Effect of C7 Modifications on Benzothiadiazine-1,1-dioxide Derivatives on Their Inhibitory Activity and Selectivity toward Aldose Reductase

Article abstract of DOI:10.1002/cmdc.201200386

The development and progression of chronic complications in diabetic patients, such as retinopathy, nephropathy, neuropathy, cataracts, and stroke, are related to the activation and/or overexpression of aldose reductase (ALR2), which is a member of the al

Full text of DOI:10.1002/cmdc.201200386

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DOI: 10.1002/cmdc.201200386
Effect of C7 Modifications on Benzothiadiazine-1,1-dioxide
Derivatives on Their Inhibitory Activity and Selectivity
toward Aldose Reductase
Shuzhen Zhang, Xin Chen, Shagufta Parveen, Saghir Hussain, Yanchun Yang, Chaojun Jing,
and Changjin Zhu*[a]
The development and progression of chronic complications in
diabetic patients, such as retinopathy, nephropathy, neuropa-
thy, cataracts, and stroke, are related to the activation and/or
overexpression of aldose reductase (ALR2), which is a member
of the aldo–keto reductase superfamily. A structure–activity re-
lationship study focused on the C7 position of 1,2,4-benzothia-
diazine-1,1-dioxide derivatives was pursued in an attempt to
discover ALR2 inhibitors with enhanced potency and selectivi-
ty. These studies led to a series of new C7-substituted com-
pounds, which were evaluated for their inhibitory activity
against ALR2; they exhibited IC50 values in the range of 2.80–
45.13 nm. Two compounds with a C7-dimethylcarbamoyl and
a C7-diethylcarbamoyl substituent, respectively, were found to
be the most active and presented excellent selectivity for ALR2
over aldehyde reductase (ALR1). The structure–activity relation-
ship analyses and molecular modeling studies presented
herein highlight the importance of hydrophobic and bulky
groups at the C7 position for inhibitory activity and selectivity
toward ALR2.
Introduction
Diabetes mellitus as a complex metabolic disorder remains
a life-threatening disease that is spreading around the world.
Diabetic patients have a high risk for onset of chronic compli-
cations including retinopathy, nephropathy, neuropathy, cata-
racts, and stroke. The development and progression of these
complications are related to the activation and/or overexpres-
sion of the enzyme aldose reductase (ALR2, EC 1.1.1.21), which
is a member of the aldo–keto reductase superfamily.[1] During
periods of normal blood sugar levels, glucose is preferentially
metabolized through the glycolytic pathway, while during hy-
perglycemia conditions, the polyol metabolic pathway is acti-
vated and leads to the accumulation of sorbitol. ALR2, which
catalyzes the reduction of excess glucose to sorbitol in the
presence of NADPH as a cofactor, is the first and rate-determin-
ing enzyme in the polyol pathway (Figure 1). Thus, inhibition
of ALR2 has received considerable attention for the prevention
and treatment of diabetic complications.
shown in Figure 2.[2] However, only epalrestat has reached the
market, in Japan in 1992 and recently in China and India, for
the treatment of diabetic neuropathy.[3] Many ARIs that ap-
peared to be promising have yet to be clinically successful be-
cause of undesirable effects. The side effects are believed to
result from a lack of selectivity relative to aldehyde reductase
(ALR1, EC 1.1.1.2) which plays a detoxification role in specifical-
ly metabolizing toxic aldehydes such as hydroxynonenal (HNE),
3-deoxyglucosone, and methylglyoxal.[4] ALR2 and ALR1 share
a high degree of homology in their primary and secondary
structures, substrate specificities, and kinetic mechanisms.[5]
Our effort has focused on the development of more selective
and effective ARIs. We have recently designed a number of
potent ARIs based on the scaffolds of 1,2,4-benzothiadiazine-
1,1-dioxide, pyrido[2,3-e][1,2,4]thiadiazine-1,1-dioxide, 1,2-ben-
zothiazine-1,1-dioxide and quinoxalinone, and we found that
substitutions at the C7 position of the benzothiadiazine scaf-
fold greatly increase the activity.[6] Herein, we report further
modifications at the C7 position and discuss structure–activity
relationships (SAR).
A large number of ALR2 inhibitors (ARIs) have been devel-
oped in the past few decades, and some typical ARIs are
Results and Discussion
Chemistry
Figure 1. The polyol pathway of glucose metabolism.
2-Benzyl-1,1-dioxido-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetic
acid derivatives with diverse substitutions at the C7 position
(8a–d and 16a–l) were prepared following the synthetic path-
way depicted in Scheme 1.[6a] Reaction of commercially avail-
able 4-substituted anilines 1a–b with chlorosulfonyl isocyanate
formed ureas, which were then sulfonylated intramolecularly in
[a] S. Zhang, X. Chen, S. Parveen, S. Hussain, Y. Yang, C. Jing, Prof. Dr. C. Zhu
Department of Applied Chemistry, Beijing Institute of Technology
Zhongguancun South Street, 100081 Beijing (China)
E-mail: zcj@bit.edu.cn
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ing from modifications to the C7
position of the 1,1-dioxido-2H-
benzo[e][1,2,4]thiadiazin-4(3H)-yl-
acetic acid framework containing
an N2 side chain of 2-fluoro-4-
bromobenzyl or 2,4,5-trifluoro-
benzyl. This synthetic work led
to two series of compounds, in-
cluding derivatives of 2-(2-
fluoro-4-bromobenzyl)-1,1-dioxi-
do-2H-benzo[e][1,2,4]thiadiazin-
4(3H)-ylacetic acid (8a–b and
16a–f) and 2-(2,4,5-trifluoroben-
zyl)-1,1-dioxido-2H-benzo[e]-
[1,2,4]thiadiazin-4(3H)-ylacetic
acid (8c–d and 16g–l) as shown
in Table 1. All of these com-
pounds were tested for their po-
tential inhibitory activity toward
ALR2 isolated from rat lenses
and ALR1 isolated from rat kid-
neys. IC50 values were deter-
mined by linear regression analy-
sis of the log of the concentration–response curve.[6c] In order
Figure 2. Structures of aldose reductase inhibitors.
the presence of anhydrous aluminum chloride to produce 2H-
benzo[e][1,2,4]thiadiazin-3(4H)-one-1,1-dioxides (2a–b). Amino-
benzenesulfonamides 3a–b were obtained by the decarboxyla-
tion of 2a–b using sulfuric acid. They were then reacted with
triethyl orthoformate to give 4a–b, which were alkylated with
methyl bromoacetate to form methyl esters 5a–b. Reduction
of the double bond at the 2,3 positions of 5a–b with sodium
borohydride gave 6a–b. A benzyl group was introduced at the
N2 position with benzylbromide, and the products (7a–d)
were then treated with aqueous sodium hydroxide to provide
compounds 8a–d. Alternatively, 2a was used in the prepara-
tion of compounds 16a–l and was oxidized by means of po-
tassium permanganate to form carboxylic acid 9. Preparation
of 11 from 9 was completed by using the same method as for
preparation of 4a–b by 2a–b. Acid 11 was converted into
ester 12a by treatment with methanol and sulfuric acid
(step k). Conversely, 11 was treated with thionyl chloride
(step l) and then with secondary amines in the presence of
triethylamine (step m) to form amides 12b–f. Alkylation at the
N2 and N4 positions of 12a–f led to 15a–l by using the same
method as for the modification of 4a–b to produce 7a–d. The
ester groups in the N4 side chain of 15a–l and in the C7 side
chain of 15a and 15g were hydrolyzed with sodium hydroxide
to yield 16a–l.
to study the SAR, compounds 17a–f (reported previously[6a]
)
were retested along with the newly synthesized compounds in
the present study (Table 1).
ALR2 inhibition
Among all of the compounds synthesized in the present study
and previously, as shown in Table 1, 16l and 16k (with IC50
values of 2.80 and 3.20 nm, respectively) appeared to have the
most potent inhibitory activity against ALR2. Further SAR anal-
ysis found that C7-carbamoyl derivatives 16 f (IC50 =4.57 nm)
and 16e (IC50 =6.43 nm) were the most active compounds in
the N2-2-fluoro-4-bromobenzyl series (8a–b, 16a–f, and 17a–
c).
The
C7-chloro
derivative
17b,
C7-N,N-methyl-
(phenyl)carbamoyl derivative 16d, and C7-methyl derivative
8a also exhibited considerable ALR2 inhibitory activity. The
ranking order of the compounds displaying the highest levels
of ALR2 inhibitory activity in this series is 16 f > 16e > 17b >
16d > 8a > 17c > 8b, therefore giving the relative prefer-
ence for increased ALR2 inhibition with a diethylcarbamoyl C7
substituent, followed by dimethylcarbamoyl, chloro, N,N-
methyl(phenyl)carbamoyl, methyl, bromo, and methoxy
groups.
Highly similar results to the compounds in the N2-2-fluoro-
4-bromobenzyl series were also obtained for compounds in
the N2-2,4,5-trifluorobenzyl series (8c–d, 16g–l, and 17d–f).
The C7-carbamoyl derivatives 16l (IC50 =2.80 nm) and 16k
(IC50 =3.20 nm) were the most active compounds. In addition,
C7-chloro derivative 17e, C7-methyl derivative 8c, and C7-N,N-
methyl(phenyl)carbamoyl derivative 16j showed excellent
ALR2 inhibitory activity. The ranking for the inhibitory activity
of the most potent compounds in this series is 16l > 16k >
17e > 8c > 16j > 17 f > 8d, indicating the preferred order
Biological activity and SAR studies
With regard to the design of effective ARIs based on benzo-
thiadiazine-1,1-dioxide and pyridothiadiazine-1,1-dioxide core
structures, an N4-acetate group and a substituted N2-benzyl
group have proven to be essential side chains.[6a,b] 2-Fluoro-4-
bromobenzyl and 2,4,5-trifluorobenzyl turned out to be two of
the most potent structures among the substituted N2-benzyl
groups. The present study then focused on derivatives result-
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ferent effects on ALR2 inhibition
despite their similarity in size.
