Synthesis and effects of some novel tetrahydronaphthalene derivatives on proliferation and nitric oxide production in lipopolysaccharide activated Raw 264.7 macrophages

Article abstract of DOI:10.1016/j.ejmech.2010.11.021

In this study, novel N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalene-2-yl)-carboxamide (6-15) and 5,5,8,8-tetramethyl-5,6,7,8- tetrahydronaphthalene-2-carboxamide (16-32) derivatives were synthesized and their in vitro effects at 5 μM and 50 μM concentrations on proliferation and nitric oxide (NO) production in lipopolysaccharide (LPS) activated RAW 264.7 macrophage cells were determined. Compounds 12, 17, 24 and 26 were found to decrease nitrite levels in a dose-dependent manner in LPS-activated cells. At the tested concentrations, these compounds did not exhibit cytotoxic effects. Interestingly, compound 27 which contains nitroxide free radical was the most active compound in this series showing 59.2% nitrite inhibition in LPS-activated macrophage cells.

Full text of DOI:10.1016/j.ejmech.2010.11.021

European Journal of Medicinal Chemistry 46 (2011) 468e479
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and effects of some novel tetrahydronaphthalene derivatives on
proliferation and nitric oxide production in lipopolysaccharide activated
Raw 264.7 macrophages
,
b
b
a
A. Selen Gurkan a , Arzu Z. Karabay , Zeliha Buyukbingol , Erdem Buyukbingol
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey
b Department of Biochemistry, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, novel N-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalene-2-yl)-carboxamide (6e15)
and 5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carboxamide (16e32) derivatives were
Received 27 August 2010
Received in revised form
8 November 2010
Accepted 15 November 2010
Available online 26 November 2010
synthesized and their in vitro effects at 5 mM and 50 mM concentrations on proliferation and nitric oxide
(NOꢀ) production in lipopolysaccharide (LPS) activated RAW 264.7 macrophage cells were determined.
Compounds 12, 17, 24 and 26 were found to decrease nitrite levels in a dose-dependent manner in
LPS-activated cells. At the tested concentrations, these compounds did not exhibit cytotoxic effects.
Interestingly, compound 27 which contains nitroxide free radical was the most active compound in this
series showing 59.2% nitrite inhibition in LPS-activated macrophage cells.
Keywords:
Nitric oxide
RAW 264.7 macrophage cells
Retinoids
Ó 2010 Elsevier Masson SAS. All rights reserved.
Tempamine
1. Introduction
[1]. The use of retinoids in many biological models of carcinogen-
esis has suggested that their action may be related to their anti-
Free radicals and oxidative stress lead to uncontrolled reactions
resulting in the oxidative damage of biological macromolecules
including lipids, proteins and DNA. Protection of these molecules
from oxidative stress is very important in preventing the progres-
sion of a number of diseases, such as cancer, diabetes, and
inflammatory diseases. Antioxidant vitamins are very effective in
inhibiting above-mentioned disorders which generally occur via
free radical involvements. Retinoids, synthetic and naturally
occurring derivatives of vitamin A (retinol) and its most active
metabolite all-trans-retinoic acid (ATRA), have been known to
expose diverse pharmacological effects such as mediation of cell
growth and differentiation in both normal and neoplastic cells, and
modulation of programmed cell death known as apoptosis [1].
These properties confer a significant therapeutic potential in the
fields of oncology and dermatology [2,3] as well as in other free
radical-induced diseases. Retinoid analogues have received
considerable attention as agents that may be useful for both
prevention and treatment of some cancers [4] due to their potent
growth inhibiting activities on cancer cell lines in vitro and in vivo
oxidant properties [5]. Retinoids have been shown to scavenge
several free radicals and inhibit microsomal lipid peroxidation
[6,7]. However, beneficial usage of these compounds is restricted
due to their undesirable side effects [8,9]. For optimal results in
disease prevention, which would include reduced side effects and
modulating the effectiveness of their beneficial applications, new
retinoidal compounds should be then required for those aspects
indicated above.
On the other hand, macrophages are the main immune system
cells responsible for the defensive environment of vertebrate
animals, acting against pathogens and other antigenic materials. It
is reported that one of the mechanisms of macrophages involves
the release of various inflammatory molecules including the nitric
oxide (NOꢀ) free radical [10,11]. Overproduction of the inflamma-
tory mediators is involved in many diseases, such as rheumatoid
arthritis, chronic hepatitis, and pulmonary fibrosis [12e14].
Inflammation that is mediated by tissue-resident macrophages is
an essential component of many metabolic, endocrine and
cardiovascular disorders. When activated, macrophages transcrip-
tionally express inducible nitric oxide synthase (iNOS) enzymes
which cause longterm and extensive NOꢀ production [10,11].
Abnormal release of NOꢀ may lead to inflammation and tissue
injury; highly synthesized NOꢀ reacts with DNA and causes cells
* Corresponding author. Tel.: þ90 3122033080; fax: þ90 3122131081.
E-mail address: sgurkan@pharmacy.ankara.edu.tr (A.S. Gurkan).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.021

A.S. Gurkan et al. / European Journal of Medicinal Chemistry 46 (2011) 468e479
469
to undergo apoptosis [15e17]. Therefore, pharmacological inter-
ventions that aim to control potentially harmful pro-inflammatory
activity of macrophages present promising treatment options. RAW
264.7 macrophages undergo apoptosis due to high levels of NOꢀ
production by iNOS activation when treated with lipopolysaccha-
ride (LPS) in a concentration- and time-dependent manner. The use
of iNOS inhibitors and scavengers of NOꢀ produced by these acti-
vated cells blocks cell injury and death [18,19] and brings benefit to
the treatment of inflammatory diseases characterized with high
NOꢀ levels.
In the present study, we aim to reveal more active and less toxic
novel retinoid analogues compared to ATRA. We have prepared
a series of novel retinoid compounds consisting of a tetrahy-
dronaphthalene ring system which is integrated with several
moieties including alpha-lipoic acid, caffeic acid, ferulic acid, trolox
and tempamine residues which have been reported to possess free
radical scavenging activities [20,21]. We investigated the effects of
these retinoid analogues in vitro on proliferation and NOꢀ produc-
tion in LPS-activated RAW 264.7 macrophages with comparison to
known antioxidant compounds.
1,1,4,4,6-pentamethyl-7-aminonaphthalene (5) [24]. The crude
amine was used without purification. Trolox acetate was also
synthesized for the preparation of 14. A mixture of trolox (2 mmol),
pyridine (5.1 ml), and acetic anhydride (4.3 ml) was stirred for 2 h
at room temperature. The mixture was diluted with water (10 ml),
and extracted with diethylether. The combined organic layers were
washed with saturated copper sulfate solution and brine. It was
dried with sodium sulfate and the solvent evaporated in vacuo to
give acetyltrolox, which was used without further purification [25].
The acetylation process was confirmed by mass spectrum, 293
(M þ 1) and 291 (Mꢁ1). The final products N-(3,5,5,8,8-pentam-
ethyl-5,6,7,8-tetrahydronaphthalene-2-yl)-carboxamides (6e15),
and 5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carbox-
amides (16e32) were prepared by the amidification of compound 5
with appropriate acids, and compound 3 with several amines,
respectively, by using different methods and various amidification
agents which are stated in Experimental section.
