Synthesis of tropolone derivatives and evaluation of their in vitro neuroprotective activity

Article abstract of DOI:10.1016/j.ejmech.2009.12.006

β-Thujaplicin (hinokitiol or 2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one), a natural tropolone, shows numerous activities while its synthetic analogues were found to exhibit anticancer and anti-ischemic activity. However, the ability of tropolone derivatives to protect neuronal cells from oxidative stress-induced cell damage has not been studied so far. As an ongoing effort toward highly effective antioxidants with potential neuroprotective activity, we have synthesized 7-substituted derivatives of β-thujaplicin and its methoxy analogue. The substituents were heterocycles (piperazine, morpholine) or heteroaromatics (triazoles, pyridine). Only the piperazine derivatives of β-thujaplicin were able to protect HT22 hippocampal neurons from oxidative stress-induced cell death.

Full text of DOI:10.1016/j.ejmech.2009.12.006

European Journal of Medicinal Chemistry 45 (2010) 1107–1112
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of tropolone derivatives and evaluation of their in vitro
neuroprotective activity
,
a
b
b
b
Maria Koufaki a , Elissavet Theodorou , Xanthippi Alexi , Faidra Nikoloudaki , Michael N. Alexis
*
a Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., 116 35 Athens, Greece
b Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., 116 35 Athens, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
b
-Thujaplicin (hinokitiol or 2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one), a natural tropolone,
Received 9 April 2009
Received in revised form
30 July 2009
Accepted 3 December 2009
Available online 31 December 2009
shows numerous activities while its synthetic analogues were found to exhibit anticancer and anti-
ischemic activity. However, the ability of tropolone derivatives to protect neuronal cells from oxidative
stress-induced cell damage has not been studied so far. As an ongoing effort toward highly effective
antioxidants with potential neuroprotective activity, we have synthesized 7-substituted derivatives of
b
-thujaplicin and its methoxy analogue. The substituents were heterocycles (piperazine, morpholine) or
heteroaromatics (triazoles, pyridine). Only the piperazine derivatives of -thujaplicin were able to
protect HT22 hippocampal neurons from oxidative stress-induced cell death.
Ó 2010 Elsevier Masson SAS. All rights reserved.
b
Keywords:
Tropolone
Oxidative stress
Neuronal cell damage
1. Introduction
piperazine and benzothiazoline
found to exhibit anti-ischemic activity, while
b
-thujaplicin analogues were
-thujaplicin
b
Tropolones represent a group of compounds the chemical
structure of which suggests that they may display good antioxidant
as well as metal chelating properties [1]. Natural tropolones (Fig. 1)
are mainly of plant origin and have been proposed to be responsible
for the resistance to fungal decay and insect attack on the heart-
analogues bearing 2-pyrimidyl or thiazolyl-methyl-piperazine
moieties were active against urinary frequency and incontinence
[9–11].
Although tropolone derivatives possess antioxidant and iron-
chelating properties, their ability to protect neuronal cells from
oxidative stress-mediated damage has not been studied so far.
The central nervous system is particularly vulnerable to damage
by ROS, due to several factors, including low levels of the natural
antioxidant glutathione (GSH) in neurons, membranes rich in
polyunsaturated fatty acids, the presence of excitatory amino acids
such as glutamate and increased requirement for oxygen because of
the high metabolic activities of the brain [12,13]. In addition, there
is evidence that increased concentrations of iron in the brain
contribute to neurodegenerative processes. High concentrations of
reactive iron can increase oxidative stress and induce neuronal
vulnerability [14,15].
Recently we have shown that the presence of an antioxidant-
iron-chelating moiety such as catechol, significantly increased the
activity of chroman or 1,2-dithiolane derivatives against glutamate
toxicity [16,17].
Based on the above data tropolone represents an ideal chemical
entity for the development of antioxidants with potential neuro-
protective activity. The interesting biological activities of the
wood of several cupressaceous trees. b-Thujaplicin (2-hydroxy-4-
isopropyl-2,4,6-cycloheptatrien-1-one or hinokitiol), one of the
many tropolones purified from the woods of Chamaecyparis and
Thujopsis (hinoki and hiba), shows numerous activities, including
antimicrobial, antifungal, antitumor, anti-ischemic and potent iron-
chelating activities [2].
