Highly selective aldose reductase inhibitors. 1. 3-(Arylalkyl)-2,4,5- trioxoimidazolidine-1-acetic acids

Article abstract of DOI:10.1021/jm9508393

A series of 3-(arylalkyl)-2,4,5-trioxoimidazolidine-1-acetic acids (1) was prepared and tested for aldose reductase (AR) and aldehyde reductase (ALR) inhibitory activities. These compounds showed strong inhibitory activity against AR without significant inhibitory activity for ALR. The ratio of IC₅₀(ALR)IC₅₀(AR) was > 1000 in some compounds. On the basis of pharmacological tests such as the recovery of reduced motor nerve conduction velocity and toxicological profile, 3-(3-nitrobenzyl)-2,4,5- trioxoimidazolidine-1-acetic acid (NZ-314) was selected as the candidate for clinical development.

Full text of DOI:10.1021/jm9508393

1924
J . Med. Chem. 1996, 39, 1924-1927
High ly Selective Ald ose Red u cta se In h ibitor s. 1.
3-(Ar yla lk yl)-2,4,5-tr ioxoim id a zolid in e-1-a cetic Acid s
Akira Ishii,* Takayuki Kotani, Yasuhiro Nagaki, Yoji Shibayama, Yoshio Toyomaki, Nobuhisa Okukado,
Kazuharu Ienaga, and Kaoru Okamoto
Institute of Bio-Active Science, Nippon Zoki Pharmaceutical Company, Ltd., Kinashi, Yashiro-cho, Kato-gun 673-14, J apan
Received November 14, 1995X
A series of 3-(arylalkyl)-2,4,5-trioxoimidazolidine-1-acetic acids (1) was prepared and tested
for aldose reductase (AR) and aldehyde reductase (ALR) inhibitory activities. These compounds
showed strong inhibitory activity against AR without significant inhibitory activity for ALR.
The ratio of IC50(ALR)/IC50(AR) was >1000 in some compouds. On the basis of pharmacological
tests such as the recovery of reduced motor nerve conduction velocity and toxicological profile,
3-(3-nitrobenzyl)-2,4,5-trioxoimidazolidine-1-acetic acid (NZ-314) was selected as the candidate
for clinical development.
In tr od u ction
Recently, much attention has been paid to the aldose
reductase inhibitor (ARI) due to its therapeutic potential
for reduction of diabetic complications such as neuropa-
thy, nephropathy, retinopathy, keratopathy, angiopathy,
and cataract.1-4 Although the mechanisms through
which hyperglycemia causes tissue damage are a matter
of controversy, accumulating experimental evidences
indicate that increased aldose reductase (AR) (EC
1.1.1.21) activity initiates biochemical changes which
result in the onset of secondary diabetic complications.
AR is an enzyme in the polyol pathway that utilizes
NADPH to catalyze the reduction of aldose to the sugar
alcohol alditol. Many of aldose reductase inhibitors
have also been observed to inhibit aldehyde reductase
(ALR) (EC 1.1.1.2).5 This suggests that both AR and
ALR possess certain structural similarities capable of
binding these ARIs.6 Though it is not clear how ALR
works in diabetic patients, ALR would be one of the
important enzymes for reduction of many aldehydes and
would have functions such as counteraction, excretion
of drugs, synthesis of ascorbic acid, and metabolism of
4-hydroxylactic acid.7 AR can exist only in Mesangium
cells or renal medullas in human kidney where over 100-
fold expression of ALR can be observed.8 This means
F igu r e 1.
that ARI in kidney would be consumed by ALR before
they can react with AR unless they have different
affinity.4 To apply ARI for the treatment of diabetes
complications clinically, long term administration of a
drug is required.9 For such reasons, we must pay a lot
of attention to adverse effects, and the drug that inhibits
AR selectively is truly expected. Therefore, the selective
inhibition against AR seems to be important to avoid
F igu r e 2.
undesired adverse effects. In fact, various potential
ARIs were developed (Figure 1), but many of them were
far from ideal. Here, we describe a number of 2,4,5-
trioxoimidazolidine-1-acetic acids and their inhibitory
activities against rat lens AR and rat kidney ALR. In
particular, the unique parabanic acid derivatives 3 were
novel AR inhibitors having a desired effect in recovery
of motor nerve conduction velocity without significant
inhibitory activity against ALR (Figure 2).
Ch em istr y
All of the parabanic acid derivatives 1 in this paper
were prepared according to method A or B. Method A
was applicable to the synthesis of most 3-(arylmethyl)-
2,4,5-trioxoimidazolidine-1-acetic acid derivatives 1.
