Synthesis of potential aldose reductase inhibitors based on minimal pharmacophore requirements

Article abstract of DOI:10.1211/0022357011776180

A series of 17 compounds were synthesized based on the premise that the minimal pharmacophore for aldose reductase inhibition requires the presence of both an aryl group and polar group connected by a linking structure. Three groups of compounds were synt

Full text of DOI:10.1211/0022357011776180

Journal of
Pharmacy and Pharmacology
JPP 2001, 53: 831–839
# 2001 The Authors
Received August 29, 2000
Accepted February 26, 2001
ISSN 0022-3573
Synthesis of potential aldose reductase inhibitors
based on minimal pharmacophore requirements
Martin Schlitzer, Labaniel Rodriguez and Peter F. Kador
Abstract
A series of 17 compounds were synthesized based on the premise that the minimal
pharmacophore for aldose reductase inhibition requires the presence of both an aryl group and
polar group connected by a linking structure. Three groups of compounds were synthesized,
the first possessing an aniline-4-(2h-6h-methylbenzothiazole) or 2-aminobenzothiazole group as
the aryl group, the second possessing a 2-naphthyl as the aryl group and the third possessing
either a 4-(2-phenylthiazole) or 2-(5-2h-nitrophenylfuran) as the aryl group. In all three of these
groups the carboxylate or its methyl ester are linked to the aryl group through various lengths
of methylene carbons and amide or cinnamide groups. Optimal activity was observed when the
carboxylic group was separated from the aryl group by a linking structure of five atoms in
length. Both a double bond and an amide moiety are well tolerated in the linking structure.
Introduction
Although progress has been made in the treatment of diabetes mellitus with insulin
or oral anti-hyperglycaemics, this current therapy cannot prevent the development
of long-term, diabetes-related complications. These complications, which include
neuropathy, nephropathy and retinopathy, not only considerably diminish the
quality of life of diabetics but also reduce their lifespan. In tissues possessing
insulin-independent glucose uptake, elevated blood-glucose concentrations lead to
an increased flow of glucose into the sorbitol pathway. In the sorbitol pathway
glucose is reduced to sorbitol by the enzyme aldose reductase using NADPH as
hydride donor. Under diabetic conditions the increased flow of glucose through the
sorbitol pathway results in elevated intracellular sorbitol concentrations in tissues
developing diabetic complications and altered redox changes associated with the
increased utilization of nucleotides. This intracellular increase of sorbitol can lead
to osmotic effects and eventually the loss of membrane integrity. Both membrane
permeability changes and redox changes have been linked to the molecular cause of
the clinically manifest symptoms (Kador et al 1985; Kador 1988; Tomlinson et al
1992; Yabe-Nishimura 1998). Therefore, inhibition of aldose reductase has been
considered an important target for drug development. The efforts of several
industrial and academic research groups have resulted in the identification of
numerous structurally diverse aldose reductase inhibitors (Larson et al 1988;
Sarges 1989), which include the carboxylic acids and hydantoins summarized in
Figure 1.
Institut fur Pharmazeutische
Chemie, Philipps-Universitat
Marburg, Marbacher Weg 6, D-
35032 Marburg, Germany
Martin Schlitzer
National Eye Institute, National
Institutes of Health, Bethesda,
MD 20892, USA
Labaniel Rodriguez, Peter F.
Kador
Correspondence: M. Schlitzer,
Institut fur Pharmazeutische
Chemie, Philipps-Universitat
Marburg, Marbacher Weg 6,
D-35032 Marburg, Germany
Acknowledgement: We thank
Mr Oβwald and Mr Weiser for
technical assistance.
831

832
Martin Schlitzer et al
COOH
O
O
HOOC
N
N
COOH
O
S
N
S
S
O
CF3
Tolrestat
Alrestatin
Epalrestat
COOH
COOH
Br
CF3
N
N
S
N
N
N
O
O
Ponalrestat
F
Zopolrestat
O
H
N
O
H
N
HN
HN
O
O
F
F
O
Sorbinil
Figure 1 Representative examples of known aldose reductase inhibitors.
AI1567
Material and Methods
O
NO2
OH
O
N
H
Chemistry
X
O
1H NMR and 13C NMR spectra were recorded on a
Jeol JMN-GX-400 and a Jeol JMN-LA-500 spectro-
meter. Mass spectra were obtained with a Vacuum
Generators VG 7070 H using a Vector 1 data acquisition
system from Teknivent or an AutoSpec mass spectro-
meter from Micromass. High resolution mass spectro-
scopy was carried out as an additional structural proof
Linker
R-Aryl
Figure 2 Pharmacophore template for the design of aldose re-
ductase inhibitors exemplified with compound 16.
