Isoxazole substituted chromans against oxidative stress-induced neuronal damage

Article abstract of DOI:10.1016/j.bmc.2011.06.074

We have previously reported that catechol-bearing regioisomers of 5-isoxazolyl-6-hydroxy-chroman display higher in vitro neuroprotective activity, compared to hybrids with other nitrogen heterocycles, but their activity is hampered by cytotoxicity at high

Full text of DOI:10.1016/j.bmc.2011.06.074

Bioorganic & Medicinal Chemistry 19 (2011) 4841–4850
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Isoxazole substituted chromans against oxidative stress-induced
neuronal damage
Maria Koufaki a, , Alexandra Tsatsaroni a, Xanthippi Alexi b, Helène Guerrand a, Sofia Zerva b
⇑
Michael N. Alexis b,
⇑
a National Hellenic Research Foundation, Institute of Organic and Pharmaceutical Chemistry, 48 Vas. Constantinou Ave., 11635 Athens, Greece
b National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, 48 Vas. Constantinou Ave., 11635 Athens, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously reported that catechol-bearing regioisomers of 5-isoxazolyl-6-hydroxy-chroman dis-
play higher in vitro neuroprotective activity, compared to hybrids with other nitrogen heterocycles, but
their activity is hampered by cytotoxicity at higher concentrations. In an effort to discover non-cytotoxic
isoxazole substituted chromans of high neuroprotective activity, 20 new 3- and 5-substituted (chroman-
5-yl)-isoxazoles and (chroman-2-yl)-isoxazoles were synthesized using the copper(I)-catalysed cycload-
dition reaction between in situ generated nitrile oxides and terminal acetylenes. An additional aim was to
further explore the effect of the isoxazole ring substituents on the neuroprotective activity. The activity of
these compounds against oxidative stress-induced death (oxytosis) of neuronal HT22 cells was evaluated
and interesting SARs for this group of analogues were derived. The vast majority of new chroman ana-
Received 18 May 2011
Revised 24 June 2011
Accepted 26 June 2011
Available online 29 June 2011
Keywords:
Antioxidants
Chromans
Click chemistry
Isoxazoles
logues displayed high in vitro neuroprotective activity displaying EC50 values below 1 lM and lacked
cytotoxicity. The position of substituents on the isoxazole ring influences the activity of the regioisomers,
with the 3-aryl-5-(chroman-5-yl)-isoxazoles, 17 and 18 and bis-chroman 20 displaying higher neuropro-
Oxidative stress
Neuronal damage
tective activity (EC50 ꢀ0.3
l
M) compared to other (chroman-5-yl) and (chroman-2-yl)-isoxazoles.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
against oxytosis of HT22 hippocampal neurons (EC50 = 0.3–0.6
but they were cytotoxic at concentrations higher than 1 M.
lM),
l
The 1-benzopyran ring system constitutes the basic skeleton
of a variety of natural compounds with interesting biological
activities.1,2 In addition, several biologically active synthetic 3,4-
dihydro-2H-1-benzopyrans (chromans)3,4 have been reported,
suggesting that derivatives of this benzopyran motif may be capa-
ble of interacting with a variety of cellular targets. Our group has
long been involved with the design and synthesis of cardioprotec-
tive5 and neuroprotective chroman derivatives.6 We have recently
reported the synthesis of 1,2-dithiolane/chroman hybrids6b and
chroman/catechol6c hybrids bearing heterocyclic rings such as
isoxazole, 1,2,4- and 1,3,4-oxadiazole, 1,2,3-triazole and tetrazole
and the study of the influence of these heterocycles on the activity
of the compounds at the cellular level. Interestingly, we found that
the two isoxazole analogues we synthesized displayed higher
neuroprotective activity than chromans bearing other nitrogen
heterocycles.6c Specifically, the regioisomers of 5-isoxazolyl-6-
hydroxy-chroman bearing a catechol moiety displayed strong activity
A number of analogues based on the isoxazole pharmacophore
have been reported to exhibit a wide range of biological activities
including, antithrombotic,7 antiviral,8 antiapoptotic9 and antipara-
sitic10 properties. In this light it appears that the isoxazole scaffold
can be incorporated in the design for a wide range of bioactive
agents. We therefore set out to synthesize new analogues to ex-
plore the effect of various isoxazole ring substituents on the ability
of chroman derivatives to protect glutamate-challenged HT22 hip-
pocampal neurons from oxytosis. The specific aim of this study was
to discover new 2- or 5-isoxazolyl-chromans of even higher neuro-
protective activity and much lower cytotoxicity compared to the
catechol-bearing regioisomers of 5-isoxazolyl-chroman previously
reported.6c To this end we designed and synthesized 20 new ana-
logues in which isoxazole moieties bear aliphatic or aromatic sub-
stituents. The copper(I)-catalysed cycloaddition reaction between
in situ generated nitrile oxides and terminal acetylenes was ap-
plied in the synthesis of new compounds.
2. Chemistry
Isoxazoles were obtained by 1,3-dipolar cycloaddition of nitrile
oxides to terminal acetylenes. The appropriate aldoximes were
transformed to the corresponding nitrile oxides using
⇑
Corresponding authors. Tel.: +30 210 7273818; fax: +30 210 7273831 (M.K.);
Tel.: + 30 210 7273741; fax: +30 210 7273677 (M.N.A.).
E-mail addresses: mkoufa@eie.gr (M. Koufaki), mnalexis@eie.gr (M.N. Alexis).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.074

4842
M. Koufaki et al. / Bioorg. Med. Chem. 19 (2011) 4841–4850
chloramine-T trihydrate, which acts as both a halogenating agent
and a base.11 In the presence of a catalytic amount of copper(I), ob-
tained from comproportionation of Cu metal and CuSO4ꢁ5H2O, the
in situ generated nitrile oxides reacted with the acetylene interme-
diates to afford the corresponding isoxazoles. Scheme 1 shows the
synthesis of 5-substituted chromans: cycloaddition reaction of
chroman oxime I6c with aliphatic or aromatic alkynes and subse-
quent deprotection using BF3ꢁSMe2 gave isoxazole derivatives 5–9.
The 1H NMR spectrum of compounds 5–7 and 9 showed a singlet
at ꢀ8.4 ppm indicating a hydrogen bond between the 6-OH and the
nitrogen of the isoxazole. For the synthesis of the regioisomers 14–
18, 5-ethynyl-benzopyran II6b reacted with the appropriate oximes
to afford the isoxazole analogues 10–13 which were deprotected
using BF3ꢁSMe2. Bis-chroman 19 was prepared from benzopyran
oxime I and 5-ethynyl-benzopyran II and after deprotection,
gave 20.
The yields of cycloaddition depend on electronic and steric
properties of oximes and alkynes. Aryl alkynes or oximes bearing
electron donating substituents (compounds 3, 13, 37) led to higher
yields. Steric hindrance caused by 2-substituted chromans (com-
pounds 22–24, 29, 30) and the bis-chromans (19, 33) resulted in
lower yields.
