Synthesis and structure based optimization of 2-(4-phenoxybenzoyl)-5- hydroxyindole as a novel CaMKII inhibitor

Article abstract of DOI:10.1016/j.bmc.2012.09.048

Based on 2-(4-phenoxybenzoyl)-5-hydroxyindole (2), a novel structural class of CaMKII inhibitors were synthesized and further optimized. The strong acidity of the hydroxyl group and the lipophilic group at the 4 and 6-positions were found to be necessary for strong CaMKII inhibition. Compound 25 was identified as a promising compound with 50-fold more potent inhibitory activity for CaMKII than 2. Compound 25 also showed high selectivity for CaMKII over off-target kinases.

Full text of DOI:10.1016/j.bmc.2012.09.048

Bioorganic & Medicinal Chemistry 20 (2012) 6840–6847
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure based optimization of
2-(4-phenoxybenzoyl)-5-hydroxyindole as a novel CaMKII inhibitor
Masafumi Komiya a, , Shigehiro Asano b, Nobuyuki Koike b, Erina Koga b, Junetsu Igarashi a,
⇑
Shogo Nakatani b, Yoshiaki Isobe a
a Research Division, Dainippon Sumitomo Pharma Co., Ltd, Enoki 33-94, Suita, Osaka 564-0053, Japan
b Research Division, Dainippon Sumitomo Pharma Co., Ltd, 3-1-98 Kasugade Naka, Konohana-ku, Osaka 554-0022, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on 2-(4-phenoxybenzoyl)-5-hydroxyindole (2), a novel structural class of CaMKII inhibitors were
synthesized and further optimized. The strong acidity of the hydroxyl group and the lipophilic group
at the 4 and 6-positions were found to be necessary for strong CaMKII inhibition. Compound 25 was iden-
tified as a promising compound with 50-fold more potent inhibitory activity for CaMKII than 2. Com-
pound 25 also showed high selectivity for CaMKII over off-target kinases.
Received 8 August 2012
Revised 23 September 2012
Accepted 24 September 2012
Available online 2 October 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
CaMKII
Indole
Kinase
Inhibitor
Calmodulin
1. Introduction
The CaM-competitive inhibitor KN-9316 and the autocamtide-2-
related inhibitory peptide (AIP)17 are well-known CaMKII inhibi-
Calcium (Ca2+) is an important intracellular messenger, control-
ling a diverse range of cellular processes, such as apoptosis, ion
channel and cell cycle regulation, and cellular response to oxidative
stress.1,2 A rise in intracellular Ca2+ concentration leads to binding
of Ca2+ ions to calmodulin (CaM), which in turn binds to and acti-
vates Ca2+/CaM-dependent protein kinases (CaMKs). CaMKs, which
are ubiquitous serine/threonine kinases classified into three sub-
types (I, II, and IV), modulate many cellular functions in response
to intracellular Ca2+ levels.3,4 CaMKII, a member of CaMKs family,
tors. Recent studies have reported Ca2+/CaM antagonists18 and
CaM non-competitive inhibitors.19 Based on these reports, we con-
sidered CaMKII to be a good target for anti-inflammatory agents.
Our work started with high throughput screening against CaM-
KII. A description of this approach, which led to the discovery of 1
and its subsequent preliminary optimization to 2 (Fig. 1), was re-
cently published.20 In particular, we showed that a hydroxyl group
in the indole moiety is important for CaMKII inhibition. Here we
report in detail the structure–activity relationship (SAR) of this
series compounds.
assembles into a complex of dodecamers with four isoforms (
, and d), having each a subunit composed of three main parts; cat-
alytic, regulatory and association domains.5–7 Upon binding Ca2+
a, b,
c
/
CaM in the presence of ATP/Mg2+, CaMKII undergoes a rapid auto-
phosphorylation at the Thr286/Thr287 located within the autoinhib-
itory domain. The activated CaMKII maintains considerable enzyme
activity even without Ca2+/CaM.8–11 This autophosphorylation has
been reported to cause a dramatic increase in the affinity of the en-
zyme for Ca2+/CaM.12 CaMKII is well known for its modulating ef-
fects on synaptic plasticity and other processes like learning and
memory.13 In addition, CaMKII plays a role in osteoclasts differenti-
ation and bone resorption,14 and active CaMKII is known to enhance
proliferation and cytotoxic activity of T cells.15
O O
N
HO
N
S
OMe
O
Me
Cl
KN-93
O
HN
HN
OH
OH
O
1
2
⇑
Corresponding author. Tel.: +81 6 6337 5815; fax: +81 6 6337 6010.
E-mail address: masafumi-komiya@ds-pharma.co.jp (M. Komiya).
Figure 1. Structures of KN-93, 1 and 2.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.048

M. Komiya et al. / Bioorg. Med. Chem. 20 (2012) 6840–6847
6841
4
5
O
2. Chemistry
4
R1
R1
5
a
N
H
HN
The synthetic routes to the indole compound are shown in
Schemes 1–3. Benzylation of the commercially available 3, fol-
lowed by reaction with diethyl oxalate in the presence of potas-
sium ethoxide and reduction of the nitro group with iron under
acidic conditions provided 6. Reduction of the ester group of 6 with
LiAlH4, followed by oxidation of the formed hydroxyl group with
MnO2 afforded the aldehyde 7. The aldehyde 7 was reacted with
4-lithium diphenylether, which was generated by halogen–lithium
exchange reaction using n-BuLi. Oxidation of the formed hydroxyl
group, followed by deprotection of the benzyl group with BBr3
gave the desired 9 (Scheme 1).
O
1
1
13
14
10
11
R =5-OTBS
R =5-OTBS
1
1
R =5-OMe
R =5-OMe
15 R1=4-OTBS
12 R1=4-OTBS
O
4
R1
b,c
13,15
N
5
O
Boc
1
16
17
R =5-OH
R =4-OH
1
The other indole derivatives 2, 14, 22–25, 32, 34–41 were pre-
pared from the commercially available indoles 10–11 or from the
reported compound 12.21 According to the reported method,22 pro-
tection of NH group in 10–12 using carbon dioxide, followed by
treatment with tert-butyllithium afforded 2-lithium carbanion.
Nucleophilic addition of the carbanion to 4-phenoxybenzaldehyde,
followed by oxidation of the hydroxyl group with MnO2 produced
the 2-acylindole derivatives 13–15. Protection of NH group in 13
and 15 with di-tert-butyl dicarbonate, followed by deprotection
of TBS group afforded compounds 16–17. Halogenation of 16 with
N-bromosuccinimide or N-chlorosuccinimide, followed by depro-
tection of the Boc group with trifluoroacetic acid gave 22–25
(Scheme 2). Reaction of the hydroxyindole derivative 16 with ethyl
bromoacetate or ethyl 2-bromo-2-methylpropanoate, followed by
deprotection of the Boc group and hydrolysis of the ester afforded
32 and 34. Alkylation of 16–17 with 1-bromo-2-chloroethane or
1-bromo-3-chloropropane, followed by nucleophilic addition of
the corresponding amines provided various amine derivatives
(35–40). In the case of 41, compound 16 was reacted with N-(2-
bromoethyl)phthalimide, and the phthalimide moiety, as a protec-
tive group, was removed by methylamine (Scheme 3).
O
O
R2
OH
R3
R2
OH
R3
d
e
16
N
NH
O
O
Boc
2
3
2
3
18
19
20
R =Cl, R =H
R =R =Cl
22
23
24
R =Cl, R =H
2
3
2
3
R =R =Cl
2
3
2
3
R =Br, R =H
R =Br, R =H
21 R2=R3=Br
25 R2=R3=Br
Scheme 2. Reagents and conditions: (a) (i) n-BuLi, CO2(s), THF, À78 °C, 1 h; (ii)
t-BuLi; (iii) 4-phenoxybenzaldehyde, À78 °C to rt, 2 h; (iv) MnO2, acetone, rt,
overnight, 13:83%, 14:56%, 15:75%; (b) Boc2O, THF, rt, 1 h; (c) tetrabutylammonium
fluoride, THF, rt, 1 h, 16:97%, 17:56% in two steps; (d) N-chlorosuccinimide or
N-bromosuccinimide, THF, 0 °C to rt, 1 h, 18:33%, 19:23%, 20:43%, 21:29%; (e)
trifluoroacetic acid, CHCl3, rt, 1 h, 22:75%, 23:74%, 24:67%, 25:98%.
