Synthesis of novel benzo-fused heteroaryl derivatives as Ca 2+/calmodulin-dependent protein kinase II inhibitors

Article abstract of DOI:10.1248/cpb.c13-00493

Based on the structure activity relationship of 2-(4-phenoxybenzoyl)-5- hydroxyindole (1), a novel structural class of Ca²⁺ /calmodulin-dependent protein kinase II (CaMKII) inhibitors were synthesized. We show in this study that the acidic prot

Full text of DOI:10.1248/cpb.c13-00493

1094
Note
Chem. Pharm. Bull. 61(10) 1094–1097 (2013)
Vol. 61, No. 10
Synthesis of Novel Benzo-Fused Heteroaryl Derivatives as Ca2+/
Calmodulin-Dependent Protein Kinase II Inhibitors
,a
Masafumi Komiya,* Shigehiro Asano,b Nobuyuki Koike,b Erina Koga,b Junetsu Igarashi,a
Shogo Nakatani,b and Yoshiaki Isobeb
a Research Division, Dainippon Sumitomo Pharma Co., Ltd.; 33–94 Enoki, Suita, Osaka 564–0053, Japan: and
b Research Division, Dainippon Sumitomo Pharma Co., Ltd.; 3–1–98 Kasugade Naka, Konohana-ku, Osaka 554–
0022, Japan.
Received June 19, 2013; accepted July 11, 2013
Based on the structure activity relationship of 2-(4-phenoxybenzoyl)-5-hydroxyindole (1), a novel struc-
tural class of Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitors were synthesized. We show
in this study that the acidic proton at the N(1)-position of the indole moiety is not essential for CaMKII in-
hibitory activity. Among the synthesized compounds, we found the benzofuran and benzothiazole derivative
as promising scaffolds for the developement of potent CaMKII inhibitors. In particular, compounds 8 and 14
inhibited CaMKII with IC50 values of 24nM and 32nM, respectively.
Key words Ca2+/calmodulin-dependent protein kinase II; inhibitor; kinase; indole; calmodulin
Calcium (Ca2+) is an important intracellular messenger, proton at the N(1)-position of the indole moiety was under
controlling a diverse range of cellular processes, such as apo- examination. Here, we report in detail on our findings, in par-
ptosis, ion channel and cell cycle regulation, and cellular re- ticular the structure–activity relationship (SAR) of a series of
sponse to oxidative stress.1,2) A rise in intracellular Ca2+ con- benzofuran and benzothiazole derivatives.
centration leads to binding of Ca2+ ions to calmodulin (CaM),
Chemistry The synthetic routes to the benzofuran and
which in turn binds to and activates Ca2+/CaM-dependent benzothiazole compounds are shown in Charts 1 and 2. The
protein kinases (CaMKs). CaMKs, which are ubiquitous ser- benzofuran derivatives 5–8 were prepared from the 4-phen-
ine/threonine kinases classified into three subtypes (І, II, and oxyacetophenone 4, which was treated with bromine to give a
IV), modulate many cellular functions in response to changes phenacyl bromide. Subsequent condensation with 2-hydroxy-
in intracellular Ca2+ levels.3,4) CaMKII, a member of the 5-methoxybenzaldehyde under basic conditions22) furnished
CaMKs family, assembles into a complex of dodecamers with the benzofuran compound 5. Deprotection of the methyl group
four isoforms (α, β, γ, and δ), having each a subunit composed of 5, followed by bromination afforded compounds 7 and 8
of three main parts; catalytic, regulatory and association do- (Chart 1).
mains.5–7) Upon binding to Ca2+/CaM in the presence of ATP/
The benzothiazole derivatives 12–14 were prepared from 9,
Mg2+, CaMKII undergoes a rapid autophosphorylation at the which was reacted with SOCl2 to provide 10. According to a
Thr286/Thr287 located within the autoinhibitory domain. The reported method,23) the anion of 4-phenoxyphenylacetonitrile
transformed CaMKII maintains considerable enzyme activ- was reacted with the chloride 10 to afford the diarylacetoni-
ity even without Ca2+/CaM.8–11) This autophosphorylation has trile. Oxidation of the acetonitrile moiety with sodium perox-
been reported to cause a dramatic increase in the affinity of ide and aqueous NH4OAc solution gave 11. Demethylation of
the enzyme for Ca2+/CaM.12) CaMKII is well known for its 11, followed by bromination of 12 afforded compounds 13 and
modulating effects on synaptic plasticity and other processes 14 (Chart 2).