Thus, condensation of the amines 2 with urea gave the
alkylated ureas 3, which were treated with oxalyl
chloride to give 1-(arylmethyl)-2,4,5-imidazolidinetrione
4. The acetates 5 obtained by the treatment of 4 with
ethyl bromoacetate (8) were hydrolyzed in acidic condi-
tion to give the desired carboxylic acids 1 in good yields.
X Abstract published in Advance ACS Abstracts, March 15, 1996.
0022-2623/96/1839-1924$12.00/0 © 1996 American Chemical Society

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9 1925
Ta ble 1. Physical Data for Substituted Parabanic Acids 1
Sch em e 1a
A
compd
X
n
R
methoda
mp (°C)
formulab
1a
5c
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
H
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
3
4
2
2
2
2
2
2
H
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
A
A
B
A
B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
207.5-209.5 C12H10N2O5
124.5-125.5 C14H13N3O7
3-NO2
2-NO2
3-NO2
4-NO2
2-Cl
197-197.6
192-194
180-181
227-228
207-208
185-186
C12H9N3O7
C12H9N3O7
C12H9N3O7
C12H9ClN2O5
C12H9ClN2O5
C12H9ClN2O5
3-Cl
4-Cl
3,4-Cl2
2,4-Cl2
2-MeO
3-MeO
3,4-(MeO)2
2-Me
4-Me
4-Br
4-F
H
H
H
196.5-197.5 C12H8Cl2N2O5
230.5-232
185-186
189-190
163-164
198-199
192-193.5
C12H8Cl2N2O5
C13H12N2O6
C13H12N2O6
C14H14N2O7
C13H12N2O5
C13H12N2O5
200.5-201.5 C12H9BrN2O5
173-174 C12H9FN2O5
154.5-155.5 C13H12N2O5
145.5-146.5 C14H14N2O5
1s
1t
102-103
148-149
141-142
158-159
136-138
139-142
172-173
C15H16N2O5
C13H11ClN2O5
C15H16N2O7
C14H14N2O5
C13H10Cl2N2O5
C13H11N3O7
C13H11BrN2O5
2-Cl
1u
1v
1w
1x
1y
3,4-(MeO)2
4-Me
3,4-Cl2
3-NO2
4-Br
a
b
Synthetic method shown in Scheme 1. Elemental analyses
were within (0.4% of the calculated values.
Ta ble 2. Aldose Reductase Inhibitory Activity of 1 and 5
% inhib for AR
% inhib for AR
in vitroa at
in vitroa at
compd
5.0 × 10-7
M
compd
5.0 × 10-7
M
1a
5c
1b
17
31
66
75
26
32
31
56
77
68
16
44
29
1m
1n
1o
1p
1q
1r
41
30
65
9
30
16
25
37
42
42
57
36
47
1c (NZ-314)
1d
1e
1f
1g
1h
1i
1j
1k
1l
a
Reagents: (a) urea, concentrated HCl, H2O, reflux; (b)
1s
1t
(COCl)2, THF, 0 °C; (c) BrCH2CO2Et (8), KOH-EtOH or KHCO3-
acetone; (d) 2- or 4-nitrobenzyl bromide, NaH, DMF; (e) concen-
trated HCl, AcOH, reflux.
1u
1v
1w
1x
1y
In the case of 2- and 4-nitrobenzyl derivatives 1b,d ,
method B was adopted. Condensation of ethyl ure-
idoacetate (7) with oxalyl chloride gave ethyl 2,4,5-
trioxoimidazolidine-1-acetate (9), which was then treated
with 2- or 4-nitrobenzyl bromide to give the ester 5b or
5d , respectively. The acetates 5 were treated in the
same manner as described above to give the acids 1.
a
Inhibition in a partially purified rat lens aldose reductase.