For example, 16b and 16c ex-
hibited significantly lower activi-
ty than 16d, and 16h and 16i
showed lower activity than 16j.
The decreased inhibitory effect
for the C7-piperidine-1-carbonyl
and C7-morpholine-4-carbonyl
substituents might result from
that fact that their structures are
more constrained than that of
the
C7-N,N-methyl(phenyl)car-
bamoyl substituent. This is also
evidenced by the fact that the
most potent compounds, includ-
ing 16 f, 16e, 16l, and 16k,
have the most flexible C7-carba-
moyl substituents in each series
compared with the other C7-car-
bamoyl derivatives. Moreover,
the chloro, methyl, and N,N-
methyl(phenyl)carbamoyl
C7
substituents are very different in
size but showed similar activity.
These results suggest that the
effect of C7 substituents de-
pends more on the hydropho-
bicity and flexibility of the sub-
stituents than on the size.
Furthermore, all compounds
in the N2-2,4,5-trifluorobenzyl
series were more potent than
their counterparts in the N2-2-
fluoro-4-bromobenzyl
series,
demonstrating that the N2-2,4,5-
trifluorobenzyl group is favora-
ble for the design of ALR2 inhibi-
tors based on a benzothiadia-
zine-1,1-dioxide core structure.
This is consistent with recently
reported study results.[6a,b]
Scheme 1. Reagents and conditions: a) ClSO2NCO, CH3NO2, À408C, 0.5 h; b) AlCl3, 1108C, 1 h, 64–70%; c) 50%
H2SO4, 1408C, 6 h; d) NaOH(aq), 08C, 0.5 h, 46–56%; e) HC(OEt)3, reflux, 2 h, 86–92%; f) BrCH2COOCH3, K2CO3,
CH3CN, 758C, 2 h, 68–84%; g) NaBH4, CH3OH, 08C, 5 min, 56–75%; h) Bn-Br, K2CO3, CH3CN, 708C, 2 h, 69–86%;
i) 1,4-dioxane, NaOH, RT, 2 h, 69–88%; j) NaOH, KMnO4, 808C, 7 h, 68%; k) CH3OH, H2SO4, reflux, 5 h, 41%; l) SOCl2,
758C, overnight; m) Et3N, secondary amines, CH3CN, RT, 0.5 h, 67–78%.
ALR1 inhibition
of effect of the C7 substituents as diethylcarbamoyl > dime-
thylcarbamoyl
>
chloro
>
methyl
>
N,N-methyl-
All of the synthetic compounds were evaluated for ALR1 inhib-
ition at a concentration of 5 mm, as shown in Table 1. Notably,
C7-carbamoyl derivatives (16b–f and 16h–l) showed low in-
hibitory potency within both of the two series, whereas the
C7-halogen derivatives (17a–c and 17d–f) displayed higher in-
hibitory activities. The inhibitory activities of C7-carboxy deriva-
tives were similar to those shown by the C7-carbamoyl deriva-
tives. The activity of C7-methyl and C7-methoxy derivatives
was moderate. Among the C7-carbamoyl derivatives, C7-piperi-
dine-1-carbonyl and C7-morpholine-4-carbonyl compounds
(16b–c and 16h–i) were the least active with regard to ALR1
inhibition in each of the two series, respectively, although they
(phenyl)carbamoyl > bromo > methoxy.
In addition, both of the C7-carboxy derivatives (16a and
16g) had the lowest ALR2 inhibitory activity in each series. C7-
piperidine-1-carbonyl derivatives 16b and 16h and C7-mor-
pholine-4-carbonyl derivatives 16c and 16i also showed rela-
tively low ALR2 inhibitory activity. Therefore, it is likely that this
decrease in ALR2 inhibitory activity was caused by the hydro-
philicity of the C7 substituents. Furthermore, it was found that
the C7-piperidine-1-carbonyl and C7-morpholine-4-carbonyl
substituents,
compared
with
the
C7-N,N-methyl-
(phenyl)carbamoyl substituent in both series, yielded quite dif-
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Molecular docking
Table 1. Biological data for the synthesized compounds.
Docking studies were performed
to examine the manner by
which newly prepared com-
pounds interact with ALR2,
which may explain the above-
described SARs and mechanistic
details of the biological activity.
The most active compound, 16l,
was docked into the conforma-
tion of human ALR2 from the
complex with NADP+/IDD594
(PDB code: 1US0) as shown in
Figure 3.
Compd
R1
R2
ALR2 IC50 [nm][b]
ALR1 Inhib. [%][c]
8a
8b
16a
CH3
OMe
OH
2-F-4-Br
2-F-4-Br
2-F-4-Br
8.23 (6.96–9.51)
20.17 (17.02–23.31)
45.13 (41.02–49.25)
23.4
22.3
18.4
16b
16c
2-F-4-Br
2-F-4-Br
25.97 (21.83–30.10)
35.07 (27.19–42.95)
14.9
12.2
Docking results revealed that
inhibitor 16l fit tightly within
the active sites of ALR2. The car-
boxy group was bound to an
anion-binding pocket through
three tight hydrogen bonds with
the hydroxy group of Tyr48
(3.30 ꢁ), the Ne2 atom of His110
(2.70 ꢁ), and the Ne1 of Trp 111
(2.82 ꢁ), which are three key resi-
dues in binding and catalysis.[7]
The carboxy head also formed
a stabilizing electrostatic interac-
tion with the positively charged
nicotinamide moiety of the co-
factor NADP+ (N–O=4.15 ꢁ).
The substituted benzyl ring fit
well in the specificity pocket
formed by Leu300, Cys303,
Cys298, Thr113, Phe122, and
Trp111, among which Trp111 p-
stacked perfectly against the
benzyl ring. In addition, the ben-
16d
2-F-4-Br
8.20 (4.45–11.95)
17.8
16e
16 f
2-F-4-Br
2-F-4-Br
6.43 (5.02–7.85)
4.57 (3.94–5.19)
18.3
15.6
17a
17b
17c
8c
8d
16g
F
Cl
Br
CH3
OMe
OH
2-F-4-Br
2-F-4-Br
2-F-4-Br
2,4,5-F3
2,4,5-F3
2,4,5-F3
25.83 (18.62–33.04)
7.30 (5.81–8.79)
9.50 (7.71–11.29)
4.36 (3.09–5.64)
8.43 (6.50–10.36)
15.97 (12.72–19.21)
25.6
30.2
28.3
46.1
38.3
25.1
16h
16i
2,4,5-F3
2,4,5-F3
11.10 (8.00–14.20)
13.20 (7.75–18.65)
14
22
16j
2,4,5-F3
5.50 (3.76–7.24)
35.8
16k
16l
2,4,5-F3
2,4,5-F3
3.20 (2.12–4.28)
2.80 (2.14–3.46)
25
22.8
17d
17e
17 f
F
Cl
Br
2,4,5-F3
2,4,5-F3
2,4,5-F3
13.77 (9.29–18.24)
3.87 (2.83–4.90)
7.70 (4.65–10.75)
14.32 (12.26–16.08)
57.8
52.9
50.8
79.2
zothiadiazine-1,1-dioxide
core
was trapped in the hydrophobic
cage formed by the side chains
of Trp20, Phe122, Trp79, and
Trp219, which in turn led to the
placement of the relatively bulky
7-diethylcarbamoyl substituent
Epalrestat
[a] Compounds prepared previously.[6a] [b] IC50 (95% CL) values represent the concentration of the tested com-
pounds required to decrease enzymatic activity by 50%. [c] Inhibitory effects were evaluated at a concentration
of 5 mm.
did not exhibit the highest ALR2 inhibition, as described
above. The C7-diethylcarbamoyl and C7-dimethylcarbamoyl
compounds (16e–f and 16k–l) that were the most potent in-
hibitors of ALR2 exhibited low inhibitory activity against ALR1.
Accordingly, it is likely that bulky and rigid substituents at the
C7 position greatly decrease the ALR1 inhibitory activity of the
compounds. These results, together with the data from the
compounds in the ALR2 inhibition assay, suggest that the C7-
carbamoyl derivatives are highly selective inhibitors for ALR2
over ALR1. In particular, the C7-diethylcarbamoyl derivatives
(16 f and 16l) proved to be potent and selective ALR2 inhibi-
tors.
in the deep cavity of the hydrophobic cage formed by
Phe121, Phe122, Gln49, and Val47. The ability of the core, to-
gether with the bulky 7-substituent, to be accommodated by
the hydrophobic cage is likely due to the relatively open space
of the pocket,[2c] suggesting that this pocket may also play an
important role in ligand binding. This binding behavior is dif-
ferent from that of compound 17e (reported previously[6a]) in
which the phenyl ring of the benzothiadiazine-1,1-dioxide core
penetrates the specificity pocket, while the benzyl ring occu-
pies the hydrophobic cage, which consequently may explain
the higher ALR2 inhibitory activity of 16l as compared with
17e.
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C7 substituent both on activity and selectivity. Consequently,
the present study determined the important role of the side
chain at the C7 position of benzothiadiazine-1,1-dioxide. This
side chain, together with the other two essential side chains of
the N4-carboxylic acid and the N2-benzyl determined by our
recent studies,[6a] provides a structural basis for effective ARIs,
as well as insight into the pharmacophore requirements for
ALR2 inhibition.
