Synthesis of a second generation chroman/catechol hybrids and evaluation of their activity in protecting neuronal cells from oxidative stress-induced cell death

Article abstract of DOI:10.1016/j.bmc.2010.04.042

A new generation of chroman/catechol hybrids bearing heterocyclic five-membered rings, such as 1,2,4-oxadiazole 1,3,4-oxadiazole, 1,2,3-triazole, tetrazole and isoxazole, were designed and synthesized. The activity of the new derivatives against oxidative stress induced neuronal damage, was evaluated using glutamate-challenged hippocampal HT22 cells. Compound 3 in which a 3,4-dimethoxyphenyl moiety, is directly attached to the 1,2,4-oxadiazole ring was the most active among the 2-substituted chroman analogues, with EC₅₀ = 254 ± 65 nM. Concerning the 5-subtituted chroman analogues, isoxazole derivative 29 exhibited the strongest activity (EC₅₀ = 245 ± 38 nM). However, 29 was cytotoxic at concentrations higher than 1 μM, while the triazole analogue 24 (EC₅₀ = 801 ± 229 nM), was non-toxic at all concentrations tested.

Full text of DOI:10.1016/j.bmc.2010.04.042

Bioorganic & Medicinal Chemistry 18 (2010) 3898–3909
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of a second generation chroman/catechol hybrids and evaluation
of their activity in protecting neuronal cells from oxidative stress-induced
cell death
a
b
b
Maria Koufaki a, , Elissavet Theodorou , 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:
A new generation of chroman/catechol hybrids bearing heterocyclic five-membered rings, such as 1,2,
4-oxadiazole 1,3,4-oxadiazole, 1,2,3-triazole, tetrazole and isoxazole, were designed and synthesized.
The activity of the new derivatives against oxidative stress induced neuronal damage, was evaluated
using glutamate-challenged hippocampal HT22 cells.
Compound 3 in which a 3,4-dimethoxyphenyl moiety, is directly attached to the 1,2,4-oxadiazole ring
was the most active among the 2-substituted chroman analogues, with EC50 = 254 65 nM. Concerning
the 5-subtituted chroman analogues, isoxazole derivative 29 exhibited the strongest activity
Received 20 October 2009
Revised 24 March 2010
Accepted 15 April 2010
Available online 21 April 2010
Keywords:
Chroman
Catechol
Bioisosterism
Oxidative stress
Neuronal damage
(EC50 = 245 38 nM). However, 29 was cytotoxic at concentrations higher than 1
analogue 24 (EC50 = 801 229 nM), was non-toxic at all concentrations tested.
lM, while the triazole
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
HT22 hippocampal neurons do not express functional ionotro-
pic glutamate receptors. When challenged with glutamate these
cells undergo oxidative stress and oxytosis, a form of cell death
involving glutamate blockage of cystine/glutamate antiporters,
inhibition of cystine uptake and depletion of intracellular gluta-
thione (GSH) as a consequence.6 The exact steps in the HT22 cell
death pathway have been the subject of extensive research in
recent years but are yet poorly understood.7,8 Nevertheless, the
ability of HT22 cells to undergo glutamate-induced oxytosis
independently of ionotropic glutamate receptor signalling ren-
ders them a valuable tool for high throughput screening of
chemical libraries and natural products for potentially neuropro-
tective agents. It was recently reported that flavonoids screened
positive against oxidative toxicity in HT22 cells were also tested
positive in reducing stroke-induced behavioural defects in
rabbits.9
In search of novel antioxidants, we have previously synthesized
hybrids containing the chroman moiety of vitamin E and a catechol
group (Fig. 1) and evaluated their activity against oxidative stress
induced cellular damage. Specifically, the ability of the new mole-
cules to protect cultured cells from H2O2-induced DNA damage
was evaluated using single cell gel electrophoresis (comet assay),
while their in vitro neuroprotective activity was assessed using
glutamate-challenged HT22 cells. The inference from this previous
study was that the activity of the hybrids against DNA damage
could be attributed to their iron-chelating properties, while their
The brain is particularly vulnerable to oxidative stress because
of its high rate of oxidative metabolism (20% of total oxygen uptake
in spite of its relatively small size) and reduced capacity for cellular
regeneration compared to other organs. Neurodegenerative dis-
eases of distinct pathophysiology are known to share common bio-
chemical characteristics such as mitochondrial dysfunction and
oxidative stress-induced cell damage.1
The oxidative stress-dependent neuronal death is due to multi-
factorial events. For instance, elevated levels of the excitatory
amino acid glutamate are thought to cause oxidative stress by a
non-receptor-mediated oxidative pathway which blocks cystine
uptake and results in depletion of intracellular glutathione (GSH).
There is also evidence that brain iron misregulation and oxidative
stress, result in hydroxyl radical (ꢀOH) generation from H2O2 and
stimulation of inflammatory processes, triggering a cascade of events
leading to apoptotic cell death in neurodegenerative disorders.2,3
Hence, developing of agents that are able to modulate multiple
mechanisms of free radical production and scavenging, without
dangerously hampering any essential physiological mechanism
based on free radical cellular signalling, is a promising approach
against neurodegeneration.4,5
* Corresponding author. Tel.: +30 210 7273818; fax: +30 210 7273831.
E-mail address: mkoufa@eie.gr (M. Koufaki).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.042

M. Koufaki et al. / Bioorg. Med. Chem. 18 (2010) 3898–3909
3899
MeO
OMe
HO
OH
HO
HO
H
N
O
C
O
N
N
C
O
O
O
I
II
OH
OH
O
OH
HN
HO
HO
HO
H
N
OH
OMe
OMe
O
O
O
O
IV
V
III
Figure 1. First generation of chroman/catechol hybrids.
HT22-protective activity could be due not only to their antioxidant
properties but perhaps also to their capacity to interfere with other
cell signaling cascades implicated in oxytosis.10
The present work involves the design and synthesis of new hy-
brids in which chroman and catechol moieties are connected
trough heterocyclic five-membered rings, such as 1,2,4-oxadiazole
1,3,4-oxadiazole, 1,2,3-triazole, tetrazole and isoxazole. These het-
erocycles are potential pharmacophores and have been utilized as
ester or amide bioisosteres in the search for compounds with supe-
rior pharmacokinetic profiles.11
1,3,4-Oxadiazole analogues were synthesized from hydrazide 5
which was reacted with activated 3,4-dimethoxybenzoic acid or
3,4-dimethoxyphenylacetic acid to give intermediates 6 and 7.
Cyclodehydration in boiling POCl3 produced the 2-substituted
chroman analogues 8 and 9.
Analogues bearing 1,2,4-oxadiazoles at position 5 of the chro-
man moiety were prepared from N-hydroxy-amidine 1013 (Scheme
2), following similar procedure as for 2-substituted derivatives and
14
were deprotected using BF3ꢀSMe2 to afford the final analogues
19–22.
The neuroprotective activity of the new hybrids was evaluated
using glutamate-challenged HT22 cells.
Scheme 3 depicts the synthesis of triazole 24 and its isomer 27.
CuI-catalyzed ‘click’ cycloaddition15,16 between (3,4-dihydro-6-
methoxy-2,2,7,8-tetramethyl-2Y-benzopyran-5-yl) methylazide,
and 4-ethynyl-1,2-dimethoxybenzene, in the presence of
CuSO4ꢀ5H2O and sodium ascorbate, afforded the 1,2,3-triazole
analogue 23, which was deprotected to produce the dihydroxy
derivative 24. Similarly, alkyne 25 (prepared by treatment of the
2. Chemistry
The synthesis of analogues bearing oxadiazoles at position 2 of
the chroman moiety is depicted in Scheme 1. Specifically for the
preparation of 1,2,4-oxadiazole derivatives, N-hydroxysuccinim-
idyl-trolox ester reacted with the appropriate N-hydroxy-ami-
corresponding chromanaldehyde with Bestmann-Ohira reagent17
)
reacted with 3,4-dimethoxy-benzylazide to afford compound 26,
which was treated with BF3ꢀSMe2 to produce 27.
dines12 to give the acyl amidoximes
1 and 2. Subsequent
3,5-Disubstituted isoxazoles 29 and 32 were obtained as shown
in Scheme 4. The appropriate aldoximes were transformed to the
intramolecular cyclization in the presence of tetrabutylammonium
fluoride produced the 1,2,4-oxadiazole analogues 3 and 4.
HO
O
COON
OMe
OMe
MeO
O
HO
OMe
HO
NH2
a
O
+
b
O
N
O
N
MeO
MeO
NOH
NH2
n=0,1
n
O
n
n
O
O
N
3 n=0
4 n=1
1 n=0
2 n=1
1,2,4-oxadiazole analogues
HO
HO
HO
d
c
O
O
O
O
CONHNH2
O
NHNH
n
OMe
OMe
OMe
OMe
N
N
n
5
O
6
7
n=0
8 n=0
9 n=1
n=1
Scheme 1. Synthesis of 2-substituted chromans. Reagents and conditions: (a) CH2Cl2, rt; (b) n-Bu4NF, THF; (c) CDI, THF, 3,4-dimethoxybenzoic acid or 3,4-
dimethoxyphenylacetic acid; (d) POCl3.

