Acetic acid derivatives of 3,4-dihydro-2 H-1,2,4-benzothiadiazine 1,1-dioxide as a novel class of potent aldose reductase inhibitors

Article abstract of DOI:10.1021/jm100962a

A series of novel benzothiadiazine 1,1-dioxide derivatives were synthesized and tested for their inhibitory activity against aldose reductase. Of these derivatives, 17 compounds, having a substituted N2-benzyl group and a N4-acetic acid group on the benzothiadiazine, were found to be potent and selective aldose reductase inhibitors in vitro with IC₅₀ values ranging from 0.032 to 0.975 μM. 9m proved to be the most active in vitro. The eight top-scoring compounds coming from the in vitro test for ALR2 inhibition activity were then tested in vivo, whereby three derivatives, 9i, 9j, and 9m, demonstrated a significantly preventive effect on sorbitol accumulation in the sciatic nerve in the 5-day streptozotocin-induced diabetic rats in vivo. Structure-activity relationship and molecular docking studies highlighted the importance of substitution features of N4-acetic acid group and halogen-substituted N2-benzyl group in the benzothiadiazine scaffold and indicated that substitution with hallogen at C-7 had a remarkably strong effect on ALR2 inhibition potency.

Full text of DOI:10.1021/jm100962a

8330 J. Med. Chem. 2010, 53, 8330–8344
DOI: 10.1021/jm100962a
Acetic Acid Derivatives of 3,4-Dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxide as a Novel Class
of Potent Aldose Reductase Inhibitors
Xin Chen,† Changjin Zhu,*,† Fan Guo,† Xiaowei Qiu,† Yanchun Yang,† Shuzhen Zhang,† Minlan He,† Shagufta Parveen,†
Chaojun Jing,† Yan Li,‡ and Bing Ma*,†
†Department of Applied Chemistry, School of Chemical Engineering and Environment, Beijing Institute of Technology, Zhongguancun South Street,
100081 Beijing, China, and ‡School of Life Science and Biotechnology, Beijing Institute of Technology, Zhongguancun South Street,
100081 Beijing, China
Received July 28, 2010
A series of novel benzothiadiazine 1,1-dioxide derivatives were synthesized and tested for their
inhibitory activity against aldose reductase. Of these derivatives, 17 compounds, having a substituted
N2-benzyl group and a N4-acetic acid group on the benzothiadiazine, were found to be potent and
selective aldose reductase inhibitors in vitro with IC50 values ranging from 0.032 to 0.975 μM. 9m proved
to be the most active in vitro. The eight top-scoring compounds coming from the in vitro test for ALR2
inhibition activity were then tested in vivo, whereby three derivatives, 9i, 9j, and 9m, demonstrated a
significantly preventive effect on sorbitol accumulation in the sciatic nerve in the 5-day streptozotocin-
induced diabetic rats in vivo. Structure-activity relationship and molecular docking studies highlighted
the importance of substitution features of N4-acetic acid group and halogen-substituted N2-benzyl
group in the benzothiadiazine scaffold and indicated that substitution with hallogen at C-7 had a
remarkably strong effect on ALR2 inhibition potency.
Introduction
the complications.3,4 Patients with all forms of diabetes of
sufficient duration, including insulin-dependent diabetes mel-
litus and non-insulin-dependent diabetes mellitus, are vulner-
able to these complications, which cause serious morbidity.
Increasing evidence suggests that the enzyme aldose reductase
(ALR2,a EC 1.1.1.21) may provide a common biochemical
link in the pathogenesis of many of diabetic complications5-7
and that hyperactivity of the polyol metabolic pathway cat-
alyzed by the enzyme in individuals with high blood glucose
levels contributes to the progression of diabetic compli-
cations.8-10
Aldose reductase, a member of the aldo-keto reductase
(AKR) superfamily of enzymes,11 together with sorbitol
dehydrogenase forms the polyol pathway. In this pathway
aldose reductase initially catalyzes the NADPH-dependent
reduction of the aldehyde form of glucose to form sorbitol.
Sorbitol dehydrogenase in turn, utilizing NADþ, oxidizes this
intermediate polyol to fructose (Figure 1).8 Therefore, aldose
reductase have been a potential target for drug design and
aldose reductase inhibitors (ARIs) have received attention as
possible therapeutic drugs for the prevention and treatment of
diabetic complications.4,11 Since tetramethyleneglutaric acid
was identified as the first ARI in the 1960s,12 a number of
structurally different ARIs including at least two chemical
classes of structures have been developed (Figure 2). The first
chemical class is carboxylic acids such as alrestatin,13 tolre-
stat,14 epalrestat,15 zopolrestat,16 zenarestat,17 ponalrestat,18
and recently developed lidorestat,19 naphtho[1,2-d]isothiazole
derivatives,20 and acetic acid derivatives of oxadiazole.21
Epalrestat is the only ARI as a drug commercially available
in Japan and recently in China, and lidorestat and derivatives
of oxadiazole have been identified as a highly potent and
Diabetes mellitus is a chronic disease that requires long-term
medical attention to limit the development of its devastating
complications and to manage them when these are active. This
disease is a major threat to global public health that is rapidly
getting worse, as it afflicted over 171 million people worldwide
in 2000 and the incidence is expected to grow steadily to 366
million by 2030.1 China is among the top 3 countries and may
bear a higher diabetes-related burden than any other country.
A very recent report showed that the prevalence of diabetes
and prediabetes was 9.7% and 15.5%, respectively, accounting
for 92.4 million adults with diabetes and 148.2 million adults
with prediabetes.2 These estimates showed approximately 2
times higher factor of than those reported in 2003. It is a
disproportionately expensive disease; in 2002, the per capita
cost of health care was $13,243 for people with diabetes while it
was $2560 for those without diabetes.1
The complications that are specific to diabetes include
retinopathy, nephropathy, and neuropathy.3 Also, a substan-
tial increase in atherosclerotic disease of large vessels, includ-
ing cardiac, cerebral, and peripheral vascular disease, is seen in
*To whom correspondence should be addressed. For C.Z.: phone,
phone: þ86-10-68918506; fax, þ86-10-68918506; e-mail, zcj@bit.edu.
cn. For B.M.: phone, +86-10-68918506; fax, +86-10-68918506; e-mail,
mabing@bit.edu.cn.
a Abbreviations: ALR2, aldose reductase; ALR1, aldehyde reduc-
tase; ARI, aldose reductase inhibitor; AKR, aldo-keto reductase;
AGEs, advanced glycation end products; AMPA, 2-amino-3-(3-hydroxy-
5-methylisoxazol-4-yl)propionate; DMSO, dimethylsulfoxide; HNE,
hydroxynonenal; NADPH, β-nicotinamide adenine dinucleotide phos-
phate reduced form; NADPþ, β-nicotinamide adenine dinucleotide
phosphate; NADH, β-nicotinamide adenine dinucleotide reduced form;
NADþ, β-nicotinamide adenine dinucleotide; STZ, streptozotocin;
SAR, structure-activity relationship.
r
pubs.acs.org/jmc
Published on Web 11/09/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8331
selective ARIs with a favorable pharmacokinetic profile,19,21
although poor tissue penetration has been observed as the
major shortcoming in some of this class.22,23 The second
chemical class is spirohydantoins involving sorbinil24 and
fidarestat.25 Fidarestat and the spirohydantoin-like spiro-
imide ARI ranirestat (AS-3201)26 have been identified as
powerful ARIs with remarkable efficacy and safety,25,27,28
although the hypersensitivity has been observed for some of
the spirohydantoins.22,23 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-one29 and flavinoid bioisoster pyridopyrimi-
dinones (PPP)30 have been reported for their excellent selec-
tivity and potent inhibitory activity both in vitro and in vivo.
Despite these excellent efforts, to date, none of the ARIs have
been approved for clinical use worldwide. Many ARIs that
had promising activity against ALR2 during in vitro and in
vivo studies with animal models failed to be advanced through
late stage of clinical trials 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. Both
ALR1 and ALR2 are members of the aldo-keto reductase
(AKR) superfamily and present in a number of tissues. They
share a high degree of sequence (∼65%) and structural
homology31 with the majority of the differences present at
the C-terminal end of the enzyme proteins,32 which is the
region containing the least conserved residues and lining the
hydrophobic pocket of the active site called the “specificity
pocket”.33 The“specificitypocket”is responsible forsubstrate
and inhibitor specificity in the AKRs.34 ALR1 plays a detox-
ification role, as it specifically metabolizes toxic aldehydes such
as hydroxynonenal (HNE), 3-deoxyglucosone, and methyl-
glyoxal, which arise in 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).35-37 Thus, ALR1 inhibition
may account for some of the undesirable side effects associated
with the present ARIs. It is believable that the development of
more structurally diverse ARIs and in turn the identification of
molecularly targeted candidates that specifically block ALR2
are important approaches in the search of new drugs.
In the present study, we have developed a new class of
potent ARIs having a benzothiadiazine 1,1-dioxide scaffold
and N4-acetic acid and substituted N2-benzyl residues. Since
the 1950s, various pharmacological investigations of newly
synthesized benzothiadiazines demonstrated interesting phar-
macological activities and showed great potential for devel-
opment of new medications for treating diseases. They were
studied extensively for their diuretic, hypoglycemic, antihy-
pertensive, and potassium channels opener activity.38,39 Apart
from the above-mentioned activities, benzothiadiazine deriv-
atives also showed some interesting efficacy as highly potent
HIV-1 non-nucleoside reverse transcriptase inhibitors40 and
potentiators of AMPA receptors (Figure 3).41-46 Therefore,
various benzothiadiazine compounds are of considerable
interest for their diverse pharmaceutical uses. The above
considerations prompted us to design and synthesize a new
series of substituted benzothiadiazine 1,1-dioxide derivatives
and to test their ALR2 inhibition activity both in vitro and
in vivo. The present work describes the synthesis of the
benzothiadiazine 1,1-dioxide derivatives as well as SAR and
molecular docking studies on their ALR2 inhibition activity.
Chemistry
All compounds including 5a, 6-10, and 13-15 (Figure 4)
described in this study have been obtained by synthesis either
Figure 1. Polyol pathway of glucose metabolism.
Figure 2. Chemical structures of aldose reductase inhibitors.

8332 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Chen et al.
starting from aniline or substituted aniline (1a-d), as shown
in Schemes 1 and 2.
N4-Acetic Acid Derivatives of 1,2,4-Benzothiadiazine 1,1-
Dioxide. Compounds 2a-d were synthsized in three steps
from aniline and 4-substituted aniline (1a-d) as shown in
Scheme 1.47 N-Acylation of anilines 1a-d with chlorosulfo-
nyl isocyanate in nitromethane formed ureas, which were
then sulfonylated intramolecularly in the presence of alumi-
num chloride to produce 3-oxo-1,2,4-benzothiadiazine 1,1-
dioxides by ring closure. Removal of the carbonyl group of
these products by treatment with sulfuric acid and then
sodium hydroxide yielded the corresponding aminoben-
zenesulfonamides 2a-d.
Compounds 5a-d served as intermediates for the synthe-
sis of novel N4-carboxylic acid derivatives of benzothiadia-
zine 1,1-dioxide 6-10. The preparation of the intermediates
5a-d from the aminobenzenesulfonamides 2a-d was carried
out using the synthetic procedure described by Francotte and
co-workers.46 Compounds 2a-d reacted with triethyl ortho-
formate to give corresponding 3a-d by ring closure. To
introduce an alkylcarboxylic acid residue as a side chain at
the N4 position, compounds 3a-d were alkylated with the
methyl bromoacetate in the presence of potassium carbonate
in acetonitrile, yielding compounds 4a-d. Saturation of the
double bond in the 2,3-positions of 4a-d was achieved in
2-propanol by means of sodium borohydride to give the
methyl esters 5a-d. The C-7 unsubstituted ester 5a was
hydrolyzed with aqueous sodium hydroxide or aminolyzed
Figure 3. Chemical structures of 1,2,4-benzothiadiazine 1,1-diox-
ide derivatives according to Francotte and co-workers.46
Figure 4. Substituted 1,2,4-benzothiadiazine 1,1-dioxide derivatives (5a, 6-10, and 13-15).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8333
Scheme 1a
a Reagents and conditions: (a) ClSO2NCO, CH3NO2, -40 °C; (b) AlCl3, 110 °C; (c) 50% H2SO4, 140 °C; (d) aq NaOH; (e) HC(OEt)3, reflux;
(f) BrCH2COOCH3, K2CO3, CH3CN, 70 °C; (g) NaBH4, 2-propanol; (h) Bn-Br, K2CO3, CH3CN, 70 °C; (i) 1,4-dioxane, NaOH; (j) NH3, CH3OH.