In addition, we endeavoured to synthesize the 2-subtituted
chromans on solid support. Thus, trolox ethyl ester was attached
to bromo Wang resin.14 The loading to the resin did not exceed
50%. The yield of the loading was even lower using Wang15 or Mer-
rifield resins. Although reduction to the chroman-2-methanol
using LiAlH4 was quantitative, subsequent alkylation17 with
16
propargyl bromide to the corresponding propargyl ether proceeded
with low yield, suggesting that the synthesis in solution was the
preferred procedure for the synthesis of these chromans.
The synthesis of 2-substituted chromans is depicted in
Scheme 2. Chroman-2-methanol IV,12 obtained from the reduc-
tion with LiAlH4 of 6-methoxy-trolox methyl ester III,6a was
used as starting material. Oxidation using pyridinium chloro-
chromate (PCC), followed by reaction with hydroxylamine gave
oxime 21, which reacted with the appropriate alkyne to pro-
duce isoxazoles 22–24 which upon treatment with BF3ꢁSMe2
gave compounds 25–27. Reaction of aldehyde V with dimethyl-
(diazomethyl)phosphonate (generated in situ from dimethyl-1-
3. Results and discussion
Oxidative stress is implicated in neurodegenerative diseases
such as Alzheimer and Parkinson diseases and is mediated via a
series of disturbances to the redox homeostasis of the cells. The
mouse hippocampal cell line HT22 has been used to elucidate
sequential cellular events during oxidative stress-induced cell
death (oxytosis) caused by glutamate-induced depletion of intra-
cellular glutathione.18–21 Glutamate-challenged HT22 cells suffer
oxytosis within 24 h as a result of accumulation of reactive oxygen
species (ROS). Administration of antioxidants has been shown to
prevent oxytosis, and the HT22 cells have been extensively used
for the identification of novel neuroprotective agents that were
subsequently tested positive in reducing stroke-induced brain
damage.22,23 Figures 1 and 2 show representative assessments of
the neuroprotective activity of 5- and 2-substituted chromans,
respectively. The efficacy of neuroprotection, defined as the per-
centage of glutamate-challenged HT22 cells rescued from oxytosis
diazo-2-oxopropylphosphonate (Bestmann–Ohira reagent13
) by
treatment with K2CO3, MeOH) afforded alkyne 28 which in turn
gave isoxazoles 29 and 30 and, upon deprotection, analogues 31,
32. Cycloaddition of oxime 21 to ethynyl-benzopyran II gave
33 which, after deprotection, afforded the bis-chroman 34.
Alkylation of chroman-2-methanol with propargyl bromide
produced 35 which reacted with aromatic oximes to give
compounds 36, 37 and, after deprotection of methoxy groups,
compounds 38–40.
R1
O
N
R1
N
O
NOH
MeO
RO
RO
MeO
c
a
O
O
O
O
II 6b
I 6c
1
1
10
R = Me, R = Ph
1
2
R = Me, R =Ph
11 R = Me, R1 =4-F-Ph
1
R = Me, R =4-NO2-Ph
1
3 R = Me, R1 =4-OMe-Ph
12
13
R = Me, R =4-NO2-Ph
1
1
R = Me, R =4-OMe-Ph
4
R = Me, R =CH2CH2CH3
b
b
R = H, R1 = Ph
1
14
5
6
R = H, R =Ph
15 R = H, R1 =4-F-Ph
1
R = H, R =4-NO2-Ph
R = H, R1 = 4-NO2-Ph
7 R = H, R1 =4-OMe-Ph
16
17 R = H, R1 =4-OMe-Ph
1
8
R = H, R =4-OH-Ph
R = H, R1=4-OH-Ph
9 R = H, R1 = CH2CH2CH3
18
1
1
B
R = H, R =3,4-OH-Ph
A
R = H, R =3,4-OH-Ph
OR
O
NOH
N
O
MeO
MeO
d
+
19
20
RO
R = Me
R = H
b
O
O
O
Scheme 1. Reagents and conditions: (a) alkyne, TsN(Cl)Naꢁ3H2O, CuSO4ꢁ5H2O, Cu0, t-BuOH/H2O, rt, 12 h, (b) BF3ꢁSMe2 (20 equiv), CH2Cl2, at 0 °C for 1 h and an additional 1 h
at rt (deprotection of 3 gave a mixture of analogues 7 and 8) (c) oxime, TsN(Cl)Naꢁ3H2O, CuSO4ꢁ5H2O, Cu0, t-BuOH/H2O, rt, 12 h, (d) TsN(Cl)Naꢁ3H2O, CuSO4ꢁ5H2O, Cu0,
t-BuOH/H2O, rt, 12 h.

M. Koufaki et al. / Bioorg. Med. Chem. 19 (2011) 4841–4850
4843
MeO
MeO
MeO
b
MeO
a
O
O
OH
O
III 6a
O
V 12
O
IV 12
OMe
c
d
RO
RO
MeO
f
e
NOH
N
O
R2
O
O
O
O
O
N
28
21
R2
2
22
R = Me, R = Ph
2
29
R = Me, R = Ph
23 R = Me, R2 =4-OMe-Ph
30 R = Me, R2 =4-OMe-Ph
2
24
25
R = Me, R =cyclopentyl
g
g
31 R = H, R2 = Ph
2
R = H, R = Ph
2
32
R = H, R =4-OMe-Ph
26 R = H, R2 =4-OMe-Ph
2
27
R = H, R =cyclopentyl
RO
N
O
MeO
MeO
+
O
h
NOH
O
21
O
33
34
R = Me
R = H
RO
R3
O
II
g
g
3
36
R = Me, R = Ph
37 R = Me, R3 =4-OMe-Ph
RO
MeO
f
O
O
38 R = H, R3 = Ph
O
O
O
N
3
39
R = Me, R =4-OH-Ph
35
40 R = H, R3 =4-OMe-Ph
Scheme 2. Reagents and conditions: (a) LiAlH4, THF, 70 °C, (b) PCC, anhyd CH2Cl2, (c) NH2OHꢁHCl, anhyd pyr, (d) Bestmann–Ohira reagent, K2CJ3, MeOH, (e) alkyne,
TsN(Cl)Naꢁ3H2O, CuSO4ꢁ5H2O, Cu0, t-BuOH/H2O, rt, 12 h, (f) oxime, TsN(Cl)Naꢁ3H2O, CuSO4ꢁ5H2O, Cu0, t-BuOH/H2O, rt, 12 h, (g) BF3ꢁSMe2, CH2Cl2, at 0 °C for 1 h and an
additional 1 h at rt (deprotection of 36 gave a mixture of analogues 39 and 40) (h) TsN(Cl)Naꢁ3H2O, CuSO4ꢁ5H2O, Cu0, t-BuOH/H2O, DMF, rt, 12 h.