+
except for those compounds in which the M signal was
Examination of a number of these structurally diverse either too weak or not recordable. IR spectra were
inhibitors has resulted in the hypothesis that the mini- recorded on a Nicolet 510P FTIR spectrometer. Micro-
mum pharmacophore requirements for an aldose re- analyses were obtained from a CH analyser according
ductase inhibitor consist of a hydrophobic aromatic to Dr Salzer from Labormatic and from a Hewlett
structure (R-Aryl) and a polar or ionic group (X) linked Packard CHN-analyser type 185. Column chroma-
by a flexible chain of suitable length (linker) (Kador et al tography was carried out using silica gel 60 (0.062–
1985; Lee et al 1994, 1998). To test this basic premise a 0.200 mm) from Merck.
number of compounds were prepared according to the
suggested general lead structure the basic features of resolution mass spectroscopy; EI, electron impact ion-
which are outlined in Figure 2. ization.
Abbreviations: MS, mass spectroscopy; HRMS, high

833
Synthesis of aldose reductase inhibitors
Method 1. Preparation of amides of dicarbonic
acid methyl esters 1, 2, and 5
aromatic), 7.29 (m, 1H, aromatic), 3.61 (s, 3H, OCH3),
2.77 (t, J l 7 Hz, 2H, CH2-CH2), 2.67 (t, J l 7 Hz, 2H,
CH2-CH2); 13H NMR (d6-DMSO): δ 172.4, 170.9 (2i
CO), 157.7, 148.4, 131.4, 125.9, 123.3, 121.5, 120.4
(aromatic C), 51.3 (OCH3), 30.0, 28.0 (CH2-CH2); MS
An equimolar mixture of an aromatic amine and ma-
lonic acid methyl ester chloride or succinic acid methyl
esterchloridewasrefluxedindrytetrahydrofuran(THF)
for 2 h. The reaction mixture was evaporated to dryness
and the resulting residue was purified by recrystal-
lization.
+
(EI): m\z (%) l 264 (8) [M ], 177 (100), 150 (79);
HRMS calculated for C12H12N2O3S: 264.0569; found
264.0571;
elemental
analysis
calculated
for
C12H12N2O3S: C 54.53, H 4.58, N 10.60; found C 54.92,
H 4.61, N 10.29.
N-[4-(6-Methylbenzothiazole-2-yl)-phenyl]-malonamic
acid methyl ester (1)
Method 2. Preparation of amides 3, 4, 6 and 8
from amines and succinic anhydride or glutaric
anhydride
Recrystallization from dioxane\methanol. Yield 0.52 g
(76%), white crystals; mp 187mC. IR: ν l 3020, 1750,
1
1
−
1685, 1590, 1525 cm ; H NMR (d6-DMSO): δ 10.69
(s, 1H, NH), 8.36–8.14 (m, 2H, aromatic), 7.90–7.88 (m,
2H, aromatic), 7.81–7.79 (m, 2H, aromatic), 7.34–7.32
(m, 1H, aromatic), 3.68 (s, 3H, OCH3), 3.57 (s, 2H,
CH2), 2.45 (s, 3H, Ar-CH3); 13H NMR (d6-DMSO):
δ l 167.8, 165.6, 164.3 (2iCO and benzothiazole-C-2),
151.6, 141.3, 134.9, 134.3, 127.9, 127.7, 122.0, 121.6,
119.3 (aromatic), 51.8 (OCH3), 43.4 (CH2), 20.9 (Ar-
Anequimolarmixtureofanaromaticamineandsuccinic
anhydride or glutaric anhydride was refluxed in dry
THF for 2 h. The reaction mixture was evaporated to
dryness and the resulting residue was purified by re-
crystallization.
N-[4-(6-Methylbenzothiazole-2-yl)-phenyl]-succinamic
acid (3)
Recrystallization from dioxane. Yield 0.384 g (64%),
+
CH3); MS: m\z (%) l 340 (100) [M ], 266 (33), 240
(87); HRMS calculated for C18H16N2O3S: 340.0882;
found 340.0892; elemental analysis calculated for
C18H16N2O3S: C 63.51, H 4.74, N 8.23; found C 63.84, H
5.09, N 7.98.
