Synthesis and pharmacological characterization of 1-benzyl-4-aminoindole- based thyroid hormone receptor β agonists

Article abstract of DOI:10.1016/j.bmc.2013.11.001

A series of 1-benzylindole-based TRβ agonists were prepared and evaluated. Compounds 11b′ and 11c′ were found to have cholesterol-lowering in a rat model with marginal effects on cardiac function and HPT axis. The present work illustrates the potential use of indoles as inner ring isosteres.

Full text of DOI:10.1016/j.bmc.2013.11.001

Bioorganic & Medicinal Chemistry 22 (2014) 488–498
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological characterization
of 1-benzyl-4-aminoindole-based thyroid hormone
receptor b agonists
Naoki Takahashi a, Koji Maeda a, Yukiyasu Asano b, Nobuhide Watanabe a,
⇑
a Drug Discovery Laboratories, Sanwa Kagaku Kenkyusho Co., Ltd, 363 Shiosaki, Hokusei-cho, Inabe-city, Mie 511-0406, Japan
b Product Planning II, R&D Planning Department, R&D Strategy Center, Sanwa Kagaku Kenkyusho Co., Ltd, 35 Higashisotobori-cho, Higashi-ku, Nagoya 461-8631, Japan
a r t i c l e i n f o
a b s t r a c t
A series of 1-benzylindole-based TRb agonists were prepared and evaluated. Compounds 11b0 and 11c0
were found to have cholesterol-lowering in a rat model with marginal effects on cardiac function and
HPT axis. The present work illustrates the potential use of indoles as inner ring isosteres.
Ó 2013 Elsevier Ltd. All rights reserved.
Article history:
Received 3 October 2013
Revised 31 October 2013
Accepted 1 November 2013
Available online 13 November 2013
Keywords:
Thyroid hormone receptor b agonists
Dyslipidemia
Hypothalamic/pituitary/thyroid axis
Thyroid stimulating hormone
1. Introduction
The efficacy and safety of the thyromimetic dextrothyroxine so-
dium (D-T4) were investigated in a large clinical trial using coro-
Management of dyslipidemia is established as reliable clinical
intervention to reduce the morbidity and mortality associated with
atherosclerotic cardiovascular diseases (CVD). Among a number of
therapeutic approaches, statins are the mainstay of treatment for
dyslipidemia in CVD patients.1 Statins, however, are not equally
effective in all patients, and are less effective in lowering triglycer-
ides than low-density lipoprotein (LDL) cholesterol.2 In addition,
the withdrawal of cerivastatin in 2001 has led to recommendations
for the appropriate use of statins, including cautions, contraindica-
tions, and safety monitoring for statin therapy.3 Therefore, the
development of new lipid-lowering drugs with novel mechanisms
of action and better safety profiles is still desirable.4
nary heart disease (CHD) patients with hyperlipidemia.6
Treatment with D-T4 decreased cholesterol and triglyceride levels
by 12% and 15–20%, respectively, compared to a concomitant in-
crease for the placebo group. Although these beneficial effects
lasted over three years, treatment with D-T4 resulted in a signifi-
cantly higher overall mortality than that with the placebo. In addi-
tion, the risk of death increased in patients with a history of
myocardial infarction or angina pectoris. The clinical trial was
eventually discontinued, however, intensive efforts for the devel-
opment of safer thyromimetics are continuing (Fig. 1).7 Recently,
I
Thyroid hormones (TH) are involved in growth, development,
and metabolism via their two homologous receptors, TRa and TRb,
HO
I
I
CO2H
NH2
HO
Me
Me
O
CO2H
O
which belong to the nuclear receptor superfamily of ligand-depen-
dent transcription factors.5 TH synthesis and secretion are strictly
regulated by the hypothalamic/pituitary/thyroid (HPT) axis, and
their receptors are ubiquitously expressed in human and rodents.
Plasma levels of circulating TH thereby link to various pathological
states. For example, hyperthyroidism is characterized by decreased
serum cholesterol and triglycerides levels, body weight loss, tachy-
cardia, arrhythmia, muscle wasting, and other complaints.5a
I
Me
Me
D-T4
GC-1
H
O
N
O
H
H
HO
Me
Br
O
N
O
N
Cl
N
CO2H
N
N
CN
O
Me
O
Me
Br
Me
Cl
KB2115
MGL-3196
⇑
Corresponding author. Tel.: +81 (594) 72 6221; fax: +81 (594) 82 0072.
E-mail address: no_watanabe@skk-net.com (N. Watanabe).
Figure 1. Structures of representative TR agonists.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.001

N. Takahashi et al. / Bioorg. Med. Chem. 22 (2014) 488–498
489
a new clue was provided from studies on phenotypes of TR-defi-
cient mice.8 While TR
1-deficient mice had low heart rate and
low body temperature, TR -deficient (e.g., deletion of TR 1 and
TR 2) mice displayed severe hypothyroidism, intestinal malforma-
tion, and growth-retardation. As for mice with TRb-deficiency, they
exhibited a modest increase in cholesterol level and had impaired
T3-dependent regulation of cholesterol metabolism. The mice also
had goiter, overproduced thyroxine (T4) and thyroid stimulating
hormone (TSH), and thus developed resistance to TH (RTH). It
the outer ring, and successfully identified optimal groups, such as
the pyridazinonyl group in SK&F L-94901,16 that efficiently main-
tain the ‘active’ conformation. On the other hand, the explorations
of these optimal groups resulted in a considerable reduction in
intellectual property space around the outer ring, making modifi-
cation around the inner ring an attractive option for further
improvement.17 From this point of view, the structure GC-1 is quite
fascinating because it is rationally simplified and amenable to
modification. A crystal structure of GC-1 in complex with the
TRb-ligand binding domain reveals that the pockets for the 3,5-
dimethylphenyl outer ring are not fully filled.14b We therefore
envisaged that when the outer ring is connected at the 1-position,
a larger planar 2-methylindole might not only restrict free rotation
of the outer ring, but also make better fit into the pockets.
To begin with, we prepared a series of compounds with various
linkage lengths between the indole core and the terminal carbox-
ylic acid (Scheme 1). The outer ring moiety of the benzyl chlorides
1, was prepared from 2-isopropylphenol by four steps and imme-
diately used without purification due to their instability. N-alkyl-
ation of the indoles 2 with 1 proceeded smoothly by use of
sodium hydride as base. Alkaline hydrolysis of 3 gave the desired
acids 4 in excellent yields. Elongation of 4a,b with glycine ethyl es-
ter was carried out under standard coupling conditions, followed
by hydrolysis to give the desired acids 5a,b. The binding affinity
and transcriptional activity of 4 and 5 are shown in Table 1. The
binding assay was carried out by radio-active ligand displacement
in a range test compound concentrations at radiolabelled endoge-
nous ligand thyronine (T3) concentration of 0.16 nM, using recom-
a
a
a
a
should be noted here that while TR
sults in exacerbated hypothyroidism, TR
tion can be lethal after weaning. These findings indicate that the
non-binding TR 2 receptor is crucial to survival.
a1 and TRb double mutation re-
a
2 and TRb double muta-
a
Given that TRb selective modulation may produce beneficial ef-
fects on lipid metabolism without affecting cardiac functions, sev-
eral TRb-selective agonists were developed.9 For instance, GC-1,
which has a 10-fold selectivity for TRb over TRa, dose-dependently
lowered plasma cholesterol in cholesterol-fed rats without signifi-
cant tachycardia.10 In the clinical trials with healthy subjects, GC-1
was well tolerated and decreased LDL cholesterol level by up to
41% at the dose of 100 lg, QD over 2 weeks. KB2115, another
TRb-selective hepato-specific agonist,11 decreased total and LDL
cholesterol levels without affecting heart rate in obese subjects
with 255 mg/dl of mean total plasma cholesterol. In particular,
KB2115 decreased LDL cholesterol level by as much as 40% when
given at the dose of 100
hypercholesterolemia, co-treatment with statin and KB2115
(100 g daily for 12 weeks) further decreased serum LDL choles-
lg daily for 1 week. In patients with
l
terol level by 32%. This co-treatment also decreased triglycerides
and Lp(a) lipoprotein without significant heart, bone, or pituitary
adverse effects. Encouraged by these clinical data, current medici-
nal chemistry for thyromimetics is focusing on the design of more
TRb selective hepato-specific modulators that can activate only the
target gene responsible for the beneficial effects in the liver.12 Very
recently, MGL-3196 has emerged as a new TRb modulator with
promising phase 1 trial results that support the high potential of
this research area.13
binant hTR
activity was measured using monkey kidney CV-1 cells transiently
transfected with hTR 1 or hTRb1 and an alkaline phosphatase re-
porter gene. In this assay, T3 showed strong binding affinity for
TR 1 and TRb1 with IC50 values of 3.2 and 2.9 nM, respectively
and consistent transcriptional activity for both receptors. On the
whole, the 2-methylindoles 4 and 5 preferred TRb1 to TR 1,
a1 and hTRb1 ligand binding domains. Transcriptional
a
a
a
although they were at least 100-fold less potent in binding to both
TRs than T3. The length of the linkage seems to have an impact on
Herein, we report the synthesis and pharmacological profile of
1-benzyl-4-aminoindoles as new TRb selective agonists.
the binding affinity to TRa1 rather than TRb1, wherein 5b showed
the highest TRb selectivity with a ratio of 19. The reporter assay re-
sults of 4c–e also indicated a similar preference of all compounds
for TRb1, although the observed agonist activity of 4c–e was higher
than expected from their binding affinity. Malm and co-workers
have also reported a similar discrepancy, albeit in the opposite
sense, between the binding affinity and the functional activity
and suggested that this discrepancy might be due to the lack of ac-
tive transporters in the cell-based reporter assay.14d,17a In our case,
there may be a favorable access of 4c–e to target organelles, such
as the nucleus.
2. Results and discussion
Until comprehensive studies with TR-deficient mice and X-ray
structural analysis of TR-ligand complexes,8,14 it was believed that
thyromimetics selective delivery to the liver was a key factor for
dissociation of these agents beneficial lipid-lowering effects from
the broad hormonal effects, and that this delivery was partly
attributed to thyromimetics constrained conformations with both
a perpendicular arrangement of the two aromatic rings and a distal
position of the 30-substituent to the inner aromatic ring (Fig. 2).15
Early medicinal chemical efforts for thyromimetic were thereby
centered on the design of isosteres of the 30-iodo substituent in
Crystallographic studies have demonstrated that the two sub-
type receptors differ in a single amino acid residue, that is, Asn
for TRb and Ser for TRa, in the ligand-binding pocket. Furthermore,
these two residues form a hydrogen bond network with two
outer ring
HO
HO
Br
CO2H
NH2
Me
HO
Me
3'
O
Me
Me
inner ring
Br
5
Me
N
HN
1'
OCH2CO2H
R
N
Me
O
3
Me
L-94901
putative 'active' conformation
Figure 2. Structure of L-94901 and representation of a putative ‘active’ conformation of TR agonists.

490
N. Takahashi et al. / Bioorg. Med. Chem. 22 (2014) 488–498
PGO
HO
Me
a, b, c, d
a
: PG = Bn
Me
Cl
b: PG = TIPS
Me
Me
1
BnO
Me
a
: n = 0
Me
HN
Me
(CH2)n
CO2R
(CH2)n
CO2Et
b: n = 1
c
d
: n = 2
: n = 3
e
N
Me
e: n = 4
2
3
HO
HO
Me
N
Me
N
Me
(CH2)n
H
N
Me
(CH2)n
CO2H
f
g
CO2H
Me
Me
O
4
5a, 5b
Scheme 1. Synthesis of 5. Reagents and conditions: (a) NBS, MeCN; (b) PG = Bn: BnCl, NaHCO3, NaI, MeCN, 60 °C, PG = TIPS: TIPSCI, imidazole, DMF; (c) (i) tBuLi, THF, ꢀ78 °C,
(ii) NaOH, MeOH, 60 °C; (d) SOCl2, CH2Cl2; (e) (i) NaH, DMF, 0 °C, (ii) compound 1a, 0 °C; (f) (i) H2/Pd-C, EtOH/EtOAc, (ii) NaOH, MeOH, 60 °C; (g) (i) glycine ethyl ester HCl,
WSC, HOBt, Et3N, DMF, (ii) NaOH, MeOH, 60 °C.
