Design and synthesis of 8-hydroxyquinoline-based radioprotective agents

Article abstract of DOI:10.1016/j.bmc.2014.06.017

In radiation therapy, adverse side effects are often induced due to the excessive cell death that occurs in radiosensitive normal cells. The radiation-induced cell death of normal cells is caused, at least in part, by apoptosis, which undergoes via activation of p53 and increase in the p53 protein, a zinc-containing transcriptional factor, in response to cellular damage. Therefore, radioprotective drugs that can protect normal cells from radiation and thus suppress adverse side effects would be highly desirable. We report herein on the radioprotective activity of 8-hydroxyquinoline (8HQ) derivatives that were initially designed so as to interact with the Zn ²⁺ in p53. Indeed, the 5,7-bis(methylaminosulfonyl)-8HQ and 8-methoxyquinoline derivatives considerably protected MOLT-4 cells against γ-ray radiation (10 Gy), accompanied by a low cytotoxicity. However, mechanistic studies revealed that the interaction of these drugs with p53 is weak and the mechanism for inhibiting apoptosis appears to be different from that of previously reported radioprotectors such as bispicen, which inhibits apoptosis via the denaturation of p53 as well as by blocking both transcription-dependent and -independent apoptotic pathways.

Full text of DOI:10.1016/j.bmc.2014.06.017

Bioorganic & Medicinal Chemistry 22 (2014) 3891–3905
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of 8-hydroxyquinoline-based radioprotective
agents
a
b
c
b
b
d
Shinya Ariyasu , Akiko Sawa , Akinori Morita , Kengo Hanaya , Misato Hoshi , Ippei Takahashi ,
e
a,b,
⇑
Bing Wang , Shin Aoki
a
Center for Technologies Against Cancer, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan
Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan
Department of Radiological Science, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima-shi, Hiroshima, Japan
Radiation Risk Reduction Research Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
In radiation therapy, adverse side effects are often induced due to the excessive cell death that occurs in
radiosensitive normal cells. The radiation-induced cell death of normal cells is caused, at least in part, by
apoptosis, which undergoes via activation of p53 and increase in the p53 protein, a zinc-containing tran-
scriptional factor, in response to cellular damage. Therefore, radioprotective drugs that can protect nor-
mal cells from radiation and thus suppress adverse side effects would be highly desirable. We report
herein on the radioprotective activity of 8-hydroxyquinoline (8HQ) derivatives that were initially
Received 16 May 2014
Revised 5 June 2014
Accepted 6 June 2014
Available online 18 June 2014
Keywords:
2+
designed so as to interact with the Zn in p53. Indeed, the 5,7-bis(methylaminosulfonyl)-8HQ and 8-
8
-Hydroxyquinoline
methoxyquinoline derivatives considerably protected MOLT-4 cells against
c-ray radiation (10 Gy),
Radioprotector
accompanied by a low cytotoxicity. However, mechanistic studies revealed that the interaction of these
drugs with p53 is weak and the mechanism for inhibiting apoptosis appears to be different from that of
previously reported radioprotectors such as bispicen, which inhibits apoptosis via the denaturation of
p53 as well as by blocking both transcription-dependent and -independent apoptotic pathways.
Ó 2014 Elsevier Ltd. All rights reserved.
Mechanistic study
Radiation
1
. Introduction
1
Radiation therapy is a major form of cancer therapy, in which
Generation of reactive
oxygen speices (ROS)
tumor cells are forced to undergo cell death by strong and spatially
controlled radiation. Generally, this therapy is thought to be less
invasive than surgery. However, adverse side effects such as death
of bone marrow or intestinal cells are sometimes induced, due to
the apoptosis of radiosensitive normal cells that are in close prox-
imity to the tumor tissue. Therefore, the development of radiopro-
tective drugs that can protect normal cells against radiation and
thus suppress adverse side effects are urgently needed.
p53-independent
cell death
DNA-damages
Activation of p53
2
Transcription-
dependent
pathway
Transcription-
independent
pathway
The radiation-induced cell death of normal cells is caused, at
Specific DNA-binding
of p53
Interaction of p53
least in part, by the induction of apoptosis by the p53 protein
with Bcl-2 family
on mitochondria
3
(
Fig. 1), which is a zinc-containing transcription factor. The p53
protein is known as a tumor-suppressor factor and is frequently
4
Transcription of
apoptosis-related proteins
(PUMA etc)
mutated or inactivated in various cancer cells. In the case of crit-
ical DNA-damage in radiosensitive normal cells by radiation, the
activation of p53 proteins induce apoptosis as a result of an acute
p53-dependent apoptosis
Figure 1. Possible radiation-induced apoptosis pathways.
⇑
Corresponding author. Tel./fax: +81 4 7121 3670.
E-mail address: shinaoki@rs.noda.tus.ac.jp (S. Aoki).
http://dx.doi.org/10.1016/j.bmc.2014.06.017
968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
0

3
892
(
S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
a) Radical scavengers
HO
R2
R3
N
R1
O
OR4
Vitamin E
1
R : H, Me
2
R : H, SO NH , SO NHMe, SO NMe , SO NEt
SH
2
2
2
2
2
2
2
O
H
+
COO-
HO
H
H
N
H N
N
S
3
N
N
H
P
N
H
NH2
N
NH
S
S
NH2
HO
S
COO-
O
O
O
2
O
2
O
2
Glutathione
Amifostine
H
N
NH
OEt
H
N
S
N
(
b) p53 inhibitors
O
S
S
2
O
O
O
2
2
S
N
O
NH
O
3
N
R : H, Br, I, SO NH , SO NHMe, SO NMe , SO NEt
2
2
2
2
2
2
2
O
S
O
O
H
N
H
N
NH2
NaO
V
ONa
ONa
Vanadate
N
NH
S
S
NH2
S
O
O
2
2
Pifithrin μ
O
2
H
N
NH
Pifithrin α
OEt
H
N
S
N
O
S
S
2
O
O
O
2
2
N
N
O
N
4
R : H, Me, Et, SO Me, SO Ph
2
2
H
H
N
N
N
N
N
Figure 3. General structure of 8HQ derivatives in this work.
N
N
radiation reaction.5 The mechanisms responsible for p53-induced
apoptosis are classified into a transcription-dependent pathway
and a transcription-independent pathway, as indicated in Figure 1.6
In the transcription-dependent pathway, p53 binds to DNA and
induces apoptosis. In the transcription-independent pathway, on
N
Bispicen
TPEN
Figure 2. Chemical structures of reported radioprotectors and related compounds,
a) radical scavengers and (b) p53 inhibitors.
(
R2
R2
SO Cl
2
MeNH
2
or
3
R I, K
2
CO
3
HSO
3
Cl
Me
NH
2
THF
R1
N
R1
THF
R1
N
R1
N
N
3
OH
OH
OH
OR
1
1
1
1
2
1
2
3
1
: R = H
3: R = H (from 1)
5: R = H, R = SO
: R = Me, R = SO
: R = H, R = SO
: R = Me, R = SO
2
NHMe (from 3)
9: R = Me, R = SO NMe , R = Me (from 8)
2
2
1
2
: R = Me
4: R = Me (from 2)
1
2
1
2
3
6
7
8
2
NHMe (from 4) 10: R = Me, R = SO
NMe
, R = Et (from 8)
2
2
1
2
2
NMe (from 3)
2
NMe (from 4)
2
MeI, K
2
3
CO /THF
1
2
2
O
X
N
N
OMe
8 (from 2)
) HSO
) MeNH
O
SO NMe
2 2
SO NMe
2
2
1
NBS: X = Br
NIS: X = I
3
R I, K
2
CO
3
1
2
3
Cl
/THF
MeCN
R1
THF
R1
2
X
N
X
N
OR3
OH
SO NHMe
1
1
3
2
11: R = H, X =Br (from 7)
14: R = Me, R = Me, X = Br (from 12)
1
1
3
1
1
2: R = Me, X =Br (from 8)
15: R = Me, R = Et, X = Br (from 12)
MeI, K
2
CO
3
3: R = Me, X =I (from 8)
1
16: R = Me, R = Me, X = I (from 13)
1
3
MeSO
2
Cl or PhSO
N, DMAP
2
Cl
1
3
N
Et
3
17: R = Me, R = Et, X = I (from 13)
THF
OMe
1
9
SO
2
NMe
N
2
Br
3
OR
3
2
2
0: R = SO
1: R = SO
2
Me (from 12)
Ph (from 12)
3
2
Scheme 1. Synthesis of 5-aminosulfonyl-8HQ derivatives.

S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
3893
the other hand, p53 binds to the Bcl-2 family proteins on
mitochondria, resulting in the induction of apoptosis.
ligand, unlike bispicen, which is a tetradentate ligand.15 Therefore,
it was hypothesized that 8HQ derivatives are capable of coordinat-
ing to metal ions in metalloproteins without eliminating metal
As of this writing, inhibitors of radiation-induced apoptosis
1
6
(
radioprotectors) are roughly classified into two categories. The
ions from metalloproteins. In our previous study, for instance,
we reported that 8HQ derivatives are potent and selective inhibi-
tors of an aminopeptidase from Aeromonas proteolytica (AAP) (EC
7
first class includes radical scavengers such as vitamin E, glutathi-
one, and amifostine (Fig. 2), which scavenge reactive oxygen spe-
cies (ROS) generated by radiation. Another class includes pifithrin
8
9
2
+
17
3.4.11.10), a dinuclear Zn -containing peptidase.
An X-ray
1
0
11
12
a
,
pifithrin
l
and vanadate (Fig. 2), which are reported to
co-crystal structure of AAP with 8HQ itself revealed that 8HQ
2
+
exhibit radioprotective effects by the temporary inhibition of p53
derivatives coordinate to two Zn ions in the active site of AAP.
In addition, it has recently been proposed that 8HQ-based com-
pounds could function as potent protease-inhibitors and are poten-
tial candidate drugs for use in the treatment of AIDS, cancer,
neurodegenerative diseases (Alzheimer’s and Parkinson’s disease),
with negligible cancer formation in normal cells.5
The p53 protein is a zinc(II)-containing protein and its function
2
+
can be inactivated by the elimination of zinc(II) ions (Zn ) from
2
+
4,13
p53 using Zn chelators.
In a previous study, we evaluated
2+
18
the radioprotective activity of various Zn chelators such as TPEN
tetrakis(2-pyridylmethyl)ethylenediamine) and bispicen (N,N -bis
parasitic or bacterial infections.
0
(
Based on this background, we initiated the development of
8HQ-based radioprotective drugs in our laboratory. We designed
and prepared 8HQ derivatives that mainly contained sulfonamide
groups (Fig. 3), which are frequently found in a wide variety of clin-
(
2-pyridylmethyl)-1,2-ethanediamine) and found that bispicen
has higher radioprotective activity than the other tested
1
4
candidates. Mechanistic analyses revealed that bispicen induces
a conformational change in p53, resulting in inhibition of its com-
plexation with DNA and the inhibition of both the p53-mediated
transcription-dependent and independent pathways. Although this
radioprotective mechanism is similar to that for vanadate, bispicen
exhibits a high cytotoxicity, possibly due to nonspecific interac-
tions with metals of various metalloproteins and/or free metals
in normal cells by virtue of its strong metal-chelation properties.
It is well known that 8-hydroxyquinoline (8HQ) derivatives also
function as good ligands against various metal ions as a bidentate
1
9
ical drugs, and carried out evaluations of their radioprotective
activities. The finding indicated that 8HQ containing two meth-
ylaminosulfonyl groups at 5- and 7-positions of the quinoline ring
had the optimal combination of radioprotective activity and a low
cytotoxicity. In addition, 8-methoxyquinoline derivatives also act
as low cytotoxic radioprotectors. In this study, we report on the
synthesis and radioprotective activity of 8HQ-based radioprotector
candidates and a discussion of some aspects of the mechanism
underlying their radioprotective action.
R2
SO
2
NHMe
N
R2
N
OSO
2
Me
MeHNO
2
S
Me
2
4
4
2: R = SO
2
2
NHMe (from 27)
NMe (from 28)
OMe
1 (from 27)
2
3: R = SO
2
4
TMSCHN
2
MeSO
Et
2 2 2
Cl/CH Cl
N, DMAP
THF, MeOH
MeI, K CO3
3
2
THF
R2
R2
SO
2
Cl
3
amine
R I, K CO
2
3
THF
ClO
2
S
N
R1
R2
N
R1
R2
N
R1
OH
OH
OR3
1
1
2
36: R = H, R2 = SO
1
, R3 = Me (from 25)
2
2
2: R1 = H
24: R = H, R = SO NHMe (from 22)
2
NMe
2
2
3: R = Me
1
2
37: R1 = Me, R2 = SO NMe , R = Me (from 28)
3
2
5: R = H, R = SO
2
NMe
2
(from 22)
2
2
1
2
38: R1 = Me, R2 = SO NMe , R = Et (from 28)
3
2
6: R = Me, R = SO NH (from 23)
2
2
2
2
2
2
2
2
2
1
2
2
7: R = Me, R = SO
NHMe (from 23)
9: R = Me, R2 = SO
1
3
3
4
NEt
NEt
2
, R = Me (from 29)
, R = Et (from 29)
1
2
2
8: R = Me, R = SO
NMe
NEt
2
(from 23)
(from 23)
0: R = Me, R2 = SO
1
3
2
1
2
2
9: R = Me, R = SO
2
NH
NH
H
N
33: R1 = Me, R2
=
0: R1 = Me, R =
2
N
N
3
S
S
O
2
2
O
2
H
N
OEt
1
2
2
34: R = Me, R =
3
1: R1 = Me, R =
S
(from 23)
S
O
O
O
2
2
H
N
H
N
3
2: R1 = Me, R2
=
S
NH2
35: R = Me, R =
1
2
O
2
S
O
N
O
Scheme 2. Synthesis of 5,7-bis(aminosulfonyl)-8HQ derivatives.

