Discovery of 3H-imidazo[4,5-c]quinolin-4(5H)-ones as potent and selective dipeptidyl peptidase IV (DPP-4) inhibitors: Use of a carboxylate prodrug to improve bioavailability

Article abstract of DOI:10.1016/j.bmc.2014.12.051

We have previously reported a novel series of 3H-imidazo[4,5-c]quinolin-4(5H)-ones with potent dipeptidyl peptidase IV (DPP-4) inhibitory activity. However, these compounds showed poor oral absorption. We attempted in this study esterification of the carb

Full text of DOI:10.1016/j.bmc.2014.12.051

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of 3H-imidazo[4,5-c]quinolin-4(5H)-ones as potent
and selective dipeptidyl peptidase IV (DPP-4) inhibitors: Use
of a carboxylate prodrug to improve bioavailability
⇑
Yohei Ikuma , Hitoshi Hochigai, Hidenori Kimura, Noriko Nunami, Tomonori Kobayashi,
Katsuya Uchiyama, Takashi Umezome, Yasumitsu Sakurai, Naoyuki Sawada, Jun Tadano, Eiji Sugaru,
Michiko Ono, Yuko Hirose, Hiroyuki Nakahira
Drug Research Division, Sumitomo Dainippon Pharma Co., Ltd, 33-94 Enoki-cho, Suita, Osaka 564-0053, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously reported a novel series of 3H-imidazo[4,5-c]quinolin-4(5H)-ones with potent dipep-
tidyl peptidase IV (DPP-4) inhibitory activity. However, these compounds showed poor oral absorption.
We attempted in this study esterification of the carboxylic acid moiety to improve the compounds 1–4
plasma concentrations. Our efforts yielded 10h with a 5-methyl-2-oxo-1,3-dioxol-4-yl methyl ester as
an S9/plasma-cleavable functionality. Compound 10h showed significantly high oral absorption and
potent DPP-4 inhibition in vivo and decreased Zucker fatty rats glucose levels in the oral glucose toler-
ance test. Optimization of the ester moiety revealed that rapid conversion to the carboxyl form in both
liver S9 fractions and serum was important for prodrugs not to be detected in the plasma after oral
administration. In particular, lability in the serum was found to be an important characteristic. Through
our investigation, we were able to develop a novel efficient synthetic method for construction of 3H-imi-
dazo[4,5-c]quinolin-4(5H)-ones using intramolecular radical cyclization.
Received 19 November 2014
Revised 19 December 2014
Accepted 20 December 2014
Available online xxxx
Keywords:
Dipeptidyl peptidase IV
Type 2 diabetes mellitus
3H-Imidazo[4,5-c]quinolin-4(5H)-ones
Prodrug
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
meal ingestion. GLP-1 stimulates insulin biosynthesis and thus
contributes to maintenance of postprandial glycemic control.3,4
Diabetes is a major metabolic disorder that affects approxi-
mately 382 million people worldwide (2013 statistics), and this
number is predicted to increase to 592 million by 2035.1 Forty-
However, active GLP-1 (7-36 amide) is rapidly degraded by dipep-
tidyl peptidase IV (DPP-4), a serine protease, to give inactive GLP-1
(9-36 amide) in vivo, which limits its ability to normalize blood
glucose levels.5–8 Thus, inhibition of DPP-4 would help maintain
appropriate levels of active GLP-1 in plasma, which in turn would
stimulate insulin secretion. In fact, development of DPP-4 inhibi-
tors is emerging as a promising approach for the treatment of
patients with T2DM with low risk of hypoglycemia.9,10 Clinical
proof-of-concept studies have shown that DPP-4 inhibitors are
more efficient and safer than conventional antidiabetic agents.11,12
Based on these findings, a number of DPP-4 inhibitors, including
sitagliptin,13 vildagliptin,14 saxagliptin,15 alogliptin,16 and linaglip-
tin (Fig. 1)17 have already been approved for the treatment of
T2DM.
Recently, we have reported a novel series of 3H-imidazo[4,5-
c]quinolin-4(5H)-ones as DPP-4 inhibitors (Fig. 1).18,19 In this ser-
ies, compounds 1–4 with a carboxyl group at the 8- or 7-position
showed more potent DPP-4 inhibitory activity in vitro than mar-
keted DPP-4 inhibitors with excellent selectivity against various
DPP-4 homologues (Table 1). However, these compounds had poor
two percent of people treated for type
2 diabetes mellitus
(T2DM), which is the most common form of diabetes, do not reach
their blood glucose goals, thereby risking organ damage, blindness
and even death.2 Currently used antidiabetic agents, such as perox-
isome proliferator-activated receptor
nylurea derivatives, biguanides, and
c
(PPAR
c) agonists, sulpho-
a-glucosidase inhibitors can
produce beneficial effects in patients with T2DM by effectively
enhancing insulin secretion and/or decreasing glucose absorption.
However, these agents are often associated with undesired side
effects, including hypoglycemia, weight gain, gastrointestinal dis-
orders, and lactic acidosis. There remain therefore important
unmet medical needs in the treatment of T2DM.
Glucagon-like peptide
1 (GLP-1) is an incretin hormone
secreted from the intestine in response to glucose absorption after
⇑
Corresponding author. Tel.: +81 6 6337 5903; fax: +81 6 6337 6051.
E-mail address: yohei-ikuma@ds-pharma.co.jp (Y. Ikuma).
http://dx.doi.org/10.1016/j.bmc.2014.12.051
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ikuma, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.051

2
Y. Ikuma et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Cl
Cl
Cl
Cl
O
O
O
O
F
NH2
F
N
N
N
N
N
N
N
N
MeN
MeN
MeN
HO2C
MeN
HO2C
N
N
N
N
NH2
NH2
NH2
CO2H
H2N
CO2H
1
2
3
4
Me
F
F
CN
O
NC
NH2
F
O
N
O
CN
O
MeN
O
H
N
N
N
N
N
N
N
N
N
N
N
HO
N
HO
N
O
N
N
O
NH2
Me
Me
CF3
NH2
Sitagliptin
Vildagliptin
Saxagliptin
Alogliptin
Linagliptin
Figure 1. 3H-Imidazo [4,5-c]quinolin-4(5H)-ones (1–4) and representative marketed DPP-4 inhibitors.
oral absorption because of low membrane permeability caused by
the formation of a zwitterion between the carboxyl group and the
amino group.20 In fact, a pharmacokinetic (PK) study in rats
showed that compounds 1 and 4 had bioavailability (BA) values
of <0.1% and 2.7%, respectively. Therefore, we had to consider other
strategies to improve these compounds bioavailability. In this
study, we describe a rational ester introduction to 1–4 to construct
prodrugs that would allow improvement of these compounds
plasma concentrations with focus on in vitro metabolic character-
ization. We also present a novel efficient synthetic method for the
preparation of 3H-imidazo[4,5-c]quinolin-4(5H)-ones using intra-
molecular radical cyclization. Our investigation led to 10h as an
orally active DPP-4 inhibitor with a 5-methyl-2-oxo-1,3-dioxol-4-
yl methyl (Dox) ester.21,22
All of the substrate 12 was consumed following treatment with
sodium nitrite in AcOH at room temperature to give the diazonium
salt 11. Compound 9a was obtained in 51% yield by adding Cu to
the reaction mixture (entry 1). When CuCl (I) was used as an addi-
tive instead of Cu, no difference in the yield of 9a was seen,
although reaction rate was slower (entry 2). The additive CuCl2
(II) gave lower yield than Cu or CuCl (I) (entry 3). No change in
the yield of 9a was observed at 80 °C using sodium nitrite and
Cu (entry 4). Using isoamyl nitrile as a diazotization reagent
slightly reduced the yield of 9a compared to sodium nitrite in
AcOH (entry 5). As for solvent effect, no diazonium salt 11 was
detected in toluene, and 9a was produced at room temperature
without addition of Cu (entry 6). While AcOH helped stabilize
the diazonium salt 11, toluene did not have such effect. It was thus
believed that thermal homolysis of the diazonium salt intermedi-
ate26 in toluene led to generation of a radical and subsequently
cyclization reaction to give 9a. Furthermore, reaction in toluene
resulted in fewer side-products compared to AcOH. We next exam-
ined the effect of temperature on the reaction yield using toluene
as a solvent. Carrying out the reaction at 80 °C significantly
improved the yield of 9a (entry 7). Using isoamyl nitrite, we sought
to optimize the reaction solvent at 80 °C without use of additive.
Although carrying out the reaction in THF gave 9a in a yield similar
to that obtained in toluene, the use of xylene, pyridine or acetoni-
trile gave unsatisfactory result (entries 8–11). Among the test-
solvents, 1,4-dioxane provided the best yield of 9a (entry 12).
Using intramolecular radical cyclization, we could develop a novel
and efficient 4 steps synthetic method for construction of 3H-imi-
dazo[4,5-c]quinolin-4(5H)-ones, including the key intermediate 9a
(52% total yield).
2. Chemistry
The prodrug esters 10a–10l were prepared as shown in
Scheme 1. Compounds 9a–9l were synthesized by alkylation with
various alkyl halides using the previously reported intermediates
5, 6, 7, or 8.19 Removal of the tert-butoxy carbonyl group yielded
the prodrugs 10a–10l. Compound 9a was thought to be a key
intermediate because the orally active DPP-4 inhibitor 10h was
synthesized via 9a. Although preparation of 9a from diphenyl
cyanocarbonimidate has already been reported,18,19 the proposed
method not only requires 8 steps, but provides an unsatisfactory
total yield of 19.9%. An efficient synthetic route to 9a was therefore
necessary. First we designed a new synthetic route for preparation
of 3H-imidazo[4,5-c]quinolin-4(5H)-ones from 12 using intramo-
lecular radical cyclization via the diazonium salt 11 (Scheme 2).23–25
The methyl 4-methylaminobenzoate was converted to 13 in
quantitative yield by alkylation with chloroacetyl chloride
(Scheme 3). Replacement of one phenoxy group in the diphenyl
cyanocarbonimidate with (R)-tert-butoxycarbonylaminopiperi-
dine, followed by treatment with 2-chlorobenzylamine at 80 °C
provided 14 in high yield. N-Alkylation of 14 with 13 at 40 °C affor-
ded a mixture of 15 and 12, and subsequent heating at 60 °C to
promote cyclization gave 12 (one pot) in 75% yield. Next, we
attempted intramolecular radical cyclization of 12 (Scheme 4).
