Synthesis and biological evaluation of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins as potent NADPH oxidase (NOX) inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.06.056

We report the synthesis of novel 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins 3, and their biological evaluation using NADPH oxidase (NOX) 1 and 4. Based on structural and pharmacophore analyses of known inhibitors such as hydroxypyrazole 2, we e

Full text of DOI:10.1016/j.bmc.2016.06.056

Accepted Manuscript
Synthesis and biological evaluation of 3-substituted 5-benzylidene-1-methyl-2-
thiohydantoins as potent NADPH oxidase (NOX) inhibitors
Yun Soo Bae, Sun Choi, Jung Jae Park, Jung Hee Joo, Minghua Cui, Hyunsung
Cho, Won Jae Lee, Sang Hyup Lee
PII:
S0968-0896(16)30479-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.06.056
BMC 13112
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
2 June 2016
28 June 2016
29 June 2016
Please cite this article as: Bae, Y.S., Choi, S., Park, J.J., Joo, J.H., Cui, M., Cho, H., Lee, W.J., Lee, S.H., Synthesis
and biological evaluation of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins as potent NADPH oxidase
(
NOX) inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.06.056
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*
To whom correspondence should be addressed:
Sang Hyup Lee
College of Pharmacy and Innovative Drug Center,
Duksung Women’s University,
Seoul 132-714, Republic of Korea.
Phone: +82-2-901-8393.
Email: sanghyup@duksung.ac.kr.
Synthesis and biological evaluation of 3-substituted 5-
benzylidene-1-methyl-2-thiohydantoins as potent NADPH
oxidase (NOX) inhibitors
a,1
b,1
c
a
b
Yun Soo Bae, Sun Choi, Jung Jae Park, Jung Hee Joo, Minghua Cui,
c
d
c,
Hyunsung Cho, Won Jae Lee, Sang Hyup Lee, *
a
Department of Life Science, College of Natural Sciences, Ewha Women’s University,
Seoul 120-750, Republic of Korea
b
College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Women’s
University, Seoul 120-750, Republic of Korea
c
College of Pharmacy and Innovative Drug Center, Duksung Women’s University, Seoul
32-714, Republic of Korea
1
d
Department of Life Science, Seoul National University, Seoul 151-747, Republic of
Korea
1
These authors contributed equally to this paper.
1

Abstract
We report the synthesis of novel 3-substituted 5-benzylidene-1-methyl-2-
thiohydantoins 3, and their biological evaluation using NADPH oxidase (NOX) 1 and 4.
Based on structural and pharmacophore analyses of known inhibitors such as
hydroxypyrazole 2, we envisioned interesting 2-thiohydantoin compounds, 3-substituted
5
-benzylidene-1-methyl-2-thiohydantoins 3 that would be expected to well match the
structural features in 2. Efficient synthesis of eighteen target compounds 3 were achieved
through the synthetic pathway of 4  11  3, established after consideration of several
plausible synthetic pathways. The inhibitory activities of compounds 3 against NOX 1
and 4 were measured, with some of the target compounds showing similar or higher
activities compared with reference 2; in particular, compounds 3bz, 3cz, and 3ez were
found to be promising inhibitors of both NOX 1 and 4 with modest isozyme selectivities,
which highlights the significance of the 2-thiohydantoin substructure for inhibition of
NOX 1 and 4. This marks the first time these compounds have been applied to the
inhibition of NOX enzymes.
Keywords
thiohydantoin, benzylidene, aluminum chloride, NADPH oxidase (NOX)
2

1. Introduction
The 5-benzylidene-2-thiohydantoin compounds have been found to display a
1
2
3
range of biological activities, such as antimycobacterial, antiviral, anticonvulsant, and
4
antitumor properties. Among various thiohydantoin compounds we were interested in 3-
substituted 5-benzylidene-1-methyl-2-thiohydantoins as potential inhibitors of NADPH
oxidase (NOX) 1 and 4. NOX enzymes are known to generate reactive oxygen species
(
ROS) that play a significant role in many kinds of pathogenesis such as inflammation
5
and vascular and fibrotic disorders. NOX 1 seems to play a significant role in CNS
3
diseases. NOX 4 appears to be the most abundant isoform in endothelial and vascular
6
,7
smooth muscle cells, and a strong correlation between expression of NOX 4 and total
8
NOX activities in human coronary arteries was observed. Various natural and synthetic
9
compounds display NOX inhibitory activities, and the general results were reviewed in
10
9,11
detail by Borbely et. al. A triazolo[4,5-d]pyrimidine derivative 1 (VAS2870) was
1
2
found to inhibit oxLDL-induced superoxide release from human endothelial cells. More
1
3-16
significantly, a hydroxypyrazole derivative 2
was recently disclosed to have potent
1
5
activities against NOX enzymes.
Although the results thus far seem reasonably good and a substantial amount of
information has been accumulated, there are still no potent and selective inhibitors of
NOX 1 and 4. Therefore, with the aim to develop promising inhibitors against NOX 1
and 4, we performed preliminary studies through analysis of the structural features of
3

known inhibitors such as 1 and 2, library searches, and molecular modeling, which
eventually guided us to focus on the thiohydantoin substructure. Here, we present studies
on the synthesis of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins 3 and their
inhibitory activities against NOX 1 and 4, which is the first application of these target
compounds to the inhibition of NOX enzymes.
2. Results and Discussion
2
.1. Synthesis of 2-thiohydantoins 3
We began a program to develop potent inhibitors of NOX 1 and 4 starting from
known analogues such as hydroxypyrazole 2. Based on the structural analysis of 2 and
related derivatives (data not shown), we obtained information on the structural features
and important pharmacophores. For example, a consecutive arrangement of rigid ring
structures, a hydrophilic region including hydrogen bond acceptors (P1), hydrophobic
side chains (S1) near N(3), and extra hydrophobic pockets (S2) near C(5) may be
important for activity (Fig. 1). Through these studies, we envisioned a structurally similar
moiety, thiohydantoin, and consequently, suggested novel 3-substituted 5-benzylidene-1-
methyl-2-thiohydantoins 3 as NOX inhibitors. When we compared thiohydantoins 3 with
1
the reference 2, we recognized that the thiohydantoin core, ring substituent R at N(3),
and benzylidene group at C(5) in 3 would correlate well with the hydroxypyrazole,
4

pyridinyl, and phenyl groups in 2, respectively. In particular, we tried to align the
thiohydantoin ring to best overlap with the hydroxypyrazole ring according to the
pharmacophore analysis. Thus, we introduced ring substituents such as cycloalkyl or aryl
group at N(3) and incorporated the benzylidene group at C(5) for a better match with the
reference compound, to finally afford the target thiohydantoins 3. Although the
benzylidene group appeared to be slightly different from the phenyl group in 2, we
believed that a large group could be accommodated in this region (S2) according to the
1
5
previous studies.
Figure 1. Pharmacophores of 2 and comparison with 3.
In order to establish an efficient synthetic method of target compounds 3, we
evaluated several retrosynthetic pathways, as shown in Scheme 1. First, a starting
17
material sarcosine (4) could be transformed to thiourea 5, which could be further
1
7
converted to 2-thiohydantoin 6. Alternatively, compound 6 could be prepared from
hydantoin 7. Compound 6 needs to undergo both C(5)-functionalization and N(3)-
alkylation to yield target compounds 3. Thus, C(5)-functionalization using the
corresponding benzaldehydes 8 would give 5-benzylidene-2-thiohydantoins 9, and N(3)-
5