Experimental Section
Chemistry
General: Melting points were recorded on an X-4 microscopic
melting point apparatus and were uncorrected. All reactions were
1
routinely checked by TLC on Merck 60F254 silica gel. H NMR spec-
tra were recorded at 400 MHz, while 13C NMR spectra were record-
ed at 100 MHz in [D6]DMSO (unless otherwise stated). Data for
1H NMR are presented as follows: chemical shift (d, ppm), multiplic-
ity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet),
coupling constant J (Hz), and integration. Data for 13C NMR are re-
ported in chemical shifts (d, ppm). A Hitachi d-2000 Elite HPLC
system was used to determine the purity of the final acetic acid
derivatives with the following method: Inertsil ODS-2 250 mm ꢂ
10 mm, 5 mm column; mobile phase: CH3CN (0.2% TFA)/CH3OH=
1:1 for 8 min; room temperature; flow rate: 1 mLminÀ1; detection
at l 254 nm.
The p-toluidine (1a), 4-methoxyaniline (1b), methyl bromoacetate,
4-bromo-2-fluorobenzyl bromide, 2,4,5-trifluorobenzyl bromide, pi-
peridine, morpholine, dimethylamine, diethylamine and N-methyla-
niline used to obtain the target inhibitors were from Alfa Aesar.
d,l-Glyceraldehyde, sodium d-glucuronate, and NADPH were from
Sigma–Aldrich. All other chemicals were of reagent grade. Wistar
rats (200–250 g) were supplied by Vital River, Beijing, China.
General procedure for the synthesis of 2a–b: A three-necked
flask (500 mL) was charged with chlorosulfonyl isocyanate (13 mL,
150 mmol) and MeNO2 (150 mL). The flask was cooled in a dry ice/
CH3CN bath, and
a solution of the corresponding aniline
1 (135 mmol) in MeNO2 (100 mL) was then added dropwise to the
flask with vigorous stirring. After the addition was complete, the
reaction mixture was stirred for an additional 30 min. Anhydrous
aluminum chloride (20 g, 150 mmol) was then added to the result-
ing suspension, and the mixture was stirred for 1 h at 1108C. The
resulting mixture was then poured into ice, and the precipitate
was collected by filtration and washed with cold H2O, then dry
Et2O, to yield the final product.
Figure 3. Docking of inhibitor 16l into the active site of ALR2. Ligand 16l
and NADP are shown as stick models, while protein structures are shown as
a) ribbon diagrams and b) in surface representation. The docked pose of 16l
is shown in cyan (C), red (O), blue (N), yellow (S), and grey (F); hydrogen
bonds are shown as yellow dashed lines.
Conclusions
7-Methyl-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one-1,1-dioxide
(2a): White solid (yield: 18.3 g, 64%): mp: >3008C; 1H NMR
(400 MHz, [D6]DMSO): d=11.10 (s, 1H), 7.58 (s, 1H), 7.51 (d, J=
8 Hz, 1H), 7.18 (d, J=8.4 Hz, 1H), 2.34 ppm (s, 3H).
A group of new derivatives of benzothiadiazine-1,1-dioxide
were synthesized with modifications at the C7 position and
showed ALR2 inhibitory activity, with IC50 values ranging from
2.80 nm to 45.13 nm. This allowed us to investigate the effect
of various C7 substituents of the benzothiadiazine-1,1-dioxide
core on inhibitory activity against aldose reductase and selec-
tivity. SAR analysis, combined with docking studies, demon-
strated that bulky hydrophobic groups were favorable for C7
substitution in the design of ARIs. In particular, we found that
a flexible amide group at the C7 position not only increased
ALR2 inhibition activity but also greatly decreased the inhibi-
tion against ALR1, demonstrating a pronounced effect of the
7-Methoxy-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one-1,1-dioxide
(2b): Brown solid (yield: 21.5 g, 70%): mp: >3008C; 1H NMR
(400 MHz, [D6]DMSO): d=11.08 (s, 1H), 7.24 (m, 3H), 3.81 ppm (s,
3H).
7-Carboxy-2H-benzo[e][1,2,4]thiadiazin-3(4H)-one-1,1-dioxide
(9): Compound 2a (10 g, 47 mmol) was first suspended in H2O
(600 mL) and then dissolved during the addition of saturated aque-
ous NaOH (10 mL). KMnO4 (24.5 g, 155 mmol) was added portion-
wise at 08C, and the reaction mixture was stirred at 808C for 7 h.
The insoluble material was removed by filtration, and the filtrate
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was acidified to pH 1 with concentrated HCl. The precipitate was
collected by filtration, washed with H2O, and dried to yield the
product as a white solid (yield: 7.7 g, 68%): mp: >3008C; H NMR
(400 MHz, [D6]DMSO): d=12.77 (s, 1H), 9.83 (s, 1H), 8.02 (s, 1H),
7.84 (dd, J1 =2 Hz, J2 =8.4 Hz, 1H), 7.01 ppm (d, J=8.4 Hz, 1H).
solvent was evaporated in vacuo, and the residue was washed
with H2O and dried to yield the product.
1
(1,1-Dioxido-4H-benzo[e][1,2,4]thiadiazin-7-yl)(piperidin-1-yl)me-
thanone (12b): Light-brown solid (yield: 2.3 g, 78%): mp: >3008C;
1H NMR (400 MHz, [D6]DMSO): d=12.45 (s, 1H), 8.03 (s, 1H), 7.72
(m, 2H), 7.37 (d, J=8.4 Hz, 1H), 3.57 (s, 2H), 3.33 (s, 2H), 1.56 ppm
(d, 6H).
General procedure for the synthesis of 3a–b, 10: Compound 2
or 9 (28 mmol) was added to 50% aqueous H2SO4 (120 mL) and
stirred at 1408C for 6 h. The resulting mixture was cooled to room
temperature and then poured over ice. Saturated aqueous NaOH
(60 mL) was added to neutralize the mixture. The precipitate was
collected by suction filtration, washed with cold H2O, and dried in
vacuo to give desired product 3 or 10.
(1,1-Dioxido-4H-benzo[e][1,2,4]thiadiazin-7-yl)(morpholino)me-
thanone (12c): Light-brown solid (yield: 2.2 g, 76%): mp: 298–
1
2998C; H NMR (400 MHz, [D6]DMSO): d=12.45 (s, 1H), 8.03 (s, 1H),
7.83 (s, 1H), 7.72 (d, J=7.2 Hz, 1H), 7.38 (d, J=8.4 Hz, 1H),
3.4 ppm (d, 8H).
2-Amino-5-methylbenzenesulfonamide (3a): White solid (yield:
2.9 g, 56%): mp: 107–1088C; 1H NMR (400 MHz, [D6]DMSO): d=
7.35 (s, 1H), 7.14 (s, 2H), 7.05 (d, J=8 Hz, 1H), 6.70 (d, J=8.4 Hz,
1H), 5.63 (s, 2H), 2.17 ppm (s, 3H).
N-Methyl-N-phenyl-4H-benzo[e][1,2,4]thiadiazine-7-carboxa-
mide-1,1-dioxide (12d): Light-brown solid (yield: 2.1 g, 67%): mp:
289–2918C; H NMR (400 MHz, [D6]DMSO): d=12.35 (s, 1H), 7.94 (s,
1
1H), 7.65 (s, 1H), 7.50 (m, 1H), 7.24 (m, 5H), 7.13 (d, J=8.4 Hz, 1H),
2-Amino-5-methoxybenzenesulfonamide (3b): Brown solid (yield:
2.9 g, 52%); mp: 118–1198C; 1H NMR (400 MHz, [D6]DMSO): d=
7.22 (s, 2H), 7.10 (s, 1H), 6.93 (m, 1H), 6.76 (d, J=8.8 Hz, 1H), 5.43
(s, 2H), 3.68 ppm (s, 3H).
3.38 ppm (s, 3H).
N,N-Dimethyl-4H-benzo[e][1,2,4]thiadiazine-7-carboxamide-1,1-
dioxide (12e): Light-brown solid (yield: 1.9 g, 76%): mp: 252–
1
2538C; H NMR (400 MHz, [D6]DMSO): d=12.46 (s, 1H), 8.03 (s, 1H),
4-Amino-3-sulfamoylbenzoic acid (10): White solid (yield: 2.8 g,
46%); mp: 240–2418C; 1H NMR (400 MHz, [D6]DMSO): d=8.18 (d,
J=2 Hz, 1H), 7.75 (m, 1H), 7.38 (s, 2H), 6.81 (d, J=8.8 Hz, 1H),
6.54 ppm (s, 2H).
7.74 (m, 2H), 7.37 (d, J=8.4 Hz, 1H), 2.96 ppm (d, 6H).
N,N-Diethyl-4H-benzo[e][1,2,4]thiadiazine-7-carboxamide-1,1-di-
oxide (12 f): Light-brown solid (yield: 2.1 g, 74%): mp: 210–2118C;
1H NMR (400 MHz, [D6]DMSO): d=12.47 (s, 1H), 8.04 (s, 1H), 7.73
(m, 1H), 7.68 (m, 1H), 7.66 (m, 1H), 3.42 (s, 2H), 3.18 (s, 2H),
1.13 ppm (d, 6H).
General procedure for the synthesis of 4a–b, 11: The appropriate
2-aminophenylsulfonamide 3 or 10 (10 mmol) was suspended in
triethylorthoformate (30 mL) and stirred at reflux for 2 h. The mix-
ture was then cooled to room temperature, and the precipitate
was collected by filtration, washed with Et2O, and dried in vacuo to
yield the product.
