A novel serine racemase inhibitor suppresses neuronal over-activation in vivo

Article abstract of DOI:10.1016/j.bmc.2017.05.011

Serine racemase (SRR) is an enzyme that produces D-serine from L-serine. D-Serine acts as an endogenous coagonist of NMDA-type glutamate receptors (NMDARs), which regulate many physiological functions. Over-activation of NMDARs induces excitotoxicity, which is observed in many neurodegenerative disorders and epilepsy states. In our previous works on the generation of SRR gene knockout (Srr-KO) mice and its protective effects against NMDA- and Aβ peptide-induced neurodegeneration, we hypothesized that the regulation of NMDARs’ over-activation by inhibition of SRR activity is one such therapeutic strategy to combat these disease states. In the previous study, we performed in silico screening to identify four compounds with inhibitory activities against recombinant SRR. Here, we synthesized 21 derivatives of candidate 1, one of four hit compounds, and performed screening by in vitro evaluations. The derivative 13J showed a significantly lower IC₅₀ value in vitro, and suppressed neuronal over-activation in vivo.

Full text of DOI:10.1016/j.bmc.2017.05.011

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A novel serine racemase inhibitor suppresses neuronal over-activation
in vivo
b
c
c
d
Hisashi Mori a,d, , Ryogo Wada , Satoyuki Takahara , Yoshikazu Horino , Hironori Izumi ,
⇑
Tetsuya Ishimoto d, Tomoyuki Yoshida a,d, Mineyuki Mizuguchi a,d, Takayuki Obita d, Hiroaki Gouda e,
,
⇑
Shuichi Hirono f, Naoki Toyooka b,c,
⇑
a Graduate School of Innovative Life Science, University of Toyama, Toyama 930-0194, Japan
b Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555, Japan
c Graduate School of Innovative Life Science, University of Toyama, Toyama 930-8555, Japan
d Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan
e School of Pharmacy, Showa University, Tokyo 142-8555, Japan
f School of Pharmacy, Kitasato University, Tokyo 108-8641, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Serine racemase (SRR) is an enzyme that produces D-serine from L-serine. D-Serine acts as an endogenous
Received 12 April 2017
Revised 2 May 2017
Accepted 5 May 2017
Available online xxxx
coagonist of NMDA-type glutamate receptors (NMDARs), which regulate many physiological functions.
Over-activation of NMDARs induces excitotoxicity, which is observed in many neurodegenerative disor-
ders and epilepsy states. In our previous works on the generation of SRR gene knockout (Srr-KO) mice and
its protective effects against NMDA- and Ab peptide-induced neurodegeneration, we hypothesized that
the regulation of NMDARs’ over-activation by inhibition of SRR activity is one such therapeutic strategy
to combat these disease states. In the previous study, we performed in silico screening to identify four
compounds with inhibitory activities against recombinant SRR. Here, we synthesized 21 derivatives of
candidate 1, one of four hit compounds, and performed screening by in vitro evaluations. The derivative
13J showed a significantly lower IC50 value in vitro, and suppressed neuronal over-activation in vivo.
Ó 2017 Elsevier Ltd. All rights reserved.
Keywords:
Serine racemase
D-Serine
Arc-Luc Tg mice
NMDA receptors
Over-action
In vivo assay
1. Introduction
NMDA- and Ab peptide-induced neurodegeneration.4 Thus, the
regulation of NMDARs’ over-activation by the inhibition of SRR
In mammals, a relatively high amount of free d-serine, which is
not used for protein synthesis, is detected mainly in the forebrain.1
d-Serine is produced from l-serine by serine racemase (SRR), a
pyridoxal phosphate (PLP)-dependent enzyme.2 d-Serine acts as
an endogenous coagonist of NMDA-type glutamate receptors
(NMDARs). Full activation of NMDARs requires simultaneous bind-
ing of the agonist glutamate and coagonist d-serine or glycine.
NMDARs are involved in many physiological and pathological
states including neural network formation, synaptic plasticity,
learning and memory, neurodegenerative disorders, and psychi-
atric disorders.3 Over-activation of NMDARs induces excitotoxicity,
which is observed in many neurodegenerative disorders and epi-
lepsy states. We have generated SRR gene knockout (Srr-KO) mice
and revealed the protective phenotypes of the Srr-KO mice against
activity is one such therapeutic strategy to combat these disease
states. Malonate was reported as the most popular SSR inhibitor,5
followed by several other inhibitors such as dipeptides,6 hydrox-
amic acid derivatives,7 and malonate-based derivatives.8 However,
the in vivo effect of these inhibitors has not been reported.
Previously, we identified SRR inhibitor candidates using in silico
screening based on the reported three-dimensional structure of
SRR.9 Table 1 shows the inhibitory activities of these compounds
using recombinant mouse SRR. In our previous study, we also
reported novel chemicals possessing SRR inhibitory activities com-
parable to malonate, based on the candidate 4 (Fig. 1).10
In this study, we attempted to obtain novel compounds with
more potent SRR inhibitory activities than malonate, based upon
the remaining candidates. We specifically focused on derivatives
of compound 1, as its inhibitory activity was the most potent
among the four compounds. Next, to extend our study in vivo, we
employed the Arc-Luc transgenic (Tg) mouse strain.11 Using the
Arc-Luc Tg hairless (HL) mouse strain, we can examine the change
in neuronal-activity-dependent expression of the Arc gene with
⇑
Corresponding authors at: Graduate School of Innovative Life Science, Univer-
sity of Toyama, Toyama 930-0194, Japan (H. Mori). Graduate School of Science and
Engineering, University of Toyama, Toyama 930-8555, Japan (N. Toyooka).
E-mail address: toyooka@eng.u-toyama.ac.jp (N. Toyooka).
http://dx.doi.org/10.1016/j.bmc.2017.05.011
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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H. Mori et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 1
potent candidate 1 based on the central structure of acyl hydrazino
thiourea moiety 1D (Fig. 3) of 1.
Results of in vitro assay of 1–4 using mouse SRR. The
remaining activity of SRR with the inhibitors (1 mM)
was evaluated with the percentage of the D-serine
production compared with that without inhibitors.
Compounds 13A–U were synthesized as shown in Scheme 1. The
aldehydes 5a–h were converted to unsaturated carboxylic acids
7a–h by the Doebner condensation or the Horner-Wadsworth-
Emmons reaction of the corresponding aldehydes 5a–h followed
by hydrolysis of the resulting esters 6g, h. Conversion of 7a–h to
the corresponding acid chlorides, which were treated with NH4SCN
in the presence of catalytic amounts of PEG-40014, afforded the
isothiocyanates 8a–h. The isothiocyanates 10c, i were also prepared
from the carboxylic acids 9c, i in the same manner as that for 8a–h.
However, the hydrazides 12j–r were prepared from the carboxylic
acids 11j–r via the corresponding esters with hydrazine. Finally,
the addition reaction of the hydrazides 12j–r to isothiocyanates
8a–h and 10c, i gave rise to the desired thioureas 13A–U.