The inhibitory activity against CaMKII of the synthesized com-
pounds is summarized in Table 1. First, we examined the effect
of a substituent on the indole at the 4-position. Introduction of a
methyl group, as an electron-donating group, resulted in twofold
drop in the activity, while
a chloro group, as an electron-
withdrawing group, showed an activity comparable to that of the
unsubstituted 2, suggesting that substitution at the 4-position is
tolerated. Although a methyl and chloro groups are considered to
be of similar molecular size, they exert mutually opposite elec-
tronic effects. This led us to hypothesize that a hydroxyl group is
related to inhibitory activity against CaMKII. Based on this hypoth-
esis, we focused on the acidity of the hydroxyl group in the indole
moiety. We first prepared the di-chloro compound 23 to increase
the acidity of the hydroxyl group (calculated pKa value of the
hydroxyl group in 23 decreased over one log-fold compared to
22). Compound 23 inhibited CaMKII with an IC50 value of 61 nM,
which was 10-fold more potent than that of 22. Next we replaced
the chloro group in 22 with a bromo one. Surprisingly, the mono-
bromo compound 24 showed potent inhibitory activity against
CaMKII with an IC50 value of 40 nM, which was 15-fold more
potent than that of the mono-chloro compound 22. However, the
calculated pKa value of the hydroxyl group was similar. The most
potent inhibition of CaMKII was seen with the di-bromo compound
25 (IC50 = 12 nM). As the calculated pKa values of 23 and 25 were
similar (data not shown), we guessed that a strong acidic proton
might be important for interaction with CaMKII protein and that
lipophilic groups should be placed at the 4 and 6-positions. As ref-
erence, we prepared the methoxy 14, as a no-acidic proton com-
pound. Compound 14 showed complete loss of inhibitory activity
against CaMKII, indicating an acidic proton is essential for strong
CaMKII inhibitory activity. Next, we introduced an acidic proton
via a carboxylate at the 5-position. The inhibitory activity of com-
pound 32 was equal to that of the lead compound 2, while that of
compound 34 was moderate. These results indicated that various
acidic substituents can be acceptable at the 5-position. Interest-
ingly, the ethylaminoethoxy compound 35 also showed moderate
3. Results and discussion
Although compound 2 inhibited CaMKII with an IC50 value of
0.61 lM, this inhibitory activity was not considered high enough.
In our last paper, a 4-phenoxybenzoyl group was identified as
the best substituent at the 2-position of the indole in 2, while a hy-
droxyl group at the 5-position was considered as important for
CaMKII inhibitory activity. To improve compound 2 inhibitory
activity for CaMKII, we designed further structural modifications,
in particular, modification of the substituent around the hydroxyl
group.
Me
O
Me
Me
BnO
Me
HO
Me
BnO
Me
a
b
OEt
O
NO2
NO2
NO2
5
3
4
Me
BnO
BnO
d,e
c
CHO
CO2Et
N
H
N
H
6
7
O
Me
f,g
R1
HN
O
1
8
9
R =OBn
h
1
R =OH
Scheme 1. Reagents and conditions: (a) BnCl, K2CO3, DMF, 100 °C, 2 h, 88%; (b)
(CO2Et)2, KOEt, Et2O–EtOH, rt, overnight, 36%; (c) Fe, AcOH, EtOH, reflux, 3 h, 75%;
(d) LiAlH4, THF, 0 °C, 1 h; (e) MnO2, acetone, rt, overnight, 77% in two steps;
(f) 4-bromodiphenylether, n-BuLi, THF, -20 °C, 2 h; (g) MnO2, acetone, rt, 2 h, 38% in
two steps; (h) BBr3, CHCl3, rt, 1 h, 43%.

6842
M. Komiya et al. / Bioorg. Med. Chem. 20 (2012) 6840–6847
O
O
4
4
R1
5
a
R1
5
b or c
16,17
N
NH
O
O
Boc
1
1
26
27
31
32
R =5-OCH2CO2Et
R =5-OCH2CO2Et
d
1
1
R =5-OC(Me)2CO2Et
R =5-OCH2CO2H
28 R1=5-O(CH2)2Cl
29 R1=4-O(CH2)2Cl
30 R1=4-O(CH2)3Cl
33 R1=5-OC(Me)2CO2Et
d
34 R1=5-OC(Me)2CO2H
1
35
36
37
R =5-O(CH2)2NHEt
1
R =4-O(CH2)2NHEt
1
R =4-O(CH2)2(piperidyl)
38 R1=4-O(CH2)3(piperidyl)
39 R1=4-O(CH2)2(piperazyl)
1
40
R =4-O(CH2)2(4'-hydroxypiperidyl)
O
NH2
O
e,f
16
NH
O
41
Scheme 3. Reagents and conditions: (a) alkyl bromide, K2CO3, DMF, 40 °C or 100 °C, 2 h, 26:72%, 27:79%, 28:47%, 29:83%, 30:69%; (b) trifluoroacetic acid, CHCl3, rt, 1 h,
31:88%, 33:95%; (c) amines, NaI, DMF, 100 °C , 6 h, 35:26%, 36:37%, 37:80%, 38:64%, 39:69%, 40:89%; (d) 1 M NaOH aq, THF, MeOH, rt, 2 h, 32:67%, 34:48%; (e) N-(2-
bromoethyl)phthalimide, K2CO3, NaI, DMF, 100 °C , 3 h; (f) MeNH2, MeOH, 50 °C, 1 h, 52% in two steps.
Table 1
Table 2
Inhibitory activity of indole derivatives against CaMKII
Selected compounds inhibitory activity against CaMKII and their selectivity for
CaMKII over other kinases
O
Compd
CaMKII IC50
(l
M)
Selectivity (fold)
4 R2
P38
a
MLCK
Akt1
PKCc
5 R1
HN
O
6
2
24
25
0.61
0.040
0.012
<1
115
450
<1
12
63
295
1500
>5000
93
R3
550
1250
41 decreased the inhibitory activity for CaMKII, while a tertiary
amine as in compounds 37 and 38, slightly improved the activity.
CaMKII inhibitory activity was improved in the piperazine 39 and
hydroxypiperidine 40 compared to 36, suggesting that a bipolar
group is beneficial for the activity. However, these amine com-
pounds showed cytotoxicity in mouse bone marrow cells, forcing
us to stop further examination of this series of compounds.
Based on the above structure–activity relationships, we focused
on compounds 2, 24 and 25, and further examined their selectivity
for CaMKII. As shown in Table 2, compound 2 exhibited no higher
Compd
R1
R2
R3
CaMKII IC50 (lM)
2
9
OH
OH
OH
OH
OH
OH
OMe
OCH2CO2H
OC(Me)2CO2H
O(CH2)2NHEt
H
H
H
H
H
H
Cl
H
Br
H
H
H
H
H
H
0.61
1.3
Me
Cl
Cl
Br
Br
H
H
H
H
22
23
24
25
14
32
34
35
36
41
0.64
0.061
0.040
0.012
>10
0.51
1.5
1.5
1.1
2.6
O(CH2)2NHEt
O(CH2)2NH2
selectivity for CaMKII over p38a and MLCK, whereas compound 24
and 25 showed good selectivity for CaMKII over all the other se-
lected kinases. In particular, compound 25 was considered as a
promising compound considering the side-effects associated with
O
N
37
38
39
H
H
H
H
H
H
0.82
0.80
0.27
interaction with p38a and MLCK.
O
O
N
N
N
4. Conclusion
NH
O
40
H
H
0.42
Based on our previous work, we carried out an optimization
study to find strong and selective CaMKII inhibitors. This optimiza-
tion focused on acidity of the acid proton in the indole. Introduc-
tion of a halogen atom at the 4 and 6-positions of the indole was
effective in improving both CaMKII inhibitory activity and kinase
selectivity. In particular, the di-bromo compound 25 showed
strong CaMKII inhibitory activity with an IC50 value of 12 nM as
well as good selectivity for this kinase.