General procedure for the synthesis of 5a–b, 13a–f: The corre-
sponding compound 4 or 12 (20 mmol) was dissolved in CH3CN
(60 mL). K2CO3 (3 g, 21.7 mmol) and methyl bromoacetate (3.25 g,
20 mmol) were successively added to this solution, and the mixture
was stirred at 758C for 2 h. The solvent was evaporated in vacuo
and the residue was washed with H2O, dried, and then recrystal-
lized from EtOAc to give pure product.
7-Methyl-4H-benzo[e][1,2,4]thiadiazine-1,1-dioxide (4a): White
solid (yield: 1.8 g, 90%): mp: 278–2798C; 1H NMR (400 MHz,
[D6]DMSO): d=12.23 (s, 1H), 7.96 (s, 1H), 7.63 (m, 1H), 7.50 (m,
1H), 7.23 (d, J=8.4 Hz, 1H), 2.38 ppm (s, 3H).
Methyl 2-(7-methyl-1,1-dioxido-4H-benzo[e][1,2,4]thiadiazin-4-
yl)acetate (5a): White solid (yield: 4.5 g, 84%): mp: 178–1798C;
1H NMR (400 MHz, [D6]DMSO): d=8.04 (s, 1H), 7.71 (s, 1H), 7.54
(dd, J1 =1.6 Hz, J2 =8.8 Hz, 1H), 7.23 (d, J=8.8 Hz, 1H), 5.06 (s, 2H),
3.72 (s, 3H), 2.40 ppm (s, 3H).
7-Methoxy-4H-benzo[e][1,2,4]thiadiazine-1,1-dioxide (4b): White
solid (yield: 2 g, 92%): mp: 244–2468C; 1H NMR (400 MHz,
[D6]DMSO): d=12.22 (s, 1H), 7.92 (s, 1H), 7.29 (m, 1H), 7.28 (m,
1H), 7.23 (m, 1H), 3.83 ppm (s, 3H).
4H-Benzo[e][1,2,4]thiadiazine-7-carboxylic acid-1,1-dioxide (11):
White solid (yield: 1.9 g, 86%): mp: >3008C; 1H NMR (400 MHz,
[D6]DMSO): d=12.59 (s, 1H), 8.25 (s, 1H), 8.18 (d, J=8.4 Hz, 1H),
8.07 (s, 1H), 7.43 (d, J=8.8 Hz, 1H), 3.4 ppm (s, 1H).
Methyl 2-(7-methoxy-1,1-dioxido-4H-benzo[e][1,2,4]thiadiazin-4-
yl)acetate (5b): White solid (yield: 4.1 g, 72%): mp: 163–1648C;
1H NMR (400 MHz, [D6]DMSO): d=8.02 (s, 1H), 7.32 (m, 3H), 5.08 (s,
2H), 3.86 (s, 3H), 3.70 ppm (s, 3H).
4H-Benzo[e][1,2,4]thiadiazine-7-carboxylate-1,1-dioxide
(12a):
Methyl 4-(2-methoxy-2-oxoethyl)-4H-benzo[e][1,2,4]thiadiazine-
7-carboxylate-1,1-dioxide (13a): White solid (yield: 4.9 g, 78%):
mp: 205–2068C; 1H NMR (400 MHz, [D6]DMSO): d=8.33 (s, 1H),
8.21 (m, 2H), 7.54 (d, J=8.8 Hz, 1H), 5.15 (s, 2H), 3.90 (s, 3H),
3.73 ppm (s, 3H).
Compound 11 (10 mmol, 2.3 g) was suspended in a solution of
H2SO4 (1.3 mL) and CH3OH (20 mL), and the mixture was stirred at
reflux for 5 h. The solvent was removed in vacuo, and the residue
was suspended in H2O and adjusted to pH 8 with aqueous NaOH
(10% w/v). The precipitate was collected by filtration, washed with
H2O, and dried to yield the product (yield: 984 mg, 41%): mp:
Methyl
2-(1,1-dioxido-7-(piperidine-1-carbonyl)-4H-benzo[e]-
1
[1,2,4]thiadiazin-4-yl)acetate (13b): White solid (yield: 5.7 g, 78%):
mp: 243–2448C; 1H NMR (400 MHz, [D6]DMSO): d=8.13 (s, 1H),
7.86 (s, 1H), 7.74 (d, J=8.4 Hz, 1H), 7.43 (d, J=8.4 Hz, 1H), 5.12 (s,
2H), 3.74 (s, 3H), 3.60 (s, 2H), 3.32 (s, 2H), 1.55 ppm (d, 6H).
235–2368C; H NMR (400 MHz, [D6]DMSO): d=12.63 (s, 1H), 8.27 (s,
1H), 8.20 (m, 1H), 8.08 (s, 1H), 3.89 ppm (s, 3H).
General procedure for the synthesis of 12b–f: Compound 11
(10 mmol, 2.3 g) was suspended in SOCl2 (10 mL) and stirred at
reflux overnight. The remaining SOCl2 was removed by distillation
in vacuo. The residue was then dissolved in CH3CN (20 mL), to
which a solution of the corresponding secondary amine (12 mmol),
Et3N (20 mmol), and CH3CN (5 mL) was added dropwise. The result-
ing mixture was stirred at room temperature for 30 min, then the
Methyl
2-(7-(morpholine-4-carbonyl)-1,1-dioxido-4H-benzo[e]-
[1,2,4]thiadiazin-4-yl)acetate (13c): White solid (yield: 5 g, 68%):
mp: 142–1448C; 1H NMR (400 MHz, [D6]DMSO): d=8.12 (s, 1H),
7.93 (s, 1H), 7.77 (s, 1H), 7.44 (d, J=8.4 Hz, 1H), 5.13 (s, 2H), 3.74
(s, 3H), 3.60 ppm (s, 8H).
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Methyl 2-(7-(methyl(phenyl)carbamoyl)-1,1-dioxido-4H-benzo[e]-
[1,2,4]thiadiazin-4-yl)acetate (13d): White solid (yield: 5.4 g, 70%):
mp: 190–1918C; 1H NMR (400 MHz, [D6]DMSO): d=8.04 (s, 1H),
7.72 (s, 1H), 7.56 (m, 1H), 7.27 (m, 5H), 7.21 (d, J=8.8 Hz, 1H), 5.01
(s, 2H), 3.69 ppm (s, 3H).
68%): mp: 176–1788C; H NMR (400 MHz, [D6]DMSO): d=8.27 (m,
1H), 7.60 (m, 1H), 7.46 (m, 1H), 6.78 (d, J=8.8 Hz, 1H), 4.81 (s, 2H),
4.40 (s, 2H), 3.68 (s, 3H), 2.96 ppm (d, 6H).
Methyl
2-(7-(diethylcarbamoyl)-1,1-dioxido-2H-benzo[e]-
[1,2,4]thiadiazin-4(3H)-yl)acetate (14 f): White solid (yield: 2.1 g,
1
60%): mp: 167–1688C; H NMR (400 MHz, CDCl3): d=7.74 (s, 1H),
Methyl
2-(7-(dimethylcarbamoyl)-1,1-dioxido-4H-benzo[e]-
7.44 (d, J=8.8 Hz, 1H), 6.63 (d, J=8.8 Hz, 1H), 5.55 (s, 1H), 4.87 (d,
J=7.6 Hz, 2H), 4.16 (s, 2H), 3.70 (s, 3H), 3.41 (m, 4H), 1.19 ppm (m,
6H).
[1,2,4]thiadiazin-4-yl)acetate (13e): White solid (yield: 5.1 g, 78%):
mp: 179–1808C; 1H NMR (400 MHz, [D6]DMSO): d=8.13 (s, 1H),
7.90 (d, J=1.6 Hz, 1H), 7.79 (m, 1H), 7.43 (d, J=8.8 Hz, 1H), 5.13 (s,
2H), 3.74 (s, 3H), 2.96 ppm (d, 6H).
General procedure for the synthesis of 7a–b and 15a–l: A mix-
ture of corresponding acetate 6 or 14 (5 mmol), K2CO3 (1.38 g,
10 mmol), and 2-fluoro-4-bromobenzyl bromide (1.3 g, 5 mmol) in
CH3CN (20 mL) was stirred at 708C for 2 h. After evaporation of the
solvent in vacuo, the residue was washed with H2O, dried, and re-
crystallized from EtOAc to give the product.
Methyl
2-(7-(diethylcarbamoyl)-1,1-dioxido-4H-benzo[e]-
[1,2,4]thiadiazin-4-yl)acetate (13 f): White solid (yield: 5.2 g, 74%):
mp: 205–2068C; 1H NMR (400 MHz, [D6]DMSO): d=8.13 (s, 1H),
7.83 (d, J=1.6 Hz, 1H), 7.73 (dd, J1 =1.6 Hz, J2 =8.6 Hz, 1H), 7.43
(d, J=8.8 Hz, 1H), 5.13 (s, 2H), 3.74 (s, 3H), 3.44 (s, 2H), 3.16 (s,
2H), 1.11 ppm (d, 6H).
Methyl 2-(2-(4-bromo-2-fluorobenzyl)-7-methyl-1,1-dioxido-2H-
benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate (7a): The mixture was
purified using a petroleum ether (PE)/EtOAc (1:1) eluent to obtain
7a as a white solid (yield: 1.9 g, 86%): mp: 145–1468C; 1H NMR
(400 MHz, [D6]DMSO): d=7.58 (m, 1H), 7.44 (m, 3H), 7.26 (m, 1H),
6.69 (d, J=8.8 Hz, 1H), 4.86 (s, 2H), 4.32 (s, 2H), 4.25 (s, 2H), 3.66
(s, 3H), 2.25 ppm (s, 3H).