like learning and memory.13) In addition, CaMKII plays a role
in osteoclasts differentiation and bone resorption,14) and active
Results and Discussion
CaMKII is known to enhance T cells proliferation and cyto-
The inhibitory activity against CaMKII of the synthesized
toxic activity.15)
compounds is summarized in Table 1.24) According to the
The CaM-competitive inhibitor KN-9316) and the auto- results of our previous paper,20) a substitution at the N(1)-
camtide-2-related inhibitory peptide (AIP)17) are well-known position of the indole moiety should be well tolerated. As ex-
CaMKII inhibitors. Recent studies have also reported a num- pected, the benzofuran 6 showed moderate inhibitory activity
ber of Ca2+/CaM antagonists18) and CaM non-competitive against CaMKII, confirming the assumption that the proton of
inhibitors.19) Based on these reports, we considered CaMKII the indole at the 1-position is not essential for activity. How-
to be a good target for the development of anti-inflammatory ever, like the indole derivatives, the methoxy compound 5, as
agents.
Our work started with high throughput screening against
CaMKII. A description of this approach, which led to the
discovery of 1 and its subsequent preliminary optimization
to 2 and 3 (Fig. 1), was recently published.20,21) We showed
that a hydroxyl group in the indole moiety of 1 is essential for
CaMKII inhibition and indicated that the effect of an acidic
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: masafumi-komiya@ds-pharma.co.jp
Fig. 1. Structures of 1, 2, and 3
© 2013 The Pharmaceutical Society of Japan

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(a) SOCl2, DMF(cat.), 70°C, 10h, 70%; (b) NaHMDS, 4-phenoxyphenylaceto-
nitrile, THF, rt, 3h then NH4OAc, Na2O2, rt, overnight, 51%; (c) BBr3, CH2Cl2, rt,
overnight, 65%; (d) NBS, THF, −10°C to rt, 1h, 13: 31%, 14: 21%.
Reagents and conditions: (a) Br2, AcOH, CHCl3, rt, 1h; (b) 2-hydroxy-5-meth-
oxybenzaldehyde, K2CO3, MeCN, 70°C, 5h, 73% in two steps; (c) BBr3, CHCl3, rt,
1h, 83%. (d) NBS, THF, 0°C to rt, 1h, 7: 53%, 8: 30%.
Chart 2
Chart 1
Table 1. Inhibitory Activity of Fused Heteroaryl Derivatives against CaMKII
Compd.
X
Y
R1
R2
R3
CaMKII IC50 (µM)
1
2
NH
NH
NH
O
CH
CH
CH
CH
CH
CH
CH
N
H
Br
Br
H
H
H
OH
OH
OH
OMe
OH
OH
OH
OH
OH
OH
0.61
0.040
0.012
>10
1.1
3
Br
H
5
6
O
H
H
7
O
Br
Br
H
H
0.26
8
O
Br
H
0.024
1.1
12
13
14
S
S
N
Br
Br
H
0.17
S
N
Br
0.032
a no-acidic proton compound, showed diminished CaMKII in-
hibitory activity. Introduction of a bromo group into 6 resulted
Experimental
Melting points (mp) were determined on an electrothermal
in improved activity, suggesting that benzofuran derivatives apparatus without correction. IR spectra were recorded on a
can be potent CaMKII inhibitors. Elsewhere, the mono-bromo JEOL JIR-SPX60 spectrometer. NMR spectra were recorded
compound 7 showed better membrane permeability than the on a JEOL JNM-LA300 spectrometer. Chemical shifts (δ) are
indole 2 (data not shown). The benzothiazole 12 also showed given in parts per million, and tetramethylsilane (TMS) was
moderate CaMKII inhibitory activity, suggesting that a thia- used as the internal standard for spectra obtained in DMSO-d6
zole moiety can be well tolerated. Finally, the mono-bromo and CDCl3. All J values are given in Hz. Mass spectra were
compound 13 and di-bromo compound 14 showed strong recorded on a Waters ACQUITY UPLC/MS system. Elemen-
CaMKII inhibitory activity, suggesting that benzothiazole de- tal analysis was performed on a CE Instrument EA1110 and
rivatives can also be potent CaMKII inhibitors.
