Design and synthesis of 4-alkyl-2-amino(acetamino)-6-aryl-1,3-thiazine derivatives as influenza neuraminidase inhibitors

Article abstract of DOI:10.1002/ardp.201300122

With a convenient and economical method, two series of 1,3-thiazine derivatives 1 and 2 were synthesized, and their neuraminidase (NA) inhibitory activities were evaluated. The pharmacological results showed that most of the compounds have potent NA inhibitory activity. Especially, 1g exhibited the best activity against influenza virus A (H1N1) NA (IC₅₀ = 29.06 μg/mL), and its crystal structure was determined by single-crystal X-ray diffraction. The preliminary biological assay indicated that 1,3-thiazine could be used as a core structure to design novel influenza NA inhibitors. Two series of novel 1,3-thiazine analogs were synthesized and their neuraminidase (NA) inhibitory activities were evaluated. Most of the compounds have potent NA inhibitory activity. Compound 1g exhibited the best activity against influenza virus A (H1N1) NA with an IC₅₀ of 29.06 μg/mL, and its crystal structure was determined by single-crystal X-ray diffraction.

Full text of DOI:10.1002/ardp.201300122

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–10
1
Full Paper
Design and Synthesis of 4-Alkyl-2-amino(acetamino)-6-aryl-1,3-
thiazine Derivatives as Influenza Neuraminidase Inhibitors
Wan Li1, Lin Xia1, Aixi Hu1, Ailin Liu2, Junmei Peng1, and Weiqing Tan1
1
College of Chemistry and Chemical Engineering, Hunan University, Changsha, China
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing,
2
China
With a convenient and economical method, two series of 1,3-thiazine derivatives 1 and 2 were
synthesized, and their neuraminidase (NA) inhibitory activities were evaluated. The pharmacological
results showed that most of the compounds have potent NA inhibitory activity. Especially, 1g exhibited
the best activity against influenza virus A (H1N1) NA (IC50 ¼ 29.06 mg/mL), and its crystal structure
was determined by single-crystal X-ray diffraction. The preliminary biological assay indicated that
1,3-thiazine could be used as a core structure to design novel influenza NA inhibitors.
Keywords: Crystal structure / Neuraminidase inhibitor / Synthesis / 1,3-Thiazine
Received: April 13, 2013; Revised: June 18, 2013; Accepted: June 28, 2013
DOI 10.1002/ardp.201300122
Introduction
ring may not be appropriate for NA inhibitors [6]. Recently,
the H7N9 avian influenza virus infected humans for the first
time, killing 37 people, and infected a further 132 people in
China [7]. Peramivir is a kind of new potent NA inhibitors
which is effective against the HXNY-type influenza virus, and
has a significant therapeutic effect in patients against H7N9.
Avian influenza virus is variable, but the treatment medicines
are restricted. Therefore, there is a great need to design,
screen, and identify new agents for the chemotherapy of
influenza virus infection, and develop more effective drugs.
In 2001, Wang et al. derived an “airplane” model of the NA
active site (Fig. 1), and summarized the basic structural
requirements of a potent NA inhibitor [8]. Luo et al. proposed
that the enzymatic active site contains four main well-
conserved binding sites arranged in a propeller fashion [9].
Each monomeric subunit has an active site cavity lined with
10 conserved residues and four H2O molecules. The positively
charged site a consists of Arg118, Arg292, and Arg371; the
inhibitors bind to the active site with carboxylic acid, and are
located on one side of the active site. Opposite to the triad
guanidine, there is a hydrophobic pocket formed by side
chains of Trp178, Ile222, and part of Arg224 (Fig. 1) as site c.
The negatively charged site b consists of Glu119, Glu227, and
Asp151 and can interact with the guanidino group, while site
d, consisting of Glu276 and Glu277, binds to hydrophobic side
chains. In the two-dimensional sense, sites c and a are situated
The influenza virus is still the major source of pandemic
affecting humans. The development of vaccines to combat
influenza is restricted as they are not compatible with the
high mutability of the virus [1]. Effective chemotherapy for
influenza virus is also limited due to newly discovered drug
resistance in mutant strains. Neuraminidase (NA) is one of two
glycoproteins expressed on the virus surface. It has been
shown that NA is required for elution of newly synthesized
virons from infected cells, and that it facilitates the movement
of the virus through the mucus of the respiratory tract. Since
the structure of the NA active site is highly conserved, the NA
inhibitors are well-tolerated against all influenza types.
Zanamivir and oseltamivir are two NA inhibitors, which
were approved by the FDA. Zanamivir displays excellent
antiviral activity but it has very low bioavailability through
oral administration [2]. At the same time, it has been reported
that oseltamivir would cause vomiting and nausea [3], and has
drug-resistant mutant from infected humans [4]. The core
structures are cyclohexene and 3,4-dihydropyran, respectively
[5]. The previously reported data indicated that six-membered
aromatic flat structures such as benzene ring and pyridine
Correspondence: Dr. Aixi Hu, College of Chemistry and Chemical
Engineering, Hunan University, Changsha 410082, China.
E-mail: axhu@hnu.edu.cn
Fax: þ86 731 88713642
Additional correspondence: Prof. Ailin Liu, E-mail: liuailin@imm.ac.cn
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W. Li et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–10
us to explore novel 1,3-thiazine derivatives as NA inhibitors.
We chose 1,3-thiazine as the core structure for further
modification and optimization.
According to the structure–activity relationship (SAR) of
NA, we hope that 5-carboxylate group of the compounds binds
to the triad guanidine groups of three arginine residues,
Arg118, Arg371, and Arg292 in the active site a. Facing the 5-
carboxylate group in the structure of 1,3-thiazine, the 2-
amino (acetamido) group binds to the hydrophobic pocket c
formed by the side chains of Trp178 and Ile 222 [19, 20]. The
substituent of aromatic ring can bind to the residue in the site
b, and the 4-methyl group of 1,3-thiazine can bind to the
hydrophobic pocket d. In view of this SAR, we hope that the
newly synthesized 1,3-thiazine derivatives can show excellent
NA inhibitory activity.
We have synthesized two series of 1,3-thiazine analogs, 2-
amino-5-carboxylates-1,3-thiazine derivatives (2a–2t) and 2-
acetamido-5-carboxylates-1,3-thiazine (1a–1t). Their NA inhib-
itory activity was evaluated in vitro. The structure of the crystal
of compound 1g was determined by single-crystal X-ray
diffraction. All the newly synthesized compounds were
characterized by 1H NMR, 13C NMR, MS, and elemental
analysis. The overall strategy for the synthesis of 1a–1t is
outlined in Scheme 1.
Figure 1. “Airplane” model of the NA active site.
at the head and tail of the “airplane” respectively, while sites b
and d are situated at the two wings of the “airplane”.
1,3-Thiazine, an important type of heterocycle, shows a
wide variety of pharmacological properties. Many 1,3-thiazine
derivatives have been reported as sedatives, hypnotics, Results and discussion
intravenous anesthetics, and anticancer reagents [10–13].