100
100
A
25
B
5
6
7
8
26
27
34
31
32
38
39
40
80
60
40
20
0
80
60
40
20
0
9
14
15
16
17
18
20
-20
-20
0.01
0.1
1
10
0.01
0.1
1
10
Concentration (µΜ)
Concentration (µΜ)
Figure 1. Protection of HT22 cells from oxytosis by 5-substituted chroman
analogues. Cells were challenged with 5 mM glutamate in the presence of vehicle
or increasing concentrations of analogues for 24 h and relative numbers of living
cells were assessed as described in Section 5. Values are mean SEM of three
independent experiments carried out in triplicate with an intra-assay variation
similar to that shown for analogue 18. A, B: Catechol moiety-bearing regioisomers
of 5-isoxazolyl-6-hydroxy-chroman previously reported to display cytotoxicity as
well as strong activity against oxytosis of HT22 cells.6c
Figure 2. Protection of HT22 cells from oxytosis by 2-substituted chroman
analogues. Cells were challenged with 5 mM glutamate in the presence of vehicle
or increasing concentrations of analogues for 24 h and relative numbers of living
cells were assessed as described in Experimental part. Values are mean SEM of
three independent experiments carried out in triplicate with an intra-assay
variation similar to that shown for analogue 25.
centration for 50% efficacy (EC50), are listed in Tables 1 and 2 along
with test compound cytotoxicity. The latter is defined as% loss of
cell viability among not challenged cells in the presence of
in the presence of 10
lM test compound compared to vehicle, and
the potency of neuroprotection, defined as the test compound con-

4844
M. Koufaki et al. / Bioorg. Med. Chem. 19 (2011) 4841–4850
10
l
M test compound compared to vehicle. The activity and cyto-
However, whether and how these substituents may differentially
impact on the various pathways of ROS production and/or oxytosis
modulation is presently unclear.
toxicity of new compounds is compared to that of 5-isoxazolyl-6-
hydroxy-chroman regioisomers bearing a catechol moiety previously
reported (compounds A and B, Table 1).6c
The data of Table 1 show that the potency of 5-substituted chro-
mans depends on the type of regioisomer as well as the substituent.
With respect to the nature of the substituentin 5-chromanyl-isoxaz-
4. Conclusion
The majority of the new chroman analogues displayed high
in vitro neuroprotective activity, with EC50 values below 1 lM,
oles, the hydroxyphenyl analogue 18 (EC50 = 0.33
methoxyphenyl derivative 17 (EC50 = 0.32 M) and the bis-chroman
derivative 20 (EC50 = 0.30 M) display similar potency (p >0.05; t-
test) with their catechol counterpart B (EC50 = 0.25 M) but are
not cytotoxic. Similarly, among the 3-chromanyl isoxazoles, the
hydroxyphenyl analogue 8 (EC50 = 0.49 M) and the methoxy-
phenyl derivative (EC50 = 0.70 M) display similar potency
(p >0.05; t-test) with their catechol counterpart A (EC50 = 0.60 M)
lM), the
l
and lacked cytotoxicity. With respect to 5-substituted chromans,
the nature of the substituents on the isoxazole ring influences
the activity of the regioisomers, with 3-aryl-5-chromanyl-isox-
azoles displaying higher potency than their 5-aryl-3-chroma-
nyl-isoxazole isomers. With respect to 2-substituted chromans,
3-aryl-5-chromanyl-isoxazoles and 5-aryl-3-chromanyl-isoxazoles
display largely similar potency and both are nearly equipotent
with 5-subsituted chromans bearing 5-aryl-isoxazole moieties. In
5-subsituted chromans bearing 3-aryl-isoxazole substituents, the
presence of an electron donating substituent or a second antioxi-
dant moiety (analogues 17, 18 and 20) seems requisite for high po-
tency. The 3,5-bis(chroman-5-yl)isoxazole analogue 20, in
particular, is more potent than its 5-(chroman-5-yl)-3-(chroman-
2-yl)isoxazole congener 34 by a factor of 2.4.
l
l
l
7
l
l
and are also not cytotoxic. The 5-chromanyl-isoxazole 17 is more
potent (p = 0.028; t-test) that the respective 3-chromanyl isoxazoles
7 by a factor of about 2, possibly reflecting a negative role of the
hydrogen bond between the 6-hydroxy group and the isoxazole
nitrogen in 3-chromanyl-isoxazoles. However, the potencies of 8
and 18 are not significantly different (p >0.05; t-test). Similarly, 5-
chromanyl-3-phenyl-isoxazole analogue 14 (EC50 = 0.73
plays similar potency (p >0.05; t-test) with its 3-chromanyl isomer
5 (EC50 = 0.61 M), indicating that presence of a strong electron
lM) dis-
l
5. Experimental section
5.1. Chemistry
donating substituent on the phenyl group or a second antioxidant
moiety are requisite for 5-chromanyl analogues of comparatively
higher potency. Nonetheless, while 5-chromanyl-3-nitrophenyl
isoxazole 16 (EC50 = 0.51
and 18, its 3-chromanyl isomer 6 (EC50 = 4.68
(p <0.000; t-test) than 7 and 8 by a factor of 6.7 and 9.6, respectively,
indicating that the nature as well as the position of the substituent
has a strong influence on the in vitro neuroprotective activity of 5-
substituted chromans.
l
M) displays similar potency with 17
Melting points were determined on a Buchi 510 apparatus and
are uncorrected. 1H NMR spectra were recorded on Varian spec-
trometers operating at 300 MHz or 600 MHz and 13C spectra were
recorded at 75 MHz using CDCl3 as solvent. Silica gel plates Mache-
rey-Nagel Sil G-25 UV254 were used for thin layer chromatography.
Chromatographic purification was performed with silica gel (200–
400 mesh). The purity of the tested compounds was determined by
HPLC (Thermo Scientific HPLC Spectra System) column Nucleosil
l
M) is less potent
On the other hand, 5-phenyl-isoxazole 25 (EC50 = 0.53
plays similar potency (p >0.05; t-test) with its 3-phenyl-isoxazole
isomer 31 (EC50 = 0.52 M), the 3-phenyl-isoxazyl-methyloxy ana-
logue 38 (EC50 = 0.54 M) and 5-chromanyl analogue 5. Thus, chro-
lM) dis-
l
100-5 150 ꢂ 4.6 mm C18 5
l, Macherey-Nagel. Mass spectra were
l
obtained on TSQ 7000 Finigan instrument or HPLC–MSn Fleet-Ther-
mo, in the ESI mode. HRMS spectra were recorded, in the ESI mode,
on UPLC-MSn Orbitrap Velos-Thermo.
mans substituted at position 2 by 5-aryl-isoxazole moieties are by
and large as potent as their 3-aryl-isoxazole isomers (Table 2) and
nearly as potent as chromans substituted at position 5 by 5-aryl-
isoxazole moieties (Table 1). However, methoxyphenyl analogue
5.1.1. General procedure for the synthesis of isoxazoles
To the appropriate oxime (0.1 mmol) dissolved in 1 mL of
t-BuOH/H2O (1/1), chloramine-T trihydrate (0.1 mmol) was added
in small portions over 5 min. Then, CuSO4ꢁ5H2O (0.01 mmol), cop-
per turnings (catalytic amount ꢀ0.5 mg) and the requisite alkyne
(0.1 mmol) were added and the mixture was stirred overnight.