3
894
S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
2
2
. Results and discussion
derivatives 22 and 23 (Scheme 2) were prepared according to pro-
2
0
cedures in the literature, and were then reacted with the corre-
sponding amines (NH methylamine, dimethylamine,
.1. Synthesis of HQ derivatives as candidate radioprotective
3
,
drugs
diethylamine, n-propylamine, b-alanine ethyl ester, piperazine,
homopiperazine and 3-amino-5-methylisoxazole) to afford 5–8
and 24–35. The 7-halo-8HQ derivatives 11–13 were prepared by
the reaction of 7 or 8 with N-bromosuccinimide (NBS) or N-iodo-
succinimide (NIS). Methylation and ethylation of the 8-hydroxy
The synthesis of the 8HQ derivatives is shown in Schemes 1 and
. The 5-chlorosulfonyl-8HQ derivatives 3 and 4 (Scheme 1),
2
8
-methoxy derivative 18, and the 5,7-bis(chlorosulfonyl)-8HQ
Cl
2
SO NMe2
(
a)
(b)
(c)
N
N
Me NO S
N
2
2
OH
OH
4
OH
25
1
4
1
00
100
100
80
8
0
80
6
4
2
0
0
0
0
60
40
20
60
40
20
0
0
0
100
200
300
400
0
100
200
300
400
0
100
200
300
400
Dose (μM)
Dose (μM)
Dose (μM)
Cl
SO NMe2
2
(d)
(e)
(f)
Me
2
NO
2
S
2 2
SO NMe
OH
OH
OH
47
4
5
4
6
1
00
100
100
80
8
0
80
6
4
2
0
0
0
0
60
40
20
0
60
40
20
0
0
100
200
300
400
0
100
200
300
400
0
100
200
300
400
Dose (μM)
Dose (μM)
Dose (μM)
N
N
H
H
N
N
N
N
(
g)
(h)
(i)
N
(j)
N
N
N
N
N
4
8
49
Bispicen
TPEN
1
00
100
1
00
100
80
8
0
80
8
0
6
4
2
0
0
0
0
60
40
20
0
6
4
2
0
0
0
0
60
40
20
0
0
100
200
300
400
0
100
200
300
400
0
100
200
300 400
0
100
200
Dose (μM)
300
400
Dose (μM)
Dose (μM)
Dose (μM)
(
HO
m)
(
HO
k)
HO
H
OH
O
(l)
HO
O
O
O
O
NaO
OH
O
OH
Vitamin E
Vitamin C
Curcumin
1
00
100
80
100
80
8
0
6
4
2
0
0
0
0
60
40
20
0
60
40
20
0
0
100
200
300 400
0
100
200
300 400
0
100
200
300 400
Dose (μM)
Dose (μM)
Dose (μM)
Figure 4. Radioprotective activity and cytotoxicity of 8HQ derivatives (1 (a), 44 (b) and 25 (c)), naphthol derivatives (45 (d), 46 (e) and 47 (f)), quinoline 48 (g), isoquinoline
9 (h), Zn-chelators (bispicen (i) and TPEN (j)), well-known antioxidants (vitamin E (k), vitamin C (l) and curcumin (m)) based on a dye-exclusion test. The percentage of cell
death of MOLT-4 cells with or without -ray irradiation (10 Gy) is shown as solid and open circles, respectively.
4
c

S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
3895
group of 8, 12, 13, 25, 28 and 29 were carried out by reacting them
incubated in the presence of 8HQ derivatives (0–320
in a humidified atmosphere containing 5% CO
to -ray irradiation (10 Gy). The irradiated MOLT-4 cells from this
procedure were incubated in a medium and 5% CO at 37 °C for
18 h, and the numbers of dead cells were then counted using an
l
M) for 1 h
with MeI or EtI to afford 9, 10, 14–17 and 36–40. For the synthesis
of 8-methoxy derivative 41, 27 was reacted with MeI under con-
ventional reaction conditions (Scheme 2). However, the main prod-
2
and then subjected
c
2
uct of this reaction was 37, due to the methylation of the SO
groups. Alternatively, 41 was obtained by the reaction of 27 with
trimethylsilyl diazomethane (TMSCHN ). The reaction of 12, 27
2
NHMe
2
1
erythrosine B dye-exclusion test (Figs. 4–8). The cytotoxicity of
the 8HQ derivatives themselves was also evaluated by the
2
and 28 with methanesulfonyl chloride and benzenesulfonyl chlo-
ride afforded their corresponding sulfonates, 20, 21, 42 and 43.
dye-exclusion test without c-ray radiation.
The results shown in Figs. 4–8 are summarized below.
2
.2. Evaluation of the radioprotective activity and cytotoxicity
(1) In Figure 4, the radioprotective activity of the 8-hydroxy-
quinoline (8HQ) derivatives, naphthol derivatives, quinoline
and isoquinoline were tested for comparison. The radiopro-
tective activity of 8HQ itself 1 and 5-chloro-8HQ 44 (Fig. 4a
and b) showed more potent radioprotective activity than
that of bispicen (Fig. 4i), TPEN (Fig. 4j), vitamin E (Fig. 4k),
vitamin C (Fig. 4l) and curcumin (Fig. 4m) at low concentra-
of 8HQ derivatives
The radioprotective activities of the 8HQ derivatives were
evaluated by a radiation assay using MOLT-4 cells as a model of
normal cells. The reason for this is because the p53 protein in
MOLT-4 cells, which are leukemia cells, is normally activated and
1
4
induces apoptosis upon
c-ray radiation. MOLT-4 cells were first
tions (<50
lM), while 1 and 44 are cytotoxic. The introduc-
R2
X
N
R1
OR3
1
(
d) 6: R = Me
(c) 5: R1 = R3 = H
2
(
b) 2: R1 = Me
R = SO NHMe
2
1
2
3
2
3
2
(
a) 1: R = R = R = H
R
= R = H
R
= SO NHMe
3
2
R
= H
X = H
X = H
X = H
X = H
1
00
100
80
100
80
100
80
8
0
6
4
2
0
0
0
60
40
20
0
60
40
20
60
40
20
0
0
0
0
100
200
300
400
400
400
0
100
200
300
400
400
400
0
100
200
300
400
0
100
200
300
400
Dose (μM)
Dose (μM)
Dose (μM)
Dose (μM)
1
1
(
f) 8: R = Me
(h) 10: R = Me
2
1
3
2
(
e) 7: R1 = R3 = H
R = SO
2
NMe
2
(g) 9: R = R = Me
R
R
= SO NMe
= Et
2 2
2
3
2
3
R
= SO NMe
R
= H
R = SO
2
NMe
2
2
2
X = H
X = H
X = H
X = H
1
00
100
80
100
80
100
80
8
0
6
4
2
0
0
0
0
60
40
20
0
60
40
20
0
60
40
20
0
0
100
200
Dose (μM)
300
0
100
200
Dose (μM)
300
0
100
200
300
400
0
100
200
Dose (μM)
300
400
Dose (μM)
(j) 19: R1 = R3 =Me
(
i) 18: R1 = R3 = Me
2
2
R
= H
R
= SO NHMe
2
X = H
X = H
1
00
100
8
0
8
6
4
2
0
0
0
0
0
60
40
20
0
0
100
200
300
0
100
200
300
Dose (μM)
Dose (μM)
Figure 5. Radioprotective activity and cytotoxicity of 5-aminosulfonyl-8HQ derivatives 1 (a), 2 (b), 5 (c), 6 (d), 7 (e), 8 (f), 9 (g), 10 (h), 18 (i) and 19 (j) based on a dye-
exclusion test. The percentage of cell death of MOLT-4 cells with or without -ray irradiation (10 Gy) is shown as solid and open circles, respectively.
c

3
896
S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
R2
X
N
R1
OR3
(c) 13: R1 = Me
(
b) 12:R1 = Me
1
3
2
3
2
(d) 14: R1 = R3 = Me
2
(
a) 11: R = R = H
R
R
= SO NMe
= H
R
R
= SO NMe
= H
2
2
2
2
2
3
R = SO NMe
R = SO NMe
2 2
2
2
X = Br
X = Br
X = I
X = Br
1
00
100
100
80
100
80
8
0
8
6
4
2
0
0
0
0
60
40
20
0
60
40
20
60
40
20
0
0
0
0
100
200
300
300
300
400
400
400
0
100
200
300
400
0
100
200
300
400
0
100
200
Dose (μM)
300
400
Dose (μM)
Dose (μM)
Dose (μM)
1
(
e) 15: R1 = Me
(g) 17: R1 = Me
(h) 20: R = Me
2
3
(f) 16: R1 = R3 = Me
2
2
R
R
= SO NMe2
= Et
R
= SO
= Et
2
NMe
2
R
R
= SO NMe
= SO Me
2
2
2 2
2
3
3
R
= SO NMe
2
2
R
X = Br
X = I
X = I
X = Br
1
00
100
80
100
80
100
8
0
8
0
6
4
2
0
0
0
0
60
40
20
0
60
40
20
0
60
40
20
0
0
100
200
Dose (μM)
0
100
200
Dose (μM)
300
400
0
100
200
Dose (μM)
300
400
0
100
200
Dose (μM)
300
400
(
i) 21: R1 = Me
2
3
R
R
= SO NMe2
= SO Ph
2
2
X = Br
1
00
8
0
6
4
2
0
0
0
0
0
100
200
Dose (μM)
Figure 6. Radioprotective activity and cytotoxicity of 5-aminosulfonyl-7-halo-8HQ derivatives 11 (a), 12 (b), 13 (c), 14 (d), 15 (e), 16 (f), 17 (g), 20 (h) and 21 (i) based on a
dye-exclusion test. The percentage of cell death of MOLT-4 cells with or without
c
-ray irradiation (10 Gy) is shown as solid and open circles, respectively.
tion of dimethylaminosulfonyl groups at 5- and 7-positions
somehow reduced its cytotoxicity (Fig. 4c). In contrast, the
naphthol compounds 45–47, quinoline 48 and isoquinoline
(3) Among 8HQ derivatives having no protecting groups at 8-OH
group shown in Figs. 5–8, the 5,7-bis(methylaminosulfonyl)-
8HQ derivatives 24 (Fig. 7a) and 27 (Fig. 7d) show the highest
radioprotective activity in a dose-dependent manner with
low cytotoxicity. 8HQs containing hydrophilic groups such
as 26 (Fig. 7c) and 32 (Fig. 8c) have weak radioprotective
activity, possibly because they are not efficiently incorpo-
rated into the cells. Interestingly, the water-soluble 33
(Fig. 8d) and 34 (Fig. 8e) had a better radioprotective activity.
(4) The cytotoxicity of 28 (Fig. 7e), 29 (Fig. 7f) and 30 (Fig. 8a) is
higher than that of 27 (Fig. 7d), indicating that more hydro-
phobic 8HQ derivatives tend to be more cytotoxic.
4
9 show lower solubility in the cell culture medium than
the corresponding 8HQ derivatives and their radioprotective
activity was weak (Fig. 4d–h). Considering the water solubil-
ity and easy availability of the 8HQ derivatives22 together
with the versatility of quinoline sulfonamides found in a
lot of drug candidates,19 we selected 8HQ as a scaffold of
radioprotective drugs.
(
2) The methyl group at the 2 position of 8HQ had negligible
effects on its radioprotective activity and cytotoxicity. For
example, the activity of 1 (Fig. 5a) is almost equal to that
of 2 (Fig. 5b). A similar relationship was observed in the case
of 7 (Fig. 5e) and 8 (Fig. 5f), 11 (Fig. 6a) and 12 (Fig. 6b), 24
(5) The aforementioned points (1–4) indicate that an ade-
quate balance between hydrophilicity and hydrophobicity
is important for achieving effective radioprotecting
activity.
(Fig. 7a) and 27 (Fig. 7d).

