Small-molecular inhibitors of Ca2+-induced mitochondrial permeability transition (MPT) derived from muscle relaxant dantrolene

Article abstract of DOI:10.1016/j.bmc.2012.08.062

A structure consisting of substituted hydantoin linked to a 5-(halophenyl)furan-2-yl group via an amide bond was identified as a promising scaffold for development of low-molecular-weight therapeutic agents to treat vascular dysfunction, including ischemia/reperfusion injury. Among the compounds synthesized, 5-(3,5-dichlorophenyl)-N-{2,4-dioxo-3-[(pyridin-3-yl)methyl] imidazolidin-1-yl}-2-furamide (17) possessed the most potent inhibitory activity against Ca²⁺-induced mitochondrial swelling. The structural development, synthesis and structure-activity relationship of these compounds are described.

Full text of DOI:10.1016/j.bmc.2012.08.062

Bioorganic & Medicinal Chemistry 20 (2012) 6384–6393
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Small-molecular inhibitors of Ca2+-induced mitochondrial permeability
transition (MPT) derived from muscle relaxant dantrolene
Shinpei Murasawa a, Katsuya Iuchi b,c,d, Shinichi Sato a,e, Tomomi Noguchi-Yachide a, Mikiko Sodeoka b,c
,
a
f,
Tsutomu Yokomatsu f, Kosuke Dodo b,c, , Yuichi Hashimoto , Hiroshi Aoyama
⇑
⇑
a Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b Sodeoka Live Cell Chemistry Project, ERATO, JST, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
c Advanced Science Institute (ASI), RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
d Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-machi, Nakahara-ku, Kawasaki-city, Kanagawa 211-8533, Japan
e Department of Chemistry, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan
f School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2012
Revised 29 August 2012
Accepted 29 August 2012
Available online 14 September 2012
A structure consisting of substituted hydantoin linked to a 5-(halophenyl)furan-2-yl group via an amide
bond was identified as a promising scaffold for development of low-molecular-weight therapeutic agents
to treat vascular dysfunction, including ischemia/reperfusion injury. Among the compounds synthesized,
5-(3,5-dichlorophenyl)-N-{2,4-dioxo-3-[(pyridin-3-yl)methyl]imidazolidin-1-yl}-2-furamide (17) pos-
sessed the most potent inhibitory activity against Ca2+-induced mitochondrial swelling. The structural
development, synthesis and structure–activity relationship of these compounds are described.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Ischemia/reperfusion
Mitochondrial swelling
Mitochondrial permeability transition
(MPT)
Cyclophilin A
Cyclophilin D
Dantrolene
1. Introduction
of over 1200,20 so modification to isolate the CypD-inhibitory
activity would be difficult. Therefore, there is a need for low-
The mitochondrial permeability transition (MPT) is a rapid per-
meabilization of the mitochondrial inner membrane, which leads
to mitochondrial swelling, dysfunction and cell death.1–3 It is
thought to play an important role in ischemia/reperfusion injury
in a variety of pathological conditions, including trauma,4,5 heart
attack6,7 and stroke.6,8–10 MPT is a consequence of opening of the
MPT pore, and one of the components of this pore has been iden-
tified as the peptidylpropyl cis–trans isomerase (PPIase) cyclophilin
D (CypD).11 It has been reported that cyclosporin A (CsA, Fig. 1) is a
CypD inhibitor,12 and therefore CsA may inhibit MPT and show
cytoprotective effects in various diseases. Indeed, CsA was found
to ameliorate ischemia/reperfusion injury in cellular and animal
models.13–16 However, although CsA is already in clinical use as a
potent immunosuppressant, it is associated with serious adverse
effects, including neurotoxicity and nephrotoxicity.17–19 Further,
CsA is a lipophilic, cyclic undecapeptide with a molecular weight
molecular-weight CypD inhibitors as candidate drugs to treat
ischemia/reperfusion injury.
To address this issue, we focused on dantrolene (1,21 Fig. 2), an
aminohydantoin derivative condensed with a nitro-substituted
arylfuran component that has been used clinically to treat malig-
nant hyperthermia, neuroleptic malignant syndrome, heat stroke,
and muscle spasticity.22 Although dantrolene (1) primarily acts
on skeletal muscles, it also shows protective effects against ische-
mic damage to neurons and myocardial tissues.23–25 The major tar-
get of dantrolene was reported to be ryanodine receptor RyR1,26
but direct effects on mitochondria were also reported,27 suggesting
the existence of a mitochondrial target of dantrolene. On the other
hand, compound 3 (Fig. 2) was reported to show an inhibitory ef-
fect on HIV-1 replication through binding to cyclophilin
A
(CypA).28 We were struck by the similarity between the hydan-
toin-type structure of dantrolene and its major metabolite 2,29
and the oxalyl diamide structure of 3, both of which seemed anal-
ogous to parabanic acid (also known as oxalyl urea) (Fig. 3). Based
on (a) the CypA-inhibitory activity of 3,28 (b) the existence of a
mitochondrial target of dantrolene,27 (c) the reported 75% identity
⇑
Corresponding authors. Tel.: +81 48 487 8091; fax: +81 48 487 8092 (K.D.); tel.:
+81 42 676 3246; fax: +81 42 676 3243 (H.A.).
E-mail addresses: dodo@riken.jp (K. Dodo), aoyamahr@toyaku.ac.jp (H. Aoyama).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.062

S. Murasawa et al. / Bioorg. Med. Chem. 20 (2012) 6384–6393
6385
2. Results and discussion
The X-ray crystal structure of CypD in the complex with CsA,31
a
cyclic peptide consisting of hydrophobic residues, shows two char-
acteristic gorge areas around the CsA-binding site. One is sur-
rounded with hydrophobic amino acid residues such as Phe and
Leu. The other is surrounded with polar amino acid residues such
as Ser, Thr, and Gln. On the basis of the design rationale described
in the introduction, together with the above structural and chem-
ical information, we designed and synthesized a series of mole-
cules possessing chloro-substituted phenylfurans as
a
hydrophobic core bound to hydantoin at the 1-position via an imi-
no or amido linkage, and having a 3-aminobenzyl group as a
hydrogen-bond acceptor and/or donor at the 3-position of hydan-
toin (8–11). Compounds with a methyl group at the 3-position of
hydantoin (4–7) were also synthesized for comparison.
Figure 1. Structure of cyclosporin A (CsA).
The synthetic routes are shown in Schemes 1 and 2. Briefly, 3,5-
dichloroaniline was coupled with 2-furaldehyde by Meerwein ary-
lation reaction to give 5-(3,5-dichlorophenyl)-2-furaldehyde (19b).
Compound 19b and commercially available 5-(3-chlorophenyl)-2-
furaldehyde (19a) were condensed with 1-aminohydantoin hydro-
chloride. The resulting imino compounds 20a and 20b were N-
alkylated with methyl iodide or N-Boc-3-aminobenzyl bromide32
under basic conditions to give 4, 5, 21a, and 21b. Finally, deprotec-
tion of the amino group of 21a and 21b with HCl gave 8 and 9. As
for the amido-type compounds, aldehydes 19a and 19b were oxi-
dized under Pinnick oxidation conditions to give carboxylic acids
22a and 22b, which were condensed with 1-aminohydantoin
hydrochloride by using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride (DMT-MM)33 as a condensation
in the PPIase domain between CypA and CypD,30 and (d) the struc-
tural similarity of dantrolene and 3 noted above, we speculated
that dantrolene might inhibit CypD and consequently inhibit
MPT. Indeed, we found that dantrolene inhibited Ca2+-induced
mitochondrial swelling, which we employed as a surrogate of MPT.
In this paper, we describe the synthesis of a series of small-
molecular inhibitors of Ca2+-induced MPT with dantrolene-derived
structures consisting of substituted hydantoin linked to a 5-(hal-
ophenyl)furan-2-yl group via an amide bond, based on the ratio-
nale described above. We also report the results of assay of their
MPT-inhibitory activity and analysis of their structure–activity
relationship.
Figure 2. Structures of dantrolene (1), 5-hydroxydantrolene (2), and cyclophilin inhibitor (3).
Figure 3. Illustration of the design strategy employed in this study (see text).

6386
S. Murasawa et al. / Bioorg. Med. Chem. 20 (2012) 6384–6393
Scheme 1. Synthesis of hydantoin analogs with imino linkage (4, 5, 8, and 9).
Scheme 2. Synthesis of hydantoin analogs with amido linkage (6, 7, 10, and 11).
agent and N-methylmorpholine (NMM) as a base. The resulting
amido compounds 23a and 23b were N-alkylated under the same
conditions as described for the imino compounds to afford 6, 7, 10,
and 11.