1
alkylation using alkyl halide (R X) would lead to the generation of compounds 3.
Alternatively, N(3)-alkylation of 6 would give the key intermediate, 3-substituted
thiohydantoins 11, and further C(5)-functionalization would provide 3. Additionally, the
key intermediates 11 could also be synthesized from coupling of 4 and alkyl
isothiocyanates 10.
In order to investigate the feasibility of these synthetic pathways, we briefly
checked their practical applicability by carrying out several model reactions. By treating
1
7
4
and ammonium thiocyanate (NH
4
SCN) as previously described, we obtained the
thiohydantoin 6 in relatively low yield (30~40%). We then applied another method in
18
which hydantoin 7 was treated with phosphorous pentasulfide (P
2
S ) to give compound
5
6
in moderate yield (~60%). Next, we attempted N(3)-alkylation to convert 6 to 11 using
1
9
an analogous procedure ; however, the N(3)-alkylation of 6 with alkyl halide (e.g.,
cyclohexyl bromide) did not provide good results, as several side products were
generated along with the desired product. We found that the reaction proceeded with the
formation of several polar spots on TLC; the side product(s) might be generated by S-
2
0,21
alkylation, which derives some support from previous work.
If this is the case, then
N(3)-alkylation could be a problematic step in the whole pathway. In addition, we
believed that, in order to avoid interference of N(3)H in C(5)-functionalization, it would
be more advantageous to perform the N(3)-alkylation prior to C(5)-functionalization
rather than the reverse order of transformations.
6

Scheme 1. Retrosynthetic analysis of 2-thiohydantoins 3.
Considering the efficiency of each pathway and the preliminary results of the
model reactions, we adopted the pathway of 4  11  3. Therefore, for the synthesis of
2
2,23
key intermediates 11, we employed the previous procedures
As shown in Scheme 2, sarcosine (4) was treated with alkyl isothiocyanates 10 to provide
-substituted 1-methyl-2-thiohydantoins 11 in modest yields. In these reactions,
with minor modifications.
3
cyclohexyl (a), phenyl (b), 4-methylphenyl (c), 4-ethylphenyl (d), 3-chlorophenyl (e),
1
and indan-5-yl group (f) were used as a set of R groups at N(3). For the synthesis of
indan-5-yl isothiocyanate (10f), N-indan-5-yl-O-phenyl thiocarbamate, prepared from 5-
aminoindane and phenyl chlorothionoformate, was treated with trichlorosilane and
2
4
triethylamine to afford 10f in 50% yield (for two steps). The reactions to synthesize 2-
thiohydantoins 11 generally provided good results (more than 90% yield), except in the
cases of the cyclohexyl (11a, 66% yield) and 3-chlorophenyl groups (11e, 74% yield),
7

probably due to their electronic and steric effects on the reactivity of nitrogen in
25
22,26
27
28
isothiocyanate. As a result, the six key intermediates, 11a, 11b,
11c, 11d, 11e,
2
2
and 11f were efficiently synthesized.
We then investigated several methods to perform the condensations of key
intermediates 11 with aldehydes 8 to provide the products 3. Here, three aromatic
aldehydes, 2,4-dihydroxybenzaldehyde (8x), 2,3-dihydroxybenzaldehyde (8y), and 4-
(
N,N-dimethylamino)benzaldehyde (8z) were employed. At first, the method using melt
1
9
sodium acetate in acetic acid was applied to our substrates, which gave unsuccessful
results probably due to the different structures in the thiohydantoins and aldehydes. Next,
2
9
microwave-assisted synthesis was found to give moderate results with a limited range
of substrates. Furthermore, the condensation reactions were conducted by simply using a
secondary amine such as piperidine or pyrrolidine; however, we were only partially
successful in obtaining the product with some unhindered aldehydes in moderate to low
yield (less than 60% yield). Acid catalysts have also been used for similar reactions with
different substrates (not thiohydantoins), and furthermore, Greg et al. achieved improved
2
3,30
results by employing Lewis acid catalysts
compared to the same reactions without
acid catalysts. Therefore, we investigated reaction conditions using our substrates and
o
acid catalysts in the presence of piperidine at 25–80 C. First, reactions of 11a and 8x
using acid catalysts such as SnCl
4
, TiCl
4
, AlCl
3
, and MgBr
or TiCl
2
to afford 3ax were examined.
When a catalytic amount (0.1–0.5 equiv.) of SnCl
4
4
was employed, the starting
materials 11a and 8x gradually disappeared; however, although several side products
were generated, product 3ax was not formed. When MgBr was used, the results of the
reaction were inconclusive; a small amount of product formed but subsequently
2
8

disappeared. When AlCl was applied, the product 3ax was formed in good to modest
3
yields (~60%) with an appropriate reaction time and temperature. Considering the
varying results with substrate and reaction conditions, we believed that polar hydroxy
groups might affect the function of the acid catalyst, presumably by lowering its activity.
Thus, the reaction was tested using 8z, that does not contain a hydroxy group, to afford
3
az in an improved yields (~85%) compared to 3ax (~60%).
Scheme 2. Synthesis of 2-thiohydantoins 3.
Having chosen to use AlCl , we synthesized the target compounds 3 with minor
3
modifications to the reaction conditions and work-up procedures. Thus, AlCl (0.1
3
o
equiv.) in the presence of piperidine (1.5 equiv.) at 65–80 C was used for the
condensation of 11 and 8 to synthesize the target compounds 3. In general, the reaction
mixtures were directly applied to a column chromatography after completion of the
reaction. However, in some cases (e.g., 3ey and 3ez) the reaction mixtures were subjected
to work-up procedures to give the crude products that were subsequently applied to
column chromatography to afford 3. Notably, compounds 3 contain an exocyclic C=C
bond and could therefore exist as Z/E-isomers; however, information on the isomeric
mixture of 5-benzylidene-2-thiohydantoins has not been revealed except in a few
9

2
9,31
cases.
In our experiments, we observed the formation of a single isomer in some cases
1
13
and a mixture of isomers in the other cases based on H and C NMR analyses. However,
the identification of isomer structures and their separation would require further intensive
work, which was not within the scope of our present studies. Consequently, we have
achieved the efficient synthesis of eighteen 2-thiohydantoin compounds 3. Among these,
2
3
compound 3fz was previously reported and compound 3bz was simply introduced
3
0
without any characterization. Compounds 3ax, 3az, 3by, 3fx, and 3fy were known as
2
8,32
members of chemical libraries,
but no information on their preparation, isomer ratio,
and spectral data were disclosed. To the best of our knowledge, the other eleven
compounds have not been previously identified with appropriate characterization.
2
3
.2. Evaluation of NOX 1 and 4 inhibitory activities and SAR studies
We evaluated the NOX 1 and 4 inhibitory activities of the synthesized compounds
along with hydroxypyrazole derivative 2 as a reference. This marks the first time these
compounds have been applied to the inhibition of NOX enzymes. To obtain specific
inhibition of NOX 1 and 4 enzymes, we established transgenic fly lines overexpressing
human NOX 1 or human NOX 4 in a Drosophila DUOX knockdown condition according
3
3
to our previous procedure. Transgenic flies were homogenated with ice-cold PBS
containing protease inhibitors, and the membranes enriched human NOX 1 or 4 were
harvested. The membranes with human NOX 1 or 4 served as ROS production
monitoring by lucigenin chemiluminescence in the absence or presence of chemical
3
4
compounds. The results of inhibitory activities for all of the thiohydantoins against
NOX 1 and 4 were shown in Table 1. We first evaluated 3ax, 3ay, and 3az (3a series)
1
0

1
containing a cyclohexyl group at N(3) as an aliphatic chain (R ) and three benzylidene
groups at C(5), respectively. For NOX 1, compound 3ay that contains a 2,3-
dihydroxybenzylidene group exhibited higher activity than 3ax and 3az, but, lower
activity than reference 2. Inhibitory activities of the compounds in the 3a series also
displayed similar trends for NOX 4. Next, we evaluated 3bx, 3by, 3bz (3b series), 3cx,
3
cy, 3cz (3c series), and 3dx, 3dy, 3dz (3d series) containing phenyl, 4-methylphenyl,
1
and 4-ethylphenyl groups at N(3) as nonpolar aromatic groups (R ), and the same three
benzylidene groups at C(5), respectively. In the 3b series, while compounds 3bx and 3by
displayed moderate to low activities for NOX 1 and 4, compound 3bz with a 4-(N,N-
dimethylamino)benzylidene group showed excellent activities that were similar or higher
compared to reference 2 [~3.5 fold (NOX 4)]. Trends in the 3c series were similar to the
3
b series, but compound 3cz exhibited higher activities in both NOX 1 and 4 than
reference 2 [~1.4 fold (NOX 1) and ~1.3 fold (NOX 4)]. A similar trend was observed in
the 3d series. Compound 3dz showed similar or higher activities compared to reference 2
and notably, 3dx containing a 2,4-dihydroxybenzylidene group also displayed higher
activity than 2 against NOX 4. We then evaluated 3ex, 3ey, and 3ez (3e series) containing
3
-Cl-phenyl group at N(3) as a slightly different group to see the effect of bulky meta-
substituents. Surprisingly, among the compounds tested, compound 3ez displayed the
highest activities that were higher than reference 2 against both NOX 1 and 4 [~4.1 fold
(
NOX 1) and ~3.7 fold (NOX 4)]. Finally, we evaluated 3fx, 3fy, and 3fz (3f series)
containing indan-5-yl group at N(3) to assess the effect of an additional fused ring. In
general, these compounds showed good to modest activities, and compound 3fy
displayed higher activities than 2 for NOX 4. As for isozyme selectivities between NOX
1
1

