Synthesis, structure-activity relationships, and anticonvulsant activities of 2-amino-4H-pyrido[3,2-e][1,3]thiazin-4-one derivatives as orally active AMPA receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2015.02.033

As part of a program aimed at discovering orally active 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor antagonists, we screened our compound library and identified 2-[allyl(4-methylphenyl)amino]-4H-pyrido[3,2-e][1,3]thiazin-4-on

Full text of DOI:10.1016/j.bmc.2015.02.033

Bioorganic & Medicinal Chemistry 23 (2015) 1788–1799
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, structure–activity relationships, and anticonvulsant
activities of 2-amino-4H-pyrido[3,2-e][1,3]thiazin-4-one derivatives
as orally active AMPA receptor antagonists
⇑
Hiroshi Inami , Jun-ichi Shishikura, Tomoyuki Yasunaga, Kazushige Ohno, Hiroshi Yamashita, Kota Kato,
Shuichi Sakamoto
Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
As part of a program aimed at discovering orally active 2-amino-3-(3-hydroxy-5-methyl-4-isoxa-
zolyl)propionic acid (AMPA) receptor antagonists, we screened our compound library and identified 2-
[allyl(4-methylphenyl)amino]-4H-pyrido[3,2-e][1,3]thiazin-4-one (7) as a lead compound that inhibited
kainate-induced neurotoxicity mediated by AMPA receptors in rat hippocampal cultures. Structure–
Received 11 December 2014
Revised 16 February 2015
Accepted 17 February 2015
Available online 25 February 2015
activity relationship studies of
a series of 2-amino-4H-pyrido[3,2-e][1,3]thiazin-4-one derivatives
revealed that substituents on the phenyl ring attached to the 2-amino group and the 4H-pyrido[3,2-
e][1,3]thiazin-4-one ring system play an important role in inhibitory activity against kainate-induced
neurotoxicity. Several analogs bearing a phenyl group with a 4-substituent or five- or six-membered ring
fused at the 3,4-positions exhibited potent inhibitory activity against kainate-induced neurotoxicity.
Further, some of these compounds exhibited significant suppression of maximal electroshock seizure
in mice following oral administration. Of these compounds, 2-[(4-chlorophenyl)(methyl)amino]-4H-
pyrido[3,2-e][1,3]thiazin-4-one (16i) (YM928) demonstrated the most potent inhibitory effect with an
ED50 value of 7.4 mg/kg.
Keywords:
Noncompetitive
AMPA receptor antagonist
Kainate
Neurotoxicity
Anticonvulsant
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Quinoxalinediones are the most representative class of com-
petitive antagonist4 with a number of reported compounds, such
Glutamate is a major excitatory neurotransmitter in the central
nervous system (CNS) of vertebrates and plays an essential role in
a variety of physiological functions via the activation of postsynaptic
ionotropic and metabotropic receptors. Overstimulation of the
ionotropic receptors leads to neuronal cell damage and death in epi-
lepsy, cerebral ischemia, and CNS trauma, as well as in various forms
of neurodegeneration such as Parkinson’s disease and amyotrophic
lateral sclerosis.1 Antagonists of ionotropic glutamate receptors are
therefore considered as promising candidates for the development
of novel therapeutic agents.2,3 The ionotropic 2-amino-3-(3-hy-
droxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor has
attracted interest and a number of competitive and noncompetitive
antagonists for this receptor have been identified during the past
quarter-century (Fig. 1).4–8
as NBQX (1),9 YM90K (2),10 zonampanel (3)11 and others.12–14
Due to their neuroprotective effects in models of cerebral ischemia,
some of these compounds have advanced to clinical trials.
Presumably due to their highly polar properties, however, these
compounds have rarely exhibited acceptable efficacy following
oral administration, which hampers their clinical use for CNS
diseases other than stroke. Several different series of compounds
have also been described as competitive AMPA receptor antago-
nists,15–24 some of which have displayed oral activity in models
of seizure or pain.16,17,21–23 However, none of these have been
marketed for use as therapeutic agents.
Approaches to develop novel AMPA receptor antagonists have
yielded other classes of compounds that act noncompetitively at
the receptor. These compounds are considered to have the advan-
tage of maintaining efficacy even in the presence of the elevated
levels of glutamate observed with some neurological disorders.25,26
Further, these agents are potentially orally active, as the allosteric
binding site for these compounds appears less polar than the
glutamate binding site.27,28 The most representative class of non-
competitive antagonists are the 2,3-benzodiazepines,29,30 such as
⇑
Corresponding author. Tel.: +81 29 829 6171; fax: +81 29 852 5387.
E-mail address: hiroshi.inami@astellas.com (H. Inami).
Present address: Research & Development Headquarters, Kyorin Pharmaceutical
Co., Ltd, 6, Kanda Surugadai 4-Chome, Chiyoda-ku, Tokyo 101-8311, Japan.
http://dx.doi.org/10.1016/j.bmc.2015.02.033
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

H. Inami et al. / Bioorg. Med. Chem. 23 (2015) 1788–1799
1789
O
O
R
N
N
H
N
O
N
N
O
O
N
O
O
H2NO2S
O2N
O2N
N
H
N
H
NH2
NBQX (1)
YM90K (2) R = H (hydrochloride)
Zonampanel (3) R = CH2COOH
Talampanel (4)
Cl
O
N
F
N
O
N
CN
N
N
N
CP-465022 (5)
Perampanel (6)
Figure 1. Structures of AMPA receptor antagonists.
talampanel (4),31 which exhibit potent activity in various animal
seizure models following oral administration. Talampanel (4) has
been developed for the treatment of epilepsy, other neurode-
generative disorders, and brain tumors, but it has not yet reached
the market. Several recent reports have been published on differ-
ent series of noncompetitive antagonists, such as CP-465022
(5)32 and perampanel (6).33,34 Perampanel (6) was developed by
Eisai and launched on the market in 2012 as the first AMPA recep-
tor antagonist for the treatment of epilepsy. However, despite
intense research to develop AMPA receptor antagonists, there are
only a few drug candidates currently in clinical development.
In an effort to develop a novel class of orally active AMPA
receptor antagonists, we screened our compound library to iden-
tify inhibitors of kainate-induced neurotoxicity mediated by
AMPA receptors in primary rat hippocampal cultures.35 As a result,
we identified 2-[allyl(4-methylphenyl)amino]-4H-pyrido[3,2-
e][1,3]thiazin-4-one (7) as a lead compound with an IC50 value of
O
S
N
N
N
7
Figure 2. Structure of compound 7.
2-amino-4H-pyrido[3,2-e][1,3]thiazin-4-ones 13–15 in 80–92%
overall yields. Alkylation of these compounds with appropriate
alkyl iodides in the presence of lithium hydride gave the desired
N,N-disubstituted
2-amino-4H-pyrido[3,2-e][1,3]thiazin-4-ones
16a–d in 4–11% yields along with corresponding N3-alkylated
analogs 17a–d as dominant products in 44–86% yields.
Compounds 16a–d and 17a–d were easily separated by column
chromatography and structures were confirmed by carbonyl
absorption in their infrared (IR) spectra as previously described
9.0 lM (Fig. 2). We then modified the 2-amino group to give the
optimal compound 2-[(4-chlorophenyl)(methyl)amino]-4H-pyr-
ido[3,2-e][1,3]thiazin-4-one (16i) (YM928). As we previously
reported, functional characterization of this compound showed
that it did not displace [3H]AMPA binding to rat forebrain mem-
brane, and that it blocked AMPA-induced intracellular calcium
influx and inward currents in primary rat hippocampal cultures.
These results demonstrate that this compound is a noncompetitive
AMPA receptor antagonist.36 We also previously reported the
anticonvulsant activity of 16i in various animal seizure models
following oral administration.37–39 Here, we describe the synthesis,
structure–activity relationships (SARs), and anticonvulsant activi-
ties in a maximal electroshock seizure (MES) test in mice of a series
of 2-amino-4H-pyrido[3,2-e][1,3]thiazin-4-one derivatives, includ-
ing 16i.
by Košcik et al.40 N,N-Disubstituted 2-amino-4H-pyrido[3,2-
ˇ
e][1,3]thiazin-4-ones were amenable to direct and efficient
preparation by the addition of appropriate secondary amines to
the isothiocyanate 9 at ambient temperature. Compounds 18–21
and 16e–k, m, o–u were synthesized by this method in 18–90%
yields. The secondary amines used in this step were either com-
mercially available or prepared via known procedures.41 Aniline
derivative 16l was obtained by hydrogenation of 16k in 72% yield.
Hydrolysis of 16m gave the benzoic acid derivative 16n in 91%
yield.
As shown in Scheme 3, 4H-pyrido[3,4-e][1,3]thiazin-4-one
derivative 24 was synthesized from 4-chloronicotinic acid (22) in
26% yield, and a 7-methyl analog of 16i (25) was synthesized from
2-chloro-6-methylnicotinic acid (23) in 39% yield by a similar
method to that described in Scheme 2.
