Synthesis and aldose reductase inhibitory activities of novel N-nitromethylsulfonanilide derivatives

Article abstract of DOI:10.1016/S0968-0896(00)00158-9

A novel series of 14 N-nitromethylsulfonanilide derivatives were synthesized and evaluated for their ability to inhibit recombinant aldose reductase. Computational docking simulations provided a good explanation for the observed structure-activity relatio

Full text of DOI:10.1016/S0968-0896(00)00158-9

Bioorganic & Medicinal Chemistry 8 (2000) 2167±2173
Synthesis and Aldose Reductase Inhibitory Activities of Novel
N-Nitromethylsulfonanilide Derivatives
Jun Inoue,a Ying-She Cui,a,* Osamu Sakai,a Yoshikuni Nakamura,a
Hiromi Kogisoa and Peter F. Kadorb
aResearch Laboratory, Kobe Creative Center, Senju Pharmaceutical Co., Ltd, 1-5-4 Murotani, Nishi-Ku, Kobe, Hyogo 651-2241, Japan
bLaboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA
Received 26 April 2000; accepted 3 June 2000
AbstractÐA novel series of 14 N-nitromethylsulfonanilide derivatives were synthesized and evaluated for their ability to inhibit
recombinant aldose reductase. Computational docking simulations provided a good explanation for the observed structure±activity
relationships. Kinetic analysis of (2-¯uoro-5-methyl-N-methyl)-N-nitromethylsulfonanilide, 11, one of the most potent compounds
in this series with an IC50=0.35 mM, showed uncompetitive inhibition. Subsequent in vitro culture studies of rat lenses with 11
indicated that this series of aldose reductase inhibitors are e€ective in either preventing or retarding sugar cataract formation
associated with diabetes. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
synthesized and identi®ed as potential inhibitors,2,4 7
many of these compounds have failed clinically due to
inadequate ecacy, adverse pharmacokinetics properties,
or toxic side-e€ects. To date, two general series of ARIs
have been developedÐthose possessing a carboxylic
acid group and those containing a cyclic imide ringÐthe
latter of which possesses more favorable pharmacoki-
netics properties.8,9
Aldose reductase (alditol; NADP+ oxidoreductase,
E.C. 1.1.1.21, ALR2), the ®rst enzyme in the polyol
pathway, utilizes NADPH to reduce the aldehyde form
of glucose to its respective sugar alcohol, sorbitol. Sub-
sequently, sorbitol dehydrogenase (l-iditol; NAD+ 5-
oxidoreductase, E.C. 1.1.1.14, SD), the second enzyme
in the pathway, utilizes NAD+ to convert sorbitol to
fructose.1 In diabetes, the increased ¯ux of glucose
through the polyol pathway can result in altered cellular
redox potentials and increased intracellular levels of sor-
bitol which results in hyperosmotic stress, cellular swelling
and increased membrane permeability and perturbation
of membrane transport processes. These changes can
initiate cellular lesions associated with late-onset diabetic
complications such as neuropathy, nephropathy, kerato-
pathy, angiopathy, and cataract.1 3 Experimental studies
indicate that aldose reductase inhibitors (ARIs), which
block the ¯ux of the glucose through polyol pathway
and prevent the intracellular accumulation of sorbitol,
can prevent, retard, or reverse these complications of
chronic diabetes.1,2 Over the past 30 years, world-wide
e€orts have been made in the development of clinically
useful ARIs. While a number of ARIs have been
The use of sulfonylnitromethanes as potential ARIs were
®rst reported by Brittain et al.10 Based on published
pharmacophore requirements,11,12 a new series of N-
nitromethylsulfonanilide derivatives were designed and
synthesized. They were subsequently evaluated for their
in vitro ability to inhibit aldose reductase and the
observed structure±activity relationships were analyzed
using computational docking simulation.
Results and Discussion
The general route for the synthesis of the N-nitro-
methylsulfonanilide derivatives is illustrated in Scheme
1. The methanesulfonyl group was introduced into the
starting arylamine or N-alkylaniline by treating methane-
sulfonyl chloride in the presence of triethylamine. The
resulting methanesulfonamide was treated with n-butyl-
lithium at
10ꢀC, followed by reaction with iso-
amylnitrate at 30ꢀC to a€ord the nitromethylsulfon-
amide.