In addition, the natural tropolone analogues,
manicol and goupiolone display antiviral [3] and anticancer activ-
ities [4]. -Thujaplicin and purpurogallin were found to be able to
b-thujaplicinol,
b
protect cells from H2O2-induced DNA damage, while methoxy-
tropone derivatives, such as the known potent anti-mitotic agent
colchicine [5] and the purpurogallin trimethyl ether, did not show
any protective effects [6].
Synthetic mono- and bistropolones, synthesized from b-thuja-
plicin [7] possess antitumor activity. Morpholinomethyl-analogues
of tropolone and 3,7-dibromo-tropolone showed potent inhibitory
effect in the hepatitis C virus helicase assay [8]. Moreover, synthetic
natural
prompted us to synthesize new analogues and to evaluate their in
vitro neuroprotective activity. -Thujaplicin was chosen as starting
b-thujaplicin and its 7-substituted synthetic derivatives
* Corresponding author. Fax: þ30 210 7273831.
E-mail address: mkoufa@eie.gr (M. Koufaki).
b
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.006

1108
M. Koufaki et al. / European Journal of Medicinal Chemistry 45 (2010) 1107–1112
O
H3CO
OH
O
OH
O
O
HO
HO
OH
OH
H3CO
H3CO
N
H
O
Purpurogallin
Tropolone
β-Thujaplicin
H3CO
Colchicine
OH
O
O
HO
OH
OH
O
O
HO
OH
HO
H3CH2CO
thujaplicinol
goupiolone
manicol
Fig. 1. Natural tropolone analogues.
compound for the synthesis of derivatives bearing, morpholine,
piperazine, pyridine and triazole as substituents.
were prepared (Scheme 2) and their activity to protect HT22 cells
from oxytosis was evaluated. Using equimolar amounts of tropo-
lone and morpholine, compound 7 was obtained instead of 3,5,
7-trimopholinmethyl tropolone described in the literature [8].
Mitsunobu reaction using hydroxymethyl analogue 10 and
2-hydroxypyridine or 2-hydroxy-5-methylpyridine afforded
compounds 11, 12 (Scheme 3). Attempts to deprotect the methox-
ytropone derivatives using 10% NaOH/MeOH, 10% KOH/MeOH, 10%
HCl/THF or BF3.SMe2/CH2Cl2 did not result to the desired tropolone
analogues. For the synthesis of triazole derivatives (Scheme 4),
hydroxymethyl analogue 10 was converted to azide 13 using DPPA
and DBU and then, by ‘‘click’’ cycloaddition with the appropriate
alkyne, to triazoles 14 and 15 [22,23].
The in vitro neuroprotective activity of the new compounds was
studied using HT22 cells, a neuronal cell line deriving from mouse
hippocampus. While these cells lack glutamate receptors, their
exposure to high concentrations of glutamate is known to block
cystine uptake, leading to depletion of intracellular cysteine and
loss of GSH [18]. This loss is followed by an early increase in ROS
and a subsequent massive production of ROS by mitochondria as
a consequence, eventually causing oxytosis, a type of oxidative
stress-induced programmed cell death that is distinct from
apoptosis. Glutamate-induced cell death of HT22 cells is thought to
faithfully mimic cytotoxicity due to oxidative stress as implicated in
Alzheimer’s disease and other neurodegenerative disorders [19,20].
3. Results and discussion
2. Chemistry
HT22 cells suffer cell death from oxidative stress within 24 h
following exposure to 1–5 mM glutamate. These cells lack iono-
tropic glutamate receptors (that could mediate excitotoxicity) and
are known to succumb to acute oxidative stress due to glutamate
inhibition of cystine uptake and the ensuing depletion of intracel-
lular GSH [18]. The new compounds were tested at concentrations
0.01–10 mM. Fig. 2 shows a representative assessment of the effi-
cacy and potency of the tropolone derivatives to protect glutamate-
challenged HT22 cells. Piperazine analogues 1–6 were more active
Piperazine analogues 1–5 were prepared according to proce-
dures described in the literature [11]. Specifically, Mannich reaction
using
b-thujaplicin, formaldehyde and monosubstituted pipera-
zines afforded compounds 1–5 (Scheme 1). Compound 2 is repor-
ted in the patent literature [10]. Monosubstituted piperazines were
prepared using the appropriate halide and an excess of piperazine,
while the synthesis of chroman analogues was reported in previous
publications of our group [21]. Compound 6 was prepared using an
excess of
b
-thujaplicin. Since morpholine analogues have previ-
than the parent compound b-thujaplicin in protecting HT22 cells,
ously shown promising biological activity [8], compounds 7 and 8
with EC50 values ranging from 0.08 to 1.7 mM. Since vitamin E has
N
N
O
O
N
N
O2N
N
N
O
OH
R
1
3
2
O2N
R:
O
HO
OH
HO
a
N
O
C
O
N
N
N
O
N
1-5
H
O
4
5
HO
b
O
HO
O
N
N
6
Scheme 1. Reagents and conditions: a) CH3COOH, MeOH, HCHO 37%, RH, 60 ꢀC, 2 h; b) CH3COOH, MeOH, HCHO 37%, piperazine, 60 ꢀC, 3 h.

