Curcumin analogs as potent aldose reductase inhibitors

Article abstract of DOI:10.1002/ardp.200500205

In the present study, curcuminoids isolated from curcuma longa were demonstrated to possess inhibitory activities on bovine lens aldose reductase. In order to find more potent aldose reductase inhibitor, curcumin analogs were synthesized and evaluated for their ability to inhibit bovine lens aldose reductase enzyme. The results indicated that the compounds with tetrahydroxyl groups, 2,6-bis(3,4-dihydroxybenzylidene)cyclohexanone (A₂), 2,5-bis(3,4-dihydroxybenzylidene)cyclopentanone (B₂), 1,5-bis(3,4-dihydroxyphenyl)-1,4-pentadiene-3-one (C₂), and 3,5-bis(3,4-dihydroxybenzylidene)-4-piperidone (D₂) showed remarkably potent inhibitory effects on aldose reductase with IC₅₀ of 2.9 μM, 2.6 μM, 3.4 μM, and 4.9 μM, respectively. The structure-activity relationship revealed that the curcumin analogs with ortho-dihydroxyl groups could form a more tight affinity with aldose reductase to exert more potential inhibitory activities.

Full text of DOI:10.1002/ardp.200500205

Arch. Pharm. Chem. Life Sci. 2006, 339, 123–128
Z.-Y. Du et al.
123
Full Paper
Curcumin Analogs as Potent Aldose Reductase Inhibitors
Zhi-Yun Du1,2, Ya-Dan Bao1, Zhong Liu1, Wei Qiao2, Lin Ma2, Zhi-Shu Huang1, Lian-Quan Gu1,2
Albert S. C. Chan3
,
1 School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China
2 School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, China
3 Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong
Kong, China
In the present study, curcuminoids isolated from curcuma longa were demonstrated to possess
inhibitory activities on bovine lens aldose reductase. In order to find more potent aldose reduc-
tase inhibitor, curcumin analogs were synthesized and evaluated for their ability to inhibit
bovine lens aldose reductase enzyme. The results indicated that the compounds with tetrahydro-
xyl groups, 2,6-bis(3,4-dihydroxybenzylidene)cyclohexanone (A2), 2,5-bis(3,4-dihydroxybenzylide-
ne)cyclopentanone (B2), 1,5-bis(3,4-dihydroxyphenyl)-1,4-pentadiene-3-one (C2), and 3,5-bis(3,4-
dihydroxybenzylidene)-4-piperidone (D2) showed remarkably potent inhibitory effects on aldose
reductase with IC50 of 2.9 lM, 2.6 lM, 3.4 lM, and 4.9 lM, respectively. The structure-activity
relationship revealed that the curcumin analogs with ortho-dihydroxyl groups could form a
more tight affinity with aldose reductase to exert more potential inhibitory activities.
Keywords: Curcuminoids / Curcumin analogs / Aldose reductase inhibitors / Diabetic complication /
Received: September 8, 2005; accepted: December 1, 2005
DOI 10.1002/ardp.200500205
cellular swelling and increases membrane permeability
and perturbation of membrane transport process under
hyperglycemic conditions. These changes can initiate cel-
lular lesions associated with late onset diabetic complica-
tions such as neuropathy, nephropathy, retinopathy, cat-
aract, and cardio-vascular disease [1–3]. Therefore, aldose
reductase inhibitor has received much attention as an
attractive strategy to prevent or delay the onset and to
minimize the seriousness of chronic diabetic complica-
tions [4].
Over the past 30 years, a considerable number of com-
pounds with different chemical structures have been
synthesized and shown to be effect aldose reductase inhi-
bitors. In general, two main classes of orally active aldose
reductase inhibitors were synthesized and tested clini-
cally, cyclic imides and carboxylic acids. The carboxylic
acid derivatives proofed to be very active in vitro, but gen-
erally less active in vivo, probably owning to their lower
pKa values that resulted in less favorable pharmacoki-
netics. Cyclic imides derivatives were known to cause
side effects from skin rash and hypersensitivity to liver
toxicity. The only aldose reductase inhibitor available as
Introduction
Aldose reductase (EC 1.1.1.21, ALR2) is the first enzyme of
the polyol pathway that catalyzes the conversion of glu-
cose to sorbitol, which is in turn converted to fructose by
sorbitol dehydrogenase (EC 1.1.1.14, SD). The polyol path-
way can be found in many tissues such as lens, retina,
nervous, and kidney tissue. Under normal physiological
conditions, this pathway plays a minor role in the glu-
cose metabolism of most tissues. However, in diabetes,
the increased flux of blood glucose through the polyol
pathway can result in significant quantities of sorbitol
accumulated in cells because of sobitol's slow metabo-
lism by sorbitol dehydrogenase and poor penetration
through the cellular membranes. As the polyol pathway
can alter cellular redox potentials and increases intracel-
lular levels of sorbitol, it results in hyperosmotic stress,
Correspondence: Lian-Quan Gu, Zhi-Shu Huang School of Pharmaceu-
tical Sciences, Sun Yat-sen University, Guangzhou 510980, China.