*Corresponding author. Tel.: +81-78-997-1010; fax: +81-78-997-
1016; e-mail: yingshe-cui@senju.co.jp
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00158-9

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J. Inoue et al. / Bioorg. Med. Chem. 8 (2000) 2167±2173
Scheme 1.
The newly synthesized compounds were evaluated for
their ability to inhibit the in vitro reduction of glycer-
aldehydes by recombinant human muscle aldose reductase
which was spectrophotometrically followed by following
the consumption of NADPH cofactor at 340 nm.
Appropriate controls were employed to negate potential
changes in the absorption of nucleotide and/or protein
modi®cation reagents or aldose reductase inhibitors at
340 nm in the absence of substrate. The in vitro inhibi-
tory activities of these compounds compared to SG21013
are summarized in Table 1.
reported by Harrison et al.14 and deposited in the Protein
Data Bank (code 2ACQ). Minimization of the mole-
cules was conducted by using the Tripos force ®eld with
the Powell method,15 and docking simulations were
performed by using the Tripos docking programs
FlexXTM and FlexiDockTM 16
The enzyme±inhibitor
.
interactions in FlexX include both polar (hydrogen
bond and charge±charge) and non-polar (hydrophobic)
terms. Many of these interactions are geometrically
quite restrictive, which allows FlexX to accurately place
inhibitors and their fragments. Directly docking a
potential inhibitor into the active site of the enzyme
which covers all possible docking combinations of inhi-
bitor conformations and binding modes, and an induced
®t could be explored by FlexiDock, which allow both
the inhibitor and receptor binding pocket to `¯ex'; dur-
ing docking. Finally the docking results were minimized
with the Tripos force ®eld, de®ning monomers outside
5 angstrom area around the inhibitor as an aggregate.
The results of 11, one of the most potent compounds of
this study, are shown in Figure 1.
To explain the observed structure±activity relationships,
these compounds were docked into the crystal structure
of aldose reductase with NADP+ glucose-6-phosphate
.
Table 1. Inhibitory activities of novel nitromethylsulfonanilide ARIs
The nitromethylsulfonyl moiety was positioned for
potential reversible participation (H-bonds) with nucleo-
philic residues on the aldose reductase in the charge
transfer pocket of the inhibitor binding site (distance
from NO2 to Tyr 48-OH=3.204 A, angle=163ꢀ; distance
from SO2 to Trp 111-NH=2.758 A, angle=161ꢀ, distance
from SO2 to His 110-E NH=2.941 A, angle=125ꢀ). It
could be considered that the nitromethylsulfonyl group,
like hydantoin or carboxyl groups, is a functional moi-
ety which is susceptible to reversible nucleophilic attack
by aldose reductase.11,16
Compound
R1
R2
R3
R4
R5
R6
IC50 (mM)a
1
2
3
4
5
6
7
8
H
Me
Et
H
Me
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
i-Pr
6.4
0.7
5.9
4.9
0.6
20.3
2.0
1.2
0.3
2.8
0.35
2.7
Cl
OMe
OMe
H
H
H
H
H
H
H
H
Me OMe
H
Me
H
Me
H
Me
Me
F
F
Me
i-Pr
OMe
OMe
Me
Me
H
The benzene ring of the inhibitor was located between
Val 47 and Trp 20, to keep hydrophobic interaction
with lipophilic region of the enzyme (shortest distance
from Val 47=3.810 A; distance from Trp 20=3.908 A).
The hydrophobic interaction of N-methyl moiety of 11
with Phe 122, Trp 20 and Trp 219 (shortest distances are
4.813, 4.353 and 4.056 A, respectively), no such inter-
action in the case of N-methyl unsubstituted analogue
10 (Fig. 2), led to an enhancement of inhibition activity
of 11 compared with 10. In addition, the FlexiDock
9
10
11
12
13
14
SG210c
H
H
>100b
>100b
0.1
aInhibitory activity against recombinant human muscle cell AR.
bNo signi®cant activity at 1Â10 M.