M. Koufaki et al. / European Journal of Medicinal Chemistry 45 (2010) 1107–1112
1109
4. Experimental section
O
O
OH
OH
N
a
O
4.1. Chemistry
7 R1 = H
8 R1 = isopropyl
R1
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
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 MS facility of
the University of Notre Dame, IN, USA.
R1
Scheme 2. Reagents and conditions: a) CH3COOH, MeOH, HCHO 37%, morpholine,
60 ꢀC, 2 h.
been reported to protect HT22 cells from oxytosis under similar
conditions with an EC50 of 0.2 mM [24], 4 and 5 behave by
comparison as potent antioxidants with higher neuroprotective
activity in vitro.
The nature of piperazine substituent evidently influences the
activity. Thus, compounds 4 and 5 bearing the antioxidant chroman
moiety were active at nanomolar concentrations. Compounds 4 and
5 were 20 and 80-fold more active than the chroman/caffeic acid
counterparts previously reported by our group [16]. Moreover
4.1.1. General procedure for the synthesis of piperazine derivatives
1–5
To a solution of
b
-thujaplicin (0.61 mmol) in methanol (C ¼ 3.5 M),
wereaddedtheappropriatemonosubstitutedpiperazine(0.63 mmol)
and acetic acid (0.63 mmol). The mixture was stirred at 60 ꢀC, form-
aldehyde 37% (0.63 mmol) was added and stirring was continued at
60 ꢀC for 2 h. Upon completion of the reaction the mixture was
neutralized by adding sat. aqueous NaHCO3 and extracted with
CH2Cl2. The organic layer was dried and the solvent evaporated.
chroman/
plicin derivative 6 (EC50 ¼ 0.92 ꢁ 0.13
b
-thujaplicin hybrid 4 is more active than the bis-thuja-
M).
m
Compound 3 was almost as potent as 6 and somewhat more
potent than 1 and 2. It seems that delocalization of the nitrogen
lone pair of piperazine to the nitrosubstituted aromatic ring in 2
results in lower activity of this compound compared to
compound 3.
4.1.1.1. 4-(6-Hydroxy-4-isopropyl-7-oxo-1,3,5-cycloheptatrienyl-met
hyl)-1-piperazine carboxylic ester (1). Purification by column
chromatography (AcOEt/pet. ether 80:20). Yield: 200 mg (99%),
Morpholine analogues were also active in protecting HT22 cells
from oxytosis, but they were less active than the piperazine
derivatives. The presence of the isopropyl group seems to be
important for the activity of morpholine analogues since tropolone
brown oil. 1H NMR
trop), 6.99 (d, J ¼ 10.4 Hz, 1H, H5 trop), 4.12–4.09 (m, 2H, –CH2–
CH3), 3.69 (s, 2H, –CH2–), 3.49 (m, 4H, piper), 2.89–2.83 (m, 1H,
–CH– trop), 2.48 (m, 4H, piper), 1.26–1.24 (m, 9H, 3CH3–). 13C NMR
d
: 7.76 (d, J ¼ 10.4 Hz,1H, H6 trop), 7.31 (s,1H, H3
analogue 7, was not active while
protective with EC50 ¼ 2.56 ꢁ 0.21
b
-thujaplicin analogue 8 was
mM. Table 1 summarizes data
from several independent assessments and ranks the tropolone
d
: 171.5, 167.9, 158.9, 155.5, 138.2, 134.2, 126.3, 120.9, 61.4, 58.9,
53.0, 43.7, 38.6, 23.5, 14.7. MS m/z: 335 [M þ H]þ.
derivatives potency-wise relative to b-thujaplicin and tropolone.
