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Novel Aldose Reductase Inhibitors Derived from 6-[[Diphenylmethyleneamino]oxy]hexanoic Acid.

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202
Arch. Pharm. Chem. Life Sci. 2007, 340, 202 – 208
Full Paper
Novel Aldose Reductase Inhibitors Derived from
6-[[(Diphenylmethylene)amino]oxy]hexanoic Acid
Dietmar Rakowitz, Grete Piccolruaz, Cornelia Pirker, and Barbara Matuszczak
Institute of Pharmacy, University of Innsbruck, Innsbruck, Austria
Starting from the recently identified aldose reductase inhibitor 6-[[(diphenylmethylene)amino]oxy]hexanoic acid 1, the following systematic structural modifications were performed: (a) formal substitution of the phenyl rings, (b) isosteric replacement of the benzene core by the heteroarenes pyridine and thiophene, (c) formal reduction of the aromatic substructure and subsequent diminution of the cyclohexyl ring, (d) introduction of methylene spacer between C=N and
the phenyl rings, and finally (e) formal ring closure in order to get derivatives of the tricycles
fluorenone, xanthone, and thioxanthone, respectively. Out of these series, compounds 22 – 24
bearing disubstituted phenyl rings exhibit the highest inhibitory activity (IC50 value approx.
3 lM) which lie almost in the range of the reference sorbinil.
Keywords: Aldose reductase inhibitors / Benzophenone oxime congeners / 6-[[(Diphenylmethylene)amino]oxy]hexanoic acids / SAR /
Received: October 10, 2006; accepted: November 8, 2006
DOI 10.1002/ardp.200600166
Introduction
Known as one of the most common chronic disorders,
diabetes mellitus is currently affecting more than 150
million people worldwide. Its rapidly increasing prevalence is a major cause for concern as the number of people with diabetes mellitus is expected to exceed 300 million by 2025 making diabetes fated to become one of the
world most important and costly diseases. Much of this
increase will occur in developing countries and will be
due to population growth, ageing, unhealthy diets, obesity, and sedentary lifestyles. The number of deaths
attributed to diabetes is likely to be around four million
per year [1]. Diabetes mellitus is characterized by chronic
hyperglycaemia and the development of diabetes-specific
microvascular pathology in the retina, renal glomerulus,
and peripheral nerves. As a consequence, diabetes mellitus is a leading cause of blindness, endstage renal disease,
and a variety of debilitating neuropathies [2]. This may
Correspondence: Dietmar Rakowitz, Institute of Pharmacy, University of
Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
E-mail: dietmar.rakowitz@uibk.ac.at
Fax: +43-512-5072940
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2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
arise due to the saturation of the“normal” glucose metabolism which results in a dramatic increase in flux
through the polyol pathway. Inhibition of the key
enzyme (i. e. aldose reductase, EC 1.1.1.21, ALR 2) has been
recognized as one possibility for preventing the onset or
progression of long-term diabetic complications (for a
representative choice of the latest results see [3 – 7]). However, so far only a very small number of aldose reductase
inhibitors (ARIs) have met the criteria of sufficient
potency, oral activity, and an acceptable side-effect profile. Therefore, the area still requires further efforts, in
particular with respect to the discovery of new lead structures.
As part of our program to develop novel ARIs [8 – 17], xcarboxyalkylated derivatives of benzophenone oxime
(see Fig. 1) and their corresponding diaza isosters were
identified as inhibitors of aldose reductase [8, 9]. The
activity of such compounds, however, might be
explained with the presence of structural requisites
essential for aldose reductase inhibition (i. e. an acidic
function and a lipophilic moiety) [18 – 23]. Due to the promising results, we became interested to study the influence of structural features on the biological activity.
Thus, starting from the most active compound 6-[[(diphe-
Arch. Pharm. Chem. Life Sci. 2007, 340, 202 – 208
Aldose Reductase Inhibitors
203
Figure 1. Chemical structure of the lead compound 1.
Scheme 1. Reaction sequence to prepare compounds 1 – 24.
Table 1. Aldose reductase inhibition of compounds 1 – 24.
Compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17b)
18b)
19
20b)
21b)
22
23
24
Figure 2. Envisaged chemical modifications of the lead compound 1.
nylmethylene)amino]oxy]hexanoic acid (1 [9]), the following modifications were envisaged (Fig. 2): (a) formal introduction of substituents into the phenyl rings, (b) isosteric
replacement of the benzene core by the heteroarenes pyridine and thiophene, (c) formal reduction of the aromatic
substructure and subsequent diminution of the cyclohexyl ring, (d) introduction of methylene spacer between
C=N and the phenyl rings, and finally (e) formal ring closure in order to get derivatives of the tricycles fluorenone, xanthone, and thioxanthone, respectively.
Results and discussion
The chosen potential ARIs were synthesized as shown in
Scheme 1: O-Alkylation of the oximes using ethyl 6-bromohexanoate gave the intermediates which were subsequently hydrolyzed under basic conditions. The structures of the novel compounds were confirmed by elemental analysis, IR, and 1H-NMR spectroscopy as well as MS
data.
Inhibitory activities of the substituted hexanoic acids
1 – 14 were evaluated in a spectrometric assay with D,Lglyceraldehyde as the substrate and NADPH as the cofactor. According to the in vitro results obtained (see
Table 1), the desired compounds show satisfactory inhibitory activity, hence, the following conclusions can be
drawn: Introduction of substituents into the benzene
cores leads to the higher active compounds 2 – 6 com-
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2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
a)
b)
Aldose reductase inhibitiona)
32.2
12.0
5.80
7.58
11.6
14.4
28.6
16.4
30.3
86.3
23.4
11.5
11.8
15.9
20.2
16.5
45%
26%
4.80
41%
32%
2.97
2.99
2.92
(31.7
(9.36
(5.48
(6.74
(8.19
(14.3
(21.9
(14.7
(24.2
(66.8
(21.1
(10.6
(10.6
(13.9
(19.2
(13.3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
32.7)
15.6)
6.13)
8.53)
16.5)
14.5)
37.4)
18.3)
38.0)
111.0)
26.0)
12.4)
13.2)
18.0)
21.3)
20.5)
(3.84 – 5.99)
(2.56 – 3.44)
(2.56 – 3.50)
(2.23 – 3.82)
IC50 values (95% confidence limits) in lM or enzyme inhibition at 6.25 lM
IC50 value not detectable due to the poor solubility in the test
system.
pared to the benzophenone oxime derivative 1. Whereas
formal replacement of the benzene core by 2-pyridyl 7
does not cause a significant change of the in vitro activity,
the corresponding analogue of the p-electron rich thiophene 8 shows a two-fold increase in inhibitory activity.
Formal reduction of the phenyl residues leads to a compound 9 with comparable activity, however, diminution
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204
D. Rakowitz et al.
of the cycloalkyl to give compound 10 results in a considerable loss of activity. Furthermore, formal introduction
of a methylene spacer between benzene core and the C=N
substructure (i. e. compound 11) does not cause a significant change in activity. Interestingly, cyclization of the
diphenylmethylene substructure of compound 1 yielding the rigid analogues 12 – 14 is accompanied with an
increase in activities by a factor of two to three.
So far, from the structural variations elaborated above,
the bis(4-methoxyphenyl) derivative 3 emerged as the
most active compound (IC50 = 5.8 lM). In this context, formal shift of the methoxy groups from position 4 to 2 and
introduction of other alkoxy as well as an additional substituent became an object of our interest. According to
the in vitro results obtained for compounds 15 – 24 (see
Table 1), it was found that the 2-substituted congeners
are less active, e. g. IC50 16.5 lM (16) vs. 4.80 lM (19). Concerning the nature of the alkoxy substituent, nearly the
same activities were obtained for 15 – 18, by contrast,
within the series of 4,49-disubstituted compounds, derivatives bearing propoxy (20) or butoxy (21), respectively,
are less active. However, congeners 22 – 24 bearing disubstituted phenyl rings exhibit the highest activities (IC50 L
3 lM) which lie almost in the same range of the reference
sorbinil (IC50 L 1.2 lM).
