of the C3 substituents, C5 linker,
carboxylate group, and N1 carbocycles were employed to explore the structure–activity
relationships (SAR) in these new indoleꢀbased inhibitors. Our preliminary SAR investigation
indicated that the phenyl group at the N1 position was necessary for activity; thus, it was not
modified. The analogs were prepared from two brominated indoles, 5ꢀbromoindole and 5ꢀbromoꢀ
2ꢀmethylindole, which are commercially available (Scheme 1).
_
a
Scheme 1. Preparation of 13a b and 20
R
R
R
X
Br
b
Br
d
f
N
N
N
N
R
Ph
CO R'
CO H
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R = H
9a R = Ph, X = O
9b R = Bn, X = O
11a R = Ph, R' = Me
11b R = Bn, R' = Me
13a R = Ph
13b R = Bn
a
8 R = Ph
c
e
1
0a R = Ph, X = H, H
10b R = Bn, X = H, H
12a R = Ph, R' = H
2b R = Bn, R' = H
1
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To perform the pilot syntheses for the attachment of a benzyl group at the C3 position, the N1
phenyl group was installed early in the synthesis. 5ꢀBromoindole (7) was Nꢀarylated by
iodobenzene via a coupling reaction catalyzed by ligandꢀfree copper to afford the intermediate
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39
(
8), which then underwent FriedelꢀCrafts acylation with benzoyl chloride at position 3.
Reduction of the carbonyl group of the C3ꢀacylated intermediate (9a) to methylene was carried
out in a stepwise manner using NaBH and Et SiH; the C3ꢀbenzyl derivative (10a) was then
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obtained via the corresponding alcohol intermediate. Methyl acrylate was introduced at
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position 5 by the Heck reaction, and the C5 propenoate side chain was formed. Hydrolysis of
the methyl propenoate intermediate (11a) afforded the propenoic acid (12a), and subsequent
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catalytic hydrogenation led to the corresponding propanoic acid (13a). A similar synthetic
sequence was used to prepare the C3ꢀphenethyl analog (13b), except that phenylacetyl chloride
was used instead of benzoyl chloride during FriedelꢀCrafts acylation. The C5ꢀpropenoic acid
derivative (12b) was produced as the precursor to 13b in this process. The C2ꢀmethyl analog
(20) was also prepared by employing the same reaction sequence beginning with 5ꢀbromoꢀ2ꢀ
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methylindole (14) instead of 5ꢀbromoindole (7), as depicted in Scheme 1. In this case, an
unexpected styryl intermediate (17) was accidentally produced via the dehydration of the alcohol
intermediate during the carbonyl reduction step, owing to the workꢀup with aqueous
hydrochloric acid. Nevertheless, the side product (17) was conveniently converted to the desired
2ꢀmethylindole derivative (20) via the Heck reaction, hydrolysis, and concurrent hydrogenation.
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To impart versatility to the C3ꢀphenethyl group in 13b, a C3ꢀunsubstituted intermediate (21)
was generated by installing a C5ꢀpropenoate onto the previously prepared N1ꢀphenylated
intermediate (8) by the Heck reaction. The unsaturated ester derivative (21) was employed to
prepare the unsaturated acid (6) and for the syntheses of the various C3ꢀacylated intermediates
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(
22a–e). In our study, we found that the HuangꢀMinlon conditions, a modified form of the
WolffꢀKishner reduction, not only reduced the carbonyl group to methylene but also
simultaneously hydrogenated the cinnamic double bond; thus, this procedure was applied to
generate analogs 23a and 23c from their precursor acids. Furthermore, because the Huangꢀ
Minlon modification uses aqueous KOH, which hydrolyzes esters, we applied it directly to the
precursor methyl propenoates (22b, 22d, and 22e) to successfully obtain the desired 3ꢀphenethyl
propanoic acids (23b, 23d, and 23e) in only one step (Scheme 2).
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As shown in Scheme 2, we isolated several final compounds as tromethamine salts because in
general this salt significantly improved the solubility of this series of compounds in aqueous
media, which was important for each biological assay. In addition, this salt was convenient for
transforming the oily compounds to solids, which facilitated handling. Naturally, tromethamine
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itself has no inhibitory activity for cPLA α enzyme at this assay level (inꢀhouse data).
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The carboxylic acid function of 13b was examined by derivatizing it to form bioisosteres such
as hydroxamic acid, acylꢀcyanamide, and tetrazole. Hydroxamic acid 25 was prepared via the
treatment of 13b with Oꢀbenzylhydroxylamine to form an amide followed by debenzylation.
Acylꢀcyanamide derivative 26 was also synthesized via condensation with cyanamide. Tetrazole
29 was synthesized from 13b in three steps: (i) conversion to primary amide 27; (ii) dehydration
to nitrile intermediate 28; and (iii) reaction with sodium azide by Sharpless’s zinc bromide
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procedure. Trifluoromethylketone 30 was successfully prepared via the temporary formation of
the corresponding carboxylic chloride intermediate and subsequent treatment with trifluoroacetic
44
anhydride (Scheme 3).
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Etherꢀlinker derivatives 34, 36, and 38 were generated from the common 5ꢀhydroxyindole
derivative (32), which was derivatized from the 5ꢀbromoindole analog (10b) via the 5ꢀmethoxy
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intermediate (31). The 5ꢀhydroxyindole (32) was alkylated with ethyl bromoacetate and tꢀbutyl
2ꢀbromopropionate under SchottenꢀBaumann conditions resulting in Oꢀacetates 33 and 35,
respectively, which were derivatized to the corresponding Oꢀacetic acids (34 and 36) by the
appropriate hydrolysis method. The Oꢀpropionic acid derivative (38) was synthesized from 32 by
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a reaction with methyl acrylate under Naꢀhydroquinone conditions and subsequent NaOH
hydrolysis (Scheme 4).