The rank order of relative potencies is 4 ꢂ 5 > 6 ꢂ 3 >1 ꢂ 2 ꢂ 8 >
b
-thujaplicin > tropolone.
4.1.1.2. 1-(4-Isopropyl-6-hydroxy-7-oxo-1,3,5-cycloheptatrienyl-met
hyl)-4-(4-nitrophenyl) piperazine (2). HRMS: Calcd for C21H26 N3O4
[M þ H]þ 384.1923, found 384.1922.
Although biologically active natural and synthetic methoxy-
tropone derivatives have been reported in the literature, our
methoxytropone derivative 11 did not show any activity against
oxytosis at the concentrations tested and 14 was only weakly
effective in the same assay.
Thus, the presence of tropolone scaffold results in compounds
with high potency against oxidative stress-induced cell death of
4.1.1.3. 4-(6-Hydroxy-4-isopropyl-7-oxo-1,3,5-cycloheptatrienyl-met
hyl)-1-(4-nitrobenzyl) piperazine (3). Purification by column chro-
matography (AcOEt/pet. ether 80:20). Yield: 17 mg (14%), yellow
viscous oil. 1H NMR
d
: 8.17 (d, J ¼ 8.5 Hz, 2H, ArH), 7.51 (d,
HT22 cells. Whether the mechanism of action of
b-thujaplicin-
J ¼ 8.5 Hz, 2H, ArH), 7.33 (s, 1H, H3), 6.99 (d, J ¼ 9.8 Hz, 2H), 3.79 (s,
related compounds involves metal chelation between the carbonyl
and the hydroxyl groups in their tropolone skeletons is presently
a matter of conjecture.
2H, –CH2–), 3.62 (s, 2H, –CH2–), 2.89–2.87 (m, 1H, –CH–), 2.66–2.57
(m, 8H), 1.28 (s, 3H, CH3–), 1.25 (s, 3H, CH3–). 13C NMR
d: 171.5,
168.0, 158.8, 147.2, 146.2, 138.6, 134.3, 129.5, 126.2, 123.5, 123.2,
121.0, 62.1, 58.9, 53.1, 38.6, 29.7, 29.3, 23.4, 14.1. HRMS: Calcd for
C22H28 N3O4 [M þ H]þ 398.2080, found 398.2073.
In conclusion, we synthesized 7-substituted
b-thujaplicin
analogues and tested their activity against oxidative stress-induced
cell death of HT22 cells. All tropolone derivatives were more active
than the parent compound
combining two biologically active moieties, tropolone and 6-
hydroxy-chroman, were active at concentrations below 100 nM.
b-thujaplicin. Compounds 4 and 5,
4.1.1.4. N-(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopy
ran-2-carbonyl)-4-(6-hydroxy-4-isopropyl-7-oxo-1,3,5-cycloheptatri
en-methyl)-piperazine (4). Yield: 151 mg (99%), orange oil. 1H NMR
R2
N
O
O
O
O
MeO
HO
HO
MeO
OH
OH
O
a
c
b
10
9
11 R2 = H
12
R2 =CH3
Scheme 3. Reagents and conditions: a) KOH 25%, HCHO 37%, 60 ꢀC, 24 h; b) (MeO)2SO2, K2CO3, acetone, reflux, 2 h; c) DIAD, Ph3P, THF, 2-hydroxypyridine or 2-hydroxy-6-
methylpyridine, rt, 24 h.

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M. Koufaki et al. / European Journal of Medicinal Chemistry 45 (2010) 1107–1112
O
MeO
N
O
O
N
MeO
MeO
N
R
OH
N3
a
b
R
n
14 R=H n =1
15 R=OMe n = 0
13
10
Scheme 4. Reagents and conditions: a) (PhO)2PON3, DBU, THF, 0 ꢀC for 2 h and rt for 24 h; b) propyn-2-ylbenzene or 4-ethynyl-1,2-dimethoxybenzene, CuSO4$5H2O, sodium-
L(þ)ascorbate, t-BuOH/H2O, rt, 24 h.