1
and 4, compounds 3cz and 3ez showed modest selectivities for NOX 1 (2.3 and 2.4 fold,
respectively), and compounds 3bz, 3dx and 3fy showed modest selectivities for NOX 4
2.2, 3.5 and 2.8 fold, respectively).
(
Table 1
Inhibitory activities of compounds 3 for NOX 1 and 4
Inhibitory activity (Ki, µM)
Compound
NOX 1
NOX 4
3
ax
3ay
az
bx
3by
bz
cx
3cy
cz
dx
3dy
dz
ex
3ey
26.0 ± 1.2
7.39 ± 3.6
12.3 ± 19.3
33.8 ± 4.7
49.7 ± 4.4
1.95 ± 0.07
27.1 ± 7.6
50.3 ± 33
1.02 ± 0.07
8.40 ± 0.27
27.9 ± 6.1
4.39 ± 0.06
34.5 ± 3.3
7.65 ± 1.4
0.35 ± 0.04
6.58 ± 0.33
6.63 ± 6.0
37.6 ± 0.76
6.35 ± 2.4
35.6 ± 14
3
a series
3
3
27.7 ± 12
3
b series
24.0 ± 6.3
0.88 ± 0.01
34.6 ± 4.4
27.0 ± 2.8
2.33 ± 0.05
2.39 ± 0.02
26.4 ± 5.7
2.63 ± 0.13
33.8 ± 8.1
9.31 ± 0.10
0.84 ± 0.06
6.83 ± 8.6
2.36 ± 1.9
3
3
3
c series
3
3
3
d series
3
3
3
e series
3
3
ez
fx
3
f series
3fy
fz
3
8.04 ± 2.5a
13.1 ± 6.3a
a
2
1.45 ± 0.15
3.12 ± 0.52
a
ref 15
From the inhibition results, it was evident that the three types of substituents at
C(5) strikingly affected the activities. While compounds containing a
dihydoxybenzylidene group (x or y) at C(5) gave low to modest activities, compounds
containing a 4-(N,N-dimethylamino)benzylidene group (z) provided the highest activities
in most of the series. Considering these results, a nonpolar hydrogen bond acceptor (e.g.
N,N-dimethylamino group) at C(5) was highly preferable rather than a polar hydrogen
1
2

bond donor (e.g. dihydroxy group). However, the six types of substituents at N(3) did not
seem to significantly affect the activities, and the 3-chlorophenyl (3e series) and indan-5-
yl (3f series) groups gave slightly better results than the other series. Consequently, it was
found that compounds 3bz, 3cz and 3ez displayed the highest level of activities against
both NOX 1 and 4, similar or higher compared to reference 2, along with modest isozyme
selectivity. Thus, we have successfully developed improved, promising inhibitors of
NOX 1 and 4 using a combination of a 4-(N,N-dimethylamino)benzylidene group at C(5)
and a phenyl, 4-methylphenyl, or 3-chlorophenyl group at N(3), respectively.
3. Conclusion
In this study, we synthesized novel 3-substituted 5-benzylidene-1-methyl-2-
thiohydantoins 3 and investigated their inhibitory activities against NOX 1 and 4.
Compounds 3 were designed based on an analysis of the structures of existing inhibitors
such as reference 2 and information on their pharmacophores, and thus, were expected to
match the structural features found in 2. We achieved the efficient synthesis of target
compounds 3 through the synthetic pathway of 4  11  3, to provide eighteen 2-
thiohydantoin compounds 3. We then measured the inhibitory activities of compounds 3
against NOX 1 and 4, and found that compounds 3bz, 3cz and 3ez showed the highest
levels of activity with modest isozyme selectivities that were similar or higher compared
to reference 2, which suggests they are promising compounds as inhibitors of NOX 1
and/or 4. This marks the first time these compounds have been applied to the inhibition of
NOX enzymes. Thus, the targeted 2-thiohydantoin moiety was demonstrated to be a
meaningful substructure for the inhibition of NOX 1 and 4.
1
3

4. Experimental section
4
.1. General information
1
13
H (300 MHz) and C (75 MHz) NMR spectra were recorded on a Bruker DRX
00 spectrometer. Mass spectra were obtained by the CI, EI or FAB ionization method.
3
FT-IR spectra were run on a Perkin-Elmer Spectrum GX spectrometer. Melting points
were determined in open capillary tubes using a Buchi B-545 melting point apparatus and
were uncorrected. Thin layer chromatography was performed with general purpose silica
gel plates (20 x 20 cm; Aldrich No. Z12272-6). Deionized water was obtained with a
Milli-Q (18 Ms) water system (Millipore). The solvents and reactants were of the
highest commercial grade available and were used without further purification unless
noted.
2
2,23
4
.2. General procedure
for preparation of 3-substituted 1-methyl-2-
thiohydantoins 11
To a stirred solution of sarcosine (4, 180 mg, 2.0 mmol) in anhydrous ethanol (6
mL) was added the appropriate isothiocyanate (10, 2.0 mmol). The reaction mixture was
refluxed for 5 h and then was allowed to cool to room temperature. The solvent was
removed in vacuo, and the resulting residue was purified by column chromatography
(
EtOAc/hexanes) to afford the title compound.
4
.2.1. 3-Cyclohexyl-1-methyl-2-thiohydantoin (11a). Use of cyclohexyl isothiocyanate
o
(
10a) and the general procedure (85 C, 6 h) afforded the title compound as a yellow
o
1
solid: yield, 281 mg (66%); Mp 160–162 C; R
f
0.36 (1:2 EtOAc/hexanes); H NMR (300
MHz; DMSO-d ) δ: 0.93–1.35 (m, 3 H, c-Hx), 1.50–1.65 (m, 3 H, c-Hx), 1.68–1.84 (m, 2
6
1
4