Table 1
Potency of compounds 4 and 5 for TRs
HO
Me
N
R
Me
Me
Binding assaya (IC50
,
lM)
Reporter assay (EC50, lM)
Compd
R
TR
—
1.3
3.4
>10
8.2
>10
a
TRb
a
/b
TR
a
TRb
a/b
T3
0.0032
0.0029
—
0.76
0.29
1.4
1.1
—
2.6
>2.1
>7.1
19
0.0021
0.45
0.039
0.18
0.10
—
0.002
0.15
0.037
0.037
0.062
—
1.0
3.0
1.0
4.8
1.6
—
4b
4c
4d
4e
5b
5a
CH2CO2H
CH2CH2CO2H
CH2CH2CH2CH2CO2H
CH2CH2CH2CH2CH2CO2H
CH2CONHCH2CO2H
CONHCH2CO2H
0.43
1.1
>9.0
—
—
—
a
Values are the means of quadruplicate experiments.
neighboring Arg residues, that is, the carboxylate of a ligand and
water molecules in the individual binding pockets. Therefore, it is
proposed that this subtle difference renders the selectivity in bind-
ing largely dependent on the position of the ligand carboxylate.
The proposed relationship fairly rationalizes our results with the
2-methylindoles 4 and 5,18 and thereby necessitates further opti-
mization around the carboxylic acid side chain. To this end, we
chose the nitroindoles 6 as a starting precursor with a nitro group
that can be easily connected to a carboxylic acid side chain after
conversion into an amino group (Scheme 2). Thus, 6 was obtained
according to a procedure described in the literature (see, Section 3),
and introduction of the outer ring moiety was as described above.
Reduction of 7 smoothly proceeded by catalytic hydrogenation.
Condensation of 7 with diethyl oxalate or ethyl malonyl chloride
and subsequent removal of the silyl protecting group followed by
hydrolysis gave the desired acids 11. Similarly, the 5-subsutituted
congener 11d was prepared from the commercially available 2-
methyl-5-nitroindole. At this stage, to see whether the observed
discrepancy of the results with 4 was a case only with the monkey
cell line, the transcriptional activity of 11 was evaluated using
HepG2 cells as source of a human cell line. Unexpectedly, only a
small gap between the binding and reporter assays was observed
regardless of the cell line used, although HepG2-based assay
provided better agreement with the binding affinity (Table 2). A
short explanation is worthwhile here. First, a marked difference
can be seen between 11b and 11d. Compound 11b showed the
highest TRb1 binding affinity with an IC50 value of 40 nM, whereas
11d affinity for this receptor was the worst. Apparently, the chain
extension at the 4-position is far more favorable for better binding
affinity than that at the 5-position. Second, absence of the
2-methyl substituent (11a) resulted in a more than fourfold de-
crease in binding affinity, which actually supports our working
hypothesis for restricted rotation of the outer ring. Interestingly,
a positive substituent effect of a methyl group was also observed
with 7-methyl analogue 11c. In this line, extension with one meth-
ylene spacer unit was evaluated in both the 2- and 7-methyl ana-
logues 11b0, c0. Gratifyingly, both analogues produced significant
improvement in TRb1 selectivity. Although the HepG2-based assay
showed an apparent difference in TRb selectivity between 11b0 and
11c0, we considered these two analogs to be virtually the same in
terms of potency and selectivity. In contrast, the two analogues
showed a considerable decrease in TRb1 binding affinity compared
to 11b,c, and therefore no further extension was attempted.
To test in vivo potency of 11b0 and 11c0, the two analogs were
orally administered to cholesterol-fed rats (Table 3). Cholesterol
feeding continued for 1 week before drug treatment with average

N. Takahashi et al. / Bioorg. Med. Chem. 22 (2014) 488–498
491
TIPSO
Me
TIPSO
Me
R2
R2
N
R2
N
HN
a
X
Y
X
Y
b
Me
Me
X
Y
R1
R1
R1
7
6
8
1
: R = H, R2 = H, X = NH2, Y = H
: R = H, R2 = H, X = NO2, Y = H
1
a
a
b: R1 = H, R2 = Me, X = NH2, Y = H
c: R1 =Me, R2 = H, X = NH2, Y = H
d: R1 = H, R2 = Me, X = H, Y = NH2
b: R1 = H, R2 = Me, X = NO2, Y = H
c: R1 =Me, R2 = H, X = NO2, Y = H
d: R1 = H, R2 = Me, X = H, Y = NO2
TIPSO
HO
R2
R2
Me
Me
c
d
N
Me
N
X
X
Y
Me
R1
R1
Y
9
10
1
: R = H, R2 = H, X = NHCOCO2Et, Y = H
a
b: R1 = H, R2 = Me, X = NHCOCO2Et, Y = H
c: R1 =Me, R2 = H, X = NHCOCO2Et, Y = H
b': R1 = H, R2 = Me, X = NHCOCH2CO2Et, Y = H
c': R1 =Me, R2 = H, X = NHCOCH2CO2Et, Y = H
: R = H, R2 = Me, X = H, Y = NHCOCO2Et
1
d
HO
R2
Me
e
N
Me
X
Y
R1
11
1
: R = H, R2 = H, X = NHCOCO2H Y = H
a
b: R1 = H, R2 = Me, X = NHCOCO2H, Y = H
c: R1 =Me, R2 = H, X = NHCOCO2H, Y = H
b': R1 = H, R2 = Me, X = NHCOCH2CO2H, Y = H
c': R1 =Me, R2 = H, X = NHCOCH2CO2H, Y = H
1
: R = H, R2 = Me, X = H, Y = NHCOCO2H
d
Scheme 2. Synthesis of 11. Reagents and conditions: (a) (i) NaH, DMF, 0 °C, (ii) 1b, 0 °C; (b) H2/Pd-C, EtOH, (c) 9a, 9b, 9c, 9d: diethyl oxalate, 100 °C, 9b0 and 9d0: ethyl malonyl
chloride, Et3N, CH2Cl2; (d) TBAF, THF; (e) NaOH, EtOH.
Table 2
Potency of compound 11 for TRs
HO
R2
Me
N
Me
X
Y
R1
Substituents
Binding assaya (IC50
,
l
M)
Reporter assaya (EC50
CV-1
,
l
M)
HepG2
Compd
X
Y
R1
R2
TR
a
TRb
a/b
TRb
a/b
TRb
a/b
11b
11d
11c
11a
11b0
11c0
NHCOCO2H
H
NHCOCO2H
NHCOCO2H
NHCOCH2CO2H
NHCOCH2CO2H
H
H
H
Me
H
H
Me
Me
H
H
Me
H
0.23
0.23
0.19
1.2
5.2
5.2
0.04
1.5
0.054
0.17
0.23
0.24
5.7
0.15
3.5
7.0
21
26
0.59
—
0.50
1.9
1.7
3.1
2.2
—
1.5
2.7
4.5
3.4
0.11
—
0.19
0.55
0.22
1.3
1.5
—
2.5
2.2
16
7.2
NHCOCO2H
H
H
H
H
Me
a
Values are the means of quadruplicate experiments.
plasma cholesterol level reaching increased to 190 49 mg/dl,
compared to 66 11 mg/dl in normal rats. We found that adminis-
tration of 11b0 or 11c’ at the dose of 30 or 6 mg/kg QD, respectively,
produced a significant reduction in total cholesterol level. Similar
cholesterol-lowering effects were observed with T3 at the dose of
50 l
g/kg QD. Differences in in vivo potency between 11b0, c0 and

492
N. Takahashi et al. / Bioorg. Med. Chem. 22 (2014) 488–498
Table 3
Cholestrol-lowering effects of 11b0 and 11c0 in cholesterol-fed ratsa
HO
R2
N
Me
CO2H
H
N
Me
O
R1
R1, R2
Total Cho (mg/dl)
LDL-Cho (mg/dl)
8.0
51 15
28 5⁄
20 2⁄
25 7⁄
Free Cho (mg/dl)
HDL-Cho (mg/dl)
TG (mg/dl)
Normal
Control
T3
11c0
11b0
66 11
2
39
36
9
9
28
22
22
20
23
3
6
2
1
7
100 40
62 25
190 49
123 15⁄
94 5⁄
22 2⁄
15 1⁄
19 4⁄
61
27 7⁄
44
8
Me, H
H, Me
111 19⁄
9
a
Cholesterol-fed rats were orally treated once daily for 1 week with 11b0 (30 mg/kg/day, n = 13), T3 as a positive control (50
lg/kg/day, n = 13), or the vehicle or to be the
untreated (control) .
*
P<0.05 compared to the treated control.
Table 4
a
Effects of 11b0 and 11c0 on heart rate, heart weight, TSH, T3, and T4
Heart rate (bpm)
Heart weight (g)
TSH (
llU/ml)
Total-T4
(l
g/dl)
Total-T3 (ng/dl)
Normal
Control
T3
Low-dose 11c0
High-dose 11c0
Low-dose 11b0
High-dose 11b0
404.8 37.2
383.0 26.7
430.0 19.3
401.8 22.3
424.3 36.5
403.2 32.5
415.8 35.8
1.08 0.04
1.14 0.12
1.25 0.13
1.12 0.06
1.18 0.08
1.13 0.10
1.24 0.07
2.8 1.6
3.0 1.0
0.1 0⁄
2.7 1.8
1.3 0.6
3.1 1.3
0.9 0.7
4.4 1.2
3.8 0.9
68
62
6
7
0.9 0.4⁄
2.4 1.0⁄
1.9 0.5⁄
2.8 0.5
1.7 0.3⁄
31 9⁄
61 14
53
63 11
53
8
8
a
Cholestrol-fed rats were orally treated once daily for 1 week with 11b0 (30 and 150 mg/kg/day, n = 13), 11c0 (6 and 150 mg mg/kg/day, n = 13), T3 as a positive control
g/kg/day, n = 13), or the vehicle or to be left untreated (control).
P<0.05 compared to the treated control.
(50
l
*
T3 are at least partly attributed to their binding affinity. Also, these
cholesterol-lowering was found to be largely due to a decrease in
LDL cholesterol, which is consistent with the reported results for
GC-1 or T-681, another liver-selective thyromimetic, in mice.19
As for plasma triglyceride level, 11c0 produced a significant reduc-
tion, while 11b0 induced a none significant downward trend. No
change in triglyceride level was observed following treatment with
T3. Briefly, the safety profiles were examined (Table 4). Although
heart rate and heart weight increased following treatment with
T3, 11b0, or 11c0, the increase in heart rate produced by the two
analogues was less than 15%, which is considered the upper limit
for clinical use,20 even at the highest dose of 150 mg/kg. Unlike
T3 or GC-1,20,21 11b0 and 11 c0 had no effect on TSH at their choles-
terol-lowering doses. This lack of effect on TSH seems to be a char-
acteristic feature of 11b0 and 11c0, because TRb is thought to
mediate cholesterol metabolism and TSH regulation. In addition,
both analogues produced no significant reduction in plasma T3
and T4 levels at their cholesterol-lowering doses. These results
demonstrate that 11b0 and 11c0 had marginal effects on cardiac
function and HPT axis in cholesterol-fed rats.
