Synthesis and biological evaluation of [1,2,4]triazino[4,3-a] benzimidazole acetic acid derivatives as selective aldose reductase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2009.11.031

The acetic acid derivatives of [1,2,4]triazino[4,3-a]benzimidazole (TBI) were synthesized and tested in vitro and in vivo as selective aldose reductase (ALR2) inhibitors. Compound PS11 showed highest inhibitory activity (IC₅₀) 0.32 μM and was f

Full text of DOI:10.1016/j.ejmech.2009.11.031

European Journal of Medicinal Chemistry 45 (2010) 909–914
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of [1,2,4]triazino[4,3-a] benzimidazole
acetic acid derivatives as selective aldose reductase inhibitors
,
b
Prasanta Kumar Sahoo a , Pritishova Behera
*
a Sri Vasavi Institute of Pharmaceutical Sciences, Pedatadepalli, Tadepalligudem, West Godavari District 534101, Andhra Pradesh, India
b K. G. R. L. College of Pharmacy, Bhimabharam, West Godavari District, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The acetic acid derivatives of [1,2,4]triazino[4,3-a]benzimidazole (TBI) were synthesized and tested in
vitro and in vivo as selective aldose reductase (ALR2) inhibitors. Compound PS11 showed highest
inhibitory activity (IC50) 0.32 mM and was found to be effective in preventing cataract development in
severely galactosemic rats when administered as an eyedrop solution. All the compounds investigated
were selective for ALR2, since none of them inhibited appreciably aldehyde reductase, sorbitol dehy-
drogenase, or glutathione reductase.
Received 5 October 2009
Received in revised form
10 November 2009
Accepted 13 November 2009
Available online 4 December 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Benzimidazole
Aldose reductase
Acetic acid derivative
ALR1
ALR2
1. Introduction
classes: (i) acetic acid derivatives, including alrestatin, tolrestat,
zopolrestat, and epalrestat; (ii) cyclic imides such as sorbinil; (iii)
Despite of recent advances in the chemistry and molecular
pharmacology of antidiabetic drugs, diabetes still remains a life-
threatening disease. Tissues capable of insulin-independent
glucose uptake develop structural and functional damage (reti-
nopathy, nephropathy, cataract, keratopathy, neuropathy, and
angiopathy) in more than 50% of diabetics. A strict glycemic control
prevents or at least delays the onset of such complications.
However, close control is difficult to maintain and because there is
a parallel risk of severe hypoglycemia and obesity in intensive
insulin-treated patients, considerable efforts have been made to
find novel, effective antidiabetic agents acting by mechanisms
independent of the control of blood glucose [1–3].
Several experimental data have revealed a link between glucose
metabolism via the polyol pathway and long-term diabetic
complications. Aldose reductase (alditol/NADP þ oxidoreductase,
EC 1.1.1.21, ALR2), the first enzyme of the pathway, catalyzes the
NADPH-dependent reduction of glucose to sorbitol [2–4]. There-
fore, inhibition of ALR2 has become an attractive therapeutic
strategy, and a large number of ALR2 inhibitors (ARIs) have been
identified to date. Currently known ARIs can be divided into three
flavonoids, whose prototype is quercetin [5]. Structural require-
ments for ALR2 inhibitory activity are a planar cyclic moiety and an
acidic function ionized at the ALR2 active site [6–9]. One of the most
important requirements of clinically useful ARIs is ALR2 selectivity
over closely related enzymes such as aldehyde reductase (ALR1),
sorbitol dehydrogenase (SD), and glutathione reductase (GR). ALR1
belongs to the aldo-keto reductase family like ALR2 and exhibits
the highest homology in structure (51% sequence identity) and
activity with ALR2 [10]. SD is the second enzyme in the polyol
pathway, and its inhibition increases sorbitol levels, thus reducing
the effects of ALR2 inhibition [11]. GR maintains physiological levels
of glutathione, which in turn prevents glycosylation of cellular
protein and protects against oxidative stress. The intracellular
accumulation of oxidized glutathione, resulting from inhibition of
GR, leads to a structural modification of ALR2 that reduces its
sensitivity to ARI action [12,13]. In addition to problems related to
selectivity among homologous enzymes, ARI-based therapy suffers
from many limitations due to pharmacokinetic problems, revers-
ibility of diabetic neuropathy, adverse reactions, and poor repro-
ducibility of clinical measurements [14]. Only epalrestat is on the
Japanese market nowadays. For these reasons, there is still a need
to identify and develop clinically effective and well-tolerated ARIs.