S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
3897
R2
R2
N
R1
OR3
(
c) 26: R1 = Me
(d) 27: R1 = Me
a) 24: R1 = R3 = H
(b) 25: R1 = R3 = H
R
R
2
3
= SO
= H
NH
2
R
R
2
= SO
= H
(
2
2
NHMe
2
2
3
R
= SO NHMe
R
= SO NMe
2
2
2
1
00
100
80
100
100
80
60
40
20
0
8
0
8
6
4
2
0
0
0
0
0
60
40
20
0
60
40
20
0
0
100
200
300
400
0
100
200
Dose (μM)
300
400
400
400
400
0
100
200
300
300
300
400
400
400
0
100
200
300
300
300
400
400
400
Dose (μM)
Dose (μM)
Dose (μM)
(f) 29: R1 = Me
(g) 36: R1 = H
(
e) 28: R1 = Me
2
3
2
2
(h) 37: R = R3 = Me
1
R
R
= SO
= H
2
NMe
2
R
= SO NEt
= H
R
= SO NMe
= Me
2
2
2
2
3
3
2
R
R
R
= SO
2
NMe
2
1
00
100
80
100
100
80
60
40
20
0
8
0
8
0
6
4
2
0
0
0
0
60
40
20
0
60
40
20
0
0
100
200
Dose (μM)
300
400
0
100
200
300
0
100
200
0
100
200
Dose (μM)
Dose (μM)
Dose (μM)
1
(
i) 38: R1 = Me
(k) 40: R = Me
2
(j) 39: R1 = R3 = Me
2
(l) 41: R1 = R3 = Me
R2 = SO
NHMe
2
R
= SO
= Et
2
NMe
2
R
= SO
= Et
2
NEt
2
3
R2 = SO
3
R
2
NEt
2
R
1
00
100
80
100
80
100
80
60
40
20
0
8
0
6
4
2
0
0
0
0
60
40
20
0
60
40
20
0
0
100
200
Dose (μM)
300
400
0
100
200
Dose (μM)
300
0
100
200
Dose (μM)
0
100
200
Dose (μM)
(n) 43: R1 = Me
(
m) 42: R1 = Me
2
2
R
= SO NHMe
= SO Me
R
= SO
2
NMe
Me
2
2
3
3
R
R = SO
2
2
1
00
100
8
0
8
6
4
2
0
0
0
0
0
60
40
20
0
0
100
200
300
400
0
100
200
Dose (μM)
300
Dose (μM)
Figure 7. Radioprotective activity and cytotoxicity of 5,7-bis(alkylaminosulfonyl)-8HQ derivatives 24 (a), 25 (b), 26 (c), 27 (d), 28 (e), 29 (f), 36 (g), 37 (h), 38 (i), 39 (j), 40 (k),
1 (l), 42 (m) and 43 (n) based on a dye-exclusion test. The percentage of cell death of MOLT-4 cells with or without -ray irradiation (10 Gy) is shown as solid and open
circles, respectively.
4
c
(
6) More interestingly, 8-alkoxyquinoline derivatives such as 9
Fig. 5g), 10 (Fig. 5h), 14 (Fig. 6d), 16 (Fig. 6f), 19 (Fig. 5j),
7 (Fig. 7h), 38 (Fig. 7i), 39 (Fig. 7j) and 41 (Fig. 7l) effectively
by a methyl group contributes to a reduction of cytotoxicity,
possibly due to the avoidance of interaction with various
intracellular metalloproteins. For instance, the cytotoxicity
of 19 (Fig. 5j) is lower than that of 6 (Fig. 5d). Similar results
were obtained in the case of 9 (Fig. 5g) and 8 (Fig. 5f), 14
(
3
suppressed the radiation-induced apoptosis of MOLT-4 cells.
Besides, the protection of the hydroxyl group at 8 position

3
898
S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
R4
R4
N
OH
(
a)
(b)
(c)
H
N
H
N
H
N
3
0: R4
=
3
1: R4
=
OEt
32: R4
=
S
S
S
NH2
O
O
O
2
2
2
O
1
00
100
100
80
60
40
20
0
8
0
8
0
6
4
2
0
0
0
0
60
40
20
0
0
100
200
Dose (μM)
300
400
0
100
200
Dose (μM)
300
400
0
100
200
300
400
Dose (μM)
(
d)
(e)
H
N
NH
(f)
35: R4
=
3: R4
=
34: R4
=
NH
N
3
N
S
S
S
O
2
O
O
2
N O
2
1
00
100
100
80
60
40
20
0
8
0
0
0
0
0
80
60
40
20
0
6
4
2
0
100
200
Dose (μM)
300
400
0
100
200
300
400
0
100
200
300
400
Dose (μM)
Dose (μM)
Figure 8. Radioprotective activity and cytotoxicity of 5,7-bis(alkylaminosulfonyl)-8HQ derivatives 30 (a), 31 (b), 32 (c), 33 (d), 34 (e) and 35 (f) based on a dye-exclusion test.
The percentage of cell death of MOLT-4 cells with or without -ray irradiation (10 Gy) is shown as solid and open circles, respectively.
c
(
(
Fig. 6d) and 12 (Fig. 6b), 16 (Fig. 6f) and 13 (Fig. 6c), 37
Fig. 7h) and 28 (Fig. 7e), 39 (Fig. 7j) and 29 (Fig. 7f). These
(
a) 100
80
Treatment with 27 at 3hr
after irradiation
results suggest that it is unlikely that these drugs directly
bind to Zn in p53 or other Zn enzymes.
2
+
2+
6
0
(
(
7) 8-Ethoxyquinolines exhibit higher cytotoxicities than
Treatment with 27 at 1hr
before irradiation
8
(
-methoxy derivatives. For instance, the cytotoxicity of 15
Fig. 6e) is higher than that of 14 (Fig. 6d). Similar results
were obtained in the case of 17 (Fig. 6g) and 16 (Fig. 6f),
0 (Fig. 7k) and 39 (Fig. 7j).
40
2
0
0
without irradiation
4
8) Radioprotective activity can also be improved by introducing
a methanesulfonyl group on the hydroxyl group at the 8-posi-
tion. The radioprotective activities of the 8-methanesulfonyl
derivatives 20 (Fig. 6h), 42 (Fig. 7m) and 43 (Fig. 7n) are higher
than that of the corresponding 8-hydroxy derivatives 12
0
100
200
300
400
Dose (μM)
(
b) 100
80
Treatment with 37 at 3hr
after irradiation
(Fig. 6b), 27 (Fig. 7d) and 28 (Fig. 7e). Because it was observed
6
4
2
0
0
0
0
that the sulfonate S–O bond of 43 is slowly hydrolyzed after
Treatment with 37 at 1hr
before irradiation
17b
incubation at 37 °C for 18 h (<20%),
its toxicity at 200–
3
00 M (Fig. 7e) might be due to the toxicity of its hydrolysis
l
without irradiation
product 28.
(
9) From these results, two compounds, 27 and 37, were used
for further study including the mechanistic study of their
radioprotective activity, as described below.
0
100
200
300
400
Dose (μM)
Figure 9. Radioprotective effect of 27 (a) and 37 (b) after being irradiated by
c
-ray
(
10 Gy) and then treated with these drugs. The percentage of cell death of MOLT-4
2
2
.3. Apotosis inhibition by the treatment of MOLT-4 cells with
7 and 37 after irradiation
cells dosed 8HQ derivatives at 1 h before
irradiation or without irradiation are shown as solid circles, solid triangles and open
circles, respectively.
c-ray irradiation (10 Gy), at 3 h after
MOLT-4 cells were treated with 27 and 37 after
10 Gy). As shown in Figure 9, cell death of MOLT-4 cells was con-
c-ray radiation
drugs prior to the c-ray irradiation (solid circles in Fig. 9). These
(
results suggest that 8HQ derivatives might be useful for the
apoptosis-inhibition of cells (or animals) after being exposed to
irradiation.
siderably suppressed by the addition of these compounds 3 h after
irradiation (solid triangles in Fig. 9), although these effect was
somewhat smaller than that when cells were treated with these

S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
3899
(
a)
1
00
p53-dependent apoptosis pathway. This selectivity might be
important for the clinical uses of 8HQ-based radioprotectors.
Next, the radical scavenging activity of 8HQ derivatives was
examined by the spectral changes in DPPH (2,2-diphenyl-l-pic-
8
0
6
0
2
4
rylhydrazyl) free radical (Fig. 11). Positive control compounds
such as vitamin C, vitamin E and curcumin, which are well-known
as antioxidants, rapidly reacted with DPPH free radicals and the
absorption of DPPH was decreased. In contrast, bispicen slightly
scavenged DPPH radicals, and 27 and 37 show negligible radical
scavenging activity.
4
0
20
0
Jurkat
γ-ray - + +
MOLT-4
+ + -
-
-
27
-
-
+
+
- - + +
We previously reported that bispicen, which inhibits the p53-
transcription dependent pathway, induces conformational changes
in the p53 protein, resulting in the inhibition of DNA-binding of
(
b) 100
80
1
4
p53. The inhibition activity of the HQ derivatives against this
pathway was evaluated by circular dichroism (CD) spectroscopy
60
(
Fig. 12) and electrophoretic mobility shift assay (EMSA) (Fig. 13).
The CD spectra of a recombinant p53 protein in the presence of
4
0
20
bispicen or 8HQ derivatives were measured in order to investigate
conformational changes in the p53 proteins. As shown in Figure 12,
while a considerable CD spectral change was measured upon the
0
Jurkat
γ-ray - + +
MOLT-4
+ + -
-
-
1
4
37
-
-
+
+
-
-
+
+
addition of bispicen, the 8HQ derivatives 27 or 37 negligibly
Figure 10. p53-dependency of radioprotective activity of (a) 27 and (b) 37 based on
a dye-exclusion test using Jurkat and MOLT-4 cells. White and black bars show the
+
10
percentage of cell death in the absence of 8HQ derivatives without and with
irradiation, respectively. Gray and pale gray bars show the percentage of cell death
in the presence of 8HQ derivatives with and without -ray irradiation, respectively.
Dose of -ray were 40 Gy against Jurkat cells and 10 Gy against MOLT-4 cells,
respectively.
c-ray
0
p53 +bispicen
c
c
·
·
-
10
20
30
-
p53 +27 or 37
p53 only
2
.4. Mechanistic studies of the radioprotective effect of 8HQ
derivatives
-
×
A mechanistic study of the radioprotective effect of 27 and 37
-40
205
was carried out. First, the p53-dependency of the radioprotective
activity of 27 and 37 was investigated by a dye-exclusion test using
Jurkat cells, which are p53-inactive leukemia cell line,23 and MOLT-
cells. As shown in Figure 10, 27 and 37 protect MOLT-4 cells from
-ray radiation-induced p53-dependent apoptosis. In contrast,
both 8HQ derivatives negligibly suppressed the cell death of Jurkat
cells, which is generally thought to be a p53-independent event.
These results suggest that radioprotective 8HQ’s inhibit the
215
225
235
245
Wavelength (nm)
Figure 12. CD spectra of recombinant p53 in PBS buffer (pH 7.4) in the presence of
radioprotectors. CD spectrum of p53 alone is shown as a plain curve. CD spectra of
p53 in the presence of bispicen or 8HQ derivatives (27 or 37) are shown as dashed
4
c
and bold curves, respectively ([p53] = 20 nM, [radioprotector] = 2
lM).
(
a)
Lane
1
2
+
+
3
4
FITC-DNA (10.0 nM)
recombinant p53 (12.5 nM)
radioprotector (250 μM)
+
+
+
+
+
N
N
O2N
NO2
27
37
NO2
,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical
2
0.6
0.5
0.4
0.3
0.2
0.1
2
7 or 37
(
b)
bispicen
1
0
.8
0.6
.4
vitamin C, E
curcumin
0
0
300
600
time (sec)
900
1200
0.2
0
1
2
3
4
Figure 11. Scavenging of DPPH free radicals by radioprotectors in MeOH followed
by UV–vis spectroscopy (initial [DPPH] = 50 M). The change in absorbance at
16 nm by the addition of well-known antioxidants (vitamin C, E and curcumin),
bispicen and 8HQ derivatives (27 or 37) are shown as plain, dashed and bold curves,
respectively. The scavenging reactions were initiated by the addition of each
Lane
l
5
Figure 13. EMSA of recombinant p53 with p53-probe DNA in the presence of 8HQ
derivatives. (a) Electrophoretic image of the mixture containing p53, DNA and 8HQ
derivatives (27 or 37). (b) Relative binding ratio of DNA with recombinant p53. The
condition of electrophoresis was constant-voltage at 230 V at 4 °C.
radioprotectors (initiate concentration is 200 lM in total).