For example, 1,3-thiazine nucleus is the active core of
cephalosporins, which are the most widely used b-lactam
antibiotics [14]; 5-ethyl-6-phenyl-1,3-thiazine-2,4-dione has
been demonstrated to be an anesthetic agent; 3-(2-amino-
ethyl)-1,3-thiazine-2,4-dione hydrochloride has been reported
to have antiradiation activity [15]; and 5,6-dihydro-4H-1,3-
thiazines have antimycobacterial (M. tuberculosis H37Rv)
activity in vitro [16]. The 6-aryl-1,3-thiazine scaffold has
hitherto drawed less attention, despite its structural similari-
ty to the dihydropyrimidinones [17, 18]. While compounds of
thiazine exhibit a wide spectrum of bioactivity, which inspire
Chemistry
The reaction started with the Knoevenagel reaction. b-keto
esters were reacted with different aromatic aldehydes to yield
unsaturated ketones 3a–3t, and the water generated was
removed by toluene to facilitate the reaction. We tried various
catalysts and found that piperidine is the most effective
catalyst in this reaction.
The unsaturated ketones (3a–3t) were mixed with the
appropriate thioureas and catalyzed by hydrochloric acid at
reflux in ethanol to give 2-amino-4-alkyl-6-aryl-6H-1,3-thiazine-
5-carboxylate (2a–2t) [21–23]. We found that the substituent R
O
O
O
O
RO
N
RO
N
O
b
a
c
H
OR
X
X
S
NH2
S
N
H
X
X
O
3a–3t
2a–2t
1a–1t
a: X = 3-NO2, R = Me;
e: X = 3-NO2, R = Et;
i: X = 3,4-diMeO, R = Et;
b: X = 2-Cl-5-NO2, R = Et; c: X = 2-NO2, R = Me;
d: X = 4-NO2, R = Me;
h: X = 4-Cl, R = Et;
f: X = 2-EtO, R = Et;
j: X = 2-NO2, R = Et;
n: X = 4-F, R = Et;
g: X = 2-MeO, R = Et;
k: X = 4-NO2, R = Et;
o: X = 4-MeO, R = Et;
l: X = 4-N(CH3)2, R = Et;
p: X = 4-CH3, R = Et;
m: X = 4-HO/4-AcO, R = Et;
q: X = 3-Et-4-HO/AcO, R = Et; r: X = 3-NO2, R = t-Bu;
s: X = 2-NO2, R = t-Bu; t: X = 3-NO2, R = (CH2)2OCH3;
Scheme 1. Reagents and conditions: (a) CH3COCH2CO2R, piperidine, toluene, 54–94%; (b) (1) CS(NH2)2, concentrated HCl, EtOH, reflux,
3–7 h; (2) 10% NaOH, 1 h, 26–91%; (c) Ac2O, DMAP, THF, 45–91%.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–10
Thiazines as Neuraminidase Inhibitors
3
ꢀ
with a ¼ 12.8634(7), b ¼ 16.1726(8), c ¼ 8.5390(4) A , Z ¼ 4,
ꢀ
V ¼ 1713.03(15) A 3, Mr ¼ 348.41, Dc ¼ 1.351 mg/m3,
S
¼ 1.05, m ¼ 0.21 mmꢁ1, F(000) ¼ 736. The phenyl ring (plane
1) and thiazine ring (plane 2) form two planes of the molecule.
The dihedral angle between plane 1 and 2 is 87.80°, which is
almost perpendicular. As shown in Fig. 2, an intermolecular
hydrogen bond N(2)–H ꢂ ꢂ ꢂ O(2) can be seen between the
acidamide and cꢀarbethoxyl groups, the distance and angle of
which are 3.01 A and 163.7°, respectively, belonging to the
typical V-type hydrogen bond. The intermolecular hydrogen
bonds play an important role in stabilizing the crystal.
In the crystal structure of compound 1g (Fig. 3), 1,3-thiazine
ring is in an half-chair conformation. The groups 2-acetamido,
4-methyl, and 5-carboxylate are all in the position of e, while
the 2-methoxyphenyl group is in the position of a, and the
position of 2-acetamido, 5-carboxylate is consistent with the
design. According to the SAR, the crystal structure of 1g could
occupy the four active sites of NA very well.
Figure 2. X-ray crystal structure of compound 1g with atom labels
and 50% probability displacement ellipsoids.
has great influence on the cyclization reaction. The
compounds 2a–2t with methyl have the highest yield, the
compounds with ethyl have higher yield, and the yields of
the compounds with methoxyethyl are lower than those of
the compounds with ethyl, but higher than those of the
compounds with tert-butyl, while the compounds with tert-
butyl have the lowest productivity. The order of yield is
Me > Et > (CH2)2OCH3 > t-Bu. This is because steric hin-
drance increases with increasing size of the R group.
Compounds 2a–2t were acylated by Ac2O to give 1a–1t. The
substituents R and X have little influence on the reaction.
Generally speaking, this method is high-yield, safe, operation-
ally simple, and cost-effective, meeting the requirements of
contemporary organic synthesis.
Biological in vitro evaluation
Preliminary results showed that most of the compounds (40
compounds) displayed good inhibitory activity (Table 1);
compounds 1g and 1t have the best inhibitory activities
(IC50 ¼ 29.06, 29.38 mg/mL, respectively), at the concentra-
tion of 40 mg/mL, which is similar to oseltamivir. At the same
time, compounds 1k and 1r have relatively good activities
(IC50 ¼ 31.80, 35.17 mg/mL, respectively, which are indicated
in bold in Table 2). Generally, the inhibitory activities of most
compounds still have a certain margin compared with
oseltamivir.
From Table 2, we find that all potent analogs (1g, 1k, 1r, and
1t) contain the acetamido group. According to the SAR, the 2-
amino (acetamido) would bind to the hydrophobic pocket
resulting from the movement of Ile222 and Trp178. As shown
in Table 2, the compounds of series 1 with the acetamido
group have better activity than the compounds 2 with amino
group, which may be due to the acetamide group possessing
better hydrophobicity than amino group.
Crystal structure
The data obtained from X-ray investigation are in good
agreement with the structure of compound 1g. X-ray analysis
reveals that crystal of compound 1g is made up of monoclinic
unit cells, each containing four molecules, space group Pbca
Figure 3. Hydrogen bonding diagram of com-
pound 1g. H atoms have been omitted for clarity.
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W. Li et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–10
Table 1. The IC50 of compounds 1g, 1k, 1r, and 1t against NA.
bic alkoxy group on benzene ring show better inhibitory
activity, such as compounds 1f, 1g, and 1i, and the 2-methoxy
is the most suitable. Accordingly, 1g has the best inhibitory
activity (IC50 ¼ 29.06 mg/mL). Secondly, Table 2 shows the
compounds (1a–1t) with a nitro group on benzene ring. We
find that compounds with nitro group in meta and para
positions of benzene ring (1k, 1r, 1t) have better inhibitory
activity than those with nitro group in ortho position (1c, 1j,
1s), and most of nitrobenzenes have potent inhibitory activity.
On the other hand, we find that the F and Cl of the benzene
ring (1h, 1n) have little effect on the NA inhibitory activity.
Also, as shown in Table 2, 2b, 2d, 2g, and 2k have better
inhibitory activity in series 2; accordingly, 1b, 1d, 1g, and 1k
have comparatively good inhibitory activity.
Compound 1g has the best NA inhibitory activity with
acetamido and methyl groups as hydrophobic side binds to
the NA active sites c and d, respectively. This agrees with the
fact that compounds with acetamido group showed better
activity than those with amino group. The –COOC2H5 group of
1g interacts with the positively charged site a by interaction
with Arg292, Arg371, and Arg118. 6-Benzene ring with 2-
methoxy group binds to the negatively charged region b by
Compounds
IC50 (mg/mL)
1g
1k
1r
1t
29.06
31.80
35.17
29.38
0.434
Oseltamivir
Opposite to the hydrophobic pocket, 5-carboxylate group
binds to the three arginine residues (site a), Arg118, Arg371,
and Arg292. From the preliminary results we find that 1a, 1e,
1r, and 1t have a nitro group on benzene ring in meta, when R
is tert-butyl; it exhibited the best inhibitory activity (1t,
IC50 ¼ 29.38 mg/mL), and methoxyethane has relatively good
activity. This may be due to the large hydrophobic group
occupying the active site a better. So we conclude that the
5-carboxylate and the acetamido groups of the compounds are
favorable for the inhibitory activity.
