New analogues of butylated hydroxytoluene as anti-inflammatory and antioxidant agents

Article abstract of DOI:10.1016/j.bmc.2006.04.030

Amine or amide derivatives bearing the 2,6-di-tert-butyl phenol moiety are synthesised. Almost all are antioxidants, reduce acute inflammation and inhibit COX-1 and lipoxygenase activity. The most potent anti-inflammatory, COX-1 inhibitor and antioxidant agent, with low toxicity, is 2,6-di-tert-butyl-4-thiomorpholin-4-ylmethyl-phenol.

Full text of DOI:10.1016/j.bmc.2006.04.030

Bioorganic & Medicinal Chemistry 14 (2006) 5616–5624
New analogues of butylated hydroxytoluene as anti-inflammatory
and antioxidant agents
George N. Ziakas, Eleni A. Rekka,* Antonios M. Gavalas, Phaedra T. Eleftheriou and
Panos N. Kourounakis
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki, Thessaloniki 54 124, Greece
Received 9 March 2006; revised 8 April 2006; accepted 13 April 2006
Available online 11 May 2006
Abstract—Amine or amide derivatives bearing the 2,6-di-tert-butyl phenol moiety are synthesised. Almost all are antioxidants,
reduce acute inflammation and inhibit COX-1 and lipoxygenase activity. The most potent anti-inflammatory, COX-1 inhibitor
and antioxidant agent, with low toxicity, is 2,6-di-tert-butyl-4-thiomorpholin-4-ylmethyl-phenol.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
hypersensitivity, elevated blood pressure and increased
risk of cardiovascular events.9
Non-steroidal anti-inflammatory drugs (NSAIDs) are
the most widely used agents for the treatment of inflam-
mation and highly prescribed medications.1 However,
they possess major and even life-threatening side effects,
such as damage to the gastric mucosa, prolongation of
bleeding time and renal failure.2,3 It has been reported
that there are about 17,000 deaths in the United States
each year as a result of NSAID-related gastrointestinal
complications.2
A strategy recently followed involves combined COX
and 5-lipoxygenase (5-LOX) inhibitors, interfering with
the biosynthesis of both prostaglandins and leukotri-
enes. These agents are expected to possess potent anti-
inflammatory activity with reduced prothrombotic effect
and a good gastrointestinal safety profile.10
It is well known that reactive oxygen species are impli-
cated in inflammation.11 Superoxide anion radical has
proinflammatory actions, such as endothelial cell dam-
age and increased microvascular permeability,12 forma-
tion of chemotactic factors,13,14 recruitment of
neutrophils at sites of inflammation,15 lipid peroxidation
and DNA damage.16 Furthermore, since COX catalysis
involves radical intermediates, a radical scavenger
would be considered to interfere with the COX reaction.
Linkage of the antioxidant di-tert-butyl phenol sub-
structure with thiazolone, oxazolone17 or thiadiazole
moieties18 gave non-ulcerogenic, orally active COX-2
inhibitors.
A number of approaches have been pursued to minimize
these adverse effects, such as entering coating, parenteral
administration, combination of conventional NSAIDs
with a proton pump inhibitor or prodrug formulations,
with moderate results.4
Cyclooxygenase (COX)-2 selective inhibitors have been
evolved as agents that would treat inflammatory diseas-
es via inhibition of the COX isoform responsible for the
inflammatory response, without affecting the COX-1
mediated production of cytoprotective prostaglandins
in the gastrointestinal tract.5–7 Yet, emerging data sug-
gest that selective COX-2 inhibitors could still cause
serious side effects such as gastrointestinal toxicity,8
In addition, derivatives bearing the 3,5-di-tert-butyl-4-
hydroxybenzilidene moiety have been found to be dual
inhibitors of COX-2 and 5-LOX and to suppress inter-
leukin-1 production.19 Considering the above studies,
we designed some new derivatives of 2,6-di-tert-butyl
phenol (compounds 8, 10–14) (Chart 1) which are
amines or amides of thiomorpholine, morpholine and
proline residues. We also included in our series
Keywords: 2,6-Di-tert-butylphenol derivatives; Antioxidants; Anti-
inflammatory agents; Cyclooxygenase, lipoxygenase inhibition.