3900
M. Koufaki et al. / Bioorg. Med. Chem. 18 (2010) 3898–3909
O
N
O
NH2
NH2
NOH
N
R'
R
C
O
R
N
C
O
N
N
MeO
MeO
HO
MeO
c
a
b
O
O
O
O
15-18
19-22
10
11-14
HO
HO
HO
HO
MeO
MeO
MeO
MeO
R' :
R:
19
12, 16
14, 18
20
11, 15
13, 17
HO
HO
HO
MeO
MeO
MeO
MeO
HO
21
22
Scheme 2. 5-Substituted chromans, 1,2,4-oxadiazole analogues. Reagents and conditions: (a) RCOOH, BOP, DMF, CH2Cl2; (b) n-Bu4NF, THF; (c) BF3.SMe2, CH2Cl2.
RO
OR
N3
a
MeO
N
+
N
N
MeO
O
RO
OMe
23 R = Me
24 R = H
b
O
OR
OR
N3
N N
N
MeO
a
+
RO
O
MeO
26 R = Me
27 R = H
b
OMe
O
25
Scheme 3. 5-Substituted chromans, 1,2,3-triazole analogues. Reagents and conditions: (a) CuSO4ꢀ5H2O, sodium ascorbate, t-BuOH, H2O; (b) BF3ꢀSMe2, CH2Cl2.
corresponding nitrile oxides using chloramine-T trihydrate, which
acts as both a halogenating agent and a base.18 In the presence of a
catalytic amount of copper(I), obtained from comproportionation
of Cu metal and CuSO4ꢀ5H2O, the in situ generated nitrile oxides re-
acted with 25 or 4-ethynyl-1,2-dimethoxybenzene, respectively,
furnishing the 3,5-disubstituted isoxazoles 28 and 31 which were
deprotected to afford 29 and 32.
The synthesis of tetrazole derivatives is depicted in Scheme 5.
Acylation of the appropriate amines synthesized by our group19,20
with benzotriazol-1-yloxytris(dimethylamino)phosphonium hexa-
fluorophosphate (BOP) activated 3-(3,4-dimethoxyphenyl)propa-
noic acid, as previously reported,10 afforded amides 33 and 34,
which in turn were converted to thioamides 35 and 36 by treat-
ment with Lawesson’s reagent. Tetrazoles 37 and 38 were obtained
by treatment of thioamides with trimethylsilyl azide (TMSN3), in
the presence of triphenylphosphine and diisopropylazodicarboxyl-
ate (DIAD).21 Analogue 39 was obtained by deprotection of 37 with
BF3ꢀSMe2.
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.6–8 Although HT22 cells lack ionotropic
glutamate receptors that could mediate excitotoxicity, they
undergo oxytosis within 24 h following exposure to 1–5 mM gluta-
mate. Recent findings suggest that oxytosis faithfully mimics
oxidative cytotoxicity in cerebral ischemia, Alzheimer’s disease
and other neurodegenerative disorders with an oxidative stress
component.6,8,22
Among the 2-substituted chroman analogues (Table 1) com-
pound 3 in which the 3,4-dimethoxyphenyl moiety is directly at-
tached to the 1,2,4-oxadiazole ring, showed the highest activity
(EC50 = 254 65 nM) while its 3,4-dimethoxybenzyl derivative 4
was less active (EC50 = 741 183 nM). Compound 4 was almost
equipotent with the piperazine analogue II of the first generation

M. Koufaki et al. / Bioorg. Med. Chem. 18 (2010) 3898–3909
3901
OH
HO
OMe
MeO
a
NOH
MeO
N
O
N
+
b
O
OMe
O
OMe
HO
MeO
25
O
O
29
28
OH
HO
OMe
MeO
O
NOH
O
HC
N
a
b
N
MeO
+
HO
MeO
MeO
O
OMe
O
O
30
32
31
Scheme 4. 5-Substituted chromans, isoxazole analogues. Reagents and conditions: (a) TsN(Cl)Naꢀ3H2O, CuSO4ꢀ5H2O, Cu0, t-BuOH/H2O; (b) BF3ꢀSMe2, CH2Cl2.
OMe
OMe
OMe
OMe
O
NHC
S
NHC
NH2
n
n
n
MeO
MeO
MeO
b
a
O
O
O
33 n=1
34 n=2
35 n=1
36 n=2
n=1, 2
OMe
OH
MeO
HO
N
N
N
N
N
N
N
n
N
n
MeO
c
d
HO
O
O
37 n=1
38 n=2
39 n=1
Scheme 5. 5-Substituted chromans, tetrazole analogues. Reagents and conditions: (a) 3-(3,4-dimethoxyphenyl)propanoic acid, DMF, Et3N, BOP, CH2Cl2; (b) Lawesson’s
reagent, THF, reflux; (c) DIAD, TMSN3, Ph3P, THF; (d) BF3ꢀSMe2, CH2Cl2.
analogues and more active than the caffeic acid amide I and the
3,4-dimethoxyphenethyl derivative III (prepared from the N-
hydroxysuccinimidyl-trolox ester and 3,4-dimethoxyphenethyl-
amine). Replacement of the 1,2,4-oxadiazole ring by 1,3,4-oxadia-
zole, in the phenyl derivatives 3 and 8 strongly affected the
neuroprotective activity (compound 8 was inactive), with the ben-
zyl derivatives 4 and 9 being almost equipotent. It seems that the
presence of conjugated system in 8 reduces its activity. Other fac-
tors, not directly related to oxidative stress pathways but differen-
tially affected by 1,2,4-oxadiazole versus 1,3,4-oxadiazole
derivatives,23 might indirectly influence cell fate. Although the
activity of our compounds at the cellular level depends on the nat-
ure of heterocycle and its substituents, whether and how these
may differentially impact on the various pathways of ROS produc-
tion and/or oxytosis modulation is presently unexplored.
Concerning the 5-substituted chroman analogues (Table 2), sty-
rene analogue 21 was the most active of 1,2,4-oxadiazole deriva-
tives with EC50 = 637 321 nM. 3,4-Dihydroxyphenyl derivative
19 was less active (EC50 = 1340 214 nM), while the more flexible
3,4-dihydroxyphenethyl derivative 22 exhibited the lowest activity
(EC50 = 2552 258 nM) compared to the other 1,2,4-oxadiazole
analogues as well as to the 3,4-dihydroxyphenethyl derivative V
of the first generation hybrids. In addition, 22 was equipotent to
its tetrazole analogue 39 (EC50 = 2823 437 nM).

