1,2-Benzothiazine 1,1-dioxide carboxylate derivatives as novel potent inhibitors of aldose reductase

Article abstract of DOI:10.1016/j.bmc.2011.07.051

Due to the importance of aldose reductase (ALR2) as a potential drug target in the treatment of diabetic complications, there are increasing interests in design and synthesis of ALR2 inhibitors. Here, we prepared 1,2-benzothiazine 1,1-dioxide acetic acid derivatives and investigated their inhibition activity. Most of these derivatives were found to be active with IC₅₀ values ranging from 0.11 μM to 10.42 μM, and compound 8d, 2-[2-(4-bromo-2- fluorobenzyl)-1,1-dioxido-2H-1,2-benzothiazin-4(3H)-ylidene]acetic acid, showed the most potent inhibition activity. Further, SAR and docking studies suggest that in comparison with the α,β-unsaturated derivatives, the saturated carboxylic acid derivatives had a greater binding affinity with the enzyme and thus an enhanced inhibition activity. Therefore, development of more powerful ARIs based on benzothiazine 1,1-dioxide by stereo-controlled synthesis could be expected.

Full text of DOI:10.1016/j.bmc.2011.07.051

Bioorganic & Medicinal Chemistry 19 (2011) 7262–7269
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,2-Benzothiazine 1,1-dioxide carboxylate derivatives as novel potent
inhibitors of aldose reductase
Xin Chen a, Shuzhen Zhang a, Yanchun Yang a, Saghir Hussain a, Minlan He a, Dequan Gui a,
b,
Bing Ma a, Chaojun Jing a, Zhixin Qiao b, Changjin Zhu a, , Qun Yu
⇑
⇑
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
a r t i c l e i n f o
a b s t r a c t
Article history:
Due to the importance of aldose reductase (ALR2) as a potential drug target in the treatment of diabetic
complications, there are increasing interests in design and synthesis of ALR2 inhibitors. Here, we pre-
pared 1,2-benzothiazine 1,1-dioxide acetic acid derivatives and investigated their inhibition activity.
Received 18 June 2011
Accepted 25 July 2011
Available online 30 July 2011
Most of these derivatives were found to be active with IC50 values ranging from 0.11 lM to 10.42 lM,
and compound 8d, 2-[2-(4-bromo-2-fluorobenzyl)-1,1-dioxido-2H-1,2-benzothiazin-4(3H)-ylidene]ace-
tic acid, showed the most potent inhibition activity. Further, SAR and docking studies suggest that in
comparison with the a,b-unsaturated derivatives, the saturated carboxylic acid derivatives had a greater
binding affinity with the enzyme and thus an enhanced inhibition activity. Therefore, development of
Keywords:
1,2-Benzothiazine 1,1-dioxide
Aldose reductase
Inhibitor
more powerful ARIs based on benzothiazine 1,1-dioxide by stereo-controlled synthesis could be
expected.
Ó 2011 Published by Elsevier Ltd.
1. Introduction
ALR1, an enzyme that is closely related to ALR221 and plays a detox-
ification role as it specifically metabolizes toxic aldehydes such as
Diabetes mellitus is a chronic disease caused by deficiency in
production of insulin by pancreas, and by resistance to insulin’s ef-
fects. Such a deficiency results in increased concentrations of glu-
cose in the blood, which in turn triggers the polyol pathway and
then damages many of the body’s systems such as eyes, kidneys,
nerves, heart, and blood vessels. Increasing evidence reveals a link
between glucose metabolism via the polyol pathway and long-
term diabetic complications.1–3 Aldose reductase (EC 1.1.1.21,
ALR2) catalyses the NADPH-dependent reduction of glucose to
sorbitol in the first step of this metabolic pathway.4–6 Therefore,
it has received great attention as a target enzyme in the search
for drugs able to prevent or delay the onset and progression of
diabetic complications, independently of glycaemic levels.
Animal model studies demonstrated that diabetic complications
can be ameliorated by the use of aldose reductase inhibitors (ARIs).7
In the past years, a range of structurally different compounds have
been reported as ARIs (Fig. 1), such as alrestatin,8 tolrestat,9 epalre-
stat,10 zopolrestat,11 zenarestat,12 ponalrestat,13 lidorestat,14 naph-
tho[1,2-d]isothiazole derivatives,15 sorbinil,16 fidarestat17 and
ranirestat (AS-3201).18 However, many of the clinically tested ARIs
proved to be inadequateas drug candidates because of adverse phar-
macokinetics, toxic side effects or low efficacy.19,20 Inhibition of
hydroxynonenal (HNE), 3-deoxyglucosone, and methylglyoxal,22–24
may account for some of the undesirable side effects associated with
the present ARIs. Thus, we are putting a lot of effort into the
development of a larger variety of ARIs that block ALR2 strongly
and specifically. In the last few years, we have designed two classes
of compounds based on 1,2,4-benzothiadiazine 1,1-dioxide and
pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide, respectively, and
numerous compounds of each class have been demonstrated to be
potent and selective ARIs (Fig. 1).25,26 Both core structures have a
nitrogen atom at the position-4, which is connected to the carboxyl-
ate group essential for the ARIs and therefore determines the
orientation of the side chain (Fig. 1). Accordingly, it is interesting
to detect effect of the substitution of a carbon atom for the N-4 of
the scaffold on the aldose reductase inhibition and then our further
efforts at a new design based on benzothiazine core resulted in the
formation of a novel series of 1,2-benzothiazine 1,1-dioxide deriva-
tives. Here, we report their preparation, aldose reductase inhibition
activity, and docking studies.
2. Chemistry
2-[2-Benzyl-1,1-dioxido-2H-1,2-benzothiazin-4(3H)-ylidene]
acetic acid (8a–f) and 2-(2-benzyl-1,1-dioxido-3,4-dihydro-
2H-1,2-benzothiazin-yl) acetic acid (9a–d) were prepared
following the synthetic pathway depicted in Scheme 1 from com-
mercially available sodium saccharin 1. By reacting 1 with methyl
⇑
Corresponding authors. Tel./fax: +86 10 68918506 (C.Z.); tel./fax: +86 10
66932965 (Q.Y.).
E-mail addresses: zcj@bit.edu (C. Zhu), yuq@nic.bmi.ac.cn (Q. Yu).