Scheme 2a
with ammonia to generate acid product 6 and amide 7,
respectively. In the synthesis of the other end products,
introduction of a benzyl group as another side chain at the
N2 position of esters 5a-d was achieved by the alkylation
reaction with the appropriate benzyl bromide to afford the
corresponding methyl esters 8a-q, which were then con-
verted to the carboxylic acids 9a-q and amides 10a-q,
respectively, by using the same procedures as those for the
formation of the carboxylic acid 6 and the amide 7.
N2-Carboxylic Acid Derivatives of 1,2,4-Benzothiadiazine
1,1-Dioxide. The same synthetic methods as described above
were applied for the preparation of the expected compounds
13-15 starting from compound 3a (Scheme 2). Alkylation of
compound 3a at the N4 position with the appropriate benzyl
bromide produced 11a-d, which were then hydrogenated
with sodium borohydride to give compounds 12a-d.
A second alkylation at the N2 position by the reaction of
12a-d with the methyl bromoacetate afforded the methyl
esters 13a-d. Saponification of the esters with aqueous
sodium hydroxide led to carboxylic acids 14a-d, and ami-
nolysis of the esters 13a and 13b with ammonia provided
amides 15a and 15b, respectively.
a Reagents and conditions: (a) Bn-Br, K2CO3, CH3CN, 70 °C;
(b) NaBH4, 2-propanol; (c) BrCH2COOCH3, K2CO3, CH3CN, 70 °C;
(d) 1,4-dioxane, NaOH; (e) NH3, CH3OH.
Results and Discussion
Inhibition of Enzymes in Vitro. To ascertain which struc-
tural features are required for ALR2 inhibition activity and
selectivity of substituted benzothiadiazine 1,1-dioxide deriv-
atives, the structurally diverse compounds described above
(Figure 4) were designed and synthesized for testing (Tables 1
and 2). The all synthesized compounds including 5a, 6-10,

8334 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Chen et al.
Table 1. In Vitro Data for N2-Benzyl-N4-carboxyl Derivatives of
Benzothiadiazine 1,1-Dioxide
Table 2. In Vitro Data for N2-Carboxyl-N4-benzyl Derivatives of
Benzothiadiazine 1,1-Dioxide
compd
R
ALR2 IC50 (μM)a
substituent
R1 R2
2-F,4-Br 0.125 (0.093-0.157) 70.3 (51.5-89.2)
IC50 (μM) a
14a
14b
14c
14d
2-F,4-Br
3-NO2
4-CF3
58.4 (46.3-70.4)
74.5 (58.9-90.1)
41.3% b
compd
9a
9b
ALR2
ALR1
H
H
H
H
H
F
3-NO2
4-CF3
0.219 (0.153-0.286) 55.8 (34.7-76.8)
2,4,5-F3
14.0 (9.25-18.7)
a IC50 (95% CL) values represent the concentration required to
produce 50% enzyme inhibition. b The inhibitory effect was estimated
at 10-4 M.
9c
9d
0.975 (0.760-1.189) 42% c
2,4,5-F3 0.111(0.095-0.127)
H 101.5 (85.9-117.2)
b
b
9e
9f
9g
2-F,4-Br 0.090 (0.064-0.116) 121.9 (108.1-135.8)
F
3-NO2
4-CF3
0.108 (0.088-0.129) 61.9 (51.9-71.9)
0.931 (0.721-1.141) 40% c
ranirestat, minalrestat, zenarestat, and ponalrestat (Figure 2).
This modification was supported by docking studies. It was
found that 9a showed a significantly enhanced ALR2 inhibi-
tion effect compared with the parent compound 6, and the
activity (IC50 = 0.125 μM) was approximately 3 orders of
9h
9i
F
F
2,4,5-F3 0.066 (0.054-0.077) 80.9 (62.5-99.2)
9j
Cl 2-F,4-Br 0.041 (0.034-0.048) 58.9 (47.2-70.6)
Cl 3-NO2
Cl 4-CF3
9k
9l
0.094 (0.068-0.120) 82.5 (69.8-95.2)
0.595 (0.465-0.725) 45% c
9m
9n
Cl 2,4,5-F3 0.032 (0.024-0.040) 50.6 (36.0-65.2)
Br 2-F,4-Br 0.071 (0.052-0.090) 69.4 (57.7-81.1)
Br 3-NO2
Br 4-CF3
magnitude greater than that of 6 (IC50 = 0.125 versus IC50
=
93.9 μM). By taking into account the key structural features
required for ALR2 inhibition activity, we sought to design
and synthesize variants that display a greater activity. There-
fore, further substitutions at the N2-benzyl ring combined
with substitutions at the 7-position resulted in the prepara-
tion of a series of acetic acid derivatives 9b-q. We also
obtained the corresponding ester derivatives 8a-q and
amide derivatives 10a-q (Figure 4) in order to investigate
structure-activity relationships.
As expected above and shown (Table 1), all of the N2-
benzyl and N4-acetic acid substituted derivatives (9a-q)
except 9e showed significantly inhibitory activity against
ALR2. Of these acid derivatives, 2-(2,4,5-trifluorobenzyl)-
4-carboxymethyl-7-cholro-3,4-dihydro-2H-1,2,4-benzothia-
diazine 1,1-dioxide 9m was the most active compound having
an IC50 value of 0.032 μM. Four compounds 9i, 9j, 9n, and 9q
also demonstrated potent ALR2 inhibition activity with IC50
values of 0.066, 0.041, 0.071, and 0.052 μM, respectively.
However, only compound 9e exhibited an almost negligible
ALR2 inhibition activity (IC50 = 101.5 μM), and the
remaining 11 compounds including 9c, 9h, 9p, 9l, 9b, 9a,
9o, 9d, 9g, 9k, and 9f showed varying levels of efficiency with
IC50 values ranging from 0.975 to 0.090 μM.
We will now discuss SAR relative to substituents at the
N2-benzyl ring in the N4-acetic acid substituted derivatives
9a-q, which could be arranged according to 7-substituent in
series: 9a-e without 7-substitution, 9f-i with the 7-fluoro
substituent, 9j-m with the 7-chloro substituent, and 9n-q
with the 7-bromo substituent (Table 1). By comparison of the
compounds in series 9a-e, compound 9e without substitu-
tion at the N2-benzyl ring greatly lost ALR2 inhibition
activity (IC50 = 101.5 μM) while compounds with substitu-
tions of 2-F with 4-Br (9a), 3-NO2 (9b), 4-CF3 (9c), and 2,4,5-
F3 (9d) at the benzyl ring all retained potent activities
(IC50 = 0.125, 0.219, 0.975, 0.111 μM). The 2,4,5-F3-benzyl
derivative 9d was found to be the most active within this
series. Similarly, comparing compounds in each of the
remaining series 9f-i, 9j-m, and 9n-q revealed that com-
pounds 9i, 9m, and 9q all having 2,4,5-trifluoro substituents
9o
9p
0.116 (0.089-0.143) 102.9 (86.5-119.3)
0.674 (0.525-0.824) 109.6 (87.5-131.8)
9q
fidarestat
Br 2,4,5-F3 0.052 (0.038-0.067) 54.1 (36.6-71.6)
0.029 (0.020-0.037)
b
a IC50 (95% CL) values represent the concentration required to
produce 50% enzyme inhibition. b Not determined. c The inhibitory
effect was estimated at 10-4 M.
and 13-15 were tested for their potential inhibitory effect on
ALR2 isolated from rat lenses. Only significantly active
compounds coming from in vitro test for ALR2 inhibition
activity were then subjected to test for their inhibition
activity against aldehyde reductase (ALR1), isolated from
rat kidneys, in order to evaluate their selectivity for ALR2
inhibition. The results are expressed as IC50 (μM) or the
percentage of enzyme inhibition (%)summarized in Tables1,
2, and S3-S5.
For the building of target ARIs based on the benzothia-
diazine 1,1-dioxide scaffold, initially, carboxylic acid deriv-
atives (ester 5a, acid 6, and amide 7) were prepared by
introducing an acetic acid group as only a side chain to the
N4 position because carboxylic acid residue such as acetic
acid group in well-known ARIs (Figure 2)13-19 is believed to
hold an important role in the interaction with ALR2 enzyme,
especially in physiological conditions.48 Recently successful
examples of the carboxylic acid ARIs have been reported by
Van Zandt, La Motta, and their co-workers.19,21
Of the three benzothiadiazine 1,1-dioxide derivatives, the
acid 6 showed a little inhibition activity against ALR2 with
IC50 value of 93.9 μM, whereas both the ester 5a and the
amide 7 were inactive (Table S3), indicating that simply
retaining the carboxylic acid residue as in 6 is not sufficient
for ALR2 inhibition activity. Therefore, we assumed that an
additional substituent at the N2 position of the acid 6 as a
second side chain could improve ALR2 inhibition potency.
This consideration led to the modification, the acid deriv-
ative 9a, by attaching a 2-fluoro-4-bromobenzyl residue to
the N2 position of 6 because this substituent as a side chain
had already been employed to design potent ARIs such as

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8335
at the benzyl ring were the most active within each subseries,
respectively. Moreover, it was found that effects of substitu-
tion 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 as deduced from the comparison of compounds in
7-unsubstituted derivative series (9d > 9a > 9b > 9c),
7-fluoro substituted derivative series (9i > 9f > 9g > 9h),
7-chloro derivative series (9m > 9j > 9k > 9l), and 7-bromo
derivative series (9q > 9n > 9o > 9p). These results suggest
that apart from the acetic acid residue at the N4 position, the
N2 benzyl residue with halogen substituents at the benzyl
ring is essential for the activity of ARIs based on the
benzothiadiazine 1,1-dioxide scaffold.
Further SAR studies on substituents at the 7-position in
the derivatives 9a-q except for 9e indicated that ALR2
inhibition activity of the derivatives obtained from the
introduction of the N4-acetic acid group combined with
substitutions at the N2-benzyl ring could be further in-
creased by substitutions with halogen atoms at the 7-posi-
tion. It was evidenced by comparing biological activities of
compounds in the 2-F,4-Br-benzyl derivative series including
9f with 7-F group, 9j with 7-Cl, 9n with 7-Br, and 9a with
7-unsubstitution, in which 9j together with 9n and 9f proved
to be more potent than 9a (IC50 = 0.041, 0.071, 0.090 μM vs
IC50 = 0.125 μM). Identically, comparing the activities of
compounds in the 3-NO2-benzyl derivative series including
9g with 7-F, 9k with 7-Cl, 9o with 7-Br, and 9b with
7-unsubstitution revealed that 9k, 9o, and 9g were more
on ALR2 inhibition activity, compounds 13-15 (Figure 4)
were prepared by repositioning of the carboxyl and the
benzyl residues on the benzothiadiazine 1,1-dioxide scaffold
of the corresponding compounds 8-10. Of these com-
pounds, however, only the N2-acetic acid derivatives 14a,
14c, and 14d showed a somewhat inhibitory activity against
ALR2 (IC50 = 58.4 μM, 74.5 μM, and 14.0 μM), while the
remaining N2-esters 13a-d and the N2-amides 15a and 15d
exhibited negligible activity as shown in Tables 2 and S5,
indicating that the N2-acetic acid-N4-benzyl substitutions
were not desirable. These results suggest that positions of
carboxylic acid and benzyl residues as side chains on the
benzothiadiazine 1,1-dioxide scaffold greatly influence bio-
logical activity in which substitution of the carboxylic acid
residue at the N4 position combined with substitution of the
benzyl residue at the N2 position drastically increases the
ALR2 inhibition activity other than substitutions of the two
residues at reversed positions. Comparable results were
found in the cases of compounds 9a-d versus their corre-
sponding compounds 14a-d (9a with N2-(2-F,4-Br-benzyl),
IC50 = 0.125 μM vs 14a with N4-(2-F,4-Br-benzyl), IC50
=
58.4 μM; 9b with N2-(3-NO2-benzyl), IC50 = 0.219 μM vs
14b with N4-(3-NO2-benzyl), IC50 = 74.5 μM; 9c with
N2-(4-CF3-benzyl), IC50 = 0.975 μM vs 14c with N4-(4-
CF3-benzyl), inhibition of 43% at 10-4 μM; 9d with
N2-(2,4,5-F3-benzyl), IC50 = 0.111 μM vs 14d with
N4-(2,4,5-F3-benzyl), IC50 = 14.0 μM).