4
cRef 13.

J. Inoue et al. / Bioorg. Med. Chem. 8 (2000) 2167±2173
2169
Figure 1. Stereoview of the most stable docking model for (2-¯uoro-5-methyl-N-methyl)-N-nitromethylsulfonanilide, 11 within the substrate pocket of
aldose reductase. Intermolecular hydrogen bonds (distances less than 2.8 A between the hydrogen bonded to a H-bond donor and an acceptor) are shown
in dotted yellow lines. Carbon atoms are shown in white except those of the surrounding amino acids (green) and NADPH (brown). Hydrogen atoms are
shown in cyan except those of the amino acids (green). Nitrogens are blue, oxygens are red, sulfur is yellow, and ¯uorine is light green.
Figure 2. Detailed view of the most stable docking model for 11 (right) and 10 (left) within the substrate pocket of aldose reductase.
energy of 11 ( 682.9 kcals/mol) was much lower than
that of 10 ( 470.3 kcals/mol). As a scoring function in
FlexiDock, better ®ts simply give a lower energy of
interaction. Actually, all of the compounds with a methyl
moiety substituted on the nitrogen atom which bears the
nitromethylsulfonyl group, exhibited better inhibition
activity than the respective non-methyl substituted ones
(activities: 2,>1; 5>4; 7>6; 9>8; 11>10). While the
methyl group was replaced with an ethyl moiety, the
activity decreased (activity: 2>3). Similar results were
observed when bulky substituents were substituted on
the benzene ring (activity: 12>>13). This result might
be assigned to the bulky substituents interfering with the
H-bond interactions between the nitromethylsulfonyl
moiety and proton donor moieties of the enzyme.
Kinetic studies of the inhibition of recombinant aldose
reductase by (2-¯uoro-5-methyl-N-methyl)-N-nitro-
methylsulfonanilide, 11 was conducted with dl-glycer-
aldehyde as substrate.17 Parallel slopes were obtained

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J. Inoue et al. / Bioorg. Med. Chem. 8 (2000) 2167±2173
Figure 3. Lineweaver±Burke plot (A) and Hanes±Woolf plot (B) of the inhibition of human recombinant AR by 11 with varying concentrations
of dl-glyceraldehyde as substrate. The concentrations of 11 were: *, 0 mM; &, 0.02 mM; ~, 0.04 mM; ^, 0.06 mM. S represents the substrate,
dl-glyceraldehyde, while V is the measured decrease per minute in the UV absorption of NADPH at 340 nm.
on a Lineweaver±Burke plot (Fig. 3A) when the con-
centration of inhibitor was varied indicating that the
observed inhibition of 11 was uncompetitive with glycer-
aldehyde as substrate. This uncompetitive inhibition was
con®rmed by a Hanes±Woolf plot of [S]/V versus [S] (Fig.
3B).
were photographed under a dissecting microscope. As
illustrated in Figure 4, lenses remained clear when incu-
bated in normal medium (upper-left, normal lens) while
similar lenses cultured in the presence of 30% xylose
formed lens opacities associated with sugar cataract
formation (upper-right, cataract lens). Addition of 1 and
10mM of 11 to the xylose medium resulted in a dose-
dependent reduction lens opacities caused by sugar catar-
act formation (Fig. 4, lower-left and right). Lens density
(2-Fluoro-5-methyl-N-methyl)-N-nitromethylsulfonani-
lide, 11 was also evaluated for its ability to inhibit sugar
cataract formation inducted in intact rat lenses cultured
in vitro in media containing 30% xylose.18 After 48 h
culture in the xylose medium, the changes in lens opacity
Figure 5. Measured opacities (optical densities) in lenses resulting
from xylose induced sugar cataract formation and its reduction by
treatment with 11. Data are means
**P<0.01 relative to xylose.
Æ
S.D. (n=5). *P<0.05,
Figure 4. Representative appearance of lenses cultured for 2 days with
and without 30% xylose and treatment with 11. The upper-left is a
normal lens cultured without xylose. The upper-right is a lens cultured
in medium containing 30% xylose while the lower left and right lenses
are lenses cultured in medium containing 30% xylose and 1 and 10 mM
of 11, respectively. Darker areas indicate increased opacity due to
sugar cataract accumulation.