OH
n.d. = Not determined.
inhibitory activity for CaMKII (IC50 = 1.5 lM), suggesting that this
compound inhibits CaMKII by interacting with other amino acids
in CaMKII. The solubility of the above halogen-atom substituted
compounds 23–25 was poor (<0.001 mg/mL at pH 7.4), indicating
that further modification is necessary to identify orally available
drug candidates. Generally, an amino group is useful to improve
compounds water solubility. To confirm this, we carried out a pre-
liminary examination. As the inhibitory activity of 36, containing
an ethylaminoethoxy at the 4-position, was slightly stronger than
that of 35, the 4-position was chosen for further modification. A
primary amine with the same ethylene chain length as compound
5. Experimental
5.1. Chemistry
Melting points (Mp) were determined on an electrothermal
apparatus without correction. IR spectra were recorded on a JEOL
JIR-SPX60 spectrometer as ATR. NMR spectra were recorded on a

M. Komiya et al. / Bioorg. Med. Chem. 20 (2012) 6840–6847
6843
JEOL JNM-LA300 spectrometer. Chemical shifts (d) are given in
parts per million, and TMS was used as the internal standard for
spectra obtained in DMSO-d6 and CDCl3. All J values are given in
Hz. Mass spectra were recorded on a Waters ACQUITY UPLC/MS
system. Elemental analysis was performed on a CE Instrument
EA1110 and a Yokokawa analytical system IC7000. Reagents and
solvents were used as obtained from commercial suppliers without
further purification. Column chromatography was carried out
using a Yamazen W-prep system, and performed using prepacked
silica gel. Reaction progress was determined by TLC analysis on sil-
ica gel coated glass plate. Visualization was done with UV light
(254 nm). All reactions were carried out under a nitrogen atmo-
sphere unless otherwise mentioned.
chromatography on silica gel (EtOAc/hexane) to provide compound
7 (66.0 mg, 77%) as a yellow solid.
1H NMR (CDCl3, 300 MHz) d 2.50 (3H, s), 5.10 (2H, s), 7.15–7.48
(8H, m), 8.90 (1H, br s), 9.83 (1H, s); MS (ESI) m/z 266 (M+1).
5.1.5. (5-(Benzyloxy)-4-methyl-1H-indol-2-yl)(4-phenoxy-
phenyl)methanone (8)
A solution of 4-bromodiphenylether (375 mg, 1.50 mmol) in
THF (1.0 ml) was cooled to À20 °C and n-BuLi in hexane (2.64 M,
0.571 ml, 1.50 mmol) was added. The mixture was stirred at
À20 °C for 1 h and a solution of 7 (50.0 mg, 0.188 mmol) in THF
(0.5 ml) was added. The mixture was allowed to rise to room tem-
perature for 1 h and then quenched with saturated NH4Cl solution.
The aqueous layer was extracted with EtOAc and the combined
organic layers were washed with brine. The organic fraction was
dried over MgSO4 and the solvent was evaporated under reduced
pressure. The residue was purified by flash chromatography on
silica gel (EtOAc/hexane) to provide secondary alcohol intermedi-
ate. The intermediate was dissolved in acetone (1.0 ml) and
MnO2 (49.0 mg, 0.564 mmol) was added. The mixture was stirred
at room temperature for 2 h and filtered through celite. The filtrate
was evaporated under reduced pressure. The residue was purified
by flash chromatography on silica gel (EtOAc/hexane) to provide
compound 8 (30.9 mg, 38%) as a yellow solid.
5.1.1. 1-(Benzyloxy)-2,3-dimethyl-4-nitrobenzene (4)
A mixture of 3 (1.67 g, 10.0 mmol), K2CO3 (2.07 g, 15.0 mmol)
and BnCl (1.39 g, 11.0 mmol) in DMF (17 ml) was stirred at
100 °C for 2 h. The mixture was cooled and diluted with EtOAc,
then washed with water and brine. The organic fraction was dried
over MgSO4 and the solvent was evaporated under reduced pres-
sure. The residue was purified by flash chromatography on silica
gel (EtOAc/hexane) to provide compound 4 (2.26 g, 88%) as a yel-
low solid.
1H NMR (CDCl3, 300 MHz) d 2.28 (3H, s), 2.46 (3H, s), 5.15 (2H,
s), 6.80 (1H, d, J = 9.0 Hz), 7.32–7.44 (5H, m), 7.76 (1H, d,
J = 9.0 Hz); MS (ESI) m/z 258 (M+1).
1H NMR (CDCl3, 300 MHz) d 2.48 (3H, s), 5.06 (2H, s), 7.08–7.49
(15H, m), 7.99–8.03 (2H, m), 9.14 (1H, br s); MS (ESI) m/z 434
(M+1).
5.1.2. Ethyl 3-(3-(benzyloxy)-2-methyl-6-nitrophenyl)-2-
oxopropanoate (5)
5.1.6. (5-Hydroxy-4-methyl-1H-indol-2-yl)(4-phenoxy
phenyl)methanone (9)
To a solution of 4 (1.86 g, 7.24 mmol) in Et2O (3.7 ml) and EtOH
(37 ml) was added diethyl oxalate (2.12 g, 14.5 mmol) and potas-
sium ethoxide (670 mg, 7.96 mmol). The mixture was stirred at
room temperature overnight and then quenched with saturated
NH4Cl solution. The aqueous layer was extracted with EtOAc and
the combined organic layers were washed with brine. The organic
fraction was dried over MgSO4 and the solvent was evaporated un-
der reduced pressure. The residue was purified by flash chromatog-
To a solution of 8 (18.0 mg, 0.042 mmol) in CHCl3 (1.0 ml) at
0 °C was added BBr3 in CH2Cl2 (1.0 M, 0.5 ml, 0.500 mmol). The
mixture was stirred at room temperature for 1 h and then
quenched with saturated NaHCO3 solution. The aqueous layer
was extracted with EtOAc and the combined organic layers were
washed with brine. The organic fraction was dried over MgSO4
and the solvent was evaporated under reduced pressure. The resi-
due was purified by flash chromatography on silica gel (EtOAc/hex-
ane) to provide compound 9 (6.0 mg, 43%) as a white solid.
Mp = 143–144 °C. 1H NMR (CDCl3, 300 MHz) d 2.44 (3H, s), 4.58
(1H, s), 6.95 (1H, d, J = 8.8 Hz), 7.07–7.14 (5H, m), 7.18–7.26 (2H,
m), 7.40–7.45 (2H, m), 8.01 (2H, m), 9.16 (1H, br s); 13C NMR
(DMSO-d6, 75 MHz) d 11.9, 109.6, 110.2, 113.7, 117.2, 120.1,
124.8, 128.7, 130.4, 131.5, 132.8, 134.1, 148.1, 155.2, 160.6,
184.8; IR (ATR) 3292, 1622 cmÀ1; MS (ESI) m/z 344 (M+1); Anal.
Calcd for C22H17NO30.75H2O: C, 74.04; H, 5.22; N, 3.92. Found: C,
74.09; H, 5.17; N, 3.80.
raphy on silica gel (EtOAc/hexane) to provide compound
(933 mg, 36%) as a yellow oil.
5
1H NMR (CDCl3, 300 MHz) d 1.42 (3H, t, J = 7.2 Hz), 2.26 (3H, s),
4.41 (2H, q, J = 7.2 Hz), 4.52 (2H, s), 5.18 (2H, s), 6.94 (1H, d,
J = 9.2 Hz), 7.34–7.45 (5H, m), 8.01 (1H, d, J = 9.2 Hz); MS (ESI)
m/z 358 (M+1).
5.1.3. Ethyl 5-(benzyloxy)-4-methyl-1H-indole-2-carboxylate
(6)
A mixture of 5 (850 mg, 2.39 mmol), AcOH (287 mg, 4.78 mmol)
and Fe (664 mg, 12.0 mmol) in EtOH (9.0 ml) was stirred under re-
flux for 3 h. The mixture was cooled and filtered through celite. The
filtrate was evaporated under reduced pressure. The residue was
purified by flash chromatography on silica gel (EtOAc/hexane) to
provide compound 6 (553 mg, 75%) as a white solid.
1H NMR (CDCl3, 300 MHz) d 1.42 (3H, t, J = 7.2 Hz), 2.47 (3H, s),
4.40 (2H, q, J = 7.2 Hz), 5.08 (2H, s), 7.08 (1H, m), 7.18–7.48 (7H, m),
8.85 (1H, br s); MS (ESI) m/z 310 (M+1).
5.1.7. (5-(tert-Butyldimethylsilyloxy)-1H-indol-2-yl)(4-phenoxy
phenyl)methanone (13)
According to a literature procedure, a solution of 10 (4.95 g,
20.0 mmol) in THF (100 ml) was cooled to À78 °C and n-BuLi in
hexane (2.64 M, 9.09 ml, 24.0 mmol) was added. The mixture
was stirred at À78 °C for 1 h and then excess of carbon dioxide
(dry ice, 10 g) was added. The solution was allowed to rise to room
temperature for 1 h. The mixture was diluted with EtOAc and
washed with water and brine. The organic fraction was dried over
MgSO4 and the solvent was evaporated under reduced pressure.