3. Results and discussion
Regarding the toxic side effects, the usage of natural and
synthetic retinoids is limited. Therefore, new retinoid derivatives
are need to be synthesized that have increased beneficial properties
and reduced adverse effects. Thus, in this study, we aimed to
synthesize novel retinoid compounds which are expected to have
certain antioxidative properties. To achieve this, two groups of novel
twenty-seven retinoid derivatives expected to possess antioxidant
activity with regard to nitric oxide radical inhibition were designed
and synthesized with the substituents at 2nd and/or 3rd positions of
the tetrahydronaphthalene ring. N-(3,5,5,8,8-Pentamethyl-5,6,7,8-
tetrahydronaphthalene-2-yl)-carboxamide derivatives (6e15) were
synthesized in five steps while the other seventeen compounds,
5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carboxamide
derivatives (16e32), were synthesized in four steps (Scheme 1). The
compounds (6e15) which have a methyl group at the 3rd position of
the tetrahydronaphthalene moiety are linked to an amide group
with appropriate substituents at the second position of the tetra-
hydronaphthalene ring. The amide bond formation was established
with the carboxylic acid compounds. Some of these compounds
2. Chemistry
The synthetic procedures for the preparation of the compounds
(16e32) are shown in Scheme 1. Commercially available
2,5-dimethyl-2,5-hexanediol and appropriate carboxylic acid or
amine compounds served as starting materials. 2,5-Dichloro-2,5-
dimethyl hexane (1) was prepared in 55% yield by passing dry
hydrochloride gas over 2,5-dimethyl-2,5-hexanediol, as described
by Boehm et al., 1994 [22]. Toluene was alkylated by 1 in
dichloromethane catalyzed with aluminum chloride to produce
1,2,3,4-tetrahydro-1,1,4,4,6-pentamethylnaphthalene (2), in 68%
yield [22]. Then, 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naph-
thalene carboxylic acid (3) was obtained by oxidation of 2 by
KMnO4 in alkali medium, in 57% yield [23]. A mixture of HNO3
and H2SO4 was added to 2 at ꢁ10 ꢂC to give 1,2,3,4-tetrahydro-
1,1,4,4,6-pentamethyl-7-nitronaphthalene (4) in 35% yield. This
nitro compound was hydrogenated by the usual method (35 psi H2
and 10% Pd/C in EtOH, 61% yield) to produce 1,2,3,4-tetrahydro-
CH3
CH3
OH
OH
Cl
Cl
dry HCl (g)
abs. Et-OH
AlCl3
2
1
HNO3 , H2SO4
KMnO4
COOH
CH3
NO2
CH3
NH2
Pd.C / H2
3
4
5
i, ii
Ar(R)-NH2
i, ii
Ar'(R')-COOH
O
C
O
C
N
H
Ar(R)
N
H
Ar'(R')
(6 - 15)
(16 - 32)
Scheme 1. Synthesis of novel retinoid compounds 6e32. i: Coupling agent; N,N0-CDI (method I), EDCI and HOBT (method II). ii: a) SOCl2, b) pyridine and triethylamine (method III);
a) SOCl2, b) NaHCO3 (method IV); a) oxalyl chloride, b) DMAP and pyridine (method V).

470
A.S. Gurkan et al. / European Journal of Medicinal Chemistry 46 (2011) 468e479
showstrong antioxidant properties, such as alpha-lipoic acid, caffeic
acid, ferulic acid, and trolox [20]. A different substitution pattern
exists in compounds 16e32 in which the second position of the
tetrahydronaphthalene ring bears the substituents instead of the
methyl group as seen in the first set of compounds. One of these
linked amine compounds is a cyclic, stable nitroxide free radical
tempamine which is known to possess antioxidant properties [21].
Thus, the first group of compounds indicate that the NHeCO moiety
binds to tetrahydronaphthalene ring at 3rd position whereas the
second substitution pattern occurred with the reverse amide
formation (COeNH) at the 2nd position of the ring.
Synthesized compounds were purified by column chromatog-
raphy and recrystallization using appropriate solvent systems
stated in Table 1. Expected chemical structures of the compounds
have been proven by mass, IR and NMR spectral findings indicated
in Experimental section and elemental analyses (Table 1).
In order to determine whether compounds 9, 10, 11, and 12 are
trans or cis configured, a standard three-pulse NOESY NMR
sequence [26] was applied in phase sensitive hypercomplex mode
(StateseHaberkorn method) [26] by using a relaxation delay of 1 s,
an acquisition time of 0.16 s, 512 increments in t1 dimension, 1024
data points in t2 dimension, 8 transients to collect FIDs in t2
dimension, and a mixing time of 200 ms. All NOESY FIDs were
acquired over 16 ppm spectral width and processed by Varian NMR
v6.1C. The NOESY FIDs were zero-filled to 2K data points with
a linear prediction function in both dimensions, apodized with
a 90ꢂ sine-square and 6 Hz Gaussian functions in both dimensions
and Fourier transformed in quadrature mode in the t2 dimension
and in hypercomplex mode in the t1 dimension. NOESY spectra
were displayed and analyzed either in phase sensitive or magni-
tude modes.
The proximity of the proton pairs Hi and Hj was estimated from
2D NOESY cross-peak intensities by the NMR ISPA (distance
constraints via isolated spin pair approximation) method [27]
which is expressed as follows,
rij ¼ rref (aref/aij)1/6
(1)
in which rij is the distance between Hi and Hj to be estimated; aij
is the cross-peak intensity for Hij, rref and aref are a known distance
and a cross-peak intensity for a proton pair, respectively. The rref
was chosen to be the distance between either of the trans ethylene
ꢀ
protons and one of the phenyl protons, 2.08 and 2.53 A, whose
distances were obtained from the crystal structure of cinnamic acid
in complex with tyrosine ammonia-lyase [28]. Apparently, the
ꢀ
distance between the ethylene protons (rij) exceeds 3 A when the
Table 1
Isolation methods and some physico-chemical properties of compounds 6e32.
O
C
O
C
N Ar(R)
H
N
H
Ar'(R')
(16-32)
(6-15)
No
eR0 (eAr0) eR (eAr)
Isolation
Yield (%)
85
M.p. (ꢂC)
87
Elemental analyses (exp.)
S
S
6
n-Hexane:AcOEt (5:1), (4:1), cc
n-Hexane:AcOEt (3:1), cc
C23H35NOS2: C: 67.93%; H: 8.55%; N: 3.55%; S: 15.70%.
7
8
63
69
145e147
126e128
C25H33NO$0.1H2O: C: 82.12%; H: 9.11%; N: 3.99%.
OCH3
OCH3
n-Hexane:CHCl3 (1:1), cc
C27H37NO3: C: 76.29%; H: 8.77%; N: 3.41%.
9
Petroleum ether:AcOEt (3:1), (1:4), cc
29
27
189e190
C24H29NO: C: 83.00%; H: 8.16%; N: 3.91%.
OH
OH
C
24H29NO3$0.05(CH3)2NCHO$0.75H2O: C: 73.15%; H:
10
n-Hexane:AcOEt (3:1), (1:1), cc
198
7.85%; N: 3.55%.
OCH3
OH
11
n-Hexane:AcOEt (3:1), (1:1), cc
48
214
C25H31NO3: C: 76.11%; H: 8.07%; N: 3.54%.

A.S. Gurkan et al. / European Journal of Medicinal Chemistry 46 (2011) 468e479
471
Table 1 (continued )
No
eR0 (eAr0) eR (eAr)
OCH3
OCH3
Isolation
Yield (%)
37
M.p. (ꢂC)
Elemental analyses (exp.)
12
Petroleum ether:AcOEt (3:1), (1:4), cc
175e179
C26H33NO3: C: 76.24%; H: 8.25%; N: 3.37%.
CH3
CH3
OH
H3C
C
29H39NO3$0.2H2O: C: 76.85%; H: 8.88%;
13
14
15
n-Hexane:AcOEt (5:1), (3:1), cc
n-Hexane:AcOEt (5:1), (3:1), cc
n-hexane:CHCl3 (1:1), cc
5
4
182e183
188e189
150e151
O
CH3
N: 3.14%.
CH3
O
O
C
CH3
H3C
C
31H41NO4$0.33H2O: C: 74.73%; H: 8.56%;
O
CH3
N: 2.93%.
CH3
H3C
H3C
CH3
C
30H41NO$0.5H2O: C: 81.83%; H: 9.60%;
18
N: 3.17%.
CH3
C
25H27NO$1.75H2O: C: 77.28%; H: 7.63%;
16
17
n-Hexane:AcOEt (20:1), cc
n-hexane:AcOEt (3:1), cc
10
32
118
236
N: 3.49%.