The reaction mixture was poured onto ice/water and after addition
of dilute NH4OH (to remove all copper salts), it was extracted with
AcOEt. The organic layer was washed with satd aqueous NaCl,
dried and evaporated to dryness.
26 (EC50 = 1.07
t-test) than its isomer 32 (EC50 = 0.42
chroman derivative 26 is significantly less potent (p <0.025; t-test)
than its regioisomer 34. Protection of the 6-hydroxy group strongly
influences the activity. Thus, compound 39 (EC50 = 7.11
significantly less potent (p <0.000) than 40 (EC50 = 0.69 M).
l
M) is again significantly less potent (p <0.002;
l
M). In addition, the bis-
lM) is
l
On the whole it appears that the bioactivities of the new ana-
logues cannot be analysed by considering only hydrogen bonding
and/or electronic properties of the substituents. Other factors not
directly related to oxidative stress pathways but differentially af-
fected by regioisomers, such as intracellular and/or intraorganellar
bioavailability21,24 or interaction with signalling entities,25 may
indirectly influence cell fate. Interestingly, it has been reported
that the potential of antioxidants to protect glutamate-challenged
HT22 cells from oxytosis depends on their accessibility to specific
mitochondrial sites of ROS production under oxidative stress con-
ditions as well as their intracellular bioavailability, which is also
known to vary considerably among structurally related com-
pounds.21,24 In addition, depending on the considered structure,
delocalization of the phenoxy radical, for example, between regioi-
somers 20 and 34, may occur differently and this may also inter-
fere with the antioxidant properties. Between regioisomers 20
and 34 delocalization of phenoxy radical seems to be more efficient
in compound 20. Clearly, the activity of our compounds at the cel-
lular level depends on the nature of the heterocycle substituents.
5.1.1.1. 5-(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-1-
benzopyran-5-yl)-3-phenyl-isoxazole (10). Purification by column
chromatography (pet. ether–AcOEt 9:1). Yield 38%, yellowish solid,
mp 75–77 °C. 1H NMR d: 7.89 (m, 2H), 7.48 (m, 3H), 6.76 (s, 1H),
3.52 (s, 3H, –OCH3), 2.72 (t, 2H, J = 6.8 Hz, –CH2), 2.23 (s, 3H,
Ar–CH3), 2.18 (s, 3H, Ar–CH3), 1.73 (t, 2H, J = 6.8 Hz, –CH2), 1.34
(s, 6H, CH3). 13C NMR d: 168.2, 162.4, 149.6, 148.3, 129.9, 129.3,
128.9, 126.8, 118.2, 118.1, 102.7, 73.6, 61.5, 32.5, 26.9, 21.4, 12.5,
12.4. MS m/z: 386.24 (M+Na)+
5.1.2. General procedure for deprotection of methoxy groups
A solution of protected compound (1 equiv) in anhyd CH2Cl2
(C = 0.026 M), was cooled at 0 °C and BF3ꢁSMe2 (20 equiv) was
added. The mixture was stirred at 0 °C for 1 h and an additional

M. Koufaki et al. / Bioorg. Med. Chem. 19 (2011) 4841–4850
4845
Table 1
Efficacy and potency of 5-substituted chroman analogues to protect glutamate-challenged HT22 cells from oxytosis
Compound
Efficacya (%)
Potencyb
(l
M)
Cytotoxicityc (%)
OH
OH
O
O
N
N
HO
A
70
2
0.60 0.19
0.61 0.08
4.68 0.27
52
36
ns
3
7
O
HO
HO
5
6
79 4*
O
NO2
OCH3
OH
O
N
79
77
81
5
4
5
O
O
O
N
N
HO
HO
7
8
0.70 0.15
ns
ns
O
0.49 0.11
O
N
O
HO
9
B
77 1*
1.76 0.10
33 13
O
OH
OH
OH
73
76
5
5
0.25 0.04
82
ns
1
N
O
O
OH
N
14
0.73 0.07
O
O
F
OH
15
16
77
3
0.52 0.08
ns
N
O
O
OH
NO2
76 3*
0.51 0.17
26
7
N
O
O
(continued on next page)

4846
M. Koufaki et al. / Bioorg. Med. Chem. 19 (2011) 4841–4850
Table 1 (continued)
Compound
Efficacya (%)
Potencyb
(l
M)
Cytotoxicityc (%)
OCH3
OH
OH
17
18
75
74
79
3
4
4
0.32 0.03
0.33 0.12
0.30 0.07
ns
N
N
O
O
O
O
O
OH
ns
ns
O
OH
O
20
N
HO
a
Defined as % protection of viability of glutamate-challenged HT22 cells from oxytosis at 10 lM test compound unless defined otherwise. Cell viability was assessed using
the MTT assay (Section 5.2.1). Efficacy (%) of neuroprotection was calculated as described in Section 5. Values are mean SEM of at least three independent experiments such
as those shown in Figures 1 and 2.
b
Defined as the test compound concentration, that is, able to protect viability of glutamate-challenged HT22 cells to a level equal to 50% of that of non-challenged cells.
Values are mean SEM of at least three independent experiments similar to those shown in Figures 1 and 2.
c
Defined as % loss of cell viability in the presence of 10 lM test compound compared to vehicle in the absence of glutamate as assessed using the Trypan blue exclusion
assay (Section 5.2.2). Values are mean SEM of at least three independent experiments carried out in triplicate. ns = non-significant (p >0,05; t-test).
*
Assessed at 3
Assessed at 1
l
l
M due to cytotoxicity at test compound concentrations P10
M due to cytotoxicity at test compound concentrations P3
l
M.
lM.
1 h at rt. The excess of BF3ꢁSMe2 was removed under argon, water
was added and the mixture was extracted with AcOEt. The organic
layer was washed with satd aqueous NaCl, dried and concentrated
in vacuo.
7.72 min, detection at 254 nm, purity 99.8%. 1H NMR d: 8.44 (s,
1H), 7.79 (d, 2H, J = 8.7 Hz), 7.01 (d, 2H, J = 8.7 Hz), 6.66 (s, 1H),
3.88 (s, 3H), 2.84 (t, 2H, J = 6.7 Hz), 2.24 (s, 3H), 2.18 (s, 3H), 1.76
(t, 2H, J = 6.7 Hz), 1.36 (s, 6H). 13C NMR d: 169.5, 161.7, 161.3,
146.7, 145.2, 128.8, 127.6, 123.6, 119.8, 115.5, 114.4, 110.0, 99.8,
72.8, 55.4, 32.9, 27.0, 22.8, 12.4. HRMS: calcd for C23H26NO4
(M+H)+ 380.1856. Found: 380.1847.
