Design and synthesis of novel neuroprotective 1,2-dithiolane/chroman hybrids

Article abstract of DOI:10.1016/j.bmc.2009.07.010

Novel 1,2-dithiolane/chroman hybrids bearing heterocyclic rings such as 1,2,4- and 1,3,4-oxadiazole, 1,2,3-triazole and tetrazole were designed and synthesized. The neuroprotective activity of the new analogues was tested against oxidative stress-induced cell death of glutamate-challenged HT22 hippocampal neurons. Our results show that bioisosteric replacement of amide group in 2-position of the chroman moiety, by 1,3,4-oxadiazole did not affect activity. However, analogue 5 bearing the 1,2,4-oxadiazole moiety showed improved neuroprotective activity. The presence of nitrogen heterocycles strongly influences the neuroprotective activity of 5-substituted chroman derivatives, depending on the nature of heterocycle. Replacement of the amide group of the first generation analogues by 1,2,4-oxadiazole or 1,2,3-triazole resulted in significant improvement of the activity against glutamate induced oxidative stress.

Full text of DOI:10.1016/j.bmc.2009.07.010

Bioorganic & Medicinal Chemistry 17 (2009) 6432–6441
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of novel neuroprotective 1,2-dithiolane/chroman hybrids
a
b
b
Maria Koufaki a, , Christina Kiziridi , Xanthippi Alexi , Michael N. Alexis
*
a Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., 11635, Athens, Greece
b Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 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:
Novel 1,2-dithiolane/chroman hybrids bearing heterocyclic rings such as 1,2,4- and 1,3,4-oxadiazole,
1,2,3-triazole and tetrazole were designed and synthesized. The neuroprotective activity of the new ana-
logues was tested against oxidative stress-induced cell death of glutamate-challenged HT22 hippocampal
neurons. Our results show that bioisosteric replacement of amide group in 2-position of the chroman
moiety, by 1,3,4-oxadiazole did not affect activity. However, analogue 5 bearing the 1,2,4-oxadiazole
moiety showed improved neuroprotective activity. The presence of nitrogen heterocycles strongly influ-
ences the neuroprotective activity of 5-substituted chroman derivatives, depending on the nature of het-
erocycle. Replacement of the amide group of the first generation analogues by 1,2,4-oxadiazole or 1,2,3-
triazole resulted in significant improvement of the activity against glutamate induced oxidative stress.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 13 May 2009
Revised 29 June 2009
Accepted 5 July 2009
Available online 9 July 2009
Keywords:
1,2-Dithiolane
Chroman
Bioisosterism
Oxidative stress
Neuronal damage
1. Introduction
Based on our experience on bioactive dithiolane analogues, we
designed and synthesized novel 1,2-dithiolane/chroman hybrids
An increasing number of studies published the last decade have
reported that -lipoic acid (LA) is beneficial in a number of oxida-
tive stress models of cell death. The potential of LA to work as a
therapeutic agent appears to lie not only in its actions as a direct
scavenger of ROS/RNS, but also in its ability to affect signaling
cascades.1
containing heterocyclic rings such as 1,2,4- and 1,3,4-oxadiazole,
1,2,3-triazole and tetrazole. The neuroprotective activity of the
new analogues was evaluated using glutamate-challenged hippo-
campal HT22 cells.
a
2. Chemistry
In addition, LA represents an ideal chemical entity for the
development of multifunctional compounds.2 The design and
synthesis of hybrid molecules encompassing two pharmacophores
in one molecular scaffold is a well established approach to the
synthesis of more potent drugs with dual activity.3 Using this
approach, several research groups have recently designed and
synthesized hybrid molecules by coupling LA with other bioactive
molecules. These efforts resulted in new molecules with antioxi-
dant activity hyphenated with a wide variety of other activities
such as: protection against reperfusion arrhythmias,4,5 nitric oxide
synthase inhibition,6 erythrocyte protection from hemolysis,7
antiproliferative activity,8,9 acetylcholinesterase inhibition,10
butyrylcholinesterase inhibition,11 as well as radioprotective,12
and anti-inflammatory activity.13,14
The synthesis of 1,2,4-oxadiazole analogues is depicted in
Scheme 1. For the preparation of racemic 2-substituted chroman
analogue 5, mesylate 116 was converted to nitrile 2 using NaCN,
DMSO and then, to the N-hydroxy-amidine 3 by treatment with
hydroxylamine hydrochloride and Et3N. Reaction of 3 with N-
hydroxysuccinimidyl-trolox ester gave the acyl amidoxime 4 and
subsequent intramolecular cyclization in the presence of tetrabu-
tylammonium fluoride produced the 1,2,4-oxadiazole analogue 5.
5-Substituted chroman analogue 10 was prepared from bromide
17
65 using similar procedure and was deprotected using BF3ꢀS(Me)2
to afford the final analogue 11.
1,3,4-Oxadiazole analogues were synthesized as shown in
Scheme 2. Hydrazide 12 was prepared from the trolox methyl ester
and then reacted with N-hydroxysuccinimide-activated LA to give
13. Cyclodehydration in boiling POCl3 produced the 2-substituted
chroman analogue 14. For the synthesis of 5-substituted chroman
derivatives, LA was converted to the corresponding ethyl ester 15
and then to hydrazide 16 by treatment with hydrazine. Reaction
of 16 with 3,4-dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-1-
benzopyran-5-carboxylic acid in the presence of benzotriazol-1-
Moreover, combination of 1,2-dithiolane-3-alkyl group with the
catechol moiety through five-membered heterocyclic rings, as bio-
isosters,15 of the amide bond led to compounds which exhibited
strong neuroprotective activity.16
* Corresponding author. Tel.: +30 210 7273818; fax: +30 210 7273831.
E-mail address: mkoufa@eie.gr (M. Koufaki).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.010

M. Koufaki et al. / Bioorg. Med. Chem. 17 (2009) 6432–6441
6433
NOH
NH2
b
a
CN
OSO2Me
S
S
S
S
S
S
3
2
1
HO
HO
d
c
H2N
O
O
O
S
S
N
O
N
S
S
N
O
5
4
NOH
CN
Br
NH2
MeO
MeO
MeO
e
a
b
O
O
O
6
7
8
O
N
S
S
N
O
NH2
S
S
RO
MeO
d
O
O
10 R = OMe
11 R = OH
9
f
Scheme 1. Synthesis of 1,2,4-oxadiazole analogues. Reagents and conditions: (a) NaCN, DMSO; (b) NH2OHꢀHCl, Et3N, EtOH, (c) N-hydroxysuccinimidyl-trolox ester, CH2Cl2;
(d) TBAF, THF; (e) lipoic acid, DCC, CH2Cl2; (f) BF3ꢀS(Me)2, CH2Cl2.
yloxytris(dimethylamino) phosphonium hexafluorophosphate
(BOP) and Et3N, gave hydrazide 17 which was cyclized in boiling
POCl3 to afford 18. Analogue 19 was obtained by treatment of 18
with BF3ꢀS(Me)2.
controls in order to investigate the influence of the replacement
of amide group by nitrogen heterocycles on the activity against
oxidative stress-induced cell death in HT22 cells.
CuI-catalyzed ‘click’ cycloaddition18–20 between 3-(5-azidopen-
tyl)-1,2-dithiolane and alkynes 23, 27, 28 as well as between
(3,4-dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-benzopyran-5-yl)-
methylazide, and alkyne 34, in the presence of CuSO4ꢀ5H2O and
sodium ascorbate, afforded the 1,2,3-triazole analogues 24, 29,
30, 35 (Scheme 3).
3. Results and discussion
The mouse hippocampal cell line HT22 has been used to eluci-
date sequential cellular events during programmed cell death from
oxidative stress (oxytosis) caused by glutamate-induced depletion
of intracellular glutathione.30 Although HT22 cells lack ionotropic
glutamate receptors that could mediate excitotoxicity, they under-
go oxytosis within 24 h following exposure to 1–5 mM glutamate.