S. Murasawa et al. / Bioorg. Med. Chem. 20 (2012) 6384–6393
6387
Table 1
drial swelling at 25
l
M in this assay system. Based on these results,
Effects of linkage structure (X–Y) and substituents (R1, R2, and R3) of hydantoin
analogs on the inhibition of Ca2+-induced mitochondrial swelling
inhibition by the synthesized compounds (4–11) and dantrolene
(1) was determined at 40 min after CaCl2 treatment (Table 1).
Among compounds 4–11, the 3,5-dichlorophenyl compounds all
exhibited similar or higher inhibitory activity than the 3-chloro-
phenyl compounds, suggesting that an increase of the hydropho-
bicity around the phenyl group bound to the furan ring is
favorable for the activity. As for linkage structure, among com-
pounds with a methyl group at the 3-position of hydantoin, the
imino-linked compounds tended to be more potent than the ami-
do-linked compounds. On the other hand, compounds with a 3-
aminobenzyl group at the 3-position of hydantoin exhibited the
opposite tendency, that is, the amido-linked compounds were
more potent than the imino-linked compounds. These results sug-
gest that the site to which these compounds bind can accommo-
date and interact with a polar group beyond the 3-position of
hydantoin.
Focusing on amido-linked compound 11, which showed the
greatest Ca2+-induced mitochondrial swelling-inhibitory activity
among these compounds, the effect of substituents on the phenyl
ring was examined. Several compounds halogenated on the phenyl
ring were prepared in a manner similar to that shown in Scheme 2,
with some modifications34 as shown in Scheme 3, and their mito-
chondrial swelling-inhibitory activities were measured at a fixed
Compd
R1 R2 R3
XÀY
Inhibition ratioa (%)
Dantrolene (1) 4-NO2
4
5
6
7
8
9
10
11
Na
HC@N
HC@N
HC@N
CO–NH 2.0
CO–NH 8.5
13
6.0
23
H
Cl
H
Cl
H
Cl
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CH3
CH3
CH3
CH3
m-NH2C6H4CH2 HC@N
m-NH2C6H4CH2 HC@N
14
14
m-NH2C6H4CH2 CO–NH 16
m-NH2C6H4CH2 CO–NH 35
Cl
a
The inhibitory activity of test compounds was evaluated as inhibition (%) by
measuring the change of absorbance at 540 nm in MPT assay in the presence of the
test compound at 25 M, 40 min after incubation. In the absence of test compound,
l
ÀCaCl2 is equivalent to 100% inhibition and +CaCl2 is equivalent to 0% inhibition
(see Fig. 4).
MPT in isolated mitochondria is a well-characterized Ca2+-in-
duced phenomenon that results in large-amplitude swelling,
which can be monitored based on the decrease of absorbance at
540 nm. Therefore, we adopted MPT assay to evaluate the biologi-
cal activity of our compounds. MPT-inhibitory activity was evalu-
ated in terms of Ca2+-induced mitochondrial swelling in the
presence and absence of the synthesized compounds. In isolated
mitochondria from HL-60 cells, CaCl2 induced a rapid decrease of
concentration of 50 lM (Table 2). Compounds with a bromo group
and trifluoromethyl group, 13 and 14, showed decreased activity in
comparison with the chlorinated compounds 11 and 12. In con-
trast, dehalogenated compound 15 showed almost the same activ-
ity as chlorinated compound 12. These results suggest that the
effect of steric hindrance around the phenyl ring is greater than
that of hydrophobicity.
OD540, which was inhibited by 5 lM CsA (a known MPT inhibitor),
Next, the effect of N-substituents of hydantoin was examined.
Compounds bearing an anilyl or a pyridyl group were synthesized
in a manner similar to that shown in Scheme 2, as illustrated in
indicating that this assay is suitable for detection of MPT-inhibi-
tory activity. Dantrolene (1) also inhibited Ca2+-induced mitochon-
Scheme 3. Synthesis of hydantoin analogs 12–15.

6388
S. Murasawa et al. / Bioorg. Med. Chem. 20 (2012) 6384–6393
ilar to RyR1 is expressed on the mitochondrial inner membrane.35
On the other hand, our previous structure–activity relationship
study of the inhibitory effect of dantrolene analogs on RyR1 open-
ing demonstrated that replacement of the imino bond of dantrolene
(1) with an amide bond resulted in a marked decrease of the activity
(data not shown). Thus, it seems unlikely that the MPT-inhibitory
activities of our compounds involve inhibition of mRyR function.
We are currently examining the interaction between our com-
pounds and CypD. But, whether our compounds act on CypD or
not, they represent a novel type of MPT inhibitor, and appear to
be candidate therapeutic agents for ischemia/reperfusion injury.
Table 2
Effects of halogenated phenyl group combining to furanyl group of hydantoin analogs
on the inhibition of Ca2+-induced mitochondrial swelling
Compd
R1
R2
R4
Inhibition ratioa (%)
b
11
12
13
14
15
Cl
H
H
H
H
Cl
H
Br
CF3
H
H
Cl
H
H
H
48 (35)
20
11
6.5b
19
3. Conclusion
a
The inhibitory activity of test compounds was evaluated as inhibition (%) by
measuring the change of absorbance at 540 nm in MPT assay in the presence of the
test compound at 50 M, 40 min after incubation. In the absence of test compound,
We have designed and synthesized a series of small-molecular
inhibitors of Ca2+-induced MPT based on a structural analysis of
dantrolene (1), CypA inhibitor 3 and CypD, and examined their
structure–function relationship. Among them, compound 17 was
the most potent inhibitor of Ca2+-induced MPT, its efficacy at
l
ÀCaCl2 is equivalent to 100% inhibition and +CaCl2 is equivalent to 0% inhibition
(see Fig. 4).
b
At 25 lM..
50 lM concentration being similar to that of CsA at 5 lM. Further
structure–activity relationship studies based on 17 are under way,
and work is also in progress to identify the molecular target(s) of
these compounds.
Scheme 4. The N-aminobenzylated compounds 11 and 16 showed
similar mitochondrial swelling-inhibitory activity with inhibition
rates of 46–48% (Table 3). On the other hand, N-pyridylmethylated
compounds 17 and 18 showed quite different behaviors, that is, the
3-pyridylmethyl compound 17 was a potent inhibitor, while the 4-
pyridylmethyl compound 18 was markedly less potent. The reason
for these differences among the four compounds is thought to be
related to the characteristics of the amino group. Generally speak-
ing, primary amines possess both hydrogen bond-donating and -
accepting abilities, while tertiary amines, including heterocyclic
aromatic amine such as pyridine, act only as hydrogen bond accep-
tors. Thus, in the case of aminobenzylated 11 and 16, hydrogen
bond-donating ability of the amino group may predominate,
whereas 17 and 18 possess only hydrogen bond-accepting ability.
From the viewpoint of interaction of the compounds with amino
acid residues of their binding site, interaction via hydrogen bond
donation is rather non-specific, whereas hydrogen bond-accepting
interaction is much more specific. This may explain why the 3-pyr-
idylmethyl compound 17 showed the most potent inhibitory activ-
ity (71% after 40 min incubation).
4. Experimental
4.1. Chemistry
4.1.1. General
1H NMR spectra were recorded on a JEOL JNMGX500 (500 MHz)
spectrometer. Chemical shifts (d) are expressed in parts per million
(ppm) relative to chloroform or tetramethylsilane as an internal ref-
erence. Coupling constants are given in Hz. The abbreviations s, d, t,
dd, dt, br, br s, and m signify singlet, doublet, triplet, double doublet,
double triplet, broad, broad singlet, and multiplet, respectively. Fast
atom bombardment mass spectra (FAB-MS) and high-resolution
mass spectra (HRMS) were recorded on a JEOL JMS-DX303 spec-
trometer with m-nitrobenzyl alcohol matrix. Melting points (Mp)
were determined by using a Yanagimoto hot-stage melting point
apparatus. Elemental analyses were carried out in the Microanalyt-
ical Laboratory, Faculty of Pharmaceutical Science, The University
of Tokyo, and were within 0.4% of theoretical values.
Finally, we compared the effect of 17 with that of CsA. As shown
in Figure 4 and 17 at 50
lM showed similar inhibitory activity to
CsA at 5 M. Thus, the MPT-inhibitory activity of dantrolene has
been greatly increased by the structural conversion to 17.
l
4.1.2. Synthesis of 5-aryl-2-furaldehydes by Meerwein arylation
reaction
The mechanism of action of these compounds remains to be
examined. Although the design of our compounds, including 17,
was partly based on the idea that they might inhibit MPT via inhi-
bition of CypD, we cannot rule out the possibility that they have a
different target(s). This possibility may be supported by the unex-
pected finding that one of the lead compounds, the CypA inhibitor
3, did not show MPT-inhibitory activity (data not shown). For
example, it has recently been reported that a channel (also known
as mitochondrial ryanodine receptor, mRyR) having a function sim-
4.1.2.1. 5-(3,5-Dichlorophenyl)-2-furaldehyde (19b): General
procedure A.
