Discovery, synthesis and biological evaluation of isoquinolones as novel and highly selective JNK inhibitors (2)

Article abstract of DOI:10.1016/j.bmc.2008.02.027

3-Metoxycarbonyl isoquinolone derivative 1 has been identified as a potent JNK inhibitor and significantly inhibited cardiac hypertrophy in a rat pressure-overload model. Herein, a series of isoquinolones with an imidazolylmethyl or a pyrazolylmethyl group at the 2-position were designed based on X-ray crystallographic analysis of the complex between the isoquinolone compound and JNK3, as wells as the relationship between compound lipophilicity (log D) and activity in a cell-based assay. The compounds prepared showed potent JNK1 inhibitory activities in a cell-based assay. Among them the isoquinolone derivative possessing 5-[(cyclopropylamino)carbonyl]-1-methyl-1H-pyrazole (16e) exhibited significant anti-hypertrophic activity at doses of more than 1 mg/kg (po) in a pressure-overload model.

Full text of DOI:10.1016/j.bmc.2008.02.027

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4715–4732
Discovery, synthesis and biological evaluation of isoquinolones
as novel and highly selective JNK inhibitors (1)
Yasutomi Asano,a,* Shuji Kitamura,a Taiichi Ohra,a Kazuyoshi Aso,a Hideki Igata,c
Tomoko Tamura,c Tomohiro Kawamoto,b Toshimasa Tanaka,b Satoshi Sogabe,b
Shin-ichi Matsumoto,b Masashi Yamaguchi,b Hiroyuki Kimurab and Fumio Itoha
aMedicinal Chemistry Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 17-85,
Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
bDiscovery Research Center, Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 17-85,
Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
cPharmacology Research Laboratories I, Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 17-85,
Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
Received 27 November 2007; revised 7 February 2008; accepted 8 February 2008
Available online 13 February 2008
Abstract—A novel series of 4-phenylisoquinolones were synthesized and evaluated as c-Jun N-terminal kinase (JNK) inhibitors. Ini-
tial modification at the 2- and 3-positions of the isoquinolone ring of hit compound 4, identified from high-throughput screening, led
to the lead compound 6b. The optimization was carried out using a JNK1-binding model of 6b and several compounds exhibited
potent JNK inhibition. Among them, 11g significantly inhibited cardiac hypertrophy in rat pressure-overload models without affect-
ing blood pressure and the concept of JNK inhibitors as novel therapeutic agents for heart failure was confirmed.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
matory diseases, neurological diseases, metabolic dis-
eases and cancer has also been reported.2
The c-Jun N-terminal kinases (JNKs)1 form one sub-
family of the mitogen-activated protein kinase (MAPK)
group of serine–threonine protein kinases. JNK iso-
forms are created by alternative splicing of messenger
RNA transcripts derived from three genes (JNK1,
JNK2, JNK3). JNK1 and JNK2 exhibit a broad tissue
distribution, whereas JNK3 is predominantly localized
in the brain and testes. JNK activation leads to the
phosphorylation of a number of transcription fac-
tors—most notably the c-Jun component of AP-1—
and cellular proteins, particularly those associated with
apoptosis (for example, Bcl2, p53 and so on). In this
way, JNKs can serve as critical mediators of a variety
of extra-cellular stimuli. Furthermore, activation of
JNK in many diseases such as autoimmune and inflam-
Numerous companies have been pursuing discovery
programs aimed at identifying small-molecule inhibitors
of this enzyme. Recent progress in the X-ray crystal
structure analysis of the members of the MAPK families
such as ERK,3 p384 and JNK5 has also contributed to
design of potent and selective JNK inhibitors. Several
JNK inhibitors have been reported in the literature
(Fig. 1). Celgene (Signal Pharmaceutical) reported the
discovery of a series of pyrazoloanthrone derivatives 1
(SP600125)6 as JNK inhibitors. Serono reported benzaz-
ole-acetonitrile derivatives 2 (AS601245)7 which demon-
strated efficacy by oral administration in an
experimental model of rheumatoid arthritis. AstraZene-
ca also disclosed a series of aminoindazoles, represented
by 3,8 as potent JNK inhibitors with good pharmacoki-
netic profiles. Additionally, some short JNK-inhibitory
peptides derived from JNK-interacting protein
1
Keywords: JNK; JNK inhibitor; Isoquinolone; SAR; Modeling; Heart
failure; Cardiac hypertrophy; X-ray crystallography.
(JIP1), the protein ‘scaffold’ in the JNK pathway, have
been identified to investigate the therapeutic potential
of the JNK inhibitors.9
*
Corresponding author. Tel.: +81 6 6300 6836; fax: +81 6 6300
6306; e-mail: Asano_Yasutomi@takeda.co.jp
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.027

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Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
Figure 1. The structures of JNK inhibitors.
Recently, several reports have indicated that JNK plays
important roles in the progression of heart failure. The
development of cardiac hypertrophy in a murine pres-
sure-overload model was suppressed without affecting
blood pressure by JNK inhibition using dominant nega-
tive SEK-1, a kinase upstream of JNK.10 Additionally,
the MKK-7 over-expressing mice caused heart failure.11
Moreover, JNK was activated in the heart of the pa-
tients with heart failure following myocardial infarc-
tion.12 This accumulated evidence suggested that JNK
inhibitors could be useful in the treatment of heart fail-
ure. On the other hand, some angiotensin II type 1
receptor blockers (ARB) also attenuate pressure-over-
load-induced cardiac hypertrophy in rats. However, this
suppressive effect of ARB is accompanied by lowering
blood pressure.13 Therefore, we launched an investiga-
tion of JNK inhibitors as novel therapeutic agents for
heart failure that should not affect blood pressure.
position of 5a was converted to a benzyl (6b), phenethyl
(6c) or phenylpropyl (6d) group. The inhibitory activity
of 6b was most potent (IC50 = 86 nM) after the initial
modification. Compound 6b was selected as a lead com-
pound and further optimization was carried out.
The binding mode of lead compound 6b to JNK1 was
predicted by a docking study using a JNK1 model based
on crystallographic data for JNK3. The amino acid res-
idues near the ATP-binding site of JNK1 and JNK3 are
well conserved and only two amino acid residues are dif-
ferent. Thus, Ser125 and Leu144 of JNK3 are substi-
tuted to Gly87 and Ile106 in JNK1, respectively. As
for Ser125, the side chain is oriented toward the outside
of the ATP pocket (solvent). Leu144 is situated at the
back of the pocket and is out of range of hinge binders.
It was assumed that compound 6b was an ATP compet-
itive inhibitor. As shown in Figure 3, the docking study
suggested that compound 6b filled the adenine-binding
site by forming a hydrogen bond between the carbonyl
oxygen of the isoquinolone group and the backbone
NH group of Met111 in the hinge region of JNK1.
Asp169 is close to the methoxycarbonyl group at the
3-position and the phenyl group at the 4-position. In
addition to Asp169, other amino acid residues such as
Lys55, Gln37 and Arg174 are near the phenyl group
at the 4-position. The bromine atom at the 6-position
occupies the hydrophobic pocket. Asn114 and Gln117
were found in front of the benzyl group, and it was
thought that functionality at the 4-position of the benzyl
Isoquinolone derivative 4 was identified from high-
throughput screening (HTS) for JNK1 inhibitors. The
hit compound 4 showed relatively potent enzymatic
inhibitory activity with an IC50 value of 1400 nM for
JNK1, and it was also selective over two other closely
related MAP kinases, p38 MAP kinase and ERK1
(Fig. 2).
During an initial SAR study of the hit compound 4, the
hydroxymethyl group at the 3-position and the cyclo-
hexylmethyl group at the 2-position were modified due
to their metabolic instability and the possibility of
enhancing JNK-inhibitory activity (Table 1). Substitu-
tions of the hydroxymethyl group for carboxylic acid
(5a) and carboxylic acid methyl ester (6a) at the 3-posi-
tion were performed. As a result, compounds 5a and 6a
showed increased activity compared to the hit com-
pound 4. In addition, the cyclohexylmethyl group at 2-
Table 1. Effect of substitutions on the isoquinolone ring
Compound R1
R2
a
IC50 (nM)
JNK1 p38a
CH2-c-Hex CH2OH 1400
ERK1
4
>1100 >1100
>1100 >1100
>1100 >1100
>1100 >1100
>1100 >1100
>1100 >1100
5a
6a
6b
6c
6d
CH2-c-Hex CO2H
CH2-c-Hex CO2Me
860
450
86
CH2Ph
(CH2)2Ph
(CH2)3Ph
CO2Me
CO2Me
CO2Me
1100
190
Figure 2. Structure and enzymatic inhibitory activity of hit compound
4.
a Enzymatic inhibitory activity.

Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
4717
Figure 3. Predicated binding mode of lead compound 6b to JNK1.
group could suitably form hydrogen bonds with Asn114
or Gln117.
2. Chemistry
Initial modification at the 2- and 3-positions of isoqui-
nolone ring was carried out as shown in Scheme 1. Fri-
edel–Crafts reaction of commercially available 4-
bromophthalic anhydride 7 with benzene and subse-
quent alkylation with diethyl bromomalonate followed
by treatment with acetic acid and concentrated hydro-
chloric acid gave 1-oxoisochromene 8. Reaction of 8
with the appropriate amines followed by treatment with
4 N hydrogen chloride in ethyl acetate afforded isoqui-
nolones 5. Methyl esters 6 were obtained by methylation
of carboxylic acids 5 under basic condition. Conversion
of carboxylic acid 5a to the acid chloride followed by
reduction with sodium borohydride yielded alcohol 4.
From consideration of binding model described
above, we planned the following strategy to increase
the potency of compound 6b: (1) installation of
hydrogen bond-forming functionality at the 3- or 4-
position of the benzyl group in anticipation of inter-
action with Asn114 and/or Gln117, (2) optimization
of the 6-position substituent aimed at forming hydro-
phobic interaction with the hydrophobic pocket, (3)
introduction of substituents onto the pendant phenyl
ring at the 4-position in order to form interactions
with polar amino acid residues such as Asp169,
Lys55, Gln37 and Arg174. In this report we describe
the design, synthesis, SAR, and pharmacological
properties of the isoquinolone derivatives, and also
a co-crystal structure of an isoquinolone derivative
with JNK3.
Installation of various functional groups onto the 2-ben-
zyl group was performed as shown in Scheme 2. Reac-
tion of 1-oxoisochromene 8 with ammonia followed by
Scheme 1. Modification of substituent at 2 and 3-position. Reagents and conditions: (a) AlCl3, benzene, rt; (b) K2CO3, BrCH(CO2Et)2, rt; (c) concd
HCl, AcOH, 120 ꢁC; (d) RNH2, MeOH, rt; (e) 4 N-HCl in AcOEt, rt; (f) MeI, K2CO3, DMF, rt; (g) (COCl)2, DMF, THF, rt; (h) NaBH4, THF, 1,2-
dimethoxyethane, 0 ꢁC.

