Synthesis of antioxidants for prevention of age-related macular degeneration

Article abstract of DOI:10.1021/np300769c

Photooxidation of A2E may be involved in diseases of the macula, and antioxidants could serve as therapeutic agents for these diseases. Inhibitors of A2E photooxidation were prepared by Mannich reaction of the antioxidant quercetin. These compounds contai

Full text of DOI:10.1021/np300769c

Note
pubs.acs.org/jnp
Synthesis of Antioxidants for Prevention of Age-Related Macular
Degeneration
Dharati Joshi,† James Field,† John Murphy,† Mohammed Abdelrahim,† Heike Schonherr,‡
̈
Janet R. Sparrow,‡ George Ellestad,† Koji Nakanishi,† and Arie Zask*,†
†Department of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027, United States
‡Department of Ophthalmology, Columbia University, 630 West 168th Street, New York, New York 10032, United States
S
* Supporting Information
ABSTRACT: Photooxidation of A2E may be involved in diseases of the macula,
and antioxidants could serve as therapeutic agents for these diseases. Inhibitors of
A2E photooxidation were prepared by Mannich reaction of the antioxidant
quercetin. These compounds contain water-solubilizing amine groups, and several
were more potent inhibitors of A2E photooxidation than quercetin.
A2E, a pigment of the lipofuscin of retinal pigment cells, is
thought to play a role in diseases of the retina such as macular
degeneration, and methods to prevent its formation, or
ameliorate its destructive effects, have been proposed as
therapeutic approaches.1−3 A2E is a pyridinium bis-retinoid4,5
and is formed intracellularly from retinal and phosphatidyle-
thanolamine6 (Figure 1). Studies have shown that A2E
photosensitizes singlet oxygen production upon irradiation in
the visible region, leading to formation of autoxidation
products, including the nonaoxirane shown in Figure 1,
resulting from epoxidation of the A2E olefins.7,8 These
oxidation products have been proposed to lead to cellular
damage and death.2,9 Antioxidants, such as anthocyanins
isolated from bilberry, vitamin E, and resveratrol, can inhibit
A2E autoxidation.9 In an effort to prepare more stable and
effective antioxidants than the anthocyanins, a recent study
from our laboratories utilized quercetin linked to antioxidants
such as curcumin and caffeic acid to inhibit A2E oxidation.10
The Mannich reaction is a versatile reaction that leads to the
incorporation of amines into organic molecules. Amines have
been used extensively as water-solubilizing groups in drugs to
improve physicochemical properties (e.g., solubility) leading to
improved bioavailability and formulation. We have used the
Mannich reaction to prepare compounds that combine multiple
antioxidants with water-solubilizing amine groups. These
substances have been evaluated in noncellular and intracellular
assays of A2E photooxidation and shown to prevent irradiation-
induced destruction of A2E.
photooxidation of A2E.9,10 We prepared previously analogues
wherein quercetin, caffeic acid, and curcumin were linked
through aliphatic groups for this purpose.10 A different
approach to covalent modification is utilized in the present
study, where the Mannich reaction is used to connect
antioxidants through amine linkers. The chemistry in this
approach is straightforward and leads to analogues containing
water-solubilizing amines, which are found in many therapeutic
agents and confer desirable physicochemical properties and
improved bioavailability and formulation.
Quercetin11 (1) and sesamol12 (2) can undergo regiose-
lective Mannich reactions under certain conditions, and we
were able to selectively synthesize dimers of quercetin (3 and
4) and sesamol (5 and 6), by utilizing diamines (piperazine or
N1,N2-dimethylethylenediamine, respectively) in the Mannich
reaction of quercetin and sesamol with formaldehyde (Scheme
1). Reaction of quercetin with morpholine and formaldehyde
regioselectively gave the Mannich adduct 7 (Scheme 2).11
Mannich reaction of quercetin (1) with dopamine occurred
with concomitant Pictet−Spengler reaction of dopamine and
formaldehyde to give compound 8 (Scheme 3). Whereas
sesamol (2) and quercetin (1) underwent Mannich reaction
regioselectively at room temperature with stoichiometric
quantities of base and formaldehyde, chalcone 9 required
elevated temperatures and excess reagents and gave equimolar
mixtures of regioisomers 10a and 10b when morpholine was
used as the amine (Scheme 4). When treated with piperazine
and formaldehyde, three possible regioisomeric dimers of the
chalcone were obtained (11a−c), as expected.