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M. Koufaki et al. / Bioorg. Med. Chem. 18 (2010) 3898–3909
Table 1
Efficacy and potency of 2-substituted chroman analogues to protect glutamate-challenged HT22 cells from oxytosis
Relative potencyb
Efficacyc
a
Compound
EC50 (nV)
OMe
HO
N
254 65
2.6
100% (full)
OMe
O
O
N
3
OMe
OMe
HO
HO
741 183
0.9
60.1
0.8
75% (full)
N
O
O
O
N
N
4
8
OMe
P10000
ns
O
N
OMe
OMe
HO
HO
OMe
843 175
80% (full)
94% (full)
O
O
O
N
N
9
OH
OH
H
1140 350
0.6
N
O
I
MeO
OMe
OMe
HO
HO
650 13
1
100% (full)
O
O
C
O
N
N
C
O
II
MeO
1707 321
0.4
92% (full)
C
O
N
H
III
a
EC50 values are test compound concentrations able to maintain the 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 2 and 3.
b
Relative potencies were calculated by [EC50
Hybrids exhibiting statistically significant effects in protecting cell viability at 10 lM were classified as exhibiting full, partial or weak neuroprotective efficacy depending
compound/EC50 compound]. For the 2-substituted chroman analogues, the reference compound was hybrid II.
reference
c
on whether their % neuroprotective effect was, respectively, 67–100%, 34–66% and 633%. Values are the mean of at least three independent experiments similar to those
shown in Figures 2 and 3. ns = non-significant.
Interestingly, triazole analogue 24 was ꢁ10 times more active
(EC50 = 801 229) than its isomer 27 (EC50 = 7093 929 nM) and
ꢁ2 times more potent than its 1,2,4-oxadiazole counterpart 19.
Isoxazole derivatives 29 and its regioisomer 32 exhibited the
strongest activity with EC50 = 245 38 nM and 584 33 nM,
respectively.
Comparing 1,2,4-oxadiazole derivatives, 2-substituted chro-
mans bearing the protected catechols showed highest activity than
the 5-substituted chromans with the free catechols. Although in li-
poic acid conjugates bearing nitrogen heterocycles, the presence of
a catechol moiety was requisite for activity,12 replacement of the
dithiolane by a chroman scaffold strongly enhanced the neuropro-
tective activity even when the catechol group was masked. More-
over, the presence of catechol moiety in compounds 21, 22, 29 and
32 is associated with cytotoxicity. Catechols can be converted in
two one-electron oxidative steps to redox-active electrophilic o-
quinones that in the presence of NAD(P)H are readily recycled in
a non-enzymatic way back to catechols, thus amplifying ROS pro-
duction and cell damage.24 The prooxidant catechol-o-quinone re-
dox cycling is apparently inhibited by catechol O-methyl-
transferases,25 explaining in part why 3 is a better choice than
any of the catechol derivatives of Table 2.

M. Koufaki et al. / Bioorg. Med. Chem. 18 (2010) 3898–3909
3903
Figure 2. Protection of HT22 cells from oxytosis 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 Section 5. Values are mean SEM of three independent experiments
with an intra-assay variation similar to that shown for 3.
Figure 3. Protection of HT22 cells from oxytosis by 5-substituted chroman analogues. Cells were treated and data are presented as described in the legend to Figure 2.
further investigation and the synthesis of new derivatives is cur-
rently in progress.
4. Conclusion
Our results show that chroman/catechol hybrids bearing five
membered heterocycles exhibited considerably higher in vitro
neuroprotective activity compared to the majority of the first gen-
eration hybrids, with compounds 3, 4, 9, 21, 24, 29 and 32 display-
5. Experimental part
5.1. Chemistry
ing EC50 values below 1
with isoxazole and oxadiazole moieties (21, 22, 29 and 32) dis-
played cytotoxicity at concentrations higher than 1–3 M. How-
ever, chroman analogues bearing isoxazole substituents merit
lM. Some 5-substituted chroman hybrids
Melting points were determined on a Buchi 510 apparatus and
are uncorrected. NMR spectra were recorded on a Varian 300
spectrometer operating at 300 MHz for 1H and 75.43 MHz for 13C
l

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M. Koufaki et al. / Bioorg. Med. Chem. 18 (2010) 3898–3909
Table 2
Efficacy and potency of 5-substituted chroman analogues to protect glutamate-challenged HT22 cells from oxytosis
Relative potencyb
Efficacyc
a
Compound
EC50 (nV)
OH
OH
O
N
N
HO
1340 214
0.7
95% (full)
O
19
OH
OH
O
N
N
HO
*
73% (full)
637 321
2552 258
801 229
1.5
0.4
1.2
O
21
OH
OH
O
N
N
HO
*
72% (full)
O
22
N
O
N
N
OH
OH
HO
77% (full)
24
OH
OH
N
N
N
7093 929
0.1
86% (full)
HO
O
27
HO
OH
N
O
245 38
3.8
73%} (full)
HO
O
29

M. Koufaki et al. / Bioorg. Med. Chem. 18 (2010) 3898–3909
3905
Table 2 (continued)
Compound
HO
Relative potencyb
Efficacyc
a
EC50 (nV)
OH
O
N
582 33
1.6
87%} (full)
HO
O
32
HO
HO
N
N
N
N
2823 437
0.3
66% (full)
HO
O
39
OH
OH
O
HN
HO
1230 260
0.8
100% (full)
O
IV
OH
OH
930 190
1
100% (full)
HO
O
V
a,b,c
as for legend to Table 1.
*
Assessed at 3
Assessed at 1
lM 21 or 22.
}
lM 29; ns = non-significant.
spectra with CDCl3 as solvent. Silica gel plates Macherey-Nagel Sil
G-25 UV254 were used for thin layer chromatography. Chromato-
graphic purification was performed with silica gel (200–400 mesh).
Mass spectra were recorded on TSQ 7000 Finigan instrument in the
ESI mode. HRMS were recorded in FAB mode, at the University of
Notre Dame, IN, USA.
NMR d: 172.0, 158.0, 151.5, 149.0, 146.3, 145.7, 123.3, 122.2,
122.1, 119.9, 119.6, 117.7, 110.7, 109.9, 78.1, 56.2, 56.1, 31.4,
26.2, 21.3, 12.5, 12.0, 11.6. MS m/z: 429.5 [M+H]+.
5.1.2. N-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2Y-1-benzo-
pyran-2-carbonyloxy)-N0-(3,4-dimethoxyphenyl)
acetimidamide (2)
5.1.1. N -(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2Y-1-ben-
zopyran-2-carbonyloxy)-N0-(3,4-dimethoxy)benzimidamide (1)
To a solution of N0-hydroxy-3,4-dimethoxybenzimidamide12
(80 mg, 0.41 mmol) in 4 mL anhyd CH2Cl2 were added N-hydroxy-
succinimidyl trolox ester (142 mg, 0.41 mmol) and the mixture
was stirred at rt overnight. CH2Cl2 and water were then added,
the organic layer was washed with satd aqueous NaCl, dried with
Na2SO4, the solvent evaporated and the residue was purified by
flash chromatography (CH2Cl2/MeOH, 97/3). Yield: 125 mg (72%),
white solid, mp 218–220 °C. 1H NMR d: 7.12 (s, 1H, ArH), 7.04 (d,
J = 8.4 Hz, 1H, ArH), 6.72 (d, J = 8.4 Hz, 1H, ArH), 4.92 (br s, 1H,
–OH), 4.51 (s, 2H, –NH2), 3.82 (s, 3H, CH3O–), 3.77 (s, 3H, CH3O–),
2.61–2.48 (m, 3H), 2.19 (s, 3H, FrCH3), 2.12 (s, 3H, FrCH3), 2.01
(s, 3H, FrCH3), 1.91–1.82 (m, 1H, CH–), 1.71 (s, 3H, CH3). 13C
This compound was prepared according to the procedure de-
scribed for 1. Yield: 72%, yellowish gummy solid. 1H NMR d: 6.77–
6.75 (m, 1H), 6.71 (br s, 2H), 4.06 (br s, 1H), 3.84 (s, 3H), 3.82 (s,
3H), 3.46 (s, 2H), 2.63–2.54 (m, 3H), 2.15 (s, 3H), 2.11 (s, 3H), 2.03
(s, 3H), 1.94–1.82 (m, 1H), 1.72 (s, 3H). 13C NMR d: 171.9, 158.8,
149.5, 148.7, 146.3, 145.7, 127.2, 121.6, 121.5, 112.1, 111.5, 78.1,
56.2, 56.1, 36.9, 31.3, 25.2, 21.3, 12.5, 12.0, 11.5. HRMS: calcd for
C24H31N2O6 [M+H]+ 443.2182, found 443.2169.
5.1.3. 3-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2Y-1-benzo-
pyran-2-yl)-5-(3,4-dimethoxyphenyl)-1,2,4-oxadiazole (3)
Compound 1 (40 mg, 0.093 mmol) in 3.72 mL anhyd THF, was
treated with 0.1 mL TBAF. After 2 h the solvent was evaporated