N
C
22H25N3O$0.25 (CH3)2NCHO: C: 74.82%;
N
H
H: 7.27%; N: 12.22%.
S
N
C
18H22N2OS$0.1C6H14$0.1H2O: C: 68.56%;
18
19
n-Hexane:AcOEt (3:1), cc
n-Hexane:AcOEt (2:1), cc
48
25
144
187
N
H: 7.43%; N: 8.45%; S: 9.65%.
C
19H23N3O$0.25H2O: C: 72.82%; 7.32%;
N: 13.47%.
N
NH
C
19H23N3O2$0.25(CH3)2NCHO: C: 69.19%;
20
21
n-hexane:AcOEt (3:1), cc
n-hexane:AcOEt (3:1), cc
21
75
320
H: 6.95%; N: 12.96%.
N
O
143e144
C22H27NO: C: 82.24%; H: 8.58%; N: 4.45%.
F
22
23
n-Hexane:AcOEt (3:1), cc
70
38
139
160
C22H25F2NO: C: 73.83%; H: 7.17%; N: 4.06%.
F
Cl
n-hexane:AcOEt (3:1), cc
C22H25Cl2NO: C: 67.40%; H: 6.27%; N: 3.69%.
Cl
(continued on next page)

472
A.S. Gurkan et al. / European Journal of Medicinal Chemistry 46 (2011) 468e479
Table 1 (continued )
No
eR0 (eAr0) eR (eAr)
Isolation
Yield (%)
54
M.p. (ꢂC)
Elemental analyses (exp.)
N
C
27H36N2O$0.2H2O: C: 79.45%; H: 8.85%;
24
n-Hexane:AcOEt (3:1), cc
165e166
N: 7.19%.
O
C
C
23H34N2O3$0.2H2O: C: 70.84%; H: 8.77%;
N
OEt
25
26
n-Hexane:AcOEt (3:1), (1:1), cc
80
46
69e70 bubl.
N: 6.99%.
CH3
H3C
H3C
NH
C
24H38N2O$0.1H2O: C: 77.32%; H: 9.96%;
n-Hexane:AcOEt (20:1), cc
213
N: 7.53%.
CH3
CH3
CH3
N
O
27
n-Hexane:AcOEt (20:1), cc
29
210
C24H37N2O2$: C: 74.33%; H: 9.57%; N: 7.16%.
CH3
CH3
C
20H30N2O$0.5H2O: C: 73.86%; H: 9.32%;
28
29
30
n-Hexane:AcOEt (3:1), cc
n-Hexane:AcOEt (3:1), cc
n-Hexane, recrystallization
32
27
32
199
N
N: 8.59%.
202e203
242e244
C21H32N2O: C: 76.74%; H: 10.01%; N: 8.35%.
N
O
C
19H28N2O2$0.1H2O: C: 71.73%; H: 9.10%;
N
N: 8.89%.
O
C
21H32N2O2$0.25H2O: C: 72.27%; H: 9.39%;
31
32
n-Hexane, recrystallization
n-Hexane:AcOEt (3:1), cc
67
92
142e144
157e158
N
N: 8.03%.
C
18H25NO$0.1H2O: C: 78.91%; H: 8.88%;
N: 5.11%.
given parameters aref, aij, and rref are applied in Eq. (1), proving that
the ethylene protons of the compounds 9, 10, 11, and 12 are trans
configured.
d6 in an NMR tube at 20 ꢂC. The progress of the reduction was
monitored visually by the disappearance of the light pink color of
compound 27, indicating the formation of N-hydroxylamine.
Stable free radical compound 27 was also proven by Electron
Paramagnetic Resonance (EPR) spectra. EPR is a spectroscopic
technique that directly detects chemical species with unpaired
electron(s). Using persistent nitroxide spin probes, it has been
widely utilized to investigate the organizational and dynamic
properties of biomolecules for several decades [32e34]. Direct
evaluations and single domain simulations were performed for the
new TEMPO-labeled retinoid derivative 27 and tempamine (Fig. 1).
Determination of the feature of compound 27 using 1H NMR was
difficult because of the paramagnetic nature of the nitroxide radical.
It is reported that the reduction of similarly behaved nitroxide
radicals by certain agents that transform the paramagnetic behavior
to a diamagnetic feature could allow the more specifically deter-
mined features obtained from 1H NMR spectra [29e31]. Therefore,
the conversion of paramagnetic nitroxide to diamagnetic hydrox-
ylamine was achieved by the reduction of the compound with
sodium dithionide in DMSO-d6, which then yielded 5,5,8,8-tetra-
methyl-N-(1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)-5,6,7,8-
tetrahydronaphthalene-2-carboxamide. A small amount of sodium
dithionite in D2O was added to a solution of compound 27 in DMSO-
The rotational correlation times (s), which is related to the mobility
of the probe, and nitrogen hyperfine splitting constants (aN), which
is related to the polarity of the environment, of these spin probes
were calculated by EPRSIM program [35] shown in Table 2. As

A.S. Gurkan et al. / European Journal of Medicinal Chemistry 46 (2011) 468e479
473
Table 3
The effects of the standards on the nitrite levels in RAW 264.7 macrophage cells.
Compds
% Inhibition of nitrite levels
5
m
M
50 mM
ATRA
57.2 ꢃ 0. 5
25.2 ꢃ 3.8
23.2 ꢃ 1.4
24.8 ꢃ 1.4
33.4 ꢃ 1.9
26.9 ꢃ 5.1
66.0 ꢃ 1.1
46.0 ꢃ 1.2
44.2 ꢃ 0.2
39.1 ꢃ 8.9
39.3 ꢃ 2.5
45.6 ꢃ 1.3
a
-Lipoic acid
Caffeic acid
Ferulic acid
Trolox
Tempamine
dependent manner [37], in this study, the RAW 264.7 macrophage
system was utilized to achieve the inhibition of NOꢀ production.
The synthesized retinoid compounds were tested in vitro on
LPS-activated RAW 264.7 macrophage cells for their nitric oxide
radical inhibition activities. Moreover, the MTT test was used in
order to investigate the effectiveness of the compounds on the
viability of the cells. Consequently, our results showed that ATRA
and the other standard compounds inhibit the nitrite production
in LPS induced macrophages at concentrations of 5 mM and 50 mM
(Table 3) without showing any effect on the cell viability. Biolog-
ical activity findings of the targeted compounds 6e32 are
summarized in Table 4.
Compounds 10, 11, 17, 26, 27, 29, and 31 have been found
to provide 40% inhibition of nitrite dose-dependently, when
compared with the antioxidant reference compounds ATRA, alpha-
lipoic acid, ferulic acid, caffeic acid, trolox and tempamine (Table 4).
However, three compounds (compounds 6, 14 and 16) in Table 4
show less inhibition at 50 mM than at 5 mM. Among these three
compounds, compounds 14 and 16 very slightly affect NOꢀ
production. Since the activity was not potent we could not achieve
a consistent inhibition of NOꢀ production dose-dependently.
Fig. 1. Experimental (black) and simulated (red) EPR spectra of compound 27 and
4-amino-TEMPO (tempamine) in THF. A) 0.1 mM, B) 1 mM (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this
article).
Table 4
The effects of the targeted compounds (6e32) on nitrite levels in RAW 264.7
macrophage cells (Nitrite levels in LPS treated group is represented as 100% and
equal to 15.1 ꢃ0.1
m
M
where nitrite accumulation in untreated group is
2.9 ꢃ 0.036
mM).
a result of these calculations it was shown that there is no signifi-
cant change on nitrogen hyperfine splitting constants of two spin
probes. This shows that these two spin probes give information
approximately from the same environment. The rotational corre-
lation time of the new TEMPO-labeled retinoid derivative 27 is
higher than the rotational correlation time of tempamine, indi-
cating more restricted motion for this new probe.