Irradiation of A2E at its absorption maximum of 440 nm
leads to singlet oxygen generation and subsequent oxidation of
A2E. The epoxide oxidation products of A2E are hypothesized
to act as destructive agents within cells, causing cell damage and
death, and may be responsible for several diseases of the retina.
A potential treatment for retinal damage would be to inhibit the
oxidation of A2E with antioxidants, and several natural products
and their synthetic derivatives have been shown to inhibit
Only antioxidants containing quercetin (3, 4, 7, and 8)
showed sufficient antioxidant ability to inhibit noncellular and
Special Issue: Special Issue in Honor of Lester A. Mitscher
Received: November 2, 2012
Published: January 24, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Formation of A2E from retinal and phosphatidylethanolamine and oxidation to A2E nonaoxirane.
Scheme 1. Synthesis of Homodimeric Antioxidants of Quercetin (1) and Sesamol (2)
were tested at 50, 100, and 200 μM concentrations and
compared with quercetin at the same concentration (Figure
2B). A 400 μM quercetin sample was also tested to allow for
comparison with the quercetin dimers 3 and 4 at 200 μM.
Interestingly, while 7 and 8 showed inhibition and dose−
response curves comparable to that of quercetin, compounds 3
and 4 gave shallow dose−response curves, so that the inhibition
of A2E oxidation at 50 μM was comparable to that of quercetin
at 200 μM. This is twice the antioxidant capacity that would be
expected on the basis of the stoichiometry of two quercetin
units per molecule for 3 and 4. Similarly, single concentration
determinations at 100 μM showed that compounds 3 and 4
outperformed quercetin in inhibiting A2E photooxidation
(Figure 2A). That compounds 3 and 4 contain a diamine
(piperazine or N1,N2-dimethylethane-1,2-diamine, respectively)
may explain the increase in antioxidant ability. Phenolic
compounds have been shown to be better antioxidants as the
corresponding phenolate ions at basic pH.14 Hence, the basicity
of the diamine group in proximity to the phenolic hydroxy
groups may give rise to the increased antioxidant effect seen in
the present investigation.
Scheme 2. Regioselective Formation of Morpholine/
Quercetin Adduct 7
Scheme 3. Synthesis of Quercetin/Tetrahydroisoquinoline
Conjugate 8
To evaluate the efficacy of these compounds in inhibiting
intracellular photooxidation of A2E, human adult retinal
pigment epithelium (RPE) cells (ARPE-19 cell line) were
allowed to accumulate A2E by incubation with 10 μM A2E.
Subsequently, cells were allowed to accumulate antioxidants 3,
4, 7, 8, and quercetin (1) by treatment of the cells with a 20
intracellular photooxidation of A2E (Figure 2). While sesamol
and chalcone 9 are known antioxidants, they were ineffective
inhibitors of A2E photooxidation at 200 μM. This finding is
consistent with the lack of ortho phenolic groups in sesamol
and chalcone 9 and the correlation of ortho phenolic groups
with antioxidant capacity.13 The quercetin-containing analogues
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Note
Scheme 4. Mannich Reaction of Chalcone 9
Figure 2. Protective effects of antioxidants on the photooxidation of A2E. (A) Comparison of the inhibition of in vitro (noncellular) A2E
photooxidation by quercetin (1) and compounds 3, 4, 7, and 8 at 100 μM. (B) Inhibition of in vitro (noncellular) A2E photooxidation upon varying
the concentrations of antioxidants 3, 4, 7, 8, and quercetin (1). (C) Comparison of the cellular (RPE cells accumulated antioxidant from a 20 μM
concentration in medium) inhibition of A2E photooxidation by compounds 3, 4, 7, 8, and quercetin (1).
Column chromatography was performed on silica gel (particle size
40−63 μm) (Sorbent Technologies, Atlanta, GA, USA), and TLC
plates (w/UV 254) were used for fraction and compound detection.
The spots were visualized using UV light at 254 nm. All final
compounds were >95% pure as determined by analytical reversed-
phase HPLC.
μM solution. Irradiation of the cells at 430 nm led to inhibition
of A2E photooxidation as determined by quantitation of
cellular A2E levels. The results obtained (Figure 2C) are very
similar to those obtained in vitro (noncellular) (Figure 2A),
indicating that the compounds are able to penetrate the cells to
provide protection to intracellular A2E upon irradiation.
In conclusion, the Mannich reaction has been instrumental in
the design and synthesis of novel antioxidants. Several of these
compounds containing quercetin were potent inhibitors of A2E
photooxidation and superior to quercetin itself. The water-
solubilizing amine groups in these antioxidants were designed
to provide drug-like physicochemical properties and may be
responsible for the improved antioxidant activity.