E-mail: cedc42@zsu.edu.cn
Fax: +86 20 8411-0272
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

124
Z.-Y. Du et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 123–128
phenolic compounds like curcumin prompted us to
study a series of curcumin analogs (Scheme 1).
Results and discussion
Initially, in the course of a screening study on aldose
reductase inhibitors from plant sources, we found that
the natural curcuminoids 1–3 produced remarkable
inhibitory activities on bovine lens aldose reductase
(Table 1). Among the curcuminoids, 1 exhibited the high-
est inhibitory activity with the lowest IC50 value, 6.8 lM,
which was approximately 5-fold lower than that of the
well known aldose reductase inhibitor from natural
resource, quercetin, followed by compounds 3 and 2.
Based on the structure of curcumin 1, a series of curcu-
min analogs were designed and synthesized. Curcumin
analogs (A, B, C, and D) were synthesized through con-
densation of the appropriate ketone with a variety of aro-
matic aldehydes under acidic condition (Scheme 1). Com-
paring the curcumin analogs, almost all compounds
with p-hydroxyl groups (R2 = OH) showed an inhibitory
Figure 1. Chemical structures of curcuminoids.
a drug was epalrestat developed by Ono Pharmaceutical
[5–7].
Turmeric (curcuma longa) was widely used in Chinese
traditional medicine, such as prescriptions against the
diabetic complications [8]. In the course of a screening
study on aldose reductase inhibitors from plant sources,
we found that the curcuminoids (Fig. 1) isolated from
curcuma longa demonstrated remarkable inhibitory
activities on bovine lens aldose reductase. The interesting
discovery of the aldose reductase inhibitory activity of
Scheme 1. Synthesis of curcumin
analoges.
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2006, 339, 123–128
Table 1. Aldose reductase inhibitory activity.
Curcumin Analogs as Potent Aldose Reductase Inhibitors
125
acidity with respect to carboxylic acids, which poten-
tially made for better pharmacokinetic properties [9].
Most of the aldose reductase inhibitors from natural
sources belong to phenolic compounds [10]. The struc-
ture studies for the inhibition of aldose reductase by phe-
nolic compounds indicated that the acidity constants of
hydroxylated compounds play an important role [11],
and ortho-dihydroxyl phenolic compounds show marked
inhibitory activities [12]. Therefore, we assumed that the
inhibitory mode of curcuminoids and curcumin analogs
was similar to that of flavones and chalcones.
In conclusion, a number of curcumin analogs were
designed, synthesized, and tested for their inhibitory
activities against aldose reductase. The findings indi-
cated that the para-hydroxyl groups present on the phe-
nolic ring of curcumin analogs play a very important
role on aldose reductase inhibitory activity. The ortho-
dihydroxyl groups of curcumin analogs can form a more
tight interaction with the enzyme to exert more poten-
tial inhibitory activities. This study suggest that curcu-
min analogs with dihydroxyl groups would be the lead
compounds suitable for developing patent aldose reduc-
tase inhibitors.
Compound R1
R2
R3
IC50 [%
inhibition]a)
1
2
3
OCH3
H
H
OCH3
OCH3
H
6.8
15.9
13.7
27.5
2.9
26.4
35.8%
NIb)
24.9
21.0
27.4
2.6
25.8
38.2%
NIb)
23.4
20.8
33.1
3.4
35.9
31.0%
NIb)
25.3
31.1
4.9
29.9
29.2%
NIb)
A1
A2
A3
A4
A5
A6
A7
B1
H
OH
OH
OH
OH
OH
OCH3
OH
OH
H
H
H
OCH3
C(CH3)3
OCH3
OCH3
Br
C(CH3)3
H
OCH3
OCH3
H
H
OH
B2
OH
OH
H
B3
B4
B5
B6
B7
C1
OCH3
C(CH3)3
OCH3
OCH3
Br
OH
OH
OCH3
OH
OH
H
C(CH3)3
H
OCH3
OCH3
H
H
OH
C2
OH
OH
H
C3
C4
C5
C6
OCH3
C(CH3)3
OCH3
OCH3
H
OH
OH
OCH3
OH
OH
OH
OH
OH
OCH3
OH
H
C(CH3)3
H
OCH3
H
H
H
C(CH3)3
H
OCH3
OCH3
D1
D2
D3
D4
D5
D6
D7
Quercetin
OH
OCH3
C(CH3)3
OCH3
OCH3
Br
We are indebted to the National Nature Science Foundation of
China (20472117), the Science Foundation of Zhuhai
(PC20041131), the Guangzhou city Science Foundation, the
Guangdong Provincial Science Foundation, and the Hong
Kong Polytechnic University Area of Strategic Development
Fund for financial support of this study.