In accordance with the hitherto described enzyme-ARI
interactions [18 – 23], we assume that the carboxylic acid
substructure will interact in its anionic form with Tyr48,
His110, and Trp111. Moreover, lipophilic interactions
between the hydrophobic ring system(s) and appropriate
amino acid side chains (e. g. those of Phe122 and/or Trp20)
should play an important role for the effect of these compounds as enzyme inhibitors. In summary, from the
results obtained, it is impossible to rationalize which
structural features are important since except for size
and electronical effects, respectively, there are more factors to consider. For example, both electron-withdrawing
substituents like chloro or fluoro as well as electrondonating groups (e. g. alkoxy) enhance the amount of
enzyme inhibition. The complex situation is also demonstrated by the fact that, by instance, compounds 1 and 9
exhibit nearly the same activities, although their chemical constitutions differ completely concerning steric
requirements, flexibility, and their dimensions as well as
electronic distribution.
Conclusion
In conclusion, the substituted benzophenone oximes 1 –
24 represent an interesting new family of compounds to
inhibit the enzyme aldose reductase. A marked increase
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2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2007, 340, 202 – 208
in activity could be obtained by introduction of substituents into the aromatic core as well as by formal cyclization of this substructure leading to a rigidized system.
Out of these series, compounds 22, 23, and 24 bearing
2,4-dimethoxy-, 3,5-dimethoxy-, or 4-methoxy-3-methylsubstituted phenyl rings, respectively, exhibit the highest enzyme inhibition. Thus, based on these results,
synthesis and evaluation not only of additional symmetric but also of asymmetric congeners will be performed.
The authors wish to acknowledge Schlachthof Salzburg-Bergheim (Austria) (KR Ing. Sebastian Grießner and Mag. Erika
Sakoparnig) for providing calf lenses.
Experimental
Chemistry
Melting points were determined with a Kofler hot-stage microscope (C. Reichert, Vienna, Austria) and are uncorrected. Solvents were purified by distillation and stored over molecular
sieves (3 ). Light petroleum refers to the fraction of bp. 40 –
608C. Infrared spectra (KBr pellets) were recorded on a Mattson
Galaxy Series FTIR 3000 spectrophotometer (Mattson Instruments, Inc., Madison, WI, USA). Mass spectra were obtained on a
Finnigan MAT SSQ 7000 spectrometer (EI, 70 eV or CI, 200 eV,
reactant gas: methane; Thermo Electron Corporation, Bremen,
Germany). All 1H-NMR spectra were recorded in CDCl3 solution
in 5 mm tubes at 308C on a Varian Gemini 200 spectrometer
(199.98 MHz; Varian Inc., Palo Alto, CA, USA) with the deuterium
signal of the solvent as the lock and TMS as internal standard.
Chemical shifts are expressed in parts per million (ppm).
Reactions were monitored by thin layer chromatography
using Polygramm SIL G/UV254 (Macherey-Nagel) plastic-backed
plates (0.25 mm layer thickness) and visualized using an UV
lamp. Column chromatography was conducted on Merck silica
gel 60 (230 – 400 mesh) using dichloromethane, mixtures of
dichloromethane/light petroleum, dichloromethane/ethyl acetate, or ethyl acetate/10% triethylamine (only in the case of the 2pyridyl substituted starting material). Elemental analyses were
performed by Mag. J. Theiner, “Mikroanalytisches Laboratorium”, Faculty of Chemistry, University of Vienna, Austria, and
the data for C and H are within l 0.4% of the calculated values.
Not commercially available ketones were prepared starting
from the appropriate dihydroxybenzophenone by alkylation
according to [24]. The oximes were synthesized in analogy to
[25].
General procedure for O-substitution of several oximes
Method A: used for the synthesis of the starting material for
compounds 2 – 14.
Powdered potassium hydroxide (2 equiv.) was added to a solution of the appropriate oxime (1 equiv.) in dry dimethyl sulfoxide under an atmosphere of nitrogen. After stirring for 30 min
at room temperature, ethyl 6-bromohexanoate (1 equiv.) was
added and stirring was continued until TLC indicated no further
conversion. Then the mixture was poured into 2N HCl and
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Arch. Pharm. Chem. Life Sci. 2007, 340, 202 – 208
Aldose Reductase Inhibitors
205
Table 2. Data of the target compounds 2 – 11 and 15 – 24.