3. Results and discussion
Although compound 1–4 had excellent clearance in human and
rat microsomes (<0.01/<0.01 mL/min/mg protein, respectively),
they showed poor oral absorption. To overcome this problem, we
considered the use of prodrugs. Initially, we used 1 and 3, both
of which have a carboxyl group at a different position, as represen-
tative active metabolites and evaluated the metabolic properties of
the methyl esters 10a and 10j in vitro (Table 2). Compound 10a
was rapidly metabolized in the rat liver S9 fractions and showed
moderate serum stability. On the other hand, 10j was stable in
the rat liver S9 fractions, but labile in serum. In each case, only
the active metabolite 1 or 3 was generated, and no other byprod-
ucts were obtained. Although 10a and 10j had different metabolic
properties, they were expected not to remain in the plasma after
oral administration in rats. While 1 and 3 membrane permeability,
as determined by the parallel artificial membrane permeability
assay (PAMPA; pH 5.0/7.4, 10ꢀ6 cm/s), was poor (<0.1/<0.1,
Table 1
Compounds 1–4 inhibitory activity for DPP-4 and DPP-4 homologues (IC50 nM)
DPP-4
FAPa
DPP-2
DPP-8
DPP-9
1
2
3
4
5.8
4.8
1.6
0.48
>100,000
>100,000
20,400
>100,000
>100,000
>10,000
>10,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
30,600
Please cite this article in press as: Ikuma, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.051

Y. Ikuma et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
Cl
Cl
O
Cl
O
O
a
b
N
N
N
N
N
N
MeN
MeN
MeN
N
N
N
NHBoc
NHBoc
NH2
HCl
CO2H
CO2R
CO2R
9a-9h
10a-10h
5
Me
Me
O
O
N
R
Me
a
Me2N
Me2N
O
O
O
O
N
e
O
O
h
N
Me
b
c
d
f
g
Cl
Cl
Cl
O
O
F
NHBoc
F
N
N
N
N
O
MeN
MeN
O
F
N
N
a
a
b
b
N
N
MeN
N
NH2
HCl
Me
Me
O
NHBoc
O
9i
O
O
O
O
CO2H
O
O
6
10i
O
Cl
Cl
Cl
O
O
O
N
N
N
N
N
N
MeN
HO2C
MeN
RO2C
MeN
RO2C
N
N
N
NHBoc
NHBoc
NH2
HCl
9j-9k
10j-10k
7
O
O
O
R
Me
j
Me
k
Cl
Cl
Cl
O
O
O
F
F
F
a
N
b
N
N
MeN
MeN
N
N
MeN
HO2C
O
O
O
O
N
N
N
N
O
O
NHBoc
NH2
HCl
O
O
NHBoc
Me
Me
O
O
8
9l
10l
Scheme 1. Preparation of the prodrugs 10a–10l. Reagents and conditions: (a) R-halide, K2CO3, DMF, 34%-quant.; (b) HCl, 1,4-dioxane, 70%-quant.
Cl
Cl
Cl
MeO2C
O
O
MeO2C
N
N
N
N
O
OPh
NCN
PhO
MeN
N
N
N
N
N
Me
X- +
N
N
N
N
Me
NHBoc
NHBoc
H2N
NHBoc
CO2Me
9a
11
12
Scheme 2. New synthetic route for the preparation of 3H-imidazo[4,5-c]quinolin-4(5H)-ones by intramolecular radical cyclization.
Me
NHMe
a
N
Cl
O
MeO2C
MeO2C
H
13
MeO2C
Cl
O
MeO2C
N
b
c
O
OPh
NCN
PhO
Me
N
N
N
N
NCN
N
N
NHBoc
NHBoc
N
N
Cl
NCN
Me
Cl
H2N
NHBoc
14
15
12
Scheme 3. Synthesis of 12. Reagents and conditions: (a) chloroacetyl chloride (1.2 equiv), pyridine (1.0 equiv), THF, 0 °C, quant.; (b) (R)-3-tert-butoxycarbonylaminopi-
i
peridine (1.0 equiv), PrOH then 2-chlorobenzylamine (1.5 equiv), 80 °C, 95%; (c) 13 (1.1 equiv), Cs2CO3 (2.0 equiv), MeCN, 40 °C then 60 °C, 75%.
respectively), that of 10a and 10j was markedly increased by ester-
ification, exhibiting high PAMPA values of 34.1/47.4 and 36.2/41.4,
respectively. Next, the oral absorption and plasma concentration of
both compounds 1 and 3 and their respective prodrugs 10a and 10j
were evaluated in rats (Fig. 2). The BA value for 1 after oral admin-
istration of 10a was only 2.4%. Contrary to our expectation, much
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4
Y. Ikuma et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Cl
Cl
Diazotization reagent
solvent, temp.
Cl
O
MeO2C
MeO2C
Additive
N
N
O
O
MeN
N
N
N
N
N
N
N
N
N
Me
NHBoc
Me
X- +
H2N
N
N
NHBoc
NHBoc
CO2Me
9a
12
11
Entry
1
2
3
4
5
6
7
8
Diazotization reagent (equiv.) Additive (equiv.)
Solvent
AcOH
AcOH
AcOH
AcOH
Temp.
rt
rt
rt
80 °C
rt
Time (h)
Yield (%)
51
NaNO2 (3.0)
NaNO2 (3.0)
NaNO2 (3.0)
NaNO2 (3.0)
isoamyl nitrite (2.0)
isoamyl nitrite (3.0)
isoamyl nitrite (5.0)
isoamyl nitrite (5.0)
isoamyl nitrite (5.0)
isoamyl nitrite (5.0)
isoamyl nitrite (5.0)
isoamyl nitrite (5.0)
5
26
26
3
1.5
22
3
3
3
3
3
Cu (1.0)
CuCl (1.0)
CuCl2 (1.0)
Cu (1.0)
Cu (1.0)
none
43
29
53
AcOH
36
toluene
toluene
xylene
pyridine
acetonitrile
THF
33a
71
rt
none
none
none
none
80 °C
80 °C
80 °C
80 °C
80 °C
80 °C
59
34
51
64
9
10
11
12
none
none
1,4-dioxane
3
73
a The yield was determined by HPLC using external standard method aꢀer quenching the reacꢁon
Scheme 4. Intramolecular radical cyclization of 12.
Table 2
Compounds 10a and 10j membrane permeability and metabolic properties in the rat (r-) and human (h-) sera and liver S9 fractions
Prodrug
Active metabolite
Remaining ratioa (%)
r-serum h-liver S9
72
8.4
PAMPA Pe (10ꢀ6 cm/s) pH 5.0/7.4
r-liver S9
h-serum
10a
10j
1
3
<0.5
70
>99
>99
80
92
34.1/47.4
36.2/41.4
a
Compounds were incubated at 37 °C for 30 min.
of 10a remained in the plasma with an area under the plasma
curve (AUC) ratio 6.3-fold that of 1. Oral administration of 10j
resulted in excellent plasma concentration of compound 3 with a
BA value of 90%. Only a small amount of the prodrug 10j was
detected in the plasma, and no 10j remained in the plasma as indi-
cated by a concentration below the limit of quantification (LOQ;
1.0 ng/mL) six hours later, as indicated by a plasma AUC ratio
0.1-fold that of 3. Highly lipophilic prodrugs with an amino group
are inappropriate for plasma exposure, because they may induce
inhibition of hERG channel, mechanism-based inhibition of cyto-
chrome P450, drug-induced phospholipidosis, and hepatotoxicity
in humans.27 We considered that in addition to rapid metabolism
in the serum, adequate transformation in liver S9 fractions is cru-
cial in achieving a prodrug rapid clearance.
As high quantities of 10a and 10j were predicted to remain in
the plasma after oral administration in humans, we sought a
prodrug that can rapidly be transformed into a carboxyl form both
in the human serum and liver S9 fractions. Using the active metab-
olite 1, various esters were synthesized and evaluated for their
metabolic properties in vitro (Table 3). Although 10b with a piva-
loyloxymethyl (POM) ester28 was rapidly metabolized in human
liver S9 fractions, it showed insufficient conversion in human
serum. As alkyl amino alkyl esters are reported to be labile in
human plasma, we considered the use of the dimethyl amino ethyl
ester29 10c and the dimethyl amino propyl ester 10d. Compounds
10c and 10d were completely converted to the active metabolite 1
in human serum. However, these esters were hardly metabolized
in human liver S9 fractions and had poor membrane permeability.
Based on these findings, we prepared a series of morpholino alkyl
esters. Although compound 10e with a morpholino ethyl ester30
was sufficiently converted to 1 in human serum, it was unsatisfac-
torily transformed in human liver S9 fractions and had low
membrane permeability. Both compounds 10f31 and 10g were con-
verted to the carboxyl form in human serum. Compound 10g was
not only metabolized to a greater extent than 10e in human liver
S9 fractions, but also had good membrane permeability. However,
1 was scarcely detected in human liver S9 fractions, and other
byproducts were obtained. In conclusion, no alkyl amino alkyl
ester or morpholino alkyl ester with the desired profile could be
isolated. It is reported that the 5-methyl-2-oxo-1,3-dioxol-4-yl
methyl (Dox) ester is labile in human plasma.32 Accordingly, 10h,
which has a Dox ester, was labile in human serum and liver S9 frac-
tions and had good membrane permeability. Compound 10h was
therefore expected to have sufficient oral absorption with good
release of the active metabolite 1. Furthermore, no 10h was
1000
100
10
1
3
10a
10j
1
0.1
0
5
10
Time (h)
15
20
25
Figure 2. Time-dependent concentrations of 1 and 10a after administration of 10a,
and 3 and 10j after administration of 10j in the plasma of rats (10 mg/kg).
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Y. Ikuma et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Table 3
Ester membrane permeability and metabolic properties in the human (h-) and rat (r-) serum and liver S9 fractions
entry
Prodrug
Active metabolite
Remaining ratioa (%)
h-serum r-liver S9
PAMPA Pe(10ꢀ6 cm/s) pH 5.0/7.4
h-liver S9
r-serum
1
2
3
4
5
6
7
8
9
10b
10c
10d
10e
10f
10g
10h
10i
1
1
1
1
1
1
1
1
3
3
<0.5
>99
>99
37
48
22
7.8
3.5
17
73
<0.5
>99
>99
>99
78
30
<0.5
1.0
0.6
97
38
50
76
6.5/6.3
<0.5
<0.5
6.8
<0.1/0.6
<0.1/<0.1
0.9/7.1
<0.1/3.4
10.1/18.0
10.3/12.1
11.0/8.4
12.5/14.2
15.8/15.9
7.0
<0.5
<0.5
<0.5
<0.5
<0.5
92
<0.5
<0.5
<0.5
<0.5
10k
10l
1.0
3.6
10
13
a
Compounds were incubated at 37 °C for 30 min.
Table 4
120
Pharmacokinetic parameters of the active metabolites 1–4 after iv administration in
rats
100
80
Active
metabolite
Dose (mg/
kg)
AUC (ngꢁh/
CL (mL/min/ Vd (L/
T1/2
(h)
mL)
kg)
kg)
60
40
20
0
1
2
3
4
1.5
1
3
188
75.3
276
99.9
132
222
181
167
2.9
7.1
4.5
31.5
1.0
1.3
0.6
8.3
10h
1
Sitagliptin
15
0
5
10
20
expected to remain in human plasma. When the Dox ester was
introduced to the active metabolites 2–4, the obtained prodrugs
10i, 10k, and 10l exhibited properties similar to those of 10h and
were readily converted to the corresponding active metabolites.
Additionally, 10h, 10i, 10k, and 10l metabolic profiles in rat liver
S9 fractions were similar to those in human fractions. Compounds
10h, 10i, 10k, and 10l with a Dox ester were then considered for
further evaluation.
Time (h)
Figure 3. Time-dependent plasma DPP-4 activity after oral administration of 10h
(3 mg/kg) and sitagliptin (3 mg/kg) in SD rats.
ties in human are similar to those in rats. Thus, 10h was selected
for further evaluation, including assessment of its in vivo inhibi-
tory activity for DPP-4 and its effect on plasma glucose level in oral
glucose tolerance test (OGTT).
To determine plasma concentrations of the prodrugs and the
corresponding active metabolites, we performed PK studies in rats.