The residue was dissolved in THF (100 ml) and the solution was
cooled to À78 °C. t-BuLi in hexane (1.57 M, 14.0 ml, 22.0 mmol)
was added and stirred at À78 °C for 1 h, and then a solution of 4-
phenoxybenzaldehyde (4.36 g, 22.0 mmol) in THF (5.0 ml) was
added at À78 °C. The solution was allowed to rise to room temper-
ature for 1 h and then quenched with saturated NH4Cl solution.
The aqueous layer was extracted with EtOAc and the combined or-
ganic layers were washed with brine. The organic fraction was
5.1.4. 5-(Benzyloxy)-4-methyl-1H-indole-2-carbaldehyde (7)
To a solution of LiAlH4 (36.8 mg, 0.969 mmol) in THF (2.0 ml) at
0 °C was added a solution of 6 (100 mg, 0.323 mmol) in THF
(0.5 ml). The mixture was stirred at 0 °C for 1 h and then quenched
with water and 1 N NaOH solution. The mixture was filtered
through celite. The filtrate was evaporated under reduced pressure.
The residue was dissolved in acetone (2.0 ml) and MnO2 (84.3 mg,
0.969 mmol) was added. The mixture was stirred at room temper-
ature overnight and filtered through celite. The filtrate was evapo-
rated under reduced pressure. The residue was purified by flash

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M. Komiya et al. / Bioorg. Med. Chem. 20 (2012) 6840–6847
dried over MgSO4 and the solvent was evaporated under reduced
pressure. The residue was purified by flash chromatography on
silica gel (EtOAc/hexane) to provide secondary alcohol intermedi-
ate. The intermediate was dissolved in acetone (100 ml) and
MnO2 (7.00 g, 60.0 mmol) was added. The mixture was stirred at
room temperature overnight and filtered through celite. The
filtrate was evaporated under reduced pressure. The residue was
purified by flash chromatography on silica gel (EtOAc/hexane) to
provide compound 13 (7.40 g, 83%) as a white solid.
pressure. The residue was purified by flash chromatography on
silica gel (EtOAc/hexane) to provide compound 18 (25.0 mg, 33%)
as a white solid.
1H NMR (CDCl3, 300 MHz) d 1.40 (9H, s), 5.50 (1H, s), 6.88 (1H,
s), 7.01–7.26 (6H, m), 7.39–7.45 (2H, m), 7.88–7.93 (2H, m), 8.02
(1H, m); MS (ESI) m/z 464 (M+1).
5.1.13. tert-Butyl 4,6-dichloro-5-hydroxy-2-(4-phenoxy
benzoyl)-1H-indole-1-carboxylate (19)
1H NMR (CDCl3, 300 MHz) d 0.18 (6H, s), 1.01 (9H, s), 6.91–7.13
(6H, m), 7.19–7.45 (5H, m), 7.98–8.03 (2H, m), 9.24 (1H, br s); MS
(ESI) m/z 444 (M+1).
To a solution of 16 (100 mg, 0.233 mmol) in THF (1.6 ml) and
i-PrOH (0.4 ml) was added N-chlorosuccinimide (78.0 mg,
0.583 mmol). The mixture was stirred at room temperature for
1 h and the solvent was evaporated under reduced pressure. The
residue was purified by flash chromatography on silica gel
(EtOAc/hexane) to provide compound 19 (23.0 mg, 23%) as a white
solid.
5.1.8. (5-Methoxy-1H-indol-2-yl)(4-phenoxyphenyl) methanone
(14)
Compound 14 was prepared from 11 using the procedure for 13
with 56% as a white solid.
1H NMR (CDCl3, 300 MHz) d 1.39 (9H, s), 5.82 (1H, s), 6.87 (1H,
s), 7.01–7.11 (4H, m), 7.20–7.26 (1H, m), 7.38–7.45 (2H, m), 7.88–
7.92 (2H, m), 8.21 (1H, s); MS (ESI) m/z 498 (M+1).
Mp = 155–156 °C. 1H NMR (CDCl3, 300 MHz) d 3.86 (3H, s),
7.03–7.14 (7H, m), 7.20–7.26 (1H, m), 7.37–7.45 (3H, m), 8.00
(2H, m), 9.30 (1H, br s); 13C NMR (CDCl3, 75 MHz) d 55.7, 102.7,
111.7, 113.1, 117.4, 118.2, 120.1, 124.5, 128.0, 130.1, 131.5,
132.4, 132.8, 134.8, 154.8, 155.6, 161.5, 185.6; IR (ATR) 3280,
1618 cmÀ1; MS (ESI) m/z 344 (M+1); Anal. Calcd for C22H17NO3:
C, 76.95; H, 4.99; N, 4.08. Found: C, 77.00; H, 4.93; N, 4.10.
5.1.14. tert-Butyl 4-bromo-5-hydroxy-2-(4-phenoxybenzoyl)-
1H-indole-1-carboxylate (20) and tert-Butyl 4,6-dibromo-5-
hydroxy-2-(4-phenoxybenzoyl)-1H-indole-1-carboxylate (21)
To an ice cooled solution of 16 (4.30 g, 10.0 mmol) in THF
(50 ml) was added N-bromosuccinimide (1.82 g, 10.2 mmol). The
mixture was stirred at room temperature for 1 h and the solvent
was evaporated under reduced pressure. The residue was purified
by flash chromatography on silica gel (EtOAc/hexane) to provide
compound 20 (2.37 g, 43%) as a white solid and compound 21
(1.72 g, 29%) as a white solid.
5.1.9. (4-(tert-Butyldimethylsilyloxy)-1H-indol-2-yl)(4-phenoxy
phenyl)methanone (15)
Compound 15 was prepared from 12 using the procedure for 13
with 75% as a yellow solid.
1H NMR (DMSO-d6, 300 MHz) d 0.21 (6H, s), 0.98 (9H, s), 6.50
(1H, m), 6.96 (1H, s), 7.09–7.29 (7H, m), 7.46–7.51 (2H, m), 7.95
(2H, m), 11.99 (1H, br s); MS (ESI) m/z 444 (M+1).
Compound 20: 1H NMR (CDCl3, 300 MHz) d 1.39 (9H, s), 5.44
(1H, s), 6.83 (1H, s), 7.01–7.26 (6H, m), 7.42–7.46 (2H, m), 7.88–
7.93 (2H, m), 8.04 (1H, m); MS (ESI) m/z 510 (M+1).
5.1.10. tert-Butyl 5-hydroxy-2-(4-phenoxybenzoyl)-1H-indole-
1-carboxylate (16)
Compound 21: 1H NMR (CDCl3, 300 MHz) d 1.38 (9H, s), 5.83
(1H, s), 6.82 (1H, s), 7.01–7.11 (4H, m), 7.20–7.45 (3H, m), 7.86–
7.91 (2H, m), 8.41 (1H, m); MS (ESI) m/z 587 (M+1).
To a solution of 13 (6.40 g, 14.4 mmol) in CH2Cl2 (72 ml) was
added Boc2O (3.46 g, 15.8 mmol) and DMAP (2.10 g, 17.3 mmol).
The mixture was stirred at room temperature for 1 h and diluted
with CHCl3, then washed with water and brine. The organic frac-
tion was dried over MgSO4 and the solvent was evaporated under
reduced pressure. The residue was dissolved in THF (72 ml) and
TBAF in THF (1.0 M, 14.4 ml, 14.4 mmol) was added. The mixture
was stirred at room temperature for 1 h and diluted with EtOAc,
and then washed with water and brine. The organic fraction was
dried over MgSO4 and the solvent was evaporated under reduced
pressure. The residue was purified by flash chromatography on
silica gel (EtOAc/hexane) to provide compound 16 (6.02 g, 97%)
as a white solid.
5.1.15. (4-Chloro-5-hydroxy-1H-indol-2-yl)(4-phenoxy
phenyl)methanone (22)
To a solution of 18 (17.0 mg, 0.037 mmol) in CHCl3 (1.0 ml) was
added trifluoroacetic acid (1.0 ml). The mixture was stirred at room
temperature for 1 h and the solvent was evaporated under reduced
pressure. The residue was purified by flash chromatography on sil-
ica gel (EtOAc/hexane) to provide compound 22 (10.0 mg, 75%) as a
white solid.