Recent findings suggest that oxytosis faithfully mimics cytotoxicity
due to oxidative stress in Alzheimer’s disease and other neurode-
generative disorders.31,32
Reduction of trolox ethyl ester 20 to the alcohol 21, followed by
Swern oxidation21,22 gave aldehyde 22 which, in turn reacted with
Bestmann–Ohira reagent23,24 in the presence of K2CO3 to afford al-
kyne 23. Alkynes 27 and 28 were prepared by treatment of the
appropriate aldehydes25,26 with Bestmann–Ohira reagent in the
presence of K2CO3. Alkyne 34 was prepared from lipolol which
was oxidized to the corresponding aldehyde 33 using Pfitzner–
Moffatt conditions.27,28
Figure 1 shows the neuroprotective activity of the 2-substituted
chroman derivatives against oxytosis of glutamate-challenged hip-
pocampal HT22 cells. Amide analogue I and its isostere 45, have
comparable EC50 values (1.24 0.38 and 1.59 0.53
tively) (Table 1, column 2). Bioisosteric replacement of amide
group of analogue 45 by 1,3,4-oxadiazole (analogue 14, EC50
1.65 0.36 M) had no effect in antioxidant activity. However,
analogue 5 bearing the 1,2,4-oxadiazole moiety showed improved
neuroprotective activity (EC50 = 0.93 0.19 M). Differences in
activity between 1,2,4-oxadiazoles and 1,3,4-oxadiazoles were also
observed by other researchers.33 Specifically, C-glucosylated 1,3,4-
oxadiazoles proved practically inactive while the 1,2,4-oxadiazole
series displayed inhibition of glycogen phosphorylase in the micro-
molar range. Although in this case the nature of the heterocycle
influences the interaction with the enzyme and thus the inhibitory
lM, respec-
The synthesis of tetrazole derivatives is depicted in Scheme 4.
Acylation of amines 36 and 37 with N-(lipoyloxy)succinimide,
according to procedures reported in previous publications of our
group,5,26 afforded amides 38 and 39, which in turn were con-
verted to thioamides 40 and 41 by treatment with Lawesson’s re-
agent. Tetrazoles 42 and 43 were obtained by treatment of
thioamides with trimethylsilyl azide (TMSN3), in the presence of
triphenylphosphine and diisopropylazodicarboxylate (DIAD).29
Analogue 44 was obtained by treatment of 42 with BF3ꢀS(Me)2.
Amide analogues I and II which were previously synthesized in
our laboratory5 as well as compound 45, synthesized from acti-
vated trolox and 5-(1,2-dithiolan-3-yl)pentanamine, were used as
=
l
l

6434
M. Koufaki et al. / Bioorg. Med. Chem. 17 (2009) 6432–6441
HO
HO
O
a
H
N
O
N
H
O
CONHNH2
S S
O
12
13
HO
b
O
O
S
S
S
N
N
14
COOEt
CONHNH2
COOH
c
d
S
S
S
S
S
16
lipoic acid
15
S
S
O
H
N
O
N
H
N
N
S
S
MeO
b
e
O
18R = OMe
19 R = OH
f
O
RO
17
O
Scheme 2. Synthesis of 1,3,4-oxadiazole analogues. Reagents and conditions: (a) N-(lipoyloxy)succinimide, CH2Cl2; (b) POCl3, 100 °C; (c) SOCl2, EtOH; (d) NH2NH2ꢀH2O,
MeOH; (e) 3,4-dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-1-benzopyran-5-carboxylic acid, BOP, Et3N, DMF, CH2Cl2; (f) BF3ꢀS(Me)2, CH2Cl2.
activity, prevention of oxytosis of HT22 cells depends on inhibition
of the increase in ROS production caused by the decrease of cellular
GSH level upon treatment with glutamate.30
tuted chroman 31 bearing 1,2,3-triazole moiety was active at low
M concentrations, while the 2-substituted analogue 24, bearing
the same moiety, was 10 times less potent (Table 1, column 3)
l
In general, apart from 24, the 2-substituted derivatives were
and only weakly effective at 10 lM (Table 1, column 4).
fully effective against oxytosis at concentrations P3 lM (Fig. 1
and Table 1, column 4).
5. Experimental section
5.1. Chemistry
The presence of nitrogen heterocycles strongly influences the
neuroprotective activity of 5-substituted chroman derivatives
(Fig. 2 and Table 1). Replacement of the amide group of analogue
II (EC50 = 2.10 0.40 lM) by 1,2,4-oxadiazole or 1,2,3-triazole re-
sulted in significant improvement of the activity against glutamate
induced oxidative stress. Thus, analogues 11 and 31 exhibited
EC50 = 0.57 0.10 and 0.90 0.04 lM, respectively. The 1,3,4-oxa-
diazole analogue 19, in which the heteroaromatic ring is directly
connected to the chroman moiety, was weakly effective at the con-
centrations tested (Fig. 2, Table 1, column 4). The presence of
methylene group between the chroman and the five membered
rings seems to increase the activity of these hybrids. Moreover,
the less flexible among these analogues, tetrazole 44 was less ac-
Melting points were determined on a Buchi 510 apparatus and
are uncorrected. NMR spectra were recorded on a Varian 300 spec-
trometer operating at 300 MHz for 1H and 75.43 MHz for 13C spec-
tra with CDCl3 as solvent. Silica gel plates Macherey–Nagel Sil G-25
UV254 were used for thin layer chromatography. Chromatographic
purification was performed with silica gel (200–400 mesh). Mass
spectra were recorded on TSQ 7000 Finigan instrument in the ESI
mode. HRMS were in FAB mode.
5.1.1. 1,2-Dithiolan-3-hexanenitrile (2)
tive (EC50 = 3.04 1.15 lM) than the amide counterpart II.
To a solution of analogue 1 (0.100 g, 0.37 mmol) in 2 mL anhyd
DMSO, was added NaCN (0.091 g, 1.85 mmol) and the mixture was
stirred at rt overnight. After completion of the reaction, cold water
was added and the mixture was extracted with Et2O, the organic
layer was washed with satd aqueous NaCl, dried with Na2SO4, fil-
tered, the solvent evaporated and the residue was purified by
flash-column chromatography (pet. ether/AcOEt 80:20). Yield:
0.050 g (66%), yellow gummy solid. 1H NMR d: 3.73–3.68 (m,
1H), 3.24–3.20 (m, 1H), 2.98–2.89 (m, 2H), 2.62–2.58 (m, 1H),
2.33 (t, J = 7.0 Hz, 2H), 1.79–1.60 (m, 4H), 1.49–1.41 (m, 2H),
1.39–1.21 (m, 2H). 13C NMR d: 119.9, 42.6, 39.2, 34.0, 28.4, 25.9,
25.5, 22.0, 17.3.
4. Conclusions
Our results show that the bioisosteric replacement of the amide
group by five-membered nitrogen heterocycles influences the neu-
roprotective activity of the new compounds, in a manner depend-
ing on the position of the substitution as well as the nature of
heterocycle. Thus, the 2-substituted analogue 5 and the 5-substi-
tuted analogue 11, both bearing 1,2,4-oxadiazole rings, exhibited
the strongest neuroprotective activity, with 11 being ꢁ2 times
more potent than 5 (Table 1, column 3). Similarly, the 5-substi-

M. Koufaki et al. / Bioorg. Med. Chem. 17 (2009) 6432–6441
6435
HO
HO
HO
b
a
O
CHO
O
COOEt
O
CH2OH
21
20
22
HO
HO
d
c
S
S
O
O
N
N
N
24
23
S
S
N
N
N
CHO
n
n
n
MeO
RO
MeO
d
c
O
O
O
29R=OMe, n=0
30 R=OMe, n=1
25n=0
26 n=1
27n=0
28 n=1
e
31 R=OH, n=1
CHO
c
f
OH
N3
S
S
S
S
S
S
34
33
32
N
N
N
S
S
MeO
g
MeO
34
O
O
35
Scheme 3. Synthesis of 1,2,3-triazole analogues. Reagents and conditions: (a) LiAlH4, THF, 70 °C; (b) (COCl)2, DMSO, Et3N, CH2Cl2, ꢂ60 °C; (c) Bestmann–Ohira reagent, K2CO3,
CH3OH; (d) 3-(5-azidopentyl)-1,2-dithiolane, CuSO4ꢀ5H2O, sodium ascorbate, t-BuOH, H2O; (e) BF3ꢀS(Me)2, CH2Cl2; (f) DMSO, N,N0-diisopropyl carbodiimide, Cl2CHCOOH;
(g) CuSO4ꢀ5H2O, sodium ascorbate, t-BuOH, H2O.
H
N
n
H
N
n
S
S
NH2
n
S
S
S
O
O
MeO
MeO
MeO
a
b
O
O
38 n=1
39 n=2
40 n=1
41 n=2
36 n=1
37 n=2
S
S
S
S
N
N
N
d
N
N
n
c
N
n
N
N
HO
MeO
O
O
42 n=1
43 n=2
44 n=1
Scheme 4. Synthesis of tetrazole analogues. Reagents and conditions: (a) N-(lipoyloxy)succinimide, CH2Cl2; (b) Lawesson’s reagent, THF; (c) TMSN3, DIAD, Ph3P, THF; (d)
BF3ꢀS(Me)2, CH2Cl2.