As shown in Table 1, the N2-benzy-N4-carboxylic acid
derivatives 9a-q except for 9e with significantly potent
ALR2 inhibitory activity were tested for their inhibition
ability against ALR1. All of the tested compounds were
found to be slightly active to inactive (IC50 > 50 μM),
demonstrating their selectivity for ALR2.
active than 9b (IC50 = 0.094, 0.116, and 0.108 μM vs IC50
=
0.219 μM). Again, comparison of the activities of com-
pounds in the 4-CF3-benzyl derivative series including 9h
with 7-F, 9l with 7-Cl, 9p with 7-Br, and 9c with 7-unsub-
stitution revealed that 9l, 9p, and 9h were more active than 9c
(IC50 = 0.595, 0.674, and 0.931 μM vs 0.975 μM). Regarding
the 2,4,5-F3-benzyl derivative series, there were four individ-
uals including substitution at C-7 with F 9i, Cl 9m, Br 9q, and
unsubstituted 9d. Within this series, 9m was the most active
Biological Activity in Vivo. Of the 16 significantly potent
ARIs identified in the in vitro test as described above, the
eight top ranked compounds were then subjected to test for
oral in vivo activity in streptozotocin diabetic rat model as
measured by the ability of the inhibitors to normalize
sorbitol that had become elevated in diabetic nerve. In
particular, we focused our attention on the comparison of
2,4,5-F3-benzyl derivative series containing 9i, 9m, and 9q.
As shown in Table 3, three compounds 9i, 9j, and 9m showed
significant ALR2 inhibition activity in vivo, as they normal-
ized elevated sorbitol levels of the sciatic nerve of diabetic
rats by 68%, 61%, and 62%, respectively. Although no
statistically significant data from in vivo investigation were
available for the other test compounds, a trend toward the
normalization of sorbitol levels was observed. Particular
interest is that all three in vivo active compounds were most
readily distinguished by their trifluoro substituents on the
N2-benzyl ring. The finding is consistent with previous
results in which 2-fluoro substituted derivatives were found
to have the best oral activities among tolrestat analogues
containing 2-halo and other substituents.49 It seems there-
fore likely that the significant in vivo potency of the com-
pounds 9i and 9m may be greatly attributed to the presence of
the trifluoro substituents. This can be explained by assuming
that multifluoro substituted compounds are more favorable
for tissue penetration than less fluoro substituted com-
pounds in the present study and subsequently show more
potent activity in vivo. The assumption is strengthened by
comparing in vivo activities of 2,4,5-F3-benzyl derivative
series including 9i, 9m, and 9q. Within these three com-
pounds, 9i showed the most potent activity in vivo (68%)
(IC50 = 0.032 μM) while 9d was the least active (IC50
=
0.111 μM). Consequently, effects of substitutions at C-7 on
ALR2 inhibition activity showed the rank order to be Cl >
Br > F > H as deduced from the above comparisons (9j >
9n > 9f > 9a, 9k > 9o ∼ 9g > 9b, 9l > 9p > 9h > 9c, and
9m > 9q > 9i > 9d) and also showed that C-7 chloro
substituted derivatives 9j, 9k, 9l, and 9m were the most active
in each series, respectively, while unsubstituted 9a, 9b, 9c,
and 9d showed the lowest activity. However, unlike the
substitutions at the N4 position and at the benzyl ring, the
7-halogen was not essential for the activity.
In contrast to the compounds in the N4-acetic acid
derivative series 9a-q, except for 9e, which presented potent
ALR2 inhibition activities as described above, all of com-
pounds in the corresponding N4-acetic acid ester derivative
series 8a-q and the N4-amide derivative series 10a-q re-
vealed little or no inhibitory activity against ALR2 (Table
S4). In this case, substitutions neither at the N2-benzyl ring
nor at the 7-position could obviously influence biological
activity. It is possible to conclude from the results given
above that esterification and amidation of the N4-acetic acid
group result in a considerable loss of ALR2 inhibition
activity, and subsequently the carboxylic acid anion may
be the key structural feature required for ALR2 inhibition
activity of benzothiadiazine 1,1-dioxide derivatives.
In order to investigate the influence of positions of car-
boxyl and benzyl groups in the benzothiadiazine 1,1-dioxide

8336 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Chen et al.
Table 3. Biochemical and Nerve Sorbitol Assessment with 9f, 9g, 9i, 9j, 9k, 9m, 9n, and 9q in 5-Day STZ-Induced Diabetic Rat Screening Modela
group
blood glucose (mmol/L)
nerve sorbitol(μmol/g)
% improvementb
nondiabetic control
diabetic control
6.6 ( 0.43
0.1062 ( 0.0102
0.2055 ( 0.0542
0.1585 ( 0.0211
0.1622 ( 0.0301
0.1378 ( 0.0154*
0.1455 ( 0.0114*
0.1610 ( 0.0099
0.1444 ( 0.0240*
0.1634 ( 0.0170
0.1546 ( 0.0184
0.1039 ( 0.0105**
22.2 ( 2.72
17.9 ( 1.18
19.8 ( 2.99
18.4 ( 0.98
18.2 ( 0.91
19.7 ( 1.09
17.9 ( 0.84
18.4 ( 1.39
20.1 ( 2.25
18.5 ( 3.25
9f
9g
47.3
43.6
68.2
60.7
45.0
61.5
42.4
51.2
100
9i
9j
9k
9m
9n
9q
fidarestat
a Five days of diabetes followed by 5 days of drug treatment at 100 (mg/kg)/day: (/) p < 0.05, (//) p < 0.01 compared to untreated diabetic control
group. b Percent improvement of drug-treated group relative to the difference between nondiabetic and diabetic control groups.
Figure 5. Docking of the inhibitor 9m into the active site of ALR2. (a) Protein structure is shown as a cartoon diagram with selected and
labeled residues shown in line representation, and ligand 9m and NADP are shown as stick models. Docked pose of 9m is shown in cyan (C), red
(O), blue (N), yellow (S), green (Cl), and gray (F). The hydrogen bonds are shown as yellow dashed lines. (b) Protein residues are in surface
representation.
while 9m showed the decreased activity in vivo (62%),
although it was more potent in vitro than 9i (IC50 = 0.032
μM vs 0.066 μM). 9q was inactive in vivo probably because of
its greater molecular weight than those of 9i and 9m,
although it also contained trifluoro substituents on the N2-
benzyl ring.
identified in the present study had more similarity with the
ligand tolrestat compared to the other ligands, and therefore,
the tolrestat bound conformation (2FDZ)52 was used for the
docking. Compound 9m, the most active in vitro as described
above, was docked using the FlexX docking into the con-
formation 2FDZ of ALR2 as shown in Figure 5. Docking
results reveal that the inhibitor 9m is tightly bound in the
active site of ALR2. The carboxylate group is well inserted in
the anion binding site, hydrogen-bonding with Tyr48,
His110, and Trp111 side chains and engaging a stabilizing
electrostatic interaction with the positively charged nicotin-
Given that the N4-acid derivative 9i appeared to have the
most in vivo potency, we evaluated in vivo activity of its
bioisosteric N4-amide counterpart 10i. However, no activity
in vivo was found for the amide 10i. Also, we investigated the
in vivo activity of the N4-ester 8m, which might be consid-
ered to be a prodrug of its acid counterpart 9m having the
most potent in vitro activity, but did not find improvement in
the in vivo activity over 9m (data not shown). However, we
have noted that ester and amide derivatives in some cases
could function as prodrugs of corresponding acid ARIs
reported respectively by La Motta et al.21 and Wrobel
et al.49 In the present study, the possibility of the N4-amide
and N4-ester compounds of benzothiadiazine 1,1-dioxide as
prodrugs of the N4-acid derivatives may be excluded.
Docking Studies. To better understand the inhibitory
mechanism of the newly synthesized ARIs and to design
new compounds, docking experiments were performed.
Comparison of different aldose reductase-inhibitor X-ray
complexes by Sotriffer and co-workers showed that there are
three main kinds of binding conformations of ALR2,50 best
represented by the complexes with ligands sorbinil (PDB
entry code 1AH051), tolrestat (PDB entry code 2FDZ52), and
IDD594 (PDB entry code 1US053). The potent derivatives
amide moiety of the cofactor NADPþ (N-O = 4.71 A,
˚
Figure 5). Further, N2-nitrogen is hydrogen-bonded to the
Trp20 side chain. Moreover, the 7-chloro substituted phenyl
ring of benzothiadiazine 1,1-dioxide core penetrates the
crevice formed between the Leu300 and Phe122 residues of
the specificity pocket, while the 7-chloro atom at the phenyl
ring is well placed in the deep hole of the pocket formed by
the side chains of Phe115, Trp79, and Val130 (Figure 5). In
addition, these interactions together with the presence of the
benzothiadiazine ring well orient the substituted benzyl ring
in a hydrophobic pocket mainly formed by the side chains of
the following ALR2 residues: Phe121, Phe122, Trp20, Val47,
and Tyr48.
Conclusions
In this paper we described the synthesis, biological activity,
and structure-activity relationships of a series of benzothia-
diazine 1,1-dioxide derivatives bearing different substituent

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8337
groups at the N2, N4, and 7 positions of the benzothiadiazine
scaffold. Most of the derivatives (9a-d and 9f-q) bearing a
N4-acetic acid group and a N2-benzyl group with different
substituents at the benzyl ring showed strong and selective in
vitro inhibition effect on ALR2 with IC50 values in the range
of 0.032-0.975 μM, whereby three of them (9i, 9j, and 9m)
showed more prominent ALR2 inhibitory activity both in
vitro and in vivo, indicating high ALR2 binding affinity as
well as sufficient intracellular availability. On the other hand,
the SAR analysis seemed to corroborate the importance of the
structural requirements of the N4-acetic acid and N2-benzyl
substituents in the benzothiadiazine 1,1-dioxide structure in
order to achieve good activity. Either conversion of the acid
group into the corresponding ester and amide groups or
removal of substituents at the benzyl ring diminished in vitro
activity. The ester and amide compounds were not yet active
in vivo more than their acid counterparts, making them
unavailable as prodrugs. Furthermore, the positions of the
acetic acid group and the substituted benzyl group on ben-
zothiadiazine scaffold played a role in their ALR2 inhibitory
activity, whereby N4-acetic acid group combined with N2-
benzyl group retained the potent activity while exchange of
their positions led to drastic loss of the activity. Consequently,
it is suggested that an acid group at the N4 position and a
substituted benzyl group at the N2 position are minimum
requirements for ALR2 inhibitory activity of benzothiadia-
zine 1,1-dioxide derivatives. In addition, substitutions at the
7-position are not essential but greatly increase the activity.
These results were also supported by molecular docking
studies in which the inhibitor is tightly anchored in the active
site with its acid residue trapped in the anion-binding pocket,
with the halogen-substituted phenyl ring of the benzothiadia-
zine core fit in the specificity pocket, and with its benzyl group
inserted in a hydrophobic pocket. The results from the present
study have encouraged us to continue our investigations into
the design of more potent and selective analogues by con-
ducting appropriate modifications based on the benzothia-
diazine scaffold.
4-(trifluoromethyl)benzyl bromide, 2,4,5-trifluorobenzyl bro-
mide, used to obtain the target inhibitors, were from Alfa Aesar.