*
Corresponding author. Tel.: +302310997614; fax: +302310997622;
e-mail: rekka@pharm.auth.gr
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.030

G. N. Ziakas et al. / Bioorg. Med. Chem. 14 (2006) 5616–5624
5617
S
S
S
N
N
O
N
OH
OH
OH
10
9
8
O
O
OEt
OEt
N
O
N
OH
OH
12
11
N
N
O
OH
OH
COOCH3
COOCH3
13
14
Chart 1. Structure of amine and amide derivatives (8–14) of butylated hydroxytoluene (BHT).
compound 9, that has been prepared earlier.20 We have
previously found that some substituted morpholines
O
OH
OH
N
O
b
possess anti-inflammatory21 and antioxidant proper-
ties.22,23 Thus, in this study, an effort was made to com-
bine and increase these activities with the antioxidant
potential that the phenolic moiety would convey, since
butylated hydroxytoluene (BHT) is a well-known anti-
oxidant with low toxicity. These compounds were tested
in vitro and in vivo for the evaluation of their antioxi-
dant and anti-inflammatory activities. Finally, the toxic-
ity of 8, one of the most active compounds, was
examined, in relation to the parent molecule, BHT.
HN
+
Br
4
c
O
OCH2CH3
O
d
N
OCH2CH3
HN
6
5
Scheme 2. Synthesis of the substituted morpholine. Reagents and
conditions: (b) Ethyl ether/acetone, room temperature, 12 h; (c)
Ethanol, hydrobromic acid/ethyl ether, reflux, 3 h; (d) Ethanol, Pd/C
under hydrogen, room temperature, 3 h.
2. Results and discussion
2.1. Chemistry
Preparation of the intermediate amines 3 and 6 is dem-
onstrated in Schemes 1 and 2. 3-Phenylthiomorpholine
(3) was prepared by the hydrogenation of the intermedi-
ate imine, obtained from the reaction of 2-aminoetha-
nethiol with 2-bromo-1-phenylethanone in alkaline
environment. 2-Ethoxy-2-phenyl morpholine (6) was
obtained after hydrogenolysis of the 4-benzyl analogue.
The final reaction for the synthesis of compounds 8–14
is demonstrated in Scheme 3. For the amides 9, 12 and
14, direct amidation of the carboxylic group of 3,5-di-
tert-butyl-4-hydroxybenzoic acid in the presence of
2,2-dicyclohexylcarbodiimide
accomplished.
could
not
be
2.2. In vivo experiments
2.2.1. Carrageenan-induced inflammation. The effect of
compounds 8–14 and BHT on carrageenan induced
edema is presented in Table 1. They showed a
significant anti-inflammatory action administered at
a
dose of 0.28 mmol/kg, with the exception of
SH
Br
S
N
S
+
a
NH2
O
N
H
3
Scheme 1. Synthesis of the substituted thiomorpholine. Reagents and conditions: (a) Potassium hydroxide, methanol, room temperature under
nitrogen, 2 h, sodium borohydride, 1 h.

5618
G. N. Ziakas et al. / Bioorg. Med. Chem. 14 (2006) 5616–5624
X
X
N
A
e
HO
Z +
HN
HO
Y
B
A
C
B
C
8-14
Z=CH2Clf
Z=COClg
8: Y: CH2, X: CH2S, A, B, C: H
9: Y: C=O, X: CH2S, A, B, C: H
10: Y: CH2, X: CH2S, A, B: H, C: Ph
11: Y: CH2, X: CH2O, A: Ph, B: OCH2CH3, C: H
12: Y: C=O, X: CH2O, A: Ph, B: OCH2CH3, C: H
13: Y: CH2, X: CH2, A, B: H, C: COOCH3
14: Y: C=O, X: CH2, A, B: H, C: COOCH3
Scheme 3. Synthesis of the final compounds. Reagents and conditions: (e) Ethyl ether, triethylamine, room temperature, 12 h; (f) Prepared from 3,5-
di-tert butyl-4-hydroxybenzyl alcohol, hydrochloric acid, in petroleum ether, room temperature, 12 h; (g) Prepared from 3,5-di-tert-butyl-4-
hydroxybenzoic acid sodium salt, thionyl chloride, in petroleum ether, room temperature, 2–3 h.
Table 1. Effect of compounds on hind paw edema induced by
carrageenan in rats
2.3. In vitro experiments
2.3.1. Inhibition of cyclooxygenase activity (isoforms 1
and 2). Cyclooxygenase, one of the two activities of
prostaglandin endoperoxide synthase, is the key enzyme
in the conversion of arachidonic acid (AA) to prosta-
glandins (PGs) and other eicosanoids, important inflam-
matory mediators, also playing a major role in gastric
mucosa protection, platelet aggregation and kidney
function. It has been established that there are two dis-
tinct isoforms of COX. COX-1 is the constitutive isoen-
zyme found in most tissues under physiological
conditions which maintains cellular homeostasis.
COX-2 is highly inducible in response to proinflamma-
tory stimuli, resulting in exaggerated PG release.
Compound
Percent paw
weight increasea
Percent edema
inhibitiona
Control
43.22
23.56
21.12
29.58
40.60
24.87
26.98
27.96
43.10
—
8
9
45.49*
51.13*
31.56*
6.06
10
11
12
13
14
BHT
42.46*
37.58*
35.31*
0.00
a The effect on edema is expressed as percent weight increase of hind
paw and as percent of inhibition of edema in comparison to controls.