0968-0896/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2011.07.051

X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 7262–7269
7263
O
O
O
OH
O
N
HN
N
NH
NH
O
O
HN
N
HN
Cl
CO2H
O
S
O
O
O
HN N
O
Br
F
O
N
OH
OH
O
S
N
O
NH2
O
O
O
O
F
Sorbinil
Fidarestat
ppp
Epalrestat
Ranirestat
6-(5-Chloro-3-methylbenzofuran-
2-sulfonyl)-2H-pyridazin-3-one
CO2H
O
CO2H
S
N
F
N
N
OH
O
CO2H
R1 = H, Cl, Br
X
N
S
N
S
N
R2 = 2-F,4-Br; 3-NO2; 4-CF3; 2,4,5-F3
X = C, N
R2
F
N
O
HO
O
R1
O
CF3
Tolrestat
F
Acetic acid derivatives
Lidorestat
Acetic acid derivative
of oxadiazole
of benzothiadiazine and pyridothiadiazine
Figure 1. Chemical structures of aldose reductase inhibitors.
OH
O
O
O
O
O
b
d-f
a
c
O
N
O
NH
S
NH
NH
S
S
S
O
O
O
O
O
O
O
O
O
1
2
4
3
O
N
O
N
O
O
h
g
R
R
R
N
S
S
S
O
O
O
O
O
O
6a: R = 2,4,5-F3
6b: R = 3,5-CF3
6c: R = 4-CF3
6d: R = 2-F,4-Br
6e: R = 3-NO2
6f: R = 2-F
7
7a: R = 2,4,5-F3
7b: R = 3,5-CF3
7c: R = 4-CF3
7d: R = 2-F
6
5
5a: R = 2,4,5-F3
5b: R = 3,5-CF3
5c: R = 4-CF3
5d: R = 2-F,4-Br
5e: R = 3-NO2
i
i
h
O
O
N
OH
OH
R
R
N
S
S
O
O
O
O
9a: R = 2,4,5-F3
9b: R = 3,5-CF3
9c: R = 4-CF3
9d: R = 2-F
8a: R = 2,4,5-F3
8b: R = 3,5-CF3
8c: R = 4-CF3
8d: R = 2-F,4-Br
8e: R = 3-NO2
8f: R = 2-F
9
8
Scheme 1. Reagents and conditions: (a) BrCH2COOCH3, DMF, 60 °C; (b) NaOMe, MeOH, 55 °C; (c) HCl, reflux; (d) Dean and Stark apparatus, ethane-1,2-diol, p-TsOH, PhH,
reflux; (e) substituted Bn–Br, K2CO3, CH3CN, 70 °C; (f) HCl, H2O, THF, reflux; (g) Ph3P@CHCOOCH3, PhCH3, reflux; (h) H2, 10% Pd/C, EtOH, AcOEt; (i) 1,4-dioxane, NaOH.
bromoacetate under mild conditions, methyl [1,1-dioxido-3-oxo-
1,2-benzisothiazol-2(3H)-yl]acetate 2 was obtained in excellent
yield.27 Gabriele-Colman type ring expansion of the five-membered
isothiazole ring of 2 to the six-membered thiazine ring, having
synchronous ring cleavage and ring closure steps, in an inert
atmosphere yielded methyl 4-hydroxy-2H-1,2-benzothiazine-3-
carboxylate 1,1-dioxide 3.28,29 Subsequently, 3 was refluxed with
concentrated hydrochloric acid to get 2H-1,2-benzothiazin-4(3H)-
one 1,1-dioxide (4). N-Substituted ketones 5a–e were achieved by
alkylation of 4 in three steps with commercially available benzylic
bromides30 and then followed by a reaction with methyl 2-(tri-
phenylphosphoranylidene) acetate to give a series of 2-[2-benzyl-
1,1-dioxido-2H-1,2-benzothiazine-4(3H)-ylidene]carboxylic acid
methyl esters 6a–e.31 1,4-Hydrogenation of the ester 6d catalyzed
by Pd/C resulted in a removal of bromine of the benzyl group leading
to the formation of ester 6f. The esters 6a–f were then converted into
by Pd/C catalyzed 1,4-hydrogenation to form the esters 7a–c and 7d
in good yields,32 which were then converted to acids 9a–d by hydro-
lysis. However, saturated acids corresponding to 8d–e could be
achieved neither by the 1,4-hydrogenation of precursors 6d–e due
to the presence of bromine and nitro groups which are more sensi-
tive to the reduction reaction, nor via other synthetic procedures.
All acids thus prepared were characterized as stereoisomeric
mixtures.
3. Results and discussion
3.1. Aldose reductase inhibition and SAR studies
All newly synthesized acetic acid derivatives of 1,2-benzothi-
azine 1,1-dioxide were tested for their potential inhibitory effect
on ALR2 isolated from rat lenses. In order to evaluate their selectiv-
ity for ALR2 inhibition, these compounds, which showed potent
ALR2 inhibition activity, were also subjected to test for their inhi-
bition activity against ALR1, isolated from rat kidneys. IC50 values
the desired
sodium hydroxide. In order to obtain the corresponding saturated
products, the ,b-double bond of esters 6a–c and 6f were saturated
a,b-unsaturated acids 8a–f by hydrolysis with aqueous
a

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X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 7262–7269
were determined by linear regression analysis of the log of the con-
centration–response curve. Their effectiveness was evaluated with
respect to epalrestat as a 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.
As shown in Table 1, most of 1,2-benzothiazine 1,1-dioxide
acetic acid derivatives (8a–f, 9a–d) showed significantly ALR2
inhibitory activity. Of these acid derivatives, 2-[2-(4-bromo-
2-fluorobenzyl)-1,1-dioxido-2H-1,2-benzothiazin-4(3H)-ylidene]
carboxylic acid (8d) was the most active compound having an IC50
In further SAR study at the C-4 position of benzothiazine
derivatives, it was found that saturated carboxylic acids showed
a significantly enhanced ALR2 inhibition activity over their corre-
sponding
a,b-unsaturated compounds. This was evidenced by
comparing biological activities to each other for four pairs of com-
pounds containing 8a and 9a, 8b and 9b, 8c and 9c, and 8f and 9d
(Table 1) although saturated compounds corresponding to 8d–e
could not be synthetically obtained and thus used for the evidence.
The compound of 9 series in each pair proved an increased activity
than the other, for example, 9a (IC50 = 0.34
lM) was more active
value of 0.11
pound, producing 32% inhibition at 10
9a were shown to be potent ARIs, displaying IC50 values in the
range between 0.14 and 0.59 M. The remaining five compounds
l
M. In contrast, 8b was the lowest effective com-
than 8a (IC50 = 0.59 M). This result indicates that the acetic acid
l
lM dose. Acids 8a, 8e and
side chain linking to the saturated C-4 may take a more favorable
orientation on the saturated C-4 for the interaction with the anion
site of the enzyme.
l
including 8c, 8f, 9b, 9c, and 9d displayed varying levels of effi-
ciency with IC50 values in the low micromolar ranging from
As shown in Table 1, compounds 8a, 8d, 8e and 9a with signif-
icantly potent ALR2 inhibition activity (IC50 = 0.59, 0.11, 0.14 and
1.73
lM to 10.42
lM.