As mentioned above, the ATRA was used as a reference for
those synthesized compounds to make comparison for better
understanding of the activities of the compounds. According to
our findings, the nitrite inhibition pattern of ATRA is consistent
with the similar results reported from various studies [36,37].
Although the activity of ATRA against NOꢀ production was shown
in different type of macrophage cells (J774A.1) with a dose-
Compds
% Inhibition of nitrite levels
5
mM
50 mM
6
7
8
9
28.5 ꢃ 0.6
22.8 ꢃ 0.9
NI
NI
NIa
NI
NI
NI
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
23.3 ꢃ 2.3
17.8 ꢃ 2.6
24.1 ꢃ 2.6
NI
46.2 ꢃ 1.4
48.0 ꢃ 2.6
32.8 ꢃ 1.9
10.5 ꢃ 1.5
4.4 ꢃ 0.8
NI
15.8 ꢃ 1.7
NI
10.5 ꢃ 2.1
15.6 ꢃ 3.9
NI
5.7 ꢃ 0.5
45.7 ꢃ 0.7
33.5 ꢃ 1.9
18.6 ꢃ 2.6
21.1 ꢃ 1.7
15.9 ꢃ 4.9
7.7 ꢃ 1.3
6.6 ꢃ 1.5
22.5 ꢃ 3.4
30.0 ꢃ 1.1
42.9 ꢃ 8.7
59.2 ꢃ 0.3
10.5 ꢃ 1.0
43.6 ꢃ 1.6
34.3 ꢃ 2.4
40.1 ꢃ 3.7
38.2 ꢃ 0.7
NI
11.5 ꢃ 2.1
NI
Table 2
NI
NI
Rotational correlation time and hyperfine splitting constant parameters of
compound 27 and 4-amino-TEMPO in THF at 0.1 mM and 1 mM concentration
calculated by EPRSIM program.
21.0 ꢃ 3.8
NI
19.1 ꢃ 3.4
18.2 ꢃ 3.4
NI
25.7 ꢃ 3.1
5.8 ꢃ 0.2
14.3 ꢃ 3.3
20.5 ꢃ 6.6
Concentration
Sample
Rotational
correlation
time, sc (ns)
Hyperfine splitting
constant, a (mT)
0.1 mM
1 mM
4-Amino-TEMPO
27
4-Amino-TEMPO
27
0.016
0.047
0.025
0.051
1.543
1.545
1.540
1.545
a
No inhibition.

474
A.S. Gurkan et al. / European Journal of Medicinal Chemistry 46 (2011) 468e479
Compound 6, which bears a powerful antioxidant alpha-lipoic
acid residue, showed decreased nitrite levels in the cells that were
occurred in both concentrations (5 M and 50 M). The inhibitory
effect of this compound was unexpectedly found to be lower at
the 50 M concentration than the one obtained from 5 M (Table
4). Therefore, we could not achieve a dose-dependent response
for this compound. At 5 M concentration, a more potent effect
was achieved, because of the probability that at higher concen-
trations, some antioxidants [38e40] may also act as pro-oxidants
and this may lead to diminishment of their effects in the bio-
logical system.
Nitrite levels in macrophage cells were reduced significantly by
the implementation of compounds 10, 11 and 27, which consist of
residues of known antioxidant structures, i.e. caffeic acid, ferulic
acid, and tempamine. Compounds 6 and 14 are expected to show
higher efficacy in decreasing the macrophage nitric oxide levels due
to their constitution of antioxidant residues (alpha-lipoic acid and
acetyltrolox). The results were surprisingly disappointing as they
exerted slight decreases in the nitrite levels. Moreover, the trolox
m
m
m
m
m
derivative (compound 13) showed no inhibition at the 5
concentration whereas very little inhibition was observed at the
50 M concentration. The finding that such antioxidant-residue-
mM
m
Even though, the consequent toxic effects on the viability of the
cells were elevated with increasing doses of the compounds 7, 8,
and 9 (cinnamic acid derivative), observation on the nitrite inhi-
bition was not obtained from these compounds. The latter has
showed a completely different activity profile compared to those
having the cinnamic acid residue (compounds 10, 11, and 12) and
also showed no inhibition in contrast to compounds 10, 11, and 12
which reduced the level of nitrite. The caffeic acid derivative
(compound 10) was found to possess more likely an identical
inhibition pattern obtained from caffeic acid which is attributed as
a potent antioxidant [41]. Approximately, a 3-fold increase in nitrite
inhibition was observed with the increasing doses of compound 11;
the ferulic acid residue was utilized because of its potency to
address antioxidant [42] and antiinflammatory [43] activities. The
results obtained from compounds 10, 11, and 12 seemed to show no
meaningful indications regarding the number of hydroxy groups
within their molecular structures.
bearing compounds had no effect on the inhibition of nitrite
formation in the macrophage cells is also surprising. Compound 15
(bis-retionid-bearing compound) did not exhibit inhibition; these
results are similar to those obtained from compounds 7, 8, and 9.
These results are thought to be associated with their lipophilic
structures. Compound 15, which contains two tetrahydronaph-
thalene rings, as explained above, showed no nitrite inhibition but
some cell viability decreasing effect. The role of the tetrahy-
dronaphthalene ring system most likely influenced the pattern of
such biological activity, where in the retinoid moiety might be
related to the features of nitrite inhibition and cell viability.
Cyclic stable nitroxide free radical 2,2,6,6-tetramethylpiper-
idine-N-oxyl (TEMPO) and its derivative tempamine possess anti-
cancer and antioxidant properties due to its antitumor activities
and protective effects against oxidative damage [21,44,45]. New
TEMPO-labeled retinoid derivative 27 displayed 18.2% and 59.2%
nitrite inhibition at 5 mM and 50 mM concentrations, respectively
Fig. 2. (A) Effects of compounds 6e32 (5
mean ꢃ S.D. Compounds 10, 11, 12, 17, 24, 26, 27, 29 and 31 are not toxic to cells at 5
from cytotoxicity. (B) Effects of compounds 6e32 (50 M) on cell viability. The bar chart represents absorbance values of control, LPS and compound 6e32 (50
mean ꢃ S.D. Compounds 10, 11, 27 and 29 show cytotoxicity at 50 M concentration which can lead to diminished nitrite values. (*): p < 0.001 vs. LPS treated. **p < 0.01 vs. LPS
treated. ***p < 0.05 vs. LPS treated. (control: no stimulation. LPS: cells stimulated with LPS).
m
M) on cell viability. The bar chart represents MTT assay absorbance values of control, LPS and compounds 6e32 (5
M concentration which means that their inhibitory effect on nitrite production doesn’t stem
M) treated groups as
mM) treated groups as
m
m
m
m

A.S. Gurkan et al. / European Journal of Medicinal Chemistry 46 (2011) 468e479
475
and therefore seemed to be the most active compound in this series
d scale (ppm) from internal standard TMS. Mass spectra were
with 59.2% nitrite inhibition. However it exhibited significant toxic
effect at 50 mM which can be related with this high nitrite inhibition
while tempamine did not show any cytotoxicity to RAW 264.7
macrophages at the concentrations tested.
The amount of nitrite might be related to decreased cell viability
or the strength of compounds on the nitrite levels in the macro-
phage cells. The cinnamic acid derivative, compound 12, and the
benzimidazole containing compound, 17, were found to decrease
nitrite levels significantly in LPS-activated cells (Table 4) and were
effective without showing cytotoxicity to the cells at tested
concentrations (Fig. 2(A) and (B)). Of all of the novel synthesized
compounds, compound 12 showed the greatest increase in cell
recorded on a Waters ZQ micromass LC-MS spectrometer (Waters
Corporation, Milford, MA, USA) by the method of ESI and IR
spectra were recorded on a Jasco FTIR-420 spectrophotometer
(JASCO Research Ltd., Victoria, British Columbia, Canada).