All compounds were administered ip at a dose of 0.28 mmol/kg of
body weight. Each value represents the mean obtained from 6 to 8
animals in two independent experiments.
The effect of compounds 8–14 (concentration of 10 lM)
and indomethacin (IND) (concentration of 1 lM for
COX-1 and 10 lM for COX-2) on both COX isoforms
has been examined. The concentration of the substrate,
arachidonic acid, was 0.1 lM for both COX isoforms
and 1 lM for IND. At higher concentrations of arachi-
donic acid there was no significant inhibition. For com-
pound 8, more concentrations were tested for the
determination of its IC50 value towards COX-1.
* P < 0.001 (Student’s t-test).
compound 11, the amine derivative of substituted
morpholine, and the parent molecule (BHT) which
were not active. Under the same conditions, indo-
methacin produced 54% edema inhibition.24 Com-
pounds
8
and 9, which are derivatives of
Compounds 8–14 and BHT had negligible effect on
COX-2. However, as shown in Table 2, most
thiomorpholine, were the most potent anti-inflamma-
tory agents of this series, with 9 being the most ac-
tive. Compound 12, which is the amide derivative of
substituted morpholine, followed closely the activity
of compounds 8 and 9, while the amine (compound
11) had no action. The addition of a phenyl moiety
Table 2. In vitro evaluation of anti-inflammatory activity
Compounda Percent cyclooxygenase Lipoxygenase inhibition
inhibition COX-1b
IC50 (lM)
to the thiomorpholine ring of compound
8 de-
8
74
50
32
5
70
300
112
30
creased its activity (compound 10). Compounds 13
and 14, the derivatives of substituted proline, dem-
onstrated an anti-inflammatory activity similar to
that of compound 10.
9
10
11
12
8
100
230
250
192
1.3
—
13
14
24
25
—
—
59
2.2.2. Toxicity. Acute toxicity of BHT and compound 8
was estimated after the ip administration of equimolar
doses (1 mmol/kg body weight) to rats. In both groups,
all animals survived and looked normal both
macroscopically and by autopsy. These results indicate
that the synthesised compounds seem to possess a wide
safety margin.
BHT
NDGA
INDc
a Compound concentration 10 lM.
b Arachidonic acid 0.1 lM.
c IND 1 lM, arachidonic acid 1 lM.

G. N. Ziakas et al. / Bioorg. Med. Chem. 14 (2006) 5616–5624
5619
compounds showed significant inhibition of the COX-1
isoform with the exception of 11 and 12, the substituted
morpholine derivatives. Compound 8, the thiomorpho-
line amine derivative, was the most potent inhibitor,
with an IC50 value of 6.5 lM for COX-1 inhibition.
Compound 9, the thiomorpholine amide analogue, close-
ly followed 8. Phenyl substitution on the thiomorpholine
ring (10) reduced activity, in agreement with their in vivo
results. The same effect of ring substitution on COX-1
activity may also explain the lack of inhibitory action of
the morpholine derivatives 11 and 12. Proline derivatives
showed some activity against COX-1 isoform, although
diminished, compared to the thiomorpholine derivatives.
When arachidonic acid was used at a concentration of
100 lM, which is slightly higher than the saturating sub-
strate concentration, no inhibition was observed, under
the same experimental conditions. These results indicate
that the examined compounds act as competitive inhibi-
tors of COX-1, since inhibition can be overcome by
increasing substrate concentration.
ation of polyunsaturated fatty acids containing 1(Z),
4(Z) pentadiene system, to form hydroperoxides, which
are leukotriene precursors. The dioxygenase activity of
LOX displays a rather broad substrate specificity. The
IC50 values of compounds 8–14, BHT and nord-
ihydroguaiaretic acid (NDGA) towards soybean LOX
after 7 min of incubation are given in Table 2. The time
course of lipoxygenase activity, as affected by various
concentrations of four most potent compounds (8, 10,
11, and 12), is depicted in Figure 1. Most compounds
were more potent inhibitors than BHT, with the excep-
tion of 9, 13 and 14, which were the amide derivative of
thiomorpholine and the derivatives of substituted pro-
line, respectively. The most potent inhibitor was com-
pound 11, the amine derivative of substituted
morpholine, followed by compound 8, the amine deriv-
ative of thiomorpholine, and compounds 10 and 12
which had similar activity. These results indicate that
the amine derivatives are more potent inhibitors than
the respective amide analogues. In general, the substitut-
ed morpholine derivatives appeared to be the most po-
tent, followed by the thiomorpholine derivatives, while
the addition of a phenyl moiety to the thiomorpholine
ring slightly reduced activity.