For the synthesis of 4H-pyrido[4,3-e][1,3]thiazin-4-one deriva-
tive 30, N-carbamothioyl-3-fluoroisonicotinamide 28 was
prepared from 3-fluoroisonicotinic acid (26) and cyclized in the
presence of sodium hydride at 110 °C in a total yield of 29% as
shown in Scheme 4. In a similar manner, benzothiazinone deriva-
tive 31 was synthesized starting from 2-fluorobenzoic acid (27)
2. Chemistry
The synthesis of 2-amino-4H-pyrido[3,2-e][1,3]thiazin-4-one
derivatives is outlined in Schemes 1 and 2.40 The reaction of the
acid chloride prepared from 2-chloronicotinic acid (8) with
ammonium thiocyanate gave 2-chloronicotinoyl isothiocyanate
(9). Addition of appropriate primary anilines to the isothiocyanate
9
gave N-carbamothioyl-2-chloronicotinamides 10–12, which
when cyclized in refluxing toluene gave N-monosubstituted

1790
H. Inami et al. / Bioorg. Med. Chem. 23 (2015) 1788–1799
R1
O
O
S
COOH
Cl
a, b
c
NCS
N
H
N
H
N
N
Cl
N
Cl
8
9
10 R1 = Br
11 R1 = CF3
12 R1 = OMe
O
O
S
O
R2
N
N
N
N
R2
d
e
N
N
N
S
NH
R1
N
S
+
R1
R2 = Me
R1
13 R1 = Br
14 R1 = CF3
15 R1 = OMe
16a R1 = Br
17a R1 = Br
R2 = Me
R2 = Me
17b R1 = CF3
R2 = Me
16b R1 = CF3
16c R1 = OMe R2 = Me
16d R1 = OMe R2 = Et
17c R1 = OMe R2 = Me
17d R1 = OMe R2 = Et
Scheme 1. Reagents and conditions: (a) SOCl2, cat. DMF; (b) NH4SCN, acetone; (c) primary aniline, acetone; (d) toluene; (e) LiH, DMF, then alkyl iodide.
in a total yield of 64%, and thienothiazinone derivative 34 was
synthesized starting from 3-chlorothiophene-2-carboxylic acid
(32) in a total yield of 42%.
As shown in Scheme 5, benzoxazinone derivative 36 was
synthesized via O-cyanation of methyl salicylate (35), followed
by a treatment with 4-chloro-N-methylaniline in the presence of
benzoic acid in 71% yield in accordance with a known procedure.42
O
S
Y
N
COOH
Cl
Me
a, b, c
R
X
N
Y
R
X
3. Results and discussion
Cl
22 R = H X = CH Y = N
23 R = Me X = N Y = CH
24 R = H X = CH Y = N
25 R = Me X = N Y = CH
We used kainate-induced neurotoxicity in primary rat
hippocampal cultures to assess the inhibitory activities of the syn-
thesized compounds. The structures of the compounds and the
results of the assay are summarized in Tables 1–5. Inhibitory
activities are presented as the concentration required for 50%
Scheme 3. Reagents and conditions: (a) SOCl2, cat. DMF; (b) NH4SCN, acetone; (c)
4-chloro-N-methylaniline, acetone.
inhibition of the neurotoxicity response (IC50; lM) or percent
O
N
O
a, b, c, d
COOH a, b, c
Cl
N
R2
N
S
N
Me
N
S
N
R
N
R1
R2 = Me
18 R = CH2Ph
8
16e R1 = H
19
R = 3-Thienyl
R = Pyridin-3-yl
16f R1 = 2-Me
R2 = Me
R2 = Me
R2 = Me
R2 = Me
R2 = Me
R2 = Me
R2 = Me
R2 = Me
R2 = Me
R2 = Me
R2 = Me
R2 = Me
R2 = Et
R2 = Et
R2 = Et
20a
16g
R1 = 4-Me
21 R = c-Hex
16h R1 = 4-F
16i
16j
R1 = 4-Cl
R1 = 4-OCF3
16k R1 = 4-NO2
e
f
16l
R1 = 4-NH2
16m
16n
16o
16p
16q
16r
R1 = 4-COOEt
R1 = 4-COOH
R1 = 4-cHex
R1 = 3,4-(CH2)3-
R1 = 3,4-CH=CHS-
R1 = 3-OMe
16s
16t
R1 = 3,4-(OMe)2
R1 = 3,4-OCH2O-
16u
R1 = 3,4-O(CH2)2O- R2 = Et
Scheme 2. Reagents and conditions: (a) SOCl2, cat. DMF; (b) NH4SCN, acetone; (c) secondary amine, acetone; (d) HCl in EtOAc, EtOH for 20; (e) H2, Raney-nickel, DMF; (f) aq
NaOH, EtOH. a HCl salt.

H. Inami et al. / Bioorg. Med. Chem. 23 (2015) 1788–1799
1791
O
S
N
Cl
O
F
S
X
Me
COOH
F
a, b, c
d
N
N
H
N
Me
X
X
Cl
26 X = N
27 X = CH
28 X = N
29 X = CH
30 X = N
31 X = CH
O
N
S
Cl
S
O
COOH
Cl
Me
S
a, b, c
d
S
N
S
N
H
N
Me
Cl
Cl
34
32
33
Scheme 4. Reagents and conditions: (a) SOCl2, cat. DMF; (b) NH4SCN, acetone; (c) 4-chloro-N-methylaniline, acetone; (d) NaH, DMF, THF.
O
Table 2
Inhibitory activities of N-substituted 2-(methylamino)-4H-pyrido[3,2-e][1,3]thiazin-
4-one derivatives against kainate-induced neurotoxicity
N
Me
COOMe
OH
a, b
O
N
O
N
Me
N
S
N
R
Cl
35
36
Compound
R
Anti-kainate toxicity
% inhibition (at 10 lM)
Scheme 5. Reagents and conditions: (a) BrCN, Et3N, acetone; (b) 4-chloro-N-
methylaniline, PhCOOH, acetone.
16e
18
Ph
CH2Ph
43
0
19
3-Thienyl
Pyridin-3-yl
c-Hex
28
18
16
Table 1
20a
21
Inhibitory activities of N-substituted 2-[(4-methylphenyl)amino]-4H-pyrido[3,2-
e][1,3]thiazin-4-one derivatives against kainate-induced neurotoxicity
a
HCl salt.
O
N
R
We next assessed the effect of a phenyl moiety (Table 2). The
phenyl derivative 16e exhibited moderate activity with 43%
inhibition at 10 M. Insertion of a methylene group between the
N
S
N
l
nitrogen atom and the benzene ring (18) resulted in a loss of this
activity. The thienyl (19), pyridyl (20), and cyclohexyl (21) analogs
each exhibited somewhat weak activity compared to 16e. In this
study, aromatic or aliphatic rings directly attached to the amino
group appeared to be tolerated, and the phenyl group was found
to be relatively favorable for potent activity.
We then examined the effect of substituents on the phenyl
group (Table 3). The 2-methylphenyl analog 16f had substantially
reduced activity, whereas the 4-methylphenyl analog 16g was
nearly as potent as the unsubstituted phenyl analog 16e. Based
on this result, we focused on the effect of 4-substituents.
Introduction of halogen atoms resulted in an increase in activity,
which was particularly evident in the potent inhibition exerted by
Compound
R
Anti-kainate toxicity
IC50 (lM)
a
7
16g
Allyl
Me
9.0
10
6.0
Talampanel (4)
a
Concentration necessary for 50% inhibition.
inhibition of the response at a concentration of 10
compound.
lM test
the chlorophenyl (16i; IC50 = 2.0
IC50 = 1.0 M) derivatives. The (trifluoromethyl)phenyl derivative
16b also exhibited potent activity, with an IC50 value of 2.0 M,
which was 5-fold more potent than the methylphenyl derivative
16g. Introduction of the methoxy group (16c) led to an approxi-
mately 3-fold increase in inhibitory activity compared to 16e, and
the trifluoromethoxy group (16j) had an effect comparable to that
lM) and bromophenyl (16a;
We first examined the effect of an allyl substituent on the 2-
amino group of compound 7 (Table 1). Compound 16g bearing a
methyl group retained activity comparable to that of 7, indicating
that the allyl substituent is not critical for inhibitory activity. We
therefore conducted further investigations using N-methyl
analogs, which were readily synthesized from commercially
available N-methylamines.
l
l

1792
H. Inami et al. / Bioorg. Med. Chem. 23 (2015) 1788–1799
Table 3
Table 5
Inhibitory activities of 2-[methyl(substituted phenyl)amino]-4H-pyrido[3,2-
e][1,3]thiazin-4-one derivatives against kainate-induced neurotoxicity
Inhibitory activities of aryl-fused 2-[(4-chlorophenyl)(methyl)amino]-4H-1,3-thiazin-
4-one derivatives and benzoxazinone analog 36 against kainate-induced
neurotoxicity
O
Me
R
N
N
Me
N
S
N
R
Cl
Compound
R
Anti-kainate toxicity
Compound
R
Anti-kainate toxicity
IC50 (lM)
a
a
IC50
(lM)
(43%)b
(4%)b
10
7.2
2.0
O
16e
16f
16g
16h
16i
H
2-Me
4-Me
4-F
N
N
16i
2.0
N
S
4-Cl
O
16a
16b
16c
16j
16k
16l
16m
16n
16o
16p
16q
4-Br
1.0
2.0
3.4
2.1
4-CF3
4-OMe
4-OCF3
4-NO2
4-NH2
4-COOEt
4-COOH
4-cHex
31
34
30
24
25
36
6.5
S
O
(38%)b
(19%)b
(29%)b
(0%)b
(29%)b
1.3
S
N
5.4
S
3,4-(CH2)3–
3,4-CH@CHS–
O
0.40
(4%)b
(2%)b
(0%)b
a
N
Concentration necessary for 50% inhibition.
Percent inhibition at 10 lM concentration.
b
N
N
S
O
N
Table 4
Inhibitory activities of 2-[(alkoxyphenyl)(ethyl)amino]-4H-pyrido[3,2-e][1,3]thiazin-
4-one derivatives against kainate-induced neurotoxicity
S
O
O
N
N
Me
N
O
S
Et
N
S
N
(28%)b
N
R
O
a
Concentration necessary for 50% inhibition.
Percent inhibition at 10 lM concentration.
Compound
R
Anti-kainate toxicity
IC50 (lM)
b
a
16r
16d
16s
16t
16u
3-OMe
4-OMe
3,4-(OMe)2
3,4-OCH2O–
3,4-O(CH2)2O–
3.8
2.8
the binding space around the 4-position is limited in size.