In summary, a series of 1-benzylindole-based TRb agonists were
prepared and evaluated. Compounds 11b0 and 11c0 were found to
have cholesterol-lowering effect in a rat model with marginal ef-
fects on cardiac function and HPT axis. Considering the previous
work by Malm and co-workers,17a in which 1H-indole-2-carboxylic
acid was designed as an inner ring isostere and found to be an excel-
lent scaffold for TRb selective ligand, our present work here addi-
tionally illustrates the potential use of indoles as inner ring
isosteres. Further optimization of 11b0 and 11c0 requires under-
standing the mechanisms of TRb selectivity and separation of these
analogues cholesterol-lowering effect from cardiac and HPT axis
toxicity. PK-PD relationship studies on this series of compounds
should be informative and the results would be reported elsewhere.
3. Experimental section
3.1. Chemistry
3.1.1. General
All reagents and solvents were purchased commercially and
used without further purification. The starting ethyl esters of
2-methylindole-3-carboxylic acid and 2-methylindole-3-acetic
acid, and the 2-methyl-5-nitroindole are commercially available.
Other 3-indoyl acid esters22 and nitroindoles,23 and the 3-isopro-
pyl-4-methoxybenzylalcohol24 were prepared according to the lit-
erature. Flash chromatography was performed using Silica Gel 60
(particle size 0.040–0.050 mm, Kanto Kagaku) or Hi-Flash columns
(silica gel, particle size 0.070 mm, Yamazen Science). Melting
points were determined on a cover glass with a Yanaco MP-J3 mi-
cro melting point apparatus and are given as uncorrected values.
1H NMR spectra were recorded on a Bruker AVANCE III spectrom-
eter operating at 400 MHz and 25 °C with tetramethylsilane as
internal standard. Data are reported as follows: chemical shift in
ppm (d) relative to trimethylsilane, integration, multiplicity
(s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = mul-
tiplet), and coupling constant (Hz). Infrared spectra (IR) were re-
corded on
a JASCO FT/IR-4200 spectrometer with a single-
reflection diamond ATR unit. LC/MS spectra were determined on
a Waters ZMD2000 equipped with a Waters 2690 injector and a
PDA detector operating at 210–400 nm and interfaced with a
Micromass ZMD mass spectrometer. High-resolution mass spectra
(HRMS) were obtained using a Thermo LTQ Orbitrap.

N. Takahashi et al. / Bioorg. Med. Chem. 22 (2014) 488–498
493
3.1.2. 4-Bromo-2-isopropylphenol
(1H, d, J = 8.0 Hz), 7.12 (1H, dd, J = 2.0, 8.4 Hz), 7.23–7.43 (6H,
m); MS (ESI) m/z 239 (M+HꢀH2O)+, 255 (MꢀH)ꢀ.
To a solution of 2-isopropylphenol (50.0 g, 367 mmol) in MeCN
(500 mL) at 0 °C was added NBS (71.9 g, 404 mmol). After stirring
at room temperature for 4 h, the reaction mixture was concen-
trated under reduced pressure. The residue was suspended in hex-
ane and the precipitates were filtered off. The filtrate was washed
with water and brine, and then dried over Na2SO4. After filtration,
the solvent was concentrated under reduced pressure to obtain the
title compound (84.0 g, quantitative yield) as a pale yellow oil: 1H
NMR (CDCl3) d 1.10 (6H, d, J = 7.0 Hz), 3.17 (1H, m), 6.61 (1H, d,
J = 8.4 Hz), 7.15 (1H, dd, J = 2.2, 8.4 Hz), 7.27 (1H, d, J = 2.2 Hz);
MS (ESI) m/z 213, 215 (MꢀH)ꢀ.
3.1.7. (4-Chloromethyl-2-isopropylphenoxy)triisopropylsilane
(1b)
Thionyl chloride (1.6 mL, 22 mmol) was added dropwise to a
solution of (3-isopropyl-4-triisopropylsilanyloxyphenyl)methanol
(4.6 g, 14 mmol) in CH2Cl2 (46 mL), and the mixture was stirred
at room temperature for 2 h. The mixture was next concentrated
under reduced pressure to give 1b, which was used immediately
without further purification. 1-Benzyloxy-4-chloromethyl-2-iso-
propylbenzene (1a) was prepared from the corresponding alcohol
in a similar manner and immediately used the next reaction.
3.1.3. 1-Benzyloxy-4-bromo-2-isopropylbenzene
To a solution of 4-bromo-2-isopropylphenol (2.2 g, 7 mmol) in
MeCN (10.0 mL) at room temperature were added benzyl chloride
(1.7 g, 13 mmol), sodium bicarbonate (1.0 g, 12 mmol) and sodium
iodide (0.2 g, 1 mmol). After stirring at 60 °C for 18 h, the reaction
mixture was cooled to room temperature, and concentrated under
reduced pressure. The residue was taken up with water and ex-
tracted with EtOAc, and the organic layers were washed with
water and brine, and then dried over Na2SO4. After filtration, the
solvent was concentrated under reduced pressure, and the residue
was purified by flash chromatography on silica gel and eluted with
hexane/EtOAc (1/0–9/1) to give the title compound (2.7 g, 86%) as a
colorless oil: 1H NMR (CDCl3) d 1.21 (6H, d, J = 7.2 Hz), 3.37 (1H, m),
6.76 (1H, d, J = 8.4 Hz), 7.23 (1H, dd, J = 2.8, 8.4 Hz), 7.31–7.43 (6H,
m); MS (ESI) m/z 303, 305 (MꢀH)ꢀ.
3.1.8. Ethyl [1-(4-benzyloxy-3-isopropylbenzyl)-2-methyl-1H-
indol-3-yl]acetate (3b)
To a suspension of NaH (60% in oil, 87.1 mg, 2.2 mmol) in DMF
(2.0 mL) was added dropwise a solution of ethyl (2-methylindol-3-
yl)acetate (430.0 mg, 2.0 mmol) in DMF (2.0 mL) at 0 °C. After stir-
ring at 0 °C for 1 h, a DMF solution (2.0 mL) of freshly prepared 1a
(ca. 2.2 mmol) was added dropwise to the mixture at 0 °C, and the
resulting mixture was stirred for an additional 3 h. The reaction
was quenched with ice water, and then extracted with EtOAc.
The organic layers were successively washed with water and brine,
and dried over Na2SO4. After evaporation of the solvent, the residue
was purified by flash chromatography on silica gel and eluted with
hexane/EtOAc (9/1) to give the title compound (387 mg, 43%) as a
white solid: mp 76–77 °C; 1H NMR (CDCl3) d 1.18 (6H, d, J = 6.9 Hz),
1.22 (3H, t, J = 7.1 Hz), 2.35 (3H, s), 3.35 (1H, m), 3.73 (2H, s), 4.14
(2H, q, J = 7.1 Hz), 5.00 (2H, s), 5.25 (2H, s), 6.57 (1H, dd, J = 2.2,
8.4 Hz), 6.72 (1H, d, J = 8.4 Hz), 7.02 (1H, d, J = 2.2 Hz), 7.09–7.13
(2H, m), 7.24 (1H, m), 7.32 (1H, m), 7.36–7.40 (4H, m), 7.58 (1H,
m); MS (ESI) m/z 456 (M+H)+; HRMS (ESI) m/z calcd for
3.1.4. 4-Bromo-2-isopropyl-1-triisopropylsilanyloxybenzene
To a solution of 4-bromo-2-isopropylphenol (84.8 g, 394 mmol)
in DMF (848 mL) were added triisopropylsilyl chloride (101 mL,
472 mmol) and imidazole (53.7 g, 789 mmol). After stirring at
room temperature for 24 h, the reaction mixture was diluted with
hexane, washed with water and brine, and dried over Na2SO4. The
solvent was then concentrated under reduced pressure, and the
residue was crystallized with MeCN to give the title compound
(98.0 g, 67%) as a white solid: mp 59–60 °C; 1H NMR (CDCl3) d
1.10 (18H, d, J = 7.3 Hz), 1.18 (6H, d, J = 7.0 Hz), 1.28 (3H, m),
3.32 (1H, m), 6.63 (1H, d, J = 8.4 Hz), 7.12 (1H, dd, J = 2.6, 8.4 Hz),
7.25 (1H, d, J = 2.6 Hz); MS (ESI) m/z 370, 372 (M+H)+.
C
30H34NO3 (M+H)+ 456.2533, found 456.2528.
3.1.9. Ethyl 1-(4-benzyloxy-3-isopropylbenzyl)-2-methyl-1H-
indole-3-carboxylate (3a)
The title compound was prepared according to the procedure
described for 3b, using ethyl 2-methyl-1H-indole-3-carboxylate
as a white solid (88%): mp 89–91 °C, 1H NMR (CDCl3) d 1.18 (6H,
d, J = 6.9 Hz), 1.26 (3H, t, J = 7.1 Hz), 2.75 (3H, s), 3.35 (1H, m),
4.43 (2H, q, J = 7.1 Hz), 5.00 (2H, s), 5.29 (2H, s), 6.58 (1H, dd,
J = 1.9, 8.4 Hz), 6.73 (1H, d, J = 8.4 Hz), 7.04 (1H, d, J = 1.9 Hz),
7.13–7.32 (4H, m), 7.36–7.40 (4H, m), 8.17 (1H, d, J = 7.5 Hz); MS
(ESI) m/z 442 (M+H)+; HRMS (ESI) m/z calcd for C29H32NO3
(M+H)+ 442.2377, found 442.2378.
3.1.5. (3-Isopropyl-4-triisopropylsilanyloxyphenyl)methanol
To a solution of 4-bromo-2-isopropyl-1-triisopropylsilanyloxy-
benzene (23.6 g, 64 mmol) in THF (236 mL) was added dropwise
a solution of tBuLi (1.5 M in pentane, 100 mL, 150 mmol) at
ꢀ78 °C over 1 h. After stirring at the same temperature for 1 h,
paraformaldehyde (3.9 g) was added to the mixture, and the
resulting mixture was stirred at ꢀ78 °C for an additional 1 h, and
then warmed to room temperature for 3 h. The reaction mixture
was subsequently diluted with Et2O (300 mL), successively washed
with 1 M hydrochloric acid, and brine, and dried over Na2SO4. After
evaporation of the solvent, the residue was purified by flash chro-
matography on silica gel and eluted with hexane/EtOAc (1/0–4/1)
to give the title compound (9.4 g, 46%) as a colorless oil: 1H NMR
(CDCl3) d 1.12 (18H, d, J = 7.3 Hz), 1.21 (6H, d, J = 6.9 Hz), 1.32
(3H, m), 3.37 (1H, m), 4.59 (2H, d, J = 5.5 Hz), 6.75 (1H, d,
J = 8.1 Hz), 7.02 (1H, dd, J = 1.2, 8.0 Hz), 7.20 (1H, d, J = 1.2 Hz);
MS (ESI) m/z 305 (MꢀH2O)+, 321 (MꢀH)ꢀ.
3.1.10. Ethyl [1-(4-benzyloxy-3-isopropylbenzyl)-2-methyl-1H-
indol-3-yl]propanoate (3c)
The title compound was prepared according to the procedure
described for 3b, using ethyl (2-methylindol-3-yl)propanoate as a
foam (17%): 1H NMR (CDCl3) d 1.18 (6H, d, J = 6.9 Hz), 1.26 (3H, t,
J = 7.0 Hz), 2.32 (3H, s), 2.62 (2H, t, J = 7.8 Hz), 3.08 (2H, t,
J = 7.8 Hz), 3.35 (1H, m), 4.11 (2H, q, J = 7.0 Hz), 5.00 (2H, s), 5.22
(2H, s), 6.54 (1H, dd, J = 2.2, 8.4 Hz), 6.71 (1H, d, J = 8.4 Hz), 7.00
(1H, d, J = 2.2 Hz), 7.05–7.12 (2H, m), 7.22 (1H, m), 7.30 (1H, m),
7.34–7.41 (4H, m), 7.54 (1H, m); MS (ESI) m/z 470 (M+H)+; HRMS
(ESI) m/z calcd for C31H36NO3 (M+H)+ 470.2690, found 470.2690.