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Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
Scheme 2. Introduction of substituent on the benzene ring of 2-benzyl group. Reagents and conditions: (a) NH3, MeOH, rt; (b) 4 N-HCl in AcOEt,
rt; (c) H2SO4, MeOH, reflux; (d) ArCH2X, basic resine, DMF, rt; (e) NaH, ArCH2Br, DMF, rt; (f) ArCH2NH2, MeOH, rt; (g) NaOH, H2O, MeOH,
THF, rt; (h) RCl, Et3N, THF, rt; (i) succinic anhydride, Et3N, THF, rt.
dehydration under acidic conditions and esterification
afforded isoquinolone 9. Isoquinolones 11b–d with sub-
stitutions at the 2-position were obtained by the reaction
of isoquinolone 9 with the appropriate benzyl halide.
Carboxylic acid 8 was esterified to give methyl ester
10, which was reacted with benzylamine derivatives fol-
lowed by dehydration under acidic conditions to afford
isoquinolones 11a, e, g, h. Alkaline hydrolysis of esters
11e yielded carboxylic acids 11f. Sulfonylation or acyla-
tion of amino derivative 11h yielded sulfonyl or acyl
derivatives 12a–d.
13e. Chlorine compound 19b and fluorine compound
16a were prepared by a similar reaction shown in
Schemes 1 and 2 using commercially available phthalic
anhydrides 14a (Cl) and 14b (F) as starting materials.
Reaction of fluorine compound 16a with sodium meth-
oxide followed by methylation of the carboxylic acid
afforded 6-methoxy compound 17.
Introduction of substituents into the pendant phenyl
ring at the 4-position was carried out as shown in
Scheme 4. Successive treatment of 4-chlorophthalic
anhydride 14a with sodium methoxide, methyl N-(4-
(methylsulfonyl)benzyl)glycinate and then sodium meth-
oxide yielded isoquinolone 20, which was reacted with
sodium hydride and then N-phenyltrifluoromethanesulf-
onimide to give trifluoromethanesulfonate 21. Suzuki
coupling reaction of 21 with the appropriate phenyl
boronic acids provided isoquinolones 22a and 22c, hav-
ing a substituted pendant phenyl group at the 4-posi-
tion. Reduction of aldehydes 22a and 22c using
Conversion of the substituent at the 6-position of isoqu-
inolone derivatives is shown in Scheme 3. Catalytic
hydrogenation of bromides 6b and 11g gave 13a and
19a, respectively. Suzuki coupling of 6b with the appro-
priate alkylboronic acids yielded 6-alkylated compounds
13b, c, respectively. Diester 13d was also synthesized
from 6b by methoxycarbonylation using palladium cat-
alyst. Alkaline hydrolysis of 13d afforded carboxylic acid

Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
4719
Scheme 3. Conversion of substituent at 6-position. Reagents and conditions: (a) 10% Pd–C, 1 atm H2 gas, methanol, THF, rt, to 13a; (b) Rb(OH)2,
Pd(PPh3)4, K2CO3, toluene, THF, 100 ꢁC, to 13b,c; (c) Pd(OAc)2, DPPF, Et3N, 1 atm CO2 gas, MeOH, DMF, 50 ꢁC, to 13d; (d) 8 N NaOH, THF,
rt; (e) AlCl3, benzene, rt; (f) K2CO3, BrCH(CO2Et)2, rt; (g) concd HCl, AcOH, 120 ꢁC; (h) PhCH2NH2, MeOH, rt; (i) 4 N HCl in AcOEt, rt; (j) 28%
NaOMe in MeOH, reflux; (k) MeI, K2CO3, DMF, rt; (l) concd H2SO4 MeOH, reflux; (m) ArCH2NH2, MeOH, reflux.
sodium hydroborate gave hydroxymethyl derivatives
22b and 22d, respectively.
pound 6b. In contrast, a methoxy group at the 2- or 3-
position of the benzyl group (11a and 11b) did not in-
crease the activity compared to 6b. In a similar manner,
methoxy carbonyl or carboxyl groups at the 4-position
increased activity (IC50 values for 11e and 11f were 14
and 20 nM, respectively), while the methoxy carbonyl
group at the 3-position showed no increase (11d,
IC50 = 89 nM). Introduction of substituents at the 4-po-
sition that can interact as hydrogen bond acceptors,
such as the methylsulfonyl group (11g, IC50 = 30 nM)
also increased potency compared to 6b. In the same
way, substitution with an amino group at the 4-position
increased JNK1-inhibitory activity (11h, IC50 = 43 nM)
compared to 6b, and furthermore methylsulfonylation
of the amino group of 11h further increased potency
(12a, IC50 = 12 nM). Replacement of the methyl group
of 12a by a phenyl group maintained potent activity
3. Results and discussion
The synthesized isoquinolone derivatives were evaluated
for their ability to inhibit JNK1 using a radiometric as-
say and results are shown in Tables 2–4.
Initially, various hydrogen bond-forming functional
groups were installed into the benzyl group at the iso-
quinoline 2-position with the aim of increasing interac-
tions with Asn114 and/or Gln117 (Table 2).
A
methoxy group, acting as a hydrogen bond acceptor at
the benzyl 4-position (11c, IC50 = 42 nM) increased
JNK-inhibitory activity twofold compared to lead com-

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Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
Scheme 4. Introduction of substituent on the benzene ring at 4-position of isoquinolone ring. Reagents and conditions: (a) NaOMe, MeOH, rt; (b)
ArNHCH2CO2Me, WSC, HOBt–H2O, Et3N, THF, CH3CN, rt; (c) 28% NaOMe in MeOH, MeOH, rt; (d) NaH, PhN(SO2CF3)2, DMF, rt; (e)
ArB(OH)2, Pd(PPh4)3, Na2CO3, H2O, toluene, EtOH, 80 ꢁC; (f) NaBH4, MeOH, THF, 0 ꢁC.
Table 2. Substituent effect on the benzene ring of 2-benzyl group
Table 3. Substituent effect on 6-position of isoquinolone ring
Compound
R
JNKla IC50 (nM)
6b
H
2-OMe
86
100
79
Compound
R1
R2
JNKIa IC50 (nM)
11a
11b
11e
11d
11e
11f
11g
11h
12a
12b
12c
12d
13a
13b
13c
6b
H
H
95
61
3-OMe
4-OMe
Me
n-Bu
Br
H
42
89
H
1500
86
3-CO2Me
4-CO2Me
H
14
20
17
OMe
CO2H
H
H
140
8800
59
4-CO2H
4-SO2Me
4-NH2
13e
19a
19b
11g
H
30
43
SO2Me
SO2Me
SO2Me
Cl
Br
28
30
4-NHSO2Me
4-NHSO2Ph
4-NHSO2(C6H4-4-CO2H)
4-NHCO(CH2)2CO2H
12
48
a Inhibitory activity against JNK1.
3.3
9.2
a Inhibitory activity against JNK1.
tency (13c, IC50 = 1500 nM). Installation of more polar
substituents like methoxy or carboxyl (17 and 13e) re-
sulted in significant loss of potency. Furthermore, sub-
stituents that enhanced JNK-inhibitory activity in the
benzyl series such as hydrogen and halogens were also
introduced into the 4-methylsulfonylbenzyl series. The
compounds prepared showed potent activity, in particu-
lar 6-chloro and 6-bromo derivatives 19b and 11g exhib-
ited highly potent inhibition (IC50 values of 28 and
30 nM, respectively).
(12b, IC50 = 48 nM). Interestingly, introduction of a car-
boxyl group into the terminal benzene ring of 12b in-
creased the potency further and compound 12c
exhibited the most potent JNK1 inhibition
(IC50 = 3.3 nM). Furthermore, compound 12d possess-
ing a methylene linker instead of a phenylene linker also
showed similarly potent activity (IC50 = 9.2 nM).
Next, the substituent at the 6-position of the isoquino-
lone ring was modified for a series of compounds with
benzyl or 4-methylsulfonyl benzyl at the 2-position (Ta-
ble 3). Compounds with small lipophilic substituents
such as hydrogen, methyl, or bromine showed potent
activity (IC50 values of 13a, 13b, and 6b were 95, 61
and 86 nM, respectively). Substitution with relatively
hindered lipophilic groups such as n-butyl reduced po-
Lastly, various substituents on the pendant phenyl ring
at the 4-position of isoquinolone ring were investigated,
expecting interaction with polar amino acid residues of
JNK1 such as Asp169, Lys55, Gln37 and Arg174. We
prepared several compounds with substituted phenyl
groups, and among them the 3- and 4-hydroxymethyl
groups showed the same level of potency as non-substi-

Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
Table 4. Substituent effect on pendant phenyl ring
4721
Compound
R
JNKla IC50 (nM)
19b
23b
23a
H
28
63
21
4-CH2OH
3-CH2OH
a Inhibitory activity against JNK1.
tuted compound 19b. However, no remarkable improve-
ment in potency was observed and it is unclear whether
these substituents interact with the protein.
From the results of optimization studies of isoquinolone
6b, we found that installation of hydrogen bond accept-
ing and/or donating groups such as methanesulfonyl
and carboxyl groups at 4-position of the 2-benzyl group
increased JNK1-inhibitory activity, and that small lipo-
philic substituents such as chlorine and bromine atoms
were favorable for substituents at the 6-position. How-
ever, no interaction with JNK1 was indicated by the
modification of substituents on the phenyl group at
the isoquinolone 4-position.
Due to its promising in vitro activities (including inhibi-
tion of anisomycin-induced c-Jun phosphorylation in
H9c2 cells derived from rat myocardia), ADME profile
and phamacokinetic parameters, compound 11g was se-
lected for further investigation of biological activity (Ta-
ble 5).
Compound 11g was evaluated for selectivity with re-
spect to the JNK isoforms (JNK1-3) and other serine–
threonine kinases. It showed similarly potent inhibition
against all JNK subtypes (IC50 values: 30, 34 and 30 nM
for JNK1, JNK2 and JNK3, respectively) and showed
extremely high selectivity for JNK over other serine–
threonine kinases tested (Table 6).
Compound 11g was evaluated for suppressive effects on
the development of cardiac hypertrophy in a rat pres-
sure-overload model. It was orally administered to aor-
tic-banded rats, once a day for 7 days in total. One day
after final administration, the suppression of cardiac
hypertrophy was evaluated by left ventricular weight/
body weight ratio (LVW/BW). Compound 11g exhibited
significant suppression of cardiac hypertrophy at a dose
of 10 mg/kg (Fig. 4). Additionally, the extent of c-Jun
phosphorylation in the heart and the extent of cardiac
hypertrophy in 11g-treated and untreated pressure-over-
load rats were examined (Fig. 4). Compound 11g also
inhibited c-Jun phosphorylation at doses of 3 and