5.1.2.1. 3,4-Dihydro-2,2,7,8-tetramethyl-5-(5-phenylisoxazol-3-
yl)-2H-1-benzopyran-6-ol (5). Purification by column chroma-
tography (pet. ether–AcOEt 9:1). Yield 49%, yellow solid, mp
155–157 °C. HPLC: 8:2 MeOH–H2O, 0.05% TFA, flow rate: 1.5 mL/
min, tR: 6.97 min, detection at 254 nm, purity 97.9%. 1H NMR d:
8.50 (s, 1H), 7.87–7.84 (m, 2H), 7.51–7.48 (m, 3H), 6.79 (s, 1H),
2.84 (t, 2H, J = 6.7 Hz), 2.24 (s, 3H), 2.18 (s, 3H), 1.77 (t, 2H,
J = 6.7 Hz), 1.36 (s, 6H). 13C NMR d: 169.5, 161.7, 146.7, 145.2,
136.5, 130.5, 129.1, 128.9, 126.0, 123.7, 115.5, 109.9, 101.1, 72.8,
5.1.2.4. 3,4-Dihydro-2,2,7,8-tetramethyl-5-(5-(4-hydroxyphenyl)-
isoxazol-3-yl)-2H-1-benzopyran-6-ol (8). Yield: 51%, yellowish
solid, mp 217–219 °C. HPLC: 7:3 MeOH–H2O, flow rate: 1 mL/
min, tR: 2.93 min, detection at 254 nm, purity 99.9%. 1H NMR
(CDCl3, MeOD) d: 7.66 (d, 2H, J = 8.7 Hz), 6.88 (d, 2H, J = 8.7 Hz),
6.57 (s, 1H), 2.76 (t, 2H, J = 6.6 Hz), 2.17 (s, 3H), 2.12 (s, 3H), 1.70
(t, 2H, J = 6.6 Hz), 1.30 (s, 6H). 13C NMR (CDCl3, MeOD) d: 170.0,
161.5, 159.1, 146.2, 145.2, 128.6, 127.7, 127.6, 123.5, 118.8,
115.9, 115.8, 110.4, 99.4, 72.9, 32.8, 26.8, 22.6, 12.3. HRMS: calcd
for C22H24NO4 (M+H)+ 366.1700. Found: 366.1691.
32.9, 27.0, 22.8, 12.4. HRMS: calcd for:
350.1751. Found: 350.1736.
C
22H24NO3 (M+H)+
5.1.2.2. 3,4-Dihydro-2,2,7,8-tetramethyl-5-(5-(4-nitrophenyl)-
isoxazol-3-yl)-2H-1-benzopyran-6-ol (6). Purification by column
chromatography (pet. ether–AcOEt 8.5:1.5). Yield: 59%, yellow-or-
ange solid 203–205 °C. HPLC: 8:2 MeOH–H2O, flow rate: 1.5 mL/
min, tR: 5.35 min, detection at 297 nm, purity 98.8%. 1H NMR d:
8.38 (d, 2H, J = 8.8 Hz) 8.04 (d, 2H, J = 8.8 Hz), 8.13 (s, 1H), 6.98
(s, 1H), 2.83 (t, 2H, J = 6.6 Hz), 2.24 (s, 3H), 2.19 (s, 3H), 1.77 (t,
2H J = 6.6 Hz), 1.36 (s, 6H). 13C NMR d: 166.9, 162.0, 148.6, 146.6,
145.4, 132.5, 129.4, 126.8, 126.7, 124.5, 123.9, 115.4, 109.4,
103.8, 72.9, 32.8, 29.7, 26.9, 22.9, 12.5, 12.4. HRMS: calcd for
5.1.2.5. 3,4-Dihydro-2,2,7,8-tetramethyl-5-(5-propylisoxazol-3-
yl)-2H-1-benzopyran-6-ol (9). Purification by column chroma-
tography (pet. ether–AcOEt 8.5:1.5). Yield: 25%, yellowish solid,
mp 160–162 °C. HPLC: 8:2 MeOH–H2O, flow rate: 1.5 mL/min, tR:
5.23 min, detection at 254 nm, purity 98.9%. 1H NMR d: 8.54 (s,
1H), 6.26 (s, 1H), 2.83–2.74 (m, 4H), 2.22 (s, 3H), 2.16 (s, 3H),
1.84–1.72 (m, 4H), 1.34 (s, 6H), 1.03 (t, 3H, J = 7.3 Hz). 13C NMR
d: 173.2, 161.1, 146.7, 145.1, 128.6, 123.6, 115.5, 110.0, 102.6,
32.9, 28.6, 26.9, 22.8, 20.9, 13.7, 12.3. HRMS: calcd for C19H26NO3
(M+H)+ 316.1907. Found: 316.1895.
C
22H23N2O5 (M+H)+ 395.1607. Found: 395.1582.
5.1.2.3. 3,4-Dihydro-2,2,7,8-tetramethyl-5-(5-(4-methoxyphenyl)-
isoxazol-3-yl)-2H-1-benzopyran-6-ol (7). According to the gen-
eral procedure, described above, a mixture of analogues 7 and 8
was obtained which was purified by column chromatography
(pet. ether–AcOEt 8.5:1.5). Yield: 36%, yellowish solid, mp 160–
162 °C. HPLC: 8:2 MeOH–H2O, flow rate: 1.5 mL/min, tR:
5.1.2.6. 3,4-Dihydro-2,2,7,8-tetramethyl-5-(3-phenylisoxazol-5-
yl)-2H-1-benzopyran-6-ol (14). Purification by column chroma-
tography (pet. ether–AcOEt 9:1). Yield: 93%, yellow solid, mp
123–125 °C. HPLC: 8:2 MeOH–H2O, flow rate: 1.5 mL/min, tR:
4.93 min, detection at 254 nm, purity 95%. 1H NMR d: 7.89–7.87

M. Koufaki et al. / Bioorg. Med. Chem. 19 (2011) 4841–4850
4847
Table 2
Efficacy and potency of 2-substituted chroman analogues to protect glutamate-challenged HT22 cells from oxytosis
b
Compound
Efficacya (%)
TC50
(lM)
Cytotoxicityc (%)
HO
HO
HO
HO
25
81 3*
0.53 0.13
1.07 0.14
0.45 0.11
35
ns
ns
8
O
O
O
O
N
N
N
O
O
O
26
27
70
71
5
4
OCH3
HO
34
86
4
0.63 0.11
ns
N
O
O
HO
HO
31
32
82
87
5
8
0.52 0.09
0.42 0.08
ns
ns
O
O
N
N
OCH3
O
O
O
HO
38
39
40
87
60
74
4
6
4
0.54 0.09
7.11 1.7
0.69 0.04
ns
ns
ns
O
O
N
H3CO
HO
OH
O
O
O
N
OCH3
O
O
O
N
a,b,c
As for legend to Table 1.
Assessed at 3 M due to cytotoxicity at 10 lM test compound.