We also synthesized 35 and 37 derivatives of the candidates 2
and 3, respectively. Like the chemical structure of 1, both 2 and 3
possess several hydrogen bonding and two aromatic groups in
their central and edge parts, respectively. Therefore, we adopted
a similar strategy to that for 1 for designing derivatives of 2 and
3. With the in vitro screening of these derivatives, we identified
the derivatives 14 and 15 as the possible inhibitors of SSR.
(Scheme 2) The derivative 14 possesses an amide linkage instead
of ester functionality in 2, and the derivative 15 possesses
phenylenediamine structure instead of ethylenediamine unit in 3.
As shown in Fig. 4, the derivatives 14 and 15 showed almost same
IC50 value (0.27 mM and 0.28 mM, respectively).
Compound
Activity [%]
1
2
3
4
27
32
36
35
bioluminescent signals. The expression of Arc is dependent on the
activity of NMDARs,12 thus, we can monitor the activity of NMDAR
in the Arc-Luc Tg HL mice with the change in the bioluminescent
signal from a live mouse. Using this technique, we successfully
identified a novel SRR inhibitor suppressing the Arc expression
in vivo.
2. Results and discussion
2.1. Modeling of interaction mode of 1 with SRR
First, in order to obtain a strategy for designing derivatives of 1,
the interaction mode of 1 with SRR was investigated by the previ-
ously constructed procedure,10 which consisted of the molecular-
docking calculation and molecular mechanics Poisson–Boltzmann
surface area (MM-PBSA) free energy analysis.13 The resulting bind-
ing mode of 1 with SRR is shown in Fig. 2.
2.3. Evaluation of SRR inhibitory activities of derivatives of 13A–U, 14,
and 15
SRR exists in a dimeric form. Therefore, we denote one chain as
‘‘A” and the other chain as ‘‘B.” The central part of 1, the acyl hydra-
zino thiourea moiety, is suggested to be significantly important to
interact with SRR, as it formed a total of four hydrogen bonds with
N86, D238, and N316 of the A chain of SRR (Fig. 2A). The thiophene
moiety of 1 was located in the malonate binding site of the A chain
of SRR to create van der Waals interactions with PLP-K56, S83, S84,
G85, N86, and G239. On the other hand, the phenyl moiety of 1 ori-
ented in the opposite direction and created van der Waals interac-
tions with R277, E276, and K279 of the B chain of SRR. This means
the phenyl moiety of 1 is located in the interface region of the
dimer. As shown in Fig. 2B, available spaces are visible around
the thiophene and phenyl moieties of 1. Therefore, we replaced
these two moieties with larger substituents to design more potent
derivatives of 1, aiming at the increase of the van der Waals
interaction.
The synthesized derivatives 13A–U, 14, and 15 were assayed
in vitro with recombinant human SRR with C2D2 mutations for
which the X-ray crystal structure was reported9 and that we used
previously (Table 2).10
According to the in vitro evaluations of all synthesized deriva-
tives, we selected three potent derivatives 13J, 14, and 15.
Although compounds 13A-B are also highly potent inhibitors, the
structures of these compounds are very similar to that of the orig-
inal compound 1. From the structural diversity, we expected to be
able to identify a novel inhibitor. For these reasons, we examined
the inhibitory effects of 13J, 14, and 15 in more detail, and deter-
mined the IC50 values of these compounds. Among three com-
pounds, two of them (14, 15) showed similar IC50 values to that
of malonate, one of the standard inhibitors of SRR (Fig. 4). Another
compound named 13J showed a significantly lower IC50 value than
that of malonate (Fig. 4).
2.2. Chemistry
13J possesses the phenyl and 3,5-dibromophenyl moieties,
which are larger than the thiophene and phenyl moieties of 1,
respectively.
According to the above information obtained by the interaction
mode of 1 with SRR, we designed the derivatives of the most
Fig. 1. Structure of four small molecules with inhibitory activity against SRR from eighteen virtual hits.
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H. Mori et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
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Fig. 2. Stereoview of modeled binding mode of 1 with SRR. (A) Hydrogen-bonding interactions are indicated by dashed lines. (B) 1 and SRR are shown in space-filling
representation and surface display, respectively.
affinity KA-type glutamate receptor, which is abundant in the
dentate gyrus and CA3 regions in the hippocampus.15 We reported
the increase in bioluminescence signal intensity after KA injection
in Arc-Luc Tg mice.11
We examined the effect of 13J treatment on changes in biolumi-
nescence signal intensity in regions of interest (ROIs) of the brain
after KA injection. We first measured bioluminescence signal
Fig. 3. The central structure of acyl hydrazino thiourea moiety of 1.
intensity before starting the experiment and administrated orally
13J or vehicle (PEG-400). Three hours after the administration of
Fig. 5 shows the modeled binding mode of 13J with SRR. 13J is
13J or vehicle, we measured the bioluminescence signals as a basal
suggested to bind to SRR in a similar manner to that of 1. The acyl
level and injected KA intraperitoneally (i.p.). We measured the bio-
hydrazino thiourea moiety of 13J retained four hydrogen bonds
with SRR. The phenyl and 3,5-dibromophenyl moieties of 13J were
located in the malonate binding site and the dimer interface
luminescence signals 2 h, 4 h, and 6 h after the KA treatment
(Fig. 6A). The bioluminescence signal intensity in the brain of the
control mice significantly increased after KA injection. In contrast,
in the 13J administered mice, the intensities of detected biolumi-
nescence signals 2 h after KA injection were restrained by about
1.4-fold, as compared with mice treated with vehicle (Fig. 6B, C).
These results suggest the inhibitory effect of 13J treatment on
the over-activation of NMDARs in vivo.
region, respectively, as observed in the binding mode of
(Fig. 2). As expected, 13J showed more favorable van der
Waals interaction energy (–43.74 kcal/mol) than that of
1
1
(–39.07 kcal/mol), which led to the superior binding free energy
(–25.86 kcal/mol) of 13J compared to that of 1 (–22.22 kcal/mol).
This energy analysis may explain the more potent SRR inhibitory
activity of 13J over 1.
3. Conclusion
2.4. In vivo assay of 13J
We synthesized 21 derivatives for 1, 35 derivatives for 2, and
37 derivatives for 3, respectively, to obtain novel compounds
with more potent SRR inhibitory activities than malonate. Among
the synthesized derivatives, three derivatives (13J, 14, and 15)
showed relatively strong inhibitory effects on recombinant
human SRR with C2D2 mutations. In particular, compound 13J
showed a significantly lower IC50 value compared to those of
14, 15, and malonate. We also demonstrated that compound
13J suppressed neuronal activity-dependent Arc expression
in vivo. These results suggest that compound 13J will regulate
Then, we performed an in vivo assay on 13J to examine the
effect on over-activation of NMDARs in the brain. To examine the
over-activation of the brain in mice, we used Arc-Luc Tg mice.11a
In this study, to detect the brain activity with the
bioluminescence signal more easily, we crossed the original Arc-
Luc Tg mice with a hairless (HL) mouse strain to establish the
Arc-Luc Tg HL mice strain.11b Acute injection of kainate (KA) to
the Arc-Luc HL Tg mice induces limbic epileptic seizure and
induction of Arc expression owing to the activation of the high
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H. Mori et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Scheme 1. Synthesis of the derivatives 13A–U: (a) malonic acid, pyridine, aniline (cat.), toluene, reflux, 18 h; (b) triethyl phosphonoacetate, NaH, THF, 0 °C, 30 min; r.t., 18 h;
(c) LiOHꢂH2O, MeOH:H2O = 3:1, reflux, 18 h; (d) SOCl2, CH2Cl2, r.t., 18 h; (e) NH4SCN, PEG-400 (cat.), CH2Cl2, r.t., 2 h; (f) SOCl2, benzene, r.t., 18 h; (g) HCl (cat.), EtOH, reflux, 18
h; (h) N2H4ꢂH2O, EtOH, reflux, 18 h; (i) CH2Cl2, r.t., 18 h.