The preparation of the C6ꢀpropanoic acid derivative (44) is illustrated in Scheme 5; it began
with 6ꢀbromoindole (39), which was functionalized with phenylacetyl and methyl acrylate
groups at positions 3 and 6, respectively; subsequent ester hydrolysis generated the propenoic
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acid (43). The HuangꢀMinglon modification mentioned above was then applied to successfully
produce the corresponding C6 positional isomer (44).
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59
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As shown in Scheme 6, the N1ꢀbenzyl derivative (50) was synthesized starting from 5ꢀ
bromoindole (7), similar to 13b, except that the first step comprised Nꢀbenzylation instead of Nꢀ
phenylation.
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5
5
5
5
5
5
5
6
An approach that leads to lateꢀstage intermediates for varying the N1ꢀsubstituents via alkylation
or palladiumꢀmediated coupling is depicted in Scheme 7. The synthetic route to key
intermediates 54a and 54b began with C3 functionalization of 5ꢀbromoindole (7) with a
phenylacetyl group, and involved a combination of 1ꢀphenylacetylꢀ1,2,3ꢀbenzotriazole as an
0
1
2
3
4
5
6
7
8
9
0
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5
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9
0
4
7
acylating agent and TiCl as a Lewis acid to improve both the yield and handling. In addition,
4
the stepwise reduction method of carbonyl to methylene that was mentioned above was
integrated to provide direct lithium aluminum hydride reduction in diethyl ether without reducing
the yield. The Heck reaction was performed on the prepared 3ꢀphenethylindole (52) with both
methyl and ethyl acrylates to produce propenoate intermediates 53a and 53b, respectively, which
were converted to the corresponding propanoate derivatives 54a and 54b by following
hydrogenation. The analogs with a variety of aryl substituents at the N1ꢀposition (56b–q) were
48
successfully prepared via BuchwaldꢀHartwig crossꢀcoupling of propanoate derivatives 54a and
54b with the corresponding aryl bromides and subsequent debenzylation and/or ester hydrolysis.
Scheme 7. Preparation of 56b, 56d and 56f_qa
Br
X
a
c
d
N
Br
HN
HN
H
N
7
H
CO R
CO R
2
2
51 X = O
2 X = H,H
53a R = Me
53b R = Et
54a R = Me
54b R = Et
b
5
e
f
N
N
R'
R'
CO R
CO H
2
2
55a R = Me, R' = 4-OBn
5b R = Me, R' = 4-OH
56b R' = 4-OH
d
5
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56
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Biology. The enzyme assay used crudely purified human cPLA α from the cytoplasm fraction
2
4
b
14
of U937 with a radioꢀlabeled Lꢀαꢀ1ꢀpalmitoylꢀ2ꢀ[ C] arachidonyl phosphatidyl choline
substrate presented in a liposome. The inhibitory potency was evaluated by measuring the
1
4
radioactive index of [ C]ꢀAA released because of the enzyme reaction. The cellꢀbased assay
used mouse peritoneal exudate cells (PECs). In this assay, prostaglandin E was generated, a
2
reflection of the release of AA because of the activity of the cPLA α enzyme, and then
2
metabolized by the COX pathway. This assay reveals that the inhibitors possess sufficient
membrane permeability to reach cPLA α in the cytosol.
2
Structure–Activity Relationships. As shown in Table 1, the C3ꢀbenzyl derivative (13a) was
substantially more potent (IC : 0.18 ꢀM) than both compound 5 and 6. Extension of the linker
5
0
of the C3 substituent from methylene to ethylene enhanced the inhibitory potency. Thus, with an
IC50 value of 0.075 ꢀM against the isolated enzyme, 3ꢀ(3ꢀphenethylꢀ1ꢀphenylꢀ1Hꢀindolꢀ5ꢀ
yl)propanoic acid (13b) showed a greater potency than compound 13a. Because of its high
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5
5
5
5
5
5
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potency (IC of less than 0.1 ꢀM), we positioned this 3ꢀphenethyl derivative (13b) as the lead
5
0
compound of the series. Because the C2 methyl analog (20) was less potent than the C2
unsubstituted analog (13b), C2 was not substituted in the remaining analogs.
0
1
2
3
4
5
6
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9
0
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9
0
Next, we investigated the effect of introducing substituents onto the phenyl moiety of the C3ꢀ
phenethyl group. Substitution of the 4ꢀposition with a fluorine atom (24a) or chlorine atom (24b)
decreased the cPLA α inhibitory potency (IC : 0.57 ꢀM and 1.2 ꢀM, respectively) relative to
2
50
that of the 4ꢀunsubstituted analog (13b). These results indicate that the electronꢀwithdrawing
character of the substituents inversely affects the potency. On the other hand, the potency of the
4ꢀmethoxy analog (24c), which possesses electronꢀdonating character because of the resonance
effect, was even lower (IC : 0.72 ꢀM) than that of the 4ꢀunsubstituted analog (13b) and was
5
0
similar to that of the 4ꢀfluoro analog (24a). These results suggest that substitution at the 4ꢀ
position adversely affects the inhibitory potency irrespective of the electronic character of the
substituents. Moreover, shifting the methoxy moiety of compound 24c to the 3 position (24d)
and adding another methoxy group to the 3 position (24e) further lowered the enzyme inhibition
(
IC : 1.0 and 1.4 ꢀM, respectively). Therefore, we concluded that any substitution on the phenyl
5
0
group of the C3ꢀphenethyl moiety in compound 13b decreased the potency; thus, it is likely that
there is spatially strict recognition by the enzyme pocket around the C3ꢀphenethyl moiety of this
series of compounds. Accordingly, we fixed the simple phenethyl group as the C3 substituent
and systematically varied the other portions of the indole pharmacophore.