white crystals; mp 228mC; IR: ν l 3330, 1735, 1690,
1
1
−
1590 cm ; H NMR (d6-DMSO): δ 10.23 (s, 1H, NH),
8.01–7.99 (m, 2H, aromatic), 7.89–7.87 (m, 2H, aro-
matic), 7.78–7.77 (m, 2H, aromatic), 7.34–7.32 (m, 1H,
aromatic), 2.61 (m, 2H, CH2-CH2), 2.54 (m, 2H, CH2-
CH2), 2.45 (s, 3H, Ar-CH3); 13H NMR (d6-DMSO):
δ 173.5, 170.4, 165.7 (2iCO and benzothiazole-C-2),
151.7, 141.7, 134.8, 134.3, 127.8, 127.6, 127.4, 121.9,
121.5, 119.0 (aromatic), 31.1, 28.6 (CH2-CH2), 20.9 (Ar-
N-[4-(6-Methylbenzothiazole-2-yl)-phenyl]-succinamic
acid methyl ester (2)
Recrystallization from dioxane\methanol. Yield 0.62 g
(87%), white crystals; mp 209mC; IR: ν l 3330, 1735,
+
CH3); MS (EI): m\z (%) l 340 (3) [M ], 322 (100), 240
1
1
−
1690, 1590 cm ; H NMR (d6-DMSO): δ 10.32 (s, 1H,
NH), 8.01–7.98 (m, 2H, aromatic), 7.89–7.87 (m, 2H,
aromatic), 7.78–7.76 (m, 2H , aromatic), 7.34–7.32 (m,
1H, aromatic), 3.61 (s, 3H, OCH3), 2.70–2.60 (m, 4H,
CH2-CH2), 2.45 (s, 3H, Ar-CH3); 13H NMR (d6-
DMSO): δ 172.6, 172.0, 170.1 (2iCO and benzo-
thiazole-C-2), 151.0, 141.0, 134.8, 134.3, 127.8, 127.6,
127.4, 122.0, 121.6, 119.0 (aromatic), 51.2 (OCH3), 30.9,
28.3 (CH2-CH2), 20.9 (Ar-CH3); MS: m\z (%) l 354 (2)
(45), 97 (59); elemental analysis calculated for
C18H16N2O3S: C 63.51, H 4.74, N 8.23; found C 63.87, H
4.54, N 8.58.
N-[4-(6-Methylbenzothiazole-2-yl)-phenyl]-glutaramic
acid (4)
Recrystallization from dioxane. Yield 0.560 g (69%),
white crystals; mp 231mC; IR: ν l 3310, 1705, 1665,
1
1
−
1525 cm ; H NMR (d6-DMSO): δ l 10.20 (s, 1H,
NH), 8.01–7.98 (m, 2H, aromatic), 7.89–7.87 (m, 2H,
aromatic), 7.78–7.77 (m, 2H , aromatic), 7.34–7.32 (m,
1H, aromatic), 2.45 (s, 3H, Ar-CH3), 2.41 (m, 2H, CH2-
CH2-CH2), 2.29 (m, 2H, CH2-CH2-CH2), 1.84 (m, 2H,
CH2-CH2-CH2); 13H NMR (d6-DMSO): δ l 173.9,
+
[M ], 323 (100); elemental analysis calculated for: C
64.39, H 5.12, N 7.90; found C 64.19, H 5.19, N 7.86.
N-Benzothiazole-2-yl-succinamic acid methyl ester (5)
Recrystallization from THF. Yield 0.35 g (70%), white 170.1, 165.7 (2iCO and benzothiazole-C-2), 151.7,
crystals; mp 179mC; IR: ν l 3095, 1735, 1720, 141.8, 134.8, 134.3, 127.8, 127.6, 127.4, 121.9, 121.5,
1
1560 cm ; H NMR (d6-DMSO): δ 7.96–7.93 (m, 1H, 119.1 (aromatic), 35.4, 32.9, 20.2 (CH2-CH2-CH2), 20.8
1
−
+
aromatic), 7.74–7.72 (m, 1H, aromatic), 7.42 (m, 1H, (Ar-CH3); MS: m\z (%) l 354 (45) [M ], 336 (63), 240

834
Martin Schlitzer et al
(100); HRMS calculated for C19H18N2O3S: 354.1038; C13H14N2O3S: C 56.10, H 5.07, N 10.06; found C 55.81,
found 354.1028; elemental analysis calculated for H 5.34, N 9.89.
C19H18N2O3S: C 64.39, H 5.12, N 7.90; found C 64.62, H
5.16, N 7.75.
Method 3. Preparation of N-acyl amino acids
N-Benzothiazole-2-yl-succinamic acid (6)
To 1 mmol amino acid dissolved in 4 mL water, 3 mmol
Recrystallization from dioxane\ethanol\pentane. Yield KHCO3 was added and the mixture was cooled to 0mC.
0.6 g (80%), white crystals; mp 250mC; IR: ν l 3185, The appropriate acid chloride (1 mmol) in 4 mL acetone
1
1
−
1735, 1690, 1610, 1565 cm ; H NMR (d6-DMSO): was added and the mixture was stirred for 1 h at 0mC
δ l 12.26 (s, 1H, COOH), 7.95–7.93 (m, 1H, aromatic), and then for an additional 1 h at room temperature.
7.73–7.71 (m, 1H, aromatic), 7.42 (m, 1 H, aromatic), After removal of the acetone on a rotary evaporator, the
7.31 (m, 1H, aromatic), 2.74 (t, J l 6 Hz, 2H, CH2- reaction mixture was cooled and acidified to pH 1 with
CH2), 2.59 (t, J l 6 Hz, 2H, CH2-CH2); 13H NMR (d6- concentrated hydrochloric acid. The resulting solid was
DMSO): δ 173.3, 171.2 (2iCO), 157.7, 148.5, 131.4, collected by suction and washed with water. The dried
125.9, 123.3, 121.5, 120.3 (aromatic), 30.2, 28.3 (CH2- product was purified by recrystallization.