3
900
S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
induced conformational changes of p53, possibly because the affin-
ity of 27 or 37 for zinc ions is not sufficiently strong to induce con-
formational changes in p53.
were recorded on a JASCO UV/vis spectrophotometer V-630 Bio
UV–vis Spectrophotometer, and CD spectra were recorded on a
Chirascan spectrophotometer (Applied Photophysics). IR spectra
were recorded on a Perkin–Elmer FT-IR spectrophotometer (Spec-
The effect of 27 and 37 on the DNA-binding activity of recombi-
nant p53 in vitro was evaluated by means of electrophoretic
mobility shift assay (EMSA) (Fig. 13), in which an FITC-labeled
p53-targeting DNA probe was used.14 Figure 13a shows the results
of poly-acrylamide gel electrophoresis of a mixture containing
recombinant p53, DNA, and 8HQ derivatives. Only a high mobility
band corresponding to free-DNA was detected in lane 1 and the
free-DNA band that was shifted to the low mobility area in lane 2
was assigned to a p53-DNA complex. The DNA-binding ratio esti-
mated from the intensities of the free-DNA band and the DNA-p53
band is displayed in Figure 13b. Lanes 3 and 4 in Figure 13a show
the results in the presence of 27 or 37, respectively, which show neg-
ligible inhibition of DNA-binding to p53 by these two compounds.
The results of CD spectra measurements (Fig. 12) and EMSA
1
13
trum 100) at room temperature. H (300 MHz) and C (75 MHz)
NMR spectra were recorded on a JEOL Always 300 spectrometer.
1
Tetramethylsilane was used as an internal reference for H and
1
3
C NMR measurements in CDCl
3 6 3
, DMSO-d , or CD OD. 3-(Trimeth-
ylsilyl)propionic-2,2,3,3-d acid (TSP) sodium salt was used as an
4
1
13
2
external reference for H and C NMR measurements in D O. MS
spectra were recorded on a JEOL JMS-SX102A and Agilent (Varian)
910-MS. Thin-layer (TLC) and silica gel column chromatographies
were performed using a Merck 5554 (silica gel) TLC plates and Fuji
Silysia Chemical FL-100D, respectively.
4.1.1. Synthesis of HQ derivatives
2
0a
20a
20a
20a
18,20b 22,20a 23,20a 25,20a 2820a
Compounds 3,
4,
7,
8,
2
2b
(Fig. 13) strongly suggest that the 8HQ derivatives have negligible
and 47
were prepared according to literature methods.
effects on both the conformation and the DNA-binding properties
of p53. In addition, they have negligible radical-scavenging activity
4.1.2. 8-Hydroxy-5-N-methylaminosulfonylquinoline (5)
2
0
(Fig. 11). These results suggest that mechanism for the radiopro-
Compound 3 (243 mg, 1.00 mmol) was added to 67 mM
MeNH (2.0 mmol) in THF (30 mL), and the resulting solution
tective effect of the 8HQ derivatives is different from that of
bispicen. Considering the fact that the 8HQ derivatives suppress
p53-dependent apoptosis (Fig. 10), our finding suggests that these
derivatives inhibit the p53-transcription independent pathway.
Further mechanistic analyses of 8HQ derivatives and related
in vivo testing are currently underway.
2
was stirred at room temperature for 3 h. After concentrating the
reaction mixture under reduced pressure, the resulting residue
was purified by column chromatography on silica gel with CHCl .
3
The obtained residue was recrystallized from CHCl
3
/hexanes to
give 5 (38 mg, 16% yield) as colorless needles.
1
Mp 154.0–155.0 °C. H NMR (300 MHz, CDCl
3
/TMS): d = 2.61
(
8
3H, d, J = 5.7 Hz), 7.21 (1H, d, J = 8.4 Hz), 7.64 (1H, dd, J = 4.5,
3
. Conclusions
.7 Hz), 8.26 (1H, d, J = 8.4 Hz), 8.88 (1H, d, J = 4.2 Hz), 9.02 (1H,
1
3
d, J = 8.7 Hz). C NMR (75 MHz, DMSO-d
6
): d = 28.3, 109.5, 123.2,
23.5, 124.7, 131.7, 133.3, 138.5, 146.8, 157.9. IR (neat): 3307,
921, 1715, 1570, 1504, 1314, 1289, 1199, 1152, 1069, 833, 787,
In summary, we report on the design and synthesis of some 8HQ
1
2
6
2
derivatives that contain aminosulfonyl groups, for use as radiopro-
tective drugs. Evaluations of their radioprotective activities indicate
that 5,7-bis(methylaminosulfonyl) derivatives such as 24 and 27
and some 5,7-bis(alkylaminosulfonyl)-8HQs such as 31, 33, 34
and 35 have a high radioprotective activity and a low cytotoxicity
for MOLT-4 cells. In addition, some 8-methoxy derivatives such as
ꢀ1
+
49, 527 cm
.
HRMS (FAB ) Calcd for
C
10
12
H N
2
O
3
S
(M+H),
39.0490: found 239.0489. Anal. Calcd for C10
H
11
2 3
N O S: C, 50.41;
H, 4.23; N, 11.76. Found: C, 50.43; H, 4.04; N, 11.31.
4
.1.3. 8-Hydroxy-5-N-methylaminosulfonylquinaldine (6)
-Hydroxy-5-N-methylaminosulfonylquinaldine (6) was pre-
pared as a colorless crystal (4.6 mg, 18% yield) from 4 (25 mg,
.092 mmol) and 80 mM MeNH (2.0 mmol) in THF (25 mL) in a
1
9 and 37 and 8-sulfonates 20, 42 and 43 also act as less toxic radio-
8
protectors. The findings strongly suggest that aminosulfonyl groups
are important for suppressing the radiation-induced apoptosis of
p53-active cells and for achieving a low degree of cytotoxicity. In
addition, 8HQ derivatives are useful for the apoptosis-inhibition
of MOLT-4 cells after being exposed to irradiation. The results of
mechanistic studies using 27 and 37 suggest that the mechanism
responsible for the radioprotective effects of the 8HQ derivatives
is different from that of bispicen and that it might possibly involve
the inhibition of the p53-transcription independent pathway,
suggesting the high possibility of these compounds as selective
radioprotective drugs for radiation therapy and other purpose.
The effect of these compounds on the radiation therapy of the can-
cer-bearing animals will be examined in the near future.
0
2
manner similar to that described for 5.
1
Mp 176.0–177.0 °C. H NMR (300 MHz, CDCl
3
/TMS): d = 2.60
(
3H, d, J = 5.1 Hz), 2.77 (3H, s), 7.16 (1H, d, J = 8.4 Hz), 7.50 (1H,
13
d, J = 8.7 Hz), 8.17 (1H, d, J = 8.1 Hz), 8.87 (1H, d, J = 8.7 Hz).
NMR (75 MHz, DMSO-d ): d = 24.5, 28.3, 109.2, 122.8, 123.5,
24.0, 130.5, 133.2, 137.9, 157.1, 157.6. IR (neat): 3294, 2919,
C
6
1
1
(
ꢀ
1
567, 1504, 1312, 1152, 1116, 1064, 829, 672, 563 cm . HRMS
+
FAB ) Calcd for C11
13
H N
2
O
3
S (M+H), 253.0647: found 253.0646.
Anal. Calcd for C11
C, 52.38; H, 4.39; N, 11.18.
H
13
N
2
O
3
S: C, 52.37; H, 4.79; N, 11.10. Found:
4
.1.4. 5-N,N-Dimethylaminosulfonyl-8-methoxyquinaldine (9)
CO (299.4 mg, 2.16 mmol) and MeI (449.5 L, 7.22 mmol)
were added to a solution of 8 (192.4 mg, 0.72 mmol) in MeCN
30 mL), and the resulting solution was stirred for 2 h at 80 °C.
After concentrating the reaction mixture under reduced pressure,
the residue was dissolved in CHCl and the organic solution
washed with water. The organic layer was dried over Na SO
filtered, and concentrated under reduced pressure. The obtained
4
4
. Experimental section
K
2
3
l
.1. General information
(
All reagents and solvents purchased were of the highest com-
3
mercial quality and were used without further purification.
Recombinant FLAG-p53 was synthesized by a silkworm-baculovi-
rus expression system (Procube-T system; Sysmex, Hyogo, Japan)
2
4
,
residue was recrystallized from AcOEt/hexanes to give
9
1
38
14
using the Ala
version of a FLAG-p53 as template, and purified
(
105.6 mg, 52% yield) as colorless crystals.
Mp 128.0–129.0 °C. H NMR (300 MHz, CDCl
1
by a FLAG-affinity purification. All aqueous solutions were pre-
pared using deionized and distilled water. Melting points were
determined on a Yanaco Melting Point Apparatus and a Büchi
3
/TMS): d = 2.73
(
6H, s), 2.82 (3H, s), 4.16 (3H, s), 7.08 (1H, d, J = 8.4 Hz), 7.47 (1H,
13
d, J = 9.3 Hz), 8.15 (1H, d, J = 8.4 Hz), 8.96 (1H, d, J = 8.7 Hz).
C
5
10 Melting Point Apparatus and are uncorrected. UV–vis spectra
3
NMR (75 MHz, CDCl ): d = 25.5, 37.3, 56.6, 105.6, 123.4, 124.0,