General procedure for the synthesis of 6a–b and 14a–f: NaBH4
(1.5 g, 40 mmol) was added to a solution of the appropriate ace-
tate 5 or 13 (10 mmol) in CH3OH. The reaction mixture was stirred
at room temperature for 5 min, then the residue was poured into
ice, and the alkaline suspension was acidified with 0.1n HCl. The
resulting precipitate was collected by filtration, washed with H2O,
dried, and recrystallized from EtOAc to give the desired product.
Methyl 2-(2-(4-bromo-2-fluorobenzyl)-7-methoxy-1,1-dioxido-2H-
benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate (7b): The mixture was
purified using a PE/EtOAc (1:1) eluent to obtain 7b as a white solid
Methyl
2-(7-methyl-1,1-dioxido-2H-benzo[e][1,2,4]thiadiazin-
4(3H)-yl)acetate (6a): White solid (yield: 2.0 g, 75%): mp: 181–
1828C; 1H NMR (400 MHz, [D6]DMSO): d=8.05 (m, 1H), 7.22 (m,
1H), 7.18 (m, 1H), 6.64 (d, J=8.8 Hz, 1H), 4.72 (d, J=8 Hz, 2H),
4.30 (s, 2H), 3.65 (s, 3H), 2.22 ppm (s, 3H).
1
(yield: 1.8 g, 78%): mp: 163–1648C; H NMR (400 MHz, [D6]DMSO):
d=7.59 (d, J=10 Hz, 1H), 7.45 (m, 2H), 7.11 (m, 2H), 6.77 (d, J=
10 Hz, 1H), 4.84 (s, 2H), 4.32 (s, 2H), 4.26 (s, 2H), 3.75 (s, 3H),
3.66 ppm (s, 3H).
Methyl 2-(7-methoxy-1,1-dioxido-2H-benzo[e][1,2,4]thiadiazin-
4(3H)-yl)acetate (6b): White solid (yield: 2.1 g, 72%): mp: 127–
1288C; H NMR (400 MHz, [D6]DMSO): d=8.10 (t, J=8 Hz, 1H), 7.04
(m, 2H), 6.73 (d, J=8.8 Hz, 1H), 4.70 (d, J=8 Hz, 2H), 4.30 (s, 2H),
3.72 (s, 3H), 3.65 ppm (s, 3H).
Methyl
2-(7-methyl-1,1-dioxido-2-(2,4,5-trifluorobenzyl)-2H-
1
benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate (7c): The mixture was
purified using a PE/EtOAc (1:1) eluent to obtain 7c as a white solid
(yield: 1.7 g, 83%): mp: 144–1458C; H NMR (400 MHz, [D6]DMSO):
1
d=7.53 (m, 2H), 7.47 (m, 1H), 7.28 (m, 1H), 6.67 (d, J=9.6 Hz, 1H),
Methyl
4-(2-methoxy-2-oxoethyl)-3,4-dihydro-2H-benzo[e]-
4.89 (s, 2H), 4.33 (s, 2H), 4.25 (s, 2H), 3.67 (s, 3H), 2.25 ppm (s, 3H).
[1,2,4]thiadiazine-7-carboxylate-1,1-dioxide (14a): White solid
(yield: 2.4 g, 75%): mp: 133–1348C; H NMR (400 MHz, [D6]DMSO):
d=8.41 (t, J=7.6 Hz, 1H), 8.07 (s, 1H), 7.85 (d, J=9.2 Hz, 1H), 6.87
(d, J=9.2 Hz, 1H), 4.84 (d, J=7.6 Hz, 2H), 4.45 (s, 2H), 3.82 (s, 3H),
3.68 ppm (s, 3H).
1
Methyl
2-(7-methoxy-1,1-dioxido-2-(2,4,5-trifluorobenzyl)-2H-
benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate (7d): The mixture was
purified using a PE/EtOAc (1:1) eluent to obtain 7d as a white solid
(yield: 1.7 g, 80%): mp: 157–1588C; H NMR (400 MHz, [D6]DMSO):
1
d=7.37 (m, 1H), 7.03 (m, 1H), 6.95 (m, 1H), 6.53 (d, J=8.8 Hz, 1H),
Methyl
2-(1,1-dioxido-7-(piperidine-1-carbonyl)-2H-benzo[e]-
4.84 (s, 2H), 4.37 (s, 2H), 4.00 (s, 2H), 3.80 (s, 3H), 3.75 ppm (s, 3H).
[1,2,4]thiadiazin-4(3H)-yl)acetate (14b): White solid (yield: 2.5 g,
68%): mp: 206–2078C; 1H NMR (400 MHz, [D6]DMSO): d=8.26 (s,
1H), 7.55 (s, 1H), 7.41 (m, 1H), 6.78 (d, J=8 Hz, 1H), 4.80 (s, 2H),
4.40 (s, 2H), 3.68 (s, 3H), 3.60 (s, 2H), 3.44 (s, 2H), 1.55 ppm (d,
6H).
Methyl
2-(4-bromo-2-fluorobenzyl)-4-(2-methoxy-2-oxoethyl)-
3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine-7-carboxylate-1,1-di-
oxide (15a): The mixture was purified using a CH2Cl2/CH3OH (60:1)
eluent to obtain 15a as a white solid (yield: 1.9 g, 75%): mp: 177–
1788C; 1H NMR (400 MHz, [D6]DMSO): d=8.14 (m, 1H), 7.95 (m,
1H), 7.59 (d, J=9.6, 1H), 7.43 (m, 2H), 6.94 (d, J=9.2, 1H), 4.99 (s,
2H), 4.45 (s, 2H), 4.26 (s, 2H), 3.83 (s, 3H), 3.69 ppm (s, 3H).
Methyl
2-(7-(morpholine-4-carbonyl)-1,1-dioxido-2H-benzo[e]-
[1,2,4]thiadiazin-4(3H)-yl)acetate (14c): White solid (yield: 2.6 g,
70%): mp: 180–1818C; 1H NMR (400 MHz, [D6]DMSO): d=8.29 (t,
J=8 Hz, 1H), 7.59 (dd, J1 =2 Hz, J2 =12 Hz, 1H), 7.45 (dd, J1 =2 Hz,
J2 =8.8 Hz, 1H), 6.79 (d, J=8.8 Hz, 1H), 4.80 (d, J=8.4 Hz, 2H), 4.41
(s, 2H), 3.68 (s,3H), 3.59 (s, 4H), 3.49 ppm (s, 4H).
Methyl 2-(2-(4-bromo-2-fluorobenzyl)-1,1-dioxido-7-(piperidine-
1-carbonyl)-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate (15b):
The mixture was purified using a CH2Cl2/CH3OH (60:1) eluent to
obtain 15b as a white solid (yield: 2.2 g, 81%): mp: 89–908C;
1H NMR (400 MHz, [D6]DMSO): d=7.60 (m, 2H), 7.17 (m, 3H), 6.83
(d, J=8.8 Hz, 1H), 4.94 (s, 2H), 4.40 (s, 2H), 4.28 (s, 2H), 3.69 (s,
3H), 3.45 (s, 4H), 1.57 ppm (d, 6H).
Methyl 2-(7-(methyl(phenyl)carbamoyl)-1,1-dioxido-2H-benzo[e]-
[1,2,4]thiadiazin-4(3H)-yl)acetate (14d): White solid (yield: 2.2 g,
1
56%): mp: 192–1938C; H NMR (400 MHz, [D6]DMSO): d=8.19 (m,
1H), 7.48 (m, 1H), 7.32 (m, 2H), 7.20 (m, 4H), 6.54 (d, J=9.2 Hz,
1H), 4.72 (d, J=8, 2H), 4.30 (s, 2H), 3.63 (s, 3H), 3.35 ppm (s, 3H).
Methyl 2-(2-(4-bromo-2-fluorobenzyl)-7-(morpholine-4-carbon-
yl)-1,1-dioxido-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate
(15c): The mixture was purified using a CH2Cl2/CH3OH (60:1) eluent
to obtain 15c as a white solid (yield: 1.94 g, 70%): mp: 152–
Methyl
2-(7-(dimethylcarbamoyl)-1,1-dioxido-2H-benzo[e]-
[1,2,4]thiadiazin-4(3H)-yl)acetate (14e): White solid (yield: 2.2 g,
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1538C; H NMR (400 MHz, [D6]DMSO): d=7.68 (s, 1H), 7.58 (m, 1H),
(m, 1H), 6.59 (d, J=9.2 Hz, 1H), 4.91(s, 2H), 4.32 (s, 2H), 4.05 (s,
7.52 (m, 1H), 7.46 (m, 2H), 6.84 (d, J=8.8 Hz, 1H), 4.94 (s, 2H), 4.40
2H), 3.19 (s, 3H), 3.08 ppm (s, 6H).
(s, 2H), 4.28 (s, 2H), 3.69 (s, 3H), 3.60 (s, 4H), 3.51 ppm (s, 4H).