1
3
H, c-Hx), 1.98–2.20 (m, 2 H, c-Hx), 3.18 (s, 3 H, N(1)CH
3
), 4.17 (s, 3 H, C(5)CH
2
); C
NMR (75 MHz; DMSO-d
6
) δ: 24.8 (c-Hx), 25.5 (c-Hx), 28.2 (c-Hx), 33.9 (N(1)CH
3
),
+
5
3.5 (C(5)), 55.0 (N(3)CH), 171.3 (C=O), 182.1 (C=S). MS m/z 212 [M] . HRMS (EI+)
+
calcd for C10
H
16
N OS [M] : 212.0983; found 212.0982.
2
22
4
.2.2. 3-Phenyl-1-methyl-2-thiohydantoin (11b). Use of phenyl isothiocyanate (10b)
o
and the general procedure (85 C, 2 h) afforded the title compound as a yellow solid:
o
1
yield, 375 mg (91%); Mp 163–165 C; R
CDCl ) δ: 3.34 (s, 3 H, N(1)CH ), 4.16 (s, 2 H, C(5)H
.48 (m, 3 H, Ph); C NMR (75 MHz; CDCl ) δ: 34.7 (N(1)CH
f
0.25 (1:2 EtOAc/hexanes); H NMR (300 MHz;
3
3
2
), 7.18–7.26 (m, 2 H, Ph), 7.34–
1
3
7
1
3
3
), 54.7 (C(5)), 128.7,
+
29.5, 129.6, 133.7 (Ph), 170.0 (C=O), 183.6 (C=S). MS m/z 206 [M] . HRMS (EI+)
+
calcd for C10
H
10
N
2
OS [M] : 206.0513; found 206.0515.
27
4
.2.3. 3-(4-Methylphenyl)-1-methyl-2-thiohydantoin (11c). Use of 4-methylphenyl
o
isothiocyanate (10c) and the general procedure (85 C, 2 h) with recrystallization
(
EtOAc/hexanes) instead of column chromatography afforded the title compound as a
o
1
yellow solid: yield, 405 mg (92%); Mp 147–149 C; R
NMR (300 MHz; CDCl ) δ: 2.32 (s, 3 H, PhCH ), 3.31 (s, 3 H, N(1)CH
C(5)H ), 7.10 (d, J= 6.3 Hz, 2 H, Ph), 7.22 (d, J= 6.3 Hz, 2 H, Ph); C NMR (75 MHz;
CDCl
f
0.29 (1:2 EtOAc/hexanes); H
3
3
3
), 4.13 (S , 2 H,
1
3
2
3
) δ: 21.4 (PhCH
3
), 34.4 (N(1)CH
3
), 54.5 (C(5)), 128.1, 130.0, 130.8, 139.5 (Ph),
+
+
1
2
4
70.0 (C=O), 183.4 (C=S). MS m/z 220 [M] . HRMS (EI+) calcd for C11
H
12
N OS [M] :
2
20.0670; found 220.0668.
.2.4. 3-(4-Ethylphenyl)-1-methyl-2-thiohydantoin (11d). Use of 4-ethylphenyl
o
isothiocyanate (10d) and the general procedure (85 C, 1 h) afforded the title compound
o
1
as a yellow solid: yield, 422 mg (90%); Mp 115–117 C; R
NMR (300 MHz; CDCl ) δ: 1.28 (t, J= 7.6 Hz, 3 H, PhCH
PhCH ), 3.42 (s, 3 H, N(1)CH ), 4.23 (s, 2 H, C(5)H ), 7.22 (d, J= 8.2 Hz, 2 H, Ph), 7.34
d, J= 8.2 Hz, 2 H, Ph); C NMR (75 MHz; CDCl ) δ: 15.3 (PhCH CH ), 28.8 (PhCH ),
f
0.16 (1:2 EtOAc/hexanes); H
3
2
CH ), 2.71 (q, J= 7.6 Hz, 2 H,
3
2
3
2
1
3
(
3
2
3
2
1
5

3
4.5 (N(1)CH
3
), 54.4 (C(5)), 128.2, 128.9, 131.0, 145.6 (Ph), 170.0 (C=O), 183.6 (C=S).
+
+
MS m/z 234 [M] . HRMS (EI+) calcd for C12
H
14
N OS [M] : 234.0826; found 234.0825.
2
4
.2.5. 3-(3-Chlorophenyl)-1-methyl-2-thiohydantoin (11e). Use of 3-chlorophenyl
o
isothiocyanate (10e) and the general procedure (85 C, 3 h) with recrystallization
(
EtOAc/hexanes) instead of column chromatography afforded the title compound as a
o
1
yellow solid: yield, 356 mg (74%); Mp 192–194 C; R
NMR (300 MHz; CDCl ) δ: 3.41 (s, 3 H, N(1)CH ), 4.24 (s, 2 H, C(5)H
H, Ph); C NMR (75 MHz; CDCl ) δ: 34.5 (N(1)CH
f
0.16 (1:2 EtOAc/hexanes); H
3
3
2
), 7.47–7.21 (m,
1
3
4
1
3
3
), 54.4 (C(5)), 126.8, 128.8, 129.6,
+
30.2, 134.4, 134.7 (Ph), 169.5 (C=O), 182.6 (C=S). MS m/z 240 [M] . HRMS (EI+)
+
calcd for C10
H
9
ClN
2
OS [M] : 240.0124; found 240.0122.
22
4
.2.6. 3-(Indan-5-yl)-1-methyl-2-thiohydantoin (11f). Use of indan-5-yl
o
isothiocyanate (10f) and the general procedure (85 C, 1 h) afforded the title compound
o
1
as a yellow solid: yield, 473 mg (96%); Mp 158-160 C; R
NMR (300 MHz; CDCl ) δ: 2.07–2.28 (m, 2 H, CH -indanyl), 2.87–3.01 (m, 4 H, CH
indanyl), 3.43 (s, 3 H, N(1)CH ), 4.23 (s, 2 H, C(5)H ), 7.04 (dd, J= 7.8, 1.2 Hz, 1 H, Ph-
indanyl), 7.12 (s, 1 H, Ph-indanyl), 7.34 (d, J= 7.8 Hz, 1 H, Ph-indanyl); C NMR (75
MHz; CDCl ) δ: 25.8, 33.1, 33.2 (CH -indanyl), 34.7 (N(1)CH ), 54.7 (C(5)), 124.6,
25.3, 126.5, 131.6, 145.9, 146.2 (Ph-indanyl), 170.4 (C=O), 184.0 (C=S). MS m/z 246
f
0.14 (1:2 EtOAc/hexanes); H
3
2
2
-
3
2
1
3
3
2
3
1
+
+
[
M] . HRMS (EI+) calcd for C13
H
14
N OS [M] : 246.0826; found 246.0828.
2
2
3
4
3
.3. General procedure for 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins
To a stirred solution of thiohydantoin (11, 0.20 mmol), aldehyde (8, 0.24 mmol)
and piperidine (30 μL, 0.30 mmol) in dioxane (0.6 mL) was added aluminum chloride
o
(
2.7 mg, 0.020 mmol). The stirred reaction mixture was heated to 65–80 C until
1
6

completion of the reaction. In cases where the reaction was incomplete, more aluminum
chloride (0.1–0.2 equiv.) was added along with the corresponding amount of piperidine in
the same ratio, and the reaction mixture was refluxed for additional time. After cooling to
room temperature, the reaction mixture was directly subjected to column chromatography
(
gradient elution with appropriate EtOAc/hexanes mixtures) to give the title compounds 3.
In the cases of 3ey and 3ez, the reaction mixture was worked-up prior to column
chromatography. The reaction mixture was evaporated to approximately half of the
original volume. H
with EtOAc (2  50 mL). The combined organic layers were washed with saturated
aqueous NaHCO and brine (50 mL), dried (MgSO ), and concentrated in vacuo to give
2
O (50 mL) was slowly added, and the resulting mixture was extracted
3
4
an oily residue. Purification of the residue by column chromatography (gradient elution
with appropriate EtOAc/hexanes mixtures) afforded the title compound.
4
.3.1. 3-Cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (3ax).
o
Use of thiohydantoin 11a and aldehyde 8x in the general procedure (80 C, 3 h) afforded
o
the title compound as a red solid: yield, 43 mg (64%); Mp 205–207 C; R
f
0.13 (1:2
-1 1
EtOAc/hexanes); IR (KBr) 3434, 2090, 1637, 1384, 1096 cm ; H NMR (300 MHz;
DMSO-d ) δ: 1.10–1.35 (m, 3 H, c-Hx), 1.56–1.70 (m, 3 H, c-Hx), 1.75–1.87 (m, 2 H, c-
Hx), 2.15–2.29 (m, 2 H, c-Hx), 3.53 (s, 3 H, N(1)CH ), 4.53–4.66 (m, 1 H, N(3)CH),
.28 (dd, J= 8.8, 2.2 Hz, 1 H, Ph), 6.37 (d, J= 2.2Hz, 1 H, Ph), 6.97 (s, 1 H, CH=C), 8.43
6
3
6
13
(
d, J= 8.8 Hz, 1 H, Ph), 10.10 (br s, 2 H, OH); C NMR (75 MHz; DMSO-d
Hx), 25.6 (c-Hx), 28.2 (c-Hx), 31.0 (N(1)CH ), 54.8 (N(3)CH), 101.7, 107.0, 110.8,
16.9, 125.2, 132.1, 158.8, 161.4 (Ph, CH=C), 161.2 (C=O), 174.2 (C=S). MS m/z 332
6
) δ: 24.9 (c-
3
1
+
+
[
M] . HRMS (EI+) calcd for C17
H
20
N
2
O S [M] : 332.1194; found 332.1195.
3
4
.3.2. 3-Cyclohexyl-5-(2,3-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (3ay).
o
Use of thiohydantoin 11a and aldehyde 8y in the general procedure (80 C, 9 h) afforded
1
7