Next, we investigated the function of the propanoic acid moiety in position 5 of the indole
core, as shown in Table 2. The importance of the carboxylic acid for the activity of 13b became
evident when this group was replaced with other functional groups. Replacement of the
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carboxylic acid with bioisosteres such as hydroxamic acid (25) and tetrazole (29) resulted in
lowered potency (IC : 16 ꢀM and 13 ꢀM, respectively); even the most active bioisostere, acylꢀ
5
0
cyanamide (26), resulted in a 13ꢀfold lower potency (IC : 0.98 ꢀM) than 13b against the
5
0
10
11
12
13
14
15
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19
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24
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50
51
52
53
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55
56
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isolated enzyme. The amide derivative (27) was inactive against the isolated enzyme at 100 ꢀM
concentration, which indicates that acidic functionality is necessary. The arachidonyl
trifluoromethylketone (AACOCF ), which is an analog derived from AA by replacing the
3
carboxylic acid group with trifluoromethylketone, has been reported as a slow, tight binding
28a
inhibitor of cPLA2. Thus, we made the same replacement in compound 13b; however, the
resultant compound (30) had a lower potency (IC : 1.2 ꢀM) than 13b. These results indicate that
5
0
no surrogates for the carboxylic acid function were effective for increasing activity, which
suggests that this enzyme strictly recognizes the ‘carboxylic acid’, and that the carboxylic acid
4
9
moiety of 13b might not interact with a serine residue of the active site (Serꢀ228). The
excellent study using a combination of deuterium exchange mass spectrometry (DXMS) and
molecular dynamics (MD) simulations suggests that the pyrrolidineꢀderived inhibitor
pyrrophenone is mainly bound to a hydrophobic pocket near the interfacial binding surface of the
50
enzyme. We now consider that our compounds may similarly interact with the enzyme using
their own hydrophobic groups. The exact binding mode will be investigated in the future.
Modification of the C5ꢀlinker was then investigated. The compound with a trans double bond
linking, the propenoic acid (12b), had lower potency (IC : 0.32 ꢀM) than 13b, which contains a
50
singleꢀbond linker. Replacement of the twoꢀcarbon linker with an etheric linker dramatically
reduced the potency of the compound (34) (IC : 3.9 ꢀM). Another analog with a methyl branch
5
0
(
36) was not tolerated (IC : 15 ꢀM); however, a longer linker (38) restored the potency (IC :
50
50
1.7 ꢀM), although the resultant compound was still less potent than that with the two carbon
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5
5
6
linker (13b). Thus, we recognized that modifications that affect the carboxylic acid functionality
or linker of the propanoic acid moiety in position 5 are not beneficial. Compound 44, which
contains the positional isomer of the propanoic acid moiety, had a 5.3ꢀfold lower potency (IC :
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
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0
0.40 ꢀM) than the lead compound (13b), which illustrates the increased potency resulting from
positioning of this functional group at the 5ꢀposition rather than the 6ꢀposition of the indole core.
These results indicate that this enzyme also recognizes the space surrounding the C5ꢀpropanoic
acid moiety of these indole inhibitors.
Accordingly, we considered that an unsubstituted C2, a phenethyl group at C3, and a propanoic
acid moiety at C5 of the indole template are essential for high cPLA α inhibitory potency;
2
therefore, this structure was maintained while the N1 substituents of the indole core were varied.
This new series of cPLA α inhibitors are characterized by the Nꢀphenyl conjugated system at
2
position 1 of the indole pharmacophore. The 1ꢀbenzyl derivative (50), which has an interrupted
Nꢀphenyl conjugated system, had significantly lower potency (IC : 1.6 ꢀM, Table 3); therefore,
5
0
direct linkage of the phenyl group is important to the high potency of these analogs.
The next phase of optimization focused on variation of the substituents attached to the phenyl
group at position 1 of the indole core. These analogs are presented in Table 3. Our aim is to
develop a new series of orally active small cPLA α inhibitors without long alkyl chains or large
2
hydrophobic groups. Although the compounds developed hitherto are relatively small (MW <
500) and contain neither long alkyl chains nor large hydrophobic groups, they are prone to be
highly lipophilic because of the N1ꢀphenyl conjugated system and C3ꢀphenethyl group.
Therefore, because the compounds had poor cell membrane permeability, we introduced the cellꢀ
based assay (PEC), which is used to evaluate the potency, including membrane permeability, by
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monitoring the production of prostaglandin E , in addition to the enzyme assay for further
2
optimization. Concurrently, we checked the log P values of the compounds to monitor their
lipophilicities. The partition coefficients (log P) of the compounds were determined by reversed
phase HPLC according to our procedure.