Methyl 2-(7-(diethylcarbamoyl)-1,1-dioxido-2-(2,4,5-trifluoroben-
zyl)-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate (15l): The mix-
ture was purified using a CH2Cl2/CH3OH (60:1) eluent to obtain 15l
Methyl
2-(2-(4-bromo-2-fluorobenzyl)-7-(methyl(phenyl)car-
bamoyl)-1,1-dioxido-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)ace-
tate (15d): The mixture was purified using a CH2Cl2/CH3OH (60:1)
eluent to obtain 15d as a white solid (yield: 2.1 g, 72%): mp: 199–
2008C; 1H NMR (400 MHz, [D6]DMSO): d=7.56 (d, J=8 Hz, 1H),
7.49 (d, J=1.2 Hz, 1H), 7.43 (d, J=8.4 Hz, 1H), 7.33 (m, 4H), 7.21
(m, 3H), 6.61 (d, J=8.8 Hz, 1H), 4.86 (s, 2H), 4.30 (s, 2H), 4.02 (s,
2H), 3.64 (s, 3H), 3.36 ppm (s, 3H).
as
a
white solid (yield: 1.8 g, 74%): mp: 90–918C; 1H NMR
(400 MHz, CDCl3): d=7.81 (s, 1H), 7.49 (m, 1H), 7.35 (m, 1H), 6.95
(m, 1H), 6.58 (d, J=8.8 Hz, 1H), 4.91 (s, 2H), 4.34 (s, 2H), 4.05 (s,
2H), 3.78 (s, 3H), 3.43 (s, 4H), 1.23 ppm (s, 6H).
General procedure for the synthesis of 8a–8b and 16a–16 f: A
mixture of the appropriate acetate 7 or 15 (1 mmol), 1,4-dioxane
(5 mL), and saturated aqueous NaOH (1 mL) was stirred at room
temperature for 2 h. The alkaline suspension was poured into ice
and acidified with 0.1n HCl (0.6 mL). The resulting precipitate was
collected by filtration, washed with H2O, dried, and recrystallized
from CH3OH to give the pure product.
Methyl
2-(2-(4-bromo-2-fluorobenzyl)-7-(dimethylcarbamoyl)-
1,1-dioxido-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate (15e):
The mixture was purified using a CH2Cl2/CH3OH (60:1) eluent to
obtain 15e as a white solid (yield: 2.0 g, 79%): mp: 157–1588C;
1H NMR (400 MHz, [D6]DMSO): d=7.68 (m, 1H), 7.55 (m, 2H), 7.44
(m, 2H), 6.83 (d, J=8.8 Hz, 1H), 4.95 (s, 2H), 4.40 (s, 2H), 4.28 (s,
2H), 3.69 (s, 3H), 2.97 ppm (s, 6H).
2-(2-(4-Bromo-2-fluorobenzyl)-7-methyl-1,1-dioxido-2H-benzo[e]-
[1,2,4]thiadiazin-4(3H)-yl)acetic acid (8a): White solid (yield:
1
390 mg, 88%, purity: 99.90%): mp: 141–1428C; H NMR (400 MHz,
Methyl 2-(2-(4-bromo-2-fluorobenzyl)-7-(diethylcarbamoyl)-1,1-
dioxido-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate (15 f): The
mixture was purified using a CH2Cl2/CH3OH (60:1) eluent to obtain
15 f as a white solid (yield: 1.9 g, 70%): mp: 114–1168C; 1H NMR
(400 MHz, [D6]DMSO): d=7.58 (m, 2H), 7.43 (m, 3H), 6.83 (d, J=
8.8 Hz, 1H), 4.95 (s, 2H), 4.40 (s, 2H), 4.29 (s, 2H), 3.69 (s, 3H), 3.40
(s, 4H), 1.16 ppm (m, 6H).
[D6]DMSO): d=12.92 (s, 1H), 7.57 (m, 1H), 7.42 (m, 3H), 7.28 (m,
1H), 6.68 (d, J=8.4 Hz, 1H), 4.86 (s, 2H), 4.26 (s, 2H), 4.19 (s, 2H),
2.25 ppm (s, 3H); 13C NMR (100 MHz, [D6]DMSO): d=171.14, 160.45
(d, J=250 Hz), 140.28, 134.76, 132.59 (d, J=4.6 Hz), 127.70 (d, J=
3.4 Hz), 126.71, 125.06, 122.98 (d, J=14.1 Hz), 121.36 (d, J=9.5 Hz),
119.694, 118.89 (d, J=24.4), 113.62, 65.48, 49.87, 43.65, 19.57 ppm;
HRMS (ESI+) m/z calcd for [M+H]+ 443.0076, found 443.0069.
Methyl 4-(2-methoxy-2-oxoethyl)-2-(2,4,5-trifluorobenzyl)-3,4-di-
hydro-2H-benzo[e][1,2,4]thiadiazine-7-carboxylate-1,1-dioxide
(15g): The mixture was purified using a CH2Cl2/CH3OH (60:1)
eluent to obtain 15g as a white solid (yield: 1.7 g, 76%): mp: 155–
1578C; 1H NMR (400 MHz, [D6]DMSO): 8.47 (s, 1H), 8.06 (d, J=
8.8 Hz, 1H), 7.36 (m, 1H), 6.95 (m, 1H), 6.59 (d, J=8.8 Hz, 1H), 4.93
(s, 2H), 4.34 (s, 2H), 4.08 (s, 2H), 3.90 (s, 3H), 3.79 ppm (s, 3H).
2-(2-(4-Bromo-2-fluorobenzyl)-7-methoxy-1,1-dioxido-2H-
benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetic acid (8b): White solid
(yield: 376 mg, 82%, purity: 98.86%): mp: 133–1348C; 1H NMR
(400 MHz, [D6]DMSO): d=12.94 (s, 1H), 7.59 (m, 1H), 7.45 (m, 2H),
7.14 (m, 2H), 6.76 (d, J=8.8, 1H), 4.82 (s, 2H), 4.27 (s, 2H), 4.09 (s,
2H), 3.73 ppm (s, 3H); 13C NMR (100 MHz, [D6]DMSO): d=171.32,
160.48 (d, J=249.9 Hz), 150.97, 136.81, 132.63 (d, J=4.2 Hz),
127.71 (d, J=3.0 Hz), 122.94 (d, J=14.1 Hz), 121.94, 121.40 (d, J=
9.5 Hz), 119.97, 118.90 (d, J=24.4 Hz), 115.34, 108.41, 65.49, 55.76,
50.08, 43.63 ppm; HRMS (ESI+) m/z calcd for [M+H]+ 459.0026,
found 459.0017.
Methyl 2-(1,1-dioxido-7-(piperidine-1-carbonyl)-2-(2,4,5-trifluoro-
benzyl)-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate (15h): The
mixture was purified using a CH2Cl2/CH3OH (60:1) eluent to obtain
1
15h as a white solid (yield: 2.0 g, 77%): mp: 159–1608C; H NMR
(400 MHz, CDCl3): d=7.82 (s, 1H), 7.53 (d, J=8.8 Hz, 1H), 7.34 (m,
1H), 6.94 (m, 1H), 6.58 (d, J=8.8 Hz, 1H), 4.91 (s, 2H), 4.34 (s, 2H),
4.05 (s, 2H), 3.78 (s, 3H), 3.55 (s, 4H), 1.64 ppm (d, 6H).
2-(7-Methyl-1,1-dioxido-2-(2,4,5-trifluorobenzyl)-2H-benzo[e]-
[1,2,4]thiadiazin-4(3H)-yl)acetic acid (8c): White solid (yield:
340 mg, 85%, purity: 99.90%): mp: 112–1138C; H NMR (400 MHz,
1
Methyl 2-(7-(morpholine-4-carbonyl)-1,1-dioxido-2-(2,4,5-trifluor-
obenzyl)-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate (15i): The
mixture was purified using a CH2Cl2/CH3OH (60:1) eluent to obtain
15i as a white solid (yield: 1.8 g, 69%): mp: 141–1438C; 1H NMR
(400 MHz, CDCl3): d=7.84 (s, 1H), 7.55 (d, J=8.8 Hz, 1H), 7.35 (m,
1H), 6.96 (m, 1H), 6.60 (d, J=8.4 Hz, 1H), 4.92 (s, 2H), 4.34 (s, 2H),
4.06 (s, 2H), 3.79 (s, 3H), 3.71 (s, 4H), 3.66 ppm (s, 4H).
[D6]DMSO): d=7.55 (m, 2H), 7.43 (s, 1H), 7.28 (d, J=8.4 Hz, 1H),
6.68 (d, J=8.8 Hz, 1H), 4.88 (s, 2H), 4.26 (s, 2H), 4.12 (s, 2H),
2.25 ppm (s, 3H); 13C NMR (100 MHz, [D6]DMSO): d=171.27,
140.30, 134.81, 126.74, 125.07, 119.66, 119.06, 118.86, 113.62,
106.46, 106.24, 106.18, 105.97, 65.43, 49.87, 43.53, 40.15,
19.57 ppm; HRMS (ESI+) m/z calcd for [M+H]+ 401.0783, found
401.0773.
Methyl 2-(7-(methyl(phenyl)carbamoyl)-1,1-dioxido-2-(2,4,5-tri-
fluorobenzyl)-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate (15j):
The mixture was purified using a CH2Cl2/CH3OH (60:1) eluent to
obtain 15j as a white solid (yield: 1.9 g, 71%): mp: 130–1318C;
1H NMR (400 MHz, CDCl3): d=7.67 (s, 1H), 7.43 (d, J=8.0 Hz, 1H),
7.30 (m, 1H), 7.24 (m, 3H), 7.08 (m, 2H), 6.92 (m, 1H), 6.34 (d, J=
8.8 Hz, 1H), 4.83 (s, 2H), 4.21 (s, 2H), 3.96 (s, 2H), 3.74 (s, 3H),
3.49 ppm (s, 3H).