5.1.2. N-Hydroxy-1,2-dithiolan-3-hexamidine (3)
To a solution of nitrile 2 (0.050 g, 0.244 mmol) in 4 mL abs
EtOH, were added Et3N (0.1 mL), NH2OHꢀHCl (0.085 g, 1.22 mmol)
and the mixture was stirred at 40 °C overnight. The mixture was
then diluted by AcOEt, the organic layer was washed with satd
aqueous NaCl, dried with Na2SO4, filtered, the solvent evaporated

6436
M. Koufaki et al. / Bioorg. Med. Chem. 17 (2009) 6432–6441
water was added and the mixture was extracted with Et2O. The or-
ganic layer was washed with satd aqueous NaCl, dried over Na2SO4,
filtered and the solvent was evaporated in vacuo. Purification by
flash-column chromatography (pet. ether/AcOEt 80:20) afforded
compound 7 as yellowish gummy solid. Yield: 0.050 g (75%) 1H
NMR d: 3.73 (s, 3H, OMe), 3.70 (s, 2H), 2.75 (t, J = 6.8 Hz, 2H),
2.20 (s, 3H), 2.11 (s, 3H), 1.84 (t, J = 6.8 Hz, 2H), 1.32 (s, 6H).
5.1.6. N0-Hydroxy-2-(3,4-dihydro-6-methoxy-2,5,7,8-tetramethyl-
2H-benzopyran-5-yl) acetamidine (8)
To a solution of compound 7 (0.080 g, 0.31 mmol) in 6 mL abs
EtOH, were added NH2OHꢀHCl (0.107 g, 1.54 mmol), Et3N
(0.22 mL, 1.54 mmol) and the mixture was stirred at 60 °C for
24 h. After completion of the reaction, AcOEt and satd aqueous
NaCl were added, the organic layer was dried and the solvent evap-
orated. The residue was purified by flash-column chromatography
(CH2Cl2/CH3OH 95:5). Yield 0.060 g (66%) grey solid, mp 167–
171 °C. 1H NMR d: 4.93 (br s, 2H), 3.69 (s, 3H), 3.45 (s, 2H), 2.75
(t, J = 6.8 Hz, 2H), 2.20 (s, 3H), 2.09 (s, 3H), 1.74 (t, J = 6.8 Hz, 2H),
1.27 (s, 6H). 13C NMR d: 149.6, 148.7, 148.6, 128.2, 125.4, 124.0,
118.4, 73.3, 61.0, 32.9, 28.5, 27.0, 20.3, 13.1, 12.2. MS m/z: 293.5
(M+H)+.
Figure 1. Protection of HT22 cells from oxidative stress-induced cell death by 2-
substituted chroman analogues. Cells were challenged with 5 mM glutamate in the
absence or presence of increasing concentrations of the hybrids for 24 h and relative
numbers of viable cells were assessed as described in experimental section. Values
are mean SEM (shown only for 5) of three independent experiments.
5.1.7. N0-(5-(1,2-Dithiolan-3-yl)pentanoyloxy)-2-(6-methoxy-2,2,
7,8-tetramethyl-2H-benzopyran-5-yl) acetamidine (9)
and the residue was purified by flash-column chromatography
(CH2Cl2/CH3OH 95:5). Yield: 0.030 g (52%), green, gummy solid.
1H NMR d: 4.52 (br s, 1H, –OH), 3.74–3.66 (m, 1H, –CHSS–),
3.27–3.18 (m, 1H, –HCHCH2SS–), 2.99–2.91 (m, 2H, –CH2SS–),
To a solution of analogue 8 (0.040 g, 0.137 mmol) in 4 mL anhyd
CH2Cl2, were added lipoic acid (0.028 g, 0.137 mmol) and DCC
(0.034 g, 0.164 mmol) and the mixture was stirred at rt for 24 h.
CH2Cl2 and satd aq were then added, the organic layer was dried
and the solvent evaporated. Purification by flash-column chroma-
tography (CH2Cl2/CH3OH 97:3) afforded 9 as green-yellow solid,
mp 109–111 °C. Yield 0.042 g (63%) 1H NMR d: 5.18 (br s, 2H),
3.69 (s, 3H), 3.54 (m, 3H), 2.78 (t, J = 6.5 Hz, 2H), 2.46–2.35 (m,
3H), 2.18 (s, 3H), 2.08 (s, 3H), 1.94–1.85 (m, 2H), 1.77–1.67 (m,
7H), 1.49–1.47 (m, 2H), 1.26 (s, 6H). 13C NMR d: 171.2, 158.1,
149.5, 148.9, 128.3, 125.9, 123.4, 118.7, 73.4, 61.0, 56.5, 40.4,
38.7, 34.8, 33.2, 32.8, 29.0, 28.3, 27.0, 26.6, 25.0, 20.5, 13.1, 12.3.
MS m/z: 481 (M+H)+.
2.62–2.56 (m, 1Y, –HCHCH2SS–), 2.15 (t, J = 7.0 Hz, 2H,
CH2C@NOH), 1.74–1.20 (m, 8Y, (CH2)4).
–
5.1.3. N-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-benzo-
pyran-2-carbonyloxy)-1,2-dithiolan-3-hexanamidine (4)
To a solution of analogue 3 (0.035 g, 0.15 mmol) in 3 mL anhyd
CH2Cl2, was added a solution of trolox (0.037 g, 0.15 mmol) and
DCC (0.031 g, 0.15 mmol) in 3 mL anhyd CH2Cl2 and the mixture
was stirred at rt overnight. CH2Cl2, was then added and the organic
layer was washed with satd aqueous NaCl, dried with Na2SO4, fil-
tered, evaporated to dryness and the residue was purified by
flash-column chromatography (pet. ether/AcOEt 70:30). Yield
0.020 g (28%), yellow gummy solid. 1H NMR d: 4.05 (br s, 1H),
3.76–3.62 (m, 1H), 3.27–3.18 (m, 1H), 2.99–2.83 (m, 2H), 2.64–
2.52 (m, 4H), 2.20 (s, 3H, ArMe), 2.17 (s, 3H, ArMe), 2.15–2.09 (m,
2H), 2.06 (s, 3H, ArMe), 1.92–1.82 (m, 1H), 1.75–1.48 (m, 11H).
13C NMR d: 171.7, 159.8, 146.3, 145.5, 122.0, 121.6, 119.3, 117.9,
78.1, 42.9, 39.3, 34.0, 31.3, 28.8, 26.8, 26.5, 26.1, 22.2, 22.0, 21.3,
12.5, 12.0, 11.5. MS m/z: 467.3 (M+H)+.
5.1.8. 5-(4-(1,2-Dithiolan-3-yl)butyl)-3-((3,4-dihydro-6-methoxy-
2,2,7,8-tetramethyl-2H-benzopyran-5-yl)methyl)-1,2,4-oxadiazole
(10)
To a solution of analogue 9 (0.040 g, 0.083 mmol) in 2 mL anhyd
THF, was added TBAF (0.08 mL, 0.083 mmol) and the mixture was
stirred at rt for 30 min. The solvent was then evaporated and the
residue was taken up by AcOEt. The organic layer was washed with
satd aqueous NaCl, dried over Na2SO4, filtered and the solvent was
evaporated in vacuo. Purification by flash-column chromatography
(pet. ether/AcOEt 70:30) gave 10 as yellowish gummy solid. Yield
0.035 g (92%). 1Y NMR d: 4.09 (s, 2H), 3.68 (s, 3H), 3.57–3.52 (m,
1H), 3.18–3.10 (m, 2H), 2.84 (t, J = 7.5 Hz, 2H), 2.65 (t, J = 6.7 Hz,
2H), 2.47–2.41 (m, 1H), 2.20 (s, 3H), 2.10 (s, 3H), 1.94–1.67 (m,
7H), 1.57–1.46 (m, 2H), 1.28 (s, 6H). 13C NMR d: 179.3, 169.7,
149.7, 148.2, 128.2, 125.4, 123.4, 117.7, 73.0, 61.2, 56.4, 40.4,
38.7, 34.6, 33.0, 28.8, 27.1, 26.6, 26.5, 23.8, 20.7, 13.1, 12.3. MS
m/z: 463.3 (M+H)+. HRMS calcd for C24H34O3N2S2: 462.2011,
found: 462.2019.
5.1.4. 3-(6-(1,2-Dithiolan-3-yl)pentyl)-5-(2-(3,4-dihydro-6-hydro-
xy-2,5,7,8-tetramethyl-2H-benzopyran))-1,2,4-oxadiazole (5)
To a solution of analogue 4 (0.015 g, 0.031 mmol) in 2 mL anhyd
THF was added n-TBAF and the mixture was stirred at rt for 2 h.
The solvent was then evaporated and the residue was diluted in
AcOEt. The organic layer was washed with satd aqueous NaCl,
dried with Na2SO4, filtered, the solvent evaporated and the residue
was purified by flash-column chromatography (pet. ether/AcOEt
70:30). Yield: 0.007 g (50%), yellow gummy solid. 1H NMR d: 4.29
(br s, 1H), 3.75–3.66 (m, 1H), 3.29–3.19 (m, 1H) 2.99–2.85 (m,
2H), 2.71–2.57 (m, 5H), 2.21 (s, 3H), 2.17 (s, 3H), 2.06 (s, 3H),
1.77–1.56 (m, 13H). MS m/z: 449.8 (M+H)+.