3.1.11. Ethyl [1-(4-benzyloxy-3-isopropylbenzyl)-2-methyl-1H-
indol-3-yl]butanoate (3d)
3.1.6. (4-Benzyloxy-3-isopropylphenyl)methanol
The title compound was prepared according to the procedure
described in Section 3.1.5, using 1-benzyloxy-4-bromo-2-isopro-
pylbenzene as white solid: mp 56–58 °C; 1H NMR (CDCl3) d 1.22
(6H, d, J = 7.2 Hz), 3.41 (1H, m), 4.60 (2H, s), 5.07 (2H, s), 6.87
The title compound was prepared according to the procedure
described for 3b, using ethyl (2-methylindol-3-yl)butanoate as a
foam (34%): 1H NMR (CDCl3) d 1.18 (6H, d, J = 6.9 Hz), 1.23 (3H, t,
J = 7.1 Hz), 1.97 (2H, m), 2.29 (3H, s), 2.32 (2H, t, J = 7.3 Hz), 2.80

494
N. Takahashi et al. / Bioorg. Med. Chem. 22 (2014) 488–498
(2H, t, J = 7.3 Hz), 3.35 (1H, m), 4.11 (2H, q, J = 7.1 Hz), 5.00 (2H, s),
5.23 (2H, s), 6.57 (1H, dd, J = 1.4, 8.4 Hz), 6.73 (1H, d, J = 8.4 Hz),
6.97 (1H, d, J = 1.4 Hz), 7.06–7.12 (2H, m), 7.22 (1H, m), 7.31 (1H,
m), 7.34–7.41 (4H, m), 7.54 (1H, m); MS (ESI) m/z 484 (M+H)+;
HRMS (ESI) m/z calcd for C32H38NO3 (M+H)+ 484.2846, found
484.2843.
3.1.16. [1-(3-Isopropyl-4-hydroxybenzyl)-2-methyl-1H-indol-3-
yl]butanoic acid (4d)
The title compound was prepared according to the procedure
described for 4b, using 3d as a foam (29%, 2 steps): 1H NMR (CDCl3)
d 1.21 (6H, d, J = 7.0 Hz), 2.07 (2H, m), 2.30 (3H, s), 2.39 (2H, t,
J = 7.3 Hz), 2.83 (2H, t, J = 7.3 Hz), 3.17 (1H, m), 5.23 (2H, s), 6.49
(1H, dd, J = 1.2, 8.4 Hz), 6.56 (1H, d, J = 8.4 Hz), 6.96 (1H, br s),
7.10–7.14 (2H, m), 7.23 (1H, brd, J = 7.7 Hz), 7.55 (1H, d,
J = 7.6 Hz); IR (ATR) 3380, 2959, 1702 cmꢀ1; MS (ESI) m/z 366
(M+H)+, 364 (MꢀH)ꢀ; HRMS (ESI) m/z calcd for C23H28NO3
(M+H)+ 366.2064, found 366.2064.
3.1.12. Ethyl [1-(4-benzyloxy-3-isopropylbenzyl)-2-methyl-1H-
indol-3-yl]pentanoate (3e)
The title compound was prepared according to the procedure
described for 3b, using ethyl (2-methylindol-3-yl)pentanoate as a
foam (12%): 1H NMR (CDCl3) d 1.18 (6H, d, J = 6.9 Hz), 1.22 (3H, t,
J = 7.1 Hz), 1.63–1.74 (4H, m), 2.29 (3H, s), 2.31 (2H, t, J = 6.9 Hz),
2.76 (2H, t, J = 6.9 Hz), 3.35 (1H, m), 4.11 (2H, q, J = 7.1 Hz), 5.00
(2H, s), 5.22 (2H, s), 6.55 (1H, dd, J = 2.2, 8.5 Hz), 6.72 (1H, d,
J = 8.5 Hz), 6.99 (1H, d, J = 2.2 Hz), 7.03–7.11 (2H, m), 7.22 (1H, d,
J = 7.7 Hz), 7.28 (1H, m), 7.34–7.40 (4H, m), 7.53 (1H, m); MS
(ESI) m/z 498 (M+H)+; HRMS (ESI) m/z calcd for C33H40NO3
(M+H)+ 498.3003, found 498.3004.
3.1.17. [1-(3-Isopropyl-4-hydroxybenzyl)-2-methyl-1H-indol-3-
yl]pentanoic acid (4e)
The title compound was prepared according to the procedure
described for 4b, using 3e as a foam (44%, 2 steps): 1H NMR
(DMSO-d6) d 1.07 (6H, d, J = 7.0 Hz), 1.45–1.70 (4H, m), 2.22 (2H,
t, J = 7.0 Hz), 2.33 (3H, s), 2.69 (2H, t, J = 6.6 Hz), 3.21 (1H, m),
5.23 (2H, s), 6.52 (1H, dd, J = 2.2, 8.4 Hz), 6.63 (1H, d, J = 8.4 Hz),
6.89 (1H, d, J = 1.9 Hz), 6.95 (1H, t, J = 7.0 Hz), 7.01 (1H, t,
J = 6.9 Hz), 7.33 (1H, d, J = 8.0 Hz), 7.44 (1H, d, J = 7.7 Hz), 9.09
(1H, br s), 11.8 (1H, br s); IR (ATR) 3394, 2929, 1703 cmꢀ1; MS
(ESI) m/z 380 (M+H)+, 378 (MꢀH)ꢀ; HRMS (ESI) m/z calcd for
3.1.13. [1-(3-Isopropyl-4-hydroxybenzyl)-2-methyl-1H-indol-3-
yl]acetic acid (4b)
To a solution of 3b (339 mg, 0.7 mmol) in EtOAc (2.5 mL) and
ethanol (2.5 mL) was added 10% Pd-C (135 mg). The mixture was
stirred under hydrogen atmosphere at room temperature for 3 h.
The Pd catalyst was filtered off, and the filtrate was concentrated
under reduced pressure. The residue was purified by flash chroma-
tography on silica gel (hexane/EtOAc = 1/0–4/1) to give a debenzy-
lated intermediate (206 mg; MS (ESI) m/z 394 (M+H)+, 392
(MꢀH)ꢀ), which was then dissolved with methanol (2.0 mL), and
treated with 1 M NaOH (1.1 mL). The mixture was heated at
60 °C for 2 h. After evaporation, the mixture was partitioned be-
tween 1 M hydrochloric acid and EtOAc, and the organic layers
were washed with brine and dried over Na2SO4. The solvent was
evaporated, and the residue was chromatographed on silica gel
and eluted with hexane/EtOAc (1/1) to give 4b (95 mg, 38%, 2
steps) as a foam: 1H NMR (CDCl3) d 1.17 (6H, d, J = 6.9 Hz), 2.31
(3H, s), 3.12 (1H, m), 3.76 (2H, s), 5.21 (2H, s), 6.48 (1H, dd,
J = 1.8, 8.4 Hz), 6.52 (1H, d, J = 8.4 Hz), 6.94 (1H, d, J = 1.8 Hz),
7.07–7.12 (2H, m), 7.21 (1H, m), 7.57 (1H, m); IR (ATR) 3498,
2960, 1698 cmꢀ1; MS (ESI) m/z 338 (M+H)+, 336 (MꢀH)ꢀ; HRMS
(ESI) m/z calcd for C21H24NO3 (M+H)+ 338.1751, found 338.1752.
C
24H30NO3 (M+H)+ 380.2220, found 380.2223.
3.1.18. {[1-(3-Isopropyl-4-hydroxybenzyl)-2-methyl-1H-indol-
3-yl]acetylamino}acetic acid (5b)
To a solution of 4b (985 mg, 2.9 mmol) in DMF (75 mL) were
added Et3N (985 lL, 7.0 mmol), HOBt (492 mg, 3.2 mmol), glycine
ethyl ester HCl (448 mg, 3.2 mmol), and WSC (671 mg, 3.5 mmol)
at room temperature. The mixture was stirred for 48 h and parti-
tioned between water and EtOAc. The organic layers were succes-
sively washed with saturated aqueous NaHCO3, 1 M hydrochloric
acid, water, and brine, and dried over Na2SO4. After evaporation,
the residue was triturated with hexane/Et2O to give an ester inter-
mediate (1.1 g; MS (ESI) m/z 423 (M+H)+, 421 (MꢀH)ꢀ), which was
hydrolyzed in a similar manner as described in the preparation of
4b to give 5b (1.0 g, 87%) as a white solid: mp 117–120 °C; 1H NMR
(DMSO-d6) d 1.10 (6H, d, J = 6.9 Hz), 2.32 (3H, s), 3.13 (1H, m), 3.24
(2H, d, J = 4.0 Hz), 3.51 (2H, s), 5.24 (2H, s), 6.51 (1H, dd, J = 2.2,
8.4 Hz), 6.64 (1H, d, J = 8.0 Hz), 6.91–7.14 (4H, m), 7.35 (1H, d,
J = 8.0 Hz), 7.46 (1H, d, J = 7.7 Hz), 9.32 (1H, br s); IR (ATR) 3302,
2959, 1731, 1651, 1509 cmꢀ1; MS (ESI) m/z 395 (M+H)+, 393
(MꢀH)ꢀ; HRMS (ESI) m/z calcd for C23H27N2O4 (M+H)+ 395.1965,
found 395.1954.
3.1.14. 1-(3-Isopropyl-4-hydroxybenzyl)-2-methyl-1H-indole-3-
carboxylic acid (4a)
The title compound was prepared according to the procedure
described for 4b, using 3a as a white solid (38%, 2 steps): mp
198–200 °C (dec), 1H NMR (DMSO-d6) d 1.19 (6H, d, J = 6.9 Hz),
2.70 (3H, s), 3.12 (1H, m), 5.35 (2H, s), 6.52 (1H, dd, J = 1.8,
8.0 Hz), 6.64 (1H, d, J = 8.0 Hz), 7.01 (1H, d, J = 2.2 Hz), 7.10–7.16
(2H, m), 7.51 (1H, m), 8.00 (1H, m), 9.26 (1H, br s); IR (ATR)
3111, 1698 cmꢀ1; MS (ESI) m/z 324 (M+H)+, 322 (MꢀH)ꢀ; HRMS
(ESI) m/z calcd for C20H22NO3 (M+H)+ 324.1594, found 324.1597.
3.1.19. 2-[1-(4-hydroxy-3-isopropylbenzyl)-2-methyl-1H-
indole-3-carboxamido]acetic acid (5a)
The title compound was prepared according to the procedure
described for 5b, using 4a as a white solid (35%): mp 145–148 °C
(dec); 1H NMR (DMSO-d6) d 1.10 (6H, d, J = 7.0 Hz), 2.32 (3H, s),
3.13 (1H, m), 3.88 (2H, d, J = 5.2 Hz), 5.32 (2H, s), 6.51 (1H, d,
J = 8.0 Hz), 6.65 (1H, d, J = 8.0 Hz), 7.03 (1H, s), 7.07–7.18 (2H, m),
7.48 (1H, d, J = 7.3 Hz), 7.62 (1H, br s), 7.86 (1H, d, J = 7.7 Hz),
9.20 (1H, br s); IR (ATR) 3328, 2958, 1721, 1591, 1543 cmꢀ1; MS
(ESI) m/z 381 (M+H)+, 379 (MꢀH)ꢀ; HRMS (ESI) m/z calcd for
3.1.15. [1-(3-Isopropyl-4-hydroxybenzyl)-2-methyl-1H-indol-3-
yl]propanoic acid (4c)
The title compound was prepared according to the procedure
described for 4b, using 3c as a foam (40%, 2 steps): 1H NMR (CDCl3)
d 1.19 (6H, d, J = 7.0 Hz), 1.22 (3H, t, J = 7.0 Hz), 2.34 (3H, s), 2.66
(2H, t, J = 7.3 Hz), 3.08 (2H, t, J = 7.3 Hz), 3.14 (1H, m), 5.19 (2H,
s), 5.28 (1H, s), 6.43 (1H, d, J = 8.1 Hz), 6.51 (1H, d, J = 8.4 Hz),
6.97 (1H, s), 7.07–7.12 (2H, m), 7.24 (1H, m), 7.57 (1H, d,
J = 6.6 Hz); IR (ATR) 3386, 2965, 1669 cmꢀ1; MS (ESI) m/z 352
(M+H)+, 350 (MꢀH)ꢀ; HRMS (ESI) m/z calcd for C22H26NO3
(M+H)+ 352.1907, found 352.1910.