o
the title compound as a red solid: yield, 46 mg (72%); Mp 95–97 C; R
f
0.40 (1:2
EtOAc/hexanes); IR (KBr) 3433, 2930, 2854, 1635, 1471, 1402, 1372, 1283, 1188, 1146,
-1 1
1
099, 728, 530 cm ; H NMR (300 MHz; CDCl
3
) δ: 1.20–1.46 (m, 3 H, c-Hx), 1.63–1.79
(
m, 3 H, c-Hx), 1.81–1.93 (m, 2 H, c-Hx), 2.15–2.35 (m, 2 H, c-Hx), 3.32, 3.66 (2s, 3 H,
N(1)CH
3
), 4.56–4.84 (m, 1 H, N(3)CH), 6.64, 6.84 (2s, 1 H, CH=C), 6.69–6.83 (m, 1 H,
1
3
Ph), 6.87–7.08 (m, 2 H, Ph); C NMR (75 MHz; CDCl
3
) δ: 25.2 (c-Hx), 26.1 (c-Hx),
2
1
8.8 (c-Hx), 31.7, 34.5 (N(1)CH
3
), 56.4, 56.7 (N(3)CH), 109.7, 116.2, 117.8, 120.2,
20.3, 122.0, 122.4, 122.6, 124.8, 128.6, 130.8, 143.1, 143.3, 144.2, 148.7 (Ph, CH=C,
one peak was not detected and believed to overlap with the observed peaks), 163.9, 165.4
+
(
[
C=O), 176.1, 180.5 (C=S). MS m/z 332 [M] . HRMS (EI+) calcd for C17
H
20
N
2
3
O S
+
M] : 332.1194; found 332.1195.
4
.3.3. 3-Cyclohexyl-5-(4-(N,N-dimethylamino)benzylidene)-1-methyl-2-
thiohydantoin (3az). Use of thiohydantoin 11a and aldehyde 8z in the general procedure
o
(
65 C, 6 h) afforded the title compound as a red solid: yield, 59 mg (86%); Mp 215–217
o
C; R
233, 1185, 1078, 950, 875, 807, 515 cm ; H NMR (300 MHz; DMSO-d
m, 3 H, c-Hx), 1.57–1.73 (m, 3 H, c-Hx), 1.76–1.89 (m, 2 H, c-Hx), 2.12–2.35 (m, 2 H,
c-Hx), 2.98, 3.01 (2s, 6 H, N(CH ), 3.41, 3.56 (2s, 3 H, N(1)CH ), 4.41–4.67 (m, 1 H,
N(3)CH), 6.68–6.85 (m, 3 H, Ph, CH=C), 7.38 (d, J= 9.0 Hz, 1H, Ph), 8.16 (d, J= 9.0 Hz,
f
0.57 (1:2 EtOAc/hexanes); IR (KBr) 2925, 1701, 1588, 1530, 1443, 1395, 1341,
-1 1
1
6
) δ: 1.09–1.42
(
3
)
2
3
1
3
1
2
1
1
3
4
H, Ph); C NMR (75 MHz; DMSO-d
6
) δ: 24.5, 24.9 (c-Hx), 25.5, 25.6 (c-Hx), 28.2,
8.3 (c-Hx), 31.1, 35.6 (N(1)CH ), 54.8, 55.2 (N(3)CH), 111.2, 111.4, 116.8, 118.7,
3
19.9, 123.8, 124.6, 125.8, 132.0, 133.4, 150.6, 151.3 (Ph, CH=C), 161.1, 163.1 (C=O),
+
+
73.5, 178.6 (C=S). MS m/z 343 [M] . HRMS (EI+) calcd for C19
H
25
N OS [M] :
3
43.1718; found 343.1718.
.3.4. 3-Phenyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (3bx). Use
(0.020 mmol) and piperidine
of thiohydantoin 11b, aldehyde 8x, and additional AlCl
3
1
8

o
(
0.30 mmol) in the general procedure (80 C, 1 d and additionally, 10 h) afforded the title
o
compound as a red solid: yield, 37 mg (57%); Mp 263–265 C; R
f
0.25 (1:1
EtOAc/hexanes); IR (KBr) 3162, 2918, 2849, 2348, 2110, 1635, 1454, 1396, 1260, 1023,
-1 1
8
73, 749, 538 cm ; H NMR (300 MHz; DMSO-d
6
) δ: 3.61 (s, 3 H, N(1)CH
3
), 6.23 (dd,
J= 8.8, 2.2 Hz, 1 H, Ph), 6.37 (d, J= 2.2 Hz, 1 H, Ph), 7.05 (s, 1 H, CH=C), 7.28–7.63 (m,
1
3
5
H, Ph), 8.41 (d, J= 8.8 Hz, 1 H, Ph), 10.01, 10.27 (2s, 2 H, OH); C NMR (75 MHz;
DMSO-d ) δ: 30.6 (N(1)CH ), 101.7, 106.9, 110.9, 117.3, 125.2, 128.5, 128.7, 128.9,
32.0, 133.9, 158.9, 161.4 (Ph, CH=C), 160.9 (C=O), 174.1 (C=S). MS m/z 326 [M] .
6
3
+
1
+
HRMS (EI+) calcd for C17
H
14
N
2
O S [M] : 326.0725; found 326.0724.
3
4
.3.5. 3-Phenyl-5-(2,3-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (3by). Use
o
of thiohydantoin 11b and aldehyde 8y in the general procedure (80 C, 7 h) afforded the
o
title compound as a red solid: yield, 47 mg (71%); Mp 155–156 C; R
f
0.18 (1:6:12
MeOH/EtOAc/hexanes); IR (KBr) 3355, 3062, 2938, 1732, 1595, 1471, 1350, 1283,
-
1 1
1
3
233, 1154, 1052, 1004, 735, 529 cm ; H NMR (300 MHz; DMSO-d
6
) δ: 3.35, 3.63 (2s,
H, N(1)CH ), 6.88, 7.04 (2s, 1 H, CH=C), 6.56–6.87 (m, 2 H, Ph), 7.32–7.73 (m, 6 H,
3
1
3
Ph), 9.07, 9.17 (2s, 1 H, OH), 9.53, 9.65 (2s, 1 H, OH); C NMR (75 MHz; DMSO-d
6
)
δ: 30.4, 33.8 (N(1)CH ), 112.0, 116.1, 116.3, 116.7, 117.8, 118.7, 119.5, 119.6, 120.9,
3
1
1
21.0, 127.5, 127.6, 127.7, 128.4, 128.5, 128.6, 128.7, 133.5, 133.6, 144.3, 144.7, 145.1,
45.2 (Ph, CH=C, one peak was not detected and believed to overlap with the observed
+
peaks), 160.6, 162.6 (C=O), 175.4, 179.0 (C=S). MS m/z 326 [M] . HRMS (EI+) calcd
+
for C17
H
14
N
2
O
3
S [M] : 326.0725; found 326.0722.
4
.3.6. 3-Phenyl-5-(4-(N,N-dimethylamino)benzylidene)-1-methyl-2-thiohydantoin
o
(
3bz). Use of thiohydantoin 11b and aldehyde 8z in the general procedure (65 C, 4 h)
o
afforded the title compound as a red solid: yield, 61 mg (92%); Mp 238–240 C; R
f
0.36
(
1:2 EtOAc/hexanes); IR (KBr) 3748, 2368, 1715, 1581, 1530, 1501, 1478, 1393, 1344,
-1 1
1
194, 1147, 1056, 819, 763, 518 cm ; H NMR (300 MHz; DMSO-d ) δ: 3.00 (s, 6 H,
6
1
9