the binding site of AR enzyme.12 Additionally, it was
revealed that increase in the number of methylene
groups between the aryl part and the parabanic acid
unit resulted in a significant loss of AR inhibitory
activity. Introduction of electron-withdrawing groups
onto the aromatic moiety enhanced the inhibitory activ-
ity against AR. Because of the sequence homology13-15
and the nonselectivity of inhibition for both AR and
ALR,4,16-19 it has been suggested that the inhibitor
binding sites of both enzymes are structurally similar.18
Therefore, compounds 1b,c,h ,i were assayed for their
ALR inhibitory abilities. Surprisingly, all of these
compounds showed extremely weak inhibitory activity
against ALR as shown in Table 3. For example, the
ratio of IC50(ALR)/IC50(AR) of compound 1c was >1600,20
whereas that of epalrestat was 71. Since the ratio of
IC50(ALR)/IC50(AR) of EBPC was reported to be >400021
using rat lens AR and guinea pig kidney ALR, the
Resu lts a n d Discu ssion
Inhibitory activities of the parabanic acids 1 against
rat lens AR10 and rat kidney ALR11 were evaluated in
a spectrometric assay with DL-glyceraldehyde as the
substrate and NADPH as the cofactor. In vitro AR
inhibitory activity was expressed as the inhibition of the
test compounds at 5.0 × 10-7 M concentration. ALR
inhibitory activity was also measured for the compounds
with strong AR inhibitory activity.
Structure-activity relationship studies of the para-
banic acid derivatives have suggested that acetic acid
functionality is critical to the AR inhibitory activity (5c
vs 1c), possibly participating in a key interaction with

1926 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9
Notes
Ta ble 3. Ratio of AR IC50 and ALR IC50 of Compounds 1
was dried over anhydrous Na2SO4 and passed through a short
silica gel pad. Evaporation of the solvent followed by recrys-
tallization from hexane-EtOAc gave 4c as yellow crystals
IC50 (M)
ARa
ALRb
IC50(ALR)/IC50(AR)
1
compd
1c
1c
1h
(20.3 g, 82%): mp 161-163 °C; H NMR δ 4.78 (s, 2H, CH2),
7.62-8.26 (m, 4H, Ar-H), 12.08 (s, 1H, NH); IR (KBr, cm-1
)
1.3 × 10-7
6.2 × 10-8
5.0 × 10-8
1.0 × 10-7
2.1 × 10-8
1.1 × 10-7
1.0 × 10-4
>1.0 × 10-4
>1.0 × 10-4
4.6 × 10-5
1.5 × 10-6
3.2 × 10-5
769
>1613
>2000
460
1738 (CdO), 1527 (NO2), 1336 (NO2); MS m/ z 249 (M+). Anal.
(C10H7N3O5) C, H, N.
E t h yl 3-(3-Nit r ob en zyl)-2,4,5-t r ioxoim id a zolid in e-1-
a ceta te (5c). The parabanic acid 4c (n ) 1, X ) NO2; 24.9 g,
100 mmol) and ethyl bromoacetate (8; 17 mL, 154 mmol) were
added to a solution of KOH (8.0 g, 120 mmol) in EtOH (600
mL). After being refluxed for 8 h, the mixture was allowed to
cool to 0 °C and filtered. The solid was dissolved in EtOAc
and washed with H2O and brine. The organic phase was dried
over anhydrous Na2SO4 and passed through a short silica gel
pad. After being condensed, the crude solid was recrystallized
from hexane-EtOAc to give 5c as yellow crystals (17.6 g,
53%): 1H NMR δ 1.21 (t, J ) 7.2 Hz, 3H, CH3), 4.17 (q, J )
7.2 Hz, 2H, OCH2), 4.41 (s, 2H, NCH2CO2), 4.91 (s, 2H, CH2-
Ar), 7.64-8.26 (m, 4H, Ar-H); IR (KBr, cm-1) 1740 (CdO), 1720
(CdO), 1525 (NO2), 1350 (NO2); MS m/ z 335 (M+).
Meth od B: Eth yl Ur eid oa ceta te (7). A solution of
glycine ethyl ester hydrochloride (6; 1000 g, 7.16 mol) and urea
(750 g, 12.5 mol) in water (600 mL) was refluxed for 5 h. The
reaction mixture was left standing at ambient temperature.
The solid was filtered and rinsed well with H2O. Recrystal-
lization from H2O (600 mL) gave the urea 7 as white crystals
(734 g, 70%): mp 139-140 °C; 1H NMR δ 1.19 (t, J ) 7.0 Hz,
3H, CH3), 3.73 (d, J ) 6.0 Hz, 2H, NCH2CO2), 4.08 (q, J ) 7.0
Hz, 2H, OCH2), 5.67 (s, 2H, NH2), 6.25 (t, J ) 6.0 Hz, 1H,
NH); IR (KBr, cm-1) 3475 (NH2), 3375 (NH2), 1730 (CdO), 1645
(CdO).