The TBI system proved to be a suitable scaffold for several
compounds reported as selective ligands at the benzodiazepine
* Corresponding author.
E-mail address: psahoo.phar@gmail.com (P.K. Sahoo).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.031

910
P.K. Sahoo, P. Behera / European Journal of Medicinal Chemistry 45 (2010) 909–914
[15] and A1 adenosine [16] receptors. The QSAR studies of
a reported article [17] guided us to design a series of [1,2,4]tri-
azino[4,3-a]benzimidazole acetic acid derivatives as selective
aldose reductase inhibitors. The conclusion cited in that article
was ‘‘high lipophilic nature of the molecule and low electrostatic
potential energy were favorable for the selective aldose reductase
inhibitory activity. Thus, modification in structure (TBI) to improve
lipophilic character and electrostatic potential energy might result
in a more potent selective aldose reductase inhibitor’’ [17] which
lead us to model these compounds. The present paper describes
the synthesis and the biological evaluation of selective ARIs
featuring an acetic acid residue at the 2 position of the [1,2,4]tri-
azino[4,3-a]-benzimidazole (TBI) nucleus (compounds PS01–
PS12). Compound PS11, the most active ALR2 inhibitor among
those examined and were also investigated in vivo for their
effectiveness to prevent cataract development in galactosemic
rats.
derivatives C01–C12 which were cyclized to the corresponding
triazinobenzimidazoles D01–D12 by refluxing with an equimolar
amount of diethyl oxalate in ethanolic solution [20]. Reaction of
D01–D12 with ethyl bromoacetate in refluxing acetone, in the
presence of anhydrous potassium carbonate [11], gave the ester
derivatives E01–E12. These were hydrolyzed with concentrated
hydrochloric acid at 100 ꢀC to yield the designed compounds
(PS01–PS12).
3. Biochemistry and pharmacology
Compounds PS01–PS12 were evaluated for their in vitro
inhibitory activity against ALR2 as well as against ALR1, SD, and
GR. Primary in vitro screening was conducted on a water-soluble
enzymatic extract purified from rat lenses [21–24]. IC50 values
were determined by linear regression analysis of the log of the
concentration-response curve. Compounds PS01–PS12 were also
assayed for their potency to inhibit SD [25] and two other
enzymes not involved in the polyol pathway, namely, ALR1 [26]
and GR [27]. Sorbinil [28] and tolrestat [29] were used as the
reference standards. Compound PS11 was investigated in vivo for
their effectiveness to prevent cataract development in severely
galactosemic rats [28]. Their effectiveness was evaluated with
respect to tolrestat as a highly potent reversible inhibitor of
lenticular ALR2 when topically administered to rats fed with 50%
galactose diet [30].
2. Chemistry
The title compounds PS01–PS12 were synthesized as outlined
in Scheme 1. The substituted 1-alkyl-2-chlorobenzimidazoles
(Table 1) were prepared in good yields from the commercially
available 2-chlorobenzimidazole A by reaction with the appropriate
alkyl halide in the presence of sodium hydride [18]. Treatment of
B01–B12 with hydrazine hydrate [19] resulted in the hydrazino
N
N
RBr
Cl
Cl
NaH
N
N
R
B01-B12
H
A
R = alkyl, arylhydroxy, arylhalide, arylester
O
O
Hydrazine hydrate
H
N
N
D
i
e
N
t
h
y
l
o
x
a
l
a
t
e
N
N
N
NHNH2
R
D01-D12
R = alkyl, arylhydroxy, arylhalide, arylester
H
C01-C12
R = alkyl, arylhydroxy, arylhalide, arylester
Ethylbromoacetate
O
O
CH2COOEt
N
O
O
N
HCl Conc., 1000C
N
CH2COOH
12 - 72 h
N
N
N
N
N
R
E01-E12
R = alkyl, arylhydroxy, arylhalide, arylester
R
PS01-PS12
R = alkyl, arylhydroxy, arylhalide, arylester
Scheme 1.