S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
3901
1
1
9
N
O
24.3, 131.3, 133.8, 139.8, 159.0, 159.1. IR (neat): 2965, 2845,
596, 1560, 1502, 1462, 1323, 1262, 1190, 1150, 1126, 1108,
124.7, 131.7, 134.7, 136.5, 139.6, 156.9, 158.8. IR (neat): 3312,
3069, 2962, 1594, 1557, 1492, 1455, 1426, 1374, 1312, 1256,
1199, 1153, 1133, 1055, 962, 943, 820, 751, 726, 675, 627,
ꢀ1
+
52, 830, 716, 653, 610, 540 cm . HRMS (FAB ): Calcd for C13
H
17-
ꢀ1
+
O
2 3
S (M+H), 281.0960: found, 281.0960. Anal. Calcd for C13
H
16
N
2-
576 cm
392.9770: found, 392.9770. Anal. Calcd for C12
6.86; H, 3.09; N, 7.09. Found: C, 36.75; H, 3.34; N, 7.14.
.
HRMS (FAB ): Calcd for
C
12
H14IN
2
O
3
S
(M+H),
3
S: C, 55.70; H, 5.75; N, 9.99. Found: C, 55.82; H, 5.71; N, 9.84.
H13IN O S: C,
2 3
3
4
.1.5. 5-N,N-Dimethylaminosulfonyl-8-ethoxyquinaldine (10)
-N,N-Dimethylaminosulfonyl-8-ethoxyquinaldine (10) was
prepared as a colorless crystal (114.9 mg, 80% yield) from 8
130 mg, 0.49 mmol) and EtI (390 L, 4.87 mmol) in a manner sim-
ilar to that described for 9.
5
4.1.9. 7-Bromo-5-N,N-dimethylaminosulfonyl-8-
methoxyquinaldine (14)
7-Bromo-5-N,N-dimethylaminosulfonyl-8-methoxyquinaldine
(14) was prepared as colorless crystals (685 mg, 86% yield) from 12
(594 mg, 1.72 mmol) and MeI (1070 lL, 17.2 mmol) in a manner
(
l
1
Mp 63.0–64.5 °C. H NMR (300 MHz, CDCl
3
/TMS): d = 1.66 (3H, t,
J = 7.1 Hz), 2.74 (6H, s), 2.81 (3H, s), 4.41 (2H, q, J = 7.1 Hz), 7.05 (1H,
similar to that described for 9.
d, J = 9.0 Hz), 7.45 (1H, d, J = 8.7 Hz), 8.12 (1H, d, J = 8.4 Hz), 8.94
Mp 127.0–128.0 °C. 1H NMR (300 MHz, CDCl
3
/TMS): d = 2.82
(3H, s), 2.83 (6H, s), 4.26 (3H, s), 7.44 (1H, d, J = 8.7 Hz), 8.29 (1H,
1
3
(
6
1H, d, J = 8.7 Hz). C NMR (75 MHz, CDCl
5.0, 106.3, 111.0, 123.0, 123.9, 124.4, 131.4, 133.7, 158.4, 158.8.
IR (neat): 2982, 1593, 1559, 1502, 1463, 1393, 1322, 1259, 1178,
3
): d = 14.3, 25.5, 37.3,
1
3
3
s), 8.94 (1H, d, J = 8.7 Hz). C NMR (75 MHz, CDCl ): d = 25.5,
37.4, 62.7, 114.3, 123.5, 123.9, 128.2, 133.5, 134.0, 143.0, 157.4,
159.8. IR (neat): 3086, 2939, 2849, 1607, 1573, 1550, 1494, 1460,
1408, 1395, 1343, 1319, 1252, 1196, 1157, 1141, 1091, 983, 956,
1
5
154, 1127, 1106, 1030, 958, 833, 797, 716, 660, 609, 568,
ꢀ1
+
39 cm . HRMS (FAB ): Calcd for C14
H
19
N
2
O
3
S (M+H), 295.1116:
S+0.5H O: C, 55.36;
H, 6.35; N, 9.22. Found: C, 55.43; H, 6.31; N, 9.23.
ꢀ1
+
found, 295.1116. Anal. Calcd for C14
H
18
N
O
2 3
2
830, 756, 720, 592, 563 cm . HRMS (FAB ): Calcd for C13
H
H
16BrN2-
15BrN2-
O
O
3
S (M+H), 359.0065: found, 359.0065. Anal. Calcd for C13
S: C, 43.47; H, 4.21; N, 7.80. Found: C, 43.56; H, 4.13; N, 7.78.
3
4
.1.6. 7-Bromo-5-N,N-dimethylaminosulfonyl-8-
hydroxyquinoline (11)
4.1.10. 7-Bromo-5-N,N-dimethylaminosulfonyl-8-
ethoxyquinaldine (15)
7-Bromo-5-N,N-dimethylaminosulfonyl-8-ethoxyquinaldine
(15) was prepared as colorless crystals (73.5 mg, 98% yield) from
NBS (185 mg, 1.00 mmol) was added to a solution of 720 (263 mg,
.00 mmol) in MeCN (40 mL) at 0 °C, and the resulting solution was
1
stirred for 3 h at room temperature. After concentrating the reaction
mixture under reduced pressure, the residue was dissolved in CHCl
and the organic solution washed with water. The organic layer was
dried over Na SO , filtered, and concentrated under reduced pres-
3
12 (80.0 mg, 0.20 mmol) and EtI (640
similar to that described for 9.
l
L, 8.0 mmol) in a manner
Mp 114.0–118.0 °C. 1H NMR (300 MHz, CDCl
/TMS): d = 1.56
3
2
4
sure. The obtained residue was recrystallized from CHCl
3
/hexanes
(3H, t, J = 6.9 Hz), 2.78 (3H, s), 2.81 (6H, s), 4.61 (2H, q,
to give 11 (298 mg, 86% yield) as colorless needles.
J = 6.9 Hz), 7.43 (1H, d, J = 9.0 Hz), 8.29 (1H, s), 8.92 (1H, d,
J = 9.0 Hz). C NMR (75 MHz, CDCl ): d = 15.9, 25.5, 37.4, 71.7,
3
1
13
Mp: 218–220 °C. H NMR (300 MHz, CDCl
3
/TMS): d = 2.80 (6H,
s), 7.64 (1H, dd, J = 4.2, 8.7 Hz), 8.35 (1H, s), 8.88 (1H, d,
114.7, 123.4, 123.9, 127.9, 133.5, 134.0, 156.9, 159.5. IR (neat):
3087, 2977, 1804, 1609, 1580, 1552, 1495, 1443, 1386, 1311,
J = 4.2 Hz), 9.08 (1H, d, J = 8.7 Hz). 1 C NMR (75 MHz, CDCl
3
):
3
ꢀ1
d = 37.4, 102.1, 123.5, 124.3, 134.7, 136.3, 138.0, 141.2, 149.3,
1247, 1196, 1136, 1091, 1023, 957, 831, 759, 721, 689, 669 cm
.
+
1
1
C
54.2. IR (neat): 660, 721, 795, 815, 859, 961, 1134, 1188, 1307,
HRMS (FAB ): Calcd for C14
373.0222. Anal. Calcd for C14
7.51. Found: C, 45.15; H, 4.57; N, 7.65.
H
18BrN S (M+H), 373.0222: found,
2
O
3
ꢀ1
+
329, 1371, 1487, 1615, 2888, 3253 cm . HRMS(FAB ) Calcd for
12BrN S, 330.9752; found, 330.9750. Anal. Calcd for C11
S: C, 39.89; H, 3.35; N, 8.46. Found: C, 40.09; H, 3.07; N, 8.51.
H
17BrN O S: C, 45.05; H, 4.59; N,
2 3
11
H
2
O
3
H
11-
2 3
BrN O
4
.1.11. 5-N,N-Dimethylaminosulfonyl-7-iode-8-
4
.1.7. 7-Bromo-5-N,N-dimethylaminosulfonyl-8-
hydroxyquinaldine (12)
-Bromo-5-N,N-dimethylaminosulfonyl-8-hydroxyquinaldine
methoxyiodequinaldine (16)
5-N,N-Dimethylaminosulfonyl-7-iode-8-methoxyiodequinal-
dine (16) was prepared as colorless crystals (58.2 mg, 72% yield)
7
(12) was prepared as colorless needles (594 mg, 96% yield) from 8
from 13 (80.0 mg, 0.20 mmol) and MeI (250
lL, 2.0 mmol) in a
(
478 mg, 1.80 mmol) and NBS (320 mg, 1.80 mmol) in a manner
manner similar to that described for 9.
1
similar to that described for 11.
Mp 128.0–130.0 °C. H NMR (300 MHz, CDCl
3
/TMS): d = 2.79
Mp 215.0–217.0 °C. 1H NMR (300 MHz, CDCl
(3H, s), 2.81 (6H, s), 4.28 (3H, s), 7.44 (1H, d, J = 8.7 Hz), 8.45 (1H,
3
/TMS): d = 2.77
1
3
(
9H, s), 7.49 (1H, d, J = 8.7 Hz), 8.22 (1H, s), 8.93 (1H, d,
3
s), 8.93 (1H, d, J = 8.7 Hz). C NMR (75 MHz, CDCl ): d = 25.5,
1
3
J = 8.7 Hz). C NMR (75 MHz, DMSO-d
6
): d = 24.7, 37.4, 101.9,
22.4, 123.1, 124.5, 134.6, 134.7, 137.5, 153.7, 159.0. IR (neat):
326, 3075, 2928, 1600, 1560, 1494, 1459, 1425, 1314, 1244,
37.4, 62.7, 88.4, 123.7, 124.7, 128.5, 134.1, 138.5, 141.8, 159.5,
160.5. IR (neat): 2967, 2840, 1606, 1542, 1455, 1395, 1335, 1260,
1
3
1
BrN
BrN
ꢀ1
+
1201, 1144, 1092, 948, 835, 715 cm . HRMS (FAB ): Calcd for C13-
ꢀ
1
+
132, 962, 820, 759, 723, 675 cm . HRMS (FAB ): Calcd for C12
H
H
14-
H
H
16IN
15IN
2
O
O
3
S (M+H), 406.9926: found, 406.9925. Anal. Calcd for C13-
S: C, 38.44; H, 3.72; N, 6.90. Found: C, 38.59; H, 3.61; N,
2
O
O
3
S (M+H) 344.9909: found, 344.9909. Anal. Calcd for C12
S: C, 41.75; H, 3.80; N, 8.12. Found: C, 41.71; H, 3.67; N,
13-
2
3
2
3
6.96.
8
.09.
4
.1.12. 5-N,N-Dimethylaminosulfonyl-8-ethoxy-7-
4
.1.8. 5-N,N-Dimethylaminosulfonyl-8-hydroxy-7-
iodequinaldine (17)
iodequinaldine (13)
-N,N-Dimethylaminosulfonyl-8-hydroxy-7-iodequinaldine
5-N,N-Dimethylaminosulfonyl-8-ethoxy-7-iodequinaldine (17)
was prepared as colorless crystals (81.4 mg, 97% yield) from 13
5
(
13) was prepared as colorless crystals (106 mg, 52% yield) from 8
(80.0 mg, 0.20 mmol) and EtI (640
lar to that described for 9.
l
L, 8.00 mol) in a manner simi-
Mp 114.0–116.0 °C. 1H NMR (300 MHz, CDCl
/TMS): d = 1.59
3
(
80.7 mg, 0.30 mmol) and NIS (68.2 mg, 0.30 mmol) in a manner
similar to that described for 11.
Mp 230.0–232.0 °C. 1H NMR (300 MHz, CDCl
(3H, t, J = 6.9 Hz), 2.77 (3H, s), 2.80 (6H, s), 4.62 (2H, q,
3
/TMS): d = 2.77
(
9H, s), 7.49 (1H, d, J = 8.7 Hz), 8.40 (1H, s), 8.93 (1H, d,
J = 6.9 Hz), 7.43 (1H, d, J = 8.7 Hz), 8.45 (1H, s), 8.92 (1H, d,
J = 9.0 Hz). C NMR (75 MHz, CDCl ): d = 16.1, 25.5, 37.4, 71.8,
3
1
3
13
J = 8.7 Hz). C NMR (75 MHz, CDCl
3
): d = 24.7, 37.4, 123.0, 123.4,