General Synthetic Procedure for 2b-d. A solution of chloro-
sulfonyl isocyanate (26 mL, 300 mmol) in 200 mL of nitroethane
was cooled to -40 °C, and a solution of the appropriate aniline 1
(270 mmol) in 100 mL of nitroethane was then added dropwise
from a dropper funnel with stirring. After the addition was
completed, the reaction mixture was stirred for an additional
30 min and aluminum chloride (39 g, 300 mmol) was added. The
mixture was then heated to 110 °C with stirring for 1 h. The
crude material was then poured into ice, and the precipitate was
collected by suction filtration, washed with cold water and dry
ether. The residue was added to 320 mL of 50% aqueous sulfuric
acid and heated to 140 °C for 8 h. The solution was then poured
over ice and neutralized at 0 °C with saturated aqueous sodium
hydroxide. The precipitate was collected by suction filtration,
washed with cold water, and dried in vacuo to give the desired
product.
2-Amino-5-fluorobenzenesulfonamide (2b). Yield 51%; mp
1
149-152 °C; H NMR (400 MHz, DMSO-d6) δ 5.71 (s, 2H),
6.80 (m, 1H), 7.14 (m, 1H), 7.28 (m, 1H), 7.35 (s, 2H).
2-Amino-5-chlorobenzenesulfonamide (2c). Yield 62%; mp
1
155-157 °C; H NMR (400 MHz, DMSO-d6) δ 5.96 (s, 2H),
6.80 (d, J = 8.8 Hz, 1H), 7.24 (m, 1H), 7.36 (s, 2H), 7.48 (d, J =
2.4 Hz, 1H).
2-Amino-5-bromobenzenesulfonamide (2d). Yield 45%; mp
1
179-181 °C; H NMR (500 MHz, DMSO-d6) δ 6.02 (s, 2H),
6.77 (d, J = 8.5 Hz, 1H), 7.38 (m, 1H), 7.40 (s, 2H), 7.61 (d, J =
2.4 Hz, 1H).
General Synthetic Procedure for 3a-d. The appropriate
2-aminobenzenesulfonamide 2 (100 mmol) was heated in triethyl
orthoformate (150 mL) to reflux during 2 h. After the mixture
was cooled to room temperature, the desired compound was
collected by filtration, washed with diethyl ether, and dried in
vacuo.
4H-1,2,4-Benzothiadiazine 1,1-Dioxide (3a). Yield 89%; mp
226-228 °C; 1H NMR (400 MHz, DMSO-d6): δ 7.28 (m, 1H),
7.43 (m, 1H), 7.65 (m, 1H), 7.78 (m, 1H), 7.96 (s, 1H), 12.28
(s, 1H).
7-Fluoro-4H-1,2,4-benzothiadiazine 1,1-Dioxide (3b). Yield
92%; mp 261-264 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.39
(m, 1H), 7.58 (m, 1H), 7.69 (m, 1H), 7.98 (s, 1H), 12.39 (s, 1H).
7-Cholro-4H-1,2,4-benzothiadiazine 1,1-Dioxide (3c). Yield
95%; mp 244-247 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.34
(d, J = 8.8 Hz, 1H), 7.72 (m, 1H), 7.85 (d, J = 2.4 Hz, 1H), 8.01
(s, 1H), 12.42 (d, J = 4.4 Hz,1H).
7-Bromo-4H-1,2,4-benzothiadiazine 1,1-Dioxide (3d). Yield
88%; mp 280-281 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.27
(d, J = 8.8 Hz, 1H), 7.86 (m, 1H), 7.95 (d, J = 2.4 Hz, 1H), 8.02
(s, 1H), 12.43 (s, 1H).
Experimental Section
Chemistry. Melting points were recorded on a X-4 micro-
scopic melting point apparatus and were uncorrected. All reac-
tions were routinely checked by TLC on silica gel Merck
60F254. The 1H NMR spectra were recorded on a Bruker
Advance 400 spectrometer (400 MHz, Bruker (Beijing) Tech-
nologies and Services Co., Ltd.) or Bruker Advance 500 spectrom-
eter (500 MHz, Beijing Normal University, China). Chemical
shifts are given in δ 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 instrumen-
tation center of Peking University (China) and realized on an
Elementar Vario MICRO CUBE elemental analyzer.
General Synthetic Procedure for 4a-d. A mixture of the
appropriate 4H-1,2,4-benzothiadiazine 1,1-dioxide 3 (80 mmol),
potassium carbonate (12 g), and methyl bromoacetate (13.4 g,
88 mmol) in acetonitrile (180 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.
The following HPLC methods were used to determine the purity
of acetic acid derivatives using a Shimadze LC-20AT liquid chro-
matograph (Shimadze Corporation, Japan). All acetic acid deriv-
atives tested in biological assays were g95% pure. Conditions were
as follows. Method A: column, C18, 5 μm, 250 mm ꢀ 4.6 mm;
mobile phase MeCN (0.1% TFA)/MeOH = 6:4, for 6 min; room
temperature; flow rate of 1 mL/min; detection at 254 nm. Method
B: column, C18, 5 μm, 250 mm ꢀ 4.6 mm; mobile phase H2O (0.1%
TFA)/MeOH = 1:9, for 8 min; room temperature; flow rate of
1 mL/min; detection at 254 nm.
(4H-1,2,4-Benzothiadiazine 1,1-dioxide-4-yl)acetic Acid Methyl
Ester (4a). Colorless crystal, yield 83%; mp 154-156 °C;
1H NMR (500 MHz, DMSO-d6) δ 3.74 (s, 3H), 5.10 (s, 2H),
7.34 (d, J = 8.5 Hz, 1H), 7.55 (m, 1H), 7.74 (m, 1H), 7.91 (d, J =
9.0 Hz, 1H), 8.10 (s, 1H).
(7-Fluoro-4H-1,2,4-benzothiadiazine 1,1-dioxide-4-yl)acetic Acid
Methyl Ester (4b). Colorless crystal, yield 85%; mp 197-
200 °C; 1H NMR (500 MHz, DMSO-d6) δ 3.73 (s, 3H), 5.12
(s, 2H), 7.49 (m, 1H), 7.67 (m, 1H), 7.84 (m, 1H), 8.10
(s, 1H).
The 4-fluoroaniline, 1b, 4-chloroaniline, 1c, 4-bromoaniline,
1d, and 2-aminobenzenesulfonamide, 2a, methyl bromoacetate,
4-bromo-2-fluorobenzyl bromide, 3-nitrobenzyl bromide,
(7-Cholro-4H-1,2,4-benzothiadiazine 1,1-dioxid-4-yl)acetic
Acid Methyl Ester (4c). Colorless crystal, yield 79%; mp
248-250 °C; H NMR (400 MHz, DMSO-d6) δ 3.72 (s, 3H),
1

8338 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Chen et al.
5.09 (s, 2H), 7.42 (d, J = 9.2 Hz, 1H), 7.79 (m, 1H), 7.95 (d, J =
2.4 Hz, 1H), 8.10 (s, 1H).
6.55 (d, J = 8.4 Hz, 1H), 6.92 (m, 1H), 7.27 (m, 2H), 7.40 (m,
2H), 7.75 (m, 1H). Anal. (C17H16BrFN2O4S) C, H, N.
(7-Bromo-4H-1,2,4-benzothiadiazine 1,1-dioxide-4-yl)acetic
Acid Methyl Ester (4d). Colorless crystal, yield 82%; mp
214-216 °C; H NMR (500 MHz, DMSO-d6) δ 3.73 (s, 3H),
5.11 (s, 2H), 7.38 (d, J = 9.5 Hz, 1H), 7.94 (m, 1H), 8.08 (d, J =
2.0 Hz, 1H), 8.13(s, 1H).
[2-(3-Nitrobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-
dioxide-4-yl]acetic Acid Methyl Ester (8b). Colorless crystal, yield
80%; mp 163-165 °C; H NMR (400 MHz, CDCl3) δ 3.80 (s,
3H), 3.97 (s, 2H), 4.47 (s, 2H), 4.82 (s, 2H), 6.56 (d, J = 8.8 Hz,
1H), 6.95 (m, 1H), 7.45 (m, 1H), 7.57 (m, 1H), 7.80 (m, 2H), 8.18
(m, 1H), 8.26 (s, 1H). Anal. (C17H17N3O6S) C, H, N.
1
1
General Synthetic Procedure for 5a-d. To a solution of the
appropriate acetate 4 (50 mmol) in 2-propanol-acetonitrile
(1:1, 80 mL) with stirring was added NaBH4 (8 g). Then the
mixture was stirred at room temperature for 5 min. The solvent
was then removed by distillation under reduced pressure, and
the residue was suspended in water (25 mL). The alkaline sus-
pension was treated with 0.1 N HCl until the pH value indicated
that the mixture was acidic. The resulting insoluble material was
collected by filtration, washed with water, dried, and then
recrystallized in ethyl acetate to give pure desired product.
(3,4-Dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide-4-yl)ace-
tic Acid Methyl Ester (5a). Colorless crystal, yield 84%; mp
146-148 °C, 1H NMR (400 MHz, CDCl3) δ 3.75 (s, 3H), 4.16
(s, 2H), 4.85 (d, J = 7.8 Hz, 2 H), 5.26 (m, 1 H), 6.66 (d, J =
6.4 Hz,1H), 6.94 (m, 1H), 7.37 (m, 1H), 7.74 (m, 1H).
(7-Fluoro-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide-
4-yl)acetic Acid Methyl Ester (5b). Colorless crystal, yield 67%;
mp 175-176 °C, 1H NMR (500 MHz, DMSO-d6) δ 3.66 (s, 3H),
4.36 (s, 2H), 4.74 (d, J = 8.0 Hz, 2 H), 6.81 (m, 1 H), 7.31 (m,
1H), 7.44 (m, 1H), 8.27 (m, 1H).
(7-Cholro-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxid-
4-yl)acetic Acid Methyl Ester (5c). Colorless crystal, yield 71%;
mp 186-187 °C, 1H NMR (500 MHz, DMSO-d6) δ 3.67 (s, 3H),
4.38 (s, 2H), 4.76 (d, J = 8.0 Hz, 2 H), 6.81 (d, J = 9.0 Hz, 1 H),
7.44 (m, 1H), 7.57 (d, J = 2.5 Hz, 1H), 8.34 (m, 1H).
[2-(4-(Ttrifluoromethyl)benzyl)-3,4-dihydro-2H-1,2,4-benzothia-
diazine 1,1-dioxide-4-yl]acetic Acid Methyl Ester (8c). Colorless
crystal, yield 86%; mp 120-121 °C; 1H NMR (400 MHz, CDCl3)
δ 3.76 (s, 3H), 3.94 (s, 2H), 4.41 (s, 2H), 4.79 (s, 2H), 6.55 (d, J =
8.4 Hz, 1H), 6.93 (m, 1H), 7.42 (m, 1H), 7.54 (d, J = 8.0 Hz, 2H),
7.63 (d, J = 8.0 Hz, 2H), 7.78 (m, J = 8.0 Hz, 1H). Anal.
(C18H17F3N2O4S) C, H, N.
[2-(2,4,5-Trifluorobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine
1,1-dioxide-4-yl]acetic Acid Methyl Ester (8d). Colorless crystal,
yield 73%; mp 110-112 °C; 1H NMR (400 MHz, CDCl3) δ 3.77
(s, 3H), 4.01 (s, 2H), 4.11 (s, 2H), 4.89 (s, 2H), 6.56 (d, J = 8.4 Hz,
1H), 6.95 (m, 2H), 7.41 (m, 2H), 7.77 (m, 1H).
(2-Benzyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide-
4-yl)acetic Acid Methyl Ester (8e). Colorless crystal, yield 75%;
mp 80-82 °C; 1H NMR (400 MHz, CDCl3) δ 3.73 (s, 3H), 3.91
(s, 2H), 4.30 (s, 2H), 4.75 (s, 2H), 6.54 (d, J = 8.4 Hz, 1H), 6.91
(m, 1H), 7.35 (m, 6H), 7.76 (m, 1H). Anal. (C17H18N2O4S) C,
H, N.
[2-(4-Bromo-2-fluorobenzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid Methyl Ester (8f). Color-
less crystal, yield 89%; mp 108-110 °C (500 MHz, DMSO-d6)
δ 3.68 (s, 3H), 4.27 (s, 2H), 4.37 (s, 2H), 4.89 (s, 2H), 6.87
(m, 1H), 7.40 (m, 2H), 7.47 (d, J = 8.0 Hz, 1H), 7.55 (m,
1H), 7.59 (d, J = 10.0 Hz, 1H). Anal. (C17H15BrF2N2O4S)
C, H, N.
