Design and synthesis of potent and selective aldose reductase inhibitors based on pyridylthiadiazine scaffold

Article abstract of DOI:10.1016/j.ejmech.2011.01.072

A series of pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide acetic acid derivatives were synthesized and tested for their inhibitory activity against aldose reductase (ALR2). These derivatives were found to be potent aldose reductase inhibitors with IC

Full text of DOI:10.1016/j.ejmech.2011.01.072

European Journal of Medicinal Chemistry 46 (2011) 1536e1544
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of potent and selective aldose reductase inhibitors based
on pyridylthiadiazine scaffold
Xin Chen a, Yanchun Yang a, Bing Ma a, Shuzhen Zhang a, Minlan He a, Dequan Gui a,
,
b
,
c
Saghir Hussain a, Chaojun Jing a, Changjin Zhu a , Qun Yu
, Yan Liu
*
**
a Department of Applied Chemistry, Beijing Institute of Technology, No. 5, Zhongguancun South Street, 100081 Beijing, China
b Beijing Institute of Transfusion Medicine, 100850 Beijing, China
c College of Chemistry and Chemical Engineering, Xiamen University, 361005 Xiamen, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide acetic acid derivatives were synthesized and
tested for their inhibitory activity against aldose reductase (ALR2). These derivatives were found to be
potent aldose reductase inhibitors with IC50 values ranging from 0.038 mM to 11.29 mM. Most but not all
of them showed a strong ALR2 inhibition activity and significant selectivity, which were further sup-
ported by docking studies. Of these inhibitors, compound 7d exhibited highest inhibition activity.
Structureeactivity relationship studies indicate the requirement of N2-benzyl group with electron-
withdrawing substituents and N4-acetic acid group in the pyridothiadiazine scaffold.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 19 November 2010
Received in revised form
19 January 2011
Accepted 29 January 2011
Available online 1 March 2011
Keywords:
Pyrido[2,3e]-[1,2,4]-thiadiazine
Acetic acid derivative
Inhibitor
Aldose reductase
1. Introduction
In the past years, a number of structurally different ARIs
including at least two chemical classes of structures have been
Most diabetic patients suffer from so-called long-term compli-
cations such as neuropathy, nephropathy, retinopathy and cataracts
[1,2]. These complications arise from chronic hyperglycemia, which
causes damage to blood vessels and peripheral nerves, greatly
increasing the risk of heart attack. Increasing evidence reveals a link
between glucose metabolism via the polyol pathway and long-term
diabetic complications [3e5]. Aldose reductase (EC 1.1.1.21, aALR2),
the first enzyme of the polyol metabolic pathway that catalyzes the
NADPH-dependent reduction of glucose to sorbitol (Fig. 1), has
been found to be implicated in the etiology of the long-term dia-
betic complications [6e8]. Therefore, aldose reductase inhibitors
(ARIs) have been gaining attention over the last decades from the
pharmaceutical community as a promising and an attractive ther-
apeutic strategy.
identified (Fig. 2). The first chemical class is carboxylic acids such
as alrestatin [9], tolrestat [10], epalrestat [11], zopolrestat [12],
zenarestat [13], ponalrestat [14], and recently developed lidorestat
[15] and naphtho[1,2-d]isothiazole derivatives [16]. The second
chemical class is cyclic imides involving sorbinil [17], fidarestat
[18], minalrestat [19] and ranirestat (AS-3201) [20]. Very recently,
new classes of ARIs that are SO2 linker-bearing pyridazinone based
series including most potent 6-(5-chloro-3-methylbenzofuran-2-
sulfonyl)-2H-pyridazin-3-one [21] and flavinoid bioisoster pyr-
idopyrimidinones (PPP) [22] have been reported for their excellent
selectivity and potent inhibitory activity both in vitro and in vivo.
Also, phenolic compounds that may serve as bioisosters of
carboxylic acid ARIs have been shown to be an emerging class
because of their ALR2 inhibition ability and antioxidant activity
[22e24]. However, very few of the ARIs have been approved for
clinical use so far. Many of these candidates failed to gain accep-
tance because of their side effects or poor efficacy. The side effects
are widely believed to be mainly due to a lack of an ARI selectivity,
which is often determined by measuring its activity against
aldehyde reductase (EC 1.1.1.2, ALR1), an enzyme that is closely
related to ALR2 [25]. ALR1 plays a detoxification role as it specif-
ically metabolizes toxic aldehydes such as hydroxynonenal (HNE),
3-deoxyglucosone, and methylglyoxal. These aldehydes arise in
Abbreviations: ALR2, aldose reductase; ALR1, aldehyde reductase; ARI, aldose
reductase inhibitor; AGEs, advanced glycation end products; DMSO, dimethyl
sulfoxide; HNE, hydroxynonenal; NADPH,
b-nicotinamide adenine dinucleotide
phosphate reduced form; SAR, Structure-activity relationship.
* Corresponding author. Tel./fax: þ86 10 68918506.
** Corresponding author. Tel./fax: þ86 10 66932965.
E-mail addresses: zcj@bit.edu.cn (C. Zhu), yuq@nic.bmi.ac.cn (Q. Yu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.072

X. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 1536e1544
1537
4ae4c. Saturation of the double bond in the 2,3-positions of
4ae4c was achieved by means of sodium borohydride to give the
methyl esters 5ae5c. In the synthesis of the end products, intro-
duction of a benzyl group as another side chain at the N2 position
of esters 5ae5c was achieved by the alkylation reaction with the
appropriate benzyl bromide to afford the corresponding methyl
esters 6ae6o, which were then converted to the carboxylic acids
7ae7o by hydrolysis with aqueous sodium hydroxide and then
HCl [30].
Fig. 1. The polyol pathway of glucose metabolism.
large quantities from pathological conditions connected with
oxidative stress, as in hyperglycemia, and are intermediates for the
formation of the advanced glycation end products (AGEs) [26e28].
As a member of the aldo-ketoreductase super family, ALR1 shares
greater than 65% sequence homology with ALR2. Specific amino
acid changes in the C-terminal loop, a section of the protein lining
the active site cleft known as the specificity pocket, which is
primarily responsible for the various substrate and inhibitor
specificities [29]. Thus, ALR1 inhibition may account for some of
the undesirable side effects associated with the present ARIs. This
concern urged us to put a lot of effort into the development of
a larger variety of ARIs that block ALR2 strongly and specifically.
In a preceding study, we have shown a series of potent ARIs
having a benzothiadiazine as core structure, and explored the SAR
of substituents as they relate to aldose reductase inhibition potency
[30]. In the present study, further efforts at new design based on
pyridothiadiazine core resulted in the discovery of a novel series of
3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide acetic
acid derivatives as potent and selective ARIs.
3. Biological evaluation
All newly synthesized 3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-
thiadiazine 1,1-dioxide derivatives were tested for their potential
inhibitory effect on ALR2 isolated from rat lenses. In order to
evaluate their selectivity for ALR2 inhibition, all compounds were
also subjected to test for their inhibition activity against ALR1,
isolated from rat kidneys. IC50 values were determined by linear
regression analysis of the log of the concentrationeresponse curve.
Their effectiveness was evaluated with respect to fidarestat as
a highly potent inhibitor of ALR2. Table 1 lists the results of these
biological evaluations. Full details of the biological methods
employed are included in the Experimental section.