3
902
S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
8
9.0, 123.6, 124.6, 128.1, 134.0, 138.6, 141.9, 159.2, 160.0. IR
J = 8.7 Hz). 13C NMR (75 MHz, CDCl
): 24.9, 37.4, 116.9, 123.4,
3
(
1
(
4
neat): 2978, 1608, 1573, 1548, 1493, 1420, 1385, 1316, 1246,
124.1, 128.6, 128.8, 132.6, 133.5, 134.0, 138.2, 142.7, 148.2,
161.2. IR (neat): 3066, 2945, 1606, 1583, 1482, 1455, 1417, 1373,
ꢀ
1
134, 1089, 1021, 955, 832, 802, 720, 687, 665 cm . HRMS
+
ꢀ1
FAB ): Calcd for
C
14
H
18IN
H
2
O
3
S
(M+H), 421.0083: found,
S: C, 40.01; H, 4.08; N, 6.67.
1339, 1180, 1154, 1138, 1070, 966, 794, 723, 688, 650 cm . HRMS
+
21.0081. Anal. Calcd for C14 17IN
2
O
3
(FAB ) Calcd for C18
2
H18BrN O
S
5 2
(M+H) 484.9841: found 484.9841.
Found: C, 40.03; H, 3.94; N, 6.63.
4
.1.16. 5,7-Bis(N-methylaminosulfonyl)-8-hydroxyquinoline
4
.1.13. 8-Methoxy-5-N-methylaminosulfonylquinaldine (19)
(24)
2
0
1
8
(866.1 mg, 5.00 mmol) was added to chlorosulfonic acid
3 mL), and the resulting solution was stirred at room temperature
for 12 h. The reaction was quenched by pouring the reaction mix-
ture into cold brine (200 mL) and CH Cl (200 mL) in separatory
funnel. The organic layer was neutralized by K CO (1.5 g), filtered,
and concentrated under reduced pressure to give crude
-methoxy-5-sulfonylchloridequinaldine as a white solid. This
crude compound was dissolved in 400 mM MeNH (20 mmol) in
5,7-Bis(N-methylaminosulfonyl)-8-hydroxyquinoline (24) was
prepared as yellow needles (78 mg, 24% yield) from 22 (342 mg,
1.00 mmol) and 71 mM MeNH (2.1 mmol) in THF (30 mL) in a
2
(
2
2
manner similar to that described for 5.
1
2
3
Mp: 237.0–238.0 °C. H NMR (300 MHz, CD
3
OD/TMS): d = 2.52
(3H, s), 2.60 (3H, s), 7.85 (1H, dd, J = 4.4, 8.9 Hz), 8.54 (1H, s),
9.02 (1H, dd, J = 1.5, 4.5 Hz), 9.12 (1H, dd, J = 1.5, 8.7 Hz). 13
NMR (75 MHz, DMSO-d ): 28.3, 28.8, 119.2, 121.1, 125.1, 126.5,
8
C
2
6
THF (50 mL), and the resulting solution was stirred at room tem-
perature for 1 h. After concentrating the reaction mixture under
reduced pressure, the residue was dissolved in AcOEt and the
organic solution washed with water. The organic layer was dried
130.6, 134.8, 138.5, 148.7, 157.4. IR (neat): 3516, 3295, 2769,
1594, 1528, 1505, 1393, 1374, 1312, 1287, 1209, 1144, 1120,
1055, 973, 811, 723, 678, 639, 578 cm . HRMS (FAB ): Calcd for
ꢀ
1
+
C
C
11
H
H
14
N
N
3
O
O
5
S
S
2
(M+H) 332.0375; Found 332.0374; Anal. Calcd for
: C, 39.87; H, 3.95; N, 12.68. Found: C, 39.71; H,
2 4
over Na SO , filtered, and concentrated under reduced pressure.
11
13
3
5
2
The obtained residue was recrystallized EtOH to give 19
3.81; N, 11.58.
(
550.7 mg, 41% yield) as colorless crystals.
Mp 217.0–218.0 °C. H NMR (300 MHz, CDCl
1
3
/TMS): 2.59 (3H, d,
J = 5.5 Hz), 2.83 (3H, s), 4.16 (3H, s), 4.38 (1H, br), 7.06 (1H, d,
J = 8.4 Hz), 7.49 (1H, d, J = 9.0 Hz), 8.21 (1H, d, J = 8.4 Hz), 8.87
4.1.17. 5,7-Bis(aminosulfonyl)-8-hydroxyquinaldine (26)
5,7-Bis(aminosulfonyl)-8-hydroxyquinaldine (26) was prepared
as yellow crystals (68.2 mg, 39% yield) as HCl salts from 22
1
3
(
1
(
8
2
5
1H, d, J = 9.0 Hz). C NMR (75 MHz, DMSO-d
06.3, 122.7, 123.6, 125.2, 129.9, 132.9, 139.2, 158.1, 158.4. IR
neat): 3099, 2923, 2851, 1725, 1560, 1502, 1320, 1149, 1086,
6
): 24.7, 28.3, 56.0,
(178 mg, 0.50 mmol) and NH
680 L) in a manner similar to that described for 5.
Mp >250 °C. H NMR (300 MHz, DMSO-d
7.78 (1H, d, J = 8.7 Hz), 8.39 (1s, s), 8.91 (1s, d, J = 8.7 Hz). C NMR
(75 MHz, DMSO-d ): 24.0, 122.5, 123.5, 123.9, 125.7, 126.8, 134.7,
137.8, 155.0, 158.4. IR (neat): 3603, 3535, 3309, 3194, 3061, 1645,
591, 1539, 1494, 1306, 1238, 1192, 1140, 1047, 915, 775, 706,
3
aqueous solution (10.0 mmol,
l
1
6
/TMS): d = 2.77 (3H, s),
ꢀ1
+
13
42, 660, 530 cm . HRMS (FAB ) Calcd for C12
H
15
N
2
O
N
3
S (M+H)
S: C,
67.0803: found 267.0803; Anal. Calcd for
C
12
H
14
2
O
3
6
4.12; H, 5.30; N, 10.52. Found: C, 54.10; H, 5.22; N, 10.53.
1
ꢀ1
+
4
.1.14. 7-Bromo-5-N,N-dimethylaminosulfonyl-8-
651, 627, 560 cm . HRMS (FAB ); Calcd for C10
318.0218: found 318.0218.
12 3 5 2
H N O S (M+H)
methanesulfonyloxyquinaldine (20)
Methanesulfonyl chloride (66.6 L, 0.86 mmol) and 4-dimethyl-
aminopyridine (cat.) were added to a solution of 12 (248 mg,
l
4.1.18. 5,7-Bis(N-methylaminosulfonyl)-8-hydroxyquinaldine
(27)
0
.72 mmol) and Et
3 2 2
N (120 lL, 0.86 mmol) in CH Cl (10 mL) at
0
°C under an argon atmosphere, and the resulting solution was
5,7-Bis(N-methylaminosulfonyl)-8-hydroxyquinaldine (27) was
prepared as yellow needles crystals (69.1 mg, 12% yield) from 23
stirred at room temperature for 30 min. After concentrating the
reaction mixture under reduced pressure, the residue was dis-
(602 mg, 1.69 mmol) and 83 mM MeNH
2
(4.0 mmol) in THF
solved in CH
The organic layer was dried over Na
under reduced pressure. The resulting residue was purified by col-
umn chromatography on silica gel with CH Cl . The obtained resi-
/hexanes to give 20 (19 mg, 8%
2
Cl
2
and the organic solution washed with water.
(48 mL) in a manner similar to that described for 5.
1
2
SO , filtered, and concentrated
4
6
Mp 105.0–107.0 °C. H NMR (300 MHz, DMSO-d /TMS): d = 2.38
(3H, d, J = 4.8 Hz), 2.47 (3H, s), 2.79 (3H, s), 7.71 (1H, q, J = 4.5 Hz),
7.81 (1H, d, J = 9.3 Hz) 8.29 (1H, s), 8.90 (1H, d, J = 8.7 Hz). 13C NMR
2
2
due was recrystallized by CHCl
yield) as colorless needles.
3
6
(75 MHz, DMSO-d ): 23.6, 28.3, 28.8, 118.9, 120.2, 124.8, 126.1,
129.8, 134.9, 138.0, 157.2, 158.1. IR (neat): 3298, 2772, 1593,
1528, 1506, 1390, 1315, 1285, 1207, 1120, 1047, 973, 812, 722,
Mp 178.0–182.0 °C. 1H NMR (300 MHz, CDCl
/TMS): 2.79 (3H,
s), 2.85 (6H, s), 3.82 (3H, s), 7.50 (1H, d, J = 9.0 Hz), 8.31 (1H, s),
3
ꢀ1
+
637, 556 cm . HRMS (FAB ); Calcd for C12
346.0531: found 346.0531. Anal. Calcd for C12
41.73; H, 4.38; N, 12.17. Found: C, 42.06; H, 4.02; N, 12.02.
H
16
N
3
O
5
S
2
(M+H)
8
4
.97 (1H, d, J = 9.0 Hz). 13C NMR (75 MHz, CDCl
1.3, 117.5, 123.6, 124.2, 132.6, 134.1, 138.6, 142.6, 147.8, 161.3.
3
): 2.51, 3.74,
H N O S : C,
15 3 5 2
IR (neat): 3035, 2941, 1602, 1585, 1487, 1432, 1360, 1340, 1172,
ꢀ
1
1
156, 1139, 1074, 952, 819, 763, 744, 723, 708, 686, 645 cm
.
4.1.19. 5,7-Bis(N,N-diethylaminosulfonyl)-8-hydroxyquinaldine
(29)
+
2 5 2
HRMS (FAB ) Calcd for C13H16BrN O S (M+H) 422.9684: found
4
22.9680.
5,7-Bis(N,N-diethylaminosulfonyl)-8-hydroxyquinaldine (29)
was prepared as pale yellow crystals (356 mg, 38% yield) as HCl
4
.1.15. 8-Benzenesulfonyloxy-7-bromo-5-N,N-
2
salts from 23 (240 mg, 0.883 mmol) and 100 mM Et NH
dimethylaminosulfonylquinaldine (21)
-Benzensulfonyloxy-7-bromo-5-N,N-dimethylaminosulfonyl-
quinaldine (21) was prepared as colorless crystals (116 mg, 48%
yield) from 12 (173 mg, 0.50 mmol), benzenesulfonyl chloride
(2.0 mmol) in THF (20 mL) in a manner similar to that described
8
for 5.
1
Mp 104.0–105.0 °C. H NMR (300 MHz, DMSO-d
6
/TMS): d = 0.98
(6H, t, J = 6.9 Hz), 1.05 (6H, t, J = 7.2 Hz), 2.82 (3H, s), 3.26 (4H, q,
(
76.8
3
lL, 0.60 mmol) and Et N (30.2 lL, 0.60 mmol) in a manner
J = 7.2 Hz), 3.36 (4H, q, J = 7.2 Hz), 7.87 (1H, d, J = 9.3 Hz), 8.34
(1H, s), 8.83 (1H, d, J = 8.7 Hz). C NMR (75 MHz, DMSO-d ):
6
d = 13.5, 14.3, 23.4, 120.1, 121.0, 125.0, 126.6, 130.4, 135.2, 137.8,
157.5, 158.0. IR (neat): 2415, 1863, 1665, 1593, 1464, 1384,
1323, 1200, 1143, 1015, 950, 848, 772, 714, 697, 671, 636,
1
3
similar to that described for 21.
1
Mp 158.0–160.0 °C. H NMR (300 MHz, CDCl
.83 (6H, s), 7.40 (1H, d, J = 8.7 Hz), 7.60 (2H, t, J = 7.5 Hz), 7.74 (1H,
t, J = 7.4 Hz), 8.09 (2H, d, J = 7.2 Hz), 8.10 (1H, s), 8.89 (1H, d,
3
/TMS): 2.49 (3H, s)
2