N(CH
3
)
2
), 3.63 (s, 3 H, N(1)CH ), 6.72 (d, J= 9.0 Hz, 2 H, Ph), 6.89 (s, 1 H, CH=C), 7.36
3
1
3
(
(
d, J= 7.8 Hz, 2 H, Ph), 7.42–7.54 (m, 3 H, Ph), 8.18 (d, J= 9.0 Hz, 2 H, Ph); C NMR
75 MHz; DMSO-d ) δ: 30.8 (N(1)CH ), 39.6 (N(CH ), 111.0, 119.9, 124.2, 124.4,
6
3
3 2
)
1
3
4
28.5, 128.7, 128.9, 133.4, 134.0, 151.4 (Ph, CH=C), 160.9 (C=O), 173.4 (C=S). MS m/z
+
+
37 [M] . HRMS (EI+) calcd for C19
H
19
N OS [M] : 337.1248; found 337.1248.
3
.3.7. 3-(4-Methylphenyl)-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin
o
(
3cx). Use of thiohydantoin 11c and aldehyde 8x in the general procedure (80 C, 8 h)
o
afforded the title compound as a red solid: yield, 44 mg (65%); Mp 275–279 C; R
f
0.36
(
1:1 EtOAc/hexanes); IR (KBr) 3748, 3444, 2917, 2381, 2324, 1688, 1593, 1511, 1463,
1
398, 1350, 1275, 1175, 1105, 1002, 977, 947, 873, 802, 765, 677, 629, 588, 535, 499
-1 1
cm ; H NMR (300 MHz; DMSO-d
6
) δ: 2.36 (s, 3 H, PhCH
3
), 3.62 (s, 3 H, N(1)CH ),
3
6
.24 (dd, J= 8.7, 2.4 Hz, 1 H, Ph), 6.39 (d, J= 2.4 Hz, 1 H, Ph), 7.05 (s, 1 H, CH=C), 7.22
(
(
(
d, J= 8.4 Hz, 2 H, Ph), 7.30 (d, J= 8.4 Hz, 2 H, Ph), 8.42 (d, J= 8.7 Hz, 1 H, Ph), 10.00
1
3
s, 1 H, OH), 10.26 (s, 1 H, OH); C NMR (75 MHz; DMSO-d
6
) δ: 20.8 (PhCH ), 30.6
3
N(1)CH ), 101.7, 106.9, 110.9, 117.2, 125.2, 128.6, 129.2, 131.3, 132.0, 138.1, 158.8,
3
+
1
61.4 (Ph, CH=C), 161.0 (C=O), 174.3 (C=S). MS m/z 340 [M] . HRMS (EI+) calcd for
+
C
18
H
16
N
2
O S [M] : 340.0881; found 340.0878.
3
4
.3.8. 3-(4-Methylphenyl)-5-(2,3-dihydroxybenzylidene)-1-methyl-2-thiohydantoin
o
(
3cy). Use of thiohydantoin 11c and aldehyde 8y in the general procedure (80 C, 9 h)
o
afforded the title compound as a red solid: yield, 52 mg (78%); Mp 184–185 C; R
f
0.60
(
1:1 EtOAc/hexanes); IR (KBr) 3383, 3037, 2922, 2853, 2306, 2071, 1727, 1621, 1586,
-
1 1
1
514, 1472, 1394, 1349, 1282, 1204, 1154, 1051, 1003, 942, 873, 764, 663, 523 cm ; H
) δ: 2.41, 2.43 (2s, 3 H, PhCH ), 3.41, 3.76 (2s, 3 H, N(1)CH ),
) δ:
NMR (300 MHz; CDCl
3
3
3
1
3
6
2
1
.73–7.10 (m, 3 H, Ph, CH=C), 7.15–7.41 (m, 5 H, Ph); C NMR (75 MHz; CDCl
3
1.5 (PhCH ), 31.4, 34.2 (N(1)CH ), 110.9, 116.4, 116.6, 118.7, 120.0, 120.5, 122.1,
3
3
22.5, 122.6, 124.8, 128.1, 128.3, 130.1, 130.2, 130.5, 130.8, 139.6, 140.1, 142.9, 143.3,
2
0

1
44.1, 148.5 (Ph, CH=C, two peaks were not detected and believed to overlap with the
+
observed peaks), 163.7, 164.8 (C=O), 175.9, 180.2 (C=S). MS m/z 340 [M] . HRMS
+
(
EI+) calcd for C18
H
16
N
2
O
3
S [M] : 340.0881; found 340.0878.
4
.3.9. 3-(4-Methylphenyl)-5-(4-(N,N-dimethylamino)benzylidene)-1-methyl-2-
thiohydantoin (3cz). Use of thiohydantoin 11c and aldehyde 8z in the general procedure
o
(
65 C, 11 h) afforded the title compound as a red solid: yield, 46 mg (66%); Mp 241–
o
2
43 C; R
f
0.83 (1:1 EtOAc/hexanes); IR (KBr) 3748, 2917, 2853, 2382, 2091, 1714,
-
1
619, 1580, 1515, 1478, 1441, 1390, 1194, 1146, 1053, 952, 888, 819, 763, 662, 516 cm
1
1
;
H NMR (300 MHz; CDCl
3
) δ: 2.42 (s, 3 H, PhCH
3
), 3.06 (s, 6 H, N(CH
3
2
) ), 3.51, 3.57
(
2s, 3 H, N(1)CH
3
), 6.56, 7.03 (2s, 1 H, CH=C), 6.65–6.77 (m, 3 H, Ph), 7.23–7.36 (m, 3
1
3
H, Ph), 8.15 (d, J= 9.0 Hz, 2 H, Ph); C NMR (75 MHz; CDCl
3
) δ: 21.5 (PhCH ), 31.1,
3
3
1
5.9 (N(1)CH
3
), 40.3 (N(CH
3
) ), 111.6, 111.7, 118.3, 119.6, 120.3, 124.0, 125.2, 126.7,
2
28.2, 128.4, 129.9, 130.0, 131.2, 132.0, 133.8, 139.1, 139.3, 151.0, 151.8 (Ph, CH=C,
one peak was not detected and believed to overlap with the observed peaks), 161.7, 163.9
+
+
(
C=O), 174.9, 179.7 (C=S). MS m/z 351 [M] . HRMS (EI+) calcd for C20
H
21
N OS [M] :
3
3
51.1405; found 351.1407.
4
.3.10. 3-(4-Ethylphenyl)-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin
o
(
3dx). Use of thiohydantoin 11d and aldehyde 8x in the general procedure (80 C, 4 h)
o
afforded the title compound as a red solid: yield, 43 mg (63%); Mp 235–239 C; R
f
0.45
(
1:1 EtOAc/hexanes); IR (KBr) 3434, 1627, 1513, 1391, 1348, 1155, 1105, 1053, 673,
-
1 1
4
97 cm ; H NMR (300 MHz; DMSO-d
), 3.61 (s, 3 H, N(1)CH
.4 Hz, 1 H, Ph), 7.05 (s, 1 H, CH=C), 7.24 (d, J= 8.4 Hz, 2 H, Ph), 7.33 (d, J= 8.4 Hz, 2
6
) δ: 1.22 (t, J= 7.6 Hz, 3 H, PhCH
2
CH ), 2.66 (q,
3
J= 7.6 Hz, PhCH
2
3
), 6.24 (dd, J= 8.7, 2.4 Hz, 1 H, Ph), 6.39 (d, J=
2
1
3
H, Ph), 8.41 (d, J= 8.7 Hz, 1 H, Ph), 10.10 (br s, 2 H, OH); C NMR (75 MHz; DMSO-
d
6
) δ: 15.4 (PhCH
2
CH
3
), 27.8 (PhCH
2
), 30.6 (N(1)CH
3
), 101.7, 106.9, 110.9, 117.2,
1
25.2, 128.6, 129.1, 131.5, 132.0, 144.2, 158.9, 161.4 (Ph, CH=C), 161.1 (C=O), 174.3
2
1