Consistent with the SAR described above, the dihydroindenyl
(16p) and benzothienyl (16q) derivatives bearing hydrophobic
and relatively small bicyclic rings exhibited potent inhibition. In
particular, 16q exhibited the most potent inhibitory activity of
(25%)b
0.40
0.60
a
Concentration necessary for 50% inhibition.
Percent inhibition at 10 lM concentration.
b
phenyl and heteroaryl derivatives, with an IC50 value of 0.40 lM,
which was >25-fold more potent than 16e.
of the methoxy group (16c). Conversely, introduction of the amino
group (16l) moderately diminished inhibitory activity, and the
carboxylphenyl derivative 16n had markedly decreased potency
compared to 16e, suggesting that hydrophilic substituents are
unfavorable in terms of inhibitory activity. A hydrophobic effect
rather than an electronic one appears to play a dominant role in
inhibitory activity. Interestingly, an 4-amino group on the phenyl
ring of 2,3-benzodiazepine derivatives, such as talampanel (4),
enhanced antagonistic activity,29 suggesting that the phenyl rings
of 4H-pyrido[3,2-e][1,3]thiazin-4-ones and 2,3-benzodiazepines
either occupy the binding pocket in different orientations or bind
to distinct sites on the AMPA receptor. In addition, somewhat
reduced potency of the ethoxycarbonylphenyl (16m) and
cyclohexylphenyl (16o) derivatives compared to 16e suggests that
Given the improved activity of the 4-methoxyphenyl
derivative 16c, we further examined the effect of alkoxy sub-
stituents on the phenyl group (Table 4). Reduced hydrophobicity
by introduction of lower alkoxy groups might cause a decrease in
CNS penetration. The methyl substituent on the 2-amino group
was therefore replaced by a more hydrophobic ethyl substituent
in this series of compounds. Initially, the ethyl(4-methoxypheny-
l)amino derivative 16d exhibited a closely similar activity relative
to corresponding N-methyl analog 16c. Introduction of the
3-methoxy group (16r) had an effect comparable to that of the
4-methoxy group (16d). As substituents at the 3- and 4-positions
each had an independent and favorable effect on potency, the
effect of disubstitution was investigated. The activity of the resul-
tant dimethoxyphenyl derivative 16s unexpectedly decreased. In

H. Inami et al. / Bioorg. Med. Chem. 23 (2015) 1788–1799
1793
Table 6
4. Conclusions
Anticonvulsant activities of 16h, 16i, 16a, 16j, and 16t in MES test in mice
b
Compound
Anti-kainate toxicity
IC50 (lM)
ED50 (mg/kg, po)
In our search for novel and orally active AMPA receptor antago-
nists, we screened our compound library against kainate-induced
neurotoxicity and conducted subsequent SAR study for a series of
2-amino-4H-pyrido[3,2-e][1,3]thiazin-4-one derivatives as non-
competitive AMPA receptor antagonists. The study revealed that
the substitution pattern on the phenyl ring attached to the 2-
amino group was crucial for determining the inhibitory activity
of the analogs against kainate-induced neurotoxicity and sug-
gested that the hydrophobic interactions and steric hindrances of
4- (and 3-) substituents play dominant roles in inhibitory activity.
Potent inhibitory activity was exhibited by compounds bearing
chloro (16i), bromo (16a), trifluoromethyl (16b), or trifluo-
romethoxy (16j) substituents on the 4-position of the phenyl group
and compounds with a five- or six-membered ring fused at the 3,4-
positions of the phenyl group, such as dihydroindenyl (16p),
benzothienyl (16q), benzodioxolyl (16t), or dihydrobenzodioxinyl
(16u) derivatives. In addition, the 4H-pyrido[3,2-e][1,3]thiazin-4-
one ring system plays an important role in potent inhibitory activ-
ity. Further, in the MES test in mice, compounds 16h, 16i, 16a, 16j,
and 16t exhibited significant anticonvulsant activity following oral
administration. Of these compounds, 16i demonstrated the most
potent activity with an ED50 value of 7.4 mg/kg. Our results indi-
cate that the present compounds, particularly 16i (YM928), are
potential candidates for the treatment of various neurodegenera-
tive diseases based on their AMPA receptor antagonism.
a
16h
16i
16a
16j
7.2
2.0
1.0
2.1
0.40
0.71
6.0
15.3 (11.7–19.0)
7.4 (5.9–9.1)
23.1 (16.2–36.0)
25.2 (22.1–28.3)
38.9 (24.4–60.3)
>100
16t
YM90K (2)
Talampanel (4)
4.2 (2.6–7.4)
a
Concentration necessary for 50% inhibition.
b
ED50 values were calculated by the probit method (95% confidence intervals
shown in parentheses).
Table 7
Pharmacokinetic parameters of 16i in rats
Route
AUCinf
CLtot
Vdss
t1/2
Cmax
Tmax
F
(ngÁh/mL) (mL/h/kg) (mL/kg) (h)
(ng/mL) (h)
(%)
iv (1 mg/kg) 542.0
po (1 mg/kg) 472.3
1845.1
1176.0
0.44
0.58 425.1
0.25 87.1
contrast, the benzodioxolyl derivative 16t demonstrated strong
inhibitory activity, with an IC50 value of 0.40 M, which was
l
equipotent with 16q. The dihydrobenzodioxinyl derivative 16u
also exhibited relatively potent inhibitory activity nearly equal
to that of 16t. The less sterically-hindered methylenedioxy or
ethylenedioxy groups appear favorable for the establishment of
potent activity.
5. Experimental
5.1. Chemistry
We next investigated the effect of the 4H-pyrido[3,2-
e][1,3]thiazin-4-one core (Table 5). Replacement of the pyridine
ring of 16i with a benzene ring (31) or thiophene ring (34) led to
a 3-fold decrease in activity. The 4H-pyrido[4,3-e][1,3]thiazin-4-
one (30) and 4H-pyrido[3,4-e][1,3]thiazin-4-one (24) analogs
exhibited dramatic reductions in activity relative to 16i. These
results show that the addition of a nitrogen atom at the 6- or 7-
position is highly detrimental to inhibitory activity. In addition,
the fact that the 7-methyl analog of 16i (25) exhibited no effect
Melting points were determined using a Yanaco MP-S3 melting
point apparatus and are uncorrected. IR spectra were recorded on a
Horiba FT-720 spectrometer as KBr disks. 1H NMR spectra were
recorded on a JEOL JNM-EX90, a JEOL JNM-LA300, a JEOL JNM-
EX400, a JEOL JNM-A500, or a Bruker Avance III HD 500 spectrome-
ter. 13C NMR spectra were recorded on a Varian VNS-400 or a
Bruker Avance III HD 500 spectrometer. Chemical shifts were
expressed in d (ppm) values with tetramethylsilane as an internal
standard (NMR descriptions; s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet, and br, broad peak). Mass spectra were
recorded on a JEOL JMS-DX300, a JEOL JMS-LX2000, a Hewlett-
Packard 5970 MSD, a Fisons TRIO-1000, or a Finnigan MAT TSQ
700 spectrometer. For salts, assignment of ion peaks was based
on the basic component. Elemental analyses were conducted using
a Yanaco MT-5 microanalyzer (C, H, N) and a Yokogawa IC-7000S
ion chromatographic analyzer (halogen and S). Preparative column
chromatography was performed using Wakogel C-200 (Wako;
100–200 mesh).
at 10 lM suggests that the binding space around this position is
extremely limited. Substitution of the sulfur atom of 31 with an
oxygen atom (36) led to further loss of activity. In this study, the
4H-pyrido[3,2-e][1,3]thiazin-4-one ring system was the most
favorable for the generation of compounds with strong inhibitory
activity.
Finally, to investigate the in vivo activity of these 2-amino-4H-
pyrido[3,2-e][1,3]thiazin-4-one derivatives, the 4-fluorophenyl
(16h), 4-chlorophenyl (16i), 4-bromophenyl (16a), 4-(trifluo-
romethoxy)phenyl (16j), and benzodioxolyl (16t) derivatives that
exhibited good to excellent potencies regarding kainate-induced
neurotoxicity were evaluated for their anticonvulsant activity in
MES test in mice (Table 6).26 YM90K (2), one of the representative
quinoxalinedione derivatives, exhibited no effect following oral
administration. In contrast, all tested 2-amino-4H-pyrido[3,2-
e][1,3]thiazin-4-one derivatives exhibited significant anticonvul-
sant activity following oral administration (ED50 = 7.4–38.9
mg/kg). The relatively weak in vivo activity of benzodioxolyl
derivative 16t may be due in part to susceptibility of the
methylenedioxy moiety to cytochrome P450 metabolism. Of these
compounds, 16i exhibited the most potent activity with an ED50
value of 7.4 mg/kg.37 As shown in Table 7, 16i had a favorable
pharmacokinetic profile with high oral bioavailability in rats
(F = 87%).
5.1.1. 2-[(4-Bromophenyl)amino]-4H-pyrido[3,2-e][1,3]thiazin-
4-one (13)
A mixture of 2-chloronicotinic acid (8, 8.64 g, 54.8 mmol),
thionyl chloride (40 ml, 0.55 mol), and 2 drops of N,N-dimethylfor-
mamide (DMF) was heated to reflux for 1.5 h. The reaction mixture
was concentrated to dryness, and the residue was dissolved in
acetone (30 ml). This solution was added dropwise to a stirred
solution of NH4SCN (4.16 g, 54.7 mmol) in acetone (60 ml). After
stirring for 30 min at room temperature, insoluble matter was fil-
tered off, and a solution of 4-bromoaniline (9.41 g, 54.7 mmol) in
acetone (35 ml) was added dropwise to the filtrate over a period
of 10 min. After stirring for 30 min at room temperature, the reac-
tion mixture was poured into ice water. The resulting precipitate
was collected and washed with water to give 10 (19.03 g, 94%). A

1794
H. Inami et al. / Bioorg. Med. Chem. 23 (2015) 1788–1799
mixture of 10 (7.54 g, 20.3 mmol) and toluene (40 ml) was heated
to reflux for 4 h. After cooling, the resulting precipitate was col-
lected and washed with toluene to give 13 (6.66 g, 98%) as a beige
solid: 1H NMR (500 MHz, DMSO-d6) d 10.81–12.42 (m, 1H), 8.72
(br s, 1H), 8.48 (br s, 1H), 7.37–7.96 (m, 4H), 6.93 (br s, 1H); MS
(FAB) m/z 334 (M++1). Anal. Calcd for C13H8N3OSBr: C, 46.72; H,
2.41; N, 12.57; S, 9.59; Br, 23.91. Found: C, 46.58; H, 2.34; N,
12.58; S, 9.56; Br, 23.60.
