Chroman/catechol hybrids: Synthesis and evaluation of their activity against oxidative stress induced cellular damage

Article abstract of DOI:10.1021/jm0506120

Three series of chromans substituted at positions 2 or 5 by catechol derivatives were synthesized, and their activity against oxidative stress induced cellular damage was studied. Specifically, the ability of the new molecules to protect cultured cells fr

Full text of DOI:10.1021/jm0506120

300
J. Med. Chem. 2006, 49, 300-306
Chroman/Catechol Hybrids: Synthesis and Evaluation of Their Activity against Oxidative
Stress Induced Cellular Damage
Maria Koufaki,*,† Elissavet Theodorou,† Dimitrios Galaris,‡ Lambros Nousis,‡ Efrosini S. Katsanou,§ and Michael N. Alexis§
Institute of Organic and Pharmaceutical Chemistry and Institute of Biological Research and Biotechnology, National Hellenic Research
Foundation, 48 Vas. Constantinou AVe., 116 35 Athens, Greece, and Laboratory of Biological Chemistry, UniVersity of Ioannina, School of
Medicine, 45110 Ioannina, Greece
ReceiVed June 28, 2005
Three series of chromans substituted at positions 2 or 5 by catechol derivatives were synthesized, and their
activity against oxidative stress induced cellular damage was studied. Specifically, the ability of the new
molecules to protect cultured cells from H2O2-induced DNA damage was evaluated using single cell gel
electrophoresis (comet assay), while the neuroprotective activity of the new compounds against oxidative
stress induced programmed cell death was studied using glutamate-challanged hippocampal HT22 cells.
The majority of the new compounds are stronger neuroprotectants than quercetin. 5-Substituted chroman
analogues such as the caffeic acid amides 12 and 16 and the dihydrostilbene analogue 24 were the most
potent against both H2O2- and glutamate-induced damage in Jurkat T cells and HT22 cells, respectively.
Introduction
•OH, due to their extreme reactivity, interact exclusively in the
•
vicinity of the bound metal.9 Formation of OH close to DNA
Low amounts of reactive oxygen species (ROS) are continu-
ously generated in the cells of aerobic organisms, and they are
likely to play important physiological roles, especially in signal
transduction processes.1,2 However, elevated steady-state levels
of these species (a situation usually called “oxidative stress”)
has been implicated in the initiation or progression of various
pathological conditions including neurological diseases, par-
ticularly neurodegenerative diseases, as well as cardiovascular
diseases and cancer.3-6
Oxidative damage and disease progression may be retarded
by administering exogenous protective compounds, which can
act in several different ways such as chain breaking antioxidants
or free radical scavengers, inhibitors of ROS formation, transi-
tion metal chelators, etc. The protective efficacy of antioxidants
depends on the type of ROS that is generated, the place of
generation (body barriers such as blood-brain barrier reduce
the permeability of most antioxidants), and the severity of the
insult.
The central nervous system (CNS) is particularly vulnerable
to damage by ROS due to several factors, including low levels
of the natural antioxidant glutathione in neurons, membranes
rich in polyunsaturated fatty acids, the presence of excitatory
amino acids such as glutamate, and increased requirement for
oxygen because of the high metabolic activities of the brain.7a-b
Moreover, there is evidence that transition metals such as iron
are mediators of ROS production.7c Since iron is the most
abundant transition metal in the brain, it is considered a potent
potential toxin. Histological and quantitative changes in iron
have been reported in most neurodegenerative diseases.7d Iron
catalyzes the formation of hydroxyl radicals (•OH) by Fenton-
type reaction.8 The exact intracellular location of iron is likely
to be of outmost importance for the ultimate effect, because
results in its damage, including base modifications and single
and double strand breakage. However, there are indications that
location of iron at positions other than near the DNA may
contribute indirectly to DNA damage and ensuing apoptosis.10
Compounds bearing 3,4-dihydroxyphenyl (catechol) moieties
possess a wide spectrum of biological activities, which is related
to their capacity to transfer electrons, to chelate ferrous ions,
and to scavenge ROS.11 Catechol and caffeic acid derivatives
were found to be potent lipoxygenase inhibitors,12 HIV integrase
inhibitors,13 neuroprotectants,14 or cholesterol-lowering agents.15
The natural 3,4-dihydroxy stilbene analogue asriginin exhibits
strong cardioprotective activity in ischemic/reperfused rat
hearts.16 Moreover synthetic cis-3,4-dihydroxy stilbene ana-
logues showed strong antiproliferative and apoptotic activity
in HL60 leukemia cells.17 Flavonoids bearing a catechol moiety
in ring B such as quercetin, luteolin, and catechins showed
protective effects against coronary heart disease and oxidative
stress induced neuronal cell death.18-21 Hydroxy groups in
different positions of flavonoids and caffeic acid derivatives may
allow scavenging of ROS but at the same time decrease
lipophilicity and therefore the ability of polyphenols to penetrate
and remain in the cells. For instance, quercetin aglycone is taken
up by Caco-2 intestinal cells far more effectively than its more
hydrophilic glucosides. However, quercetin metabolism and
ensuing rapid efflux of the aglycone conjugates causes intra-
cellular aglycone levels to decrease rapidly.22 Thus, some of
these compounds, while effective in vitro, cannot afford
cytoprotection because they are inefficiently taken up by the
cells or are rapidly metabolized to conjugates effectively
transported out of the cells.