4. Results and discussion
2. Chemistry
As shown in Table 1, most of [2-benzyl-3,4-dihydro-2H-pyrido
[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid derivatives
(7ae7o) showed significantly inhibitory activity against ALR2. Of
these acid derivatives, [2-(2,4,5-trifluorobenzyl)-7-cholro-3,4-dihy-
dro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid
The synthetic pathways used to prepare the acetic acid deriva-
tives of 3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-diox-
ides are illustrated in Scheme 1.
7-Halogen or 7-methyl group substituted 3,4-dihydro-2H-pyr-
ido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxides were prepared starting
from the corresponding 5-substituted 2-amino-pyridine 1ae1c,
which in two steps, were initially converted into compounds
2ae2c [31]. 2ae2c were used in a ring closure reaction with
triethyl orthoformate to obtain corresponding 3ae3c. To introduce
an alkyl carboxylic acid residue as a side chain at the N4 position,
compounds 3ae3c were alkylated with the methyl bromoacetate
in the presence of potassium carbonate, yielding compounds
7d was the most active compound having an IC50 value of 0.038 mM.
9 compounds 7a, 7b, 7f, 7g, 7h, 7i, 7k, 7l, and 7n also demonstrated
potent ALR2 inhibition activity with IC50 values ranging from
0.044
7j, 7m, and 7o showed varying levels of efficiency with IC50 values in
the low micromolar range of 1.01 M, 6.54 M, 11.29 M, 1.00 M,
and 6.27 M, respectively.
mM to 0.965 mM. The remaining 5 compounds including 7c, 7e,
m
m
m
m
m
We will now discuss SAR relative to substituents at the N2-
benzyl ring in the N4-acetic acid substituted derivatives 7ae7o.
Fig. 2. Chemical structures of aldose reductase inhibitors.

1538
X. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 1536e1544
Scheme 1. Synthesis of pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide acetic acid derivatives (7aeo). Reagents and conditions: (a) ClSO3H, reflux; (b) NH3, THF; (c) HC(OCH2CH3)3,
reflux; (d) BrCH2COOCH3, K2CO3, CH3CN, 70 ꢀC; (e) NaBH4, 1,4-Dioxane; (f) Bn-Br, K2CO3, CH3CN, 70 ꢀC; (h) 1,4-Dioxane, aqueous NaOH, then aqueous HCl.
First, substituents at the N2-benzyl ring were chosen to probe the
influence of groups of electron-withdrawing, electron-releasing,
and steric bulk. In general, compact, electron-withdrawing F and Br
substituents that are reasonably lipophilic gave potent compounds,
while electron-releasing substituent (e.g., OMe) and bulky group
(e.g., CF3) had unfavorable effect on potency. Further, according to
7-substituent, compounds 7ae7o could be arranged in series
7ae7e with the 7-chloro group, 7fe7j with the 7-bromo, and
7ke7o with the 7-methyl. Comparing the compounds in series
7ae7e bearing a N2-benzyl group with different substituents at the
aromatic ring demonstrated that compounds with substitutions of
2-F,4-Br (7a), 3-NO2 (7b), 4-CF3 (7c), 2,4,5-F3 (7d), and 4-OCH3 (7e)
at the benzyl ring all retained potent activities (IC50 ¼ 0.044, 0.093,
Similarly, comparing compounds in each of the remaining series
7fe7j, and 7ke7o, revealed that compounds 7i and 7n all having
2,4,5-F3 groups at the benzyl ring were the most active within each
series (IC50 ¼ 0.066, 0.084
mM), respectively. Moreover, it was found
that effects of substitution groups at the benzyl ring on ALR2
inhibition activity were in the rank order of 2,4,5-F3 > 2-F,4-Br > 3-
NO2 > 4-CF3 > 4-OCH3 as deduced from the comparison of
compounds in 7-chloro derivative series (7d > 7a > 7b > 7c > 7e),
7-bromo derivative series (7i > 7f > 7g > 7h > 7j), and 7-fluoro
substituted derivative series (7n > 7k > 7l > 7m > 7o). These
results suggest that apart from the acetic acid residue at the N4
position, the N2-benzyl residue with compact, electron-with-
drawing substituents at the benzyl ring is essential for the activity
of ARIs based on pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide
scaffold.
1.01, 0.038, 6.54 mM). 7d that bears 2,4,5-F3 substituents at the N2-
benzyl ring was found to be the most active within this series.
Further SAR studies focused on substituents at the 7-position in
the derivatives 7ae7o. Again, substituents at the 7-position were
chosen to probe the influence of electron-withdrawing and elec-
tron-releasing groups. As shown in Table 1, 7-Cl substituted
compounds (7ae7e) and 7-Br substituted compounds (7fe7j),
especially 7ae7e, showed the most attractive activity. Among these,
Table 1
In vitro data for 3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide acetic
acid derivatives.
No.
Substituents
ALR2 IC50
(m
M)a
ALR1 (% of inhibition)b
7d was the best (IC50 ¼ 0.038
mM). Electron-releasing substituents
R1
R2
(7-CH3 in 7ke7o) had less effect on inhibition potency, and IC50
values for this group of compounds were in the range of
0.084e6.27 mM. It was evidenced by comparing biological activities
of compounds in the 2-F,4-Br-benzyl derivative series including 7a
with 7-Cl, 7f with 7-Br and 7k with 7-methyl, in which 7a together
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
2-F,4-Br
3-NO2
4-CF3
2,4,5-F3
4-OCH3
2-F,4-Br
3-NO2
0.044 (0.033e0.055)
0.093 (0.077e0.108)
1.01 (0.78e1.25)
0.038 (0.028e0.048)
6.54 (3.90e9.17)
0.069 (0.056e0.081)
0.098 (0.076e0.120)
0.965 (0.725e1.20)
0.066 (0.055e0.078)
11.29 (8.70e13.87)
0.127 (0.111e0.143)
0.143 (0.106e0.179)
1.00 (0.669e1.33)
0.084 (0.563e0.111)
6.27 (4.90e7.63)
36.6%
40.2%
42.3%
36.5%
32.3%
44.9%
31.5%
43.3%
44.2%
27.9%
24.6%
23.2%
20.5%
35.4%
17.7%
c
7f proved more potent activities than 7k (IC50 ¼ 0.044, 0.069
mM vs
IC50 ¼ 0.127
m
M). Identically, comparing the activities of compounds
4-CF3
in the 3-NO2-benzyl derivative series including 7b with 7-Cl, 7g
with 7-Br, and 7l with 7-CH3, revealed that 7b and 7g were more
Br
Br
2,4,5-F3
4-OCH3
2-F,4-Br
3-NO2
4-CF3
2,4,5-F3
4-OCH3
active than 7l (IC50 ¼ 0.094 and 0.108
m
M vs IC50 ¼ 0.219
mM).