5.1.9. 5-(4-(1,2-Dithiolan-3-yl)butyl)-3-((3,4-dihydro-6-hydroxy-
2,2,7,8-tetramethyl-2H-benzopyran-5-yl)methyl)-1,2,4-oxadiazole
(11)
To a solution of 10 (0.030 g, 0.064 mmol) in 2 mL anhyd CH2Cl2,
BF3ꢀS(Me)2 (0.1 mL, 1 mmol) was added and the mixture was stir-
red at ambient temperature overnight. The solvent was evaporated
under argon and the residue was extracted with ethyl acetate and
water. The organic layer was washed with saturated aqueous NaCl,
5.1.5. 2-(3,4-Dihydro-6-methoxy-2,5,7,8-tetramethyl-2H-benzo-
pyran-5-yl)acetonitrile (7)
To a solution of 2-(6-methoxy-2,2,7,8-tetramethylchroman-5-
yl)methyl bromide 6 (0.080 g, 0.26 mmol) in 2 mL anhyd DMSO,
NaCN (0.063 g, 1.28 mmol) was added at 0 °C and the mixture
was stirred at rt for 24 h. After completion of the reaction cold

M. Koufaki et al. / Bioorg. Med. Chem. 17 (2009) 6432–6441
6437
Table 1
Efficacy and potency of dithiolane/chroman hybrids to protect glutamate-challenged HT22 cells from oxytosis
Relative potencyb
Efficacyc (%)
a
Compound
EC50
(
lM) (mean SEM)
HO
H
1.24 0.38
1
80
N
O
O
n
n=4
O
S
S
I
HO
O
0.93 0.19
1.65 0.36
1.33
0.75
88
91
N
n=5
n=4
N
S
S
S
n
5
HO
HO
O
O
O
n
N
N
S
14
9.56
0.13
0.78
58
87
S
S
n=5
n=5
N
N
n
N
24
HO
H
S
1.59 0.53
N
S
O
n
O
45
H
N
n
O
O
S
HO
S
2.10 0.40
0.59
2.18
95
72
n=4
II
N
O
S
S
N
n
HO
0.57 0.10
n=4
O
11
S
S
n
N
O
N
>10
<0.12
20
HO
n=4
O
19
S
S
N
N
N
n
0.90 0.04
1.38
68
HO
n=5
O
31
S
n
N
S
N
N
N
3.04 1.15
0.41
100
HO
n=4
O
44
a
EC50 values are compound concentrations maintaining cell viability to 50% of non-challenged cells. Values are mean SEM of three independent experiments shown in
Figures 1 and 2.
b
Relative (to I) potencies were calculated by [EC50 I/EC50 compound].
Compounds are considered to be fully, partially or weakly neuroprotective if % efficacy at 10 lM was respectively, >66–100, >33–66 and 633%; ns = non-significant.
c

6438
M. Koufaki et al. / Bioorg. Med. Chem. 17 (2009) 6432–6441
dried and the solvent was evaporated in vacuo. The residue was
purified by flash-column chromatography (CH2Cl2/CH3OH 97:3)
to afford 11 as yellow gummy solid. Yield: 0.015 g (53%). 1H
NMR d: 4.02 (s, 2H), 3.57–3.53 (m, 1H, –CHSS–), 3.18–3.11 (m,
2H, –CH2SS–), 2.84–2.77 (m, 4H, ArCH2, OC(@N)CH2–), 2.48–2.42
(m, 1Y, –HCHCH2SS–), 2.22 (s, 3H, ArMe), 2.10 (s, 3H, ArMe),
1.93–1.67 (m, 7H), 1.59–1.49 (m, 2H), 1.28 (s, 6Y). 13C NMR d:
179.6, 168.8, 146.2, 145.5, 125.4, 125.3, 118.6, 116.4, 72.6, 56.1,
40.2, 38.5, 34.4, 32.8, 28.6, 26.7, 26.3, 26.0, 23.3, 20.7, 12.5, 12.0.
MS m/z: 448.5 (M+H)+. HRMS calcd for C23H32O3N2S2: 447.1776,
found: 447.1760.
NH2NH2ꢀH2O (0.15 mL) and the mixture was stirred at 40 °C over-
night. After completion of the reaction the mixture was extracted
with AcOEt, the organic layer was washed with satd aqueous NaCl,
dried with Na2SO4, filtered and the solvent evaporated. Purification
by flash-column chromatography (CH2Cl2/CH3OH 90:10). Yield:
0.070 g (73%), yellow solid, mp 66–68 °C. 1H NMR d: 6.80 (br s,
1H), 3.58–3.53 (m, 1H), 3.19–3.08 (m, 3H), 2.48–2.42 (m, 1H),
2.15 (t, J = 7.3 Hz, 2H), 1.95–1.86 (m, 1H), 1.72–1.64 (m, 4H),
1.51–1.42 (m, 2H).
5.1.13. N-(5-(1,2-Dithiolan-3-yl)pentanoyl)-N0-(3,4-dihydro-6-
methoxy-2,2,7,8-tetramethyl-2H-benzopyran-5-carbonyl)-
hydrazine (17)
5.1.10. N-(5-(1,2-Dithiolan-3-yl)pentanoyl)-3,4-dihydro-6-hydroxy-
2,5,7,8-tetramethyl-2H-benzopyran-2-carbohydrazide (13)
To a solution of 3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-
benzopyran-2-carbohydrazide 12 (0.080 g, 0.3 mmol) in 3 mL an-
hyd THF, was added a solution of N-hydroxysuccinimide activated
lipoic acid (0.092 g, 0.3 mmol) in 3 mL anhyd THF the mixture was
stirred at rt overnight. The solvent was then evaporated and the
residue was diluted in AcOEt. The organic layer was washed with
satd aqueous NaCl, dried with Na2SO4, filtered, the solvent evapo-
rated and the residue was purified by flash-column chromatogra-
phy (CH2Cl2/CH3OH 95:5). Yield: 0.120 g (88%), yellow solid mp
114–116 °C. 1H NMR d: 8.96 (d, J = 5.7 Hz, 1H), 8.65 (d, J = 5.7 Hz,
1H), 3.56–3.48 (m, 1H), 3.18–3.06 (m, 2H), 2.65–2.57 (m, 2 H),
2.47–2.41 (m, 1H), 2.29–2.23 (m, 2H), 2.20 (s, 3H), 2.16 (s, 3H),
2.08 (s, 3H), 1.95–1.87 (m, 3H), 1.67–1.40 (m, 9H). MS m/z: 453.5
(M+H)+.
To a solution of 3,4-dihydro-6-methoxy-2,2,7,8-tetramethyl-
2H-benzopyran-5-carboxylic acid (0.030 g, 0.113 mmol) in 2 mL
anhyd DMF, was added at 0 °C, Et3N (0.1 mL). After 30 min, a solu-
tion of BOP (0.050 g, 0.113 mmol) in 1 mL anhyd CH2Cl2 and a solu-
tion of analogue 16 (0.023 g, 0.113 mmol) in 1 mL anhyd CH2Cl2,
were added and the mixture was stirred at rt overnight. After com-
pletion of the reaction the mixture was extracted with AcOEt, the
organic layer was washed with satd aqueous NaCl, dried with
Na2SO4, filtered and the solvent evaporated and the residue was
purified by flash-column chromatography (CH2Cl2/CH3OH 95:5).
Yield: 0.020 g (40%), yellow gummy solid. 1H NMR d: 8.86 (d,
J = 4.5 Hz, 1H), 8.74 (d, J = 4.5 Hz, 1H), 3.68 (s, 3H), 3.58–3.54 (m,
1H), 3.17–3.09 (m, 2H), 2.85 (t, J = 6.7 Hz, 2H), 2.48–2.39 (m, 1H),
2.34 (t, J = 7.4 Hz, 2H), 2.13 (s, 3H), 2.09 (s, 3H), 1.93–1.84 (m, 1H),
1.73 (t, J = 6.7 Hz, 2H), 1.57–1.37 (m, 6H), 1.29 (s, 6H). 13C NMR d:
170.2, 165.2, 148.4, 148.3, 129.0, 128.7, 123.3, 117.4, 73.7, 62.5,
56.3, 40.2, 38.5, 34.6, 33.9, 32.5, 29.7, 28.8, 26.9, 25.0, 20.5, 12.3.