0.34 lM) were tested for their inhibition ability against ALR1. They
In structure–activity relationship (SAR) study at the N-2
position, our reported results from the comparison of derivatives
of benzothiadiazine and pyridothiadiazine have shown 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
(Fig. 1).25,26 Herein, it was found that the order of ALR2 inhibition
activity for the benzothiazine derivatives was shown as 2-F,4-Br >
3-NO2 > 2,4,5-F3 > 2-F > 4-CF3 > 3,5-(CF3)2 evidenced by comparing
biological activities of compounds in series 8a–f (8d > 8e >
8a > 8f > 8c > 8b) and 9a–d (9a > 9d > 9c > 9b), respectively. 2-
F,4-Br substitution (8d) on the benzyl ring structure of the benzo-
thiazine was favored for the inhibition activity over the others and
3-NO2 substitution (8e) had a close activity. Further more, both the
benzothiazine derivatives 8d–e were found to appear higher inhi-
bition efficiency whereas 2,4,5-F3 substituted benzothiazine deriv-
ative 8a displayed lower efficiency when comparing them with
their benzothiadiazine counterpart having the same substituent
at the N-2 position (Fig. 1).25 This indicates that at least in each
were found to be slightly active (IC50 >10
selectivity for ALR2.
lM), demonstrating their
3.2. Molecular modeling
To get a better understanding of the ALR2 inhibitory potency 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 on the human ALR2/
NADP+/IDD594 complex (PDB entry code 1US0) using the
automated docking program SYBYL 7.1. Optimized structures of
compounds 8a–f and 9a–d were flexibly docked to the active site,
which is comprised of three binding subsites. The first is the con-
served anion-binding site located between the nicotinamide ring
of the coenzyme and active site residues Tyr48, His110, and
Trp111. The second is the hydrophobic pocket lined by residues
Trp20, Phe122, and Trp219. The third is the specificity pocket
formed by residues Trp111, Thr113, Phe122, Ala299, and Leu300.33
Analysis of the docking results revealed that most of the com-
pounds were occupying the active site of ALR2, as the carboxyl
head of the inhibitors, except for 8e, formed hydrogen bonds with
the anion-binding site of the enzyme. However, 8e was bound to
the anion site with its nitro group but not carboxylate displaying
case of 2-F,4-Br and 3-NO2 substitution the
a,b-unsaturated (C-4
double bond) moiety of 8d–e may make orientation of the carbox-
ylic acid side chain favored over the N-4 moiety of benzothiadi-
azine derivatives ( Fig. 1)25 for the interaction between the
carboxylate and the anion site of ALR2.33
a
different pattern from typical carboxylic inhibitors. Scores
docked for 8d–e were favorable when compared to those for the
other compounds in 8 series. This is well consist with the study re-
sult from the ALR2 inhibition assay in which 8d–e showed the
most active. In addition, it was found that docking score for each
of compounds 9 was more favorable than that for the counterpart
in 8, which is in agreement with the result of the enzyme inhibi-
tion experiment (Table 1), strongly suggesting that the saturated
Table 1
Biological activity data for 1,2-benzothiazine 1,1-dioxide carboxylic acid derivatives
O
O
OH
OH
R
R
N
N
acids (9) are favored as inhibitors over the
a,b-unsaturated acids.
S
S
O
O
O
O
Analysis of docking poses for 8a and 9a may explain why saturated
acids 9 have greater inhibitory activities. In the docking of 9a, as
shown in Figure 2c and d, the carboxyl head forms tight hydrogen
8
9
a
No.
R
ALR2 IC50
(l
M)
ALR1 (% of inhibition)b
bonds with the OH of Tyr48 (3.09 Å), the Ne2 atom of His110
8a
8b
8c
8d
8e
8f
9a
9b
2,4,5-F3
3,5-(CF3)2
4-CF3
2-F,4-Br
3-NO2
2-F
2,4,5-F3
3,5-(CF3)2
4-CF3
0.59 (0.27–0.90)
32%b
18
c
c
11
26
c
28
c
(2.87 Å), and the Ne1 of Trp111 (2.70 Å), and a salt bridge with
the positively charged nicotinamide moiety of the cofactor NADP+
(N–O = 4.06 Å) within the anion-binding site. Further, the benzyl
ring gets deeply trapped in the hydrophobic cage of the specificity
6.13 (2.84–9.41)
0.11 (0.01–0.21)
0.14 (0.05–0.23)
2.13 (1.20–3.06)
0.34 (0.09–0.59)
10.42 (5.97–14.87)
5.30 (3.59–7.02)
1.73 (8.70–13.87)
0.12 (0.05–0.19)
pocket and in turn p-stacks perfectly against the indole moiety of
Trp111. These interactions anchor the inhibitor tightly within the
enzyme active site. Whereas in the docking of 8a, despite a similar
interaction with the anion-binding site was present, no such
9c
9d
Epalrestat
c
c
c
2-F
p-stacking interaction was found between the benzyl group and
a
IC50 (95% CL) values represent the concentration required to produce 50%
enzyme inhibition.
the side chain of Trp111 so that the benzyl side chain was pointed
in an opposite direction but not the deep of the specificity pocket
(Fig. 2a and b).
b
The inhibitory effect was estimated at a concentration of 10 lM.
Not determined.
c

X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 7262–7269
7265
Figure 2. Docking of the inhibitors 8a (panels a and b) and 9a (panels c and d) into the active site of ALR2. (a and c) Protein structures are shown as cartoon diagrams with
selected and labeled residues shown in line representation, and ligands and NADP are shown as stick models. Docked poses of compounds are shown in cyan (C), red (O), blue
(N), yellow (S), and gray (F). (b and d) Protein residues are in surface representation.
Varian 500-MS ion trap mass spectrometer equipped with an ESI
source (Varian, USA).
4. Conclusions
The following HPLC methods were used to determine purity of
acetic acid derivatives and epalrestat using Hitachi L2000 liquid
chromatograph (Hitachi High-Technologies Corporation, Japan).