P.K. Sahoo, P. Behera / European Journal of Medicinal Chemistry 45 (2010) 909–914
911
Table 1
Physical properties of (10-substituted[1,2,4]triazino[4,3-a]benzimidazol-3,4(10H)-dion-2-yl)acetic acids (PS01–PS12).
O
O
CH2COOH
N
N
N
N
R
Compound no.
R
Molecular weight
Yield (%)
Recrystal solvent
mp (ꢀC)
Formulaa
PS01
PS02
PS03
PS04
PS05
PS06
PS07
PS08
PS09
PS10
PS11
PS12
–CH2CH3
288
366
463
365
393
429
467
402
440
386
394
350
86
72
68
76
62
74
76
62
64
70
84
64
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
182–184
248–250
252–254
212–214
234–236
243–245
238–240
256–258
223–225
244–246
246–252
258–260
C13H12N4O4
C18H14N4O5
C18H12N4O4ClBr
C18H15N5O4
–CH2C6H4–4OH
–CH2C6H3–2-Cl,4-Br
–CH2C6H4–4NH2
–CH2C6H4–4N(CH3)2
–CH2C6H4–4-Br
–CH2C6H4–4CCl3
–CH2C6H3–2F,4-Cl
–CH2C6H3–3,5-(NO2)2
–CH2C6H3–2,4-F2
–CH2COOC6H5
C20H19N5O4
C18H13N4O4Br
C19H13N4O4Cl3
C18H12N4O4FCl
C18H12N6O8
C18H12N4O4F2
C19H14N4O6
–CH2C6H5
C18H14N4O4
a
Elemental analyses for C, H, N were within ꢁ0.4% of the calculated values.
4. Results and discussion
cataract development in severely galactosemic rats. Topical
administration can in principle achieve significant drug levels in
the lens with negligible effects on other tissues, thus avoiding
bioavailability and/or metabolism-related problems associated
with systemic administration [30]. It has been reported that esters
of acid drugs display a better corneal permeability and ocular
bioavailability by virtue of their higher lipophilicity [31–33]. The
pharmacological data are reported in Table 4. After 21 days of a 50%
galactose diet, 90% of the animals treated only with vehicle
developed nuclear cataract. Those treated with 3% ophthalmic
solution of PS11 and with 1% ophthalmic solution of tolrestat were
protected by 50%, 60%, and 53%, respectively, whereas no nuclear
cataracts were detected in rats that were administered a 3%
ophthalmic solution of tolrestat.
4.1. Biological evaluation
Table 3 presents the inhibitory activities against ALR2 of
compounds PS01–PS12 expressed as IC50 values. Several
compounds exhibited IC50 values in the low micromolar range. The
TBI derivative PS11 turned out the most potent inhibitor of the
series, with an IC50 value 0.32 mM.
4.2. Pharmacological evaluation of compound PS11
Compound PS11 was administered as an eyedrop solution in the
precorneal region to investigate its in vivo potency to prevent
Table 2
Analytical data of (10-substituted[1,2,4]triazino[4,3-a]benzimidazol-3,4(10H)-dion-2-yl)acetic acids (PS01–PS12).
Compound no.