Figure 6. Increased hydration in xylose-induced sugar cataractous lens
after 2 days of culture and the reduction of hydration in similar lenses
treated with 11. Data are means Æ S.D. (n=5). *P<0.01 relative to
normal lens, and **P<0.01 relative to untreated xylose lens.

J. Inoue et al. / Bioorg. Med. Chem. 8 (2000) 2167±2173
2171
measurements using computer imaging analysis con®rmed
that 11 was e€ective in reducing the total lens opacity
(P<0.01, Fig. 5). In addition, measurement of lens water
levels indicated that 10mM 11 signi®cantly reduced polyol
associated lens hydration (P<0.01, Fig. 6).
stirred overnight at rt. Water was added to the reaction
solution, and the mixture was extracted with CH2Cl2.
The organic layer was washed with 1N HCl and then
brine, and dried over anhydrous Na2SO4. After ®ltration,
the solvent was evaporated in vacuo.
To a dry THF solution containing 1 equiv of the above
obtained N-methylsulfonanilide (or N-methyl-N-methyl-
sulfonanilide) at 10 ꢀC under a nitrogen atmosphere
was dropwise added 2.4 equiv (or 1.2 equiv in the case of
N-methyl-N-methylsulfonanilide) of n-butyllithium (1.6
mol/L in hexane). The mixture was stirred for 10 min,
and then cooled to 30 ꢀC and isoamylnitrate (2.2 equiv,
or 1.1 equiv in the case of N-methyl-N-methylsulfonani-
lide) was then dropwise added. After stirring for 4 h at
the same temperature, the reaction was quenched with
water, and the volatiles were evaporated in vacuo. 1N
NaOH (2 equiv) was added to the reaction mixture and
the mixture was washed with ethyl ether. The aqueous
layer was acidi®ed with acetic acid (pH=5), and extrac-
ted with CH2Cl2. The organic layer was washed with
brine and dried over anhydrous Na2SO4. Following ®l-
tration, the solvent was evaporated in vacuo, and the
residue was puri®ed by column chromatography and
recrystallization to give the desired compounds.
Conclusions
This new series of N-nitromethylsulfonanilide derivatives
inhibited recombinant human muscle aldose reductase in
vitro with IC50 values ranging from 20.3 to 0.3 mM.
Computational docking simulation analysis showed that
the nitromethylsulfonyl group was positioned for poten-
tial reversible participation with nucleophilic residues on
the aldose reductase in the charge transfer pocket of the
inhibitor binding site. Increased hydrophobic interaction
of the N-methyl moiety led to enhanced inhibition and
the benzene ring of the inhibitor appears to be important
for hydrophobic interaction with a lipophilic region on
the aldose reductase.
Kinetic analysis of (2-¯uoro-5-methyl-N-methyl)-N-
nitromethylsulfonanilide, 11, one of the most potent com-
pounds with an IC50=0.35 mM, showed uncompetitive
inhibition. Compound 11 also e€ectively prevented lens
opacity formation and reduced lens hydration caused by
sugar cataract formation in rat lens cultured in vitro in
medium containing 30% xylose. These results indicate
that this series of aldose reductase inhibitors are e€ective
in delaying or preventing cataract formation in diabetes.
N-Nitromethylsulfonanilide (1). Overall yield, 7%. Col-
orless crystals, mp 91.8±92.4 ꢀC (48% EtOH). 1H NMR
(CDCl3): d 5.43 (2H, s), 7.12 (1H, br s), 7.30±7.46 (5H,
m). 13C NMR (CDCl3): d 82.9, 123.2 (2C), 127.2 (2C),
129.7, 129.9. Anal. calcd for C7H8N2O4S: C, 38.89; H,
3.73; N, 12.96. Found: C, 39.11; H, 3.69; N, 12.81.