1i
epalrestat
EBPC
71
291
a
b
IC50 value for rat lens AR. IC50 value for rat kidney ALR.
selectivity of EBPC was tested in the same conditions
as that for compounds described above. As summarized
in Table 3, the ratio of EBPC in enzyme selectivity was
291. From these results, it was found that the para-
banic acid moiety was the essential unit for AR selective
inhibitory activity.
Con clu sion
It was found that the parabanic acid moiety was
essential for AR selectivity and strong AR inhibitory
activity. In particular, 1c (NZ-314) showed strong
inhibitory activity against AR with extremely weak ALR
inhibition in vitro. These compounds are the first
examples of ARIs that have high AR selectivity. Studies
on the effect of the aryl part for enzyme selectivity are
in progress.
Eth yl 2,4,5-Tr ioxoim id a zolid in e-1-a ceta te (9). To a
suspension of the urea 7 (730 g, 5.0 mol) in THF (100 mL)
was added oxalyl chloride (515 mL, 6.0 mol) dropwise at 0 °C.
After vigorous stirring for 4 h at room temperature, the
precipitate was collected by filtration. The filtrate was
concentrated, and the residual solid was combined with the
precipitate obtained above. The solid was dissolved in MeOH
and filtered to remove the insoluble material. The filtrate was
concentrated, and the residue was recrystallized from EtOAc
Exp er im en ta l Section
Melting points (mp) were measured by a Yamato MP-21
melting point apparatus and are uncorrected. Proton nuclear
magnetic resonance (1H NMR) spectra were determined in
dimethyl sulfoxide-d6 on a Bruker AM-400 (400 MHz) spec-
trometer. Chemical shifts are reported in δ value from
internal tetramethylsilane. Splitting patterns are designated
as follows: s, singlet; d, doublet; t, triplet; q, quartet; br s,
broad singlet; and m, multiplet. Coupling constants are
reported in hertz (Hz). Infrared (IR) spectra were recorded
with a Hitachi 260-30 instrument. Mass spectra (MS) were
taken on a Hitachi M-80B mass spectrometer. Elemental
analyses (C, H, N) were carried out on a Perkin-Elmer 240C
element analyzer. Thin-layer chromatography (TLC) analyses
and chromatographic separations were performed with silica
gel 60 F254 plates (Merck Art. 5715) and silica gel 60 (Merck
Art. 7734; 70-230 mesh), respectively. Visualization was
accomplished with UV light and/or 10% phosphomolybdic acid
in ethanol. Unless otherwise noted, all commercially available
materials were used without further purification.
Gen er al P r ocedu r e for th e P r epar ation of Eth yl 3-(Ar y-
la lk yl)-2,4,5-tr ioxoim id a zolid in e-1-a ceta tes 5 (Sch em e 1).
Meth od A: 1-(3-Nitr oben zyl)u r ea (3c, n ) 1, X ) 3-NO2).
To a solution of 3-nitrobenzylamine hydrochloride (2c, n ) 1,
X ) 3-NO2; 47.2 g, 0.25 mol) and urea (60.0 g, 1.0 mol) in H2O
(100 mL) was added concentrated HCl (2 mL, 32 mmol), and
the mixture was refluxed for 3 h. The reaction mixture was
crystallized by standing at room temperature. The precipitate
was filtered and rinsed well with H2O. Recrystallization from
EtOH gave 3c as white crystals (47.2 g, 97%): mp 186-187
°C; 1H NMR δ 4.30 (d, J ) 6.2 Hz, 2H, CH2), 5.66 (s, 2H, NH2),
6.22 (t, J ) 6.2 Hz, 1H, NH), 7.62 (dd, J ) 8.8, 7.6 Hz, 1H,
Ar-H), 7.71 (d, J ) 7.6 Hz, 1H, Ar-H), 8.09 (d, J ) 8.8 Hz, 1H,
Ar-H), 8.10 (s, 1H, Ar-H); IR (KBr, cm-1) 3469 (NH2), 3319
(NH2), 1652 (CdO), 1585 (NO2), 1346 (NO2).
1
to give 9 as white crystals (850 g, 85%): mp 154-156 °C; H
NMR δ 1.21 (t, J ) 7.2 Hz, 3H, CH3), 4.16 (q, J ) 7.2 Hz, 2H,
OCH2), 4.34 (s, 2H, NCH2CO2), 12.40 (s, 1H, NH); IR (KBr,
cm-1) 1740 (CdO).