Indomethacin, used as a reference, was a potent inhibi-
tor of both COX isoforms (59% and 33% for COX-1
and COX-2, respectively) at a lower concentration
(1 lM for COX-1 and 10 lM for COX-2) using 1 lM
concentration of AA for both COX isoforms.
2.3.3. Antioxidant activity. Reactive oxygen species may
be important contributors to tissue injury in inflamma-
tory diseases. Thus, antioxidant properties may contrib-
ute to the anti-inflammatory action of compounds.
2.3.2. Inhibition of lipoxygenase activity. Lipoxygenases
(LOX) catalyse the regio- and stereo-specific dioxygen-
1.00
1.00
Compound 8
Compound 11
C
C
2
0.75
0.50
0.25
0.00
0.75
3
1
0.50
4
6
2
4
0.25
0.00
5
0
100
200
300
400
0
100
200
300
400
Incubation time (sec)
Compound 10
Incubation time (sec)
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
0.00
Compound 12
C
C
2
2
5
4
6
5
6
7
7
0
100
200
300
400
0
100
200
300
400
Incubation time (sec)
Incubation time (sec)
Figure 1. Effect of various concentrations of compounds 8, 10, 11 and 12 on lipoxygenase activity. C, control; 1, 10 lM; 2, 50 lM; 3, 60 lM; 4,
75 lM; 5, 100 lM; 6, 125 lM; 7, 150 lM.

5620
G. N. Ziakas et al. / Bioorg. Med. Chem. 14 (2006) 5616–5624
BHT, a known strong antioxidant, was used as a parent
molecule and some of the final compounds 8–14 showed
antioxidant activity similar to this molecule. The antiox-
idant activity of these compounds was estimated by their
inhibition of rat hepatic microsomal membrane lipid
peroxidation and by their interaction with the N-centred
1,1-diphenyl-2-picrylhydrazyl (DPPH) stable free radi-
cal. The IC50 values of the final compounds in the above
tests are shown in Table 3.
were very potent inhibitors of lipid peroxidation, with
the amine derivative being slightly better. Compound
12 was the only amide derivative that had an antioxi-
dant activity. The substitution of the thiomorpholine
ring with a phenyl moiety slightly reduced activity
(compound 10). Finally, compound 13, the proline
derivative, lost its activity at a concentration of
300 lM, while the amide derivative of this group was
not active (compound 14). The inhibitory activity of
compounds 8, 10, 11 and 12 was time dependent and
it is demonstrated in Figure 2.
2.3.3.1. In vitro inhibition of lipid peroxidation. Com-
pound 8, the amine derivative of thiomorpholine, was
the most potent inhibitor, with an action comparable
to that of BHT. The amide derivative of thiomorpho-
line (compound 9) showed a low action which is lost
at a concentration of 500 lM. Compounds 11 and
12, which are the substituted morpholine derivatives,
2.3.3.2. In vitro interaction with the stable radical
DPPH. Compounds 8 and 11, the thiomorpholine and
substituted morpholine derivatives, showed the greatest
interaction with DPPH, 11 being the most potent, hav-
ing activity similar to BHT, while compound 13, which
had a substituted proline moiety, showed a diminished
activity. The interaction of these two compounds with
DPPH at various concentrations, which is time depen-
dent, is shown in Figure 3. Compound 10, the derivative
of the substituted thiomorpholine, showed a very low
activity only at an equimolar dose (0.2 mM). Com-
pounds 9, 11, and 14, which are the amide derivatives,
did not interact with DPPH.
Table 3. In vitro evaluation of antioxidant activity
Compound Inhibition of lipid
Interaction with DPPH
peroxidation IC50 (lM) (200 lM) IC50 (lM)
8
9
7.5
75
>750
22.5
18.5
19.5
95
>200
>200
55
10
11
12
13
14
BHT
>200
>500
>200
45
The results on lipid peroxidation largely correlate with
the effect on LOX. Thus, compound 11 is a LOX inhib-
itor and a potent antioxidant, while 9 demonstrated
weak LOX inhibition and negligible antioxidant proper-
>1000
5
Compound 8
2
2
1
Compound 11
C
2
4
C
1
2
1
5
7
3
4
0
0
0
0
10
20
30
40
50
10
20
30
40
50
time (min)
time (min)
Compound 10
2
2
1
0
Compound 12
C
2
C
4
5
4
1
6
6
7
7
0
0
0
10
20
30
40
50
10
20
30
40
50
time (min)
time (min)
Figure 2. Time course of lipid peroxidation as affected by compounds 8, 10, 11 and 12. C, control; 1, 5 lM; 2, 10 lM; 3, 12.5 lM; 4, 15 lM; 5, 20 lM;
6, 25 lM; 7, 30 lM.

