Identification of 3-aminomethyl-1,2-dihydro-4-phenyl-1-isoquinolones: A new class of potent, selective, and orally active non-peptide dipeptidyl peptidase IV inhibitors that form a unique interaction with Lys554

Article abstract of DOI:10.1016/j.bmc.2011.06.059

The design, synthesis, and structure-activity relationships of a new class of potent and orally active non-peptide dipeptidyl peptidase IV (DPP-4) inhibitors, 3-aminomethyl-1,2-dihydro-4-phenyl-1-isoquinolones, are described. We hypothesized that the 4-ph

Full text of DOI:10.1016/j.bmc.2011.06.059

Bioorganic & Medicinal Chemistry 19 (2011) 4953–4970
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of 3-aminomethyl-1,2-dihydro-4-phenyl-1-isoquinolones:
A new class of potent, selective, and orally active non-peptide dipeptidyl
peptidase IV inhibitors that form a unique interaction with Lys554
Yoshihiro Banno a, Yasufumi Miyamoto a, Mitsuru Sasaki a, Satoru Oi a, Tomoko Asakawa a, Osamu Kataoka a,
Koji Takeuchi a, Nobuhiro Suzuki a, Koji Ikedo a, Takuo Kosaka a, Shigetoshi Tsubotani a, Akiyoshi Tani a,
Miyuki Funami a, Michiko Tawada a, Yoshio Yamamoto a, Kathleen Aertgeerts b, Jason Yano b,
Hironobu Maezaki a,
⇑
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 17-85 Jusohonmachi 2-Chome Yodogawa-ku, Yodogawa-ku, Osaka 532-8686, Japan
b Takeda San Diego, Inc., 10410, Science Center Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis, and structure–activity relationships of a new class of potent and orally active
non-peptide dipeptidyl peptidase IV (DPP-4) inhibitors, 3-aminomethyl-1,2-dihydro-4-phenyl-1-iso-
quinolones, are described. We hypothesized that the 4-phenyl group of the isoquinolone occupies the
S1 pocket of the enzyme, the 3-aminomethyl group forms an electrostatic interaction with the S2 pocket,
and the introduction of a hydrogen bond donor onto the 6- or 7-substituent provides interaction with the
hydrophilic region of the enzyme. Based on this hypothesis, intensive research focused on developing
new non-peptide DPP-4 inhibitors has been carried out. Among the compounds designed in this
study, we identified 2-[(3-aminomethyl-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-dihydro-6-isoquinoli-
nyl)oxy]acetamide (35a) as a potent, selective, and orally bioavailable DPP-4 inhibitor, which exhibited
in vivo efficacy in diabetic model rats. Finally, X-ray crystallography of 35a in a complex with the enzyme
validated our hypothesized binding mode and identified Lys554 as a new target-binding site available for
DPP-4 inhibitors.
Received 13 May 2011
Revised 20 June 2011
Accepted 20 June 2011
Available online 28 June 2011
Keywords:
Dipeptidyl peptidase IV (DPP-4)
Isoquinolone
Anti-diabetic effects
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
that increases glucose-stimulated insulin secretion. Furthermore,
numerous studies have indicated the importance of GLP-1 not only
Dipeptidyl peptidase IV (DPP-4, EC 3.4.14.5) is an extracellular
membrane-bound serine protease expressed on the cell surface
of a variety of organs, for example, intestine, kidney, and liver.1 It
catalyses the cleavage of N-terminal dipeptides from polypeptides
as a potent insulin secretagogue, but also as a multifunctional
hypoglycemic hormone responsible for the stimulation of b-cell
growth, survival, and differentiation, the inhibition of glucagon re-
lease, the retardation of gastric emptying, etc. Actually, many clin-
ical studies have shown that GLP-1 normalizes postprandial and
fasting glycemia in subjects with type 2 diabetes.17–21 On the basis
of the above evidence, GLP-1 itself is one of the logical candidates
for therapeutic agents in the treatment of diabetes.22–25 However,
active GLP-1 is rapidly metabolized by DPP-4 resulting in
GLP-1[9–36]amide.1 Therefore, alternative strategies to elicit the
beneficial anti-diabetic effects of GLP-1 are required. One of the
promising approaches is the inhibition of DPP-4 activity, which
can extend the duration of GLP-1 action through the blockade of
its degradation, resulting in improvement of the elevated glucose
levels in diabetes. Accordingly, DPP-4 inhibitors have been
expected to serve as a new type of glucose-lowering agent with
little or no adverse effects observed with sulfonylureas. Many
DPP-4 inhibitors, such as MK-403 (sitagliptin phosphate),26 BMS-
477118 (saxagliptin),27,28 LAF-237 (vildagliptin),28 and SYR-322
with L-proline or L-alanine residues situated at the penultimate
position of the substrates.2,3 The unique aminopeptidase function
of DPP-4 has been shown to modulate the biological activities of
circulating regulatory peptides involved in the endocrine, neuroen-
docrine, and immune systems in vitro.4 It has been revealed that
DPP-4 plays a principal role in the inactivation of glucagon-like
peptide-1 (GLP-1 or GLP-1[7–36]amide), which contributes to the
control of blood glucose levels in vivo.4–13
GLP-1 is one of the gastrointestinal hormones (incretins) se-
creted by intestinal L-cells in response to the ingestion of nutri-
ents.14–16 It is known as the most potent insulinotropic hormone
⇑
Corresponding author. Tel.: +81 6 6300 6263; fax: +81 6 6300 6306.
E-mail address: Maezaki_Hironobu@takeda.co.jp (H. Maezaki).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.059

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Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
Introduction of
Modification of
Hydrogen-bond Donor
Hydrophobic group
Hydrophobic
residue
O
2
MNe
Cl
O
CN
4
3
N
H2N
H2N
Acidic
S1' Pocket
S1' Pocket
residue
S2 Pocket
Modification of
Basic group
S2 Pocket
S1 Pocket
S1 Pocket
Compound 6a
(Hydrophobic pocket)
P2-P1 dipeptidy lanalogue
Chart 1. Binding mode of P2–P1 dipeptidyl analog of the substrate30 and the lead compound 6a.
(alogliptin benzoate),29 have demonstrated improved blood sugar
control in patients with type 2 diabetes. In our research for a
new non-peptide DPP-4 inhibitor, high-throughput screening of
the in-house chemical library led to the identification of an isoqu-
inolone-based lead compound 6a that showed potent inhibitory
activity against human DPP-4 with a half-maximal inhibitory con-
boxylation and dehydration. A ring-opening reaction of 3a–c with
various primary amines and subsequent recyclization under acidic
conditions furnished the 4-phenyl-1-isoquinolone-3-carboxylic
acids 4a–i. The fluorine atom of 4c was replaced with a methoxy
group to yield 4j. The reduction of 4a,b and 4d–j with sodium boro-
hydride with the corresponding acid chlorides provided hydroxy-
methyl derivatives 5a–i. Methanesulfonylation or chlorination of
5a and 5c–i followed by a reaction with ethanolic ammonia in a
sealed tube afforded the desired products 6a–h.
Modification of the basic group at the 3-position of the
4-phenyl-1-isoquinolones was conducted by the routes illustrated
in Schemes 2 and 3. The hydroxymethyl derivatives 5a and 5f were
converted to mesylates 7a and 7b, and then, the reaction of the
compound 7b with a large excess of methylamine or dimethyl-
amine afforded the corresponding secondary amine 8a or tertiary
amine 8b, respectively. Reaction of 7a with diethyl malonate in
the presence of sodium hydride gave the diester 9, which was con-
verted to propionic acid 10 by acidic hydrolysis with heating in
concentrated hydrochloric acid and consequent decarboxylation.
Curtius rearrangement reaction of the 3-carboxylic acid 10 with
diphenylphosphoryl azide (DPPA) gave isocyanate, which was re-
acted with tert-butyl alcohol in one-pot to produce a primary
amine protected by a tert-butoxycarbonyl (Boc) group. Next,
deprotection of the Boc group of 11 provided the desired 3-(2-ami-
no)ethyl analog 12. The compound 4a was derivatized to amine 13
by a similar procedure as that described above for the preparation
of 12.
centration (IC50) value of 16 lM.
In this article, we describe the synthesis and structure–activity
relationships (SARs) of the 1,2-dihydro-4-phenyl-1-isoquinolone
derivatives, starting from the lead 3-(aminomethyl)-6-chloro-2-
methyl-4-phenylisoquinolin-1(2H)-one (6a). The predicted bind-
ing mode of compound 6a to DPP-4 is illustrated with the mode
of a P2–P1 dipeptidyl analog of the substrate in Chart 1.30
A
docking study of compound 6a with the enzyme indicated that
the 3-aminomethyl and 4-phenyl moieties of the isoquinolone ring
interacted with S2 and S1 pockets of the enzyme, respectively. On
the basis of the results, we hypothesized that the 2-position of the
isoquinolone would correspond to the hydrophobic moiety of the
P2 fragment, and the introduction of a hydrogen bond donor onto
the 6- or 7-position of the isoquinolone ring would increase the
inhibitory activity by forming an interaction with the hydrophilic
region of the enzyme. In order to verify our hypothesis, we first
investigated the importance of the basic group at the 3-position
and next investigated the effect of introducing an appropriate lipo-
philic moiety at the 2-position of the isoquinolone core and a
hydrogen bond donor at the 6- or 7-position. Finally, we confirmed
the binding mode of the isoquinolone derivative by using X-ray co-
crystallography.
The synthetic routes for 6-carboxylic acid 19a, 6-carboxamides
19b–d, 6-cyano 19e, 6-acrylamide 23a, and 6-propylamide 23b are
shown in Schemes 4 and 5. The compound 5b was converted to the
aminomethyl derivative, which was then protected by a Boc group
to yield compound 14. Palladium-catalyzed methoxycarbonylation
of the bromide 14 using carbon monoxide and methanol afforded
the methyl ester 15.32 Hydrolysis of the ester 15 gave carboxylic
acid 16, which was converted to the amides 17a–c by condensation
with the corresponding amines. The cyano derivative 18 was pre-
pared by dehydration of 17a using cyanuric chloride. Removal of
the Boc group of 16, 17a–c, and 18 produced the aminomethyl
derivatives 19a–e. In addition, compound 14 was subjected to
the Heck reaction with ethyl acrylate in the presence of the palla-
dium catalyst to provide exclusively ethyl (E)-propenate 20a.33
Compound 20a was then hydrogenated over palladium-charcoal
to yield propionate 20b. Hydrolysis of 20 gave the corresponding
carboxylic acids 21, which were converted to the desired carbox-
amide 23, respectively, by amidation and subsequent deprotection.
The synthesis of 6- or 7-carbamoylmethoxy-1-isoquinolone 35
was carried out by the route depicted in Scheme 6. Commercially
available 4-hydroxyphthalic acid 24 was converted to 4-benzyl-
oxyphthalic anhydride 26 by benzylation, subsequent saponifica-
tion, and dehydration using acetic anhydride. Nucleophilic attack
These studies revealed that the isoquinolone nucleus is an
excellent template mimicking a P2–P1 dipeptidyl substrate and
discovered the potent, selective, non-peptide DPP-4 inhibitor
2-{[3-(aminomethyl)-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-di-
hydroisoquinolin-6-yl]oxy}acetamide (35a). The results of
co-crystallographic studies of 35a and the pharmacological profiles
are also reported.
2. Chemistry
Construction of the 4-phenyl-1-isoquinolone nucleus was
performed according to the previously reported procedure31
shown in Scheme 1. Initially, commercially available 4-substituted
phthalic anhydrides 1a–c were reacted with benzene under
Friedel–Crafts acylation conditions in order to afford 4-substi-
tuted-2-benzoylbenzoic acids 2a–c together with the undesired
regioisomers in an approximate 1:1 mixture. The undesired regio-
isomer, however, was easily removed by recrystallization in order
to give pure compounds 2a–c. After alkylation of 2a–c with diethyl
2-bromomalonate, the resulting diesters were converted to iso-
coumarin-3-carboxylic acids 3a–c by hydrolysis under reflux in
concentrated hydrochloric acid–acetic acid and consequent decar-

Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
4955
O
O
O
O
R2
O
OH
O
O
N
c
a
b
O
OH
OH
R1
R1
R1
R1
O
O
R1 = Cl
R1 = Br
R1 = F
1a:
1b:
1c:
R1 = Cl
R1 = Br
R1 = F
R1 = Cl
R1 = Br
R1 = F
R = Cl, R2 = Me
1
2a:
2b:
2c:
3a:
3b:
3c:
4a:
4b:
R = Br, R2 = i-Bu
1
1
4c
:
R = F,
R2 = i-Bu
R = Cl, R2 = Et
1
4d:
4e:
d
R = Cl, R2 = n-Pr
1
R = Cl, R2 = n-Bu
1
4f
:
R = Cl, R2 = i-Bu
1
4g:
4h:
4i:
R = Cl, R2 = n-Pen
1
R1 = Cl, R2 = neopentyl
R1 = MeO,R2= i-Bu
4j:
O
O
R2
R2
e
f
N
N
OH
NH2
R1
R1
1
1
R = Cl, R2 = Me
R = Cl, R2 = Me
5a
:
6a:
1
1
R = Br, R2 = i-Bu
R = Cl, R2 = Et
5b:
5c:
6b:
6c:
6d:
6e:
6f:
1
1
R = Cl, R2 = Et
R = Cl, R2 = n-Pr
1
1
R = Cl, R2 = n-Pr
R = Cl, R2 = n-Bu
5d
:
1
1
R = Cl, R2 = n-Bu
R = Cl, R2 = i-Bu
5e:
5f:
R1 = Cl, R2 = i-Bu
R = Cl, R2 = n-Pen
1
1
1
R = Cl, R2 = n-Pen
R = Cl, R2 = neopentyl
5g
:
6g:
6h:
1
1
2
R = Cl, R2 = neopentyl
R = MeO,R = i-Bu
5h:
5i:
R1 = MeO, R2= i-Bu
Scheme 1. Synthesis of compounds 6a–h. Reagents: (a) AlCl3, benzene; (b) (i) BrCH(CO2Et)2, K2CO3, DMF, acetone; (ii) concd HCl, AcOH; (c) (i) R2NH2, MeOH; (ii) 4 M HCl–
AcOEt; (d) MeONa, MeOH; (e) (i) (COCl)2, DMF, THF; (ii) NaBH4, DME, THF; (f) (i) MsCl, Et3N, CH2Cl2; (ii) 10% NH3–MeOH, THF or 10% NH3–EtOH, THF.
Me Me
O
R2
R3
N
N
Cl
b
2
3
8a
: R = Me, R = H
2
3
8b
: R = R = Me
O
O
O
R1
R1
Me
N
N
N
d
a
c
CO2Et
CO2Et
OMs
OH
Cl
Cl
Cl
1
1
5a
: R = Me
7a
: R = Me
9
5f: R1 = i-Bu
7b: R1 = i-Bu
O
O
O
Me
N
Me
Me
N
N
f
e
OH
Cl
Cl
NHBoc
Cl
NH2
HCl
O
12
10
11
Scheme 2. Synthesis of compounds 8a,b and 12. Reagents: (a) MsCl, Et3N, CH2Cl2; (b) R2R3NH, K2CO3, THF; (c) NaH, CH2(CO2Et)2, THF; (d) concd HCl, AcOH; (e) (i) DPPA, Et3N,
DMF; (ii) toluene; (iii) t-BuOH; (f) 4 M HCl–AcOEt.
of N-isobutylglycine ethyl ester provided the corresponding
carboxylic acids as a mixture of the regioisomers 27. Hence, the
mixture of the carboxylic acids 27 was then converted to the
6- or 7-benzyloxyisoquinolones 28a and 28b by alkylation
and subsequent cyclization under basic conditions. The regioisom-
ers 28a and 28b were successfully separated by column