*
l
(m, 2H), 7.51–7.48 (m, 3H), 6.71 (s, 1H), 2.73 (t, 2H, J = 6.7 Hz), 2.22
(s, 3H), 2.19 (s, 3H), 1.74 (t, 2H, J = 6.7 Hz), 1.34 (s, 6H). 13C NMR d:
167.6, 162.7, 145.8, 145.3, 130.2, 129.6, 129.0, 127.0, 123.3, 116.4,
110.2, 102.7, 73.2, 33.0, 29.7, 27.0, 22.0, 12.4, 12.2. HRMS: calcd for
5.1.2.8. 3,4-Dihydro-2,2,7,8-tetramethyl-5-(3-(4-nitrophenyl)-
isoxazol-5-yl)-2H-1-benzopyran-6-ol (16). Purification by column
chromatography (pet. ether–AcOEt 9:1). Yield: 70%, yellow solid,
mp 172–174 °C. HPLC: 8:2 MeOH–H2O, flow rate: 1.5 mL/min, tR:
4.71 min, detection at 297 nm, purity 99.4%. 1H NMR d: 8.35 (d,
2H, J = 8.8 Hz), 8.06 (d, 2H, J = 8.8 Hz), 6.80 (s, 1H), 2.74 (t, 2H,
J = 6.7 Hz), 2.22 (s, 3H), 2.19 (s, 3H), 1.75 (t, 2H, J = 6.7 Hz), 1.34
(s, 6H). 13C NMR d: 168.9, 160.8, 148.7, 145.9, 145.2, 135.0,
130.0, 127.7, 124.2, 123.2, 116.5, 110.0, 102.7, 73.2, 53.4, 32.6,
29.7, 29.6, 26.8, 21.9, 12.5, 12.2. HRMS: calcd for C22H23N2O5
(M+H)+ 395.1607. Found: 395.1584.
C
22H24NO3 (M+H)+ 350.1751. Found: 350.1736.
5.1.2.7. 3,4-Dihydro-5-(3-(4-fluorophenyl)isoxazol-5-yl)-2,2,7,8-
tetramethyl-2H-1-benzopyran-6-ol (15). Purification by column
chromatography (pet. ether–AcOEt 9:1). Yield: 99%, yellow solid,
mp 152–154 °C. HPLC: 8:2 MeOH–H2O, flow rate: 1.5 mL/min, tR:
4.70 min, detection at 254 nm, purity 100%. 1H NMR d: 7.87–7.83
(m, 2H), 7.17 (t, 2H, J = 8.4 Hz), 6.67 (s, 1H), 2.72 (t, 2H,
J = 6.7 Hz), 2.22 (s, 3H), 2.19 (s, 3H), 1.74 (t, 2H, J = 6.7 Hz), 1.34
(s, 6H). 13C NMR d: 167.8, 165.5, 162.2, 161.7, 145.8, 145.2,
129.7, 128.8, 128.7, 125.1, 125.0, 123.3116.0, 115.9, 102.5, 73.2,
32.7, 26.8, 21.9, 12.4, 12.2. 19F NMR d (ppm): ꢃ110.3. HRMS: calcd
for C22H23FNO3 (M+H)+ 368.1656. Found: 368.1642.
5.1.2.9. 3,4-Dihydro-5-(3-(4-methoxyphenyl)isoxazol-5-yl)-2,2,7,
8-tetramethyl-2H-1-benzopyran-6-ol (17). Purification by column
chromatography (pet. ether–AcOEt 9:1). Yield: 82%, yellow solid,
mp 155–157 °C. HPLC: 8:2 MeOH–H2O, flow rate: 1.5 mL/min,
tR: 5.28 min, detection at 297 nm, purity 100%. 1H NMR d: 7.80

4848
M. Koufaki et al. / Bioorg. Med. Chem. 19 (2011) 4841–4850
(d, 2H, J = 8.5 Hz), 7.00 (d, 2H, J = 8.5 Hz), 6.65 (s, 1H), 5.61 (s, 1H),
3.87 (s, 3H), 2.73 (t, 2H, J = 6.6 Hz), 2.22 (s, 3H), 2.18 (s, 3H), 1.74 (t,
2H, J = 6.6 Hz), 1.33 (s, 6H). 13C NMR d: 167.3, 162.3, 161.1, 145.7,
145.3, 129.5, 128.3, 124.8, 123.2, 121.3, 116.3, 114.4, 110.2, 102.4,
73.1, 55.4, 32.7, 26.8, 21.9, 12.4, 12.2. HRMS: calcd for C23H26NO4
(M+H)+ 380.1856. Found: 380.1844.
5.1.2.15. 3,4-Dihydro-2,5,7,8-tetramethyl-2-(3-phenyl-5-isoxaz-
olyl)-2H-1-benzopyran-6-ol (31). Purification by column chroma-
tography (pet. ether–AcOEt 9:1). Yield: 33%, yellow gummy solid.
HPLC: 8:2 MeOH–H2O, flow rate: 1 mL/min, tR: 5.68 min, detection
at 254 nm, purity 96.6%. 1H NMR d: 7.75–7.72 (m, 2H), 7.42–7.35
(m, 3H), 6.24 (s, 1H), 2.71–2.62 (m, 1H), 2.61–2.48 (m, 1H), 2.47–
2.33 (m, 1H), 2.24 (s, 3H), 2.19 (s, 3H), 2.10–2.09 (m, 1H), 2.04 (s,
3H), 1.75 (s, 3H). 13C NMR d: 175.9, 162.2, 145.4, 144.7, 129.9,
128.8, 126.8, 122.3, 121.3, 118.5, 117.1, 99.0, 74.2, 31.4, 27.3,
5.1.2.10. 3,4-Dihydro-5-(3-(4-hydroxyphenyl)isoxazol-5-yl)-2,2,7,
8-tetramethyl-2H-1-benzopyran-6-ol (18). Obtained by depro-
tection of 17. Purification by column chromatography (pet.
ether–AcOEt 9:1). Yield: 56%, yellow oil. HPLC: 8:2 MeOH–H2O,
flow rate: 1 mL/min, tR: 4.46 min, detection at 254 nm, purity
97.3%. 1H NMR d: 7.72 (d, 2H, J = 7.9 Hz), 6.89 (d, 2H, J = 7.9 Hz),
6.80 (s, 1H), 2.64 (t, 2H, J = 6.4 Hz), 2.18 (s, 3H), 2.14 (s, 3H), 1.73
(t, 2H, J = 6.4 Hz), 1.31 (s, 6H). 13C NMR d: 169.6, 163.7, 160.6,
147.0, 129.5, 129.4, 125.6, 121.7, 118.4, 116.8, 114.5, 104.1, 74.3,
33.9, 30.8, 27.1, 22.6, 12.7, 12.6. HRMS: calcd for: C22H24NO4
(M+H)+ 366.1700. Found: 366.1686.
20.7, 12.2, 11.9, 11.2. HRMS: calcd for:
350.1751. Found: 350.1740.
C
22H24NO3 (M+H)+
5.1.2.16. (3,4-Dihydro-2,5,7,8-tetramethyl-2-[3-(4-methoxyphenyl)-
isoxazol-5-yl]-2H-1-benzopyran-6-ol (32). Purification by column
chromatography (pet. ether–AcOEt 9:1). Yield: 67% yellowish oil.