E t h yl 3-(2-Nit r ob en zyl)-2,4,5-t r ioxoim id a zolid in e-1-
a ceta te (5b). A solution of the parabanic acid 9 (10.0 g, 50
mmol) in DMF (50 mL) was added to a suspension of NaH (60
wt % in oil, 2.00 g, 50 mmol) in DMF (50 mL) with the
temperature maintained below 0 °C over a period of 30 min.
After stirring for 1 h, a solution of 2-nitrobenzyl bromide (10.8
g, 50 mmol) in DMF (50 mL) was slowly added at 0 °C. The
mixture was stirred for a further 2 h at 0 °C. The resulting
mixture was poured into ice-H2O containing concentrated HCl
(1 mL) to give a solid, which was filtered and washed well with
H2O and hexane. Recrystallization from EtOH gave 5b (12.5
g, 75%) as white crystals: mp 158.5-159.5 °C; 1H NMR δ 1.21
(t, J ) 7.1 Hz, 3H, CH3), 4.17 (q, J ) 7.0 Hz, 2H, OCH2), 4.43
(s, 2H, NCH2CO2), 5.11 (s, 2H, CH2Ar), 7.61 (ddd, J ) 8.2, 7.8,
1.3 Hz, 1H, Ar-H), 7.64 (dd, J ) 7.8, 1.3 Hz, 1H, Ar-H), 7.74
(ddd, J ) 7.8, 7.8, 1.2 Hz, 1H, Ar-H), 8.13 (dd, J ) 8.2, 1.2 Hz,
1H, Ar-H); IR (KBr, cm-1) 1738 (CdO), 1527 (NO2), 1336 (NO2);
MS m/ z 335 (M+). Anal. (C14H13N3O7) C, H, N.
Hyd r olysis of Ester s 5: 3-(3-Nitr oben zyl)-2,4,5-tr iox-
oim id a zolid in e-1-a cetic Acid (1c). A mixture of ethyl 3-(3-
nitrobenzyl)-2,4,5-trioxoimidazolidine-1-acetate (5c; 3.70 g,
11.0 mol), AcOH (10 mL), and concentrated HCl (5 mL) was
refluxed for 2.5 h. The reaction mixture was condensed under
reduced pressure to give a residue, which was refluxed again
with AcOH (10 mL) and concentrated HCl (5 mL) for another
2 h. The solid obtained by condensation was dissolved in
EtOAc, washed with H2O, and extracted with 10% aqueous
Na2CO3. The aqueous layer was washed with EtOAc and
acidified with concentrated HCl. The precipitated solid
was extracted with EtOAc, washed with H2O and brine, and
dried over anhydrous Na2SO4. Concentration followed by
1-(3-Nitr oben zyl)im id a zolid in e-2,4,5-tr ion e (4c, n ) 1,
X ) 3-NO2). To a suspension of 1-(3-nitrobenzyl)urea (3c, n
) 1, X ) 3-NO2; 19.5 g, 100 mmol) in THF (300 mL) was added
oxalyl chloride (10.2 mL, 120 mmol) dropwise at 0 °C. The
mixture was warmed to room temperature and stirred vigor-
ously for 3 h. After removal of the precipitate by filtration,
the filtrate was concentrated. The residual solid was dissolved
in EtOAc and washed with H2O and brine. The organic layer

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9 1927
crystallization from Et2O gave a crude solid, which was
recrystallized from hexane-EtOAc to give 1c as yellow crystals
(2.30 g, 68%): 1H NMR δ 4.30 (s, 2H, NCH2CO2), 4.91 (s, 2H,
CH2Ar), 7.65-8.25 (m, 4H, Ar-H), 13.42 (br s, 1H, COOH); IR
(KBr, cm-1) 1740 (CdO), 1720 (CdO), 1525 (NO2), 1350 (NO2);
MS m/ z 307 (M+).
Su p p or tin g In for m a tion Ava ila ble: Melting point, 1H
NMR, IR, and MS data for compounds 1 and 3-5 (14 pages).
Ordering information is given on any current masthead page.
Refer en ces
Biologica l Meth od s: En zym e P r ep a r a tion . Rat lens AR
was prepared according to the procedure developed by Kador
with some modifications.10 Lens from male Wister rats were
homogenized in 3 vol of 10 mM phosphate buffer (pH 7.0)
containing 5 mM 2-mercaptoethanol and 1 mM EDTA at 0-4
°C with a tissue homogenizer set at a speed of 5-6 (Polytron,
Kinematica Gmbh, CH-6010). The homogenate was centri-
fuged at 18000g for 20 min. Ammonium sulfate was added to
the supernatant fraction to form a 30% saturated solution. This
solution was stirred for 1 h, allowed to stand for 20 min, and
then centrifuged at 18000g for 20 min. The recovered super-
natant fraction was then sequentially fractionated with 80%
saturated ammonium sulfate. The precipitate recovered from
the 80% saturated ammonium sulfate solution was dissolved
in the phosphate buffer described above and dialyzed for 24 h
against 10 L of the same buffer changed twice. The dialyzed
material was separated into 1.0-mL aliquots and stored at -40
°C.