+
CH2); MS: m\z (%) l 250 (9) [M ], 232 (91), 177 (71),
150 (100); HRMS calculated for C11H10N2O3S: N-2-Naphthylacetyl-β-alanine (9)
250.0412; found 250.0406; elemental analysis calculated Recrystallization from toluene\dioxane. Yield 0.50 g
for C11H10N2O3S: C 52.79, H 4.03, N 11.19; found C (97%), white crystals; mp 232mC; IR: ν l 3245, 3070,
1
1
−
52.79, H 4.01, N 10.82.
1715, 1630, 1565 cm ; H NMR (d6-DMSO): δ 12.10
(s, 1H, COOH), 8.13 (s, 1H, NH), 7.87–7.82 (m, 3H,
aromatic), 7.74 (s, 1H, aromatic), 7.51–7.40 (m, 3H,
N-Benzothiazole-2-yl-glutaramic acid (8)
Recrystallization from dioxane\ethanol. Yield 1.04 g aromatic), 3.58 (s, 2H, Ar-CH2-CO), 3.28 (q, J l 7 Hz,
(84%), white crystals; mp 232mC; IR: ν l 2960, 1690, 2H, β-ala-β-H), 2.40 (q, J l 7 Hz, 2H, β-ala-α-H); 13
1605, 1555 cm ; H NMR (d6-DMSO): δ 7.96–7.94 NMR (d6-DMSO): δ 172.7, 170.0 (2iCO), 134.0,
(m, 1H, aromatic), 7.73–7.71 (m, 1H, aromatic), 7.42 132.9, 131.7, 127.4, 127.3, 127.2, 125.9, 125.3 (aromatic),
(m, 1H, aromatic), 7.29 (m, 1H, aromatic), 2.54 (m, 2H, 42.3 (β-ala-β-C), 34.9 (β-ala-α-C) 33.8 (Ar-CH2-CO);
H
1
1
−
+
CH2-CH2-CH2), 2.29 (m, 2H, CH2-CH2-CH2), 1.86 (m, MS: m\z (%) l 257 (7) [M ], 141 (33), 59 (100);
2H, CH2-CH2-CH2); 13H NMR (d6-DMSO): δ 173.8, elemental analysis calculated for C15H15NO3 : C 70.02,
171.7 (2iCO), 157.7, 148.5, 131.4, 125.9, 123.3, 121.5, H 5.88, N 5.44; found C 69.95, H 5.82, N 5.40.
120.3 (aromatic), 34.2, 32.8, 19.8 (CH2-CH2-CH2); MS:
m\z (%) l 190 (65), 121 (100); elemental analysis cal- N-3-(2-Phenyl-thiazole-4-yl)-acryloyl-glycine (12)
culated for C12H12N2O3S: C 54.53, H 4.58, N 10.60; Recrystallization from toluene\dioxane. Yield 0.33 g
found C 54.63, H 4.44, N 10.50.
(57%), white crystals; mp 169mC; IR: ν l 3320, 1745,
1735, 1660, 1625 cm ; H-NMR (d6-DMSO): δ 12.52
(s, 1H, COOH), 8.55 (s, 1H, NH), 7.89 (m, 3H, aro-
1
1
−
N-Benzothiazole-2-yl-glutaramic acid methyl ester (7)
Compound 8 (0.925 g; 3.5 mmol) was dissolved in matic), 7.54–7.52 (m, 3H, aromatic), 7.47 (d, J l 15 Hz,
50 mL methanol. After addition of 0.7 mL thionyl chlo- 1H, vinylic), 7.00 (d, J l 15 Hz, 1H, vinylic), 3.90 (d, J
ride the mixture was heated to reflux for 2 h. After l 6 Hz, 2H, gly-α-H); 13H NMR (d6-DMSO): δ 171.1,
concentration and cooling the product precipitated as 167.5 (2 i CO), 165.3, 152.5, 132.7, 131.8, 130.5, 129.2,
white crystals. Yield 0.61 g (63%), white crystals; mp 126.3, 123.7, 121.9 (aromatic, vinylic), 40.8 (gly-α-C);
1;
142mC; IR: ν l 3100, 3045, 1725, 1715, 1565 cm 1H MS: m\z (%) l 288 (30) [M ], 215 (56), 214 (100), 187
NMR (d6-DMSO): δ 7.96–7.93 (m, 1H, aromatic), (40); HRMS calculated for C14H12N2O3S: 288.0569;
7.73–7.71 (m, 1H, aromatic), 7.42 (m, 1H , aromatic), found 288.0569; elemental analysis calculated for
7.29 (m, 1H, aromatic), 3.59 (s, 3H, OCH3), 2.55 (m, 2H, C14H12N2O3S: C 58.32, H 4.20, N 9.72; found C 58.51, H
CH2-CH2-CH2), 2.38 (m, 2 H, CH2-CH2-CH2), 1.89 (m, 4.50, N 9.36.