d
: 7.75 (d, J ¼ 10.3 Hz, 1H), 7.28–7.18 (m, 1H), 6.98 (d, J ¼ 10.3 Hz,
(0.04 mL, 0.671 mmol). The mixture was stirred at 60 ꢀC for 3 h
and worked up as described in the general procedure. Yield:
1H), 4.06 (bs, 1H), 3.70–3.39 (m, 6H), 2.91–2.69 (m, 3H) 2.56–2.50
(m, 5H), 2.15 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 1.75–1.65 (m, 1H), 1.57
267 mg (100%), yellow solid. mp: 198–200 ꢀC. 1H NMR
d: 7.79 (d,
(s, 3H), 1.27 (s, 3H), 1.25 (s, 3H). 13C NMR
d
: 171.6, 167.9, 160.1, 145.5,
J ¼ 11.0 Hz, 2H), 7.33 (s, 1H), 7.32 (s, 1H), 6.99 (d, J ¼ 11.0 Hz, 2H),
144.4, 138.8, 137.3, 126.4, 123.4, 122.4, 121.6, 121.0, 117.9, 78.9, 58.7,
53.4, 52.9, 46.0, 42.9, 38.9, 38.6, 31.4, 25.2, 23.5, 21.1, 12.3, 12.1, 11.3.
MS m/z: 495 [M þ H]þ. HRMS: Calcd for C29H39N2O5
[M þ H]þ495.2859, found 495.2862.
3.72 (s, 4H), 2.97–2.85 (m, 2H), 2.77–2.61 (bs, 8H), 1.28 (s, 6H),
1.25 (s, 6H). 13C NMR
d: 171.4, 167.9, 158.7, 138.4, 134.7, 126.4,
126.3, 121.0, 58.9, 53.4, 53.3, 51.6, 38.6, 23.5. MS m/z: 439
[M þ H]þ. HRMS: Calcd for C26H35N2O4 [M þ H]þ 439.2597, found
439.2598.
4.1.1.5. N-(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopy
ran-2-carbonyl)4-(6-hydroxy-4-isopropyl-7-oxo-1,3,5-cycloheptatrie
nyl-methyl)-1-piperazine-ethylamine (5). Purification by column
chromatography (CH2Cl2/MeOH 90:10). Yield: 60 mg (71%), yellow
4.1.2. 2-hydroxy-7-(morpholin-methyl)-cyclohepta-2,4,6-trienone (7)
Yield: 33%. HRMS calcd for C12H16NO3 [M þ H]þ 222.1130, found
222.1130.
oil. 1H NMR
d
: 7.75 (d, J ¼ 10.6 Hz, 1H), 6.99–6.96 (m, 2H), 3.61 (s,
2H), 3.26–3.22 (m, 2H), 2.90–2.89 (m, 1H), 2.62–2.21 (m, 13H),
2.19 (s, 6H), 2.07 (s, 3H), 1.81–1.75 (m, 1H), 1.51 (s, 3H), 1.27 (s, 3H),
4.1.3. 4-(4-Isopropyl-6-hydroxy-7-oxo-1,3,5-cycloheptatrienyl-
methyl)morpholine (8)
1.24 (s, 3H). 13C NMR
d
: 174.5, 172.1, 167.5, 158.9, 144.5, 139.1, 137.3,
Yield: 88%. HRMS calcd for C15H22NO3 [M þ H]þ 264.1600, found
264.1605.
135.3, 127.6, 126.4, 122.2, 120.7, 119.8, 117.7, 78.4, 58.8, 55.8, 52.8,
52.5, 38.9, 38.5, 35.7, 29.7, 29.6, 25.0, 23.5, 23.4, 20.6, 12.6, 12.3,
11.5. HRMS: Calcd for C31H44N3O5 [M þ H]þ 538.3281, found
538.3289.