22.3
20.8
37.6
OH
a)
IC50 (lM) (95% C. L.) or% inhibition at 100 lM.
NI = non inhibition at 100 lM.
b)
effect with low IC50 values varying from 2.6 lM to 35.9
lM, except for A4, B4, C4, and D4 due to their poor solubi-
lity. However, compounds without a p-hydroxyl group (R2
= OCH3) had no inhibitory effect on aldose reductase at
100 lM.
The introduction of methoxy groups or bromine atoms
at the ortho-positions of phenols led to the activities with-
out significant improvement compared with p-hydroxyl
phenolic compounds (A1, B1, C1, and D1), the introduction
of the bulky ortho tert-butyl groups (A4, B4, C4, and D4) led
to remarkably decreased activities. Whereas introduction
of hydroxyl groups at the ortho-positions of phenols led
to the compounds A2, B2, C2, and D2 produced much
higher inhibitory activity than others, the inhibitory
effects of A2, B2, C2, and D2 were approximately twofold
lower than that of curcumin, and almost tenfold lower
than that of quercetin (IC50 = 37.6 lM).
Experimental
General
Melting points were taken with a Thomas Hoover Melting appa-
ratus (Capillary method) (Thomas Hoover Capillary Apparatus,
Philadelphia, PA, USA) and were uncorrected. The 1H-NMR spec-
tra were measured on a Mercury – Plus 300 spectrometer (Var-
ian Inc., Palo Alto, CA, USA) using DMSO-d6 as solvent unless
otherwise specified. Chemical shifts for 1H-NMR were expressed
in ppm with tetramethylsilane (TMS) as an internal standard.
Multiplicities were recorded as s (singlet), brs (broad singlet), d
(doublet), t (triplet), q (quartet), m (multiplet). Mass spectra were
obtained on a LCMS-2010A spectrometer (Shimadzu, Tokyo,
Japan). Elemental analyses were performed on a Vario EL ele-
mental analyzer (Elementar Analysansysteme, Hanau, Ger-
many). Thin-layer chromatography was performed on Merk
silica gel plates DC-60 F254 (Merck, Darmstadt, Germany).
Chemistry
Recently, the inhibitory activities of phenolic com-
pounds such as flavones, chalcones, and lignans on
aldose reductase received much attention for their lower
Isolation compound 1, 2 and 3 from curcuma longa
The curcuma longa extract was isolated from curcuma longa roots
by supercritical CO2 liquid extraction system. The extract (2.0 g)
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

126
Z.-Y. Du et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 123–128
was chromatographed on
a silica gel column (Merk 70-
2,6-Bis(4-hydroxy-3-methoxybenzylidene)cyclohexanone
230 mesh, 80 g, 4.5 cm6100 cm) using chloroform/methanol/
acetic acid (95:4:1) as the eluent to give curcumin (1.32 g),
demethoxycurcumin (0.12 g), and bisdemethoxycurcumin
(0.25 g), respectively. Rf values of curcumin, demethoxycurcu-
min, and bisdemethoxycurcumin were 0.68, 0.43, and 0.21 in
hexane/chloroform/methanol (3:6:1) on TLC, respectively.
A3
Yield 98%, mp. 178–1798C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
1.71 (q, J = 6.7 Hz, 2H, C–CH2 –C), 2.87 (t, J = 6.7 Hz, 4H, H2C–C–
CH2), 3.79 (s, 6H, OCH3), 6.82 (d, J = 8.1 Hz, 2H, ArH), 7.01 (d, J = 8.1
Hz, 2H, ArH), 7.08 (s, 2H, ArH), 7.53 (s, 2H, –CH=), 9.48 (brs, 2H, –
OH). LC-MS (m/z): 366. Anal. Calcd. for C22H22O5: C 72.12, H 6.05.
Found: C 72.03.48, H 6.02.
1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-
dione 1
2,6-Bis(3,5-di-tert-butyl-4-hydroxylbenzylidene)cyclo-
Yellow needles, mp. 183–1848C (1838C [13]). 1H-NMR (DMSO-d6,
300 MHz) d (ppm): 3.83 (s, 6H), 6.04 (s, 1H), 6.75 (d, J = 15.9 Hz,
2H), 6.86 (d, J = 8.1 Hz, 2H), 7.12-7.31 (m, 4H), 7.53 (d, J = 15.9 Hz,
2H), 9.66 (brs, 2H). LC-MS (m/z): 368. Anal. Calcd. for C21H20O6: C
68.47, H 5.47. Found: C 68.32, H 5.38.
hexanone A4
1
Yield 65%, mp. 188–1908C. H-NMR (CDCl3, 300 MHz) d (ppm):
1.47 (s, 36H, C–CH3), 1.83 (q, J = 6.7 Hz, 2H, C–CH2 –C), 2.94 (t, J =
6.7 Hz, 4H, H2C–C–CH2), 5.45 (brs, 2H, –OH), 7.35 (s, 4H, ArH),
7.77 (s, 2H, –C=). LC-MS (m/z): 530. Anal. Calcd. for C36H50O3: C
81.46, H 9.49. Found: C 81.21, H 9.36.