Vitamin E is widely studied not only as an antioxidant and
oxidative stress induced programmed cell death (oxytosis)
inhibitor but also for its nonantioxidant activities, such as gene
regulation, inhibition of protein kinase C, 5-lipoxygenase, and
phospholipase A2, and activation of protein phosphatase 2A
and diacylglycerol kinase.23 However, vitamin E showed limited
efficacy in emergency reperfusion or DNA damage despite its
high activity against oxytosis,23b which might be attributed to
* Corresponding author. Tel: +30 210 7273818. Fax: +30 210 7273831.
E-mail: mkoufa@eie.gr.
† Institute of Organic and Pharmaceutical Chemistry, National Hellenic
Research Foundation.
‡ University of Ioannina.
§ Institute of Biological Research and Biotechnology, National Hellenic
Research Foundation.
10.1021/jm0506120 CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/23/2005

Chroman/Catechol Hybrids
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 301
Scheme 2a
Scheme 1a
a Reagents and conditions: (a) BOP, caffeic acid, Et3N; (b) trans-3,4-
diacetoxycinnamoyl chloride, Et3N; (c) MeONa, MeOH; (d) BF3‚SMe2
CH2Cl2.
a Reagents and conditions: (a) BOP, caffeic acid, Et3N; (b) (MeO)2SO2,
K2CO3, (c) NaOH 1 N; (d) CDI, piperazine; (e) BOP, trans-3,4-dimethoxy-
cinnamic acid, Et3N.
Scheme 3a
its highly lipophilic properties. However, amides or amines of
the synthetic vitamin E analogue trolox are more effective than
vitamin E as neuroprotectants and against reperfusion-induced
oxidative damage.24-26
As an ongoing effort27,28 toward highly effective antioxidants,
we synthesized three series of hybrids containing the chroman
moiety of vitamin E and a catechol group, and we explored
their ability to protect cells exposed to different types of
oxidative stress.
The first series of the new analogues contain caffeic acid and
trolox connected through diamine or triamine spacers. The
second series are amides of caffeic acid with 2- or 5-amino-
methylchroman and 5-aminochroman derivatives. The last series
of compounds involves chroman analogues substituted at
position 5 by catechol moieties.
The ability of the new molecules to protect cultured cells
from H2O2-induced DNA damage was evaluated using the
highly sensitive methodology of single cell gel electrophoresis
or comet assay.29 Cellular DNA is especially sensitive to the
action of H2O2, and we have shown that this DNA damage is
mediated mainly by iron, which catalyzes the formation of the
•
extremely reactive OH.10,29
a Reagents and conditions: (a) triethyl phosphite, TBAI; (b) t-BuOK,
DMF; (c) BF3‚SMe2 CH2Cl2, 2 h; (d) Pd/C 10%, H2, 50 psi; (e) BF3‚SMe2
CH2Cl2, 24 h; (f) PdCl2(PPh3)2, Et3N, CuI.
The neuroprotective activity of the new compounds was
studied using HT22 cells, a neuronal cell line derived from
murine hippocampus. Exposure of these cells to high concentra-
tions (1-5 mM) of glutamate blocks cystine uptake, which leads
to depletion of intracellular cysteine and loss of glutathione
(GSH), an early increase in ROS, and, eventually, production
of ROS by mitochondria and programmed cell death.30a
Glutamate-induced cell death of HT22 cells is thought to mimic
cytotoxicity due to oxidative stress as implicated in several
neurodegenerative diseases, such as Alzheimer’s disease and
Parkinson’s disease.30b
hydrolysis of the resulting ester 6a and coupling with piperazine
in the presence of CDI (compound 6b) and finally with caffeic
acid using BOP. The analogue 8 in which the catechol group is
protected was synthesized from the corresponding piperazine
amide and coupling with trans-3,4-dimethoxycinnamic acid
using BOP.
Amide 16 was synthesized from the appropriate chroman
amine and trans-3,4-diacetoxycinnamoyl chloride in Et3N and
THF. Deprotection of acetyl groups of compound 14 was
achieved using MeONa in MeOH to give compound 15, while
for the deprotection of the methoxy group, BF3‚SMe2 was used31
(Scheme 2).