Mp = 173–174 °C. 1H NMR (CDCl3, 300 MHz) d 5.39 (1H, s),
7.15–7.18 (6H, m), 7.20–7.33 (2H, m), 7.40–7.46 (2H, m), 8.02
(2H, m), 9.33 (1H, br s); 13C NMR (CDCl3, 75 MHz) d 109.1,
111.9, 117.0, 117.4, 120.3, 124.7, 126.7, 130.1, 131.5, 131.9,
132.6, 135.2, 146.1, 155.4, 161.9, 185.4; IR (ATR) 3282,
1H NMR (CDCl3, 300 MHz) d 1.39 (9H, s), 4.90 (1H, s), 6.77 (1H,
s), 6.94–7.10 (6H, m), 7.18–7.44 (3H, m), 7.87–7.92 (2H, m), 8.05
(1H, m); MS (ESI) m/z 430 (M+1).
1624 cmÀ1
;
MS (ESI) m/z 364 (M+1); Anal. Calcd for
C
21H14ClNO30Á3H2O: C, 68.32; H, 3.99; N, 3.79. Found: C,
5.1.11. tert-Butyl 4-hydroxy-2-(4-phenoxybenzoyl)-1H-indole-
1-carboxylate (17)
68.66; H, 4.38; N, 3.77.
Compound 17 was prepared from 15 using the procedure for 16
with 56% as a white solid.
5.1.16. (4,6-Dichloro-5-hydroxy-1H-indol-2-yl)(4-phenoxy
phenyl)methanone (23)
Compound 23 was prepared from 19 using the procedure for 22
with 74% as a white solid.
1H NMR (DMSO-d6, 300 MHz) d 1.34 (9H, s), 6.67 (1H, m), 7.09–
7.29 (7H, m), 7.44–7.49 (3H, m), 7.88 (2H, m), 10.13 (1H, br s); MS
(ESI) m/z 430 (M+1).
Mp = 206–207 °C. 1H NMR (CDCl3, 300 MHz) d 5.69 (1H, s),
7.08–7.15 (5H, m), 7.20–7.26 (1H, m), 7.40–7.46 (3H, m), 8.01
(2H, m), 9.30 (1H, s); 13C NMR (CDCl3, 75 MHz) d 108.2, 112.1,
117.3, 120.1, 123.1, 124.8, 125.5, 130.4, 131.6, 131.8, 132.0,
135.5, 143.0, 155.0, 161.0, 184.6; IR (ATR) 3334, 1618 cmÀ1; MS
(ESI) m/z 398 (M+1); Anal. Calcd for C21H13Cl2NO3: C, 63.34; H,
3.29; N, 3.52. Found: C, 63.02; H, 3.36; N, 3.60.
5.1.12. tert-Butyl 4-chloro-5-hydroxy-2-(4-phenoxybenzoyl)-
1H-indole-1-carboxylate (18)
To an ice cooled solution of 16 (70.0 mg, 0.163 mmol) in THF
(1.2 ml) and i-PrOH (0.3 ml) was added N-chlorosuccinimide
(17.4 mg, 0.130 mmol). The mixture was stirred at room tempera-
ture for 1 h and the solvent was evaporated under reduced

M. Komiya et al. / Bioorg. Med. Chem. 20 (2012) 6840–6847
6845
5.1.17. (4-Bromo-5-hydroxy-1H-indol-2-yl)(4-phenoxy
phenyl)methanone (24)
Compound 24 was prepared from 20 using the procedure for 22
with 67% as a yellow solid.
1H NMR (DMSO-d6, 300 MHz) d 1.35 (9H, s), 3.99(2H, m), 4.41 (2H,
m), 6.90 (1H, m), 7.00 (1H, m), 7.08–7.16 (4H, m), 7.28 (1H, m), 7.41–
7.47 (3H, m), 7.66 (1H, m), 7.88 (2H, m); MS (ESI) m/z 492 (M+1).
5.1.23. tert-Butyl 4-(3-chloropropoxy)-2-(4-phenoxybenzoyl)-
1H-indole-1-carboxylate (30)
Mp = 192–193 °C. 1H NMR (CDCl3, 300 MHz) d 5.38 (1H, s), 7.04
(1H, m), 7.10–7.15 (5H, m), 7.20–7.26 (1H, m), 7.33–7.46 (3H, m),
8.02 (2H, m), 9.36 (1H, br s); 13C NMR (DMSO-d6, 75 MHz) d 99.1,
109.7, 112.8, 117.3, 117.5, 120.1, 124.8, 128.4, 130.4, 131.5,
132.3, 132.5, 134.8, 148.2, 155.1, 160.9, 184.8; IR (ATR) 3280,
1612 cmÀ1; MS (ESI) m/z 408 (M+1); Anal. Calcd for C21H14BrNO3:
C, 61.78; H, 3.46; N, 3.43. Found: C, 61.72; H, 3.55; N, 3.54.
Compound 30 was prepared from 17 and 1-bromo-3-chloropro-
pane using the procedure for 28 with 69% as a colorless oil.
1H NMR (DMSO-d6, 300 MHz) d 1.34 (9H, s), 2.17–2.26 (2H, m),
3.85 (2H, m), 4.23 (2H, m), 6.87 (1H, m), 7.08–7.15 (5H, m), 7.25
(1H, m), 7.37–7.50 (3H, m), 7.63 (1H, m), 7.88 (2H, m); MS (ESI)
m/z 506 (M+1).
5.1.18. (4,6-Dibromo-5-hydroxy-1H-indol-2-yl)(4-phenoxy
phenyl)methanone (25)
Compound 25 was prepared from 21 using the procedure for 22
with 98% as a yellow solid.
5.1.24. Ethyl 2-(2-(4-phenoxybenzoyl)-1H-indol-5-yloxy)acetate
(31)
To a solution of 26 (40.0 mg, 0.078 mmol) in CHCl3 (0.5 ml) was
added trifluoroacetic acid (1.0 ml). The mixture was stirred at room
temperature for 1 h and the solvent was evaporated under reduced
pressure. The residue was purified by flash chromatography on sil-
ica gel (EtOAc/hexane) to provide compound 31 (28.0 mg, 88%) as a
yellow solid.
Mp = 205–206 °C. 1H NMR (CDCl3, 300 MHz) d 6.85 (1H, s),
7.12–7.28 (5H, m), 7.47 (2H, m), 7.66 (1H, s), 7.98 (2H, m), 9.47
(1H, s), 12.20 (1H, br s); 13C NMR (DMSO-d6, 75 MHz) d 103.5,
109.9, 113.0, 115.7, 117.3, 120.2, 124.8, 128.0, 130.4, 131.6,
132.0, 132.7, 135.4, 144.5, 155.0, 161.0, 184.8; IR (ATR) 3317,
1610 cmÀ1; MS (ESI) m/z 488 (M+1); Anal. Calcd for C21H13Br2NO3:
C, 51.78; H, 2.69; N, 2.88. Found: C, 52.07; H, 2.79; N, 2.99.
1H NMR (DMSO-d6, 300 MHz) d 1.20 (3H, t, J = 7.2 Hz), 4.16 (2H,
q, J = 7.2 Hz), 4.74 (2H, s), 6.99–7.28 (8H, m), 7.39–7.50 (3H, m),
7.98 (2H, m), 11.85 (1H, s); MS (ESI) m/z 416 (M+1).
5.1.19. tert-Butyl 5-(2-ethoxy-2-oxoethoxy)-2-(4-phenoxy
benzoyl)-1H-indole-1-carboxylate (26)
5.1.25. 2-(2-(4-Phenoxybenzoyl)-1H-indol-5-yloxy)acetic acid
(32)
A
mixture of 16 (50.0 mg, 0.116 mmol), K2CO3 (32.0 mg,
To a solution of 31 (28.0 mg, 0.0674 mmol) in THF (0.5 ml) and
MeOH (0.5 ml) was added 2 N NaOH solution (0.078 ml,
0.156 mmol). The mixture was stirred at room temperature for
2 h and the solvent was evaporated under reduced pressure. The
residue was dissolved in water (1.0 ml) and neutralized with 2 N
HCl solution. The resulting solid was filtered and dried to afford
compound 32 (20.0 mg, 67%) as a yellow solid.
0.232 mmol) and ethyl bromoacetate (39.0 mg, 0.232 mmol) in
DMF (1.0 ml) was stirred at 40 °C for 2 h. The mixture was cooled
and diluted with EtOAc, then washed with water and brine. The or-
ganic fraction was dried over MgSO4 and the solvent was evapo-
rated under reduced pressure. The residue was purified by flash
chromatography on silica gel (EtOAc/hexane) to provide compound
26 (43.0 mg, 72%) as a colorless oil.