A mixture of 3,5-dichloroaniline (1.94 g,
12.0 mmol) and 2 N HCl (45 mL) was heated at 110 °C to get a clear
solution. This was cooled to 0 °C, and to it was added a solution of
NaNO2 (829 mg, 12.0 mmol) in cold H2O (3.0 mL) at 0 °C. The mix-
ture was stirred for 20 min at 0 °C, then a solution of 2-furaldehyde
(1.15 g, 12.0 mmol) in cold acetone (3.0 mL) was added to it at 0 °C.
Stirring was continued for 15 min at 0 °C, then a solution of CuCl2
(408 mg, 2.40 mmol) in cold H2O (3.0 mL) was added at 0 °C. After
Scheme 4. Synthesis of hydantoin analogs 16–18.

S. Murasawa et al. / Bioorg. Med. Chem. 20 (2012) 6384–6393
6389
Table 3
4.1.2.3. 5-(3-Bromophenyl)-2-furaldehyde (19d).
Prepared
Effects of aminobenzyl and azabenzyl group substituted at 1-position of hydantoin
from 3-bromoaniline in accordance with procedure A. Orange oil
(8%); 1H NMR (500 MHz, DMSO-d6) d 9.66 (1H, s) 7.95 (1H, t,
J = 1.8 Hz), 7.73 (1H, dd, J = 7.8, 1.8 Hz), 7.50 (1H, dt, J =7.8,
1.8 Hz), 7.31 (1H, d, J = 3.6 Hz), 7.30 (1H, t, J = 7.8 Hz), 6.84 (1H,
d, J = 3.6 Hz); MS (FAB) m/z 251 (M+H)+.
analogs on the inhibition of Ca2+-induced mitochondrial swelling
4.1.3. Synthesis of 5-aryl-2-furaldehydes by Suzuki–Miyaura
cross-coupling reaction
4.1.3.1. 5-(4-Chlorophenyl)-2-furaldehyde (19e): General proce-
Compd
Ar
Inhibition ratioa (%)
48
11
dure B.
2.50 mmol) in a mixture of DME (7.5 mL) and EtOH (5.0 mL) were
added PdCl2(PPh3)2 (36.5 mg, 52.0 mol), 5-formyl-2-furanboronic
To a solution of 1-bromo-4-chlorobenzene (478 mg,
l
16
46
acid (365 mg, 2.61 mmol), and 2 M aqueous Na2CO3 solution. The
reaction mixture was stirred for 2 h at 60 °C, then cooled to room
temperature, and volatile materials were removed under reduced
pressure. To the obtained residue was added H2O and the resulting
mixture was extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy (hexane/EtOAc = 5:1) to afford 19e (323 mg, 63%) as a yellow
solid. 1H NMR (500 MHz, CDCl3) d 9.64 (1H, s), 7.73 (2H, d,
J = 8.5 Hz), 7.40 (2H, d, J = 8.5 Hz), 7.30 (1H, d, J = 3.6 Hz), 6.81
(1H, d, J = 3.6 Hz); MS (FAB) m/z 207 (M+H)+.
17
18
71
8.7
a
The inhibitory activity of test compounds was evaluated as inhibition (%) by
measuring the change of absorbance at 540 nm in MPT assay in the presence of the
test compound at 50 M, 40 min after incubation. In the absence of test compound,
l
ÀCaCl2 is equivalent to 100% inhibition and +CaCl2 is equivalent to 0% inhibition
(see Fig. 4).
4.1.3.2. 5-Phenyl-2-furaldehyde (19f).
Prepared from bro-
mobenzene in accordance with procedure B. Orange oil (75%); 1H
NMR (500 MHz, DMSO-d6) d 9.64 (1H, s), 7.81 (2H, d, J = 7.3 Hz),
7.45–7.36 (3H, m), 7.30 (1H, d, J = 3.6 Hz), 6.83 (1H, d, J = 3.6 Hz);
MS (FAB) m/z 173 (M+H)+.
4.1.4. Synthesis of 5-aryl-2-furancarboxylic acid by Pinnick
oxidation reaction
4.1.4.1. 5-(3-Chlorophenyl)-2-furancarboxylic acid (22a): Gen-
eral procedure C.
To a solution of 5-(3-chlorophenyl)-2-fur-
aldehyde (19a) (206 mg, 1.00 mol) in a mixture of tert-BuOH
(37.5 mL) and H2O (7.5 mL) was added NaH2PO4 (213 mg,
1.50 mmol), 2-methyl-2-butene (0.78 mL, 7.50 mmol), and NaClO2
(228 mg, 2.50 mmol) and the mixture was stirred for 11 h at room
temperature. The volatile materials were removed under reduced
pressure, and then H2O and 1 N HCl were added. The mixture
was extracted with EtOAc. The combined organic layer was washed
with brine, dried over MgSO4, and concentrated to afford 22a
(214 mg, 97%) as a white solid. 1H NMR (500 MHz, CDCl3) d 7.77
(1H, s) 7.66 (1H, dd, J = 7.2, 1.8 Hz), 7.38 (1H, d, J = 3.6 Hz), 7.35
(1H, t, J = 7.3 Hz), 7.32 (1H, dd, J = 7.3, 1,8 Hz), 6.79 (1H, d,
J = 3.6 Hz); MS (FAB) m/z 223 (M+H)+.
Figure 4. Mitochondrial swelling induced by CaCl2 (250
presence of CsA (5 M), 11 and 17 (50 M).
lM) in the absence or
l
l
4.1.4.2.
(22b).
5-(3,5-Dichlorophenyl)-2-furancarboxylic
acid
Prepared from 19b in accordance with procedure C.
further stirring for 14 h at room temperature, the reaction mixture
was filtered and extracted with EtOAc. The organic layer was
washed with saturated aqueous NaHCO3 solution and brine, dried
over MgSO4, and concentrated. The resulting residue was purified
by silica gel chromatography (hexane/EtOAc = 15:1) to afford 19b
(920 mg, 32%) as a yellow solid. 1H NMR (DMSO-d6, 500 MHz) d
9.64 (1H, s), 7.91 (2H, d, J = 1.8 Hz), 7.69 (1H, t, J = 1.8 Hz), 7.67
(1H, d, J = 4.2 Hz), 7.52 (1H, d, J = 4.2 Hz); MS (FAB) m/z 241
(M+H)+.
White solid (96%); 1H NMR (500 MHz, CDCl3) d 7.65 (2H, d,
J = 1.8 Hz), 7.37 (1H, d, J = 3.6 Hz), 7.33 (1H, t, J = 1.8 Hz), 6.81
(1H, d, J = 3.6 Hz); MS (FAB); m/z 257 (M+H)+.
4.1.4.3. 5-[3-(Trifluoromethyl)phenyl]-2-furancarboxylic acid
(22c).
Prepared from 19c in accordance with procedure C.
White solid (99.7%); 1H NMR (500 MHz, CDCl3) d 8.00 (1H, s)
7.97 (1H, d, J = 7.9 Hz), 7.60 (1H, d, J = 7.9 Hz), 7.55 (1H, t,
J = 7.9 Hz), 7.39 (1H, d, J = 3.6 Hz), 6.85 (1H, d, J = 3.6 Hz); MS
(FAB) m/z 257 (M+H)+.
4.1.2.2.
(19c).
5-[3-(Trifluoromethyl)phenyl]-2-furaldehyde
Prepared from 3-(trifluoromethyl)aniline in accordance
with procedure A. Yellow solid (91%); 1H NMR (500 MHz, DMSO-
d6) d 9.67 (1H, s), 8.03 (1H, s), 7.98 (1H, d, J = 7.9 Hz), 7.63 (1H, d,
J = 7.9 Hz), 7.56 (1H, t, J = 7.9 Hz), 7.32 (1H, d, J = 3.6 Hz), 6.91
(1H, d, J = 3.6 Hz); MS (FAB) m/z 241 (M+H)+.
4.1.4.4.
(22d).