−
+
2H, CH2-CH2-CH2); 13H NMR (d6-DMSO): δ l 172.7,
171.5 (2iCO), 157.7, 148.4, 131.3, 125.8, 123.2, 121.4, N-3-(2-Phenyl-thiazole-4-yl)-acryloyl-β-alanine (13)
120.3 (aromatic), 51.1 (OCH3), 34.0, 32.4, 19.7 (CH2- Recrystallization from toluene\dioxane. Yield 0.41 g
+
CH2-CH2); MS: m\z (%) l 278 (47) [M ], 150 (100); (68%), white crystals; mp 148mC; IR: ν l 3315, 2960,
1
HRMS calculated for C13H14N2O3S: 278.0725; found 1715, 1650, 1600 cm ; H NMR (d6-DMSO): δ 12.18
1
−
278.0729;
elemental
analysis
calculated
for (s, 1H, COOH), 8.30 (t, J l 5 Hz, 1H, NH), 7.98–7.95

835
Synthesis of aldose reductase inhibitors
1
1
−
(m, 3H, aromatic), 7.56–7.52 (m, 3H, aromatic), 7.44 (d, 3030, 1715, 1655, 1625, 1530, 1210 cm ; H NMR (d6-
J l 15 Hz, 1H, vinylic), 6.95 (d, J l 15 Hz, vinylic), 3.39 DMSO): δ 12.70 (s, 1H, COOH), 8.59 (d, J l 8 Hz, 1H,
(q, J l 7 Hz, 2H, β-ala-β-H), 2.50 (q, J l 7 Hz, 2H, β- NH), 7.91 (m, 2H, aromatic), 7.76 (m, 1H, aromatic),
ala-α-H); 13H NMR (d6-DMSO): δ 172.6, 167.4 (2i 7.62 (m, 1H, aromatic), 7.30–7.17 (m, 6H, aromatic,
CO), 165.9, 152.5, 132.6, 131.1, 130.4, 129.1, 126.2, vinylic), 7.01 (d, J l 3 Hz, 1H, furylic), 6.92 (d, J l 3
124.1, 121.6 (aromatic, vinylic), 34.9, 33.7 (ala-C); MS: Hz, 1H, furylic), 6.44 (d, J l 16 Hz, 1H, vinylic), 4.55
+
m\z (%) l 302 (52) [M ], 215 (56), 214 (100), 187 (57); (d, J l 6 Hz, 1H, phe-α-H), 3.12 (m, 1H, phe-β-H), 2.92
HRMS calculated for C15H14N2O3S: 302.0725; found (m, 1H, phe-β-H); 13H NMR (d6-DMSO): δ l 172.9,
302.0711;
elemental
analysis
calculated
for 164.4 (2iCO), 152.1, 149.1, 146.9, 137.6, 132.5, 129.6,
C15H14N2O3S: C 59.59, H 4.67, N 9.27; found C 59.16, H 128.9, 128.1, 126.3, 125.8, 124.0, 122.2, 119.9, 115.9,
5.03, N 9.22.
112.3 (aromatic, vinylic), 53.7 (phe-α-C), 36.7 (phe-β-
+
C); MS: m\z (%) l 406 (37) [M ], 297 (44), 242 (100),
212 (54); HRMS calculated for C22H18N2O6 : 406.1165;
found 406.1171; elemental analysis calculated for
C22H18N2O6 : C 65.02, H 4.46, N 6.89; found C 65.18, H
4.55, N 6.82.
N-3-(2-Phenyl-thiazol-4-yl)-acryloyl-alanine (14)
Recrystallization from toluene\dioxane. Yield 0.55 g
(91%), white crystals; mp 159mC; IR: ν l 3300, 3095,
1
1
−
1715, 1665, 1630 cm ; H NMR (d6-DMSO): δ 12.45
(s, 1H, COOH), 8.52 (d, J l 7 Hz, 1H, NH), 8.01–7.96
(m, 3H, aromatic), 7.53 (m, 3H, aromatic), 7.46 (d, J l
15 Hz, 1H, vinylic), 7.01 (d, J l 15 Hz, vinylic), 4.35 (m,
1H, ala-α-H), 2.50 (d, J l 8 Hz, 3H, ala-β-H); 13H
NMR (d6-DMSO): δ 173.9, 167.4 (2iCO), 164.6, 152.5,
132.6, 130.6, 129.2, 126.3, 123.8, 121.8 (aromatic, vin-
ylic), 47.6 (ala-α-C), 17.1 (ala-β-C); MS: m\z (%) l
Method 4. Preparation of N-acyl amino acid
methyl esters
The appropriate carboxylic acid was dissolved in dry
dimethylformamide (DMF) in a flame-dried flask under
an Ar atmosphere. After addition of 3 mmol N-methyl-
morpholine the solution was cooled to k15mC and
1.3 mmol isobutyl chloroformate was added. After
5 min, a solution containing 1 mmol of the amino acid
ester hydrochloride and 1 mmol of N-methylmorpho-
line in dry DMF was added. The mixture was left to
warm to room temperature overnight and then poured
into brine (400–800 mL). The aqueous mixture was
extracted with ethyl acetate (3i100 mL) and the com-
bined organic extracts were washed successively with
0.67  citric acid, sat. NaHCO3 solution and NaCl
solution and dried with MgSO4. The residue obtained
after filtration and removal of the solvent was purified
as described below.