No
R
Formula (MW)
Yield
Purification
(recryst. solvent)
Properties Mp
IR (cm – 1)
MS
1
2
4-CH3-C6H4
C21H25NO3 (339.44)
1710 (mCO)
339 (M+)
3
4-OCH3-C6H4
C21H25NO560.5 H2O
(380.44)
77% DIPE
yellowish crystals
94 – 968C
78% EtOH
colourless crystals
52 – 568C
4
4-N(CH3)2-C6H4
C23H31N3O3 (397.52)
73% EtOH
yellow crystals
146 – 1488C
1707 (mCO)
397 (M+)
5
4-Cl-C6H4
C19H19Cl2NO3 (380.27)
80% DIPE
colourless crystals
84 – 868C
1703 (mCO)
380 (M+)
6
4-F-C6H4
C19H19F2NO3 (347.36)
35% DIPE
colourless crystals
49 – 518C
1700 (mCO)
347 (M+)
7
C5H4N
C17H19N3O360.1
EtOH60.1 DIPE (328.18)
44% DIPE/EtOH
colourless crystals
91 – 938C
1700 (mCO)
314 (M+1)+
8
C4H3S
C15H17NO3S2 (323.43)
36% DIPE/PE
colourless crystals
43 – 448C
1698 (mCO)
324 (M+1)+
9
C6H11
C19H33NO3 (323.48)
1702 (mCO)
323 (M+)
10
C3H5
C13H21NO3 (239.32)
11
C6H5CH2
C21H25NO3 (339.44)
93% PE
colourless crystals
34 – 368C
92%
yellow solid
27 – 288C
71% PE
colourless crystals
42 – 448C
15
2-OCH3-C6H4
C21H25NO5 (371.44)
35% DIPE
colourless crystals
104 – 1068C
1697 (mCO)
372 (M+1)+
16
2-OC2H5-C6H4
C23H29NO5 (399.49)
42% DIPE
light yellow crystals
102 – 1048C
1716 (mCO)
400 (M+1)+
7.38 – 7.09 (m, 8H, phenyl-H), 4.16 (t, J = 6.6 Hz, 2H,
NOCH2), 2.39 – 2.31 (m, 8H, CH2CO, 26CH3), 1.80 –
1.59 (m, 4H, 26CH2), 1.48 – 1.36 (m, 2H, CH2)
7.41 – 7.26 (m, 4H, phenyl-H), 6.92 – 6.80 (m, 4H, phenyl-H), 4.12 (t, J = 6.6 Hz, 2H, NOCH2), 3.81 (s, 3H,
OCH3), 3.79 (s, 3H, OCH3), 2.26 (t, J = 7.4 Hz, 2H,
CH2CO), 1.76 – 1.50 (m, 4H, 26CH2), 1.42 – 1.20 (m,
2H, CH2)
7.39 – 7.26 (m, 4H, phenyl-H), 6.73 – 6.63 (m, 4H, phenyl-H), 4.14 (t, J = 6.6 Hz, 2H, NOCH2), 2.99 (s, 6H,
26CH3), 2.96 (s, 6H, 26CH3), 2.35 (t, J = 7.3 Hz, 2H,
CH2CO), 1.81 – 1.60 (m, 4H, 26CH2), 1.50 – 1.38 (m,
2H, CH2)
7.43 – 7.36 (m, 4H, phenyl-H), 7.32 – 7.24 (m, 4H, phenyl-H), 4.17 (t, J = 6.6 Hz, 2H, NOCH2), 2.35 (t, J = 7.3
Hz, 2H, CH2CO), 1.78 – 1.58 (m, 4H, 2 x CH2), 1.47-1.35
(m, 2H, CH2)
7.48 – 7.30 (m, 4H, phenyl-H), 7.15 – 6.97 (m, 4H, phenyl-H), 4.17 (t, J = 6.6 Hz, 2H, NOCH2), 2.35 (t, J = 7.3
Hz, 2H, CH2CO), 1.79 – 1.59 (m, 4H, 26CH2), 1.48 –