G. N. Ziakas et al. / Bioorg. Med. Chem. 14 (2006) 5616–5624
5621
non-toxic, thus possibly possessing a wide safety
margin. Consequently, their design could offer more
alternatives to the medicinal treatment of inflammatory
conditions.
Compound 8
3
2
1
0
C
1
2
3
5
4. Experimental
4.1. Materials
6
All commercially available chemicals are of the appro-
priate purity. For the in vivo experiments, Fischer-344
rats (150–250 g) were used.
30
60
90
120
150
180
210
240
time (sec)
4.2. Synthesis
Compound 11
3
Melting points (mp) were obtained on a MEL-TEMP II
(Laboratory Devices) apparatus and are uncorrected.
Infrared (IR) spectra were recorded on a Perkin-Elmer
597 infrared spectrophotometer. Proton nuclear mag-
netic resonance (1H NMR) spectra were obtained with
a Bruker 400 MHz spectrometer. Chemical shifts are
reported in parts per million (d) relative to tetramethylsi-
lane (TMS), and signals are given as follows: s, singlet;
d, doublet; t, triplet; m, multiplet. Elemental analyses
are performed with a Perkin-Elmer 2400 CHN analyzer.
C
2
1
0
1
2
3
4
5
6
30
60
90
120
150
180
210
240
time (min)
4.2.1. Synthesis of the intermediates (1–7).
4.2.1.1. 2,6-Di-tert-butyl-4-chloromethyl-phenol (1)26.
Concentrated hydrochloric acid (1 mL, 0.001 mol) was
added to a slurry of 2,6-di-tert-butyl-4-hydroxymethyl-
phenol (0.786 g, 0.003 mol) in petroleum ether (40 mL)
and stirred at ambient temperature overnight under
nitrogen. The resulting two layers were separated and
the petroleum ether layer was washed with water, dried
(MgSO4) and evaporated to give 0.704 g of the chloride.
Yellow oil, yield 83%. IR (neat): 3625, 2950, 1430, 1230,
700 cmÀ1.1H NMR (CDCl3): d 1.45 (s, 18 H, CH3) d
4.55 (s, 2H, CH2Cl) d 5.25 (s, 1H, OH) d 7.15 (s, 2H
arom).
Figure 3. Time dependent interaction of DPPH (200 lM) with
compounds 8 and 11. C, control; 1, 25 lM; 2, 30 lM; 3, 50 lM; 4,
75 lM; 5, 100 lM; 6, 200 lM.
ties. In general, amines are more potent antioxidants
than their amide counterparts, in agreement with their
effect on LOX activity. Thus, these compounds may be
able to offer protection against free radical attack by
direct inhibition of lipid peroxidation as well as by
inhibiting LOX, which is considered to play a significant
contributory role in the generation of cellular oxidative
stress.25
4.2.1.2. 3,5-Di-tert-butyl-4-hydroxy-benzoyl chloride
(2)27. 3,5-Di-tert-butyl-4-hydroxy-benzoic acid (5 g,
0.020 mol) was added to a solution of NaOH (0.8 g,
0.020 mol) in 95% aqueous methanol and the mixture
was evaporated under reduced pressure, pulverised
and dried to yield 5.4 g of the corresponding salt.
The salt was treated with petroleum ether (50 mL),
cooled to 0 °C and then 10 mL of distilled thionyl
chloride was added to the mixture and the reaction
was left at room temperature for 2 h. The volatiles
were removed under reduced pressure below 50 °C
and the residue was treated with 100 mL of petroleum
ether. The mixture was filtered and the filtrate was
concentrated under reduced pressure yielding 4.7 g of
the acid chloride. White solid, yield 80%, mp 96 °C.
3. Conclusion
From the in vitro and in vivo experiments it could be
concluded that BHT has only antioxidant activity
and that substitution with a thiomorpholine, substi-
tuted thiomorpholine, morpholine or proline moiety
offered anti-inflammatory properties. The most potent
derivatives possess a thiomorpholine moiety and are
followed by the substituted morpholine compounds,
while the addition of a phenyl group to the thi-
omorpholine moiety reduced activity. Finally, the
introduction of a five-membered ring without the
presence of a sulfur or oxygen atom, like that of pro-
line, significantly reduced activity. It seems that the
in vivo anti-inflammatory activity correlates more
closely with COX inhibition, while LOX inhibitory
action and antioxidant activity appear to be
interrelated.
IR (Nujol): 3545, 1725, 1590 cmÀ1
.
4.2.1.3. 3-Phenyl-thiomorpholine (3)28. 2-Amino-etha-
nethiol hydrochloride (2.3 g, 0.02 mol) was added to a
solution of potassium hydroxide (2.2 g, 0.04 mol) in
50 mL of methanol cooled to 0–5 °C under nitrogen.