S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
3903
ꢀ
1
+
6
13 cm . HRMS (FAB ): Calcd for C18
H
28
N
3
O
5
S
2
(M+H) 430.1470:
+HCl: C, 46.39; H,
4.1.24. 5,7-Bis(homopiperazinylsulfonyl)-8-hydroxyquinaldine
(34)
5,7-Bis(homopiperazinylsulfonyl)-8-hydroxyquinaldine (34)
found, 430.1471. Anal. Calcd for C18
.06; N, 9.02. Found: C, 46.64; H, 5.87; N, 9.10.
H
28
N
3
O
5
S
2
6
was prepared as yellow crystals (182.5 mg, 60% yield) as HCl salts
from 23 (222.6 mg, 0.625 mmol) and monoBoc-protected homo-
piperazine (532.4 mg, 2.5 mmol) in MeCN (20 mL) in a manner
4
.1.20. 5,7-Bis(N-n-propylaminosulfonyl)-8-hydroxyquinaldine
(
30)
5
,7-Bis(N-n-propylaminosulfonyl)-8-hydroxyquinaldine
(30)
similar to that described for 5.
Mp >250.0 °C. H NMR (300 MHz, DMSO-d
(4H, m), 2.86 (3H, s), 3.08–3.22 (8H, m), 3.35–3.43 (4H, m), 3.60
1
was prepared as pale yellow powders (251 mg, 61% yield) from
3 (356 mg, 1.00 mmol) and PrNH
6
/TMS): d = 1.95–2.08
n
2
2
(10.0 mmol) in THF (20 mL)
in a manner similar to that described for 5.
(2H, t, J = 4.8 Hz), 3.08 (2H, t, J = 4.8 Hz), 7.92 (1H, d, J = 8.7 Hz),
8.30 (1H, s), 8.95 (1H, d, J = 8.7 Hz). C NMR (75 MHz, DMSO-
1
13
Mp 81.0–82.0 °C. H NMR (300 MHz, CDCl
3
/TMS): d = 0.80–0.91
(
(
(
1
3
1
6H, m), 1.42–1.59 (4H, m), 2.82 (3H, s), 2.92–3.00 (4H, m), 7.63
6
d ): d = 22.1, 24.6, 24.8, 43.4, 44.1, 44.4, 44.5, 45.8, 46.1, 46.2,
1
3
1H, d, J = 8.7 Hz), 8.58 (1H, s), 8.98 (1H, d, J = 8.7 Hz). C NMR
75 MHz, CDCl ): d = 11.0, 11.1, 22.9, 23.1, 24.7, 45.1, 45.3, 119.6,
24.6, 125.5, 126.5, 128.7, 134.8, 137.6, 153.5, 159.5. IR (neat):
580, 3284, 2967, 2936, 2877, 1642, 1553, 1501, 1421, 1315,
46.9, 119.1, 126.0, 127.2, 132.9, 137.3, 137.4, 156.5, 160.2, 185.2.
IR (neat): 3297, 2951, 2731, 1587, 1560, 1457, 1431, 1311, 1188,
3
ꢀ1
1135, 1010, 955, 893, 835, 785, 709, 625, 553 cm . HRMS
+
(FAB ): Calcd for
484.1687. Anal. Calcd for C20
N, 11.81. Found: C, 40.57; H, 5.71; N, 11.71.
C
20
H
30
N
5
O
5
S
2
(M+H) 484.1688: found,
ꢀ1
234, 1194, 1132, 1075, 972, 840, 782, 695, 650, 629, 557 cm
.
H N O S +3HCl: C, 40.51; H, 5.44;
29 5 5 2
+
23 3 5 2
HRMS (FAB ): Calcd for C16H N O S (M+H) 402.1157: found,
4
02.1156.
4
.1.25. 5,7-Bis(5-methylisoxazol-3-ylaminosulfonyl)-8-
4
.1.21. 5,7-Bis(ethyl 3-propanoate-1-aminosulfonyl)-8-
hydroxyquinaldine (35)
hydroxyquinaldine (31)
,7-Bis(ethyl 3-propanoate-1-aminosulfonyl)-8-hydroxyquinal-
dine (31) was prepared as yellow powders (547 mg, 56% yield)
from 23 (712 mg, 2 mmol) and b-alanine ethyl ester (2529 mg,
5,7-Bis(5-methylisoxazol-3-ylaminosulfonyl)-8-hydroxyquinal-
dine (35) was prepared as yellow powders (60.1 mg, 12% yield)
from 23 (356 mg, 1.00 mmol) and 3-amino-5-methylisoxazole
(980 mg, 10 mmol) in MeCN (30 mL) in a manner similar to that
5
1
0 mmol) in EtOH (40 mL) in a manner similar to that described
for 5.
Mp 138.0–139.0 °C. 1H NMR (300 MHz, CDCl
6H, t, J = 6.9 Hz), 2.50–2.58 (4H, m), 3.20–3.25 (4H, m), 4.05 (4H,
q, J = 6.9 Hz), 7.64 (1H, d, J = 8.7 Hz), 8.58 (1H, s), 8.94 (1H, d,
described for 5.
1
Mp 213.0–214.0 °C. H NMR (300 MHz, DMSO-d
(3H, s), 2.28 (3H, s), 2.74 (3H, s), 6.00 (2H, s), 7.97 (1H, d, J = 8.7 Hz),
8.52 (1H, s), 9.05 (1H, d, J = 8.7 Hz). C NMR (75 MHz, DMSO-d ):
6
d = 11.9, 12.0, 22.1, 94.9, 95.2, 118.9, 125.6, 127.2 132.9, 136.7,
136.8, 156.9, 157.1, 157.5, 163.8, 167.5, 169.3, 170.2. IR (neat):
3085, 2977, 2821, 1596, 1499, 1460, 1378, 1326, 1271, 1196,
6
/TMS): d = 2.25
3
/TMS): d = 1.21
1
3
(
1
3
J = 8.7 Hz). C NMR (75 MHz, CDCl
3
): d = 14.0, 14.1, 24.4, 34.0,
3
1
1
6
5
4.1, 38.8, 39.0, 60.9, 61.0, 119.6, 124.6, 126.6, 128.9, 134.9,
37.5, 154.2, 159.2, 172.2. IR (neat): 3222, 2905, 1730, 1554,
503, 1375, 1314, 1192, 1141, 1093, 1048, 1022, 942, 852, 787,
64, 554 cm . HRMS (FAB ): Calcd for C20
18.1267: found, 518.1268.
ꢀ1
1142, 1029, 1011, 974, 928, 887, 803, 782, 660, 634, 547 cm .
+
HRMS (FAB ): Calcd for C18
480.0648. Anal. Calcd for C18
N, 14.08. Found: C, 43.35; H, 3.60; N, 14.41.
H
H
18
N
5
O
7
S
2
(M+H) 480.0648: found,
+H O: C, 43.46; H, 3.85;
ꢀ1
+
H
28
N
3
O
S
9 2
(M+H)
17
N
5
O
7
S
2
2
4
.1.22. 5,7-Bis(2-aminoethylaminosulfonyl)-8-
4.1.26. 5,7-Bis(N,N-dimethylaminosulfonyl)-8-
methoxyquinoline (36)
5,7-Bis(N,N-dimethylaminosulfonyl)-8-methoxyquinolinol (36)
was prepared as orange crystals (87.1 mg, 32% yield) from 25
hydroxyquinaldine (32)
,7-Bis(2-aminoethylaminosulfonyl)-8-hydroxyquinaldine (32)
was prepared as yellow powders (547 mg, 56% yield) as HCl salts
from 23 (214 mg, 0.6 mmol) and monoBoc-protected ethylenedia-
mine (385 mg, 2.4 mmol) in MeCN (10 mL) in a manner similar to
5
(267 mg, 0.74 mmol) and MeI (461
lL, 7.4 mmol) in a manner sim-
ilar to that described for 9.
1
that described for 5.
Mp >250.0 °C. H NMR (300 MHz, DMSO-d
6
/TMS): d = 2.64 (6H,
1
Mp 239.0–240.0 °C. H NMR (300 MHz, D
2
O/TSP): d = 2.95 (3H,
s), 2.78 (6H, s), 4.79 (3H, s), 8.02 (1H, dd, J = 6.0, 8.9 Hz), 8.37 (1H,
1
3
s), 3.13–3.24 (8H, m), 7.98 (1H, d, J = 9.0 Hz), 8.48 (1H, s), 9.18
s), 8.93 (1H, d, J = 6.0 Hz), 9.30 (1H, d, J = 8.9 Hz). C NMR (75 MHz,
CDCl ): d = 36.8, 37.8, 51.0, 102.9, 121.7, 124.2, 132.8, 136.4, 137.9,
141.9, 146.9, 167.1. IR (neat): 3066, 2969, 1606, 1561, 1524, 1478,
1
3
(
1H, d, J = 9.0 Hz). C NMR (75 MHz, DMSO-d
6
): d = 23.4, 36.7,
6.8, 39.9, 40.6, 119.2, 119.9, 125.3, 126.9, 130.6, 136.5, 137.9,
58.0, 158.4. IR (neat): 2905, 2052, 1590, 1315, 1136, 1085,
3
3
1
1
ꢀ1
1410, 1312, 1244, 1129, 1042, 946, 823, 761, 701, 602, 558 cm
.
ꢀ1
+
+
031, 968, 781, 646, 560 cm . HRMS (FAB ): Calcd for C14
(M+H) 404.1062: found, 404.1061.
H
22
N
5
O
5-
HRMS (FAB ): Calcd for C14
374.0844. Anal. Calcd for C14
1.25. Found: C, 44.95; H, 5.00; N, 11.19.
H
20
N
3
O
5
S
2
(M+H) 374.0844: found,
S
2
19 3 5 2
H N O S : C, 45.03; H, 5.13; N,
1
4
.1.23. 5,7-Bis(piperazinylsulfonyl)-8-hydroxyquinaldine (33)
,7-Bis(piperazinylsulfonyl)-8-hydroxyquinaldine (33) was pre-
pared as yellow powders (53.6 mg, 24% yield) as HCl salts from 23
178 mg, 0.50 mmol) and monoBoc-protected piperazine (777 mg,
.95 mmol) in MeCN (10 mL) in a manner similar to that described
for 5.
Mp >250 °C. H NMR (300 MHz, D
.38 (8H, m), 3.51 (4H, t, J = 5.4 Hz), 3.63 (4H, t, J = 5.4 Hz), 8.02
5
4.1.27. 5,7-Bis(N,N-dimethylaminosulfonyl)-8-
methoxyquinaldine (37)
5,7-Bis(N,N-dimethylaminosulfonyl)-8-methoxyquinaldine (37)
was prepared as colorless crystals (357 mg, 70% yield) from 28
(
3
(513 mg, 1.37 mmol) and MeI (853 lL, 13.7 mmol) in a manner
1
2
O/TSP): d = 2.99 (3H, s), 3.32–
similar to that described for 9.
3
Mp 190.0–191.0 °C. 1H NMR (300 MHz, CDCl
3
/TMS): d = 2.83
(3H, s), 2.88 (6H, s), 2.93 (6H, s), 4.45 (3H, s), 7.55 (1H, d,
1
3
(
(
1
2
1
1H, d, J = 9.0 Hz), 8.50 (1H, s), 9.22 (1H, d, J = 9.0 Hz). C NMR
75 MHz, DMSO-d ): d = 21.7, 41.9, 42.2, 42.6, 42.7, 117.6, 126.9,
27.6, 134.4, 137.2, 138.4, 156.3, 161.9, 188.5. IR (neat): 3338,
936, 2727, 2494, 1558, 1501, 1455, 1401, 1299, 1222, 1188,
140, 1064, 929, 843, 778, 712, 625, 552 cm . HRMS (FAB ): Calcd
(M+H) 456.1375: found, 456.1377.
1
3
6
J = 9.3 Hz), 8.51 (1H, s), 9.03 (1H, d, J = 9.0 Hz). C NMR (75 MHz,
CDCl ): d = 25.6, 37.5, 37.7, 64.1, 125.3, 127.0, 128.3, 129.0, 129.3,
3
134.2, 143.0, 158.8, 160.1. IR (neat): 3097, 2948, 1609, 1579,
1550, 1499, 1461, 1409, 1393, 1338, 1254, 1199, 1143, 1094,
ꢀ1
+
ꢀ1
+
for C18
H
25
N
5
O
S
5 2
960, 835, 776, 722, 617, 557 cm . HRMS (FAB ): Calcd for C15H
22-