HPLC: 8:2 MeOH–H2O, flow rate: 1.5 mL/min, tR: 3.84 min, detec-
tion at 254 nm, purity 100%. 1H NMR (600 MHz) d: 7.67 (d, 2H,
J = 8.8 Hz), 6.92 (d, 2H, J = 8.8 Hz), 6.18 (s, 1H), 4.27 (s, 1H), 3.83
(s, 3H), 2.68–2.65 (m, 1H), 2.55–2.52 (m, 1H), 2.43–2.38 (m, 1H),
2.23 (s, 3H), 2.19 (s, 3H), 2.10–2.05 (m, 1H), 2.04 (s, 3H), 1.73 (s,
3H). 13C NMR d: 175.7, 161.8, 160.9, 145.4, 144.8, 128.2, 122.3,
121.5, 121.3, 118.5, 117.1, 114.1, 98.7, 74.1, 55.3, 31.4, 27.3, 20.7,
12.2, 11.9, 11.2. HRMS: calcd for: C23H26NO4 (M+H)+ 380.1856.
Found: 380.1842.
5.1.2.11. 5,50-(isoxazole-3,5-diyl)bis(3,4-dihydro-2,2,7,8-tetra-
methyl-2H-1-benzopyran-6-ol) (20). Purification by column chro-
matography (pet. ether–AcOEt 9:1). Yield: 95%, yellow gummy
solid. HPLC: 9:1 MeOH–H2O, flow rate: 1.5 mL/min, tR: 3.61 min,
detection at 254 nm, purity 95.7%. 1H NMR d: 6.69 (s, 1H), 2.82
(t, 2H, J = 6.6 Hz), 2.71 (t, 2H, J = 6.7 Hz), 2.25 (s, 3H), 2.22 (s, 3H),
2.19 (s, 6H), 1.78–1.72 (m, 4H), 1.35 (s, 6H), 1.34 (s, 6H). 13C
NMR d: 166.7, 161.5, 146.8, 145.9, 145.3, 145.1, 129.8, 129.1,
123.8, 123.2, 116.5, 115.5, 110.0, 109.7, 106.4, 73.2, 72.9, 32.9,
32.7, 29.7, 27.0, 26.8, 22.9, 21.9, 12.5, 12.4, 12.2. HRMS: calcd
for: C29H36NO5 (M+H)+ 478.2588. Found: 478.2581.
5.1.2.17. 2-(5-(3,4-Dihydro-6-hydroxy-2,2,7,8-tetramethyl-2H-1-
benzopyran-5-yl)isoxazol-3-yl)-3,4-dihydro-2,5,7,8-tetramethyl-
2H-1-benzopyran-6-ol (34). Purification by column chromatography
(pet. ether–AcOEt 9:1). Yield: 79%, yellow oil. HPLC: 8:2 MeOH–
H2O, flow rate: 1.5 mL/min, tR: 5.86 min, detection at 254 nm,
purity 99.6%. 1H NMR (600 MHz) d: 6.25 (s, 1H, H-isoxazole), 5.55
(s, 1H, OH), 4.25 (s, 1H, OH), 2.70–2.65 (m, 3H, –CH2), 2.55–2.52
(m, 3H, –CH2), 2.20 (s, 3H, Ar–CH3), 2.17 (s, 6H, Ar–CH3), 2.14 (s,
3H, Ar–CH3), 2.06 (s, 3H, Ar–CH3), 1.74 (s, 3H, –CH3), 1.69–1.66
(m, 2H, –CH2), 1.30 (s, 6H, –CH3). 13C NMR d: 168.8, 166.5, 145.7,
145.4, 145.1, 144.9, 129.5, 123.2, 122.33, 121.2, 118.6, 117.5,
116.1, 110.0, 103.1, 73.5, 73.0, 32.7, 31.9, 27.8, 26.8, 21.8, 20.9,
5.1.2.12. 3,4-Dihydro-2,5,7,8-tetramethyl-2-(5-phenyl-3-isoxaz-
olyl)-2H-1-benzopyran-6-ol (25). Purification by column chro-
matography (pet. ether–AcOEt 9:1). Yield: 58%, yellow oil. HPLC:
8:2 MeOH–H2O, flow rate: 1.5 mL/min, tR: 4.1 min, detection at
254 nm, purity 99.0%. 1H NMR d: 7.72–7.70 (m, 2H), 7.44–7.40
(m, 3H), 6.36 (s, 1H), 2.69–2.55 (m, 3H), 2.24 (s, 3H), 2.18 (s, 3H),
2.14–2.10 (m, 1H), 2.05 (s, 3H), 1.72 (s, 3H). 13C NMR d: 169.5,
169.3, 145.3, 145.0, 130.0, 128.8, 127.4, 125.8, 122.3, 121.1,
118.6, 117.5, 98.0, 73.5, 31.6, 28.4, 20.9, 12.2, 11.9, 11.2. HRMS:
calcd for: C22H24NO3 (M+H)+ 350.1751. Found: 350.1740.
12.3, 12.2, 11.9, 11.2. HRMS: calcd for:
478.2588. Found: 478.2576.
C
29H36NO5 (M+H)+
5.1.2.18. 2-{[(3-(phenyl)isoxazol-5-yl)methoxy]methyl}-2,5,7,8-
tetramethyl-2H-1-benzopyran-6-ol (38). Purification by column
chromatography (pet. ether–AcOEt 9:1). Yield: 68% yellowish oil.
HPLC: 8:2 MeOH–H2O, flow rate: 1.5 mL/min, tR: 3.45 min, detec-
tion at 254 nm, purity 98.7%. 1H NMR (600 MHz) d: 7.80–7.78 (m,
2H), 7.47–7.44 (m, 3H), 6.51 (s, 1H), 4.79–4.72 (m, 2H), 4.24 (s, 1H),
3.60 (dd, 2H, J = 26.8, 10.0 Hz), 2.63 (t, 2H, J = 6.7 Hz), 2.16 (s, 3H),
2.12 (s, 3H), 2.11 (s, 3H), 2.03–1.98 (m, 1H), 1.81–1.76 (m, 1H), 1.31
(s, 3H). 13C NMR d: 170.0, 162.3, 145.0, 144.9, 130.0, 128.9, 128.9,
126.8, 122.5, 121.2, 118.5, 117.2, 100.8, 76.2, 74.7, 64.6, 29.7,
28.6, 22.0, 20.3, 12.2, 11.9, 11.3. HRMS: calcd for: C24H28NO4
(M+H)+ 394.2013. Found: 394.1995.
5.1.2.13. 3,4-Dihydro-2,5,7,8-tetramethyl-2-[5-(4-methoxyphenyl)-
3-isoxazolyl]-2H-1-benzopyran-6-ol (26). Purification by column
chromatography (pet. ether–AcOEt 9:1). Yield: 58%, yellow oil.
HPLC: 8:2 MeOH–H2O, flow rate: 1.5 mL/min, tR: 4.17 min, detec-
tion at 254 nm, purity 99.7%. 1H NMR (600 MHz) d: 7.64 (d, 2H,
J = 8.8 Hz), 6.93 (d, 2H, J = 8.8 Hz), 6.24 (s, 1H), 4.23 (s, 1H), 3.84
(s, 3H), 2.66–2.56 (m, 3H), 2.23 (s, 3H), 2.18 (s, 3H), 2.12–2.10
(m, 1H), 2.05 (s, 3H), 1.71 (s, 3H). 13C NMR d: 169.5, 169.2, 160.9,
145.3, 145.0, 127.4, 122.3, 121.1, 120.3, 118.6, 117.5, 114.2, 96.6,
55.3, 31.7, 28.4, 20.9, 12.2, 11.9, 11.2. HRMS: calcd for:
C
23H26NO4 (M+H)+ 380.1856. Found: 380.1844.