Elemental analyses were performed on LECO CHNS-932 instru-
ment and were within ꢃ0.4% of the theoretical values. 1H NMR,
Mass, FTIR and elemental analyses were performed at The Central
Instrumentation Laboratory of the Pharmacy Faculty of Ankara
University, Ankara, Turkey. The EPR spectra of compound 27 and
tempamine were performed on Bruker EMX-131 X-Band EPR
spectrometer, at Nuclear Magnetic Resonance Laboratory of
Physics Engineering Department, Faculty of Engineering, Hacet-
tepe University, Beytepe, Ankara, Turkey.
viability at a dose of 50 mM. Compounds 24 and 26 were also found
to decrease nitrite levels in a dose-dependent manner in LPS-
activated cells and found to be effective without showing toxic
effects on the viability of the cells. In this system, however,
decreased cell viability was observed with increasing concentra-
tions of compounds 10, 11, 13, 18, 20, 22, 27, 28, 29, 30, 31 and 32.
Therefore, the decline in nitrite levels in the presence of high
concentrations of these retinoid compounds may be associated
with the reduction in cell viability and due to cytotoxicity. Espe-
cially compound 10, which has inhibitory effects on nitrite levels at
5.1.1. General methods for the preparation of the N-(3,5,5,8,8-penta-
methyl-5,6,7,8-tetrahydronaphthalene-2-yl)-carboxamide derivatives
(6e15), and 5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-
carboxamide derivatives (16e32)
5.1.1.1. Method I. N,N0-Carbonyldiimidazole (N,N0-CDI) was used as
an amidification agent. N,N0-CDI (1.3 mmol) was added to a solu-
tion containing compound 3 (or the appropriate acid) (1.2 mmol)
in DMF (or THF) (10 ml) at 0 ꢂC. The mixture was stirred at room
temperature for 3.5 h. An appropriate amine (or the compound 5)
(1.2 mmol) was added to the reaction mixture in an ice-cooled
bath, and then stirred overnight at room temperature. The reac-
tion mixture was evaporated, extracted with ethyl acetate, and
washed with water. The organic layer was dried over Na2SO4, and
the solvent was evaporated in vacuo. The residue was purified by
silica gel column chromatography using the solvents given in
Table 1 [47,48]. Compounds 6, 7, 8, 13, 14, 17, 18, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31 and 32 were prepared according to the
method I.
both concentrations, exerted cytotoxicity at the 50
tion (Fig. 2(A) and (B)).
mM concentra-
4. Conclusion
Our results have shown that some of these novel retinoid
analogues, especially the most active compound 27, and non-toxic
compounds 12, 17, 24 and 26, have inhibitory effects on iNOS-
related NOꢀ production and can therefore be used as promising
treatment candidate compounds for diseases related with excess
NOꢀ production. Indeed, there are still some undetermined mech-
anisms that exist. It is not clearly known if this inhibitory effect is
achieved by direct scavenging of nitric oxide or inhibition of iNOS
enzyme. Therefore we also tested direct in vitro NOꢀ scavenging
activity of these novel compounds which may help us to explain the
mechanisms of their effects on NOꢀ production in biological system
[46]. Our results showed that majority of the compounds display
poor NOꢀ scavenging activity. However compounds 10 and 11 have
5.1.1.2. Method II. N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide
HCl (EDCI) and 1-hydroxybenzotriazole hydrate (HOBT) were used as
amidification agents. EDCI (1.095 mmol) and HOBT (1.095 mmol)
were added to a solution of caffeic acid (or ferulic acid) (0.91 mmol) in
dry CH2Cl2 (15 ml) at 0 ꢂC and the mixture was stirred at room
temperature for 15 min under N2. After the addition of compound 5
(1.095 mmol), the mixture was stirred overnight at the same
temperature. Cold 1% HCl aq. was added to the reaction mixture and
the whole was extracted with CHCl3. The organic layer was washed
with saturated NaHCO3 aq. and then brine, dried over Na2SO4 and
evaporated [49,50]. The residue was purified by silica gel column
chromatography using n-hexane:AcOEt (3:1 and 1:1) as eluent to
afford amides. Compounds 10 and 11 were prepared according to the
method II.
NOꢀ scavenging activity at both 5
mM and 50 mM concentrations
(data not shown) which means that only these two compounds
decrease nitrite levels by scavenging NOꢀ and/or effecting biological
pathways. These findings suggest that most of these retinoid
analogues show their effects biologically such as inhibition of target
protein (iNOS) rather than scavenging NOꢀ in the media.
Further investigation is needed to determine mechanisms
underlying this effect and the most effective concentrations.
5.1.1.3. Method III. A mixture of compound 3 (or the appropriate
acid) (2 mmol), SOCl2 (2 ml), and benzene (10 ml) was heated at
reflux for 4 h. The resulting solution was rotary evaporated to yield
acid chloride. An appropriate amine (1 mmol) was suspended
in pyridine (2 ml) and triethylamine (0.5 ml). After stirring for
20 min at room temperature, the resulting mixture was filtered.
The filtrate was cooled to ꢁ5 ꢂC, and at this temperature CH2Cl2
(5 ml) with acyl chloride (1.2 mmol) was added. After being stirred
and refluxed for 4 h, the reaction mixture was filtered to remove
white precipitation, and the filtrate was extracted with ethyl
acetate, and washed with water. The organic extracts were
combined, dried over Na2SO4, and the solvent was evaporated. The
residue was purified by silica gel column chromatography [51,52].
Compounds 9, 12, and 16 were prepared according to the method
III. Cinnamoyl chloride was commercially available for the synthesis
of compound 9.
5. Experimental section
5.1. General synthetic
All starting materials and reagents were high-grade commer-
cial products purchased from Aldrich, Merck or Fluka. The struc-
tures of all synthesized compounds were assigned on the basis of
1H NMR, Mass, and IR spectral analyses. Analytical thin-layer
chromatographies (TLC) were run on silica gel 60 F254 plates
(Merck, Germany). Column chromatographies were accomplished
on silica gel 60 (40e63 mm particle size) (Merck, Germany).
Melting points were determined with an electrothermal 9100
melting point apparatus and are uncorrected. 1H NMR (400 MHz)
spectra were recorded with a Varian Mercury-400 spectrometer
(Varian Inc., Palo Alto, CA, USA), in CDCl3, DMSO-d6 or acetone-d6,

476
A.S. Gurkan et al. / European Journal of Medicinal Chemistry 46 (2011) 468e479
5.1.1.4. Method IV. The procedure used by Ayhan-Kılcıgil and
Altanlar [53] was modified. Compound (0.32 mmol) was
(CH3)2NCHO), 2.89 (br s, 2H, Are30-OH, Are40-OH), 2.25 (s, 3H),1.68
(s, 4H), 1.26 (d, 12H).
3
refluxed in benzene (5 ml) with SOCl2 (5 ml) for 4 h at 80 ꢂC.
Then, solvent and excess of SOCl2 were evaporated completely
and the residue was dissolved in ether (10 ml). This mixture
was added to an ice-cold mixture of 5 (0.32 mmol), NaHCO3
(0.64 mmol), ether (5 ml) and water (5 ml) over a 1 h period. The
mixture was stirred overnight at room temperature. Water and
organic layers were separated. The organic layer was washed with
1 N HCl and then water, and the solvent was evaporated. The
residue was purified by column chromatography using chlor-
oform:n-hexane (1:1) as eluent. Compound 15 was prepared by
using method IV.
5.1.7. E-3-(4-Hydroxy-3-methoxyphenyl)-N-(3,5,5,8,8-pentamethyl-
5,6,7,8-tetrahydronaphthalen-2-yl)acrylamide (11)
ESIþ-MS (m/z, %): 416 (M þ Na, 100), 394 (M þ H, 15). IR (KBr,
cmꢁ1): 3306 (NH), 3050 (OH), 1659 (CO). 1H NMR
d ppm (acetone-
d6): 8.47 (br s, 1H, eNH), 7.84 (d, 1H), 7.58 (d, 1H, J ¼ 15.6 Hz), 7.22
(s, 1H), 7.16 (s, 1H), 7.11 (d, 1H, Jo ¼ 8.4 Hz), 6.86 (d, 1H, Jo ¼ 8 Hz),
6.82 (d, 1H, J ¼ 15.6 Hz), 3.89 (s, 3H), 2.25 (s, 3H), 1.68 (s, 4H), 1.26
(s, 12H).