1H NMR,
d
IR (
y
cmꢂ1
)
Elemental data (calculated/found)
C
H
N
PS01
PS02
PS03
PS04
PS05
PS06
PS07
PS08
PS09
PS10
PS11
PS12
1.13 (s, CH3), 3.10–3.90(s, 2H, CH2), 6.56–7.42
(m, 4H, ArH), 11.0 (OH)
1.37–4.32 (s, 2H, CH2), 6.41–7.42 (m, 8H, ArH), 5.00
(OH), 11.0 (OH)
3406, 1720, 1592, 1200, 900
54.17
54.15
59.02
59.00
46.63
46.62
59.18
59.16
61.06
61.00
50.37
50.25
48.79
48.74
53.68
53.62
49.10
49.05
55.96
55.94
57.87
57.81
61.71
61.65
4.20
4.18
3.85
3.82
2.61
2.58
4.14
4.10
4.87
4.82
3.05
3.00
2.80
2.76
3.00
2.98
2.75
2.70
3.13
3.12
3.58
3.54
4.03
4.00
19.44
19.42
15.29
15.26
12.08
12.06
19.17
19.15
17.80
17.76
13.05
13.00
11.98
11.94
13.91
13.88
19.09
19.04
14.50
14.47
14.21
14.18
15.99
15.94
3412, 1733, 1582, 1210, 924
1.37–4.32 (s, 2H, CH2), 6.41–7.42 (m, 7H, ArH), 11.0 (OH)
3432, 1742, 1578, 1233, 928, 642, 584
3412, 1722, 1634,1592, 1208, 908
3392, 1728, 1642, 1592, 1224, 912
3384, 1705, 1584, 1248, 926, 632
3428, 1687, 1596, 1226, 948, 592
3388, 1705, 1626, 1242, 894, 626, 568
3418, 1727, 1605,1559, 1242, 897
3402, 1722, 1592, 1208, 905, 582
3212, 2667, 1642, 1180, 890
1.37–4.32 (s, 2H, CH2), 6.41–7.42 (m, 8H, ArH), 4.00
(Aromatic C–NH), 11.0 (OH)
0.86–2.85 (s, 2H, CH3), 1.37–4.32 (s, 2H, CH2), 6.41–7.42
(m, 8H, ArH), 11.0 (OH)
1.37–4.32 (s, 2H, CH2), 6.41–7.42 (m, 8H, ArH), 11.0 (OH)
1.37–4.32 (s, 2H, CH2), 6.41–7.69 (m, 8H, ArH), 11.0 (OH)
1.37–4.32 (s, 2H, CH2), 6.41–7.42 (m, 7H, ArH), 11.0 (OH)
1.37–4.32 (s, 2H, CH2), 6.41–8.93 (m, 7H, ArH), 11.0 (OH)
1.37–4.32 (s, 2H, CH2), 6.41–7.42 (m, 7H, ArH), 11.0 (OH)
1.37–4.00 (s, 2H, CH2), 6.41–7.42 (m, 9H, ArH), 11.0 (OH)
1.37–4.32 (s, 2H, CH2), 6.41–7.42 (m, 9H, ArH), 11.0 (OH)
3406, 1660, 1540, 1210, 880

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P.K. Sahoo, P. Behera / European Journal of Medicinal Chemistry 45 (2010) 909–914
Table 3
spectra were obtained in KBr discs on a Shimadzu-8400S FTIR
spectrophotometer. Elemental analysis was performed using EL
11/carlo Ebra 1108 analyzer.1H NMR spectra were recorded on an
FT NMR (500 MHz); MeOD was used as a solvent and tetrame-
thylsilane [(CH3)4Si] as internal standard. Chemical shifts are
ALR2 inhibition data of acid derivatives PS01–PS12a.
Compound no.
R
Aldose reductase IC50b
(mM)
O
O
reported as
d (ppm). The FAB Mass spectra were recorded on
CH2COOH
N
a JEOL SX 102/DA-6000 Mass spectrometer. Elemental analyses
were performed on a Perkin–Elmer model 240c analyzer (Perkin–
Elmer, USA). The spectral data of the titled compounds (PS01-
PS12) are presented in Table 2.
N
N
N
R
The alkyl halides ethylbromide, 4-hydroxybenzyl chloride,
4-bromo-2-fluorobenzyl bromide, 4-aminobenzyl chloride, 4-bromo-
benzyl bromide, 4-(trichloromethyl)benzyl chloride, 4-chloro-2-fluo-
robenzyl bromide, 3,5-dinitrobenzyl bromide, 2,4-difluorobenzyl
PS01
PS02
PS03
PS04
PS05
PS06
PS07
PS08
PS09
PS10
PS11
PS12
Sorbinil
Tolrestat
–CH2CH3
42.40 (32.16–54.80)
4.56 (3.21–7.24)
14.34 (8.46–18.74)
32.42 (28.44–43.62)
38.42 (30.24–43.46)
4.76 (4.22–6.28)
4.24 (2.46–7.24)
36.46 (32.36–45.24)
22.84 (16.28–33.40)
8.22 (6.27–10.44)
0.32 (0.21–0.42)
0.36 (0.25–0.48)
0.62 (0.52–0.78)
0.05 (0.03–0.06)
–CH2C6H4–4OH
–CH2C6H3–2-Cl,4-Br
–CH2C6H4–4NH2
–CH2C6H4–4N(CH3)2
–CH2C6H4–4-Br
–CH2C6H4–4CCl3
–CH2C6H3–2F,4-Cl
–CH2C6H3–3,5-(NO2)2
–CH2C6H3–2,4-F2
–CH2COOC6H5
bromide, a-bromo phenyl ester and benzylbromide used to obtain
compounds PS01–PS12 respectively and 2-chlorobenzimidazole (A)
were from E. Merck, India. The compounds B01–B12 were prepared in
accordance with reported procedures of lit [34–36].