To this mixture a solution of 2-bromo-1-phenyl-etha-
All the synthesised compounds are more active anti-
inflammatory agents than BHT and they seem to be

5622
G. N. Ziakas et al. / Bioorg. Med. Chem. 14 (2006) 5616–5624
none (4 g, 0.02 mol) in 10 mL of methanol was added.
The reaction mixture was stirred below 5 °C for 1 h
and then acidified with 30% methanolic hydrochloric
acid. After stirring for an additional 1 h at 0 °C, sodium
borohydride (1.5 g, 0.04 mol) was slowly added and the
mixture was stirred for 30 min. The hydrolysis was car-
ried out with aqueous hydrochloric acid, the solvent was
evaporated to dryness and the residue was treated with
water. The aqueous layer was made alkaline with a sat-
urated solution of sodium bicarbonate and extracted
with chloroform. The extracts were dried and the sol-
vent was removed at reduced pressure to give the final
product. White solid, yield 52%, mp 62–64 °C. IR (Nu-
duced pressure. The acidification and evaporation pro-
cedures were repeated and the oily residue was dried
over P2O5 and NaOH overnight. The oil was crystallised
and the crystals were washed thoroughly with diethyl
ether. White solid, yield 29%, mp 151–152 °C. IR (Nu-
jol): 3400, 2760, 1750 cmÀ1
.
4.2.2. General procedure for the preparation of the amines
of 2,6-di-tert-butyl-4-(chloromethyl)phenol and the
amides of 3,5 di-tert-butyl-4-hydroxybenzoyl chloride
(8–14).31
A
solution
of
2,6-di-tert-butyl-4-
(chloromethyl)-phenol (1) or 3,5-di-tert-butyl-4-
hydroxybenzoyl chloride (2) (1 mol) was slowly
added to a solution of the appropriate amine (thi-
omorpholine, 3-phenyl-thiomorpholine, 2-ethoxy-2-
phenyl-morpholine, or pyrrolidine-2-carboxylic acid
methyl ester, 1 mol) and triethylamine (1.1 mol) in
diethyl ether (30 mL). The reaction mixture was stir-
red overnight at room temperature and filtered to
remove triethylamine hydrochloride. The filtrate was
then washed with a saturated aqueous NaCl solu-
tion, dried over CaCl2 and evaporated under re-
duced pressure. The final product, in the case of
amines, was either isolated as hydrochloride, recrys-
tallised from acetone/diethyl ether, or purified by
flash chromatography, eluting with petroleum ether/
ethyl acetate containing 1% triethylamine. Amides
were purified by flash chromatography, eluting with
petroleum ether/ethyl acetate.
jol): 2705, 2641, 2598, 2449 cmÀ1
.
4.2.1.4. 4-Benzyl-2-phenyl-morpholin-2-ol hydrobro-
mide 2-Bromo-1-phenyl-ethanone (5.2 g,
(4)29.
0.026 mol) was dissolved in a mixture of ethyl ether
and acetone 10:1 and added to a solution of 2-benzyla-
mino-ethanol (8.2 g, 0.054 mol) in diethyl ether. The
reaction mixture was stirred overnight at room temper-
ature. It was then filtered, the filtrate washed with a sat-
urated aqueous NaCl solution. The organic layer was
dried over potassium carbonate, concentrated and final-
ly the amine was acidified with a 10% solution of HBr in
diethyl ether to give the corresponding salt. The salt was
recrystallised from ethyl ether/acetone. White solid,
yield 88%, mp 150–151 °C. IR (Nujol): 3160, 2760,
2725, 2650, 2500, 2425, 1600 cmÀ1
.
4.2.1.5. 4-Benzyl-2-ethoxy-2-phenyl-morpholine hyd-
robromide (5)29. A solution of 4-benzyl-2-phenyl-mor-
pholin-2-ol hydrobromide (3 g, 0.0085 mol) in ethanol
(150 mL) was acidified with a 10% solution of HBr in
diethyl ether and the mixture was refluxed for 3 h. Then,
the solvent was removed under reduced pressure and the
residue was recrystallised from diethyl ether/ethanol to
give the final product. White solid, yield 93%, mp 163–
164 °C. IR (Nujol): 2725, 2675, 2650, 2625, 2580,
4.2.2.1. 2,6-Di-tert-butyl-4-thiomorpholin-4-ylmethyl-
phenol hydrochloride (8). White solid, purified by recrys-
tallisation from acetone/diethyl ether, yield 66%, mp
235–236 °C. IR (Nujol): 3624, 2700, 2650, 2470,
1
2406 cmÀ1. H NMR (CDCl3): d 1.39 (s, 18H, CH3) d
2.54–2.83 (m, 4H, CH2SCH2) d 3.63–3.70 (m, 4H,
CH2NCH2) d 4.01 (s, 2H, NCH2Ar) d 5.41 (s, 1H,
OH) d 7.27 (s, 2H arom) d 12.67 (s, 1H, HCl). Anal.