5.1.11. 5-(4-(1,2-Dithiolan-3-yl)butyl)-2-(3,4-dihydro-6-hydroxy-
2,5,7,8-tetramethyl-2H-benzopyran-2-yl)-1,3,4-oxadiazole (14)
A mixture of 0.025 g of analogue 13, and POCl3 (0.2 mL) was
heated at 100 °C for 1 h. After completion of the reaction cold
water was added and the mixture was extracted with CH2Cl2, the
organic layer was washed with satd aqueous NaCl, dried with
Na2SO4, filtered, the solvent evaporated and the residue was puri-
fied by flash-column chromatography (CH2Cl2/CH3OH 97:3). Yield:
0.009 g (38%), white gummy solid. 1H NMR d: 4.29 (d, J = 4.7 Hz,
1H), 3.52–3.47 (m, 1H), 3.17–3.08 (m, 2H), 2.77 (t, J = 7.4 Hz, 2H)
2.71–2.64 (m, 2H), 2.42–2.38 (m, 1H), 2.16 (s, 3H), 2.15 (s, 3H),
2.05 (s, 3H), 1.89–1.36 (m, 12H). MS m/z: 435.2 (M+H)+. HRMS
calcd for C22H30O2N3S2: 434.1698, found: 434.1671.
5.1.14. 2-(4-(1,2-Dithiolan-3-yl)butyl)-5-(3,4-dihydro-6-methoxy-
2,2,7,8-tetramethyl-2H-benzopyran-5-yl)-1,3,4-oxadiazole (18)
A mixture of 0.012 g of analogue 17, and POCl3 (0.2 mL) was
heated at 100 °C for 1 h. After completion of the reaction cold
water was added and the mixture was extracted with CH2Cl2, the
organic layer was washed with satd aqueous NaCl, dried with
Na2SO4, filtered, the solvent evaporated and the residue was puri-
fied by flash-column chromatography (CH2Cl2/CH3OH 97:3). Yield:
0.008 g (67%), yellow gummy solid. 1H NMR d: 3.64 (s, 3H), 3.62–
3.58 (m, 1H), 3.10–3.01 (m, 2H), 2.94 (t, J = 6.4 Hz, 2H), 2.68 (t,
J = 6.7 Hz, 2H), 2.48–2.40 (m, 1H), 2.21 (s, 3H), 2.16 (s, 3H), 1.95–
1.88 (m, 1H), 1.75 (t, J = 6.7 Hz, 2H), 1.57 (m, 6H), 1.32 (s, 6H).13C
NMR d: 150.4, 149.9, 148.3, 139.5, 130.2, 129.0, 118.6, 115.0,
73.7, 62.0, 56.2, 40.2, 38.5, 34.4, 32.3, 29.7, 28.6, 26.9, 25.3, 21.2,
12.5, 12.4. MS m/z: 449.5 (M)+. HRMS calcd for C23H32O3N2S2:
449.1933, found: 449.1956.
5.1.12. (1,2-Dithiolan-3-yl)pentanoyl-hydrazide (16)
To a solution of 1,2-dithiolan-3-yl pentanoic acid ethyl ester 15
(0.110 g, 0.47 mmol) in 3 mL anhyd CH3OH, were added
5.1.15. 2-(4-(1,2-Dithiolan-3-yl)butyl)-5-(3,4-dihydro-6-hydroxy-
2,2,7,8-tetramethyl-2H-benzopyran-5-yl)-1,3,4-oxadiazole (19)
This compound was prepared according to the procedure de-
scribed for 11 using analogue 18 (0.007 g, 0.016 mmol). Purifica-
tion by flash-column chromatography (CH2Cl2/CH3OH 97:3).
Yield: 0.004 g (60%), yellowish gummy solid. 1H NMR d: 3.64–
3.57 (m, 1H), 3.20–3.12 (m, 2H), 3.04–2.95 (m, 4H), 2.50–2.46
(m, 1H), 2.25 (s, 3H), 2.19 (s, 3H), 1.96–1.57 (m, 9H), 1.32 (s, 6H).
MS m/z: 436.4 (M+H)+. HRMS calcd for C22H30O3N2S2: 435.1776,
found: 435.1755.
5.1.16. General procedure for the preparation of alkynes (23, 27
and 28)
To a solution of aldehyde (1 mmol) in 6 mL anhyd CH3OH, were
added K2CO3 (2 mmol) and the Bestmann-Ohira reagent
(1.4 mmol) and the mixture was stirred at rt for 24 h. The solvent
was then evaporated and the residue was taken up by Et2O. The
organic layer was washed with satd aqueous NaCl, dried with
Figure 2. Protection of HT22 cells from oxidative stress-induced cell death by 5-
substituted chroman analogues.

M. Koufaki et al. / Bioorg. Med. Chem. 17 (2009) 6432–6441
6439
Na2SO4, filtered and the solvent evaporated. Purification by flash-
column chromatography (pet. ether/ether, 90:10) afforded the de-
sired compounds.
5.1.17.3. 1-(5-(1,2-Dithiolan-3-yl)pentyl)-4-((3,4-dihydro-6-
methoxy-2,2,7,8-tetramethyl-2H-benzopyran-5-yl)methyl)-1H-
1,2,3-triazole (30). The compound was synthesized according to
the general procedure for the preparation of 1,2,3-triazoles using
analogue 28 (0.030 g, 0.12 mmol) and 3-(5-azidopentyl)-1,2-
dithiolane (0.055 g, 0.24 mmol). Yield: 0.016 g (28%) yellow vis-
cous oil. 1H NMR d: 7.07 (s, 1H), 4.22 (t, J = 6.7 Hz, 2H), 4.07 (s,
2H), 3.64 (s, 3H), 3.54–3.50 (m, 1H), 3.17–3.10 (m, 2H), 2.65 (t,
J = 6.2 Hz, 2H), 2.47–2.40 (m, 1H), 2.20 (s, 3H), 2.10 (s, 3H),
1.89–1.81 (m, 1H), 1.71 (t, J = 6.2 Hz, 2H), 1.63–1.41 (m, 8H),
1.26 (s, 6H). 13C NMR d: 149.3, 148.3, 128.2, 127.0, 124.6, 121.4,
121.3, 117.4, 73.0, 61.2, 56.3, 50.1, 40.2, 38.5, 34.6, 32.7, 30.0,
28.6, 26.8, 26.5, 26.2, 23.1, 20.4, 12.9, 12.0. MS m/z: 477.5
(M+H)+. HRMS calcd for C25H37O2N3S2: 476.2405, found:
476.2402.
5.1.16.1. 2-Ethynyl-3,4-dihydro-2,5,7,8-tetramethyl-2H-6-benzo-
pyranol (23). This compound was synthesized according to the
general procedure for the preparation of alkynes using analogue
22 (0.070 g, 0.3 mmol). Yield: 0.061 g (88%). 1H NMR d: 3.08–3.03
(m, 1H), 2.85–2.79 (m, 1H), 2.37 (m, 1H), 2.33 (m, 1H), 2.03 (s, 3H),
1.99 (s, 6H), 1.87 (s, 1H), 1.61 (s, 3H).
5.1.16.2. 3,4-Dihydro-5-ethynyl-6-methoxy-2,2,7,8-tetramethyl-
2H-benzopyran (27). According to the general procedure for the
preparation of alkynes using the 3,4-dihydro-6-methoxy-2,2,7,8-tet-
ramethyl-2H-benzopyran-5-carboxaldehyde (0.124 g, 0.5 mmol)
compound 27 was obtained as white gummy solid. Yield: 0.050 g
(41%). 1H NMR d: 3.80 (s, 3H), 3.43 (s, 1H, ArC„CH), 2.82 (t,
J = 6.8 Hz, 2H), 2.17 (s, 3H), 2.11 (s, 3H), 1.78 (t, J = 6.8 Hz, 2H), 1.30
(s, 6H).
5.1.18. 1-(5-(1,2-Dithiolan-3-yl)pentyl)-4-((3,4-dihydro-6-
hydroxy-2,2,7,8-tetramethyl-2H-benzopyran-5-yl)methyl)-1H-
1,2,3-triazole (31)
This compound was prepared according to the procedure de-
scribed for 11, using analogue 30 (0.012 g, 0.025 mmol). Purifica-
tion by flash-column chromatography (pet. ether/AcOEt, 50:50).
Yield: 0.005 g (50%) yellow gummy solid. 1H NMR d: 7.22 (s, 1H),
5.26 (s, 1H), 4.23 (t, J = 6.7 Hz, 2H) 3.94 (s, 2H), 3.51–3.47 (m,
1H), 3.13–3.06 (m, 2H), 2.69 (t, J = 6.6 Hz, 2H), 2.45–2.35 (m, 1H),
2.17 (s, 3H), 2.05 (s, 3H), 1.85–1.31 (m, 11H), 1.23 (s, 6H). 13C
NMR d: 147.0, 145.7, 145.6, 124.7, 124.3, 121.6, 120.3, 115.3,
72.4, 56.3, 50.3, 40.2, 38.4, 34.6, 33.0, 29.9, 29.6, 28.5, 26.6, 26.2,
22.5, 21.0, 12.4, 11.9. MS m/z: 463.4 (M+H)+. HRMS calcd for
C24H35O2N3S2: 462.2249, found: 462.2221.