N-Methyl-N-nitromethylsulfonanilide (2). Overall yield,
17%. Colorless crystals, mp 50.0±51.0 ꢀ C (95% EtOH). 1H
NMR (CDCl3): d 3.47 (3H, s), 5.45 (2H, s), 7.37±7.54 (5H,
m). 13C NMR (CDCl3): d 41.0, 83.7, 127.6 (2C), 128.9,
129.9 (2C), 139.3. Anal. calcd for C8H10N2O4S: C, 41.73;
H, 4.38; N, 12.17. Found: C, 41.79; H, 4.36; N, 12.22.
Experimental
Chemistry
Melting points (mp) were obtained using a Yanaco Micro
Melting Point Apparatus and were uncorrected. Elemental
analyses were measured with a Perkin Elmer CHNS/
02400 analyzer and mass spectra analyses were performed
by Takeda Analytical Research Laboratories, Ltd. All
indicated analyses were within Æ 0.4% of theoretical
values. NMR spectra were measured using a Varian
N-Ethyl-N-nitromethylsulfonanilide (3). Overall yield,
1
13%. A yellow oil. H NMR (CDCl3): d 1.14 (3H, t,
J=7.2 Hz), 3.84 (2H, q, J=7.2 Hz), 5.44 (2H, s), 7.39±
7.49 (5H, m). 13C NMR (CDCl3): d 14.9, 49.3, 84.2, 129.4,
129.6 (2C), 130.1 (2C), 137.0. Anal. calcd for C9H12
N2O4S: C, 44.26; H, 4.95; N, 11.47. Found: C, 44.30; H,
4.95; N, 11.52.
1
Gemini-300 spectrometer (300MHz for H, 75MHz for
13C, and 282 MHz for 19F, respectively), and the chemical
shifts are expressed in d (ppm) units with tetra-
methylsilane as an internal standard for 1H and 13C
NMR, and with tri¯uoromethylbenzene as internal
standard for 19F NMR. Unless stated otherwise, column
chromatography was conducted on silica gel Merk-60
with a mixture of EtOAc and hexane as eluent.
(4-Chloro)-N-nitromethylsulfonanilide (4). Overall yield,
4%. Colorless crystals, mp 117.7±118.3 ꢀC (79% EtOH).
1H NMR (CDCl3): d 5.43 (2H, s), 7.11 (1H, br s), 7.31±
7.34 (2H, m), 7.38±7.43 (2H, m). 13C NMR (CDCl3) d
82.5, 125.0 (2C), 130.2 (2C), 132.7, 133.7. Anal. calcd
for C7H7ClN2O4S: C, 33.54; H, 2.81; N, 11.18. Found:
C, 33.68; H, 2.77; N, 11.13.
Starting anilines or N-alkylanilines were commercially
available or prepared according to a published method.19
(4-Chloro-N-methyl)-N-nitromethylsulfonanilide (5). Over-
all yield, 17%. Colorless crystals, mp 106.0±106.5ꢀC
(EtOH/CH2Cl2, 3:1, v/v ). 1H NMR (CDCl3): d 3.44 (3H,
s), 5.44 (2H, s), 7.41±7.48 (4H, m). 13C NMR (CDCl3): d
40.9, 83.7, 128.9 (2C), 130.1 (2C), 134.9, 137.8. Anal.
calcd for C8H9ClN2O4S: C, 36.30; H, 3.43; N, 10.58.
Found: C, 36.37; H, 3.36; N, 10.54.
General preparation of N-nitromethylsulfonanilide
Methanesulfonyl chloride (1.1 equiv) was added drop-
wise to a stirred solution containing 1.0 equiv aniline (or
N-alkylaniline) and triethylamine (1.5 equiv) in CH2Cl2
(or DMF) under ice±water cooling and the mixture was

2172
J. Inoue et al. / Bioorg. Med. Chem. 8 (2000) 2167±2173
(2,4-Dimethoxy)-N-nitromethylsulfonanilide (6). Overall
yield, 8%. Colorless crystals, mp 91.0±91.8 ꢀC (95%
EtOH). 1H NMR (CDCl3): d 3.81 (3H, s), 3.85 (3H, s), 5.44
(2H, s), 6.47±6.50 (2H, m), 7.02 (1H, br s), 7.39 (1H, m).