10
11
12
13
14
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24
25
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50
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56
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It was immediately evident that compounds with substituents at the 4ꢀposition of the N1ꢀphenyl
group were the most potent, with the potency decreasing as the substituents moved further away,
e.g., from the 3ꢀposition to the 2ꢀposition. The phenolic analogs were the representative example
in this SAR. The 4ꢀhydroxy analog (56b) showed a substantially higher potency with an IC of
5
0
0.011 ꢀM and was the first compound that was more potent than the lead compound (13b) in the
enzyme assay. In contrast, the 3ꢀhydroxy (56d) and 2ꢀhydroxy (56f) derivatives had substantially
lower potency (IC : 0.19 ꢀM and 0.78 ꢀM, respectively). The PEC experiments showed a
5
0
reversed order of potency for the 3ꢀ and 2ꢀanalogs (IC : 2.3 ꢀM and 1.2 ꢀM, respectively);
5
0
nevertheless, both analogs were less potent than the 4ꢀanalog (IC : 0.54 ꢀM). A similar
5
0
regioisomeric effect was also observed for the other substituents (data not shown); as a result,
further optimization via the substituents of the N1ꢀphenyl moiety focused on the 4ꢀposition.
Although the 4ꢀhydroxy derivative (56b) was more potent (IC : 0.54 ꢀM) than compound 13b
5
0
(
IC : 1.3 ꢀM) in the PEC assay, the 4ꢀmethoxy analog (56g) had a 2ꢀfold lower potency in the
5
0
enzyme assay (IC : 0.026 ꢀM) and was equipotent in the PEC assay (IC : 0.52 ꢀM). This result
5
0
50
indicates that methyl masking of the hydroxyl group enhances the membrane permeability.
Elongation of the alkyl chain from methyl to ethyl (56h) restored the potency in the enzyme
assay (IC : 0.015 ꢀM) and enhanced the potency in the PEC assay (IC : 0.10 ꢀM). Further
5
0
50
elongation of the alkyl chain to nꢀpropyl and isopropyl (56i and 56j, respectively) resulted in
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major advances, as these compounds showed nanoꢀmolar activities in the enzyme assay (IC :
5
0
0.007 ꢀM); however, there was a concomitant 2ꢀ to 9ꢀfold decrease in their potency in the PEC
assay (IC : 0.21 and 0.89 ꢀM, respectively) relative to that of the ethyl analog (56h). Thus,
5
0
0
1
2
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0
further alkyl elongation was not investigated. Interestingly, the enzymatic activities and log P
values (5.9 and 5.8, respectively) of the nꢀpropoxy (56i) and isopropoxy (56j) analogs were
similar, while the PEC activities differed by a magnitude of four. This result indicates that alkyl
branching of this moiety adversely affects the membrane permeability. Introduction of a
hydroxyl group to the end of the ethyl residue of the ethoxy analog (56h) resulted in compound
5
6k, which was equipotent to 56h in the enzyme assay (IC : 0.014 ꢀM) and 8ꢀfold less potent in
50
the PEC assay (IC : 0.80 ꢀM); this indicates that lowering the lipophilicity (log P value: 4.5) by
5
0
introducing the hydroxyl group decreases the membrane permeability. The 4ꢀdifluoromethoxy
analog (56l) had a similar cPLA α inhibitory potency (IC : 0.028 ꢀM) and slightly lower
2
50
activity in the PEC assay (IC : 0.85 ꢀM) compared to the corresponding 4ꢀmethoxy analog
5
0
(56g). In contrast to compound 56l, the 4ꢀtrifluoromethoxy analog (56m) resulted in a 5ꢀfold
lower enzymatic activity (IC : 0.14 ꢀM), although it represented an important advancement in
5
0
the cellular assay (IC : 0.18 ꢀM). These results indicate that the strong electronꢀwithdrawing
5
0
group (OCF ) was not tolerated by the isolated enzyme, though the log P value in this range (8–
3
9) contributed to the membrane permeability. With an IC value of 0.020 ꢀM, the 4ꢀphenoxy
5
0
derivative (56n) was wellꢀtolerated in the enzyme assay. It is interesting that this compound,
with an IC value of 0.50 ꢀM, was equipotent to the 4ꢀhydroxy analog (56b) and 4ꢀmethoxy
50
analog (56g) in the PEC assay despite the substantial difference in their HPLCꢀderived log P
values (8.9 vs. 4.4 and 4.6, respectively). This also means that high lipophilicity based on the 4ꢀ
phenoxy group does not significantly decrease the membrane permeability. Thus, most of the
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compounds of this 4ꢀOꢀsubstituted series have much higher inhibitory potencies in the enzymatic
and/or cellular assays than the lead compound (13b). Hence, these compounds are prospective
candidates for further investigation.
10
11
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Substituents linked to the 4ꢀposition of the N1ꢀphenyl moiety without oxygen were further
examined. The pꢀmethyl analog (56o) was equipotent to 13b in the enzyme assay (IC : 0.077
5
0
ꢀ
M) and had higher potency in the PEC assay (IC : 0.86 ꢀM). The 4ꢀchloro (56p) and 4ꢀfluoro
5
0
analogs (56q) had higher potencies in both the enzyme assay (IC : 0.036 and 0.058 ꢀM,
5
0
respectively) and cellular assay (IC : 0.71 and 0.28 ꢀM, respectively).