5.1.16.3. 3,4-Dihydro-6-methoxy-5-(prop-2-ynyl)-2,2,7,8-tetra-
methyl-2H-benzopyran (28). This compound was synthesized
according to the general procedure for the preparation of alkynes
using
3,4-dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-benzopy-
ran-5-acetaldehyde (0.100 g, 0.38 mmol).Yield: 0.040 g (41%) yel-
low gummy solid. 1H NMR d: 3.74 (s, 3H), 3.54 (d, J = 2.7 Hz, 2H),
2.82 (t, J = 6.8 Hz, 2H), 2.20 (s, 3H), 2.11 (s, 3H), 1.97 (t, J = 2.7 Hz,
1H), 1.83 (t, J = 6.8 Hz, 2H), 1.33 (s, 6H).
5.1.17. General procedure for the preparation of 1,4-substituted
1,2,3-triazoles (24, 29 and 30)
To a solution of the appropriate alkyne (0.1 mmol) in 2 mL t-
BuOH/H2O (2:1), were added azide (0.2 mmol) in 1 mL t-BuOH/
H2O (2:1), CuSO4ꢀ5H2O (0.03 mmol) and sodium ascorbate
(0.06 mmol) and the mixture was stirred at rt for 24 h. AcOEt
and aqueous NH4OH. The organic layer was washed with satd
aqueous NaCl, dried with Na2SO4, filtered and the solvent evapo-
rated. Purification by flash-column chromatography (pet. ether/
AcOEt, 50:50) afforded the desired compounds.
5.1.19. 1,2-Dithiolan-3-pentanal (33)
0.170 g of 5-(1,2-dithiolan-3-yl)-pentanol were diluted in an-
hyd DMSO (0.38 mL, 5.3 mmol). To this solution were added
N,N0-diisopropylcarbodiimide
(0.41 mL,
2.65 mmol)
and
Cl2CHCOOH (0.05 mL, 0.53 mmol) and the mixture was stirred at
rt for 4 h. After completion of the reaction the mixture was ex-
tracted with AcOEt, the organic layer was washed with satd aque-
ous NaCl, dried with Na2SO4, filtered and the solvent evaporated.
Purification by flash-column chromatography (pet. ether/AcOEt
80:20) afforded the desired aldehyde. Yield: 0.090 g (54%), yellow
oil. 1H NMR d: 9.74 (s, 1H), 3.56–3.51 (m, 1H), 3.16–3.08 (m, 2H),
2.47–2.41 (m, 2H), 1.89–1.85 (m, 1H), 1.72–1.40 (m, 7H). 13C
NMR d: 202.5, 56.5, 43.9, 40.5, 38.7, 34.9, 29.0, 22.0.
5.1.17.1. 1-(5-(1,2-Dithiolan-3-yl)pentyl)-4-(3,4-dihydro-6-hydro-
xy-2,5,7,8-tetramethyl-2H-benzopyran-2-yl)-1H-1,2,3-triazole
(24). This compound was synthesized according to the general
procedure for the preparation of 1,2,3-triazoles, using analogue 23
(0.015 g, 0.065 mmol) and 3-(5-azidopentyl)-1,2-dithiolane
(0.015 g, 0.065 mmol). Yield: 0.012 g (40%). 1H NMR d: 7.34 (s, 1H),
4.25 (t, J = 7.2 Hz, 2H), 3.58–3.49 (m, 1H), 3.19–3.05 (m, 2H), 2.97
(d, J = 11.7 Hz, 1H), 2.68 (d, J = 11.7 Hz, 1H), 2.47–2.41 (m, 2H),
2.12 (s, 3H), 1.93 (s, 3H), 1.88 (s, 3H), 1.71–1.65 (m, 5H), 1.57 (s,
3H), 1.46–1.33 (m, 5H). HRMS calcd for C23H34O2N3S2 (M+H)+:
448.2092, found: 448.2113.
5.1.20. 3-(Hexyn-5-yl)-1,2-dithiolane (34)
This compound was synthesized according to the general proce-
dure for the preparation of alkynes using analogue 33 (0.062 g,
0.32 mmol). Yield: 0.017 g (27%) yellow gummy solid. 1H NMR d:
3.61–3.56 (m, 1H), 3.20–3.08 (m, 2H), 2.50–2.40 (m, 1H), 2.23–
2.20 (m, 2H), 1.98–1.89 (m, 2H), 1.73–1.65 (m, 6H).
5.1.17.2. 1-(5-(1,2-Dithiolan-3-yl)pentyl)-4-(3,4-dihydro-6-
methoxy-2,2,7,8-tetramethyl-2H-benzopyran-5-yl)-1H-1,2,3-
triazole (29). The compound was synthesized according to the
general procedure for the preparation of 1,2,3-triazoles using ana-
logue 27 (0.022 g, 0.09 mmol) and 3-(5-azidopentyl)-1,2-dithio-
lane (0.042 g, 0.18 mmol). Yield: 0.010 g (24%) yellow viscous oil.
1H NMR d: 7.66 (s, 1H, ArC@CH–), 4.42 (t, J = 7.1 Hz, 2H), 3.63–
3.52 (m, 1H), 3.33 (s, 3H), 3.22–3.09 (m, 2H), 2.74 (t, J = 6.8 Hz,
2H), 2.48–2.42 (m, 1H), 2.20 (s, 3H), 2.14 (s, 3H), 1.92–1.83 (m,
1H), 1.69 (t, J = 6.8 Hz, 2H), 1.67–1.38 (m, 8H), 1.32 (s, 6H). 13C
NMR d: 151.7, 151.5, 149.1, 148.4, 128.1, 127.0, 123.7, 118.4,
73.4, 60.8, 56.3, 53.4, 50.2, 40.2, 38.5, 34.7, 32.9, 30.1, 28.6, 27.0,
26.2, 22.0, 12.5, 12.2. MS m/z: 463.4 (M+H)+. HRMS calcd for
C24H35O2N3S2: 462.2249, found: 462.2249.
5.1.21. 4-(4-(1,2-Dithiolan-3-yl)butyl)-1-((3,4-dihydro-6-
methoxy-2,2,7,8-tetramethyl-2H-benzopyran-5-yl)methyl)-1H-
1,2,3-triazole (35)
The compound was synthesized according to the general proce-
dure for the preparation of 1,2,3-triazoles using analogue 34
(0.010 g, 0.054 mmol) and 3,4-dihydro-6-methoxy-2,2,7,8-tetra-
methyl-2H-benzopyran-5-yl)methylazide (0.030 g, 0.11 mmol).
Yield: 0.005 g (21%) yellowish gummy solid. 1H NMR d: 7.25 (s,
1H), 5.51 (s, 2H), 3.67 (s, 3H), 3.55–3.50 (m, 1H), 3.16–3.08 (m,
2H), 2.69 (t, J = 7.5 Hz, 2H), 2.62 (t, J = 6.5 Hz, 2H), 2.44–2.39 (m,
1H), 2.22 (s, 3H), 2.11 (s, 3H), 1.90–1.81 (m, 1H), 1.74–1.43 (m,
8H), 1.25 (s, 6H).

6440
M. Koufaki et al. / Bioorg. Med. Chem. 17 (2009) 6432–6441
5.1.22. N-((3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-benzo-
pyran-5-yl)methyl)-(1,2-dithiolan-3-yl)pentanthioamide (40)
5.1.26. N-(2-(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-
benzopyran-5-yl)ethyl)-(1,2-dithiolan-3-yl)pentanethioamide
(41)
To
a
solution of N-((3,4-dihydro-6-methoxy-2,2,7,8-tetra-
pen-
methyl-2H-benzopyran-5-yl)methyl)-(1,2-dithiolan-3-yl)
Prepared from 39 (0.110 g, 0.24 mmol) using the procedure de-
scribed for 40. Purification by flash-column chromatography
(CH2Cl2/CH3OH 97:3) Yield: 0.080 g (71%), yellow gummy solid
1H NMR d: 8.67 (br s, 1H), 3.71 (s, 3H), 3.66–3.63 (m, 2H), 3.53–
3.50 (m, 1H), 3.16–3.07 (m, 2H), 2.92 (m, 2H), 2.66 (t, J = 6.7 Hz,
2H), 2.58 (t, J = 7.5 Hz, 2H), 2.43–2.37 (m, 1H), 2.18 (s, 3H), 2.08
(s, 3H), 1.88–1.63 (m, 7H), 1.50–1.35 (m, 8H). 13C NMR d: 204.5,
149.0, 148.7, 128.0, 126.3, 125.0, 116.9, 73.0, 60.9, 60.4, 56.3,
47.5, 46.6, 40.2, 38.4, 34.5, 32.6, 28.3, 26.9, 24.0, 20.3, 12.8, 12.0.