1.37 (m, 2H, CH2)
8.74 – 8.70 (m, 1H, pyridyl-H), 8.58 – 8.54 (m, 1H, pyridyl-H), 7.85 – 7.66 (m, 3H, pyridyl-H), 7.58 – 7.53 (m,
1H, pyridyl-H), 7.35 – 7.21 (m, 2H, pyridyl-H), 4.24 (t, J
= 6.4 Hz, 2H, NOCH2), 2.29 (t, J = 7.3 Hz, 2H, CH2CO),
1.79 – 1.56 (m, 4H, 26CH2), 1.45 – 1.20 (m, 2H, CH2)
7.56 (d, J = 5.0 Hz, 1H, thienyl-H), 7.49 (d, J = 3.0 Hz,
1H, thienyl-H), 7.36 (d, J = 5.0 Hz, 1H, thienyl-H), 7.29
(d, J = 3.0 Hz, 1H, thienyl-H), 7.07 (br s, 2H, thienyl-H),
4.33 (t, J = 6.2 Hz, 2H, NOCH2), 2.39 (t, J = 7.1 Hz, 2H,
CH2CO), 1.87 – 1.54 (m, 6H, 36CH2)
3.97 (t, J = 6.4 Hz, 2H, NOCH2), 2.84 – 2.69 (m, 1H, CH),
2.37 (t, J = 7.5 Hz, 2H, CH2CO), 2.18 – 2.06 (m, 1H, CH),
1.76 – 1.19 (m, 26H, 136CH2)
4.00 (t, J = 6.4 Hz, 2H, NOCH2), 2.40 – 2.33 (m, 3H, CH,
CH2CO), 1.74 – 1.59 (m, 4H, 2x CH2), 1.49 – 1.35 (m, 2H,
CH2), 0.95 – 0.57 (m, 9H, 46CH2, CH)
7.33 – 7.09 (m, 10H, phenyl-H), 4.15 (t, J = 6.4 Hz, 2H,
NOCH2), 3.55 (s, 2H, CH2), 3.41 (s, 2H, CH2), 2.36 (t, J =
7.4 Hz, 2H, CH2CO), 1.80 – 1.62 (m, 4H, 26CH2), 1.51 –
1.39 (m, 2H, CH2)
7.45 (dd, J = 7.4 Hz, J = 1.8 Hz, 1H, phenyl-H), 7.33 –
7.24 (m, 2H, phenyl-H), 7.18 (dd, J = 7.2 Hz, J=1.8 Hz,
1H, phenyl-H), 6.98 – 6.81 (m, 4H, phenyl-H), 4.15 (t, J
= 6.6 Hz, 2H, NOCH2), 3.75 (s, 3H, OCH3), 3.61 (s, 3H,
OCH3), 2.33 (t, J = 7.3 Hz, 2H, CH2CO), 1.76 – 1.57 (m,
4H, 26CH2), 1.45-1.29 (m, 2H, CH2)
7.51 (dd, J = 7.4 Hz, J = 1.8 Hz, 1H, phenyl-H), 7.30 –
7.20 (m, 3H, phenyl-H), 6.97 – 6.75 (m, 4H, phenyl-H),
4.15 (t, J = 6.6 Hz, 2H, NOCH2), 3.91 (q, J = 7.0 Hz, 2H,
OCH2), 3.80 (q, J = 7.0 Hz, 2H, OCH2), 2.32 (t, J = 7.3 Hz,
2H, CH2CO), 1.78 – 1.57 (m, 4H, 26CH2), 1.46 – 1.30
(m, 2H, CH2), 1.19 (t, J = 7.0 Hz, 3H, CH3), 1.07 (t, J = 7.0
Hz, 3H, CH3)
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2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1705 (mCO)
371 (M+)
1708 (mCO)
239 (M+)
1718 (mCO)
339 (M+)
H-NMR (CDCl3)
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206
D. Rakowitz et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 202 – 208
Table 2. Continued ...