4722
Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
Table 6. Inhibitory activities of 11g against several serine–threonine kinases
Kinase
IC50 (nM)
% Inhibition 10 lM
Kinase
IC50 (nM)
% Inhibition 10 lM
JNK1
JNK2
JNK3
p38a
30
34
IKKb
TAK1
PKCh
MEKK
>11,000
>11,000
>11,000
>11,000
20
12
8
30
>11,000
>11,000
18
2
8
ERK1
Figure 4. Inhibitory effect of 11g on the c-jun phosphorylation in hearts and cardiac hypertorphy in aortic-banded rats. Data are represented as
means SEM. *p < 0.025 versus Vehicle (Williams test).
10 mg/kg even after 24 h of final administration. From
these results, we concluded that inhibition of JNK activ-
ity in the heart led to the suppression of cardiac hyper-
trophy in our rat model.
tion of a hydrogen bond acceptor at the 4-position of
the 2-benzyl group led to potent JNK inhibition, sug-
gesting that these functional groups might interact with
Lys68. The crystal structure also showed a small hydro-
phobic space around the 6-bromine atom of 12d and it
could explain the decrease in inhibitory activity found
for isoquinolones containing a hindered substituent or
hydrophilic functional group at this position.
The effects of 11g on systolic blood pressure and cardiac
hypertrophy in aortic-banded rats (30 mg/kg, po) were
also checked. As shown in Figure 5, compound 11g sig-
nificantly inhibited cardiac hypertrophy, however, it did
not affect systolic blood pressure even at a dose of
30 mg/kg. These results indicated the possibility of
JNK inhibitor as novel therapeutic agents for heart fail-
ure without hypotensive activity.
4. Conclusion
With the goal of discovering novel JNK inhibitors, we
carried out the initial optimization of isoquinolone
derivatives based on the hit compound 4 found by
HTS. As a result, we discovered that a hydrogen
bond-accepting functional group at the 4-position of
the benzyl group led to an increase of JNK inhibition.
Co-crystallization of isoquinolone-based JNK inhibitor
and JNK3 was investigated and the crystal structure of
the complex between 12d and JNK3 was obtained. It
showed that compound 12d interacts with JNK3 in the
ATP-binding site (Fig. 6). The binding mode of 12d
was slightly different from that of binding model shown
in Figure 3. Met149 on the main chain of the hinge re-
gion of JNK3 (corresponding to Met111 for JNK1)
forms a hydrogen bond with 1-position carbonyl oxygen
of 12d as predicted by the original binding model. An
unexpected electrostatic interaction is observed between
the 4-carboxyl group on the benzyl group of 12d and
Lys68 (corresponding to Lys30 of JNK1, that is posi-
tioned out of Fig. 3), but no interaction is observed with
Asn152 (corresponding to Asn114 of JNK1). Introduc-
Among the isoquinolone derivatives synthesized, meth-
ylsulfonyl derivative 11g showed potent inhibitory activ-
ity against JNK1 and was found to be a JNK selective
and ATP competitive inhibitor. From an X-ray crystal
structure analysis of the complex of 12d and JNK3,
interactions between the isoquinolone derivatives and
the surrounding amino acid residue of JNK3 were clar-
ified. Methylsulfonyl derivative 11g showed significant
suppression of cardiac hypertrophy in a rat model after
oral administration, as well as no hypotensive effect at a

Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
4723
Figure 5. Inhibitory effect of 11g on the c-jun phosphorylation in hearts and cardiac hypertroph in aortic-banded rats. Data are represented as
means SEM. *p < 0.025 versus Vehicle (Student’s t test).
Figure 6. The co-crystal structure of 12d and JNK3.
dose of 30 mg/kg. These results suggested that JNK
inhibitor can be a novel therapeutic agent for heart fail-
ure without affecting blood pressure.
i.d. · 50 mm) with acetonitrile/water mobile phase, and
a HP-1100 (Agilent Technologies) apparatus for moni-
toring at 220 nm. Preparative reversed-phase high-per-
formance
liquid
chromatography
(Preparative
RPHPLC) was performed on a Gilson, Inc. high-
throughput purification system with acetonitrile/water
mobile phase. Reaction progress was determined by
thin-layer chromatography (TLC) analysis on silica gel
60 F254 plates (Merck). Chromatographic purification
was carried out on silica gel columns (Kieselgel 60,
0.063–0.22 mm, Merck) or on Purif-Pack (SI 60 lm or
NH 60 lm, Fuji Silysia, Ltd). Reagents and solvents
were obtained from commercial sources and used with-
out further purification. Abbreviations are used as fol-
lows: CDCl3, deuterated chloroform; DMSO-d6,
dimethyl sulfoxide-d6; AcOEt, ethyl acetate; DMF,
N,N-dimethylformamide; MeOH, methanol; THF, tet-
rahydrofuran; EtOH, ethanol; DMSO, dimethyl
sulfoxide.
5. Experimental
5.1. Chemistry
Melting points were determined on a Yanagimoto micro
melting point apparatus and were not corrected. Proton
nuclear magnetic resonance (1H NMR) spectra were re-
corded on Varian Gemini-200 (200 MHz), Varian Mer-
cury-300 (300 MHz), or Bruker DPX-300 (300 MHz)
instruments. Chemical shifts are reported as d values
(ppm) down field from internal TMS of the indicated or-
ganic solutions. Peak multiplicities are expressed as fol-
lows: Abbreviations are used as follows: s, singlet; d,
doublet; t, triplet; q, quartet; dd, doublet of doublet;
dt, doublet of triplet; br s, broad singlet; m, multiplet.
Coupling constants (J values) are given in hertz (Hz).
Elemental analyses were carried out by Takeda Analyt-
ical Laboratories. LC/MS (ESI+) was performed on a
Micromass ZMD instrument, using a CAPCELL PAK
UG-120 ODS (Shiseido Co., Ltd) column (2.0 mm
5.1.1. 6-Bromo-1-oxo-4-phenyl-1H-isochromene-3-car-
boxylic acid (8). To a solution of 4-bromophthalic acid
anhydride 7 (100 g, 0.441 mol) in benzene (500 ml) was
added aluminum chloride (118 g, 0.885 mol), and the
mixture was stirred at room temperature for 1 h and

4724
Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
subsequently refluxed for 30 min. After cooling the reac-
tion mixture, it was diluted with AcOEt (200 ml) and
poured into ice water. Concentrated hydrochloric acid
(50 ml) was added and the mixture was stirred at room
temperature for 30 min. The organic layer was separated
and washed successively with 4 N hydrochloric acid,
water and brine, dried over anhydrous sodium sulfate
and concentrated under reduced pressure until crystals
precipitated. The crude crystals were collected by filtra-
tion, and recrystallized from toluene–AcOEt to give 2-
benzoyl-4-bromobenzoic acid (59.0 g, 44%) as colorless
crystals. To a solution of 2-benzoyl-4-bromobenzoic
acid (57.6 g, 0.189 mol) in acetone (500 ml) was added
potassium carbonate (26.1 g, 0.189 mol), and the mix-
ture was stirred at room temperature for 10 min to allow
precipitation of potassium salt. Then diethyl bromomal-
onate (54.2 g, 0.226 mol) and DMF (25 ml) were added,
and the mixture was stirred at room temperature for
20 h. The reaction mixture was concentrated under re-
duced pressure, and AcOEt (200 ml) and water
(200 ml) were added to the residue. The mixture was stir-
red at room temperature for 30 min. The organic layer
was separated and washed with water and brine, dried
over anhydrous sodium sulfate and concentrated under
reduced pressure. A mixed solution of acetic acid
(200 ml) and concentrated hydrochloric acid (200 ml)
was added to the obtained residue, and the mixture
was stirred at 120 ꢁC for 15 h. After cooling, the reaction
mixture was concentrated under reduced pressure, and
the residue was dissolved in AcOEt, washed with water
and brine, dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The obtained res-
idue was crystallized from diisopropyl ether and col-
lected by filtration to give 8 (53.0 g, 81%) as colorless
Compound 5a: mp 221–223 ꢁC. 1H NMR (DMSO-d6) d:
0.80–1.30 (6H, m), 1.50–1.80 (4H, m), 1.80–2.00 (1H,
m), 3.93 (2H, d, J = 7.2 Hz), 7.14 (1H, d, J = 1.8 Hz),
7.30–7.40 (2H, m), 7.45–7.60 (3H, m), 7.74 (1H, dd,
J = 1.8, 8.4 Hz), 8.25 (1H, d, J = 8.4 Hz). LC/MS
(ESI+): 440, 442. Anal. Calcd for C23H22BrNO3: C,
62.74; H, 5.04; N, 3.18. Found: C, 62.40; H, 5.15; N,
3.14.
1
Compound 5c: H NMR (DMSO-d6) d: 2.95–3.10 (2H,
m), 4.05–4.20 (2H, m), 7.17 (1H, d, J = 1.8 Hz), 7.20–
7.40 (7H, m), 7.45–7.55 (3H, m), 7.78 (1H, dd, J = 1.8,
8.8 Hz), 8.28 (1H, d, J = 8.8 Hz). LC/MS (ESI+): 448,
450.
1
Compound 5d: H NMR (DMSO-d6) d: 2.06 (2H, m),
2.66 (2H, t, J = 7.2 Hz), 4.01 (2H, t, J = 7.8 Hz), 7.10–
7.40 (7H, m), 7.45–7.55 (4H, m), 7.74 (1H, dd, J = 1.8,
8.7 Hz), 8.24 (1H, d, J = 8.7 Hz). LC/MS (ESI+): 462,
464.
5.1.3. 6-Bromo-2-cyclohexylmethyl-3-hydroxymethyl-4-
phenylisoquinolin-1(2H)-one (4). To a solution of 5a
(200 mg, 0.454 mmol) in THF (5 ml) were added oxalyl
chloride (0.119 ml, 1.41 mmol) and DMF (0.2 ml), and
the mixture was stirred at room temperature for 1 h.
The solvent was evaporated under reduced pressure. A
solution of the residue in THF (3 ml) was added drop-
wise to a suspension of sodium borohydride (52 mg,
1.37 mmol) in 1,2-dimethoxyethane. The mixture was
stirred under ice-cooling for 1 h. and poured into 1 N
hydrochloric acid and the mixture was extracted with
AcOEt. The extract was washed successively with satu-
rated aqueous sodium hydrogen carbonate and satu-
rated brine, and dried over anhydrous magnesium
sulfate. The solvent was evaporated under reduced pres-
sure and the residue was purified by silica gel column
chromatography (hexane/AcOEt) to give 4 (10.0 mg,
5.2%). Mp 181–182 ꢁC. 1H NMR (CDCl3) d: 1.00–
1.30(4H, m), 1.60–2.00 (7H, m), 4.22 (2H, d,
J = 6.8 Hz), 4.45 (2H, s), 7.12 (1H, d, J = 1.8 Hz),
7.25–7.35 (2H, m), 7.40–7.55 (4H, m), 8.27 (1H, d,
J = 8.8 Hz). LC/MS (ESI+): 426, 428. Anal. Calcd for
C23H24BrNO2: C, 64.79; H, 5.67; N, 3.29. Found: C,
64.97; H, 5.74; N, 3.40.
1
crystals. H NMR (CDCl3) d: 7.18–7.30 (3H, m), 7.46–
7.56 (3H, m), 7.77 (1H, dd, J = 1.8, 8.4 Hz), 8.26 (1H,
d, J = 8.4 Hz).
5.1.2. 2-Benzyl-6-bromo-1-oxo-4-phenyl-1,2-dihydroiso-
quinoline-3-carboxylic acid (5), typical procedure. To a
solution of 8 (1.00 g, 29.0 mmol) in MeOH (20 ml) was
added benzylamine (3.16 ml, 28.9 mmol), and the mix-
ture was stirred at room temperature for 2 h. The sol-
vent was evaporated under reduced pressure and 1 N
hydrochloric acid was added. The mixture was extracted
with AcOEt. The extract was washed successively with
1 N hydrochloric acid and saturated brine and anhy-
drous magnesium sulfate was added to dry the mixture.
The solvent was evaporated under reduced pressure, and
4 N hydrogen chloride–AcOEt solution (17 ml) was
added to the obtained residue, and the mixture stirred
at room temperature for 18 h. The solvent was evapo-
rated under reduced pressure, and addition of water
gave crystal precipitation. The obtained crystals were
collected by filtration, washed with water and ether
and dried under reduced pressure at 50 ꢁC to give 5b
5.1.4. Methyl 2-benzyl-6-bromo-1-oxo-4-phenyl-1,2-dihy-
droisoquinoline-3-carboxylate (6b), typical procedure. A
mixture of 5b (150 mg, 0.345 mmol), potassium carbon-
ate (95.0 mg, 0.687 mmol), iodomethane (0.043 ml,
0.69 mmol) and DMF (5 ml) was stirred at room tem-
perature for 3 h. After diluting with water, the mixture
was extracted with ether and the extract was washed
with saturated brine, and dried over anhydrous magne-
sium sulfate. The solvent was evaporated under reduced
pressure. AcOEt and hexane were added to the residue,
and the mixture was crystallized to give 6b (96.0 mg,
1
(921 mg, 73%). H NMR (DMSO-d6) d: 5.28 (2H, s),
1
7.15–7.40 (8H, m), 7.45–7.55 (3H, m), 7.77 (1H, dd,
J = 1.8, 8.4 Hz), 8.26 (1H, d, J = 8.4 Hz). LC/MS
(ESI+): 434, 436.
62%). Mp 142.5–143.5 ꢁC. H NMR (CDCl3) d: 3.19
(3H, s), 5.42 (2H, s), 7.20–7.30 (7H, m), 7.35–7.50
(4H, m), 7.66 (1H, dd, J = 2.0, 8.8 Hz), 8.41 (1H, d,
J = 8.4 Hz). LC/MS (ESI+): 448, 450. Anal. Calcd for
C24H18BrNO3: C, 64.30; H, 4.05; N, 3.12. Found: C,
64.54; H, 4.02; N, 3.13.
The following compounds (5a, 5c, 5d) were prepared
from 8 in a manner similar to that used for 5b.

Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
4725
The following compounds (6a, 6c, 6d) were prepared in
a manner similar to that used for 6b.
were suspended in DMF (2 ml), and the suspension
was shaken at room temperature for 15 h. using a shak-
ing apparatus. After filtration, the solvent was evapo-
rated under reduced pressure, and the obtained residue
was purified by preparative RPHPLC to give 11c
(10 mg, 14%). Mp 198–199 ꢁC. 1H NMR (CDCl3) d:
3.24 (3H, s), 3.76 (3H, s), 5.35 (2H, s), 6.81 (2H, d,
J = 8.7 Hz), 7.13–7.32 (4H, m), 7.33–7.49 (4H, m), 7.64
(1H, dd, J = 1.8, 8.5 Hz), 8.40 (1H, d, J = 8.5 Hz). LC/
MS (ESI+): 478, 480. Anal. Calcd for C25H20BrNO4:
C, 62.77; H, 4.21; N, 2.93. Found: C, 62.90; H, 4.22;
N, 2.88.
1
Compound 6a: mp 181–183 ꢁC. H NMR (CDCl3) d:
0.95–1.30 (6H, m), 1.60–1.90 (5H, m), 3.46 (3H, s),
3.96 (2H, d, J = 7.4 Hz), 7.25–7.40 (3H, m), 7.40–7.50
(3H, m), 7.63 (1H, dd, J = 1.8, 8.4 Hz), 8.36 (1H, d,
J = 8.4 Hz). LC/MS (ESI+): 454, 456. Anal. Calcd for
C24H24BrNO3: C, 63.44; H, 5.32; N, 3.08. Found C,
63.40; H, 5.46; N, 3.00.
1
Compound 6c: mp 179–180 ꢁC. H NMR (CDCl3) d:
3.12 (2H, t, J = 8.1 Hz), 3.50 (3H, s), 4.16 (2H, t,
J = 8.1 Hz), 7.20–7.35 (7H, m), 7.38 (1H, d,
J = 1.8 Hz), 7.45–7.50 (3H, m), 7.66 (1H, dd, J = 1.8,
8.4 Hz), 8.39 (1H, d, J = 8.4 Hz). LC/MS (ESI+): 462,
464. Anal. Calcd for C25H20BrNO3: C, 64.95; H, 4.36;
N, 3.03. Found: C, 64.73; H, 4.46; N, 3.08.
11b was prepared from 9 in a manner similar to that
used for 8c.
1
Compound 11b: mp 127–129 ꢁC. H NMR (CDCl3) d:
3.22 (3H, s), 3.75 (3H, s), 5.38 (2H, s), 6.72–6.88 (3H,
m), 7.20 (1H, t, J = 7.8 Hz), 7.24–7.33 (2H, m), 7.34–
7.50 (4H, m), 7.65 (1H, dd, J = 1.9, 8.3 Hz), 8.40 (1H,
d, J = 8.3 Hz). LC/MS (ESI+): 478, 480. Anal. Calcd
for C25H20BrNO4: C, 62.77; H, 4.21; N, 2.93. Found:
C, 62.82; H, 4.35; N, 3.03.
1
Compound 6d: mp 176.5–177 ꢁC. H NMR (CDCl3) d:
2.05–2.25 (2H, m), 2.74 (2H, t, J = 7.2 Hz), 3.74 (3H,
s), 3.95–4.10 (2H, m), 7.15–7.40 (8H, m), 7.45–7.50
(3H, m), 7.65 (1H, dd, J = 1.8, 8.4 Hz), 8.37 (1H, d,
J = 8.4 Hz). LC/MS (ESI+): 476, 478. Anal. Calcd for
C26H22BrNO3: C, 65.55; H, 4.66; N, 2.94. Found: C,
64.35; H, 4.77; N, 3.01.
5.1.7. Methyl 6-bromo-2-[3-(methoxycarbonyl)benzyl]-1-
oxo-4-phenyl-1,2-dihydroisoquinoline-3-carboxylate (11d).
To a solution of 9 (1.30 g, 3.63 mmol) in DMF (20 ml)
was added sodium hydride (60% in mineral oil,
216 mg, 5.40 mmol), and the mixture was stirred at
room temperature for 20 min. Methyl 3-(bromo-
methyl)benzoate (990 mg, 4.32 mmol) was added, and
the mixture was stirred at room temperature for 5 h.
The solvent was evaporated under reduced pressure,
and 1 N hydrochloric acid was added. The mixture
was extracted with AcOEt. The extract was washed with
saturated brine and dried over anhydrous sodium sul-
fate. The solvent was evaporated under reduced pres-
sure, and the obtained residue was purified by silica
gel column chromatography (hexane/AcOEt) to give
5.1.5. Methyl 6-bromo-1-oxo-4-phenyl-1,2-dihydroiso-
quinoline-3-carboxylate (9). To a solution of 8 (15.0 g,
43.5 mmol) in MeOH (100 ml) was added 12% ammo-
nia/MeOH solution (60 ml), and the mixture was stirred
at room temperature for 24 h. The reaction mixture was
concentrated under reduced pressure, and 1 N hydro-
chloric acid was added to the obtained residue. The mix-
ture was extracted with AcOEt. The organic layer was
dried and concentrated under reduced pressure and
4 N hydrochloric acid AcOEt solution (30 ml) was
added to the obtained oily substance. The mixture was
stirred at room temperature for 2 h. The reaction mix-
ture was concentrated under reduced pressure. The ob-
tained crystal was washed with water, and dried under
reduced pressure to give 6-bromo-1-oxo-4-phenyl-1,2-
dihydroisoquinoline-3-carboxylic acid as colorless crys-
tals. To the obtained crystals was added a solution of
concentrated sulfuric acid (40 ml) in MeOH (400 ml),
and the mixture was refluxed for 20 h. The reaction mix-
ture was concentrated under reduced pressure, and the
residue was neutralized with aqueous potassium carbon-
ate solution and extracted with CHCl3. The organic
layer was washed with water and brine, dried over anhy-
drous sodium sulfate, and concentrated under reduced
pressure to give 9 (6.51 g, 42%) as colorless crystals.
1H NMR (CDCl3) d: 3.64 (3H, s), 7.20–7.27 (2H, m),
7.32 (1H, d, J = 1.8 Hz), 7.45–7.55 (3H, m), 7.71 (1H,
dd, J = 1.8, 8.4 Hz), 8.37 (1H, d, J = 8.4 Hz), 9.35 (1H,
br s).
1
11d (719 mg, 39%). Mp 151–152 ꢁC. H NMR (CDCl3)
d: 3.23 (3H, s), 3.89 (3H, s), 5.44 (2H, s), 7.26–7.53 (8H,
m), 7.99 (1H, dd, J = 1.8, 8.4 Hz), 7.91–7.95 (2H, m),
8.40 (1H, d, J = 8.7 Hz). LC/MS (ESI+): 506, 508. Anal.
Calcd for C26H20BrNO5: C, 61.67; H, 3.98; N, 2.77.
Found: C, 61.66; H, 4.00; N, 2.67.
5.1.8. Methyl 6-bromo-1-oxo-4-phenyl-1H-isochromene-
3-carboxylate (10). To a solution of 8 (10 g, 29.0 mmol)
in methanol (200 ml) was added sulfuric acid (20 ml)
and the mixture was refluxed for 2 h. The reaction mix-
ture was concentrated, and the residue was neutralized
with aqueous sodium hydrogen carbonate. The resulting
mixture was extracted with AcOEt. The organic layer
was washed with water and brine, dried over sodium sul-
fate and concentrated. The residue was crystallized from
isopropyl ether to give 10 (9.76 g, 94%) as colorless crys-
1
tals. H NMR (CDCl3) d: 3.72 (3H, s), 7.22–7.30 (3H,
m), 7.50–7.57 (3H, m), 7.75 (1H, dd, J = 1.8, 8.4 Hz),
8.26 (1H, d, J = 8.4 Hz).
5.1.6. Methyl 6-bromo-2-(4-methoxybenzyl)-1-oxo-4-phe-
nyl-1,2-dihydroisoquinoline-3-carboxylate (11c), typical
procedure. Compound 9 (54 mg, 0.15 mmol), 4-meth-
oxybenzylbromide (34 mg, 0.17 mmol) and 2-tert-butyli-
mino-2-diethylamino-1,3-dimethyl-perhydro-1,3,2-dia-
zaphosphorine resin (2.2 mmol/g, 0.11 g, 0.24 mmol)
5.1.9. Methyl 6-bromo-2-(4-methylsulfonylbenzyl)-1-oxo-
4-phenyl-1,2-dihydroisoquinoline-3-carboxylate (11g),
typical procedure. A mixture of 10 (575 mg, 1.60 mmol),