5
0
Mouse Tetradecanoyl Phorbol Acetate (TPA)-induced Ear Edema Model. Phorbol ester
12ꢀOꢀtetradecanoylphorbolꢀ13ꢀacetate, TPA) was applied to the ear to elicit acute inflammation
(
with ear edema as a main symptom. In the present test model, it is known that AA metabolites,
51
which constitute various lipid mediators, are involved in inflammation. Therefore, it is possible
to evaluate the in vivo antiꢀinflammatory activity of antiꢀinflammatory compounds that act on the
AA cascade using this model. In this test, the compounds were administered orally or
intraperitoneally, and the difference of the ear thickness before and after the application of TPA
was evaluated. Based on the results, the activity of each test compound in suppressing the
increase in the ear thickness elicited by the application of TPA was evaluated. The suppression
rates of the compounds are shown in Table 3. We used this model as a compound screening
procedure with consideration of the results of the PEC assays and found that six compounds (56b,
56k–n, and 56q) showed significant efficacy at an oral dose of 50 mg/kg, while the lead
compound (13b) was not effective in the same dose. In particular, compound 56n inhibited ear
swelling by 30%, which is the maximal response generally seen in this model. The other tested
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1
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2
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2
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2
2
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5
6
compounds (56g–i, 56p) were not effective in this model despite intraperitoneal dosing.
Compounds 56b, 56k–n, and 56q, which showed high potency in this in vivo assay, were further
characterized in other assays.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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0
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0
1
2
3
4
5
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8
9
0
Selectivity Assays. To examine the selectivity of these compounds, their inhibition of PLA2
1
4
subtypes were determined using Lꢀαꢀ1ꢀpalmitoylꢀ2ꢀ[ C] arachidonyl phosphatidyl choline
liposomes to assess their activity against type II sPLA . sPLA is a secretory protein with a mass
2
2
of approximately 14 kD and a structure similar to that of its other isoforms; His 48 in the active
2
+
site and Ca loop are wellꢀconserved. cPLA is a cytosolic protein with mass 85 kD and an
2
active site that essentially consists of Ser 228 and Asp 549 (also known as the “catalytic
4
9b
diad”). Thus, because the molecular size and active site structures of these PLA s are quite
2
different, we used sPLA s derived from insects and reptiles: sPLA ꢀIA (cobra venom), sPLA ꢀ
2
2
2
IIA (crotalus venom), sPLA ꢀIII (bee venom), and sPLA ꢀIB (porcine pancreas), instead of those
2
2
from mammals. As a result, while each of the selected six compounds (56b, 56kꢀn, and 56q)
inhibited human cPLA α with IC values less than 0.14 ꢀM, none of them inhibits these sPLA s
2
50
2
at 10 ꢀM in this assay (data not shown).
Whole Blood Assays. To assess the effect of serum albumin binding and speciesꢀderived
differences of the selected inhibitors, inhibition by the inhibitors in guinea pig whole blood
(GWB) and human whole blood (HWB) assays were examined. Both GWB and HWB assays
require calcium ionophore (A23187) for consistent stimulation, which generates LTs and PGs. In
these whole blood assays, we monitored the TXB generation by the COXꢀ1/thromboxane
2
synthase pathway to evaluate the inhibitory activities of the cPLA α inhibitors selected above.
2
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Compounds 56k and 56n had relatively high potency (IC < 1.0 ꢀM) in both the GWB and
5
0
HWB assays. Similarly, compounds 56b, 56l, and 56q had high potencies (IC < 1.0 ꢀM) in the
5
0
HWB assay; however, both 56b and 56q had substantially lower activities (10ꢀ and 7ꢀfold
decrease) in the GWB assay. This result indicates that there are speciesꢀderived differences in the
reaction of guinea pigs and humans to compounds 56b and 56q; thus, they were not suitable for
further development. Compound 56m showed a wellꢀbalanced inhibitory potency that was
relatively low (IC > 1.0 ꢀM) toward both whole blood assays. Accordingly, 56k, 56l, and 56n
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5
0
were chosen for further study (Table 3).
Pharmacokinetics. The oral bioavailabilities (F) of the three selected analogs (56k, 56l, and
5
6n) in mice, dogs and monkeys were determined. Based on the data calculated with an
intravenous dose of 1 mg/kg and oral dose of 10 mg/kg, these three compounds offered good oral
bioavailabilities (F > 30%) and showed high blood concentration time courses (AUC and Cmax
after oral administration in all animal species (Table 4). According to this result, all of them (56k,
6l, 56n) have good potential for oral treatment of the diseases in which cPLA α activation is
)
5
2
involved.
Guinea Pig Ovalbumin (OVA)-induced Asthma Model. Inhalation of an antigen by asthma
patients produces an immediate asthmatic response (IAR), which peaks 15–30 min after
inhalation; this response disappears within 2 h. However, it has been reported that 60% of
patients relapse into a late asthmatic response (LAR), which peaks 4–12 h after inhalation. LAR
is accompanied by airway hyperreactivity (AHR), which is caused by nonspecific stimuli and
52
believed to lead to severe and chronic asthma. Because it is sustained over a long time period,
LAR is very similar to naturally occurring asthma attacks, especially intractable asthma attacks.
Therefore, it is vitally important for asthma treatment to suppress the pathophysiology of LAR.
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In this guinea pig model of asthma, we examined the efficacy of the selected three compounds
(
56k, 56l, and 56n) against antigen (OVA)ꢀinduced IAR, LAR, and AHR. An antigen–antibody
reaction was elicited in passively sensitized guinea pigs by inhaling the antigen resulting in
increased specific airway resistance (sRaw) immediately after and 4 to 8 h after inhalation,
which indicates the biphasic response, i.e., both IAR and LAR. The AHR was observed the
following day. Dexamethasone, which is a steroid that is frequently used for asthma treatment,
showed a significant effect against these sequential responses.
0
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Each of the selected cPLA α inhibitors (56k, 56l, and 56n) was administered once a day from
2
the day after the final sensitization to the day before the antigenꢀchallenge at doses of 5 and 20
mg/kg; additionally, on the day of the challenge, they were administered orally 1 h before and 8
h after the challenge.