6.2. General procedure for the synthesis of 1-alkyl-2-
chlorobenzimidazoles (B01–B12)
–CH2C6H5
–
–
Sodium hydride (1.2 mmol, 50% dispersion in mineral oil) was
added portionwise, under a nitrogen atmosphere, to an ice-cooled
solution of 2-chlorobenzimidazole (A, 0.153 g, 1 mmol) in 5 mL of
freshly distilled DMF. Once hydrogen evolution had ceased, the
appropriate alkyl halide (1.2 mmol) was added dropwise and the
reaction mixture was maintained under stirring at room tempera-
ture until the disappearance of the starting material (2–18 h, TLC
analysis). The solution was then slowly poured onto crushed ice,
and the solid precipitate was collected, washed with water, and
recrystallized.
All compounds did not inhibit ALR1, SD and GR up to a concentration of 10ꢂ3 M.
IC50 (95% CL) values represent the concentration required to produce 50%
a
b
enzyme inhibition.
Table 4
Effect of treatment with opthalmic solution of PS11 and Tolrestat on development of
Nuclear Cataract in severely Galactosemic Rats.
Day of treatment
Rats with nuclear cataract (%)
Control
PS11 (3%)
Tolrestat (1%)
Tolrestat (3%)
12
13
14
15
16
17
18
19
20
21
10
20
20
30
50
50
70
80
90
90
0
0
0
0
0
0
20
30
30
40
40
50
50
0
0
0
0
0
0
0
0
0
0
6.3. General procedure for the synthesis of 1-alkyl-2-
hydrazinobenzimidazoles (C01–C12)
0
10
10
30
30
40
50
The appropriate 1-alkyl-2-chlorobenzimidazole (B01–B12,
1 mmol) was heated at 160 ꢀC in a Pyrex capped tube with 0.1 mL of
hydrazine hydrate for 5 h. After the mixture was cooled, a white
solid separated, which was collected and recrystallized.
6.4. General procedure for the synthesis of 10-alkyl[1,2,4]-
5. Conclusions
triazino[4,3-a]benzimidazol-3,4(10H)-diones (D01–D12)
Compound PS11, the most potent derivative of the series,
exhibited an ALR2 inhibitory activity ((IC50) 0.32 M) similar to that
of sorbinil (0.65 M) and proved to be effective in preventing
cataract development in severely galactosemic rats when admin-
istered as an eyedrop solution.
A solution of the appropriate 1-alkyl-2-hydrazinobenzimidazole
(C01–C12, 1 mmol) and diethyl oxalate (0.16 mL, 1.2 mmol) in 5 mL
of absolute ethanol was heated under reflux until the disappear-
ance of the starting material (3–20 h, TLC analysis). After the
mixture was cooled, a yellow solid separated, which was collected
and recrystallized.
m
m
6. Experimental section
6.5. General procedure for the synthesis of ethyl (10-
Alkyl[1,2,4]triazino[4,3-a]benzimidazol-3,4(10H)-dion-2-yl)acetates
(E01–E12)
6.1. Chemistry
The chemicals and solvents used for the experimental work
were commercially procured from E. Merck, India, CDH, s.d. Fine
Chem. and Qualigens, India. Silica gel G (160–120 mm) used for
analytical chromatography (TLC) was obtained from E. Merck.