Calcd for C19H32ClNOS: C, 63.75; H, 9.01; N, 3.91.
Found: C, 63.46; H, 9.24, N, 3.87.
1600 cmÀ1
.
4.2.1.6. 2-Ethoxy-2-phenyl-morpholine hydrobromide
(6)29. 4-Benzyl-2-ethoxy-2-phenyl-morpholine hydrobr-
omide (2.7 g, 0.007 mol) was dissolved in absolute etha-
nol (150 mL), palladised charcoal (0.2 g, 10%) was
added and hydrogen was introduced with shaking. The
hydrogenation was stopped when the theoretical volume
of hydrogen was consumed. The solution was filtered,
the filtrate concentrated and alkalified with potassium
carbonate to obtain the free base, which was purified
by flash chromatography, eluting with ethyl acetate con-
taining 1% triethylamine. The purified base was then
acidified with a 10% solution of HBr in ethyl ether to ob-
tain the corresponding salt. White solid, yield 64%, mp
125–126 °C. IR (Nujol): 2760, 2725, 2650, 2500, 2425,
4.2.2.2.
(3,5-Di-tert-butyl-4-hydroxy-phenyl)-thio-
morpholin-4-yl-methanone (9)20. White solid, purified
by flash chromatography, eluting with petroleum
ether/ethyl acetate 6:1, yield 49%, mp 181–182 °C. IR
(Nujol): 3538, 1636, 1606 cmÀ1. H NMR (DMSO-d6):
1
d 1.45 (s, 18H, CH3) d 2.58–2.70 (m, 4H, CH2SCH2) d
3.75–3.90 (m, 4H, CH2NCH2) d 6.70 (s, 1H, OH) d
7.20 (s, 2H arom). Anal. Calcd for C19H29NO2S: C,
68.02; H, 8.71; N, 4.18. Found: C, 68.14; H, 9.13; N,
4.27.
4.2.2.3. 2,6-Di-tert-butyl-4-(3-phenyl-thiomorpholin-4-
ylmethyl)-phenol hydrochloride (10). White solid, puri-
fied by recrystallisation from acetone/diethyl ether,
yield 83%, mp 194–195 °C IR (Nujol): 3624, 2705,
1600 cmÀ1
.
2662, 2534, 2448, 1611 cmÀ1 1H NMR (CDCl3): d
.
4.2.1.7. Pyrrolidine-2-carboxylic acid methyl ester
(7)30. Pyrrolidine-2-carboxylic acid (11.5 g, 0.1 mol)
was dissolved in 125 mL of anhydrous methanol and
cooled to 0 °C. The solution was acidified with the addi-
tion of a 30% methanolic solution of HCl and stirred
overnight. The solution was then concentrated under re-
1.36 (s, 18H, CH3) d 2.56–2.62 (t, J = 3.0 Hz, 2H,
NCH2CH2S) d 2.93–2.96 (d, 1H, NCH(Ar)CH2S) d
3.73–3.87 (m, 3H, NCH2CH2S, NCH(Ar)CH2S) d
4.02–4.08 (t, J = 3.0 Hz, 3H, NCH(Ar)CH2S,
NCH2Ar) d 5.38 (s, 1H, OH) d 6.95–7.48 (m, 7H
arom)
d 12.86 (s, 1H, HCl). Anal. Calcd for

G. N. Ziakas et al. / Bioorg. Med. Chem. 14 (2006) 5616–5624
5623
C25H35NOS (base): C, 75.52; H, 8.87; N, 3.52. Found:
C, 75.05; H, 8.80; N, 3.17.
as control. The tested compounds (dissolved or sus-
pended in water with a few drops of Tween 80)
were given ip (0.28 mmol/kg of body weight) 5 min
prior to the carrageenan injection. After 3.5 h, the
hind paws were excised and weighed separately.
The produced edema was estimated as paw weight
increase.21
4.2.2.4. 2,6-Di-tert-butyl-4-(2-ethoxy-2-phenyl-mor-
pholin-4-ylmethyl)-phenol hydrochloride (11). White sol-
id, purified by flash chromatography, eluting with
petroleum ether, or by recrystallisation from ace-
tone/diethyl ether, yield 82%, mp 124–125 °C. IR
(Nujol): 3645, 2726, 2598, 2350, 1606 cmÀ1
.