2-(7-Methoxy-1,1-dioxido-2-(2,4,5-trifluorobenzyl)-2H-benzo[e]-
[1,2,4]thiadiazin-4(3H)-yl)acetic acid (8d): White solid (yield:
324 mg, 78%, purity: 98.24%): mp: 146–1478C; H NMR (400 MHz,
[D6]DMSO): d=7.55 (m, 2H), 7.13 (m, 2H), 7.76 (d, J=7.2 Hz, 1H),
4.87 (s, 2H), 4.21 (s, 2H), 4.19 (s, 2H), 3.74 ppm (s, 3H); 13C NMR
(100 MHz, [D6]DMSO): d=171.39, 151.00, 136.79, 121.99, 119.93,
119.07, 118.88, 115.32, 108.42, 106.45, 106.24, 106.18, 105.96, 65.40,
55.76, 49.97, 43.47 ppm; HRMS (ESI+) m/z calcd for [M+H]+
417.0732, found 417.0721.
1
Methyl 2-(7-(dimethylcarbamoyl)-1,1-dioxido-2-(2,4,5-trifluoro-
benzyl)-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetate (15k): The
mixture was purified using a CH2Cl2/CH3OH (60:1) eluent to obtain
2-(4-Bromo-2-fluorobenzyl)-4-(carboxymethyl)-3,4-dihydro-2H-
benzo[e][1,2,4]thiadiazine-7-carboxylic acid-1,1-dioxide (16a):
White solid (yield: 331 mg, 70%, purity: 99.90%): mp: 173–1748C;
1H NMR (400 MHz, [D6]DMSO): d=13.05 (s, 2H), 8.11 (d, J=2 Hz,
1
15k as a white solid (yield: 1.8 g, 78%): mp: 110–1118C; H NMR
(400 MHz, CDCl3): d=7.86 (s, 1H), 7.57 (m, 1H), 7.34 (m, 1H), 6.95
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1H), 7.95 (m, 1H), 7.59 (m, 1H), 7.44 (m, 2H), 6.89 (d, J=9.2 Hz,
1H), 4.97 (s, 2H), 4.31 (s, 2H), 4.28 ppm (s, 2H); 13C NMR (100 MHz,
[D6]DMSO): d=170.46, 166.00, 160.47 (d, J=250.4 Hz), 145.55,
134.41, 132.63 (d, J=4.2 Hz), 127.71 (d, J=3.4 Hz), 127.12, 122.67
(d, J=14.5 Hz), 121.55 (d, J=9.9 Hz), 119.15 (d, J=19.5 Hz), 118.88,
118.81, 113.77, 65.71, 50.05, 44.19 ppm; HRMS (ESI+) m/z calcd for
[M+H]+ 472.9818, found 472.9820.
119.06 (d, J=10.3 Hz), 118.77, 113.42, 65.57, 49.92, 44.03, 13.41;
HRMS (ESI+) m/z calcd for [M+H]+ 528.0604, found 528.0596.
4-(Carboxymethyl)-2-(2,4,5-trifluorobenzyl)-3,4-dihydro-2H-
benzo[e][1,2,4]thiadiazine-7-carboxylic acid-1,1-dioxide (16g):
White solid (yield: 297 mg, 69%, purity: 98.84%): mp: 244–2458C;
1H NMR (400 MHz, [D6]DMSO): d=12.96 (s, 1H), 8.12 (s, 1H), 7.95
(d, J=8.8 Hz, 1H), 7.57 (m, 2H), 6.89 (d, J=8.8 Hz, 1H), 4.99 (s, 2H),
4.32 (s, 2H), 4.27 ppm (s, 2H); 13C NMR (100 MHz, [D6]DMSO): d=
170.56, 165.97, 145.54, 134.42, 127.11, 120.17, 119.23, 118.99,
118.81, 113.73, 106.49, 106.28, 106.21, 106.00, 65.53, 49.93,
43.96 ppm; HRMS (ESI+) m/z calcd for [M+H]+ 431.0525, found
431.0512.
2-(2-(4-Bromo-2-fluorobenzyl)-1,1-dioxido-7-(piperidine-1-car-
bonyl)-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetic acid (16b):
White solid (yield: 470 mg, 87%, purity: 98.95%): mp: 136–1378C;
1H NMR (400 MHz, [D6]DMSO): d=13.12 (s, 1H), 7.59 (m, 2H), 7.44
(m, 3H), 6.81 (d, J=9.2 Hz, 1H), 4.94 (s, 2H), 4.29 (s, 2H), 4.28 (s,
2H), 3.52 (s, 2H), 3.46 (s, 2H), 1.56 ppm (d, 6H); 13C NMR (100 MHz,
[D6]DMSO): d=170.77, 167.46, 160.48 (d, J=250.3 Hz), 143.20,
132.69, 127.67, 124.73, 122.67 (d, J=15.2 Hz), 121.54, 119.08,
119.00, 118.76, 113.34, 109.43, 65.57, 50.04, 44.01, 29.48, 25.63,
24.05 ppm; HRMS (ESI+) m/z calcd for [M+H]+ 540.0604, found
540.0602.
2-(1,1-Dioxido-7-(piperidine-1-carbonyl)-2-(2,4,5-trifluorobenzyl)-
2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetic acid (16h): White
solid (yield: 383 mg, 77%, purity: 99.98%): mp: 218–2198C;
1H NMR (400 MHz, [D6]DMSO): d=7.56 (m, 4H), 6.80 (d, J=8.8 Hz,
1H), 4.95 (s, 2H), 4.28 (s, 2H), 4.26 (s, 2H), 3.56 (s, 4H), 1.61 ppm
(d, 6H); 13C NMR (100 MHz, [D6]DMSO): d=170.85, 167.48, 160.09,
157.07, 143.17, 132.76, 124.72, 120.21, 119.17, 118.98, 113.33,
106.45, 106.16, 105.96, 65.45, 49.94, 43.85, 25.56, 24.00 ppm; HRMS
(ESI+) m/z calcd for [M+H]+ 498.1311, found 498.1296.
2-(2-(4-Bromo-2-fluorobenzyl)-7-(morpholine-4-carbonyl)-1,1-di-
oxido-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetic acid (16c):
White solid (yield: 428 mg, 79%, purity: 98.23%): mp: 137–1388C;
1H NMR (400 MHz, [D6]DMSO): d=7.68 (s, 1H), 7.54 (m, 2H), 7.42
(m, 2H), 6.81 (d, J=9.2 Hz, 1H), 4.94 (s, 2H), 4.29 (s, 2H), 4.27 (s,
2H), 3.70 (s, 4H), 3.45 ppm (s, 4H); 13C NMR (100 MHz, [D6]DMSO):
d=170.73, 167.70, 160.52 (d, J=249.9 Hz), 143.44, 132.88 (d, J=
35.4 Hz), 127.74, 125.30, 123.92, 122.71 (d, J=13.8 Hz), 121.55 (d,
J=9.5 Hz), 119.14 (d, J=16.4 Hz), 118.81, 113.37, 109.50, 66.41 (2C),
66.10 (2C), 65.63, 50.00, 44.10 ppm; HRMS (ESI+) m/z calcd for
[M+H]+ 542.0397, found 542.0397.
2-(7-(Morpholine-4-carbonyl)-1,1-dioxido-2-(2,4,5-trifluoroben-
zyl)-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetic acid (16i): White
solid (yield: 364 mg, 73%, purity: 98.34%): mp: 138–1398C;
1H NMR (400 MHz, [D6]DMSO): d=7.68 (s, 1H), 7.55 (m, 3H), 6.82
(d, J=8.8 Hz, 1H), 4.95 (s, 2H), 4.28 (s, 2H), 4.27 (s, 2H), 3.58 ppm
(d, 8H); 13C NMR (100 MHz, [D6]DMSO): d=170.82, 167.61, 143.39,
133.04, 125.23, 123.89, 120.09, 119.08, 118.98, 113.35, 106.49,
106.21, 106.00, 66.03, 65.43, 49.95, 43.82 ppm; HRMS (ESI+) m/z
calcd for [M+H]+ 500.1103, found 500.1086.
2-(2-(4-Bromo-2-fluorobenzyl)-7-(methyl(phenyl)carbamoyl)-1,1-
dioxido-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetic acid (16d):
White solid (yield: 483 mg, 86%, purity: 99.62%): mp: 152–1538C;
1H NMR (400 MHz, [D6]DMSO): d=7.54 (m, 1H), 7.49 (m, 1H), 7.42
(m, 3H), 7.35 (m, 2H), 7.31 (m, 3H), 6.58 (d, J=9.2 Hz, 1H), 4.85 (s,
2H), 4.16 (s, 2H), 4.15 (s, 2H), 3.36 ppm (s, 3H); 13C NMR (100 MHz,
[D6]DMSO): d=170.68, 167.44, 160.42 (d, J=249.9 Hz), 144.80,
143.19, 134.12, 132.54 (d, J=4.6 Hz), 129.39 (2C), 127.67 (d, J=
3.0 Hz), 126.91 (2C), 126.71 (d, J=13.8 Hz), 124.06, 122.72 (d, J=
13.7 Hz), 121.46 (d, J=9.5 Hz), 119.01, 118.76, 118.31, 112.65, 65.53,
49.93, 43.99, 38.35 ppm; HRMS (ESI+) m/z calcd for [M+H]+
562.0448, found 562.0446.
2-(7-(Methyl(phenyl)carbamoyl)-1,1-dioxido-2-(2,4,5-trifluoro-
benzyl)-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetic acid (16j):
White solid (yield: 410 mg, 79%, purity: 99.99%): mp: 240–2418C;
1H NMR (400 MHz, [D6]DMSO): d=7.54 (m, 3H), 7.31 (m, 6H), 6.59
(d, J=8.0 Hz, 1H), 4.87 (s, 2H), 4.16 (s, 4H), 3.36 ppm (s, 3H);
13C NMR (100 MHz, [D6]DMSO): d=170.78, 167.39, 144.78, 143.17,
134.14, 129.37, 126.88, 126.77, 126.61, 124.02, 118.00 (d, J=
20.9 Hz), 118.21, 112.61, 106.20 (dd, J1 =21.3 Hz, J2 =28.2 Hz), 65.38,
49.92, 43.75, 38.33 ppm; HRMS (ESI+) m/z calcd for [M+H]+
520.1154, found 520.1143.