4956
Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
chromatography. The 4-hydroxy-1-isoquinolone derivatives 28
were converted to triflates 29. Suzuki coupling reaction of the tri-
flates with phenylboronic acid was followed by saponification to
give 4-phenyl-1-isoquinolone-3-carboxylic acids 31. Compounds
31 were converted to Boc-protected derivatives 32 by using Gab-
riel synthesis. Catalytic hydrogenation of 32, followed by the reac-
tion with iodoacetamide gave carboxamides 34. The target amines
35a, 35b, and 36 were readily obtained by the removal of the Boc
group of 34a, 34b, and 33a, respectively.
The synthesis of 7-carboxamide derivative 41 is shown in
Scheme 7. Alkoxycarbonylation of triflate 37, obtained from the
7-hydroxy derivative 33b, was employed in a similar method to
that used for the preparation of compound 15 in Scheme 4 to give
methyl ester 38. Compound 38 was then converted to the final
product 41 by the same procedure described in Scheme 4.
3. Results and discussion
3.1. In vitro enzyme inhibition studies
The 4-phenyl-1-isoquinolones described above were evaluated
for their in vitro inhibition of human DPP-4, and the results are ex-
pressed as IC50 values.
First, a 3-substituent of the 4-phenyl-1-isoquinolone analog
was investigated in order to confirm the role of the basic function
of the lead compound 6a, as presented in Table 1. Replacement of
the aminomethyl moiety of 6a with a hydroxymethyl group was
unfavorable to its activity (5a vs 6a). Shortening of the linker
length between the C3 of the isoquinolone core and the nitrogen
atom led to a decrease in activity (13 vs 6a). In addition, elongation
of the linker length reduced the inhibitory activity (12 vs 6a).
N-Methylation or N,N-dimethylation of 6e resulted in a marked
decrease in activity. These results indicated the importance of
the unsubstituted aminomethyl group as the optimal basic moiety
in this system. Hence, the aminomethyl group was selected for
optimization in view of its inhibitory activity.
Next, the effect of the 2-substituent on the 4-phenyl-1-isoqui-
nolone core was evaluated, as shown in Table 2. Introduction of a
sterically bulky group resulted in further enhancement of the
activity (6e and 6g vs 6a). These results suggest that the 2-alkyl
substituent interacts with the hydrophobic residue in the target
enzyme, which supports our hypothesis described above (Chart
1). Accordingly, the isobutyl group was chosen as the 2-substituent
for the subsequent SAR studies.
O
O
Me
Me
N
N
a
OH
Cl
Cl
NH2
O
4a
13
Scheme 3. Synthesis of compound 13. Reagents: (a) (i) DPPA, Et3N, DMF; (ii)
toluene; (iii) t-BuOH; (iv) 4 M HCl–AcOEt.
Me Me
Me Me
Me Me
N
O
O
O
N
N
a
b
OH
NHBoc
MeO
NHBoc
Br
Br
O
5b
14
15
Me Me
Me Me
N
O
O
R1
N
N
d
c
HO
NHBoc
NHBoc
R2
O
O
17a: R1 = R2 = H
16
17b: R1 = H, R2 = Me
17c: R1 = R2 = Me
e
Me Me
O
N
NHBoc
NC
18
Me Me
N
Me Me
O
O
f
N
NHBoc
NH2
R
19a
: R = CO2H
16:R = CO2H
R
HCl
17a
19b: R = CONH2
: R = CONH2
19c: R = CONHMe
19d: R = CONMe2
17b: R = CONHMe
17c:R = CONMe2
19e
18:
: R = CN
R = CN
Scheme 4. Synthesis of compounds 19a–e. Reagents: (a) (i) SOCl2, pyridine, THF, toluene; (ii) 2 M NH3–EtOH; (iii) Boc2O, DMAP, THF; (b) CO, Pd(OAc)2, dppp, Et3N, DMSO,
MeOH; (c) NaOH, MeOH, THF; (d) HOBtꢀNH3 or R1R2NH and HOBt, WSC, DMF; (e) cyanuric chloride, DMF; (f) 4 M HCl–AcOEt, THF.

Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
4957
Me Me
N
Me Me
O
Me Me
N
O
O
a
O
N
c
O
NHBoc
NHBoc
NHBoc
Br
EtO
X
HO
X
14
20a: X = CH=CH-(E)
20b: X = CH2CH2
21a: X = CH=CH-(E)
21b: X = CH2CH2
b
Me Me
Me Me
N
O
O
d
e
O
N
O
NHBoc
NH2
H2N
X
H2N
X
HCl
22a:
23a:
X = CH=CH-(E)
X = CH=CH-(E)
22b: X = CH2CH2
23b: X = CH2CH2
Scheme 5. Synthesis of compounds 23a and 23b. Reagents: (a) ethyl acrylate, Pd(OAc)2, Et3N, DMF; (b) 5% Pd–C, H2, EtOH, THF; (c) NaOH, MeOH, THF; (d) WSC, HOBtꢀNH3,
DMF; (e) 4 M HCl–AcOEt.
O
O
O
Me
O
a
b
c
OH
OH
OH
OH
7
O
N
Me
CO2Et
BzlO
HO
BzlO
BzlO
6
CO2H
O
O
O
24
25
26
27
Me Me
O
Me Me
Me Me
O
O
N
7
e
f
d
BzlO
N
N
OEt
BzlO
BzlO
OEt
OEt
6
O
OH
O
OTf O
29a: 6-substituted
28a: 6-substituted
28b: 7-substituted
29b: 7-substituted
30a: 6-substituted
30b: 7-substituted
Me Me
Me Me
O
Me Me
O
O
N
N
N
HO
h, j, k,m
g
BzlO
n
BzlO
NHBoc
OH
NHBoc
O
31a: 6-substituted
31b: 7-substituted
32a: 6-substituted
32b: 7-substituted
33a: 6-substituted
33b: 7-substituted
33a
For
p
Me Me
Me Me
N
Me Me
O
O
O
o
p
H2N
H2N
O
N
N
O
O
NHBoc
NH2
NH2
HCl
O
HO
HCl
34a: 6-substituted
34b: 7-substituted
35a: 6-substituted
35b: 7-substituted
36
Scheme 6. Synthesis of compounds 35a,b and 36. Reagents: (a) (i) PhCH2Br, K2CO3, DMF; (ii) NaOH, MeOH, THF; (b) Ac2O; (c) (CH3)2CHCH2NHCH2CO2Et,THF; (d) (i) EtI,
K2CO3, DMF; (ii) 20% EtONa in EtOH; (e) NaH, PhNTf2, DMF; (f) PhB(OH)2, Na2CO3, Pd(PPh3)4, toluene, EtOH, H2O; (g) NaOH, EtOH, THF; (h) (i) (COCl)2, DMF, THF; (ii) NaBH4,
DME; (j) SOCl2, toluene; (k) potassium phthalimide, DMF; (m) (i) H2NNH2ꢀH2O, EtOH; (ii) Boc2O, THF; (n) 5% Pd–C, H2, EtOH, THF; (o) ICH2CONH2, DBU, DMF; (p) 4 M
HCl–AcOEt.
As mentioned above, we hypothesized that the introduction of
an appropriate hydrogen bond donor into the 6- or 7-position of
the 4-phenyl-1-isoquinolone would provide an interaction with
the S10 pocket. Effects of the 6- or 7-substituents were thus inves-
tigated. The results are summarized in Table 3. Replacement of the
6-chloro group of 6e with a hydrophilic group, such as a carboxy
(19a), a carbamoyl (19b), a cyano (19e), a hydroxy (36), and a
methoxy group (6h), tended to reinforce the inhibitory potency.

4958
Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
Me Me
N
Me Me
O
Me Me
N
O
O
O
HO
TfO
N
MeO
a
b
NHBoc
NHBoc
NHBoc
37
33b
38
Me Me
N
Me Me
Me Me
N
O
O
O
O
O
O
HO
H2N
N
H2N
e
d
c
NH2
HCl
NHBoc
NHBoc
39
40
41
Scheme 7. Synthesis of compound 41. Reagents: (a) NaH, PhNTf2, DMF; (b) CO, Pd(OAc)2, dppp, Et3N, DMSO, MeOH; (c) NaOH, MeOH, THF; (d) WSC, HOBtꢀNH3, DMF; (e) 4 M
HCl–AcOEt.
Table 1
Table 3
SAR summary for the 3-substituents on the isoquinolone derivatives
SAR summary for the 6- or 7-substituents on the isoquinolone derivatives
Me Me
O
R2
R1
N
NH2
Compound
R1
R2
DPP-IV inhibition
(IC50, l
M)a
Compound
R
DPP-IV inhibition (IC50, l
M)a
5a
6a
12
13
CH2OH
>100
16
87
6e
19a
19b
19e
41
19c
19d
36
Cl
CO2H
CONH2
CN
H
H
H
H
1.6
CH2NH2
CH2CH2NH2
NH2
0.98
0.36
1.1
2.4
1.9
>100
H
CONH2
H
H
H
H
H
OCH2CONH2
H
H
6e
8a
8b
CH2NH2
CH2NHMe
CH2NMe2
1.6
>100
>100
CONHMe
CONMe2
OH
OMe
OCH2CONH2
H
4.0
0.79
0.87
0.24
1.1
0.28
1.5
a
Inhibitory activity against human DPP-4.
6h
35a
35b
23a
23b
CH@CHCONH2 (E)
CH2CH2CONH2
Table 2
SAR summary for the 2-substituents on the isoquinolone derivatives
a
Inhibitory activity against human DPP-4.
O
R
N
NH2
Cl
Compound
R
DPP-IV inhibition (IC50, l
M)a
6b
6c
6d
6e
6f
Et
n-Pr
n-Bu
i-Bu
n-Pen
Neopentyl
8.3
2.4
2.4
1.6
3.8
1.9
6g
a
Inhibitory activity against human DPP-4.
Remarkably, compound 19b with a hydrogen-bond donating car-
bamoyl group exhibited submicromolar inhibitory activity. A sig-
nificant potency-enhancing effect was achieved by the insertion
of an oxymethylene linker between the carbamoyl moiety and
the C6 of the isoquinolone core (35a), which led to a fivefold
improvement in potency when compared with 6e. A similar
improvement was found with an unsaturated ethylene derivative
(23a). In contrast, compounds bearing a bulkier substituent, such
Figure 1. X-ray structure of compound 35a in complex with human DPP-4. The
amino acids (black carbons, blue texts) in the binding sites of 35a (green carbons)
are indicated. Major interactions are depicted as dotted lines (hydrophobic, blue;
hydrophilic, red).

Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
4959
Table 4
Selectivity for other peptidases
Enzyme DPP-4 (human) DPP-4 (rat plasma) DPP-2 (rat spleen) Cathepsin C
Prolyl endopeptidase Proline iminopeptidase Leucine aminopeptidase
a
a
a
IC50
(l
M)
IC50
(
lM)
IC50 (lM)
(bovine spleen) (Flavobacterium sp.) (Bacillus sp.) (porcine kidney)
a
a
a
a
IC50
(
lM)
IC50
(l
M)
IC50
(l
M)
IC50 (lM)
19b
23a
35a
0.36
0.28
0.24
0.55
0.27
0.15
38
74
61
>30
170
>100
>300
>300
>300
>300
>300
>300
>300
>300
>300
a
IC50 values shown are means of duplicate measurements.
as a mono- or dialkylcarbamoyl (19c and 19d), and the insertion of
a saturated linker (23b) led to a slight decrease in activity com-
pared to 19b. Moreover, transposition of the 6-carbamoyl and
the 6-carbamoylmethoxy groups onto the 7-position (35b and
41, respectively) caused a deleterious effect on the activity. These
results suggest the importance of a hydrogen-bond donating moi-
ety linked through an appropriate linker to the C6 of the isoquino-
lone core and show that the carbamoylmethoxy group of 35a and
the (E)-2-carbamoylethenyl group of 23a are the optimal substitu-
ents at the 6-position.
and its amide NH2 groups are assumed to interact weakly with
Trp629. The most interesting interaction is the 3.1–3.4 Å hydrogen
bond formed between the carbonyl oxygen on the 6-carbamoyl-
methoxy moiety and the side chain of Lys554 residue. This crystal-
lography identified a potential and useful binding site that has not
been reported or targeted before. Thus, this study indicates that
the binding mode of the isoquinolone derivative to DPP-4 hypoth-
esized by our docking study is consistent with that observed in the
co-crystals of 35a.
Consequently, the carbamide (19b), oxyacetamide (35a), and
acrylamide (23a) analogs were further evaluated for in vitro en-
zyme selectivity. As presented in Table 4, compounds 19b, 23a,
and 35a displayed high selectivity for DPP-4 over closely related
peptidases, such as cathepsin C, prolyl endopeptidase, proline imi-
nopeptidase, and DPP-2. We selected compounds 23a and 35a on
the basis of safety pharmacology. The compounds 23a and 35a
analogs showed excellent oral bioavailability in rats (data not
shown). Thus, 23a and 35a are promising compounds for exerting
potent in vivo effects when administered orally.
The X-ray structure of compound 35a in complex with human
DPP-4 was obtained (Fig. 1). The 3-aminomethyl group interacts
with the Glu-motif (Glu205 and Glu206) that are located on the
a-helix that protrudes from the b-propeller domain into the active
site. The bulky end of the 2-isobutyl group makes a hydrophobic
interaction with Phe357, and the 4-phenyl ring occupies the
well-defined hydrophobic S1 pocket. In addition, the 6-carbamoyl-
methoxy moiety exerts van der Waals interactions with Tyr547,
3.2. In vivo studies
The in vivo hypoglycemic effects of compounds 23a and 35a
were evaluated in an oral glucose tolerance test (OGTT) using fe-
male Wistar fatty rats, which are one of the type 2 diabetic animal
models with obesity, and the results are shown in Figure 2. Oral
administration of 23a and 35a at a dose of 1 mg/kg significantly re-
duced plasma glucose levels in female Wistar fatty rats.
The dose-dependent effects of compound 35a in the OGTT are
shown in Figure 3. The results indicate that compound 35a pro-
duced a dose-dependent improvement of glucose tolerance in Wis-
tar fatty rats after oral administration and its minimum effective
dose was 0.3 mg/kg ( Fig. 3 A). Simultaneously, plasma insulin
levels increased in a dose-dependent manner, which was consis-
tent with the glucose-lowering effect of 35a (Fig. 3 B). Therefore,
the potent, selective, and orally active DPP-4 inhibitor 35a is ex-
pected to provide a potential therapeutic efficacy for the treatment
of type 2 diabetes.
Figure 2. Oral glucose tolerance test in female Wistar fatty rats. Plasma glucose
levels of female Wistar fatty rats (n = 6) administered 1 mg/kg of compounds or
0.5% MC 60 min before oral glucose load (1 g/kg). Data points indicate means, and
the error bars indicate SD (n = 6). ⁄p <0.05, ⁄⁄p <0.01 versus control by Dunnet test.
Figure 3. Oral glucose tolerance test of compound 35a in female Wistar fatty rats. Plasma glucose (A) and plasma insulin levels (B) of female Wistar fatty rats administered
0.03–1 mg/kg of compound 35a or 0.5% MC 60 min before oral glucose load (1 g/kg). Data points indicate means, and the error bars indicate SD (n = 6). ⁄: p <0.025 versus
control by Williams test.