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M. Koufaki et al. / Bioorg. Med. Chem. 18 (2010) 3898–3909
and the residue was diluted by AcOEt. The organic layer was
washed with satd aqueous NaCl, dried with Na2SO4, the solvent
evaporated and the residue was purified by flash chromatography
(AcOEt/pet. ether, 95/5) to afford compound 3 as yellow foam.
Yield: 38 mg (100%). 1H NMR d: 7.65 (d, J = 8.4 Hz, 1H), 7.53 (s,
1H), 6.91 (d, J = 8.4 Hz, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 2.72–2.67
(m, 3H, CH2 and CHH), 2.22 (s, 3H), 2.15 (s, 3H), 1.82 (s, 3H),
1.49–1.45 (m, 1H, CH), 1.25 (s, 3H). 13C NMR d: 181.1, 167.9,
151.4, 149.1, 145.7, 144.8, 123.0, 121.4, 120.9, 119.3, 118.4,
116.7, 110.9, 109.8, 73.7, 56.0, 55.9, 53.4, 31.4, 26.9, 20.5, 12.2,
11.9. MS m/z: 411 [M+H]+. HRMS: calcd for C23H26N2O5 [M]+
410.1842, found 410.1846.
20.6, 12.1, 11.7, 11.2. MS m/z: 425.3 [M+H]+. HRMS: calcd for
C24H28N2O5 [M]+ 424.1998, found 424.1981.
5.1.8. J-(3,4-Dimethoxybenzoyl)-(3,4-dihydro-6-methoxy-2,2,7,
8-tetramethyl-2Y-1-benzopyran-5-acetamidoxime) (11)
To a solution of compound 1013 (40 mg, 0.14 mmol) in 4 ml
anhyd CH2Cl2 were added 3,4-dimethoxybenzoic acid (25 mg,
0.14 mmol), DCC (34 mg, 0.16 mmol) and the mixture was stir-
red at rt overnight. CH2Cl2 was then added, the organic layer
was washed with satd aqueous NaCl, dried and concentrated.
The residue was purified by flash chromatography (CH2Cl2/
MeOH, 95/5). Yield: 56 mg (90%), yellow solid, mp 198–200 °C.
1H NMR d: 7.63 (d, J = 8.4 Hz, 1H), 7.53 (s, 1H), 6.87 (d,
J = 8.4 Hz, 1H), 5.29 (br s, 2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.73
(s, 3H), 3.64 (s, 2H), 2.87 (t, J = 6.8 Hz, 2H), 2.21 (s, 3H), 2.10
(s, 3H), 1.79 (t, J = 6.8 Hz, 2H), 1.29 (s, 6H). 13C NMR d: 165.2,
158.7, 149.2, 141.5, 128.1, 123.3, 123.1, 112.1, 110.3, 73.3,
60.8, 56.1, 34.9, 29.7, 26.8, 25.5, 24.7, 20.3, 12.9, 12.1. MS m/z:
457.6 [M+H]+.
5.1.4. 5-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2Y-1-benzo-
pyran)-3-(3,4-dimethoxybenzyl)-1,2,4-oxadiazole (4)
This compound was prepared according to the procedure de-
scribed for 3. Yield: 64%, yellow solid, mp 170–172 °C. 1H NMR d:
6.79 (br s, 3H), 3.98 (s, 2H), 3.85 (s, 3H), 3.81 (s, 3H), 2.63–2.56
(m, 4H), 2.19 (s, 3H), 2.15 (s, 3H), 2.03 (s, 3H), 1.75 (s, 3H). 13C
NMR d: 181.3, 169.4, 148.9, 148.0, 145.6, 144.8, 127.8, 122.9,
121.0, 118.3, 116.7, 112.1, 111.2, 73.6, 55.9, 55.8, 31.9, 31.4, 26.8,
20.5, 12.2, 11.9, 11.2. MS m/z: 425.3 [M+H]+. HRMS: calcd for
C24H28N2O5 [M]+ 424.1998, found 424.1978.
5.1.9. J-(3,4-Dimethoxyphenylacetyl)-(3,4-dihydro-6-methoxy-
2,2,7,8-tetramethyl-2Y-1-benzopyran-5-acetamidoxime) (12)
In 2 mL anhyd DMF were added, 3,4-dimethoxyphenylacetic
acid (34 mg, 0.17 mmol) and 0.05 mL anhyd Et3N. After 30 min
the mixture was cooled and a solution of BOP (75 mg, 0.17 mmol)
in 2 mL anhyd CH2Cl2 followed by a solution of 10 (50 mg,
0.17 mmol) in 3 mL anhyd CH2Cl2 were added. The mixture was
stirred at 0 °C for 1 h and at rt overnight. The reaction mixture
was then acidified by 10% HCl, diluted by water and extracted with
AcOEt. The organic layer was washed with satd aqueous NaHCO3,
satd aqueous NaCl, dried and concentrated. Analogue 12 was ob-
tained as a yellow gummy solid after purification by flash chroma-
tography (CH2Cl2/MeOH, 97/3). Yield: 76 mg (93%). 1H NMR d: 6.86
(s, 1H), 6.83 (br s, 2H), 3.89 (s, 6H), 3.73 (s, 3H), 3.71 (s, 2H), 3.57 (s,
2H), 2.84 (t, J = 6.5 Hz, 2H), 2.24 (s, 3H), 2.14 (s, 3H), 1.81 (t,
J = 6.5 Hz, 2H), 1.33 (s, 6H). 13C NMR d: 169.3, 158.7, 149.4,
149.2, 149.0, 148.4, 128.4, 126.8, 126.0, 123.4, 121.7, 118.7,
112.6, 111.5, 73.5, 61.1, 56.2, 40.5, 32.9, 28.4, 27.1, 20.6, 13.2,
12.4. MS m/z: 471.4 [M+H]+ HRMS: calcd for C26H35N2O6 [M+H]+
471.2495, found 471.2481.
5.1.5. N-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2Y-1-benzo-
pyran-2-carbonyl)-N0-(3,4-dimethoxyphenyl-carbonyl)
hydrazine (6)
CDI (26.8 mg, 0.165 mmol) was added to a solution of 3,4-
dimethoxybenzoic acid (27 mg, 0.15 mmol) in 2.9 mL anhyd THF
and the mixture was stirred at rt for 90 min. A solution of 5
(40 mg, 0.15 mmol) in 2.9 mL anhyd THF was then added and the
new mixture was stirred at rt overnight. THF was evaporated and
the residue was taken up by AcOEt and washed with satd aqueous
NaCl. The organic layer was dried and evaporated to dryness and
the residue was purified by flash chromatography (CH2Cl2/CH3OH,
97/3) affording 6 as yellow solid. Yield: 50 mg (77%), mp 198–
200 °C. 1H NMR d: 9.32 (br s, 1H), 9.17 (br s, 1H), 7.75–7.56 (m,
1H), 7.38 (s, 1H), 6.98–6.81 (m, 1H), 3.89 (s, 3H), 3.86 (s, 3H),
2.66–2.56 (m, 2H), 2.25 (s, 3H), 2.17 (s, 3H), 1.54–1.43 (m, 5H),
1.25 (s, 3H). MS m/z: 429.7 [M+H]+.
5.1.10. J-(3,4-Dimethoxyphenylpropenoyl)-(3,4-dihydro-6-
methoxy-2,2,7,8-tetramethyl-2Y-1-benzopyran-5-acetamid-
oxime) (13)
5.1.6. 2-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2Y-1-benzo-
pyran)-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole (8)
A mixture of compound 6 (39 mg, 0.09 mmol) and 0.18 mL
POCl3 was refluxed for 3 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 and
evaporated to dryness. The residue was purified by flash chroma-
tography (CH2Cl2/CH3OH, 95/5) affording 8 as yellowish gummy
solid. Yield: 23 mg (64%). 1H NMR d: 7.92 (d, J = 8.4 Hz, 1H), 7.70
(s, 1H), 6.97 (d, J = 8.4 Hz, 1H), 3.98 (s, 3H), 3.96 (s, 3H), 2.71 (br
s, 3H), 2.51–2.45 (m, 1H), 2.17 (s, 3H), 2.07 (s, 3H), 2.00 (s, 3H),
1.73 (s, 3H). 13C NMR d: 164.8, 154.2, 153.5, 148.8, 141.9, 127.8,
125.5, 124.3, 121.8, 116.9, 112.4, 110.4, 93.3, 82.7, 56.1, 56.0,
29.7, 13.1, 12.2, 11.9. HRMS: calcd for C23H26N2O5 [M]+ 410.1842,
found 410.1849.
According to the procedure described for 12 and using ana-
logue 10 (40 mg, 0.14 mmol), 3,4-dimethoxyphenylpropenoic
acid (29 mg, 0.14 mmol) and BOP (61 mg, 0.14 mmol), compound
13 was obtained as a yellow gummy solid after purification by
flash chromatography (CH2Cl2/MeOH, 97/3). Yield: 18 mg (27%).
1H NMR d: 7.69 (d, J = 15.9 Hz, 1H), 7.11 (d, J = 8.3 Hz, 1H),
7.05 (s, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.36 (d, J = 15.9 Hz, 1H),
5.29 (br s, 2H), 3.91 (s, 6H), 3.71 (s, 3H), 2.84 (t, J = 6.8 Hz,
2H), 2.21 (s, 3H), 2.09 (s, 3H), 1.77 (t, J = 6.8 Hz, 2H), 1.28 (s,
6H). 13C NMR d: 165.3, 158.3, 151.4, 149.5, 149.4, 148.9, 145.2,
128.3, 127.7, 125.9, 123.4, 122.8, 118.8, 114.2, 111.3, 109.9,
73.5, 61.1, 56.2, 56.1, 32.8, 28.4, 27.1, 20.5, 13.1, 12.3. MS m/z:
483.2 [M+H]+.
5.1.7.2-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2Y-1-benzo-
pyran)-5-(3,4-dimethoxybenzyl)-1,3,4-oxadiazole (9)
5.1.11. 5-(3,4-Dimethoxyphenyl)-3-[(3,4-dihydro-6-methoxy-
2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)methyl]-1,2,4-
oxadiazole (15)
To a solution of acylated amidine 11 (46 mg, 0.11 mmol) in an-
hyd THF (C = 0.025 M), was added TBAF (0.04 mL, 0.11 mmol) and
the mixture was stirred at rt for 2 h. After dilution with water
the mixture was extracted with AcOEt, the organic layer was
washed with satd aqueous NaHCO3, satd aqueous NaCl, dried and
Compound 7 (77 mg, 0.17 mmol) was treated as described for
the synthesis of 8. Yield: 10 mg (14 %), white gel. 1H NMR d:
6.79–6.67 (m, 3H), 4.08 (s, 2H), 3.88 (s, 3H), 3.78 (s, 3H), 2.82–
2.63 (m, 4H), 2.13 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 1.73 (s, 3H).
13C NMR d: 168.8, 165.9, 149.1, 148.3, 145.7, 144.6, 126.2, 122.8,
120.8, 118.5, 117.2, 111.6, 111.3, 72.2, 55.9, 55.8, 31.4, 30.8, 26.6,