NMDARs’ over-activation, and this compound will provide a ther-
apeutic strategy to combat neurodegenerative disorders using the
inhibition of SSR to reduce the production of d-serine, the coago-
nist of NMDARs.
4.2. General procedure for the Doebner condensation
To a stirred solution of aldehyde 5a-f (1.00 mmol) in toluene
(5 mL) were added malonic acid (156 mg, 1.50 mmol), pyridine
(0.12 mL, 1.54 mmol), and aniline (0.01 mL, 0.12 mmol), and the
resulting mixture was refluxed for 18 h. After cooling, the reaction
mixture was diluted with AcOEt and 10% HCl aq. and organic phase
was separated, dried over Na2SO4, and concentrated in vacuo. The
4. Experimental section
4.1. Chemistry
residue
was
chromatographed
on
SiO2
(Hexane:Ace-
tone = 100:1 ꢁ 40:1) to give corresponding carboxylic acid 7a–f.
According to this procedure, the following known carboxylic
acids were prepared. 3-(4-Chlorophenyl)acrylic acid (7a: 89%),16
3-p-Tolylacrylic acid (7b: 100%),17 3-(4-Fluoro-phenyl)acrylic acid
(7c: 86%),18 3-(4-Isopropylphenyl)acrylic acid (7d: 90%),19 3-(2,6-
Dimethoxyphenyl)acrylic acid (7e: 100%),20 3-(4-Trifluo-
romethylphenyl)acrylic acid (7f: 100%).21
General: Flash chromatography was performed with Kanto
Kagaku silica gel 60N (63–210 mm). NMR spectra were recorded
on a JEOL JNM-A 400, JEOL JNM-ECX 500 spectrometer in the sol-
vent indicated. Chemical shifts (d) are given in ppm downfield from
TMS and referenced with CHCl3 (7.26 ppm) or DMSO (2.49 ppm) as
an internal standard. Peak multiplicities are designated by the fol-
lowing abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad and coupling constants are given in (J) Hz.
Infrared spectra were obtained with a SHIMADZU FTIR-8400 spec-
trometer using film KBr pellet technique. High resolution mass
spectral data was obtained on a JEOL MStation JMS-700. All com-
mercial reagents were used as received unless otherwise noted.
The purities (=95%) of compounds 13A–U, 14, and 15 were ana-
lyzed by HPLC analysis. Analytical HPLC performed on JASCO PU-
2082 plus intelligent HPLC pump with GL Science InertSustainÒC18
4.3. General procedure for the Horner-Wadsworth-Emmons reaction
To a stirred suspension of NaH (133 mg, 3.32 mmol) in THF
(5 mL) was added triethyl phosphonoacetate (0.66 mL, 3.32 mmol)
at 0 °C, and the resulting mixture was stirred at 0 °C. After stirring
for 30 min., a solution of aldehyde 5g, h (2.77 mmol) in THF (5 mL)
was added to the reaction mixture, and the resulting mixture was
stirred at room temperature for 18 h. The reaction was quenched
by adding H2O, and the aqueous mixture was extracted with
(5
lL, 4.6 ꢀ 250 mm) column and 100% MeCN as mobile phase
with flow rate of 1–2 mL/min at 30–50 °C.
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H. Mori et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Scheme 2. Synthesis of the derivatives 14, 15.
5.38 mmol), and the resulting mixture was refluxed for 2 h. After
cooling, the solvent was removed, and then the residue was acidi-
fied with 10% HCl aq. The aqueous mixture was extracted with
AcOEt, and the organic extracts were dried over Na2SO4, and evap-
orated to give the carboxylic acid 7g, h.
According to this procedure, the following known carboxylic
acids were prepared. 3-(3,5-Dibromophenyl)acrylic acid (7g:
100%),22 3-(2,4,6-Trifluorophenyl)acrylic acid (7h: 99%).23
4.4. General procedure for the synthesis of isothiocyanate (8a–h, 10c, i)
To a stirred solution of carboxylic acid 7a–h or 9c, i (0.55 mmol)
in CH2Cl2 for 7a–h or benzene for 9c, i (3 mL) was added SOCl2
(0.05 mL, 0.66 mmol), and the resulting mixture was stirred at
room temperature for 18 h. The solvent was removed, and then
to the residue were added CH2Cl2 (3 mL), NH4SCN (63 mg,
0.82 mmol), and PEG-400 (1 drop), and the resulting mixture was
stirred at room temperature for 2 h. The solvent was removed,
and the residue was chromatographed on SiO2 (Hexane:Ace-
tone = 50:1 ꢁ 40:1) to give corresponding isothiocyanate 8a–h
and 10c, i which were used in the next step immediately.
According to this procedure, the following known isothio-
cyanates were prepared. 3-(4-Chlorophenyl)-acryloyl isothio-
cyanate (8a: 85%),24 3-p-Tolylacryloyl isothiocyanate (8b: 60%),25
(4-Fluorophenyl)acetyl isothiocyanate (10c: 48%),26 Phenylacetyl
isothiocyanate (10i: 75%).27
Fig. 4. Dose-response curves of compounds inhibition against the SRR activity.
Curves of malonate (h), 14 (4), 15 (}), and 13J (s) are indicated. Data are
presented as mean SD (n = 5–15 in each concentration). The IC50 values of
malonate, 14, 15, and 13J are 0.77 mM, 0.27 mM, 0.28 mM, and 0.14 mM,
respectively. The IC50 value of 13J is significantly lower than that of malonate
(P = 6.97 ꢀ 10ꢃ7).
CH2Cl2, and the organic phase was separated, the aqueous mixture
was extracted with CH2Cl2. The organic phase and extracts were
combined, dried over Na2SO4, and concentrated in vacuo. The
residue was chromatographed on SiO2 (Hexane:Acetone =
90:1 ꢁ 80:1) to give corresponding ethyl ester 6g, h. To a stirred
solution of 6g, h (2.69 mmol), obtained above, in the mixed solvent
(20 mL) with MeOH-H2O (3: 1) was added LiOHꢂH2O (226 mg,
4.4.1. 3-(4-Fluorophenyl)acryloyl isothiocyanate (8c)
Yield 88%; 1H NMR (400 MHz, CDCl3) d: 6.43 (1H, d, J = 15.8 Hz),
7.13 (2H, t, J = 8.6 Hz), 7.57 (2H, dd, J = 8.6, 5.3 Hz), 7.73 (1H, d,
J = 15.8 Hz).