1-(4-Hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-
1,6-heptadiene-3,5-dione 2
2,6-Bis(3,4-dimethoxybenzylidene)cyclohexanone A5
Yield 95%, mp. 138–1408C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
1.73 (q, J = 6.7 Hz, 2H, –C–CH2 –C–), 2.91 (t, J = 6.7 Hz, 4H, –
CH2 –C–CH2 –), 3.79 (s, 12H, OCH3), 7.02 (s, 2H, ArH), 7.13 (d, J =
8.1, 4H, ArH), 7.58 (s, 2H, –CH=). LC-MS (m/z): 394. Anal. Calcd.
for C24H26O5: C 73.08, H 6.64. Found: C 69.90, H 6.57.
Yellow needles, mp. 180–1818C (181–1828C [13]). 1H-NMR
(DMSO-d6, 300 MHz) d (ppm): 3.83 (s, 3H), 6.03 (s, 1H), 6.68 (d, J =
15.9 Hz, 1H), 6.75 (d, J = 15.9 Hz, 1H), 6.80 (d, J = 8.1 Hz, 2H), 7.13
(d, J = 8.1 Hz, 1H), 7.31 (d, J = 1.5 Hz, 1H), 7.53 (d, J = 15.9 Hz, 2H),
7.55 (d, J = 8.1 Hz, 2H), 9.66 (brs, 1H), 10.05 (brs, 1H). LC-MS (m/z):
338. Anal. Calcd. for C20H18O5: C 70.99, H 5.36. Found: C 70.78, H
5.23.
2,6-Bis(4-hydroxy-3,5-dimethoxybenzylidene)cyclo-
hexanone A6
1,7-Bis(4-hydroxyphenyl)-1,6-heptadiene-3,5-dione 3
Light red needles, mp. 232–2338C (232–2348C [13]). 1H-NMR
(DMSO-d6, 300 MHz) d (ppm): 6.02 (s, 1H), 6.68 (d, J = 15.9 Hz, 2H),
6.80 (d, J = 8.1 Hz, 4H), 7.52 (d, J = 15.9 Hz, 4H), 7.55 (d, J = 8.1 Hz,
2H), 10.05 (brs, 2H). LC-MS (m/z): 308. Anal. Calcd. for C21H20O6: C
74.01, H 5.23. Found: C 73.87, H 5.16.
Yield 62%, mp. 134–1358C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
1.74 (q, J = 6.7 Hz, 2H, –C–CH2 –C–),2.93 (t, J = 6.7 Hz, 4H, –CH2 –
C–CH2 –),3.71 (s, 12H, OCH3), 6.83 (s, 4H, ArH), 7.55 (s, 2H, –CH=),
8.89 (brs, 2H, –OH). LC-MS (m/z): 426. Anal. Calcd. for C24H26O7: C
67.59, H 6.15. Found: C 67.48, H 6.09.
2,6-Bis(3-bromo-4-hydroxy-5-methoxybenzylidene)cyclo-
General procedure for the preparation of A1 – 7, B1 – 7, C1 – 6
and D1 – 7
,
hexanone A7
Yield 51%, mp. 218–2208C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
1.73 (q, J = 6.7 Hz, 2H, –C–CH2 –C–), 2.88 (t, J = 6.7 Hz, 4H, –
CH2 –C–CH2 –), 3.86 (s, 6H, OCH3), 7.13 (s, 2H, ArH), 7.27 (s, 2H,
ArH), 7.50 (s, 2H, –CH=), 9.94 (brs, 2H, –OH). LC-MS (m/z): 522.
Anal. Calcd. for C22H20Br2O5: C 50.41, H 3.85. Found: C 50.22, H
3.76.
Curcumin analogs were synthesized as previously described [14,
15] but with modification. A mixture of the appropriate alde-
hyde (0.01 mol) and the ketone (0.005 mol) was dissolved in gla-
cial acetic acid saturated with anhydrous hydrogen chloride and
heated in a water bath at 25–308C for 2 h. After standing for
2 days, the mixture was treated with cold water and filtered. The
solid obtained was then washed and dried. The crude product
was recrystallized from appropriate solvents (methanol or etha-
nol).
2,5-Bis(4-hydroxybenzylidene)cyclopentanone B1
1
Yield 90%, mp. A3008C. H-NMR (DMSO-d6, 300 MHz) d (ppm):
3.01 (s, 4H, –CH2 –CH2 –), 6.85 (d, J = 8.1 Hz, 4H, ArH), 7.31 (s, 2H,
–CH=), 7.51 (d, J = 8.1 Hz, 4H, ArH), 10.01 (brs, 2H, –OH). LC-MS
(m/z): 292. Anal. Calcd. for C19H16O3: C 78.06, H 5.52. Found: C
77.93, H 5.46.