C
22H25N2O4 (M+H)+ 381.1809, found 381.1812.
3.1.20. 1-(3-Isopropyl-4-triisopropylsilanyloxybenzyl)-4-nitro-
1H-indole (7a)
To a suspension of NaH (60% in oil, 59.2 mg, 1.5 mmol) in
DMF (1.0 mL) was added dropwise a solution of 4-nitro-1H-in-
dole (200 mg, 1.2 mmol) in DMF (1.0 mL) at 5 °C. After stirring
at the same temperature for 1 h, a DMF solution (2.0 mL) of 1b
(ca. 1.5 mmol) was added. The resulting mixture was stirred

N. Takahashi et al. / Bioorg. Med. Chem. 22 (2014) 488–498
495
for an additional 3 h, and the reaction was quenched with ice
3.1.25. 1-(3-Isopropyl-4-triisopropylsilanyloxybenzyl)-2-
water, and then extracted with EtOAc. The organic layers were
successively washed with water and brine, and dried over Na2-
SO4. After evaporation of the solvent, the resulting residue was
purified by flash chromatography on silica gel (hexane/
EtOAc = 9/1) to give the title compound (481 mg, 84%) as a yel-
low solid: mp 98–100 °C; 1H NMR (CDCl3) d 1.08 (18H, d,
J = 7.4 Hz), 1.09 (6H, d, J = 6.6 Hz), 1.27 (3H, m), 3.34 (1H, m),
5.29 (2H, s), 6.67 (1H, s), 7.05 (1H, s), 7.26 (3H, m), 7.38 (1H,
d, J = 6.2 Hz), 7.66 (1H, d, J = 8.4 Hz), 8.13 (1H, d, J = 8.1 Hz);
MS (ESI) m/z 467 (M+H)+; HRMS (ESI) m/z calcd for C27H39N2O3Si
(M+H)+ 467.2724, found 467.2720.
methyl-1H-indol-4-ylamine (8b)
The title compound was prepared according to the procedure
described for 8a, using 7b as a foam (54%): 1H NMR (CDCl3) d
1.09 (18H, d, J = 7.3 Hz), 1.16 (6H, d, J = 7.0 Hz), 1.27 (3H, m),
2.36 (3H, s), 3.33 (1H, m), 3.49 (1H, br s), 5.16 (2H, s), 6.21 (1H,
s), 6.38 (1H, d, J = 7.3 Hz), 6.48 (1H, dd, J = 2.2, 8.1 Hz), 6.59 (1H,
d, J = 8.4 Hz), 6.75 (1H, d, J = 8.1 Hz), 6.94 (1H, t, J = 7.9 Hz), 7.01
(1H, d, J = 1.8 Hz); MS (ESI) m/z 451 (M+H)+; HRMS (ESI) m/z calcd
for C28H43N2OSi (M+H)+ 451.3139, found 451.3134.
3.1.26. 1-(3-Isopropyl-4-triisopropylsilanyloxybenzyl)-7-
methyl-1H-indol-4-ylamine (8c)
3.1.21. 1-(3-Isopropyl-4-triisopropylsilanyloxybenzyl)-2-
methyl-4-nitro-1H-indole (7b)
The title compound was prepared according to the procedure
described for 8a, using 7c as a beige solid (71%): mp 121–123 °C;
1H NMR (CDCl3) d 1.08 (18H, d, J = 7.3 Hz), 1.15 (6H, d, J = 6.9 Hz),
1.26 (3H, m), 2.48 (3H, s), 3.33 (1H, m), 3.81 (2H, br s), 5.47 (2H,
s), 6.31 (1H, d, J = 7.7 Hz), 6.42 (1H, d, J = 2.3 Hz), 6.47 (1H, dd,
J = 2.2, 8.4 Hz), 6.62 (1H, d, J = 8.1 Hz), 6.69 (1H, d, J = 7.7 Hz),
6.94 (1H, d, J = 2.2 Hz), 6.96 (1H, d, J = 2.9 Hz); MS (ESI) m/z 451
(M+H)+; HRMS (ESI) m/z calcd for C28H43N2OSi (M+H)+ 451.3139,
found 451.3136.
The title compound was prepared according to the procedure
described for 7a, using 2-methyl-4-nitro-1H-indole as a yellow so-
lid (45%): mp 116–118 °C; 1H NMR (CDCl3) d 1.07 (18H, d,
J = 7.4 Hz), 1.14 (6H, d, J = 7.0 Hz), 1.26 (3H, m), 2.46 (3H, s), 3.32
(1H, m), 5.29 (2H, s), 6.42 (1H, dd, J = 2.2, 8.4 Hz), 6.62 (1H, d,
J = 8.1 Hz), 6.94 (1H, d, J = 2.2 Hz), 7.08 (1H, s), 7.13 (1H, t,
J = 8.1 Hz), 7.54 (1H, d, J = 8.1 Hz), 8.10 (1H, d, J = 8.0 Hz); MS
(ESI) m/z 481 (M+H)+; HRMS (ESI) m/z calcd for C28H41N2O3Si
(M+H)+ 481.2881, found 481.2875.
3.1.27. 1-(3-Isopropyl-4-triisopropylsilanyloxybenzyl)-2-
methyl-1H-indol-5-ylamine (8d)
3.1.22. 1-(3-Isopropyl-4-triisopropylsilanyloxybenzyl)-7-
methyl-4-nitro-1H-indole (7c)
The title compound was prepared according to the procedure
described for 8a, using 7d as a beige solid (74%): mp 105–106 °C;
1H NMR (CDCl3) d 1.07 (18H, d, J = 7.3 Hz), 1.15 (6H, d, J = 6.9 Hz),
1.25 (3H, m), 2.33 (3H, s), 3.32 (1H, m), 3.46 (1H, br s), 5.15 (2H,
s), 6.12 (1H, s), 6.44 (1H, dd, J = 2.2, 8.4 Hz), 6.55 (1H, dd, J = 2.2,
8.4 Hz), 6.58 (1H, d, J = 8.4 Hz), 6.87 (1H, d, J = 2.2 Hz), 6.98 (1H,
d, J = 2.2 Hz), 7.03 (1H, d, J = 8.7 Hz); MS (ESI) m/z 451 (M+H)+;
HRMS (ESI) m/z calcd for C28H43N2OSi (M+H)+ 451.3139, found
451.3134.
The title compound was prepared according to the procedure
described for 7a, using 7-methyl-4-nitro-1H-indole as a yellow so-
lid (82%): mp 102–104 °C; 1H NMR (CDCl3) d 1.08 (18H, d,
J = 6.9 Hz), 1.13 (6H, d, J = 7.4 Hz), 1.26 (3H, m), 2.63 (3H, s), 3.34
(1H, m), 5.55 (2H, s), 6.42 (1H, dd, J = 2.4, 8.3 Hz), 6.65 (1H, d,
J = 8.3 Hz), 6.86 (1H, d, J = 2.4 Hz), 6.94 (1H, d, J = 8.1 Hz), 7.29
(1H, d, J = 3.1 Hz), 7.33 (1H, d, J = 3.1 Hz), 8.04 (1H, d, J = 8.1 Hz);
MS (ESI) m/z 481 (M+H)+; HRMS (ESI) m/z calcd for C28H41N2O3Si
(M+H)+ 481.2881, found 481.2880.
3.1.28. Ethyl N-[1-(3-isopropyl-4-triisopropylsilanyloxybenzyl)-
1H-indol-4-yl]oxamate (9a)
3.1.23. 1-(3-Isopropyl-4-triisopropylsilanyloxybenzyl)-2-
methyl-5-nitro-1H-indole (7d)
A mixture of 8a (139.0 mg, 0.3 mmol) in diethyl oxalate
(3.0 mL) was stirred at 100 °C for 3 h. The reaction mixture was
cooled to room temperature and concentrated under reduced pres-
sure. The resulting residue was purified by flash chromatography
on silica gel, (hexane/EtOAc = 1/0–4/1) to give the title compound
(126 mg, 74%) as a pale yellow solid: mp 115–116 °C; 1H NMR
(CDCl3) d 1.06 (18H, d, J = 7.3 Hz), 1.13 (3H, m), 1.35 (3H, t,
J = 7.3 Hz), 3.31 (1H, m), 4.33 (2H, q, J = 7.3 Hz), 5.21 (2H, s), 6.51
(1H, d, J = 3.3 Hz), 6.64 (1H, d, J = 8.5 Hz), 6.68 (1H, dd, J = 2.2,
8.4 Hz), 7.01 (1H, d, J = 2.2 Hz), 7.11 (1H, d, J = 3.3 Hz), 7.17 (1H,
d), 7.90 (1H, m), 9.16 (1H, br s); MS (ESI) m/z 537 (M+H)+; HRMS
(ESI) m/z calcd for C31H45N2O4Si (M+H)+ 537.3143, found 537.3152.
The title compound was prepared according to the procedure
described for 7a, using 2-methyl-5-nitro-1H-indole as a yellow so-
lid (34%): mp 118–120 °C; 1H NMR (CDCl3) d 1.07 (18H, d,
J = 7.3 Hz), 1.14 (6H, d, J = 6.9 Hz), 1.26 (3H, m), 2.41 (3H, s), 3.32
(1H, m), 5.26 (2H, s), 6.45 (1H, dd, J = 2.2, 8.5 Hz), 6.48 (1H, s),
6.63 (1H, d, J = 8.0 Hz), 6.92 (1H, d, J = 2.2 Hz), 7.23 (1H, s), 8.02
(1H, dd, J = 2.2, 8.8 Hz), 8.50 (1H, d, J = 2.2 Hz); MS (ESI) m/z 481
(M+H)+; HRMS (ESI) m/z calcd for C28H41N2O3Si (M+H)+
481.2881, found 481.2877.