The synthesis of the compounds of the third series is depicted
in Scheme 3. 3,4-Dimethoxy benzyl alcohol was converted to
the corresponding bromide using PBr3 and then to phosphonate
ester using triethyl phosphite. Horner-Emmons reaction of 18
with aldehyde 19 in the presence of t-BuOK in DMF32a gave
one geometrical isomer of the stilbene analogue 20. These
Chemistry
Amide analogues of caffeic acid 2-5, 7, 8, 10, and 12
(Schemes 1 and 2) were synthesized from the corresponding
aminoamides 1 or amines 9 and 11, prepared according to our
previously described methodology,27,28 and caffeic acid using
BOP (benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium
hexafluorophosphate) as condensing agent. The methoxy-
chroman derivative 7 was obtained by methylation of trolox
using dimethyl sulfate and potassium carbonate, followed by

302 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Koufaki et al.
Table 1. Lipophilicity (ClogP) Data and Antioxidant Potency of the
Chroman/Catechol Hybrids as Assessed Chemically as Well as
Biologically Using Jurkat and HT22 Cells
analogues (compounds 2-5) of the first series are very strong
antioxidants with TEAC values of 6.24-6.55 µM, while the
TEAC value of the protected analogue 8 is 1.26 ( 0.04 µM.
The most active compounds from the second series are 10 and
15 with TEAC values of 6.53 µM. Among the compounds of
the third series, benzofuran analogue 21 is the strongest
antioxidant with TEAC ) 5.90 ( 0.34 µM.
The oxidative insult by hydrogen peroxide has been widely
used to assess cytoprotection. The effects of continuously
generated H2O2 (by the action of added glucose oxidase) on
DNA damage was assessed by single cell gel electrophoresis
or “comet assay” using Jurkat T-cells.
The IC50 values of the analogues bearing di- or triamine
spacers are between 20 and 75 µM. The most active is
compound 5 with IC50 ) 20 ( 3 µM. while compounds 3, 4,
and 7 have similar activity (IC50 ) 50 µM). Protection of the
chroman hydroxy group did not diminish the activity of the
piperazine analogue, while protection of the catechol group
abolishes the cytoprotective activity.
Among the amides of the second series, the most active is
compound 12 with IC50 ) 1.0 ( 0.1 µM. Compound 16 is also
very active against DNA damage with IC50 ) 5.0 ( 2.0 µM,
while its methoxy analogue 15 has IC50 ) 20 ( 1 µM. The
2-substituted chroman 10 has IC50 ) 25 ( 2 µM.
IC50 (µM)b
Jurkat cells
EC50 (µM)c
HT-22 cells
compound ClogPa
TEAC
2
3
4
5
7
8
10
12
15
16
20
21
22
23
24
27
3.6
3.5
4.1
4.1
4.8
5.0
4.3
4.8
4.4
4.5
7.0
5.3
6.7
5.8
5.2
6.3
1.3
1
6.50 ( 0.15
6.33 ( 0.18
6.55 ( 0.24
6.24 ( 0.35
6.12 ( 0.21
1.26 ( 0.04
6.53 ( 0.18
3.80 ( 0.23
6.53 ( 0.12
4.28 ( 0.22
75 ( 8
50 ( 6
50 ( 6
20 ( 3
50 ( 8
d
25 ( 2
1 ( 0.1
20 ( 1
5 ( 2
7.36 ( 0.24
>10
1.48 ( 0.18
6.35 ( 0.04
2.11 ( 0.39
0.65 ( 0.13
1.14 ( 0.35
2.15 ( 0.59
>10
1.23 ( 0.26
>10
5.90 ( 0.34
0.05 ( 0.01
1.60 ( 0.08
3.74 ( 0.12
10 ( 2
d
10 ( 2
5 ( 2
1.00 ( 0.21
4.59 ( 0.45
1.17 ( 0.15
0.93 ( 0.19
>10
2.98 ( 0.31
>10000
quercetin
caffeic acid
trolox
4.30 ( 0.16
1.2
1
5 ( 1
d
d
3.1
a Calculated with the program ChemDraw Ultra 8.0.1. b IC50 values are
test compound concentration promoting a 50% reduction of the H2O2-
induced DNA single-strand breakage. c EC50 values are test compound
concentrations required to achieve viability of the glutamate-exposed cells
equal to 50% of that of the nonexposed cells (see legend to Figure 1). Values
are mean ( SEM of at least three independent experiments. d Inactive.
Both benzofuran analogue 21 and the trihydroxy analogue
24 from the third series are strong inhibitors of DNA damage
with IC50 ) 10 ( 2 µM and IC50 ) 5 ( 2 µM respectively.
The trimethoxy analogue 22 is inactive.
reaction conditions usually afford the trans isomer as the major
product. In the NMR spectrum, olephinic protons appear as a
singlet at 7.05 ppm, which is in accordance with previously
observed chemical shift for the trans isomer.32b Thus, we assume
that 20 should be trans.