3
904
S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
N
3
O
H
5
S
2
(M+H) 388.1001: found, 388.1003. Anal. Calcd for
: C, 46.50; H, 5.40; N, 10.84. Found: C, 46.63; H,
3 3
hexanes/CHCl . The obtained residue was recrystallized by CHCl /
C
5
15
21
N
3
O
5
S
2
hexanes to give from hexanes/CHCl
as colorless crystals.
3
to give 41 (7.9 mg, 16% yield)
.40; N, 10.69.
Mp 175.0–176.0 °C. 1H NMR (300 MHz, CDCl
/TMS): d = 2.65
3
4
.1.28. 5,7-Bis(N,N-dimethylaminosulfonyl)-8-
(3H, d, J = 5.4 Hz), 2.75 (3H, d, J = 5.1 Hz), 2.83 (3H, s), 4.50 (3H,
ethoxyquinaldine (38)
,7-Bis(N,N-dimethylaminosulfonyl)-8-ethoxyquinaldine (38)
was prepared as colorless crystals (103 mg, 47% yield) from 28
200 mg, 0.54 mmol) and EtI (864 L, 10.8 mmol) in a manner
similar to that described for 9.
Mp 172.0–173.0 °C. 1H NMR (300 MHz, CDCl
3H, t, J = 6.9 Hz), 2.82 (3H, s), 2.87 (6H, s), 2.93 (6H, s), 4.78 (2H,
q, J = 7.0 Hz), 7.54 (1H, d, J = 9.0 Hz), 8.52 (1H, s), 9.02 (1H, d,
s), 4.97 (1H, q, J = 5.1 Hz), 5.15 (1H, d, J = 5.4 Hz), 7.59 (1H, d,
1
3
5
J = 9.0 Hz), 8.58 (1H, s), 9.00 (1H, d, J = 9.3 Hz). C NMR (75 MHz,
CDCl ): d = 25.5, 29.3, 29.6, 64.7, 125.6, 126.6, 127.5, 128.7, 129.7,
133.8, 142.3, 158.0, 160.2. IR (neat): 3288, 2952, 1550, 1501,
3
(
l
ꢀ1
+
1459, 1391, 1316, 1148, 1092, 834, 633, 559 cm . HRMS (FAB )
3
/TMS): d = 1.64
Calcd for C13
Calcd for C13
43.54; H, 4.66; N, 11.52.
H
H
18
N
3
O
5
S
2
(M+H) 360.0688: found 360.0686; Anal.
: C, 43.44; H, 4.77; N, 11.69. Found: C,
(
17
N
3
O
5
S
2
1
3
J = 9.3 Hz). C NMR (75 MHz, CDCl
3
/TMS): d = 15.8, 25.5, 37.5,
3
1
1
6
4
4
7.8, 73.5, 125.2, 126.9, 127.7, 128.9, 129.2, 132.2, 143.1, 158.6,
4.1.32. 5,7-Bis(N-methylaminosulfonyl)-8-
methanesulfonyloxyquinaldine (42)
5,7-Bis(N-methylaminosulfonyl)-8-methanesulfonyloxyquinal-
dine (42) was prepared as colorless needles (19 mg, 8% yield) from
59.8. IR (neat): 3099, 2970, 1609, 1578, 1550, 1497, 1458, 1428,
338, 1277, 1251, 1220, 1198, 1143, 1093, 1015, 963, 777, 722,
ꢀ
1
+
95, 617, 558 cm . HRMS (FAB ): Calcd for C16
H
24
N
H
3
O
5
S
2
(M+H),
: C,
02.1157: found, 402.1158. Anal. Calcd for C16
23
N
3
O
5
S
2
27 (200 mg, 0.59 mmol), methanesulfonyl chloride (76.8
0.60 mmol) and Et N (98.5 L, 0.71 mmol) in a manner similar to
that described for 20.
lL,
7.86; H, 5.77; N, 10.47. Found: C, 47.68; H, 5.75; N, 10.32.
3
l
1
4
.1.29. 5,7-Bis(N,N-diethylaminosulfonyl)-8-
3
Mp 205.0–206.0 °C. H NMR (300 MHz, CDCl /TMS): 2.74 (3H, d,
methoxyquinaldine (39)
,7-Bis(N,N-diethylaminosulfonyl)-8-methoxyquinaldine (39)
was prepared as colorless crystals (23.9 mg, 54% yield) from 29
46.6 mg, 0.10 mmol) and MeI (62.3 L, 1.0 mmol) in a manner
similar to that described for 9.
Mp 117.0–118.0 °C. 1H NMR (300 MHz, CDCl
.22 (12H, m), 2.81 (3H, s), 3.39–3.46 (8H, m), 4.43 (3H, s), 7.52
1H, d, J = 8.7 Hz), 8.49 (1H, s), 8.93 (1H, d, J = 8.7 Hz). 13C NMR
75 MHz, CDCl ): d = 13.8, 14.3, 41.2, 42.1, 120.5, 124.5, 125.9,
26.1, 128.7, 134.6, 138.1, 153.6, 159.3. IR (neat): 3336, 2979,
J = 5.1 Hz), 2.81 (3H, d, J = 5.1 Hz), 2.83 (3H, s), 4.06 (3H, s), 4.66
5
(1H, br), 5.80 (1H, br), 7.61 (1H, d, J = 8.7 Hz), 8.68 (1H, s), 9.02
1
3
(1H, d, J = 9.0 Hz).
6
C NMR (75 MHz, DMSO-d ): 25.2, 29.5,
(
l
29.7, 42.1, 127.2, 127.4, 128.4, 132.4, 134.5, 134.8, 143.0,
148.1, 163.4. IR (neat): 3368, 3281, 1594, 1495, 1353, 1331,
ꢀ1
+
3
/TMS): d = 1.12–
1169, 1151, 1078, 968, 796, 953, 559 cm . HRMS (FAB ) Calcd
1
(
(
1
1
for C13
for C13
H, 3.81; N, 9.89.
H
H
18
N
3
O
7
S
3
(M+H) 424.0307: found 424.0309. Anal. Calcd
: C, 36.87; H, 4.05; N, 9.92. Found: C, 36.73;
17
N
3
O
7
S
3
3
ꢀ1
+
463, 1316, 11134, 1009, 719, 615, 556 cm . HRMS (FAB ); Calcd
4.1.33. 5,7-Bis(N,N-dimethylaminosulfonyl)-8-
for C19
Calcd for C19
H
30
N
3
O
H
5
S
2
(M+H) 444.1627: found: found 441.1628. Anal;
: C, 51.45; H, 6.59; N, 9.47. Found: C,
methanesulfonyloxyquinaldine (43)
29
N
3
O
5
S
2
5,7-bis(N,N-dimethylaminosulfonyl)-8-methanesulfonyloxy-
quinaldine (43) was prepared as colorless crystals (53.3 mg, 59%
yield) from 28 (74.6 mg, 0.20 mmol), methanesulfonyl chloride
5
1.43; H, 6.42; N, 9.53.
4
.1.30. 5,7-Bis(N,N-diethylaminosulfonyl)-8-ethoxyquinaldine
(20.2
3
lL, 0.40 mmol) and Et N (55.8 lL, 0.40 mmol) in a manner
(
40)
5
similar to that described for 20.
1
,7-Bis(N,N-diethylaminosulfonyl)-8-ethoxyquinaldine
(40)
Mp 190.0–191.0 °C. H NMR (300 MHz, CDCl
3
/TMS): 2.84 (3H, s)
was prepared as colorless crystals (118 mg, 55% yield) from 29
200 mg, 0.47 mmol) and EtI (752 L, 9.4 mmol) in a manner
similar to that described for 9.
Mp 103.0–104.0 °C. 1H NMR (300 MHz, CDCl
6H, t, J = 7.2 Hz), 1.18 (6H, t, J = 6.9 Hz), 1.63 (3H, t, J = 7.1 Hz),
.79 (3H, s), 3.42 (8H, q, J = 7.1 Hz), 4.77 (2H, q, J = 7.1 Hz), 7.51
1H, d, J = 8.7 Hz), 8.50 (1H, s), 8.92 (1H, d, J = 8.7 Hz). 13C NMR
75 MHz, CDCl ): d = 14.0, 14.4, 15.8, 25.5, 41.6, 42.2, 73.3, 124.9,
26.3, 127.4, 130.6, 131.6, 134.0, 143.2, 157.7, 159.6. IR (neat):
978, 2938, 1610, 1580, 1549, 1498, 1426, 1382, 1329, 1250,
2.90 (6H, s), 2.93 (6H, s), 3.85 (3H, s), 7.58 (1H, d, J = 8.7 Hz), 8.54
(1H, s), 9.04 (1H, d, J = 8.7 Hz). C NMR (75 MHz, DMSO-d ):
6
1
3
(
l
24.9, 37.5, 37.7, 41.7, 126.0, 126.7, 128.0, 130.0, 132.2, 133.9,
142.4, 147.8, 161.6. IR (neat): 2950, 1618, 1589, 1552, 1498,
1459, 1364, 1336, 1285, 1171, 1139, 1078, 957, 812, 772, 720,
3
/TMS): d = 1.14
(
2
ꢀ1
+
622, 554 cm . HRMS (FAB ) Calcd for C15
452.0620: found 452.0614. Anal. Calcd for C15
39.52; H, 4.57; N, 9.12. Found: C, 39.90; H, 4.69; N, 9.31.
H
22
N
3
O
7
S
3
(M+H)
(
(
1
2
1
H N O S : C,
22 3 7 3
3
4.1.34. Cell culture and c-ray irradiation
ꢀ1
201, 1141, 1092, 1016, 942, 775, 707, 618, 558 cm . HRMS
MOLT-4 cells were cultured in RPMI 1640 medium supple-
mented with 10% fetal bovine serum (FBS) and antibiotics
(100 U/ml penicillin and 0.1 mg/ml streptomycin). Cells were
maintained at 37 °C in a humidified atmosphere containing 5%
+
(
FAB ): Calcd for
C
20
H
32
3
N O
S
5 2
(M+H), 458.1783: found,
4
9
58.1783. Anal; Calcd for C20
.18. Found: C, 52.75; H, 7.03; N, 9.18.
H N O S : C, 52.50; H, 6.83; N,
31 3 5 2
2
CO and treated with HQ derivatives, Bispicen or TPEN for 1 h
4
.1.31. 5,7-Bis(N-methylaminosulfonyl)-8-methoxyquinaldine
(stock solutions of these compounds (20 mM) in DMSO were pre-
5
(
41)
TMSCHN
50 mg, 0.145 mmol) in THF (5 mL) and MeOH (5.5 mL) in Erlen-
pared and added to cell-culture medium). Next, cells (5 ꢁ 10
2
(740
lL, 0.445 mmol) was added to a solution of 27
cells/mL) in tissue culture flasks were irradiated at room tempera-
1
37
(
ture with a
Csc-ray source (Gammacell 40, Nordion Interna-
1
2,14
meyer flask, and the resulting solution was vigorously stirred for
min at room temperature in the dark. The reaction was quenched
tional) at a dose rate of 0.815 Gy/min.
1
by pouring AcOH (1 mL) into the reaction mixture. After concen-
trating the reaction mixture under reduced pressure, the resulting
residue was purified by silica gel column chromatography with
4.1.35. Dye-exclusion test using erythrosine B dye
The suspension culture of MOLT-4 cells (1 ꢁ 10 cells/mL,
100 L) were mixed with 25 of 1% erythrosine in
6
l
l
L
B

S. Ariyasu et al. / Bioorg. Med. Chem. 22 (2014) 3891–3905
3905
phosphate-buffered saline (PBS) at 18 h after
c-ray irradiation. A
References and notes
cell that lost its ability to exclude erythrosin B was defined as a
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stained cells.
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respectively.
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.3. Evaluation of radical scavenging ability by using of DPPH
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(
(
2
l
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1
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1
l
M zinc(II) chelators (or 2
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2
1
We thank Dr. Ikuo Nakanishi (Research Center for Charged Par-
ticle Therapy, National Institute of Radiological Sciences) for his
helpful discussions and Ms. Miki Miyauchi, Mr. Kei Tamura and
Mr. Yuichi Tamura for the preparation and the spectroscopic mea-
surement of some compounds. We thank Ms. Fukiko Hasegawa
Chem. 2007, 31, 927.
1. For the screening of many drug candidates, WST (water soluble tetrazolium
salts) assay (Refs. 12, 14) may be useful, in which the UV/vis absorption at
450 nm of the assay product is measured. However, an erythrosine B dye-
exclusion test was carried out in this work, because the absorption at 450 nm
of the WST assay product is overlapped with that of some 8HQ derivatives.
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(
Faculty of Pharmaceutical Sciences, Tokyo University of Science)
2
for mass spectroscopy and Ms. Tomoko Matsuo (Research Institute
for Science and Technology, Tokyo University of Science) for ele-
mental analyses. This study was supported by grants-in-aid from
the Ministry of Education, Culture, Sports, Science, and Technology
MEXT) of Japan (Nos. 23790023 for S. Ariyasu and Nos. 18390009,
9659026, 22390005, 22659005 and 24659058 for S. Aoki) and
Academic Frontier’ project for private universities: matching fund
subsidy from MEXT, 2009–2014.
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(
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