Analytical reversed-phase HPLC measurements were carried out on
an Alliance System (Waters Corp., Milford, MA, USA) equipped with
a 2695 separation module, a 2996 photodiode array detector, and a
2475 multi-λ fluorescence detector. For chromatographic separation,
an analytical scale Atlantis dC18 (3 μm, 4.6 mm × 150 mm, Waters)
column was utilized with an acetonitrile and water gradient and 0.1%
trifluoroacetic acid (85−100%, 0.8 mL/min 25 min; 100% acetonitrile,
0.8 mL/min 15 min; monitoring at 430 nm; 20 μL injection volume).
Peak area was determined using Empower (Waters) software.
Alternatively, analytical reversed-phase HPLC measurements were
carried out on a JASCO System equipped with an MD-1510
multiwavelength detector. For chromatographic separation, an
analytical-scale Thermo Scientific Hypersil Gold C18 (150 × 4.6
mm) column was utilized with an acetonitrile and water gradient and
0.1% trifluoroacetic acid and 80 μL injection volume monitoring at
440 nm. Peak area was determined using ChromNAV (JASCO)
software.
EXPERIMENTAL SECTION
■
General Experimental Procedures. A2E was prepared from
retinal and ethanolamine by the method described in the literature.15
1H NMR spectra were recorded with a Bruker DRX300 or Bruker
Avance III 500 instrument. 13C NMR spectra were recorded with a
Bruker Avance III 500 instrument. Chemical shift values are given in δ
(ppm) using the peak signals of the solvent DMSO-d6 (δH 2.50 and δC
39.50) as references, and coupling constants are reported in Hz.
FABMS (3KV Xe beam) data were measured with an HX110 JEOL
Ltd. (Tokyo Japan) double focusing sector type mass spectrometer.
8,8′-(Piperazine-1,4-diylbis(methylene))bis(2-(3,4-dihydrox-
yphenyl)-3,5,7-trihydroxy-4H-chromen-4-one (3). To a solution
of quercetin dihydrate (200 mg, 0.59 mmol) and piperazine (26 mg,
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Note
0.30 mmol) in ethanol (5 mL) was added 37% aqueous formaldehyde
(0.044 mL, 0.59 mmol). A precipitate formed slowly over 30 min.
After 48 h the reaction mixture was filtered to provide the product as a
102.86, 111.65, 114.59, 121.76, 128.95, 129.08, 130.78, 132.34, 134.59,
143.92, 163.23, 164.17, 192.06; MS of 10b m/z MH+ 354.
(E)-1-(2-Hydroxy-3-((4-(4-hydroxy-2-methoxy-5-((E)-3-phe-
nylprop-2-enoyl)benzyl)piperazin-1-yl)methyl)-4-methoxy-
phenyl)-3-phenylprop-2-en-1-one (11a), (2E,2′E)-1,1′-(3,3′-(Pi-
perazine-1,4-diylbis(methylene))bis(2-hydroxy-4-methoxy-
3,1-phenylene))bis(3-phenylprop-2-en-1-one) (11b), and
(2E,2′E)-1,1′-(5,5′-(Piperazine-1,4-diylbis(methylene))bis(2-hy-
droxy-4-methoxy-5,1-phenylene))bis(3-phenylprop-2-en-1-
one) (11c). To a solution of 9 (63 mg, 0.247 mmol) in 2-propanol
(3.0 mL) were added paraformaldehyde (12.0 mg, 0.40 mmol) and
anhydrous piperazine (15.0 mg, 0.174 mmol). The mixture was
refluxed for 5 h and then stirred at room temperature overnight. The
resulting precipitate was collected to give a mixture of regioisomers as
a yellow powder (41 mg): MS m/z MH+ 619.
Noncellular Photooxidation of A2E Assay. To test the effect of
antioxidants on A2E photooxidation, 100 μL of A2E stock (200 μM in
DMSO) was added to 200 μL of DPBS as described in the literature.9
To these samples was added 1.5 μL of antioxidant (20 mM in DMSO)
to obtain a final concentration of 100 μM. These mixtures were
irradiated (430 nm; light intensity of 2.8 mW/cm2) for 5 min. Samples
were analyzed by reversed-phase HPLC with sample preparation and
injection performed under dim red light. To test the effects of varying
concentrations of antioxidants on A2E photooxidation, the appropriate
volume of antioxidant solution (20 mM in DMSO) was added to the
solution prepared by adding 100 μL of A2E stock solution (200 μM in
DMSO) to 200 μL of DPBS, and the samples were irradiated as
described above. Samples were diluted with EtOH (0.5 mL) and
analyzed by reversed-phase HPLC. A2E loss relative to unirradiated
samples was interpreted as percent photooxidation.