13C NMR (CDCl3): d 55.6, 55.9, 83.5, 99.2, 104.9, 116.2,
126.4, 152.9, 160.0. Anal. calcd for C9H12N2O6S: C, 39.13;
H, 4.38; N, 10.14. Found: C, 39.45; H, 4.39; N, 10.09.
(95% EtOH). 1H NMR (CDCl3): d 1.26 (12H, d,
J=6.9 Hz), 3.40 (2H, m), 5.63 (2H, s), 6.48 (1H, br s),
7.21±7.26 (2H, m), 7.38 (1H, m). 13C NMR (CDCl3): d
24.0 (4C), 28.9 (2C), 86.6, 124.5 (2C), 127.9, 130.0, 148.5
(2C). Anal. calcd for C13H20N2O4S: C, 51.98; H, 6.71;
N, 9.33. Found: C, 52.19; H, 6.75; N, 9.45.
4,5-Dimethyl-2-(nitromethylsulfonylamino)thiazole (14).
Overall yield, 3%. Colorless crystals, mp 183.0±184.0 ꢀC
(EtOH). 1H NMR (DMSO-d6): d 2.05 (3H, s), 2.11 (3H,
s), 5.96 (2H, s), 12.89 (1H, br s). 13C NMR (DMSO-d6): d
10.6, 10.8, 88.0, 114.0, 128.8, 167.9. Anal. calcd for
C6H9N3O4S2: C, 28.68; H, 3.80; N, 16.16. Found: C,
28.29; H, 3.59; N, 16.55.
(2,4-Dimethoxy-N-methyl)-N-nitromethylsulfonanilide (7).
Overall yield, 8%. Colorless crystals, mp 115.0±116.0 ꢀC
(EtOH/CH2Cl2, 1:1, v/v). 1H NMR (CDCl3): d 3.33 (3H,
s), 3.82 (3H, s), 3.86 (3H, s), 5.54 (2H, s), 6.48±6.51 (2H,
m), 7.28 (1H, m). 13C NMR (CDCl3): d 39.7, 55.6, 55.7,
85.5, 99.6, 105.2, 120.2, 132.4, 156.6, 161.6. Anal. calcd
for C10H14ClN2O6S: C, 41.37; H, 4.86; N, 9.65. Found:
C, 41.72; H, 4.98; N, 9.88.
Modeling and docking simulation
(5-Methoxy-2-methyl)-N-nitromethylsulfonanilide (8).
Overall yield, 6%. Colorless crystals, mp 90.0±91.0 ꢀC
(95% EtOH). H NMR (CDCl3): d 2.33 (3H, s), 3.80
Docking simulation were performed with the sybyl 6.6
software package20 running on a Silicon Graphics Iris
workstation.
1
(3H, s), 5.54 (2H, s), 6.78 (1H, m), 6.83 (1H, br s), 7.09 (1H,
m), 7.19 (1H, m). Anal. calcd for C9H12N2O5S: C, 41.53;
H, 4.65; N, 10.76. Found: C, 41.87; H, 4.74; N, 10.41.
Biology
Enzyme assay. Aldose reductase activity was determined
spectrophotometrically by monitoring the oxidation of
NADPH in a reaction mixture containing 100 mM
sodium phosphate bu€er, pH 6.2, 10mM dl-glycer-
aldehyde, 0.3 mM b-NADPH, 0.01 unit/mL recombinant
human muscle aldose reductase, and various concentra-
tions of inhibitor. (The enzyme was puri®ed from the
culture medium of the baculaovirus-insect cell line 32d
gene expression system in which the c-DNA containing
the entire protein coding region of human aldose
reductase is expressed and the enzyme retains the same
properties exhibited by human muscle and retina.) Non-
inhibitor samples were run as controls and non-enzyme
samples were run as Blanks.
(5-Methoxy-2-methyl-N-methyl)-N-nitromethylsulfonani-
lide (9). Overall yield, 4%. Colorless oil. 1H NMR
(CDCl3): d 2.33 (3H, s), 3.34 (3H, s), 3.81 (3H, s), 5.57
(2H, br s), 6.89 (1H, m), 7.05 (1H, m), 7.24 (1H, m). 13C
NMR (CDCl3): d 17.2, 40.9, 55.5, 84.7, 113.3, 114.9,
130.3, 132.6, 138.7, 158.8. HRMS: C10H14N2O5S (M+),
calcd 274.0623; found 274.0604.