a Yokokawa analytical system IC7000. Reagents and solvents
were used as obtained from commercial suppliers without
further purification. Column chromatography was carried out
Conclusion
In this study, we clarified the effect of an acidic proton at using a Yamazen W-prep system, and performed using pre-
the N(1)-position of the indole moiety of 1 on CaMKII inhibi- packed silica gel. Reaction progress was determined by TLC
tion by synthesizing and evaluating a series of benzofuran and analysis on silica gel coated glass plate. Visualization was
benzothiazole derivatives. We found that the acidic proton of done with UV light (254nm). All reactions were carried out
the NH group is not essential for CaMKII inhibitory activ- under a nitrogen atmosphere unless otherwise mentioned.
ity. We also found that some of the synthesized compounds
(5-(Methoxy)benzofuran-2-yl)(4-phenoxyphenyl)metha-
can serve as promising scaffolds for the discovery of potent none (5) To a solution of 4 (500mg, 2.36mmol) in CHCl3
CaMKII inhibitors. In particular, the di-bromo compounds 8 (5.9mL) and AcOH (5.9mL) was added Br2 (377mg,
and 14, which showed strong CaMKII inhibitory activity with 2.36mmol) in CHCl3 (0.5mL). The mixture was stirred at
IC50 values of 24nM and 32nM, respectively, and are therefore room temperature for 1h and the solvent was evaporated
worthy of further optimization.
under reduced pressure. According to a literature procedure,
the residue was dissolved in MeCN (11.8mL), and 2-hydroxy-
5-methoxybenzaldehyde (359mg, 2.36mmol) and K2CO3

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Vol. 61, No. 10
(978mg, 7.08mmol) were added. The mixture was stirred at at 70°C for 10h and cooled. The solvent was evaporated under
70°C for 5h and cooled. The mixture was diluted with EtOAc reduced pressure. The residue was purified by flash chro-
and washed with water and brine. The organic fraction was matography on silica gel (CHCl3) to provide compound 10
dried over MgSO4 and the solvent was evaporated under (705mg, 70%) as a white solid.
reduced pressure. The residue was purified by flash chroma-
tography on silica gel (EtOAc–hexane) and crystallized from 7.44 (1H, m), 7.62 (1H, m); MS (ESI) m/z 200 (M+1).
EtOAc to provide compound 5 (592mg, 73%) as a white solid. (5-Methoxybenzo[d]thiazol-2-yl)(4-phenoxyphenyl)meth-
1H-NMR (CDCl3, 300MHz) δ: 3.88 (3H, s), 7.05 (1H, m),
1H-NMR (CDCl3, 300MHz) δ: 3.80 (3H, s), 7.10–7.30 anone (11) According to a literature procedure, a mixture
(7H, m), 7.46–7.51 (2H, m), 7.65–7.70 (2H, m), 8.05 (2H, m); of 10 (600mg, 3.01mmol) and 4-phenoxyphenylacetonitrile
13C-NMR (DMSO-d6, 75MHz) δ: 55.6, 104.4, 113.0, 116.4, (755mg, 3.61mmol) and sodium hexamethyldisilazide in
117.2, 118.3, 120.1, 124.9, 127.4, 130.4, 131.2, 131.9, 150.3, toluene (0.99M, 7.59mL, 7.51mmol) in THF (30mL) was
152.3, 154.9, 156.2, 161.4, 181.8; MS (electrospray ionization stirred at room temperature for 3h. Then, to the mixture was
(ESI)) m/z 345 (M+1); Anal. Calcd for C22H16O4: C, 76.73; H, added saturated NH4OAc solution (20mL) and Na2O2 (937mg,