CH3
CH3
CH3
CH3
CH3
Similar results were obtained when we compared the activities of
compounds in the 2,4,5-F3-benzyl derivative series including 7d, 7i
and 7n, (IC50 ¼ 0.038, 0.066
mM, and 0.084 mM). However, when we
investigated the activities of compounds in the 4-CF3-benzyl
derivative series including 7c, 7h and 7m, it was found that, unlike
in other series, variation of the substituents at the 7-position did not
Fidarestat
0.029 (0.020e0.037)
a
IC50 (95% CL) values represent the concentration required to produce 50%
enzyme inhibition.
b
have an impact on the activity (IC50 ¼ 1.01, 0.965 and 1.00
mM). The
The inhibitory effect was estimated at a concentration of 50
Not determined.
mM.
c
changes in the inhibition activity of the 3 compounds 7e, 7j, and 7o

X. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 1536e1544
1539
Fig. 3. Compound 7d bound into the active site of ALR2. (a) Protein structure is shown as cartoon diagrams with the side chains of the key receptor residues in proximity of the
docked ligands are labeled. Ligand 7d, and NADP are shown as stick models. Docked pose of 7d is shown in cyan (C), red (O), blue (N), yellow (S), green (Cl), and grey (F). Nonpolar
hydrogens have been removed for clarity. Hydrogen bonds are represented by yellow lines. (b) Protein residues are in surface representation. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
in the 4-OCH3-benzyl derivative series could be ignored because
they all showed relatively weak potency as shown in Table 1.
All 3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-diox-
ides acetic acid derivatives 7ae7o investigated were selective for
values in the range from 0.038 mM to 11.29 mM. A strong ALR2
inhibitory effect was produced by most but not all of the derivatives.
The lead candidate, [2-(2,4,5-trifluorobenzyl)-7-cholro-3,4-dihydro-
2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl]acetic acid, 7d,
ALR2, since none of them inhibited appreciably ALR1 (IC50 > 50
m
M,
inhibits ALR2 with an IC50 of 0.038 mM. These derivatives may serve
Table 1).
as useful lead compounds in search for more powerful and selective
ARIs. SAR studies demonstrated that the N2-benzyl group with
electron-withdrawing substituents and N4-acetic acid group were
essential for the design of ARIs based on the pyridothiadiazine scaf-
fold, and that halogen substituents at the 7-position, although not
essential, could further elevate the ALR2 inhibition activity of this
class of ARIs. A significant difference between the binding modes of
inhibitor 7d docked with ALR2 and ALR1 was found, and gives an
insight into the mechanism of ALR2 inhibition activity and selectivity
of the inhibitors.
5. Molecular modeling
To get a better comprehension of the high ALR2 inhibitory
potency and selectivity of the newly synthesized compounds at
a molecular level and to propose a binding mode that explains the
above-described SARs, docking experiments were performed.
Compound 7d, the most active as described above, was docked into
the binding pocket of the human ALR2/NADPþ/lidorestat complex
(PDB entry code 1Z3N) as shown in Fig. 3. Also, the docking
behavior of 7d with ALR1/NADPþ/fidarestat complex (PDB entry
code 3H4G) was investigated.
Docking results reveal that the inhibitor 7d is tightly bound in
the active site of ALR2. The carboxylate group entered into the
anion binding site, hydrogen-bonding with Tyr48, His110, and
Trp111, which are three key residues in binding and catalysis
[32,33], and engaging a stabilizing electrostatic interaction with the
positively charged nicotinamide moiety of the cofactor NADPþ
7. Experimental section
7.1. Chemistry
Melting points were recorded on an X-4 microscopic melting
point apparatus and were uncorrected. All reactions were routinely
checked by TLC on silica gel Merck 60F254. NMR spectra were
recorded on a Bruker Advance 400 spectrometer (Bruker (Beijing)
ꢀ
(NeO ¼ 4.10 A). The pyridyl ring of pyrido[2,3-e]-[1,2,4]-thiadiazine
1,1-dioxide core gets tightly trapped in the hydrophobic cage of the
specificity pocket formed by Trp20, Leu300, Trp111, and Trp219. In
addition, these interactions together with the presence of the
pyrido[2,3-e]-[1,2,4]-thiadiazine ring will orient the substituted
benzyl ring in a hydrophobic pocket mainly formed by the side
chains of Trp20 and Pro218. However, docking of 7d with ALR1
showed a reversed pattern compared with that of the compound
with ALR2, in which the hydrophobic benzyl group but not the
carboxylate group inserted into the anion binding site. This docking
difference of 7d between ALR2 and ALR1 may account for the ALR2
selectivity of pyridylthiadiazine derivatives observed in the bio-
logical assay.
Technologies & Services Co., Ltd), chemical shifts are given in d:
units (ppm) relative to internal standard TMS and refer to CDCl3 or
DMSO-d6 solutions. Elemental analyses (C, H, N) were performed at
the analytical instrumentation center of Peking University (China)
and realized on an Elementar Vario MICRO CUBE elemental
analyzer.
The following HPLC methods were used to determine purity of
final compounds and fidarestat using Shimadze LC-20AT liquid
chromatograph (Shimadze Corporation, Japan). All final
compounds and fidarestat tested in biological assays were ꢁ95%
pure. Method: column, C18, 5
m
, 250 mm ꢂ 4.6 mm. Mobile phase:
H2O (0.1% TFA)/MeOH ¼ 1/9, for 8 min; room temperature; flow
rate of 1 mL/min; detection at 254 nm.
6. Conclusions
The 2-amino-5-chloropyridine (1a), 2-amino-5-bromopyridine
(1b), 2-amino-5-methyl pyridine (1c), methyl bromoacetate,
4-bromo-2-fluorobenzyl bromide, 3-nitrobenzyl bromide, 4-(tri-
fluoromethyl)benzyl bromide, 2,4,5-trifluorobenzyl bromide and
4-methoxybenzyl bromide, used to obtain the target inhibitors,
were from Alfa Aesar.
Efforts to develop novel aldose reductase inhibitors for treatment
of chronic diabetic complications resulted in the discovery of a series
of
3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine
1,1-dioxide
derivatives as novel, highly potent and selective ARIs having IC50

1540
X. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 1536e1544
7.2. General procedure for the synthesis of 2-aminopyridine-3-
sulfonamides (2ae2c)
7.4.1. (7-Cholro-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-
4-yl) acetic acid methyl ester (4a)
Colorless crystal (8.9 g, 77%); m.p. 142e144 ꢀC; 1H NMR
Chlorosulfonic acid (50 mL) was cooled on a dry ice-methanol
bath and then carefully supplemented with the appropriate 2-
aminopyridine (1ae1c, 0.1 mol). The mixture was heated under
reflux for 3 h. After cooling, the reaction mixture was poured on ice
under stirring, and the resulting white precipitate was collected by
filtration and washed with cold water. The solid was suspended
under stirring in 100 mL THF and treaded with NH3 (gas) for 30 min.
Then the mixture was stirred at room temperature for 18 h. After
evaporation of the solvent under reduced pressure, the crude solid
was washed with water, collected by filtration and dried in vacuo.
(400 MHz, CDCl3) d: 3.80 (s, 3H), 4.82 (s, 2H), 7.74 (s, 1H), 8.28 (d,
J ¼ 2.4 Hz, 1H), 8.53 (d, J ¼ 2.4 Hz, 1H).
7.4.2. (7-Bromo-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-
4-yl) acetic acid methyl ester (4b)
Colorless crystal (9.6 g, 85%); m.p. 152e154 ꢀC; 1H NMR
(400 MHz, CDCl3) d: 3.80 (s, 3H), 4.82 (s, 2H), 7.76 (s, 1H), 8.41 (d,
J ¼ 2.4 Hz, 1H), 8.63 (d, J ¼ 2.0 Hz, 1H).