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Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
5.3. 4-Fluoro-2-(phenylcarbonyl)benzoic acid (2c)
4. Conclusion
Compound 2c was prepared from 4-fluorophthalic anhydride in
a manner similar to that described for the synthesis of 2a in 33%
yield as a white solid. 1H NMR (200 MHz, CDCl3) d 7.03–7.72 (7H,
m), 8.10 (1H, dd, J = 5.2, 8.8 Hz).
Starting from the lead compound 6a, a series of 1,2-dihydro-4-
phenyl-1-isoquinolones was designed, synthesized, and evaluated
as DPP-4 inhibitors. Significant improvement in in vitro activity
for this class has been accomplished by the introduction of an
appropriate lipophilic side chain and a hydrogen bond donor into
the 2- and 6-position of the isoquinolone nucleus, respectively.
These studies revealed that the isoquinolone core is an excellent
template mimicking the dipeptidyl backbone and side-chains.
Finally, we discovered a potent, selective, and orally bioavailable
non-peptide DPP-4 inhibitor, 2-{[3-(aminomethyl)-2-(2-methyl-
propyl)-1-oxo-4-phenyl-1,2-dihydroisoquinolin-6-yl]oxy}acet-
amide (35a). The co-crystallographic analysis of 35a in complex
with DPP-4 validated our hypothesized binding mode, and more-
over identified Lys554 as a new target-binding site available for
DPP-4 inhibitors.
5.4. 6-Chloro-1-oxo-4-phenyl-1H-isochromene-3-carboxylic
acid (3a)
A mixture of 2a (1.3 g, 5 mmol), potassium carbonate (0.71 g,
5.1 mmol), diethyl bromomalonate (1.3 g, 5.4 mmol), acetone
(35 mL) and N,N-dimethylformamide (1 mL) was stirred at room
temperature for 1.5 h. The solvent was evaporated under reduced
pressure. The residue was poured into water and extracted with
AcOEt. The AcOEt extract was washed with water, dried over anhy-
drous MgSO4, and concentrated in vacuo. The residue was added a
mixture of acetic acid (25 mL) and concentrated hydrochloric acid
(25 mL), and the mixture was stirred at 110 °C for 3 h. After evap-
oration of the solvent, the residue was poured into water and ex-
tracted with AcOEt. The extract was washed with brine, dried
with anhydrous MgSO4 and concentrated in vacuo to give 3a
(0.89 g, 59%) as crystals. 1H NMR (200 MHz, CDCl3) d 7.07 (1H, d,
J = 1.8 Hz), 7.21–7.25 (2H, m), 7.47–7.53 (3H, m), 7.61 (1H, dd,
J = 1.8, 8.4 Hz), 8.35 (1H, d, J = 8.4 Hz).
5. Experimental section
Melting points were determined with a Yanagimoto melting
point apparatus or a Büchi melting point apparatus B-545 and
are uncorrected. 1H NMR spectra were obtained at 200 or
300 MHz on a Varian Gemini-200 or a Varian Ultra-300 spec-
trometer. Chemical shifts are given in d values (ppm) using tet-
ramethylsilane as the internal standard. Peak multiplicities are
expressed as follows. Abbreviations are used as follows: s, sin-
glet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet;
br s, broad singlet; m, multiplet. Elemental analyses and high-
resolution mass spectra (HRMS) experiments were carried out
by Takeda Analytical Laboratories Ltd. Reactions were followed
by TLC on Silica Gel 60 F 254 precoated TLC plates (E. Merck)
or NH TLC plates (Fuji Silysia Chemical Ltd). Chromatographic
separations were carried out on Silica Gel 60 (0.063–0.200 or
0.040–0.063 mm, E. Merck) using the indicated eluents. Yields
are unoptimized. Chemical intermediates were characterized by
1H NMR.
5.5. 6-Bromo-1-oxo-4-phenyl-1H-isochromene-3-carboxylic
acid (3b)
A mixture of 2b (25 g, 82 mmol), potassium carbonate (12 g,
87 mmol), diethyl bromomalonate (22 g, 92 mmol), acetone
(450 mL) and N,N-dimethylformamide (8 mL) was stirred at room
temperature for 15 h. The solvent was evaporated under reduced
pressure. The residue was poured into water and extracted with
AcOEt. The AcOEt extract was washed with brine, dried over anhy-
drous MgSO4, and concentrated in vacuo. The residue was crystal-
lized from hexanes, and collected by filtration. The obtained
crystals were added to a mixture of acetic acid (235 mL) and con-
centrated hydrochloric acid (360 mL), and the mixture was stirred
at 120 °C for 8 h. The reaction mixture was cooled and concen-
trated. The residue was poured into water and extracted with
AcOEt. The extract was washed with brine, dried with anhydrous
MgSO4, and concentrated under reduced pressure. The precipitated
crystals were collected by filtration, washed with IPE and dried to
give 3b (17 g, 60%) as crystals. 1H NMR (200 MHz, CDCl3) d 7.20–
7.28 (3H, m), 7.47–7.55 (3H, m), 7.77 (1H, dd, J = 1.8, 8.6 Hz),
8.26 (1H, d, J = 8.6 Hz). Mp 205–206 °C.
5.1. 4-Chloro-2-(phenylcarbonyl)benzoic acid (2a)
To a solution of 4-chlorophthalic anhydride (5.0 g, 27.4 mmol)
in benzene (25 mL) was added aluminum chloride (7.3 g,
54.7 mmol) in small portions at 0 °C, and the resulting mixture
was stirred at room temperature for 40 h. The mixture was
quenched with water at 0 °C and extracted with AcOEt. The organic
layer was washed with brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The resulting solid was filtered, washed
with hexane–AcOEt and dried to give 2a (6.7 g, 47%) as white crys-
tals. 1H NMR (200 MHz, CDCl3) d 7.31–7.73 (7H, m), 8.02 (1H, d,
J = 8.4 Hz).
5.6. 6-Fluoro-1-oxo-4-phenyl-1H-isochromene-3-carboxylic
acid (3c)
Compound 3c was prepared in a manner similar to that de-
scribed for 3a in 69% yield as a white solid. 1H NMR (200 MHz,
CDCl3) d 6.73 (1H, dd, J = 1.8, 9.8 Hz), 7.26–7.57 (6H, m), 8.41
(1H, dd, J = 5.4, 8.8 Hz).
5.2. 4-Bromo-2-(phenylcarbonyl)benzoic acid (2b)
To a solution of 4-bromophthalic anhydride (50 g, 220 mmol) in
benzene (500 mL) was added aluminum chloride (60 g, 450 mmol)
in small portions at 0 °C, and the resulting mixture was stirred at
room temperature for 24 h. The mixture was poured into ice-
water, and extracted with AcOEt and THF. The organic layer was
washed with brine, dried over anhydrous MgSO4, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (hexane/AcOEt = 70/30), and the residue was recrystallized
from AcOEt–IPE to afford 2b (33 g, 49%) as white crystals. 1H
5.7. 6-Chloro-2-methyl-1-oxo-4-phenyl-1,2-dihydroisoquino-
line-3-carboxylic acid (4a)
A mixture of 3a (6.0 g, 20 mmol), 40% methylamine in MeOH
(40 mL, 392 mmol) and MeOH (80 mL) was stirred at room
temperature for 4 h. After evaporation of the solvent, 4 M hydrogen
chloride in AcOEt (98 mL) was added to the residue. The mixture
was stirred at room temperature for 24 h. After evaporation of
NMR (200 MHz, CDCl3)
d 7.36–7.73 (7H, m), 7.94 (1H, d,
J = 8.4 Hz). Mp 185–187 °C.

Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
4961
the solvent, the resulting crystals were filtered, washed sequen-
5.14. 6-Chloro-1-oxo-2-pentyl-4-phenyl-1,2-dihydroiso-
quinoline-3-carboxylic acid (4h)
tially with water and Et2O, and dried in vacuo to give 4a (6.0 g,
96%) as crystals. 1H NMR (300 MHz, CDCl3) d 3.61 (3H, s), 7.17
(1H, d, J = 1.9 Hz), 7.30–7.34 (2H, m), 7.40–7.55 (4H, m), 8.36
(1H, d, J = 4.8 Hz).
A white solid (91%). 1H NMR (300 MHz, CDCl3) d 0.84–0.95 (3H,
m), 1.24–1.42 (4H, m), 1.76–1.86 (2H, m), 3.94–4.10 (2H, m), 7.15
(1H, d, J = 2.3 Hz), 7.31–7.38 (2H, m), 7.42–7.49 (4H, m), 8.37 (1H,
d, J = 8.7 Hz).
5.8. 6-Bromo-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-dihydro-
isoquinoline-3-carboxylic acid (4b)
5.15. 6-Chloro-2-(2,2-dimethylpropyl)-1-oxo-4-phenyl-1,2-
dihydroisoquinoline-3-carboxylic acid (4i)
A solution of 3b (8.0 g, 23 mmol) and isobutylamine (23 mL,
230 mmol) in MeOH (120 mL) was stirred at room temperature
for 15 h. The solvent was evaporated under reduced pressure,
and the residue was acidified with concentrated hydrochloric acid
and extracted with AcOEt. The extract was washed with brine,
dried over anhydrous MgSO4, and concentrated under reduced
pressure. To the residue was added 4 M hydrogen chloride in
AcOEt (150 mL), and the resulting mixture was stirred at room
temperature for 3 h. The solvent was evaporated under reduced
pressure, and the precipitated crystals were collected by filtration
and washed with water. The crystals were washed with water and
dried to give 4b (8.1 g, 87%) as crystals. 1H NMR(200 MHz, CDCl3) d
0.92 (6H, d, J = 6.6 Hz), 2.21 (1H, m), 3.99 (2H, d, J = 7.6 Hz), 7.32–
7.38 (3H, m), 7.42–7.47 (3H, m), 7.60 (1H, dd, J = 2.0, 8.4 Hz), 8.26
(1H, d, J = 8.4 Hz). Mp 233–235 °C.
A white solid (82%). 1H NMR (300 MHz, CDCl3) d 1.02 (9H, s),
1.74 (1H, t, J = 6.2 Hz), 4.51 (2H, d, J = 6.0 Hz), 6.94 (1H, d,
J = 1.9 Hz), 7.27–7.30 (2H, m), 7.35 (1H, dd, J = 1.9, 8.7 Hz), 7.48–
7.54 (3H, m), 8.33 (1H, d, J = 8.7 Hz).
5.16. 6-Methoxy-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-
dihydroisoquinoline-3-carboxylic acid (4j)
A mixture of 4c (5.0 g, 14.7 mmol) and 28% sodium methoxide
in MeOH (40.0 g, 207 mmol) was refluxed for 15 h. After evapora-
tion of the solvent, the residue was poured into water, and ex-
tracted with AcOEt. The extract was washed with brine, dried
with anhydrous MgSO4, and concentrated in vacuo to give 4j
(4.0 g, 77%) as crystals. 1H NMR (300 MHz, CDCl3) d 0.94 (1H, d,
J = 6.6 Hz), 2.22–2.36 (1H, m), 3.71 (3H, s), 4.02 (2H, d, J = 7.8 Hz),
6.50 (1H, d, J = 2.2 Hz), 7.06 (1H, dd, J = 2.6, 8.8 Hz), 7.39–7.48
(5H, m), 8.39 (1H, d, J = 8.8 Hz).
Compounds 4c–i were prepared in a manner similar to that de-
scribed for 4b.
5.9. 6-Fluoro-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-dihydro-
isoquinoline-3-carboxylic acid (4c)
5.17. 6-Chloro-3-(hydroxymethyl)-2-methyl-4-phenyliso-
A white solid (35%). 1H NMR (300 MHz, CDCl3) d 0.95 (1H, d,
J = 6.6 Hz), 2.22–2.36 (1H, m), 4.03–4.07 (2H, m), 6.76 (1H, dd,
J = 2.6, 10.2 Hz), 7.14–7.47 (5H, m) 7.95 (1H, dd, J = 4.8, 8.6 Hz),
8.48 (1H, dd, J = 6.0, 8.8 Hz).
quinolin-1(2H)-one (5a)
To a solution of compound 4a (17.8 g, 56.7 mmol) in THF
(270 mL) was added oxalyl chloride (10 mL, 117 mmol) and N,N-
dimethylformamide (5 drops). The mixture was stirred at room
temperature for 2 h. The reaction mixture was concentrated under
reduced pressure, and the residue was dissolved in THF (130 mL).
The resulting mixture was added dropwise to a suspension of so-
dium tetrahydroborate (6.5 g, 172 mmol) in 1,2-dimethoxyethane
(170 mL) at 0 °C. The obtained mixture was stirred at 0 °C for 2 h.
The reaction mixture was poured into 1 M hydrochloric acid and
extracted with AcOEt. The extract was washed with brine, dried
over anhydrous MgSO4, and concentrated in vacuo. The resulting
crystals were washed with IPE to give 5a (14.0 g, 82%) as crystals.
1H NMR (300 MHz, CDCl3) d 1.97 (1H, br s), 3.82 (3H, s), 4.45 (1H, d,
J = 4.8 Hz), 6.98 (1H, d, J = 2.0 Hz), 7.28–7.55 (7H, m), 8.37 (1H, d,
J = 8.6 Hz). ESI-HRMS calcd for C17H14ClN2O m/z calcd 300.0786
(M+H), found 300.0771 (M+H).
5.10. 6-Chloro-2-ethyl-1-oxo-4-phenyl-1,2-dihydroisoquino-
line-3-carboxylic acid (4d)
Colorless crystals (61%). 1H NMR (300 MHz, CDCl3) d 1.43 (3H, d,
J = 7.0 Hz), 4.15 (2H, q, J = 7.0 Hz), 7.16 (1H, d, J = 1.8 Hz), 7.30–7.49
(6H, m), 8.42 (1H, dd, J = 2.4, 8.6 Hz). Mp 242–243 °C.
5.11. 6-Chloro-1-oxo-4-phenyl-2-propyl-1,2-dihydroisoquino-
line-3-carboxylic acid (4e)
Colorless crystals (70%). 1H NMR (300 MHz, CDCl3) d 0.96 (3H, t,
J = 7.3 Hz), 1.75–1.93 (2H, m), 3.96–4.07 (2H, m), 7.15 (1H, d,
J = 1.9 Hz), 7.33–7.36 (2H, m), 7.43–7.50 (4H, m), 8.40 (1H, d,
J = 8.7 Hz).
Compounds 5b–5i were prepared in a manner similar to that
described for 5a.
5.12. 2-Butyl-6-chloro-1-oxo-4-phenyl-1,2-dihydroisoquino-
line-3-carboxylic acid (4f)
5.18. 6-Bromo-3-(hydroxymethyl)-2-(2-methylpropyl)-4-
phenylisoquinolin-1(2H)-one (5b)
A white solid (79%). 1H NMR (300 MHz, CDCl3) d 0.94 (3H, t,
J = 7.3 Hz), 1.35–1.46 (2H, m), 1.70–1.87 (2H, m), 3.97–4.09 (2H,
m), 7.15 (1H, d, J = 1.9 Hz), 7.31–7.39 (2H, m), 7.42–7.49 (4H, m),
8.37 (1H, d, J = 8.7 Hz).
Colorless crystals (74%). 1H NMR (300 MHz, CDCl3) d 0.97 (6H, d,
J = 6.6 Hz), 2.22 (1H, m), 2.35 (1H, t, J = 5.8 Hz), 4.21 (2H, d,
J = 7.6 Hz), 4.44 (2H, d, J = 5.8 Hz), 7.10 (1H, d, J = 1.8 Hz), 7.30–
7.35 (2H, m), 7.47 (1H, dd, J = 1.8, 8.4 Hz), 7.50–7.56 (3H, m),
8.20 (1H, d, J = 8.4 Hz). Mp 176–177 °C.
5.13. 6-Chloro-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-di-
hydroisoquinoline-3-carboxylic acid (4g)
5.19. 6-Chloro-2-ethyl-3-(hydroxymethyl)-4-phenylisoquinolin-
A white solid (82%). 1H NMR (300 MHz, CDCl3) d 0.92 (6H, d,
J = 6.8 Hz), 2.16–2.26 (1H, m), 3.99 (2H, d, J = 7.5 Hz), 7.14 (1H, d,
J = 1.9 Hz), 7.31–7.38 (2H, m), 7.42–7.50 (4H, m), 8.37 (1H, d,
J = 8.7 Hz).
1(2H)-one (5c)
A white solid (71%). 1H NMR (300 MHz, CDCl3) d 1.42 (3H, t,
J = 7.0 Hz), 1.71 (1H, t, J = 5.7 Hz), 4.33–4.50 (4H, m), 6.98 (1H, d,