First we examined the PK profiles of the active metabolites 1–4
after intravenous (iv) administration (Table 4). Compounds 1–4
had very high clearance values exceeding liver blood flow, and 3
showed a short half-life of 0.6 h. Better exposure (AUC) and long
half-life were seen with 1 and 4, respectively. We also determined
plasma concentration of the active metabolites 1–4 after oral
administration of their prodrugs in rats (Table 5). Remarkably,
the plasma concentration of 1 after oral administration of 10h sig-
nificantly improved with a BA value of 35%. Furthermore and as
expected, no 10h remained in the plasma as indicated by a concen-
tration below the limit of quantification (LOQ: 1.0 ng/mL) at all
sampling points. On the other hand, the prodrugs 10i, 10k and
10l gave unsatisfactory results. The BA values of 2, 3 and 4 after
oral administration of 10i, 10k and 10l were 11.7%, 4.0%, 4.8%,
respectively. No marked prodrug effect on oral absorption was
observed. Because the solubility of 10h at pH 2.5 was markedly
better than that of the other prodrugs, 1 might show the best oral
absorption. In humans, 1 is expected to show sufficient plasma
concentration after oral administration of 10h, and no 10h is
expected to remain in the plasma, because 10h metabolic proper-
To assess the potency of single oral administration of 10h
(3 mg/kg), DPP-4 activity in SD rats was evaluated over a time-
period of 0–24 h (Fig. 3). Compound 10h showed potent DPP-4
inhibition with 87% and 64% inhibition of plasma DPP-4 activity
at 1 and 10 h, respectively. This effect was superior to that of sitag-
liptin at the same dose (3 mg/kg). For OGTT (Fig. 4), ZF rats aged 11
weeks were fasted overnight and were orally administered either
the vehicle or 10h at different doses (0.3 or 1 mg/kg). After one
hour (t = 0), the rats were orally administered glucose (2 g/kg),
and plasma glucose levels were measured against time. Compound
10h showed a dose-dependent reduction in blood glucose levels
(Fig. 4), and a significant reduction of glucose levels was observed
at 1 mg/kg against glucose tolerance. These findings suggest that
the therapeutic effect of 10h is mediated via increase in GLP-1 level
following inhibition of DPP-4. Thus, compound 10h is a promising
agent for T2DM.
4. Conclusion
In summary, to identify an orally active 3H-imidazo[4,5-c]quin-
olin-4(5H)-one as a DPP-4 inhibitor, we pursued a prodrug strategy
Table 5
In vitro activity of DPP-4 and pharmacokinetic parameters after oral administration in rats and solubility of the prodrugs 10h, 10i, 10k, and 10l
Prodrug
h-DPP-4 IC50 (nM)
Active metabolite
Dose (mg/kg)
Cmax (ng/mL)
Tmax (h)
AUC (ngꢁhr/mL)
BA (%)
Solubility (mg/mL) pH 7.4/5.5/2.5
10h
10i
10k
10l
6.0
4.7
1.4
3.4
1
2
3
4
20
10
20
10
94.1
14.8
81.7
nda
2
2
728
35
<0.001/0.003/0.210
<0.001/0.002/0.025
0.002/0.002/0.054
<0.001/0.001/0.047
72.7
61.9
43.9
11.7
4.0
4.8
0.25
nda
a
Not determined.
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6
Y. Ikuma et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
A
B
350
300
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
250
200
150
**
100
50
-60
0
60
Time after glucose loading (min)
vehicle 0.3 mg/kg 1.0 mg/kg
120
vehicle
0.3 mg/kg 1.0 mg/kg
Figure 4. (A) Effect of 10h on plasma glucose concentration in OGTT in male Zucker fatty rats. (B) Changes in AUC of plasma glucose from 0 min to 120 min. Each value
represents the mean and SEM, n = 10 rats in each group. ⁄⁄P <0.0125 versus vehicle control.
using 1-4 as active metabolites. Optimization of the ester moiety
showed that for the ester not to remain in the plasma after oral
administration, rapid metabolism into the carboxyl form in both
liver S9 fractions and serum was important. In particular, lability
in the serum was found to be crucial. We were able to identify
10h having a 5-methyl-2-oxo-1,3-dioxol-4-yl methyl ester in the
C-8 carboxylate of 1 as a prodrug with good oral absorption and
efficient release of 1 in rats. Compound 10h exhibited potent
DPP-4 inhibitory activity and potent antidiabetic effect in vivo.
Through our investigation, we have developed a novel efficient
synthetic method for construction of 3H-imidazo[4,5-c]quinolin-
4(5H)-ones using intramolecular radical cyclization. The potent
and orally active DPP-4 inhibitor 10h is expected to be potentially
useful for the treatment of T2DM.
to room temperature, the reaction mixture was quenched with
saturated NH4Cl aqueous solution, and extracted with EtOAc. The
organic layer was washed with saturated NH4Cl aqueous solution
twice and brine, dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by silica-gel column
chromatography to give methyl 2-{(3R)-3-[(tert-butoxycar-
bonyl)amino]piperidin-1-yl}-3-(2-chloro-5-fluorobenzyl)-5-methyl-
4-oxo-4,5-dihydro-3H-imidazo[4,5-c]quinoline-8-carboxylate
(306.0 mg, yield 75%) as a white powder. A mixture of above-
product (216.5 mg, 0.362 mmol) and 1M NaOH (1 mL) aqueous
solution in THF (1 mL) and MeOH (1 mL) was stirred at room tem-
perature for 6 h. The mixture was acidified with 5% KHSO4 aqueous
solution and extracted with CHCl3. The organic layer was washed
with brine, dried over Na2SO4, and concentrated under reduced
pressure to give 6 (165.7 mg, yield 78%). 1H NMR (400 MHz,
DMSO-d6) d 8.65 (d, J = 2.0 Hz, 1H), 8.04 (dd, J = 2.0, 9.0 Hz, 1H),
7.64 (d, J = 9.0 Hz, 1H), 7.56–7.52 (m, 1H), 7.18–7.13 (m, 1H),
6.88 (d, J = 7.7 Hz, 1H), 6.59 (dd, J = 2.5, 9.3 Hz, 1H), 5.48 (d,
J = 18.0 Hz, 1H), 5.42 (d, J = 18.0 Hz, 1H), 3.62 (s, 3H), 3.48–3.44
(m, 1H), 3.38–3.27 (m, 2H), 2.89–2.84 (m, 1H), 2.75–2.70 (m, 1H),
1.79–1.71 (m, 2H), 1.61–1.58 (m, 1H), 1.38–1.32 (m, 10H); 13C
NMR (100 MHz, DMSO-d6) d 166.9, 161.0 (d, 1J(C, F) = 243 Hz),
158.0, 154.7, 154.0, 141.5, 140.0, 137.7 (d, 3J(C, F) = 7.2 Hz), 131.0
(d, 3J(C, F) = 7.9 Hz), 128.7, 126.2 (d, 4J(C, F) = 3.0 Hz), 124.1, 123.5,
118.8, 116.0, 115.7 (d, 2J(C, F) = 23.0 Hz), 115.7, 114.0 (d, 2J(C,
F) = 25.0 Hz), 79.1, 54.9, 50.2, 46.5, 46.3,305, 28.9, 28.1, 23.2; HRMS
(ESI) [M+H]+ calcd for C29H32O5N5ClF 584.2071, found 584.2068; IR
(ATR): 1700, 1652, 1648, 1569, 1533, 1500, 1469, 1429, 1388,
5. Experimental section
5.1. Chemistry
5.1.1. Generals
1H NMR and 13C NMR spectra were recorded on a JEOL JNM-
LA300 spectrometer and a Brucker AVANCE 400 spectrometer in
the stated solvents using tetramethylsilane as an internal standard.
Chemical shift (d) are expressed in parts per million. IR spectra
were recorded on a JEOL JIR-SPX60 spectrometer as ATR. High res-
olution MS spectra were recorded on a Thermo Fisher Scientific
LTQ orbitrap Discovery MS equipment. Elemental analysis was
conducted at Sumitomo Analytical Center Inc. Reactions were fol-
lowed by TLC on silica gel 60 F254 using precoated TLC plates (E.
Merck). Column chromatography was carried out on a Yamazen
W-prep system using prepacked silica gel or amino silica gel or
performed on silica gel 60 (230–400 or 70–230 mesh, Merck).
Unless otherwise noted, all materials were obtained from commer-
cial suppliers and used without further purification. All solvents
were of the commercially available grade. Reactions requiring
anhydrous conditions were performed under nitrogen atmosphere.
Compounds 1, 2, 3, 4, 5, 7, 9a, 9j, 10a and 10j were synthesized
as previously reported.18,19
1365, 1309, 1267, 1241, 1222, 1164, 1112 cmꢀ1
.
5.1.3. 2-{(3R)-3-[(tert-Butoxycarbonyl)amino]piperidin-1-yl}-
3-(2-chloro-5-fluorobenzyl)-5-methyl-4-oxo-4,5-dihydro-3H-
imidazo[4,5-c]quinoline-7-carboxylic acid (8)
A mixture of methyl 2-{(3R)-3-[(tert-butoxycarbonyl)amino]
piperidin-1-yl}-3-(5-fluoro-2-methylbenzyl)-5-methyl-4-oxo-4,
5-dihydro-3H-imidazo[4,5-c]quinoline-7-carboxylate19 (195 mg,
0.326 mmol) and 1M NaOH (2 mL) aqueous solution in THF
(2 mL) and MeOH (2 mL) was stirred at 50 °C for 4 h. The mixture
was acidified with 5% KHSO4 aqueous solution and extracted with
CHCl3. The organic layer was washed with brine, dried over
Na2SO4, and concentrated under reduced pressure to give 8
(171.7 mg, yield 90%) as a white amorphous. 1H NMR (400 MHz,
DMSO-d6) d 8.18 (d, J = 8.1 Hz, 1H), 8.06 (d, J = 1.1 Hz, 1H), 7.88
(dd, J = 1.1, 8.1 Hz, 1H), 7.56–7.53 (m, 1H), 7.18–7.13 (m, 1H),
6.93 (d, J = 7.7 Hz, 1H), 6.59 (dd, J = 2.5, 9.4 Hz, 1H), 5.52 (d,
J = 17.6 Hz, 1H), 5.46 (d, J = 17.6 Hz, 1H), 3.66 (s, 3H), 3.48–3.25
(m, 3H), 2.87–2.82 (m, 1H), 2.76–2.71 (m, 1H), 1.79–1.69 (m,
5.1.2. 2-{(3R)-3-[(tert-Butoxycarbonyl)amino]piperidin-1-yl}-3-
(2-chloro-5-fluorobenzyl)-5-methyl-4-oxo-4,5-dihydro-3H-
imidazo[4,5-c]quinoline-8-carboxylic acid (6)
A mixture of methyl 2-{(3R)-3-[(tert-butoxycarbonyl) amino]
piperidin-1-yl}-5-methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-c]
quinoline-8-carboxylate19 (312.5 mg, 0.686 mmol), K2CO3 (284 mg,
2.05 mmol), and 2-chloro-5-fluorobenzyl bromide (185
lL,
1.37 mmol) in DMF (3 mL) was stirred at 60 °C for 3 h. After cooling
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Y. Ikuma et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
2H), 1.60–1.57 (m, 1H), 1.40–1.35 (m, 1H), 1.33 (s, 9H); 13C NMR
(100 MHz, DMSO-d6) d 167.3, 161.2 (d, 1J(C, F) = 243 Hz), 158.2,
154.9, 154.3, 141.0, 137.9 (d, 3J(C, F) = 7.4 Hz), 137.0, 131.2 (d,
3J(C, F) = 8.3 Hz), 130.3, 126.4 (d, 4J(C, F) = 2.8 Hz), 122.9, 122.3,
120.1, 119.9, 116.6, 116.0 (d, 2J(C, F) = 23.0 Hz), 114.3 (d, 2J(C,
F) = 25.0 Hz), 77.9, 55.1, 50.5, 46.7, 46.6, 29.7, 28.8, 28.4, 23.4;
HRMS (ESI) [M+H]+ calcd for C29H32O5N5ClF 584.2071, found
584.2071; IR (ATR): 1712, 1660, 1646, 1623, 1508, 1473, 1456,
1423, 1400, 1365, 1349, 1313, 1290, 1251, 1240, 1220, 1149,
J = 7.4 Hz, 1H), 5.79 (d, J = 17.2 Hz, 1H), 5.65 (d, J = 17.2 Hz, 1H),
5.13 (br s, 1H), 4.44 (t, J = 6.4 Hz, 2H), 3.82 (br s, 1H), 3.76 (s, 3H),
3.45 (dd, J = 3.2, 12.3 Hz, 1H), 3.17–3.08 (m, 3H), 2.54 (t, J = 7.2 Hz,
2H), 2.33 (s, 6H), 2.08–2.01 (m, 2H), 1.79–1.72 (m, 2H), 1.62–1.60
(m, 2H), 1.43 (s, 9H); 13C NMR (100 MHz, CDCl3) d 166.0, 158.3,
154.9, 154.9, 142.2, 140.3, 134.9, 131.8, 129.4, 129.0, 128.5, 127.1,
126.4, 124.6, 124.1, 119.3, 116.7, 114.6, 79.1, 63.3, 56.2, 55.2, 51.3,
46.3, 46.0, 45.4, 29.5, 29.1, 28.3, 27.0, 22.1; HRMS (ESI) [M+H]+ calcd
for C34H44O5N6Cl 651.3056, found 651.3051; IR (ATR): 1716, 1654,
1122, 1112, 1068, 1049, 1027 cmꢀ1
.