3.1.24. 1-(3-Isopropyl-4-triisopropylsilanyloxybenzyl)-1H-
indol-4-ylamine (8a)
3.1.29. Ethyl N-[1-(3-isopropyl-4-triisopropylsilanyloxybenzyl)-
2-methyl-1H-indol-4-yl]oxamate (9b)
To a solution of 7a (311 mg, 0.7 mmol) in THF (5.0 mL) and
ethanol (5.0 mL) was added 10% Pd-C (93.3 mg). The mixture
was stirred under hydrogen atmosphere at room temperature
for 3 h. The Pd catalyst was filtered off, and the filtrate was con-
centrated under reduced pressure. The residue was purified by
flash chromatography on silica gel (hexane/EtOAc = 1/0–4/1) to
give the title compound (257 mg, 82%) as a foam: 1H NMR
(CDCl3) d 1.07 (18H, d, J = 7.3 Hz), 1.15 (6H, d, J = 6.9 Hz), 1.24
(3H, m), 3.33 (1H, m), 3.92 (2H, br s), 5.19 (2H, s), 6.39 (1H, d,
J = 7.3 Hz), 6.41 (1H, d, J = 3.3 Hz), 6.64 (1H, d, J = 8.1 Hz), 6.70
(1H, dd, J = 2.2, 8.5 Hz), 6.81 (1H, d, J = 8.0 Hz), 6.98–7.02 (2H,
m), 7.05 (1H, s); MS (ESI) m/z 437 (M+H)+, 435 (MꢀH)ꢀ; HRMS
The title compound was prepared according to the procedure
described for 9a, using 8b as a foam (81%): 1H NMR (CDCl3) d
1.07 (18H, d, J = 7.3 Hz), 1.15 (6H, d, J = 7.3 Hz), 1.26 (3H, m),
1.46 (3H, t, J = 7.1 Hz), 2.40 (3H, s), 3.33 (1H, m), 4.45 (2H, q,
J = 7.1 Hz), 5.23 (2H, s), 6.33 (1H, s), 6.45 (1H, dd, J = 2.4, 8.3 Hz),
6.61 (1H, d, J = 8.3 Hz), 6.96 (1H, d, J = 2.4 Hz), 7.13 (2H, m),
7.86–7.90 (1H, m), 9.13(1H, s); MS (ESI) m/z 551 (M+H)+; HRMS
(ESI) m/z calcd for C32H47N2O4Si (M+H)+ 551.3300, found 551.3301.
3.1.30. Ethyl N-[1-(3-isopropyl-4-triisopropylsilanyloxybenzyl)-
7-methyl-1H-indol-4-yl]oxamate (9c)
(ESI) m/z calcd for
C
27H41N2OSi (M+H)+ 437.2983, found
The title compound was prepared according to the procedure
437.2984.
described for 9a, using 8c as a yellow solid (85%): mp

496
N. Takahashi et al. / Bioorg. Med. Chem. 22 (2014) 488–498
136–138 °C; 1H NMR (CDCl3) d 1.07 (18H, d, J = 7.3 Hz), 1.15 (6H, d,
J = 7.0 Hz), 1.25 (3H, m), 1.46 (3H, t, J = 7.1 Hz), 2.55 (3H, s), 3.32
(1H, m), 4.45 (2H, q, J = 7.1 Hz), 5.23 (2H, s), 6.33 (1H, s), 6.44
(1H, dd, J = 2.2, 8.4 Hz), 6.61 (1H, d, J = 8.1 Hz), 6.96 (1H, d,
J = 2.2 Hz), 7.12–7.15 (2H, m), 7.91 (1H, d, J = 2.2 Hz), 9.14 (1H,
s); MS (ESI) m/z 551 (M+H)+; HRMS (ESI) m/z calcd for C32H47N2O4-
Si (M+H)+ 551.3300, found 551.3301.
381(M+H)+, 379 (MꢀH)ꢀ; HRMS (ESI) m/z calcd for C22H25N2O4
(M+H)+ 381.1809, found 381.1809.
3.1.35. Ethyl N-[1-(4-hydroxy-3-isopropylbenzyl)-2-methyl-1H-
indol-4-yl]oxamate (10b)
The title compound was prepared according to the procedure
described for 10a, using 9b as a yellow solid (23%): mp 143–
145 °C; 1H NMR (CDCl3) d 1.19 (6H, d, J = 6.9 Hz), 1.46 (3H, t,
J = 6.9 Hz), 2.40 (3H, s), 3.15 (1H, m), 4.45 (2H, q, J = 6.9 Hz), 4.66
(1H, s), 5.24 (2H, s), 6.34 (1H, s), 6.50 (1H, dd, J = 2.2, 8.1 Hz),
6.59 (1H, d, J = 8.1 Hz), 6.95 (1H, d, J = 2.2 Hz), 7.09–7.15 (2H, m),
7.87 (1H, dd, J = 1.8, 6.6 Hz), 9.12 (1H, br s); IR (ATR) 3339, 2959,
1687, 1538 cmꢀ1; MS (ESI) m/z 395 (M+H)+, 393 (MꢀH)ꢀ; HRMS
(ESI) m/z calcd for C23H27N2O4 (M+H)+ 395.1965, found 395.1954.
3.1.31. Ethyl N-[1-(3-isopropyl-4-triisopropylsilanyloxybenzyl)-
2-methyl-1H-indol-5-yl]oxamate (9d)
The title compound was prepared according to the procedure
described for 9a, using 8d as a white solid (75%): mp 150–
151 °C; 1H NMR (CDCl3) d 1.07 (18H, d, J = 7.4 Hz), 1.15 (6H, d,
J = 7.0 Hz), 1.26 (3H, m), 1.42 (3H, t, J = 7.1 Hz), 2.52 (3H, s), 3.31
(1H, m), 4.42 (2H, q, J = 7.1 Hz), 5.20 (2H, s), 6.31 (1H, s), 6.43
(1H, dd, J = 2.2, 8.4 Hz), 6.60 (1H, d, J = 8.4 Hz), 6.95 (1H, d,
J = 2.2 Hz), 7.20 (1H, d, J = 8.8 Hz), 7.26 (1H, dd, J = 2.2, 8.8 Hz),
7.91 (1H, d, J = 2.2 Hz), 8.89 (1H, br s); MS (ESI) m/z 551 (M+H)+;
HRMS (ESI) m/z calcd for C32H47N2O4Si (M+H)+ 551.3300, found
551.3303.
3.1.36. Ethyl N-[1-(4-hydroxy-3-isopropylbenzyl)-7-methyl-1H-
indol-4-yl]oxamate (10c)
The title compound was prepared according to the procedure
described for 10a, using 9c as a yellow solid (12%): mp 138–
139 °C; 1H NMR (CDCl3) d 1.19 (6H, d, J = 7.0 Hz), 1.46 (3H, t,
J = 7.1 Hz), 2.55 (3H, s), 3.16 (1H, m), 4.45 (2H, q, J = 7.1 Hz), 4.69
(1H, s), 5.51 (2H, s), 6.48 (1H, dd, J = 2.2, 8.1 Hz), 6.54 (1H, d,
J = 3.3 Hz), 6.62 (1H, d, J = 8.1 Hz), 6.88 (1H, d, J = 2.6 Hz), 6.90
(1H, d, J = 8.4 Hz), 7.08 (1H, d, J = 3.3 Hz), 7.80 (1H, d, J = 7.7 Hz),
9.14 (1H, br s); IR (ATR) 3316, 2958, 1764, 1682, 1541 cmꢀ1; MS
(ESI) m/z 395 (M+H)+, 393 (MꢀH)ꢀ; HRMS (ESI) m/z calcd for
3.1.32. Ethyl N-[1-(3-isopropyl-4-triisopropylsilanyloxybenzyl)-
2-methyl-1H-indol-4-yl]malonamate (9b’)
Ethyl malonyl chloride (248
ture of 8b (829.8 mg, 1.8 mmol) and Et3N (284
l
L, 1.9 mmol) was added to a mix-
L, 2.0 mmol) in
l
CH2Cl2 (40 mL) at 0 °C. The mixture was stirred at room tempera-
ture for 30 min and diluted with EtOAc. The resulting mixture was
washed with water and brine, and dried over Na2SO4. After evapo-
ration of the solvent, the residue was purified by flash chromatog-
raphy on silica gel (hexane/EtOAc = 4/1) to give the title compound
(737.6 mg, 71%) as a foam: 1H NMR (CDCl3) d 1.07 (18H, d,
J = 7.0 Hz), 1.15 (6H, d, J = 6.8 Hz), 1.25 (3H, m), 1.35 (3H, t,
J = 7.1 Hz), 2.39 (3H, s), 3.31 (1H, m), 3.55 (2H, s), 4.31 (2H, q,
J = 7.1 Hz), 5.21 (2H, s), 6.37 (1H, s), 6.45 (1H, d, J = 8.4 Hz), 6.59
(1H, d, J = 8.4 Hz), 6.97 (1H, s), 7.05–7.11 (2H, m), 7.82 (1H, d,
J = 7.3 Hz), 9.66 (1H, s); MS (ESI) m/z 565 (M+H)+; HRMS (ESI) m/
z calcd for C33H49N2O4Si (M+H)+ 565.3456, found 565.3455.
C
23H27N2O4 (M+H)+ 395.1965, found 395.1956.
3.1.37. Ethyl N-[1-(4-hydroxy-3-isopropylbenzyl)-2-methyl-1H-
indol-4-yl]malonamate (10b0)
The title compound was prepared according to the procedure
described for 10a, using 9b as a foam (88%): 1H NMR (CDCl3) d
1.20 (6H, d, J = 7.0 Hz), 1.35 (3H, t, J = 7.4 Hz), 2.39 (3H, s), 3.16
(1H, m), 3.55 (2H, s), 4.30 (2H, q, J = 7.4 Hz), 4.83 (1H, s), 5.22
(2H, s), 6.37 (1H, s), 6.49 (1H, dd, J = 2.2, 8.0 Hz), 6.58 (1H, d,
J = 8.4 Hz), 6.96 (1H, d, J = 2.2 Hz), 7.02–7.11 (2H, m), 7.81 (1H, d,
J = 6.6 Hz), 9.68 (1H, s); IR (ATR) 3312, 2960, 1717, 1659,
1551 cmꢀ1; MS (ESI) m/z 409 (M+H)+, 407 (MꢀH)ꢀ; HRMS (ESI)
m/z calcd for C24H29N2O4 (M+H)+ 409.2122, found 409.2113.
3.1.33. Ethyl N-[1-(3-isopropyl-4-triisopropylsilanyloxybenzyl)-
7-methyl-1H-indol-4-yl]malonamate (9c’)
The title compound was prepared according to the procedure
described for 9b’, using 8c as a white solid (62%): mp 118–
120 °C; 1H NMR (CDCl3) d 1.08 (18H, d, J = 4.9 Hz), 1.14 (6H, d,
J = 7.0 Hz), 1.26 (3H, m), 1.35 (3H, m), 2.53 (3H, s), 3.33 (1H, m),
3.55 (2H, s), 4.30 (2H, q, J = 7.3 Hz), 5.50 (2H, s), 6.42 (1H, dd,
J = 2.2, 8.4 Hz), 6.59–6.62 (2H, m), 6.86 (1H, d, J = 7.6 Hz), 6.91
(1H, d, J = 2.2 Hz), 7.05 (1H, d, J = 3.3 Hz), 7.73 (1H, d, J = 7.7 Hz),
9.69 (1H, s)); MS (ESI) m/z 565 (M+H)+, 563 (MꢀH)ꢀ; HRMS (ESI)
m/z calcd for C33H49N2O4Si (M+H)+ 565.3456, found 565.3455.
3.1.38. Ethyl N-[1-(4-hydroxy-3-isopropylbenzyl)-7-methyl-1H-
indol-4-yl]malonamate (10c0)
The title compound was prepared according to the procedure
described for 10a, using 9c as a white solid (87%): mp 141–
142 °C; 1H NMR (CDCl3) d 1.19 (6H, d, J = 7.0 Hz), 1.34 (3H, t,
J = 7.1 Hz), 2.52 (3H, s), 3.16 (1H, m), 3.55 (2H, s), 4.30 (2H, q,
J = 7.1 Hz), 4.93 (1H, s), 5.49 (2H, s), 6.46 (1H, dd, J = 2.0, 8.2 Hz),
6.59 (1H, d, J = 3.3 Hz), 6.60 (1H, d, J = 8.1 Hz), 6.85 (1H, d,
J = 7.7 Hz), 6.90 (1H, d, J = 1.2 Hz), 7.04 (1H, d, J = 3.3 Hz), 7.72
(1H, d, J = 8.1 Hz), 9.70 (1H, s); IR (ATR) 3262, 2957, 1719, 1652,
1621, 1556 cmꢀ1; MS (ESI) m/z 409 (M+H)+, 407 (MꢀH)ꢀ; HRMS
(ESI) m/z calcd for C24H29N2O4 (M+H)+ 409.2122, found 409.2120.