7.83 (d, J = 8.5 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H), 7.40 (dd, J = 7.9,
4.9 Hz, 1H), 3.68 (s, 3H); 13C NMR (125 MHz, CDCl3) d 169.73,
164.69, 155.57, 152.89, 144.21, 138.20, 132.32, 132.05, 131.79,
131.53, 128.89, 127.75, 127.72, 127.69, 127.66, 126.69, 124.53,
123.56, 122.36, 120.19, 119.92, 40.50; MS (FAB) m/z 338 (M++1).
Anal. Calcd for C15H10N3OSF3: C, 53.41; H, 2.99; N, 12.46; S, 9.51;
F, 16.90. Found: C, 53.60; H, 3.06; N, 12.28; S, 9.29; F, 16.73.
Compound 17b: White solid; 84%; IR 1685, 1595, 1574, 1408,
Compounds 14 and 15 were prepared in a similar manner.
1350, 1323, 1163, 1124, 1103, 1065, 852, 758 cmÀ1 1H NMR
;
(500 MHz, CDCl3) d 8.59 (dd, J = 4.9, 1.8 Hz, 1H), 8.56 (dd, J = 7.9,
1.8 Hz, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.31 (dd, J = 7.9, 4.3 Hz, 1H),
7.00 (d, J = 8.6 Hz, 2H), 3.72 (s, 3H); 13C NMR (100 MHz, CDCl3) d
162.17, 153.93, 153.81, 150.15, 148.49, 138.75, 128.33, 126.93,
126.89, 126.85, 126.81, 126.61, 126.28, 125.63, 122.92, 122.05,
121.02, 120.40, 31.93; MS (EI) m/z 337 (M+). Anal. Calcd for
5.1.2. 2-{[4-(Trifluoromethyl)phenyl]amino}-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (14)
White solid; 80% from 8; 1H NMR (500 MHz, CDCl3) d 8.61–8.66
(m, 1H), 8.40–8.60 (m, 2H), 7.65 (d, J = 8.5 Hz, 2H), 7.31–7.38 (m,
1H), 7.09 (d, J = 9.0 Hz, 2H); MS (FAB) m/z 324 (M++1). Anal.
Calcd for C14H8N3OSF3: C, 52.01; H, 2.49; N, 13.00; S, 9.92; F,
17.63. Found: C, 51.95; H, 2.54; N, 13.02; S, 9.98; F, 18.18.
C15H10N3OSF3Á0.1H2O: C, 53.13; H, 3.03; N, 12.39; S, 9.46; F,
16.81. Found: C, 53.00; H, 2.88; N, 12.39; S, 9.45; F, 17.05.
5.1.3. 2-[(4-Methoxyphenyl)amino]-4H-pyrido[3,2-e][1,3]
thiazin-4-one (15)
5.1.6. 2-[(4-Methoxyphenyl)(methyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16c) and 2-[(4-methoxyphenyl)imino]-3-
methyl-2,3-dihydro-4H-pyrido[3,2-e][1,3]thiazin-4-one (17c)
The title compounds were prepared from 15 in the same way as
16a and 17a. Compound 16c: White solid; 8%; IR 1647, 1572, 1527,
Pale yellow solid; 81% from 8; 1H NMR (500 MHz, DMSO-d6) d
10.66–11.96 (m, 1H), 8.70 (br s, 1H), 8.47 (d, J = 7.9 Hz, 1H),
7.42–7.80 (m, 2H), 6.90–7.18 (m, 3H), 3.78 (s, 3H); MS (EI) m/z
285 (M+).
1502, 1446, 1392, 1304, 1250, 1171, 1138, 760 cmÀ1 1H NMR
;
(500 MHz, CDCl3) d 8.67 (dd, J = 7.9, 1.8 Hz, 1H), 8.57 (dd, J = 4.9,
1.8 Hz, 1H), 7.32–7.40 (m, 1H), 7.22–7.30 (m, 2H), 7.03 (d,
J = 9.2 Hz, 2H), 3.88 (s, 3H), 3.62 (s, 3H); 13C NMR (125 MHz,
CDCl3) d 169.95, 165.70, 160.49, 156.26, 152.62, 138.05, 133.55,
129.42, 123.17, 119.90, 115.63, 55.66, 40.75; MS (ESI) m/z 300
(M++1). Anal. Calcd for C15H13N3O2SÁ0.2H2O: C, 59.47; H, 4.46; N,
13.87; S, 10.58. Found: C, 59.67; H, 4.36; N, 13.66; S, 10.51.
Compound 17c: Pale yellow solid; 44%; mp 155–157 °C (from ethyl
acetate); IR 1674, 1595, 1574, 1508, 1458, 1408, 1350, 1306, 1246,
1196, 1024, 841, 756 cmÀ1; 1H NMR (500 MHz, CDCl3) d 8.50–8.60
(m, 2H), 7.22–7.31 (m, 1H), 6.93 (d, J = 9.2 Hz, 2H), 6.85 (d,
J = 7.9 Hz, 2H), 3.83 (s, 3H), 3.71 (s, 3H); 13C NMR (125 MHz,
CDCl3) d 162.34, 156.93, 154.67, 153.60, 148.03, 140.24, 138.57,
121.72, 121.70, 120.52, 114.88, 55.52, 32.05; MS (EI) m/z 299
(M+). Anal. Calcd for C15H13N3O2S: C, 60.18; H, 4.38; N, 14.04; S,
10.71. Found: C, 60.29; H, 4.28; N, 14.01; S, 10.70.
5.1.4. 2-[(4-Bromophenyl)(methyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16a) and 2-[(4-bromophenyl)imino]-3-
methyl-2,3-dihydro-4H-pyrido[3,2-e][1,3]thiazin-4-one (17a)
To a stirred solution of 13 (900 mg, 2.69 mmol) in DMF (15 ml)
was added lithium hydride (24.0 mg, 3.02 mmol), and the mixture
was stirred for 30 min at room temperature under an argon atmo-
sphere. To this was added methyl iodide (0.184 ml, 2.96 mmol),
and the resulting mixture was stirred for 5 h. Additional lithium
hydride (3.0 mg, 0.38 mmol) and methyl iodide (0.022 ml,
0.35 mmol) were added, and the mixture was stirred for 1 h. The
mixture was poured into ice water, and the resulting precipitate
was collected and washed with water. The crude product thus
obtained was chromatographed (CHCl3) to give 16a (107 mg,
11%) as a white solid and 17a (803 mg, 86%) as a white solid.
Compound 16a: IR 1641, 1568, 1520, 1483, 1444, 1392, 1308,
1138, 1014, 762 cmÀ1 1H NMR (500 MHz, CDCl3) d 8.56–8.61 (m,
;
1H), 8.55 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 8.5 Hz, 2H), 7.26–7.33 (m,
1H), 6.79 (d, J = 8.6 Hz, 2H), 3.70 (s, 3H); 13C NMR (125 MHz,
CDCl3) d 169.78, 164.90, 155.79, 152.79, 140.02, 138.13, 133.82,
129.91, 124.01, 123.42, 119.89, 40.55; MS (FAB) m/z 348 (M++1).
Anal. Calcd for C14H10N3OSBrÁ0.25H2O: C, 47.67; H, 3.00; N,
11.91; S, 9.09; Br, 22.65. Found: C, 47.82; H, 2.79; N, 11.84; S,
8.93; Br, 22.50. Compound 17a: IR 1678, 1591, 1574, 1450, 1410,
5.1.7. 2-[Ethyl(4-methoxyphenyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16d) and 3-ethyl-2-[(4-methoxyphenyl)
imino]-2,3-dihydro-4H-pyrido[3,2-e][1,3]thiazin-4-one (17d)
The title compounds were prepared from 15 in the same way as
16a and 17a using ethyl iodide instead of methyl iodide.
Compound 16d: White solid; 9%; IR 1647, 1577, 1502, 1439,
1390, 1302, 1248, 1022, 766 cmÀ1 1H NMR (500 MHz, CDCl3) d
;
1346, 1203, 1097, 823, 754 cmÀ1
;
1H NMR (500 MHz, CDCl3) d
8.66 (dd, J = 7.9, 1.8 Hz, 1H), 8.55 (dd, J = 4.9, 1.8 Hz, 1H), 7.35
(dd, J = 7.9, 4.9 Hz, 1H), 7.22 (d, J = 8.5 Hz, 2H), 7.03 (d, J = 9.2 Hz,
2H), 4.12–4.20 (m, 2H), 3.89 (s, 3H), 1.23–1.30 (m, 3H); 13C NMR
8.59 (dd, J = 4.3, 1.8 Hz, 1H), 8.55 (dd, J = 7.9, 1.8 Hz, 1H), 7.50 (d,
J = 8.5 Hz, 2H), 7.23–7.34 (m, 1H), 6.79 (d, J = 8.6 Hz, 2H), 3.70 (s,
3H); 13C NMR (100 MHz, CDCl3) d 162.22, 154.16, 153.75, 148.34,
146.06, 138.69, 132.67, 122.52, 121.93, 120.43, 117.78, 31.96; MS
(EI) m/z 347 (M+). Anal. Calcd for C14H10N3OSBr: C, 48.29; H,
2.89; N, 12.07; S, 9.21; Br, 22.95. Found: C, 47.95; H, 2.78; N,
12.00; S, 9.31; Br, 23.57.