+
+
(
C=S). MS m/z 354 [M] . HRMS (EI+) calcd for C19
H
18
N
2
O S [M] : 354.1038; found
3
3
54.1035.
.3.11. 3-(4-Ethylphenyl)-5-(2,3-dihydroxybenzylidene)-1-methyl-2-thiohydantoin
(0.040 mmol) and
4
(
3dy). Use of thiohydantoin 11d, aldehyde 8y, and additional AlCl
3
o
piperidine (0.60 mmol) in the general procedure (80 C, 4 h and additionally, 4 h)
o
afforded the title compound as a red solid: yield, 38 mg (53%); Mp 163–168 C; R
f
0.51
(
1:1 EtOAc/hexanes); IR (KBr) 3368, 2964, 1721, 1648, 1586, 1514, 1470, 1398, 1349,
-
1 1
1
282, 1169, 1051, 1002, 939, 827, 763, 731, 692, 521 cm ; H NMR (300 MHz; CDCl
CH ), 2.53–2.71 (m, 2 H, PhCH ), 3.31, 3.64 (2s, 3 H,
), 6.62–7.03 (m, 4 H, Ph, CH=C), 7.12–7.32 (m, 4 H, Ph); C NMR (75 MHz;
) δ: 15.2 (PhCH CH ), 28.7 (PhCH ), 31.3, 34.3 (N(1)CH ), 111.4, 116.3, 116.6,
3
)
δ: 1.14–1.23 (m, 3 H, PhCH
N(1)CH
CDCl
2
3
2
1
3
3
3
2
3
2
3
1
1
18.5, 119.8, 120.4, 121.9, 122.2, 122.3, 124.6, 128.1, 128.2, 128.8, 128.9, 130.2, 130.3,
30.8, 143.0, 143.3, 144.2, 145.6, 146.1, 148.1 (Ph, CH=C, one peak was not detected
and believed to overlap with the observed peaks), 163.9, 164.6 (C=O), 175.9, 180.0
+
+
(
C=S). MS m/z 354 [M] . HRMS (EI+) calcd for C19
H
18
N
2
O S [M] : 354.1038; found
3
3
54.1041.
.3.12. 3-(4-Ethylphenyl)- 5-(4-(N,N-dimethylamino)benzylidene)-1-methyl-2-
4
thiohydantoin (3dz). Use of thiohydantoin 11d and aldehyde 8z in the general procedure
o
(
65 C, 11 h) afforded the title compound as a red solid: yield, 145 mg (95%); Mp 160-
o
1
1
1
62 C; R
f
0.66 (1:1 EtOAc/hexanes); IR (KBr) 3435, 1710, 1621, 1581, 1514, 1477,
-1 1
388, 1344, 1193, 1145, 1052, 951, 887, 817, 515 cm ; H NMR (300 MHz; CDCl
.27 (t, J= 7.6 Hz, 3 H, PhCH CH ), 2.71 (q, J= 7.6 Hz, 2 H, PhCH ), 3.03 (s, 6 H,
), 3.68 (s, 3 H, N(1)CH ), 6.52 (s, 1 H, CH=C), 6.65 (d, J= 9.0 Hz, 2 H, Ph),
.23–7.36 (m, 4 H, Ph), 8.14 (d, J= 9.0 Hz, 2 H, Ph); C NMR (75 MHz; CDCl
PhCH CH ), 28.7 (PhCH ), 30.1 (N(1)CH ), 40.2 (N(CH ), 111.6, 120.2, 124.0, 125.1,
28.4, 128.7, 131.3, 133.7, 145.2, 151.8 (Ph, CH=C), 161.7 (C=O), 174.8 (C=S). MS m/z
3
) δ:
2
3
2
N(CH
3
)
2
3
1
3
7
3
) δ: 15.3
(
2
3
2
3
)
3 2
1
2
2

+
+
3
65 [M] . HRMS (EI+) calcd for C21
H
23
N OS [M] : 365.1561; found 365.1564.
3
4
.3.13. 3-(3-Chlorophenyl)-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin
o
(
3ex). Use of thiohydantoin 11e and aldehyde 8x in the general procedure (80 C, 5 h)
o
afforded the title compound as a red solid: yield, 51 mg (70%); Mp 225–228 C; R
f
0.17
(
1:2 EtOAc/hexanes); IR (KBr) 3748, 3610, 2916, 2382, 2307, 1832, 1700, 1593, 1481,
-1 1
1
390, 1275, 1163, 975, 852, 748, 617, 501 cm ; H NMR (300 MHz; DMSO-d ) δ: 3.62
6
(
s, 3 H, N(1)CH
3
), 6.25 (dd, J= 8.7, 1.8 Hz, 1 H, Ph), 6.38 (d, J= 1.8 Hz, 1 H, Ph), 7.06 (s,
1
1
1
H, CH=C), 7.30–7.62 (m, 4 H, Ph), 8.41 (d, J= 8.7 Hz, 1 H, Ph), 10.03 (s, 1 H, OH),
13
0.29 (s, 1 H, OH); C NMR (75 MHz; DMSO-d
6
) δ: 30.6 (N(1)CH ), 101.7, 107.0,
3
10.8, 117.6, 125.1, 127.9, 128.7, 129.0, 130.2, 132.1, 132.7, 135.2, 158.9, 161.5 (Ph,
+
CH=C), 160.6 (C=O), 173.7 (C=S). MS m/z 360 [M] . HRMS (EI+) calcd for
+
C
17
H13ClN
2
O
3
S [M] : 360.0335; found 360.0336.
4
.3.14. 3-(3-Chlorophenyl)-5-(2,3-dihydroxybenzylidene)-1-methyl-2-thiohydantoin
o
(
3ey). Use of thiohydantoin 11e and aldehyde 8y in the general procedure (80 C, 3 h)
with work-up and column chromatography afforded the title compound as a red solid:
o
yield, 47 mg (65%); Mp 186–188 C; R
f
0.23 (1:2 EtOAc/hexanes); IR (KBr) 3433, 1637,
-
1
1
1
3
478, 1392, 1350, 1284, 1159, 1053 cm ; H NMR (300 MHz; CDCl
3
) δ: 3.42, 3.76 (2s,
1
3
H, N(1)CH
3
), 6.77–7.09 (m, 4 H, Ph, CH=C), 7.26–7.48 (m, 4 H, Ph); C NMR (75
), 111.5, 116.2, 116.6, 118.6, 119.5, 120.2, 121.8,
MHz; CDCl
3
) δ: 31.2, 34.1 (N(1)CH
3
1
1
1
3
4
21.9, 122.2, 124.4, 126.6, 126.7, 127.8, 128.6, 129.4, 129.8, 129.9, 130.0, 130.1, 133.5,
34.3, 134.6, 134.7, 143.0, 143.2, 143.8, 147.8, 161.1 (Ph, CH=C), 162.9, 163.9 (C=O),
+
+
74.9, 179.1 (C=S). MS m/z 360 [M] . HRMS (EI+) calcd for C17
H13ClN
2
O S [M] :
3
60.0335; found 360.0333.
.3.15. 3-(3-Chlorophenyl)-5-(4-(N,N-dimethylamino)benzylidene)-1-methyl-2-
thiohydantoin (3ez). Use of thiohydantoin 11e and aldehyde 8z in the general procedure
o
(
65 C, 10 h) with work-up and column chromatography afforded the title compound as a
2
3