The above results may be taken to suggest that the presence
of the catechol group is necessary for the activity of our
compounds against DNA damage. The 5-substituted chroman
analogues exhibited the highest cytoprotective activity. All these
analogues probably act by a mechanism that inhibits the
formation of hydroxyl radical (i.e., iron chelation). The results
of our recent study36a strongly support the idea of intracellular
binding of redox active iron as the basis for the protective
capacity of flavonoids. In addition, previous literature data11,36b
suggest that the presence of the catechol moieties in poly-
phenolic compounds is responsible for the protection offered
against oxidative stress induced DNA damage. Moreover, we
have observed that many strong antioxidants, commercially
available or compounds synthesized in our laboratory, lacking
catechol moieties, failed to protect cells from H2O2-induced
DNA damage. Thus, we can deduce that the protective effects
of our catechol derivatives are probably mediated by an iron-
binding mechanism.
The mouse hippocampal cell line HT22 has been used
to elucidate sequential cellular events during the programmed
cell death cascade triggered by glutamate-induced oxidative
stress.23c,30a Although HT22 cells lack ionotropic glutamate
receptors that could mediate excitotoxicity, they are killed by
oxytosis within 24 h following exposure to 1-5 mM glutamate.23c
Several natural (e.g., flavonoids) and synthetic (e.g., tyrphostins)
polyphenols have tested positive against glutamate oxytosis of
HT22 cells19,37a as assessed using the MTT to colored formazan
conversion assay.19,23b,37a Importantly, however, their HT22
protective effect was harnessed at concentrations g1 µM. The
majority of the new analogues are stronger neuroprotectants than
quercetin (Figure 1). In addition, they are not toxic to the cells
up to the higher concentrations tested. Compounds 2 and 5 from
the first series exhibit similar albeit rather weak neuroprotective
activity with EC50 ) 7.36 ( 0.24 and 6.35 ( 0.04 µM,
respectively. Piperazine analogues 4 and 7 are better neuro-
protectants than those containing ethylenediamine moieties, and
Reaction of 20 with BF3‚SMe2 afforded the furan analogue
21 instead of the deprotected stilbene analogue. This is not
surprising since BF3‚SMe2 is a Lewis acid and could catalyze
this cyclization reaction. The absence of optical rotation means
that 21 is racemic. The same furan analogue was obtained by
using 9-iodo-9-BBN for the cleavage of methoxy groups.
Attempts to deprotect the methoxy groups using a large excess
of EtSNa in DMF gave a complex mixture of products.
Hydrogenation of 20 using Pd/C gave the trimethoxy dihydro-
stilbene analogue 22. Deprotection of the catechol group was
achieved using BF3‚SMe2 for 2 h (compound 23). Under the
same reaction conditions but for 24 h, the trihydroxy analogue
24 was obtained.
Alkyne analogue 27 was obtained from Sonogashira reaction
of bromochroman 26 and alkyne 25 in the presence of
(PPh3)2PdCl2, CuI, and triethylamine.33 Alkyne was prepared
from 3,4-dimethoxybenzaldehyde and dimethyl-1-diazo-2-oxo-
propylphosphonate.34 Attempts to deprotect methoxy groups
using BF3‚SMe2 result in an unstable product.
Results and Discussion
Our goal was to examine the influence on antioxidant activity
(assessed chemically and biologically) of catechol groups at
position 2 or 5 of the chroman nucleus. In addition, we explored
the differences in activity when the hydroxy groups of catechol
or chroman moieties are free or protected. The results are
reported in Table 1.
Values of trolox equivalent activity concentration (TEAC)
for the new compounds were determined according to Rice-
Evans and Miller.35 The TEAC value of an antioxidant was
calculated experimentally using 0.3 mM trolox solution and
normalizing data to 1.0 mM trolox (Table). The trihydroxy

Chroman/Catechol Hybrids
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 303
5-Substituted chromans of the second and third series, such as
the catechol derivative 12 and the trihydroxy analogues 16 and
24, are strong antioxidants against ROS produced chemically
and highly potent against H2O2- and glutamate-induced damage
in Jurkat T cells and HT22 cells, respectively.