Thin layer chromatography was used to detect the completion of
reaction and purity of the compounds synthesized. Melting
points were determined in an open glass capillary using
a Kjeldahl flask containing paraffin and are uncorrected. IR
A
suspension of D01-D12 (1 mmol), ethyl bromoacetate
(0.13 mL, 1.2 mmol), and anhydrous potassium carbonate (0.166 g,
1.2 mmol) in 5 mL of acetone was heated under reflux until the
disappearance of the starting material (7–24 h, TLC analysis). After
the mixture was cooled, the solid precipitate was collected,
washed with water to remove the inorganic salts, and
recrystallized.

P.K. Sahoo, P. Behera / European Journal of Medicinal Chemistry 45 (2010) 909–914
913
6.6. General procedure for the synthesis of (1-alkyl-3-oxalo-1H,3H-
benzimidazol-2-ylidenehydrazino)acetic acids (PS01–PS12)
340 nm, which is due to the oxidation of NADPH or the reduction of
NADþ catalyzed by ALR2, ALR1, and GR or SD, respectively. The
change in pyridine coenzyme concentration per minute was
determined using a Beckman DU-64 kinetics software program
(Solf Pack TM module).
A suspension of the appropriate ester derivative (E01–E12,
1 mmol) in 3 mL of 3% NaOH was left under stirring at room
temperature until a solution was achieved (1–48 h). The solution
was then filtered and acidified with concentrated HCl under ice-
cooling. The white solid precipitate was collected, washed with
water, and recrystallized.
ALR2 activity was assayed at 30 ꢀC in a reaction mixture con-
taining 0.25 mL of 10 mM D,L-glyceraldehyde, 0.25 mL of 0.104 mM
NADPH, 0.25 mL of 0.1 M sodium phosphate buffer (pH) 6.2), 0.1 mL
of enzyme extract, and 0.15 mL of deionized water in a total volume
of 1 mL. All the above reagents, except D,L-glyceraldehyde, were
7. Biology
incubated at 30 ꢀC for 10 min; the substrate was then added to start
the reaction, which was monitored for 5 min. Enzyme activity was
calibrated by diluting the enzymatic solution in order to obtain an
average reaction rate of 0.011 (0.0010 AU/min for the sample.
ALR1 activity was determined at 37 ꢀC in a reaction mixture
7.1. Materials and methods
Aldose reductase (ALR2) and aldehyde reductase (ALR1) were
obtained from albino rats, 120–140 g body weight. In ordered to
minimize cross contamination between ALR2 and ALR1 in the
enzyme preparation, rat lens, in which ALR2 is the predominant
enzyme, and kidney, where ALR1 shows the highest concentration,
were used for isolation of ALR2 and ALR1, respectively. Glutathione
reductase (GR) type IV from baker’s yeast (100–300 U/mg), sorbitol
dehydrogenase (SD) from sheep liver (10 U/mg of protein), pyridine
containing 0.25 mL of 20 mM sodium D-glucuronate, 0.25 mL of
0.12 mM NADPH, 0.25 mL of dialyzed enzymatic solution, and
0.25 mL of 0.1 M sodium phosphate buffer (pH) 7.2 in a total volume
of 1 mL. The enzyme activity was calibrated by diluting the dialyzed
enzymatic solution in order to obtain an average reaction rate of
0.015 ꢁ 0.0010 AU/min for the sample.
SD activity [25] was determined at 37 ꢀC in a reaction mixture
containing 0.25 mL of 10 mM sorbitol, 0.25 mL of 0.47 mM NADþ,
0.25 mL of 3.75 mU/mL enzymatic solution, 0.25 mL of 100 mM
Tris–HCl buffer (pH ¼ 8) in a total volume of 1 mL. All the reagents
were incubated at 37 ꢀC for 1 min, after which the reaction was
monitored for 3 min.
coenzymes,
D,L-glyceraldehyde, glutathione disulfide, sodium
D-glucuronate and sorbitol was obtained. Sorbinil was a gift sample
from Pfizer India Ltd. Tolrestat was obtained from Ranbaxy Labo-
ratories Ltd., India. All other chemicals were of reagent grade.
7.2. Enzyme preparation (aldose reductase (ALR2))
GR activity [27] was determined at 37 ꢀC in a mixture containing
0.25 mL of 1 mM glutathione disulfide, 0.25 mL of 0.36 mM NADPH,
0.25 mL of 4.5 mU/mL enzymatic solution, 0.25 mL of 0.125 sodium
phosphate buffer (pH ¼ 7.4) supplemented with 6.3 mM EDTA
potassium salt, in a total volume of 1 mL.