7.4.3. (7-Methyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-
4-yl) acetic acid methyl ester (4c)
7.2.1. 2-Amino-5-chloropyridine-3-sulfonamide (2a)
Colorless crystal (8.6 g, 80%); m.p. 160e163 ꢀC; 1H NMR
Pale yellow powder (11.4 g, 55%); m.p. 213e216 ꢀC; 1H NMR
(400 MHz, CDCl3) d: 2.44 (s, 3H), 3.78 (s, 3H), 4.81 (s, 2H), 7.73 (s,
1H), 8.12 (m, 1H), 8.41 (d, J ¼ 1.6 Hz, 1H).
(400 MHz, DMSO-d6)
1H), 8.18 (d, J ¼ 2.4 Hz, 1H).
d
: 6.67 (s, 2H), 7.57 (s, 2H), 7.82 (d, J ¼ 2.8 Hz,
7.2.2. 2-Amino-5-bromopyridine-3-sulfonamide (2b)
7.5. General procedure for the synthesis of (3,4-dihydro-2H-pyrido
[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl) acetic acid methyl esters
(5ae5c)
Pale yellow powder (11.8 g, 47%); m.p. 184e187 ꢀC; 1H NMR
(400 MHz, DMSO-d6)
d: 6.68 (s, 2H), 7.56 (s, 2H), 7.92 (d, J ¼ 2.4 Hz,
1H), 8.23 (d, J ¼ 2.8 Hz, 1H).
To a solution of the appropriate (4H-pyrido[2,3-e]-[1,2,4]-thia-
diazine 1,1-dioxide-4-yl) acetic acid methyl ester (4ae4c, 25 mmol)
in 1,4-dioxane (10 mL) with stirring was added NaBH4 (4 g). The
mixture was stirred at room temperature for 5 min. Then the
reaction mixture was poured on ice under stirring, and treated with
0.1 N HCl until the pH value was adjusted to acidic. The resulting
insoluble material was collected by filtration, washed with water,
dried and then recrystallized in ethyl acetate to give pure desired
product as a colorless crystal.
7.2.3. 2-Amino-5-methylpyridine-3-sulfonamide (2c)
Brown powder (9.5 g, 51%); m.p. 177e180 ꢀC; 1H NMR
(400 MHz, DMSO-d6) d: 3.29 (s, 3H), 6.24 (s, 2H), 7.37 (s, 2H), 7.68
(d, J ¼ 2.0 Hz, 1H), 7.98 (d, J ¼ 2.0 Hz, 1H).
7.3. General procedure for the synthesis of 4H-pyrido[2,3-e]-[1,2,4]-
thiadiazine 1,1-dioxides (3ae3c)
The appropriate 2-aminopyridine-3-sulfonamide (2ae2c,
45 mmol) was heated in triethyl orthoformate (50 mL) to reflux
during 2 h. After cooling to room temperature, the desired
compound was collected by filtration, washed with diethyl ether
and dried in vacuo.
7.5.1. (7-Cholro-3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine
1,1-dioxide-4-yl) acetic acid methyl ester (5a)
Colorless crystal (5.0 g, 69%); m.p. 132e135 ꢀC; 1H NMR
(400 MHz, CDCl3)
5.37 (m, 1H), 7.86 (d, J ¼ 2.4 Hz, 1H), 8.20 (d, J ¼ 2.8 Hz, 1H).
d
: 3.73 (s, 3H), 4.41 (s, 2H), 4.85 (d, J ¼ 8.4 Hz, 2H),
7.3.1. 7-Chloro-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide (3a)
Pale yellow powder (8.9 g, 91%); m.p. >300 ꢀC; 1H NMR
7.5.2. (7-Bromo-3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine
1,1-dioxide-4-yl) acetic acid methyl ester (5b)
(400 MHz, DMSO-d6)
d
: 8.06 (s, 1H), 8.56 (d, J ¼ 2.4 Hz, 1H), 8.76 (d,
J ¼ 2.4 Hz, 1H), 12.98 (s, 1H).
Colorless crystal (6.6 g, 78%); m.p. 142e144 ꢀC; 1H NMR
(400 MHz, CDCl3)
2H), 5.18 (m, 1H), 8.05 (d, J ¼ 2.4 Hz, 1H), 8.32 (d, J ¼ 2.4 Hz, 1H).
d
: 3.76 (d, 3H), 4.44 (s, 2H), 4.88 (d, J ¼ 8.4 Hz,
7.3.2. 7-Bromo-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide (3b)
Pale yellow powder (9.3 g, 88%); m.p. >300 ꢀC; 1H NMR
(400 MHz, DMSO-d6)
(d, J ¼ 2.8 Hz, 1H), 13.0 (s, 1H).
d
: 8.07 (s, 1H), 8.668 (d, J ¼ 2.4 Hz, 1H), 8.87
7.5.3. (7-Methyl-3,4-dihydro-2H-pyrido[2,3-e]-[1,2,4]-thiadiazine
1,1-dioxide-4-yl)acetic acid methyl ester (5c)
Colorless crystal (5.3 g, 78%); m.p. 136e139 ꢀC; 1H NMR
7.3.3. 7-Methyl-4H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide
(3c)
(400 MHz, CDCl3) d: 2.26 (s, 3H), 3.74 (s, 3H), 4.45 (s, 2H), 4.85 (d,
Pale yellow powder (8.3 g, 94%); m.p. >300 ꢀC; 1H NMR
J ¼ 8.4 Hz, 2H), 5.09 (m, 1H), 7.79 (d, J ¼ 2.4 Hz, 1H), 8.13 (d,
J ¼ 2.0 Hz, 1H).
(400 MHz, DMSO-d6) d: 2.38 (s, 3H), 7.99 (s, 1H), 8.17 (m, 1H), 8.54
(d, J ¼ 2.0 Hz, 1H), 12.75 (s, 1H).
7.4. General procedure for the synthesis of (4H-pyrido[2,3-e]-
[1,2,4]-thiadiazine 1,1-dioxide-4-yl) acetic acid methyl esters
(4ae4c)
7.6. General procedure for the synthesis of [2-benzyl-3,4-dihydro-
2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid
methyl esters (6ae6o)
A mixture of the appropriate 4H-pyrido[2,3-e]-[1,2,4]-thiadia-
zine 1,1-dioxide (3ae3c, 40 mmol), potassium carbonate (6 g), and
methyl bromoacetate (6.7 g; 44 mmol) in acetonitrile (120 mL) was
heated at 70 ꢀC for 2 h. After evaporation of the solvent under
reduced pressure, the crude solid was washed with water, dried,
and then recrystallized in ethyl acetate to give pure product as
a colorless crystal.
A mixture of the appropriate (3,4-dihydro-2H-pyrido[2,3-e]-
[1,2,4]-thiadiazine 1,1-dioxide-4-yl) acetic acid methyl ester
(5ae5c, 2 mmol), potassium carbonate (1 g), and the appropriate
benzyl bromide (2.1 mmol) in acetonitrile (30 mL) was heated at
70 ꢀC for 2 h. After evaporation of the solvent under reduced
pressure, the crude solid was washed with water, dried, and then
recrystallized in ethyl acetate to give pure product.

X. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 1536e1544
1541
7.6.1. [2-(4-Bromo-2-fluorobenzyl)-7-cholro-3,4-dihydro-2H-
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl
ester (6a)
2H), 6.97 (m, 1H), 7.35 (m, 1H), 8.08 (d, J ¼ 2.4 Hz, 1H), 8.35 (d,
J ¼ 2.4 Hz, 1H).
Colorless crystal (0.81 g, 84%); m.p. 128e131 ꢀC; 1H NMR
7.6.10. [2-(4-methoxybenzyl)-7-bromo-3,4-dihydro-2H-pyrido
[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl ester
(6j)
(400 MHz, CDCl3) d: 3.75 (s, 3H), 4.29 (s, 2H), 4.37 (s, 2H), 4.88 (s,
2H), 7.29 (m, 1H), 7.32 (m, 1H), 7.36 (m, 1H), 7.96 (d, J ¼ 2.4 Hz, 1H),
8.26 (d, J ¼ 2.4 Hz, 1H).
Colorless crystal (0.68 g, 75%); m.p. 137e139 ꢀC; 1H NMR
(400 MHz, CDCl3) d: 3.74 (s, 3H), 3.82 (s, 3H), 4.18 (s, 2H), 4.23 (s,
7.6.2. [2-(3-Nitrobenzyl)-7-cholro-3,4-dihydro-2H-pyrido[2,3-e]-
[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl ester (6b)
Colorless crystal (0.45 g; 2.1 mmol) (0.62 g, 73%); m.p.
2H), 4.74 (s, 2H), 6.90 (m, 2H), 7.28 (m, 2H), 8.08 (d, J ¼ 2.4 Hz, 1H),
8.34 (d, J ¼ 2.4 Hz, 1H).
139e142 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 3.79 (s, 3H), 4.27 (s, 2H),
7.6.11. [2-(4-Bromo-2-fluorobenzyl)-7-methyl-3,4-dihydro-2H-
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl
ester (6k)
4.49 (s, 2H), 4.82 (s, 2H), 7.58 (m,1H), 7.80 (d, J ¼ 3.6 Hz,1H), 7.98 (d,
J ¼ 1.6 Hz, 1H), 8.20 (d, J ¼ 8.4 Hz, 1H), 8.29 (s, 1H). 8.30 (s, 1H).
Colorless crystal (0.91 g, 77%); m.p. 146e148 ꢀC; 1H NMR
7.6.3. [2-(4-(Trifluoromethyl)benzyl)-7-cholro-3,4-dihydro-2H-
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl
ester (6c)
(400 MHz, CDCl3) d: 2.28 (s, 3H), 3.73 (s, 3H), 4.29 (s, 2H), 4.37 (s,
2H), 4.86 (s, 2H), 7.29 (m, 1H), 7.31 (m, 1H), 7.38 (m, 1H), 7.81 (d,
J ¼ 1.6 Hz, 1H), 8.15 (d, J ¼ 1.6 Hz, 1H).
Colorless crystal (0.72 g, 80%); m.p. 165e167 ꢀC; 1H NMR
(400 MHz, CDCl3) d: 3.76 (s, 3H), 4.23 (s, 2H), 4.42 (s, 2H), 4.80 (s,
7.6.12. [2-(3-Nitrobenzyl)-7-methyl-3,4-dihydro-2H-pyrido[2,3-e]-
[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl ester (6l)
Colorless crystal (0.75 g, 82%); m.p. 172e174 ꢀC; 1H NMR
2H), 7.55 (d, J ¼ 8.0 Hz, 2H), 7.64 (d, J ¼ 8.0 Hz, 2H), 7.98 (d,
J ¼ 2.4 Hz, 1H), 8.27 (d, J ¼ 2.4 Hz, 1H).
(400 MHz, CDCl3) d: 2.29 (s, 3H), 3.78 (s, 3H), 4.27 (s, 2H), 4.49 (s,
2H), 4.81 (s, 2H), 7.57 (m, 1H), 7.80 (s, 1H), 7.83 (s, 1H), 8.18 (d,
J ¼ 1.6 Hz, 1H). 8.20 (s, 1H), 8.29 (s, 1H).
7.6.4. [2-(2,4,5-Trifluorobenzyl)-7-cholro-3,4-dihydro-2H-pyrido
[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl ester
(6d)
Colorless crystal (0.62 g, 71%); m.p. 131e134 ꢀC; 1H NMR
7.6.13. [2-(4-(Trifluoromethyl)benzyl)-7-methyl-3,4-dihydro-2H-
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl
ester (6m)
(400 MHz, CDCl3) d: 3.76 (s, 3H), 4.31 (s, 2H), 4.37 (s, 2H), 4.90 (s,
2H), 6.95 (m, 1H), 7.35 (m, 1H), 7.96 (d, J ¼ 2.4 Hz, 1H), 8.27 (d,
J ¼ 2.4 Hz, 1H).
Colorless crystal (0.73 g, 85%); m.p. 103e106 ꢀC; 1H NMR
(400 MHz, CDCl3) d: 2.28 (s, 3H), 3.74 (s, 3H), 4.22 (s, 2H), 4.42 (s,
7.6.5. [2-(4-Methoxybenzyl)-7-cholro-3,4-dihydro-2H-pyrido[2,3-
e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl ester (6e)
Colorless crystal (0.65 g, 79%); m.p. 128e130 ꢀC; 1H NMR
2H), 4.78 (s, 2H), 7.55 (d, J ¼ 8.4 Hz, 2H), 7.63 (d, J ¼ 8.4 Hz, 2H), 7.83
(m, 1H), 8.17 (d, J ¼ 1.6 Hz, 1H).
(400 MHz, CDCl3) d: 3.74 (s, 3H), 3.82 (s, 3H), 4.18 (s, 2H), 4.23 (s,
2H), 4.75 (s, 2H), 6.90 (d, J ¼ 8.8 Hz, 2H), 7.30 (d, J ¼ 8.8 Hz, 2H), 7.97
7.6.14. [2-(2,4,5-Trifluorobenzyl)-7-methyl-3,4-dihydro-2H-pyrido
[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl ester
(6n)
(d, J ¼ 2.4 Hz, 1H), 8.26 (d, J ¼ 2.4 Hz, 1H).
Colorless crystal (0.61 g, 74%); m.p. 129e132 ꢀC; 1H NMR
7.6.6. [2-(4-Bromo-2-fluorobenzyl)-7-bromo-3,4-dihydro-2H-
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl
ester (6f)
(400 MHz, CDCl3) d: 2.28 (s, 3H), 3.75 (s, 3H), 4.31 (s, 2H), 4.37 (s,
2H), 4.89 (s, 2H), 6.96 (m, 1H), 7.35 (m, 1H), 7.81 (d, J ¼ 2.4 Hz, 1H),
Colorless crystal (0.91 g, 87%); m.p. 114e117 ꢀC; 1H NMR
8.16 (d, J ¼ 2.4 Hz, 1H).