Although in some cases there is a correlation between
cytoprotective activity and lipophilicity, this is not applicable
for all these compounds. This is expected, however, considering
that the antioxidant potential of a compound at the cellular level
does not depend only on its capacity to scavenge ROS but also
on the cellular substrate. The level and activity of scavenging
entities and their distribution between different cellular compart-
ments is highly dependent on cell-type specific metabolism of
the antioxidants as well. For instance, Murota et al.22 have
reported that uptake of quercetin by Caco-2 intestinal cells is
accompanied by extensive conjugation during or after adsorption
and is followed by rapid efflux of the conjugates, that Caco-2
uptake of the more hydrophilic quercetin glycosides lagged
substantially behind that of quercetin aglycone, and that
conjugation and efflux of the glycosides was far less effective
by comparison. Thus, it appears that, while free hydroxy groups
contribute to antioxidant activity, they are subject to conjugation,
which in turn provides for rapid efflux of the conjugates. In
this light, an attempt to stage a structure-activity relationship
(SAR) discussion based only on log P values would appear
unjustified. However, we could deduce an optimum ClogP range
of 4.1-6.0 for the chromans exhibiting improved neuroprotec-
tive capacity. Higher or lower values result in decreased activity
(Table 1). Moreover, it appears that a combination of ClogP
values 4.5-5.8 and substitution by catechol moieties at position
5 of benzopyran ring results in highly active compounds against
oxidative stress induced cellular damage.
Although the activity against DNA damage can be largely
attributed to the iron chelating properties of the chroman/
catechol hybrids, their neuroprotective activity may be due not
only to their antioxidant properties but perhaps also to their
capacity to maintain intracellular GSH at a high enough level
or to interfere with other cell signaling cascades implicated in
oxytosis.38 For instance, Herrera et al.38a reported recently that
the sesquiterpene lactone parthenolide may increase GSH levels
in hippocampal HT22 cells by activating the antioxidant/
electrophilic response element (ARE) in the promoter of the
gene coding of glutamyl cystine synthase gene, the rate-limiting
enzyme for GSH synthesis. Torres et al.38b have shown that
epicatechin-cysteine conjugates can rescue HT22 cells from
glutamate oxytosis by maintaining glutathione levels rather than
by scavenging ROS. Sagara et al.37a have reported that tyr-
phostins protect HT22 cells from glutamate oxytosis by three
distinct mechanisms, depletion of ROS, inhibition of ROS
production by mitochondria, and increasing cellular glutathione
levels. Ishige et al.19 have reported that flavonoids protect HT22
cells from glutamate by increasing intracellular GSH, as well
as scavenging ROS and preventing the influx of Ca2+ in the
presence of high levels of ROS. Whether chroman/catechol
prevention of HT22 oxytosis involves, in addition to ROS
depletion, maintenance of GSH levels, inhibition of ROS
production, or interference with other cell signaling cascades
implicated in oxytosis is not known, however.
Figure 1. Inhibition of glutamate-induced oxytosis of HT22 cells by
chroman/catechol hybrids. HT22 cells were exposed for 24 h to 5 mM
glutamate in the absence or presence of increasing concentrations of
caffeic acid (C), quercetin (Q), and the indicated hybrids, and changes
in cell viability were assessed using MTT conversion to colored
formazan as a measure of the number of living cells. Effects of the
hybrids on the viability of HT22 cells that were not exposed to
glutamate were similarly assessed. Direct interference of the hybrids
with the MTT conversion could be excluded (see Experimental Section).
Neuroprotection refers to the number of viable glutamate-exposed cells
as a percentage of the respective number of viable nonexposed cells at
each of the test compound concentrations tested. Data (mean ( SEM
of 3-7 independent experiments) were curve-fitted using SigmaPlot
9.0. SEM values were similar to those depicted for Q. EC50 values
were calculated as described in the legend of Table 1.
their activity was somewhat better than that of quercetin (EC50
) 2.98 ( 0.31 µM). Surprisingly, analogue 8, bearing the
protected catechol group, is the most active of the new
compounds.
Trihydroxy amides 10 and 16 exhibited strong neuroprotective
activity with EC50 ) 1.14 ( 0.35 and 1.23 ( 0.26 µΜ,
respectively. The methoxy chroman analogue 12 is slightly less
active with EC50 ) 2.15 ( 0.59 µM. Analogue 15 may be taken
to be weakly active, although it is also significantly toxic to
the cells at concentrations higher than 1 µM.
The furan analogue 21, the methoxy analogue 23, and the
trihydroxy analogue 24 of the third series possess strong
neuroprotective activity with EC50 ) 1.00 ( 0.21, 1.17 ( 0.15,
and 0.93 ( 0.19 µM, respectively. Protection of all the hydroxy
groups diminishes (compound 22) or abolishes (compound 20)
neuroprotection. The presence of a hydroxy group on the
diphenylacetylene analogue 27 does not improve the neuro-
protective activity.
Although compounds 2 and 3 are very strong antioxidants,
they show decreased activity against DNA damage and weak
neuroprotective activity. Piperazine analogues show high cyto-
protective activity. Protection of the chroman hydroxy group
of compound 4 (compound 7) does not significantly compromise
cytoprotective activity. However, protection of the catechol
group of compound 4 abolishes its activity against DNA damage
but increases its neuroprotective activity. Thus, the presence of
the catechol group is requisite for activity against H2O2-induced
cellular damage, probably because of its iron chelating proper-
ties, while for neuroprotective activity of this series of com-
pounds, the chroman hydroxy group appears to be most effective
when combined with a protected catechol group. These data
may be taken to indicate that increased compound lipophilicity
may facilitate uptake of 8 and allow for chroman accumulation
in HT22 cells to levels higher than can be achieved with 4.