No
R
Formula (MW)
Yield
Purification
(recryst. solvent)
Properties Mp
IR (cm – 1)
MS
1
17
2-OC3H7-C6H4
C25H33NO5 (427.54)
36% DIPE/PE
colourless crystals
95 – 968C
1704 (mCO)
428 (M+1)+
18
2-OC4H9-C6H4
C27H37NO5 (455.60)
35% DIPE/PE
colourless crystals
79 – 808C
1704 (mCO)
456 (M+1)+
19
4-OC2H5-C6H4
C23H29NO5 (399.49)
43% DIPE/PE
colourless crystals
92 – 948C
1706 (mCO)
400 (M+1)
20
4-OC3H7-C6H4
C25H33NO5 (427.54)
35% DIPE
colourless crystals
88 – 898C
1708 (mCO)
428 (M+1)+
21
4-OC4H9-C6H4
C27H37NO5 (455.60)
30% DIPE/PE
colourless crystals
66 – 678C
1706 (mCO)
456 (M+1)+
22
2,4-OCH3-C6H3
C23H29NO7 (431.49)
58% DIPE/EA
colourless crystals
108 – 1098C
1700 (mCO)
432 (M+1)
23
3,5-OCH3-C6H3
C23H29NO7 (431.49)
86% EtOH
colourless crystals
96 – 988C
1706 (mCO)
431 (M+)
24
4-OCH3-3-CH3-C6H3
C23H29NO5 (399.49)
56% DIPE/PE
colourless crystals
87 – 898C
1704 (mCO)
400 (M+1)+
7.49 (dd, J = 7.3 Hz, J = 1.9 Hz, 1H, phenyl-H), 7.31 –
7.19 (m, 3H, phenyl-H), 6.96 – 6.76 (m, 4H, phenyl-H),
4.14 (t, J = 6.6 Hz, 2H, NOCH2), 3.80 (t, J = 6.6 Hz, 2H,
OCH2), 3.71 (t, J = 6.6 Hz, 2H, OCH2), 2.32 (t, J = 7.5 Hz,
2H, CH2CO), 1.77 – 1.21 (m, 10H, 56CH2), 0.86 (t, J =
7.5 Hz, 3H, CH3), 0.80 (t, J = 7.3 Hz, 3H, CH3)
7.49 (dd, J = 6.2 Hz, J = 1.4 Hz, 1H, phenyl-H), 7.29 –
7.20 (m, 3H, phenyl-H), 6.95 – 6.75 (m, 4H, phenyl-H),
4.14 (t, J=6.6 Hz, 2H, NOCH2), 3.83 (t, J = 6.5 Hz, 2H,
OCH2), 3.74 (t, J = 6.4 Hz, 2H, OCH2), 2.32 (t, J = 7.3 Hz,
2H, CH2CO), 1.77-1.10 (m, 14H, 76CH2), 0.86 (t, J = 7.3
Hz, 3H, CH3), 0.84 (t, J = 7.1 Hz, 3H, CH3)
7.42 – 7.27 (m, 4H, phenyl-H), 6.94 – 6.79 (m, 4H, phenyl-H), 4.18-3.99 (m, 6H, 26OCH2, NOCH2), 2.35 (t, J =
7.3 Hz, 2H, CH2CO), 1.80 – 1.59 (m, 4H, 26CH2), 1.49 –
1.33 (m, 5H, CH2, CH3), 1.26 (t, J = 7.1 Hz, 3H, CH3)
7.42 – 7.26 (m, 4H, phenyl-H), 6.94 – 6.80 (m, 4H, phenyl-H), 4.15 (t, J = 6.6 Hz, 2H, NOCH2), 3.96 (t, J = 6.6
Hz, 2H, OCH2), 3.92 (t, J = 6.5 Hz, 2H, OCH2) 2.35 (t, J =
7.5 Hz, 2H, CH2CO), 1.91 – 1.60 (m, 8H, 46CH2), 1.49 –
1.37 (m, 2H, CH2), 1.05 (t, J = 7.5 Hz, 3H, CH3), 1.03 (t, J
= 7.5 Hz, 3H, CH3)
7.41 – 7.26 (m, 4H, phenyl-H), 6.94 – 6.79 (m, 4H, phenyl-H), 4.15 (t, J = 6.4 Hz, 2H, NOCH2), 4.00 (t, J = 6.4
Hz, 2H, OCH2), 3.97 (t, J = 6.6 Hz, 2H, OCH2) 2.35 (t, J =
7.4 Hz, 2H, CH2CO), 1.82-1.37 (m, 14H, 76CH2), 0.99
(t, J = 7.1 Hz, 3H, CH3), 0.97 (t, J = 7.4 Hz, 3H, CH3)