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Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
J = 1.9 Hz), 7.28–7.31 (2H, m), 7.40 (1H, dd, J = 2.1, 8.5 Hz), 7.46–
7.57 (3H, m), 8.41 (1H, d, J = 8.7 Hz). Mp 166–167 °C.
under reduced pressure to give (6-chloro-2-methyl-1-oxo-
4-phenyl-1,2-dihydroisoquinolin-3-yl)methyl methanesulfonate.
A mixture of the (6-chloro-2-methyl-1-oxo-4-phenyl-1,2-dihydro-
isoquinolin-3-yl)methylmethanesulfonate, 10% NH3 in MeOH
(40 mL), MeOH (10 mL) and THF (50 mL) was heated in a sealed
tube at 150 °C for 6 h. After evaporation of the solvent, the reaction
mixture was poured into water and extracted with Et2O. The ex-
tract was washed with brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The residue was collected by filtration,
washed with water and Et2O to give 6a (3.20 g, 54%) as crystals.
1H NMR (200 MHz, CDCl3) d 1.41 (2H, br s), 3.66 (2H, s), 3.85
(3H, s), 6.92 (1H, d, J = 2.3 Hz), 7.22–7.29 (2H, m), 7.35–7.41 (1H,
m), 7.43–7.57 (3H, m), 8.36–8.46 (1H, m). ESI-HRMS calcd for
5.20. 6-Chloro-3-(hydroxymethyl)-4-phenyl-2-propyliso-
quinolin-1(2H)-one (5d)
A white solid (74%). 1H NMR (300 MHz, CDCl3) d 1.04 (3H, t,
J = 7.5 Hz), 1.68 (1H, t, J = 5.8 Hz), 1.75–1.91 (2H, m), 4.21–4.32
(2H, m), 4.38–4.47 (2H, m), 6.97 (1H, d, J = 1.9 Hz), 7.27–7.31
(2H, m), 7.39 (1H, dd, J = 2.3, 8.7 Hz), 7.47–7.58 (3H, m), 8.40
(1H, d, J = 8.7 Hz).
5.21. 2-Butyl-6-chloro-3-(hydroxymethyl)-4-phenylisoquinolin-
1(2H)-one (5e)
C17H15ClN2O m/z 299.0946 (M+H), found 299.0946 (M+H).
5.27. 3-(Aminomethyl)-2-ethyl-6-chloro-4-phenylisoquinolin-
1(2H)-one (6b)
A white solid (62%). 1H NMR (300 MHz, CDCl3) d 0.99 (3H, t,
J = 7.3 Hz), 1.40–1.54 (2H, m), 1.68 (1H, t, J = 6.0 Hz), 1.72–1.86
(2H, m), 4.24–4.37 (2H, m), 4.43 (2H, d, J = 6.0 Hz), 6.97 (1H, d,
J = 1.9 Hz), 7.28–7.31 (2H, m), 7.39 (1H, dd, J = 2.1, 8.5 Hz), 7.47–
7.57 (3H, m), 8.40 (1H, d, J = 8.7 Hz).
To a solution of 6-chloro-2-ethyl-3-(hydroxymethyl)-4-phenyl-
isoquinolin-1(2H)-one (0.25 g, 0.8 mmol) in CH2Cl2 (5 mL) was
added triethylamine (0.2 mL, 1.4 mmol) and methanesulfonyl
chloride (0.1 mL, 1.3 mmol) at 0 °C, and the resulting mixture
was stirred at room temperature for 30 min. The reaction mixture
was poured into 1 M hydrochloric acid and extracted with CH2Cl2.
The extract was washed with brine, dried over anhydrous MgSO4,
and concentrated in vacuo. A mixture of the residue, 10% NH3 in
EtOH (10 mL), and THF (10 mL) was heated in a sealed tube at
130 °C for 3 h. The reaction mixture was poured into water and ex-
tracted with AcOEt. The extract was washed with brine, dried over
anhydrous MgSO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/
AcOEt = 50/50) to give 6b (0.08 g, 38%) as crystals. 1H NMR
(200 MHz, CDCl3) d 1.39 (2H, br s), 1.42 (3H, t, J = 7.0 Hz), 3.64
(2H, s), 4.43 (2H, q, J = 7.0 Hz), 6.91 (1H, d, J = 2.2 Hz), 7.23–7.31
(2H, m), 7.38 (1H, dd, J = 2.2, 8.4 Hz), 7.45–7.57 (3H, m), 8.41
(1H, d, J = 8.4 Hz). Mp 126–127 °C. Anal. Calcd for C18H17ClN2Oꢀ1/
4H2O: C, 68.62; H, 5.51; N, 8.89. Found: C, 68.61; H, 5.40; N,
8.84. ESI-HRMS calcd for C18H17ClN2O m/z 313.1102 (M+H), found
313.1085 (M+H).
5.22. 6-Chloro-3-(hydroxymethyl)-2-(2-methylpropyl)-4-
phenylisoquinolin-1(2H)-one (5f)
A white solid (77%). 1H NMR (300 MHz, CDCl3) d 0.99 (6H, d,
J = 6.4 Hz), 1.70 (1H, t, J = 5.8 Hz), 2.17–2.37 (1H, m), 4.21 (2H, d,
J = 7.5 Hz), 4.45 (2H, d, J = 5.3 Hz), 6.96 (1H, d, J = 1.9 Hz), 7.28–
7.32 (2H, m), 7.38 (1H, dd, J = 1.9, 8.7 Hz), 7.47–7.56 (3H, m),
8.38 (1H, d, J = 8.7 Hz).
5.23. 6-Chloro-3-(hydroxymethyl)-2-pentyl-4-
phenylisoquinolin-1(2H)-one (5g)
A white solid (71%). 1H NMR (300 MHz, CDCl3) d 0.87–0.99 (3H,
m), 1.30–1.50 (4H, m), 1.66 (1H, t, J = 5.8 Hz), 1.75–1.85 (2H, m),
4.22–4.37 (2H, m), 4.43 (2H, d, J = 6.0 Hz), 6.97 (1H, d, J = 1.9 Hz),
7.27–7.31 (2H, m), 7.39 (1H, dd, J = 2.1, 8.5 Hz), 7.47–7.56 (3H,
m), 8.40 (1H, d, J = 8.7 Hz).
5.24. 6-Chloro-2-(2,2-dimethylpropyl)-3-(hydroxymethyl)-4-
phenylisoquinolin-1(2H)-one (5h)
5.28. 3-(Aminomethyl)-6-chloro-2-propyl-4-phenylisoquinolin-
1(2H)-one (6c)
A white solid (71%). 1H NMR (300 MHz, CDCl3) d 0.96 (9H, s),
4.14 (2H, br s), 7.11 (1H, d, J = 1.9 Hz), 7.30–7.33 (2H, m), 7.42–
7.50 (4H, m), 8.35 (1H, d, J = 8.7 Hz).
Compound 6c was prepared in a manner similar to that de-
scribed for 6a in 80% yield as a white solid. 1H NMR (200 MHz,
CDCl3) d 1.04 (3H, t, J = 7.4 Hz), 1.17 (2H, br s), 1.71–1.93 (2H,
m), 3.63 (2H, s), 4.28 (2H, t, J = 7.6 Hz), 6.90 (1H, d, J = 2.2 Hz),
7.21–7.32 (2H, m), 7.37 (1H, dd, J = 2.2, 8.4 Hz), 7.41–7.58 (3H,
m), 8.40 (1H, d, J = 8.4 Hz). Mp 146–147 °C. Anal. Calcd for
5.25. 3-(Hydroxymethyl)-6-methoxy-2-(2-methylpropyl)-4-
phenylisoquinolin-1(2H)-one (5i)
C
19H19ClN2O: C, 69.83; H, 5.86; N, 8.57. Found: C, 69.97; H, 5.90;
A white solid (63%). 1H NMR (200 MHz, CDCl3) d 0.99 (6H, d,
J = 6.8 Hz), 1.81 (1H, t, J = 6.0 Hz), 2.20–2.34 (1H, m), 3.67 (3H, s),
4.20 (2H, d, J = 7.6 Hz), 4.45 (2H, d, J = 5.7 Hz), 6.34 (1H, d,
J = 2.6 Hz), 7.00 (1H, dd, J = 2.5, 8.9 Hz), 7.28–7.36 (2H, m), 7.44–
7.57 (3H, m), 8.38 (1H, d, J = 8.7 Hz).
N, 8.49. ESI-HRMS calcd for C19H19ClN2O m/z 327.1259 (M+H),
found 327.1247 (M+H).
5.29. 3-(Aminomethyl)-2-butyl-6-chloro-4-phenylisoquinolin-
1(2H)-one (6d)
Compound 6d was prepared in a manner similar to that de-
scribed for 6a in 32% yield as a white solid. 1H NMR (200 MHz,
CDCl3) d 1.00 (3H, t, J = 7.4 Hz), 1.23 (2H, br s), 1.38–1.60 (2H, m),
1.68–1.84 (2H, m), 3.63 (2H, s), 4.33 (2H, t, J = 7.6 Hz), 6.90 (1H,
d, J = 2.0 Hz), 7.22–7.31 (2H, m), 7.38 (1H, dd, J = 2.0, 8.8 Hz),
7.45–7.58 (3H, m), 8.41 (1H, d, J = 8.8 Hz). Anal. Calcd for
5.26. 3-(Aminomethyl)-6-chloro-2-methyl-4-phenylisoquinolin-
1(2H)-one (6a)
To a solution of 5a (6.0 g, 20 mmol) in CH2Cl2 (200 mL) was
added triethylamine (5.6 mL, 40 mmol) and methanesulfonyl chlo-
ride (1.9 mL, 24.5 mmol). The mixture was stirred at room temper-
ature for 30 min. The reaction mixture was poured into 1 M
hydrochloric acid and extracted with CH2Cl2. The extract was
washed with brine, dried over anhydrous MgSO4, and concentrated
C20H21ClN2O: C, 70.48; H, 6.21; N, 8.22. Found: C, 70.27; H, 6.18;
N, 8.09. ESI-HRMS calcd for C20H21ClN2O m/z 341.1415 (M+H),
found 341.1391 (M+H).

Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
4963
5.30. 3-(Aminomethyl)-6-chloro-2-(2-methylpropyl)-4-
phenylisoquinolin-1(2H)-one (6e)
CDCl3) d 0.97 (6H, d, J = 7.0 Hz), 2.06 (6H, s), 2.13–2.33 (1H, m),
3.28 (2H, s), 4.35 (2H, d, J = 7.4 Hz), 6.91 (1H, d, J = 1.6 Hz), 7.16–
7.22 (2H, m), 7.38 (1H, dd, J = 1.6, 8.4 Hz), 7.44–7.52 (3H, m),
8.42 (1H, d, J = 8.4 Hz). ESI-HRMS calcd for C22H25ClN2O m/z
369.1728 (M+H), found 369.1728 (M+H).
Compound 6e was prepared in a manner similar to that de-
scribed for 6a in 42% yield as a white solid. 1H NMR (200 MHz,
CDCl3) d 1.00 (6H, d, J = 6.6 Hz), 1.18 (2H, br s), 2.12–2.38 (1H,
m), 3.66 (2H, s), 4.20 (2H, d, J = 7.4 Hz), 6.90 (1H, d, J = 1.6 Hz),
7.21–7.31 (2H, m), 7.37 (1H, dd, J = 1.6, 8.4 Hz), 7.45–7.58 (3H,
m), 8.40 (1H, d, J = 8.4 Hz). Mp 123–124 °C. Anal. Calcd for
5.36. Diethyl [(6-chloro-2-methyl-1-oxo-4-phenyl-1,2-dihydro-
isoquinolin-3-yl)methyl]-propanedioate (9)
C
20H21ClN2O: C, 70.48; H, 6.21; N, 8.22. Found: C, 70.35; H, 6.07;
To a solution of 7a (2.69 g, 8.4 mmol) and ethyl malonate
(1.35 g, 8.4 mmol) in THF (35 mL) was added sodium hydride
(0.34 g, 8.5 mmol) at 0 °C, and the resulting mixture was stirred
at room temperature for 48 h. The mixture was quenched with
water at 0 °C and extracted with AcOEt. The extract was washed
with brine, dried over anhydrous MgSO4, and concentrated under
reduced pressure. The residue was crystallized from AcOEt–IPE to
give 9 (3.25 g, 87%) as crystals. 1H NMR (200 MHz, CDCl3) d 1.13
(6H, t, J = 7.2 Hz), 3.30–3.47 (3H, m), 3.69 (3H, s), 4.06 (2H, q,
J = 7.2 Hz), 6.88 (1H, d, J = 2.2 Hz), 7.19–7.23 (2H, m), 7.37 (1H,
dd, J = 2.2, 8.4 Hz), 7.45–7.54 (3H, m), 8.39 (1H, d, J = 8.4 Hz). Mp
120–121 °C. Anal. Calcd for C24H24ClNO5: C, 65.23; H, 5.47; N,
3.17. Found: C, 65.12; H, 5.37; N, 3.38.
N, 8.10. ESI-HRMS calcd for C20H21ClN2O m/z 341.1415 (M+H),
found 341.1395 (M+H).
5.31. 3-(Aminomethyl)-6-chloro-2-pentyl-4-phenylisoquinolin-
1(2H)-one (6f)
Compound 6f was prepared in a manner similar to that de-
scribed for 6a in 56% yield as a white solid. 1H NMR (200 MHz,
CDCl3) d 0.93 (3H, t, J = 7.4 Hz), 1.20–1.53 (6H, m), 1.70–1.90 (2H,
m), 3.63 (2H, s), 4.31 (2H, t, J = 8.0 Hz), 6.90 (1H, d, J = 2.2 Hz),
7.22–7.31 (2H, m), 7.37 (1H, dd, J = 2.2, 8.4 Hz), 7.45–7.58 (3H,
m), 8.40 (1H, d, J = 8.4 Hz). Anal. Calcd for C21H23ClN2O: C, 71.07;
H, 6.53; N, 7.89. Found: C, 70.82; H, 6.34; N, 7.72. ESI-HRMS calcd
for C21H23ClN2O m/z 355.1572 (M+H), found 355.1550 (M+H).
5.37. 3-(6-Chloro-2-methyl-1-oxo-4-phenyl-1,2-dihydroiso-
quinolin-3-yl)propanoic acid (10)
5.32. 3-(Aminomethyl)-6-chloro-2-(2,2-dimethylpropyl)-4-
phenylisoquinolin-1(2H)-one (6g)
A mixture of 9 (3.25 g, 7.4 mmol), concentrated hydrochloric
acid (35 mL, 401 mmol) and acetic acid (35 mL, 611 mmol) was
heated at 120 °C for 2 h. After evaporation of the solvent, the resi-
due was extracted with AcOEt. The extract was washed with brine,
dried over anhydrous MgSO4, and concentrated under reduced
pressure. The residue was crystallized from AcOEt–IPE to give 10
(2.32 g, 92%) as crystals. 1H NMR (200 MHz, CDCl3) d 2.39–2.47
(2H, m), 2.81–2.89 (2H, m), 3.71 (3H, s), 6.84 (1H, d, J = 2.2 Hz),
7.21–7.26 (2H, m), 7.35 (1H, dd, J = 2.2, 8.4 Hz), 7.46–7.55 (3H,
m), 8.35 (1H, d, J = 8.8 Hz).
Compound 6g was prepared in a manner similar to that de-
scribed for 6a in 81% yield as a white solid. 1H NMR (200 MHz,
CDCl3) d 1.02 (9H, s), 1.23 (2H, br s), 3.71 (2H, s), 4.30 (2H, br s),
6.90 (1H, d, J = 2.2 Hz), 7.20–7.30 (2H, m), 7.37 (1H, dd, J = 2.2,
8.4 Hz), 7.42–7.68 (3H, m), 8.39 (1H, d, J = 8.4 Hz). Mp 173–
174 °C. Anal. Calcd for C21H23ClN2O: C, 71.07; H, 6.53; N, 7.89.
Found: C, 70.89; H, 6.54; N, 7.61. ESI-HRMS calcd for C21H23ClN2O
m/z 355.1572 (M+H), found 355.1539 (M+H).
5.33. 3-(Aminomethyl)-6-methoxy-2-(2-methylpropyl)-4-
5.38. tert-Butyl [2-(6-chloro-2-methyl-1-oxo-4-phenyl-1,2-di-
phenylisoquinolin-1(2H)-one (6h)
hydroisoquinolin-3-yl)ethyl]carbamate (11)
Compound 6h was prepared in a manner similar to that de-
scribed for 6a in 84% yield as white needles (84%). 1H NMR
(200 MHz, CDCl3) d 1.00 (6H, d, J = 6.6 Hz), 1.26 (2H, br s), 2.15–
2.38 (1H, m), 3.65 (2H, s), 3.67 (3H, s), 4.18 (2H, d, J = 7.2 Hz),
6.30 (1H, d, J = 2.4 Hz), 7.02 (1H, dd, J = 2.4, 8.8 Hz), 7.23–7.34
(2H, m), 7.39–7.57 (3H, m), 8.41 (1H, d, J = 8.8 Hz). Anal. Calcd
for C21H24N2O2: C, 74.97; H, 7.19; N, 8.33. Found: C, 74.73; H,
7.40; N, 8.32. ESI-HRMS calcd for C21H24N2O2 m/z 337.1911
(M+H), found 337.7879 (M+H).
To a solution of 10 (0.15 g, 0.4 mmol) in N,N-dimethylformam-
ide (3 mL) was added DPPA (0.1 mL, 0.5 mmol) and triethylamine
(0.07 mL, 0.5 mmol), and the resulting mixture was stirred at room
temperature for 5 h. The mixture was poured into water and ex-
tracted with AcOEt. The extract was washed with brine, dried over
anhydrous MgSO4, and concentrated under reduced pressure. The
residue was dissolved in toluene (8 mL), and the solution was re-
fluxed for 1.5 h. The solution was added tert-BuOH (5 mL), and
the resulting mixture was stirred at 85 °C for 15 h. After evapora-
tion of the solvent, the residue was extracted with AcOEt. The ex-
tract was washed with brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (hexane/AcOEt = 80/20) to give 11 (0.10 g,
57%) as needles. 1H NMR (200 MHz, CDCl3) d 1.39 (9H, s), 2.69–
2.83 (2H, m), 3.14–3.28 (2H, m), 3.81 (3H, s), 4.51 (1H, br s), 6.85
(1H, d, J = 1.9 Hz), 7.21–7.25 (2H, m), 7.33–7.39 (1H, m), 7.45–
7.57 (3H, m), 8.40 (1H, d, J = 8.7 Hz). Anal. Calcd for
5.34. 6-Chloro-3-[(methylamino)methyl]-2-(2-methylpropyl)-4-
phenylisoquinolin-1(2H)-one (8a)
Compound 8a was prepared in a manner similar to that de-
scribed for 6b in 38% yield as a white solid.
1H NMR (200 MHz, CDCl3) d 1.00 (6H, d, J = 6.6 Hz), 2.16–2.29
(4H, m), 3.45 (2H, s), 4.22 (2H, d, J = 7.2 Hz), 6.91 (1H, d,
J = 1.8 Hz), 7.21–7.26 (2H, m), 7.38 (1H, dd, J = 1.8, 8.4 Hz), 7.48–
7.52 (3H, m), 8.41 (1H, d, J = 8.4 Hz). ESI-HRMS calcd for
C23H25N2O3ꢀ1/2H2O: C, 65.47; H, 6.21; N, 6.64. Found: C, 65.74;
H, 6.17; N, 7.13.
C21H23ClN2O m/z 355.1572 (M+H), found 355.1542 (M+H).
5.39. 3-(2-Aminoethyl)-6-chloro-2-methyl-4-phenylisoquinolin-
5.35. 6-Chloro-3-[(dimethylamino)methyl]-2-(2-methylpropyl)-
1(2H)-one hydrochloride (12)
4-phenylisoquinolin-1(2H)-one (8b)
To a solution of 11 (0.07 g, 0.17 mmol) in AcOEt (2 mL) was
added 4 M hydrogen chloride in AcOEt (2 mL), and the resulting
mixture was stirred at room temperature for 8 h. After evaporation
Compound 8b was prepared in a manner similar to that de-
scribed for 6b in 38% yield as a white solid. 1H NMR (200 MHz,