(2-Fluoro-5-methyl)-N-nitromethylsulfonanilide (10). Over-
all yield, 5%. Colorless crystals, mp 93.3±93.8 ꢀC (86%
1
EtOH). H NMR (CDCl3): d 2.35 (3H, s), 5.54 (2H, s),
7.05±7.08 (2H, m), 7.10 (1H, m), 7.35 (1H, m). 13C NMR
(CDCl3): d 20.9, 83.7, 115.9 (1C, d, J=77.7 Hz), 122.0 (1C,
d, J=48.6 Hz), 124.8, 128.8 (1C, d, J=29.1 Hz), 135.3 (1C,
d, J=15.6 Hz), 152.9 (1C, d, J=972.6 Hz). 19F-NMR
(CDCl3): d 132.8. Anal. calcd for C8H9FN2O4S: C, 38.71;
H, 3.65; N, 11.29. Found: C, 38.95; H, 3.62; N, 11.22.
The reduction was initiated by the addition of NADPH
solution and incubated for 5 min. The absorbency of the
plate was read on a microplate reader (Labsystems
Multiscan Multisoft) at 340 nm.11 Appropriate controls
were employed to negate potential changes in the
absorption of nucleotide and/or protein modi®cation
reagents or aldose reductase inhibitors at 340 nm in the
absence of substrate. Under these conditions the
NADPH oxidation was linear with time. Each inhibitor
concentration was tested in duplicate, and an IC50 value
for each compound was obtained from linear regression
analyses of the log concentration versus percent inhibition
plots of the data.21
(2-Fluoro-5-methyl-N-methyl)-N-nitromethylsulfonanilide
(11). Overall yield, 4%. Colorless crystals, mp 76.0±
77.0 ꢀC (95% EtOH). H NMR (CDCl3): d 2.35 (3H, s),
1
3.41 (3H, s), 5.56 (2H, s), 7.08 (1H, m), 7.19 (1H, m), 7.26
(1H, m). 13C NMR (CDCl3): d 20.6, 40.3, 85.4, 116.6 (1C,
d, J=20.0 Hz), 126.2 (1C, d, J=12.2 Hz), 131.7 (1C, d,
J=7.8 Hz), 131.7, 135.4, 157.0 (1C, d, J=248.2 Hz). 19F
NMR (CDCl3): d 126.5. Anal. calcd for C9H11
FN2O4S: C, 41.22; H, 4.23; N, 10.68. Found: C, 41.24;
H, 4.24; N, 10.57.
Kinetic studies. A mixture of 11 (2 mL), 0.25 M sodium
phosphate bu€er (98 mL), varying concentrations of dl-
glyceraldehyde (50 mL), recombinant aldose reductase
(0.05 unit/mL, 50 mL), and 1.5 mM NADPH (50 mL) was
incubated for 5 min. The velocity (V) of decrease in the
UV absorption of NADPH at 340 nm was recorded on
the microplate reader as described above. Lineweaver±
Burke plots of 1/S (concentration of dl-glyceraldehyde
substrate, mM) versus 1/V (UV absorbency of NADPH
per minute) were obtained at measuring di€erent con-
centrations of 11 and dl-glyceraldehyde.
(2,6-Dimethyl)-N-nitromethylsulfonanilide (12). Overall
yield, 5%. Colorless crystals, mp 125.8±126.6 ꢀC (95%
1
EtOH). H NMR (CDCl3): d 2.42 (6H, s), 5.61 (2H, s),
6.50 (1H, br s), 7.12±7.22 (3H, m). 13C NMR (CDCl3): d
19.2, 87.0, 129.0 (2C), 129.1 (2C), 131.2, 137.8. Anal.
calcd for C9H12N2O4S: C, 44.26; H, 4.95; N, 11.47.
Found: C, 44.61; H, 4.99; N, 11.39.