1508, 1457, 1272, 1220, 1166, 1112, 1033 cmꢀ1
.
5.1.4. [(2,2-Dimethylpropanoyl)oxy]methyl 2-{(3R)-3-[(tert-
butoxycarbonyl)amino]piperidin-1-yl}-3-(2-chlorobenzyl)-5-
methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-c]quinoline-8-
carboxylate (9b)
5.1.7. 2-(Morpholin-4-yl)ethyl 2-{(3R)-3-[(tert-
butoxycarbonyl)amino]piperidin-1-yl}-3-(2-chlorobenzyl)-5-
methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-c]quinoline-8-
carboxylate (9e)
A mixture of 5 (217.3 mg, 0.384 mmol), K2CO3 (159.2 mg,
1.15 mmol), and chloromethyl pivalate (112 lL, 0.766 mmol) in
Compound 9e was prepared from 5 in a manner similar to that
described for compound 9b with a yield of 73% as a white amor-
phous. 1H NMR (300 MHz, CDCl3) d 8.94 (d, J = 2.0 Hz, 1H), 8.15
(dd, J = 2.0, 8.9 Hz, 1H), 7.46 (d, J = 8.9 Hz, 1H), 7.41 (dd, J = 1.1,
7.9 Hz, 1H), 7.20–7.18 (m, 1H), 7.15–7.11 (m, 1H), 6.68 (d,
J = 7.4 Hz, 1H), 5.78 (d, J = 17.2 Hz, 1H), 5.65 (d, J = 17.2 Hz, 1H),
5.12–5.10 (m, 1H), 4.55 (br s, 2H), 3.80–3.75 (m, 6H), 3.72–3.68
(m, 1H), 3.46 (dd, J = 3.2, 12.3 Hz, 1H), 3.15–3.08 (m, 3H), 2.88
(m, 2H), 2.66–2.57 (m, 5H), 1.82–1.72 (m, 2H), 1.62–1.60 (m,
2H), 1.43 (s, 9H); 13C NMR (100 MHz, CDCl3) d 166.0, 158.4,
155.0, 155.0, 142.2, 140.4, 134.9, 131.9, 129.5, 129.1, 128.5,
127.1, 126.5, 124.8, 123.9, 119.5, 116.8, 114.7, 79.2, 66.9, 62.2,
57.1, 55.2, 53.8, 51.4, 46.4, 46.0, 29.5, 29.2, 28.3, 22.2; HRMS
(ESI) [M+H]+ calcd for C35H44O6N6Cl 679.3005, found 679.3003;
IR (ATR): 1708, 1691, 1654, 1569, 1498, 1465, 1430, 1388, 1351,
DMF (2 mL) was stirred at room temperature for 12 h. The reaction
mixture was quenched with saturated NH4Cl aqueous solution, and
extracted with EtOAc. The organic layer was washed with satu-
rated NH4Cl aqueous solution twice and brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue was puri-
fied by silica-gel column chromatography to give 9b (89.0 mg,
yield 34%) as a white amorphous. 1H NMR (300 MHz, CDCl3) d
8.99 (d, J = 2.1 Hz, 1H), 8.17 (dd, J = 2.1, 9.0 Hz, 1H), 7.45 (d,
J = 9.0 Hz, 1H), 7.40–7.38 (m, 1H), 7.18–7.09 (m, 2H), 6.66 (d,
J = 7.1 Hz, 1H), 6.04 (s, 2H), 5.76 (d, J = 16.8 Hz, 1H), 5.63 (d,
J = 16.8 Hz, 1H), 5.12 (br s, 1H), 3.79 (br s, 1H), 3.74 (s, 3H), 3.45
(dd, J = 3.1, 12.3 Hz, 1H), 3.16–3.08 (m, 3H), 1.79–1.61 (m, 4H),
1.41 (s, 9H), 1.22 (s, 9H); 13C NMR (100 MHz, CDCl3) d 177.2,
164.7, 158.5, 155.0, 142.1, 140.9, 134.9, 131.9, 129.5, 129.4,
128.6, 127.1, 126.5, 125.4, 125.4, 122.7, 119.5, 116.8, 114.8, 79.9,
79.2, 55.3, 51.4, 46.4, 46.0, 31.5, 29.2, 28.3, 26.8, 22.6; HRMS
(ESI) [M+H]+ calcd for C35H43O7N5Cl 680.2846, found 680.2845;
IR (ATR): 1733, 1716, 1683, 1652, 1558, 1540, 1508, 1473, 1457,
1309, 1274, 1218, 1164, 1116, 1066, 1039 cmꢀ1
.
5.1.8. 3-(Morpholin-4-yl)propyl 2-{(3R)-3-[(tert-
butoxycarbonyl)amino]piperidin-1-yl}-3-(2-chlorobenzyl)-5-
methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-c]quinoline-8-
carboxylate (9f)
1363, 1270, 1218, 1157, 1078, 1033 cmꢀ1
.
Compound 9f was prepared from 5 in a manner similar to that
described for compound 9b with a yield of 86% as a white amor-
phous. 1H NMR (400 MHz, CDCl3) d 8.92 (d, J = 2.0 Hz, 1H), 8.15
(dd, J = 2.0, 8.9 Hz, 1H), 7.45 (d, J = 8.9 Hz, 1H), 7.42–7.40 (m, 1H),
7.22–7.18 (m, 1H), 7.15–7.11 (m, 1H), 6.68 (d, J = 7.5 Hz, 1H),
5.77 (d, J = 17.2 Hz, 1H), 5.64 (d, J = 17.2 Hz, 1H), 5.13–5.11 (m,
1H), 4.56 (t, J = 6.4 Hz, 2H), 3.79 (br s, 5H), 3.75 (s, 3H), 3.46 (dd,
J = 3.2, 12.3 Hz, 1H), 3.16–3.07 (m, 3H), 2.59 (br s, 6H), 2.10 (br s,
2H), 1.81–1.71 (m, 2H), 1.61–1.59 (m, 2H), 1.42 (s, 9H); 13C NMR
(100 MHz, CDCl3) d 166.1, 158.4, 155.0, 155.0, 142.2, 140.4,
134.9, 131.9, 129.5, 129.0, 128.5, 127.1, 126.5, 124.6, 124.0,
119.4, 116.7, 114.7, 79.1, 66.7, 63.3, 55.5, 55.2, 53.6, 51.4, 46.3,
46.0, 29.5, 29.2, 28.3, 25.8, 22.2; HRMS (ESI) [M+H]+ calcd for
C36H46O6N6Cl 693.3167, found 693.3155; IR (ATR): 1716, 1652,
5.1.5. 2-(Dimethylamino)ethyl 2-{(3R)-3-[(tert-
butoxycarbonyl)amino]piperidin-1-yl}-3-(2-chlorobenzyl)-5-
methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-c]quinoline-8-
carboxylate (9c)
Compound 9c was prepared from 5 in a manner similar to that
described for compound 9b with a yield of 85% as a white amor-
phous. 1H NMR (300 MHz, CDCl3) d 8.93 (d, J = 2.0 Hz, 1H), 8.15
(dd, J = 2.0, 9.0 Hz, 1H), 7.43 (d, J = 9.0 Hz, 1H), 7.40–7.37 (m, 1H),
7.20–7.09 (m, 2H), 6.66 (d, J = 7.3 Hz, 1H), 5.76 (d, J = 16.8 Hz,
1H), 5.61 (d, J = 16.8 Hz, 1H), 5.14–5.11 (m, 1H), 4.51 (t,
J = 5.7 Hz, 2H), 3.81 (br s, 1H), 3.73 (s, 3H), 3.42 (dd, J = 3.3,
12.5 Hz, 1H), 3.15–3.06 (m, 3H), 2.82 (t, J = 5.7 Hz, 2H), 2.40 (s,
6H), 1.80–1.58 (m, 3H), 1.41 (m, 10H); 13C NMR (100 MHz, CDCl3)
d 166.1, 158.5, 155.1, 155.0, 142.3, 140.5, 134.9, 131.9, 129.5,
129.2, 128.6, 127.1, 126.5, 124.9, 124.0, 119.5, 116.8, 114.7, 79.2,
62.9, 57.8, 55.2, 51.4, 46.4, 46.0, 45.8, 29.5, 29.2; HRMS (ESI)
[M+H]+ calcd for C33H42O5N6Cl 637.2900, found 637.2892; IR
(ATR): 1712, 1652, 1569, 1506, 1457, 1388, 1363, 1311, 1272,
1558, 1506, 1457, 1363, 1272, 1220, 1166, 1112, 1066 cmꢀ1
.
5.1.9. 2-[(2R,6S)-2,6-Dimethylmorpholin-4-yl]ethyl 2-{(3R)-3-
[(tert-butoxycarbonyl)amino]piperidin-1-yl}-3-(2-
chlorobenzyl)-5-methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-
c]quinoline-8-carboxylate (9g)
1234, 1166, 1112, 1066, 1049 cmꢀ1
.