5.1.2.19. 5-{[(3,4-Dihydro-6-methoxy-2,5,7,8-tetramethyl-2H-1-
benzopyran-2-yl)methoxy]methyl}-3-(4-hydroxyphenyl) isox-
azole (39). According to the general procedure described above a
mixture of analogues 39 and 40 was obtained which was purified
by column chromatography (pet. ether–AcOEt 8:2). Yield: 29% yel-
low oil. HPLC: 8:2 MeOH–H2O, flow rate: 1.5 mL/min, tR: 3.78 min,
detection at 254 nm, purity 97.4%. 1H NMR (600 MHz, CDCl3) d:
7.67 (d, 2Y, J = 8.3 Hz), 6.90 (d, 2H, J = 8.3 Hz), 6.46 (s, 1H), 5.53
(s, 1H), 4.76–4.70 (m, 2H), 3.63 (s, 3H), 3.65–3.56 (dd, 2H), 2.60
(t, 2H, J = 6.7 Hz), 2.18 (s, 3H), 2.13 (s, 3H), 2.09 (s, 3H), 2.03–1.98
(m, 1H), 1.80–1.75 (m, 1H), 1.31 (s, 3H). 13C NMR d: 169.8, 161.9,
157.3, 149.7, 147.3, 128.4, 128.0, 125.9, 122.8, 121.5, 117.5,
5.1.2.14. 3,4-Dihydro-2,5,7,8-tetramethyl-2-[5-(cyclopentyl-3-
isoxazolyl]-2H-1-benzopyran-6-ol (27). Purification by column
chromatography (pet. ether–AcOEt 9:1). Yield: 64%, yellow oil.
HPLC: 8:2 MeOH–H2O, flow rate: 1 mL/min, tR: 6.01 min, detection
at 286 nm, purity 100%. 1H NMR (600 MHz) d: 5.83 (s, 1Y), 4.25 (s,
1Y), 3.11–3.08 (m, 1Y), 2.65–2.62 (m, 2H), 2.51–2.47 (m, 1H), 2.18
(s, 6H), 2.09–2.07 (m, 1H), 2.06 (s, 3H), 2.05–2.00 (m, 2H), 1.74–
1.66 (m, 5H), 1.64 (s, 3H), 163–1.62 (m, 1H). 13C NMR d: 176.9,
168.5, 145.2, 145.0, 122.3, 121.1, 118.5, 117.5, 97.7, 73.4, 37.5,
31.8, 31.7, 31.6, 28.0, 25.2, 20.8, 12.2, 11.8, 11.2. HRMS: calcd
for: C21H28NO3 (M+H)+ 342.2064. Found: 342.2051.

M. Koufaki et al. / Bioorg. Med. Chem. 19 (2011) 4841–4850
4849
115.8, 100.7, 76.2, 75.0, 64.6, 60.4, 28.3, 22.1, 20.1, 12.6, 11.9, 11.7.
HRMS: calcd: C25H30NO5 (M+H)+ 424.2118. Found: 424.2103.
of the Total Cell Count (TCC) was used to measure% cytotoxicity as
calculated by: (SCCCompound ꢃ SCCVehicle)/TCC. Innate cytotoxicity of
HT22 cell culture (i.e., SCCVihecle/TCC) was <5%. Values are mean
SEM of at least three independent experiments, each carried out
in triplicate. In a few cases, cytotoxicity was assessed using the
LDH release assay with largely similar results.
5.1.2.20. 2-{[(3-(4-methoxyphenyl)isoxazol-5-yl)methoxy]-
methyl}-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol (40). Yield:
8% yellow oil. HPLC: 8:2 MeOH–H2O, flow rate: 1.5 mL/min, tR:
3.53 min, detection at 260 nm, purity 99.8%. 1H NMR (600 MHz)
d: 7.72 (d, 2Y, J = 8.7 Hz, ArH), 6.97 (d, 2H, J = 8.7 Hz, ArH), 6.45
(s, 1H, H-isoxazole), 4.76–4.69 (m, 2H, –O–CH2–), 4.20 (s, 1H, OH),
3.86 (s, 3H, –OCH3), 3.59 (dd, 2H, J = 27.3, 10.0 Hz, –CH2–O), 2.62
(t, 2H, J = 6.8 Hz, –CH2), 2.16 (s, 3H, Ar–CH3), 2.12 (s, 3H, Ar–CH3),
2.10 (s, 3H, Ar–CH3), 2.02–1.98 (m, 1H, –CHH), 1.80–1.76 (m, 1H,
–CHH), 1.30 (s, 3H, –CH3). 13C NMR d: 169.7, 161.9, 161.0, 145.1,
144.9, 128.2, 122.5, 121.5, 121.2, 118.5, 117.3, 114.3, 100.6, 74.7,
64.6, 55.3, 29.7, 28.6, 22.0, 20.3, 12.2, 11.9, 11.3. HRMS: calcd
for: C25H30NO5 (M+H)+ 424.2118. Found: 424.2104.
5.2.3. Statistics
The statistical significance of the differences observed was
determined using t-test. Differences were considered significant
for values of p <0.05.
Acknowledgment
This work was supported in part by ‘EURODESY’ MEST-CT2005-
020575.
5.2. Biology
Supplementary data
5.2.1. Evaluation of the activity of chroman analogues to
prevent oxytosis of HT22 hippocampal neurons
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.074.
The new compounds were tested as previously described.6,26
Briefly, HT22 cells were plated in a 96-well flat bottom plate at a
References and notes
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absence of chroman analogues was calculated by CDVehicle
[(dODVehicle ꢃ dODGlutamate) ꢄ 100/dODVehicle, whereas cell death in
their presence was calculated by CDCompound = [(dODCompound
Neuroprotection (%)
=
ꢃ
dODCompound+Glutamate) ꢄ 100/dODCompound
.
was calculated by [(CDVehicle ꢃ CDCompound) ꢄ 100/CDVehicle. Values
are mean SEM of at least three independent experiments, each
carried out in triplicate.
5.2.2. Evaluation of cytotoxicity of chroman analogues
Cytotoxicity was assessed as already described.26 Briefly, HT22
cells were plated in a 24-well flat bottom plates at a density of
10,000 cells per well in DMEM-Hepes-GlutaMAX medium contain-
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the cells were incubated with increasing concentrations (0.03–
10 lM) of chroman analogues or vehicle (60.1% DMSO) in fresh
medium for 24 h. After the 24-h incubation, cell culture superna-
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penetrate dead cells. Therefore, the Stained Cell Count (SCC) in the
absence and presence of chroman analogues, given as percentages
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