All acetic acid derivatives and epalrestat tested in biological assays
The present effort to develop novel ARIs based on 1,2-benzo-
thiazine 1,1-dioxide resulted in the formation of two groups of
compounds containing
acid (acetic acid) derivatives. They showed ALR2 inhibition activity
with IC50 values in the range from 0.11 M to10.42 M. The most
a,b-unsaturated and saturated carboxylic
were P95% pure. Method: column, C18, 5
l, 250 mm  4.6 mm;
l
l
mobile phase: H2O (0.1% TFA)/MeOH = 1:9, for 6 min; room tem-
active was found to be 2-[2-(4-bromo-2-fluorobenzyl)-1,1-dioxi-
perature; flow rate: 1 mL/min; detection: 250 nm.
do-2H-1,2-benzothiazin-4(3H)-ylidene] carboxylic acid (8d) which
The sodium saccharin, methyl bromoacetate, 4-bromo-2-fluorob-
enzyl bromide, 3-nitrobenzyl bromide, 4-(trifluoromethyl)benzyl
bromide, 2,4,5-trifluorobenzyl bromide and 3,5-bis(trifluoro-
methyl)benzyl bromide, used to obtain the target inhibitors, were
from Alfa Aesar.
is a,b-unsaturated. However, SAR combined with the docking stud-
ies demonstrated that the saturated carboxylic acid derivatives had
a greater binding affinity with the enzyme and thus an enhanced
inhibition activity in comparison with the
a,b-unsaturated deriva-
tives although saturated compound corresponding to
a,b-unsatu-
rated 8d was not obtained. Consequently, we suggest that the
configuration of C-4 plays an important role for the ALR2 inhibition
of benzothiazine-based ARIs and the acetic acid side chain in the
saturated compounds may take a favorable orientation on the
C-4 position for the binding interaction or cause the benzyl side
chain to point in the deep direction of specificity pocket. Given that
all carboxylic acid compounds prepared in the present study were
the stereoisomeric mixtures, we speculate that single stereoiso-
mers provided in future by stereo-controlled synthetic strategy
may be more powerful and selective ARIs.
5.2. Methyl [1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl]
acetate (2)
A
mixture of sodium saccharin (60.3 g, 250 mmol), DMF
(150 ml) and methyl bromoacetate (38.3 g, 250 mmol) was taken
in a round bottom flask and was heated at 60 °C for 1 h. Contents
were then cooled to room temperature and poured over ice cooled
water (1000 ml) resulting in the formation of a white solid, which
was filtered and washed with cold water. The solid was dried and
recrystallized from methyl alcohol to get the product as a white
solid (40.2 g, 61%); mp 113–115 °C; 1H NMR (400 MHz, DMSO-
d6) d: 3.75 (s, 3H), 4.69 (s, 2H), 8.06 (m, 1H), 8.11 (m, 1H), 8.19
(d, J = 6.0 Hz, 1H), 8.40 (d, J = 6.0 Hz, 1H).
5. Experimental section
5.1. Chemistry
5.3. Methyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-
Melting points were recorded on a X-4 microscopic melting
point apparatus and were uncorrected. All reactions were routinely
checked by TLC on silica gel Merck 60F254. The NMR spectra were
recorded on a Bruker Advance 400 spectrometer (400 MHz, Bruker
(Beijing) Tech. and Serv. Co., Ltd) or Bruker Advance 500 spectrom-
eter (500 MHz, Beijing Normal University, China); chemical shifts
are given in d units (ppm) relative to internal standard TMS and
refer to CDCl3 or DMSO-d6 solutions. MS was performed with a
dioxide (3)
Sodium metal (6.9 g, 300 mmol) and dry methanol (300 ml) was
allowed to reflux until all the metal dissolved. To this solution, 2
(30.6 g, 120 mmol) was added in a single portion under inert con-
ditions. Temperature of the mixture was maintained at 60 °C for
30 min till the completion of precipitation. The contents were then
suddenly poured over an ice-water mixture. HCl (10%) was added

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X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 7262–7269
to the mixture till the pH became approximately 3. The precipi-
tates formed were filtered and washed with cold water. The solid
was dried and recrystallized from methyl alcohol to get the prod-
uct as a white solid (16.9 g, 55%); mp 163–165 °C; 1H NMR
(500 MHz, CDCl3) d: 3.96 (s, 3H), 6.24 (s, 1H), 7.76 (m, 2H), 7.93
(m, 1H), 8.11 (d, J = 7.5 Hz, 1H), 11.44 (s, 1H).
5.6. General synthetic procedure for 6a–e
To a stirred solution of triphenylphosphine (52.4 g, 0.2 mol) in
benzene (300 mL) was added dropwise a solution of methyl bro-
moacetate (30.4 g, 0.2 mol) in benzene (100 mL) over 1 h. The reac-
tion was allowed to stir for 4 h. The solid phosphonium bromide
product was filtered and washed with hexane to provide a white
solid. The crude solid was dissolved in water (600 mL) and sodium
hydroxide (20.2 g, 0.2 mol) was added. After 1 h, the precipitate
was filtered and washed with water to provide a white solid. The
crude product was recrystallized from benzene/petroleum ether
to provide methyl 2-(triphenylphosphoranylidene) acetate
(57.4 g, 86% for two steps).
A solution containing the appropriate 5 (3 mmol) and methyl
(triphenylphosphoranylidene) acetate (4.5 mmol) in toluene
(30 mL) was heated at 100 °C for 2 h. After evaporation of the sol-
vent, the residue was purified by column chromatography (ethyl
acetate–hexane; 1:3) to give the product.
5.4. 2H-1,2-Benzothiazin-4(3H)-one 1,1-dioxide (4)
A mixture of 3 (10.2 g, 40.0 mmol) was added to 150 mL of con-
centrated hydrochloric acid and refluxed for 8 h. The crude mate-
rial was then poured into ice-water mixture and the precipitate
was collected by suction filtration, washed with cold water and
dried in vacuo to give the desired product (5.3 g, 67%); mp
160–162 °C; 1H NMR (500 MHz, CDCl3) d: 3.96 (d, J = 7.5 Hz, 2H),
5.43 (d, J = 6.5 Hz, 1H), 7.76 (m, 1H), 7.82 (m, 1H), 7.91 (m, 1H),
8.11(d, J = 7.5 Hz, 1H).
5.5. General synthetic procedure for 5a–e
5.6.1. 2-[2-(2,4,5-Trifluorobenzyl)-1,1-dioxido-2H-1,2-benzo-
thiazin-4(3H)-ylidene]acetic acid methyl ester (6a)
White powder (0.65 g, 55%); mp 103–104 °C; 1H NMR
(400 MHz, DMSO-d6) d: 3.60 (s, 3H), 3.73 (s, 2H), 4.94 (s, 2H),
7.01 (s, 1H), 7.42 (m, 1H), 7.54 (m, 1H), 7.60 (m, 2H), 7.74 (m,
1H), 7.90 (d, J = 8.0 Hz, 1H).