2-(2-(4-Bromo-2-fluorobenzyl)-7-(dimethylcarbamoyl)-1,1-dioxi-
do-2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetic acid (16e): White
solid (yield: 410 mg, 82%, purity: 98.53%): mp: 149–1508C;
1H NMR (400 MHz, [D6]DMSO): d=13.08 (s, 1H), 7.67 (m, 1H), 7.58
(m, 2H), 7.43 (m, 2H), 6.81 (d, J=8.8 Hz, 1H), 4.94 (s, 2H), 4.29 (s,
2H), 4.28 (s, 2H), 2.98 ppm (s, 6H); 13C NMR (100 MHz, [D6]DMSO):
d=170.73, 168.53, 160.48 (d, J=219.9 Hz), 143.20, 133.03, 132.65
(d, J=4.2 Hz), 127.70 (d, J=3.4 Hz), 125.07, 124.81, 122.72 (d, J=
14.5 Hz), 121.50 (d, J=9.6 Hz), 118.91 (d, J=23.7 Hz), 113.20,
109.46, 65.58, 49.95, 44.04 ppm; HRMS (ESI+) m/z calcd for [M+
H]+ 500.0291, found 500.0280.
2-(7-(Dimethylcarbamoyl)-1,1-dioxido-2-(2,4,5-trifluorobenzyl)-
2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetic acid (16k): White
solid (yield: 334 mg, 73%, purity: 99.49%): mp: 224–2258C;
1H NMR (400 MHz, [D6]DMSO): d=7.67 (s, 1H), 7.55 (m, 3H), 6.81
(d, J=8.4 Hz, 1H), 4.95 (s, 2H), 4.21 (s, 4H), 2.97 ppm (s, 6H);
13C NMR (100 MHz, [D6]DMSO): d=170.87, 168.51, 143.19, 133.07,
125.07, 124.85, 118.97, 113.22, 65.45, 49.91, 43.85 ppm; HRMS
(ESI+) m/z calcd for [M+H]+ 458.0998, found 458.0992.
2-(2-(4-Bromo-2-fluorobenzyl)-7-(diethylcarbamoyl)-1,1-dioxido-
2H-benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetic acid (16 f): White
solid (yield: 449 mg, 85%, purity: 99.78%): mp: 247–2488C;
1H NMR (400 MHz, [D6]DMSO): d=13.10 (s, 1H), 7.58 (m, 2H), 7.45
(m, 3H), 6.80 (d, J=9.6 Hz, 1H), 4.94 (s, 2H), 4.29 (s, 2H), 4.26 (s,
2H), 3.33 (s, 2H), 3.16 (s, 2H), 1.23 (s, 3H), 1.13 ppm (s, 3H);
13C NMR (100 MHz, [D6]DMSO): d=170.76, 168.42, 160.48 (d, J=
249.9 Hz), 142.96, 132.64 (d, J=4.6 Hz), 132.11, 127.67 (d, J=
3.4 Hz), 125.72, 124.06, 122.69 (d, J=14.4 Hz), 121.49 (d, J=9.9 Hz),
2-(7-(Diethylcarbamoyl)-1,1-dioxido-2-(2,4,5-trifluorobenzyl)-2H-
benzo[e][1,2,4]thiadiazin-4(3H)-yl)acetic acid (16l): White solid
(yield: 388 mg, 80%, purity: 99.91%): mp: 223–2248C; 1H NMR
(400 MHz, [D6]DMSO): d=7.52 (m, 4H), 6.81 (d, J=8.0 Hz, 1H), 4.96
(s, 2H), 4.29 (s, 2H), 4.27 (s, 2H), 3.33 (s, 4H), 1.12 ppm (s, 6H);
13C NMR (100 MHz, [D6]DMSO): d=170.92, 168.44, 142.99, 132.17,
125.72, 124.07, 120.14, 119.21, 119.16, 119.04, 113.43, 106.20 (dd,
J1 =20.9 Hz, J2 =27.8 Hz), 65.47, 49.97, 43.85 ppm; HRMS (ESI+) m/
z calcd for [M+H]+ 486.1311, found 486.1299.
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replicates at each concentration. The 95% confidence limits (95%
CL) were calculated from t values for nÀ2, where n is the total
number of determinations.
Biology
General: ALR2 was prepared according to the method of Hayman
and Kinoshita[8] and La Motta et al.[2i] Lenses freshly acquired from
rats were homogenized (Glas-Potter) in three volumes of cold de-
ionized H2O in an ice bath. The homogenate was then centrifuged
at 12000 rpm (rotor type: 12154-H) at 0–48C for 30 min. The super-
natant was precipitated with (NH4)2SO4, first at 40% and then at
50% saturation. The combined supernatant from the first two pre-
cipitations was then precipitated with (NH4)2SO4 at 75%. The final
precipitate obtained from the 75% saturated fraction, which was
determined to possess ALR2 activity, was re-dissolved in 0.05m
NaCl and then dialyzed overnight in 0.05m NaCl. The dialyzed ma-
terial was used in the enzymatic assay.
Docking studies
Docking studies were performed using Molegro Virtual Docker, ver-
sion 5.0. The crystal structure of human aldose reductase with
bound inhibitor IDD594 (PDB code: 1US0) retrieved from the RCSB
Protein Data Bank was used for docking. All solvent molecules
within the protein structure were removed for the docking proce-
dure. All structural parameters of ligands such as bond orders, hy-
bridization, explicit hydrogen atoms, and charges were assigned
when necessary in the Molegro Virtual Docker software. To obtain
better potential binding sites in the protein, detection of possible
binding cavities was implemented, and five cavities were obtained.
The cavity around the anion binding site (volume of ~124 ꢁ3) was
chosen for docking calculations. All docking calculations were car-
ried out using the grid-based MolDock score (GRID) function with
a grid resolution of 0.30 ꢁ. The best ligand poses were chosen on
the basis of the MolDock score and ReRank score. Docking calcula-
tions were performed with a dual processor Windows 7-based
computer with 4 GB RAM, and each docking process lasted 4–
6 min.
ALR1 was prepared in accordance with a previously reported meth-
od.[2i] Kidneys were removed from rats and homogenized (Glas-
Potter) in three volumes of 10 mm sodium phosphate buffer
pH 7.2 containing 0.25m sucrose, 2.0 mm EDTA di-potassium salt,
and 2.5 mm b-mercaptoethanol. The homogenate was centrifuged
at 12000 rpm (rotor type: 12154-H) at 0–48C for 30 min, and the
supernatant was subjected to a 40–75% (NH4)2SO4 fractionation,
following the same procedure previously described for ALR2. The
precipitate obtained from the 75% (NH4)2SO4 saturation, possess-
ing ALR1 activity, was re-dissolved in 50 volumes of 10 mm sodium
phosphate buffer pH 7.2 containing 2.0 mm EDTA di-potassium salt
and 2.0 mm b-mercaptoethanol and was dialyzed overnight using
the same buffer. The dialyzed material was used in the enzymatic
assay.
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (Nos. 51103012 and 21272025), the Research
Fund for the Doctoral Program of Higher Education of China (No.
20111101110042), and the Beijing Natural Science Foundation
(No. 7102091).
Enzyme activity was assayed spectrophotometrically on a Shimadzu
UV-1800 UV spectrophotometer by measuring the decrease in ab-
sorption of NADPH at l 340 nm, which accompanies the oxidation
of NADPH catalyzed by ALR2 and ALR1.
Enzymatic assays: Measurement of ALR2 activity was carried out at
308C in a reaction mixture containing 0.10 mm NADPH (0.25 mL),
0.1m sodium phosphate buffer (pH 6.2, 0.25 mL), enzyme extract
(0.1 mL), deionized H2O (0.15 mL), and 10 mm d,l-glyceraldehyde
(0.25 mL) as substrate in a final volume of 1 mL. With the excep-
tion of d,l-glyceraldehyde, the reaction mixture was incubated at
308C for 10 min. The substrate was then added to initiate the reac-
tion, which was monitored for 4 min. The ALR1 activity assay was
performed at 378C in a reaction mixture containing 0.12 mm
NADPH (0.25 mL), enzyme extract (0.1 mL), 0.1m sodium phos-
phate buffer (pH 7.2, 0.25 mL), deionized H2O (0.15 mL) and 20 mm
sodium d-glucuronate (0.25 mL) as substrate in a final volume of
1 mL. With the exception of the sodium d-glucuronate, the reac-
tion mixture was incubated at 378C for 10 min. The substrate was
then added to initiate the reaction, which was monitored for
4 min.
Keywords: aldehyde reductase
·
aldose reductase
·
benzothiadiazines · inhibitors · structure–activity relationships
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The inhibitory activity of the newly synthesized compounds
against ALR2 and ALR1 was assayed by adding 5 mL of the inhibitor
solution to the reaction mixture described above. All compounds
were dissolved in dimethyl sulfoxide (DMSO) and diluted with de-
ionized H2O. To correct for the non-enzymatic 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 com-
pounds was routinely estimated at a concentration of 10À4 m (the
concentration refers to that of the compound in the reaction mix-
ture). The compounds found to be active were tested at additional
concentrations between 10À4and 10À8 m. Each dose–response
curve was generated using at least four concentrations of com-
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Received: August 22, 2012
Revised: October 11, 2012
Published online on November 7, 2012
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