(400 MHz, CDCl3) d: 3.75 (s, 3H), 4.28 (s, 2H), 4.37 (s, 2H), 4.88 (s,
2H), 7.26 (m, 1H), 7.30 (m, 1H), 7.34 (m, 1H), 8.07 (d, J ¼ 2.0 Hz, 1H),
7.6.15. [2-(4-Methoxybenzyl)-7-methyl-3,4-dihydro-2H-pyrido[2,3-
e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl ester (6o)
Colorless crystal (0.51 g, 65%); m.p. 143e145 ꢀC; 1H NMR
8.34 (d, J ¼ 2.4 Hz, 1H).
7.6.7. [2-(3-Nitrobenzyl)-7-bromo-3,4-dihydro-2H-pyrido[2,3-e]-
[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl ester (6g)
Colorless crystal (0.72 g, 76%); m.p. 176e178 ꢀC; 1H NMR
(400 MHz, CDCl3) d: 2.27 (s, 3H), 3.73 (s, 3H), 3.82 (s, 3H), 4.19 (s,
2H), 4.24 (s, 2H), 4.73 (s, 2H), 6.90 (m, 2H), 7.30 (d, J ¼ 8.8 Hz, 2H),
7.82 (d, J ¼ 2.0 Hz, 1H), 8.15 (d, J ¼ 1.6 Hz, 1H).
(400 MHz, CDCl3) d: 3.79 (s, 3H), 4.27 (s, 2H), 4.49 (s, 2H), 4.82 (s,
2H), 7.58 (m, 1H), 7.80 (d, J ¼ 3.6 Hz, 1H), 8.10 (d, J ¼ 2.4 Hz, 1H), 8.10
(m, 1H). 8.29 (s, 1H), 8.37 (d, J ¼ 2.0 Hz, 1H).
7.7. General procedure for the synthesis of [2-benzyl-3,4-dihydro-
2H-pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acids
(7ae7o)
7.6.8. [2-(4-(Trifluoromethyl)benzyl)-7-bromo-3,4-dihydro-2H-
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl
ester (6h)
A mixture of the appropriate (2-benzyl-3,4-dihydro-2H-pyrido
[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl) acetic acid methyl ester
(6ae6o, 1 mmol), 1,4-dioxane (8 mL) and saturated aqueous
sodium hydroxide (10 mL) was stirred at room temperature for 3 h.
The alkaline suspension was adjusted to acidic with 0.1 N HCl and
extracted 3-fold with ethyl acetate (3 ꢂ 30 mL). The combined
organic layers were dried over MgSO4 and filtered. The filtrate was
concentrated to dryness under reduced pressure and the residue of
the desired compound was recrystallized in methanol to give pure
product.
Colorless crystal (0.77 g, 78%); m.p. 88e90 ꢀC; 1H NMR
(400 MHz, CDCl3) d: 3.76 (s, 3H), 4.22 (s, 2H), 4.42 (s, 2H), 4.80 (s,
2H), 7.55 (d, J ¼ 8.0 Hz, 2H), 7.64 (d, J ¼ 8.0 Hz, 2H), 8.10 (d,
J ¼ 2.4 Hz, 1H), 8.36 (d, J ¼ 2.0 Hz, 1H).
7.6.9. [2-(2,4,5-Trifluorobenzyl)-7-bromo-3,4-dihydro-2H-pyrido
[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid methyl ester (6i)
Colorless crystal (0.63 g, 66%); m.p. 126e129 ꢀC; 1H NMR
(400 MHz, CDCl3) d: 3.75 (s, 3H), 4.31 (s, 2H), 4.37 (s, 2H), 4.90 (s,

1542
X. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 1536e1544
7.7.1. [2-(4-Bromo-2-fluorobenzyl)-7-cholro-3,4-dihydro-2H-
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7a)
Colorless crystal (0.30 g, 64%); m.p. 190e192 ꢀC; 1H NMR
136.89, 135.83, 130.76, 123.76, 123.47, 117.98, 107.48, 64.53, 50.43,
48.61; Anal. (C15H13BrN4O6S) C, H, N.
(400 MHz, DMSO-d6)
d
: 4.30 (s, 2H), 4.31 (s, 2H), 4.98 (s, 2H), 7.40
7.7.8. [2-(4-(Trifluoromethyl)benzyl)-7-bromo-3,4-dihydro-2H-
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7h)
Colorless crystal (0.28 g, 58%); m.p. 177e179 ꢀC; 1H NMR
(m, 1H),7.45 (m, 1H), 7.56 (m, 1H), 8.21 (d, J ¼ 2.4 Hz, 1H), 8.44 (d,
J ¼ 2.4 Hz, 1H), 12.85 (s, 1H); 13C NMR (400 MHz, DMSO-d6)
d:
171.71, 152.06, 150.05, 148.44, 139.06, 135.85, 134.43, 130.74, 123.73,
123.44, 119.89, 117.31, 64.59, 50.44, 48.94; Anal. (C15H12BrClFN3O4S)
C, H, N.
(400 MHz, DMSO-d6) d: 4.29 (s, 2H), 4.40 (s, 2H), 5.00 (s, 2H), 7.57
(d, J ¼ 8.0 Hz, 2H), 7.73 (d, J ¼ 8.4 Hz, 2H), 8.27 (d, J ¼ 2.4 Hz, 1H),
8.50 (d, J ¼ 2.0 Hz, 1H), 12.85 (s, 1H); 13C NMR (400 MHz, DMSO-d6)
d: 171.48, 154.14, 150.19, 141.49, 136.86, 129.71 (2C), 128.87, 126.11,
7.7.2. [2-(3-Nitrobenzyl)-7-cholro-3,4-dihydro-2H-pyrido[2,3-e]-
[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7b)
126.07, 123.57, 117.96, 107.44, 64.44, 50.52, 48.60; Anal.
(C16H13BrF3N3O4S) C, H, N.
Colorless crystal (0.29 g, 70%); m.p. 171e172 ꢀC; 1H NMR
(400 MHz, DMSO-d6)
d
: 4.23 (s, 2H), 4.47 (s, 2H), 5.03 (s, 2H), 7.66
7.7.9. [2-(2,4,5-Trifluorobenzyl)-7-bromo-3,4-dihydro-2H-pyrido
[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7i)
Colorless crystal (0.26 g, 55%); m.p. 159e162 ꢀC; 1H NMR
(m, 1H), 7.82 (d, J ¼ 7.6 Hz,1H), 8.15 (m, 1H), 8.19 (s, 1H), 8.20 (s, 1H),
8.41 (d, J ¼ 2.4 Hz, 1H), 12.85 (s, 1H); 13C NMR (400 MHz, DMSO-d6)
d: 171.55, 152.09, 149.99, 134.52, 133.32, 128.41, 123.22, 123.07,
(400 MHz, DMSO-d6)
(m, 1H), 7.59 (m, 1H), 8.27 (d, J ¼ 2.4 Hz, 1H), 8.50 (d, J ¼ 2.0 Hz, 1H),
12.88 (s, 1H); 13C NMR (400 MHz, DMSO-d6)
: 171.62, 154.20,
d: 4.30 (s, 2H), 4.31 (s, 2H), 4.99 (s, 2H), 7.53
120.16, 119.74, 119.50, 117.36, 64.29, 48.75, 44.96; Anal.
(C15H13ClN4O6S) C, H, N.
d
150.15, 136.94, 120.54, 119.97, 119.77, 117.84, 107.53, 107.23, 106.95,
106.73, 64.15, 48.63, 44.73; Anal. (C15H11BrF3N3O4S) C, H, N.