o
red solid: yield, 61 mg (79%); Mp 148~150 C; R
435, 1725, 1631, 1594, 1480, 1386, 1370, 1193, 1154, 1052, 950, 823, 517 cm ; H
NMR (300 MHz; DMSO-d ) δ: 3.01 (s, 6 H, N(CH ), 3.64 (s, 3 H, N(1)CH ), 6.75 (d,
J= 9.0 Hz, 2 H, Ph), 6.91 (s, 1 H, CH=C), 7.32–7.45 (m, 1 H, Ph), 7.50–7.61 (m, 3 H, Ph),
f
0.39 (1:2 EtOAc /hexanes); IR (KBr)
-1 1
3
6
3
)
2
3
1
3
8
1
1
.17 (d, J= 9.0 Hz, 2 H, Ph); C NMR (75 MHz; DMSO-d
6
) δ: 30.6 (N(1)CH ), 111.0,
3
19.6, 124.1, 124.3, 127.8, 128.5, 129.0, 130.1, 132.5, 133.3, 135.2, 151.3 (Ph, CH=C),
+
60.4, (C=O), 172.8 (C=S). MS m/z 371 [M] . HRMS (EI+) calcd for C19
H
18ClN OS
3
+
[
M] : 371.0859; found 371.0857.
4
.3.16. 3-(Indan-5-yl)-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (3fx).
o
Use of thiohydantoin 11f and aldehyde 8x in the general procedure (80 C, 1 h) afforded
o
the title compound as a yellow solid: yield, 47 mg (63%); Mp 228–229 C; R
f
0.29 (1:2
-1
1
EtOAc/hexanes); IR (KBr) 3735, 3434, 1633, 1396, 1348, 704, 611, 518 cm ; H NMR
(
300 MHz; DMSO-d
indanyl), 3.60 (s, 3 H, N(1)CH
H, Ph), 7.03 (s, 1 H, CH=C), 7.04 (d, J= 7.8 Hz, 1 H, Ph-indanyl), 7.15 (s, 1 H, Ph-
6
) δ: 1.96–2.12 (m, 2 H, CH
2
-indanyl), 2.82–2.94 (m, 4 H, CH
2
-
3
), 6.23 (dd, J= 9.0, 2.1 Hz, 1 H, Ph), 6.36 (d, J= 2.1 Hz, 1
1
3
indanyl), 7.31 (d, J= 7.8 Hz, 1 H, Ph-indanyl), 8.38 (d, J= 9.0 Hz, 1 H, Ph); C NMR (75
MHz; DMSO-d ) δ: 25.4 (CH -indanyl), 30.8 (N(1)CH ), 32.2, 32.4 (CH -indanyl), 101.9,
07.1, 111.1, 117.3, 124.4, 124.8, 125.4, 126.8, 132.0, 132.2, 144.5, 159.0, 161.5 (Ph,
6
2
3
2
1
Ph-indanyl, CH=C, one peak was not detected and believed to overlap with the observed
+
peaks), 161.3 (C=O), 174.6 (C=S). MS m/z 366 [M] . HRMS (EI+) calcd for
+
C
20
H
18
N
2
O
3
S [M] : 366.1038; found 366.1035.
4
.3.17. 3-(Indan-5-yl)-5-(2,3-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (3fy).
o
Use of thiohydantoin 11f and aldehyde 8y in the general procedure (80 C, 5 h) afforded
o
the title compound as a yellow solid: yield, 51 mg (83%); Mp 110–113 C; R
f
0.45 (1:1
-1 1
EtOAc/hexanes); IR (KBr) 3433, 2102, 1637, 1384, 1051, 531 cm ; H NMR (300 MHz;
DMSO-d ) δ: 1.98–2.13 (m, 2 H, CH -indanyl), 2.82–2.95 (m, 4 H, CH -indanyl), 3.33,
6
2
2
2
4

3
.61 (2s, 3 H, N(1)CH
m, 4 H, Ph, Ph-indanyl); C NMR (75 MHz; DMSO-d
4.2 (N(1)CH ), 32.2, 32.4 (CH -indanyl), 112.3, 116.4, 116.5, 117.0, 118.2, 119.1, 119.9,
3
), 6.55–6.83 (m, 2 H, Ph), 6.85, 7.01 (2s, 1 H, CH=C), 7.02–7.67
1
3
(
6
) δ: 25.4 (CH -indanyl), 30.8,
2
3
1
1
1
3
2
20.0, 121.3, 121.4, 124.5, 124.6, 124.7, 124.8, 126.6, 126.8, 128.1, 129.1, 131.9, 132.0,
44.5, 144.6, 144.7, 144.8, 144.9, 145.1, 145.4, 145.5 (Ph, Ph-indanyl, CH=C), 161.1,
+
63.2 (C=O), 176.0, 179.7 (C=S). MS m/z 366 [M] . HRMS (EI+) calcd for
+
C
20
H
18
N
2
O
3
S [M] : 366.1038; found 366.1035.
4
.3.18. 3-(Indan-5-yl)-5-(4-(N,N-dimethylamino)benzylidene)-1-methyl-2-
2
3
thiohydantoin (3fz). Use of thiohydantoin 11f and aldehyde 8z in the general
o
procedure (65 C, 8 h) afforded the title compound as a yellow solid: yield, 46 mg (61%);
o
Mp 129-131 C; R
f
0.67 (1:1 EtOAc /hexanes); IR (KBr) 3455, 1715, 1635, 1599, 1489,
-
1 1
1
1
387, 1353, 1159, 1122, 1050, 951, 822, 512 cm ; H NMR (300 MHz; DMSO-d
6
) δ:
.98–2.13 (m, 2 H, CH -indanyl), 2.80–2.95 (m, 4 H, CH -indanyl), 3.00 (s, 6 H,
2
2
N(CH
3
)
2
), 3.63 (s, 3 H, N(1)CH ), 6.72 (d, J= 9.0 Hz, 2 H, Ph), 6.88 (s, 1 H, CH=C), 7.05
3
(
d, J= 7.9 Hz, 1 H, Ph-indanyl), 7.16 (s, 1 H, Ph-indanyl), 7.32 (d, J= 7.9 Hz, 1 H, Ph-
1
3
indanyl), 8.17 (d, J= 9.0 Hz, 2 H, Ph); C NMR (75 MHz; DMSO-d
6
) δ: 25.1 (CH
2
-
indanyl), 30.6 (N(1)CH ), 31.9, 32.1 (CH -indanyl), 111.0, 119.8, 123.9, 124.1, 124.3,
3
2
1
24.5, 126.5, 131.9, 133.2, 144.2, 151.2 (Ph, Ph-indanyl, CH=C, one peak was not
detected and believed to overlap with the observed peaks), 160.9 (C=O), 173.6 (C=S).
+
+
MS m/z 377 [M] . HRMS (EI+) calcd for C22
H
23
N OS [M] : 377.1561; found 377.1565.
3
4
.4. General procedure for evaluation of NOX 1 and 4 inhibitory activities
We established transgenic fly lines overexpressing human NOX 1 or human NOX 4 in a
Drosophila DUOX (dDUOX) knockdown condition according to the previous
3
3
procedure. The genotypes of lines used in this study were human NOX 1 (UAS-
2
5

hNOX1/UAS-dDUOX-RNAi; Da-GAL4/+) and human NOX 4 transgenic fly (UAS-
hNOX4/UAS-dDUOX-RNAi; Da-GAL4/+). We detected human NOX1 or human NOX4
isozyme in each transgenic fly using realtime quantitative PCR (qPCR). Transgenic flies
were homogenated with ice-cold PBS containing protease inhibitors and membranes
enriched human NOX 1 or NOX 4 were harvested. Membranes containing human NOX1
or NOX4 served to monitor ROS production by lucigenin chemiluminescence in the
absence or presence of chemical compound. The reaction medium consisted in Hepes-
buffered salt solution (128 mM NaCl, 2.5 mM KCl, 1.2 mM MgSO
4
, 1.0 mM KH
2
PO
4
,
1
.75 µM CaCl , 0.03 Na EDTA, 5.5 mM glucose, and 20 mM HEPES, pH 7.4), 400 µM
2
2
3
4
lucigenin (10,10-dimethyl-bis-9,9-bisacridinium nitrate) and 500 µM NADPH.
Acknowledgements
This work was supported by the National Research Foundation of Korea (NRF)
grant (2012R1A5A1048236, 2012R1A1A2001033 and 2011-0028885), by Redoxomics
grant (2012M3A9C5048708) funded by the Ministry of Education, Science and
Technology (MEST), and by Priority Research Centers Program through NRF funded by
MEST (2016R1A6A1A03007648).
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2
8

Graphical abstract
2
9