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H. Mori et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 2
Results of in vitro assay of 13A–U, 14, and 15 using human SRR. The remaining activity of SRR with the inhibitors (1 mM) was evaluated with the percentage of the D-serine
production compared to that without inhibitors.
Compound
Activity [%]
Compound
Activity [%]
Compound
Activity [%]
Mal
1
63
37
23
24
22
28
27
39
45
13H
13I
13J
13K
13L
13M
13N
13O
44
38
20
46
32
31
54
44
13P
13Q
13R
13S
13T
13U
14
61
62
53
61
55
49
41
77
13A
13B
13C
13D
13E
13F
13G
15
Fig. 5. Stereoview of modeled binding mode of 13J with SRR. (A) Hydrogen-bonding interactions are indicated by dashed lines. (B) 13J and SRR are shown in the space-filling
representation and surface display, respectively.
4.4.2. 3-(4-Isopropylphenyl)acryloyl isothiocyanate (8d)
4.4.6. 3-(2,4,6-Trifluorophenyl)acryloyl isothiocyanate (8h)
Yield 67%; 1H NMR (400 MHz, CDCl3) d: 6.74–6.79 (3H, m), 7.78
(1H, d, J = 16.1 Hz).
Yield 79%; 1H NMR (400 MHz, CDCl3) d: 1.25 (6H, d, J = 7.1 Hz),
2.93 (1H, sept, J = 7.1 Hz), 6.45 (1H, d, J = 15.7 Hz), 7.27 (2H, d,
J = 8.2 Hz), 7.48 (2H, d, J = 8.2 Hz), 7.73 (1H, d, J = 15.7 Hz).
4.5. General procedure for the synthesis of acyl hydrazino thiourea
(13A-O)
4.4.3. 3-(2,6-Dimethoxyphenyl)acryloyl isothiocyanate (8e)
Yield 27%; 1H NMR (400 MHz, CDCl3) d: 3.89 (6H, s), 6.55 (2H, d,
J = 8.5 Hz), 6.99 (1H, d, J = 15.9 Hz), 7.32 (1H, t, J = 8.5 Hz), 8.23 (1H,
d, J = 15.9 Hz).
To a stirred solution of carboxylic acid 11j–r (1 mmol) in EtOH
(3 mL) was added conc. HCl (1 drop), and the resulting mixture
was refluxed for 18 h. After cooling, the solvent was removed, and
then the residue was added sat. NaHCO3 aq., and the aqueous mix-
ture was extracted with CH2Cl2. The organic extracts were dried
over Na2SO4, and the solvent was removed to afford the corre-
sponding ethyl ester, which was used for the next reaction without
further purification. To a stirred solution of ethyl ester (1 mmol),
obtained above, in EtOH (0.5 mL) was added hydrazine monohy-
drate (0.05 mL, 1 mmol), and the resulting mixture was refluxed
for 18 h. The solvent was removed to give the corresponding acyl-
4.4.4. 3-(4-Trifluoromethylphenyl)acryloyl isothiocyanate (8f)
Yield 85%; 1H NMR (400 MHz, CDCl3) d: 6.57 (1H, d, J = 15.9 Hz),
7.66–7.71 (4H, m), 7.78 (1H, d, J = 15.9 Hz).
4.4.5. 3-(3,5-Dibromophenyl)acryloyl isothiocyanate (8g)
Yield 63%; 1H NMR (400 MHz, CDCl3) d: 6.45 (1H, d, J = 15.8 Hz),
7.58–7.62 (3H, m), 7.74 (1H, s).
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H. Mori et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
Fig. 6. Effect of 13J treatment on changes in bioluminescence signal intensity in the brain of Arc-Luc Tg HL mice after KA injection. (A) The protocol for in vivo imaging using
Arc-Luc Tg HL mice. (B) Representative bioluminescence images (pseudocolored, 9000–25,000 counts) show the regions of interest (ROIs) of vehicle (PEG-400) administered
mice (upper) and 13J administered mice (lower). (C) Fold change of bioluminescence signal intensity in vehicle and 13J administered mice after KA injection. The fold changes
of the bioluminescence signal intensity are calculated from the measured photon intensity of ROIs at 0 h and each time point. The data are represented as mean SD.
*P < 0.05; two-tailed Student’s t test.
hydrazide, which was used for the next reaction without further
purification. To a stirred solution of acylhydrazide, obtained above,
in CH2Cl2 (1 mL) was added a solution of isothiocyanate 8a–h or
10c, i (1 mmol) in CH2Cl2 (5 mL), and the resulting mixture was stir-
red at room temperature for 18 h. The insoluble solid was corrected
by filtration, and dried to give acyl hydrazino thiourea 13A–U.
13C NMR (100 MHz, DMSO-d6) d: 33.93, 116.25 (d, J = 22.3 Hz),
119.25, 125.23, 126.69, 126.72, 130.63 (d, J = 8.3 Hz), 130.73 (d,
J = 3.3 Hz), 136.22, 143.42, 163.46 (d, J = 248.9 Hz), 165.73,
166.31, 176.78; IR (KBr): 1628, 1651, 1680, 3186 cmꢃ1; MS (EI):
m/z 363 (M+); HRMS: calcd for C16H14FN3O2S2 363.0511, found
363.0513; Mp: 215–216 °C (from Acetone). Purity (HPLC) = 96.28%.
4.5.1. 3-(4-Chlorophenyl)-N-[N0-(2-thiophen-2-ylacetyl)-
hydrazinocarbothioyl]acrylamide (13A)
4.5.4. 3-(4-Isopropylphenyl)-N-[N0-(2-thiophen-2-ylacetyl)-
hydrazinocarbothioyl]acrylamide (13D)
Yield 64%; 1H NMR (400 MHz, DMSO-d6) d: 3.85 (2H, s), 6.95–
7.01 (3H, m), 7.38 (1H, dd, J = 5.0, 1.3 Hz), 7.53 (2H, d, J = 8.5 Hz),
7.64 (2H, d, J = 8.5 Hz), 7.73 (1H, d, J = 15.9 Hz), 11.16 (1H, br),
11.66 (1H, br), 12.57 (1H, br); 13C NMR (100 MHz, DMSO-d6) d:
33.90, 120.18, 125.18, 126.65, 126.67, 129.20, 129.90, 132.97,
135.26, 136.19, 143.14, 165.56, 166.26, 176.63; IR (KBr): 1630,
Yield 50%; 1H NMR (400 MHz, DMSO-d6) d: 1.20 (6H, d,
J = 6.8 Hz), 2.91 (1H, sept, J = 6.8 Hz), 3.85 (2H, s), 6.93–6.98 (3H,
m), 7.34 (2H, d, J = 8.1 Hz), 7.38 (1H, dd, J = 5.0, 1.3 Hz), 7.54 (2H,
d, J = 8.1 Hz), 7.70 (1H, d, J = 15.9 Hz), 11.14 (1H, br), 11.62 (1H,
br), 12.59 (1H, br); 13C NMR (100 MHz, DMSO-d6) d: 23.56, 33.40,
33.89, 118.37, 125.18, 126.64, 126.67, 127.12, 128.40, 131.73,
136.20, 144.63, 151.64, 165.92, 166.24, 176.78; IR (KBr): 1605,
1626, 1682, 3210 cmꢃ1; MS (EI): m/z 387 (M+); HRMS: calcd for
1655, 1684, 3211 cmꢃ1; MS (EI): m/z 379 (M+); HRMS: calcd for
35
C
H ClN3O2S2 379.0216, found 379.0193; Mp: 232–233 °C (from
16 14
EtOH). Purity (HPLC) ꢄ 98%.