3.1.34. Ethyl N-[1-(4-hydroxy-3-isopropylbenzyl)-1H-indol-4-
yl]oxamate (10a)
A mixture of 9a (112.0 mg, 0.2 mmol) and tetrabutylammonium
fluoride (1 M in THF, 230
l
L, 0.2 mmol) in THF (1.0 mL) was stirred
3.1.39. Ethyl N-[1-(4-hydroxy-3-isopropylbenzyl)-2-methyl-1H-
indol-5-yl]oxamate (10d)
at room temperature for 30 min. The reaction mixture was then di-
luted with EtOAc, successively washed with water and brine, and
dried over Na2SO4. After evaporation of the solvent, the residue
was purified by flash chromatography on silica gel hexane/
EtOAc = 1/0–4/1) to give the title compound (33.1 mg, 42%) as a
white solid: mp 145–146 °C; 1H NMR (CDCl3) d 1.20 (6H, d,
J = 6.9 Hz), 1.45 (3H, t, J = 7.3 Hz), 3.17 (1H, m), 4.45 (2H, q,
J = 7.3 Hz), 5.24 (2H, s), 6.54 (1H, d, J = 3.3 Hz), 6.65 (1H, d,
J = 4.5 Hz), 6.74 (1H, dd, J = 2.2, 10.2 Hz), 7.04 (1H, d, J = 2.2 Hz),
7.13 (1H, d, J = 3.3 Hz), 7.19–7.20 (2H, m), 7.92 (1H, m), 9.18 (1H,
br s); IR (ATR) 3359, 2960, 1761, 1692, 1536 cmꢀ1; MS (ESI) m/z
The title compound was prepared according to the procedure
described for 10a, using 9c as a pale yellow solid (30%): mp 126–
129 °C; 1H NMR (CDCl3) d 1.18 (6H, d, J = 7.0 Hz), 1.41 (3H, t,
J = 7.0 Hz), 2.35 (3H, s), 3.18 (1H, m), 4.40 (2H, q, J = 7.0 Hz), 5.45
(2H, s), 5.57 (1H, s), 6.28 (1H, s), 6.45 (1H, dd, J = 1.9, 8.1 Hz),
6.61 (1H, d, J = 8.5 Hz), 6.93 (1H, d, J = 1.9 Hz), 7.15 (1H, d,
J = 8.8 Hz), 7.24 (1H, dd, J = 2.2, 9.1 Hz), 7.89 (1H, s), 8.92 (1H, br
s); IR (ATR) 1736, 1683 cmꢀ1; MS (ESI) m/z 395 (M+H)+, 393
(MꢀH)ꢀ; HRMS (ESI) m/z calcd for C23H27N2O4 (M+H)+ 395.1965,
found 395.1959.

N. Takahashi et al. / Bioorg. Med. Chem. 22 (2014) 488–498
497
3.1.40. N-[1-(4-Hydroxy-3-isopropylbenzyl)-1H-indol-4-
yl]oxamic acid (11a)
3.1.45. N-[1-(4-Hydroxy-3-isopropylbenzyl)-2-methyl-1H-indol-
5-yl]oxamic acid (11d)
A mixture of 10a (33.0 mg, 0.1 mmol) and 1 M NaOH (173
l
L,
The title compound was prepared according to the procedure
described for 11a, using 10d as a foam (quant.): 1H NMR (DMSO-
d6) d 1.08 (6H, d, J = 7.0 Hz), 2.35 (3H, s), 3.12 (1H, m), 5.22 (2H,
s), 6.23 (1H, s), 6.52 (1H, dd, J = 1.5, 8.0 Hz), 6.64 (1H, d,
J = 8.0 Hz), 6.94 (1H, d, J = 1.5 Hz), 7.33 (2H, s), 7.88 (1H, s), 9.14
(1H, s), 10.39 (1H, s); MS (ESI) m/z 367 (M+H)+, 365 (MꢀH)ꢀ; HRMS
(ESI) m/z calcd for C21H23N2O4 (M+H)+ 367.1652, found 367.1655.
0.2 mmol) in EtOH (0.5 mL) was stirred at room temperature for
1 h. The reaction mixture was concentrated under reduced pres-
sure, and the residue was taken up with water, acidified with
1 M hydrochloric acid, and extracted with EtOAc. The organic layer
was washed with water and brine, and dried over Na2SO4. After
evaporation of the solvent, the residue was crystallized with
Et2O/hexane to give the title compound (22 mg, quant.) as an off-
white solid: mp 153–155 °C (dec); 1H NMR (DMSO-d6) d 1.11
(6H, d, J = 6.9 Hz), 3.12 (1H, m), 5.27 (2H, s), 6.56 (1H, d,
J = 3.0 Hz), 6.67 (1H, d, J = 8.4 Hz), 6.81 (1H, dd, J = 1.2, 8.4 Hz),
7.09 (1H, t, J = 7.7 Hz), 7.13 (1H, d, J = 1.2 Hz), 7.36 (1H, d,
J = 8.1 Hz), 7.42 (1H, d, J = 7.7 Hz), 7.44 (1H, d, J = 3.0 Hz), 9.27
(1H, s), 10.35 (1H, s); IR (ATR) 1748, 1691 cmꢀ1; MS (ESI) m/z
353 (M+H)+, 351 (MꢀH)ꢀ; HRMS (ESI) m/z calcd for C20H21N2O4
(M+H)+ 353.1496, found 353.1498.
3.2. Biology
3.2.1. TH receptor binding assay
Human TR
in Escherichia coli as a fused protein (recombinant human TR
recombinant human TRb1) having TR 1 and TRb1 ligand binding
a1 and TRb1 ligand binding domains were expressed
a
1 or
a
domains at the C-terminus of His-Patch Thioredoxin using the
His-Patch Thioredoxin fusing protein expressing system (Invitoro-
gen). The fused protein was produced by culturing Escherichia coli
(JM 109) transformed with the prepared plasmid, and adding IPTG
during proliferation to induce expression. 125I-T3 (Perkin Elmer,
Cat. No. NEX110X, AS62450) was added to the binding solution
3.1.41. N-[1-(4-Hydroxy-3-isopropylbenzyl)-2-methyl-1H-indol-
4-yl]oxamic acid (11b)
The title compound was prepared according to the procedure
described for 11a, using 10b as a foam (quant.): 1H NMR (DMSO-
d6) d 1.09 (6H, d, J = 6.9 Hz), 2.37 (3H, s), 3.13 (1H, m), 5.25 (2H,
s), 6.34 (1H, s), 6.53 (1H, dd, J = 2.2, 8.4 Hz), 6.64 (1H, d,
J = 8.4 Hz), 6.98 (1H, d, J = 2.2 Hz), 7.01 (1H, t, J = 7.7 Hz), 7.26
(1H, d, J = 8.1 Hz), 7.36 (1H, d, J = 7.7 Hz), 9.15 (1H, s), 10.19 (1H,
s); IR (ATR) 1741, 1685, 1552 cmꢀ1; MS (ESI) m/z 367 (M+H)+,
365 (MꢀH)ꢀ; HRMS (ESI) m/z calcd for C21H23N2O4 (M+H)+
367.1652, found 367.1656.
(0.16 nM) containing recombinant human TRa1 or recombinant
human TRb1, and the mixture was allowed to stand at room tem-
perature for 1–3 h to form a complex of recombinant human TRb1
or recombinant human TR
(60 L) was then added to all wells of a 96-well plate and supple-
mented with a dilution series solution (90 L), made so that the
a
1 and 125I-T3. The complex solution
l
l
test compound or non-labeled T3 represents 1.67-fold the final
concentration in the binding solution. The plate was incubated at
25 °C for 2–3 h, so that the formation of a complex with 125I-T3
may be inhibited by a test compound or non-labeled T3. Separately,
3.1.42. N-[1-(4-Hydroxy-3-isopropylbenzyl)-7-methyl-1H-indol-
4-yl]oxamic acid (11c)
SephadexG25 (80 lL) was added to each well of a MultiScreen-HV
The title compound was prepared according to the procedure
described for 11a, using 10b as a yellow solid (95%): mp 145–
146 °C (dec); 1H NMR (DMSO-d6) d 1.08 (6H, d, J = 6.6 Hz), 2.46
(3H, s), 3.13 (1H, m), 5.43 (2H, s), 6.40 (1H, dd, J = 2.4, 8.2 Hz),
6.56 (1H, d, J = 3.3 Hz), 6.65 (1H, d, J = 8.1 Hz), 6.78 (1H, d,
J = 8.1 Hz), 6.87 (1H, d, J = 2.2 Hz), 7.30 (1H, d, J = 7.3 Hz), 7.32
(1H, d, J = 3.3 Hz), 9.22 (1H, s), 10.15 (1H, s); IR (ATR) 1752,
1692, 1548 cmꢀ1; MS (ESI) m/z 367 (M+H)+, 365 (MꢀH)ꢀ; HRMS
(ESI) m/z calcd for C21H23N2O4 (M+H)+ 367.1652, found 367.1654.
(MILLIPORE, Cat. No. MANVN4550, Lot. No. F4SN86613) plate and
supplemented with sufficient amount of the binding solution to
form a gel. The mixture was then centrifuged at 600g for 1 min
to prepare a column of SephadexG25. Twenty-five lL of the mixed
solution containing the test compound or non-labeled T3 was
passed through the SephadexG25 column and immediately centri-
fuged at 600g for 1 min. Similarly, 25 lL of the untreated binding
solution was passed through the SephadexG25 column and imme-
diately centrifuged at 600g for 1 min. The separated solution con-
taining 125I-T3 complex and passed through the column twice
was recovered into an Isoplate (PS) (Perkin Elmer, Cat. No. 1450–
3.1.43. N-[1-(4-Hydroxy-3-isopropylbenzyl)-2-methyl-1H-indol-
4-yl]malonic acid (11b0)
514). Optiphase Super Mix (200 lL, Perkin Elmer, Cat. No. 1200–
The title compound was prepared according to the procedure
described for 11a, using 10b’ as a beige solid (quant.): mp 139–
141 °C (dec); 1H NMR (DMSO-d6) d 1.09 (6H, d, J = 7.0 Hz), 2.37
(3H, s), 2.88 (2H, s), 3.12 (1H, m), 5.23 (2H, s), 6.39 (1H, s), 6.49
(1H, dd, J = 2.0, 8.2 Hz), 6.63 (1H, d, J = 8.1 Hz), 6.91 (1H, t,
J = 7.9 Hz), 6.95 (1H, s), 7.03 (1H, d, J = 8.1 Hz), 7.78 (1H, d,
J = 7.7 Hz), 9.14 (1H, s); IR (ATR) 1761, 1692, 1574 cmꢀ1; MS (ESI)
m/z 381 (M+H)+, 379 (MꢀH)ꢀ; HRMS (ESI) m/z calcd for
439) was then added to each well containing the separated solu-
tion, and the radioactivity was measured with 1450 MICROBETA
TRILUX (Perkin Elmer). The amount of each recombinant human
TRa
1 or recombinant human TRb1 complex and 125I-T3 remaining
at each concentration of test compound or T3 (i.e., remaining com-
plex amount) was obtained by conversion of the value acquired by
subtracting the remaining radioactivity from the measured radio-
activity. Binding was calculated by the following equation:Binding
rate = remaining complex amount/total complex amount
Results are reported as mean of the binding rates ꢁ 100 (IC50
values) obtained from four separate experiments. IC50 values were
obtained by linear approximation using the binding rate and a log-
arithmic value of each test compound or T3 concentration, and by
determining the concentration at binding rate = 0.5. For linear
approximation, only concentrations of test compound or T3 with
a binding rate of 0.1–0.9 were used.
C
22H25N2O4 (M+H)+ 381.1809, found 381.1813.