(1) Kador, P. F. The Role of Aldose Reductase in the Development
of Diabetic Complications. Med. Res. Rev. 1988, 8, 325-352.
(2) Kador, P. F.; Kinoshita, J . H.; Sharpless, N. E. Aldose Reductase
Inhibitors: A Potential New Class of Agents for the Pharmaco-
logical Control of Certain Diabetic Complications. J . Med. Chem.
1985, 28, 841-849.
(3) Kador, P. F.; Robinson, W. G.; Kinoshita, J . H. The Pharmacol-
ogy of Aldose Reductase Inhibitors. Annu. Rev. Pharmacol.
Toxicol. 1985, 25, 691-714.
(4) Tanimoto, T.; Nishimura, C. Biochemistry and Clinical Signifi-
cance of Aldose Reductase. Peripheral Nerve 1993, 4, 149-158.
(5) Srivastava, S. K.; Petrash, J . M.; Sadana, I. J .; Ansari, N. H.;
Partridge, C. A. Susceptibility of Aldehyde and Aldose Reduc-
tases of Human Tissues to Aldose Reductase Inhibitors. Curr.
Eye Res. 1982, 2, 407-410.
(6) Poulsom, R. Inhibition of Hexonate Dehydrogenase and Aldose
Reductase from Bovine Retina by Sorbinil, Statil, M79175 and
Valproate. Biochem. Pharmacol. 1986, 35, 2955-2959.
(7) Tanimoto, T.; Nishiyama, C. Molecularbiology of Aldose Reduc-
tase. Exp. Med. 1991, 9, 541-547.
Rat kidney ALR was prepared with the procedure developed
by Takahashi with some modifications.11 Kidneys from male
Wister rats were homogenized with 10 mM phosphate buffer
(pH 7.4) containing 0.25 M sucrose and 1 mM EDTA in a
homogenizer (Polytron, Kinematica Gmbh, CH-6010). The
homogenate was centrifuged at 10000g for 20 min, and the
supernatant (crude extract) was collected. The crude extract
was subjected to fractionation with ammonium sulfate between
40% and 70% saturation. The precipitate was collected by
centrifugation at 10000g for 10 min, dissolved in a minimal
volume of 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA
and 1 mM 2-mercaptoethanol, and dialyzed for 24 h against
10 L of the same buffer changed twice. The dialyzed material
was applied to a column of DEAE-cellulose (DE-52; Whatman)
equilibrated with the same Tris buffer. The column was
developed with a linear gradient of 0 and 0.3 M NaCl in the
same buffer. The major peak was separated from the minor
peak and concentrated to 5 mL by Amicon ultrafiltration with
a PM-10 membrane. This concentrated fraction was applied
to a column of Sephadex G-100 equilibrated with a same Tris
buffer, and the major peak was collected. This collected
fraction was applied to a column of Blue Sepharose CL-6B,
equilibrated with the same Tris buffer. The column was
washed with the same buffer and then eluted with the Tris
buffer containing 5 mM NADPH. The fractions with ALR
activity were collected after dialysis of this elute and stored
at -40 °C.
En zym e Assa y. Both reductases activities were assayed
spectrophotometrically on a Hitachi U-3300 spectrophotometer
by measuring the decrease in absorption of NADPH at 340
nm. AR activity was determined by the procedure developed
by Kador10 with some modifications and assayed in a reaction
mixture containing 100 mM phosphate buffer (pH 6.3), 0.3 M
ammonium sulfate, 1 mM EDTA, 0.2 mM NADPH, and 10 mM
DL-glyceraldehyde in a final volume of 2.5 mL.
ALR activity was determined by the procedure developed
by Takahashi11 with some modifications and assayed in a
reaction mixture containing 100 mM phosphate buffer (pH
6.0), 0.2 mM NADPH, and 8 mM n-octyl aldehyde in a final
volume of 2.5 mL. The water-insoluble substrate such as
n-octyl aldehyde was dispersed in 3-fold molar of Triton X-100.
The test substrates were dissolved in propylene glycol.
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