+
302 (38) [M ], 215 (83), 214 (100), 187 (80); HRMS
calculated for C15H14N2O3S: 302.0725; found 302.0714;
elemental analysis calculated for C15H14N2O3S: C 59.59,
H 4.67, N 9.27; found C 59.58, H 4.52, N 9.53.
N-3-[5-(2-Nitrophenyl)-furan-2-yl]-acryloyl-glycine (16)
Recrystallization from toluene\dioxane. Yield 0.20 g
(42%), yellow crystals; mp 202mC; IR: ν l 3390, 3085,
1
1
−
2940, 1725, 1655, 1625, 1530, 1240 cm ; H NMR (d6-
DMSO): δ l 12.53 (s, 1H, COOH), 8.57 (t, J l 7 Hz,
1H, NH), 7.91 (m, 2H, aromatic), 7.75 (m, 1H, aro-
matic), 7.61 (m, 1H, aromatic), 7.26 (d, J l 16 Hz, 1H,
vinylic), 7.04 (d, J l 3 Hz, 1H, furylic), 6.94 (d, J l 3
Hz, 1H, furylic), 6.46 (d, J l 16 Hz, 1H, vinylic), 3.87
(d, J l 6 Hz, 2H, gly-α-H). 13H NMR (d6-DMSO): δ
171.1, 164.7 (2iCO), 152.0, 149.1, 146.9, 132.3, 129.6,
128.8, 125.8, 123.9, 122.0 (aromatic, vinylic), 40.8 (gly-
N-3-(2-Naphthyl)-acryloyl-glycine methyl ester (10)
Recrystallization from toluene. Yield 0.30 g (66%),
white crystals; mp 115mC; IR: ν l 3290, 3055, 1745,
+
α-C). MS (EI): m\z (%) l 316 (48) [M ], 242 (41),
1
1
−
1660, 1625 cm . H NMR (CDCl3): δ 7.87–7.77 (m,
5H, aromatic), 7.62 (m, 1H, aromatic), 7.50–7.44 (m,
2H, aromatic, vinylic), 6.58 (d, J l 16 Hz, 1 H, vinylic),
6.36 (s, 1H, NH), 4.20 (d, J l 5 Hz, 2H, gly-α-H), 3.78
(s, 3H, OCH3); 13H NMR (CDCl3): δ 170.6, 166.1
(2iCO), 142.0, 134.1, 133.4, 132.1, 129.6, 128.6, 128.5,
127.8, 127.1, 126.7, 123.6, 120.0 (aromatic, vinylic), 52.5
170 (83), 139 (82), 110 (100); HRMS calculated for
C15H12N2O6 : 316.0695; found 316.0697; elemental an-
alysis calculated for C15H12N2O6 : C 56.97, H 3.82, N
8.86; found C 56.75, H 3.83, N 8.55.
(p)-N-3-[5-(2-Nitrophenyl)-furan-2-yl]-acryloyl-
phenylalanine (17)
+
(OCH3), 401.6 (gly-α-C); MS: m\z (%) l 269 (52) [M ],
Recrystallization from toluene\dioxane. Yield 0.37 g 181 (100), 152 (64); HRMS calculated for C16H15NO3 :
(61%), yellow crystals; mp 180mC; IR: ν l 3385, 3065, 269.1052; found 269.1050; elemental analysis calculated

836
for C16H15NO3 : C 71.36, H 5.61, N 5.20; found C 70.99, substrate on a Shimadzu UV2100U spectrophotometer.
Martin Schlitzer et al
H 5.61, N 5.24.
Each 1-mL cuvette contained enzyme (0.0025 units),
0.1  Na,K phosphate buffer, pH 6.2, 0.3 m NADPH
with or without 10 m substrate and the inhibitor. One
unit of enzyme activity was defined as the activity
consuming 1 µ of NADPH per min. Compounds were
dissolved in either weak base or DMSO (up to 5% (v\v)
total solution). Appropriate controls containing enzyme
solution with NADPH and inhibitor (with or without
DMSO), but no substrate, were run to negate potential
changes in the absorption of nucleotide at 340 nm not
due to direct enzymatic reaction. Activity with inhibitor
dissolved in DMSO was directly compared with activity
with similar enzyme solution containing DMSO but no
inhibitor. All measurements were conducted in triplicate
and the activity reported reflects the average of three
independent measurements. IC50 values were calculated
by linear regression.