A purified rat lens extract was prepared in accordance with the
method of Hayman and Kinoshita [37] with slight modifications.
Lenses were quickly removed from normal killed rats and
homogenized in three volumes of cold, deionized water. The
homogenate was centrifuged at 12,000 rpm at 0–4 ꢀC for 30 min.
Saturated ammonium sulfate solution was added to the superna-
tant fraction to form a 40% solution, which was stirred for 30 min at
0–4 ꢀC and then centrifuged at 12,000 rpm for 15 min. Following
this same procedure, the recovered supernatant was subsequently
fractionated with saturated ammonium sulfate solution, using first
a 50% and then a 75% of salt saturation. The precipitate recovered
from the 75% saturated fraction, containing ALR2 activity, was
redissolved in 0.05 M NaCl and dialyzed overnight in 0.05 M NaCl.
The dialyzed material was used for the enzymatic assay.
7.5. Enzymatic inhibition
The inhibitory activity of the newly synthesized compounds
against ALR2, ALR1, SD, and GR was assayed by adding 0.1 mL of the
inhibitor solution to the reaction mixture described above. All the
inhibitors were dissolved in water, and the solubility was facilitated
by adjustment to a favorable pH. After complete dissolution, the pH
was readjusted to 7. To correct for the nonenzymatic oxidation of
NADPH or reduction of NADþ and for the absorption by the
compounds tested, a reference blank containing all the above assay
components except the substratewas prepared. The inhibitory
effect of the new derivatives was routinely estimated at a concen-
tration of 10–5 M. Those compounds found to be active were tested
at additional concentrations between 10–5 and 10–8 M. The
determination of the IC50 values was performed by using linear
regression analysis of the log of the dose-response curve, which
was generated using at least four concentrations of inhibitor,
causing an inhibition between 20% and 80% with two 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.
7.3. Aldehyde reductase (ALR1)
Rat kidney ALR1 was prepared in accordance with a previously
reported method [26]. Kidneys were quickly removed from normal
killed rats and homogenized (Glas-Potter) in three volumes of
10 mM sodium phosphate buffer, pH ¼ 7.2, containing 0.25 M
sucrose, 2.0 mM EDTA dipotassium salt, and 2.5 mM
b-mercap-
toethanol. The homogenate was centrifuged at 12,000 rpm at
0–4 ꢀC for 30 min, and the supernatant was subjected to a 40–75%
ammonium sulfate fractionation, following the same procedure
previously described for ALR2. The precipitate obtained from the
75% ammonium sulfate saturation, containing ALR1 activity, was
redissolved in 50 volumes of 10 mM sodium phosphate buffer,
pH ¼ 7.2, containing 2.0 mM EDTA dipotassium salt and 2.0 mM
8. Pharmacology
b
-mercaptoethanol and dialyzed overnight using the same buffer.
8.1. Materials and methods
The dialyzed material was used in the enzymatic assay.
Experiments were carried out using albino rats, 45–55 g body
weight. The galactose diet consisted of a pulverized mixture of 50%
7.4. Enzymatic assays
D-galactose and 50% TRM, and the control diet consisted of normal
The activity of the four test enzymes was determined spectro-
photometrically by monitoring the change in absorbance at
TRM. Both control and experimental rats had access to food and
water ad libitum.

914
P.K. Sahoo, P. Behera / European Journal of Medicinal Chemistry 45 (2010) 909–914
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Animals were randomly divided into groups of equal average
body weight with 15 rats per group. The test compound PS11 and
tolrestat were administered four times daily as eyedrops of
appropriate concentrations. The vehicle in which ARIs were con-
tained was administered with the same dose regimen to the control
group, which was given access to the galactose diet, and to the
group fed with normal diet, which was included to record the
aspect of normal lenses. Groups treated with the tested compounds
were predosed 1 day before switching their diet to galactose con-
taining chow.
Lenses were examined using slit-lamp microscopy, after dilating
the pupils with 1% atropine, to establish their status of integrity.
Nuclear cataracts, which appeared as a pronounced central opacity
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The authors are thankful to the Director, Jeypore College of
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