5.1.8. E-3-(3,4-Dimethoxyphenyl)-N-(3,5,5,8,8-pentamethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)acrylamide (12)
5.1.1.5. Method V. A solution of 3 (0.45 mmol) and oxalyl chloride
(0.25 ml) in CH2Cl2 (2 ml) was stirred for 1 h. After removal of the
solvent, a solution of the appropriate amine derivative (0.74 mmol)
and 4-dimethylaminopyridine (DMAP) (10 mg) in pyridine (2 ml)
was added to the residue, and the mixture was stirred overnight at
the room temperature. The reaction mixture was poured into 2 N
HCl (20 ml), and extracted with CH2Cl2. The organic layer was dried
over Na2SO4, and concentrated [51]. The residue was purified by
column chromatography using n-hexane:AcOEt (2:1) as eluent.
Compound 19 was prepared by using method V.
ESIþ-MS (m/z, %): 408 (M þ H, 100). IR (KBr, cmꢁ1): 3356 (NH),
1661 (CO). 1H NMR
d ppm (CDCl3): 7.93 (br s, 1H, eNH), 7.72 (d,
1H, J ¼ 15.2 Hz), 7.13e7.01 (4H), 6.88 (d, 1H, Jo ¼ 8.4 Hz), 6.46 (d,
1H, J ¼ 15.6 Hz), 3.93 (d, 6H), 2.27 (s, 3H), 1.68 (s, 4H), 1.28
(d, 12H).
5.1.9. 6-Hydroxy-2,5,7,8-tetramethyl-N-(3,5,5,8,8-pentamethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)chroman-2-carboxamide (13)
ESIþ-MS (m/z, %): 450 (M þ H, 100). IR (KBr, cmꢁ1): 3480 (NH),
3414 (OH), 1669 (CO). 1H NMR
d ppm (CDCl3): 8.16 (br s, 1H, eNH),
Isolation methods and some physical data of the synthesized
compounds 6e32 are summarized in Table 1.
8.04 (s, 1H), 7.02 (s, 1H), 4.30 (s, 1H, eOH), 2.65 (m, 2H), 2.50 (m,
1H), 2.24 (s, 3H), 2.18 (s, 3H), 2.08 (s, 3H), 1.97 (s, 3H), 1,92 (m, 1H),
1.64 (d, 4H), 1.57 (s, 3H), 1.28 (s, 6H), 1.22 (d, 6H).
5.1.2. 5-(1,2-Dithiolan-3-yl)-N-(3,5,5,8,8-pentamethyl-5,6,7,8-tetra-
hydronaphthalen-2-yl)pentanamide (6)
5.1.10. 2,5,7,8-Tetramethyl-2-(3,5,5,8,8-pentamethyl-5,6,7,8-tetra-
hydronaphthalen-2-ylcarbamoyl)chroman-6-yl acetate (14)
ESIþ-MS (m/z, %): 514 (M þ Na, 100), 492 (M þ H, 22). IR (KBr,
ESIþ-MS (m/z, %): 406 (M þ H, 100). IR (KBr, cmꢁ1): 3279 (NH),
1652 (CO). 1H NMR
d ppm (CDCl3): 7.70 (s, 1H), 7.09 (s, 1H), 6.88 (br
s, 1H, eNH), 3.59 (m, 1H), 3.15 (m, 2H), 2.47 (m, 1H), 2.39 (t, 2H),
2.20 (s, 4H), 1.92 (m, 1H), 1.75 (m, 4H), 1.66 (s, 3H), 1.54 (m, 2H), 1.25
(d, 12H).
cmꢁ1): 3428 (NH), 1746 (CO), 1683 (CO). 1H NMR
d ppm (CDCl3):
8.15 (br s, 1H, eNH), 8.04 (s, 1H), 7.03 (s, 1H), 2.65 (br s, 2H), 2.50 (br
s, 1H), 2.34 (s, 3H), 2.23 (s, 3H), 2.05 (s, 3H), 1.97 (s, 7H), 1.65 (s, 7H),
1.29 (s, 6H), 1.22 (d, 6H).
5.1.3. N-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalen-2-
yl)-4-phenylbutanamide (7)
5.1.11. 5,5,8,8-Tetramethyl-N-(3,5,5,8,8-pentamethyl-5,6,7,8-tetra-
hydronaphthalen-2-yl)-5,6,7,8-tetrahydronaphthalene-2-
carboxamide (15)
ESIþ-MS (m/z, %): 364 (M þ H, 100). IR (KBr, cmꢁ1): 3215 (NH),
1658 (CO). 1H NMR
d ppm (CDCl3): 7.69 (s, 1H), 7.32e7.20 (m, 5H),
7.09 (s, 1H), 6.81 (br s, 1H, eNH), 2.73 (t, 2H), 2.37 (t, 2H), 2.19 (s,
3H), 2.09 (m, 2H), 1.66 (s, 4H), 1.26 (d, 12H).
ESIþ-MS (m/z, %): 432 (M þ H, 100). IR (KBr, cmꢁ1): 3289 (NH),
1645 (CO). 1H NMR
d ppm (CDCl3): 7.97 (br s, 1H, eNH), 7.91 (s, 1H),
7.58 (s, 1H), 7.54 (dd, 1H, Jo ¼ 9.6 Hz), 7.40 (d, 1H, Jo ¼ 8 Hz), 7.13 (s,
5.1.4. 4-(3,4-Dimethoxyphenyl)-N-(3,5,5,8,8-pentamethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)butanamide (8)
1H), 2.28 (s, 3H), 1.72 (s, 4H), 1.68 (s, 4H), 1.32 (d, 12H), 1.26 (d, 12H).
ESIþ-MS (m/z, %): 446 (M þ Na, 100), 424 (M þ H, 83). IR (KBr,
5.1.12. 5,5,8,8-Tetramethyl-N-(naphthalen-1-yl)-5,6,7,8-tetrahydro-
naphthalene-2-carboxamide (16)
cmꢁ1): 3384 (NH), 1692 (CO). 1H NMR
d ppm (CDCl3): 7.71 (s, 1H),
7.09 (s, 1H), 6.83 (br s, 1H, eNH), 6.80 (d, 1H), 6.75 (d, 1H), 6.74
(s, 1H), 3.87 (d, 6H), 2.68 (t, 2H), 2.38 (t, 2H), 2.19 (s, 3H), 2.07 (m,
2H), 1.66 (s, 4H), 1.26 (d, 12H).
ESIþ-MS (m/z, %): 358 (M þ H, 100). IR (KBr, cmꢁ1): 3269 (NH),
1643 (CO). 1H NMR
d ppm (CDCl3): 8.25 (br s, 1H, eNH), 7.99 (d, 2H,
Jo ¼ 10.8), 7.88 (dd, 2H, Jo ¼ 9.2 Hz, Jm ¼ 2.4 Hz), 7.72 (d, 1H,
Jo ¼ 8 Hz), 7.68 (dd, 1H, Jo ¼ 9.6 Hz, Jm ¼ 1.4 Hz), 7.47e7.51 (m, 2H),
7.43 (d, 2H, Jo ¼ 8 Hz), 1.73 (s, 4H), 1.33 (d, 12H).