Compound 9g was prepared from 5 in a manner similar to that
described for compound 9b with a yield of 49% as a white amor-
phous. 1H NMR (400 MHz, CDCl3) d 8.92 (d, J = 2.0 Hz, 1H), 8.15
(dd, J = 2.0, 9.0 Hz, 1H), 7.45 (d, J = 9.0 Hz, 1H), 7.40 (d, J = 7.9 Hz,
1H), 7.21–7.17 (m, 1H), 7.14–7.10 (m, 1H), 6.67 (d, J = 7.4 Hz,
1H), 5.77 (d, J = 17.2 Hz, 1H), 5.63 (d, J = 17.2 Hz, 1H), 5.12–5.10
(m, 1H), 4.53 (m, 2H), 3.81–3.75 (m, 6H), 3.44 (dd, J = 3.0,
12.3 Hz, 1H), 3.14–3.07 (m, 3H), 2.90–2.82 (m, 4H), 1.94 (t,
J = 6.6 Hz, 2H), 1.79–1.71 (m, 2H), 1.61–1.59 (m, 2H), 1.42 (s, 9H),
1.17 (d, J = 6.3 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) d 166.0,
5.1.6. 3-(Dimethylamino)propyl 2-{(3R)-3-[(tert-
butoxycarbonyl)amino]piperidin-1-yl}-3-(2-chlorobenzyl)-5-
methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-c]quinoline-8-
carboxylate (9d)
Compound 9d was prepared from 5 in a manner similar to that
described for compound 9b with a yield of 70% as a white amor-
phous. 1H NMR (400 MHz, CDCl3) d 8.94 (d, J = 2.1 Hz, 1H), 8.16
(dd, J = 2.1, 9.0 Hz, 1H), 7.45 (d, J = 9.0 Hz, 1H), 7.41 (dd, J = 1.1,
7.9 Hz, 1H), 7.22–7.18 (m, 1H), 7.15–7.11 (m, 1H), 6.68 (d,
Please cite this article in press as: Ikuma, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.051

8
Y. Ikuma et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
158.5, 155.1, 155.0, 142.3, 140.5, 134.9, 132.0, 129.6, 129.2, 128.6,
127.2, 126.5, 124.8, 123.9, 119.6, 116.8, 114.8, 79.3, 62.2, 59.6,
56.7, 55.3, 51.5, 46.4, 46.0, 31.5, 29.2, 28.4, 22.6, 22.2, 19.1; HRMS
(ESI) [M+H]+ calcd for C37H48O6N6Cl 707.3318, found 707.3308; IR
(ATR): 1716, 1654, 1569, 1558, 1506, 1457, 1363, 1309, 1272,
678.2325, found 678.2325; IR (ATR): 1824, 1716, 1652, 1558,
1508, 1473, 1307, 1216, 1166, 1097, 1049, 1033 cmꢀ1
.
5.1.13. (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-{(3R)-3-[(tert-
butoxycarbonyl)amino]piperidin-1-yl}-3-(2-chloro-5-
fluorobenzyl)-5-methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-
c]quinoline-7-carboxylate (9l)
1236, 1220, 1166, 1114, 1066 cmꢀ1
.
5.1.10. (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-{(3R)-3-[(tert-
butoxycarbonyl)amino]piperidin-1-yl}-3-(2-chlorobenzyl)-5-
methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-c]quinoline-8-
carboxylate (9h)
Compound 9l was prepared from 8 in a manner similar to that
described for compound 9b with a quantitative yield as a white
amorphous. 1H NMR (400 MHz, CDCl3) d 8.38 (d, J = 8.2 Hz, 1H),
8.14 (d, J = 1.2 Hz, 1H), 7.99 (dd, J = 1.2, 8.2 Hz, 1H), 7.40–7.36 (m,
1H), 6.95–6.90 (m, 1H), 6.45–6.42 (m, 1H), 5.90 (s, 1H), 5.71 (d,
J = 17.0 Hz, 1H), 5.62 (d, J = 17.0 Hz, 1H), 5.15 (s, 2H), 3.82 (br s,
1H), 3.81 (s, 3H), 3.47 (dd, J = 3.2, 12.4 Hz, 1H), 3.21–3.10 (m,
3H), 2.28 (s, 3H), 1.84–1.56 (m, 4H), 1.46 (s, 9H); 13C NMR
(100 MHz, DMSO-d6) d 166.5, 162.3 (d, 1J(C, F) = 245 Hz), 159.0,
155.8, 155.6, 152.7, 141.9, 141.1, 137.9, 137.9 (d, 3J(C,
F) = 6.8 Hz), 134.1, 131.6 (d, 3J(C, F) = 8.2 Hz), 129.0, 127.4 (d,
4J(C, F) = 2.7 Hz), 123.8, 123.7, 121.6, 121.2, 117.3, 116.4 (d, 2J(C,
F) = 22.8 Hz), 114.5 (d, 2J(C, F) = 24.6 Hz), 79.8, 55.7, 55.2, 52.2,
47.0, 46.6, 32.2, 29.9, 29.1, 23.3, 10.1; HRMS (ESI) [M+H]+ calcd
for C34H36O8N5ClF 696.2231, found 696.2238; IR (ATR): 1824,
1716, 1652, 1558, 1540, 1508, 1473, 1457, 1363, 1307, 1216,
Compound 9h was prepared from 5 in a manner similar to that
described for compound 9b with a yield of 96% as a white amor-
phous. 1H NMR (400 MHz, CDCl3) d 8.96 (br s, 1H), 8.15 (dd, J = 2.1,
8.9 Hz, 1H), 7.46 (d, J = 8.9 Hz, 1H), 7.41 (dd, J = 1.0, 7.9 Hz, 1H),
7.22–7.18 (m, 1H), 7.15–7.11 (m, 1H), 6.69 (d, J = 7.5 Hz, 1H), 5.77
(d, J = 17.2 Hz, 1H), 5.64 (d, J = 17.2 Hz, 1H), 5.14 (s, 2H), 5.11 (br s,
1H), 3.82 (br s, 1H), 3.75 (s, 3H), 3.47 (dd, J = 3.3, 12.3 Hz, 1H),
3.16–3.09 (m, 3H), 2.28 (s, 3H), 1.81–1.72 (m, 2H), 1.61–1.60 (m,
2H), 1.43 (s, 9H); 13C NMR (100 MHz, DMSO-d6) d 165.1, 158.1,
154.9, 154.0, 152.1, 141.3, 140.6, 140.5, 135.2, 133.7, 131.2, 129.4,
128.9, 128.8, 127.7, 126.9, 123.5, 122.3, 119.0, 116.2, 116.0, 77.9,
55.1, 54.7, 50.6, 46.7, 46.2, 29.9, 29.0, 28.4, 23.5, 9.1; HRMS (ESI)
[M+H]+ calcd for C34H37O8N5Cl 678.2325, found 678.2325; IR
(ATR): 1822, 1716, 1673, 1654, 1506, 1434, 1394, 1315, 1270,
1164, 1103, 1049, 1033 cmꢀ1
.
1216, 1160, 1105, 1093, 1066, 1049 cmꢀ1
.
5.1.14. [(2,2-Dimethylpropanoyl)oxy]methyl 2-[(3R)-3-
aminopiperidin-1-yl]-3-(2-chlorobenzyl)-5-methyl-4-oxo-4,5-
dihydro-3H-imidazo[4,5-c]quinoline-8-carboxylate
5.1.11. (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-{(3R)-3-[(tert-
butoxycarbonyl)amino]piperidin-1-yl}-3-(2-chloro-5-
hydrochloride (10b)
fluorobenzyl)-5-methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-
c]quinoline-8-carboxylate (9i)
To a solution of 9b (80.0 mg, 0.118 mmol) in 1,4-dioxane (2 mL)
was added 4N HCl-1,4-dioxane (2 mL). The reaction mixture was
stirred at room temperature for 2 h and concentrated under reduced
pressure to give 10b (76.3 mg, quantitative yield) as a white amor-
phous. 1H NMR (400 MHz, DMSO-d6) d 8.68 (d, J = 2.1 Hz, 1H), 8.41
(br s, 3H), 8.07 (dd, J = 2.1, 8.9 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.50
(d, J = 7.9 Hz, 1H), 7.32–7.28 (m, 1H), 7.23–7.19 (m, 1H), 6.69 (d,
J = 7.3 Hz, 1H), 6.01 (s, 2H), 5.61 (d, J = 17.2 Hz, 1H), 5.55 (d,
J = 17.2 Hz, 1H), 3.73–3.65 (m, 1H), 3.63 (s, 3H), 3.49–3.45 (m, 1H),
3.33–3.23 (m, 1H), 3.10–3.07 (m, 1H), 2.89–2.82 (m, 1H), 1.97–
1.96 (m, 1H), 1.78–1.76 (m, 1H), 1.62–1.48 (m, 2H), 1.16 (s, 9H);
13C NMR (75 MHz, DMSO-d6) d 176.5, 164.1, 157.6, 154.0, 140.7,
140.6, 134.9, 130.9, 129.4, 129.0, 128.9, 127.6, 126.9, 123.7, 121.8,
119.1, 116.3, 115.8, 80.0, 59.2, 52.1, 50.9, 46.3, 46.1, 29.1, 27.2,
26.5, 22.0; HRMS (ESI) [M+H]+ calcd for C30H35O5N5Cl 580.2319,
found 580.2312; IR (ATR): 1704, 1672, 1616, 1594, 1508, 1442,
Compound 9i was prepared from 6 in a manner similar to that
described for compound 9b with a yield of 87% as a white amor-
phous. 1H NMR (300 MHz, CDCl3) d 8.95 (d, J = 2.2 Hz, 1H), 8.16
(dd, J = 2.2, 9.0 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 7.40–7.36 (m, 1H),
6.95–6.88 (m, 1H), 6.44–6.42 (m, 1H), 5.70 (d, J = 17.2 Hz, 1H),
5.60 (d, J = 17.2 Hz, 1H), 5.14 (s, 2H), 5.10 (br s, 1H), 3.81 (br s,
1H), 3.76 (s, 3H), 3.48 (dd, J = 2.9, 12.1 Hz, 1H), 3.12–3.06 (m,
3H), 2.28 (s, 3H), 1.85–1.75 (m, 3H), 1.42 (s, 9H), 1.27–1.15 (m,
1H); 13C NMR (100 MHz, CDCl3)
d
166.2, 162.3 (d, 1J(C,
F) = 245 Hz), 159.2, 155.7, 155.7, 152.9, 143.0, 141.6, 141.0, 138.0
(d, 3J(C, F) = 7.1 Hz), 134.4, 131.6 (d, 3J(C, F) = 8.2 Hz), 130.1,
127.5 (d, 4J(C, F) = 3.2 Hz), 125.9, 123.5, 120.1, 117.5, 116.4 (d,
2J(C, F) = 22.8 Hz), 115.6, 114.6 (d, 2J(C, F) = 24.5 Hz), 80.3, 56.2,
54.9, 52.1, 47.1, 46.9, 30.3, 30.0, 29.0, 23.2, 10.2; HRMS (ESI)
[M+H]+ calcd for C34H36O8N5ClF 696.2231, found 696.2231; IR
(ATR): 1818, 1716, 1652, 1569, 1506, 1473, 1386, 1305, 1272,
1375, 1317, 1245, 1218, 1114, 1049 cmꢀ1
.
1218, 1166, 1091, 1049 cmꢀ1
.