As seen in Table 5, these three compounds showed good efficacy in this model. At each dosing,
compound 56k showed wellꢀbalanced efficacy for both IAR and LAR while compound 56l had
extremely LARꢀbiased efficacy. The response to compound 56n was also LARꢀbiased; however,
it showed considerable efficacies against both asthmatic responses at each dosing. The AHR to
aerosolized acetylcholine was measured 24 h after the antigen challenge and compared to control
values for saline administration. Compound 56n was more effective for attenuating AHR than
the other two inhibitors, as measured by the acetylcholine challenge.
CONCLUSION
Based on the hit compound 5 from our compound library, we designed a new series of Nꢀarylꢀ
indole compounds, which led to the lead compound 13b (3ꢀ(3ꢀphenethylꢀ1ꢀphenylꢀ1Hꢀindolꢀ5ꢀ
yl)propanoic acid), which had an IC value of 0.075 ꢀM in the isolated enzyme assay. Neither
5
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the introduction of substituents on the phenyl moiety of the C3ꢀphenethyl group nor modification
of the linker or carboxylic acid group of C5ꢀpropanoic acid improved the potency. While the N1ꢀ
phenyl group was indispensable to the inhibitory potency, the introduction of substituents at the
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4
ꢀposition of the N1ꢀphenyl group resulted in greater improvement of the in vitro potency than
introduction at the 2ꢀ or 3ꢀposition. Intensive synthetic development of the 4ꢀposition resulted in
preferable inhibitors with substantial improvements in activities in the enzymatic and PEC
assays. We utilized a mice TPAꢀinduced ear edema model as a primary in vivo model for
compound screening to confirm in vivo efficacy. Six compounds (56b, 56k–n, and 56q) were
selected as potential antiꢀinflammatory agents and analyzed for subtype selectivity against type II
sPLA ; they demonstrated low activity toward the enzyme. The inhibitors were further checked
2
by two whole blood assays (GWB and HWB) and, as a result, the three inhibitors with the high
potencies (56k, 56l, and 56n) were selected for further pharmacological evaluation. The oral
efficacies of these three compounds were determined using a guinea pig OVAꢀinduced asthma
model: Compound 56n showed particularly superior inhibitory effects against all evaluations of
IAR, LAR, and AHR. These results revealed that compound 56n, 3ꢀ[1ꢀ(4ꢀphenoxyphenyl)ꢀ3ꢀ(2ꢀ
phenylethyl)ꢀ1Hꢀindolꢀ5ꢀyl]propanoic acid 2ꢀaminoꢀ2ꢀ(hydroxymethyl)propaneꢀ1,3ꢀdiol salt
(ASB14780), is a very promising candidate as a new drug for the treatment of asthma and other
pulmonary diseases. The promising antiꢀinflammatory properties of 56n (ASB14780) will be
reported in due course.
EXPERIMENTAL SECTION
General Methods. Starting materials were obtained from commercial suppliers and used
without further purification. All commercially available solvents and reagents were also used
without further purification unless otherwise stated. Reactions were conducted at room
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temperature and all organic extracts were dried over Na SO unless otherwise noted.
2
4
Evaporations were conducted in vacuo. Flash column chromatography was performed using
Merck Silica gel 60 (230–400 mesh ASTM) silica gel or Moritex PurifꢀPack packed with SI 30
0
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ꢀm silica gel. Preparative thinꢀlayer chromatography (PTLC) was performed using Merck PLC
1
Silica gel 60 F254 plates. H NMR spectra were recorded on a 400 MHz Bruker ARX400
spectrometer. Chemical shifts (δ) from the internal tetramethylsilane (TMS) standard are
expressed in parts per million unless otherwise noted. MS spectra were measured using positiveꢀ
mode electronspray ionization (ES+), and the purities of the target compounds were determined
with simultaneous UV detection at 254 nm using an Agilent Technologies 1100 HPLC
instrument equipped with LC/MSD VL or 6120 Quadrupole LC/MS. The column used was a
YMCꢀPack Pro C18 (ODS, 50 × 4.6 mmI.D., particle size 3 ꢀm) with a temperature of 40°C and
a flow rate of 1.0 mL/min. Mobile phase A consisted of CH CN/H O/TFA (5:95:0.1, v/v/v).
3
2
Mobile phase B consisted of CH CN/TFA (100:0.09, v/v), which was increased linearly from
3
10% to 100% over 8 min, after which the column was equilibrated to 10% for 1.2 min; all final
compounds were ≥95% pure. Highꢀresolution mass spectra were measured using a JEOL JMSꢀ
700 instrument with a mꢀnitrobenzyl alcohol matrix. Yields were not optimized.
General Procedure for Indole Coupling with Iodobenzene (A). A mixture of the indole (1.0
equiv.), copper(I) bromide (0.1 equiv.), potassium carbonate (4.0 equiv.), iodobenzene (13
equiv.), 85% potassium hydroxide (1.0 equiv.), and copper(II) acetate (0.1 equiv.) was stirred
overnight at 140–150°C. After the mixture was cooled to room temperature, the insolubles were
removed via filtration. The filtrate was evaporated to a residue that was purified by silica gel
chromatography.
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General Procedure for Indole Coupling with Aryl Bromide (B). Aryl bromide (2.0 equiv.),
palladium(II) acetate (0.1 equiv.), 2ꢀ(diꢀtꢀbutylphosphino)biphenyl (0.2 equiv.), and tripotassium
phosphate (4.0 equiv.) were added to a solution of the indole (1.0 equiv.) in dimethoxyethane
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(
0.15 M). The resulting suspension was stirred overnight in a pressureꢀresistant sealed vessel at
110°C. The mixture was cooled to room temperature, filtered, and evaporated to a residue that
was purified by silica gel chromatography.