Between series 1 and 2, we can find that the substituents of
benzene ring (X) also have great influence on the inhibitory
activity. Firstly, it seems that the compounds with hydropho-
Table 2. The inhibitory activities of compounds 2a–t and 1a–t against NA in vitro.
O
O
RO
N
RO
N
O
S
NH2
S
N
H
X
X
2a–2t
1a–1t
Inhibition rate/%
Inhibition rate/%
X
R
Compounds
(40 mg/mL)
Compounds
(40 mg/mL)
3-NO2
2-Cl-5-NO2
2-NO2
4-NO2
3-NO2
2-EtO
2-MeO
4-Cl
3,4-diMeO
2-NO2
Me
Et
Me
Me
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
2a
2b
2c
2d
2e
2f
2g
2h
2i
22.84
34.37
20.79
37.83
9.79
19.28
23.78
14.11
17.06
20.79
35.59
21.22
23.93
14.87
13.36
8.03
1a
1b
1c
1d
1e
1f
1g
1h
1i
24.47
39.94
34.15
46.53
16.21
37.18
68.08
39.94
29.65
10.27
60.40
19.86
25.62
37.04
10.25
17.48
23.49
52.30
18.04
59.81
72.6
2j
2k
2l
1j
1k
1l
4-NO2
4-N(CH3)2
4-HO/4-AcO
4-F
4-MeO
4-CH3
3-Et-4-HO/AcO
3-NO2
2-NO2
3-NO2
Oseltamivir
2m
2n
2o
2p
2q
2r
2s
2t
1m
1n
1o
1p
1q
1r
1s
1t
25.47
17.66
34.59
11.98
t-Bu
t-Bu
(CH2)2OCH3
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–10
Thiazines as Neuraminidase Inhibitors
5
interacting with Asp151, Glu 227, and Glu119. The result
indicated that the structure of compound 1g could bind with
the active site of NA very well, and 1g can be used as a lead
compound for further design of NA inhibitors.
(Germany) instrument. The EI-MS spectra were recorded on a
Finnigan LCQ-advantage mass spectrometer.
General procedures for the synthesis of (E,Z)-alkyl
2-benzylidene-3-oxobutanoate (3a–3t)
A mixture of aromatic aldehydes (0.01 mol), alkyl acetoacetate
(0.01 mol), and piperidine (0.2 mL) in toluene (20 mL) was heated
to reflux and the generated water was removed by Dean-Stark
trap. The reaction was stopped until there was no water
generated. Then the organic layer was concentrated under
reduced pressure to get the crude product, and the crude product
was purified by silica gel chromatography eluted with PE/
EtOAc ¼ 2:1 to give products (3a–3t).
Conclusion
In summary, we have designed and synthesized two series of
novel 1,3-thiazine analogs as NA inhibitors. The synthesis
method is high-yield, safe, operationally simple, and cost-
effective, meeting the requirements of contemporary organic
synthesis. Preliminary bioassay showed that most of the
compounds have influenza NA inhibitory activity; the
compounds of series 1 have better activity than compounds
2 and the most potent compounds of the series are
compounds 1g, 1k, 1r, and 1t. From the figure of SAR
(Fig. 4), we can find that 5-carboxylate group, 2-acetamido
group, and the substituent of benzene ring (X) of the
compounds are necessary for the NA inhibitory activity,
and compound 1g with acetamido groups at C-2 and –
COOC2H5 at C-5 as hydrophobic side chain showed the best
inhibitory activity (IC50 ¼ 29.06 mg/mL); thus, 1g can be used
as core structure for further research, and this makes it
possible to find a proper compound as NA inhibitor.
General procedures for the synthesis of 2a–2t
3a (3 mmol), thiourea (3.2 mmol), and 0.35 mL HCl (38%) were
dissolved in ethanol (40 mL), and refluxed for 3–7 h with Dean-
Stark trap removal of water. After completion of the reaction,
most of the solvent was distilled under reduced pressure; then the
reaction solution was neutralized with NaOH solution (10%),
cooled to get methyl 2-amino-4-methyl-6-(3-nitrophenyl)-6H-1,3-thiazine-5-
carboxylate (2a) as a yellow solid, yield 91%, mp 162–165°C. 1H
NMR (400 MHz, CDCl3): 2.54 (s, 3H, CH3), 3.66 (s, 3H, CH3), 6.00 (s,
1H, thiazine 6-H), 7.84–8.12 (m, 4H, C6H4). Anal. calcd. for
C13H13N3O4S: C, 50.81; H, 4.26; N, 13.67. Found C, 50.60; H, 4.23;
N, 13.65.
Ethyl 2-amino-6-(2-chloro-5-nitrophenyl)-4-methyl-6H-1,3-
thiazine-5-carboxylate (2b)
A yellow solid, yield 67%, mp 206–208°C. 1H NMR (400 MHz,
CDCl3): 1.12 (t, J ¼ 7.2 Hz, 3H, CH3), 2.58 (s, 3H, CH3), 4.07 (q,
J ¼ 7.2 Hz, 2H, CH2), 5.85 (s, 1H, thiazine 6-H), 7.78 (d, J ¼ 2.8 Hz,
1H, C6H3 3-H), 7.90 (d, J ¼ 9.2 Hz, 1H, C6H3 6-H), 8.21 (dd,
J ¼ 9.2 Hz, J ¼ 2.8 Hz, 1H, C6H3 4-H), 9.20 (bs, 2H, NH2). Anal.
calcd. for C14H14ClN3O4S: C, 47.26; H, 3.97; N, 11.81. Found C,
47.21; H, 3.95; N, 11.78.
Experimental
Chemistry
All chemicals were of analytical reagents grade and were used
directly without further purification. All reactions were moni-
tored by thin-layer chromatography on 0.25-mm silica gel plates
(60GF-254) and visualized with UV light. Flash chromatography
was performed on silica gel (200–400 mesh) using commercially
available petroleum ether and ethyl acetate. Melting point (mp)
was measured on an X-4 electrothermal digital melting point
apparatus and uncorrected. 1H and 13C NMR spectra were
recorded on a Bruker advanced instrument with TMS as internal
standard at 400/100(125) MHz with chemical shifts (d) expressed
in ppm. Elemental analyses were performed on a Vario EL III
Methyl 2-amino-4-methyl-6-(2-nitrophenyl)-6H-1,3-
thiazine-5-carboxylate (2c)
1
A yellow solid, yield 67%, mp 196°C. H NMR (400 MHz, CDCl3):
2.54 (s, 3H, CH3), 3.63 (s, 3H, CH3), 6.07 (s, 1H, thiazine 6-H),
7.22–7.99 (m, 4H, C6H4). Anal. calcd. for C13H13N3O4S: C, 50.81; H,
4.26; N, 13.67. Found C, 50.62; H, 4.24; N, 13.64.
Figure 4. The structure–activity relationship of
compounds 1 and 2.