(7-Bromo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide-
4-yl)acetic Acid Methyl Ester (5d). Colorless crystal, yield 77%;
mp 178-182 °C; (400 MHz, DMSO-d6) δ 3.65 (s, 3H), 4.35 (s,
2H), 4.75 (d, J = 8.0 Hz, 2 H), 6.79 (d, J = 9.2 Hz, 1 H), 7.41 (m,
1H), 7.54 (d, J = 2.4 Hz, 1H), 8.27 (m, 1H).
[2-(3-Nitrobenzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-benzothiadia-
zine 1,1-dioxide-4-yl]acetic Acid Methyl Ester (8g). Colorless
crystal, yield 81%; mp 149-150 °C; 1H NMR (500 MHz,
DMSO-d6) δ 3.68(s, 3H), 4.34(s, 2H), 4.42 (s, 2H), 4.94 (s, 2H),
6.87 (m, 1H), 7.40 (m, 1H), 7.56 (m, 1H), 7.69 (m, 1H), 7.83 (d,
J= 7.5 Hz, 1H), 8.20 (m, 2H). Anal. (C17H16FN3O6S) C, H, N.
[2-(4-(Trifluoromethyl)benzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-
benzothiadiazine 1,1-dioxide-4-yl]acetic Acid Methyl Ester (8h).
(3,4-Dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide-4-yl)-
acetic Acid (6). A mixture of 5a (0.51 g, 2 mmol), 1,4-dioxane
(8 mL), and saturated aqueous sodium hydroxide (8 mL) was
stirred at room temperature for 2 h. The alkaline suspension was
adjusted to be acidic with 0.1 N HCl and extracted 3-fold with
ethyl acetate (3 ꢀ 20 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
as a colorless crystal (0.40 g, 83%): mp 171-173 °C; 1H NMR
(400 MHz, DMSO-d6) δ 4.20 (s, 2H), 4.75 (d, J = 8.0 Hz, 2H),
6.70 (m, 1H), 6.82 (m, 1H), 7.37 (m, 1H), 7.54 (m, 1H), 8.06
(m, 1H), 12.8 (s, 1H). Anal. (C9H10N2O4S) C, H, N.
1
Colorless crystal, yield 77%; mp 102-104 °C; H NMR (500
MHz, DMSO-d6) δ 3.68 (s, 3H), 4.35 (s, 2H), 4.37 (s, 2H), 4.91 (s,
2H), 6.87 (m, 1H), 7.41 (m, 1H), 7.55 (m, 1H), 7.60 (d, J = 8.0 Hz,
2H), 7.76 (d, J = 8.0 Hz, 2H). Anal. (C18H16F4N2O4S) C, H, N.
[2-(2,4,5-Trifluorobenzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid Methyl Ester (8i). Col-
orless crystal, yield 84%; mp 147-149 °C; 1H NMR (500 MHz,
DMSO-d6) δ 3.68 (s, 3H), 4.27 (s, 2H), 4.38 (s, 2H), 4.90 (s, 2H),
6.87 (m, 1H), 7.40 (m, 1H), 7.54 (m, 2H), 7.61 (m, 1H). Anal.
(C17H14F4N2O4S) C, H, N.
(3,4-Dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide-4-yl)acet-
amide (7). A solution of 5a (0.51 g, 2 mmol) in 25 mL of methanol
with stirring was treaded with NH3 (gas) for 30 min. Then the
mixture was stirred at room temperature for 18 h. After evapora-
tion of the solvent under reduced pressure, the crude solid was
recrystallized in ethyl acetate-dichloromethane (1:1) to give pure
product as a colorless crystal (0.36 g, 75%): mp 225-227 °C;
1H NMR (500 MHz, DMSO-d6) δ 3.95 (s, 2H), 4.80 (s, 2H), 6.60
(d, J = 8.5 Hz, 1H), 6.83 (m, 1H), 7.28 (s, 1H), 7.40 (m, 1H), 7.55
(m, 1H), 7.57 (m, 1H), 7.93 (s, 1H). Anal. (C9H11N3O3S) C, H, N.
General Synthetic Procedure for 8a-q. A mixture of appro-
priate acetate 5 (10 mmol), potassium carbonate (3 g), and
appropriate benzyl bromide (11 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.
[2-(4-Bromo-2-fluorobenzyl)-7-cholro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid Methyl Ester (8j). Color-
less crystal, yield 75%; mp 155-157 °C; H NMR (500 MHz,
1
DMSO-d6) δ 3.68 (s, 3H), 4.27 (s, 2H), 4.38 (s, 2H), 4.90 (s, 2H),
6.86 (d, J = 9.5 Hz, 1H), 7.40 (m, 1H), 7.47 (d, J = 11.5 Hz, 1H),
7.50 (m, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.67 (d, J = 1.0 Hz, 1H).
Anal. (C17H15BrClFN2O4S) C, H, N.
[2-(3-Nitrobenzyl)-7-cholro-3,4-dihydro-2H-1,2,4-benzothiadia-
zine 1,1-dioxide-4-yl]acetic Acid Methyl Ester (8k). Colorless
crystal, yield, 80%; mp 169-170 °C; 1H NMR (500 MHz,
DMSO-d6) δ 3.69 (s, 3H), 4.36 (s, 2H), 4.41 (s, 2H), 4.96 (s,
2H), 6.86 (d, J = 9.0 Hz, 1H), 7.50 (m, 1H), 7.68 (m, 2H), 7.83
(d, J = 7.5 Hz, 1H), 8.20 (m, 2H). Anal. (C17H16ClN3O6S) C,
H, N.
[2-(4-(Trifluoromethyl)benzyl)-7-cholro-3,4-dihydro-2H-1,2,4-
benzothiadiazine 1,1-dioxide-4-yl]acetic Acid Methyl Ester (8l).
Colorless crystal, yield 83%; mp 114-116 °C; 1H NMR
(500 MHz, DMSO-d6) δ 3.68 (s, 3H), 4.36 (s, 4H), 4.93 (s, 2H),
6.86 (d, J = 9.0 Hz, 1H), 7.50 (d, J = 9.0 Hz, 1H), 7.58
[2-(4-Bromo-2-fluorobenzyl)-3,4-dihydro-2H-1,2,4-benzothia-
diazine 1,1-dioxide-4-yl]acetic Acid Methyl Ester (8a). Colorless
crystal, yield 79%; mp 128-131 °C; 1H NMR (400 MHz,
CDCl3) δ 3.75(s, 3H), 4.01 (s, 2H), 4.36 (s, 2H), 4.86 (s, 2H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8339
(d, J = 8.0 Hz, 2H), 7.68 (s, 1H), 7.57 (d, J = 8.0 Hz, 2H). Anal.
(C18H16ClF3N2O4S) C, H, N.
2H), 6.47 (d, J = 8.4 Hz, 1H), 6.80 (m, 1H), 7.23 (m, 5H), 7.64
(d, J = 8.0 Hz, 1H), 8.69 (s, 1H). Anal. (C16H16N2O4S) C, H, N.
[2-(4-Bromo-2-fluorobenzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid (9f). Colorless crystal,
yield 78%; mp 167-169 °C; 1H NMR (500 MHz, DMSO-d6) δ
4.24 (s, 2H), 4.28 (s, 2H), 4.88 (s, 2H), 6.83 (m, 1H), 7.40 (m, 2H),
7.45 (d, 1H), 7.52 (m, 1H), 7.60 (m, 1H). Anal. (C16H13BrF2-
N2O4S) C, H, N.
[2-(2,4,5-Trifluorobenzyl)-7-cholro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic acid Methyl Ester (8m).
1
Colorless crystal, yield 80%; mp 191-193 °C; H NMR (500
MHz, DMSO-d6) δ 3.68 (s, 3H), 4.26 (s, 2H), 4.39 (s, 2H), 4.93 (s,
2H), 6.87 (d, J = 9.0 Hz, 1H), 7.52 (m, 2H), 7.62 (m, 1H), 7.67 (s,
1H). Anal. (C17H14ClF3N2O4S) C, H, N.
[2-(4-Bromo-2-fluorobenzyl)-7-bromo-3,4-dihydro-2H-1,2,4-ben-
zothiadiazin 1,1-dioxide-4-yl]acetic Acid Methyl Ester (8n). Color-
less crystal, yield 88%; mp 150-152 °C; H NMR (500 MHz,
DMSO-d6) δ 3.68 (s, 3H), 4.26 (s, 2H), 4.37 (s, 2H), 4.90 (s, 2H),
6.80 (d, J = 9.5 Hz, 1H), 7.40 (m, 1H), 7.46 (d, J = 8.0 Hz, 1H),
7.61 (m, 2H), 7.75 (d, J = 1.5 Hz, 1H). Anal. (C17H15Br2FN2O4S)
C, H, N.
[2-(3-Nitrobenzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-benzothiadia-
zine 1,1-dioxide-4-yl]acetic Acid (9g). Colorless crystal, yield 86%;
mp 178-180 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.21 (s, 2H),
4.43 (s, 2H), 4.94 (s, 2H), 6.83 (m, 1H), 7.39 (m, 1H), 7.54 (m, 1H),
7.68 (m, 1H), 7.82 (d, J= 7.5 Hz, 1H), 8.18 (d, J= 8.5 Hz, 1H), 8.21
(s, 1H). Anal. (C16H14FN3O6S) C, H, N.
1
[2-(4-(Trifluoromethyl)benzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid (9h). Colorless crystal,
[2-(3-Nitrobenzyl)-7-bromo-3,4-dihydro-2H-1,2,4-benzothiadiazine
1,1-dioxide-4-yl]acetic Acid Methyl Ester (8o). Colorless crystal, yield
1
yield 79%; mp 191-193 °C; H NMR (500 MHz, DMSO-d6) δ
1
87%; mp 174-176 °C; H NMR (500 MHz, DMSO-d6) δ 3.69
4.22 (s, 2H), 4.37 (s, 2H), 4.90 (s, 2H), 6.83 (m, 1H), 7.40 (m, 1H),
7.53 (m, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.75 (d, J = 8.0 Hz, 2H).
Anal. (C17H14F4N2O4S) C, H, N.
(s, 3H), 4.33 (d, J = 9.5 Hz, 2H), 4.41 (s, 2H), 4.96 (s, 2H), 6.80 (d,
J= 9.0 Hz, 1H), 7.60(m, 1H), 7.68 (m, 1H), 7.76 (d, J= 1.5 Hz, 1H),
7.82 (d, J = 7.5 Hz, 1H), 8.20 (m, 2H). Anal. (C17H16BrN3O6S)
C, H, N.
[2-(2,4,5-Trifluorobenzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid (9i). Colorless crystal,
yield 92%; mp 146-149 °C; 1H NMR (500 MHz, DMSO-d6) δ
4.24 (s, 2H), 4.27 (s, 2H), 4.89 (s, 2H), 6.84 (m, 1H), 7.40 (m, 1H),
7.52 (m, 2H), 7.60 (m, 1H). Anal. (C16H12F4N2O4S) C, H, N.
[2-(4-Bromo-2-fluorobenzyl)-7-cholro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid (9j). Colorless crystal,
[2-(4-(Trifluoromethyl)benzyl)-7-bromo-3,4-dihydro-2H-1,2,4-
benzothiadiazine 1,1-dioxide-4-yl]acetic Acid Methyl Ester (8p).
1
Colorless crystal, yield 92%; mp 117-118 °C; H NMR (500
MHz, DMSO-d6) δ 3.68 (s, 3H), 4.35(s, 4H), 4.93(s, 2H), 6.80 (d,
J = 9.5 Hz, 1H), 7.60 (m, 3H), 7.56 (m, 3H). Anal.
(C18H16BrF3N2O4S) C, H, N.
1
yield 86%; mp 179-181 °C; H NMR (500 MHz, DMSO-d6) δ
[2-(2,4,5-Trifluorobenzyl)-7-bromo-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid Methyl Ester (8q).