(100 MHz, CDCl3)
d 170.04, 165.43, 160.59, 156.42, 152.54,
137.99, 131.76, 130.46, 123.10, 120.05, 115.57, 55.66, 47.44,
12.87; MS (EI) m/z 313 (M+). Anal. Calcd for C16H15N3O2S: C,
61.32; H, 4.82; N, 13.41; S, 10.23. Found: C, 61.29; H, 4.71; N,
13.40; S, 10.18. Compound 17d: Slightly yellow solid; 66%; IR
1670, 1599, 1574, 1506, 1448, 1412, 1369, 1323, 1242, 1196,
5.1.5. 2-{Methyl[4-(trifluoromethyl)phenyl]amino}-4H-
pyrido[3,2-e][1,3]thiazin-4-one (16b) and 3-methyl-2-{[4-
(trifluoromethyl)phenyl]imino}-2,3-dihydro-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (17b)
The title compounds were prepared from 14 in the same way as
16a and 17a. Compound 16b: White solid; 4%; IR 1647, 1570, 1516,
1394, 1327, 1300, 1180, 1128, 1068, 762 cmÀ1; 1H NMR (500 MHz,
CDCl3) d 8.68 (dd, J = 7.9, 1.8 Hz, 1H), 8.60 (dd, J = 4.9, 1.8 Hz, 1H),
1099, 756 cmÀ1 1H NMR (500 MHz, CDCl3) d 8.56 (dd, J = 4.9,
;
1.8 Hz, 1H), 8.53 (dd, J = 7.9, 1.8 Hz, 1H), 7.23–7.30 (m, 1H), 6.93
(d, J = 9.2 Hz, 2H), 6.84 (d, J = 9.2 Hz, 2H), 4.40–4.50 (m, 2H), 3.83
(s, 3H), 1.33–1.43 (m, 3H); 13C NMR (125 MHz, CDCl3) d 161.82,
156.89, 154.71, 153.52, 147.03, 140.34, 138.53, 121.68, 121.66,
120.69, 114.88, 55.53, 40.30, 12.54; MS (EI) m/z 313 (M+). Anal.
Calcd for C16H15N3O2S: C, 61.32; H, 4.82; N, 13.41; S, 10.23.
Found: C, 61.48; H, 4.76; N, 13.47; S, 10.15.

H. Inami et al. / Bioorg. Med. Chem. 23 (2015) 1788–1799
1795
5.1.8. 2-[Benzyl(methyl)amino]-4H-pyrido[3,2-e][1,3]thiazin-4-
one (18)
1H), 7.48–7.59 (m, 3H), 7.31–7.40 (m, 3H), 3.66 (s, 3H); 13C NMR
(125 MHz, CDCl3) 169.90, 165.14, 156.08, 152.66, 141.03,
d
2-Chloronicotinoyl chloride was prepared from 2-chloron-
icotinic acid (8, 1.76 g, 11.2 mmol) and dissolved in acetone
(10 ml). This solution was added dropwise to a stirred solution of
NH4SCN (891 mg, 11.7 mmol) in acetone (15 ml) over a period of
5 min. After stirring for 15 min at room temperature, insoluble
matter was filtered off, and a solution of N-methylbenzylamine
(1.42 g, 11.7 mmol) in acetone (15 ml) was added dropwise to
the filtrate. After stirring overnight at room temperature, the reac-
tion mixture was poured into ice water. The resulting precipitate
was collected, washed with water, and recrystallized from ethyl
ether/ethanol to give 18 (1.53 g in two crops, 48%) as a white solid:
mp 112–113 °C; 1H NMR (500 MHz, DMSO-d6, 80 °C) d 8.71 (dd,
J = 4.9, 1.8 Hz, 1H), 8.50 (dd, J = 7.9, 1.8 Hz, 1H), 7.52–7.58 (m,
1H), 7.26–7.41 (m, 5H), 4.98 (s, 2H), 3.24 (s, 3H); 13C NMR
(125 MHz, DMSO-d6, 80 °C) d 167.49, 163.24, 154.54, 152.84,
137.38, 135.71, 128.68, 127.59, 127.44, 123.90, 120.11, 53.50,
36.57; MS (FAB) m/z 284 (M++1). Anal. Calcd for C15H13N3OS: C,
63.58; H, 4.62; N, 14.83; S, 11.32. Found: C, 63.62; H, 4.53; N,
14.92; S, 11.48.
138.08, 130.53, 129.89, 128.17, 123.25, 119.94, 40.65; MS (EI) m/z
269 (M+). Anal. Calcd for C14H11N3OS: C, 62.43; H, 4.12; N, 15.60;
S, 11.91. Found: C, 62.44; H, 4.14; N, 15.61; S, 11.96.
5.1.13. 2-[Methyl(2-methylphenyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16f)
Slightly yellow solid; 79%; mp 208–209 °C (from EtOH); 1H
NMR (500 MHz, CDCl3) d 8.68 (dd, J = 7.9, 1.8 Hz, 1H), 8.56 (dd,
J = 4.9, 1.8 Hz, 1H), 7.33–7.47 (m, 4H), 7.25 (d, J = 7.9 Hz, 1H),
3.59 (s, 3H), 2.27 (s, 3H); 13C NMR (125 MHz, CDCl3) d 169.94,
165.22, 156.31, 152.62, 139.40, 138.16, 136.78, 132.20, 130.38,
128.74, 128.23, 123.23, 120.03, 39.29, 17.24; MS (FAB) m/z 284
(M++1). Anal. Calcd for C15H13N3OS: C, 63.58; H, 4.62; N, 14.83; S,
11.32. Found: C, 63.62; H, 4.59; N, 14.78; S, 11.34.
5.1.14. 2-[Methyl(4-methylphenyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16g)
White solid; 33% from 2-chloronicotinoyl chloride; mp 200–
201 °C (from EtOH); 1H NMR (300 MHz, CDCl3) d 8.67 (dd, J = 7.9,
1.8 Hz, 1H), 8.56 (dd, J = 4.7, 1.8 Hz, 1H), 7.30–7.39 (m, 3H), 7.22
(d, J = 8.3 Hz, 2H), 3.63 (s, 3H), 2.45 (s, 3H); 13C NMR (125 MHz,
CDCl3) d 169.94, 165.33, 156.21, 152.62, 140.20, 138.41, 138.04,
131.10, 127.85, 123.17, 119.93, 40.66, 21.31; MS (FAB) m/z 284
(M++1). Anal. Calcd for C15H13N3OS: C, 63.58; H, 4.62; N, 14.83; S,
11.32. Found: C, 63.65; H, 4.76; N, 14.84; S, 11.30.
Compounds 19–21 and 16e–k, m, o–u were prepared in a simi-
lar manner.
5.1.9. 2-[Methyl(3-thienyl)amino]-4H-pyrido[3,2-e][1,3]thiazin-
4-one (19)
White solid; 26% from 2-chloronicotinoyl chloride; mp 231–
232 °C (from CHCl3/EtOH); 1H NMR (400 MHz, CDCl3) d 8.67 (dd,
J = 8.0, 2.0 Hz, 1H), 8.59 (dd, J = 4.8, 2.0 Hz, 1H), 7.48–7.52 (m,
1H), 7.43 (dd, J = 3.2, 1.2 Hz, 1H), 7.38 (dd, J = 8.0, 4.4 Hz, 1H),
7.05 (dd, J = 5.2, 1.2 Hz, 1H), 3.62 (s, 3H); 13C NMR (125 MHz,
CDCl3) d 169.81, 165.46, 156.11, 152.73, 139.00, 138.12, 127.65,
125.39, 124.02, 123.30, 119.95, 40.45; MS (EI) m/z 275 (M+).
Anal. Calcd for C12H9N3OS2: C, 52.34; H, 3.29; N, 15.26; S, 23.29.
Found: C, 52.57; H, 3.24; N, 15.53; S, 23.37.
5.1.15. 2-[(4-Fluorophenyl)(methyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16h)
White solid; 83%; mp 184–185 °C (from EtOH); 1H NMR
(400 MHz, CDCl3) d 8.64–8.71 (m, 1H), 8.58 (dd, J = 4.8, 2.0 Hz,
1H), 7.32–7.42 (m, 3H), 7.20–7.28 (m, 2H), 3.64 (s, 3H); 13C NMR
(125 MHz, CDCl3)
d 169.82, 165.27, 164.00, 162.00, 155.88,
152.75, 138.14, 136.97, 130.34, 130.27, 123.38, 119.88, 117.72,
117.54, 40.72; MS (FAB) m/z 288 (M++1). Anal. Calcd for
5.1.10. 2-[Methyl(pyridin-3-yl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one hydrochloride (1:1) (20)
C14H10N3OSF: C, 58.53; H, 3.51; N, 14.63; S, 11.16; F, 6.61.
Found: C, 58.42; H, 3.35; N, 14.61; S, 11.20; F, 6.80.
The free base of 20 was converted to its hydrochloride salt by
crystallization from a mixture of ethanol and 4 M HCl in ethyl acet-
ate: White solid; 59%; 1H NMR (400 MHz, DMSO-d6) d 8.99 (s,1H),
8.85 (d, J = 4.8 Hz, 1H), 8.73 (dd, J = 4.0, 1.6 Hz, 1H), 8.53 (dd, J = 8.4,
1.6 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 7.71–8.26 (m, 2H), 7.61 (dd,
J = 8.0, 4.8 Hz, 1H), 3.60 (s, 3H); 13C NMR (125 MHz, DMSO-d6) d
167.86, 163.95, 154.49, 153.26, 147.10, 146.81, 139.32, 138.92,
137.54, 125.89, 124.20, 119.30; MS (FAB) m/z 271 (M++1). Anal.