5.1.5. E-N-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydronaphthalen-2-
yl)cinnamamide (9)
ESIþ-MS (m/z, %): 411 (M þ Na þ CH3CN, 100), 370 (M þ Na, 91),
5.1.13. N-(1H-Benzo[d]imidazol-2-yl)-5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalene-2-carboxamide (17)
348 (M þ H, 27). IR (KBr, cmꢁ1): 3244 (NH), 1658 (CO). 1H NMR
d
ppm (CDCl3): 7.93 (br s, 1H, eNH), 7.77 (d, 1H, J ¼ 15.2 Hz), 7.55
ESIþ-MS (m/z, %): 348 (M þ H, 100). IR (KBr, cmꢁ1): 3350 (NH),
(m, 2H), 7.38 (m, 3H), 7.13 (s, 1H), 7.08 (s, 1H), 6.59 (d, 1H,
1667 (CO). 1H NMR
(dd, 1H, Jo ¼ 8.4 Hz, Jm ¼ 2 Hz), 7.50 (d, 1H, Jo ¼ 8 Hz), 7.23 (br s, 2H),
7.09e7.07 (m, 2H), 2.78, 2.94 (s, s, (CH3)2NCHO), 1.71 (m, 4H), 1.32
(s, 6H), 1.24 (s, 6H).
d
ppm (acetone-d6): 8.16 (d, 1H, Jm ¼ 2 Hz), 7.91
J ¼ 15.2 Hz), 2.27 (s, 3H), 1.68 (s, 4H), 1.28 (d, 12H).
5.1.6. E-3-(3,4-Dihydroxyphenyl)-N-(3,5,5,8,8-pentamethyl-5,6,7,8-
tetrahydronaphthalen-2-yl)acrylamide (10)
ESIþ-MS (m/z, %): 402 (M þ Na, 100), 380 (M þ H, 35.5). IR (KBr,
5.1.14. 5,5,8,8-Tetramethyl-N-(thiazol-2-yl)-5,6,7,8-tetrahydro-
naphthalene-2-carboxamide (18)
cmꢁ1): 3522 (NH), 3235 (OH), 1652 (CO). 1H NMR
d ppm (acetone-
d6): 8.36 (br s, 1H, eNH), 8.02 (s, (CH3)2NCHO), 7.84 (d, 1H), 7.52
(d, 1H, J ¼ 15.2 Hz), 7.16 (s, 1H), 7.11 (s, 1H), 6.99 (d, 1H, Jo ¼ 8.4 Hz),
6.85 (d, 1H, Jo ¼ 7.6 Hz), 6.75 (d, 1H, J ¼ 15.2 Hz), 2.78, 2.94 (s, s,
ESIþ-MS (m/z %): 315 (M þ H, 100). IR (KBr, cmꢁ1): 3147 (NH),
1667 (CO). 1H NMR
d ppm (CDCl3): 10.88 (br s,1H, eNH), 7.95 (d,1H,
Jm ¼ 2 Hz), 7.69 (dd, 1H, Jo ¼ 8 Hz, Jm ¼ 2.4 Hz), 7.44 (d, 1H,

A.S. Gurkan et al. / European Journal of Medicinal Chemistry 46 (2011) 468e479
477
Jo ¼ 8 Hz), 7.26 (d, 1H, Jo ¼ 3.6 Hz), 6.98 (d, 1H, Jo ¼ 3.6 Hz), 1.72
Jo ¼ 8 Hz), 7.34 (d, 1H, Jo ¼ 8.2 Hz), 5.82 (d, 1H), 4.45 (m, 1H), 2.03 (d,
(s, 4H), 1.32 (d, 12H).
1H), 2.00 (d, 1H), 1.70 (s, 4H), 1.29 (d, 12H), 1.25 (d, 12H), 1.08 (t, 2H).
5.1.15. 5,5,8,8-Tetramethyl-N-(pyrimidin-2-yl)-5,6,7,8-tetrahydro-
naphthalene-2-carboxamide (19)
5.1.23. 5,5,8,8-Tetramethyl-N-(2,2,6,6-tetramethylpiperidin-1-oxyl-
4-yl)-5,6,7,8-tetrahydronaphthalene-2-carboxamide (27)
ESIþ-MS (m/z, %): 310 (M þ H, 100). IR (KBr, cmꢁ1): 3211 (NH),
ESIþ-MS (m/z, %): 386 (M þ H, 100). IR (KBr, cmꢁ1): 3283 (NH),
1670 (CO). 1H NMR
s, 1H, eNH), 7.89 (s, 1H), 7.66 (d, 1H, Jo ¼ 8.4 Hz), 7.42 (d, 1H,
d
ppm (CDCl3): 8.67 (d, 2H, Jo ¼ 4.8 Hz), 8.62 (br
1628 (CO). 1H NMR
d
ppm (DMSO-d6 þ Na2S2O4 þ D2O): 8.44 (d,
1H, eNH), 7.78 (s, 1H), 7.57 (d, 1H, Jo ¼ 8 Hz), 7.41 (d, 1H, Jo ¼ 8 Hz),
4.36 (m, 1H), 1.96 (d, 2H), 1.66 (s, 4H), 1.60 (d, 2H), 1.43 (d, 12H), 1.25
(d, 12H).
Jo ¼ 8.8 Hz), 7.06 (t, 1H), 1.72 (s, 4H), 1.32 (d, 12H).
5.1.16. 5,5,8,8-Tetramethyl-N-(2-oxo-1,2-dihydropyrimidin-4-yl)-
5,6,7,8-tetrahydronaphthalene-2-carboxamide (20)
5.1.24. 5,5,8,8-Tetramethyl-N-(piperidin-1-yl)-5,6,7,8-tetrahydro-
naphthalene-2-carboxamide (28)
ESIþ-MS (m/z, %): 326 (M þ H, 100). IR (KBr, cmꢁ1): 3216 (NH),
3145 (NH), 1710 (CO),1698 (CO). 1H NMR
d
ppm (DMSO-d6): 8.01 (d,
ESIþ-MS (m/z, %): 315 (M þ H, 100). IR (KBr, cmꢁ1): 3191 (NH),
1H, Jm ¼ 1.2 Hz), 7.93 (s, (CH3)2NCHO), 7.84 (d, 1H, Jo ¼ 6.8 Hz), 7.72
(dd, 1H, Jo ¼ 8 Hz, Jm ¼ 2 Hz), 7.43 (d, 1H, Jo ¼ 8 Hz), 7.22 (d, 1H,
Jo ¼ 5.2 Hz), 2.70, 2.88 (s, s, (CH3)2NCHO), 1.64 (s, 4H), 1.27 (s, 6H),
1.23 (s, 6H).
1641 (CO). 1H NMR
d ppm (CDCl3): 7.70 (s, 1H), 7.41 (d, 1H,
Jo ¼ 8.4 Hz), 7.32 (d, 1H, Jo ¼ 8.4 Hz), 6.73 (br s, 1H, eNH), 2.87 (t,
4H), 1.75 (m, 4H), 1.68 (s, 4H), 1.44 (m, 2H), 1.28 (d, 12H).
5.1.25. N-(Azepan-1-yl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-
naphthalene-2-carboxamide (29)
5.1.17. N-Benzyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-
2-carboxamide (21)
ESIþ-MS (m/z, %): 329 (M þ H, 100). IR (KBr, cmꢁ1): 3214 (NH),
ESIþ-MS (m/z, %): 322 (M þ H, 100). IR (KBr, cmꢁ1): 3295 (NH),
1636 (CO). 1H NMR
d
ppm (CDCl3): 7.72 (d, 1H, Jm ¼ 2 Hz), 7.40 (dd,
1625 (CO). 1H NMR
1H, Jo ¼ 8.4 Hz, Jm ¼ 2 Hz), 7.36e7.28 (5H), 7.34 (d, 1H, Jo ¼ 8.4 Hz),
6.36 (br t, 1H, eNH), 4.65 (d, 2H), 1.69 (s, 4H), 1.31 (s, 6H), 1.28
(s, 6H).
d
ppm (CDCl3): 7.81 (d, 1H, Jm ¼ 1.6 Hz), 7.46 (dd,
1H, Jo ¼ 8 Hz, Jm ¼ 2 Hz), 7.33 (d, 1H, Jo ¼ 8.4 Hz), 3.19 (t, 4H), 1.74
(m, 4H), 1.68 (s, 4H), 1.65 (m, 4H), 1.29 (d, 12H).