M. Koufaki et al. / Bioorg. Med. Chem. 18 (2010) 3898–3909
3907
concentrated. Analogue 15 was obtained as a white gummy solid
after purification by flash chromatography (pet. ether/AcOEt, 80/
20). Yield: 27 mg (56%). 1H NMR d: 7.71 (d, J = 8.4 Hz, 1H), 7.56
(s, 1H), 6.94 (d, J = 8.4 Hz, 1H), 4.17 (s, 2H), 3.95 (s, 6H), 3.71 (s,
3H), 2.73 (t, J = 6.8 Hz, 2H), 2.21 (s, 3H), 2.11 (s, 3H), 1.77 (t,
J = 6.8 Hz, 2H), 1.29 (s, 6H). 13C NMR d: 175.5, 170.6, 152.9,
150.1, 149.4, 148.5, 128.5, 125.6, 123.8, 122.2, 118.2, 117.2,
111.2, 110.7, 73.2, 61.5, 56.4, 56.3, 32.9, 27.1, 24.1, 20.8, 13.1,
12.3. MS m/z: 439.1 [M+H]+.
7 mg (64%). 1H NMR d: 7.61 (d, J = 16.3 Hz, 1H), 7.1–6.89 (m, 2H),
6.75 (s, 1H), 6.72 (d, J = 16.3 Hz, 1H), 4.07 (s, 2H), 3.51 (s, 3H),
2.83 (t, J = 6.7 Hz, 2H), 2.26 (s, 3H), 2.12 (s, 3H), 1.81 (t, J = 6.7 Hz,
2H), 1.31 (s, 6H). 13C NMR d: 175.2, 169.2, 146.4, 145.5, 143.8,
143.3, 127.6, 125.4, 125.3, 122.8, 116.5, 115.7, 114.1, 113.9, 72.7,
32.8, 29.7, 26.7, 23.4, 22.7, 20.8, 12.0. HRMS: calcd for
C24H26N2O5 [M]+ 422.1842, found 422.1842.
5.1.17. 5-[2-(3,4-Dihydroxyphenyl)ethyl]-3-[(3,4-dihydro-6-
hydroxy-2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)-methyl]-
1,2,4-oxadiazole (22)
5.1.12. 5-[2-(3,4-Dimethoxyphenyl)vinyl]-3-[(3,4-dihydro-6-
methoxy-2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)methyl]-
1,2,4-oxadiazole (17)
According to the procedure described for 15 and using 13
(16 mg, 0.04 mmol) and TBAF (0.012 mL, 0.04 mmol), analogue
17 was obtained, as a yellow gummy solid. Yield: 13 mg (87%).
1H NMR d: 7.74 (d, J = 16.3 Hz, 1H), 7.33 (s, 1H), 7.18 (br s, 2H),
6.95 (d, J = 16.3 Hz, 1H), 4.29 (s, 2H), 3.99 (s, 6H), 3.77 (s, 3H),
2.75 (t, J = 6.5 Hz, 2H), 2.28 (s, 3H), 2.17 (s, 3H), 1.83 (t, J = 6.5 Hz,
2H), 1.35 (s, 6H). MS m/z: 465.4 [M+H]+.
Treatment of compound 18 (48 mg, 0.11 mmol) as described for
19, gave 22 as yellow gummy solid. Yield: 25 mg (58%). 1H NMR d:
6.72 (d, J = 8.1 Hz, 1H), 6.63 (s, 1H), 6.54 (d, J = 8.1 Hz, 1H), 4.03 (s,
2H), 3.10–2.92 (m, 4H), 2.75 (t, J = 6.8 Hz, 2H), 2.09 (s, 3H), 2.06 (s,
3H), 1.77 (t, J = 6.8 Hz, 2H), 1.27 (s, 6H). 13C NMR d: 179.4, 168.6,
146.5, 145.1, 143.7, 142.6, 131.6, 125.4, 120.5, 118.8, 116.7,
115.4, 72.8, 32.8, 31.5, 29.7, 28.5, 26.7, 23.2, 20.7, 13.5, 12.5. MS
m/z: 425.3 [M+H]+. HRMS: calcd for C24H28N2O5 [M]+ 424.1998,
found 424.1997.
5.1.13. 5-[2-(3,4-Dimethoxyphenethyl)]-3-[(3,4-dihydro-6-met-
hoxy-2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)methyl]-1,2,4-
oxadiazole (18)
5.1.18. 4-(3,4-Dimethoxyphenyl)-1-[(3,4-dihydro-6-methoxy-
2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)-methyl]-1,2,3-
triazole (23)
According to the procedure described for 15 and using 14
(94 mg, 0.19 mmol) and TBAF (0.07 mL, 0.19 mmol), compound
18 was obtained as a yellow oil. Yield: 75 mg (86%). 1H NMR d:
6.73–6.67 (m, 3H), 4.08 (s, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 3.65 (s,
3H), 3.09–3.04 (m, 4H), 2.64 (t, J = 6.7 Hz, 2H), 2.20 (s, 3H), 2.09
(s, 3H), 1.75 (t, J = 6.7 Hz, 2H), 1.28 (s, 6H). 13C NMR d: 178.9,
169.9, 149.9, 149.1, 148.4, 147.9, 132.3, 128.5, 125.6, 123.6,
120.4, 117.9, 111.7, 111.5, 73.2, 61.4, 56.1, 55.9, 32.9, 32.4, 28.9,
27.1, 23.8, 20.7, 13.9, 13.1, 12.3. MS m/z: 467.6 [M+H]+.
A
solution of 4-ethynyl-1,2-dimethoxybenzene (40 mg,
0.25 mmol), of azidomethyl-chroman (137 mg, 0.50 mmol),
CuSO4ꢀ5H2O (18.7 mg, 0.075 mmol) and sodium ascorbate
(30 mg, 0.15 mmol) in 4.16 mL t-BuOH/H2O (2:1) was stirred at
rt for 24 h. After cooling at 0 °C, aqueous NY4JY was added and
the mixture was exracted with AcOEt. The organic layer was
washed with satd aqueous NaCl, dried with Na2SO4, filtered and
the solvent evaporated. Compound 27 was obtained as a yellow
oil. Yield: 110 mg (100%). 1H NMR d: 7.58 (s, 1H), 7.43 (s, 1H),
7.21 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 5.57 (s, 2H), 3.92
(s, 3H), 3.87 (s, 3H), 3.69 (s, 3H), 2.69 (t, J = 6.8 Hz, 2H), 2.23
(s, 3H), 2.12 (s, 3H), 1.73 (t, J = 6.8 Hz, 2H), 1.25 (s, 6H). 13C NMR
d: 150.1, 149.2, 148.9, 148.6, 147.6, 127.9, 123.8, 122.2, 118.8,
118.2, 118.1, 111.2, 108.8, 73.3, 61.7, 55.9, 45.7, 32.3, 26.7, 19.9,
12.9, 12.2. MS m/z: 438.4 [M+H]+.
5.1.14. 5-[(3,4-Dihydroxyphenyl)]-3-[(3,4-dihydro-6-hydroxy-
2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)-methyl]-1,2,4-
oxadiazole (19)
A
solution of 15 (25 mg, 0.06 mmol) in anhyd CH2Cl2
(C = 0.026 M), was cooled at 0 °C and BF3ꢀSMe2 (0.19 mL, 1.8 mmol)
was added. The mixture was stirred at 0 °C for 1 h and an addi-
tional 1 h at rt. Excess of BF3ꢀSMe2 was evaporated under argon,
water was added and the mixture was extracted with AcOEt. The
organic layer was washed with satd aqueous NaCl, dried and con-
centrated. Analogue 19 was obtained as a white gel after purifica-
tion by flash chromatography (CH2Cl2/MeOH, 97/3). Yield: 9 mg
(40%). 1H NMR d: 7.59 (br s, 2H), 6.97 (br s, 1H), 4.09 (s, 2H),
2.84 (t, J = 6.7 Hz, 2H), 2.25 (s, 3H), 2.12 (s, 3H), 1.82 (t, J = 6.7 Hz,
2H), 1.31 (s, 6H). MS m/z: 397.3 [M+H]+.
5.1.19. 4-(3,4-Dihydroxyphenyl)-1-[(3,4-dihydro-6-hydroxy-
2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)-methyl]-1,2,3-
triazole (24)
To
a solution of protected triazole analogue 23 (38 mg,
0.08 mmol) in 3.5 mL anhyd CH2Cl2 were added at 0 °C, BF3ꢀSMe2
(0.28 mL, 2.61 mmol) and the mixture was stirred at 0 °C for 1 h
and overnight at rt. Workup as described for 19 and purification
by flash chromatography (CH2Cl2/MeOH, 95/5) afforded 24 as a
yellowish solid, mp 265–268 °C. Yield: 26 mg (77%). 1H NMR d:
7.63 (s, 1H), 7.18 (s, 1H), 6.91 (d, J = 8.1 Hz, 1H), 6.75 (d,
J = 8.1 Hz, 1H), 5.52 (s, 2H), 2.72 (t, J = 6.5 Hz, 2H), 2.15 (s, 3H),
2.07 (s, 3H), 1.72 (t, J = 6.5 Hz, 2H), 1.22 (s, 6H). 13C NMR d:
147.6, 146.0, 145.8, 145.1, 144.6, 127.1, 123.6, 122.3, 119.4,
117.8, 117.6, 117.4, 115.3, 112.3, 72.9, 45.8, 32.5, 26.5, 20.1, 12.4,
12.2. MS m/z: 395.4 [M]+. HRMS: calcd for C22H26N3O4 [M+H]+
396.1923, found 396.1964.
5.1.15. 5-[1-(3,4-Dihydroxyphenyl)methyl]-3-[(3,4-dihydro-6-
hydroxy-2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)-methyl]-
1,2,4-oxadiazole (20)
Oxadiazole analogue 16 (9.0 mg, 0.02 mmol) was treated as de-
scribed for 19. Compound 20 was obtained as a yellow gummy so-
lid after purification by flash chromatography (CH2Cl2/MeOH, 97/
3). Yield: 5 mg (64%). 1H NMR d: 6.81–6.79 (m, 2H), 6.71 (d,
J = 7.5 Hz, 1H), 4.05 (s, 2H), 4.02 (s, 2H), 2.78 (t, J = 6.8 Hz, 2H),
2.23 (s, 3H), 2.11 (s, 3H), 1.78 (t, J = 6.8 Hz, 2H), 1.29 (s, 6H). MS
m/z: 411.2 [M+H]+.
5.1.20. 1-(3,4-Dimethoxybenzyl)-4-(3,4-dihydro-6-methoxy-
2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)-1,2,3-triazole (26)
A solution of 5-ethynyl-3,4-dihydro-6-methoxy-2,2,7,8-tetra-
methyl-2H-benzopyran 25 (20 mg, 0.08 mmol), 4-azidomethyl-
1,2-dimethoxy-benzene (31 mg, 0.16 mmol), CuSO4ꢀ5H2O (6.0 mg,
0.02 mmol), and sodium ascorbate (10 mg, 0.05 mmol) in t-
BuOH/H2O (2:1) (1.3 mL) was treated as described for 23. Purifica-
tion by flash chromatography (cyclohexane/AcOEt, 70/30) afforded
compound 26, as a yellow gummy solid. Yield: 34 mg (97%). 1H
5.1.16. 5-[2-(3,4-Dihydroxyphenyl)vinyl]-3-[(3,4-dihydro-6-
methoxy-2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)-methyl]-
1,2,4-oxadiazole (21)
Analogue 17 (13 mg, 0.03 mmol) was treated as described for
19. Compound 21 was obtained as a yellow gummy solid after
purification by flash chromatography (CH2Cl2/MeOH, 95/5). Yield:

3908
M. Koufaki et al. / Bioorg. Med. Chem. 18 (2010) 3898–3909
NMR d: 7.60 (s, 1H), 6.68–6.81 (m, 3H), 5.52 (s, 2H), 3.87 (s, 3H),
3.82 (s, 3H), 3.27 (s, 3H), 2.75–2.71 (m, 1H), 2.16 (s, 3H), 2.12 (s,
3H), 1.69–1.65 (m, 3H), 1.30 (s, 3H), 1.24 (s, 3H). 13C NMR d:
149.4, 149.3, 149.1, 148.3, 128.1, 127.5, 126.5, 123.6, 120.6,
120.4, 118.3, 111.2, 110.9, 73.3, 60.6, 55.9, 53.9, 32.8, 29.7, 26.9,
21.9, 12.5, 12.2. MS m/z: 438.4 [M+H]+.
gave isoxazole 32 as a white gummy solid. Yield: 9 mg (32%). 1H
NMR d: 7.45 (s, 1H), 7.32 (d, J = 8.3 Hz, 1H), 6.97 (d, J = 8.3 Hz,
1H), 6.64 (s, 1H), 2.81 (t, J = 6.7 Hz, 2H), 2.23 (s, 3H), 2.18 (s, 3H),
1.75 (t, J = 6.7 Hz, 2H), 1.35 (s, 3H), 1.25 (s, 3H) HRMS: calcd for
C22H23NO5 [M]+ 381.1576, found 381.1606.
5.1.26. N-(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-2Y-1-
benzopyran-5-methyl)-3-(3,4-dimethoxyphenyl-propane-
thioamide) (35)
5.1.21. 1-(3,4-Dihydroxybenzyl)-4-(3,4-dihydro-6-hydroxy-
2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)-1,2,3-triazole (27)
Triazole analogue 26 (18 mg, 0.04 mmol) was treated as de-
scribed for 24 to produce deprotected derivative 27, as a yellowish
gummy solid. Yield: 13 mg (82%). 1H NMR d: 7.65 (s, 1H), 6.81–6.71
(m, 3H), 5.42 (s, 2H), 2.60 (t, J = 6.1 Hz, 2H), 2.16 (s, 3H), 2.13 (s,
3H), 1.72 (t, J = 6.1 Hz, 2H), 1.26 (s, 3H), 1.22 (s, 3H). HRMS: calcd
for C22H25N3O4 [M]+ 395.1845, found 395.1833.
To a solution of carboxamide 33 (76 mg, 0.17 mmol) in 6 mL
THF was added Lawesson’s reagent (69 mg, 0.17 mmol) and the
mixture was refluxed for 6 h. The solvent was evaporated and
the residue was purified by flash chromatography (pet. ether/
AcOEt, 60/40), to afford thioamide 35, as an off white gummy so-
lid. Yield: 75 mg (96%). 1H NMR d: 6.71 (s, 1H), 6.68 (br s, 2H),
4.71 (d, J = 4.6 Hz, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 3.58 (s, 3H),
3.05 (t, J = 7.2 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H), 2.56 (t, J = 6.7 Hz,
2H), 2.16 (s, 3H), 2.09 (s, 3H), 1.72 (t, J = 6.7 Hz, 2H), 1.28 (s,
6H). 13C NMR d: 202.9, 150.2, 148.8, 148.4, 147.4, 132.8, 128.5,
126.7, 123.6, 120.2, 117.6, 111.6, 111.1, 73.3, 61.2, 49.0, 42.6,
35.0, 32.5, 26.8, 21.0, 20.3, 14.2, 12.6, 12.1. MS m/z: 458.5
[M+H]+.
5.1.22. 3-(3,4-Dimethoxyphenyl)-5-(3,4-dihydro-6-methoxy-
2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)isoxazole (28)
3,4-Dimethoxybenzaldehyde oxime (11 mg, 0.06 mmol) was
added in 1 mL of t-BuOH/H2O (1/1), followed by addition of chlora-
mine-T trihydrate (18 mg, 0.06 mmol) in small portions over
5 min, CuSO4ꢀ5H2O (2 mg, 0.02 mmol), copper turnings (catalytic
amount) and alkyne 25 (15 mg, 0.06 mmol) and the mixture was
stirred overnight. The reaction mixture was poured into ice/water
and after addition of dilute NH4OH, was extracted with AcOEt. The
organic layer was washed with satd aqueous NaCl, dried and evap-
orated to dryness. Compound 28 was obtained as yellow gummy
solid, after purification by flash chromatography (pet. ether/AcOEt,
80/20). Yield: 20 mg (81%). 1H NMR d: 7.52 (s, 1H), 7.39 (d,
J = 8.3 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.72 (s, 1H), 3.98 (s, 3H),
3.96 (s, 3H), 3.53 (s, 3H), 2.72 (t, J = 6.6 Hz, 2H), 2.24 (s, 3H), 2.18
(s, 3H), 1.74 (t, J = 6.6 Hz, 2H), 1.35 (s, 6H). 13C NMR d: 168.1,
162.2, 150.5, 149.3, 148.3, 129.0, 128.9, 112.1, 119.9, 118.2,
111.1, 109.3, 102.5, 73.6, 61.6, 56.1, 56.0, 32.6, 26.9, 21.4, 12.6,
12.4. MS m/z: 424.5 [M+H]+.
5.1.27. N-(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-2Y-1-
benzopyran-5-ethyl)-3-(3,4-dimethoxy-phenylpropanethio-
amide) (36)
Amide 34 (22 mg, 0.05 mmol) was treated as described for 35.
Yield: 19 mg (86%). 1H NMR d: 6.79–6.72 (m, 3H), 3.85 (s, 6H),
3.65–3.63 (m, 5H), 3.03–2.98 (m, 2H), 2.89–2.85 (m, 4H), 2.68 (t,