Mp = 166–167 °C. 1H NMR (DMSO-d6, 300 MHz) d 4.62 (2H, s),
6.98–7.18 (7H, m), 7.23–7.28 (1H, m), 7.39 (1H, d, J = 8.8 Hz),
7.45–7.50 (3H, m), 7.97 (2H, m), 11.83 (1H, s); 13C NMR (DMSO-
d6, 75 MHz) d 65.1, 103.6, 111.2, 113.7, 117.2, 117.6, 120.1, 124.8,
127.2, 130.4, 131.5, 132.6, 133.6, 134.7, 152.5, 155.1, 161.7,
170.5, 184.9; IR (ATR) 3288, 1747, 1622 cmÀ1; MS (ESI) m/z 388
(M+1); Anal. Calcd for C23H17NO50.25H2O: C, 70.49; H, 4.50; N,
3.57. Found: C, 70.42; H, 4.43; N, 3.63.
1H NMR (CDCl3, 300 MHz) d 1.30 (3H, t, J = 7.2 Hz), 1.39 (9H, s),
4.28 (2H, q, J = 7.2 Hz), 4.68 (2H, s), 6.82 (1H, s), 6.99–7.24 (7H, m),
7.37–7.44 (2H, m), 7.87–7.91 (2H, m), 8.12 (1H, m); MS (ESI) m/z
516 (M+1).
5.1.20. tert-Butyl 5-(1-ethoxy-2-methyl-1-oxopropan-2-yloxy)-
2-(4-phenoxybenzoyl)-1H-indole-1-carboxylate (27)
Compound 27 was prepared from 16 and ethyl 2-bromoisobu-
tyrate using the procedure for 26 with 79% as a colorless oil.
1H NMR (DMSO-d6, 300 MHz) d 1.18 (3H, t, J = 7.2 Hz), 1.32 (9H,
s), 1.52 (6H, s), 4.17 (2H, q, J = 7.2 Hz), 7.01–7.27 (8H, m), 7.46 (2H,
m), 7.86–7.96 (3H, m); MS (ESI) m/z 544 (M+1).
5.1.26. Ethyl 2-methyl-2-(2-(4-phenoxybenzoyl)-1H-indol-5-
yloxy)propanoate (33)
Compound 33 was prepared from 27 using the procedure for 31
with 95% as a white solid.
1H NMR (DMSO-d6, 300 MHz) d 1.18 (3H, t, J = 7.2 Hz), 1.48 (6H,
s), 4.16 (2H, q, J = 7.2 Hz), 4.74 (2H, s), 6.92 (1H, m), 7.08–7.50
(10H, m), 7.97 (2H, m), 11.87 (1H, s); MS (ESI) m/z 444 (M+1).
5.1.21. tert-Butyl 5-(2-chloroethoxy)-2-(4-phenoxybenzoyl)-
1H-indole-1-carboxylate (28)
A
mixture of 16 (50.0 mg, 0.116 mmol), K2CO3 (32.0 mg,
5.1.27. 2-Methyl-2-(2-(4-phenoxybenzoyl)-1H-indol-5-yloxy
)propanoic acid (34)
Compound 34 was prepared from 33 using the procedure for 32
with 48% as a light brown amorphous.
0.580 mmol) and 1-bromo-2-chloroethane (49.9 mg, 0.348 mmol)
in DMF (0.58 ml) was stirred at 100 °C for 2 h. The mixture was
cooled and diluted with EtOAc, then washed with water and brine.
The organic fraction was dried over MgSO4 and the solvent was
evaporated under reduced pressure. The residue was purified by
flash chromatography on silica gel (EtOAc/hexane) to provide com-
pound 28 (27.0 mg, 47%) as a colorless oil.
1H NMR (DMSO-d6, 300 MHz) d 1.47 (6H, s), 6.94–7.50 (11H, m),
7.97 (2H, m), 11.85 (1H, s); 13C NMR (DMSO-d6, 75 MHz) d 25.1,
79.0, 111.0, 111.3, 113.2, 117.2, 120.0, 121.2, 124.7, 127.1, 130.4,
131.5, 132.5, 134.2, 134.9, 149.2, 155.1, 160.7, 175.2, 184.9; IR
(ATR) 3340, 1718, 1618 cmÀ1; MS (ESI) m/z 416 (M+1); Anal. Calcd
for C25H21NO50.75H2O: C, 70.00; H, 5.29; N, 3.27. Found: C, 69.80;
H, 5.02; N, 3.33.
1H NMR (DMSO-d6, 300 MHz) d 1.32 (9H, s), 3.97 (2H, m), 4.37
(2H, m), 7.03–7.28 (8H, m), 7.47 (2H, m), 7.87 (2H, m), 8.02 (1H,
m); MS (ESI) m/z 492 (M+1).
5.1.28. (5-(2-(Ethylamino)ethoxy)-1H-indol-2-yl)(4-phenoxy
phenyl)methanone (35)
A mixture of 28 (23.0 mg, 0.0468 mmol), EtNH2 in water (12 M,
0.039 ml, 0.468 mmol) and NaI (7.01 mg, 0.0468 mmol) in DMF
5.1.22. tert-Butyl 4-(2-chloroethoxy)-2-(4-phenoxybenzoyl)-
1H-indole-1-carboxylate (29)
Compound 29 was prepared from 17 using the procedure for 28
with 83% as a colorless oil.

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M. Komiya et al. / Bioorg. Med. Chem. 20 (2012) 6840–6847
(0.23 ml) was stirred at 100 °C for 6 h. The mixture was cooled and
diluted with EtOAc, then washed with water and brine. The organic
fraction was dried over MgSO4 and the solvent was evaporated un-
der reduced pressure. The residue was purified by flash chromatog-
raphy on silica gel (MeOH/CHCl3) to provide compound 35
(4.89 mg, 26%) as a light brown amorphous.
(M+1); Anal. Calcd for C27H27N3O31Á5H2O: C, 69.21; H, 6.45; N,
8.97. Found: C, 69.20; H, 6.28; N, 9.13.
5.1.33. (4-(2-(4-Hydroxypiperidin-1-yl)ethoxy)-1H-indol-2-yl)
(4-phenoxyphenyl)methanone (40)
Compound 40 was prepared from 29 and 4-hydroxypiperidine
using the procedure for 35 with 89% as a yellow solid.
1H NMR (DMSO-d6, 300 MHz) d 1.02 (3H, t, J = 7.2 Hz), 2.58 (2H,
q, J = 7.2), 2.86 (2H, t, J = 5.7 Hz), 3.99 (2H, t, J = 5.7 Hz), 6.96–7.27
(8H, m), 7.36–7.50 (3H, m), 7.96 (2H, m), 11.81 (1H, s); 13C NMR
(DMSO-d6, 75 MHz) d 15.2, 43.5, 48.2, 67.8, 103.4, 111.1, 113.6,
117.2, 117.8, 120.0, 124.7, 127.3, 130.4, 131.5, 132.6, 133.4,
134.6, 153.3, 155.2, 160.6, 184.9; IR (ATR) 3288, 1620 cmÀ1; MS
(ESI) m/z 401 (M+1); Anal. Calcd for C25H24N2O3: C, 70.98; H,
6.04; N, 7.00. Found: C, 74.71; H, 6.20; N, 6.98.
Mp = 174–175 °C. 1H NMR (DMSO-d6, 300 MHz) d 1.34–1.38
(2H, m), 1.61–1.73 (2H, m), 2.11–2.17 (2H, m), 2.71–2.81 (4H,
m), 3.41 (1H, m), 4.15–4.19 (2H, m), 4.52 (1H, m), 6.56 (1H, m),
6.99–7.50 (8H, m), 7.45–7.50 (2H, m), 7.94–7.97 (2H, m), 11.96
(1H, s); 13C NMR (DMSO-d6, 75 MHz) d 34.5, 51.4, 56.5, 66.0,
66.2, 100.6, 105.5, 108.7, 117.3, 118.6, 120.0, 124.7, 126.9, 130.4,
131.4, 132.6, 133.2, 139.3, 153.5, 155.1, 160.6, 184.7; IR (ATR)
1616 cmÀ1
28H28N2O4Á2H2O: C, 73.09; H, 6.22; N, 6.09. Found: C, 73.25; H,
6.20; N, 6.20.