4.25 (s, 2H), 4.27 (s, 2H), 4.90 (s, 2H), 6.85 (m, 1H), 7.42 (m, 1H),
7.52 (m, 1H), 7.58 (m, 1H), 7.65 (d, J = 2.5 Hz, 1H), 7.69 (s, 1H).
Anal. (C16H13BrClFN2O4S) C, H, N.
1
Colorless crystal, yield 79%; mp 124-126 °C; H NMR (500
MHz, DMSO-d6) δ 3.68 (s, 3H), 4.26 (s, 2H), 4.38 (s, 2H), 4.92 (s,
2H), 6.81 (d, J = 9.0 Hz, 1H), 7.52 (m, 1H), 7.61 (m, 2H), 7.75 (d,
J = 1.5 Hz, 1H). Anal. (C17H14BrF3N2O4S) C, H, N.
General Synthetic Procedure for 9a-q. A mixture of the
appropriate acetate 8 (1 mmol), 1,4-dioxane (5 mL), and satu-
rated aqueous sodium hydroxide (5 mL) was stirred at room
temperature for 2 h. The alkaline suspension was adjusted to be
acidic with 0.1 N HCl and extracted 3-fold with ethyl acetate
(3 ꢀ 20 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 com-
pound was recrystallized in methanol to give pure product.
[2-(4-Bromo-2-fluorobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadia-
zine 1,1-dioxide-4-yl]acetic Acid (9a). Colorless crystal, yield 79%;
mp 200-203 °C; 1H NMR (400 MHz, DMSO-d6) δ 4.21 (s, 2H),
4.27 (s, 2H), 4.89 (s, 2H), 6.76 (d, J = 8.8 Hz, 1H), 6.87 (m, 1H),
7.44 (m, 3H), 7.59 (m, 2H), 12.96 (s, 1H). Anal. (C16H14Br-
FN2O4S) C, H, N.
[2-(3-Nitrobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-
dioxide-4-yl]acetic Acid (9b). Colorless crystal, yield 85%; mp
166-169 °C; 1H NMR (400 MHz, DMSO-d6) δ 4.20 (s, 2H), 4.42
(s, 2H), 4.95 (s, 2H), 6.76 (d, J = 8.4 Hz, 1H), 6.89 (m, 1H), 7.46
(m, 1H), 7.65 (m, 2H), 8.17 (m, 1H), 8.20 (m, 2H), 12.98 (s, 1H).
Anal. (C16H15N3O6S) C, H, N.
[2-(4-(Ttrifluoromethyl)benzyl)-3,4-dihydro-2H-1,2,4-benzothia-
diazine 1,1-dioxide-4-yl]acetic Acid (9c). Colorless crystal, yield
83%; mp 152-155 °C; 1H NMR (400 MHz, CDCl3) δ3.98 (s, 2H),
4.39 (s, 2H), 4.79 (s, 2H), 6.58 (d, 1H), 6.96 (m, 1H), 7.43 (m, 1H),
7.51 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 8.0 Hz, 2H), 7.78 (m, 1H).
Anal. (C17H15F3N2O4S) C, H, N.
[2-(3-Nitrobenzyl)-7-cholro-3,4-dihydro-2H-1,2,4-benzothiadia-
zine 1,1-dioxide-4-yl]acetic Acid (9k). Colorless crystal, yield 78%;
mp 201-204 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.15 (s, 2H),
4.42 (s, 2H), 4.96 (s, 2H), 6.80 (d, J = 9.5 Hz, 1H), 7.48 (m, 1H),
7.64 (d, J = 2.5 Hz, 1H), 7.67 (m, 1H), 7.82 (d, J = 7.5 Hz, 1H),
8.17 (d, J = 8.5 Hz, 1H), 8.21 (s, 1H). Anal. (C16H14ClN3O6S) C,
H, N.
[2-(4-(Trifluoromethyl)benzyl)-7-cholro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid (9l). Colorless crystal, yield
1
85%; mp 197-199 °C; H NMR (500 MHz, DMSO-d6) δ 4.21
(s, 2H), 4.37 (s, 2H), 4.93 (s, 2H), 6.83 (d, J = 9.0 Hz, 1H), 7.52
(m, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 2.0 Hz, 1H), 7.74
(d, J= 8.0 Hz, 2H). Anal. (C17H14ClF3N2O4S) C, H, N.
[2-(2,4,5-Trifluorobenzyl)-7-cholro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid (9m). Colorless crys-
tal, yield 76%; mp 140-142 °C; 1H NMR (400 MHz, DMSO-d6)
δ 4.23 (s, 2H), 4.25 (s, 2H), 4.90 (s, 2H), 6.83 (d, J = 8.8 Hz, 1H),
7.49 (m, 1H), 7.55 (m, 1H), 7.62 (m, 1H), 7.64 (s, 1H), 13.0 (s,
1H). Anal.(C16H12ClF3N2O4S) C, H, N.
[2-(4-Bromo-2-fluorobenzyl)-7-bromo-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid (9n). Colorless crystal,
1
yield 83%; mp 176-179 °C; H NMR (500 MHz, DMSO-d6)
δ 4.19 (s, 2H), 4.28 (s, 2H), 4.89 (s, 2H), 6.76 (d, J = 9.0 Hz, 1H),
7.43 (m, 2H), 7.59 (m, 2H), 7.72 (d, J = 1.0 Hz, 1H). Anal.
(C16H13Br2FN2O4S) C, H, N.
[2-(3-Nitrobenzyl)-7-bromo-3,4-dihydro-2H-1,2,4-benzothiadia-
zine 1,1-dioxide-4-yl]acetic Acid (9o). Colorless crystal, yield 90%;
mp 212-215 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.22 (s, 2H),
4.42 (s, 2H), 4.96 (s, 2H), 6.77 (d, 1H), 7.61 (m, 1H), 7.68
(m, 1H), 7.75 (d, J = 2.5 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H),
8.18 (d, J = 7.5 Hz, 1H), 8.21 (s, 1H), 13.10 (s, 1H). Anal.
(C16H14BrN3O6S) C, H, N.
[2-(4-(Trifluoromethyl)benzyl)-7-bromo-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid (9p). Colorless crystal, yield
80%; mp 192-193 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.16 (s,
2H), 4.37 (s, 2H), 4.91 (s, 2H), 6.76 (d, J = 9.5 Hz, 1H), 7.58 (s, 1H),
7.61 (m, 2H), 7.73 (m, 2H), 7.75 (s, 1H). Anal. (C17H14BrF3N2O4S)
C, H, N.
[2-(2,4,5-Trifluorobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine
1,1-dioxide-4-yl]acetic Acid (9d). Colorless crystal, yield 85%; mp
1
172-174 °C; H NMR (400 MHz, DMSO-d6) δ 4.21 (s, 2H),
4.25 (s, 2H), 4.90 (s, 2H), 6.75 (d, J = 8.4 Hz, 1H), 6.88 (m, 1H),
7.45 (m, 1H), 7.50 (m, 1H), 7.56 (m, 1H), 7.62 (m, 1H). Anal.
(C16H13F3N2O4S) C, H, N.
(2-Benzyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide-4-
yl)acetic Acid (9e). Colorless crystal, yield 90%; mp 173-175 °C;
1H NMR (400 MHz, CDCl3) δ 3.83 (s, 2H), 4.18 (s, 2H), 4.62 (s,

8340 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Chen et al.
1
[2-(2,4,5-Trifluorobenzyl)-7-bromo-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetic Acid (9q). Colorless crystal,
yield 89%; mp 197-201 °C; 1H NMR (500 MHz, DMSO-d6) δ
4.25 (s, 2H), 4.26 (s, 2H), 4.91 (s, 2H), 6.78 (d, J = 9 Hz, 1H),
7.52 (m, 1H), 7.60 (m, 2H), 7.74 (m, 1H). Anal. (C16H12-
BrF3N2O4S) C, H, N.
General Synthetic Procedure for 10a-q. Appropriate acetate 8
(1 mmol) was dissolved in 25 mL of methanol and was treaded
with NH3 (gas) for 30 min with stirring. The mixture was then
stirred at room temperature for 18 h. After evaporation of the
solvent under reduced pressure, the crude solid was recrystallized
in ethyl acetate-dichloromethane (1:1) to give pure product.
[2-(4-Bromo-2-fluorobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadia-
zine 1,1-dioxide-4-yl]acetamide (10a). Colorless crystal, yield 89%;
mp 166-168 °C; 1H NMR (400 MHz, CDCl3) δ 3.90 (s, 2H), 4.33
(s, 2H), 4.86 (s, 2H), 5.55 (s, 1H), 5.92 (s, 1H), 6.63 (d, J = 8.4 Hz,
1H), 6.97 (m, 1H), 7.29 (m, 2H), 7.33 (m, 2H), 7.43 (d, J = 6.0 Hz,
1H). Anal. (C16H15BrFN3O3S) C, H, N.
yield 93%; mp 188-191 °C; H NMR (500 MHz, DMSO-d6)
δ 4.05 (s, 2H), 4.31 (s, 2H), 4.91 (s, 2H), 6.72 (d, J = 9.0 Hz,
1H), 7.30 (s, 1H), 7.44 (m, 2H), 7.53 (d, J = 9.0 Hz, 1H), 7.58
(d, J = 9.5 Hz, 1H), 7.64 (s, 1H), 7.66 (s, 1H). Anal. (C16H14-
BrClFN3O3S) C, H, N.
[2-(3-Nitrobenzyl)-7-cholro-3,4-dihydro-2H-1,2,4-benzothiadiazine
1,1-dioxide-4-yl]acetamide (10k). Yellow crystal, yield 80%; mp
186-188 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.01 (s, 2H), 4.46
(s, 2H), 4.98 (s, 2H), 6.70 (d, J = 9.5 Hz, 1H), 7.31 (s, 1H), 7.53
(d, J = 9.0 Hz, 1H), 7.65 (s, 2H), 7.67 (d, J = 8.0 Hz, 1H), 7.84 (d,
J = 7.5 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.23 (s, 1H). Anal.
(C16H15ClN4O5S) C, H, N.
[2-(4-(Trifluoromethyl)benzyl)-7-cholro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetamide (10l). Colorless crystal, yield
89%; mp 176-178 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.02 (s,
2H), 4.40 (s, 2H), 4.93 (s, 2H), 6.72 (d, J = 9.5 Hz, 1H), 7.29 (s, 1H),
7.53 (m, 1H), 7.59 (d, J= 8.0 Hz, 2H), 7.64 (d, J= 2.0 Hz, 2H), 7.74
(s, J = 8.5 Hz, 2H). Anal. (C17H15ClF3N3O3S) C, H, N.
[2-(2,4,5-Trifluorobenzyl)-7-cholro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetamide (10m). Colorless crys-
tal, yield 94%; mp 157-159 °C; 1H NMR (500 MHz, DMSO-d6)
δ 4.05 (s, 2H), 4.30 (s, 2H), 4.92 (s, 2H), 6.71 (d, J = 9.5 Hz, 1H),
7.34 (s, 1H), 7.52 (m, 1H), 7.58 (m, 2H), 7.60 (d, J = 4.0 Hz, 1H),
7.63 (s, 1H). Anal. (C16H13ClF3N3O3S) C, H, N.
[2-(3-Nitrobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-di-
oxide-4-yl]acetamide (10b). Yellow crystal, yield 90%; mp
1
172-175 °C; H NMR (400 MHz, CDCl3) δ 3.89 (s, 2H), 4.45
(s, 2H), 4.82 (s, 2H), 5.55 (s, 1H), 5.75 (s, 1H), 6.64 (d, J = 8.4 Hz,
1H), 6.97 (m, 1H), 7.44 (m, 1H), 7.57 (m, 1H), 7.79 (s, 1H), 7.80
(s, 1H), 8.19 (s, 1H), 8.27 (s, 1H). Anal. (C16H16N4O5S) C, H, N.