5-(3-Bromophenyl)-2-furancarboxylic
Prepared from 19d in accordance with procedure C.
acid
White solid (quant.); 1H NMR (500 MHz, CDCl3) d 7.93 (1H, t,
J = 1.8 Hz), 7.71 (1H, dd, J = 7.9, 1.2 Hz), 7.48 (1H, dd, J = 7.9,

6390
S. Murasawa et al. / Bioorg. Med. Chem. 20 (2012) 6384–6393
1.2 Hz), 7.39 (1H, d, J = 3.6 Hz), 7.29 (1H, t, J = 7.9 Hz), 6.79 (1H, d,
J = 3.6 Hz); MS (FAB) m/z 267 (M+H)+.
J = 3.7 Hz), 7.39 (1H, d, J = 3.7 Hz), 4.18 (2H, s); MS (FAB) m/z 354
(M+H)+.
4.1.4.5.
(22e).
5-(4-Chlorophenyl)-2-furancarboxylic
acid
4.1.6.4. 5-(3-Bromophenyl)-N-(2,4-dioxoimidazolidin-1-yl)-2-
Prepared from 19e in accordance with procedure C.
furamide (23d).
Prepared from 22d in accordance with pro-
White solid (80%); 1H NMR (500 MHz, CDCl3) d 7.72 (2H, d,
J = 8.5 Hz), 7.40 (2H, d, J = 8.5 Hz), 7.37 (1H, d, J = 3.6 Hz), 6.76
(1H, d, J = 3.6 Hz); MS (FAB) m/z 223 (M+H)+.
cedure E. White solid (51%); 1H NMR (500 MHz, methanol-d4) d
8.11 (1H, s), 7.83 (1H, d, J = 7.9 Hz), 7.53 (1H, d, J = 7.9 Hz), 7.36
(1H, t, J = 7.9 Hz), 7.32 (1H, d, J = 3.6 Hz), 7.05 (1H, d, J = 3.6 Hz),
4.21 (2H, s); MS (FAB) m/z 364 (M+H)+.
4.1.4.6. 5-Phenyl-2-furancarboxylic acid (22f).
Prepared
from 19f in accordance with procedure C. White solid (94%); 1H
NMR (500 MHz, CDCl3) d 7.79 (2H, d, J = 7.3 Hz), 7.44–7.34 (4H,
m), 6.77 (1H, d, J = 3.6 Hz); MS (FAB) m/z 189 (M+H)+.
4.1.6.5. 5-(4-Chlorophenyl)-N-(2,4-dioxoimidazolidin-1-yl)-2-
furamide (23e).
Prepared from 22e in accordance with proce-
dure E. White solid (89%); 1H NMR (500 MHz, DMSO-d6) d 11.32
(1H, s), 10.95 (1H, s), 7.97 (2H, d, J = 8.4 Hz), 7.56 (2H, d,
J = 8.4 Hz), 7.36 (1H, d, J = 3.6 Hz), 7.23 (1H, d, J = 3.6 Hz), 4.15
(2H, s); MS (FAB) m/z 320 (M+H)+.
4.1.5. Synthesis of 5-aryl-2-furaldimines
4.1.5.1. 1-{[5-(3-Chlorophenyl)furfurylidene]amino}imidazoli-
dine-2,4-dione (20a): General procedure D.
of 5-(3-chlorophenyl)-2-furaldehyde (19a) (127 mg, 500
DMF (1.0 mL) was added 1-aminohydantoin hydrochloride
(79.4 mg, 525 mol) and the mixture was stirred for 5 h at room
temperature. The reaction was quenched by the addition of H2O,
and the resulting precipitate was collected by filtration with suc-
tion and washed with H2O on a funnel. The collected solid was
dried in vacuo to afford 20a (166 mg, 90%) as a pale yellow solid.
1H NMR (500 MHz, DMSO-d6) d 11.28 (1H, s), 7.82 (2H, d,
J = 1,8 Hz), 7.73 (1H, d, J = 7.9 Hz), 7.73 (1H, s), 7.49 (1H, d,
J = 7.9 Hz), 7.40 (1H, dd, J = 7.9, 1.8 Hz), 7.26 (1H, J = 3.6 Hz), 6.97
(1H, J = 3.6 Hz), 4.35 (2H, s); MS (FAB) m/z 304 (M+H)+.
To a solution
lmol) in
4.1.6.6.
(23f).
5-Phenyl-N-(2,4-dioxoimidazolidin-1-yl)-2-furamide
Prepared from 22f in accordance with procedure E.
l
White solid (72%); 1H NMR (500 MHz, DMSO-d6) d 11.31 (1H, s),
10.90 (1H, s), 7.93 (2H, d, J = 7.9 Hz), 7.48 (2H, dd, J = 7.9, 7.3 Hz),
7.39 (1H, t, J = 7.3 Hz), 7.36 (1H, d, J = 3.7 Hz), 7.18 (1H, d,
J = 3.7 Hz), 4.16 (2H, s); MS (FAB) m/z 286 (M+H)+.
4.1.7. N-Alkylation of hydantoin with NaH as a base
4.1.7.1. 1-{[5-(3-Chlorophenyl)furfurylidene]amino}-3-methy-
limidazolidine-2,4-dione (4): General procedure F.
solution of 20a (39.8 mg, 131 mol) in DMF (1.0 mL) was added
To a
l
NaH (6.3 mg, 0.14 mmol) at 0 °C, and the mixture was stirred for
4.1.5.2. 1-{[5-(3,5-Dichlorophenyl)furfurylidene]amino}imidaz-
10 min at room temperature. It was cooled to 0 °C, then iodometh-
olidine-2,4-dione (20b).
Prepared from 19b in accordance
ane (16.5 lL, 262 lmol) was added to it and stirring was continued
with procedure D. Pale orange solid (88%); 1H NMR (500 MHz,
DMSO-d6) d 11.30 (1H, s), 7.79 (2H, d, J = 1,8 Hz), 7.73 (1H, s),
7.57 (1H, t, J = 1,8 Hz), 7.38 (1H, d, J = 4.2 Hz), 6.98 (1H,
J = 4.2 Hz), 4.32 (2H, s); MS (FAB) m/z 338 (M+H)+.
for 6 h at room temperature. The reaction was quenched by the
addition of H2O, and the resulting precipitate was collected by fil-
tration with suction and washed with H2O on a funnel. The col-
lected solid was recrystallized from EtOH to afford 4 (5.6 mg,
13%) as a yellow solid. Mp 236–237 °C; 1H NMR (500 MHz,
DMSO-d6) d 7.82 (1H, d, J = 1.8 Hz), 7.79 (1H, s), 7.73 (1H, d,
J = 7.9 Hz), 7.49 (1H, t, J = 7.9 Hz), 7.40 (1H, dd, J = 7.9, 1.2 Hz),
7.26 (1H, d, J = 3.6 Hz), 6.98 (1H, d, J = 3.6 Hz), 4.37 (2H, s), 2.93
(3H, s); MS (FAB) m/z 318 (M+H)+; HRMS (FAB) calcd for
C15H13ClN3O3 318.0645, found 318.0652 (M+H)+.
4.1.6. Synthesis of 5-aryl-2-furamides
4.1.6.1. 5-(3-Chlorophenyl)-N-(2,4-dioxoimidazolidin-1-yl)-2-
furamide (23a): General procedure E.
methylmorpholine (14.5 L, 115
mol), DMT-MM33 (54.0 mg,
192 mol), and 22a (21.4 mg, 96.1 mol) in MeOH (1.0 mL) was
added a solution of 1-aminohydantoin hydrochloride (16.0 mg,
105 mol) in H2O (0.1 mL), and the mixture was stirred at room
To a mixture of N-
l
l
l
l
4.1.7.2.
1-{[5-(3,5-Dichlorophenyl)furfurylidene]amino}-3-
Prepared from 20b in
l
methylimidazolidine-2,4-dione (5).
temperature. Stirring was continued for 19 h, then H2O was added
to the reaction mixture, and the whole was extracted with EtOAc.
The organic layer was washed with brine, dried over MgSO4, and
concentrated. The resulting residue was purified by silica gel chro-
matography (hexane/EtOAc = 1:1) to afford 23a (17.2 mg, 56%) as a
white solid. 1H NMR (500 MHz, DMSO-d6) d 11.33 (1H, s) 10.99
(1H, s), 8.10 (1H, d, J = 1.8 Hz), 7.90 (1H, d, J = 7.9 Hz), 7.50 (1H, t,
J = 7.9 Hz), 7.44 (1H, dd, J = 7.9, 1.8 Hz), 7.36 (1H, d, J = 3.6 Hz),
7.30 (1H, d, J = 3.6 Hz); 4.16 (2H, s), MS (FAB) m/z 320 (M+H)+.
accordance with procedure F. Pale yellow solid (50%); Mp 191–
192 °C; 1H NMR (500 MHz, DMSO-d6) d 7.80 (1H, s), 7.79 (2H, d,
J = 1.8 Hz), 7.58 (1H, d, J = 1,8 Hz), 7.39 (1H, d, J = 3.6 Hz), 7.01
(1H, d, J = 3.6 Hz), 4.37 (2H, s), 2.93 (3H, s); MS (FAB) m/z 352
(M+H)+ Anal. Calcd for C15H11Cl2N3O3 1/5H2O; C, 50.64; H, 3.23;
.