;
MS (ESI) m/z 457 (M+1); Anal. Calcd for
5.1.29. (4-(2-(Ethylamino)ethoxy)-1H-indol-2-yl)(4-phenoxy
phenyl)methanone (36)
C
Compound 36 was prepared from 29 using the procedure for 35
with 37% as a yellow solid.
5.1.34. (4-(2-Aminoethoxy)-1H-indol-2-yl)(4-phenoxyphenyl)
Mp = 143–144 °C. 1H NMR (DMSO-d6, 300 MHz) d 1.01 (3H, t,
J = 7.2 Hz), 2.59 (2H, q, J = 7.2), 2.93 (2H, t, J = 5.7 Hz), 4.13 (2H, t,
J = 5.7 Hz), 6.55 (1H, m), 7.05–7.28 (8H, m), 7.48 (2H, m), 7.97
(2H, m), 11.97 (1H, s); 13C NMR (DMSO-d6, 75 MHz) d 15.2, 43.5,
48.0, 67.6, 100.3, 105.4, 109.0, 117.3, 118.6, 120.0, 124.7, 126.9,
130.4, 131.4, 132.6, 133.2, 139.3, 153.7, 155.1, 160.6, 184.7; IR
methanone (41)
A
mixture of 16 (100 mg, 0.233 mmol), K2CO3 (64.0 mg,
0.932 mmol) and N-(2-bromoethyl)phthalimide (237 mg,
0.932 mmol) in DMF (1.2 ml) was stirred at 100 °C for 3 h. The mix-
ture was cooled and diluted with EtOAc, then washed with water
and brine. The organic fraction was dried over MgSO4 and the sol-
vent was evaporated under reduced pressure. The residue was dis-
solved in MeOH (1.0 ml), and 40% MeNH2 solution (1.0 ml) was
added. The mixture was stirred at 50 °C for 1 h and the solvent
was evaporated under reduced pressure. The residue was purified
by flash chromatography on silica gel (MeOH/CHCl3) to provide
compound 41 (43.0 mg, 52%) as a brown oil.
(ATR) 1606 cmÀ1
25H24N2O3: C, 74.98; H, 6.04; N, 7.00. Found: C, 75.00; H, 6.09;
N, 7.10.
; MS (ESI) m/z 401 (M+1); Anal. Calcd for
C
5.1.30. (4-Phenoxyphenyl)(4-(2-(piperidin-1-yl)ethoxy)-1H-
indol-2-yl)methanone (37)
Compound 37 was prepared from 29 and piperidine using the
procedure for 35 with 80% as a yellow amorphous.
1H NMR (DMSO-d6, 300 MHz) d 2.46 (2H, m), 2.88 (2H, m), 3.98
(2H, m), 6.46 (1H, m), 6.99–7.23 (8H, m), 7.43 (2H, m), 7.93 (2H,
m); 13C NMR (DMSO-d6, 75 MHz) d 41.0, 70.2, 100.2, 105.4,
109.0, 117.3, 118.6, 120.1, 124.7, 126.9, 130.4, 131.4, 132.6,
133.2, 139.3, 153.8, 155.1, 160.6, 184.7; IR (ATR) 3300, 1616 cm
À1; MS (ESI) m/z 373 (M+1); Anal. Calcd for C23H20N2O30.75H2O:
C, 71.58; H, 5.62; N, 7.26. Found: C, 71.65; H, 5.56; N, 7.10.
1H NMR (DMSO-d6, 300 MHz) d 1.30–1.51 (6H, m), 2.44–2.49
(4H, m), 2.71 (2H, m), 4.17 (2H, m), 6.56 (1H, m), 6.99–7.27 (8H,
m), 7.47 (2H, m), 7.95 (2H, m), 11.97 (1H, s); 13C NMR (DMSO-d6,
75 MHz) d 23.9, 25.6, 54.4, 57.3, 65.9, 100.6, 105.5, 108.8, 117.2,
118.6, 120.0, 124.7, 126.9, 130.3, 131.3, 132.6, 133.2, 139.3,
153.6, 155.1, 160.6, 184.8; IR (ATR) 3300, 1614 cmÀ1; MS (ESI)
m/z 441 (M+1); Anal. Calcd for C28H28N2O30Á4H2O: C, 75.11; H,
6.48; N, 6.26. Found: C, 75.21; H, 6.41; N, 6.31.
5.2. Biology
5.2.1. CaMKII activity assay
5.1.31. (4-Phenoxyphenyl)(4-(3-(piperidin-1-yl)propoxy)-1H-
indol-2-yl)methanone (38)
Compound 38 was prepared from 30 and piperidine using the
procedure for 35 with 64% as a yellow solid.
CaMKII was purified according to the method of Brickey et al.
(Biochem. Biophys. Res. Commun. 1990, 173, 578) with some modi-
fications. cDNA encoding CaMKII was sub-cloned into the transfer
vector pFastBac1. Bacmids were generated in DH10Bac cells, and
CaMKII recombinant baculovirus stocks were prepared according
to the protocol of Bac-to-Bac Baculovirus Expression System (invit-
rogen, USA). Sf9 cells were maintained in Sf900 II medium and in-
fected with CaMKII recombinant baculovirus stocks. The cells were
harvested 3 days post-infection and CaMKII was affinity purified
using Calmodulin Sepharose 4B (GE healthcare).
Mp = 131–132 °C. 1H NMR (DMSO-d6, 300 MHz) d 1.30–1.49
(6H, m), 1.88 (2H, m), 2.28–2.43 (6H, m), 4.10 (2H, t, J = 6.4 Hz),
6.53 (1H, m), 7.00–7.29 (8H, m), 7.47 (2H, m), 7.95 (2H, m),
11.96 (1H, s); 13C NMR (DMSO-d6, 75 MHz) d 24.1, 25.6, 26.3,
54.1, 55.2, 66.0, 100.4, 105.4, 108.7, 117.2, 118.6, 120.0, 124.7,
126.9, 130.3, 131.4, 132.6, 133.2, 139.3, 153.7, 155.1, 160.6,
184.7; IR (ATR) 3286, 1604 cmÀ1; MS (ESI) m/z 455 (M+1); Anal.
Calcd for C29H30N2O3: C, 76.63; H, 6.65; N, 6.16. Found: C, 76.53;
H, 6.71; N, 6.21.
Autocamtide-2 and CaM were purchased from Millipore, and
c-
33P ATP was obtained from GE Healthcare. ATP was purchased
from Sigma–Aldrich. The test substances and purified CaMKII were
added to the assay buffer containing 25 mM Tris–HCl, pH 7.5,
2 mM dithiothreitol, 10 mM MgCl2, 0.1% CHAPS, and 1 mM CaCl2.
5.1.32. (4-Phenoxyphenyl)(4-(2-(piperazin-1-yl)ethoxy)-1H-
indol-2-yl)methanone (39)
Compound 39 was prepared from 29 and piperadine using the
procedure for 35 with 69% as a yellow amorphous.
Autocamtide-2 was diluted to a final concentration of 1
assay buffer containing CaM (0.5 M) and ATP (final concentration
0.1 M and 350–1500 cpm/pmol
33P ATP) were added to the di-
lM with
l
l
c-
1H NMR (DMSO-d6, 300 MHz) d 2.41–2.54 (4H, m), 2.69–2.76
(6H, m), 4.19 (2H, t, J = 6.4 Hz), 6.56 (1H, m), 7.00–7.29 (8H, m),
7.47 (2H, m), 7.95 (2H, m), 11.96 (1H, s); 13C NMR (DMSO-d6,
75 MHz) d 45.6, 54.6, 57.3, 65.7, 100.6, 105.5, 108.7, 117.3, 118.6,
120.0, 124.7, 126.9, 130.4, 131.3, 132.6, 133.2, 139.3, 153.5,
155.1, 160.6, 184.7; IR (ATR) 3317, 1608 cmÀ1; MS (ESI) m/z 442
luted test substances and the whole was incubated for 90 min at
24 °C. After incubation, the reaction was terminated by transfer
of a 16 lL aliquot onto the appropriate area of a P30 Filtermat
(PerkinElmer, USA), and the labeled substrate was captured by a
negatively charged filtermat. Phosphorylation was linear with re-
spect to time under these conditions. The filtermat was washed

M. Komiya et al. / Bioorg. Med. Chem. 20 (2012) 6840–6847
6847
three times, each for 5 min with 75 mM phosphoric acid, dried, and
then spotted by MicroScintO (PerkinElmer, USA). The radioactivity
was counted in TopCount NXT (PerkinElmer, USA). CPM counts
were calculated for transfer of a phosphate group per minute per
one mg CaMKII.
strate was captured by a negatively charged filtermat. The filter-
mat was washed three times, each for 5 min with 75 mM
phosphoric acid, dried, and then spotted by MicroScintO
(PerkinElmer, USA). The radioactivity was counted in TopCount
NXT (PerkinElmer, USA).