[2-(4-(Ttrifluoromethyl)benzyl)-3,4-dihydro-2H-1,2,4-benzothia-
diazine 1,1-dioxide-4-yl]acetamide (10c). Colorless crystal, yield
[2-(4-Bromo-2-fluorobenzyl)-7-bromo-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetamide (10n). Colorless crystal,
yield 91%; mp 203-205 °C; H NMR (500 MHz, DMSO-d6) δ
1
1
73%; mp 171-172 °C; H NMR (400 MHz, DMSO-d6) δ 4.00
(s, 2H), 4.40 (s, 2H), 4.92 (s, 2H), 6.68 (d, J = 8.4 Hz, 1H), 6.87
(m, 1H), 7.22 (s, 1H), 7.46 (m, 1H), 7.59 (d, J = 8.4 Hz, 2H),
7.60(m, 1H), 7.62 (m, 1H), 7.73 (d, J = 8.0 Hz, 2H). Anal.
(C17H16F3N3O3S) C, H, N.
4.04 (s, 2H), 4.31 (s, 2H), 4.91 (s, 2H), 6.66 (d, J= 9.5 Hz, 1H), 7.30
(s, 1H), 7.43 (s, 1H), 7.44 (s, 1H), 7.59 (d, J = 10.5 Hz, 1H), 7.64
(m, 2H), 7.72 (d, J = 2.5 Hz, 1H). Anal. (C16H14Br2FN3O3S)
C, H, N.
[2-(3-Nitrobenzyl)-7-bromo-3,4-dihydro-2H-1,2,4-benzothiadia-
zine 1,1-dioxide-4-yl]acetamide (10o). Yellow crystal, yield 75%;
mp 166-168 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.01 (s, 2H),
4.46 (s, 2H), 4.97 (s, 2H), 6.65 (d, J = 9.0 Hz, 1H), 7.31 (s, 1H),
7.62 (m, 2H), 7.67 (m, 1H), 7.73 (d, J = 2.5 Hz, 1H), 7.83 (d, J =
7.5 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.23 (s, 1H). Anal.
(C16H15BrN4O5S) C, H, N.
[2-(4-(Trifluoromethyl)benzyl)-7-bromo-3,4-dihydro-2H-1,2,4-
benzothiadiazine 1,1-dioxide-4-yl]acetamide (10p). Colorless
crystal, yield 92%; mp 188-192 °C; 1H NMR (500 MHz,
DMSO-d6) δ 4.02 (s, 2H), 4.40 (s, 2H), 4.93 (s, 2H), 6.66
(d, J = 9.5 Hz, 1H), 7.29 (s, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.61
(s, 1H), 7.64 (m, 1H), 7.73 (d, J = 2.5 Hz, 1H), 7.73 (d, J =
8.0 Hz, 2H). Anal. (C17H15BrF3N3O4S) C, H, N.
[2-(2,4,5-Trifluorobenzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetamide (10d). Colorless crystal,
yield 77%; mp 208-211 °C; 1H NMR (400 MHz, DMSO-d6) δ
4.02 (s, 2H), 4.29 (s, 2H), 4.91 (s, 2H), 6.66 (d, J = 8.4 Hz, 1H),
6.86 (m, 1H), 7.25 (s, 1H), 7.45 (m, 1H), 7.54 (m, 1H), 7.60 (m,
2H), 7.64 (s, 1H).
(2-Benzyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide-4-
yl)acetamide (10e). Colorless crystal, yield 72%; mp 144-146 °C;
1H NMR (400 MHz, DMSO-d6) δ 3.96 (s, 2H), 4.26 (s, 2H), 4.83
(s, 2H), 6.66 (d, J = 8.4 Hz, 1H), 6.85 (m, 1H), 7.29 (m, 1H), 7.31
(m, 1H), 7.34 (m, 2H), 7.36 (m, 1H), 7.43 (m, 2H). Anal.
(C16H17N3O3S) C, H, N.
[2-(4-Bromo-2-fluorobenzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetamide (10f). Colorless crystal,
yield 74%; mp 123-125 °C; 1H NMR (500 MHz, DMSO-d6) δ
4.03 (s, 2H), 4.31 (s, 2H), 4.90 (s, 2H), 6.72 (m, 1H), 7.28 (s, 1H),
7.40 (m, 1H), 7.45 (m, 2H), 7.51 (m, 1H), 7.58 (d, J = 9.0 Hz, 1H),
7.65 (s, 1H). Anal. (C16H14BrF2N3O3S) C, H, N.
[2-(2,4,5-Trifluorobenzyl)-7-bromo-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetamide (10q). Colorless crystal,
1
yield 81%; mp 153-155 °C; H NMR (500 MHz, DMSO-d6)
δ 4.05 (s, 2H), 4.30 (s, 2H), 4.92 (s, 2H), 6.66 (d, d, J = 9.0 Hz,
1H), 7.34 (s, 1H), 7.63 (m, 2H), 7.65 (m, 1H), 7.68 (s, 1H), 7.72
(d, d, J = 2.0 Hz, 1H). Anal. (C16H13BrF3N3O3S) C, H, N.
General Synthetic Procedure for 11a-d. A mixture of 3a (20
mmol), potassium carbonate (3.0 g), and appropriate benzyl
bromide (22 mmol) in acetonitrile (50 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 recrys-
tallized in ethyl acetate.
4-(4-Bromo-2-fluorobenzyl)-4H-1,2,4-benzothiadiazine 1,1-
Dioxide (11a). Colorless crystal, yield 69%; mp 207-208 °C; 1H
NMR (400 MHz, CDCl3) δ 5.14 (s, 2H), 7.01 (m, 2H), 7.29 (m,
1H), 7.47 (m, 1H), 7.56 (m, 1H), 7.78 (s, 1H), 8.04 (m, 1H).
4-(3-Nitrobenzyl)-4H-1,2,4-benzothiadiazine 1,1-Dioxide
(11b). Colorless crystal, yield 84%; mp 223-225 °C; 1H NMR
(400 MHz, CDCl3) δ 5.55 (s, 2H), 7.41 (d, J = 8.4 Hz, 1H), 7.51
(m, 1H), 7.69 (m, 3H), 7.91 (m, 1H), 8.17 (d, J = 8.0 Hz, 1H),
8.27 (s, 1H), 8.41 (s, 1H).
[2-(3-Nitrobenzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-benzothiadia-
zine 1,1-dioxide-4-yl]acetamide (10g). Yellow crystal, yield 89%;
mp 180-183 °C; 1H NMR (500 MHz, DMSO-d6) δ 4.00 (s, 2H),
4.46 (s, 2H), 4.96 (s, 2H), 6.70 (m, 1H), 7.29 (s, 1H), 7.41 (m, 1H),
7.53 (m, 1H), 7.63 (s, 1H), 7.68 (m, 1H), 7.83 (d, J = 8.0 Hz, 1H),
8.18 (d, J = 1.5 Hz, 1H), 8.21 (s, 1H). Anal. (C16H15FN4O5S) C,
H, N.
[2-(4-(Trifluoromethyl)benzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetamide (10h). Colorless crystal,
1
yield 94%; mp 103-105 °C; H NMR (500 MHz, DMSO-d6) δ
4.01(s,2H),4.41(s,2H),4.92(s,2H),6.72(m,1H),7.27(s, 1H),7.42
(m, 1H), 7.52 (m, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.63 (m, 1H), 7.76
(d, J = 8.0 Hz, 2H). Anal. (C17H15F4N3O3S) C, H, N.
[2-(2,4,5-Trifluorobenzyl)-7-fluoro-3,4-dihydro-2H-1,2,4-benzo-
thiadiazine 1,1-dioxide-4-yl]acetamide (10i). Colorless crystal,
yield 80%; mp 175-177 °C; 1H NMR (500 MHz, DMSO-d6)
δ 4.04 (s, 2H), 4.30 (s, 2H), 4.91 (s, 2H), 6.70 (m, 1H), 7.31
(s, 1H), 7.42 (m, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.61 (m, 2H),
7.68 (s, 1H). Anal. (C16H13F4N3O3S) C, H, N.
4-[4-(Trifluoromethyl)benzyl]-4H-1,2,4-benzothiadiazine 1,1-
Dioxide (11c). Colorless crystal, yield 59%; mp 216-219 °C;
1H NMR (400 MHz, CDCl3) δ 5.24 (s, 2H), 6.95 (m, 1H), 7.36
[2-(4-Bromo-2-fluorobenzyl)-7-cholro-3,4-dihydro-2H-1,2,4-ben-
zothiadiazine 1,1-dioxide-4-yl]acetamide (10j). Colorless crystal,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8341
(d, J = 8.0 Hz, 2H), 7.47 (m, 2H), 7.66 (d, J = 8.4 Hz, 2H), 7.79
(s, 1H), 8.05 (m, 1H).
temperature for 2 h. The alkaline suspension was adjusted to be
acidic with 0.1 N HCl and extracted 3-fold with ethyl acetate
(3 ꢀ 20 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 com-
pound was recrystallized in methanol.
[4-(4-Bromo-2-fluorobenzyl)-3,4-dihydro-2H-1,2,4-benzothia-
diazine 1,1-dioxide-2-yl]acetic Acid (14a). Colorless crystal, yield
69%; mp 170-173 °C; 1H NMR (400 MHz, DMSO-d6) δ 3.77
(s, 2H), 4.69 (s, 2H), 5.10 (s, 2H), 6.85 (m, 2H), 7.20 (m, 1H), 7.40
(m, 2H), 7.59 (m, 2H), 12.90 (s, 1H). Anal. (C16H14BrFN2O4S)
C, H, N.
4-(2,4,5-Trifluorobenzyl)-4H-1,2,4-benzothiadiazine 1,1-
Dioxide (11d). Colorless crystal, yield 76%; mp 203-206 °C; 1H
NMR (400 MHz, DMSO-d6) δ 5.40 (s, 2H), 7.43 (d, J = 8.4 Hz,
1H), 7.51 (m, 1H), 7.61 (m, 2H), 7.71 (m, 1H), 7.89 (m, 1H), 8.30
(d, J = 1.6 Hz, 1H).
General Synthetic Procedure for 12a-d. To a solution of
the appropriate 11 (14 mmol) in 2-propanol-acetonitrile (1:1,
20 mL) with stirring was added NaBH4 (2.0 g). Then the mixture
was stirred at room temperature for 5 min. The solvent was
removed by distillation under reduced pressure, and the residue
was suspended in Cl2H2 (10 mL). The alkaline suspension was
treated with 0.1 N HCl until the pH value indicated that the
mixture was acidic. The resulting insoluble material was col-
lected by filtration, washed with water, dried, and then recrys-
tallized in ethyl acetate.
[4-(3-Nitrobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-di-
oxide-2-yl]acetic Acid (14b). Colorless crystal, yield 77%; mp
1
160-163 °C; H NMR (400 MHz, DMSO-d6) δ 3.78 (s, 2H),
4.83 (s, 2H), 5.15 (s, 2H), 6.87 (m, 2H), 7.40 (m, 1H), 7.62 (m, 2H),
7.70 (m, 1H), 8.13 (m, 2H), 12.79 (s, 1H). Anal. (C16H15N3O6S) C,
H, N.
4-(4-Bromo-2-fluorobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadia-
zine 1,1-Dioxide (12a). Colorless crystal, yield 70%; mp 165-
[4-(4-(Ttrifluoromethyl)benzyl)-3,4-dihydro-2H-1,2,4-benzothia-
diazine 1,1-dioxide-2-yl]acetic Acid (14c). Colorless crystal, yield
1
168 °C; H NMR (400 MHz, DMSO-d6) δ 4.62 (s, 2H), 4.84
1
88%; mp 88-91 °C; H NMR (400 MHz, DMSO-d6) δ 3.79
(d, J = 8.4 Hz, 2H), 6.66 (d, J = 8.8 Hz, 1H), 6.79 (m, 1H), 7.30
(m, 2H), 7.38 (m, 1H), 7.57 (m, 2H), 8.18 (m, 1H).
(s, 2H), 4.78 (s, 2H), 5.14 (s, 2H), 6.76 (d, J = 8.4 Hz, 1H), 6.85
(m, 1H), 7.37 (m, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.59 (m, 1H), 7.71
(d, J = 8.4 Hz, 2H). Anal. (C17H15F3N2O4S) C, H, N.