N, 11.81. Found: C, 50.63; H, 3.19; N, 11.67.
4.1.7.3.
5-(3,5-Dichlorophenyl)-N-{2,4-dioxo-3-[(pyridin-3-
(17). Prepared
yl)methyl]imidazolidin-1-yl}-2-furamide
from 23b and 3-(chloromethyl)pyridine hydrochloride instead of
iodomethane in accordance with procedure F. White solid (47%);
Mp 254–254 °C; 1H NMR (500 MHz, CDCl3) d 8.91 (1H, s), 8.67
(1H, s), 8.54 (1H, d, J = 4.8 Hz), 7.74 (1H, d, J = 7.8 Hz), 7.50 (2H,
d, J = 1.8 Hz), 7.32 (1H, t, J = 1.8 Hz), 7.27 (1H, dd, J = 7.8, 4.8 Hz),
7.23 (1H, d, J = 3.6 Hz), 6.76 (1H, d, J = 3.6 Hz), 4.76 (2H, s), 4.23
(2H, s); MS (FAB) m/z 445 (M+H)+ Anal. Calcd for C20H14Cl2N4O4:
C, 53.95; H, 3.17; N, 12.58. Found: C, 53.66; H, 3.21; N, 12.30.
4.1.6.2. 5-(3,5-Dichlorophenyl)-N-(2,4-dioxoimidazolidin-1-yl)-
2-furamide (23b).
Prepared from 22b in accordance with
procedure E. White solid (75%); 1H NMR (500 MHz, DMSO-d6) d
11.35 (1H, s) 11.00 (1H, s), 8.10 (2H, d, J = 1.8 Hz), 7.63 (1H, t,
J = 1.8 Hz), 7.43 (1H, d, J = 3.6 Hz), 7.38 (1H, d, J = 3.6 Hz), 4.17
(2H, s); MS (FAB) m/z 354 (M+H)+.
4.1.6.3. 5-[3-(Trifluoromethyl)phenyl]-N-(2,4-dioxoimidazoli-
din-1-yl)-2-furamide (23c).
Prepared from 22c in accordance
4.1.8. N-Alkylation of hydantoin with carbonates as base
4.1.8.1. 5-(3-Chlorophenyl)-N-(2,4-dioxo-3-methylimidazolidin-
with procedure E. White solid (52%); 1H NMR (500 MHz, DMSO-d6)
d 11.35 (1H, s), 11.06 (1H, s), 8.35 (1H, s), 8.24 (1H, d, J = 7.3 Hz),
7.75 (1H, d, J = 8.5 Hz), 7.72 (1H, dd, J = 8.5, 7.3 Hz), 7.40 (1H, d,
1-yl)-2-furamide (6): General procedure G.
To a solution of
23a (25.5 mg, 80.0 mol) in DMF (1.0 mL) was added K2CO3
l

S. Murasawa et al. / Bioorg. Med. Chem. 20 (2012) 6384–6393
6391
(6.2 mg, 45
ture was stirred for 10 min at room temperature. To this was added
iodomethane (5.2 L, 84 mol) at 0 °C, and the whole was stirred
l
mol) at 0 °C under an argon atmosphere, and the mix-
procedure G. White solid (34%); 1H NMR (500 MHz, CDCl3) d 9.35
(1H, s), 7.78 (1H, s), 7.77 (1H, d, J = 7.9 Hz), 7.56 (1H, d,
J = 7.9 Hz), 7.43 (1H, t, J = 7.9 Hz), 7.35 (1H, br s), 7.34 (1H, s),
7.18 (1H, t, J = 7.9 Hz), 7.04 (1H, d, J = 3.6 Hz), 7.00 (1H, d,
J = 7.9 Hz), 6.68 (1H, s), 6.64 (1H, d, J = 3.6 Hz), 4.68 (2H, s), 4.09
(2H, s), 1.48 (9H, s); MS (FAB) m/z 558 (M)+.
l
l
for 4 h at room temperature. The reaction was quenched by the
addition of H2O and the resulting mixture was extracted with
EtOAc. The organic layer was washed with H2O and brine, dried
over MgSO4, and concentrated under reduced pressure. The residue
was purified by silica gel chromatography (hexane/EtOAc = 3:2) to
afford 6 (12.1 mg, 45%) as a white solid. Mp 228–229 °C; 1H NMR
(500 MHz, CDCl3) d 9.05 (1H, s), 7.70 (1H, d, J = 1.8 Hz), 7.32 (1H,
dd, J = 7.2, 1.8 Hz), 7.38–7.33 (2H, m) 7.19 (1H, d, J = 3.6 Hz), 6.71
(1H, d, J = 3.6 Hz), 4.24 (2H, s), 3.15 (3H, s); MS (FAB) m/z 334
(M+H)+; HRMS (FAB) calcd for C15H13ClN3O4 334.0595, found
334.0601 (M+H)+.
4.1.8.8. 5-(3-Bromophenyl)-N-{3-[3-(tert-butoxycarbonylami-
no)benzyl]-2,4-dioxoimidazolidin-1-yl}-2-furamide
(24d).
Prepared from 23d with 3-(tert-butoxycarbonylami-
no)benzyl bromide32 and Na2CO3 instead of iodomethane and
K2CO3 in accordance with procedure G. Pale pink solid (27%); 1H
NMR (500 MHz, CDCl3) d 9.04 (1H, s) 7.76 (1H, t, J = 1.8 Hz), 7.55
(1H, d, J = 7.3, Hz), 7.43 (1H, d, J = 7.9, Hz), 7.38 (1H, d, J = 7.9,
Hz), 7.30 (1H, s), 7.24–7.19 (2H, m), 7.11 (1H, d, J = 3.6 Hz), 7.02
(1H, d, J = 7.3 Hz), 6.64 (1H, s), 6.63 (1H, d, J = 3.6 Hz), 4.69 (2H,
s), 4.12 (2H, s), 1.49 (9H, s); MS (FAB) m/z 569 (M+H)+.
4.1.8.2. 5-(3,5-Dichlorophenyl)-N-(2,4-dioxo-3-methylimidaz-
olidin-1-yl)-2-furamide (7).
Prepared from 23b in accor-
dance with procedure G. White solid (15%); Mp 226–228 °C; 1H
NMR (500 MHz, CDCl3) d 9.05 (1H, s), 7.54 (2H, d, J = 7.3 Hz), 7.32
(1H, t, J = 1.8 Hz), 7.13 (1H, d, J = 3.6 Hz), 6.70 (1H, d, J = 3.6 Hz),,
4.22 (2H, s), 3.13 (3H, s); MS (FAB) m/z 367 (M+H)+; HRMS (FAB)
calcd for C15H12Cl2N3O4 368.0205, found 368.0209 (M+H)+.
4.1.8.9. N-{3-[3-(tert-Butoxycarbonylamino)benzyl]-2,4-dioxo-
imidazolidin-1-yl}-5-(4-chlorophenyl)-2-furamide
(24e).
Prepared from 23e with 3-(tert-butoxycarbonylami-
no)benzyl bromide32 and Na2CO3 instead of iodomethane and
K2CO3 in accordance with procedure G. White solid (45%); 1H
NMR (500 MHz, CDCl3) d 8.69 (1H, s), 7.57 (1H, d, J = 8.5 Hz), 7.39
(1H, d, J = 6.7 Hz), 7.37–7.30 (4H, m), 7.25–7.23 (1H, m), 7.16
(1H, t, J = 3.6 Hz), 7.03 (1H, d, J = 6.7 Hz), 6.64 (1H, d, J = 3.6 Hz),
6.55 (1H, s), 4.70 (2H, s), 4.16 (2H, s), 1.49 (9H, s); MS (FAB) m/z
525 (M+H)+.
4.1.8.3. 3-[3-(tert-Butoxycarbonylamino)benzyl]-1-{[5-(3-chlo-
rophenyl)furfurylidene]amino}imidazolidine-2,4-dione
(21a).