C19H21N3O2S2 387.1075, found 387.1078; Mp: 201–202 °C (from
AcOEt). Purity (HPLC) ꢄ 98%.
4.5.2. N-[N0-(2-Thiophen-2-ylacetyl)hydrazinocarbothioyl]-3-p-
tolylacrylamide (13B)
4.5.5. 3-(2,6-Dimethoxyphenyl)-N-[N0-(2-thiophen-2-ylacetyl)-
hydrazinocarbothioyl]acrylamide (13E)
Yield 77%; 1H NMR (400 MHz, DMSO-d6) d: 2.33 (3H, s), 3.85
(2H, s), 6.92–6.98 (3H, m), 7.27 (2H, d, J = 7.9 Hz), 7.38 (1H, dd,
J = 5.1, 1.0 Hz), 7.51 (2H, d, J = 7.9 Hz), 7.69 (1H, d, J = 15.9 Hz),
11.14 (1H, br), 11.60 (1H, br), 12.59 (1H, br); 13C NMR (100 MHz,
DMSO-d6) d:21.09, 33.92, 118.26, 125.20, 126.66, 126.69, 128.30,
129.75, 131.32, 136.22, 140.95, 144.69, 165.96, 166.26, 176.69; IR
(KBr): 1630, 1661, 1684, 3227 cmꢃ1; MS (EI): m/z 359 (M+); HRMS:
calcd for C17H17N3O2S2 359.0763, found 359.0766; Mp: 212–213 °C
(from EtOH). Purity (HPLC) ꢄ 98%.
Yield 59%; 1H NMR (400 MHz, DMSO-d6) d: 3.87 (8H, s), 6.71
(2H, d, J = 8.3 Hz), 6.96–6.98 (2H, m), 7.34–7.38 (3H, m), 8.06
(1H, d, J = 15.9 Hz), 11.12 (1H, br), 11.67 (1H, br), 12.73 (1H, br);
13C NMR (100 MHz, DMSO-d6) d: 33.90, 55.92, 104.07, 110.99,
120.95, 125.18, 126.62, 126.67, 132.55, 135.32, 136.23, 136.86,
159.82, 166.17, 167.53, 176.86; IR (KBr): 1611, 1653, 1670,
3219 cmꢃ1; MS (EI): m/z 405 (M+); HRMS: calcd for C18H19N3O4S2
405.0804, found 405.0826; Mp: 220–221 °C (from Hexane and
Acetone). Purity (HPLC) ꢄ 98%.
4.5.3. 3-(4-Fluorophenyl)-N-[N0-(2-thiophen-2-ylacetyl)-
hydrazinocarbothioyl]acrylamide (13C)
4.5.6. N-[N0-(2-Thiophen-2-ylacetyl)hydrazinocarbothioyl]-3-(2,4,6-
trifluorophenyl)acrylamide (13F)
Yield 89%; 1H NMR (400 MHz, DMSO-d6) d: 3.85 (2H, s), 6.93
(1H, d, J = 15.9 Hz), 6.96–6.99 (2H, m), 7.31 (2H, t, J = 8.8 Hz),
7.38 (1H, dd, J = 4.9, 1.5 Hz), 7.69 (2H, dd, J = 8.8, 5.6 Hz), 7.74
(1H, d, J = 15.9 Hz), 11.15 (1H, br), 11.64 (1H, br), 12.58 (1H, br);
Yield 69%; 1H NMR (400 MHz, DMSO-d6) d: 3.85 (2H, s), 6.96–
6.98 (2H, m), 7.20 (1H, d, J = 16.0 Hz), 7.36–7.40 (3H, m), 7.60
(1H, d, J = 16.0 Hz), 11.17 (1H, br), 11.87 (1H, br), 12.57 (1H, br);
Please cite this article in press as: Mori H., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.05.011

8
H. Mori et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
13C NMR (100 MHz, DMSO-d6) d: 33.90, 101.73 (td, J = 26.9, 2.5 Hz),
108.66 (td, J = 14.9, 5.0 Hz), 124.65, 125.19, 126.65, 126.68, 129.11,
136.19, 161.39 (ddd, J = 253.9, 15.7, 9.1 Hz), 163.01 (dt, J = 251.4,
16.5 Hz), 165.39, 166.27, 176.44; IR (KBr): 1624, 1653, 1684,
3190 cmꢃ1; MS (EI): m/z 399 (M+); HRMS: calcd for C16H12F3N3O2-
S2 399.0323, found 399.0315; Mp: 207–208 °C. Purity (HPLC) ꢄ
98%.
3172 cmꢃ1; MS (EI): m/z 349 (M+); HRMS: calcd for C15H12FN3O2S2
349.0355, found 349.0347; Mp: 217–218 °C. Purity (HPLC) ꢄ 98%.
4.5.12. 3-(2,6-Dimethoxyphenyl)-N-(N0-phenylacetylhydrazino-
carbothioyl)acrylamide (13L)
Yield 73%; 1H NMR (400 MHz, DMSO-d6) d: 3.62 (2H, s), 3.89
(6H, s), 6.71 (2H, d, J = 8.3 Hz), 7.25–7.39 (7H, m), 8.05 (1H, d,
J = 15.9 Hz), 11.07 (1H, s), 11.65 (1H, s), 12.70 (1H, br); 13C NMR
(100 MHz, DMSO-d6) d: 39.59, 55.94, 104.09, 111.02, 121.00,
126.63, 128.29, 129.21, 132.57, 135.19, 135.31, 159.84, 167.26,
167.53, 177.00; IR (KBr): 1609, 1660, 1668, 3227 cmꢃ1; MS (EI):
m/z 399 (M+); HRMS: calcd for C20H21N3O4S 399.1253, found
399.1246; Mp: 211–212 °C. Purity (HPLC) ꢄ 98%.