General Procedure for Indole Coupling with Aryl Bromide (C). Aryl bromide (2.0 equiv.),
palladium(II) acetate (0.2 equiv.), 2ꢀ(diꢀtꢀbutylphosphino)biphenyl (0.4 equiv.), and tripotassium
phosphate (4.0 equiv.) were added to a solution of the indole (1.0 equiv.) in nꢀbutyl acetate (0.05
M). The resulting suspension was stirred overnight at 110–120°C. The mixture was cooled to
room temperature, filtered, and evaporated to a residue that was purified by silica gel
chromatography.
39
General Procedure for Acylation of Indoles via Friedel-Crafts Reaction. Acyl chloride
(3.0 equiv.) was added to a suspension of aluminum chloride (6.0 equiv.) in CH Cl (0.5 M), and
2
2
the mixture was stirred at room temperature for 1 h. A solution of the indole (1.0 equiv.) in
CH Cl (0.24 M) was then added dropwise, and the mixture was stirred at room temperature for
2
2
3
h. The reaction mixture was poured into ice water and extracted with CH Cl . The organic
2 2
layer was successively washed with a saturated aqueous sodium hydrogen carbonate solution and
brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica
gel chromatography.
General Procedure for Stepwise Reduction of 3-Acylated Indoles from Carbonyl to
Methylene. A suspension of the 3ꢀacylated indole (1.0 equiv.) in MeOH (0.26 M) was stirred
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under ice cooling while sodium borohydride (30 equiv.) was added, and the mixture was stirred
at room temperature for 1 h. The reaction mixture was quenched with 1 N HCl and extracted
with CH Cl . The organic layer was washed with brine, dried over anhydrous sodium sulfate,
2
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and concentrated to afford a crude alcohol.
The alcohol was dissolved in CH Cl (0.06 M), and the mixture was cooled to −78°C. Tin(IV)
2
2
chloride (1.2 equiv.) and triethyl silane (1.2 equiv.) were consecutively added to the mixture,
which was then stirred for 1 h. The reaction mixture was quenched with MeOH, poured into
H O, and extracted with CH Cl . The organic layer was successively washed with a saturated
2
2
2
aqueous sodium hydrogen carbonate solution and brine, dried over anhydrous sodium sulfate,
and evaporated to a residue that was purified by silica gel chromatography.
General Procedure for Installation of Methyl Acrylates via Heck Reaction. Methyl acrylate
(3.0 equiv.), palladium(II) acetate (0.1 equiv.), and tris(2ꢀmethylphenyl)phosphine (0.2 equiv.)
were added to a solution of bromoindole (1.0 equiv.) in triethylamine (0.12 M), and the mixture
was heated with stirring in a sealed tube at 120°C for 5 h. The mixture was cooled to room
temperature, filtered, and evaporated to a residue that was purified by silica gel chromatography.
General Procedure for Installation of Ethyl Acrylates via Heck Reaction. Ethyl acrylate
(3.0 equiv.), palladium(II) acetate (0.1 equiv.), and tris(2ꢀmethylphenyl)phosphine (0.2 equiv.)
were added to a solution of bromoindole (1.0 equiv.) in triethylamine (0.11 M), and the mixture
was stirred overnight at 110°C. The mixture was cooled to room temperature, filtered, and
evaporated to a residue that was purified by silica gel chromatography.
General Procedure for Ester Hydrolysis. Aqueous NaOH (1 N, 2.0 mmol) and MeOH (for
the methyl ester) or EtOH (for the ethyl ester) (0.5 M) were added to a solution of the ester (1.0
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mmol) in THF (0.5 M). The mixture was stirred overnight at room temperature or heated at 45°C
until the ester starting material was consumed. The mixture was then diluted with H O, acidified
2
to pH 1 using 1 N HCl, and extracted with EtOAc. The organic extracts were washed with H O
2
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and brine, dried over anhydrous sodium sulfate, and concentrated to afford the carboxylic acid,
which was further purified by silica gel chromatography as required.
General Procedure for Hydrogenation or Hydrogenolysis. 10% Palladium carbon (18 mg)
was added to a solution of olefin or Oꢀbenzylated compound (1.0 mmol) in MeOH or EtOH
(0.01 M), and the mixture was stirred overnight under a hydrogen atmosphere at room
temperature. The insolubles were removed via filtration, and the filtrate was evaporated to a
residue that was purified by silica gel chromatography.
General Procedure for Reduction and Hydrolysis via Huang-Minlon Modification of
Wolff-Kishner Reaction. A mixture of keto olefin ester or keto olefin acid (1.0 equiv.),
potassium hydroxide (1.3 equiv.), hydrazine monohydrate (2.4 equiv.), and ethylene glycol (0.3
M) was heated at 120°C for 2–3 h and 200°C for 4–5 h. The reaction mixture was cooled to
room temperature, acidified to pH 1 using 1 N HCl, and extracted with CH Cl . The organic
2
2
extracts were washed with H O and brine, dried over anhydrous sodium sulfate, and
2
concentrated. The residue was purified by silica gel chromatography.
General Procedure for Carboxamide-forming Reaction. Amine (1.5 mmol), triethylamine
(1.0 mL), and 25% propylphosphonic acid anhydride (cyclic trimer) in EtOAc (ca. 0.85 M, 2.0
mL) were added to a solution of the acid (1.0 mmol) in DMF (14 mL). The mixture was stirred at
room temperature for 24 h. The reaction mixture was poured into H O and extracted with EtOAc.