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W. Li et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–10
Methyl 2-amino-4-methyl-6-(4-nitrophenyl)-6H-1,3-
J ¼ 8.0 Hz, J ¼ 0.9 Hz, 1H, C6H4 3-H), 7.40 (dt, J ¼ 8.0 Hz,
J ¼ 0.9 Hz, 1H, C6H4 5-H), 7.52 (dd, J ¼ 8.0 Hz, J ¼ 1.2 Hz, 1H,
C6H4 4-H), 7.67 (bs, 1H, NH). Anal. calcd. for C14H15N3O4S: C, 52.33;
H, 4.70; N, 13.08. Found C, 52.23; H, 4.65; N, 13.04.
thiazine-5-carboxylate (2d)
A white solid, yield 89%, mp 200°C. 1H NMR (400 MHz, DMSO-d6):
2.54 (s, 3H, CH3), 3.66 (s, 3H, CH3), 5.85 (s, 1H, thiazine 6-H), 7.59 (d,
J ¼ 8.8 Hz, 2H, C6H4 2,6-H), 8.23 (d, J ¼ 8.8 Hz, 2H, C6H4 3,5-H).
Anal. calcd. for C13H13N3O4S: C, 50.81; H, 4.26; N, 13.67. Found C,
50.71; H, 4.25; N, 13.60.
Ethyl 2-amino-4-methyl-6-(4-nitrophenyl)-6H-1,3-thiazine-
5-carboxylate (2k)
A yellow solid, yield 72%, mp 162–163°C. 1H NMR (400 MHz,
CDCl3): 1.24 (t, J ¼ 6.8 Hz, 3H, CH3), 2.50 (s, 3H, CH3), 4.15 (q,
J ¼ 6.8 Hz, 2H, CH2), 5.39 (s, 1H, thiazine 6-H), 7.35 (d, J ¼ 8.8 Hz,
2H, C6H4 2,6-H), 8.14 (d, J ¼ 8.8 Hz, 2H, C6H4 3,5-H). Anal. calcd.
for C14H15N3O4S: C, 52.33; H, 4.70; N, 13.08. Found C, 52.10; H,
4.64; N, 13.03.
Ethyl 2-amino-4-methyl-6-(3-nitrophenyl)-6H-1,3-thiazine-
5-carboxylate (2e)
A white solid, yield 90%, mp 162–165°C. 1H NMR (400 MHz,
CDCl3): 1.14 (t, J ¼ 7.2 Hz, 3H, CH3), 2.55 (s, 3H, CH3), 4.08 (q,
J ¼ 7.2 Hz, 2H, CH2), 6.05 (s, 1H, thiazine 6-H), 7.23–7.98 (m, 4H,
C6H4). Anal. calcd. for C14H15N3O4S: C, 52.33; H, 4.70; N, 13.08.
Found C, 52.14; H, 4.75; N, 13.05.
Ethyl 2-amino-6-(4-(dimethylamino)phenyl)-4-methyl-6H-
1,3-thiazine-5-carboxylate (2l)
Ethyl 2-amino-6-(2-ethoxyphenyl)-4-methyl-6H-1,3-
A white solid, yield 75%, mp 160°C carbonize. 1H NMR (400 MHz,
CDCl3): 1.25 (t, J ¼ 7.2 Hz, 3H, CH3), 2.93 (s, 6H, 2 ꢃ CH3), 4.16 (q,
J ¼ 7.2 Hz, 2H, CH2), 5.33 (s, 1H, thiazine 6-H), 6.65 (d, J ¼ 8.8 Hz,
2H, C6H4 2,6-H), 7.71 (d, J ¼ 8.8 Hz, 2H, C6H4 3,5-H). Anal. calcd.
for C16H21N3O2S: C, 60.16; H, 6.63; N, 13.16. Found C, 60.05; H,
6.59; N, 13.12.
thiazine-5-carboxylate (2f)
A pale yellow solid, yield 77%, mp 135–138°C. 1H NMR (400 MHz,
CDCl3): 1.18 (t, J ¼ 7.2 Hz, 3H, CH3), 1.46 (t, J ¼ 6.8 Hz, 3H, CH3),
2.50 (s, 3H, CH3), 4.08 (q, J ¼ 7.2 Hz, 2H, CH2), 4.11 (q, J ¼ 6.8 Hz,
2H, CH2), 5.15 (bs, 2H, NH2), 5.72 (s, 1H, thiazine 6-H), 6.75 (d,
J ¼ 7.8 Hz, 1H, C6H4 3-H), 6.80 (t, J ¼ 8.0 Hz, 1H, C6H4 4-H), 6.85 (t,
J ¼ 7.8 Hz, 1H, C6H4 6-H), 7.02 (d, J ¼ 8.0 Hz, 1H, C6H4 5-H). Anal.
calcd. for C16H20N2O3S: C, 59.98; H, 6.29; N, 8.74. Found C, 59.70;
H, 6.25; N, 8.70.
Ethyl 2-amino-6-(4-hydroxyphenyl)-4-methyl-6H-1,3-
thiazine-5-carboxylate (2m)
A white solid, yield 73%, mp 99–101°C. 1H NMR (400 MHz, CDCl3):
1.25 (t, J ¼ 7.2 Hz, 3H, CH3), 2.56 (s, 3H, CH3), 4.17 (q, J ¼ 7.2 Hz,
2H, CH2), 5.32 (s, 1H, thiazine 6-H), 6.70 (d, J ¼ 8.4 Hz, 2H, C6H4
2,6-H), 7.02 (d, J ¼ 8.4 Hz, 2H, C6H4 3,5-H). Anal. calcd. for
Ethyl 2-amino-6-(2-methoxyphenyl)-4-methyl-6H-1,3-
thiazine-5-carboxylate (2g)
A white solid, yield 82%, mp 158–159°C. 1H NMR (400 MHz,
CDCl3): 1.19 (t, J ¼ 7.2 Hz, 3H, CH3), 2.50 (s, 3H, CH3), 3.99 (s, 3H,
CH3), 4.11 (q, J ¼ 7.2 Hz, 2H, CH2), 5.40 (bs, 2H, NH2), 5.71 (s, 1H,
thiazine 6-H), 6.84–7.21 (m, 4H, C6H4). Anal. calcd. for
C15H18N2O3S: C, 58.80; H, 5.92; N, 9.14. Found C, 58.65; H,
5.85; N, 9.06.
C14H16N2O3S: C, 57.52; H, 5.52; N, 9.58. Found C, 57.38; H, 5.51;
N, 9.54.
Ethyl 2-amino-6-(4-fluorophenyl)-4-methyl-6H-1,3-
thiazine-5-carboxylate (2n)
A pale yellow solid, yield 86%, mp 121–122°C, 1H NMR (400 MHz,
CDCl3): 1.23 (t, J ¼ 6.8 Hz, 3H, CH3), 2.49 (s, 3H, CH3), 4.15 (q,
J ¼ 6.8 Hz, 2H, CH2), 4.80 (bs, 2H, NH2), 5.31 (s, 1H, thiazine 6-H),
6.95–7.16 (m, 4H, C6H4). Anal. calcd. for C14H15FN2O2S: C, 57.13;
H, 5.14; N, 9.52. Found C, 56.89; H, 5.18; N, 9.56.