4.5.7. 3-(4-Fluorophenyl)-N-(N0-phenylacetylhydrazino-carbothioyl)
acrylamide (13G)
Yield 89%; 1H NMR (400 MHz, DMSO-d6) d: 3.62 (2H, s), 6.93
(1H, d, J = 15.7 Hz), 7.25–7.32 (7H, m), 7.69 (2H, dd, J = 8.8,
5.6 Hz), 7.73 (1H, d, J = 15.7 Hz), 11.10 (1H, s), 11.62 (1H, s),
12.56 (1H, s); 13C NMR (100 MHz, DMSO-d6) d: 39.57, 116.20 (d,
J = 21.5 Hz), 119.25, 126.61, 128.25, 129.20, 130.57 (d, J = 9.1 Hz),
130.71 (d, J = 3.3 Hz), 135.26, 143.33, 163.41 (d, J = 248.9 Hz),
4.5.13. 3-(2,6-Dimethoxyphenyl)-N-[N0-(thiophene-2-carbonyl)-
hydrazinocarbothioyl]acrylamide (13M)
165.66, 167.29, 176.77; IR (KBr): 1629, 1651, 1681, 3191 cmꢃ1
;
Yield 26%; 1H NMR (400 MHz, DMSO-d6) d: 3.88 (6H, s), 6.72
(2H, d, J = 8.5 Hz), 7.21 (1H, t, J = 5.1 Hz), 7.37–7.42 (2H, m),
7.84–7.90 (2H, m), 8.09 (1H, d, J = 15.9 Hz), 11.07 (1H, br), 11.68
(1H, br), 12.14 (1H, br); 13C NMR (125 MHz, DMSO-d6) d: 56.43,
104.57, 109.48, 111.51, 121.58, 128.69, 130.02, 132.47, 133.07,
135.78, 160.34, 167.77, 170.80, 171.20; IR (KBr): 1595, 1609,
MS(EI): m/z 357 (M+); HRMS: calcd for C18H16FN3O2S 357.0947,
found: 357.0957; Mp: 212–213 °C. Purity (HPLC) ꢄ 98%.
4.5.8. N-[N0-(2-Thiophen-2-ylacetyl)hydrazinocarbothioyl]-3-(4-
trifluoromethylphenyl)-acrylamide (13H)
Yield 83%; 1H NMR (400 MHz, DMSO-d6) d: 3.86 (2H, s), 6.95–
6.99 (2H, m), 7.11 (1H, d, J = 15.9 Hz), 7.38 (1H, dd, J = 4.9,
1.5 Hz), 7.76–7.83 (5H, m), 11.17 (1H, br), 11.72 (1H, br), 12.53
(1H, br); 13C NMR (100 MHz, DMSO-d6) d: 33.93, 122.34, 124.00
(q, J = 272.4 Hz), 125.22, 126.02 (q, J = 4.1 Hz), 126.70, 127.83,
128.86, 130.24 (q, J = 32.0 Hz), 136.19, 138.03 (q, J = 1.7 Hz),
142.66, 165.28, 166.34, 176.62; IR (KBr): 1636, 1661, 1684,
3196 cmꢃ1; MS (EI): m/z 413 (M+); HRMS: calcd for C17H14F3N3O2-
S2 413.0479, found 413.0487; Mp: 224–225 °C. Purity (HPLC) ꢄ
98%.
1674, 3223 cmꢃ1; MS (EI): m/z 391 (M+); HRMS: calcd for C17H17
-
N3O4S2 391.0660, found 391.0657; Mp: 209–210 °C. Purity
(HPLC) ꢄ 98%.
4.5.14. 3-(3,5-Dibromophenyl)-N-{N0-[2-(4-fluorophenyl)acetyl]-
hydrazinocarbothioyl}acrylamide (13N)
Yield 28%; 1H NMR (400 MHz, DMSO-d6) d: 3.61 (2H, s), 7.06
(1H, d, J = 16.0 Hz), 7.11–7.35 (4H, m), 7.66 (1H, d, J = 16.0 Hz),
7.84 (2H, s), 7.92 (1H, s), 11.11 (1H, br), 11.56 (1H, br), 12.44
(1H, br); 13C NMR (100 MHz, DMSO-d6) d: 38.72, 115.06 (d,
J = 21.5 Hz), 122.79, 123.17, 129.86, 130.95 (d, J = 8.3 Hz), 131.45
(d, J = 2.5 Hz), 134.94, 138.34, 141.15, 165.63, 166.23 (d,
4.5.9. N-(N0-Phenylacetylhydrazinocarbothioyl)-3-(2,4,6-
trifluorophenyl)acrylamide (13I)
J = 241.5 Hz), 167.50, 176.07; IR (KBr): 1630, 1650, 1682,
Yield 75%; 1H NMR (400 MHz, DMSO-d6) d: 3.62 (2H, s), 7.19
(1H, d, J = 16.1 Hz), 7.23–7.40 (7H, m), 7.59 (1H, d, J = 16.1 Hz),
11.13 (1H, br), 11.86 (1H, br), 12.53 (1H, br); 13C NMR (100 MHz,
DMSO-d6) d: 39.54, 101.71 (td, J = 27.3, 3.3 Hz), 108.64 (td,
J = 16.5, 5.0 Hz), 124.65, 126.60, 128.25, 129.07, 129.18, 135.25,
161.36 (ddd, J = 254.7, 15.7, 9.9 Hz), 162.99 (dt, J = 252.2,
17.2 Hz), 165.35, 167.28, 176.47; IR (KBr): 1628, 1651, 1687,
3215 cmꢃ1; MS (EI): m/z 393 (M+); HRMS: calcd for C18H14F3N3O2S
393.0759, found 393.0741; Mp: 210–211 °C. Purity (HPLC) ꢄ 98%.
3196 cmꢃ1; MS (EI): m/z 515 (M+); HRMS: calcd for C18
H Br2FN3-
79
14
O2S 512.9153, found 512.9162; Mp: 195–196 °C. Purity (HPLC) ꢄ
98%.
4.5.15. 3-(3,5-Dibromophenyl)-N-[N0-(3-phenylpropionyl)-
hydrazinocarbothioyl]acrylamide (13O)
Yield 23%; 1H NMR (400 MHz, DMSO-d6) d: 2.57 (2H, t,
J = 7.8 Hz), 2.87 (2H, t, J = 7.8 Hz), 7.06 (1H, d, J = 16.0 Hz), 7.18–
7.28 (5H, m), 7.67 (1H, d, J = 16.0 Hz), 7.84 (2H, d, J = 1.5 Hz),
7.92 (1H, t, J = 1.5 Hz), 10.98 (1H, br), 11.54 (1H, br), 12.47 (1H,
br); 13C NMR (100 MHz; DMSO-d6) d: 30.57, 34.46, 122.80,
123.10, 125.99, 128.22, 128.30, 129.76, 129.51, 132.25, 138.30,
4.5.10. 3-(3,5-Dibromophenyl)-N-(N0-phenylacetylhydrazino-
carbothioyl)acrylamide (13J)
Yield 70%; 1H NMR (400 MHz, DMSO-d6) d: 3.62 (2H, s), 7.06
(1H, d, J = 15.9 Hz), 7.23–7.32 (5H, m), 7.66 (1H, d, J = 15.9 Hz),
7.84 (2H, s), 7.92 (1H, s), 11.13 (1H, br), 11.57 (1H, br), 12.47
(1H, br); 13C NMR (100 MHz, DMSO-d6) d: 39.59, 122.79, 123.12,
126.66, 128.30, 129.24, 129.80, 134.88, 135.26, 138.32, 141.06,
140.80, 168.68, 176.29, 180.24; IR (KBr): 1636, 1650, 1685,
3233 cmꢃ1; MS (EI): m/z 511 (M+); HRMS: calcd for C19
H Br2N3O2-
79
17
S 508.9409, found 508.9407; Mp: 213–214 °C. Purity (HPLC) ꢄ 98%.