N-3-(2-Phenyl-thiazole-4-yl)-acryloyl-glycine methyl
ester (11)
Recrystallization from toluene. Yield 0.20 g (57%),
white crystals; mp 128mC; IR: ν l 3370, 3090, 1740,
1
1
−
1670, 1635 cm ; H NMR (CDCl3): δ l 7.98–7.94 (m,
2H, aromatic), 7.59 (d, J l 15 Hz, 1 H, vinylic), 7.47–
7.41 (m, 3H, aromatic), 7.35 (s, 1H, thiazol), 6.95 (d,
J l 15 Hz, 1H, vinylic), 6.29 (s, 1H, NH), 4.19 (d, J l 5
Hz, 2H, gly-α-H), 3.78 (s, 3H, OCH3); 13H NMR
(CDCl3): δ 170.4, 168.6 (2iCO), 166.1, 152.8, 133.3,
130.6, 129.1, 126.8, 122.5, 121.0 (aromatic, vinylic), 52.5
+
(OCH3), 41.6 (gly-α-C); MS: m\z (%) l 302 (31) [M ],
215 (29), 214 (100); HRMS calculated for C15H14N2O3S:
301.0725; found 302.0717; elemental analysis calculated
for C15H14N2O3S: C 59.59, H 4.67, N 9.27; found C
59.22, H 4.30, N 9.58.
Results and Discussion
N-3-[5-(2-Nitrophenyl)-furan-2-yl]-acryloyl-glycine
methyl ester (15)
Recrystallization from toluene. Yield 0.30 g (61%),
The amides of the dicarbonic acid methyl esters 1, 2 and
5 were prepared by refluxing the aromatic amine with
one equivalent of malonic acid methyl ester chloride and
succinic acid methyl ester chloride, respectively (Method
1). The glutaric acid ester 7 was obtained from the
corresponding free acid 8 by treatment with methanol\
thionyl chloride. The succinic and glutaric acid amides
3, 4, 6 and 8 were prepared from the aromatic amines
and the corresponding acid anhydrides by refluxing in
THF (Method 2). For the preparation of the amino acid
derivatives carrying a free carboxyl group (9, 12, 13, 15,
16 and 17) appropriate acid chlorides were reacted with
the unprotected amino acids in water\acetone using
potassium carbonate as a base (Method 3). The N-acyl
amino acid methyl esters 10, 11 and 15 were obtained
from the acrylic acid derivatives activated as mixed
anhydrides using isobutyl chloroformate and the ap-
propriate amino acid methyl ester hydrochloride
(Method 4) (Figure 3). Arylacrylic acids were prepared
from the corresponding aldehydes via Knoevenagel
condensation (Jones 1967).
yellow crystals; mp 134mC; IR: ν l 3355, 2950, 1740,
1
1
−
1675, 1635, 1530, 1210 cm ; H NMR (CDCl3): δ
7.73–7.67 (m, 2H, aromatic), 7.58 (m, 1H, aromatic),
7.43 (m, 1H, aromatic), 7.39 (d, J l 15 Hz, 1H, vinylic),
6.71 (d, J l 4 Hz, 1H, furylic), 6.62 (d, J l 4 Hz, 1H,
furylic), 6.37 (d, J l 15 Hz, 1H, vinylic), 4.17 (d, J l 5
Hz, 2H, gly-α-H), 3.77 (s, 3H, OCH3); 13H NMR
(CDCl3): δ 170.4, 165.5 (2iCO), 152.20, 149.7, 147.6,
131.9, 129.0, 128.9, 127.9, 124.0, 123.4, 118.6, 116.0,
112.0 (aromatic, vinylic), 52.5 (OCH3), 41.6 (gly-α-C);
+
MS: m\z (%) l 330 (63) [M ], 242 (94), 170 (84), 110
(100); HRMS calculated for C16H14N2O6 : 330.0852,
found 330.0834; elemental analysis calculated for
C16H14N2O6 : C 58.18, H 4.27, N 8.48; found C 58.15, H
4.31, N 8.25.
Biological methods
Enzyme purification
Recombinant rat lens aldose reductase was purified by a
series of chromatographic procedures as previously de-
scribed (Old et al 1990).
The compounds prepared according to the proposed
lead structure (Kador et al 1985; Lee et al 1994, 1998)
and their biological activity are summarized in Figure 4
and Table 1, respectively. The compounds were assayed
against rat lens aldose reductase as previously described
Enzyme assay
The aldose reductase inhibitory assay was conducted as (Old et al 1990). In general these compounds can be
previously described (Old et al 1990). Briefly, the de- classified into three groups: one possessing an aniline-
crease in the absorption of NADPH at 340 nm was 4-(2h-6h-methylbenzothiazole) or 2-aminobenzothiazole
followed over a 4-min period with -glyceraldehyde as group as the aryl group; one possessing a 2-naphthyl

837
Synthesis of aldose reductase inhibitors
Method 1
O
O
O
O
i
Ar
+
NH2
Ar
O
N
H
O
( )n
( )n
Cl
2, 3, 6
Method 2
O
O
O
+
ii
Ar
( )n
NH2
Ar
N
H
( )n OH
4, 5, 7, 9
O
O
Method 3
R2
R2
O
O
OH
O
H2N
OH
OH
R1
N
H
+
R1
Cl
O
iii
O
O
or
or
O
H2N
OH
N
H
R1
10, 13–18
Method 4
O
O
iv
O
R1
N
H
OH
R1
O
11, 12, 16
Figure 3 Summary of methods employed for the synthesis of new aldose reductase inhibitors 2–18: (i) Tetrahydrofuran (THF), reflux, 2 h;
(ii) THF, reflux, 2 h; (iii) K2CO3, water\acetone 0mC 4 rt, 2 h; (iv) (a) iso-butyl chloroformate, N-methylmorpholine, dimethylformamide
(DMF), k15mC, 5 min., (b) glycine methyl ester hydrochloride, N-methylmorpholine, DMF, k15mC 4 rt, overnight.