(2,6-Diisopropyl)-N-nitromethylsulfonanilide (13). Over-
all yield, 11%. Colorless crystals, mp 165.0±166.0 ꢀC

J. Inoue et al. / Bioorg. Med. Chem. 8 (2000) 2167±2173
2173
In vitro inhibition of lens opacity. Sugar cataract forma-
tion was induced in lenses from 4-week-old Sprague±
Dawley rats by culturing at 37 ꢀC under 5% CO2
atmosphere in medium containing 30% xylose. Lenses
were carefully dissected from enucleated eyes by a pos-
terior approach and checked for potential dissection
damage by pre-incubation in 4 mL of Eagles essential
medium containing 10% fetal calf serum. After 24 h
remaining clear lenses were carefully transferred to
similar medium containing 30% xylose with and with-
out the aldose reductase inhibitor 11. After 48 h culture
in the xylose containing medium, each lens was photo-
graphed under a dissecting microscope and the clarity of
each lens was quanti®ed using computerized imaging
analysis (Image 1.31 software, Twilight clone BBC,
Silver Springs, MD).
3. Tomlinson D. R.; Stevens E. J.; Diemel L. T. Trends.
Pharmacol. Sci. 1994, 293.
4. Kador, P. F.; Kinoshita, J. H.; Sharpless, N. E. J. Med.
Chem. 1985, 28, 841.
5. Kador, P. F.; Robinson, W. G.; Kinoshita, J. H. Annu.
Rev. Pharmacol. Toxicol. 1985, 25, 691.
6. Sima, A. A. F.; Bril, V.; Nathaniel, T. A.; McEwen, J.;
Brown, M. B.; Lattimet, S. A.; Green, D. A. N. Engl. J. Med.
1988, 319, 548.
7. Mylary, B. L.; Larson, E. R.; Beyer, T. A.; Zembrowski,
W. J.; Aldinger, C. E.; Dee, M. F.; Siegel, T. W.; Singleton, D.
H. J. Med. Chem. 1991, 34, 108.
8. Humber L. G. In Progress in Medicinal Chemistry: The
Medicinal Chemistry of Aldose Reductase Inhibitors; Ellis G.P.;
West, G. B. Eds.; Elsevier Science: New York, 1987; Vol. 24,
pp 299±343.
9. Sarges, R. In Advances in Drug Research: Aldose Reductase
Inhibitors; Structure±Activity Relationships and Therapeutic
Potential; Academic: San Diego, 1989; Vol. 18, pp 139±175.
10. Brittain, D. R.; Brown, S. P.; Cooper, A. L.; Longridge,
J. L.; Morris, J. J.; Preston, J.; Slater, L. European Patent
Application 1991, publication number: 0 469 887 A1.
11. Kador, P. F.; Sharpless, N. E. Mol. Pharmacol. 1983, 24,
521.
Measurement of water content and hydration rate. Each
lens was weighed immediately after incubation (wet
weight, WW) and after drying at 100 ꢀC for 16 h (Wd).
The percent lens hydration was calculated using the
equation:
12. Lee, Y. S.; Pearlstein, R.; Kador, P. F. J. Med. Chem.
1994, 37, 787.
% Hydration ˆ ‰ꢀWW Wd†=WWŠ  100%:
13. Aotsuka, T.; Hosono, H.; Kurihara, T.; Nakamura, Y.;
Matsui, T.; Kobayashi, F. Chem. Pharm. Bull. 1994, 42, 1264.
14. Harrison, D. H.; Bohren, K. M.; Ringe, D.; Petsgo, G. A.;
Gabbay, K. H. Biochemistry 1994, 33, 2011.
15. Powell, M. J. D. Math. Program. 1977, 12, 241.
16. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol.
Biol. 1996, 261, 470.
Acknowledgement
The authors thank Dr. Yutaka Kawamatsu (Senju) for
his helpful advice.
17. Kinoshita, J. H.; Kador, P. F.; Datiles, M. D. J. Am. Med.
Assoc. 1981, 246, 257.
18. Azuma, M.; Inoue, E.; Oka, T. Curr. Eye Res. 1995, 14,
27.
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