4.68. Found: C, 76.68; H, 4.66.
12.0mmol). The mixture was stirred at room temperature
(5-Hydroxybenzofuran-2-yl)(4-phenoxyphenyl)metha- overnight and quenched with 10% NaHSO3 solution. The
none (6) To a solution of 5 (200mg, 0.476mmol) in aqueous layer was extracted with EtOAc and the combined
CHCl3 (2.4mL) was added BBr3 in CH2Cl2 (1.0 M, 1.40mL, organic layers were washed with brine. The organic fraction
1.40mmol) at 0°C. The mixture was stirred at room tem- was dried over MgSO4 and the solvent was evaporated under
perature for 1h and quenched with saturated aqueous sodium reduced pressure. The residue was purified by flash chroma-
bicarbonate solution. The aqueous layer was extracted with tography on silica gel (EtOAc–hexane) to provide compound
EtOAc and the combined organic layers were washed with 11 (559mg, 51%) as a yellow solid.
brine. The organic fraction was dried over MgSO4 and the
1H-NMR (CDCl3, 300MHz) δ: 3.92 (3H, s), 7.07–7.25 (6H,
solvent was evaporated under reduced pressure. The residue m), 7.39–7.46 (2H, m), 7.64 (1H, m), 7.85 (1H, m), 8.59–8.63
was purified by flash chromatography on silica gel (EtOAc– (2H, m); MS (ESI) m/z 362 (M+1).
hexane) to provide compound 6 (130mg, 83%) as a yellow
amorphous.
(5-Hydroxybenzo[d]thiazol-2-yl)(4-phenoxyphenyl)meth-
anone (12) To a solution of 11 (100mg, 0.277mmol) in
1H-NMR (CDCl3, 300MHz) δ: 5.05 (1H, s), 7.02–7.13 CH2Cl2 (2.0mL) was added BBr3 in CH2Cl2 (1.0 M, 0.830mL,
(6H, m), 7.20–7.25 (1H, m), 7.40–7.54 (4H, m), 8.09 (2H, m); 0.830mmol) at 0°C. The mixture was stirred at room tem-
13C-NMR (CDCl3, 75MHz) δ: 107.1, 113.1, 115.7, 117.3, 117.8, perature overnight and quenched with saturated NaHCO3
120.3, 124.8, 127.8, 130.1, 131.3, 132.0, 151.1, 152.3, 153.3, solution. The aqueous layer was extracted with EtOAc and
155.3, 162.1, 182.9; IR (attenuated total reflection (ATR)) the combined organic layers were washed with brine. The
1621 cm−1; MS (ESI) m/z 331 (M+1); Anal. Calcd for C21H14O4: organic fraction was dried over MgSO4 and the solvent was
C, 75.33; H, 4.36. Found: C, 75.35; H, 4.33.
evaporated under reduced pressure. The residue was purified
(4-Bromo-5-hydroxybenzofuran-2-yl)(4-phenoxyphenyl) by flash chromatography on silica gel (EtOAc–hexane) and
methanone (7) and (4,6-Dibromo-5-hydroxybenzofuran- crystallized from EtOAc to provide compound 12 (62.6mg,
2-yl)(4-phenoxyphenyl)methanone (8) To an ice cooled 65%) as a yellow solid.