7.34 (d, J = 8.4 Hz, 1H, phenyl-H), 7.07 – 7.02 (m, 1H,
phenyl-H), 6.50 – 6.38 (m, 4H, phenyl-H), 4.13 (t, J = 6.6
Hz, 2H, NOCH2), 3.80 (s, 3H, OCH3), 3.79 (s, 3H, OCH3),
3.73 (s, 3H, OCH3), 3.59 (s, 3H, OCH3), 2.33 (t, J = 7.4
Hz, 2H, CH2CO), 1.76-1.57 (m, 4H, 26CH2), 1.45 – 1.33
(m, 2H, CH2)
7.36 – 7.26 (m, 1H, phenyl-H), 7.07 – 7.02 (m, 1H, phenyl-H), 6.50 – 6.38 (m, 4H, phenyl-H), 4.13 (t, J = 6.6 Hz,
2H, NOCH2), 3.80 (s, 3H, OCH3), 3.79 (s, 3H, OCH3),
3.73 (s, 3H, OCH3), 3.59 (s, 3H, OCH3), 2.33 (t, J = 7.4
Hz, 2H, CH2CO), 1.76 – 1.57 (m, 4H, 26CH2), 1.45 –
1.33 (m, 2H, CH2)
7.32 (d, J = 2.0 Hz, 1H, phenyl-H), 7.14 (s, 1H, phenylH), 7.19 (dd, J = 8.4 Hz, J = 2.0 Hz, 2H, phenyl-H), 6.84
(d, J = 8.4 Hz, 1H, phenyl-H), 6.74 (d, J = 8.4 Hz, 1H,
phenyl-H), 4.15 (t, J = 6.6 Hz, 2H, NOCH2), 3.86 (s, 3H,
OCH3), 3.83 (s, 3H, OCH3), 2.35 (t, J = 7.5 Hz, 2H,
CH2CO), 2.21 (s, 3H, CH3), 2.19 (s, 3H, CH3), 1.80 – 1.60
(m, 4H, 2 x CH2), 1.50 – 1.34 (m, 2H, CH2)
H-NMR (CDCl3)
DIPE: diisopropyl ether; PE: petrolether; EA: ethyl acetate.
extracted with dichloromethane. The organic layer was washed
with 2N NaOH, water, and brine, dried over anhydrous sodium
sulphate and evaporated to dryness. The oily residue thus
obtained was purified by column chromatography and directly
used for further conversion.
Method B: procedure in analogy to [26], used for the synthesis
of the starting material for compounds 15 – 24.
A mixture of the appropriate oxime (1 equiv.), ethyl 6-bromohexanoate (1.1 equiv.), powdered potassium carbonate (2 equiv.),
potassium iodide (3 equiv.), and 18-crown-6 (0.5 equiv.) in
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2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
toluene was heated at 1008C until TLC indicated no further conversion. After cooling to room temperature, the mixture was filtered and the filtrate was evaporated to dryness in vacuo. The
oily residue thus obtained was purified by column chromatography and used without characterization.
General procedure for the synthesis of the target
compounds 2 – 24
A solution of the appropriate ethyl ester (1 equiv.) in ethanol
was treated with 2N NaOH (1.1 – 2.0 equiv.) and the resulting
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Arch. Pharm. Chem. Life Sci. 2007, 340, 202 – 208
Aldose Reductase Inhibitors
207
Table 3. Data of the target compounds 12 – 14.