MS m/z: 468.4 (M+H)+.
tanamide 38 (0.050 g, 0.114 mmol) in 4 mL anhyd THF,
Lawesson’s reagent (0.046 g, 0.114 mmol) was added and the mix-
ture was refluxed at 70 °C overnight. After evaporation of the sol-
vent under argon the residue was purified by flash-column
chromatography (pet. ether/AcOEt, 60:40). Yield: 0.040 g (78%),
yellowish solid mp 96–99 °C. 1H NMR d: 7.47 (s, 1H), 4.83 (d,
J = 4.3 Hz, 2H,), 3.68 (s, 3H), 3.55–3.51 (m, 1H), 3.16–3.09 (m,
2H), 2.74 (t, J = 6.6 Hz, 2H), 2.60 (t, J = 7.3 Hz, 2H), 2.46–2.40 (m,
1H), 2.19 (s, 3H), 2.11 (s, 3H), 1.89–1.79 (m, 1H), 1.77 (t,
J = 6.5 Hz, 2H), 1.68–1.42 (m, 6H), 1.29 (s, 6H). 13C NMR d: 204.1,
150.5, 148.8, 128.7, 127.0, 124.0, 117.9, 73.5, 61.5, 56.6, 46.9,
42.8, 40.4, 38.7, 34.8, 32.8, 29.2, 28.6, 27.1, 20.8, 12.9, 12.3. MS
m/z: 453.6 (M+H)+.
5.1.27. 5-(4-(1,2-Dithiolan-3-yl)butyl)-1-(2-(3,4-dihydro-6-
methoxy-2,2,7,8-tetramethyl-2H-benzopyran-5-yl)ethyl)-1H-
tetrazole (43)
To a solution of 41 (0.032 g, 0.07 mmol) in 0.5 mL anhyd THF
were added diisopropylazodicarboxylate (0.02 mL, 0.11 mmol)
and triphenylphosphine (0.029 g, 0.11 mmol) and after stirring
for 5 min trimethylsilylazide (0.02 mL, 0.11 mmol) was added.
The reaction mixture was stirred at rt for 5 h and then the solvent
was evaporated in vacuo. The residue was purified by flash-column
chromatography (pet. ether/AcOEt 50:50). Yield: 0.012 g (36%),
white gummy solid. 1H NMR d: 4.45 (t, J = 6.7 Hz, 2H), 3.65 (s,
3H), 3.53–3.48 (m, 1H), 3.12–3.09 (m, 2H), 3.06 (t, J = 6.7 Hz, 2H),
2.48–2.42 (m, 1H), 2.34 (t, J = 7.7 Hz, 2H), 2.25 (t, J = 6.9 Hz, 2H),
2.16 (s, 3H), 2.05 (s, 3H), 1.89–1.83 (m, 1H), 1.66–1.58 (m, 8H),
1.30 (s, 6H). 13C NMR d: 155.1, 149.9, 148.4, 128.4, 125.5, 124.3,
117.4, 73.1, 60.7, 56.2, 46.8, 40.2, 38.5, 34.4, 32.5, 29.7, 28.8,
27.5, 26.7, 26.5, 22.3, 20.2, 12.8, 12.1. HRMS calcd for
C24H37O4N2S2: (M+H)+ 477.2358, found: 477.2340.
5.1.23. 5-(4-(1,2-Dithiolan-3-yl)butyl)-1-((3,4-dihydro-6-methoxy-
2,2,7,8-tetramethyl-2H-benzopyran-5-yl)methyl)-1H-tetrazole (42)
To a solution of 40 (0.030 g, 0.066 mmol) in 0.5 mL anhyd THF
were added diisopropylazodicarboxylate (0.02 mL, 0.1 mmol) and
triphenylphosphine (0.026 g, 0.1 mmol) and after stirring for
5 min, trimethylsilylazide (0.02 mL, 0.1 mmol) was added. The
reaction mixture was stirred at 40 °C for 3 h and then the solvent
was evaporated in vacuo. The residue was purified by flash-column
chromatography (pet. ether/AcOEt 50:50). Yield: 0.025 g (83%)
white gummy solid. 1H NMR d: 5.49 (s, 2H), 3.60 (s, 3H), 3.52–
3.45 (m, 1H), 3.20–3.11 (m, 2H), 2.79 (t, J = 7.5 Hz, 2H), 2.57 (t,
J = 6.7 Hz, 2H), 2.46–2.38 (m, 1H), 2.19 (s, 3H), 2.10 (s, 3H), 1.89–
1.83 (m, 1H), 1.73 (t, J = 6.7 Hz, 2H), 1.62–1.44 (m, 6H), 1.20 (s,
6H). 13C NMR d: 155.3, 150.1, 148.9, 128.7, 128.5, 121.0, 118.5,
73.6, 61.9, 56.5, 43.4, 40.4, 38.7, 34.7, 32.6, 29.0, 27.1, 27.0, 23.1,
22.2, 20.6, 13.1, 12.5.
5.1.28. N-(5-(1,2-Dithiolan-3-yl)pentyl-2-(3,4-dihydro-6-hydroxy-
2,5,7,8-tetramethyl-2H-benzopyran carboxamide (45)
Trolox (0.022 g, 0.09 mmol) in 3 mL anhyd THF was activated
with CDI (0.016 g, 0.095 mmol) and to this solution, 5-(1,2-dithio-
lan-3-yl) pentanamine (0.020 g, 0.11 mmol) in 3 mL THF was
added and the mixture was stirred at rt overnight. Purification by
flash column chromatography (CH2Cl2/CH3OH 95:5) afforded 45
as yellow gummy solid. Yield: 0.025 g (66%). 1H NMR d: 6.41 (d,
J = 4.9 Hz, 1H), 4.52 (br s, 1H), 3.52–3.49 (m, 1H), 3.34–3.23 (m,
1H), 3.19–3.09 (m, 2H), 2.61–2.58 (m, 2H), 2.49–2.38 (m, 2H),
2.20 (s, 6H), 2.11 (s, 3H), 1.94–1.86 (m, 3H), 1.58–1.25 (m, 11H).
13C NMR d: 174.5, 145.8, 144.6, 122.0, 121.7, 118.4, 118.3, 78.7,
56.8, 40.5, 39.1, 38.8, 35.1, 29.8, 29.6, 26.5, 25.1, 25.0, 20.9, 12.6,
12.2, 12.0. MS m/z: 424.3 (M+H)+. HRMS calcd for C22H33ON3S2:
423.1902, found: 423.1909.
5.1.24. 5-(4-(1,2-Dithiolan-3-yl)butyl)-1-((3,4-dihydro-6-hydroxy-
2,2,7,8-tetramethyl-2H-benzopyran-5-yl)methyl)-1H-tetrazole (44)
Prepared according to the procedure described for 11, using 42
(0.020 g, 0.043 mmol). Purification by flash-column chromatogra-
phy (pet. ether/AcOEt 50:50). Yield: 0.009 g (42%), yellow gummy
solid. 1H NMR d: 5.41 (s, 2H), 3.53–3.46 (m, 1H), 3.16–3.01 (m,
2H), 2.85 (t, J = 7.5 Hz, 2H), 2.71 (t, J = 6.7 Hz, 2H), 2.42–2.35 (m,
1H), 2.09 (s, 3H), 2.06 (s, 3H), 1.89–1.77 (m, 2H), 1.73 (t,
J = 6.7 Hz, 2H), 1.65–1.42 (m, 5H), 1.20 (s, 6H). MS m/z: 449.4
(M+H)+. HRMS calcd for C22H33O2N4S2 (M+H)+: 449.2045, found:
449.2074
5.1.25. N-(2-(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-2H-
benzopyran-5-yl)ethyl)-(1,2-dithiolan-3-yl)pentanamide (39)
To a solution of 2-(3,4-dihydro-6-methoxy-2,2,7,8-tetramethyl-
2H-benzopyran-5-yl) ethylamine (0.120 g, 0.46 mmol) in 3 mL an-
hyd THF, was added N-hydroxysuccinimide activated lipoic acid
(0.138 g, 0.46 mmol) in 3 mL anhyd THF and the mixture was stir-
red at rt for 24 h. The solvent was then evaporated and the residue
was taken up by AcOEt. The organic layer was washed with satd
aqueous NaCl, dried over Na2SO4, filtered and the solvent was
evaporated in vacuo. Purification by flash-column chromatography
(CH2Cl2/CH3OH 95:5) gave 39 as yellow gummy solid. Yield:
0.110 g (53%). 1H NMR d: 6.35 (br s, 1H), 3.65 (s, 3H), 3.55–3.50
(m, 1H), 3.36–3.33 (m, 2H), 3.16–3.07 (m, 2H), 2.79 (t, J = 6.5 Hz,
2H), 2.67 (t, J = 6.5 Hz, 2H), 2.45–2.39 (m, 1H), 2.17 (s, 3H), 2.07
(s, 3H), 1.89–1.85 (m, 1H), 1.77 (t, J = 6.7 Hz, 2H), 1.66–1.61 (m,
6H), 1.30 (s, 6H). 13C NMR d: 173.1, 149.4, 148.5, 127.9, 126.7,
124.5, 117.1, 72.9, 60.8, 56.4, 40.2, 38.4, 36.4, 34.6, 32.8, 28.8,
26.9, 25.6, 25.4, 25.2, 20.2, 12.8, 11.9.