2,6-Bis(4-hydroxybenzylidene)cyclohexanone A1
Yield 95%, mp. A3008C.1H-NMR (DMSO-d6, 300 MHz) d (ppm): 1.72
(q, J = 5.7 Hz, 2H, -C-CH2-C-), 2.84 (t, J = 5.7 Hz, 4H, –CH2–C–CH2–),
6.82 (d, J = 8.1 Hz, 4H, ArH), 7.38 (d, J = 8.1 Hz, 4H, ArH), 7.52 (s,
2H, –CH=), 9.88 (brs, 2H, –OH). LC-MS (m/z): 306. Anal. Calcd. for
C20H18O3: C 78.41, H 5.92. Found: C 78.28, H 5.83.
2,5-Bis(3,4-dihydroxybenzylidene)cyclopentanone B2
Yield 85%, mp. A3008C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
3.00 (s, 4H, –CH2 –CH2 –), 6.82 (d, J = 8.1 Hz, 2H, ArH), 7.00 (d, J =
8.1 Hz, 2H, ArH), 7.10 (s, 2H, ArH), 7.23 (d, 2H, –CH=), 9.18 (brs,
2H, –OH), 9.53 (brs, 2H, –OH). LC-MS (m/z): 324. Anal. Calcd. for
C19H16O5: C 70.36, H 4.97. Found: C 70.18, H 4.89.
2,6-Bis(3,4-dihydroxybenzylidene)cyclohexanone A2
Yield 90%, mp. A3008C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
2.04 (q, J = 6.7 Hz, 2H, –C–CH2 –C–), 2.83 (t, J = 6.7 Hz, 4H, –
CH2 –C–CH2 –),6.78 (d, J = 8.1 Hz, 2H, ArH), 6.85 (d, J = 8.1 Hz, 2H,
ArH), 6.96 (s, 2H, ArH), 7.43 (s, 2H, –CH=), 9.12 (brs, 2H, –OH),
9.43 (brs, 2H, –OH). LC-MS (m/z): 338. Anal. Calcd. for C20H18O5: C
70.99, H 5.36. Found: C 70.76, H 5.27.
2,5-Bis(4-hydroxy-3-methoxybenzylidene)cyclo-
pentanone B3
Yield 94%, mp. 212–2148C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
3.06 (s, 4H, –H2C–CH2 –), 3.83 (s, 6H, OCH3), 6.87 (d, J = 8.1 Hz,
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2006, 339, 123–128
Curcumin Analogs as Potent Aldose Reductase Inhibitors
127
2H, ArH), 7.15 (d, J = 8.1 Hz, 2H, 7.23 (s, 2H, arom, ArH), 7.34 (s,
2H, –CH=), 9.64 (brs, 2H, –OH). LC-MS (m/z): 352. Anal. Calcd. for
C21H20O5: C 71.58, H 5.72. Found: C 71.50, H 5.68.
1,5-Bis(3,5-di-tert-butyl-4-hydroxylphenyl)-1,4-
pentadiene-3-one C4
Yield 81%, mp. 224–2268C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
1.40 (s, 36H, C–CH3), 7.14 (d, J = 15.9 Hz, 2H, –C=CH–), 7.50 (s,
4H, ArH), 7.64 (d, J = 15.9 Hz, 2H, –CH=C–). LC-MS (m/z): 490.
Anal. Calcd. for C33H46O3: C 80.77, H 9.45. Found: C 80.63, H 9.40.
2,5-Bis(3,5-di-tert-butyl-4-hydroxylbenzylidene)cyclo-
pentanone B4
Yield 82%, mp. 137–1388C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
1.45 (s, 36H, –C–CH3), 3.05 (4H, –H2C–CH2 –), 7.35 (brs, 2H, –
OH), 7.43 (s, 4H, ArH), 7.51 (s, 2H, –CH=). LC-MS (m/z): 516. Anal.
Calcd. for C35H48O3: C 81.35, H 9.36. Found: C 81.28, H 9.30.
1,5-Bis(3,4-dimethoxyphenyl)-1,4-pentadiene-3-one C5
Yield 98%, mp. 89–908C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
3.81 (s, 6H, OCH3), 3.84 (s, 6H, OCH3), 7.01 (d, J = 8.1 Hz, 2H, ArH),
7.21 (d, J = 15.9 Hz, 2H, –C=CH–), 7.31 (d, J = 8.1 Hz, 2H, ArH),
7.38 (s, 2H, ArH), 7.68 (d, J = 15.9 Hz, 2H, –CH=C–). LC-MS (m/z):
354. Anal. Calcd. for C21H22O5: C 71.17, H 6.26. Found: C 71.02, H
6.14.