5.1.15. 2-(Dimethylamino)ethyl 2-[(3R)-3-aminopiperidin-1-
yl]-3-(2-chlorobenzyl)-5-methyl-4-oxo-4,5-dihydro-3H-
5.1.12. (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-{(3R)-3-[(tert-
butoxycarbonyl)amino]piperidin-1-yl}-3-(2-chlorobenzyl)-5-
methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-c]quinoline-7-
carboxylate (9k)
imidazo[4,5-c]quinoline-8-carboxylate dihydrochloride (10c)
Compound 10c was prepared from 9c in a manner similar to
that described for compound 10b with a yield of 98% as a white
amorphous. 1H NMR (400 MHz, DMSO-d6) d 10.98 (s, 1H), 8.78
(d, J = 1.8 Hz, 1H), 8.51 (br s, 3H), 8.23 (dd, J = 1.8, 8.8 Hz, 1H),
7.69 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.32–7.28 (m,1H),
7.24–7.20 (m, 1H), 6.73 (d, J = 7.5 Hz, 1H), 5.64 (d, J = 17.2 Hz,
1H), 5.58 (d, J = 17.2 Hz, 1H), 4.63 (br s, 2H), 3.75–3.72 (m, 1H),
3.64 (s, 3H), 3.61–3.50 (m, 3H), 3.37–3.27 (m, 2H), 3.12–3.09 (m,
1H), 2.88 (s, 6H), 1.97 (m, 1H), 1.75 (m, 1H), 1.64–1.62 (m, 1H),
1.51–1.48 (m, 1H); 13C NMR (75 MHz, DMSO-d6) d 165.0, 157.1,
153.9, 140.4, 140.1, 134.8, 130.9, 129.4, 129.4, 129.0, 127.7,
127.0, 123.9, 122.7, 118.9, 116.0, 115.3, 59.4, 54.8, 52.1, 50.8,
46.5, 46.0, 42.5, 29.1, 27.1, 21.9; HRMS (ESI) [M+H]+ calcd for
Compound 9k was prepared from 7 in a manner similar to that
described for compound 9b with a yield of 73% as a white amor-
phous. 1H NMR (400 MHz, CDCl3) d 8.35 (d, J = 8.2 Hz, 1H), 8.13 (d,
J = 1.1 Hz, 1H), 7.96 (dd, J = 1.1, 8.2 Hz, 1H), 7.41 (dd, J = 1.0,
7.9 Hz, 1H), 7.22–7.19 (m, 1H), 7.15–7.11 (m, 1H), 6.69 (br d,
J = 7.5 Hz, 1H), 6.00 (br s, 1H), 5.78 (d, J = 17.2 Hz, 1H), 5.65 (d,
J = 17.4 Hz, 1H), 5.15 (s, 2H), 3.81 (br s, 1H), 3.80 (s, 3H), 3.44 (dd,
J = 3.3, 12.6 Hz, 1H), 3.25–3.21 (m, 1H), 3.08 (m, 2H), 2.28 (s, 3H),
1.77–1.72 (m, 3H), 1.56–1.51 (m, 1H), 1.46 (s, 9H); 13C NMR
(100 MHz, CDCl3) d 166.4, 159.0, 155.8, 155.5, 152.7, 141.6, 141.0,
137.8, 135.4, 134.1, 132.5, 130.2, 129.2, 128.7, 127.8, 127.1, 123.6,
123.5, 121.6, 121.4, 117.2, 79.7, 55.4, 55.1, 52.1, 47.0, 46.4, 30.1,
29.8, 29.1, 22.2, 10.1; HRMS (ESI) [M+H]+ calcd for C34H37O8N5Cl
C
28H34O3N6Cl 537.2375, found 537.2372; IR (ATR): 1718, 1670,
1627, 1608, 1592, 1558, 1515, 1457, 1446, 1371, 1321, 1276,
1261, 1241, 1218, 1126, 1114, 1066, 1051, 1039 cmꢀ1
.
Please cite this article in press as: Ikuma, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.051

Y. Ikuma et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
5.1.16. 3-(Dimethylamino)propyl 2-[(3R)-3-aminopiperidin-1-
yl]-3-(2-chlorobenzyl)-5-methyl-4-oxo-4,5-dihydro-3H-
white amorphous. 1H NMR (300 MHz, DMSO-d6) d 11.87 (br s,
1H), 8.72 (d, J = 2.2 Hz, 1H), 8.41 (br s, 3H), 8.21 (dd, J = 2.2,
9.0 Hz, 1H), 7.71 (d, J = 9.0 Hz, 1H), 7.51 (dd, J = 1.3, 7.9 Hz, 1H),
7.32–7.19 (m, 2H), 6.70 (d, J = 6.8 Hz, 1H), 5.65 (d, J = 17.2 Hz,
1H), 5.58 (d, J = 17.2 Hz, 1H), 4.74 (m, 2H), 4.08–4.04 (m, 3H),
3.69–3.55 (m, 7H), 3.37–3.21 (m, 2H), 3.10–3.05 (m, 1H), 2.89–
2.75 (m, 3H), 1.95 (m, 1H), 1.76 (m, 1H), 1.63–1.48 (m, 2H), 1.15
(d, J = 6.2 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) d 165.2, 157.7,
154.2, 140.8, 140.6, 135.2, 131.1, 129.6, 129.4, 129.2, 127.9,
127.1, 123.8, 122.9, 119.2, 116.2, 115.9, 68.6, 59.2, 55.5, 54.7,
52.3, 51.2, 46.5, 46.3, 29.3, 27.4, 22.1, 18.5; HRMS (ESI) [M+H]+
calcd for C32H40O4N6Cl 607.2794, found 607.2794; IR (ATR):
1718, 1673, 1652, 1635, 1558, 1540, 1508, 1457, 1324, 1272,
imidazo[4,5-c]quinoline-8-carboxylate dihydrochloride (10d)
Compound 10d was prepared from 9d in a manner similar to that
described for compound 10b with a quantitative yield as a white
amorphous. 1H NMR (300 MHz, DMSO-d6) d 10.88 (br s, 1H), 8.72
(d, J = 2.2 Hz, 1H), 8.52 (br s, 3H), 8.10 (dd, J = 2.2, 9.0 Hz, 1H), 7.65
(d, J = 9.0 Hz, 1H), 7.51 (dd, J = 1.1, 7.9 Hz, 1H), 7.32–7.27 (m, 1H),
7.24–7.19 (m, 1H), 6.72 (d, J = 6.8 Hz, 1H), 5.63 (d, J = 17.2 Hz, 1H),
5.55 (d, J = 17.2 Hz, 1H), 4.41 (t, J = 6.2 Hz, 2H), 3.77–3.73 (m, 1H),
3.63 (s, 3H), 3.33–3.21 (m, 4H), 3.11–3.07 (m, 1H), 2.90–2.83 (m,
1H), 2.78 (s, 6H), 2.24–2.19 (m, 2H), 1.97 (m, 1H), 1.75 (m, 1H),
1.65–1.61 (m, 1H), 1.51 (m, 1H); 13C NMR (75 MHz, CDCl3) d 165.5,
157.5, 154.2, 140.6, 140.5, 135.1, 131.1, 129.6, 129.2, 129.2, 127.9,
127.1, 123.7, 123.2, 119.2, 116.2, 115.8, 62.3, 53.8, 52.3, 51.1, 46.6,
46.3, 42.2, 29.3, 27.4, 23.7, 22.2; HRMS (ESI) [M+H]+ calcd for
C29H36O3N6Cl 551.2532, found 551.2531; IR (ATR): 3355, 1697,
1670, 1652, 1616, 1508, 1465, 1442, 1386, 1322, 1274, 1241, 1218,
1220, 1132, 1060, 1033 cmꢀ1
.
5.1.20. (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-[(3R)-3-
aminopiperidin-1-yl]-3-(2-chlorobenzyl)-5-methyl-4-oxo-4,5-
dihydro-3H-imidazo[4,5-c]quinoline-8-carboxylate
hydrochloride (10h)
1108, 1052, 1039 cmꢀ1
.
Compound 10h was prepared from 9h in a manner similar to
that described for compound 10b with a quantitative yield as a
5.1.17. 2-(Morpholin-4-yl)ethyl 2-[(3R)-3-aminopiperidin-1-yl]-
3-(2-chlorobenzyl)-5-methyl-4-oxo-4,5-dihydro-3H-
white amorphous. 1H NMR (300 MHz, DMSO-d6)
d 8.67 (d,
imidazo[4,5-c]quinoline-8-carboxylate dihydrochloride (10e)
Compound 10e was prepared from 9e in a manner similar to that
described for compound 10b with a quantitative yield as a white
amorphous. 1H NMR (300 MHz, DMSO-d6) d 11.77 (br s, 1H), 8.76
(d, J = 2.0 Hz, 1H), 8.47 (br s, 3H), 8.21 (dd, J = 2.0, 9.0 Hz, 1H), 7.67
(d, J = 9.0 Hz, 1H), 7.51 (dd, J = 1.3, 7.9 Hz, 1H), 7.32–7.28 (m, 1H),
7.25–7.20 (m, 1H), 6.73 (d, J = 6.4 Hz, 1H), 5.64 (d, J = 17.2 Hz, 1H),
5.57 (d, J = 17.2 Hz, 1H), 4.75–4.74 (m, 2H), 4.00–3.88 (m, 4H),
3.75–3.64 (m, 6H), 3.54–3.50 (m, 2H), 3.32–3.28 (m, 4H), 3.08 (m,
1H), 2.91–2.85 (m, 1H), 1.97 (m, 1H), 1.76 (m, 1H), 1.61–1.52 (m,
2H); 13C NMR (100 MHz, DMSO-d6) d 165.1, 157.1, 154.0, 140.6,
139.8, 135.0, 131.1, 129.6, 129.5, 129.2, 127.9, 127.2, 124.1, 122.9,
119.0, 116.2, 115.3, 63.4, 59.4, 54.6, 52.3, 51.5, 51.0, 46.8, 46.2,
29.4, 27.3, 22.1; HRMS (ESI) [M+H]+ calcd for C30H36O4N6Cl
579.2481, found 579.2464; IR (ATR): 1718, 1668, 1592, 1521,
J = 1.8 Hz, 1H), 8.40 (br s, 3H), 8.07 (dd, J = 1.8, 9.0 Hz, 1H), 7.68
(d, J = 9.0 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.32–7.27 (m, 1H),
7.23–7.19 (m, 1H), 6.69 (d, J = 7.3 Hz, 1H), 5.60 (d, J = 17.4 Hz,
1H), 5.53 (d, J = 17.4 Hz, 1H), 5.29 (s, 2H), 3.73–3.69 (m, 1H), 3.62
(s, 3H), 3.41–3.23 (m, 2H), 3.08–3.05 (m, 1H), 2.87–2.81 (m, 1H),
2.30 (s, 3H), 1.97–1.89 (m, 1H), 1.75 (m, 1H), 1.62–1.51 (m, 2H);
13C NMR (100 MHz, DMSO-d6) d 165.1, 157.8, 154.2, 152.1, 141.0,
140.7, 140.6, 135.1, 133.7, 131.1, 129.6, 129.1, 129.1, 127.8,
127.1, 123.6, 122.6, 119.3, 116.3, 116.0, 54.7, 52.3, 51.1, 46.4,
46.3, 29.3, 27.4, 22.2, 9.2; HRMS (ESI) [M+H]+ calcd for C29H29O6
N5Cl 578.1801, found 578.1782; IR (ATR): 1830, 1716, 1670,
1592, 1558, 1540, 1508, 1473, 1230, 1130, 1033 cmꢀ1
.
5.1.21. (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-[(3R)-3-
aminopiperidin-1-yl]-3-(2-chloro-5-fluorobenzyl)-5-methyl-4-
oxo-4,5-dihydro-3H-imidazo[4,5-c]quinoline-8-carboxylate
hydrochloride (10i)
1508, 1473, 1317, 1251, 1130, 1110, 1049, 1033 cmꢀ1
.