5.2. Biology
5.2.1. Evaluation of the activity of 2-dithiolane/chroman
hybrids against oxidative stress-induced cell death of HT22
hippocampal neurons
The hybrids were tested as previously described34, with minor
modifications. Briefly, HT22 cells were plated in a 96-well flat bot-
tom plate at a density of 4000 cells per well in 100 ll of DMEM-
Hepes-GlutaMAX medium containing 10% of fetal bovine serum.
24 h after plating, the cells were challenged with 5 mM glutamate
in the absence or presence of increasing concentrations of the hy-
brids in fresh medium for 24 h prior to assessing the relative num-
bers of living cells using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide]. MTT conversion to coloured forma-
zan was assessed from the difference in optical density (dOD) at
550 and 670 nm. Direct interference of the test compounds with
MTT conversion to formazan was excluded using mock cultures de-

M. Koufaki et al. / Bioorg. Med. Chem. 17 (2009) 6432–6441
6441
10. Rosini, M.; Andrisano, V.; Bartolini, M.; Bolognesi, M. L.; Hrelia, P.; Minarini, A.;
Tarozzi, A.; Melchiorre, C. J. Med. Chem. 2005, 48, 360.
prived of HT22 cells. Interference of the hybrids with mitochon-
drial conversion of MTT to formazan was excluded using the try-
pan blue exclusion assay to directly determine the number living
cells. No challenged cells served to test cytotoxicity at different hy-
brid concentrations, whereas challenged cells served to assess
neuroprotective activity by comparison. Cells exposed only to
vehicle (DMSO) or glutamate served as controls. Cell death
11. Decker, M.; Kraus, B.; Heilmann, J. Bioorg. Med. Chem. 2008, 16, 4252.
12. Venkatachalam, S. R.; Salaskar, A.; Chattopadhyay, A.; Barik, A.; Mishra, B.;
Gangabhagirathic, R.; Priyadarsini, K. I. Bioorg. Med. Chem. 2006, 14, 6414.
13. Detsi, F.; Bouloumbasi, D.; Prousis, K. C.; Koufaki, M.; Athanasellis, G.;
Melagraki, G.; Afantitis, A.; Igglessi-Markopoulou, O.; Kontogiorgis, C.;
Hadjipavlou-Litina, D. J. J. Med. Chem. 2007, 50, 2450.
14. Melagraki, G.; Afantitis, A.; Igglessi-Markopoulou, O.; Detsi, A.; Koufaki,
M.; Kontogiorgis, C.; Hadjipavlou-Litina, D. J. Eur. J. Med. Chem. 2009, 44,
3020.
15. Moreira Lima, L.; Barreiro, E. J. Curr. Med. Chem. 2005, 12, 23.
16. Koufaki, M.; Kiziridi, C.; Nikoloudaki, F.; Alexis, M. N. Bioorg. Med. Chem. Lett.
2007, 17, 4223.
17. Compton, B.; Sheng, S.; Carlson, K.; Rebacz, N.; Lee, I.; Katzenellenbogen, B.;
Katzenellenbogen, J. J. Med. Chem. 2004, 47, 5872.
(CD) in the absence of hybrids was calculated by CDvehicle
[(dODvehicle ꢂ dODglutamate) ꢃ 100/dODvehicle, whereas cell death in
their presence was calculated by CDcompound = [(dODcompound
=
ꢂ
dODcompound+glutamate) ꢃ 100/dODcompound. Neuroprotection (%) was
calculated by [(CDvehicle ꢂ CDcompound) ꢃ 100/CDvehicle
.
18. Rostovsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, B. K. Angew. Chem., Int. Ed.
2002, 41, 2596.
19. Moses, J. E.; Moorhouse, A. D. Chem. Soc. Rev. 2007, 36, 1249.
20. Tron, G. C.; Pirali, T.; Billington, R. A.; Canonico, P. L.; Sorba, G.; Genazzani, A. A.
Med. Res. Rev. 2008, 28, 278.
Acknowledgment
This work is supported in part by ‘EURODESY’ MEST-CT2005-
020575.
21. Balogh, G. T.; Vukics, K.; Könczöl, A.; Kis-Varga, A.; Gere, A.; Fischer, J. Bioorg.
Med. Chem. Lett. 2005, 15, 3012.
22. Mancuso, A. J.; Huang, S. L.; Swern, D. J. Org. Chem. 1978, 43, 2480.
23. Ohira, S. Synth. Commun. 1989, 19, 561.
References and notes
24. Muller, S.; Liepold, B.; Roth, G. J.; Bestmann, H. J. Synth. Lett. 1996, 521.
25. Koufaki, M.; Calogeropoulou, T.; Rekka, E.; Chryselis, M.; Papazafiri, P.;
Gaitanaki, C.; Makriyannis, A. Bioorg. Med. Chem. 2003, 11, 5209.
26. Koufaki, M.; Kiziridi, C.; Papazafiri, P.; Vassilopoulos, A.; Varró, A.; Nagy, Z.;
Farkas, A.; Makriyannis, A. Bioorg. Med. Chem. 2006, 14, 6666.
27. Pfitzner, K. E.; Moffatt, J. G. J. Am. Chem. Soc. 1963, 86, 3027.
28. Tronchet, J. M. J.; Koufaki, M.; Barbalat, R. F.; Geoffroy, M. Carbohydr. Lett. 1999,
3, 255.
1. Petersen Shay, K.; Moreau, R. F.; Smith, E. J.; Hagen, T. M. IUBMB Life 2008, 60, 362.
2. Bolognesi, M. L.; Minarini, A.; Tumiatti, V.; Melchiorre, C. Mini-Rev. Med. Chem.
2006, 6, 1269.
3. Meunier, B. Acc. Chem. Res. 2008, 41, 69.
4. Koufaki, M.; Calogeropoulou, T.; Detsi, A.; Roditis, A.; Kourounakis, A. P.;
Papazafiri, P.; Tsiakitzis, K.; Gaitanaki, C.; Beis, I.; Kourounakis, P. N. J. Med.
Chem. 2001, 44, 4300.
29. Athanassopoulos, C. M.; Garnelis, T.; Vahliotis, D.; Papaioannou, D. Org. Lett.
2005, 7, 561.
5. Koufaki, M.; Detsi, A.; Theodorou, E.; Kiziridi, C.; Calogeropoulou, T.;
Vassilopoulos, A.; Kourounakis, A. P.; Rekka, E.; Kourounakis, P. N.; Gaitanaki,
C.; Papazafiri, P. Bioorg. Med. Chem. 2004, 12, 4835.
6. Harnett, J. J.; Auguet, M.; Viossat, I.; Dolo, C.; Bigg, D.; Chabrier, P.-E. Bioorg.
Med. Chem. Lett. 2002, 12, 1439.
7. Durand, G.; Polidori, A.; Salles, J. P.; Prost, M.; Durand, P.; Pucci, B. Bioorg. Med.
Chem. Lett. 2003, 13, 2673.
8. Antonello, A.; Hrelia, P.; Leonardi, A.; Marucci, G.; Rosini, M.; Tarozzi, A.;
Tumiatti, V.; Melchiorre, C. J. Med. Chem. 2005, 48, 28.
30. Tan, S.; Schubert, D.; Maher, P. Curr. Top. Med. Chem. 2001, 6, 497.
31. Cumming, R. C.; Schubert, D. FASEB J. 2005, 14, 2060.
32. Lewerenz, J.; Maher, P. J. Biol. Chem. 2009, 284, 1106.
33. (a) Benltifa, M.; Vidal, S.; Fenet, B.; Msaddek, M.; Goekjian, P. G.; Praly, J.-P.;
Brunyánszki, A.; Docsa, T.; Gergely, P. Eur. J. Org. Chem. 2006, 4242; (b) Tóth,
M.; Kun, S.; Bokor, I.; Benltifa, M.; Tallec, G.; Vidal, S.; Docsa, T.; Gergely, P.;
Somsák, L.; Praly, J.-P. Bioorg. Med. Chem. 2009, 17, 4773.
34. Koufaki, M.; Theodorou, E.; Galaris, D.; Nousis, L.; Katsanou, E. S.; Alexis, M. N. J.
Med. Chem. 2006, 49, 300.
9. Antonello, A.; Tarozzi, A.; Morroni, F.; Cavalli, A.; Rosini, M.; Hrelia, P.;
Bolognesi, M. L.; Melchiorre, C. J. Med. Chem. 2006, 49, 6642.