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The organic layer was successively washed with H O and brine, dried over anhydrous sodium
2
sulfate, and concentrated. The residue was purified by silica gel chromatography.
General Procedure for Tromethamine Salt Formation. An acid (0.10 mmol) was dissolved
0
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in THF (1.5 mL) and a 0.10 M aqueous solution of tris(hydroxymethyl)aminomethane
(tromethamine, 1.0 mL) was added. The resulting solution was stirred at room temperature for a
while and concentrated to a solid, which was dried in vacuo and provided a 1:1 tromethamine
salt of the acid.
5-Bromo-1-phenyl-1H-indole (8). The coupling reaction using copper catalysts was performed
for 5ꢀbromoindole 7 (28.7 g, 146 mmol) with iodobenzene (210 mL, 1.88 mol) at 140°C
according to the general procedure A. Purification by silica gel column chromatography (5 to
1
30% EtOAc/hexane) afforded 8 (20.5 g, 52%) as a pale yellow oil. H NMR (400 MHz, CDCl )
3
δ 7.82 (d, J = 2.0 Hz, 1H), 7.46−7.56 (m, 4H), 7.26−7.43 (m, 4H), 6.63 (d, J = 3.6 Hz, 1H). MS
+
(
ES+): m/z 272.0, 274.0 (M + H) .
5-Bromo-1-phenyl-3-phenylacetyl-1H-indole (9b). The FriedelꢀCrafts acylation was applied
to 8 (2.5 g, 9.2 mmol) using phenylacetyl chloride (1.6 mL, 12 mmol) according to the general
procedure. Purification by silica gel column chromatography (10 to 50% EtOAc/hexane)
1
afforded 9b (1.77 g, 49%) as an yellow amorphous. H NMR (400 MHz, CDCl ) δ 8.68 (d, J =
3
2.0 Hz, 1H), 7.95 (s, 1H), 7.24−7.62 (m, 12H), 4.18 (s, 2H). MS (ES+): m/z 390.0, 392.0 (M +
+
H) .
5-Bromo-1-phenyl-3-(2-phenylethyl)-1H-indole (10b). The stepwise carbonyl reduction of 9b
50 mg, 0.13 mmol) was performed according to the general procedure. Purification by silica gel
(
column chromatography (10 to 40% EtOAc/hexane) afforded 10b (39 mg, 80%) as a colorless
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amorphous. H NMR (400 MHz, CDCl ) δ 7.74 (s, 1H), 7.50 (t, J = 7.9 Hz, 2H), 7.21−7.44 (m,
3
1
0H), 7.06 (s, 1H), 3.10 (s, 2H), 3.06 (d, J = 10.7 Hz, 1H), 3.06 (d, J = 3.1 Hz, 1H). MS (ES+):
+
m/z 376.1, 378.0 (M + H) .
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Methyl (2E)-3-[1-phenyl-3-(2-phenylethyl)-1H-indol-5-yl]-2-propenoate (11b). The Heck
reaction was applied to 10b (39 mg, 0.10 mmol) using methyl acrylate (0.028 mL, 0.31 mmol)
according to the general procedure. Purification by silica gel column chromatography (10 to 30%
1
EtOAc/hexane) afforded 11b (24 mg, 63%) as a pale yellow amorphous. H NMR (400 MHz,
CDCl ) δ 7.87 (d, J = 15.8 Hz, 1H), 7.76 (s, 1H), 7.41−7.55 (m, 6H), 7.21−7.41 (m, 6H), 7.10 (s,
3
1H), 6.44 (d, J = 15.8 Hz, 1H), 3.83 (s, 3H), 3.10 (dd, J = 19.8, 7.6 Hz, 2H), 3.05 (br s, 2H). MS
+
(
ES+): m/z 382.1 (M + H) .
(2E)-3-[1-Phenyl-3-(2-phenylethyl)-1H-indol-5-yl]-2-propenoic acid (12b). Hydrolysis of the
methyl ester 11b (24 mg, 0.063 mmol) was performed according to the general procedure.
Purification by silica gel column chromatography (1 to 12% MeOH/CH Cl ) afforded 12b (21
2
2
1
mg, 91%) as a pale yellow amorphous. H NMR (400 MHz, CD OD) δ 7.79 (d, J = 15.8 Hz, 1H),
3
7.70 (s, 1H), 7.41−7.55 (m, 6H), 7.36 (t, J = 7.4 Hz, 1H), 7.12−7.28 (m, 6H), 6.40 (d, J = 15.8
+
+
Hz, 1H), 2.99−3.15 (m, 4 H). MS (ES+): m/z 368.1 (M + H) . HRMS for C H NO + H calcd,
25
21
2
368.1645; found, 368.1640.
3
-[1-Phenyl-3-(2-phenylethyl)-1H-indol-5-yl]propanoic acid (13b). Hydrogenation of the
olefin acid 12b (10 mg, 0.027 mmol) was performed according to the general procedure.
Purification by silica gel column chromatography (1 to 12% MeOH/CH Cl ) afforded 13b (7.5
2
2
1
mg, 75%) as a pale yellow amorphous. H NMR (400 MHz, CD OD) δ 7.47 (dd, J = 18.6, 6.9
3
Hz, 1H), 7.38−7.54 (m, 5H), 7.14−7.33 (m, 6H), 7.11 (s, 1H), 7.06 (d, J = 8.7 Hz, 1H), 3.08 (br.
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