Cellular Photooxidation of A2E Assay. A human adult RPE cell
line (ARPE-19; American Type Culture Collection, Manassas, VA,
USA) that is devoid of endogenous A2E was cultured in 35 mm cell
culture dishes (Corning, NY, USA) to confluence for 5 days as
previously reported.17,18 The cells were allowed to accumulate
synthesized A2E15 for 18 days from a 10 μM concentration in culture
medium that included 10% fetal bovine serum (Invitrogen, Carlsbad,
CA, USA). From day 10 to 18 the cells were also incubated with 20
μM antioxidant. Subsequently, the cultures were washed 10 times with
DPBS (total 500 μL volume) and then in DPBS were irradiated at 430
nm (2.8 mW/cm2) for 10 min. The cells were then detached from the
culture dish with trypsin, washed, pelleted, and counted three times
(Bright Light Counting Chamber, Hausser Scientific Company,
Horsham, PA, USA). The cells were dried under vacuum in a
desiccator for 2 h following which 100 μL of methanol was added to
each vial for homogenization. After centrifugation, 30 μL of
supernatant solution was used for HPLC analysis. A2E loss relative
to unirradiated samples was interpreted as percent photooxidation.
1
yellow powder (185 mg, 85% yield): H NMR (DMSO-d6) δ 2.63
(8H, s, bd), 3.88 (4H, s), 6.19 (2H, s), 6.89 (2H, d, J = 9.0 Hz), 7.58
(2H, dd, J = 2.1, 9.0 Hz), 7.71 (2H, d, J = 2.1 Hz), 12.52 (2H, s, bd);
13C NMR (DMSO-d6) δ 50.9, 52.1, 98.1, 99.9, 102.8, 115.0, 115.7,
119.9, 122.2, 135.7, 145.1, 146.5, 147.7, 153.8, 159.6, 164.0, 176.0; MS
m/z MH+ = 715.
8,8′-(Ethane-1,2-diylbis(methylazanediyl))bis(methylene)-
bis(2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-
one) (4). To a solution of quercetin dihydrate (100 mg, 0.30 mmol)
and N1,N2-dimethylethane-1,2-diamine (0.036 mL, 0.33 mmol) in
ethanol (2 mL) was added 37% aqueous formaldehyde (0.041 mL,
0.33 mmol). After 48 h the resulting precipitate was collected to
provide the product as a yellow powder: 1H NMR (DMSO-d6) δ 2.29
(6H, s), 2.86 (4H, s), 3.93 (4H, s), 6.10 (2H, s), 6.86 (2H, d, J = 8.5
Hz), 7.53 (2H, d, J = 8.5 Hz), 7.67 (2H, s), 12.53 (2H, s, bd); 13C
NMR (DMSO-d6) 41.1, 50.5, 51.4, 53.9, 79.4, 98.8, 99.6, 101.8, 114.9,
115.5, 119.9, 122.3, 135.4, 145.4, 146.2, 147.7, 154.3, 160.0, 166.8,
175.6; MS m/z MH+ 717.
6,6′-(Piperazine-1,4-diylbis(methylene))dibenzo[d][1,3]-
dioxol-5-ol (5). To a solution of sesamol (200 mg, 1.44 mmol) and
piperazine (62 mg, 0.72 mmol) in ethanol was added 37% aqueous
formaldehyde (0.108 mL, 1.44 mmol) over 1 min. After 2−3 min, a
precipitate formed. After 18 h, the reaction mixture was filtered and
the resulting solid washed with ethanol. The product was obtained as a
tan solid (213 mg, 77% yield): 1H NMR (DMSO-d6) δ 2.4 (8H, s, bd),
3.50 (4H, s), 5.87 (4H, s), 6.37 (2H, s), 6.65 (2H, s); 13C NMR
(DMSO-d6) δ 51.9, 58.1, 97.7, 100.5, 108.8, 113.5, 139.5, 146.7, 151.5;
MS m/z M+ 386.