4.1.4. 4-Isopropyl-2-methoxy-7-(pyridin-2-yloxy)methyl-2,4,6-
cycloheptatrien-1-one (11)
To
a solution of 10 (50 mg, 0.31 mmol) in 0.1 mL THF
4.1.1.6. 1,4-Bis-(6-Hydroxy-4-isopropyl-7-oxo-1,3,5-cycloheptatrien
(C ¼ 3.016 M), were added 2-hydroxypyridine (36 mg, 0.37 mmol),
Ph3P (98 mg, 0.37 mmol) and DIAD (0.07 mL, 0.37 mmol). The
mixture was stirred at ambient temperature for 24 h and after
completion of the reaction, AcOEt was added and the organic layer
was washed with sat. aqueous NaCl, dried over Na2SO4 and the
solvent evaporated. The residue was purified by column chroma-
tography (AcOEt/pet. Ether 90:10). Yield: 16 mg (18%), yellow
yl-methyl) piperazine (6). To a solution of
b-thujaplicin (200 mg,
1.22 mmol) in 2.5 mL MeOH, were added piperazine (63 mg,
0.732 mmol), acetic acid (0.3 mL) and formaldehyde 37%
viscous oil. 1H NMR
3H), 6.71 (s, 1H), 5.48 (s, 2H), 3.95 (s, 3H), 2.89–2.82 (m, 1H), 1.26 (s,
3H), 1.24 (s, 3H). 13C NMR
: 178.3, 163.3, 163.0, 153.3, 147.1, 141.9,
d: 8.14 (bs,1H), 7.61–7.52 (m, 2H), 6.88–6.79 (m,
d
138.8, 134.1, 123.9, 117.1, 113.9, 110.9, 65.7, 56.1, 38.8, 23.5, 22.0. MS
m/z: 286.5 [M þ H]þ. HRMS: Calcd for C17H20NO3 [M þ H]þ
286.1443, found 286.1445.
4.1.5. 4-Isopropyl-2-methoxy-7-(pyridine-2-yloxy)methyl-2,4,6-
cycloheptatrien-1-one (12)
Prepared according to the method described for 11. Purification
by column chromatography (AcOEt/pet.ether 80:20). Yield: 30 mg
(26%), yellow viscous oil. 1H NMR
d
: 7.60 (d, J ¼ 9.5 Hz, 1H), 7.46
(t, J ¼ 7.7 Hz, 1H), 6.82 (d, J ¼ 9.5 Hz, 1H), 6.72 (bs, 2H), 6.62
(d, J ¼ 8.2 Hz, 1H), 5.45 (s, 2H), 3.95 (s, 3H), 2.89–2.81 (m, 1H), 2.39
(s, 3H), 1.26 (s, 3H), 1.25 (s, 3H). 13C NMR
d: 178.3, 162.9, 162.8, 156.6,
153.1, 142.2, 138.9, 134.1, 124.0, 116.2, 113.9, 106.8, 65.3, 56.1, 38.7,
23.5, 22.0. MS m/z: 300 [M þ H]þ.
Fig. 2. Comparative performance of tropolone analogues in protecting HT22 cells from
oxidative stress-induced cell death: HT22 cells were exposed to 5 mM glutamate in the
absence or presence of increasing concentrations of tropolone, b-thujaplicin or the
synthesized tropolone derivatives for 24 h and changes in cell viability were assessed
using MTT conversion to colored formazan to measure the relative number of living
cells. Values are mean ꢁ SEM of triplicate measurements with an intra-assay variation
similar to that shown for 5.
4.1.6. 7-Azidomethyl-4-isopropyl-2-methoxy-2,4,6-cycloheptat
rien-1-one (13)
To
a solution of 10 (200 mg, 0.96 mmol) in 1 mL THF
(C ¼ 0.96 M), were added at 0 ꢀC DPPA (0.23 mL, 1.06 mmol) and
DBU (0.14 mL, 0.96 mmol) and the mixture was stirred at 0 ꢀC for

M. Koufaki et al. / European Journal of Medicinal Chemistry 45 (2010) 1107–1112
1111
Table 1
128.8, 123.9, 120.9, 118.1, 114.3, 111.2, 108.7, 68.1, 55.9, 53.6, 38.7,
30.3, 28.9, 23.4. MS m/z: 396 [M þ H]þ.
Efficacy and potency of the tropolone analogues to protect glutamate-challenged
HT22 cells from oxidative stress-induced cell death.