To a solution of 4 (43.1 g, 220 mmol), in benzene (300 mL) were
added p-toluenesulfonic acid (PTSA) (2.6 g, 14.0 mmol) and ethyl-
ene glycol (31 mL, 550 mmol). The reaction mixture was refluxed
in a Dean and Stark apparatus for 3 h. The crude material was then
poured into ice-water mixture and the precipitate was collected by
suction filtration, washed with cold water and dried in vacuo to
leave the product, which was used without further purification. A
mixture of the dioxolane (4.9 g, 20 mmol), potassium carbonate
(3 g), and appropriate benzyl bromide (20 mmol) in acetonitrile
(50 mL) was heated at 70 °C for 2 h. After evaporation of the sol-
vent under reduced pressure, the resulting dioxolane was added
to 40 mL tetrahydrofuran and 30 mL 10% hydrochloric acid and
stirred at 80 °C for 6 h. The reaction mixture was poured into water
with vigorous stirring. After 30 min, filtration afforded the desired
product, which was used without further purification.
5.6.2. 2-[2-(3,5-Bis(trifluoromethyl)benzyl)-1,1-dioxido-2H-1,2-
benzothiazin-4(3H)-ylidene]acetic acid methyl ester (6b)
White powder (0.70 g, 49%); mp 139–141 °C; 1H NMR
(400 MHz, DMSO-d6) d: 3.59 (s, 3H), 3.72 (s, 2H), 5.15 (s, 2H),
7.08 (s, 1H), 7.56 (m, 1H), 7.62 (m, 1H), 7.76 (m, 1H), 7.91 (m,
3H), 8.05 (s, 1H).
5.6.3. 2-[2-(4-(Trifluoromethyl)benzyl)-1,1-dioxido-2H-1,2-
benzothiazin-4(3H)-ylidene]acetic acid methyl ester (6c)
White powder (0.76 g, 62%); mp 158–160 °C; 1H NMR
(400 MHz, DMSO-d6) d: 3.66 (s, 3H), 4.23 (s, 2H), 4.86 (s, 2H),
6.75 (s, 1H), 7.53 (m, 3H), 7.67 (m, 3H), 7.90 (m, 1H), 8.05 (m, 1H).
5.5.1. 2-(2,4,5-Trifluorobenzyl)-2H-1,2-benzothiazin-4(3H)-one
1,1-dioxide (5a)
Pale yellow powder (4.7 g, 70%); mp 126–128 °C; 1H NMR
(400 MHz, DMSO-d6) d: 4.48 (s, 2H), 4.54 (s, 2H), 7.50 (m, 2H),
7.84–7.96 (m, 4H).
5.6.4. 2-[2-(4-Bromo-2-fluorobenzyl)-1,1-dioxido-2H-1,2-
benzothiazin-4(3H)-ylidene]acetic acid methyl ester (6d)
White powder (0.79 g, 60%); mp 119–121 °C; 1H NMR
(400 MHz, DMSO-d6) d: 3.63 (s, 3H), 3.76 (s, 2H), 4.98 (s, 2H),
7.02 (s, 1H), 7.31 (m, 1H), 7.45 (m, 1H), 7.54 (m, 1H), 7.65 (m,
2H), 7.78 (m, 1H), 7.93 (d, J = 8.0 Hz, 1H).
5.5.2. 2-[3,5-Bis(trifluoromethyl)benzyl]-2H-1,2-benzothiazin-
4(3H)-one 1,1-dioxide (5b)
Pale yellow powder (4.9 g, 59%); mp 106–108 °C; 1H NMR
(400 MHz, DMSO-d6) d: 4.68 (s, 2H), 4.70 (s, 2H), 7.74 (m, 2H),
7.81 (m, 2H), 7.91 (m, 2H), 7.96 (m, 1H).
5.6.5. 2-[2-(3-Nitrobenzyl)-1,1-dioxido-2H-1,2-benzothiazin-
4(3H)-ylidene]acetic acid methyl ester (6e)
White powder (0.63 g, 54%); mp 137–139 °C; 1H NMR
(400 MHz, DMSO-d6) d: 3.64 (s, 3H), 3.77 (s, 2H), 5.14 (s, 2H),
7.11 (s, 1H), 7.59 (m, 1H), 7.71 (m, 2H), 7.81 (m, 2H), 8.00 (m,
1H), 8.20 (m, 2H).
5.5.3. 2-[4-(Trifluoromethyl)benzyl]-2H-1,2-benzothiazin-
4(3H)-one 1,1-dioxide (5c)
Pale yellow powder (5.0 g, 71%); mp 103–105 °C; 1H NMR
(400 MHz, DMSO-d6) d: 4.59 (s, 2H), 4.61 (s, 2H), 7.46 (m, 2H),
7.66 (m, 2H), 7.89 (m, 2H), 7.99 (m, 2H).
5.7. 2-[2-(2-Fluorobenzyl)-1,1-dioxido-2H-1,2-benzothiazin-
4(3H)-ylidene]acetic acid methyl ester (6f)
5.5.4. 2-(4-Bromo-2-fluorobenzyl)-2H-1,2-benzothiazin-4(3H)-
one 1,1-dioxide (5d)
To a suspension of 10% palladium on carbon (0.27 g) in metha-
nol (10 mL) was added a solution of 6d (0.35 g, 0.8 mmol) in EtOAc
(10 mL). The mixture was stirred at room temperature for 12 h un-
der a hydrogen atmosphere. The catalyst was filtered off through
Celite pad and the filtrate was concentrated and purified via flash
chromatograph to obtain the pure product as a white powder
(0.27 g, 92%); mp 70–72 °C; 1H NMR (400 MHz, DMSO-d6) d: 3.58
(s, 3H), 3.72 (s, 2H), 4.97 (s, 2H), 6.98 (s, 1H), 7.16 (m, 1H), 7.21
(m, 1H), 7.29 (m, 1H), 7.35 (m, 1H), 7.52 (d, J = 8.0 Hz, 1H,), 7.59
(m, 1H), 7.74 (m, 1H), 7.90 (d, J = 8.0 Hz, 1H).
Pale yellow powder (5.8 g, 75%); mp 102–105 °C; 1H NMR
(500 MHz, DMSO-d6) d: 4.49 (s, 2H), 4.50 (s, 2H), 7.35 (m, 2H),
7.43 (d, J = 9.5 Hz, 1H), 7.86 (m, 3H), 7.93 (m, 1H).