2,5-Bis(3,4-dimethoxybenzylidene)cyclopentanone B5
Yield 85%, mp. 186–1888C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
3.10 (4H, –CH2 –CH2 –), 3.81 (12, O–CH3), 7.06 (d, J = 8.1 Hz, 2H,
ArH), 7.27 (d, J = 8.1 Hz. 4H, ArH), 7.35 (s, 2H, –CH=). LC-MS (m/z):
380. Anal. Calcd. for C23H24O5: C 72.61, H 6.36. Found: C 72.52, H
6.32.
1,5-Bis(4-hydroxy-3,5-dimethoxyphenyl)-1,4-pentadiene-
3-one C6
Yield 65%, mp. 164–1668C. 1H-NMR (DMSO-d6, 300 MHz)
d
(ppm): 3.83 (s, 12H, OCH3), 7.07 (s, 4H, ArH), 7.17 (d, J = 15.9 Hz,
2H, –C=CH–), 7.63 (d, J = 15.9 Hz, 2H, –CH=C–), 9.00 (brs, 2H, –
OH). LC-MS (m/z): 386. Anal. Calcd. for C21H22O7: C 65.28, H 5.74.
Found: C 65.16, H 5.65.
2,5-Bis(4-hydroxy-3,5-dimethoxybenzylidene)cyclo-
pentanone B6
Yield 82%, mp. 226–228 8C.1H-NMR (DMSO-d6, 300 MHz) d (ppm):
3.12 (t, J = 6.7 Hz, 4H, –CH2 –CH2 –), 3.82 (s, 12H, OCH3), 6.96 (s,
4H, ArH), 7.36 (s, 2H, –CH=), 9.03 (brs, 2H, –OH). LC-MS (m/z):
412. Anal. Calcd. for C23H24O7: C 66.98, H 5.87. Found: C 66.83, H
5.82.
3,5-Bis(4-hydroxyphenyl)-4-piperidone D1
1
Yield 90%, mp. A 3008C. H-NMR (DMSO-d6, 300 MHz) d (ppm):
4.45 (s, 4H, –CH2 –), 6.91 (d, J = 8.1 Hz, 4H, ArH), 7.38 (d, J = 8.1 Hz,
4H, ArH), 7.77 (s, 2H, –CH=), 9.50 (brs, 2H, –N+ –H), 10.23 (brs,
2H, –OH). LC-MS (m/z): 343. Anal. Calcd. for C19H18ClNO3: C 66.38,
H 5.28, N 4.07. Found: C 66.18, H5.20, N 4.04.
2,5-Bis(3-bromo-4-hydroxy-5-methoxybenzylidene)cyclo-
pentanone B7
Yield 45%, mp. 280–2818C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
3.07 (4H, –CH2 –CH2 –), 3.89 (s, 6H, OCH3), 7.27 (s, 2H, ArH), 7.32
(s, 2H, ArH), 7.41 (s, 2H, –CH=), 10.09 (brs, 2H, –OH). LC-MS (m/
z):508. Anal. Calcd. for C21H18Br2O5: C 49.44, H 3.56. Found: C
49.32, H 3.52.
3,5-Bis(3,4-dihydroxybenzylidene)-4-piperidone D2
1
Yield 90%, mp. A 3008C. H-NMR (DMSO-d6, 300 MHz) d (ppm):
4.43 (s, 4H, –CH2 –), 6.82 (d, J = 8.1 Hz, 4H, ArH), 6.90 (s, 2H, ArH),
7.67 (s, 2H, –CH=), 9.37 (brs, 2H, –OH), 9.60 (brs, 2H, –N+ –H),
9.79 (brs, 2H, –OH). LC-MS (m/z): 375. Anal. Calcd. for
C19H18ClNO5: C 60.72, H 4.83, N 3.73. Found: C 60.62, H4.78, N
3.69.
1,5-Bis(4-hydroxyphenyl)-1,4-pentadiene-3-one C1
Yield 95%, mp. 243–2458C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
6.82 (d, J = 8.1 Hz, 4H, ArH), 7.08 (d, J = 15.9 Hz, 2H, –C=CH–),
7.60 (d, J = 8.1 Hz, 4H, ArH), 7.64 (d, J = 15.9 Hz, 2H, –CH=C–),
10.01 (brs, 2H, –OH). LC-MS (m/z): 266. Anal. Calcd. for C17H14O3:
C 76.68, H 5.30. Found: C 76.56, H 5.23.