3.1.44. N-[1-(4-Hydroxy-3-isopropylbenzyl)-7-methyl-1H-indol-
4-yl]malonic acid (11c0)
The title compound was prepared according to the procedure
described for 11a, using 10c0 as a white solid (quant.): mp 157–
158 °C (dec); 1H NMR (DMSO-d6) d 1.07 (6H, d, J = 6.6 Hz), 2.46
(3H, s), 3.13 (1H, m), 3.48 (2H, s), 5.48 (2H, s), 6.39 (1H, dd,
J = 2.2, 8.4 Hz), 6.54 (1H, d, J = 2.2 Hz), 6.64 (1H, d, J = 8.4 Hz),
6.82 (1H, s), 6.86 (1H, s), 7.31 (1H, d, J = 2.9 Hz), 7.74 (1H, s), 9.14
(1H, s), 9.68 (1H, s); IR (ATR) 1740, 1609, 1556 cmꢀ1; MS (ESI) m/
z 381 (M+H)+, 379 (MꢀH)ꢀ; HRMS (ESI) m/z calcd for C22H25N2O4
(M+H)+ 381.1809, found 381.1812.
3.2.2. Reporter gene assay
Cells were seeded in 96-well culture plates and grown in Dul-
becco’s Modified Eagle Medium (DMEM, Sigma, Japan) containing
10% FBS (Sigma, Japan) and an appropriate amount of penicillin/

498
N. Takahashi et al. / Bioorg. Med. Chem. 22 (2014) 488–498
3. Pasternak, R. C.; Smith, S. C., Jr.; Bairey-Merz, C. N.; Grundy, S. M.; Cleeman, J. I.;
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5. For recent comprehensive reviews, see: (a) Gessl, A.; Lemmens-Gruber, R.;
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Rev. 2001, 81, 1097.
6. The Coronary Drug Project Research Group. JAMA 1972, 220, 996.
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2010, 21, 499; (b) Webb, P. Curr. Opin. Investig. Drugs 2010, 11, 1135.
8. For reviews, see: (a) Forrest, D.; Vennström, B. Thyroid 2000, 10, 41; (b) O’Shea,
P. J.; Williams, G. R. J. Endocrinol. 2002, 175, 553; (c) Flamant, F.; Samarut, J.
Trends Endocrinol. Metab. 2003, 14, 85.
9. For reviews, see: (a) Webb, P. Expert. Opin. Investig. Drugs 2004, 13, 489; (b)
Pramfalk, C.; Pedrelli, M.; Parini, P. Biochim. Biophys. Acta 2011, 1812, 929.
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Eller, P. Recent Pat. Cardiovasc. Drug Discov. 2011, 6, 16.
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B.; Baxter, J. D. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 663; (b) Ladenson, P. W.;
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Angelin, B. N. Engl. J. Med. 2010, 362, 906.
12. Joharapurkar, A. A.; Dhote, V. V.; Jain, M. R. J. Med. Chem. 2012, 55, 5649.
13. Madrigal Pharmaceuticals website (Fort Washington, PA) (available at: http://
www.madrigalpharma.com/) (2013). See also: Chemical & Engineering News.
2013, 91, 16.
14. (a) Wagner, R. L.; Apriletti, J. W.; McGrath, M. E.; West, B. L.; Baxter, J. D.;
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K.; Kelly, A.; Cunha Lima, S. T.; Scanlan, T. S.; Apriletti, J. W.; Baxter, J. D.; West,
B. L.; Fletterick, R. J. Mol. Endocrinol. 2001, 15, 398; (c) Baxter, J. D.; Dillmann,
W. H.; West, B. L.; Huber, R.; Furlow, J. D.; Fletterick, R. J.; Webb, P.; Apriletti, J.
W.; Scanlan, T. S. J. Steroid Biochem. Mol. Biol. 2001, 76, 31; (d) Ye, L.; Li, Y. L.;
Mellström, K.; Mellin, C.; Bladh, L. G.; Koehler, K.; Garg, N.; Garcia Collazo, A.
M.; Litten, C.; Husman, B.; Persson, K.; Ljunggren, J.; Grover, G.; Sleph, P. G.;
George, R.; Malm, J. J. Med. Chem. 2003, 46, 1580; (e) Borngraeber, S.; Budny, M.
J.; Chiellini, G.; Cunha-Lima, S. T.; Togashi, M.; Webb, P.; Baxter, J. D.; Scanlan,
T. S.; Fletterick, R. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15358; (f) Martínez, L.;
Nascimento, A. S.; Nunes, F. M.; Phillips, K.; Aparicio, R.; Dias, S. M.; Figueira, A.
C.; Lin, J. H.; Nguyen, P.; Apriletti, J. W.; Neves, F. A.; Baxter, J. D.; Webb, P.; Skaf,
M. S.; Polikarpov, I. Ibid. 2009, 106, 20717.
15. (a) Koerner, D.; Schwartz, H. L.; Surks, M. I.; Oppenheimer, J. H. J. Biol. Chem.
1975, 250, 6417; (b) Dietrich, S. W.; Bolger, M. B.; Kollman, P. A.; Jorgensen, E.
C. J. Med. Chem. 1977, 20, 863; (c) Cody, V. Ibid. 1980, 23, 584; (d) Blaney, J. M.;
Jorgensen, E. C.; Connolly, M. L.; Ferrin, T. E.; Langridge, R.; Oatley, S. J.;
Burridge, J. M.; Blake, C. C. Ibid. 1982, 25, 785.
16. (a) Leeson, P. D.; Ellis, D.; Emmett, J. C.; Shah, V. P.; Showell, G. A.; Underwood,
A. H. J. Med. Chem. 1988, 31, 37; (b) Leeson, P. D.; Emmett, J. C.; Shah, V. P.;
Showell, G. A.; Novelli, R.; Prain, H. D.; Benson, M. G.; Ellis, D.; Pearce, N. J.;
Underwood, A. H. Ibid. 1989, 32, 320; (c) Yokoyama, N.; Walker, G. N.; Main, A.
J.; Stanton, J. L.; Morrissey, M. M.; Boehm, C.; Engle, A.; Neubert, A. D.; Wasvary,
J. M.; Stephan, Z. F.; Steele, R. E. Ibid. 1995, 38, 695.
17. Several pioneering works for the inner ring modification have been reported,
see: (a) GarciaCollazo, A. M.; Koehler, K. F.; Garg, N.; Färnegårdh, M.; Husman,
B.; Ye, L.; Ljunggren, J.; Mellström, K.; Sandberg, J.; Grynfarb, M.; Ahola, H.;
Malm, J. Bioorg. Med. Chem. Lett. 2006, 16, 1240; (b) Shiohara, H.; Nakamura, T.;
Kikuchi, N.; Ozawa, T.; Nagano, R.; Matsuzawa, A.; Ohnota, H.; Miyamoto, T.;
Ichikawa, K.; Hashizume, K. Bioorg. Med. Chem. 2012, 20, 3622; (c) Idem. Bioorg.
Med. Chem. 2013, 21, 592.
streptomycin (Sigma, Japan) for 24 h at 37 °C (5% CO2). The follow-
ing day, a plasmid was added to DMEM containing 6% FuGENE, and
the solution was 15 min later diluted with 20-fold volume of
DMEM containing 10% FBS. The cells were then transfected with
the prepared solution and incubated for 24 h at 37 °C (5% CO2).
The supernatant was removed and replaced with a solution of T3
or a test compound in DMEM containing 10% FBS at the required
concentrations, and the cells were incubated for 8–10 h at 37 °C
(5% CO2). SEAP activity was measured by reading p–nitrophenol
levels generated from 4-nitrophenyl phosphate with a Tecan Spec-
tra Fluor Plus reader (Tecan Group Ltd, Männedorf, Switzerland).
The assay mixtures contained 10
lL of the supernatant and
100 L of the medium (1 mM MgCl2, 0.1 M Na2CO3, pH 9.8 buffer,
l
2 mg/mL of p-nitrophenyl phosphate disodium). After a 30 min-
incubation at room temperature, the reaction was stopped with
100 lL of 0.1 N NaOH solution containing 40 mM EDTA, and the
absorbance was measured at 405 nm.
Results are reported as mean of the test compound concentra-
tion that produces half-maximal effect (EC50 values) obtained from
four separate experiments. EC50 values were calculated from max-
imal effects of T3 as 100%, by linear approximation using the activ-
ity rate, and a logarithmic value of each concentration of test
compound or T3, and by determining the concentration at
EC50 = 50%. For linear approximation, only concentrations of test
compound or T3 with a binding rate of 0.1–0.9 were used.
3.2.3. Rat model
Animals care and experimental procedures were approved by
the Animal Experimental Committee of Sanwa Kagaku Kenkyusho
Co., Ltd. Male Sprague Dawley rats (Charles River, Kanagawa, Ja-
pan) were fed a diet containing 1.5% cholesterol and 0.5% cholic
acid (Oriental Yeast, Tokyo, Japan) for 1 week, after which they
were divided into 6 groups to be orally (gavage) treated once daily
for 1 week with 11b0 (30 and 150 mg/kg/day, n = 13), 11c0 (6 and
150 mg/kg/day, n = 13), T3 as a positive control (50 lg/kg/day,
n = 13), or the vehicle (5% tragacanth, Wako, Osaka), or to be left
untreated (control). Cholesterol-feeding was continued throughout
the study. After 3–6 h of the final treatment, blood pressure and
heart rate were measured according to the method of Tail Cuff
et al. Then, the animals were sacrificed, and blood was collected
from the inferior vena cava and analyzed for lipids, TSH, T4, and
T3. Plasma total cholesterol, free cholesterol, LDL-cholesterol, free
fatty acid, and triglyceride levels were measured by enzyme as-
says, and HDL-cholesterol level was directly measured with a
chemical assay kit. TSH, T4, and T3 levels were determined by a
chemiluminescent immunoassay (CLIA).
18. Similarly, importance of the oxyacetic acid side chain in GC-1 was previously
discussed, see: Yoshihara, H. A.; Apriletti, J. W.; Baxter, J. D.; Scanlan, T. S. J.
Med. Chem. 2003, 46, 3152.
3.3. Statistical analysis
19. (a) Johansson, L.; Rudling, M.; Scanlan, T. S.; Lundåsen, T.; Webb, P.; Baxter, J.;
Angelin, B.; Parini, P. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 10297; (b) Tancevski,
I.; Demetz, E.; Eller, P.; Duwensee, K.; Hoefer, J.; Heim, C.; Stanzl, U.; Wehinger,
A.; Auer, K.; Karer, R.; Huber, J.; Schgoer, W.; Van Eck, M.; Vanhoutte, J.; Fievet,
C.; Stellaard, F.; Rudling, M.; Patsch, J. R.; Ritsch, A. PLoS One 2010, 5, e8722. In
the literatures, GC-1 and T-681 were reported to reduce HDL cholesterol in
mice, while 11b0, c0 did not affect HDL. This difference would derive from the
animal models used, because even T3, a reference compound, had no effect on
HDL in our rat model.
Differences between animal groups were analyzed by Student
t-test. P <0.05 was assumed significant. Data are given as the
mean SEM.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.11.001.
20. Grover, G. J.; Egan, D. M.; Sleph, P. G.; Beehler, B. C.; Chiellini, G.; Nguyen, N. H.;
Baxter, J. D.; Scanlan, T. S. Endocrinology 2004, 145, 1656.
21. Trost, S. U.; Swanson, E.; Gloss, B.; Wang-Iverson, D. B.; Zhang, H.; Volodarsky,
T.; Grover, G. J.; Baxter, J. D.; Chiellini, G.; Scanlan, T. S.; Dillmann, W. H.
Endocrinology 2000, 141, 3057.
Reference and notes
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24. Burnell, R. H.; Caron, S. Synth. Commun. 1991, 21, 127.