1
solution of 6 (150mg, 0.454mmol) in tetrahydrofuran (THF)
mp=140–141°C. H-NMR (CDCl3, 300MHz) δ: 4.34 (1H,
(3.0mL) was added N-bromosuccinimide (NBS) (105mg, s), 7.06–7.16 (5H, m), 7.20–7.26 (1H, m), 7.40–7.45 (2H, m),
0.590mmol). The mixture was stirred at room temperature for 7.61 (1H, d, J=2.2Hz), 7.85 (1H, d, J=8.8Hz), 8.58 (2H, m);
1h and the solvent was evaporated under reduced pressure. 13C-NMR (CDCl3, 75MHz) δ: 109.9, 117.1, 118.1, 120.5, 122.7,
The residue was purified by flash chromatography on silica 124.9, 129.2, 129.3, 130.1, 133.8, 155.0, 155.1, 155.3, 163.0,
gel (EtOAc–hexane) and crystallized from EtOAc to provide 168.8, 183.7; IR (ATR) 1633cm−1; MS (ESI) m/z 348 (M+1);
compound 7 (99.3mg, 53%) as a yellow solid. Compound 8 Anal. Calcd for C20H13NO3S: C, 69.15; H, 3.77; N, 4.03. Found:
(67.6mg, 30%) was obtained as a yellow amorphous.
7: mp=123–124°C. H-NMR (CDCl3, 300MHz) δ: 5.48 (1H,
C, 69.14; H, 3.84; N, 4.16.
(4-Bromo-5-hydroxybenzo[d]thiazol-2-yl)(4-phenoxyphe-
1
s), 7.08–7.14 (4H, m), 7.19–7.23 (2H, m), 7.41–7.51 (4H, m), 8.10 nyl)methanone (13) and (4,6-Dibromo-5-hydroxybenzo[d]
(2H, m); 13C-NMR (CDCl3, 75MHz) δ: 100.8, 112.6, 115.1, thiazol-2-yl)(4-phenoxyphenyl)methanone (14) To an ice
117.3, 117.4, 120.4, 124.8, 128.5, 130.1, 131.0, 132.0, 149.4, cooled solution of 12 (200mg, 0.576mmol) in THF (4.0mL)
150.1, 153.4, 155.2, 162.3, 182.4; IR (ATR) 3317, 1630cm−1; was added NBS (103mg, 0.576mmol). The mixture was
MS (ESI) m/z 409 (M+1); Anal. Calcd for C21H13BrO4: C, stirred at room temperature for 1h and the solvent was evapo-
61.63; H, 3.20. Found: C, 61.60; H, 3.17.
rated under reduced pressure. The residue was purified by
8: 1H-NMR (CDCl3, 300MHz) δ: 5.87 (1H, s), 7.08–7.14 flash chromatography on silica gel (EtOAc–hexane) to provide
(4H, m), 7.21–7.26 (1H, m), 7.41–7.46 (3H, m), 7.77 (1H, s), compound 13 (77.2mg, 31%) as a yellow solid and compound
8.08 (2H, m); 13C-NMR (CDCl3, 75MHz) δ: 100.9, 111.0, 14 (61.4mg, 21%) as a yellow solid.
115.1, 115.5, 117.3, 117.5, 120.4, 124.9, 128.9, 130.1, 130.8,
13: mp=161–162°C. 1H-NMR (CDCl3, 300MHz) δ: 5.83
132.0, 146.4, 149.3, 153.6, 155.2, 162.5; IR (ATR) 1637cm−1; (1H, s), 7.08–7.15 (4H, m), 7.22–7.31 (2H, m), 7.41–7.46 (2H,
MS (ESI) m/z 489 (M+1); Anal. Calcd for C21H12Br2O40.5H2O: m), 7.82 (1H, d, J=8.8Hz), 8.73 (2H, m); 13C-NMR (CDCl3,
C, 50.74; H, 2.64. Found: C, 50.40; H, 2.45.
75MHz) δ: 104.4, 117.1, 117.7, 120.6, 121.6, 124.9, 128.9,
2-Chloro-5-methoxybenzo[d]thiazole (10) To a solution 129.7, 130.1, 134.1, 152.5, 155.1, 163.2, 169.5, 182.6; IR
of 9 (1.00g, 5.07mmol) in SOCl2 (5.0mL) was added N,N- (ATR) 1631cm−1; MS (ESI) m/z 426 (M+1); Anal. Calcd for
dimethylformamide (DMF) (2 drops). The mixture was stirred C20H12BrNO3S·0.5H2O: C, 55.18; H, 3.01; N, 3.22. Found: C,

October 2013
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