Prepared from 20a and 3-(tert-butoxycarbonylami-
no)benzyl bromide32 instead of iodomethane in accordance with
procedure G. Pale yellow solid (98%); 1H NMR (500 MHz, CDCl3) d
7.96 (1H, s) 7.70 (2H, t, J = 1.8 Hz), 7.59 (1H, d, J = 7.3 Hz), 7.42 (1H,
d, J = 7.3 Hz), 7.31 (1H, t, J = 7.9 Hz), 7.25 (2H, m), 7.09 (1H, d,
J = 7.9 Hz), 6.88 (1H, d, J = 3.6 Hz), 6.75 (1H, d, J = 3.6 Hz), 6.45 (1H,
s), 4.70 (2H, s), 4.24 (2H, s), 1.48 (9H, s); MS (FAB) m/z 509 (M+H)+.
4.1.8.10. N-{3-[3-(tert-Butoxycarbonylamino)benzyl]-2,4-dioxo-
imidazolidin-1-yl}-5-phenyl-2-furamide
(24f).
Prepared
from 23f with 3-(tert-butoxycarbonylamino)benzyl bromide32
and Na2CO3 instead of iodomethane and K2CO3 in accordance with
procedure G. White solid (47%); 1H NMR (500 MHz, DMSO-d6) d
11.04 (1H, s), 9.38 (1H, s), 8.31 (1H, s), 7.93 (1H, d, J = 7.9 Hz),
7.50–7.47 (3H, m), 7.40 (1H, t, J = 7.9 Hz), 7.37 (1H, d, J = 3.6 Hz),
7.33 (1H, d, J = 7.9 Hz), 7.22 (1H, t, J = 7.9 Hz), 7.18 (1H, d,
J = 3.6 Hz), 6.88 (1H, d, J = 7.9), 4.58 (2H, s), 4.28 (2H, s), 1.46 (9H,
s); MS (FAB) m/z 491 (M+H)+.
4.1.8.4.
dichlorophenyl)furfurylidene]amino}imidazolidine-2,4-dione
(21b). Prepared from 20b and 3-(tert-butoxycarbonylami-
3-[3-(tert-Butoxycarbonylamino)benzyl]-1-{[5-(3,5-
no)benzyl bromide32 instead of iodomethane in accordance with
procedure G. White solid (85%); 1H NMR (500 MHz, CDCl3) d 7.96
(1H, s) 7.58 (2H, d, J = 1.8 Hz), 7.42–7.38 (1H, m), 7.31 (1H, s), 7.26
(1H, d, J = 1.8 Hz), 7.43 (1H, d, J = 7.3 Hz), 7.09 (1H, d, J = 7.3 Hz),
6.89 (1H, d, J = 3.6 Hz), 6.77 (1H, d, J = 3.6 Hz), 6.45 (1H, s), 4.70
(2H, s), 4.24 (2H, s), 1.48 (9H, s); MS (FAB) m/z 543 (M+H)+.
4.1.8.11. N-{3-[4-(tert-Butoxycarbonylamino)benzyl]-2,4-dioxo-
imidazolidin-1-yl}-5-(3,5-dichlorophenyl)-2-furamide
(24g).
Prepared from 23b with 4-(tert-butoxycarbonylami-
no)benzyl bromide36 and Na2CO3 instead of iodomethane and
K2CO3 in accordance with procedure G. White solid (32%); 1H
NMR (500 MHz, DMSO-d6) d 11.18 (1H, s), 9.35 (1H, s), 8.10 (2H,
d, J = 1.8 Hz), 7.63 (1H, t, J = 1.8 Hz), 7.43 (1H, d, J = 3.6 Hz), 7.41
(1H, br s), 7.39 (2H, d, J = 3.6 Hz), 7.18 (2H, d, J = 3.6 Hz), 4.57
(2H, s), 4.28 (2H, s), 1.46 (9H, s); MS (FAB) m/z 558 (M+H)+, 581
(M+Na)+.
4.1.8.5. N-{3-[3-(tert-Butoxycarbonylamino)benzyl]-2,4-dioxo-
imidazolidin-1-yl}-5-(3-chlorophenyl)-2-furamide
(24a).
Prepared from 23a and 3-(tert-butoxycarbonylami-
no)benzyl bromide32 instead of iodomethane in accordance with
procedure G. White solid (40%); 1H NMR (500 MHz, CDCl3) d 8.30
(1H, s) 7.66 (1H, d, J = 1.8 Hz), 7.55 (1H, dd, J = 7.3, 1.8 Hz), 7.40
(1H, m), 7.34 (1H, t, J = 7.3 Hz), 7.32 (1H, s), 7.34–7.22 (3H, m),
7.04 (1H, d, J = 7.3 Hz), 6.75 (1H, d, J = 3.6 Hz), 6.51 (1H, s), 4.70
(2H, s), 4.20 (2H, s), 1.49 (9H, s); MS (FAB) m/z 525 (M+H)+.
4.1.8.12.
5-(3,5-Dichlorophenyl)-N-{2,4-dioxo-3-[(pyridin-4-
(18). Prepared
yl)methyl]imidazolidin-1-yl}-2-furamide
4.1.8.6. N-{3-[3-(tert-Butoxycarbonylamino)benzyl]-2,4-dioxo-
imidazolidin-1-yl}-5-(3,5-dichlorophenyl)-2-furamide
from 23b with 4-(bromomethyl)pyridine and Na2CO3 instead of
iodomethane and K2CO3 in accordance with procedure G. White
solid (28%); Mp 275–277 °C; 1H NMR (500 MHz, DMSO-d6) d
11.26 (1H, s), 8.55 (2H, d, J = 5.4 Hz), 8.10 (2H, d, J = 1.8 Hz), 7.63
(1H, t, J = 1.8 Hz), 7.44 (1H, d, J = 3.6 Hz), 7.42 (1H, d, J = 3.6 Hz),
7.30 (2H, d, J = 5.4 Hz), 4.71 (2H, s), 4.36 (2H, s); MS (FAB) m/z
445(M+H)+; HRMS (FAB) calcd for C20H15Cl2N4O4 445.0470, found
445.0469 (M+H)+.
(24b).
Prepared from 23b and 3-(tert-butoxycarbonylami-
no)benzyl bromide32 instead of iodomethane in accordance with
procedure G. White solid (38%); 1H NMR (500 MHz, CDCl3) d 9.24
(1H, s) 7.51 (1H, s), 7.50 (1H, s), 7.34 (1H, s), 7.32 (1H, s), 7.29
(1H, t, J = 7.9 Hz), 7.18 (1H, t, J = 7.9 Hz), 7.11 (1H, d, J = 3.6 Hz),
7.00 (1H, d, J = 7.9 Hz), 6.67 (1H, s), 6.65 (1H, d, J = 3.6 Hz), 4.69
(2H, s), 4.11 (2H, s), 1.48 (9H, s); MS (FAB) m/z 558 (M)+.
4.1.9. Deprotection of Boc-protected aminobenzyl group on
hydantoin (1)
4.1.8.7. N-{3-[3-(tert-Butoxycarbonylamino)benzyl]-2,4-dioxo-
imidazolidin-1-yl}-5-[3-(trifluoromethyl)phenyl]-2-furamide
4.1.9.1.
ene]amino}imidazolidine-2,4-dione (8): General procedure
H. To a solution of 21a (29.1 mg, 57.1 mol) in 1,4-dioxane
3-(3-Aminobenzyl)-1-{[5-(3-chlorophenyl)furfurylid-
(24c).
Prepared from 23c and 3-(tert-butoxycarbonylami-
no)benzyl bromide32 instead of iodomethane in accordance with
l

6392
S. Murasawa et al. / Bioorg. Med. Chem. 20 (2012) 6384–6393
(1.8 mL) was added 4 M HCl in EtOAc (0.6 mL) at 0 °C. The mixture
was stirred for 20 h at room temperature, then cooled to 0 °C, and
the reaction was quenched by the addition of saturated aqueous
NaHCO3 solution. Volatile materials were removed under reduced
pressure. The remaining aqueous solution was extracted with
EtOAc, and the organic layer was washed with brine, dried over
MgSO4, and concentrated under reduced pressure. The resulting
residue was purified by silica gel chromatography (hexane/
EtOAc = 2:1) to afford 8 (15.6 mg, 67%) as a pale yellow solid. Mp
223–224 °C; 1H NMR (500 MHz, CDCl3) d 8.29 (1H, s), 7.97 (1H,
s), 7.79 (1H, s), 7.71 (1H, s), 7.68 (1H, d, J = 7.9 Hz), 7.60 (1H, t,
J = 7.9 Hz), 7.40–7.23 (3H, m), 7.19 (1H, d, J = 7.3 Hz), 7.07 (1H, s),
6.82 (1H, d, J = 3.6 Hz), 6.75 (1H, d, J = 3.6 Hz), 4.78 (2H, s), 4.27
(2H, s); MS (FAB) m/z 409 (M+H)+; HRMS (FAB) calcd for
7.51 (1H, t, J = 7.9 Hz), 7.45 (1H, dd, J = 7.9, 1.8 Hz), 7.38 (1H, d,
J = 3.6 Hz), 7.36 (1H, t, J = 7.9 Hz), 7.32 (1H, d, J = 3.6 Hz), 7.01–
6.90 (3H, m), 4.60 (2H, s), 4.28 (2H, s); MS (FAB) m/z 425 (M+H)+
Anal. Calcd for C21H18Cl2N4O4: C, 54.68; H, 3.93; N, 12.15. Found:
C, 54.91; H, 4.20; N, 11.88.