Compound 10i was prepared from 9i in a manner similar to that
described for compound 10b with a quantitative yield as a white
amorphous. 1H NMR (400 MHz, DMSO-d6) d 8.68 (d, J = 2.1 Hz,
1H), 8.39 (br s, 3H), 8.08 (dd, J = 2.1, 8.9 Hz, 1H), 7.69 (d,
J = 8.9 Hz, 1H), 7.59–7.55 (m, 1H), 7.21–7.16 (m, 1H), 6.65–6.62
(m, 1H), 5.58 (d, J = 17.4 Hz, 1H), 5.52 (d, J = 17.4 Hz, 1H), 5.29 (s,
2H), 3.70–3.67 (m, 1H), 3.64 (s, 3H), 3.49–3.48 (m, 1H), 3.29–
3.24 (m, 1H), 3.15–3.08 (m, 1H), 2.91–2.86 (m, 1H), 2.24 (s, 3H),
1.97 (m, 1H), 1.81–1.79 (m, 1H), 1.64–1.51 (m, 2H); 13C NMR
(100 MHz, DMSO-d6) d 165.0, 161.2 (d, 1J(C, F) = 243 Hz), 157.8,
154.2, 152.1, 141.1, 140.7, 140.6, 137.7 (d, 3J(C, F) = 7.4 Hz),
133.6, 131.3 (d, 3J(C, F) = 8.6 Hz), 129.1, 126.4 (d, 4J(C,
F) = 2.6 Hz), 123.6, 122.6, 119.3, 116.3, 116.3 (d, 2J(C,
F) = 24.7 Hz), 116.2, 114.4 (d, 2J(C, F) = 24.6 Hz), 54.7, 52.3, 51.2,
46.5, 46.3, 29.3, 27.4, 22.1, 9.1; HRMS (ESI) [M+H]+ calcd for
5.1.18. 3-(Morpholin-4-yl)propyl 2-[(3R)-3-aminopiperidin-1-
yl]-3-(2-chlorobenzyl)-5-methyl-4-oxo-4,5-dihydro-3H-
imidazo[4,5-c]quinoline-8-carboxylate dihydrochloride (10f)
Compound 10f was prepared from 9f in a manner similar to that
described for compound 10b with a quantitative yield as a white
amorphous. 1H NMR (400 MHz, DMSO-d6) d 11.51 (br s, 1H), 8.72
(d, J = 2.1 Hz, 1H), 8.47 (br s, 3H), 8.11 (dd, J = 2.1, 8.9 Hz, 1H),
7.67 (d, J = 8.9 Hz, 1H), 7.51 (dd, J = 0.9, 7.9 Hz, 1H), 7.32–7.28 (m,
1H), 7.24–7.20 (m, 1H), 6.71 (d, J = 7.4 Hz, 1H), 5.63 (d,
J = 17.2 Hz, 1H), 5.57 (d, J = 17.2 Hz, 1H), 4.20 (t, J = 6.4 Hz, 2H),
3.97–3.93 (m, 2H), 3.86–3.82 (m, 2H), 3.75–3.69 (m, 1H), 3.64 (s,
3H), 3.47–3.44 (m, 2H), 3.37–3.26 (m, 4H), 3.12–3.04 (m, 3H),
2.31–2.24 (m, 2H), 1.98–1.96 (m, 1H), 1.78–1.75 (m, 1H), 1.64–
1.51 (m, 1H), 1.50–1.48 (m, 1H); 13C NMR (100 MHz, DMSO-d6) d
165.4, 156.6, 153.9, 140.4, 139.2, 134.9, 131.1, 129.6, 129.3,
129.2, 127.9, 127.2, 123.9, 123.3, 118.7, 116.1, 114.8, 63.4, 62.5,
53.4, 52.3, 51.1, 51.0, 46.8, 46.2, 29.4, 27.2, 22.9, 22.1; HRMS
(ESI) [M+H]+ calcd for C31H38O4N6Cl 593.2643, found 593.2637;
IR (ATR): 1704, 1670, 1635, 1596, 1508, 1457, 1442, 1322, 1263,
C
29H28O6N5ClF 596.1707, found 596.1708; IR (ATR): 1816, 1716,
1668, 1592, 1558, 1540, 1521, 1508, 1457, 1429, 1373, 1303,
1255, 1224, 1191, 1110, 1049, 1033 cmꢀ1
.
1218, 1106, 1051 cmꢀ1
.
5.1.22. (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-[(3R)-3-
aminopiperidin-1-yl]-3-(2-chlorobenzyl)-5-methyl-4-oxo-4,5-
dihydro-3H-imidazo[4,5-c]quinoline-7-carboxylate
hydrochloride (10k)
Compound 10k was prepared from 9k in a manner similar to
that described for compound 10b with a yield of 95% as a white
amorphous. 1H NMR (300 MHz, DMSO-d6) d 8.42 (br s, 3H), 8.25
(d, J = 8.3 Hz, 1H), 8.06 (d, J = 1.3 Hz, 1H), 7.91 (dd, J = 1.3, 8.3 Hz,
5.1.19. 2-[(2R,6S)-2,6-Dimethylmorpholin-4-yl]ethyl 2-[(3R)-3-
aminopiperidin-1-yl]-3-(2-chlorobenzyl)-5-methyl-4-oxo-4,5-
dihydro-3H-imidazo[4,5-c]quinoline-8-carboxylate
dihydrochloride (10g)
Compound 10g was prepared from 9g in a manner similar to
that described for compound 10b with a quantitative yield as a
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10
Y. Ikuma et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
1H), 7.50 (dd, J = 1.1, 7.7 Hz, 1H), 7.32–7.27 (m, 1H), 7.24–7.21 (m,
1H), 6.71 (d, J = 6.6 Hz, 1H), 5.62 (d, J = 17.4 Hz, 1H), 5.54 (d,
J = 17.4 Hz, 1H), 5.28 (s, 2H), 3.69 (br s, 1H), 3.66 (s, 3H), 3.27–
3.21 (m, 2H), 3.03 (m, 1H), 2.82–2.79 (m, 1H), 2.24 (s, 3H), 1.97
(m, 1H), 1.90 (m, 1H), 1.74–1.55 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) d 165.3, 157.7, 154.1, 152.1, 140.7, 140.1, 137.1, 135.1,
133.6, 131.1, 129.5, 129.1, 128.3, 127.9, 127.1, 122.9, 122.7,
120.5, 120.2, 116.7, 55.0, 52.3, 51.1, 46.5, 46.4, 29.0, 27.4, 22.2,
9.2; HRMS (ESI) [M+H]+ calcd for C29H29O6N5Cl 578.1801, found
578.1786; IR (ATR):1810, 1733, 1681, 1621, 1560, 1508, 1473,
J = 8.5 Hz, 2H), 3.92 (s, 3H), 3.85 (br s, 2H), 3.32 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 165.8, 165.7, 146.5, 131.1, 129.8, 126.7, 52.2.
41.2. 37.6; HRMS (ESI) [M+H]+ calcd for C11H13O3NCl 242.0578,
found 242.0585; IR (ATR): 1716, 1674, 1600, 1504, 1435, 1408,
1373, 1257, 1188, 1172, 1095, 1045, 1014, 952 cmꢀ1
.
5.1.26. tert-Butyl {(3R)-1-[N-(2-chlorobenzyl)-N0-
cyanocarbamimidoyl]piperidin-3-yl}carbamate (14)
To solution of (R)-3-tert-butoxycarbonylaminopipiridine
(1.63 g, 8.14 mmol) in iPrOH (60 mL) was added diphenyl cyano-
carbonimidate (2.00 g, 8.14 mmol) at 0 °C. The mixture was stirred
at room temperature for 1 h. To the reaction mixture was added 2-
chlorobenzyl amine (1.73 g, 12.22 mmol) at rt, and the mixture
was stirred at 80 °C for 2 h. After cooling to room temperature,
the reaction mixture was concentrated in vacuo. The residue was
diluted with 5% potassium hydrogensulfate aqueous solution, and
extracted with AcOEt, washed with 5% potassium hydrogensulfate
aqueous solution, 1N NaOH aqueous solution twice, brine, dried
over Na2SO4, and concentrated under reduced pressure. The resi-
due was purified by silica-gel column chromatography to give 14
(3.04 g, yield 95%) as a white solid. 1H NMR (400 MHz, CDCl3) d:
7.46–7.44 (m, 1H), 7.38–7.36 (m, 1H), 7.28–7.25 (m, 2H), 5.78 (br
s, 1H), 4.66 (s, 1H), 4.65 (s, 1H), 3.75–3.70 (m, 1H), 3.62–3.57 (m,
2H), 3.45 (br s, 1H), 3.27 (br s, 1H), 1.90–1.88 (m, 1H), 1.78–1.74
(m, 2H), 1.63–1.59 (m, 2H), 1.41 (s, 9H); 13C NMR (100 MHz, CDCl3)
d 160.3, 155.5, 134.8, 133.3, 129.9, 129.4, 129.0, 126.9, 117.5, 79.8,
51.3, 47.4, 46.8, 45.0, 29.6, 28.1, 22.9; HRMS (ESI) [M+H]+ calcd for
1448, 1309, 1224, 1122, 1052, 1033 cmꢀ1
.
5.1.23. (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-[(3R)-3-
aminopiperidin-1-yl]-3-(2-chloro-5-fluorobenzyl)-5-methyl-4-
oxo-4,5-dihydro-3H-imidazo[4,5-c]quinoline-7-carboxylate
hydrochloride (10l)
Compound 10l was prepared from 9l in a manner similar to that
described for compound 10b with a yield of 70% as a white amor-
phous. 1H NMR (400 MHz, DMSO-d6) d 8.31 (br s, 3H), 8.26 (d,
J = 8.2 Hz, 1H), 8.09 (d, J = 1.3 Hz, 1H), 7.93 (dd, J = 1.3, 8.2 Hz,
1H), 7.59–7.56 (m, 1H), 7.22–7.17 (m, 1H), 6.68–6.63 (m, 1H),
5.60 (d, J = 17.6 Hz, 1H), 5.55 (d, J = 17.6 Hz, 1H), 5.28 (s, 2H),
3.68 (s, 3H), 3.59–3.47 (m, 1H), 3.38–3.22 (m, 2H), 3.10–3.07 (m,
1H), 2.90–2.85 (m, 1H), 2.24 (s, 3H), 1.97–1.96 (m, 1H), 1.79–
1.78 (m, 1H), 1.61–1.53 (m, 2H); 13C NMR (100 MHz, DMSO-d6) d
165.1, 161.0 (d, 1J(C, F) = 243 Hz), 157.5, 154.0, 151.9, 140.4,
140.0, 137.5 (d, 3J(C, F) = 8.2 Hz), 136.9, 133.3, 131.1 (d, 3J(C,
F) = 9.9 Hz), 128.2, 126.2 (d, 4J(C, F) = 4.9 Hz), 122.7, 122.6, 122.5,
120.3, 116.5, 115.9 (d, 2J(C, F) = 23.6 Hz), 114.3 (d, 2J(C,
F) = 25.6 Hz), 54.8, 52.0, 50.9, 46.4, 46.1, 28.8, 27.1, 21.8, 8.9; HRMS
(ESI) [M+H]+ calcd for C29H28O6N5ClF 596.1707, found 596.1707; IR
(ATR): 1810, 1718, 1683, 1635, 1508, 1473, 1307, 1218, 1112,
C
19H27O2N5Cl 392.1848, found 392.1853; IR (ATR): 2164, 1685,
1570, 1531, 1438, 1388, 1365, 1311, 1284, 1242, 1165, 1080,
1049, 1022, 948 cmꢀ1
.