6,6′-(Ethane-1,2-diylbis(methylazanediyl))bis(methylene)-
dibenzo[d][1,3]dioxol-5-ol (6). To a solution of sesamol (100 mg,
0.72 mmol) and N1,N2-dimethylethane-1,2-diamine (0.039 mL, 0.36
mmol) in ethanol was added 37% aqueous formaldehyde (0.054 mL,
0.72 mmol). The resulting precipitate was collected to provide the
product as a powder (62 mg, 44% yield): 1H NMR (DMSO-d6) δ 2.14
(6H, s), 2.54 (4H, s), 3.49 (4H, s), 5.87 (4H, s), 6.37 (2H, s), 6.67
(2H, s); 13C NMR (DMSO-d6) δ 41.0, 53.3, 58.0, 97.7, 100.4, 108.8,
114.3, 139.4, 146.6, 151.7; MS m/z MH+ 389.
8-((6,7-Dihydroxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-
2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one
(8). To a solution of quercetin dihydrate (100 mg, 0.30 mmol) and
dopamine hydrochloride (63 mg, 0.33 mmol) in ethanol (2 mL) was
added triethylamine (0.046 mL, 0.33 mmol) followed by 37% aqueous
formaldehyde (0.025 mL, 0.33 mmol). After 72 h the reaction was
filtered to provide a brown powder (68 mg). Silica gel chromatography
(20% MeOH in CH2Cl2) provided the product as a yellow solid (38
1
mg, 43%): H NMR (DMSO-d6, 300 MHz) δ 2.73 (2H, s, bd) 3.00
ASSOCIATED CONTENT
■
(2H, s, bd) 3.79 (2H, s, bd), 4.16 (2H, s, bd), 6.23 (1H, s), 6.43 (1H,
s), 6.49 (1H, s), 6.88 (1H, d, J = 8.4 Hz), 7.55 (1H, dd, J = 1.8, 8.4
Hz), 7.74 (1H, d, J = 1.8 Hz), 12.56 (1H, s, bd); MS m/z MH+ 480.
(E)-1-(2-Hydroxy-4-methoxy-5-(morpholinomethyl)phenyl)-
3-phenylprop-2-en-1-one (10a) and (E)-1-(2-Hydroxy-4-me-
thoxy-3-(morpholinomethyl)phenyl)-3-phenylprop-2-en-1-
one (10b). To a solution of 9 (0.443 g, 1.74 mmol) in 2-propanol
(5.0 mL) were added paraformaldehyde (0.131 g, 4.37 mmol) and
morpholine (330 μL, 3.52 mmol). The mixture was refluxed for 6 h
and then stirred at room temperature overnight to give a yellow
precipitate (489 mg). Silica gel chromatography (EtOAc/CH2Cl2)
gave 10a as a yellow solid (151 mg, 25% yield).16 Further elution of
the column with MeOH/EtOAc gave 10b as a yellow solid (139 mg,
23% yield). 1H NMR of 10a δ 2.41−2.38 (4H, m), 3.46 (1H, s), 3.57−
3.54 (4H, m), 3.89 (3H, s), 6.57 (1H, s), 7.59−7.48 (3H, m), 7.82
(1H, d, J = 15.5 Hz), 7.92−7.89 (2H, m), 7.97 (1H, d, J = 15.5), 8.12
S
* Supporting Information
1H and 13C NMR spectra are available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +01-646-709-7801. Fax: +01-212-932-1289. E-mail:
AZ2280@columbia.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
1
(1H, s), 13.39 (1H, s); MS of 10a m/z MH+ = 354. H NMR of 10b
J.R.S. and H.S. were supported by National Institutes of Health
grant RO1 EY12951 (J.R.S.) and by a grant from Research to
Prevent Blindness to the Department of Ophthalmology,
Columbia University.
(DMSO-d6) δ 2.41 (4H, s, bd), 3.51−3.53 (4H, m), 3.55 (2H, s), 3.90
(3H, s), 6.72 (1H, d, J = 9.1 Hz), 7.48−7.47 (3H, m), 7.82 (1H, d, J =
15.5 Hz), 7.91−7.90 (2H, m), 8.03 (1H, d, J = 15.5 Hz), 8.29 (1H, d, J
= 9.0 Hz); 13C NMR of 10b (DMSO-d6) δ 49.27, 53.06, 56.15, 66.20,
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Journal of Natural Products
Note
DEDICATION
■
Dedicated to Dr. Lester A. Mitscher, of the University of
Kansas, for his pioneering work on the discovery of bioactive
natural products and their derivatives.
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NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published on the Web on January 24, 2013,
with an error in Figure 1. The corrected version was reposted
on February 28, 2013.
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