5.1.26. 5,5,8,8-Tetramethyl-N-morpholino-5,6,7,8-tetrahydro-
naphthalene-2-carboxamide (30)
5.1.18. N-(2,4-Difluorobenzyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-
naphthalene-2-carboxamide (22)
ESIþ-MS (m/z, %): 339 (M þ Na, 100), 317 (M þ H, 71). IR (KBr,
cmꢁ1): 3189 (NH), 1639 (CO). 1H NMR
d ppm (CDCl3): 7.72 (s, 1H),
ESIþ-MS (m/z, %): 358 (M þ H, 100). IR (KBr, cmꢁ1): 3294 (NH),
7.42 (d,1H, Jo ¼ 8.4 Hz), 7.35 (d,1H, Jo ¼ 8.4 Hz), 6.76 (br s,1H, eNH),
1626 (CO). 1H NMR
1H, Jo ¼ 8 Hz, Jm ¼ 2 Hz), 7.73e7.38 (m, 1H), 7.34 (d, 1H, Jo ¼ 8.4 Hz),
6.86e6.78 (m, 2H), 6.50 (br s, 1H, eNH), 4.63 (d, 2H), 1.69 (s, 4H),
1.28 (d, 12H).
d
ppm (CDCl3): 7.79 (d, 1H, Jm ¼ 2 Hz), 7.45 (dd,
3.87 (t, 4H), 2.97 (t, 4H), 1.70 (s, 4H), 1.29 (d, 12H).
5.1.27. 5,5,8,8-Tetramethyl-N-(2-morpholinoethyl)-5,6,7,8-tetrahydro-
naphthalene-2-carboxamide (31)
ESIþ-MS (m/z, %): 345 (M þ H, 100). IR (KBr, cmꢁ1): 3289 (NH),
5.1.19. N-(2,4-Dichlorobenzyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-
naphthalene-2-carboxamide (23)
1628 (CO). 1H NMR
1H, Jo ¼ 8.4 Hz, Jm ¼ 1.8 Hz), 7.36 (d, 1H, Jo ¼ 8 Hz), 6.73 (br t, 1H,
eNH), 3.73 (t, 4H), 3.54 (q, 2H), 2.60 (t, 2H), 2.51 (t, 4H), 1.70 (s, 4H),
1.30 (d, 12H).
d
ppm (CDCl3): 7.78 (d, 1H, Jm ¼ 2 Hz), 7.45 (dd,
ESIþ-MS (m/z, %): 394 (M þ H þ 4, 18), 392(M þ H þ 2, 57), 390
(M þ H, 100). IR (KBr, cmꢁ1): 3286 (NH), 1629 (CO). 1H NMR
d ppm
(CDCl3): 7.80 (d, 1H, Jm ¼ 1.6 Hz), 7.45 (dd, 1H, Jo ¼ 8 Hz, Jm ¼ 2 Hz),
7.41 (d, 1H, Jo ¼ 8.4 Hz), 7.39 (d, 1H, Jm ¼ 2 Hz), 7.35 (d, 1H,
Jo ¼ 8.4 Hz), 7.22 (dd,1H, Jo ¼ 8.4 Hz, Jm ¼ 2 Hz), 6.55 (br t,1H, eNH),
4.68 (d, 2H), 1.69 (s, 4H), 1.29 (d, 12H).
5.1.28. N-Cyclopropyl-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-
naphthalene-2-carboxamide (32)
ESIþ-MS (m/z, %): 272 (M þ H, 100). IR (KBr, cmꢁ1): 3272 (NH),
1631 (CO). 1H NMR
d
ppm (CDCl3): 7.73 (d, 1H, Jm ¼ 2 Hz), 7.41 (dd,
5.1.20. N-(1-Benzylpiperidin-4-yl)-5,5,8,8-tetramethyl-5,6,7,8-tetra-
hydronaphthalene-2-carboxamide (24)
1H, Jo ¼ 8.4 Hz, Jm ¼ 2 Hz), 7.31 (d,1H, Jo ¼ 8 Hz), 6.28 (br s,1H, eNH),
2.87 (m, 1H), 1.68 (s, 4H), 1.28 (d, 12H), 0.84 (m, 2H), 0.61 (m, 2H).
ESIþ-MS (m/z, %): 405 (M þ H, 100). IR (KBr, cmꢁ1): 3284 (NH),
1626 (CO). 1H NMR
d
ppm (CDCl3): 7.75 (d, 1H, Jm ¼ 1.6 Hz), 7.40
5.2. General biological assays
(dd, 1H, Jo ¼ 8 Hz, Jm ¼ 2 Hz), 7.34 (d, 1H, Jo ¼ 7.6 Hz), 7.32e7.25
(5H), 5.91 (d, 1H, eNH), 4.01 (m, 1H), 3.51 (s, 2H), 2.85 (m, 2H),
2.17 (t, 2H), 2.01 (d, 2H), 1.69 (s, 4H), 1.57e1.53 (m, 2H), 1.29
(d, 12H).
5.2.1. Reagents for biological activity
RAW 264.7 cell line was a gift from Dr. Georg Bauer. (Depart-
ment of Virology, University of Freiburg, Freiburg, Germany). RPMI
1640 medium,
L-glutamine, penicillin/streptomycin and fetal
5.1.21. Ethyl 4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-
2-carboxamido)piperidine-1-carboxylate (25)
bovine serum were from PAA Laboratories; LPS, MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], and BSA
(bovine serum albumin) were from Sigma; plastic flasks and 96-
well plates were from Orange Sci.
ESIþ-MS (m/z, %): 409 (M þ Na, 100), 387 (M þ H, 96). IR (KBr,
cmꢁ1): 3305 (NH), 1699 (CO), 1634 (CO). 1H NMR
d ppm (CDCl3):
7.75 (d, 1H, Jm ¼ 1.6 Hz), 7.42 (dd, 1H, Jo ¼ 8.4 Hz, Jm ¼ 2 Hz), 7.34 (d,
1H, Jo ¼ 8.4 Hz), 5.97 (d, 1H, eNH), 4.11e4.19 (m, 5H), 2.95 (t, 2H),
2.04 (d, 2H), 1.69 (s, 4H), 1.43 (m, 2H), 1.29 (d, 12H), 1.28 (t, 3H).
Compounds 6e32 were dissolved in dimethyl sulfoxide to make
stock solutions, respectively, and kept at ꢁ30 ꢂC. The final
concentration of the vehicle in the solution never exceeded 0.1%
and had no effects on NOꢀ production and cell viability.
5.1.22. 5,5,8,8-Tetramethyl-N-(2,2,6,6-tetramethylpiperidin-4-yl)-
5,6,7,8-tetrahydronaphthalene-2-carboxamide (26)
5.2.2. Cell culture
RAW 264.7 cells were cultured in RPMI 1640 medium sup-
plemented with 10% fetal bovine serum,
ESIþ-MS (m/z, %): 371 (M þ H, 100). IR (KBr, cmꢁ1): 3290 (NH),
1627 (CO). 1H NMR
d
ppm (CDCl3): 7.78 (s, 1H), 7.42 (d, 1H,
L-glutamine and

478
A.S. Gurkan et al. / European Journal of Medicinal Chemistry 46 (2011) 468e479
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We would like to thank to Professor Hakan Goker and
Dr. Mehmet Alp from Ankara University, Central Instrumentation
Laboratory of Faculty of Pharmacy, for their support for the acqui-
sition of the NMR, mass and elemental analyses of this study. We
also thank to Professor Maral Sunnetcioglu and Dilek Yonar from
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mM: milimolar
cc: column chromatography
NI: no inhibition
SD: standard deviation
s
c: rotational correlation time
a: hyperfine splitting constant
EPR: Electron Paramagnetic Resonance
TEMPO: 2,2,6,6-tetramethylpiperidine-N-oxyl
ATRA: all-trans-retinoic acid
NOꢀ: nitric oxide
iNOS: nitric oxide synthase
LPS: lipopolysaccharide
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MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
BSA: bovine serum albumin