5.2.2. P38
P38 /SAPK2a active (p38) was purchased from Millipore. Mye-
lin basic protein (MBP) was purchased from Alexis biochemicals.
33P ATP was obtained from GE Healthcare. ATP was purchased
a
activity assay
5.2.5. PKC
PKC active (PKC
typeIII-SS from calf thymus (Histone) and ATP were purchased
from Sigma–Aldrich.
33P ATP was obtained from GE Healthcare.
c
activity assay
a
c
c) was purchased from Millipore. Histone
c-
c-
from Sigma–Aldrich.
Polylysine YSi SPA Scintillation Beads (SPA beads) were purchased
The test substances were added to the assay buffer containing
from Amersham Biosciences.
0.5
10 mM MgCl2, 0.1% CHAPS. The substrate MBP was diluted to a fi-
nal concentration of 50 g/mL with assay buffer containing ATP (fi-
nal concentration 0.1 M and 350–1500 cpm/pmol
33P ATP) was
added to the diluted test substances and the whole was incubated
for 90 min at room temperature. After incubation, the reaction was
terminated by spotting the mixture of reaction solution onto the
appropriate area of a P30 Filtermat (PerkinElmer, USA), and the
labeled substrate was captured by a negatively charged filtermat.
The filtermat was washed three times, each for 5 min with
75 mM phosphoric acid, dried, and then spotted by MicroScintO
(PerkinElmer, USA). The radioactivity was counted in TopCount
NXT (PerkinElmer, USA).
l
g/mL p38, 25 mM Tris–HCl, pH 7.5, 2 mM dithiothreitol,
The test substances were added to the assay buffer containing
0.25
lg/mL PKCc, 20 mM Tris–HCl, pH 7.5, 2.5 mM dithiothreitol,
l
7.5 mM MgCl2, 750
l
M CaCl2. The substrate Histone was diluted
l
c-
to a final concentration of 25
ATP (final concentration 0.1
l
g/mL with assay buffer containing
lM and 350–1500 cpm/pmol
c-
33P
ATP). Then prepared test substances were added to the substrate
solution and the whole was incubated for 90 min at room temper-
ature. After incubation, SPA beads were added to assay plate and
stay still standing for over 30 min. at room temperature. The radio-
activity was counted in 1450 MicroBeta TRILUX (PerkinElmer,
USA).
Acknowledgements
5.2.3. MLCK activity assay
We are grateful to Ms. K. Bando for performing the elemental
analysis, and Mr. T. Tanigawa for recording IR spectra and Melting
points.
MLCK active (MLCK), ZIPtide and calmodulin (CaM) were pur-
chased from Millipore.
c-
33P ATP was obtained from GE Health-
care. ATP was purchased from Sigma–Aldrich.
The test substances were added to the assay buffer containing
Reference and notes
0.5
10 mM MgAc, 0.1% CHAPS and 500
was diluted to a final concentration of 75
containing ATP (final concentration 0.1 M and 350–1500 cpm/
pmol g/mL CaM. Then prepared test sub-
33P ATP) and 0.125
stances were added to the substrate solution and the whole was
incubated for 90 min at room temperature. After incubation, the
reaction was terminated by spotting the mixture of reaction solu-
tion onto the appropriate area of a P30 Filtermat (PerkinElmer,
USA), and the labeled substrate was captured by a negatively
charged filtermat. The filtermat was washed three times, each for
5 min with 75 mM phosphoric acid, dried, and then spotted by
MicroScintO (PerkinElmer, USA). The radioactivity was counted in
TopCount NXT (PerkinElmer, USA).
l
g/mL MLCK, 20 mM Tris–HCl, pH 7.5, 2 mM dithiothreitol,
M CaCl2 . The substrate ZIPtide
M with assay buffer
1. Bootman, M. D.; Collins, T. J.; Peppiatt, C. M.; Prothero, L. S.; MacKenzie, L.; De
Smet, P.; Travers, M.; Tovey, S. C.; Seo, J. T.; Berridge, M. J.; Ciccolini, F.; Lipp, P.
Semin. Cell. Dev. Biol. 2001, 12, 3.
2. Howe, C. J.; Lahair, M. M.; Mccubrey, J. A.; Franklin, R. A. J. Biol. Chem. 2004, 279,
44573.
3. Hook, S. S. Annu. Rev. Pharmacol. Toxicol. 2001, 41, 471.
4. Braun, A. P.; Schulman, H. Annu. Rev. Physiol. 1995, 57, 417.
5. Hanson, P. I.; Schulman, H. Annu. Rev. Biochem. 1992, 12, 559.
6. Colbran, R. J.; Soderling, T. R. Curr. Top. Cell. Regul. 1990, 279, 181.
7. Gaertner, T. R.; Kolodziej, S. J.; Wang, D.; Kobayashi, R.; Koomen, J. M.; Stoops, J.
K.; Waxham, M. N. J. Biol. Chem. 2004, 279, 12484.
8. Schworer, C. M.; Colbran, R. J.; Keefer, J. R.; Soderling, T. R. J. Biol. Chem. 1988,
263, 13486.
9. Miller, S. G.; Patton, B. L.; Kennedy, M. B. Neuron 1988, 1, 593.
10. Thiel, G.; Czernik, A. J.; Gorelick, F.; Nairn, A. C.; Greengard, P. Proc. Natl. Acad.
Sci. U.S.A. 1988, 85, 6337.
11. Ikeda, A.; Okuno, S.; Fujisawa, H. J. Biol. Chem. 1991, 266, 11582.
12. Meyer, T.; Hanson, P. I.; Stryer, L.; Schulman, H. Science 1992, 256, 1199.
13. Wayman, G. A.; Lee, Y. S.; Tokumitsu, H.; Silva, A.; Soderling, T. R. Neuron 2008,
59, 914.
14. Ang, E. S.; Zhang, P.; Steer, J. H.; Tan, J. W.; Yip, K.; Zheng, M. H.; Joyce, D. A.; Xu,
J. J. Cell. Physiol. 2007, 212, 787.
l
l
l
c-
l
5.2.4. Akt1 activity assay
Akt1 and Crosstide were purchased from Millipore. c-
33P ATP
was obtained from GE Healthcare. ATP was purchased from
Sigma–Aldrich.
15. Lin, M. Y.; Zal, T.; Ch’en, I. L.; Gascoigne, N. R. J.; Hedrick, S. M. J. Immunol. 2005,
174, 5583.
The test substances and Akt1 were added to the assay buffer
containing 25 mM Tris–HCl, pH 7.5, 2 mM dithiothreitol, 15 mM
MgCl2, 0.1% CHAPS. The substrate Crosstide was diluted to a final
16. Sumi, M.; Kiuchi, K.; Ishikawa, T.; Ishii, A.; Hagiwara, M.; Nagatsu, T.; Hidaka, H.
Biochem. Biophys. Res. Commun. 1991, 181, 968.
17. Ishida, A.; Kameshita, I.; Okuno, S.; Kitani, T.; Fujisawa, H. Biochem. Biophys. Res.
Commun. 1995, 212, 806.
18. Colomer, J.; Schmitt, A. A.; Toone, E. J.; Means, A. R. Biochemistry 2010, 49, 4244.
19. Asano, S.; Komiya, M.; Koike, N.; Koga, E.; Nakatani, S.; Isobe, Y. Bioorg. Med.
Chem. Lett. 2010, 20, 6696.
20. Komiya, M.; Asano, S.; Koike, N.; Koga, E.; Igarashi, J.; Nakatani, S.; Isobe, Y.
Bioorg. Med. Chem. Lett. 2011, 21, 1456.
concentration of 2.5
l
M with assay buffer containing ATP (final
concentration 0.1 M and 350–1500 cpm/pmol
l
c-
33P ATP) was
added to the diluted test substances and the whole was incubated
for 90 min at room temperature. After incubation, the reaction was
21. Papageorgiou, G.; Corrie, J. E. T. J. Heterocycl. Chem. 2005, 42, 1101.
22. Katritzky, A. R.; Akutagawa, K. Tetrahedron Lett. 1985, 26, 5935.
terminated by transferring a 16
area of a P30 Filtermat (PerkinElmer, USA), and the labeled sub-
lL aliquot onto the appropriate