4726
Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
1-(4-(methylsulfonyl)phenyl)methanamine
(356 mg,
(ESI+): 506, 508. Anal. Calcd for C26H20BrNO5: C,
61.67; H, 3.98; N, 2.77. Found: C, 61.58; H, 3.99; N,
2.75.
1.92 mmol) and MeOH (4 ml) was heated under reflux
for 12 h. Concentrated sulfuric acid (0.4 ml) was added
to the reaction mixture under ice-cooling, and the mix-
ture was heated under reflux for 2 h. The reaction mix-
ture was concentrated under reduced pressure, and
water was added. The mixture was extracted with
AcOEt, and the organic layer was washed with water,
saturated aqueous sodium hydrogen carbonate solution,
water and brine, dried over anhydrous sodium sulfate,
and concentrated under reduced pressure. The residue
was purified by recrystallization from hexane–AcOEt
to give 11g (329 mg, 39%) as colorless crystals: mp
201–203 ꢁC; 1H NMR (CDCl3) d: 3.02 (3H, s), 3.26
(3H, s), 5.42 (2H, s), 7.26–7.51 (8H, m), 7.69 (1H, dd,
J = 1.8, 8.7 Hz), 7.87–7.91 (2H, m), 8.38 (1H, d,
J = 8.7 Hz); LCMS (ESI+) 526, 528; Anal. Calcd for
C25H20BrNO5S: C, 57.04; H, 3.83; N, 2.66. Found: C,
57.00; H, 3.75; N, 2.54.
5.1.11. 4-((6-Bromo-3-methoxycarbonyl-1-oxo-4-phenyl-
isoquinolin-2(1H)-yl)methyl)benzoic acid (11f). To a mix-
ture of 11e (2.00 g, 3.95 mmol) in MeOH (10 ml) and
THF (20 ml) was added 8 N aqueous sodium hydroxide
solution (1.00 ml, 8.00 mmol), and the mixture was stir-
red at room temperature for 12 h. The solvent was evap-
orated under reduced pressure, and water was added.
The mixture was acidified with 1 N hydrochloric acid,
and extracted with AcOEt. The extract was washed with
saturated brine, and dried over anhydrous sodium sul-
fate. The solvent was evaporated under reduced pres-
sure, and then the obtained residue was recrystallized
from MeOH to give 11f (1.29 g, 66%). Mp 240–241 ꢁC.
1H NMR (CDCl3) d: 3.21 (3H, s), 5.46 (2H, s), 7.25–
7.49 (8H, m), 7.68 (1H, dd, J = 1.8, 8.7 Hz), 8.02 (2H,
d, J = 7.0 Hz), 8.40 (1H, d, J = 8.4 Hz). LC/MS
(ESI+): 492, 494. Anal. Calcd for C25H18BrNO5: C,
60.99; H, 3.69; N, 2.85. Found: C, 60.76; H, 3.82; N,
2.69.
The following compounds (11a, 11h) were prepared
from 10 in a manner similar to that used for 11g.
1
Comound 11a: mp 237–239 ꢁC. H NMR (CDCl3) d:
3.22 (3H, s), 3.80 (3H, s), 5.39 (2H, s), 6.82–6.89 (2H,
m), 7.04 (1H, m), 7.19–7.48 (7H, m), 7.66 (1H, dd,
J = 1.8, 8.7 Hz), 8.37 (1H, d, J = 8.7 Hz). LC/MS
(ESI+): 478, 480. Anal. Calcd for C25H20NO4Br–H2O:
C, 60.50; H, 4.47; N, 2.82. Found: C, 60.35; H, 4.08;
N, 2.83.
5.1.12. Methyl 6-bromo-2-(4-(methylsulfonylamino)benzyl)-
1-oxo-4-phenyl-1,2-dihydroisoquinoline-3-carboxylate (12a),
typical procedure. To a solution of 11h (139 mg,
0.300 mmol) in THF (5 ml) was added triethylamine
(0.083 ml, 0.56 mmol), and then methanesulfonyl chlo-
ride (0.028 ml, 0.36 mmol) was added. The mixture
was stirred at room temperature for 3 h. The solvent
was evaporated under reduced pressure, 1 N hydrochlo-
ric acid was added, and the mixture was extracted with
CH2Cl2. The extract was washed with saturated brine,
and dried over anhydrous sodium sulfate. The solvent
was evaporated under reduced pressure, and the ob-
tained residue was purified by silica gel column chroma-
tography (hexane/AcOEt) to give 12a (90 mg, 55%). Mp
246–247 ꢁC. 1H NMR (CDCl3) d: 2.97 (3H, s), 3.27 (3H,
s), 5.34 (2H, s), 6.60 (1H, s), 7.14 (2H, d, J = 8.7 Hz),
7.26–7.49 (8H, m), 7.66 (1H, dd, J = 1.8, 8.4 Hz), 8.38
(1H, d, J = 8.4 Hz). LC/MS (ESI+): 541, 543. Anal.
Calcd for C25H21N2O5SBr: C, 55.46; H, 3.91; N, 5.17.
Found: C, 55.67; H, 4.03; N, 5.04.
Compound 11h: mp 203.5–204 ꢁC. 1H NMR (CDCl3) d:
3.24 (3H, s), 3.62 (2H, s), 5.31 (2H, s), 6.58 (2H, d,
J = 8.7 Hz), 7.07 (2H, d, J = 8.7 Hz), 7.26–7.48 (6H,
m), 7.64 (1H, dd, J = 1.8, 8.4 Hz), 8.40 (1H, d,
J = 8.4 Hz). LC/MS (ESI+): 463, 465. Anal. Calcd for
C24H19BrN2O3: C, 62.22; H, 4.13; N, 6.05. Found: C,
62.07; H, 4.07; N, 6.10.
5.1.10. Methyl 6-bromo-2-(4-methoxycarbonylbenzyl)-1-
oxo-4-phenyl-1,2-dihydroisoquinoline-3-carboxylate (11e).
To a solution of 10 (3.60 g, 10.0 mmol) in MeOH
(50 ml) was added methyl 4-(aminomethyl)benzoate
(3.16 g, 19.1 mmol), and the mixture was stirred at room
temperature for 18 h. The solvent was evaporated under
reduced pressure, and 1 N hydrochloric acid was added.
The resulting mixture was extracted with AcOEt. The
extract was washed with saturated brine, and dried over
anhydrous sodium sulfate. The solvent was evaporated
under reduced pressure, and the obtained residue was
dissolved in MeOH (100 ml). Concentrated sulfuric acid
(10 ml) was added, and the mixture was heated under re-
flux for 3 h. The solvent was evaporated under reduced
pressure, water was added under ice-cooling, and the
mixture was neutralized with potassium carbonate.
The resulting mixture was extracted with AcOEt, and
the extract was washed with saturated brine, dried over
anhydrous sodium sulfate, and concentrated under re-
duced pressure. The obtained residue was recrystallized
from MeOH to give 11e (3.39 g, 67%). Mp 172–174 ꢁC.
1H NMR (CDCl3) d: 3.18 (3H, s), 3.89 (3H, s), 5.45 (2H,
s), 7.24-7.47 (8H, m), 7.67 (1H, dd, J = 1.8, 8.4 Hz), 7.97
(2H, d, J = 8.4 Hz), 8.40 (1H, d, J = 8.4 Hz). LC/MS
The following compounds (12b, 12c) were prepared
from 11h by a manner similar to that used for 12a.
1
Compound 12b: mp 197–198 ꢁC. H NMR (CDCl3) d:
3.15 (3H, s), 5.31 (2H, s), 6.89 (1H, s), 6.98 (2H, d,
J = 8.7 Hz), 7.12 (2H, d, J = 8.7 Hz), 7.24–7.27 (2H,
m), 7.37–7.54 (7H, m), 7.65 (1H, dd, J = 1.8, 8.7 Hz),
7.74 (2H, d, J = 8.7 Hz), 8.37 (1H, d, J = 8.7 Hz). LC/
MS (ESI+): 603, 604. Anal. Calcd for C30H23N2O5SBr:
C, 59.71; H, 3.84; N, 4.64. Found: C, 59.69; H, 3.84;
N, 4.60.
1
Compound 12c: mp >285 ꢁC. H NMR (DMSO-d6) d:
3.31 (3H, s), 5.26 (2H, s), 7.10–7.37 (5H, m), 7.40–7.55
(3H, m), 7.65–7.93 (7H, m), 8.31 (1H, d, J = 8.4 Hz),
10.24 (1H, s). LCMS (ESI+): 647 (M+H), 649. Anal.
Calcd for C31H23BrN2O7S-1.7H2O: C, 54.91; H, 3.92;
N, 4.13. Found: C, 54.63; H, 3.55; N, 4.11.

Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
4727
5.1.13.
4-[(4-{[6-Bromo-3-(methoxycarbonyl)-1-oxo-4-
washed with water and saturated brine. The solvent
was dried over anhydrous sodium sulfate, and evapo-
rated under reduced pressure. The obtained residue
was purified by silica gel column chromatography (hex-
ane/AcOEt), and crystallized from hexane to give 13c
(210 mg, 74%). Mp 95–97 ꢁC. 1H NMR (CDCl3) d:
0.88 (3H, t, J = 7.3 Hz), 1.14–1.66 (4H, m), 2.61 (2H,
t, J = 7.7 Hz), 3.18 (3H, s), 5.44 (2H, s), 6.94–7.06
(1H, m), 7.14–7.50 (11H, m), 8.47 (1H, d, J = 8.4 Hz).
LC/MS (ESI+): 426. Anal. Calcd for C28H27NO3: C,
79.03; H, 6.40; N, 3.29. Found: C, 79.02; H, 6.37; N,
3.27.
phenylisoquinolin-2(1H)-yl]methyl}phenyl)amino]-4-oxobu-
tanoic acid (12d). To a solution of 11h (139 mg,
0.300 mmol) in THF (5 ml) were added triethylamine
(0.080 ml, 0.57 mmol) and succinic anhydride (45 mg,
0.45 mmol), and the mixture was stirred at room tem-
perature for 24 h. The solvent was evaporated under re-
duced pressure, and water was added. The mixture was
extracted with AcOEt. The extract was washed with sat-
urated brine, and dried over anhydrous sodium sulfate.
The solvent was evaporated under reduced pressure, and
the obtained residue was crystallized from diisopropyl
ether to give 12d (85 mg, 50%). Mp 204–205 ꢁC. 1H
NMR (CDCl3) d: 2.59–2.80 (4H, m), 3.24 (3H, s), 5.35
(2H, s), 6.37 (1H, s), 7.18–7.58 (9H, m), 7.60–7.75
(2H, m), 8.38 (1H, d, J = 8.4 Hz). LC/MS (ESI+): 563,
564. Anal. Calcd for C28H23BrN2O6: C, 59.69; H, 4.11;
N, 4.97. Found: C, 59.74; H, 4.27; N, 4.94.
5.1.17. Dimethyl 2-benzyl-1-oxo-4-phenyl-1,2-dihydroiso-
quinoline-3,6-dicarboxylate (13d). A mixture of 6b
(300 mg, 0.669 mmol), palladium(II) acetate (8.0 mg,
0.036 mmol),
1,10-bis(diphenylphosphino)ferrocene
(20.0 mg, 0.0361 mmol), triethylamine (0.230 ml,
1.65 mmol), DMF (3 ml) and MeOH (3 ml) was stirred
at 50 ꢁC under 1 atm carbon monoxide for 24 h. Water
was added to the reaction mixture, and the mixture
was extracted with AcOEt. The organic layer was
washed with water and saturated brine, dried over anhy-
drous sodium sulfate, and concentrated under reduced
pressure. The obtained residue was purified by silica
gel column chromatography (hexane ! 1:1 hexane/
AcOEt), and recrystallized (AcOEt/hexane) to give 13d
5.1.14. Methyl 2-benzyl-1-oxo-4-phenyl-1,2-dihydroiso-
quinoline-3-carboxylate (13a). A mixture of 6b (400 mg,
0.892 mmol), 10% palladium/carbon (100 mg), MeOH
(5 ml) and THF (5 ml) was stirred at room temperature
under 1 atm hydrogen pressure for 12 h. Palladium car-
bon was filtered off, and the filtrate was concentrated
under reduced pressure. The obtained residue was puri-
fied by silica gel column chromatography (AcOEt), and
recrystallized (AcOEt–hexane) to give 13a (230 mg,
1
(180 mg, 63%). Mp 164–166 ꢁC. H NMR (CDCl3) d:
1
57%). Mp 136–138 ꢁC. H NMR (CDCl3) d: 3.19 (3H,
3.20 (3H, s), 3.88 (3H, s), 5.45 (2H, s), 7.14–7.54 (10H,
m), 7.94–8.00 (1H, m), 8.16 (1H, dd, J = 1.4, 8.4 Hz),
8.58–8.68 (1H, m). LC/MS (ESI+): 428. Anal. Calcd
for C26H21NO5: C, 73.06; H, 4.95; N, 3.28. Found: C,
72.92; H, 4.86; N, 3.09.
s), 5.46 (2H, s), 7.14–7.68 (13H, m), 8.50–8.66 (1H,
m). LC/MS (ESI+): 370. Anal. Calcd for C24H19NO3:
C, 78.03; H, 5.18; N, 3.79. Found: C, 77.87; H, 5.12;
N, 3.49.
5.1.15. Methyl 2-benzyl-6-methyl-1-oxo-4-phenyl-1,2-
dihydroisoquinoline-3-carboxylate (13b). A mixture of
6b (300 mg, 0.669 mmol), methylboronic acid (200 mg,
3.34 mmol), tetrakis(triphenylphosphine)palladium(0)
(80.0 mg, 0.0692 mmol), potassium carbonate (280 mg,
2.03 mmol), toluene (6 ml) and THF (3 ml) was stirred
at 100 ꢁC under a nitrogen atmosphere for 12 h. Water
was added to the reaction mixture, and the mixture
was extracted with AcOEt. The organic layer was
washed with water and saturated brine, dried over anhy-
drous sodium sulfate, and concentrated under reduced
pressure. The obtained residue was purified by silica
gel column chromatography (hexane ! hexane/AcOEt)
and recrystallized (AcOEt/hexane) to give 13b (210 mg,
5.1.18. 2-Benzyl-3-methoxycarbonyl-1-oxo-4-phenyl-1,2-
dihydroisoquinoline-6-carboxylic acid (13e). To a solu-
tion of 13c (1.75 g, 4.23 mmol) in THF (10 ml) were
added MeOH (10 ml) and 8 N aqueous sodium hydrox-
ide solution (1.00 ml, 8.00 mmol) at room temperature,
and the mixture was stirred for 12 h. The reaction mix-
ture was concentrated under reduced pressure, and
water was added, after which conc. hydrochloric acid
(1 ml) was added. The mixture was extracted with
AcOEt. The organic layer was washed with water and
saturated brine, dried over anhydrous sodium sulfate,
and concentrated under reduced pressure. The obtained
residue was recrystallized (AcOEt/hexane) to give 13e
(1.67 g, 99%). Mp 245–248 ꢁC. 1H NMR (CDCl3) d:
3.21 (3H, s), 5.46 (2H, s), 7.16–7.56 (10H, m), 7.94–
8.06 (1H, m), 8.19 (1H, dd, J = 1.8, 8.4 Hz), 8.65 (1H,
d, J = 8.4 Hz). LCMS (ESI+): 414. Anal. Calcd for
C25H19NO5Æ0.2AcOEt: C, 71.89; H, 4.82; N, 3.25.
Found: C, 71.63; H, 4.77; N, 2.99.
1
82%). Mp 112–115 ꢁC. H NMR (CDCl3) d: 2.37 (3H,
s), 3.18 (3H, s), 5.44 (2H, s), 7.01 (1H, s), 7.14–7.54
(11H, m), 8.45 (1H, d, J = 8.2 Hz). LCMS (ESI+): 384.
Anal. Calcd for C25H21NO3: C, 78.31; H, 5.52; N,
3.65. Found: C, 78.30; H, 5.50; N, 3.57.
5.1.16. Methyl 2-benzyl-6-butyl-1-oxo-4-phenyl-1,2-dihy-
droisoquinoline-3-carboxylate (13c). A mixture of 6b
(300 mg, 0.669 mmol), n-butylboronic acid (340 mg,
3.34 mmol), tetrakis(triphenylphosphine)palladium(0)
(80.0 mg, 0.0692 mmol), potassium carbonate (280 mg,
2.03 mmol), toluene (6 ml) and THF (3 ml) was stirred
at 100 ꢁC under a nitrogen atmosphere for 12 h. Water
was added to the reaction mixture, and the mixture
was extracted with AcOEt. The organic layer was
5.1.19. 6-Chloro-1-oxo-4-phenyl-1H-isochromene-3-car-
boxylic acid (15a). To a solution of 4-chlorophthalic
anhydride (25.0 g, 0.137 mol) in benzene (200 ml) was
added aluminum chloride 14a (36.5 g, 0.274 mol), and
the mixture was stirred at room temperature for 1 h.
and subsequently refluxed for 30 min. After cooling
the reaction mixture, it was diluted with AcOEt
(200 ml), and poured into ice water. Concentrated
hydrochloric acid (20 ml) was added, and the mixture

4728
Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
was stirred at room temperature for 30 min. After sepa-
rating the organic layer, the layer was washed succes-
sively with 4 N hydrochloric acid, water and brine,
dried over anhydrous sodium sulfate and concentrated
under reduced pressure until the crystals precipitated.
The crystals were collected by filtration, and washed
with diisopropyl ether to give 2-benzoyl-4-chlorobenzoic
acid (16.0 g, 45%) as colorless crystals. To a solution of
2-benzoyl-4-chlorobenzoic acid (13.0 g, 49.9 mmol) in
acetone (200 ml) was added potassium carbonate
(6.90 g, 49.9 mmol), and the mixture was stirred at room
temperature for 10 min to allow precipitation of potas-
sium salt. Then diethyl bromomalonate (13.2 g,
55.2 mmol) and DMF (10 ml) were added, and the mix-
ture was stirred at room temperature for 20 h. The reac-
tion mixture was concentrated under reduced pressure,
and AcOEt (100 ml) and water (100 ml) were added to
the residue. The mixture was stirred at room tempera-
ture for 30 min. The organic layer was separated and
washed with water and brine, dried over anhydrous so-
dium sulfate, and concentrated under reduced pressure.
A mixed solution of acetic acid (100 ml) and conc.
hydrochloric acid (100 ml) was added to the obtained
residue, and the mixture was stirred for 20 h at 120 ꢁC.
After cooling, the reaction mixture was concentrated un-
der reduced pressure, and the residue was dissolved in
AcOEt, washed with water and brine, dried over anhy-
drous sodium sulfate, and concentrated under reduced
pressure. The obtained residue was crystallized from
diisopropyl ether and collected by filtration to give 15a
5.1.21. 2-Benzyl-6-methoxy-1-oxo-4-phenyl-1,2-dihydro-
isoquinoline-3-carboxylic acid (16b). To a solution of
sodium methoxide in MeOH (28%, 4 ml) was added
16a (350 mg, 0.937 mmol), and the mixture was heated
under reflux for 6 h. To the reaction mixture were
added water and 10% hydrochloric acid to acidify
the aqueous layer of the mixture and the mixture
was extracted with AcOEt. The organic layer was
washed with water and saturated brine and dried over
anhydrous sodium sulfate. The solvent was evaporated
under reduced pressure. The obtained residue was
crystallized (AcOEt) to give 16b (260 mg, 72%). Mp
254–258 ꢁC. 1H NMR (DMSO-d6) d: 3.69 (3H, s),
5.26 (2H, m), 6.46 (1H, d, J = 2.6 Hz), 7.12–7.58
(11H, m), 8.27 (1H, d, J = 8.8 Hz). Anal. Calcd for
C24H19NO4: C, 74.79; H, 4.97; N, 3.63. Found: C,
74.44; H, 5.02; N, 3.54.
5.1.22. Methyl 2-benzyl-6-methoxy-1-oxo-4-phenyl-1,2-
dihydroisoquinoline-3-carboxylate (17). Compound 16b
(160 mg, 0.415 mmol) was dissolved in DMF (3 ml),
and iodomethane (0.060 ml, 0.96 mmol) and potassium
carbonate (120 mg, 0.868 mmol) were added at room
temperature. The mixture was stirred for 12 h. Water
was added to the reaction mixture, and the mixture
was extracted with AcOEt. The organic layer was
washed with water and saturated brine. The solvent
was dried over anhydrous sodium sulfate, and evapo-
rated under reduced pressure, and the obtained residue
was crystallized (AcOEt/hexane) to give 17 (120 mg,
1
1
(9.78 g, 65%) as colorless crystals. H NMR (CDCl3)
72%). Mp 133–135 ꢁC. H NMR (CDCl3) d: 3.18 (3H,
d: 7.07 (1H, d, J = 1.8 Hz), 7.20–7.28 (2H, m), 7.45–
7.54 (3H, m), 7.60 (1H, dd, J = 1.8, 8.4 Hz), 8.34 (1H,
d, J = 8.4 Hz).
s), 3.73 (3H, s), 5.42 (2H, s), 6.60 (1H, d, J = 2.6 Hz),
7.12 (1H, dd, J = 2.4, 9.0 Hz), 7.14–7.50 (10H, m),
8.49 (1H, d, J = 8.8 Hz). LC/MS (ESI+): 400 (M+H).
Anal. Calcd for C25H21NO4: C, 75.17; H, 5.30; N,
3.51. Found: C, 75.19; H, 5.28; N, 3.42.
Compound 15b was prepared from 4-fluorophthalic
anhydride 14b in a manner similar to that used for 15a.
5.1.23. Methyl 6-chloro-1-oxo-4-phenyl-1H-isochromene-
3-carboxylate (18). To a solution of 15a (9.0 g, 30 mmol)
in MeOH (200 ml) was added conc. sulfuric acid (20 ml)
and the mixture was refluxed for 2 h. The reaction mix-
ture was concentrated under reduced pressure, and the
residue was neutralized with aqueous sodium hydrogen
carbonate, and extracted with AcOEt. The organic layer
was washed with water and brine, dried over anhydrous
sodium sulfate, and concentrated under reduced pres-
sure. The residue was crystallized (diisopropyl ether)
Compound 15b: 1H NMR (CDCl3) d: 6.77 (1H, dd,
J = 2.2, 9.4 Hz), 7.08–7.62 (6H, m), 8.45 (1H, dd,
J = 5.6, 8.8 Hz).
5.1.20. 2-Benzyl-6-fluoro-1-oxo-4-phenyl-1,2-dihydroiso-
quinoline-3-carboxylic acid (16a). A mixture of 15b
(300 mg,
1.06 mmol),
benzylamine
(230 mg,
2.14 mmol), triethylamine (0.300 ml, 2.15 mmol) and
MeOH (3 ml) was stirred at 50 ꢁC for 36 h. The reac-
tion mixture was concentrated under reduced pressure,
1 N hydrochloric acid was added, and the mixture was
extracted with AcOEt. The organic layer was washed
with water and saturated brine, dried over anhydrous
sodium sulfate, and concentrated under reduced pres-
sure. A 4 N solution of hydrochloric acid in AcOEt
(3 ml) was added to the residue at room temperature,
and the mixture was stirred for 12 h. The reaction
mixture was concentrated under reduced pressure,
and the obtained crystals were washed with (diisopro-
pyl ether) to give 16a (300 mg, 76%). Mp 232–236 ꢁC.
1H NMR (DMSO-d6) d: 5.28 (2H, m), 6.72 (1H, dd,
J = 2.6, 10.2 Hz), 7.12-7.72 (11H, m), 8.41 (1H, dd,
J = 6.0, 9.0 Hz). Anal. Calcd for C23H16FNO3: C,
73.99; H, 4.32; N, 3.75. Found: C, 73.77; H, 4.50;
N, 3.62.
1
to give 18 (8.7 g, 92%) as colorless crystals. H NMR
(CDCl3) d: 3.72 (3H, s), 7.08 (1H, d, J = 2.2 Hz), 7.22–
7.30 (2H, m), 7.48–7.55 (3H, m), 7.59 (1H, dd, J = 2.2,
8.4 Hz), 8.35 (1H, d, J = 8.4 Hz).
5.1.24. Methyl 2-(4-methylsulfonylbenzyl)-1-oxo-4-phe-
nyl-1,2-dihydroisoquinoline-3-carboxylate (19a). A mix-
ture of 11g (200 mg, 0.380 mmol), 10% palladium/
carbon (20 mg) and MeOH (4 ml) was stirred under a
hydrogen atmosphere for 1 h. The catalyst was removed
by filtration, and the filtrate was concentrated under re-
duced pressure to give 19a (127 mg, 75%) as a colorless
1
powder. Mp 184.5–186 ꢁC. H NMR (CDCl3) d: 3.02
(3H, s), 3.26 (3H, s), 5.45 (2H, s), 7.26–7.34 (3H, m),
7.41–7.52 (5H, m), 7.56–7.67 (2H, m), 7.89 (2H, d,
J = 8.4 Hz), 8.54 (1H, m). LC/MS (ESI+): 448. Anal.

Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
4729
Calcd for C25H21NO5S: C, 67.10; H, 4.73; N, 3.13.
Found: C, 66.88; H, 4.75; N, 3.04.
5.1.27. Methyl 6-chloro-2-[4-(methylsulfonyl)benzyl]-1-
oxo-4-{[(trifluoromethyl)sulfonyl]oxy}-1,2-dihydroiso-
quinoline-3-carboxylate (21). To a solution of 20 (5.48 g,
13 mmol) in DMF (45 ml) was added sodium hydride
(60% in mineral oil, 630 mg, 16 mmol) at 0 ꢁC and the
mixture was stirred for 30 min. Then, N-phenyltrifluo-
romethanesulfonimide (5.6 g, 16 mmol) was added and
the mixture was stirred at room temperature for 3 h.
The solvent was evaporated under reduced pressure.
The residue was partitioned between AcOEt and water,
and the organic layer was dried over anhydrous magne-
sium sulfate, and concentrated under reduced pressure.
The residue was purified by silica gel column chroma-
tography (hexane/AcOEt) to give 21 (3.46 g, 48%) as
colorless crystals. 1H NMR (CDCl3) d: 3.02 (3H, s),
3.79 (3H, s), 5.49 (2H, s), 7.40 (2H, d, J = 8.4 Hz),
7.65 (1H, dd, J = 1.8, 8.4 Hz), 7.79 (1H, d, J = 1.8 Hz),
7.89 (2H, d, J = 8.4 Hz), 8.42 (1H, d, J = 8.4 Hz).
5.1.25. Methyl 6-chloro-2-(4-methylsulfonylbenzyl)-1-
oxo-4-phenyl-1,2-dihydroisoquinoline-3-carboxylate (19b).
A mixture of 18 (200 mg, 0.635 mmol), 1-(4-(methylsul-
fonyl)phenyl)methanamine (240 mg, 1.30 mmol) and
MeOH (4 ml) was heated under reflux for 12 h. Concen-
trated sulfuric acid (0.4 ml) was added to the reaction
mixture under ice-cooling, and the mixture was heated
under reflux for 2 h. The reaction mixture was concen-
trated under reduced pressure, and water was added.
The mixture was extracted with AcOEt, and the
organic layer was washed with water, saturated aqueous
sodium hydrogen carbonate solution, water and satu-
rated brine, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. The residue was
purified by recrystallization (AcOEt/hexane) to give 19b
1
(200 mg, 65%). Mp 201–203 ꢁC. H NMR (CDCl3) d:
3.02 (3H, s), 3.26 (3H, s), 5.42 (2H, s), 7.18–7.62
(9H, m), 7.89 (2H, d, J = 8.4 Hz), 8.46 (1H, d,
J = 8.4 Hz). LC/MS (ESI+): 482, 484. Anal. Calcd for
C25H20ClNO5S: C, 62.30; H, 4.18; N, 2.91. Found: C,
62.10; H, 4.12; N, 2.88.
5.1.28. Methyl 6-chloro-4-(4-formylphenyl)-2-[4-(methyl-
sulfonyl)benzyl]-1-oxo-1,2-dihydroisoquinoline-3-carboxyl-
ate (22a). A mixture of 20 (116 mg, 0.21 mmol), sodium
carbonate (45 mg, 0.43 mmol), 4-formylphenylboronic
acid (37 mg, 0.25 mmol), tetrakis(triphenylphosphine)pal-
ladium(0) (12 mg, 0.010 mmol), toluene (2.0 ml), water
(0.4 ml)andEtOH(0.4 ml) wasstirredat80 ꢁC for 12 h un-
der an argon atmosphere. After cooling, the reaction mix-
ture was partitioned between water and AcOEt. The
organic layer was dried over anhydrous magnesium sul-
fate, and concentratedunder reducedpressure. The residue
was purified by preparative RPHPLC to give 22a (63 mg,
5.1.26. Methyl 6-chloro-4-hydroxy-2-[4-(methylsulfo-
nyl)benzyl]-1-oxo-1,2-dihydroisoquinoline-3-carboxylate
(20). To a solution (50 ml) of 4-chloro phthalic anhy-
dride 14a (12.1 g, 66.3 mmol) in MeOH was added so-
dium methoxide (3.60 g, 66.6 mmol) and the mixture
stirred at room temperature for 12 h. The solvent was
evaporated under reduced pressure, and the residue
was partitioned between AcOEt and 1 N hydrochloric
acid. The organic layer was dried over anhydrous mag-
nesium sulfate and concentrated under reduced pressure
to give the mixture of 4-chloro-2-(methoxycar-
bonyl)benzoic acid and 5-chloro-2-(methoxycar-
bonyl)benzoic acid. To a solution of this mixture in
CH3CN (70 ml) was added methyl N-[4-(methanesulfo-
nyl)benzyl]glycinate (17.0 g, 66.0 mmol), 1-ethyl-3-(3-
1
59%) as a colorless powder. H NMR (CDCl3) d: 3.02
(3H, s), 3.27 (3H, s), 5.42 (2H, s), 7.13 (1H, d,
J = 2.1 Hz), 7.44–7.52 (4H, m), 7.55 (1H, dd, J = 2.1,
8.7 Hz), 7.89 (2H, d, J = 8.7 Hz), 7.99 (2H, d,
J = 8.4 Hz), 8.47 (1H, d, J = 8.7 Hz), 10.10 (1H, s). LC/
MS (ESI+): 510, 512.
Compound 22c was prepared from 20 and 3-form-
ylphenylboronic acid in a manner similar to that used
for 22a.
dimethylaminopropyl)carbodiimide
hydrochloride
(16.5 g, 86.1 mmol) and 1-hydroxybenzotriazole mono-
hydrate (11.2 g, 73.1 mmol), and the mixture was stirred
at room temperature for 12 h. The solvent was evapo-
rated under reduced pressure, and the residue was parti-
tioned between AcOEt and 1 N hydrochloric acid. The
organic layer was dried over anhydrous magnesium sul-
fate and concentrated under reduced pressure. This
product was dissolved in MeOH (100 ml), and 28% so-
dium methoxide/MeOH solution (26.0 g, 135 mmol)
was added at room temperature with stirring. The mix-
ture was stirred at room temperature for 1 h. The reac-
tion mixture was concentrated under reduced pressure,
and the residue was partitioned between AcOEt and
1 N hydrochloric acid. The organic layer was dried over
anhydrous magnesium sulfate, and concentrated under
reduced pressure. The residue was purified by silica gel
column chromatography (hexane/AcOEt) and recrystal-
lized (hexane/AcOEt) to give 20 (5.57 g, 20%) as yellow
crystals. 1H NMR (CDCl3) d: 3.03 (3H, s), 3.76 (3H, s),
5.55 (2H, s), 7.32 (2H, d, J = 8.7 Hz), 7.69 (1H, dd,
J = 2.1, 8.7 Hz), 7.88 (2H, d, J = 8.7 Hz), 8.19 (1H, d,
J = 2.1 Hz), 8.40 (1H, d, J = 8.7 Hz), 11.29 (1H, s).
1
Compound 21c: H NMR (CDCl3) d: 3.02 (3H, s), 3.27
(3H, s), 5.41 (2H, d, J = 2.4 Hz), 7.10 (1H, d,
J = 1.8 Hz), 7.47 (2H, d, J = 8.7 Hz), 7.51–7.61 (2H,
m), 7.66 (1H, t, J = 7.5 Hz), 7.82 (1H, m), 7.89 (2H, d,
J = 8.7 Hz), 7.98 (1H, dt, J = 1.8, 7.5 Hz), 8.47 (1H, d,
J = 9.0 Hz), 10.06 (1H, s). LC/MS (ESI+): 510, 512.
5.1.29. Methyl 6-chloro-4-[4-(hydroxymethyl)phenyl]-2-
[4-(methylsulfonyl)benzyl]-1-oxo-1,2-dihydroisoquinoline-
3-carboxylate (22b). To a mixture of 22a (76 mg,
0.15 mmol), MeOH (1.0 ml) and THF (1.0 ml) was
added sodium borohydride (6.0 mg, 0.16 mmol) at
0 ꢁC with stirring, and the mixture was stirred for 1 h.
The reaction mixture was concentrated under reduced
pressure, and the residue was partitioned between 1 N
hydrochloric acid and AcOEt. The organic layer was
dried over anhydrous magnesium sulfate and concen-
trated under reduced pressure. The residue was purified
by preparative RPHPLC to give 22b (35 mg, 45%) as a
1
colorless powder. Mp 252–253 ꢁC. H NMR (CDCl3)
d: 1.84 (1H, t, J = 5.7 Hz), 3.03 (3H, s), 3.30 (3H, s),