Conclusion
We synthesized new chroman/catechol hybrids possessing
cytoproprotective activity against oxidative stress. The 5-sub-
stituted chromans 12, 16, and 24 are very potent against H2O2-
and glutamate-induced cellular damage. Compounds 21 and 23

304 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Koufaki et al.
135.5, 123.9, 120.6, 118.7, 116.6, 114.6, 113.2, 113.1, 83.2, 73.2,
37.9, 32.6, 29.7, 26.9, 20.7, 12.3, 11.6. Anal. (C21H24O4) C, H, N.
3,4-Dihydro-5-(3,4-dimethoxyphenyl)-6-methoxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran (22). A solution of compound 20 (110
mg, 0.29 mmol) in 8 mL of AcOEt was added to 11 mg of Pd/C
10%, and the mixture was hydrogenated at 50 psi and at ambient
temperature for 5 h. The mixture was then filtrated through Celite
and washed with AcOEt, and the solvent was evaporated. Yield
are less potent against DNA damage than the above three
analogues but more active than the 2-substituted chromans, and
they exhibit strong neuroprotective activity. While the activity
of the chromans against DNA damage may be largely attributed
to their iron-chelating properties, their activity against glutamate-
induced oxytosis of HT-22 cells is currently a matter of
conjecture.
1
110 mg (100%), yellow oil. H NMR δ: 6.81 (bs, 2H, ArH), 6.72
Experimental Section
(s, 1H, ArH), 3.87 (s, 6H, -Ar-OCH3), 3.72 (s, 3H, -OCH3-
chroman), 2.87-2.84 (m, 2H, -CH2-CH2-), 2.79-2.76 (m, 2H,
-CH2-CH2-), 2.62 (t, J ) 6.5 Hz, 2H, CH2), 2.23 (s, 3H, Ar-CH3),
2.12 (s, 3H, Ar-CH3), 1.75 (t, J ) 6.5 Hz, 2H, CH2), 1.28 (s, 6H,
2CH3). 13C NMR δ: 149.6, 148.8, 148.1, 147.2, 135.3, 129.7, 128.1,
123.7, 120.2, 116.9, 111.9, 111.3, 72.8, 61.1, 55.9, 55.7, 36.1, 32.9,
29.2, 26.9, 20.3, 12.8, 11.9. Anal. (C24H32O4) C, H, N.
General Procedure for the Preparation of Analogues 2-5.
To a solution of caffeic acid (49 mg, 0.27 mmol) and 0.14 mL of
triethylamine in 2 mL anhydrous DMF were added at 0 °C the
appropriate aminoamide 1 (0.27 mmol) and a solution of BOP (121
mg, 0.27 mmol) in 2 mL anhyd CH2Cl2. The mixture was stirred
at 0 °C for 1 h and at ambient temperature for 24 h. Ethyl acetate
was then added, and the mixture was washed with 1 N HCl,
saturated aqueous NaHCO3, and saturated aqueous NaCl. The
organic layer was dried and evaporated in vacuo.
1-[(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzo-
pyran-2-yl)carbonyl]-4-[3-(3,4-dihydroxyphenyl)-2-propenoyl]-
piperazine (4). Column chromatography (CH2Cl2/MeOH 90/10).
Yield 95%, yellow solid, mp 100-103 °C. Anal. (C27H32N2O6) C,
H, N.
N-[(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-1-benzo-
pyran-5-yl)methyl]-3-(3,4-dihydroxyphenyl)-2-propenamide (12).
Compound 12 was prepared from chroman-5-methylamine 1128 (87
mg, 0.48 mmol) and caffeic acid (120 mg, 0.48 mmol) using BOP
(212 mg, 0.48 mmol). Purification by column chromatography
(CH2Cl2/MeOH 90/10) afforded 113 mg (58%) of white solid, mp.
230-233 °C. Anal. (C24H29NO5) C, H, N.
N-(3,4-Dihydro-6-hydroxy-2,2,7,8-tetramethyl-2H-1-benzo-
pyran-5-yl)-3-(3,4-dihydroxyphenyl)-2-propenamide (16). Com-
pound 16 was prepared from 15 as described in the literature.31
Column chromatography using CH2Cl2/MeOH 95/5 as eluent
afforded 59% yield of a yellow viscous oil. Anal. (C22H25NO5) C,
H, N.