5.5.5. 2-(3-Nitrobenzyl)-2H-1,2-benzothiazin-4(3H)-one 1,1-
dioxide (5e)
Pale yellow powder (4.4 g, 67%); mp 143–145 °C; 1H NMR
(400 MHz, DMSO-d6) d: 4.64 (s, 2H), 4.68 (s, 2H), 7.60 (m, 1H),
7.80 (m, 1H), 7.87 (m, 2H), 7.97 (m, 2H), 8.14 (m, 2H).

X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 7262–7269
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5.8. General synthetic procedure for 7a–c
128.4, 128.1, 124.6, 124.5, 121.8, 121.7, 121.4, 113.2, 49.5, 35.2;
MS m/z: negative mode 463.9 ([MÀH]À), positive mode 466.0
([M+H]+), 488.1 ([M+Na]+).
The title compounds were obtained as described for 6f starting
from 5a–c (1 mmol).
5.9.3. 2-[2-(4-(Trifluoromethyl)benzyl)-1,1-dioxido-2H-1,2-
benzothiazin-4(3H)-ylidene]acetic acid (8c)
5.8.1. 2-[2-(2,4,5-Trifluorobenzyl)-1,1-dioxido-3,4-dihydro-2H-
1,2-benzothiazin-yl]acetic acid methyl ester (7a)
Colorless crystal (0.15 g, 77%); purity: 97.7%; mp 170–172 °C;
1H NMR (400 MHz, DMSO-d6) d: 3.59 (s, 2H), 5.03 (s, 2H), 7.00 (s,
1H), 7.52 (m, 3H), 7.62 (m, 1H), 7.77 (m, 3H), 7.93 (m, 1H), 12.46
(s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 172.3, 141.5, 132.9,
132.5, 131.5, 130.7, 128.6, 128.3 (2C), 127.9, 125.7, 125.6, 124.5,
121.5, 112.4, 109.5, 49.5, 35.3; MS m/z: negative mode 396.0
([MÀH]À), positive mode 398.0 ([M+H]+), 420.0 ([M+Na]+).
White powder (0.35 g, 88%); mp 103–105 °C; 1H NMR
(400 MHz, DMSO-d6) d: 2.63 (m, 1H), 2.79 (m, 1H), 3.52 (s, 3H),
3.58 (m, 1H), 3.61–3.70 (m, 2H), 4.10 (d, J = 14.0 Hz, 1H), 4.54 (d,
J = 14.0 Hz, 1H), 7.50 (m, 2H), 7.60 (m, 2H), 7.65 (m, 1H), 7.79 (d,
J = 8.0 Hz, 1H).
5.8.2. 2-(2-(3,5-Bis(trifluoromethyl)benzyl)-1,1-dioxido-3,4-
dihydro-2H-1,2-benzothiazin-yl) acetic acid methyl ester (7b)
White powder (0.45 g, 93%); mp 102–105 °C; 1H NMR
(400 MHz, DMSO-d6) d: 2.62 (m, 1H), 2.77 (m, 1H), 3.57 (s, 3H),
3.62 (m, 1H), 3.66 (m, 1H), 3.74 (m, 1H), 4.38 (d, J = 15.2 Hz, 1H),
4.69 (d, J = 15.2 Hz, 1H), 7.51 (m, 2H), 7.62 (m, 1H), 7.81 (d,
J = 8.0 Hz, 1H), 8.05 (s, 2H), 8.09 (s, 1H).
5.9.4. 2-[2-(4-Bromo-2-fluorobenzyl)-1,1-dioxido-2H-1,2-
benzothiazin-4(3H)-ylidene]acetic acid (8d)
Colorless crystal (0.14 g, 77%); purity: 99.5%; mp 159–161 °C;
1H NMR (400 MHz, DMSO-d6) d: 3.59 (s, 2H), 4.93 (s, 2H), 6.95 (s,
1H), 7.27 (m, 1H), 7.42 (m, 1H), 7.57 (m, 1H), 7.61 (m, 2H), 7.76
(m, 1H), 7.89 (m, 1H), 12.46 (s, 1H); 13C NMR (100 MHz, DMSO-
d6) d: 172.3, 132.8, 132.4, 131.8, 131.4, 130.7, 127.9, 124.5, 123.3,
123.1, 121.7, 121.4, 119.1, 118.8, 112.4, 44.0, 35.2; MS m/z: nega-
tive mode 423.7, 425.8 ([MÀH]À), positive mode 426.9, 427.9
([M+H]+), 448.9, 449.9 ([M+Na]+).
5.8.3. 2-[2-(4-(Trifluoromethyl)benzyl)-1,1-dioxido-3,4-
dihydro-2H-1,2-benzothiazin-yl]acetic acid methyl ester (7c)
White powder (0.35 g, 85%); mp 101–103 °C; 1H NMR
(400 MHz, DMSO-d6) d: 2.64 (m, 1H), 2.77 (m, 1H), 3.45 (s, 3H),
3.57 (m, 1H), 3.60–3.68 (m, 2H), 4.20 (d, J = 14.8 Hz, 1H), 4.59 (d,
J = 14.8 Hz, 1H), 7.51 (d, J = 6.8 Hz, 2H), 7.60 (m, 3H), 7.75 (d,
J = 7.6 Hz, 2H), 7.81 (d, J = 8.0 Hz, 1H).
5.9.5. 2-[2-(3-Nitrobenzyl)-1,1-dioxido-2H-1,2-benzothiazin-
4(3H)-ylidene]acetic acid (8e)
Colorless crystal (0.13 g, 67%); purity: 97.8%; mp 194–196 °C;
1H NMR (400 MHz, DMSO-d6) d: 3.59 (s, 2H), 5.08 (s, 2H), 7.03 (s,
1H), 7.57 (m, 1H), 7.63 (m, 2H), 7.73 (m, 2H), 7.92 (m, 1H), 8.14
(m, 1H), 8.20 (s, 1H), 12.48 (s, 1H); 13C NMR (100 MHz, DMSO-
d6) d: 172.3, 147.8, 139.0, 134.3, 132.8, 132.5, 131.4, 130.7, 130.3,
128.0, 124.6, 122.9, 122.5, 121.5, 112.6, 49.3, 35.2; MS m/z: nega-
tive mode 372.9 ([MÀH]À), positive mode 375.0 ([M+H]+), 397.0
([M+Na]+).