Ethyl 2-amino-6-(4-chlorophenyl)-4-methyl-6H-1,3-
thiazine-5-carboxylate (2h)
A white solid, yield 88%, mp 149–151°C. 1H NMR (400 MHz,
CDCl3): 1.23 (t, J ¼ 7.2 Hz, 3H, CH3), 2.48 (s, 3H, CH3), 4.15 (q,
J ¼ 7.2 Hz, 2H, CH2), 5.27 (bs, 2H, NH2), 5.28 (s, 1H, thiazine 6-H),
7.11 (d, J ¼ 8.4 Hz, 2H, C6H4 2,6-H), 7.23 (d, J ¼ 8.4 Hz, 2H, C6H4
3,5-H). EI-MS (m/z): 310.0 (Mþ); Anal. calcd. for C14H15ClN2O2S: C,
54.10; H, 4.86; N, 9.01. Found C, 54.35; H, 4.84; N, 8.98.
Ethyl 2-amino-6-(4-methoxyphenyl)-4-methyl-6H-1,3-
thiazine-5-carboxylate (2o)
A pale yellow solid, yield 80%, mp 145–146°C. 1H NMR (400 MHz,
CDCl3): 1.23 (t, J ¼ 7.2 Hz, 3H, CH3), 2.49 (s, 3H, CH3), 3.77 (s, 3H,
CH3), 4.14 (q, J ¼ 7.2 Hz, 2H, CH2), 5.29 (s, 1H, thiazine 6-H), 6.79
(d, J ¼ 8.8 Hz, 2H, C6H4 3,5-H), 7.11 (d, J ¼ 8.8 Hz, 2H, C6H4 2,6-H).
Anal. calcd. for C15H18N2O3S: C, 58.80; H, 5.92; N, 9.14. Found C,
58.61; H, 5.84; N, 9.09.
Ethyl 2-amino-6-(3,4-dimethoxyphenyl)-4-methyl-6H-1,3-
thiazine-5-carboxylate (2i)
A pale yellow solid, yield 80%, mp 102–104°C. 1H NMR (400 MHz,
CDCl3): 1.24 (t, J ¼ 7.2 Hz, 3H, CH3), 2.48 (s, 3H, CH3), 3.83 (s, 6H,
2 ꢃ OCH3), 4.15 (q, J ¼ 7.2 Hz, 2H, CH2), 5.29 (s, 1H, thiazine 6-H),
6.72–6.75 (m, 3H, C6H3). Anal. calcd. for C16H20N2O4S: C, 57.12; H,
5.99; N, 8.33. Found C, 57.01; H, 5.96; N, 8.30.
Ethyl 2-amino-4-methyl-6-(p-tolyl)-6H-1,3-thiazine-5-
carboxylate (2p)
A white solid, yield 75%, mp 142–145°C. 1H NMR (400 MHz,
CDCl3): 1.23 (t, J ¼ 7.2 Hz, 3H, CH3), 2.30 (s, 3H, CH3), 2.50 (s, 3H,
CH3), 4.14 (q, J ¼ 7.2 Hz, 2H, CH2), 5.30 (s, 1H, thiazine 6-H),
7.08–7.10 (m, 4H, C6H4). 13C NMR (100 MHz, CDCl3): 14.2, 21.1,
23.2, 42.3, 60.3, 126.2, 129.2, 137.3, 138.8, 155.6, 157.6, 166.8.
Anal. calcd. for C15H18N2O2S: C, 62.04; H, 6.25; N, 9.56. Found C,
61.85; H, 6.22; N, 9.60.
Ethyl 2-amino-4-methyl-6-(2-nitrophenyl)-6H-1,3-thiazine-
5-carboxylate (2j)
A white solid, yield 72%, mp 195–198°C. 1H NMR (400 MHz,
CDCl3): 2.43 (s, 3H, CH3), 3.52 (s, 3H, CH3), 5.80 (s, 1H, thiazine
6-H), 7.16 (dd, J ¼ 8.0 Hz, J ¼ 1.2 Hz, 1H, C6H4 6-H), 7.54 (dt,
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–10
Thiazines as Neuraminidase Inhibitors
7
Ethyl 2-amino-6-(3-ethyl-4-hydroxyphenyl)-4-methyl-6H-
165.48, 172.42; EI-MS: 397.5 (Mþ); Anal. calcd. for C16H16ClN3O5S:
C, 48.30; H, 4.05; N, 10.56. Found C, 48.01; H, 4.01; N, 10.60.
1,3-thiazine-5-carboxylate (2q)
A white solid, yield 78%, mp 125–128°C. 1H NMR (400 MHz,
DMSO-d6): 1.14 (t, J ¼ 7.2 Hz, 3H, CH3), 1.30 (t, J ¼ 6.8 Hz, 3H,
CH3), 2.35 (s, 3H, CH3), 3.93 (q, J ¼ 7.2 Hz, 2H, CH2), 4.03 (q,
J ¼ 6.8 Hz, 2H, CH2), 5.17 (s, 1H, thiazine 6-H), 6.50 (d, J ¼ 8.4 Hz,
1H, C6H3 5-H), 6.62 (d, J ¼ 8.4 Hz, 1H, C6H3 6-H), 6.75 (d,
J ¼ 1.6 Hz, 1H, C6H3 2-H), 7.50 (bs, 2H, NH2), 8.84 (bs, 1H, OH).
Anal. calcd. for C16H20N2O3S: C, 59.98; H, 6.29; N, 8.74. Found C,
59.79; H, 6.31; N, 8.76.
Methyl 2-acetamido-4-methyl-6-(2-nitrophenyl)-6H-1,3-
thiazine-5-carboxylate (1c)
A yellow solid, yield 88%, mp 179–181°C. 1H NMR (400 MHz,
CDCl3): 2.15 (s, 3H, CH3), 2.54 (s, 3H, CH3), 3.63 (s, 3H, CH3), 5.98 (s,
1H, thiazine 6-H), 7.23–8.02 (m, 4H, C6H4); 13C NMR (100 MHz,
DMSO-d6): 23.66, 35.64, 51.99, 52.19, 103.06, 125.94, 128.66,
129.53, 134.53, 135.76, 137.75, 146.92, 152.32, 155.74, 166.09;
EI-MS: 349.2 (Mþ); Anal. calcd. for C15H15N3O5S: C, 51.57; H, 4.33;
N, 12.03. Found C, 51.45; H, 4.27; N, 12.05.
tert-Butyl 2-amino-4-methyl-6-(3-nitrophenyl)-6H-1,3-
thiazine-5-carboxylate (2r)
A white solid, yield 26%, mp 190°C. 1H NMR (400 MHz, CDCl3):
1.44 (s, 9H, 3 ꢃ CH3), 2.48 (s, 3H, CH3), 5.33 (s, 1H, thiazine 6-H),
7.43–8.08 (m, 4H, C6H4). Anal. calcd. for C16H19N3O4S: C, 55.00; H,
5.48; N, 12.03. Found C, 55.20; H, 5.34; N, 12.10.