(E)-3,4-Dihydro-5-[2-(3,4-dimethoxyphenyl)ethenyl]-6-meth-
oxy-2,2,7,8-tetramethyl-2H-1-benzopyran (20). To a solution of
phosphonate 18 (694 mg, 2.41 mmol) in 4 mL of anhyd DMF was
added at 0 °C potassium tert-butoxide (270 mg, 2.41 mmol), and
the mixture was stirred at 0 °C for 1 h. Aldehyde 19 (400 mg, 1.61
mmol) in 2 mL of anhyd DMF was then added, and the mixture
was stirred at ambient temperature for 1 h, at 110 °C for 2 h, and
at ambient temperature for 24 h. The reaction mixture was then
cooled at 0 °C, and H2O and a solution of HCl (1 N) were added
until the pH was acidic, and the mixture was extracted with CH2Cl2
(3 × 50 mL). The organic layer was washed with saturated aqueous
NaCl, dried, and evaporated. The residue was purified by column
chromatography (petroleum ether/AcOEt 90/10) to give 220 mg
(36%) of 20 as white waxy solid. 1H NMR δ: 7.07-6.97 (m, 2H)
7.05 (s, 2H, CHdCH), 6.87 (d, J ) 7.9 Hz, 1H, ArH), 3.96 (s, 3H,
Ar-OCH3), 3.91 (s, 3H, Ar-OCH3), 3.63 (s, 3H, -OCH3), 2.85 (t, J
) 6.5 Hz, 2H), 2.23 (s, 3H, Ar-CH3), 2.15 (s, 3H, Ar-CH3), 1.78
(t, J ) 6.5 Hz, 2H), 1.33 (s, 6H, 2CH3). 13C NMR δ: 149.5, 149.1,
148.7, 148.1, 132.9, 131.5, 128.4, 126.6, 124.9, 121.3, 119.3, 116.7,
111.3, 108.8, 72.9, 60.1, 55.9, 33.1, 26.9, 21.9, 12.5, 12.2. Anal.
(C24H30O4) C, H, N.
1,7,8,9-Tetrahydro-2-(3,4-dihydroxyphenyl)-4,5,7,7-tetramethyl-
2H-furan[3,2-f]benzopyran (21). To a solution of stilbene 20 (30
mg, 0.08 mmol) in 8 mL of anhyd CH2Cl2 was added 0.62 mL
(5.85 mmol) of boron trifluoride dimethyl sulfide at 0 °C, and the
mixture was stirred at ambient temperature for 24 h and then treated
as described in the literature31 to give 26 mg (100%) of 21 as light
brown oil. 1H NMR δ: 6.91 (s, 1H, ArH), 6.81 (s, 2H, ArH), 5.57
(pseudo triplet, J ) 7.9 Hz and J ) 9.2 Hz, 1H, -OCHCH2-), 3.55-
3.35 (m, 1H, -OCHCHH-), 3.05-2.90 (dd, J ) 9.2 Hz and J )
15.3 Hz, 1H, OCHCHH-), 2.59-2.46 (m, 2H, CH2-chroman), 2.13
(s, 3H, Ar-CH3), 2.09 (s, 3H, Ar-CH3), 1.76 (t, J ) 6.5 Hz, 2H,
CH2), 1.30 (s, 6H, 2CH3). 13C NMR δ: 150.6, 145.8, 143.6, 143.4,
3,4-Dihydro-5-(3,4-dihydroxyphenyl)-6-methoxy-2,2,7,8-tetra-
methyl-2H-1-benzopyran (23). To a solution of 22 (40 mg, 0.11
mmol) in 8 mL of anhyd CH2Cl2 was added boron trifluoride
dimethyl sulfide (0.16 mL, 1.56 mmol) at 0 °C, and the mixture
was stirred at ambient temperature for 2 h.28 Purification by column
chromatography using CH2Cl2/MeOH 95/5 afforded 23 as colorless
oil. Yield 17 mg (46%). Anal. (C22H28O4) C, H, N.
3,4-Dihydro-5-(3,4-dihydroxyphenyl)-2,2,7,8-tetramethyl-2H-
1-benzopyran-6-ol (24). To a solution of 22 (60 mg, 0.17 mmol)
in 10 mL of anhyd CH2Cl2 was added boron trifluoride dimethyl
sulfide (0.27 mL, 2.52 mmol) at 0 °C, and the mixture was stirred
at ambient temperature for 24 h and then treated as previously
described.28 Purification by column chromatography using CH2Cl2/
MeOH 95/5 afforded 30 mg (53%) of 24 as colorless oil. Anal.
(C21H26O4) C, H, N.