7.7.3. [2-(4-(Trifluoromethyl)benzyl)-7-cholro-3,4-dihydro-2H-
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7c)
Colorless crystal (0.27 g, 61%); m.p. 169e171 ꢀC; 1H NMR
7.7.10. [2-(4-Methoxybenzyl)-7-bromo-3,4-dihydro-2H-pyrido
[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7j)
Colorless crystal (0.28 g, 63%); m.p. 144e146 ꢀC; 1H NMR
(400 MHz, DMSO-d6) d: 4.29 (s, 2H), 4.40 (s, 2H), 5.00 (s, 2H), 7.57
(d, J ¼ 8.0 Hz, 2H), 7.74 (d, J ¼ 8.4 Hz, 2H), 8.22 (d, J ¼ 2.4 Hz, 1H),
8.44 (d, J ¼ 2.8 Hz, 1H), 12.84 (s, 1H); 13C NMR (400 MHz, DMSO-d6)
(400 MHz, DMSO-d6) d: 3.74 (s, 3H), 4.18 (s, 2H), 4.24 (s, 2H), 4.88 (s,
d: 171.54, 152.08, 150.00, 141.49, 134.49, 129.71 (2C), 128.85, 126.12,
2H), 6.91 (d, J ¼ 8.4 Hz, 2H), 7.26 (d, J ¼ 8.4 Hz, 2H), 8.24 (d,
126.08, 123.58, 120.08, 117.38, 64.43, 50.50, 48.66; Anal.
(C16H13ClF3N3O4S) C, H, N.
J ¼ 2.4 Hz, 1H), 8.47 (d, J ¼ 2.4 Hz, 1H), 12.87 (s, 1H); 13C NMR
(400 MHz, DMSO-d6)
d: 171.45, 159.70, 154.00, 150.25, 136.67,
130.84 (2C), 127.68, 118.04, 114.65 (2C), 107.29, 63.56, 55.81, 50.34,
48.73; Anal. (C16H16BrN3O5S) C, H, N.
7.7.4. [2-(2,4,5-Trifluorobenzyl)-7-cholro-3,4-dihydro-2H-pyrido
[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7d)
Colorless crystal (0.23 g, 54%); m.p. 157e159 ꢀC; 1H NMR
7.7.11. [2-(4-Bromo-2-fluorobenzyl)-7-methyl-3,4-dihydro-2H-
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7k)
Colorless crystal (0.28 g, 63%); m.p. 112e115 ꢀC; 1H NMR
(400 MHz, DMSO-d6)
(m, 1H), 8.22 (d, J ¼ 2.4 Hz, 1H), 8.44 (d, J ¼ 2.4 Hz, 1H), 12.86 (s, 1H);
13C NMR (400 MHz, DMSO-d6)
: 171.75, 152.12, 150.02, 134.47,
d: 4.30 (s, 4H), 4.99 (s, 2H), 7.53 (m, 1H), 7.59
d
(400 MHz, DMSO-d6)
(m, 1H),7.45 (m, 1H), 7.55 (m, 1H), 7.90 (m, 1H), 8.23 (m, 1H), 12.73
(s, 1H); 13C NMR (400 MHz, DMSO-d6)
: 171.97, 153.69, 149.59,
d: 2.23 (s, 3H), 4.27 (s, 4H), 4.93 (s, 2H), 7.40
120.63, 120.02, 119.88, 117.09, 107.20, 106.98, 106.92, 106.70, 64.15,
48.95, 44.71; Anal. (C15H11ClF3N3O4S) C, H, N.
d
135.07, 133.28, 128.36, 123.71, 123.47, 123.33, 119.71, 119.47, 116.53,
64.20, 48.54, 44.69, 17.23; Anal. (C16H15BrFN3O4S) C, H, N.
7.7.5. [2-(4-Methoxybenzyl)-7-cholro-3,4-dihydro-2H-pyrido[2,3-
e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7e)
Colorless crystal (0.28 g, 71%); m.p. 141e143 ꢀC; 1H NMR
7.7.12. [2-(3-Nitrobenzyl)-7-methyl-3,4-dihydro-2H-pyrido[2,3-e]-
[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7l)
(400 MHz, DMSO-d6) d: 3.74 (s, 3H), 4.18 (s, 2H), 4.25 (s, 2H), 4.88 (s,
2H), 6.91 (d, J ¼ 8.8 Hz, 2H), 7.26 (d, J ¼ 8.8 Hz, 2H), 8.19 (d,
Colorless crystal (0.29 g, 72%); m.p. 152e155 ꢀC; 1H NMR
J ¼ 2.4 Hz, 1H), 8.42 (d, J ¼ 2.4 Hz, 1H), 12.80 (s, 1H); 13C NMR
(400 MHz, DMSO-d6) d: 2.23 (s, 3H), 4.26 (s, 2H), 4.43 (s, 2H), 4.99
(400 MHz, DMSO-d6)
d
: 171.48, 159.71, 151.93, 150.06, 134.29,
(s, 2H), 7.66 (m, 1H), 7.81(d, J ¼ 7.6 Hz, 1H), 7.90 (d, J ¼ 2.0 Hz, 1H),
130.84 (2C), 127.67, 119.95, 117.49, 114.65 (2C), 63.55, 55.80, 50.33,
48.76; Anal. (C16H16ClN3O5S) C, H, N.
8.15 (m, 1H), 8.20 (s, 1H), 8.22 (d, J ¼ 1.6 Hz, 1H), 12.75 (s, 1H); 13C
NMR (400 MHz, DMSO-d6) d: 172.00, 153.69, 149.57, 148.45, 139.27,
135.79, 135.04, 130.73, 123.69, 123.65, 123.38, 116.59, 64.41, 50.21,
48.45, 17.21; Anal. (C16H16N4O6S) C, H, N.
7.7.6. [2-(4-Bromo-2-fluorobenzyl)-7-bromo-3,4-dihydro-2H-
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7f)
Colorless crystal (0.41 g, 81%); m.p. 182e183 ꢀC; 1H NMR
7.7.13. [2-(4-(Trifluoromethyl)benzyl)-7-methyl-3,4-dihydro-2H-
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7m)
Colorless crystal (0.31 g, 76%); m.p. 162e164 ꢀC; 1H NMR
(400 MHz, DMSO-d6) d: 4.29 (s, 2H), 4.31 (s, 2H), 4.98 (s, 2H), 7.40 (m,
1H),7.45 (m, 1H), 7.56 (m, 1H), 8.27 (d, J ¼ 2.4 Hz, 1H), 8.50 (d,
J ¼ 2.4 Hz,1H),12.85 (s,1H); 13C NMR (400 MHz, DMSO-d6)
d
: 171.50,
(400 MHz, DMSO-d6) d: 2.21 (s, 3H), 4.07 (s, 2H), 4.42 (s, 2H), 4.89
154.15, 150.18, 136.90, 133.31, 128.40, 123.22, 123.08, 119.74, 119.50,
117.92, 107.53, 64.30, 48.70, 44.97; Anal. (C15H12Br2FN3O4S) C, H, N.