Methyl 2-acetamido-4-methyl-6-(4-nitrophenyl)-6H-1,3-
thiazine-5-carboxylate (1d)
A yellow solid, yield 61%, mp 92–94°C. 1H NMR (400 MHz, CDCl3):
2.14 (s, 3H, CH3), 2.53 (s, 3H, CH3), 3.73 (s, 3H, CH3), 5.26 (s, 1H,
thiazine 6-H), 7.34 (d, J ¼ 8.8Hz, 2H, C6H4 2,6-H), 8.13 (d,
J ¼ 8.8Hz, 2H, C6H4 3,5-H); 13C NMR (100 MHz, DMSO-d6):
20.30, 22.42, 49.67, 50.20, 101.94, 122.24, 126.38, 145.08,
148.15, 153.54, 158.98, 164.46, 170.58; EI-MS: 349.2 (Mþ); Anal.
calcd. for C15H15N3O5S: C, 51.57; H, 4.33; N, 12.03. Found C, 51.38;
H, 4.35; N, 12.10.
tert-Butyl 2-amino-4-methyl-6-(2-nitrophenyl)-6H-1,3-
thiazine-5-carboxylate (2s)
A yellow solid, yield 29%, mp 173–175°C. 1H NMR (400 MHz,
CDCl3): 1.44 (s, 9H, 3 ꢃ CH3), 2.56 (s, 3H, CH3), 6.00 (s, 1H, thiazine
6-H), 7.43–8.02 (m, 4H, C6H4). Anal. calcd. for C16H19N3O4S: C,
55.00; H, 5.48; N, 12.03. Found C, 55.15; H, 5.40; N, 12.11.
Ethyl 2-acetamido-4-methyl-6-(3-nitrophenyl)-6H-1,3-
thiazine-5-carboxylate (1e)
A white solid, yield 58%, mp 123–126°C. 1H NMR (400 MHz,
CDCl3): 1.15 (t, J ¼ 7.2 Hz, 3H, CH3), 2.15 (s, 3H, CH3), 2.54 (s, 3H,
CH3), 4.12 (q, J ¼ 7.2 Hz, 2H, CH2), 6.12 (s, 1H, thiazine 6-H), 7.21
(dd, J ¼ 8.0 Hz, J ¼ 1.2 Hz, 1H, C6H4 2-H), 7.45 (d, J ¼ 8.0 Hz,
J ¼ 1.2 Hz, 1H, C6H4 6-H), 7.64 (d, J ¼ 8.0 Hz, J ¼ 1.2 Hz, 1H, C6H4
4-H), 7.85 (dd, J ¼ 8.0 Hz, J ¼ 1.2 Hz 1H, C6H4 5-H), 8.12 (s, 1H,
NH); 13C NMR (100 MHz, DMSO-d6): 14.39, 21.33, 22.34, 45.31,
57.72, 105.67, 118.36, 118.98, 119.31, 119.71, 120.21, 121.21,
154.98, 160.58; EI-MS: 363.1 (Mþ); Anal. calcd. for C16H17N3O5S: C,
52.88; H, 4.72; N, 11.56. Found C, 52.69; H, 4.75; N, 11.54.
2-Methoxyethyl 2-amino-4-methyl-6-(3-nitrophenyl)-6H-
1,3-thiazine-5-carboxylate (2t)
A white solid, yield 42%, mp 147–149°C. 1H NMR (400 MHz,
CDCl3): 2.16 (s, 3H, CH3), 3.30 (s, 3H, CH3), 3.65 (t, J ¼ 4.8 Hz, 2H,
CH2), 4.27 (t, J ¼ 4.8 Hz, 2H, CH2), 5.30 (s, 1H, thiazine 6-H),
7.46–8.12 (m, 4H, C6H4). Anal. calcd. for C15H17N3O5S: C, 51.27; H,
4.88; N, 11.96. Found C, 51.16; H, 4.83; N, 11.92.
General procedures for the synthesis of 1a–1t
2a (1 mmol) and 1 mL acetic oxide were dissolved in THF (10 mL),
and a catalytic amount of DMAP was added to the solution,
stirred for 1 h at 60°C, then the reaction solution was poured into
ice water, the mixture was filtered, and recrystallized from
ethanol, dried to give methyl 2-acetamido-4-methyl-6-(3-nitrophenyl)-6H-
Ethyl 2-acetamido-6-(2-ethoxyphenyl)-4-methyl-6H-1,3-
thiazine-5-carboxylate (1f)
A pale yellow solid, yield 51%, mp 136–138°C. 1H NMR (400 MHz,
CDCl3): 1.21 (t, J ¼ 7.2 Hz, 3H, CH3), 1.47 (t, J ¼ 7.2 Hz, 3H, CH3),
2.14 (s, 3H, CH3), 2.55 (s, 3H, CH3), 4.10 (q, J ¼ 7.2 Hz, 2H, CH2),
4.14 (q, J ¼ 7.2 Hz, 2H, CH2), 5.66 (s, 1H, thiazine 6-H), 6.81–7.24
(m, 4H, C6H4); 13C NMR (100 MHz, DMSO-d6): 14.24, 14.98, 22.81,
23.60, 34.01, 60.41, 63.99, 103.51, 112.65, 120.47, 126.80, 128.98,
129.16, 154.49, 154.99, 166.13; EI-MS: 362.1 (Mþ); Anal. calcd. for
C18H22N2O4S: C, 59.65; H, 6.12; N, 7.73. Found C, 59.44; H, 6.10;
N, 7.75.
1,3-thiazine-5-carboxylate (1a),
a yellow solid, yield 46%, mp
112–114°C. 1H NMR (400 MHz, CDCl3): 1.86 (s, 3H, CH3), 2.54
(s, 3H, CH3), 3.63 (s, 3H, CH3), 6.07 (s, 1H, thiazine 6-H), 7.23–7.98
(m, 4H, C6H4); 13C NMR (100 MHz, DMSO-d6): 21.50, 22.56, 49.57,
50.13, 99.25, 122.37, 127.48, 135.20, 138.20, 138.55, 139.41,
145.29, 148.66, 152.41, 163.94; EI-MS: 349.2 (Mþ); Anal. calcd. for
C15H15N3O5S: C, 51.57; H, 4.33; N, 12.03. Found C, 51.51; H, 4.30;
N, 12.08.
Ethyl 2-acetamido-6-(2-methoxyphenyl)-4-methyl-6H-1,3-
Ethyl 2-acetamido-6-(2-chloro-5-nitrophenyl)-4-methyl-
thiazine-5-carboxylate (1g)
6H-1,3-thiazine-5-carboxylate (1b)
A white solid, yield 68%, mp 150–151°C. 1H NMR (400 MHz,
CDCl3): 1.20 (t, J ¼ 7.2 Hz, 3H, CH3), 2.13 (s, 3H, CH3), 2.52 (s, 3H,
CH3), 3.87 (s, 3H, CH3), 4.14 (q, J ¼ 7.2 Hz, 2H, CH2), 5.71 (s, 1H,
thiazine 6-H), 6.88–7.25 (m, 4H, C6H4); 13C NMR (125 MHz, CDCl3):
14.11, 22.14, 25.21, 35.15, 55.53, 60.65, 104.37, 110.86, 120.33,
127.04, 128.21, 128.99, 156.03, 166.19; EI-MS: 348.3 (Mþ); Anal.
calcd. for C17H20N2O4S: C, 58.60; H, 5.79; N, 8.04. Found C, 58.35;
H, 5.76; N, 8.00.