3,4-Dihydro-5-(3,4-dimethoxyphenylethynyl)-2,2,7,8-tetra-
methyl-2H-1-benzopyran-6-ol (27). A solution of 5-bromo-3,4-
dihydro-2,2,7,8-tetramethyl-2H-1-benzopyran-6-ol, 26 (44 mg, 0.15
mmol), and alkyne 25 (25 mg, 0.15 mmol) in anhyd triethylamine
(0.18 mL) was degassed in vacuo. PdCl2(PPh3)2 (1.7 mg, 0.003
mmol), CuI (1 mg, 0.006 mmol), and 1.8 mL of anhyd DMF were
then added, and the mixture was stirred at ambient temperature for
3 h. Saturated aqueous NH4Cl was added to the mixture and
extracted with diethyl ether. The organic layer was washed with
saturated aqueous NaCl and dried over Na2SO4, the solvent was
evaporated, and the residue was purified by column chromatography
(petroleum ether/AcOEt 70/30) to give 50 mg (89%) of 27 as brown
1
viscous oil. H NMR δ: 7.18 (d, J ) 8.5 Hz, 1H), 7.08 (s, 1H),
6.84 (d, J ) 8.5 Hz, 1H), 3.90 (s, 3H), 3.88 (s, 3H), 2.82 (t, J )
6.5 Hz, 2H), 2.05 (s, 6H), 1.68 (t, J ) 6.5 Hz, 2H), 1.31 (s, 6H).
13C NMR δ: 150.6, 149.3, 141.6, 140.0, 135.1, 130.3, 128.1, 125.8,
114.5, 112.0, 110.9, 81.6, 70.9, 55.9, 42.3, 29.1, 24.2, 12.6. Anal.
(C23H26O4) C, H, N.
Evaluation of the Activity of the New Compounds against
H2O2-Induced DNA Damage. Cell Culture and Treatment. The
ability of individual compounds to protect cellular DNA from H2O2-
induced damage was investigated by using Jurkat cells (a human
T-lymphocytic cell line, ATCC, clone E6-1). One hundred micro-
liters of RPMI 1640 growth medium (supplemented with 10% fetal
calf serum, penicillin 100 IU/ml, streptomycin 100 µg/mL, and
glutamine 300 µg/mL) containing 1.5 × 105 cells was placed into
each of 96 wells of ELISA plastic plates and incubated for 1 h at
37 °C, 95% air, 5% CO2. Cells were subsequently treated for 10
min with 60 ng of the enzyme glucose oxidase, which was able to
generate 11.8 ( 1.5 µM H2O2 per minute in the absence of cells.
Additions of the compounds at the indicated concentrations were
done 30 min prior to the addition of glucose oxidase. Following
the treatment, cells were collected by centrifugation (250 × g at 4
°C for 5 min) for further analysis.
Single Cell Gel Electrophoresis. The assay used was essentially
the same as previously described.29 Cells were suspended in 1%
low-melting-point agarose in PBS (pH 7.4) and pipetted onto
superfrosted glass microscope slides precoated with a layer of 1%
of normal-melting-point agarose (warmed at 37 °C prior to use).
The agarose was allowed to set at 4 °C for 10 min, and then the

Chroman/Catechol Hybrids
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 305
References
slides were immersed in lysis solution (2.5 M NaCl, 100 mM
EDTA, 10 mM Tris at pH 10, 1% Triton X-100 v/v) at 4 °C for 1
h to remove cellular proteins. Slides were then placed in single
rows in a 30-cm wide horizontal electrophoresis tank containing
0.3 M NaOH and 1 mM EDTA, pH > 13, at 4 °C for 40 min to
allow for separation of the two DNA strands (alkaline unwinding).
Electrophoresis was performed in the unwinding solution at 30 V
(1 V/cm), 300 A, for 30 min. The slides were then washed three
times for 5 min each with 0.4 M Tris, pH 7.5, at 4 °C before staining
with Hoechst 33342 (20 µg/mL).
Image Analysis and Scoring. Stained nucleoids were examined
under a UV microscope with an excitation filter of 435 nm and a
magnification of 400. The damage was not homogeneous, and visual
scoring of the cellular DNA on each slide was based on charac-
terization of 100 randomly selected nucleoids. The comet-like DNA
formations were categorized into five classes (0, 1, 2, 3, and 4)
representing an increasing extent of DNA damage seen as a “tail”.
Each comet was assigned a value according to its class. Accord-
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comets in class 0) to 400 (100% of comets in class 4). In this way,
the overall DNA damage of the cell population can be expressed
in arbitrary units. Visual scoring expressed in this way correlates
nearly linearly with other parameters such as percent of DNA in
tail estimated after computer image analysis using a specific
software package. Observation and analysis of the results were
always carried out by the same experienced person using a specific
pattern when moving along the slide.
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Acknowledgment. This work was supported in part by the
Greek General Secretariat for Research and Technology, Grants
EPAN 3.3.1. and PENED01-ED268. The technical assistance
of Ms. Aggela Nastou is gratefully acknowledged.
Supporting Information Available: Detailed experimental
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