4.5.16. 2-Phenyl-N-[N0-(2-thiophen-2-ylacetyl)hydrazino-carbothioyl]
acetamide (13P)
164.99, 167.42, 176.69; IR (KBr): 1632, 1655, 1684, 3180 cmꢃ1
;
MS (EI): m/z 495 (M+); HRMS: calcd for C18H15Br2N3O2S
494.9252, found 494.9232; Mp: 192–193 °C. Purity (HPLC) ꢄ 98%.
Yield 63%; 1H NMR (400 MHz, DMSO-d6) d: 3.74 (2H, s), 3.81
(2H, s), 6.93–6.95 (2H, m), 7.24–7.37 (6H, m), 11.06 (1H, br),
11.73 (1H, br), 12.30 (1H, br); 13C NMR (100 MHz, DMSO-d6) d:
33.94, 42.11, 125.24, 126.70, 126.74, 127.08, 128.32, 128.51,
129.41, 134.30, 166.39, 167.43, 176.90; IR (KBr): 1537, 1676,
4.5.11. 3-(4-Fluorophenyl)-N-[N0-(thiophene-2-carbonyl)-
hydrazinocarbothioyl]acrylamide (13K)
Yield 85%; 1H NMR (400 MHz, DMSO-d6) d: 6.98 (1H, d,
J = 15.7 Hz), 7.21 (1H, t, J = 4.5 Hz), 7.32 (2H, t, J = 8.7 Hz), 7.69–
7.72 (2H, m), 7.77 (1H, d, J = 15.7 Hz), 7.87 (1H, d, J = 4.5 Hz),
7.90 (1H, d, J = 4.5 Hz), 11.10 (1H, br), 11.67 (1H, br), 12.14 (1H,
br); 13C NMR (100 MHz, DMSO-d6) d: 116.24 (d, J = 22.3 Hz),
119.41, 128.21, 129.60, 130.63 (d, J = 9.1 Hz), 130.76 (d,
J = 3.3 Hz), 132.00, 136.63, 143.47, 159.48, 163.45 (d,
J = 248.9 Hz), 165.44, 177.93; IR (KBr): 1598, 1632, 1684,
1697, 3179 cmꢃ1; MS (EI): m/z 333 (M+); HRMS: calcd for C15H15
-
N3O2S2 333.0606, found 333.0607; Mp: 161–162 °C. Purity
(HPLC) ꢄ 98%.
4.5.17. N-{N0-[2-(4-Fluorophenyl)acetyl]hydrazinocarbothioyl}-2-
phenylacetamide (13Q)
Yield 55%; 1H NMR (400 MHz, DMSO-d6) d: 3.57 (2H, s), 3.74
(2H, s), 7.11 (2H, t, J = 8.7 H), 7.20–7.40 (7H, m), 10.10 (1H, br),
Please cite this article in press as: Mori H., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.05.011

H. Mori et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
11.02 (1H, br), 11.73 (1H, br); 13C NMR (100 MHz, DMSO-d6) d:
38.64, 42.07, 114.98 (d, J = 21.5 Hz), 127.01, 128.45, 129.37,
131.06 (d, J = 8.3 Hz), 131.43 (d, J = 2.5 Hz), 134.30, 161.16 (d,
J = 242.3 Hz), 167.33, 172.57, 177.11; IR (KBr): 1589, 1674, 1699,
3194 cmꢃ1; MS (EI): m/z 345 (M+); HRMS: calcd for C17H16FN3O2S
345.0947, found 345.0951; Mp: 182–183 °C. Purity (HPLC) ꢄ 98%.
solution of the amine, obtained above, in CH2Cl2 (5 mL) were added
Et3N (0.17 mL, 1.22 mmol), DMAP (41 mg, 0.33 mmol) at room
temperature, and the resulting mixture was added 2-p-toly-
lethene-sulfonyl chloride29 at 0 °C, and then the resulting mixture
was stirred at room temperature for 18 h. The solvent was
removed, and the residue was chromatographed on SiO2 (Hexane:
Acetone = 6: 1) to give sulfoneamide 14 (94 mg, 66%).
4.5.18. 2-Phenyl-N-[N0-(2-m-tolylacetyl)hydrazinocarbothioyl]-
acetamide (13R)
1H NMR (400 MHz, CDCl3) d: 2.36 (3H, s), 3.80 (2H, d, J = 6.1 Hz),
5.00 (2H, s), 5.29 (1H, br), 6.68 (1H, d, J = 15.4 Hz), 6.88 (2H, d,
J = 9.0 Hz), 7.17 (2H, d, J = 7.8 Hz), 7.25–7.45 (9H, m), 7.49 (1H, d,
J = 15.4 Hz), 8.02 (1H, br); 13C NMR (125 MHz, DMSO-d6) d:
20.99, 45.59, 69.33, 114.79, 120.80, 125.89, 127.64, 127.77,
128.31, 128.39, 129.51, 130.07, 131.90, 137.13, 139.05, 140.35,
154.38, 166.45; IR (KBr): 1142, 1316, 1511, 1662, 3287 cmꢃ1; MS
(EI): m/z 436 (M+); HRMS: calcd for C24H24N2O4S 436.1457, found
436.1457; Mp: 178–179 °C. Purity (HPLC) ꢄ 98%.
Yield 24%; 1H NMR (400 MHz, DMSO-d6) d: 2.26 (3H, s), 3.53
(2H, s), 3.74 (2H, s), 7.09–7.29 (9H, m), 11.01 (1H, br), 11.72 (1H,
br), 12.28 (1H, br); 13C NMR (100 MHz, DMSO-d6) d: 20.96, 40.08,
42.04, 126.24, 127.00, 127.22, 128.15, 128.44, 129.36, 129.78,
134.29, 135.13, 137.27, 167.33, 172.55, 176.75; IR (KBr): 1590,
1653, 1691, 3191 cmꢃ1; MS (EI): m/z 341 (M+); HRMS: calcd for
C18H19N3O2S 341.1198, found 341.1198; Mp: 149–150 °C. Purity
(HPLC) ꢄ 98%.
4.7. Synthesis of N-{2-[3-(3,4-dichlorophenyl)ureido]phenyl}-4-
methyl-benzenesulfonamide (15)
4.5.19. N-{N0-[2-(2,6-Difluorophenyl)acetyl]hydrazino-carbothioyl}-
2-phenylacetamide (13S)
Yield 88%; 1H NMR (400 MHz, DMSO-d6) d: 3.67 (2H, s), 3.74
(2H, s), 7.07 (2H, t, J = 7.4 Hz), 7.22–7.40 (6H, m), 11.07 (1H, br),
11.73 (1H, br), 12.23 (1H, br); 13C NMR (100 MHz, DMSO-d6) d:
26.73, 42.06, 110.87 (t, J = 20.7 Hz), 111.26 (dd, J = 19.0, 6.6 Hz),
127.00, 128.44, 129.35, 129.40 (t, J = 10.3 Hz), 134.29, 161.08 (dd,
J = 246.5, 8.3 Hz), 165.60, 172.53, 177.32; IR (KBr): 1628, 1663,
To a stirred solution of N-(2-aminophenyl)-4-methylbenzene-
sulfonamide30 (100 mg, 0.38 mmol) in CH2Cl2 (5 mL) was added
3,4-dichlorophenylisocyanate (72 mg, 0.38 mmol), and the reac-
tion mixture was stirred at room temperature for 18 h. The insol-
uble solid was corrected by filtration, and dried to provide
sulfonamide 15 (129 mg, 75%).