4-(3-Nitrobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-
Dioxide (12b). Colorless crystal, yield 66%; mp 154-156 °C; 1H
NMR (400 MHz, DMSO-d6) δ 4.74 (s, 2H), 4.87 (d, J = 8.0 Hz,
2H), 6.65 (d, J = 8.4 Hz, 1H), 6.78 (m, 1H), 7.29 (m, 1H), 7.55
(m, 1H), 7.63 (m, 1H), 7.78 (d, J = 8.0 Hz, 1H), 8.11 (m, 1H),
8.19 (s, 1H), 8.24 (s, 1H).
4-[4-(Trifluoromethyl)benzyl]-3,4-dihydro-2H-1,2,4-benzothiadia-
zine 1,1-Dioxide (12c). Colorless crystal, yield 70%; mp 187-188
°C; 1H NMR (400 MHz, CDCl3) δ 4.64 (s, 2H), 4.90 (s, 2H), 6.62
(d, J = 8.8 Hz, 1H), 6.89 (m, 1H), 7.31 (m, 1H), 7.35 (d, J= 8.4 Hz,
2H), 7.62 (d, J = 8.4 Hz, 2H), 7.74 (m, 1H).
4-(2,4,5-Trifluorobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine
1,1-Dioxide (12d). Colorless crystal, yield 83%; mp 158-160 °C;
1H NMR (400 MHz, CDCl3) δ 4.54 (s, 2H), 4.90 (s, 2H), 6.64 (d,
J = 8.8 Hz, 1H), 6.90 (m, 1H), 7.02 (m, 2H), 7.35 (m, 1H), 7.73
(m, 1H).
General Synthetic Procedure for 13a-d. A mixture of appro-
priate 12 (10 mmol), potassium carbonate (3 g), and methyl
bromoacetate (1.68 g, 11 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.
[4-(4-Bromo-2-fluorobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadia-
zine 1,1-dioxide-2-yl]acetic Acid Methyl Ester (13a). Colorless
crystal, yield 86%; mp 193-195 °C; 1H NMR (400 MHz, CDCl3)
δ 3.73 (s, 3H), 3.89 (s, 2H), 4.53 (s, 2H), 5.16 (s, 2H), 6.70 (d, J =
8.8 Hz, 1H), 6.89 (m, 1H), 7.05 (m, 1H), 7.32 (m, 2H), 7.35 (m, 1H),
7.71 (m, 1H). Anal. (C17H16BrFN2O4S) C, H, N.
[4-(3-Nitrobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-di-
oxide-2-yl]acetic Acid Methyl Ester (13b). Colorless crystal, yield
67%; mp 120-122 °C; 1H NMR(400 MHz, CDCl3) δ3.73 (s, 3H),
3.95 (s, 2H), 4.66 (s, 2H), 5.22 (s, 2H), 6.65 (d, J= 8.4 Hz, 1H), 6.92
(m, 1H), 7.35 (m, 1H), 7.55 (m, 2H), 7.74 (m, 1H), 8.11 (s, 1H),
8.17 (m, 1H). Anal. (C17H17N3O6S) C, H, N.
[4-(4-(Ttrifluoromethyl)benzyl)-3,4-dihydro-2H-1,2,4-benzothia-
diazine 1,1-dioxide-2-yl]acetic Acid Methyl Ester (13c). Colorless
crystal, yield 78%; mp 177-179 °C; 1H NMR (400 MHz, CDCl3)
δ 3.70 (s, 3H), 3.92 (s, 2H), 4.63 (s, 2H), 5.18 (s, 2H), 6.67 (d, J =
8.8 Hz, 1H), 6.90 (m, 1H), 7.33 (d, J = 7.6 Hz, 2H), 7.36 (m, 1H),
7.62 (d, J = 8.4 Hz, 2H), 7.73 (m, 1H).
[4-(2,4,5-Trifluorobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine
1,1-dioxide-2-yl]acetic Acid Methyl Ester (13d). Colorless crystal,
yield 72%; mp 128-130 °C; 1H NMR (400 MHz, CDCl3) δ3.74 (s,
3H), 3.91 (s, 2H), 4.53 (s, 2H), 5.18 (s, 2H), 6.69 (d, J = 8.4 Hz,
1H), 6.91 (m, 1H), 7.02 (m, 2H), 7.38 (m, 1H), 7.73 (m, 1H).
General Synthetic Procedure for 14a-d. A mixture of the
appropriate acetate 13 (1 mmol), 1,4-dioxane (8 mL), and
saturated aqueous sodium hydroxide (8 mL) was stirred at room
[4-(2,4,5-Trifluorobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine
1,1-dioxide-2-yl]acetic Acid (14d). Colorless crystal, yield 91%; mp
159-161 °C; 1H NMR (400 MHz, DMSO-d6) δ 3.75 (s, 2H), 4.67
(s, 2H), 5.09 (s, 2H), 6.87 (m, 2H), 7.41 (m, 2H), 7.58 (m, 2H). Anal.
(C16H13F3N2O4S) C, H, N.
General Synthetic Procedure for 15a, 15b. A solution of
appropriate acetate 13 (1 mmol) in 25 mL of methanol with
stirring was treated with NH3 (gas) for 30 min and then stirred at
room temperature for 18 h. After evaporation of the solvent
under reduced pressure, the crude solid was recrystallized in
ethyl acetate-dichloromethane (1:1) to give pure product.
[4-(4-Bromo-2-fluorobenzyl)-3,4-dihydro-2H-1,2,4-benzothia-
diazine 1,1-dioxide-2-yl]acetamide (15a). Colorless crystal, yield
63%; mp 159-161 °C; 1H NMR (400 MHz, CDCl3) δ 3.79
(s, 2H), 4.58 (s, 2H), 5.09 (s, 2H), 5.64 (s, 1H), 6.55 (s, 1H), 6.70
(d, J = 8.8 Hz, 1H), 6.92 (m, 1H), 7.05 (m, 1H), 7.29 (m, 2H),
7.38 (m, 1H), 7.71 (m, 1H). Anal. (C16H15BrFN3O3S) C, H, N.
[4-(3-Nitrobenzyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-di-
oxide-2-yl]acetamide (15b). Yellow crystal, yield 53%; mp
1
154-157 °C; H NMR (400 MHz, CDCl3) δ 3.83 (s, 2H), 4.71
(s, 2H), 5.29 (s, 2H), 5.52 (s, 1H), 6.47 (s, 1H), 6.65 (d, J = 8.4 Hz,
1H), 6.95 (m, 1H), 7.40 (m, 1H), 7.57 (m, 2H), 7.77 (d, J = 1.2 Hz,
1H), 8.10 (s, 1H), 8.18 (m, 1H). Anal. (C16H16N4O5S) C, H, N.
Biology, Materials, and Methods. ALR2 and ALR1 were
obtained from Wistar rats, 200-250 g, b.w., supplied by Vital
River, Beijing, China. STZ was from Amresco. Sorbitol dehy-
drogenase was from Wako, Japan. D,L-Glyceraldehyde, sodium
D-glucuronate, and NADPH were from Sigma-Aldrich. All
other chemicals were of reagent grade.
ALR1 and ALR2 were prepared in accordance with the
method of Kinoshita and Concettina La Motta.12,30 Enzyme
activity was assayed spectrophotometrically on an Unico 4802S
UV/vis double beam spectrophotometer by measuring the
decrease in absorption of NADPH at 340 nm, which accom-
panies the oxidation of NADPH catalyzed by ALR2 and ALR1.
Enzymatic Assays. ALR2 activity was performed at 32 °C
in a reaction mixture containing 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 deionized water, and 0.25 mL of
10 mM D,L-glyceraldehyde as substrate in a final volume of
1 mL. The reaction mixture except for D,L-glyceraldehyde was
incubated at 32 °C for 10 min. The substrate was then added to
start the reaction, which was monitored for 4 min.
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),

8342 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Chen et al.
0.15 mL of deionized water, and 0.25 mL of 20 mM sodium
D-glucuronate as substrate in a final volume of 1 mL. The reac-
tion mixture except for sodium D-glucuronate was incubated at
37 °C for 10 min. The substrate was then added to start the
reaction, which was monitored for 4 min.
FlexX Docking. Docking was performed using the SYBYL
7.1. The structure of the ligand was set up as follows: molecular
model of compound was constructed using standard bond
lengths and bond angles of the SYBYL fragment library,
Gasteiger-Huckel charges were assigned, and the minimization
was realized with the maximum number of iterations set to 1000.
The X-ray crystal structure of human aldose reductase holoen-
zyme in complex with the inhibitor tolrestat (PDB entry code
2FDZ)52 was retrieved from the Protein Data Bank (PDB).
The inhibitory activity of the newly synthesized compounds
against ALR2 and ALR1 was assayed by adding 5 μL of the
inhibitor solution to the reaction mixture described above. All
compounds were dissolved in dimethylsulfoxide (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 10-4 M (the concentration is referenced to that of
the compound in the reaction mixture). Those compounds
found to be active were tested at additional concentrations
between 10-4 and 10-8 M. Each dose-effect curve was gener-
ated 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 - 2, where n is the total number
of determinations.
STZ-Induced Diabetic Rat Model (General). Male Wistar rats
(200-250 g) were purchased from the Experimental Animal
Center of Peking University (Certificate SCXK-2006-0008). The
animals were allowed to acclimate for a week and then were
rendered diabetic by an intraperitoneal injection of STZ (55 mg/kg),
which had been freshly dissolved in 0.03 M citrate buffer,
pH 4.5. Four days later, tail blood glucose was measured using
a One Touch II blood glucose meter. Diabetic rats whose blood
glucose levels were g16 mmol/L were randomized according to
blood glucose levels into diabetic-control and diabetic-treat-
ment groups (n = 6-8).
˚
All the amino acid residues within 6.5 A of the ligand tolrestat
are defined as the active site. The docking and subsequent
scoring were performed, and a maximum of 30 poses for the
specified ligand was produced. The Total Score, Match Score, Lipo
Score, Ambig Score, Clash Score, Rot Score, Match, Avg Volume,
Max Volume, Frag No., Pdb Id, D_SCORE, PMF_SCORE,
G_SCORE, CHEMSCORE, and CSCORE for each conforma-
tion were all exported.
Acknowledgment. This work was supported by the Beijing
Natural Science Foundation (Grant No. 7102091). Many
thanks are due to Prof. Yufen Zhao and Dr. Yan Liu (Depart-
ment of Chemical Biology and Department of Chemistry) for
the use of the Sybyl program in their laboratory (Xiamen
University, Xiamen 361005, China).
Supporting Information Available: Elemental analysis data,
analytical HPLC purity data, and additional biochemical data.
This material is available free of charge via the Internet at http://
pubs.acs.org..
References
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Five-Day STZ-Induced Diabetic Rat Screening Model. Begin-
ning on day 4 after induction of diabetes with STZ, test
compounds suspended in a vehicle of 0.5% sodium carboxy-
methylcellulose were administered po consecutively for 5 days.
Nondiabetic and diabetic controls received vehicle during this
period. Six hours after the final dose was administered, the rats
were sacrificed and sciatic nerves were removed, weighed, and
stored at -80 °C for sorbitol analysis.
Tissue Sorbitol Analysis. The sciatic nerve sample (30-60 mg)
was placed into water (1.0 mL/40 mg of tissue), heated in a
boiling bath for 2 min, and then homogenized with a Polytron
instrument in 6% perchloricacid. The homogenate was centri-
fuged at 1050g for 15 min at 4 °C The supernatant was
neutralized with 2 M K2CO3 and used as tissue extract for the
assaying of sorbitol. Sorbitol was assayed by an enzymatic
method in which sorbitol dehydrogenase catalyzes the stoichio-
metric conversion of NADþ by sorbitol to a fluorogenic pro-
duct, NADH.54 The reaction mixture contained 30 mM glycine
buffer (pH 9.4), 1.3 mM NADþ, 1.3 U/mL sorbitol dehydro-
genase, and 1.0 mL of the tissue extract in a total volume of
3 mL. After the mixture was allowed to stand for 60 min at 37 °C,
the fuorescence intensity was measured at 365 nm excitation
wavelength and 430 nm emission wavelength using a fluoro-
spectrophotometer. The sorbitol concentration was quantitated
by comparison with standards of sorbitol. The sorbitol content
in the sciatic nerve of each animal was expressed as nmol/wet
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% inhibition ¼ ðS - TÞ=ðS - NÞ ꢀ 100
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