Compound
EC50
(mean ꢁ SEM)
(
m
M)a
Relative
Efficacy (%)c
4.2. Biology
potencyb
1
2
3
4
5
6
7
8
1.40 ꢁ 0.35
1.70 ꢁ 0.33
0.96 ꢁ 0.21
0.08 ꢁ 0.02
0.09 ꢁ 0.02
0.92 ꢁ 0.13
>10
3.4
2.8
5.0
60
53
5.2
<0.5
1.9
<0.5
<0.5
<0.5
1
88% (full)
91% (full)
100% (full)
62% (partial)
89% (full)
80% (full)
ns
87% (full)
ns
30% (weak)
ns
4.2.1. Evaluation of the activity of tropolone derivatives to protect
glutamate-challenged HT22 cells from oxidative stress-induced
cell death
Evaluation of the in vitro neuroprotective activity of the tropo-
lone derivatives was carried out as previously described [16], with
minor modifications. Briefly, HT22 cells were plated in a 96-well flat
2.56 ꢁ 0.21
bottom plate at a density of 4000 cells per well in 100 ml of DMEM-
11
14
tropolone
>10
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 test
compounds in fresh medium for an additional 24 h prior to assess-
ing MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] conversion to formazan as a means to measure relative
numbers of living cells. Cells that were not challenged with gluta-
mate served to test compound cytotoxicity at the different
concentrations tested, whereas cells exposed to glutamate in the
presence of test compounds served to assess invitro neuroprotective
activity by comparison. Cells exposed only to vehicle (DMSO) or
glutamate served as controls. Conversion to formazan was assessed
by the difference in optical density (dOD) at 550 and 670 nm. Cell
death (CD) in the absence of test compound was calculated by
>10
>10
4.80 ꢁ 0.42
b
-thujaplicin
62% (partial)
ns ¼ non-significant.
a
EC50 values are test compound concentrations that are able to maintain the
viability of glutamate-exposed cells to a level equal to 50% of that of non-exposed
cells. Values are mean ꢁ SEM of at least three independent experiments similar to
that shown in Fig. 1.
b
Relative (to b-thujaplicin) potencies were calculated by [EC50 -thujaplicin/EC50
b
compound].
c
Compounds exhibiting statistically significant effects in protecting cell viability
at 10 M were classified as exhibiting full, partial or weak in vitro neuroprotective
m
efficacy depending on whether their % neuroprotective effect was respectively,
67–100, 34–66 and ꢄ33%. Values are the mean of at least three independent
experiments similar to that shown in Fig. 1.
CDVehicle ¼ [(dODVehicle ꢃ dODGlutamate
in the presence of test compound was calculated by CDCom-
¼ [(dODCompound ꢃ dODCompoundþGlutamate
* 100/dODCompound].
* 100/
)
* 100/dODVehicle]. Cell death
2 h and at ambient temperature for 24 h. After completion of the
reaction, AcOEt was added and the organic layer was washed
with sat. aqueous NaCl, dried over Na2SO4 and the solvent
evaporated. The residue was purified by column chromatography
(AcOEt/pet. Ether 80:20). Yield: 44 mg (22%), yellow oil. 1H NMR
: 7.48 (d, J ¼ 9.1 Hz, 1H), 6.86 (d, J ¼ 9.1 Hz, 1H), 6.77 (s, 1H), 4.52
(s, 2H), 4.01 (s, 3H), 2.96–2.92 (m, 1H), 1.36 (s, 3H), 1.34 (s, 3H).
13C NMR
: 178.6, 163.9, 154.9, 140.5, 136.4, 123.9, 114.3, 56.5,
)
pound
% Neuroprotection was calculated by [(CDVehicle ꢃ CDCompound
)
CDVehicle]. Direct interference of the test compounds with MTT
conversion to formazan was excluded using mock cultures deprived
of HT22 cells. Interference of the tropolone analogues with mito-
chondrial conversion of MTT to formazan under normal growth
conditions was excluded using the trypan blue exclusion assay to
directly determine the number of living cells.
d
d
54.5, 39.2, 23.8.
Acknowledgments
4.1.7. 7-[(4-Benzyl-1H-1,2,3-triazol-1-yl)methyl]-4-isopropyl-2-
methoxy-2,4,6-cycloheptatrien-1-one (14)
To a solution of prop-2-ynylbenzene (0.021 mL, 0.17 mmol) in
a mixture of t-BuOH/H2O (4/1, 0.71 mL), were added azide 13
This work is supported in part by the Center for Drug Discovery,
Northeastern University, Boston, USA and by ‘‘ EURODESY’’ MEST-
CT2005-020575.
(40 mg, 0.17 mmol), sodium L-ascorbate (4.0 mg, 0.017 mmol) and
CuSO4 (1 mg, 0.0017 mmol) and the mixture was stirred at ambient
temperature for 24 h. After cooling at 0 ꢀC, AcOEt and H2O were
added and the organic layer was washed with ammonia, sat.
aqueous NaCl, dried over Na2SO4 and the solvent evaporated. The
residue was purified by column chromatography (AcOEt/pet.ether
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