3,5-Bis(4-hydroxy-3-methoxybenzyliden)-4-piperidone D3
Yield 98%, mp. 254–2568C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
4.49 (s, 4H, –CH2 –), 6.95 (d, J = 8.1 Hz, 4H, ArH), 7.11 (s, 2H, ArH),
7.79 (s, 2H, –CH=), 9.62 (brs, 2H, –N+ –H), 9.86 (brs, 2H, –OH). LC-
MS (m/z): 403. Anal. Calcd. for C21H22ClNO3: C 62.45, H 5.49, N
3.47. Found: C 62.31, H5.46, N 3.42.
1,5-Bis(3,4-dihydroxyphenyl)-1,4-pentadiene-3-one C2
Yield 90%, mp. 150–1548C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
6.78 (d, J = 8.1 Hz, 2H, ArH), 6.98 (d, J = 15.9 Hz, 2H, –C=CH–),
7.06 (d, J = 8.1 Hz, 2H, ArH), 7.13 (s, 2H, ArH), 7.54 (d, J = 15.9 Hz,
2H, –CH=C–), 9.11 (brs, 2H, –OH), 9.58 (brs, 2H, –OH). LC-MS (m/
z): 298. Anal. Calcd. for C17H14O5: C 68.45, H 4.73. Found: C 68.26,
H 4.67.
3,5-Bis(3,5-di-tert-butyl-4-hydroxylbenzylidene)-4-
piperidone D4
Yield 98%, mp. 231–2338C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
1.42 (s, 36H, C–CH3), 4.51 (s, 4H, –CH2 –), 7.68 (s, 4H, ArH), 7.81
(s, 2H, –CH=), 9.40 (brs, 2H, –N+ –H), 9.45 (brs, 2H, –OH). LC-MS
(m/z): 567. Anal. Calcd. for C35H50ClNO3: C 73.98, H 8.87, N 2.46.
Found: C 73.79, H8.81, N 2.41.
1,5-Bis(4-hydroxy-3-methoxphenyl)-1,4-pentadiene-3-
one C3
Yield 98%, mp. 99–1008C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
3.84 (s, 6H, OCH3), 9.60 (brs, 2H, –OH), 6.81 (d, J = 8.1 Hz, 2H,
ArH), 7.12 (d, J = 15.9 Hz, 2H, –C=CH–), 7.18 (d, J = 8.1 Hz, 2H,
ArH), 7.35 (s, 2H, ArH), 7.63 (d, J = 15.9 Hz, 2H, –CH=C–). LC-MS
(m/z): 326. Anal. Calcd. for C19H18O5: C 69.93, H 5.56. Found: C
69.79, H 5.51.
3,5-Bis(3,4-dimethoxybenzylidene)-4-piperidone D5
Yield 88%, mp. 216–2188C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
3.83 (s, 3H, O–CH3), 3.82 (s, 3H, O–CH3), 4.50 (s, 4H, –CH2 –), 7.10
(d, J = 8.1 Hz, 4H, ArH), 7.14 (s, 2H, ArH), 7.82 (s, 2H, –CH=), 9.82
(brs, 2H, –N+ –H). LC-MS (m/z): 431. Anal. Calcd. for C23H26ClNO5:
C 63.96, H 6.07, N 3.24. Found: C 63.78, H5.98, N 3.21.
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

128
Z.-Y. Du et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 123–128
required to produce 50% inhibition of the test models) were
determined from least-squares analyses of the linear portion of
the log dose-inhibition curves. Each curve was generated using
at least three concentrations of inhibitor causing an inhibition
between 20-80% with two replicates at each concentration. The
95% confidence limits (95% C. L.) were calculated from T values
for n-2, where n is the total number of determinations. The pro-
tein content of the enzyme preparation was determined using
Coomassie blue reagent (Bio-Rad, Guangzhou, China) according
to the manufacturer's instructions, using bovine serum albumin
(BSA) as a standard.
3,5-Bis(4-hydroxy-3,5-dimethoxybenzylidene)-4-
piperidone D6
Yield 88%, mp. 231–2338C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
3.82 (s, 6H, O–CH3), 4.55 (s, 4H, –CH2 –), 6.82 (s, 4H, ArH), 7.78 (s,
2H, –CH=), 9.20 (brs, 2H, –N+ –H), 9.74 (brs, 2H, –OH). LC-MS (m/
z): 463. Anal. Calcd. for C23H26ClNO7: C 59.55, H 5.65, N 3.02.
Found: C 59.42, H5.56, N 2.96.
3,5-Bis(3-bromo-4-hydroxy-5-methoxybenzylidene)-4-
piperidone D7
Yield 75%, mp. 260–2628C. 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
3.86 (s, 6H, O–CH3), 4.50 (s, 4H, –CH2 –), 7.22 (m, 4H, ArH), 7.75
(s, 2H, –CH=), 9.73 (brs, 2H, –N+ –H), 10.24 (brs, 2H, –OH). LC-MS
(m/z): 559. Anal. Calcd. for C21H20Br2ClNO5: C 44.91, H 3.59, N
2.49. Found: C 44.82, H 3.52, N 2.44.
References
[1] P. F. Kador, Med. Res. Rev. 1988, 8, 325–352.