(s, 2H), 7.60 (d, J ¼ 8.0 Hz, 2H), 7.70 (d, J ¼ 8.0 Hz, 2H), 7.83 (d,
J ¼ 2.0 Hz, 1H), 8.17 (d, J ¼ 2.0 Hz, 1H); 13C NMR (400 MHz, DMSO-
d6) d: 171.99, 153.61, 150.03, 142.19, 134.82, 129.81 (2C), 128.94,
7.7.7. [2-(3-Nitrobenzyl)-7-bromo-3,4-dihydro-2H-pyrido[2,3-e]-
[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7g)
126.05, 125.99, 122.49, 120.55, 115.87, 64.38, 50.26, 48.35, 17.19;
Anal. (C17H16F3N3O4S) C, H, N.
Colorless crystal (0.33 g, 73%); m.p. 193e195 ꢀC; 1H NMR
(400 MHz, DMSO-d6)
d
: 4.28 (s, 2H), 4.46 (s, 2H), 5.05 (s, 2H), 7.67
7.7.14. [2-(2,4,5-Trifluorobenzyl)-7-methyl-3,4-dihydro-2H-pyrido
[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7n)
Colorless crystal (0.27 g, 67%); m.p. 162e164 ꢀC; 1H NMR
(m, 1H), 7.81 (d, J ¼ 7.6 Hz, 1H), 8.17 (d, J ¼ 8.0 Hz, 1H), 8.20 (s, 1H),
8.28 (d, J ¼ 2.8 Hz, 1H), 8.48 (d, J ¼ 2.4 Hz, 1H), 12.87 (s, 1H); 13C
NMR (400 MHz, DMSO-d6)
d
: 171.49, 154.15, 150.14, 148.44, 138.96,
(400 MHz, DMSO-d6) d: 2.23 (s, 3H), 4.27 (s, 2H), 4.29 (s, 2H), 4.95

X. Chen et al. / European Journal of Medicinal Chemistry 46 (2011) 1536e1544
1543
(s, 2H), 7.51 (m, 1H), 7.58 (m, 1H), 7.892 (d, J ¼ 2.0 Hz, 1H), 8.23 (d,
ALR1 activity was performed at 37 ꢀC in a reaction mixture
containing 0.25 mL of 0.12 mM NADPH, 0.1 mL of enzyme extract,
0.25 mL of 0.1 M sodium phosphate buffer (pH 7.2), 0.15 mL of
J ¼ 2.0 Hz, 1H), 12.74 (s, 1H); 13C NMR (400 MHz, DMSO-d6)
d:
172.08, 153.73, 149.56, 135.09, 123.72, 119.89, 119.69, 116.47, 107.18,
106.97, 106.90, 106.69, 64.09, 48.47, 44.46, 17.21; Anal.
(C16H14F3N3O4S) C, H, N.
deionized water, and 0.25 mL of 20 mM sodium
substrate in a final volume of 1 mL. The reaction mixture except for
sodium
-glucuronate was incubated at 37 ꢀC for 10 min. The
D-glucuronate as
D
7.7.15. [2-(4-Methoxybenzyl)-7-methyl-3,4-dihydro-2H-pyrido
[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide-4-yl] acetic acid (7o)
Colorless crystal (0.18 g, 49%); m.p. 188e191 ꢀC; 1H NMR
substrate was then added to start the reaction, which was moni-
tored for 4 min.
The inhibitory activity of the newly synthesized compounds
(400 MHz, DMSO-d6)
d: 2.23 (s, 3H), 3.74 (s, 3H), 4.15(s, 2H), 4.22 (s,
against ALR2 and ALR1 was assayed adding 5 mL of the inhibitor
2H), 4.82 (s, 2H), 6.92 (d, J ¼ 8.4 Hz, 2H), 7.26 (d, J ¼ 8.8 Hz, 2H), 7.88
solution to the reaction mixture described above. All compounds
were dissolved in dimethyl sulfoxide (DMSO) and diluted with
deionized water. TocorrectforthenonenzymaticoxidationofNADPH,
the rate of NADPH oxidation in the presence of all of the reaction
mixture components except the substrate was subtracted from each
experimental rate. The inhibitory effect of the synthetic compounds
was routinely estimated at a concentration of 10ꢃ4 M (the concen-
tration is referred to that of the compound in the reaction mixture).
Those compounds found to be active were tested at additional
concentrations between 10ꢃ4 and 10ꢃ8 M. Each doseeeffect curve
was generated using at least four concentrations of inhibitor causing
an inhibition between 20% and 80% with three replicates at each
concentration. The 95% confidence limits (95% CL) were calculated
from t values for n ꢃ 2, where n is the total number of determinations.
(d, J ¼ 2.0 Hz, 1H), 8.21 (d, J ¼ 2.0 Hz, 1H), 12.71 (s, 1H); 13C NMR
(400 MHz, DMSO-d6)
d: 171.93, 159.64, 153.56, 149.67, 134.93,
130.80 (2C), 127.96, 123.51, 116.61, 114.67 (2C), 63.39, 55.80, 50.01,
48.55, 17.23; Anal. (C17H19N3O5S) C, H, N.
7.8. Biology, materials, and methods
Wistar rats (200e250 g, b.w.) were supplied by Vital River,
Beijing, China.
D,L-glyceraldehyde, sodium D-glucuronate, and
NADPH were from SigmaeAldrich. All other chemicals were of
reagent grade. Fidarestat was prepared in our laboratory according
to a reported method [34].
A partially purified ALR2 and ALR1 were obtained from rat
lenses and kidney, respectively.
Rat lens ALR2 was prepared in accordance with the method of
Hayman and Kinoshita [35] and La Motta et al. [22]. Lenses freshly
removed from rats were homogenized (Glas-Potter) in three
volumes of cold deionized water. The homogenate was centrifuged
at 12,000 rpm at 0e4 ꢀC for 30 min. The supernatant was precipi-
tated with ammonium sulfate at 40%, 50% and then 75% saturation.
The supernatant was retained after the first two precipitations. The
final precipitate recovered from the 75% saturated fraction, pos-
sessing ALR2 activity, was redissolved in 0.05 M NaCl and dialyzed
overnight in 0.05 M NaCl. The dialyzed material was stored
at ꢃ20 ꢀC and then used for the enzymatic assay.
7.10. Docking experiments
Conformational searches and molecular docking were per-
formed using version 7.1 of the SYBYL.
Acknowledgments
This work was supported by the Beijing Natural Science Foun-
dation (no. 7102091) and China Major Infectious Research Projects
(no. 2008ZX10001-006).
Rat kidney ALR1 was prepared in accordance with a previously
reported method [22]. Kidneys were quickly removed from rats and
homogenized (Glas-Potter) in three volumes of 10 mM sodium
phosphate buffer, pH 7.2, containing 0.25 M sucrose, 2.0 mM EDTA
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the on-line version, at doi:10.1016/j.ejmech.2011.01.072.
dipotassium salt, and 2.5 mM b-mercaptoethanol. The homogenate
was centrifuged at 12,000 rpm at 0e4 ꢀC for 30 min and the
supernatant was subjected to a 40e75% ammonium sulfate frac-
tionation, following the same procedure previously described for
ALR2. The precipitate obtained from the 75% ammonium sulfate
saturation, possessing ALR1 activity, was redissolved in 50 volumes
of 10 mM sodium phosphate buffer, pH 7.2, containing 2.0 mM
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