4964
Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
of the solvent, the resulting crystals was filtered and washed with
IPE to give 12 (0.04 g, 69%) as needles. 1H NMR (200 MHz, DMSO-
d6) d 2.87 (4H, br s), 3.67 (3H, s), 6.69 (1H, d, J = 2.3 Hz), 7.29–7.39
(2H, m), 7.47–7.65 (4H, m), 7.89 (3H, br s), 8.29 (1H, d, J = 8.7 Hz).
Anal. Calcd for C18H17ClN2OꢀHClꢀ1/2H2O: C, 60.34; H, 5.35; N, 7.82.
Found: C, 60.39; H, 5.24; N, 7.79. ESI-HRMS calcd for C18H17ClN2O
m/z 313.1102 (M+H), found 313.1073 (M+H).
duced pressure. The residue was purified by silica gel column chro-
matography (hexane/AcOEt = 70/30) to give 15 (1.92 g, 83%) as
crystals. 1H NMR (300 MHz, CDCl3) d 1.01 (6H, d, J = 6.6 Hz), 1.43
(9H, s), 2.26 (1H, m), 3.85 (3H, s), 4.09 (2H, d, J = 7.6 Hz), 4.22
(2H, d, J = 5.6 Hz), 4.47 (1H, br s), 7.23–7.28 (2H, m), 7.49–7.56
(3H, m), 7.66 (1H, d, J = 1.0 Hz), 8.05 (1H, dd, J = 1.7, 8.0 Hz), 8.52
(1H, d, J = 8.0 Hz). Mp 205–206 °C. Anal. Calcd for C27H32N2O5: C,
69.81; H, 6.94; N, 6.03. Found: C, 69.71; H, 6.80; N, 6.13.
5.40. 3-Amino-6-chloro-2-methyl-4-phenylisoquinolin-1(2H)-
one (13)
5.43. 3-{[(tert-Butoxycarbonyl)amino]methyl}-2-(2-methyl-
propyl)-1-oxo-4-phenyl-1,2- dihydroisoquinoline-6-carboxylic
acid (16)
Compound 13 was prepared in a manner similar to that de-
scribed for 12 in 38% yield as a yellow solid. 1H NMR (200 MHz,
CDCl3) d 3.66 (3H, s), 3.94 (2H, br s), 6.89 (1H, d, J = 1.9 Hz), 7.12
(1H, dd, J = 2.3, 8.7 Hz), 7.29–7.36 (2H, m), 7.40–7.50 (1H, m),
7.50–7.60 (2H, m), 8.27 (1H, d, J = 8.7 Hz). ESI-HRMS calcd for
To a solution of 15 (0.28 g, 0.6 mmol) in THF (10 mL) and
MeOH (10 mL) was added 1 M sodium hydroxide (5 mL). The
obtained mixture was stirred at room temperature for 2 h. The
reaction mixture was poured into water, acidified with 1 M hydro-
chloric acid, and extracted with AcOEt. The AcOEt extract was
washed with water, dried over MgSO4, and concentrated in vacuo.
The obtained crystals were recrystallized from AcOEt–Et2O to give
16 (1.54 g, 98%) as crystals. 1H NMR (300 MHz, CDCl3) d 0.96 (6H,
d, J = 6.6 Hz), 1.07 (3H, t, J = 7.4 Hz), 1.49 (9H, s), 1.50–1.72 (2H,
m), 1.84–1.98 (2H, m), 2.14–2.21 (1H, m), 3.90 (2H, t, J = 6.4 Hz),
4.00 (2H, d, J = 6.8 Hz), 4.55 (2H, d, J = 5.0 Hz), 5.37 (1H, br s),
8.08–8.13 (1H, m), 8.35–8.46 (2H, m). Anal. Calcd for
C16H13ClN2O m/z 283.0644 (MꢁH), found 283.0632 (MꢁH).
5.41. tert-Butyl {[6-bromo-2-(2-methylpropyl)-1-oxo-4-phenyl-
1,2-dihydroisoquinolin-3- yl]methyl}carbamate (14)
To a solution of 5b (8.7 g, 22.5 mmol) and pyridine (5 drops) in
THF (30 mL) and toluene (30 mL) was added thionyl chloride
(3.4 mL, 47.3 mmol). The obtained mixture was stirred at room
temperature for 3 h. The reaction mixture was concentrated,
poured into water and extracted with AcOEt. The extract was
washed with brine, dried over anhydrous MgSO4, and concentrated
under reduced pressure to give 6-bromo-3-(chloromethyl)-2-(2-
methylpropyl)-4-phenylisoquinolin-1(2H)-one (8.2 g, 90%) as crys-
tals. 6-bromo-3-(chloromethyl)-2-(2-methylpropyl)-4-phenyliso-
C26H30N2O5: C, 69.31; H, 6.71; N, 6.22. Found: C, 69.17; H, 6.59;
N, 6.27.
5.44. tert-Butyl {[6-carbamoyl-2-(2-methylpropyl)-1-oxo-4-
phenyl-1,2-dihydroisoquinolin-3- yl]methyl}carbamate (17a)
quinolin-1(2H)-one (10 g, 24.7 mmol) in THF (20 mL) and
a
solution of 2 M NH3 in ethanol (200 mL) were sealed in a stainless
tube and stirred at 140 °C for 5 h. The reaction mixture was cooled
and concentrated. The residue was poured into water and ex-
tracted with AcOEt. The extract was washed with brine, dried over
anhydrous MgSO4 and concentrated under reduced pressure. The
residue was crystallized from Et2O and the precipitated crystals
were collected by filtration to afford 3-(aminomethyl)-6-bromo-
2-(2-methyl)propyl-4-phenylisoquinolin-1(2H)-one (5.6 g, 53%)
A solution of 16 (0.6 g, 1.3 mmol), WSC (0.5 g, 2.6 mmol) and
HOBtꢀNH3 (0.4 g, 2.6 mmol) in N,N-dimethylformamide (3 mL)
was stirred at room temperature for 3 h. The reaction mixture
was poured into water and extracted with AcOEt. The AcOEt ex-
tract was washed with aqueous sodium hydrogen carbonate solu-
tion and brine, dried over anhydrous MgSO4, and concentrated in
vacuo. The residue was crystallized from Et2O–hexane to give
17a (0.5 g, 86%) as crystals. 1H NMR (200 MHz, CDCl3) d 1.00 (6H,
d, J = 6.6 Hz), 1.43 (9H, s), 2.24 (1H, m), 4.08 (2H, d, J = 7.2 Hz),
4.20 (2H, d, J = 5.4 Hz), 4.68 (1H, br s), 5.73 (1H, br s), 6.08 (1H,
br s), 7.24–7.29 (2H, m), 7.38 (1H, d, J = 2.0 Hz), 7.47–7.56 (3H,
m), 7.74 (1H, dd, J = 2.0, 8.8 Hz), 8.45 (1H, d, J = 8.8 Hz).
as crystals. To
a solution of 3-(aminomethyl)-6-bromo-2-(2-
methyl)propyl-4-phenylisoquinolin-1(2H)-one (5.6 g, 14.5 mmol)
and 4-dimethylaminopyridine (20 mg) in THF (50 mL) was added
di-t-butyl dicarbonate (6.3 g, 2.9 mmol). The obtained mixture
was stirred at room temperature for 1 h. The reaction mixture
was poured into water and extracted with AcOEt. The extract
was washed with brine, dried over anhydrous MgSO4, and concen-
trated under reduced pressure. The residue was crystallized from
Et2O to give 14 (6.6 g, 94%) as crystals. 1H NMR (300 MHz, CDCl3)
d 1.00 (6H, d, J = 7.0 Hz), 1.43 (9H, s), 2.23 (1H, m), 4.06 (2H, d,
J = 7.8 Hz), 4.19 (2H, d, J = 5.4 Hz), 4.50 (1H, br s), 7.08 (1H, d,
J = 2.0 Hz), 7.21–7.25 (2H, m), 7.48–7.56 (4H, m), 8.30 (1H, d,
J = 8.8 Hz).
5.45. tert-Butyl {[6-(methylcarbamoyl)-2-(2-methylpropyl)-1-
oxo-4-phenyl-1,2- dihydroisoquinolin-3-yl]methyl}carbamate
(17b)
Compound 17b was prepared in a manner similar to that de-
scribed for 17a in 45% yield as crystals. 1H NMR (200 MHz, CDCl3)
d 0.99 (6H, d, J = 6.6 Hz), 1.43 (9H, s), 2.20–2.24 (1H, m), 2.94 (3H,
d, J = 2.4 Hz), 4.08 (2H, d, J = 7.0 Hz), 4.20 (2H, d, J = 5.0 Hz), 4.72
(1H, br s), 6.19 (1H, br s), 7.24–7.34 (3H, m), 7.49–7.52 (3H, m),
7.64–7.68 (1H, m), 8.42 (1H, d, J = 8.0 Hz).
5.42. Methyl 3-{[(tert-butoxycarbonyl)amino]methyl}-2-(2-
methylpropyl)-1-oxo-4-phenyl- 1,2-dihydroisoquinoline-6-
carboxylate (15)
5.46. tert-Butyl {[6-cyano-2-(2-methylpropyl)-1-oxo-4-phenyl-
1,2-dihydroisoquinolin-3- yl]methyl}carbamate (18)
A
mixture of 14 (3.0 g, 6.2 mmol), 1,3-bis(diphenylphos-
phino)propane (0.45 g, 1.1 mmol) and triethylamine (0.69 g,
6.8 mmol) in DMSO (30 mL) and MeOH (15 mL) was stirred under
a carbon monoxide atmosphere at room temperature for 30 min.
Then the resulting mixture was added palladium acetate (0.25 g,
1.1 mmol), and the mixture was stirred under carbon monoxide
atmosphere at 80 °C for 15 h. The reaction mixture was poured into
water and extracted with AcOEt. The extract was washed with
water, dried over anhydrous MgSO4, and concentrated under re-
A mixture of 17a (0.3 g, 0.67 mmol), cyanuric chloride (0.37 g,
2 mmol) and N,N-dimethylformamide (3 mL) was stirred at 0 °C
for 30 min. The mixture was poured into water and extracted
with AcOEt. The AcOEt extract was washed with aqueous sodium
hydrogen carbonate solution and brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was purified by
silica gel column chromatography (hexane/AcOEt = 70/30) to give
18 (0.28 g, 97%) as crystals. 1H NMR (300 MHz, CDCl3) d 1.01

Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
4965
(6H, d, J = 7.0 Hz), 1.43 (9H, s), 2.24 (1H, m), 4.09 (2H, d,
J = 7.0 Hz), 4.23 (2H, d, J = 5.4 Hz), 4.43 (1H, br s), 7.21–7.29
(3H, m), 7.51–7.59 (3H, m), 7.65 (1H, dd, J = 1.4, 8.0 Hz), 8.55
(1H, d, J = 8.0 Hz).
8.48 (1H, d, J = 8.4 Hz), 8.67 (3H, br s). ESI-HRMS calcd for
21H21N3O m/z 332.1757 (M+H), found 332.1727 (M+H).
C
5.52. Ethyl (2E)-3-[3-{[(tert-butoxycarbonyl)amino]methyl}-2-
(2-methylpropyl)-1-oxo-4- phenyl-1,2-dihydroisoquinolin-6-
yl]prop-2-enoate (20a)
5.47. 3-(Aminomethyl)-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-
dihydroisoquinoline-6-carboxylic acid hydrochloride (19a)
To a solution of 14 (9.71 g, 20 mmol), ethyl acrylate (10.2 g,
100 mmol), and triethylamine (27.9 mL, 200 mmol) in N,N-dimeth-
ylformamide (100 mL) was added Pd(OAc)2 under argon atmo-
sphere, and the resulting mixture was stirred at 80 °C under
argon atmosphere for 8 h. The mixture was poured into water
and extracted with AcOEt. The AcOEt extract was washed with
aqueous sodium hydrogen carbonate solution and brine, dried over
anhydrous MgSO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/
AcOEt = 70/30) to give 20a (9.11 g, 90%) as an amorphous powder.
1H NMR (300 MHz, CDCl3) d 1.01 (6H, d, J = 7.0 Hz), 1.31 (3H, t,
J = 7.3 Hz), 1.43 (9H, s), 2.18–2.31 (1H, m), 4.08 (2H, d, J = 7.4 Hz),
4.13–4.28 (4H, m), 4.53 (1H, br s), 6.37 (1H, d, J = 16.2 Hz), 7.00
(1H, d, J = 1.4 Hz), 7.18–7.28 (2H, m), 7.44–7.67 (5H, m), 8.44
(1H, d, J = 8.0 Hz). Anal. Calcd for C30H36N2O5: C, 71.40; H, 7.19;
N, 5.55. Found: C, 71.37; H, 7.15; N, 5.43.
To a solution of 16 (0.15 g, 0.33 mmol) in THF (6 mL) was added
a solution of 4 M hydrogen chloride in dioxane (10 mL). The ob-
tained solution was stirred at room temperature for 15 h. The reac-
tion mixture was concentrated under reduced pressure, and the
residue was crystallized from Et2O to give 19a (0.09 g, 69%) as crys-
tals. 1H NMR (200 MHz, DMSO-d6) d 0.89 (6H, d, J = 6.6 Hz), 1.01
(3H, t, J = 7.4 Hz), 1.50–1.69 (2H, m), 1.80–1.93 (2H, m), 1.99–
2.12 (1H, m), 3.97 (2H, t, J = 6.4 Hz), 3.99 (2H, d, J = 7.6 Hz), 4.21
(2H, s), 8.09 (1H, dd, J = 1.4, 8.4 Hz), 8.34 (1H, d, J = 1.4 Hz), 8.38
(1H, d, J = 8.4 Hz), 8.69 (3H, br s). ESI-HRMS calcd for C21H22N2O3
m/z 349.1558 (MꢁH), found 349.1571 (MꢁH).
5.48. 3-(Aminomethyl)-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-
dihydroisoquinoline-6-carboxamide hydrochloride (19b)
Compound 19b was prepared in a manner similar to that de-
scribed for 19a in 85% yield as colorless crystals. 1H NMR
(200 MHz, DMSO-d6) d 0.92 (6H, d, J = 6.6 Hz), 2.11 (1H, m), 3.87
(2H, d, J = 5.6 Hz), 4.10 (2H, d, J = 7.2 Hz), 7.39 (1H, d, J = 1.4 Hz),
7.40–7.43 (2H, m), 7.56–7.62 (4H, m), 8.00 (1H, dd, J = 1.4,
8.4 Hz), 8.16 (1H, s), 8.37 (1H, d, J = 8.4 Hz), 8.61 (3H, br s). Mp
240–242 °C. ESI-HRMS calcd for C21H23N3O2 m/z 350.1863 (M+H),
found 350.1842 (M+H).
5.53. Ethyl 3-[3-{[(tert-butoxycarbonyl)amino]methyl}-2-(2-
methylpropyl)-1-oxo-4-phenyl- 1,2-dihydroisoquinolin-6-
yl]propanoate (20b)
A mixture of 20a (0.90 g, 1.8 mmol), 5% palladium on carbon
(0.5 g), EtOH (10 mL) and THF (10 mL) was stirred under atmo-
spheric pressure of hydrogen. After palladium on carbon was re-
moved by filtration, the filtrate was evaporated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy (hexane/AcOEt = 70/30) to give 20b (0.82 g, 90%) as an amor-
phous powder. 1H NMR (300 MHz, CDCl3) d 0.99 (6H, d, J = 6.6 Hz),
1.19 (3H, t, J = 7.1 Hz), 1.42 (9H, s), 2.18–2.29 (1H, m), 2.51 (2H, t,
J = 7.8 Hz), 2.90 (2H, t, J = 7.8 Hz), 4.01–4.12 (4H, m), 4.19 (2H, d,
J = 5.6 Hz), 4.40 (1H, br s), 6.74 (1H, d, J = 1.6 Hz), 7.21–7.26 (2H,
m), 7.31 (1H, dd, J = 1.6, 8.4 Hz), 7.46–7.55 (3H, m), 8.39 (1H, d,
J = 8.4 Hz).
5.49. 3-(Aminomethyl)-N-methyl-2-(2-methylpropyl)-1-oxo-4-
phenyl-1,2-dihydro- isoquinoline-6-carboxamide
hydrochloride (19c)
Compound 17c was prepared in a manner similar to that de-
scribed for 17a then compound 17c was converted to 19c in a man-
ner similar to that described for 19a in 70% yield as an amorphous
powder. 1H NMR (200 MHz, DMSO-d6) d 0.93 (6H, d, J = 6.6 Hz),
2.01–2.17 (1H, m), 2.72 (3H, d, J = 4.8 Hz), 3.87 (2H, s), 4.09 (2H,
d, J = 6.8 Hz), 7.39–7.43 (3H, m), 7.54–7.60 (3H, m), 7.95 (1H, dd,
J = 1.6, 8.6 Hz), 8.39 (1H, d, J = 8.6 Hz), 8.60 (3H, br s), 8.64 (1H, t,
J = 4.8 Hz). ESI-HRMS calcd for C22H25N3O2 m/z 364.2020 (M+H),
found 364.1988 (M+H).
5.54. (2E)-3-[3-{[(tert-Butoxycarbonyl)amino]methyl}-2-(2-
methylpropyl)-1-oxo-4-phenyl- 1,2-dihydroisoquinolin-6-
yl]prop-2-enoic acid (21a)
To a solution of 20a in THF (10 mL) and EtOH (10 mL) was
added 1 M sodium hydroxide, and the resulting mixture was stir-
red at room temperature for 1 h. The mixture was poured into
water, acidified with 1 M hydrochloric acid, and extracted with
AcOEt. The AcOEt extract was washed with brine, dried over anhy-
drous MgSO4, and concentrated in vacuo. The residue was crystal-
lized from AcOEt–hexane to 21a (0.56 g, 85%) as a white solid. 1H
NMR (300 MHz, CDCl3) d 0.99 (6H, d, J = 6.6 Hz), 1.48 (9H, s),
2.16–2.24 (1H, m), 4.06 (2H, d, J = 7.2 Hz), 4.15 (2H, d, J = 4.0 Hz),
5.62 (1H, br s), 6.27 (1H, d, J = 16.0 Hz), 6.82 (1H, s), 7.33–7.40
(3H, m), 7.48–7.58 (3H, m), 8.28 (1H, d, J = 8.8 Hz). Mp 172–
173 °C. Anal. Calcd for C28H32N2O5: C, 70.27; H, 7.16; N, 5.85.
Found: C, 70.08; H, 6.80; N, 5.65.
5.50. 3-(Aminomethyl)-N,N-dimethyl-2-(2-methylpropyl)-1-
oxo-4-phenyl-1,2- dihydroisoquinoline-6-carboxamide
hydrochloride (19d)
Compound 19d was prepared in a manner similar to that de-
scribed for 19a in 86% yield as a white solid. 1H NMR (200 MHz,
DMSO-d6) d 0.93 (6H, d, J = 6.6 Hz), 2.01–2.17 (1H, m), 2.78 (3H,
s), 2.91 (3H, s), 3.88 (2H, s), 4.08 (2H, d, J = 7.4 Hz), 6.84 (1H, d,
J = 1.0 Hz), 7.39–7.43 (2H, m), 7.54–7.61 (4H, m), 8.37 (1H, d,
J = 8.6 Hz), 8.50 (3H, br s). ESI-HRMS calcd for C23H27N3O2 m/z
378.2176 (M+H), found 378.2150 (M+H).
5.51. 3-(Aminomethyl)-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-
dihydroisoquinoline-6-carbonitrile hydrochloride (19e)
5.55. 3-[3-{[(tert-Butoxycarbonyl)amino]methyl}-2-(2-methyl-
propyl)-1-oxo-4-phenyl-1,2-dihydroisoquinolin-6-yl]propanoic
acid (21b)
Compound 19e was prepared in a manner similar to that de-
scribed for 19a in 88% yield as colorless crystals. 1H NMR
(300 MHz, DMSO-d6) d 0.93 (6H, d, J = 6.6 Hz), 1.98–2.19 (1H, m),
3.89 (2H, s), 4.11 (2H, d, J = 7.4 Hz), 7.22 (1H, d, J = 1.6 Hz), 7.42–
7.46 (2H, m), 7.58–7.61 (3H, m), 7.97 (1H, dd, J = 1.6, 8.4 Hz),
Compound 21b was prepared in a manner similar to that de-
scribed for 21a in 91% yield as a white solid. 1H NMR (300 MHz,
CDCl3) d 0.98 (6H, d, J = 6.9 Hz), 1.42 (9H, s), 2.15–2.27 (1H, m),

4966
Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
2.58 (2H, t, J = 7.8 Hz), 2.91 (2H, t, J = 7.8 Hz), 4.06 (2H, d,
J = 6.0 Hz), 4.19 (2H, d, J = 5.1 Hz), 4.53 (1H, br s), 6.76 (1H, s),
7.22–7.25 (2H, m), 7.31 (1H, d, J = 8.8 Hz), 7.46–7.52 (3H, m),
8.37 (1H, d, J = 8.8 Hz). Mp 182–183 °C. Anal. Calcd for
5.60. 5-(Benzyloxy)-2-benzofuran-1,3-dione (26)
To a suspension of 24 (45.5 g, 250 mmol), K2CO3 (103.7 g,
750 mmol) and N,N-dimethylformamide (500 mL) was added ben-
zylbromide (107 mL, 900 mmol), and the resulting mixture was
stirred at 70 °C for 6 h. The mixture was poured into water and ex-
tracted with AcOEt. The organic layer was washed with brine, dried
over anhydrous MgSO4, and concentrated in vacuo. The residue
was dissolved in THF (200 mL) and MeOH (200 mL), then added
to a solution of sodium hydroxide (25.0 g, 625 mmol) in water
(200 mL). The mixture was refluxed for 6 h. After being cooled,
the mixture was poured into water and extracted with Et2O. The
water layer was acidified with 1 M hydrochloric acid, and extracted
with AcOEt. The organic layer was washed with brine, dried over
anhydrous MgSO4, and concentrated in vacuo. The resulting crys-
tals were collected by filtration to give 4-(benzyloxy)benzene-
1,2-dicarboxylic acid 25 (61.0 g, 90%) as white crystals. A mixture
of 25 (61.0 g, 224 mmol) and acetic anhydride (200 mL) was stirred
at 150 °C for 2 h. After evaporation of the solvent, the resulting
crystals were collected by filtration to give 26 (51.4 g, 90%) as
white crystals. 1H NMR (200 MHz, CDCl3) d 5.23 (2H, s), 7.37–
7.49 (7H, m), 7.91 (1H, d, J = 8.8 Hz). Mp 124–125 °C. Anal. Calcd
for C15H10O4: C, 70.86; H, 3.96. Found: C, 70.79; H, 3.91.
C28H34N2O5: C, 70.12; H, 7.36; N, 5.84. Found: C, 70.21; H, 7.55;
N, 5.68.
5.56. tert-Butyl ({6-[(1E)-3-amino-3-oxoprop-1-en-1-yl]-2-(2-
methylpropyl)-1-oxo-4-phenyl- 1,2-dihydroisoquinolin-3-
yl}methyl)carbamate (22a)
A mixture of 21a (0.33 g, 0.7 mmol), WSC (0.27 g, 1.4 mmol),
HOBtꢀNH3 (0.21 g, 1.4 mmol) and N,N-dimethylformamide
(10 mL) was stirred at room temperature for 2 h. The mixture
was poured into water and extracted with AcOEt. The AcOEt ex-
tract was washed with brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The residue was crystallized from AcOEt–
IPE to give 22a (0.31 g, 94%) as a white solid. 1H NMR (300 MHz,
CDCl3) d 1.00 (6H, d, J = 6.6 Hz), 1.44 (9H, s), 2.16–2.24 (1H, m),
4.07 (2H, d, J = 7.4 Hz), 4.19 (2H, d, J = 5.6 Hz), 4.96 (1H, br s),
5.75 (1H, br s), 6.38 (1H, d, J = 15.6 Hz), 6.94 (1H, s), 7.26–7.30
(2H, m), 7.40–7.56 (5H, m), 8.29 (1H, d, J = 8.4 Hz). Mp 146–
147 °C. Anal. Calcd for C28H33N3O4ꢀ1/4H2O: C, 70.05; H, 7.03; N,
8.75. Found: C, 70.08; H, 7.09; N, 8.64.
5.61. Ethyl 6-(benzyloxy)-4-hydroxy-2-(2-methylpropyl)-1-oxo-
1,2-dihydroisoquinoline-3- carboxylate (28a) and ethyl 7-
(benzyloxy)-4-hydroxy-2-(2-methylpropyl)-1-oxo-1,2-
dihydroisoquinoline-3-carboxylate (28b)
5.57. tert-Butyl {[6-(3-amino-3-oxopropyl)-2-(2-methylpropyl)-
1-oxo-4-phenyl-1,2-dihydroisoquinolin-3-yl]methyl}carbamate
(22b)
Compound 22b was prepared in a manner similar to that de-
scribed for 22a in 94% yield as a white solid. 1H NMR (300 MHz,
CDCl3) d 0.99 (6H, d, J = 6.6 Hz), 1.42 (9H, s), 2.13–2.29 (1H, m),
2.43 (2H, t, J = 8.0 Hz), 2.92 (2H, t, J = 8.0 Hz), 4.05 (2H, d,
J = 7.2 Hz), 4.19 (2H, d, J = 5.2 Hz), 4.54 (1H, br s), 5.42 (2H, br s),
6.75 (1H, d, J = 1.4 Hz), 7.21–7.30 (3H, m), 7.46–7.57 (3H, m),
8.33–8.37 (1H, m). Mp 239–240 °C. Anal. Calcd for
A mixture of 26 (4.07 g, 16 mmol) and ethyl 2-(isobutylami-
no)acetate (2.86 g, 18 mmol) in THF (30 mL) was stirred at room
temperature for 1 h. The reaction mixture was poured into water
and extracted with AcOEt. The AcOEt extract was washed with
brine, dried over anhydrous MgSO4, and concentrated in vacuo to
give compound 27. The residue was dissolved in N,N-dimethyl-
formamide (30 mL), then iodoethane (1.5 mL, 19.2 mmol) and
K2CO3 (2.30 g, 12 mmol) were added to the mixture, and the result-
ing mixture was stirred at room temperature for 3 h. The reaction
mixture was poured into water and extracted with AcOEt. The
AcOEt extract was washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was dissolved in
ethanol (50 mL), then 20% sodium ethoxide in EtOH (10.9 g,
32 mmol) was added, and the mixture was stirred at room temper-
ature for 1 h. The reaction mixture was poured into 1 M hydrochlo-
ric acid (70 mL) and extracted with AcOEt. The AcOEt extract was
washed with brine, dried over anhydrous MgSO4, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (hexane/AcOEt = 70/30) and the component eluted earlier
was concentrated to give 28b (4.21 g, 67%) as an oil. 1H NMR
(300 MHz, CDCl3) d 0.82 (6H, d, J = 6.6 Hz), 1.45 (3H, t, J = 7.1 Hz),
1.77–1.91 (1H, m), 4.42–4.52 (4H, m), 7.31–7.58 (6H, m), 7.97
(1H, d, J = 2.6 Hz), 8.10 (1H, d, J = 8.8 Hz), 11.45 (1H, s).
C
28H35N3O4ꢀ1/4H2O: C, 69.76; H, 7.42; N, 8.72. Found: C, 69.54;
H, 7.41; N, 8.58.
5.58. (2E)-3-[3-(Aminomethyl)-2-(2-methylpropyl)-1-oxo-4-
phenyl-1,2-dihydroisoquinolin-6-yl]prop-2-enamide
hydrochloride (23a)
Compound 23a was prepared in a manner similar to that de-
scribed for 19a in 95% yield as a pale yellow solid. 1H NMR
(300 MHz, DMSO-d6) d 0.92 (6H, d, J = 6.9 Hz), 2.06–2.16 (1H,
m), 3.87 (2H, d, J = 4.8 Hz), 4.08 (2H, s), 6.56 (1H, d,
J = 16.0 Hz), 6.97 (1H, d, J = 1.2 Hz), 7.19 (1H, br s), 7.31 (1H, d,
J = 16.0 Hz), 7.42–7.44 (2H, m), 7.54–7.63 (3H, m), 7.69 (1H, br
s), 7.78 (1H, dd, J = 1.2, 8.8 Hz), 8.35 (1H, d, J = 8.8 Hz), 8.62
(3H, br s). Mp 223–225 °C. Anal. Calcd for C23H26Cl N3O2ꢀ1.5H2O:
C, 62.93; H, 6.66; N, 9.57. Found: C, 63.15; H, 6.66; N, 9.34. ESI-
HRMS calcd for
376.1989 (M+H).
C
23H25N3O2 m/z 376.2020 (M+H), found
The component eluted later was concentrated to give 28a
(0.80 g, 13%) as crystals. 1H NMR (300 MHz, CDCl3) d 0.81 (6H, d,
J = 6.6 Hz), 1.46 (3H, t, J = 7.2 Hz), 1.73–1.87 (1H, m), 4.39 (2H, d,
J = 7.2 Hz), 4.48 (2H, q, J = 7.2 Hz), 5.21 (2H, s), 7.28–7.49 (6H, m),
7.60 (1H, d, J = 2.6 Hz), 8.38 (1H, d, J = 8.8 Hz), 11.23 (1H, s). Mp
92–93 °C. Anal. Calcd for C23H25NO5: C, 70.90; H, 6.90; N, 3.31.
Found: C, 70.84; H, 6.85; N, 3.11.
5.59. 3-[3-(Aminomethyl)-2-(2-methylpropyl)-1-oxo-4-phenyl-
1,2-dihydroisoquinolin-6- yl]propanamide hydrochloride (23b)
Compound 23b was prepared in a manner similar to that de-
scribed for 19a in 95% yield as a white solid. 1H NMR (300 MHz,
DMSO-d6) d 0.91 (6H, d, J = 6.3 Hz), 2.02–2.16 (1H, m), 2.26 (2H,
t, J = 7.2 Hz), 2.76 (2H, t, J = 7.2 Hz), 3.86 (2H, s), 4.08 (2H, s), 6.72
(1H, s), 6.75 (1H, br s), 7.29 (1H, br s), 7.38–7.40 (2H, m), 7.45
(1H, d, J = 8.4 Hz), 7.55–7.59 (3H, m), 8.25 (1H, d, J = 8.4 Hz), 8.57
(3H, br s). Mp 186–187 °C. ESI-HRMS calcd for C23H27N3O2 m/z
378.2176 (M+H), found 378.2150 (M+H).
5.62. Ethyl 6-(benzyloxy)-2-(2-methylpropyl)-1-oxo-4-{[(tri-
fluoromethyl)sulfonyl]oxy}- 1,2-dihydroisoquinoline-3-
carboxylate (29a)
To a solution of 28a (3.95 g, 10 mmol) in N,N-dimethylformam-
ide (40 mL) was added portionwise sodium hydride (0.48 g,

Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
4967
12 mmol) at 0 °C, and the resulting mixture was stirred at 0 °C for
30 min. To the obtained mixture was added N-phenyltrifluorome-
thanesulfonimide (4.50 g, 12 mmol), and the resulting mixture
was stirred at room temperature for 6 h. The mixture was poured
into water and extracted with AcOEt. The AcOEt extract was
washed with water, dried over anhydrous MgSO4, and concen-
trated in vacuo. The residue was purified by silica gel column chro-
matography (hexane/AcOEt = 70/30) and concentrated to give 29a
(5.24 g, quant.) as a white solid. 1H NMR (300 MHz, CDCl3) d 0.89
(6H, d, J = 6.6 Hz), 1.43 (3H, t, J = 7.1 Hz), 1.93–2.09 (1H, m), 4.03
(2H, d, J = 7.6 Hz), 4.46 (2H, q, J = 7.1 Hz), 5.19 (2H, s), 7.22–7.49
(7H, m), 8.36 (1H, d, J = 8.8 Hz).
(20 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 1 h.
The reaction mixture was poured into 1 M hydrochloric acid and
extracted with AcOEt. The extract was washed with brine, dried
over anhydrous MgSO4, and concentrated in vacuo. The obtained
crystals were recrystallized from THF-IPE to give 6-(benzyloxy)-
3-(hydroxymethyl)-2-(2-methylpropyl)-4-phenylisoquinolin-1(2H)-
one (2.92 g, 88.5%) as crystals. To a suspension of 6-(benzyloxy)-
3-(hydroxymethyl)-2-(2-methylpropyl)-4-phenylisoquinolin-1(2H)-
one (2.89 g, 7 mmol) in toluene (30 mL) was added thionyl chlo-
ride (1.0 mL, 14 mmol). The obtained mixture was refluxed under
heating for 2 h. The reaction mixture was poured into saturated
aqueous sodium hydrogen carbonate solution and extracted with
AcOEt. The extract was washed with brine, dried over anhydrous
MgSO4, and concentrated in vacuo to give 6-(benzyloxy)-3-(chloro-
methyl)-2-(2-methylpropyl)-4-phenylisoquinolin-1(2H)-one (2.61 g,
86%) as crystals. A mixture of 6-(benzyloxy)-3-(chloromethyl)-2-
(2-methylpropyl)-4-phenylisoquinolin-1(2H)-one (2.59 g, 6 mmol)
and potassium phthalimide (1.67 g, 9 mmol) and N,N-dimethyl-
formamide (30 mL) was stirred at room temperature for 6 h.
The reaction mixture was poured into water and extracted with
AcOEt. After washing with water, the extract was dried over
anhydrous MgSO4, and concentrated in vacuo. The obtained
crystals were recrystallized from AcOEt–IPE to give 2-{[6-(benzyl-
oxy)-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-dihydroisoquinolin-
3-yl]methyl}-1H-isoindole-1,3(2H)-dione (3.04 g, 94%) as crystals.
To a solution of 2-{[6-(benzyloxy)-2-(2-methylpropyl)-1-oxo-4-
phenyl-1,2-dihydroisoquinolin-3-yl]methyl}-1H-isoindole-1,3(2H)-
dione (2.98 g, 5.5 mmol) in EtOH (30 mL) was added hydrazine
monohydrate (0.8 mL, 16.5 mmol). The obtained mixture was re-
fluxed under heating for 1 h. The reaction mixture was poured
into saturated aqueous sodium hydrogen carbonate solution and
extracted with AcOEt. The extract was washed with brine, dried
over anhydrous MgSO4, and concentrated in vacuo. The residue
was dissolved in THF (20 mL) and di-t-butyl dicarbonate
(1.9 mL, 8.3 mmol) was added thereto. The obtained mixture
was stirred at room temperature for 1 h. The reaction mixture
was poured into water and extracted with AcOEt. The extract
was washed with brine, dried over anhydrous MgSO4, and con-
centrated under reduced pressure. The obtained crystals were
recrystallized from AcOEt–IPE to give 32a (2.29 g, 81%) as crys-
tals. 1H NMR (300 MHz, CDCl3) d 0.99 (6H, d, J = 7.0 Hz), 1.42
(9H, s), 2.16–2.30 (1H, m), 4.03 (2H, d, J = 7.4 Hz), 4.17 (2H, d,
J = 5.6 Hz), 4.45 (1H, br s), 4.92 (2H, s), 6.35 (1H, d, J = 2.4 Hz),
7.09 (1H, dd, J = 2.4, 9.0 Hz), 7.17–7.37 (7H, m), 7.47–7.52 (3H,
m), 8.38 (1H, d, J = 9.0 Hz). Mp 141–142 °C. Anal. Calcd for
5.63. Ethyl 7-(benzyloxy)-2-(2-methylpropyl)-1-oxo-4-{[(tri-
fluoromethyl)sulfonyl]oxy}-1,2-dihydroisoquinoline-3-
carboxylate (29b)
Compound 29b was prepared in a manner similar to that de-
scribed for 29a in 91% yield as a white solid. 1H NMR (300 MHz,
CDCl3) d 0.90 (6H, d, J = 6.6 Hz), 1.44 (3H, t, J = 7.1 Hz), 1.96–2.07
(1H, m), 4.12 (2H, d, J = 7.2 Hz), 4.45 (2H, q, J = 7.1 Hz), 5.21
(2H, s), 7.30–7.48 (6H, m), 7.74 (1H, d, J = 9.0 Hz), 7.96 (1H, d,
J = 2.4 Hz).
5.64. 6-(Benzyloxy)-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-
dihydroisoquinoline-3-carboxylic acid (31a)
A mixture of 29a (5.24 g, 10 mmol), phenylboronic acid (1.44 g,
12 mmol), Na2CO3 (3.18 g, 30 mmol), tetrakis(triphenylphos-
phine)palladium (0.69 g, 2.25 mmol), toluene (50 mL), MeOH
(10 mL) and water (10 mL) was refluxed for 10 h under argon
atmosphere. After being cooled, the mixture was poured into water
and extracted with AcOEt. The AcOEt extract was washed with
brine, dried over anhydrous MgSO4, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(hexane/AcOEt = 70/30) and concentrated to give 30a as white
crystals. The obtained 30a was dissolved in EtOH (50 mL) and
THF (50 mL), then
a solution of sodium hydroxide (1.20 g,
30 mmol) in water (20 mL) was added. The resulting mixture
was refluxed for 3 h. The mixture was poured into water, acidified
with 1 M hydrochloric acid, and extracted with AcOEt. The AcOEt
extract was washed with brine, dried over anhydrous MgSO4, and
concentrated in vacuo to give 31a (2.95 g, 69.1%) as a white solid.
1H NMR (300 MHz, CDCl3) d 0.85 (6H, d, J = 6.6 Hz), 2.09–2.14 (1H,
m), 3.83 (2H, d, J = 7.5 Hz), 4.96 (2H, s), 6.58 (1H, d, J = 2.4 Hz),
7.09–7.44 (11H, m), 8.29 (1H, d, J = 8.7 Hz).
C32H35N2O4: C, 74.97; H, 7.08; N, 5.46. Found: C, 74.60; H, 7.13;
N, 5.45.
5.65. 7-(Benzyloxy)-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-
dihydroisoquinoline-3-carboxylic acid (31b)
5.67. tert-Butyl {[7-(benzyloxy)-2-(2-methylpropyl)-1-oxo-4-
phenyl-1,2-dihydroisoquinolin- 3-yl]methyl}carbamate (32b)
Compound 31b was prepared in a manner similar to that de-
scribed for 31a in 75% yield as an amorphous powder. 1H NMR
(300 MHz, CDCl3) d 0.90 (6H, d, J = 6.8 Hz), 2.14–2.28 (1H, m),
4.01 (2H, d, J = 7.8 Hz), 5.15 (2H, s), 7.11–7.48 (12H, m), 7.66 (1H,
d, J = 1.8 Hz).
Compound 32b was prepared in a manner similar to that de-
scribed for 32a in 75% yield as a white solid. 1H NMR (300 MHz,
CDCl3) d 1.01 (6H, d, J = 6.8 Hz), 1.43 (9H, s), 2.20–2.31 (1H, m),
4.08 (2H, d, J = 7.4 Hz), 4.19 (2H, d, J = 5.2 Hz), 4.55 (1H, br s),
5.16 (2H, s), 6.89 (1H, d, J = 8.8 Hz), 7.15 (1H, dd, J = 2.8, 8.8 Hz),
7.24–7.55 (10H, m), 7.96 (1H, d, J = 2.8 Hz). Mp 181–182 °C. Anal.
Calcd for C32H36N2O4: C, 74.97; H, 7.08; N, 5.46. Found: C, 74.94;
H, 7.14; N, 5.31.
5.66. tert-Butyl {[6-(benzyloxy)-2-(2-methylpropyl)-1-oxo-4-
phenyl-1,2-dihydroisoquinolin- 3-yl]methyl}carbamate (32a)
To a solution of 31a (3.42 g, 8 mmol) in THF (30 mL) were
added oxalyl chloride (0.84 mL, 9.6 mmol) and N,N-dimethylform-
amide (3 drops), and the mixture was stirred at room tempera-
ture for 1 h. The reaction mixture was concentrated under
reduced pressure and the residue was dissolved in THF (20 mL).
The obtained solution was added dropwise to a suspension of so-
dium tetrahydroborate (1.06 g, 28 mmol) in 1,2-dimethoxyethane
5.68. tert-Butyl {[6-hydroxy-2-(2-methylpropyl)-1-oxo-4-
phenyl-1,2-dihydroisoquinolin-3- yl]methyl}carbamate (33a)
A suspension of 32a (2.05 g, 4 mmol) and 5% palladium on car-
bon (0.6 g) in THF (10 mL) and EtOH (10 mL) was stirred under a
hydrogen atmosphere at room temperature for 2 h. The catalyst