4.1.10.2. N-[3-(3-Aminobenzyl)-2,4-dioxoimidazolidin-1-yl]-5-
[3-(trifluoromethyl)phenyl]-2-furamide
(14).
hydrochloride
Prepared from 24c in accordance with procedure I. Pale
yellow solid (29%); Mp 150.0–151.0 °C; 1H NMR (500 MHz, DMSO-
d6) d 11.23 (1H, s), 8.34 (1H, s), 8.24 (1H, d, J = 7.9 Hz), 7.75 (1H, d,
J = 7.9 Hz), 7.72 (1H, t, J = 7.9 Hz), 7.41–7.37 (3H, m), 7.15 (3H, m),
4.63 (2H, s), 4.17 (2H, s); MS (FAB) m/z 459 (M+H)+ Anal. Calcd for
.
C22H18ClF3N4O4 1/4 H2O; C, 52.92; H, 3.73; N, 11.22. Found: C,
C
21H18ClN4O3 409.1067, found 409.1060 (M+H)+.
52.88; H, 3.84; N, 11.15.
4.1.9.2. 3-(3-Aminobenzyl)-1-{[5-(3,5-dichlorophenyl)furfury-
lidene]amino}imidazolidine-2,4-dione (9). Prepared from
4.1.10.3. N-[3-(3-Aminobenzyl)-2,4-dioxoimidazolidin-1-yl]-5-
(3-bromophenyl)-2-furamide hydrochloride (13).
Prepared
21b in accordance with procedure H. Pale yellow solid (47%); Mp
194.0–194.5 °C; 1H NMR (500 MHz, CDCl3) d 7.93 (1H, s), 7.58
(2H, t, J = 1.8 Hz), 7.25 (1H, d, J = 1.8 Hz), 7.08 (1H, t, J = 7.9 Hz),
6.88 (1H, d, J = 3.6 Hz), 6.82 (1H, t, J = 7.9 Hz), 6.78 (1H, d,
J = 3.6 Hz), 6.77 (1H, d, J = 1.8 Hz), 6.59 (1H, d, J = 7.9 Hz), 4.64
(2H, s), 4.23 (2H, s), 3.74–3.60 (2H, br); MS (FAB) m/z 443
(M+H)+ Anal. Calcd for C21H16Cl2N4O3: C, 56.90; H, 3.64; N, 12.64.
Found: C, 57.20; H, 3.83; N, 12.16.
from 24d in accordance with procedure I. Pale orange solid
(57%); Mp 150.0–151.0 °C; 1H NMR (500 MHz, DMSO-d6) d 11.17
(1H, s), 8.23 (1H, t, J = 1.8 Hz), 7.94 (1H, d, J = 7.9 Hz), 7.58 (1H,
dd, J = 7.9, 1.8 Hz), 7.44 (1H, d, J = 7.9 Hz), 7.38 (1H, d, J = 3.6 Hz),
7.35–7.32 (1H, m), 7.30 (1H, d, J = 3.6 Hz), 7.12–7.05 (3H, m),
4.64 (2H, s), 4.30 (2H, s); MS (FAB) m/z 469 (M+H)+; HRMS (FAB)
calcd for C21H18BrN4O4 469.0511, found 469.0520 (M+H)+.
4.1.10.4. N-[3-(3-Aminobenzyl)-2,4-dioxoimidazolidin-1-yl]-5-
4.1.9.3.
N-[3-(3-Aminobenzyl)-2,4-dioxoimidazolidin-1-yl]-5-
Prepared from 24b
phenyl-2-furamide hydrochloride (15).
Prepared from 24f
(3,5-dichlorophenyl)-2-furamide (11).
in accordance with procedure I. Pale orange solid (23%); Mp
144.0–145.0 °C; 1H NMR (500 MHz, DMSO-d6) d 11.02 (1H, s),
8.30 (1H, s), 7.93 (2H, d, J = 7.9 Hz), 7.49 (2H, t, J = 7.9 Hz), 7.40
(1H, d, J = 7.9 Hz), 7.37 (1H, d, J = 3.6 Hz), 7.19 (1H, d, J = 3.6 Hz),
6.96 (1H, t, J = 7.9 Hz), 6.50 (1H, s), 6.47 (1H, d, J = 7.9 Hz), 6.43
(1H, d, J = 7.9 Hz), 5.20–5.06 (2H, br), 4.60 (2H, s), 4.28 (2H, s);
MS (FAB) m/z 391 (M+H)+; HRMS (FAB) calcd for C21H19N4O4
391.1406, found 391.1401 (M+H)+.
in accordance with procedure H. White solid (50%); Mp 247.0–
247.5 °C; 1H NMR (500 MHz, DMSO-d6) d 11.17 (1H, s), 8.10 (2H,
d, J = 1.8 Hz), 7.62 (1H, t, J = 1.8 Hz), 7.44 (1H, d, J = 3.7 Hz), 7.38
(1H, d, J = 3.7 Hz), 6.96 (1H, dd, J = 7.9, 7.3 Hz), 7.50 (1H, t,
J = 1.8 Hz), 6.46 (1H, dd, J = 7.9, 1.8 Hz), 6.42 (1H, d, J = 7.3 Hz),
5.09 (2H, s), 4.48 (2H, s), 4.28 (2H, s); MS (FAB) m/z 459 (M+H)+
.
Anal. Calcd for C21H16Cl2N4O4 1/4 H2O; C, 54.38; H, 3.59; N,
12.08. Found: C, 54.33; H, 3.49; N, 11.89.
4.1.10.5. N-[3-(4-Aminobenzyl)-2,4-dioxoimidazolidin-1-yl]-5-
4.1.9.4.
N-[3-(3-Aminobenzyl)-2,4-dioxoimidazolidin-1-yl]-5-
Prepared from 24e in
(3,5-dichlorophenyl)-2-furamide hydrochloride (16).
Pre-
(4-chlorophenyl)-2-furamide (12).
pared from 24g in accordance with procedure I using HCl/1,4-diox-
ane instead of HCl/EtOAc in the removal step of Boc group. Pale
orange solid (59%); Mp 213.0–214.0 °C; 1H NMR (500 MHz,
accordance with procedure H. Pale yellow solid (86%); Mp 187.0–
187.5 °C; 1H NMR (500 MHz, DMSO-d6) d 11.07 (1H, s), 7.97 (2H,
d, J = 8.5 Hz), 7.57 (2H, d, J = 8.5 Hz), 7.37 (1H, d, J = 3.6 Hz), 7.23
(1H, d, J = 3.6 Hz), 6.96 (1H, t, J = 7.9 Hz), 6.50 (1H, s), 6.46 (1H, d,
J = 7.9 Hz), 6.42 (1H, d, 7.9 Hz), 5.09 (2H, s), 4.47 (2H, s), 4.26
(2H, s); MS (FAB) m/z 425(M+H)+; HRMS (FAB) calcd for
DMSO-d6)
d 11.22 (1H, s), 9.75–8.75 (1H, br), 8.09 (2H, d,
J = 1.8 Hz), 7.63 (1H, t, J = 1.8 Hz), 7.44 (1H, d, J = 3.6 Hz), 7.40
(1H, d, J = 3.6 Hz), 7.35 (2H, d, J = 7.9 Hz), 7.21 (2H, d, J = 7.9 Hz),
4.65 (2H, s), 4.29 (2H, s); MS (FAB) m/z 459 (M+H)+; HRMS (FAB)
calcd for C21H17Cl2N4O4 459.0627, found 459.0646 (M+H)+.
C
21H18ClN4O4 425.1017, found 425.1007 (M+H)+.
4.1.10. Deprotection of Boc-protected aminobenzyl group on
hydantoin (2)
4.2. Bioassay
4.1.10.1. N-[3-(3-Aminobenzyl)-2,4-dioxoimidazolidin-1-yl]-5-
(3-chlorophenyl)-2-furamide hydrochloride (10): General pro-
4.2.1. Cell culture
HL-60 cells were maintained in RPMI1640 medium supple-
mented with 100 U/mL penicillin, 100 lg/mL streptomycin, and
cedure I.