5.8.4. 2-[2-(2-Fluorobenzyl)-1,1-dioxido-3,4-dihydro-2H-1,2-
benzothiazin-yl]acetic acid methyl ester (7d)
The title compounds were obtained as described for 7a starting
from 6f. White powder (0.34 g, 94%); mp 93–95 °C; 1H NMR
(400 MHz, DMSO-d6) d: 2.60 (m, 1H), 2.78 (m, 1H), 3.49 (s, 3H),
3.57 (m, 1H), 3.60–3.68 (m, 2H), 4.13 (d, J = 14.0 Hz, 1H), 4.57 (d,
J = 14.0 Hz, 1H), 7.23 (m, 2H), 7.39 (m, 1H), 7.42–7.52 (m, 3H),
7.61 (t, 1H), 7.80 (d, J = 7.6 Hz, 1H).
5.9.6. 2-[2-(2-Fluorobenzyl)-1,1-dioxido-2H-1,2-benzothiazin-
4(3H)-ylidene]acetic acid (8f)
5.9. General synthetic procedure for 8a–f
Colorless crystal (0.14 g, 83%); purity: 96.1%; mp 181–183 °C;
1H NMR (400 MHz, DMSO-d6) d: 3.60 (s, 2H), 4.97 (s, 2H), 6.96 (s,
1H), 7.17 (m, 2H), 7.35 (m, 2H), 7.61 (m, 2H), 7.74 (m, 1H), 7.90
(d, J = 7.6 Hz, 1H), 12.47 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d:
172.3, 132.8, 132.4, 131.5, 130.7, 130.1, 127.9, 124.6, 124.5,
123.6, 123.4, 121.3, 115.6, 115.4, 112.3, 44.3, 35.2; MS m/z: nega-
tive mode 345.9 ([MÀH]À), positive mode 370.1 ([M+Na]+).
A mixture of the appropriate 6 (0.5 mmol), 1,4-dioxane (5 mL)
and saturated aqueous sodium hydroxide (8 mL) was stirred at
room temperature for 12 h. The alkaline suspension was adjusted
to acidic with 0.1 N HCl and extracted threefold with ethyl acetate
(3 Â 30 mL). The combined organic layers were dried over MgSO4
and filtered. The filtrate was concentrated to dryness under re-
duced pressure and the residue of the desired compound was
recrystallised in methanol to give pure product.
5.10. General synthetic procedure for 9a–d
The title compounds were obtained as described for 8 starting
from 7a–d (0.5 mmol).
5.9.1. 2-[2-(2,4,5-Trifluorobenzyl)-1,1-dioxido-2H-1,2-
benzothiazin-4(3H)-ylidene]acetic acid (8a)
Colorless crystal (0.13 g, 69%); purity: 98.9%; mp 174–176 °C;
1H NMR (400 MHz, DMSO-d6) d: 3.61 (s, 2H), 4.93 (s, 2H), 6.98 (s,
1H), 7.43 (m, 1H), 7.54–7.63 (m, 3H), 7.75 (m, 1H), 7.90 (m, 1H),
12.48 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 172.3, 132.8,
132.5, 131.3, 130.7, 128.0, 124.6, 121.4, 118.4, 118.2, 112.6,
106.7, 106.5, 106.4, 106.2, 43.8, 35.2; MS m/z: negative mode
381.9 ([MÀH]À), positive mode 406.0 ([M+Na]+).
5.10.1. 2-[2-(2,4,5-Trifluorobenzyl)-1,1-dioxido-3,4-dihydro-2H-
1,2-benzothiazin-yl]acetic acid (9a)
Colorless crystal (0.11 g, 58%); purity: 99.2%; mp 75–78 °C; 1H
NMR (400 MHz, DMSO-d6) d: 2.54 (m, 1H), 2.72 (m, 1H), 3.56 (m,
1H), 3.65 (m, 2H), 4.13 (d, J = 14.0 Hz, 1H), 4.52 (d, J = 14.0 Hz,
1H), 7.47 (m, 1H), 7.51 (s, 1H), 7.58 (m, 2H), 7.62 (s, 1H), 7.79 (d,
J = 7.6 Hz, 1H), 12.38 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d:
172.4, 138.3, 136.0, 132.7, 128.9, 127.7, 123.9, 119.4, 119.3,
106.6, 106.4, 106.3, 106.1, 48.9, 43.5, 37.3, 30.6; MS m/z: negative
mode 384.0 ([MÀH]À), positive mode 408.0 ([M+Na]+).
5.9.2. 2-[2-(3,5-Bis(trifluoromethyl)benzyl)-1,1-dioxido-2H-1,2-
benzothiazin-4(3H)-ylidene]acetic acid (8b)
Colorless crystal (0.19 g, 82%); purity: 98.6%; mp 182–184 °C;
1H NMR (400 MHz, DMSO-d6) d: 3.60 (s, 2H), 5.14 (s, 2H), 7.06 (s,
1H), 7.58 (m, 1H), 7.61 (m, 1H), 7.76 (m, 1H), 7.90 (m, 1H), 7.94
(s, 2H), 8.05 (s, 1H), 12.49 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
d: 172.3, 140.4, 132.8, 132.6, 131.4, 130.9, 130.7, 130.3, 129.9,
5.10.2. 2-[2-(3,5-Bis(trifluoromethyl)benzyl)-1,1-dioxido-3,4-
dihydro-2H-1,2-benzothiazin-yl]acetic acid (9b)
Colorless crystal (0.17 g, 72%); purity: 95.0%; mp 180–182 °C;
1H NMR (400 MHz, DMSO-d6) d: 2.56 (m, 1H), 2.75 (m, 1H), 3.56

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X. Chen et al. / Bioorg. Med. Chem. 19 (2011) 7262–7269
(m, 1H), 3.72 (m, 2H), 4.44 (d, J = 15.2 Hz, 1H), 4.66 (d, J = 15.2 Hz,
1H), 7.50 (m, 2H), 7.62 (m, 1H), 7.80 (d, J = 8.0 Hz, 1H), 8.05 (s, 2H),
8.06 (s, 1H) , 12.41 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 172.4,
140.2, 138.3, 135.9, 132.7, 130.5, 130.2, 129.9, 129.1, 128.9, 127.7,
124.6, 123.9, 121.9, 121.5, 49.8, 49.3, 37.4, 30.4; MS m/z: negative
mode 466.0 ([MÀH]À), positive mode 468.1 ([M+H]+),
490.1([M+Na]+).
solution to the reaction mixture described above. All compounds
were dissolved in dimethyl sulfoxide (DMSO) and diluted with
deionized water. To correct for the nonenzymatic oxidation of
NADPH, 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 10À5
M
(the concentration is referred to that of the compound in the reac-
tion mixture). Those compounds found to be active were tested at
additional concentrations between 10À5 and 10À7 M. Each dose-
effect 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 À2, where n is the total
number of determinations.