5.1.27. Methyl 2-{(3R)-3-[(tert-
1051, 1033, 1008 cmꢀ1
.
butoxycarbonyl)amino]piperidin-1-yl}-3-(2-chlorobenzyl)-5-
methyl-4-oxo-4,5-dihydro-3H-imidazo[4,5-c]quinoline-8-
carboxylate (9a)
5.1.24. Methyl 4-[{[4-amino-2-{(3R)-3-[(tert-
butoxycarbonyl)amino]piperidin-1-yl}-1-(2-chlorobenzyl)-1H-
imidazol-5-yl]carbonyl}(methyl)amino]benzoate (12)
A
mixture of 12 (119 mg, 0.199 mmol), isoamylnitrite
(0.134 mL, 1.00 mmol) in 1–4 dioxane (2 mL) was stirred at 80 °C
for 3 h. The reaction mixture was cooled to room temperature
and concentrated under reduced pressure. The residue was puri-
fied by silica gel column chromatography to give 9a (84.7 mg, yield
73%) as a pale yellow amorphous. Mp 186–188 °C; 1H NMR
(300 MHz, CDCl3) d 8.95 (d, J = 1.8 Hz, 1H), 8.18 (dd, J = 2.1,
9.0 Hz, 1H), 7.47 (d, J = 11.6 Hz, 1H), 7.42 (d, J = 7.5 Hz, 1H), 7.27–
7.12 (m, 2H), 6.70 (d, J = 6.9 Hz, 1H), 5.78 (d, J = 17.4 Hz, 1H),
5.63 (d, J = 17.4 Hz, 1H), 5.40–5.37 (m, 1H), 3.98 (s, 3H), 3.84 (br
s, 1H), 3.76 (s, 3H), 3.48–3.31 (m, 1H), 3.24–3.18 (m, 1H), 3.10
(m, 2H), 1.78 (m, 4H), 1.44 (s, 9H); HRMS (ESI) [M+H]+ calcd for
A mixture of 14 (3.72 g, 9.50 mmol), 13 (2.53 g, 10.47 mmol) and
cesium carbonate (6.19 g, 19.00 mmol) in MeCN (10 mL) was stirred
at 40 °C for 4 h, and stirred at 60 °C for 3 h. After cooling to room
temperature, the mixture was filtered through a Celite pad. The fil-
trate was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography to give 12 (4.27 g,
yield 75%) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) d:
7.86 (d, J = 8.8 Hz, 2H), 7.39–7.37 (m, 1H), 7.25–7.21 (m, 2H), 6.91
(d, J = 6.3 Hz, 1H), 6.77 (d, J = 8.8 Hz, 2H), 5.03 (br s, 1H), 4.87 (s,
2H), 4.12 (s, 2H), 3.90 (s, 3H), 3.76–3.73 (m, 1H), 3.29 (s, 3H),
3.27–3.25 (m, 1H), 2.97–2.94 (m, 3H), 1.73–1.56 (m, 4H), 1.41 (s,
9H); 13C NMR (100 MHz, CDCl3) d 166.0, 163.0, 154.8, 148.8,
134.4, 133.2, 130.2, 130.0, 129.4, 129.3, 128.9, 126.8, 126.4, 125.5,
122.6, 103.8, 78.8, 54.8, 51.8, 51.2, 47.0, 45.7, 36.5, 29.3, 28.1,
22.1; HRMS (ESI) [M+H]+ calcd for C30H38ClN6O5 597.2587, found
597.2579; IR (ATR): 1705, 1600, 1512, 1473, 1435, 1404, 1346,
C30H35O5N5Cl 580.2321, found 580.2314; IR (ATR): 1718, 1689,
1652, 1569, 1500, 1446, 1429, 1388, 1353, 1313, 1274, 1234,
1174, 1124, 1110, 1066, 1039 cmꢀ1; Anal. Calcd for C30H34ClN5O5:
C, 62.12; H, 5.91; N, 12.09, found: C, 62.11; H, 5.88; N, 12.07.
5.2. Biological experiments
1311, 1276, 1246, 1165, 1107, 1049, 1014, 991 cmꢀ1
.
5.2.1. PD test in SD rats
SD rats (male, 11 weeks old) were fasted for 24 h prior to pre-
dose blood sampling (ꢀ0.5 h) and kept fasted until the end of blood
sampling performed 12 h after dosing. The rats received the test-
compound (or test-drug) solution orally by gavage. Blood samples
were collected 0.5 h before (ꢀ0.5 h), and 1, 2, 4, 6, 10 and 24 h after
dosing (0 h). Time-course changes in plasma DPP-4 activity after
administration of 10h (3 mg/kg) or sitagliptin (3 mg/kg) were
determined. DPP-4 activity in plasma samples was measured by
5.1.25. Methyl 4-[(chloroacetyl)(methyl)amino]benzoate (13)
To a solution of chloroacetyl chloride (1.23 g, 10.89 mmol) in
THF (5 mL) were added methyl 4-methylaminobenzoate (1.50 g,
9.08 mmol) and pyridine (718 mg, 9.08 mmol) in THF (10 mL) solu-
tion dropwise at 0 °C, and the mixture was stirred at 0 °C for 3 h.
The reaction mixture was quenched with saturated NH4Cl aqueous
solution, extracted with EtOAc, washed with saturated NaHCO3
aqueous solution, brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by silica-gel col-
umn chromatography to give 13 (2.19 g, quant.) as a white solid.
1H NMR (400 MHz, CDCl3) d 8.10 (d, J = 8.5 Hz, 2H), 7.32 (d,
the following procedure. Plasma dilution buffer (5 lL containing
80 mmol/L MgCl2, 140 mmol/L NaCl, 25 mmol/L HEPES, and 1%
(w/v%) BAS, pH 7.8) was placed into each well of a 96-well plate
Please cite this article in press as: Ikuma, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.051

Y. Ikuma et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
11
and a 96 well Half Area Black with Clear Flat Bottom 3881 (Corning
Incorporated), and 5 L of each plasma sample or distilled water
(for measurement of background reaction) was added to each indi-
5.3. Metabolic stability test
5.3.1. Materials
l
vidual well, mixed and left to stand for at least 5 min. To initiate
the reaction, 10 lL of 100 lmol/L Gly-Pro-MCA solution diluted
Human serum was purchased from COSMO BIO Co., Ltd (Tokyo,
Japan). Pooled liver S9 fractions from male Sprague–Dawley (SD)
rats, male beagle dogs and humans were purchased from Xenotech,
LLC (Lenexa, KS). Rat intestinal S9 fraction prepared without prote-
ase inhibitor was purchased from Biopredic international (Rennes,
France). b-NADPH was purchased from Oriental Yeast Co., Ltd
(Tokyo, Japan).
with substrate dilution buffer (140 mmol/L NaCl, 25 mmol/L
HEPES, 1% (w/v%) BAS, pH 7.8) was added to each well containing
a plasma sample or distilled water. Twenty minutes later, 5% acetic
acid was added to stop the reaction. Fluorescence intensity in RFUs
(excitation wavelength 360 nm/emission wavelength 460 nm) was
continuously monitored using a fluorescence microplate reader
(SpectraMaxÒ Gemini EM). Next, 20
(1.28. 2.52, 5, 10, 20, 40 mol/L; diluted with plasma dilution buf-
lL of standard AMC solution
5.3.2. Hydrolysis experiments
Hydrolysis experiments were performed in serum and in tissue
S9 fractions from rats, dogs and humans. Test-compounds were
incubated at 37 °C for 30 min in 100 lL of serum or in an S9 reac-
tion mixture consisting of 50 mM potassium phosphate buffer
(pH7.4), 3 mM NADPH and 1 mg protein/mL S9 fraction. The final
concentration of each test-compound was set to 1 lM. After prein-
cubation at 37 °C for 5 min, the hydrolysis reaction was initiated by
addition of the test-compound dissolved in acetonitrile–water
(1:1, v/v) and terminated by addition of 300 lL of ice-cold acetoni-
trile. The reaction mixture was then centrifuged at 4500 rpm for
10 min to remove precipitated proteins, and the supernatant was
filtrated using a 0.45 lm 96-well filter plate (Varian Inc., Palo Alto,
CA). The filtrate was next injected onto a high-performance liquid
chromatography/tandem mass spectrometry (HPLC/MS/MS)
system.
l
fer, substrate dilution buffer, rat plasma and DMSO) was placed
into each of the wells with no plasma sample or distilled water,
and fluorescence intensity in RFUs (excitation wavelength
360 nm/emission wavelength 460 nm) of the standard AMC solu-
tion was measured to construct a calibration curve of RFU against
AMC per well. The curve was constructed on an arithmetic scale
using unweighed linear regression in SoftMaxÒPro version 4.3.1
(Molecular Devices Corporation), within the fluorescence micro-
plate reader. The rates of AMC production in the samples and back-
ground wells were calculated using the constructed calibration
curve. The net rate of AMC production (nmol/min) in a given sam-
ple well was determined by subtraction of mean background rate
from the rate for the sample well. DPP-4 activity (mU/mL) per
plasma unit was calculated by dividing the rate of AMC production
by the volume of plasma sample used for the measurement. Dupli-
cate measurements were performed for the standard AMC solution
and for each plasma sample.
5.3.3. Analytical procedure
The concentrations of test-compounds and their sample hydrol-
ysates were measured using an HPLC-MS/MS system consisting of
a TSQ 7000 mass spectrometer (Thermo Fisher Scientific Inc., Wal-
tham, MA) with a Shimadzu 10A series HPLC system (Shimadzu
Corporation, Kyoto, Japan). Chromatography was performed using
a Symmetry C18 column (5 mm particle size, 2.1 ꢂ 150 mm,
Waters Corporation, Milford MA) warmed to 40 °C. The mobile
phase consisted of 10 mM ammonium acetate (pH 4.0, solvent A)
and acetonitrile (solvent B). Elution gradient conditions were as
follows: [min, B%] = [0, 20] ꢀ [6.5, 80] ꢀ 8.5, 80] ꢀ [8.6, 20] ꢀ [12,
20], and flow rate was 0.2 mL/min. Mass spectrometric detection
was performed using positive ionization electrospray. The selective
reaction monitoring mode (m/z: precursor ion?product ion) was
used to monitor ions.
5.2.2. Oral glucose tolerance test (OGTT) in Zucker fatty rats
Zucker fatty rats (male, 11 weeks) were fasted overnight before
each OGTT. Glucose (0.2 g/mL
D-glucose solution) was orally
loaded by gavage in a volume of 10 mL/kg (2 g of
D
-glucose/kg).
Each concentration of 10h (0.3, 1.0 mg/kg) suspension or 0.5%
Methyl cellulose (MC) solution used as a control was administrated
once 1 h prior to glucose loading. Blood samples for determination
of blood glucose concentration were taken just before administra-
tion of the test-substance or 0.5% MC solution (ꢀ1 h relative to the
time of glucose loading), just before glucose loading (taken as
0 min), and 10, 30, 60, 120 min after glucose loading. Blood glucose
concentration was measured for all the blood samples collected. A
10
diately mixed with 100
Potassium carbonate solution (50
and mixed. The deproteinized samples were stored in a refrigerator
set at 4 °C until measurement of blood glucose concentration.
Blood glucose concentration was measured using a commercially
available kit, Glucose CII test Wako (Wako pure Chemical Indus-
tries, Ltd). The deproteinized samples were centrifuged at
l
L blood sample taken from the tail vein of each rat was imme-
L of 0.62 mol/L perchloric acid solution.
L, 0.37 mol/L) was then added
Acknowledgments
l
l
Authors thank C. Matsuba for recording MS spectra and N. Fuk-
uda for his suggestion during the manuscript preparation.
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