Calcd for C13H10N4OSÁHClÁ0.6H2O: C, 49.17; H, 3.87; N, 17.64; S,
10.10; Cl, 11.16. Found: C, 49.11; H, 3.83; N, 17.75; S, 10.06; Cl,
11.01.
5.1.16. 2-[(4-Chlorophenyl)(methyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16i)
White solid; 87%; mp 189–190 °C (from EtOH); 1H NMR
(500 MHz, CDCl3) d 8.67 (dd, J = 7.9, 1.2 Hz, 1H), 8.59 (dd, J = 4.9,
1.8 Hz, 1H), 7.52 (d, J = 8.6 Hz, 2H), 7.39 (dd, J = 7.9, 4.9 Hz, 1H),
7.31 (d, J = 8.6 Hz, 2H), 3.63 (s, 3H); 13C NMR (125 MHz, CDCl3) d
169.79, 165.00, 155.81, 152.78, 139.49, 138.14, 135.97, 130.81,
129.64, 123.41, 119.89, 40.60; MS (FAB) m/z 304 (M++1). Anal.
Calcd for C14H10N3OSCl: C, 55.36; H, 3.32; N, 13.83; S, 10.56; Cl,
11.67. Found: C, 55.43; H, 3.30; N, 13.84; S, 10.55; Cl, 11.69.
5.1.11. 2-[Cyclohexyl(methyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (21)
5.1.17. 2-{Methyl[4-(trifluoromethoxy)phenyl]amino}-4H-
pyrido[3,2-e][1,3]thiazin-4-one (16j)
Off-white solid; 47%; mp 134–135 °C (from hexane/ethyl ether/
ethyl acetate); 1H NMR (400 MHz, DMSO-d6, 80 °C) d 8.66–8.72 (m,
1H), 8.47 (dd, J = 7.8, 1.8 Hz, 1H), 7.53 (dd, J = 7.8, 4.6 Hz, 1H), 4.00–
5.00 (br, 1H), 3.12 (s, 3H), 1.55–1.89 (m, 7H), 1.33–1.48 (m, 2H),
Beige solid; 81%; mp 176–177 °C (from EtOH); 1H NMR
(500 MHz, CDCl3) d 8.68 (dd, J = 7.9, 1.8 Hz, 1H), 8.59 (dd, J = 4.9,
1.8 Hz, 1H), 7.36–7.44 (m, 5H), 3.65 (s, 3H); 13C NMR (125 MHz,
CDCl3) d 169.77, 165.01, 155.73, 152.81, 149.84, 139.35, 138.20,
130.07, 123.48, 123.43, 122.82, 121.37, 119.90, 119.31, 117.26,
40.62; MS (FAB) m/z 354 (M++1). Anal. Calcd for C15H10N3O2SF3:
C, 50.99; H, 2.85; N, 11.89; S, 9.08; F, 16.13. Found: C, 50.91; H,
2.89; N, 11.98; S, 9.15; F, 16.35.
1.10–1.25 (m, 1H); 13C NMR (125 MHz, DMSO-d6, 80 °C)
d
167.40, 162.30, 154.69, 152.73, 137.26, 123.78, 120.38, 57.34,
30.96, 29.14, 25.14, 24.71; MS (FAB) m/z 276 (M++1). Anal. Calcd
for C14H17N3OS: C, 61.06; H, 6.22; N, 15.26; S, 11.64. Found: C,
61.02; H, 6.23; N, 15.39; S, 11.79.
5.1.18. 2-[Methyl(4-nitrophenyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16k)
5.1.12. 2-[Methyl(phenyl)amino]-4H-pyrido[3,2-e][1,3]thiazin-
4-one (16e)
Yellow solid; 81%; mp 223–224 °C (from EtOH); 1H NMR
(500 MHz, DMSO-d6) d 8.69–8.74 (m, 1H), 8.53 (d, J = 7.9 Hz, 1H),
8.43 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.59 (dd, J = 7.9,
White solid; 79%; mp 174–175 °C (from EtOH); 1H NMR
(500 MHz, CDCl3) d 8.67 (dd, J = 7.9, 1.8 Hz, 1H), 8.54–8.59 (m,

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H. Inami et al. / Bioorg. Med. Chem. 23 (2015) 1788–1799
4.9 Hz, 1H), 3.58 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 167.98,
163.23, 154.64, 153.28, 147.27, 146.85, 137.55, 129.71, 125.36,
124.19, 119.41; MS (FAB) m/z 315 (M++1). Anal. Calcd for
from 2-chloronicotinoyl chloride) as a white solid by the method
described for 18: mp 214–216 °C (from ethyl acetate/EtOH); 1H
NMR (400 MHz, CDCl3) d 8.68 (dd, J = 7.9, 1.8 Hz, 1H), 8.53–8.58
(m, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 2.0 Hz, 1H), 7.63 (d,
J = 5.4 Hz, 1H), 7.41 (d, J = 5.5 Hz, 1H), 7.34–7.40 (m, 1H), 7.28
(dd, J = 8.5, 2.1 Hz, 1H), 3.72 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 169.94, 165.54, 156.14, 152.66, 140.93, 140.83, 138.06, 137.53,
129.15, 124.65, 123.88, 123.42, 123.35, 123.22, 119.88, 40.94;
MS (FAB) m/z 326 (M++1). Anal. Calcd for C16H11N3OS2: C,
59.06; H, 3.41; N, 12.91; S, 19.71. Found: C, 59.03; H, 3.23; N,
12.79; S, 19.48.
C14H10N4O3S: C, 53.50; H, 3.21; N, 17.82; S, 10.20. Found: C,
53.28; H, 3.19; N, 17.84; S, 10.21.
5.1.19. Ethyl 4-[methyl(4-oxo-4H-pyrido[3,2-e][1,3]thiazin-2-yl)
amino]benzoate (16m)
White solid; 88% from 2-chloronicotinoyl chloride; 1H NMR
(500 MHz, CDCl3) d 8.67 (dd, J = 7.9, 1.8 Hz, 1H), 8.58 (dd, J = 4.9,
1.8 Hz, 1H), 8.22 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.6 Hz, 2H), 7.39
(dd, J = 7.9, 4.9 Hz, 1H), 4.40–4.48 (m, 2H), 3.67 (s, 3H), 1.40–1.47
(m, 3H); 13C NMR (125 MHz, CDCl3) d 169.77, 165.31, 164.66,
155.75, 152.81, 144.86, 138.14, 131.78, 131.75, 128.20, 123.44,
119.95, 61.57, 40.55, 14.32; MS (FAB) m/z 342 (M++1). Anal.
Calcd for C17H15N3O3SÁ0.2H2O: C, 59.19; H, 4.50; N, 12.18; S,
9.29. Found: C, 59.27; H, 4.33; N, 12.15; S, 9.39.
5.1.25. 2-[Ethyl(3-methoxyphenyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16r)
White solid; 83%; 1H NMR (500 MHz, CDCl3) d 8.66 (dd, J = 7.9,
1.8 Hz, 1H), 8.53–8.58 (m, 1H), 7.42–7.48 (m, 1H), 7.32–7.39 (m,
1H), 7.03–7.09 (m, 1H), 6.91 (d, J = 8.5 Hz, 1H), 6.81–6.86 (m,
1H), 4.13–4.25 (m, 2H), 3.86 (s, 3H), 1.29 (t, J = 7.3 Hz, 3H); 13C
NMR (125 MHz, CDCl3) d 169.98, 164.75, 161.10, 156.35, 152.57,
140.33, 138.01, 131.14, 123.14, 121.14, 120.10, 115.59, 114.74,
55.61, 47.43, 12.95; MS (FAB) m/z 314 (M++1). Anal. Calcd for
5.1.20. 2-[(4-Cyclohexylphenyl)(methyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16o)
White solid; 48% from 2-chloronicotinoyl chloride; mp 192–
195 °C (from EtOH); 1H NMR (400 MHz, CDCl3) d 8.64–8.70 (m,
1H), 8.56 (dd, J = 4.4, 2.0 Hz, 1H), 7.33–7.39 (m, 3H), 7.24 (d,
J = 8.4 Hz, 2H), 3.64 (s, 3H), 2.53–2.65 (m, 1H), 1.74–1.98 (m, 5H),
1.36–1.51 (m, 4H), 1.22–1.35 (m, 1H); 13C NMR (100 MHz, CDCl3)
d 169.93, 165.34, 156.26, 152.59, 150.21, 138.53, 138.08, 128.86,
127.87, 123.16, 119.96, 44.38, 40.60, 34.37, 26.78, 26.04; MS
(FAB) m/z 352 (M++1). Anal. Calcd for C20H21N3OS: C, 68.35; H,
6.02; N, 11.96; S, 9.12. Found: C, 68.43; H, 6.08; N, 11.89; S, 9.21.
C16H15N3O2S: C, 61.32; H, 4.82; N, 13.41; S, 10.23. Found: C,
61.39; H, 4.79; N, 13.42; S, 10.40.
5.1.26. 2-[(3,4-Dimethoxyphenyl)(ethyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16s)
White solid; 90%; 1H NMR (500 MHz, CDCl3) d 8.66 (dd, J = 7.9,
1.8 Hz, 1H), 8.53–8.59 (m, 1H), 7.32–7.39 (m, 1H), 6.98 (d,
J = 8.5 Hz, 1H), 6.90 (dd, J = 8.6, 1.8 Hz, 1H), 6.78 (d, J = 1.8 Hz,
1H), 4.02–4.33 (br, 2H), 3.96 (s, 3H), 3.91 (s, 3H), 1.23–1.34 (m,
3H); 13C NMR (125 MHz, CDCl3) d 170.03, 165.38, 156.46, 152.58,
150.33, 138.00, 131.83, 123.12, 121.81, 120.05, 111.77, 56.24,
56.19, 47.47, 12.97; MS (FAB) m/z 344 (M++1). Anal. Calcd for
5.1.21. 4-Cyclohexyl-N-methylaniline
The title compound was prepared from 4-cyclohexylaniline
using the procedure described by Crochet et al.;41 64%; 1H NMR
(90 MHz, CDCl3) d 7.05 (d, J = 8.4 Hz, 2H), 6.57 (d, J = 8.6 Hz, 2H),
2.82 (s, 3H), 2.20–2.63 (m, 1H), 0.90–2.10 (m, 10H); MS (EI) m/z
189 (M+).