5.10.3. 2-[2-(4-(Trifluoromethyl)benzyl)-1,1-dioxido-3,4-
dihydro-2H-1,2-benzothiazin-yl]acetic acid (9c)
Colorless crystal (0.14 g, 68%); purity: 98.6%; mp 177–178 °C;
1H NMR (400 MHz, DMSO-d6) d: 2.58 (m, 1H), 2.75 (m, 1H), 3.59
(m, 1H), 3.67 (m, 2H), 4.28 (d, J = 15.2 Hz, 1H), 4.55 (d,
J = 15.2 Hz, 1H), 7.47–7.52 (m, 2H), 7.61 (m, 3H), 7.74 (d,
J = 8.0 Hz, 2H), 7.81 (d, J = 8.0 Hz, 1H), 12.41 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) d: 172.4, 141.2, 138.4, 136.1, 132.7, 129.0,
128.8 (2C), 128.4, 127.7, 125.5, 125.4, 123.9, 109.4, 49.6, 49.3,
37.4, 30.3; MS m/z: negative mode 398.0 ([MÀH]À), positive mode
422.0 ([M+Na]+).
5.13. Docking experiments
Conformational searches and molecular docking were per-
formed using version 7.1 of the SYBYL
.
5.10.4. 2-[2-(2-Fluorobenzyl)-1,1-dioxido-3,4-dihydro-2H-1,2-
benzothiazin-yl]acetic acid (9d)
Acknowledgments
Colorless crystal (0.11 g, 63%); purity: 97.3%; mp 136–138 °C;
1H NMR (400 MHz, DMSO-d6) d: 2.57 (m, 1H), 2.73 (m, 1H), 3.59
(m, 1H), 3.65 (m, 2H), 4.19 (d, J = 14.4 Hz, 1H), 4.52 (d,
J = 14.4 Hz, 1H), 7.21 (m, 2H), 7.39 (m, 1H), 7.47–7.54 (m, 3H),
7.61 (m, 1H), 7.79 (d, J = 7.2 Hz, 1H), 12.39 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) d: 177.4, 138.3, 136.2, 132.6, 131.4, 130.2,
128.9, 127.7, 124.6, 123.8, 122.9, 115.6, 115.4, 49.0, 44.1, 37.5,
30.3; MS m/z: negative mode 347.9 ([MÀH]À), positive mode
350.0 ([M+H]+), 372.0 ([M+Na]+).
This work was supported by the Beijing Natural Science Foun-
dation (No. 7102091) and China Major Infectious Research Projects
(no. 2008ZX10001-006).
References and notes
1. Hers, H. G. Biochim. Biophys. Acta 1956, 22, 202.
2. van Heyningen, R. Nature (London) 1959, 184, 194.
3. Gabbay, K. H.; Merola, L. O.; Field, R. A. Science 1966, 151, 209.
4. Kinoshita, J. H.; Nishimura, C. Diabetes Metab. Rev. 1988, 4, 323.
5. Yabe-Nishimura, C. Pharmacol. Rev. 1998, 50, 21.
5.11. Biology, materials, and methods
6. Chung, S. S.; Chung, S. K. Curr. Med. Chem. 2003, 10, 1375.
7. Kador, P. F.; Kinoshita, J. H.; Sharpless, N. E. J. Med. Chem. 1985, 28, 841.
8. Dvornik, D.; Simard-Duquesne, N.; Keami, M.; Sestanj, K. Science 1973, 182,
1146.
ALR2 and ALR1 were obtained from Wistar rats, 200–250 g,
b.w., supplied by Vital River, Beijing, China.
D,L-Glyceraldehyde, so-
9. Sestanj, K.; Bellini, F.; Fung, S.; Abraham, N.; Treasurywala, A.; Humber, L.;
Simard-Duquesne, N.; Dvornik, D. J. Med. Chem. 1984, 27, 255.
10. Kikkawa, R.; Hatanaka, I.; Yasuda, H.; Kobayashi, N.; Shigeta, Y.; Terashima, H.;
Morimura, T.; Tsuboshima, M. Diabetologia 1983, 24, 290.
11. Mylari, B. L.; Larson, E. R.; Beyer, T. A.; Zembrowski, W. J.; Aldinger, C. E.; Dee,
M. F.; Siegel, T. W.; Singleton, D. H. J. Med. Chem. 1991, 34, 108.
12. Ao, S.; Shingu, Y.; Kikuchi, C.; Takano, Y.; Nomura, K.; Fujiwara, T.; Ohkubo, Y.;
Notsu, Y.; Yamaguchi, I. Metabolism 1991, 40, 77.
13. Stribling, D.; Mirrlees, D. J.; Harrison, H. E.; Earl, D. C. Metabolism 1985, 34, 336.
14. Van Zandt, M. C.; Jones, M. L.; Gunn, D. E.; Geraci, L. S.; Jones, J. H.; Sawicki, D.
R.; Sredy, J.; Jacot, J. L.; DiCioccio, A. T.; Petrova, T.; Mitschler, A.; Podjarny, A. D.
J. Med. Chem. 2005, 48, 3141.
dium -glucuronate, and NADPH were from Sigma–Aldrich. All
other chemicals were of reagent grade. Epalrestat was prepared
in our laboratory according to a reported method.
ALR1 and ALR2 were prepared in accordance with the method
of Kinoshita34 and Concettina La Motta.35 Enzyme activity was as-
sayed spectrophotometrically on an Unico 4802S UV/VIS double
beam spectrophotometer by measuring the decrease in absorption
of NADPH at 340 nm, which accompanies the oxidation of NADPH
catalyzed by ALR2 and ALR1.
D
15. Da Settimo, F.; Primofiore, G.; La Motta, C.; Sartini, S.; Taliani, S.; Simorini, F.;
Marini, A. M.; Lavecchia, A.; Novellino, E.; Boldrini, E. J. Med. Chem. 2005, 48,
6897.
5.12. Enzymatic assays
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ALR2 activity was performed at 32 °C in a reaction mixture con-
taining 0.25 mL of 0.10 mM NADPH, 0.25 mL of 0.1 M sodium
phosphate buffer (pH 6.2), 0.1 mL of enzyme extract, 0.15 mL of
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D,L
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ALR1 activity was performed at 37 °C in a reaction mixture con-
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0.25 mL of 0.1 M sodium phosphate buffer (pH 7.2), 0.15 mL of
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D-glucuronate as
D
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substrate was then added to start the reaction, which was moni-
tored for 4 min.
The inhibitory activity of the newly synthesized compounds
against ALR2 and ALR1 was assayed adding 5 lL of the inhibitor

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