A yellow solid, yield 61%, mp 150–153°C. 1H NMR (400 MHz,
CDCl3): 1.23 (t, J ¼ 6.8 Hz, 3H, CH3), 2.15 (s, 3H, CH3), 2.55 (s, 3H,
CH3), 4.15 (q, J ¼ 6.8 Hz, 2H, CH2), 5.66 (s, 1H, thiazine 6-H), 7.58
(d, J ¼ 8.8 Hz, 1H, C6H3 3-H), 7.77 (d, J ¼ 2.4 Hz, 1H, C6H3 6-H),
8.07 (dd, J ¼ 8.8 Hz, J ¼ 2.4 Hz, 1H, C6H3 4-H), 8.52 (bs, 1H,
CONH); 13C NMR (125 MHz, CDCl3): 14.06, 22.57, 23.54, 38.06,
61.80, 103.54, 122.82, 123.82, 131.16, 139.44, 1339.86, 146.93,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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8
W. Li et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–10
Ethyl 2-acetamido-6-(4-chlorophenyl)-4-methyl-6H-1,3-
Ethyl 2-acetamido-6-(4-acetoxyphenyl)-4-methyl-6H-1,3-
thiazine-5-carboxylate (1h)
thiazine-5-carboxylate (1m)
A pale yellow solid, yield 58%, mp 135–136°C. 1H NMR (400 MHz,
CDCl3): 1.21 (t, J ¼ 6.8 Hz, 3H, CH3), 2.18 (s, 3H, CH3), 2.45 (s, 3H,
CH3), 4.12 (q, J ¼ 6.8 Hz, 2H, CH2), 5.36 (s, 1H, thiazine 6-H), 7.31
(d, J ¼ 8.8 Hz, 2H, C6H4 2,6-H), 8.10 (d, J ¼ 8.8 Hz, 2H, C6H4 3,5-H),
8.54 (s, 1H, NH); 13C NMR (100 MHz, CDCl3): 14.15, 22.43, 24.83,
40.43, 60.90, 105.52, 128.16, 128.77, 133.61, 139.45, 165.92; EI-MS:
352.6 (Mþ); Anal. calcd. for C16H17ClN2O3S: C, 54.46; H, 4.86; N,
7.94. Found C, 54.25; H, 4.84; N, 7.91.
A white solid, yield 61%, mp 150–153°C. 1H NMR (400 MHz,
CDCl3): 1.23 (t, J ¼ 6.8 Hz, 3H, CH3), 2.15 (s, 3H, CH3), 2.55 (s, 3H,
CH3), 4.15 (q, J ¼ 6.8 Hz, 2H, CH2), 5.66 (s, 1H, thiazine 6-H), 7.58
(d, J ¼ 8.8 Hz, 1H, C6H3 3-H), 7.77 (d, J ¼ 2.4 Hz, 1H, C6H3 6-H),
8.07 (dd, J ¼ 8.8 Hz, J ¼ 2.4 Hz, 1H, C6H3 4-H), 8.52 (bs, 1H, NH);
13C NMR (125 MHz, CDCl3): 14.18, 21.09, 22.30, 24.73, 40.52,
60.89, 105.77, 121.67, 127.96, 138.52, 150.14, 166.09, 169.47; EI-
MS: 360.3 (Mþ); Anal. calcd. for C18H20N2O4S: C, 59.98; H, 5.59; N,
7.77. Found C, 59.79; H, 5.54; N, 7.74.
Ethyl 2-acetamido-6-(3,4-dimethoxyphenyl)-4-methyl-6H-
1,3-thiazine-5-carboxylate (1i)
Ethyl 2-acetamido-6-(4-fluorophenyl)-4-methyl-6H-1,3-
A pale yellow solid, yield 52%, mp 172–174°C. 1H NMR (400 MHz,
CDCl3): 1.25 (t, J ¼ 7.2 Hz, 3H, CH3), 2.16 (s, 3H, CH3), 2.51 (s, 3H,
CH3), 3.84 (s, 6H, 2 ꢃ OCH3), 3.85 (s, 6H, CH3), 4.15 (q, J ¼ 7.2 Hz,
2H, CH2), 5.29 (s, 1H, thiazine 6-H), 6.68–6.75 (m, 3H, C6H3); 13C
NMR (100 MHz, DMSO-d6): 12.87, 14.37, 22.71, 23.65, 55.73, 60.44,
105.36, 110.92, 111.85, 118.75, 134.52, 148.61, 148.90, 153.11,
166.28, 170.63; EI-MS: 378.2 (Mþ); Anal. calcd. for C18H22N2O5S: C,
57.13; H, 5.86; N, 7.40. Found C, 56.93; H, 5.81; N, 7.46.
thiazine-5-carboxylate (1n)
A yellow solid, yield 62%, mp 72–73°C. 1H NMR (400 MHz, CDCl3):
1.25 (t, J ¼ 7.2 Hz, 3H, CH3), 2.16 (s, 3H, CH3), 2.53 (s, 3H, CH3),
4.17 (q, J ¼ 7.2 Hz, 2H, CH2), 5.20 (s, 1H, thiazine 6-H), 6.96 (d,
J ¼ 8.8 Hz, 2H, C6H4 2,6-H), 7.16 (d, J ¼ 8.8 Hz, 2H, C6H4 3,5-H);
13C NMR (100 MHz, DMSO-d6): 12.87, 14.32, 22.76, 23.64, 60.34,
104.98, 115.32, 115.60, 128.82, 138.35, 153.67, 160.50, 162.93,
166.09; EI-MS: 336.3 (Mþ); Anal. calcd. for C16H17FN2O3S: C, 57.13;
H, 5.09; N, 8.33. Found C, 57.35; H, 5.12; N, 8.30.
Ethyl 2-acetamido-4-methyl-6-(2-nitrophenyl)-6H-1,3-
thiazine-5-carboxylate (1j)
Ethyl 2-acetamido-6-(4-methoxyphenyl)-4-methyl-6H-1,3-
A white solid, yield 64%, mp 131–132°C. 1H NMR (400 MHz,
CDCl3): 1.24 (t, J ¼ 6.8 Hz, 3H, CH3), 2.13 (s, 3H, CH3), 2.33 (s, 3H,
CH3), 2.43 (q, J ¼ 6.8 Hz, 2H, CH2), 5.80 (s, 1H, thiazine 6-H), 7.16
(dd, J ¼ 8.0 Hz, J ¼ 1.2 Hz, 1H, C6H4 6-H), 7.54 (dt, J ¼ 8.0 Hz,
J ¼ 0.9 Hz, 1H, C6H4 3-H), 7.40 (dt, J ¼ 8.0 Hz, J ¼ 0.9 Hz, 1H,
C6H4 5-H), 7.52 (dd, J ¼ 8.0 Hz, J ¼ 1.2 Hz, 1H, C6H4 4-H), 8.67 (bs,
1H, NH); 13C NMR (100 MHz, DMSO-d6): 18.05, 27.82, 28.55, 40.48,
65.50, 108.27, 130.48, 130.70, 133.55, 134.51, 139.53, 140.57,
151.81, 158.87, 160.35, 170.13; EI-MS: 363.2 (Mþ); Anal. calcd. for
thiazine-5-carboxylate (1o)
A pale yellow solid, yield 72%, mp 142–144°C. 1H NMR (400 MHz,
CDCl3): 1.24 (t, J ¼ 6.8 Hz, 3H, CH3), 2.15 (s, 3H, CH3), 2.50 (s, 3H,
CH3), 3.77 (s, 3H, CH3), 4.17 (q, J ¼ 6.8 Hz, 2H, CH2), 5.19 (s, 1H,
thiazine 6-H), 6.79 (d, J ¼ 8.8 Hz, 2H, C6H4 3,5-H), 7.11 (d,
J ¼ 8.8 Hz, 2H, C6H4 2,6-H); 13C NMR (100 MHz, DMSO-d6): 14.34,
23.57, 55.33, 60.39, 105.27, 114.17, 127.99, 134.03, 158.89, 166.19;
EI-MS: 348.0 (Mþ); Anal. calcd. for C17H20N2O4S: C, 58.60; H, 5.79;
N, 8.04. Found C, 58.24; H, 5.75; N, 8.02.