J = 6.7 Hz, 2H), 2.18 (s, 3H), 2.10 (s, 3H), 1.80 (t, J = 6.7 Hz, 2H),
1.32 (s, 6H). 13C NMR d: 203.6, 148.9, 148.8, 148.7, 147.4, 133.2,
128.0, 126.3, 125.1, 120.1, 116.9, 111.7, 111.2, 73.1, 60.8, 55.9,
55.8, 48.9, 47.5, 34.8, 32.7, 26.8, 24.0, 20.3, 12.8, 11.9. MS m/z:
471.5 [M]+.
5.1.28. 1-(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-2Y-1-
benzopyran-5-yl-methyl)-5-(3,4-dimethoxy-2-phenyl- ethyl)
tetrazole (37)
5.1.23. 3-(3,4-Dihydroxyphenyl)-5-(3,4-dihydro-6-hydroxy-
2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)isoxazole (29)
Isoxazole analogue 28 (10 mg, 0.03 mmol) was treated as de-
scribed for 24 to produce deprotected derivative 29, as an or-
ange gummy solid, after purification by flash chromatography
(CH2Cl2/MeOH, 95/5. Yield: 6 mg (67%). 1H NMR d: 7.43 (s,
1H), 6.97 (d, J = 8.3 Hz, 1H), 6.60–6.57 (m, 2H), 5.57 (br s, 2H),
2.68–2.67 (m, 2H), 2.18 (s, 3H), 2.15 (s, 3H), 1.71–1.68 (m,
2H), 1.31 (s, 6H). HRMS: calcd for C22H23NO5 [M]+ 381.1576,
found 381.1584.
To a solution of thioamide 35 (75 mg, 0.16 mmol) in 1.2 mL an-
hyd THF, were added DIAD (0.05 mL, 0.24 mmol), triphenylphos-
phine (65 mg, 0.24 mmol) and after 5 min TMSN3 (0.03 mL,
0.24 mmol). The reaction mixture was stirred at rt for overnight.
The solvent was evaporated in vacuo and the residue was purified
by flash chromatography (pet. ether/AcOEt 50:50). Yield: 76 mg
(100%), white gummy solid. 1H NMR d: 6.74 (d, J = 8.0 Hz, 1H),
6.61–6.57 (m, 2H), 5.22 (s, 2H), 3.84 (s, 3H), 3.81 (s, 3H), 3.53 (s,
3H), 3.14 (t, J = 7.6 Hz), 2.89 (t, J = 7.6 Hz), 2.53 (t, J = 6.7 Hz, 2H),
2.17 (s, 3H), 2.09 (s, 3H), 1.72 (t, J = 6.7 Hz, 2H), 1.26 (s, 3H), 1.24
(s, 3H). 13C NMR d: 154.7, 149.8, 148.9, 148.6, 147.7, 132.3,
128.4, 125.1, 120.8, 120.3, 118.3, 111.6, 111.2, 73.3, 61.5, 60.4,
55.9, 42.9, 33.3, 32.3, 26.7, 21.0, 20.4, 14.2, 12.8, 12.3. MS m/z:
467.6 [M+H]+.
5.1.24. 5-(3,4-Dimethoxyphenyl)-3-(3,4-dihydro-6-methoxy-
2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)isoxazole (31)
Oxime 30 (58 mg, 0.22 mmol) and 4-ethynyl-1,2dimethoxyben-
zene (37 mg, 0.23 mmol), were treated as described for 28. Purifica-
tion by flash chromatography (pet. ether/AcOEt, 85/15) afforded
compound 31 as a yellow solid, mp 140–142 °C. Yield: 60 mg
(65%). 1H NMR d: 7.40 (d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 6.94 (d,
J = 8.4 Hz, 1H), 6.54 (s, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.49 (s, 3H),
2.69 (t, J = 6.8 Hz, 2H), 2.21 (s, 3H), 2.15 (s, 3H), 1.69 (t, J = 6.8 Hz,
2H), 1.31 (s, 6H). 13C NMR d: 169.2, 160.8, 150.8, 149.8, 149.5,
148.6, 128.8, 127.9, 120.9, 119.9, 119.3, 118.1, 111.5, 108.9, 100.9,
73.7, 61.8, 56.3, 56.2, 32.9, 27.2, 21.8, 12.7, 12.5. MS m/z: 424.1
[M+H]+. HRMS: calcd for C25H30NO5 [M+H]+ 424.2124, found
424.2122.
5.1.29. 1-(3,4-Dihydro-6-methoxy-2,2,7,8-tetramethyl-2Y-1-
benzopyran-5-yl-ethyl)-5-(3,4-dimethoxy-2-phenylethyl)
tetrazole (38)
Thioamide 36 (19 mg, 0.04 mmol) was treated as described
for 37. Yield: 9 mg (47%), white gummy solid. 1H NMR d: 6.74
(d, J = 8.1 Hz, 1H), 6.58–6.53 (m, 2H), 4.29 (t, J = 6.9 Hz, 2H),
3.85 (s, 3H), 3.84 (s, 3H), 3.64 (s, 3H), 3.04 (t, J = 6.9 Hz, 2H),
2.91–2.86 (m, 2H), 2.66 (t, J = 7.7 Hz, 2H), 2.28 (t, J = 6.7 Hz,
2H), 2.14 (s, 3H), 2.07 (s, 3H), 1.67 (t, J = 6.7 Hz, 2H), 1.24 (s,
6H). 13C NMR d: 149.8, 148.9, 148.4, 147.7, 132.2, 128.4, 125.5,
124.3, 120.1, 117.3, 111.6, 111.3, 73.0, 60.7, 55.9, 55.8, 46.7,
32.9, 32.6, 27.4, 26.7, 24.7, 20.2, 12.7, 12.0 MS m/z: 481.4
[M+H]+. HRMS: calcd for C27H37N4O4 [M+H]+ 481.2815, found
481.2809.
5.1.25. 5-(3,4-Dihydroxyphenyl)-3-(3,4-dihydro-6-hydroxy-
2,2,7,8-tetramethyl-2Y-1-benzopyran-5-yl)isoxazole (32)
Compound 31 (32 mg, 0.08 mmol), was treated as described for
24. Purification by flash chromatography (CH2Cl2/MeOH, 95/5)