No
R
Formula (MW)
12
Yield
Purification
(recryst. solvent)
Properties Mp
IR (cm – 1)
MS
1
72% DIPE
colourless crystals
108 – 1108C
1695 (mCO)
309 (M+)
8.28 – 8.24 (m, 1H, phenyl-H), 7.78 – 7.74 (m, 1H, phenyl-H), 7.66 – 7.59 (m, 2H, phenyl-H), 7.46 – 7.23 (m,
4H, phenyl-H), 4.41 (t, J = 6.6 Hz, 2H, NOCH2), 2.40 (t, J
= 7.3 Hz, 2H, CH2CO), 1.95-1.67 (m, 4H, 26CH2),
1.62 – 1.48 (m, 2H, CH2)
56% EtOH
yellow crystals
85 – 878C
1705 (mCO)
325 (M+)
8.88 – 8.83 (m, 1H, phenyl-H), 8.09 – 8.04 (m, 1H, phenyl-H), 7.48 – 7.34 (m, 2H, phenyl-H), 7.26 – 7.10 (m,
4H, phenyl-H), 4.31 (t, J = 6.5 Hz, 2H, NOCH2), 2.39 (t, J
= 7.3 Hz, 2H, CH2CO), 1.93 – 1.67 (m, 4H, 26CH2),
1.60 – 1.48 (m, 2H, CH2)
78% DIPE/EtOH
yellowish crystals
118 – 1208C
1714 (mCO)
341 (M+)
8.33 – 8.28 (m, 1H, phenyl-H), 7.89 – 7.84 (m, 1H, phenyl-H), 7.48 – 7.25 (m, 6H, phenyl-H), 4.26 (t, J = 6.6 Hz,
2H, NOCH2), 2.38 (t, J = 7.3 Hz, 2H, CH2CO), 1.88 – 1.64
(m, 4H, 26CH2), 1.56 – 1.44 (m, 2H, CH2)
C19H19NO3 (309.37)
13
H-NMR (CDCl3)
C19H19NO4 (325.37)
14
C19H19NO3S (341.43)
DIPE: diisopropyl ether.
mixture was stirred overnight at room temperature. Then the
solvent was removed, the residue thus obtained treated with a
small amount of water, and the pH adjusted to 5 with 2N HCl.
The reaction mixture was extracted with ethyl acetate, the
organic layer washed with water and brine, dried over anhydrous sodium sulphate, and evaporated to dryness in vacuo. The
residue thus obtained was recrystallized from an appropriate
solvent to yield the analytically pure compound. For solvent,
yield, analytical and spectroscopic data see Tables 2 and 3.
final volume of 1.5 mL. All inhibitors were dissolved in DMSO;
the final concentration in the incubation mixture was 1%.
To correct for the nonenzymatic oxidation of NADPH, the rate
of NADPH oxidation in the presence of all the components
except the substrate was subtracted from each experimental
rate. Each dose-effect curve was generated using at least three
concentrations of the potential inhibitor causing an inhibition
between 20 and 80%. Each concentration was tested in duplicate
and IC50 values as well as the 95% confidence limits were
obtained by using CalcuSyn software [27] for dose effect analysis.
Biology
Enzyme inhibition studies
NADPH, D,L-glyceraldehyde, and dithiothreitol (DTT) were purchased from Sigma Chemical Co. DEAE-cellulose (DE-52) was
obtained from Whatman. The standard enzyme inhibitor Sorbinil [IC50 = 1.2 (l 0.2) lM] was a gift from Prof. Dr. Luca Costantino,
University of Modena (Italy) and was tested in parallel during
the series.
All other chemicals were commercial samples of good grade.
Calf lenses for the isolation of ALR 2 were obtained locally from
freshly slaughtered animals. The enzyme was purified by a chromatographic procedure as previously described [10]. Enzyme
activities were assayed on a Cecil Super Aurius CE 3041 spectrophotometer (Cecil Instruments, Cambridge, England) by measuring the decrease in absorption of NADPH at 340 nm which
accompanies the oxidation of NADPH catalyzed by ALR 2. The
assay was performed at 378C in a reaction mixture containing
potassium phosphate buffer pH 6.8, ammonium sulphate, the
cofactor NADPH, and D,L-glyceraldehyde acting as substrate in a
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2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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