C17H17N3O3S: C, 59.46; H, 4.99; N, 12.24; S, 9.34. Found: C,
59.27; H, 4.93; N, 12.15; S, 9.35.
5.1.27. 2-[1,3-Benzodioxol-5-yl(ethyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16t)
5.1.22. 2-[2,3-Dihydro-1H-inden-5-yl(methyl)amino]-4H-pyrido
[3,2-e][1,3]thiazin-4-one (16p)
White solid; 65%; mp 184–185 °C (from ethyl ether/EtOH); 1H
NMR (500 MHz, CDCl3) d 8.66 (dd, J = 7.9, 1.8 Hz, 1H), 8.52–8.59
(m, 1H), 7.36 (dd, J = 7.9, 4.3 Hz, 1H), 6.92 (d, J = 7.9 Hz, 1H), 6.80
(dd, J = 7.9, 1.8 Hz, 1H), 6.75 (d, J = 2.4 Hz, 1H), 6.10 (s, 2H), 4.03–
4.26 (br, 2H), 1.27 (t, J = 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3) d
169.99, 165.34, 156.37, 152.59, 149.05, 148.92, 138.02, 132.75,
123.23, 123.16, 120.07, 109.61, 109.18, 102.25, 47.45, 12.86; MS
(EI) m/z 327 (M+). Anal. Calcd for C16H13N3O3S: C, 58.70; H, 4.00;
N, 12.84; S, 9.80. Found: C, 58.62; H, 4.02; N, 12.80; S, 9.85.
Pale yellow solid; 57% from 2-chloronicotinoyl chloride; mp
157–160 °C (from EtOH); 1H NMR (400 MHz, CDCl3) d 8.67 (dd,
J = 8.0, 2.0 Hz, 1H), 8.56 (dd, J = 4.8, 2.0 Hz, 1H), 7.33–7.38 (m,
2H), 7.16 (s, 1H), 7.05–7.10 (m, 1H), 3.64 (s, 3H), 2.94–3.02 (m,
4H), 2.17 (quintet, J = 7.6 Hz, 2H); 13C NMR (100 MHz, CDCl3) d
169.96, 165.37, 156.34, 152.59, 146.95, 146.43, 139.07, 138.04,
126.06, 125.74, 123.90, 123.12, 119.95, 40.72, 32.90, 32.71,
25.63; MS (FAB) m/z 310 (M++1). Anal. Calcd for C17H15N3OS: C,
66.00; H, 4.89; N, 13.58; S, 10.36. Found: C, 65.93; H, 4.97; N,
13.50; S, 10.38.
5.1.28. 2-[2,3-Dihydro-1,4-benzodioxin-6-yl(ethyl)amino]-4H-
pyrido[3,2-e][1,3]thiazin-4-one (16u)
5.1.23. N-Methylindan-5-amine
Pale yellow solid; 73%; mp 192–193 °C (from EtOH); 1H NMR
(500 MHz, CDCl3) d 8.65 (dd, J = 7.9, 1.8 Hz, 1H), 8.56 (dd, J = 4.9,
1.8 Hz, 1H), 7.35 (dd, J = 7.9, 4.9 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H),
6.82 (d, J = 2.4 Hz, 1H), 6.78 (dd, J = 8.5, 2.4 Hz, 1H), 4.21–4.40 (m,
4H), 4.14 (q, J = 7.0 Hz, 2H), 1.27 (t, J = 7.0 Hz, 3H); 13C NMR
The title compound was prepared from indan-5-amine using
the procedure described by Crochet et al.;41 56%; 1H NMR
(90 MHz, CDCl3) d 7.04 (d, J = 8.0 Hz, 1H), 6.34–6.59 (m, 2H),
2.63–2.97 (m, 7H), 1.83–2.22 (m, 2H); MS (EI) m/z 147 (M+).
(125 MHz, CDCl3)
d 170.03, 165.30, 156.47, 152.54, 144.99,
5.1.24. 2-[1-Benzothiophen-5-yl(methyl)amino]-4H-pyrido
[3,2-e][1,3]thiazin-4-one (16q)
144.54, 138.00, 132.22, 123.09, 122.15, 120.07, 118.69, 118.11,
64.42, 64.31, 47.41, 12.89; MS (FAB) m/z 342 (M++1). Anal. Calcd
for C17H15N3O3SÁ0.25H2O: C, 59.03; H, 4.52; N, 12.15; S, 9.27.
Found: C, 58.95; H, 4.34; N, 12.08; S, 9.48.
5-Nitro-1-benzothiophene (1.70 g, 9.49 mmol) was hydro-
genated in ethanol (34 ml) under 3 atm of H2 in the presence of
5% Pd/C (0.17 g). After removal of the catalyst by filtration, the fil-
trate was concentrated in vacuo to give an amino derivative. This
compound was methylated using the procedure described by
Crochet et al.41 The crude product thus obtained was treated with
2-chloronicotinoyl isothiocyanate (9) prepared from 2-chloroni-
cotinoyl chloride (1.67 g, 9.49 mmol) to give 16q (546 mg, 18%
5.1.29. N-Ethyl-2,3-dihydro-1,4-benzodioxin-6-amine
A
stirred solution of 2,3-dihydro-1,4-benzodioxin-6-amine
(3.01 g, 19.9 mmol) in CH2Cl2 (20 ml) was cooled in an ice-water
bath and treated dropwise with trifluoroacetic anhydride
(4.10 ml, 29.4 mmol) over a period of 5 min. After stirring for

H. Inami et al. / Bioorg. Med. Chem. 23 (2015) 1788–1799
1797
30 min at room temperature, the reaction mixture was concen-
trated to dryness, and ethyl acetate was added to the residue.
The organic phase was washed with water and brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The crude product
obtained was dissolved in DMF (35 ml) and cooled in an ice-water
bath. To the solution was added lithium hydride (190 mg,
23.9 mmol), and the mixture was stirred for 15 min under an argon
atmosphere. To this was added ethyl iodide (1.93 ml, 23.9 mmol),
and the resulting mixture was stirred overnight at room tempera-
ture. After concentration, the residue was dissolved in ethyl acet-
ate, washed with water and brine, dried over anhydrous Na2SO4,
and concentrated in vacuo. The crude product obtained was dis-
solved in MeOH (50 ml) and treated with 28% NH3 (30 ml), and
the mixture was heated to reflux for 4 h. After concentration, the
residue was dissolved in ethyl acetate, washed with water and
brine, dried over anhydrous Na2SO4, and concentrated in vacuo.
The residue was chromatographed (hexane/ethyl acetate 6:1–
5:1) to give the title compound (3.33 g, 93%): 1H NMR (500 MHz,
CDCl3) d 6.68 (d, J = 8.6 Hz, 1H), 6.07–6.18 (m, 2H), 4.08–4.25 (m,
4H), 3.10–3.33 (br, 1H), 2.98–3.10 (m, 2H), 1.13–1.24 (m, 3H);
MS (EI) m/z 179 (M+).
10.56; Cl, 11.67. Found: C, 55.15; H, 3.29; N, 13.79; S, 10.70; Cl,
11.58.
5.1.33. 2-[(4-Chlorophenyl)(methyl)amino]-7-methyl-4H-
pyrido[3,2-e][1,3]thiazin-4-one (25)
The title compound was prepared in the same manner as
described for 18 using 2-chloro-6-methylnicotinic acid (23) and
4-chloro-N-methylaniline instead of 2-chloronicotinic acid (8)
and N-methylbenzylamine, respectively: Pale brown solid; 39%;
mp 194–196 °C (from EtOH); 1H NMR (500 MHz, CDCl3) d 8.53
(d, J = 7.9 Hz, 1H), 7.51 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 8.5 Hz, 2H),
7.22 (d, J = 7.9 Hz, 1H), 3.62 (s, 3H), 2.54 (s, 3H); 13C NMR
(100 MHz, CDCl3)
d 169.99, 164.93, 163.01, 155.24, 139.58,
138.22, 135.88, 130.73, 129.73, 123.41, 117.36, 40.43, 24.48; MS
(FAB) m/z 318 (M++1). Anal. Calcd for C15H12N3OSCl: C, 56.69; H,
3.81; N, 13.22; S, 10.09; Cl, 11.16. Found: C, 56.57; H, 3.78; N,
13.08; S, 10.17; Cl, 11.03.