1700, 3180 cmꢃ1; MS (EI): m/z 363 (M+); HRMS: calcd for C17H15
-
1H NMR (500 MHz, CDCl3) d: 2.40 (3H, s), 6.68 (1H, dd, J = 7.8,
1.2 Hz), 6.92 (1H, td, J = 7.8, 1.2 Hz), 7.06 (1H, br), 7.10 (1H, br),
7.18 (1H, dd, J = 9.0, 2.5 Hz), 7.20–7.27 (3H, m), 7.30 (1H, d,
J = 9.0 Hz), 7.57 (1H, br), 7.58 (1H, d, J = 2.5 Hz), 7.64 (2H, d,
J = 8.6 Hz), 7.79 (1H, dd, J = 7.8, 1.2 Hz); 13C NMR (100 MHz,
DMSO-d6) d: 21.00, 118.14, 119.16, 121.27, 122.41, 123.10,
125.35, 127.20, 127.32, 127.61, 129.52, 130.63, 131.11, 136.26,
F2N3O2S 363.0857, found 363.0851; Mp: 174–175 °C. Purity
(HPLC) ꢄ 98%.
4.5.20. N-{N0-[2-(3,5-Difluorophenyl)acetyl]hydrazino-carbothioyl}-
2-phenylacetamide (13T)
Yield 4%; 1H NMR (400 MHz, DMSO-d6) d: 3.74 (2H, s), 4.03 (2H,
s), 7.03–7.12 (3H, m), 7.23–7.34 (5H, m), 11.02 (1H, br), 11.74 (1H,
br), 12.18 (1H, br); 13C NMR (100 MHz, DMSO-d6) d: 40.79, 42.04,
102.17 (t, J = 25.6 Hz), 112.43 (dd, J = 18.6, 7.0 Hz), 126.99,
128.43, 129.35, 134.28, 139.58 (t, J = 10.3 Hz), 162.12 (dd,
J = 245.2, 13.6 Hz), 166.42, 172.43, 177.72; IR (KBr): 1627, 1653,
136.46, 140.08, 143.30, 152.18; IR (KBr): 1154, 1324, 1545, 1658,
3327 cmꢃ1; MS (EI): m/z 449 (M+); HRMS: calcd for C20
H Cl2N3O3-
35
17
S 449.0372, found 449.0364; Mp: 204–205 °C. Purity (HPLC)
= 96.51%.
1684, 3187 cmꢃ1; MS (EI): m/z 363 (M+); HRMS: calcd for C17H15
-
4.8. In vitro assay for compounds
F2N3O2S 363.0838, found 363.0849; Mp: 178–179 °C. Purity
(HPLC) ꢄ 98%.
For the assay of compounds 1–4, enzyme activities were exam-
ined as described previously10 using 1.25
l
g recombinant mouse
serine racemase (Srr) in a final volume of 125 L of 100 mM HEPES
(pH 8.0), 10 M PLP, 1 mM MgCl2, 5 mM DTT, 1 mM ATP, 20 mM l-
Ser, and 1 mM inhibitors. Reactions were proceeded more than 8 h
at 37 °C. The reaction mixture (25 L) was further incubated in
100 mM HEPES (pH 8.0), 10 M PLP, and recombinant Dsd1 in a
4.5.21. 2-(4-Fluorophenyl)-N-{N0-[2-(4-fluorophenyl)acetyl]-
hydrazinocarbothioyl}acetamide (13U)
l
l
Yield 35%; 1H NMR (400 MHz, DMSO-d6) d: 3.57 (2H, s), 3.74
(2H, s), 7.12–7.32 (8H, m), 11.03 (1H, br), 11.72 (1H, br), 12.28
(1H, br); 13C NMR (100 MHz, DMSO-d6) d: 38.60, 41.08, 114.93
(d, J = 19.8 Hz), 115.15 (d, J = 19.8 Hz), 130.38 (d, J = 2.5 Hz),
130.01 (d, J = 8.3 Hz), 131.32 (d, J = 7.4 Hz), 131.40 (d, J = 3.3 Hz),
161.13 (d, J = 242.3 Hz), 161.32 (d, J = 242.3 Hz), 167.24, 172.38,
176.98; IR (KBr): 1610, 1669, 1700, 3194 cmꢃ1; MS (EI): m/z 363
(M+); HRMS: calcd for C17H15F2N3O2S 363.0853, found 363.0857;
Mp: 178–179 °C. Purity (HPLC) ꢄ 98%.
l
l
final volume of 50
was mixed with 50
l
L for 30 min at 30 °C. The reaction mixture
l
L of 0.05% DNP in 2 M HCl aq. After the incu-
bation for 5 min at 30 °C, 100 lL of EtOH and 125 lL of 10 M NaOH
aq. were sequentially added. Absorbance at 515 nm of the resultant
hydrazine was measured after 10 min incubation at room temper-
ature. For the assay of compounds 13A–U, recombinant human SRR
(8 l
g) with C2D2 mutation7 was used as the same method as
4.6. Synthesis of N-(4-benzyloxyphenyl)-2-(2-p-tolylethene-
sulfonylamino)acetamide (14)
above. The activity of the inhibitors was evaluated with the per-
centage of the d-serine production compared with that without
inhibitors.
To a stirred solution of (p-Benzyloxyphenylcarbamoyl)-methyl-
carbamic acid tert-butyl ester28 (140 mg, 0.39 mmol) in CH2Cl2
(3 mL) was added TFA (0.30 mL, 3.95 mmol), and the resulting mix-
ture was stirred at room temperature for 18 h. The reaction was
quenched with sat. NaHCO3 aq, and the organic phase was sepa-
rated. The aqueous layer was extracted with CH2Cl2. The organic
phase and extracts were combined, dried over K2CO3, and concen-
trated in vacuo to yield corresponding amine (80 mg, 84%), which
was used for the next reaction without further purification. To a
4.9. Arc-Luc HL Tg mice
Arc-Luc HL Tg mouse strain was established as described.11b
Animal care and experimental protocols were carried out in
accordance with the Guidelines for the Care and Use of
Laboratory Animals of the University of Toyama (Authorization
No. A2011med-13). Mice were housed under standard laboratory
conditions (12 h–12 h/light–dark cycle with light on at 7:00 am,
Please cite this article in press as: Mori H., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.05.011

10
H. Mori et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
room temperature at 22 2 °C) and had free access to food and
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We thank Ms. Kiyomi Nakagawa for her experimental help. The
study was supported by a Grant from the Ministry of Education,
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.05.011.
Please cite this article in press as: Mori H., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.05.011