4968
Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
was filtered off and the filtrate was concentrated under reduced
pressure. The obtained crystals were recrystallized from AcOEt–IPE
to give 33a (1.56 g, 92.3%) as crystals. 1H NMR (300 MHz, CDCl3) d
0.97 (6H, d, J = 6.8 Hz), 1.42 (9H, s), 2.14–2.24 (1H, m), 4.02 (2H, d,
J = 7.2 Hz), 4.18 (2H, d, J = 5.4 Hz), 4.47 (1H, br s), 6.33 (1H, d,
J = 2.4 Hz), 7.03 (1H, dd, J = 2.4, 8.8 Hz), 7.20–7.27 (2H, m), 7.43–
7.46 (3H, m), 7.97 (1H, br s), 8.30 (1H, d, J = 8.8 Hz). Mp 218–
219 °C. Anal. Calcd for C25H30N2O4: C, 71.07; H, 7.16; N, 6.63.
Found: C, 70.85; H, 7.10; N, 6.62.
9.75. Found: C, 62.18; H, 6.40; N, 9.89. ESI-HRMS calcd for
22H25N3O3 m/z 380.1969 (M+H), found 380.1946 (M+H).
C
5.73. 2-{[3-(Aminomethyl)-2-(2-methylpropyl)-1-oxo-4-phenyl-
1,2-dihydroisoquinolin-7-yl]oxy}acetamide hydrochloride
(35b)
Compound 35b was prepared in a manner similar to that de-
scribed for 19a in 91% yield as a white solid. 1H NMR (300 MHz,
DMSO-d6) d 0.92 (6H, d, J = 6.6 Hz), 1.99–2.19 (1H, m), 3.85 (2H,
d, J = 4.2 Hz), 4.09 (2H, d, J = 7.0 Hz), 4.58 (2H, s), 6.85 (1H, d,
J = 8.8 Hz), 7.33 (1H, dd, J = 2.6, 8.8 Hz), 7.36–7.41 (2H, m), 7.56–
7.60 (3H, m), 7.69 (1H, br s), 7.72 (1H, d, J = 2.6 Hz), 8.64 (3H, s).
Mp 244–245 °C. ESI-HRMS calcd for C22H25N3O3 m/z 380.1969
(M+H), found 380.1943 (M+H).
5.69. tert-Butyl {[7-hydroxy-2-(2-methylpropyl)-1-oxo-4-
phenyl-1,2-dihydroisoquinolin-3- yl]methyl}carbamate (33b)
Compound 33b was prepared in a manner similar to that de-
scribed for 33a in 97% yield as a white solid. 1H NMR (300 MHz,
CDCl3) d 1.02 (6H, d, J = 7.0 Hz), 1.42 (9H, s), 2.21–2.35 (1H, m),
4.11 (2H, d, J = 7.2 Hz), 4.22 (2H, d, J = 5.2 Hz), 4.52 (1H, br s),
6.91 (1H, d, J = 8.8 Hz), 7.16 (1H, dd, J = 2.6, 8.8 Hz), 7.23–7.28
(2H, m), 7.44–7.55 (3H, m), 8.52 (1H, d, J = 2.8 Hz), 8.90 (1H, s).
Mp 232–233 °C. Anal. Calcd for C25H30N2O4: C, 71.07; H, 7.16; N,
6.63. Found: C, 70.81; H, 7.22; N, 6.35.
5.74. 3-(Aminomethyl)-6-hydroxy-2-(2-methylpropyl)-4-
phenylisoquinolin-1(2H)-one hydrochloride (36)
Compound 36 was prepared in a manner similar to that de-
scribed for 19a in 89% yield as a white solid. 1H NMR (300 MHz,
DMSO-d6) d 0.90 (6H, d, J = 6.6 Hz), 1.99–2.13 (1H, m), 3.82 (2H,
d, J = 5.6 Hz), 4.03 (2H, d, J = 7.0 Hz), 6.20 (1H, d, J = 2.4 Hz), 7.02
(1H, dd, J = 2.4, 8.6 Hz), 7.36–7.40 (2H, m), 7.47–7.60 (3H, m),
8.04 (1H, br s), 8.16 (1H, d, J = 8.6 Hz), 8.61 (3H, br s). Mp 249–
251 °C. ESI-HRMS calcd for C20H22N2O2 m/z 321.1609 (MꢁH),
found 321.1623 (MꢁH).
5.70. tert-Butyl {[6-(2-amino-2-oxoethoxy)-2-(2-methylpropyl)-
1-oxo-4-phenyl-1,2- dihydroisoquinolin-3-yl]methyl}-
carbamate (34a)
A solution of 33a (0.63 g, 1.5 mmol), 2-iodoacetamide (0.43 g,
2.3 mmol) and 1,8-diazabicyclo[5.4.0]-7-undecene (0.34 mL,
2.3 mmol) in N,N-dimethylformamide (10 mL) was stirred at
80 °C for 10 h. The reaction mixture was poured into water and ex-
tracted with AcOEt. The extract was washed with brine, dried over
anhydrous MgSO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/
AcOEt = 50/50) to give 34a (0.32 g, 44%) as crystals. 1H NMR
(300 MHz, DMSO-d6) d 0.89 (6H, d, J = 6.6 Hz), 1.38 (9H, s), 2.07–
2.21 (1H, m), 3.88 (2H, d, J = 6.6 Hz), 3.95 (2H, d, J = 4.0 Hz), 4.34
(2H, s), 6.30 (1H, d, J = 2.4 Hz), 7.13 (1H, dd, J = 2.4, 8.8 Hz), 7.34–
7.52 (8H, m), 8.24 (1H, d, J = 8.8 Hz). Mp 226–227 °C. Anal. Calcd
for C27H33N3O5: C, 67.62; H, 6.94; N, 8.76. Found: C, 67.36; H,
6.73; N, 8.60.
5.75. 3-{[(tert-Butoxycarbonyl)amino]methyl}-2-(2-methyl-
propyl)-1-oxo-4-phenyl-1,2- dihydroisoquinolin-7-yl
trifluoromethanesulfonate (37)
Compound 37 was prepared in a manner similar to that de-
scribed for 29a in 90% yield as an amorphous powder. 1H NMR
(300 MHz, CDCl3) d 1.01 (6H, d, J = 6.6 Hz), 1.43 (9H, s), 2.20–2.29
(1H, m), 4.09 (2H, d, J = 7.5 Hz), 4.23 (2H, d, J = 5.4 Hz), 4.46 (1H,
br s), 7.06 (1H, d, J = 9.0 Hz), 7.22–7.27 (2H, m), 7.36 (1H, dd,
J = 2.7, 9.0 Hz), 7.42–7.56 (3H, m), 8.34 (1H, d, J = 2.7 Hz).
5.76. Methyl 3-{[(tert-butoxycarbonyl)amino]methyl}-2-(2-
methylpropyl)-1-oxo-4-phenyl- 1,2-dihydroisoquinoline-6-
carboxylate (38)
5.71. tert-Butyl {[7-(2-amino-2-oxoethoxy)-2-(2-methylpropyl)-
1-oxo-4-phenyl-1,2- dihydroisoquinolin-3-yl]methyl}-
carbamate (34b)
Compound 38 was prepared in a manner similar to that de-
scribed for 15 in 74% yield as crystals. 1H NMR (300 MHz, CDCl3)
d 1.01 (6H, d, J = 6.6 Hz), 1.43 (9H, S), 2.05–2.28 (1H, m), 3.93
(3H, s), 4.10 (2H, d, J = 7.5 Hz), 4.22 (2H, d, J = 5.4 Hz), 4.61 (1H,
br s), 6.98 (1H, d, J = 8.7 Hz), 7.24–7.28 (2H, m), 7.46–7.57 (3H,
m), 8.02 (1H, d, J = 8.7, 1.8 Hz), 9.10 (1H, d, J = 1.8 Hz). Mp 134–
135 °C. Anal. Calcd for C27H32N2O5: C, 69.81; H, 6.94; N, 6.03.
Found: C, 69.46; H, 7.04; N, 5.81.
Compound 34b was prepared in a manner similar to that de-
scribed for 34a in 40% yield as
a
white solid. 1H NMR
(300 MHz, CDCl3) d 1.00 (6H, d, J = 6.6 Hz), 1.43 (9H, s), 2.18–
2.34 (1H, m), 4.08 (2H, d, J = 7.4 Hz), 4.20 (2H, d, J = 5.4 Hz),
4.51 (1H, br s), 4.58 (2H, s), 5.86 (1H, br s), 6.57 (1H, br s), 6.94
(1H, d, J = 8.8 Hz), 7.13 (1H, d, J = 8.8 Hz), 7.23–7.27 (2H, m),
7.49–7.52 (3H, m), 7.90 (1H, s). Mp 211–212 °C. Anal. Calcd for
C27H33N3O5: C, 67.62; H, 6.94; N, 8.76. Found: C, 67.38; H, 6.69;
N, 8.87.
5.77. 3-{[(tert-Butoxycarbonyl)amino]methyl}-2-(2-methyl-
propyl)-1-oxo-4-phenyl-1,2- dihydroisoquinoline-7-carboxylic
acid (39)
5.72. 2-{[3-(Aminomethyl)-2-(2-methylpropyl)-1-oxo-4-phenyl-
1,2-dihydroisoquinolin-6- yl]oxy}acetamide hydrochloride (35a)
Compound 39 was prepared in a manner similar to that de-
scribed for 16 in 92% yield as crystals. 1H NMR (300 MHz, CDCl3)
d 0.91 (6H, d, J = 6.6 Hz), 1.38 (9H, s), 2.11–2.26 (1H, m), 3.91
(2H, d, J = 6.6 Hz), 3.99 (2H, d, J = 4.2 Hz), 6.99 (1H, d, J = 8.8 Hz),
7.34 (1H, br s), 7.39–7.42 (2H, m), 7.46–7.56 (3H, m), 8.09 (1H,
dd, J = 2.0, 8.8 Hz), 8.87 (1H, d, J = 2.0 Hz). Mp 246 °C. Anal. Calcd
for C26H30N2O5ꢀ1/4H2O: C, 68.62; H, 6.76; N, 6.16. Found: C,
68.81; H, 6.83; N, 5.87.
Compound 35a was prepared in a manner similar to that de-
scribed for 19a in 95% yield as a white solid. 1H NMR (300 MHz,
DMSO-d6) d 0.91 (6H, d, J = 6.6 Hz), 2.01–2.18 (1H, m), 3.37 (2H,
br s), 3.85 (2H, br s), 4.05 (2H, d, J = 6.8 Hz), 4.36 (2H, s), 6.30
(1H, d, J = 2.0 Hz), 7.19 (1H, dd, J = 2.0, 8.8 Hz), 7.36–7.40 (2H, m),
7.52–7.58 (3H, m), 8.28 (1H, d, J = 8.8 Hz), 8.55 (3H, s). Mp 185–
186 °C. Anal. Calcd for C22H26ClN3O3ꢀ1/2H2O: C, 62.23; H, 6.44; N,

Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
4969
Table 5
5.78. 3-{[(tert-Butoxycarbonyl)amino]methyl}-2-(2-methyl-
propyl)-1-oxo-4-phenyl-1,2- dihydroisoquinoline-7-
carboxamide (40)
Summary of crystallographic analysis
Compound 35a
3OPM
PDB code
Data collection
Space group
Unit cell lengths (Å)
Unit cell angles (°)
Resolution (Å)
Observations
Unique
Completeness (%)
I/rI
Rsym (%)
Compound 40 was prepared in a manner similar to that de-
scribed for 17a in 96% yield as crystals. 1H NMR (300 MHz, CDCl3)
d 0.99 (6H, d, J = 6.6 Hz), 1.43 (9H, s), 2.10–2.24 (1H, m), 4.08 (2H,
d, J = 7.0 Hz), 4.22 (2H, d, J = 5.2 Hz), 4.76 (1H, br s), 5.96 (1H, br s),
6.74 (1H, br s), 7.02 (1H, d, J = 8.6 Hz), 7.25–7.30 (2H, m), 7.45–7.56
(3H, m), 8.05 (1H, dd, J = 1.4, 8.6 Hz), 8.78 (1H, d, J = 1.4 Hz). Mp
232–233 °C. Anal. Calcd for C26H31N3O4ꢀ1/2H2O: C, 68.10; H,
7.03; N, 9.16. Found: C, 68.31; H, 7.07; N, 8.75.
P21
121.8; 122.6; 144.7
90.0; 115.0; 90.0
2.72
413,990
100,745
97.5 (82.7)
10.9 (1.5)
0.114 (0.590)
Model refinement
Reflections (work/free)
Rfactor (work/free %)
Protein molecules per ASU
Solvent molecules
Mean B value (Å2)
RMSD ideal bond lengths (Å)
RMSD ideal bond angles (°)
95,647/5038
19.17/25.57
4
506
49.2
5.79. 3-(Aminomethyl)-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-
dihydroisoquinoline-7-carboxamide hydrochloride (41)
Compound 41 was prepared in a manner similar to that de-
scribed for 19a in 95% yield as a white solid. 1H NMR (300 MHz,
DMSO-d6) d 0.94 (6H, d, J = 6.6 Hz), 2.04–2.19 (1H, m), 3.89 (2H,
d, J = 4.4 Hz), 4.13 (2H, d, J = 7.0 Hz), 6.94 (1H, d, J = 8.6 Hz), 7.39–
7.44 (2H, m), 7.54–7.64 (4H, m), 8.16 (1H, dd, J = 1.8, 8.6 Hz),
8.30 (1H, br s), 8.67 (3H, br s). Mp 254–256 °C. Anal. Calcd for
0.009
1.24
Rsym
=
=
R
R
|I ꢁ <I>|/
RI, where I is the integrated intensity for a reflection. Rfac-
|FP ꢁ Fc|/ FP, where Fp and Fc are the observed and calculated structure factor
R
tor
amplitudes, while Rfree is calculated on 5% of the data excluded from refinement.
Values in parenthesis are for the highest resolution shell in parenthesis are for the
highest resolution shell.
C
21H23N3O2ꢀHClꢀH2O: C, 63.87; H, 6.38; N, 10.64. Found: C, 63.76;
H, 6.29; N, 10.30. ESI-HRMS calcd for C21H23N3O2 m/z 350.1863
(M+H), found 350.1828 (M+H).
5.80. DPP4 + compound 35a structure determination
clearly visible in the electron density maps. Data collection and
model refinement statistics are summarized in Table 5.
The cDNA encoding human DPP-4 was isolated by PCR from
spleen cDNA (Clontech) and the extracellular domain (residues
39–766) was cloned into a modified pFastBacHTb vector (Invitro-
gen). The final construct contains a baculovirus gp67 signal peptide
followed by a His6 tag fused to the coding sequence corresponding
to residues 39–766 of DPP-4. Recombinant baculovirus was gener-
ated by transposition using the Bac-to-Bac system (Gibco-BRL).
Large-scale production of recombinant protein was performed by
infection of Trichoplusia ni (Hi5) insect cells (Gibco-BRL) for 48 h
in 5 L Wave Bioreactors (Wave Biotech). The secreted glycosylated
recombinant protein was isolated from the cell culture medium by
diafiltration using cross-flow ultrafiltration followed by passage
over a nickel chelate resin (Binding buffer: 25 mM Tris, pH 7.9;
400 mM NaCl). The column was washed overnight (0.2 mL/min)
with 50 mM K2HPO4 pH 7.9; 400 mM NaCl; 20 mM imidazole–
HCl and 0.25 mM TCEP followed by 5 column volumes (1 mL/
min) of 50 mM Tris HCl pH 7.9; 400 mM NaCl and 0.25 mM TCEP.
Protein bound was eluted with 4 column volumes of 50 mM Tris–
HCl, pH 7.9, 400 mM NaCl, 200 mM imidazole–HCl and 0.25 mM
TCEP. To remove oligomeric forms, the sample was further purified
over a size exclusion column (BioSep SEC S3000, 300 ꢂ 21.2 mm,
Phenomenex) equilibrated with 25 mM Tris pH 7.6; 150 mM NaCl;
0.25 mM TCEP and 1 mM EDTA. Wild-type DPP-4 in free form was
concentrated to ꢃ14 mg/mL and crystallized at 4 °C. The reservoir
solution was 20% PEG MME 2000, 100 mM Bicine (pH 8.0–8.5).
Thick plate shaped crystals appeared in about 5 days that grew
to about 0.5 mm in longest dimension and varying width and
thickness. All protein–inhibitor complexes were obtained by soak-
ing preformed DPP-4 crystals in a solution containing 1 mM of
compound of interest. Crystals were then cryo-protected with eth-
ylene glycol and flash frozen in liquid nitrogen. X-ray diffraction
5.81. In vitro DPP-4, DPP-2, DPP-8 and DPP-9 enzyme assay
Human DPP-4 was partially purified from Caco-2 cells (ATCC
No. HTB-37). The compounds (1
were added to 79 L of assay buffer (0.25 mol/L Tris–HCl pH 7.5,
0.25% bovine serum albumin, 0.125% CHAPS) and mixed with
20 L of the DPP-4 fraction. After the mixture was incubated at
room temperature for 15 min, the reaction was initiated by adding
100
lL in DMSO) at each concentration
l
l
l
L of 1 mmol/L of Gly-Pro-pNAꢀTos as a substrate and run for
60 min at 37 °C.
Rat DPP-2 was partially purified from rat kidney according to
the method previously reported. One microliter of compounds dis-
solved in DMSO was mixed with 29
1 mol/L 3,3-dimethylglutamic acid buffer (pH 5.5), and 10
DPP-2 fraction. After the mixture was incubated at room tempera-
ture for 20 min, the reaction was initiated by adding 50 L of
l
L of distilled water, 10
lL of
l
L of the
l
1 mmol/L of H-Lys-Ala-pNAꢀ2HCl and run at 37 °C for 60 min. Hu-
man DPP-8 and DPP-9 were purified, respectively by affinity chro-
matography from the 293-F cells expressing each FLAG-tagged
protein. One microliter of compounds dissolved in DMSO was
mixed with 29
fer (pH 7.5), and 10
was incubated at room temperature for 20 min, the reaction was
initiated by adding 50
lL of distilled water, 10
lL of 1 mol/L Tris–HCl buf-
lL of the enzyme fraction. After the mixture
lL of 2 mmol/L of Gly-Pro-pNAꢀTos for
DPP-8 or 4 mmol/L of Gly-Pro-pNAꢀTos for DPP-9 and run at
37 °C for 90 min. Absorbance at 405 nm of each reaction mixture
was measured using a microplate reader at the initial time and
the end of the reaction. The well containing substrate alone was
used as a basal control. The well containing the substrate and the
enzyme without the compound was used as a total reaction.
data were collected at Advanced Light Source (ALS) beam lines
18
5.0.2 and 5.0.3, and processed using the program HKL2000
.
The
5.82. Oral glucose tolerance test in female Wistar fatty rats
structures of DPP-4 inhibitor complexes were determined by
molecular replacement using MOLREP, utilizing the previously
determined coordinates of DPP-4 with accession code 1R9M.19,20
Subsequent structure refinement and model building were per-
formed utilizing REFMAC and XtalView.21 Bound inhibitors were
Prior to the start of the study, rats were fasted overnight and di-
vided into six groups based on plasma glucose and body weight.
Each group was administered vehicle (0.5% methylcellulose) or
compound orally. One hour after drug or vehicle administration,

4970
Y. Banno et al. / Bioorg. Med. Chem. 19 (2011) 4953–4970
13. Marguet, D.; Baggio, L.; Kobayashi, T.; Bernard, A. M.; Pierres, M.; Nielsen, P. F.;
Ribel, U.; Watanabe, T.; Drucker, D. J.; Wagtmann, N. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 6874.
14. Holst, J. J. Gastroenterology 1994, 107, 1848.
15. Orskov, C. Diabetologia 1992, 35, 701.
all animals received an oral glucose load (1 g/kg). Blood samples
were collected before the glucose load (time 0), and 10, 30, 60
and 120 min after the glucose load and plasma glucose and plasma
immuno-reactive insulin (IRI) were analyzed.
16. Drucker, D. J. Diabetes 1998, 47, 159.
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The authors thank Dr. Hidefumi Yukimasa, Dr. Kaneyoshi Kato,
and Dr. Akio Miyake for helpful discussions. We also thank ALS,
which is supported by the Director, Office of Science, Office of Basic
Energy Sciences, Materials Sciences Division of the US Department
of Energy under contract DE-AC03-76SF00098 at Lawrence Berke-
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.059. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
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