5.1.34. N-[(4-Chlorophenyl)(methyl)carbamothioyl]-3-
fluoroisonicotinamide (28)
A mixture of 3-fluoroisonicotinic acid (26, 1.66 g, 11.8 mmol),
thionyl chloride (15 ml, 0.21 mol), and 2 drops of DMF was heated
to reflux for 2 h. The reaction mixture was concentrated to dryness,
and the residue was dissolved in acetone (10 ml). This solution was
5.1.30. 2-[(4-Aminophenyl)(methyl)amino]-4H-pyrido[3,2-e]
[1,3]thiazin-4-one (16l)
The nitro compound 16k (1.51 g, 4.80 mmol) was hydrogenated
in DMF (50 ml) under atmospheric pressure in the presence of
Raney-nickel (1.5 g). The reaction mixture was filtered and concen-
trated in vacuo, and the residue was recrystallized from ethanol to
give 16l (984 mg in two crops, 72%) as a slightly yellow solid: mp
249–251 °C; 1H NMR (400 MHz, CDCl3) d 8.62 (dd, J = 8.0, 2.0 Hz,
1H), 8.54–8.59 (m, 1H), 7.37 (dd, J = 8.0, 4.4 Hz, 1H), 7.06 (d,
J = 8.4 Hz, 2H), 6.77 (d, J = 8.8 Hz, 2H), 4.44 (s, 2H), 3.58 (s, 3H);
13C NMR (125 MHz, CDCl3) d 170.04, 165.97, 156.51, 152.56,
147.86, 138.01, 131.20, 129.15, 123.06, 119.92, 115.94, 40.78; MS
(FAB) m/z 285 (M++1). Anal. Calcd for C14H12N4OS: C, 59.14; H,
4.25; N, 19.70; S, 11.28. Found: C, 58.98, H, 4.32; N, 19.42; S, 11.51.
added dropwise to
a stirred solution of NH4SCN (984 mg,
12.9 mmol) in acetone (15 ml). After stirring for 5 min at 40 °C,
insoluble matter was filtered off, and a solution of 4-chloro-N-
methylaniline (1.55 g, 10.9 mmol) in acetone (10 ml) was added
dropwise to the filtrate over a period of 5 min. After stirring over-
night at room temperature, the reaction mixture was poured into
ice water and the resulting precipitate was collected. The crude
solid was chromatographed (CHCl3/MeOH 99:1–98:2) to give 28
(1.45 g, 41%): 1H NMR (400 MHz, CDCl3) d 8.63–8.85 (br, 1H),
8.43–8.61 (m, 2H), 7.52–7.75 (br, 1H), 7.39 (d, J = 8.8 Hz, 2H),
7.26 (d, J = 8.4 Hz, 2H), 3.72 (s, 3H); MS (FAB) m/z 324 (M++1).
5.1.35. N-[(4-Chlorophenyl)(methyl)carbamothioyl]-2-
fluorobenzamide (29)
5.1.31. 4-[Methyl(4-oxo-4H-pyrido[3,2-e][1,3]thiazin-2-yl)
amino]benzoic acid (16n)
The title compound was prepared in the same manner as
described for 28 using 2-fluorobenzoic acid (27) instead of 3-fluo-
roisonicotinic acid (26): 92%; 1H NMR (500 MHz, CDCl3) d 8.63–
8.87 (m, 1H), 7.72–7.93 (br, 1H), 7.40–7.57 (m, 1H), 7.36 (d,
J = 8.5 Hz, 2H), 7.15–7.34 (m, 3H), 6.99–7.15 (m, 1H), 3.73 (s,
3H); MS (FAB) m/z 323 (M++1).
To a stirred solution of the ester 16m (70 mg, 0.20 mmol) in
ethanol (0.90 ml) was added 0.25 M NaOH (0.90 ml, 0.23 mmol).
After stirring for 5 h at room temperature, the reaction mixture
was diluted with water and acidified with 0.5 M HCl. The resulting
precipitate was collected and washed successively with water and
ethyl ether to give 16n (61 mg, 91%) as a pale yellow solid: 1H NMR
(400 MHz, CDCl3) d 8.65 (dd, J = 7.6, 2.0 Hz, 1H), 8.60 (dd, J = 5.2,
2.0 Hz, 1H), 8.23 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.38–
7.44 (m, 1H), 3.67 (s, 3H); 13C NMR (125 MHz, DMSO-d6) d
167.99, 166.35, 163.15, 154.76, 153.11, 144.66, 137.43, 131.57,
131.10, 128.47, 123.97, 119.30; MS (APCI) m/z 314 (M++1). Anal.
Calcd for C15H11N3O3SÁ0.9H2O: C, 54.67; H, 3.91; N, 12.75; S,
9.73. Found: C, 54.77; H, 3.75; N, 12.67; S, 9.65.
5.1.36. 3-Chloro-N-[(4-chlorophenyl)(methyl)carbamothioyl]
thiophene-2-carboxamide (33)
The title compound was prepared in the same manner as
described for 28 using 3-chlorothiophene-2-carboxylic acid (32)
instead of 3-fluoroisonicotinic acid (26): 73%; 1H NMR (400 MHz,
CDCl3) d 8.99 (br s, 1H), 7.51 (d, J = 5.2 Hz, 1H), 7.38 (d, J = 8.8 Hz,
2H), 7.27 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 5.2 Hz, 1H), 3.71 (s, 3H);
MS (FAB) m/z 345 (M++1).
5.1.32. 2-[(4-Chlorophenyl)(methyl)amino]-4H-pyrido[3,4-e]
[1,3]thiazin-4-one (24)
5.1.37. 2-[(4-Chlorophenyl)(methyl)amino]-4H-pyrido[4,3-e]
[1,3]thiazin-4-one (30)
The title compound was prepared in the same manner as
described for 18 using 4-chloronicotinic acid (22) and 4-chloro-
N-methylaniline instead of 2-chloronicotinic acid (8) and N-
methylbenzylamine, respectively: Off-white solid; 26%; mp 238–
239 °C (from EtOH); 1H NMR (400 MHz, CDCl3) d 9.51 (s, 1H),
8.56 (d, J = 5.6 Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.4 Hz,
2H), 7.07 (d, J = 5.6 Hz, 1H), 3.61 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 167.93, 162.33, 152.13, 150.92, 143.40, 139.30, 136.20,
130.92, 129.70, 119.58, 116.99, 40.59; MS (FAB) m/z 304 (M++1).
Anal. Calcd for C14H10N3OSCl: C, 55.36; H, 3.32; N, 13.83; S,
To a stirred solution of 28 (1.23 g, 3.80 mmol) in DMF (10 ml)
was added a mixture of 60% sodium hydride in mineral oil
(167 mg, 4.18 mmol) and tetrahydrofuran (THF) (1 ml), and the
mixture was stirred for 10 min at room temperature under an
argon atmosphere. The mixture was then heated to 110 °C and stir-
red for 5 h. After cooling, the reaction mixture was concentrated to
dryness. Ice water was added to the residue, and the resulting pre-
cipitate was collected and washed with water. The crude product
was recrystallized from ethanol to give 30 (802 mg in two crops,

1798
H. Inami et al. / Bioorg. Med. Chem. 23 (2015) 1788–1799
69%) as a white solid: mp 255–256 °C; 1H NMR (400 MHz, DMSO-
d6) d 8.83 (s, 1H), 8.66 (d, J = 4.8 Hz, 1H), 8.02 (d, J = 4.8 Hz, 1H),
7.70 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 8.8 Hz, 2H), 3.51 (s, 3H); 13C
NMR (100 MHz, DMSO-d6) d 166.53, 163.19, 148.90, 148.08,
140.02, 134.55, 130.67, 130.59, 129.49, 127.54, 121.77; MS (FAB)
m/z 304 (M++1). Anal. Calcd for C14H10N3OSCl: C, 55.36; H, 3.32;
N, 13.83; S, 10.56; Cl, 11.67. Found: C, 55.31; H, 3.34; N, 14.06; S,
10.54; Cl, 11.64.
Analyzer (Hitachi). A single experiment was performed in triplicate
for each compound.
5.2.2. Maximal electroshock seizure (MES) in ICR mice26
A group of 10 male ICR mice was used. Sixty minutes after test
compounds were orally administered, electroshock stimulation
(50 mA, 0.2 s, 50 Hz) was applied to each mouse via the cornea.
Inhibition of the maximal electroshock seizure action was evalu-
ated by the presence or absence of tonic extension development
as an index, and the ED50 value was calculated by the probit
method.
Compounds 31 and 34 were prepared in a similar manner.
5.1.38. 2-[(4-Chlorophenyl)(methyl)amino]-4H-1,3-
benzothiazin-4-one (31)
Beige solid; 69%; mp 216–218 °C (from EtOH); 1H NMR
(500 MHz, DMSO-d6) d 8.23 (d, J = 7.9 Hz, 1H), 7.42–7.72 (m, 7H),
3.50 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 168.14, 163.44,
140.60, 134.52, 133.53, 132.88, 130.94, 130.82, 129.77, 128.61,
126.60, 122.15; MS (FAB) m/z 303 (M++1). Anal. Calcd for
Acknowledgments
We would like to thank Rie Tsutsumi and Mika Katoh-Sudoh for
performing the pharmacological experiments and the staff of the
Structure Analysis Department for measurement of NMR and mass
spectra and elemental analyses. We would like to thank Drs.
Hirokazu Kubota and Akira Tanaka for their valuable advice in
preparing this manuscript and Drs. Shin-ichi Tsukamoto, Tokio
Yamaguchi, and Toshiyasu Mase for their support.
C
15H11N2OSClÁ0.2H2O: C, 58.80; H, 3.75; N, 9.14; S, 10.47; Cl,
11.57. Found: C, 58.77; H, 3.58; N, 9.14; S, 10.43; Cl, 11.52.
5.1.39. 2-[(4-Chlorophenyl)(methyl)amino]-4H-thieno[2,3-e]
[1,3]thiazin-4-one (34)
References and notes
Brown solid; 57%; mp 246–247 °C dec (from EtOH); 1H NMR
(400 MHz, CDCl3) d 7.70 (d, J = 5.6 Hz, 1H), 7.53 (d, J = 8.8 Hz, 2H),
7.30 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 5.2 Hz, 1H), 3.61 (s, 3H); 13C
NMR (100 MHz, CDCl3) d 165.42, 164.85, 140.18, 135.80, 134.16,
132.82, 130.85, 129.80, 125.92, 124.39, 41.04; MS (FAB) m/z 309
(M++1). Anal. Calcd for C13H9N2OS2Cl: C, 50.56; H, 2.94; N, 9.07;
S, 20.77; Cl, 11.48. Found: C, 50.44; H, 2.84; N, 9.00; S, 20.65; Cl,
11.24.
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