[2] C. Yabe-Nishimura, Pharmacol. Rev. 1998, 50, 21–33.
[3] M. Brownlee, Nature 2001, 414, 813–820.
Aldose reductase inhibitory assays
Aldose reductase was isolated and purified from bovine lenses
according to the methods described [16] with slight modifica-
tion. Briefly, bovine lenses were obtained from freshly-slaugh-
tered animals and homogenized in 3 vol. of cold 5 lM Tris-HCl
buffer, pH 7.4, containing 5 lM b-mercaptoethanol. The homo-
genate was centrifuged at 15 000 rpm at 0–48C for 30 min.
Saturated ammonium sulfate solution was added to the super-
natant fraction to form a 40% solution, which was stirred for
30 min at 0–48C and then centrifuged at 15000 rpm for 30 min.
Following the same procedure, the recovered supernatant was
subsequently fractionated with saturated ammonium sulfate
solution using first 50% and then 75% saturation. The precipi-
tate recovered from the 75% saturated fraction, containing
aldose reductase activity, was re-dissolved in 0.05 M NaCl and
dialyzed overnight in 5 lM Tris-HCl buffer, pH 7.4, and then the
enzyme solution was subjected to ion exchange chromatogra-
phy (DE-52). All procedures were carried out at 0–48C.
[4] L. Costantino, G. Rastelli, P. Vianello, G. Cignarella, D.
Barlocco, Med. Res. Rev. 1999, 19, 3–23.
[5] S. Miyamoto, Exp. Opin. Ther. Patents 2002, 12, 621–631.
[6] L. Costantino, G. Rastelli, M. C. Gamberini, D. Barlocco,
Exp. Opin. Ther. Patents 2000, 10, 1245–1262.
[7] D. Ziegler, Nephrol. Dial. Transplant. 2004, 19, 2170–2175.
[8] Z. W. Wang, W. Y. La, Q. Yuan, Z. Y. Feng, D. C. Zheng,
Chinese J. Pr. Chinese Modern Med. 1995, 10, 600–601.
[9] L. Costantino, G. Rastelli, M. C. Gamberini, J. A. Vinson,
P. Bose, A. Iannone, M. Staffieri, L. Antolini, A. Del Corso,
U. Mura, A. Albasini, J. Med. Chem. 1999, 42, 1881–1893.
[10] J. A. de la Fuente, M. Sonia, Nat. Prod. Rep. 2003, 20, 243–
251.
[11] G. Rastelli, L. Antolini, S. Benvenuti, L. Costantino,
Bioorg. Med. Chem. 2000, 8, 1151–1158.
The activity of this preparation was determined by measuring
the amount of NADP+ converted from NADPH per unit time at
378C as reported previously [17]. One unit (U) of activity of
defined as the amount of the enzyme catalyzing the oxidation of
1 mmol of NADPH per minute under our experimental condi-
tions. Enzyme activity was measured by monitoring the change
in absorbance, which accompanies the oxidation of NADPH cata-
lyzed by ALR2, at 340 nm on a UV spectrophotometer (UV-2450,
Shimadzu, Japan). The incubation mixture contained 0.20 M Na,
K-phosphate buffer (pH 6.8), 0.30 M ammonium sulfate, 0.10 lM
NADPH, 4.7 lM DL-glyceraldehyde, and enzyme preparation,
with or without an inhibitor, at a total volume of 1.0 mL. Each
inhibitor was dissolved in dimethyl sulfoxide, which was found
to have no effect on the enzyme activity at less than 1%. Appro-
priate blanks contained all of the above mentioned compounds
except DL-glyceraldehyde was used to correct for the non-enzy-
matic oxidation of NADPH. The reaction was initiated by adding
NADPH at 378C. IC50 values (the concentration of the inhibitor
[12] F. Severi, S. Benvenuti, L. Costantino, G. Vampa, M. Mele-
gari, L. Antolini, Eur. J. Med. Chem. 1998, 33, 859–866.
[13] T. Kosuge, H. Ishida, H. Yamazaki, Chem. Pharm. Bull.
1985, 33, 1499–1502.
[14] S. S. Sardjiman, M. S. Reksohadiprodjo, L. Kakim, H. van
der Goot, H. Timmerman, Eur. J. Med. Chem. 1997, 32,
625–630.
[15] M. Artico, R. D. Santo, C. Roberta, E. Novellino, G. Greco,
S. Massa, E. Tramontano, M. E. Marongiu, A. D. Montis, P.
L. Colla, J. Med. Chem. 1998, 41, 3948–3960.
[16] S. Hayman, J. H. Kinoshita, J. Med. Chem. 1965, 240, 877–
882.
[17] L. Costantino, G. Rastelli, K. Vescovini, G. Cignarella, P.
Vianello, A. D. Corso, M. Cappiello, U. Mura, D. Barlocco,
J. Med. Chem. 1996, 39, 4396–4405.
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com