4730
Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
4.80 (2H, d, J = 5.7 Hz), 5.41 (2H, s), 7.21–7.36 (3H, m),
7.43–7.57 (5H, m), 7.89 (2H, d, J = 8.7 Hz), 8.45 (1H, d,
J = 8.7 Hz). MS (ESI+): 512, 514. Anal. Calcd for
C26H22ClNO6S: C, 60.99; H, 4.33; N, 2.74. Found: C,
61.23; H, 4.47; N, 2.67.
the abdominal aorta was exposed by median laparot-
omy. The left and right renal arteries were detached
from the back, a 22G injection needle was put along
the artery and was ligated together with the aorta using
a 2-0 silk thread just below the right renal artery. Then,
the injection needle was removed to make a stenosis in
the abdominal aorta. The abdomen was closed and
the rats were housed together. For a sham group, the
abdomen was closed without ligation (no stenosis) with
a silk thread. A compound was suspended in a 0.5%
methyl cellulose solution (2 ml/kg) and orally adminis-
tered before aortic stenosis and once a day from day 1
(the next day of stricture) to day 7 after operation. Vehi-
cle (0.5% methyl cellulose solution) was administered in-
stead of the compound to the sham group and control
group. At day 7 after the preparation of aortic stenosis,
body weight was measured and the heart was removed
and then extracted under pentobarbital (50 mg/kg, ip)
anesthesia. The right and left artium and right ventricle
were excised from the heart and the wet weight of the
left ventricle was measured. The left ventricle weight
to body weight ratio of each rat was calculated and
an increase from the average value of the sham group
was calculated. The rate of inhibition for each group
to cardiac hypertrophy was calculated based on the con-
trol group as 100%. Systolic blood pressure (SBP) was
monitered before the heart was extracted under pento-
barbital (50 mg/kg, ip) anesthesia. The venous catheter
was passed through the right carotid artery and con-
nected to a pressure transducer coupled to a polygraph
system (NEC San-ei Instruments, Nihon Kohden
Corporation).
Compound 22d was prepared from 22c and 3-form-
ylphenylboronic acid in a manner similar to that used
for 22b.
Compound 21d: mp 214–215 ꢁC. 1H NMR (CDCl3) d: 1.82
(1H, t, J = 5.4 Hz), 3.03 (3H, s), 3.29 (3H, s), 4.77 (2H, d,
J = 5.1 Hz), 5.38 (1H, d, J = 16.5 Hz), 5.45 (1H, d,
J = 16.5 Hz), 7.18–7.36 (3H, m), 7.41–7.66 (5H, m), 7.89
(2H, d, J = 8.4 Hz), 8.46 (1H, d, J = 8.4 Hz). LC/MS
(ESI+): 512, 514. Anal. Calcd for C26H22ClNO6S: C,
60.99; H, 4.33; N, 2.74. Found: C, 61.08; H, 4.46; N, 2.60.
5.2. Measurement of JNK1 activity
All test compounds were dissolved in DMSO. The ki-
nase reaction mixture was preincubated with test com-
pound for 5 min at 30 ꢁC. Kinase reactions were
performed at 30 ꢁC for 60 min in reaction buffer
(25 mM HEPES, pH 7.5, 10 mM magnesium acetate,
1 mM dithiothreitol) containing 50 ng activated human
JNK1, 0.1 lCi [c-32P]ATP, 500 nM ATP and 1 lg
GST-c-Jun (1–79) with test compounds. After incuba-
tion, the reactions were terminated by adding 10% (final
concentration) trichloroacetic acid (Wako, Japan).
Phosphorylated proteins were filtrated in GF/C filter
plates (PerkinElmer, USA) with a Cell Harvester (Perk-
inElmer, USA), and washed with 250 mM phosphoric
acid. The plates were then incubated for 60 min at
45 ꢁC, followed by the addition of 40 lL of Micro-
Scint-O (PerkinElmer, USA). The radioactivity was
counted by a TopCount scintillation counter (PerkinEl-
mer, USA). The IC50 values were calculated as the con-
centration of the test compound at which the c-Jun
phosphorylation was reduced to 50% of the control va-
lue. The IC50 values for JNK1 inhibitors were calculated
from a dose–response curve at compound concentrations
of 0.001, 0.01, 0.1, 1 and 10 lM in the presence of 0.5 lM
ATP.
5.5. Measurement of phosphorylated c-Jun in the heart of
cardiac hypertrophy model rats
The left ventricle was homogenized in 5.0 ml of ice-
cooled lysis buffer (20 mM Tris–HCl, pH 7.4, 1%
NP40, 0.1% SDS, 150 mM NaCl, 1 mM EDTA,
0.1 mg/ml PMSF, 0.3 TIU aprotinin, 1 mM sodium
orthovanadate), and centrifuged at 12,000 rpm
(15,800g), 4 ꢁC for 30 min. After centrifugation, the
supernatant was recovered and used as a tissue extract
solution. The protein amount was measured by the
Bradford method (Bio-Rad, Protein Assay). The tissue
extract solution containing 15 mg of protein was added
to 8 lg of anti-c-Jun rabbit polyclonal antibody (H79,
Santa Cruz) and 40 ll of Protein A-Agarose (Santa
Cruz), and the mixture was stirred overnight at 4 ꢁC.
The mixture was centrifuged at 2500 rpm, 4 ꢁC for
5 min, and after removal of supernatant, washed three
times with TBS buffer (50 mM Tris–HCl, pH 7.4,
150 mM NaCl, 0.1 mg/ml phenylmethanesulfonyl fluo-
ride (PMSF), 0.3 Trypsin inhibitor unit (TIU) aprotinin,
1 mM sodium orthovanadate), and dissolved in 30 ll of
Laemmli sample buffer (Bio-Rad). A 10 ll aliquot of the
dissolved sample was loaded onto a 10% separation gel
(Bio-Rad) and electrophoresed at 150 V for 60 min. The
protein on the gel was transferred onto PVDF mem-
brane (Bio-Rad) after methanol treatment by wet blot-
ting at 100 V for 1 h. The membrane was washed with
TBS-T (0.05% Tween 20), blocked with 5% skim milk/
TBS-T (blocking buffer) at room temperature for 1 h.
5.3. H9c2 cell based assay
H9c2 cells (serum free/DMEM) derived from rat myo-
cardium were seeded in 6 well plates and then incubated
with test compound for 30 min. Subsequently, they were
stimulated with anisomycin (50 ng/ml, Sigma, US) for
30 min. After being washed with iced PBS, the H9c2
cells were dissolved in 30 lL of Cell Lysis buffer (CST,
US). After centrifugation, the solution of cells was mea-
sured by a human AP-I Transcription Factor Assay Kit
(Active Motif). The IC50 values were calculated as the
concentration of test compound at which c-Jun phos-
phorylation was reduced to 50% of the control value.
5.4. Preparation of the pressure-overloaded cardiac hypertro-
phy model induced by abdominal aortic stenosis in rats
Male 9-week-old Wistar rats were used. Under xylazine
(10 mg/kg, ip) and ketamine (50 mg/kg, ip) anesthesia,

Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
4731
˚
and reacted with anti-phosphorylated c-Jun (ser63)
mouse monoclonal antibody (KM-1 diluted 250-fold
with blocking buffer, Santa Cruz) at room temperature
for 1.5 h. The membrane was washed with TBS-T
(5 min, two times), reacted with HRP labeled-anti
mouse IgG secondary antibody (Amersham) diluted
2000-fold with blocking buffer at room temperature
for 1 h. and washed with TBS-T (5 min, three times)
and TBS. The membrane was reacted with 2 ml of
ECL reactive solution (ECL Plus, Amersham) for
5 min, exposed to X-ray film for 1 min, and the film
was immersed in a developing solution for 90 s, washed
with water and immersed in a fixing solution to develop
the film. The developed X-ray film was scanned by
DeskscanII, automatically corrected, and further auto-
matically corrected by Photoshop to reverse the color
tone. The density of the band seen at the objective
molecular weight was digitalized on the histogram of
Photoshop.
c = 108.0 A and one molecule per asymmetric unit. X-
ray diffraction data were collected on beamline
BL32B2 at the facilities of the Pharmaceutical Consor-
tium at SPring-8 and processed to an Rmerge value of
˚
0.100 and completeness of 98.3 at 2.5 A resolution.
The structure was solved by molecular replacement with
CNX (Accelrys, USA) using the coordinates of the
JNK3:AMPPNP complex (PDB code: 1JNK) as a
search model. Initial difference Fourier electron density
maps were used to determine the binding conformation
of the ligand. Refinement was carried out using alternat-
ing cycles of manual rebuilding using the graphic soft-
ware QUANTA (Accelrys, USA) and computer-based
refinement using CNX (Accelrys, USA). The crystallo-
graphic R-factor/free-R were refined to 0.266/0.331, with
˚
˚
RMSD bond length/angle of 0.008 A/1.5ꢁ at 2.5 A reso-
lution. Coordinates are deposited in the Protein Data
Bank (PDB ID: 2ZDU).
5.6. Statistical analysis
References and notes
Data are expressed and presented in the figure as
means SD. A Williams’ test or Student’s t test was
used to compare the means of normally distributed con-
tinuous variables. A value of p < 0.05 was chosen as the
limit of statistical significance.
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5.7. Pharmacokinetic analysis in rats
Plasma samples were deproteinized with acetonitrile,
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centrifugation. The compound concentrations in the
supernatant were measured by HPLC-UV or LC/MS/
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and a MS/MS API 3000 triple quadruple mass spec-
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were as follows: L-column ODS (4.6 · 250 mm),
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phase, 1 ml/min flow rate, 40 ꢁC column temperature,
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mobile phase (adjusted to pH 3 with formic acid),
0.2 ml/min flow rate, 40 ꢁC column temperature, turbo
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JNK3 was cloned and purified following the published
protocol.14 Compound 12d was incubated with the en-
zyme with a threefold excess of inhibitor for 3 h in ice.
Crystals of the complex were grown by hanging-drop
co-crystallization under the following conditions: 20%
pentaerythritol ethoxylate (15/4 EO/OH) reservoir,
0.1 M ammonium sulfate in 0.1 M Bis–Tris, pH 6.5,
and the use of the micro-seeding method using crystals
of the JNK3:AMPPNP complex. Then, crystals were
transferred to the reservoir solution containing 15% gly-
col as cryoprotectant and frozen with liquid nitrogen.
They belong to the orthorhombic space group P212121,
˚
˚
with unit cell parameters a = 57.0 A, b = 70.3 A,

4732
Y. Asano et al. / Bioorg. Med. Chem. 16 (2008) 4715–4732
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