C16H17N3O5S: C, 52.88; H, 4.72; N, 11.56. Found C, 52.78; H, 4.69;
N, 11.50.
Ethyl 2-acetamido-4-methyl-6-(p-tolyl)-6H-1,3-thiazine-5-
Ethyl 2-acetamido-4-methyl-6-(4-nitrophenyl)-6H-1,3-
carboxylate (1p)
A pale yellow solid, yield 48%, mp 125–127°C. 1H NMR (400 MHz,
CDCl3): 1.24 (t, J ¼ 6.8 Hz, 3H, CH3). 2.14 (s, 3H, CH3), 2.30 (s, 3H,
CH3), 2.55 (s, 3H, CH3), 4.17 (q, J ¼ 6.8 Hz, 2H, CH2), 5.20 (s, 1H,
thiazine 6-H), 7.08–7.67 (m, 4H, C6H4); 13C NMR (100 MHz, DMSO-
d6): 12.85, 14.31, 20.77, 20.98, 23.53, 60.41, 105.04, 126.67, 129.34,
137.01, 139.11, 151.43, 155.03, 166.18, 170.48; EI-MS: 332.1 (Mþ);
Anal. calcd. for C17H20N2O3S: C, 61.42; H, 6.06; N, 8.43. Found C,
61.73; H, 5.97; N, 8.39.
thiazine-5-carboxylate (1k)
A yellow solid, yield 49%, mp 123–126°C, 1H NMR (400 MHz,
CDCl3): 1.25 (t, J ¼ 7.2 Hz, 3H, CH3), 2.16 (s, 3H, CH3), 2.53 (s, 3H,
CH3), 4.20 (q, J ¼ 7.2 Hz, 2H, CH2), 5.27 (s, 1H, thiazine 6-H),
7.27–8.15 (m, 4H, C6H4); 13C NMR (100 MHz, DMSO-d6):
13.54, 20.35, 23.47, 40.98, 60.85, 102.98, 123.65, 128.41, 146.31,
146.89, 150.64, 166.52; EI-MS: 363.1 (Mþ); Anal. calcd. for
C16H17N3O5S: C, 52.88; H, 4.72; N, 11.56. Found C, 52.60; H,
4.68; N, 11.49.
Ethyl 2-acetamido-6-(4-acetoxy-3-ethylphenyl)-4-methyl-
Ethyl 2-acetamido-6-(4-(dimethylamino)phenyl)-4-methyl-
6H-1,3-thiazine-5-carboxylate (1q)
A pale yellow solid, yield 85%, mp 122–124°C. 1H NMR (400 MHz,
CDCl3): 1.26 (t, J ¼ 7.2 Hz, 3H, CH3), 1.37 (t, J ¼ 7.2 Hz, 3H, CH3),
2.16 (s, 3H, CH3), 2.29 (s, 3H, CH3), 2.49 (s, 3H, CH3), 4.01 (q,
J ¼ 7.2 Hz, 2H, CH2), 4.18 (q, J ¼ 7.2 Hz, 2H, CH2), 5.20 (s, 1H,
thiazine 6-H), 6.70–6.90 (m, 3 H, C6H3); 13C NMR (100 MHz, DMSO-
d6): 14.34, 14.38, 20.49, 20.70, 22.88, 23.55, 60.54, 64.15, 105.02,
112,48, 112.58, 118.50, 118.72, 122.88, 122.98, 139.26, 141,04,
150.15, 166.10, 168.86; EI-MS: 388.3 (Mþ); Anal. calcd. for
6H-1,3-thiazine-5-carboxylate (1l)
A yellow solid, yield 78%, mp 118–121°C. 1H NMR (400 MHz,
CDCl3): 1.15 (t, J ¼ 7.2 Hz, 3H, CH3), 1.98 (s, 3H, CH3), 2.50 (s, 3H,
CH3), 2.85 (s, 6H, N(CH3)2), 4.07 (q, J ¼ 7.2 Hz, 2H, CH2), 5.08 (s, 1H,
thiazine 6-H), 6.60 (d, J ¼ 4.8 Hz, 2H, C6H4 3,5-H), 6.92 (d,
J ¼ 4.8 Hz, 2H, C6H4 2,6-H), 11.50 (bs, 1H, NH); 13C NMR (100 MHz,
DMSO-d6): 13.81, 14.31, 20.10, 22.27, 23.15, 60.04, 63.64, 104.50,
112.00, 118.02, 122.42, 138.73, 149.62, 153.03, 165.87, 168.34; EI-
MS: 361.2 (Mþ); Anal. calcd. for C18H23N3O3S: C, 59.81; H, 6.41; N,
11.63. Found C, 59.64; H, 6.37; N, 11.58.
C20H24N2O4S: C, 61.83; H, 6.23; N, 7.21. Found C, 61.69; H,
6.43; N, 7.24.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 1–10
Thiazines as Neuraminidase Inhibitors
9
tert-Butyl 2-acetamido-4-methyl-6-(3-nitrophenyl)-6H-1,3-
acetyl neuraminic acid (MUNANA) is the substrate of NA, and
cleavage of this substrate by NA produces a fluorescent product,
which can emit an emission wavelength of 450 nm with an
excitation wavelength of 360 nm. The intensity of fluorescence
can reflect the activity of NA sensitively [30].
In the enzyme reaction system, a certain concentration of the
test compounds with the NA enzyme was suspended in MES
buffer (pH 6.5), followed by addition of the substrate, and
incubated at 37°C for 60 min. The reaction was terminated by
adding the stop solution, and the plate was read within 20 min
after adding the stop solution detecting fluorescence. The IC50
was calculated by plotting percent inhibition versus the inhibitor
concentration, and determination of each point was performed
in duplicate. The data are expressed as the mean of three
independent experiments.
thiazine-5-carboxylate (1r)
A brownish yellow solid, yield 90%, mp 78–80°C. 1H NMR
(400 MHz, CDCl3): 1.44 (s, 9H, 3 ꢃ CH3), 2.16 (s, 3H, CH3), 2.50 (s,
3H, CH3), 5.23 (s, 1H, thiazine 6-H), 7.47–8.02 (m, 4H, C6H4); 13C
NMR (100 MHz, DMSO-d6): 21.11, 22.20, 28.11, 36.47, 78.54,
105.20, 123.10, 123.80, 124.35, 132.14, 132.87, 133.54, 154,23,
167.58; EI-MS: 391.0 (Mþ); Anal. calcd. for C18H21N3O5S: C, 55.23;
H, 5.41; N, 10.73. Found C, 55.20; H, 5.45; N, 10.71.
tert-Butyl 2-acetamido-4-methyl-6-(2-nitrophenyl)-6H-1,3-
thiazine-5-carboxylate (1s)
A brownish yellow solid, yield 91%, mp 178–182°C. 1H NMR
(400 MHz, CDCl3): 1.30 (s, 9H, 3 ꢃ CH3), 2.14 (s, 3H, CH3), 2.52 (s,
3H, CH3), 5.90 (s, 1H, thiazine 6-H), 7.23–8.02 (m, 4H, C6H4); 13C
NMR (100 MHz, DMSO-d6): 22.31, 23.00, 27.81, 35.84, 80.81,
104.69, 125.64, 125.89, 128.66, 133.65, 134.74, 136.33, 146.81,
164.73; EI-MS: 391.0 (Mþ); Anal. calcd. for C18H21N3O5S: C, 55.23;
H, 5.41; N, 10.73. Found C, 55.10; H, 5.35; N, 10.76.
The authors have declared no conflict of interest.
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