as the aryl group; and one possessing either a 4-(2- carbon (compound 3) resulted in a 5-fold decrease in
phenylthiazole) or 2-(5–2h-nitrophenylfuran) as the aryl activity. However, in the case of compounds 6 and 8, the
group. In these groups the carboxylate or its methyl shorter inhibitor having a 4-carbon linker was approxi-
ester is linked to the aryl group through various lengths mately twice as active as its homologue 8. Nevertheless,
of methylene carbons and amide or cinnamide groups. this observation may not be significant since both com-
The establishment of correct structure–activity rela- pounds display only weak inhibitory activity. In ad-
tionships among the groups was hampered by insuf- dition to a 5-atom linker both active compounds (4 and
ficient solubility of at least one compound within each 16) carry a free carboxyl group. In case of compound 16,
series of structurally related groups. Two of the com- transformation of the carboxyl group in the corre-
pounds prepared (4 and 16) where active in the low sponding methyl ester resulted in a reduction in activity
micromolar range. Both have a chain length of five by two orders of magnitude. The corresponding methyl
atoms between the aryl moiety and the carboxyl group. ester of 4 was not prepared since the already one carbon
In the case of compound 4, shortening the linker by one shorter ester 2 was too insoluble to be assayed ap-

838
Martin Schlitzer et al
Figure 4 Structure of synthesized compounds.
propriately. In the cases of compounds 11 and 12, there
In this series, these observations suggest that an
was no difference in activity between the free carboxyl appropriate aryl moiety and a polar group, possibly a
derivative and its corresponding ester. Again, the free carboxyl group, connected by a linker of five atoms
apparent activity of these two compounds suggests that seems to be the minimal structural requirement for
the interaction between the aryl and polar groups of aldose reductase inhibition while a double bond and an
these compounds and the enzyme are weak.
amide moiety are well tolerated in the linking structure.

839
Synthesis of aldose reductase inhibitors
Table 1 Inhibitory activity of compounds 2–18
Lee, Y. S., Pearlstein, R., Kador, P. F. (1994) Molecular modeling
studies of aldose reductase. J. Med. Chem. 37: 787–792
Lee, Y. S., Chen, Z., Kador, P. F. (1998) Molecular modeling studies
of the binding modes of aldose reductase inhibitors at the active site
of human aldose reductase. Bioorg. Med. Chem. 6: 1811–1819
Kador, P. F. (1988) The role of aldose reductase in the development
of diabetic complications. Med. Res. Rev. 8: 325–352
Compound
IC50 (µ)
Compound
IC50 (µ)
1
2
" 10a
" 10a
29
11
66
12
69
3
13
" 100
" 10a
100
4
5.6
" 10a
45
14
Kador, P. F., Kinoshita, J. H., Sharpless, N. E. (1985) Aldose re-
ductase inhibitors: a potential new class of agents for the phar-
macological control of certain diabetic complications. J. Med.
Chem. 28: 841–849
5
15
6
16
6.3
7
" 100
75
17
" 10a
0.02
0.07
0.01
8
Ponalrestatb
Sorbinilb
Tolrestatb
Old, S. E., Sato, S., Kador, P. F., Carper, D. A. (1990) In vitro
expression of rat lens aldose reductase in Escherichia coli. Proc. Natl
Acad. Sci. USA 87: 4942–4945
9
" 100
" 10a
10
Sarges, R. (1989) Aldose reductase inhibitors: structure-activity re-
lationships and therapeutic potential. In: Testa, B. (ed.) Advanced
Drug Research. Vol 18. Academic Press Ltd, London, pp. 139–175
Sato, S., Kador, P. F. (1990) Inhibition of aldehyde reductase by
aldose reductase inhibitors. Biochem. Pharmacol. 40: 1033–1042
Tomlinson, D. R., Willars, G. B., Carrington, A. L. (1992) Aldose
reductase inhibitors and diabetic complications. Pharmacol. Ther.
54: 151–194
aSolubility problems precluded assay at higher concentrations. bFrom
Sato & Kador (1990)
References
Jones, G. (1967) The Knoevenagel condensation. In: Cope, A. C.
(ed.) Organic Reactions. Vol. 15. John Wiley & Sons, Inc., New
York, pp 204–599
Yabe-Nishimura, C. (1998) Aldose reductase in glucose toxicity: a
potential target for the prevention of diabetic complications.
Pharmacol. Rev. 50: 21–33
Larson, E. R., Lipinski, C. A., Sarges, R. (1988) Medicinal chemistry
of aldose reductase inhibitors. Med. Res. Rev. 8: 159–186