M. Koufaki et al. / Bioorg. Med. Chem. 18 (2010) 3898–3909
3909
5.1.30. 1-(3,4-Dihydro-6-hydroxy-2,2,7,8-tetramethyl-2Y-1-
Acknowledgments
benzopyran-5-yl-methyl)-5-(3,4-dihydroxy-2-phenylethyl)
tetrazole (39)
This work was supported in part by the EST Marie Curie pro-
gram EURODESY, Contr. No. MEST-CT-2005-020575 (2006–2010).
Tetrazole analogue 37 (34 mg, 0.07 mmol) was treated as de-
scribed for 24. Purification by flash chromatography (pet. ether/
AcOEt, 50/50). Yield: 18 mg (60%), white gummy solid. 1H NMR
(CDCl3) d: 6.73 (d, J = 7.8 Hz, 1H), 6.55 (s, 1H), 6.45 (d, J = 7.8 Hz,
1H), 5.26 (s, 2H), 3.19–3.15 (m, 2H), 2.85–2.81 (m, 2H), 2.74–
2.70 (m, 1H), 2.09 (s, 6H), 1.78–1.74 (m, 3H), 1.25 (s, 6H). HRMS:
calcd for C23H28N4O4 [M]+ 424.2111, found 424.2136.
References and notes
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or modifications. Briefly, HT22 cells were plated in a 96-well flat
bottom 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 hybrids in fresh medium for 24 h prior to assessing the
relative numbers of living cells using MTT [3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide]. MTT conversion to
coloured formazan 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 deprived of HT22 cells. Interference of the hybrids
with mitochondrial conversion of MTT to formazan was excluded
using the trypan blue exclusion assay to directly determine the
number living cells. No challenged cells served to test cytotoxicity
at different hybrid concentrations, whereas challenged cells served
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(CD) in the absence of hybrids was calculated by CDVehicle
*
) 100/dODVehicle, whereas cell death in
=
[(dODVehicle ꢂ dODGlutamate
their presence was calculated by CDCompound = [(dODCompound
ꢂ
*
dODCompound+Glutamate
)
100/dODCompound
.
Neuroprotection (%)
25. Park, J. H.; Mangal, D.; Tacka, K. A.; Quinn, A. M.; Harvey, R. G.; Blair, I. A.;
Penning, T. M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6846.
was calculated by [(CDVehicle ꢂ CDCompound) * 100/CDVehicle
.