To a solution of 24a (75.2 mg, 143 lmol) in EtOAc
(1.4 mL) was added 4 N HCl in EtOAc (1.4 mL) at 0 °C, and the mix-
ture was stirred for 3 h at room temperature. The reaction was
quenched by the addition of saturated aqueous NaHCO3 solution
and the resulting mixture was extracted with EtOAc. The organic
layer was washed with brine, dried over MgSO4, and concentrated
under reduced pressure. The residue was passed through a short
silica plug (hexane/EtOAc = 1:1). The eluate was evaporated under
reduced pressure, and the resulting residue was dissolved in EtOAc
(0.9 mL). To this was added 4 N HCl in EtOAc (0.1 mL). The result-
ing precipitate was collected by filtration with suction, washed
with EtOAc in the funnel, and dried under reduced pressure to af-
ford 10 (42.1 mg, 64%) as a pale yellow solid. Mp 163–164 °C; 1H
NMR (500 MHz, DMSO-d6) d 11.18 (1H, s), 8.10 (2H, t, J = 1.8 Hz),
5% heat-inactivated fetal bovine serum (FBS). Cells were grown in
a humidified incubator at 37 °C under 5% CO2/95% air.
4.2.2. Mitochondrial swelling assay
Mitochondria were isolated from human leukemia HL-60 cells
as described previously.37 Mitochondria were incubated with test
compounds at 30 °C (0.2 mg mitochondrial protein/mL, 20 mM
Tris–MOPS, pH 7.4, 200 mM sucrose, 12.5 mM succinate, 2
rotenone, 10 M EGTA) for 10 min. Then CaCl2 solution was added
(final concentration: 250 M) and OD540 was monitored at 30 °C
lM
l
l
with a microplate reader (SpectraMax M2e, Molecular Devices
Inc.).

S. Murasawa et al. / Bioorg. Med. Chem. 20 (2012) 6384–6393
15. Yoshimoto, T.; Siesjö, B. K. Brain Res. 1999, 839, 283.
6393
Acknowledgments
16. Hausenloy, D. J.; Duchen, M. R.; Yellon, D. M. Cardiovas. Res. 2003, 60, 617.
17. Bennett, W. M.; DeMattos, A.; Meyer, M. M.; Andoh, T.; Barry, J. M. Kidney Int.
1996, 50, 1089.
18. Hauben, M. Pharmacotherapy 1996, 16, 576.
19. Gijtenbeek, J. M.; van den Bent, M. J.; Vecht, C. J. J. Neurol. 1999, 246, 339.
20. Kahan, B. D. N. Engl. J. Med. 1989, 321, 1725.
The work described in this paper was partially supported by a
Grant-in-Aid for Young Scientists (B) 24790119 from the Ministry
of Education, Culture, Sports, Science and Technology.
21. Snyder, H. R., Jr.; Davis, C. S.; Bickerton, R. K.; Halliday, R. P. J. Med. Chem. 1967,
10, 807.
22. Ward, A.; Chaffman, M. O.; Sorkin, E. M. Drugs 1986, 32, 130.
23. Yano, T.; Nakayama, R.; Imaizumi, T.; Terasaki, H.; Ushijima, K. Resuscitation
2001, 50, 117.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.08.062.
24. Li, F.; Hayashi, T.; Jin, G.; Deguchi, K.; Nagotani, S.; Nagano, I.; Shoji, M.; Chan, P.
H.; Abe, K. Brain Res. 2005, 1048, 59.
25. Acikel, M.; Buyukokuroglu, M. E.; Erdogan, F.; Aksoy, H.; Bozkurt, E.; Senocak,
H. Int. J. Cardiol. 2005, 98, 389.
References and notes
26. Fruen, B. R.; Mickelson, J. R.; Louis, C. F. J. Biol. Chem. 1997, 272, 26965.
27. Thomson, I.; Shepherd, R. M.; Fraser, R.; Kenyon, C. J. J. Steroid Biochem. Mol.
Biol. 1991, 38, 703.
28. Chen, S.; Zhao, X.; Tan, J.; Lu, H.; Qi, Z.; Huang, Q.; Zeng, X.; Zhang, M.; Jiang, S.;
Yu, L. Eur. J. Pharmacol. 2007, 565, 54.
29. Ellis, K. O.; Wessels, F. L. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1978, 301,
237.
30. Daum, S.; Schumann, M.; Mathea, S.; Aumüller, T.; Balsley, M. A.; Constant, S.
L.; de Lacroix, B. F.; Kruska, F.; Braun, M.; Schiene-Fischer, C. Biochemistry 2009,
48, 6268.
31. Kajitani, K.; Fujihashi, M.; Kobayashi, Y.; Shimizu, S.; Tsujimoto, Y.; Miki, K.
Proteins 2008, 70, 1635.
32. Brown, F. J.; Bernstein, P. R.; Cronk, L. A.; Dosset, D. L.; Hebbel, K. C.; Maduskuie,
T. P., Jr.; Shapiro, H. S.; Vacek, E. P.; Yee, Y. K.; Willard, A. K.; Krell, R. D.; Snyder,
D. W. J. Med. Chem. 1989, 32, 807.
33. Kunishima, M.; Kawachi, C.; Iwasaki, F.; Terao, K.; Tani, S. Tetrahedron Lett.
1999, 40, 5327.
34. Hosoya, T.; Aoyama, H.; Ikemoto, T.; Kihara, Y.; Hiramatsu, T.; Endo, M.; Suzuki,
M. Bioorg. Med. Chem. 2003, 11, 663.
1. Tsujimoto, Y.; Nakagawa, T.; Shimizu, S. Biochim. Biophys. Acta 2006, 1757,
1297.
2. Tsujimoto, Y.; Shimizu, S. Apoptosis 2007, 12, 835.
3. Lemasters, J. J.; Theruvath, T. P.; Zhong, Z.; Nieminen, A-. L. Biochim. Biophys.
Acta 2009, 1787, 1395.
4. Sullivan, P. G.; Rabchevsky, A. G.; Waldmeier, P. C.; Springer, J. E. J. Neurosci. Res.
2005, 79, 231.
5. Mazzeo, A. T.; Beat, A.; Singh, A.; Bullock, M. R. Exp. Neurol. 2009, 218, 363.
6. Honda, H. M.; Ping, P. Cardiovasc. Drugs Ther. 2006, 20, 425.
7. Gomez, L.; Thibault, H.; Gharib, A.; Dumont, J.-M.; Vuagniaux, G.; Scalfaro, P.;
Derumeaux, G.; Ovize1, M. Am. J. Physiol. Heart Circ. Physiol. 2007, 293, H1654.
8. Stavrovskaya, I. G.; Narayanan, M. V.; Zhang, W.; Krasnikov, B. F.; Heemskerk,
J.; Young, S. S.; Blass, J. P.; Brown, A. M.; Beal, M. F.; Friedlander, R. M.; Kristal, B.
S. J. Exp. Med. 2004, 200, 211.
9. Sims, N. R.; Muyderman, H. Biochim. Biophys. Acta 2010, 1802, 80.
10. Osman, M. M.; Lulic, D.; Glover, L.; Stahl, C. E.; Lau, T.; van Loveren, H.;
Borlongan, C. V. Neuropeptides 2011, 45, 359.
11. Tanveer, A.; Virji, S.; Andreeva, L.; Totty, N. F.; Hsuan, J. J.; Ward, J. M.;
Crompton, M. Eur. J. Biochem. 1996, 238, 166.
12. Kieffer, L. J.; Seng, T. W.; Li, W.; Osterman, D. G.; Handschumacher, R. E.;
Bayney, R. M. J. Biol. Chem. 1993, 268, 12303.
13. Shiga, Y.; Onodera, H.; Matsuo, Y.; Kogure, K. Brain Res. 1992, 595, 145.
14. Matsumoto, S.; Friberg, H.; Ferrand-Drake, M.; Wieloch, T. J. Cereb. Blood Flow
Metab. 1999, 19, 736.
35. Beutner, G.; Sharma, V. K.; Giovannucci, D. R.; Yule, D. I.; Sheu, S. S. J. Biol. Chem.
2001, 276, 21482.
36. Ma, D.; Yu, S.; Chen, L.; Chen, R.; Yu, K.; Zhang, L.; Chen, Z.; Zhong, D.; Gong, Z.;
Wang, R.; Jiang, H.; Pei, G. ChemMedChem 2007, 2, 187.
37. Trounce, I. A.; Kim, Y. L.; Jun, A. S.; Wallace, D. C. Methods Enzymol. 1996, 264,
484.