1H
4.4. Toxicity
NMR (CDCl3): d 1.31–1.50 (m, 21H, OCH2CH3,
CH3) d 2.53–3.53 (m, 4H, OCH2CH2N, NCH2C(Ar)-
OCH2CH3) d 3.71–4.85 (m, 6H, OCH2CH3, NCH2Ar,
OCH2CH2N) d 5.46 (s, 1H, OH) d 7.27–7.61 (m, 7H
A dose of 1 mmol/kg of compound 8 and BHT was
administered ip to female animals and their toxicity
was evaluated by mortality, GI ulcers and body weight
change after 24 h.
arom)
d 12.66 (s, 1H, HCl). Anal. Calcd for
C27H40ClNO3: C, 70.18; H, 8.73; N, 3.03. Found:
C, 69.81; H, 8.44; N, 3.22.
4.5. In vitro evaluation of cyclooxygenase activity
(isoforms 1 and 2)
4.2.2.5. (3,5-Di-tert-butyl-4-hydroxy-phenyl)-(2-eth-
oxy-2-phenyl-morpholin-4-yl)-methanone (12). White sol-
id, purified by flash chromatography, eluting with
petroleum ether/ethyl acetate 6:1, yield 43%, mp
The effect of compounds 8–14 on COX-1 and COX-2
activity was measured using ovine COX-1 and human
recombinant COX-2 enzymes included in the ‘‘COX
Inhibitor Screening Assay’’ kit provided by Cayman
(Cayman Chemical Co., Ann Arbor, MI, USA). The as-
say directly measures PGF2a produced by SnCl2 reduc-
tion of COX-derived PGH2. The prostanoid product is
quantified via enzyme immunoassay using a broadly
specific antibody that binds to all the major prostaglan-
din compounds.
143–144 °C. IR (Nujol): 3624, 1645, 1607 cmÀ1
.
1H
NMR (CDCl3):
d 1.05–1.18 (t, J = 6.5 Hz, 3H,
OCH2CH3) d 1.34 (s, 18H, CH3) d 2.43–2.98 (m,
4H, OCH2CH3, OCH2CH2N) d 3.83–4.25 (m, 4H,
OCH2CH2N, NCH2C(Ar)OCH2CH3) d 5.68 (s, 1H,
OH) d 7.26–7.31 (m, 7H arom). Anal. Calcd for
C27H37NO4: C, 73.77; H, 8.48; N, 3.19. Found: C,
73.35; H, 8.12; N, 2.96.
The inhibitory activity of the compounds was measured
at various concentrations of arachidonic acid (0.1–
1 lM). The compounds were added to the reaction mix-
ture at a final concentration of 10 lM, unless otherwise
mentioned. COX, 10À9 IU/ml for both isoforms.
4.2.2.6. 1-(3,5-Di-tert-butyl-4-hydroxy-benzyl)-pyrrol-
idine-2-carboxylic acid methyl ester hydrochloride (13).
White solid, purified by flash chromatography, eluting
with petroleum ether/ethyl acetate 7:1 containing 1% tri-
ethylamine, or by recrystallisation from acetone/diethyl
ether, yield 69%, mp 150–151 °C. IR (Nujol): 3646,
4.6. In vitro evaluation of lipoxygenase activity
1
2727, 2683, 2598, 1748, 1611 cmÀ1. H NMR (CDCl3):
d 1.38 (s, 18H, CH3) d 1.95–2.70 (m, 4H, CH2CH2) d
3.35–3.40 (t, J = 2.5 Hz, 1H, NCHCOOCH3) d 3.72 (s,
5H, NCH2Ar COOCH3) d 4.20–4.40 (m, 2H, NCH2) d
5.36 (s, 1H, OH) d 7.34 (s, 2H arom) d 13.00 (s, 1H,
HCl). Anal. Calcd for C21H34ClNO3Æ0.3 H2O: C,
64.78; H, 8.96; N, 3.60. Found: C, 64.71; H, 8.91; N,
3.59.
The reaction mixture contained (final concentration)
the test compounds, dissolved in propyleneglycol at
concentrations of 10–450 lM, or the solvent (control),
soybean lipoxygenase, dissolved in 0.9% NaCl solution
(250 u/mL) and sodium linoleate (100 lM), in
Tris–HCl buffer, pH 9.0. The reaction was monitored
for 7 min at 28 °C, by recording the absorbance of a
conjugated diene structure at 234 nm, due to the
formation of 13-hydroperoxy-linoleic acid. The perfor-
mance of the assay was checked using nord-
ihydroguaiaretic acid as a reference.32
4.2.2.7. 1-(3,5-Di-tert-butyl-4-hydroxy-benzoyl)-pyr-
rolidine-2-carboxylic acid methyl ester (14). White solid,
purified by flash chromatography, eluting with petro-
leum ether/ethyl acetate 10:1, or by recrystallisation
from acetone/petroleum ether, yield 61%, mp 135–
4.7. In vitro lipid peroxidation
136 °C. IR (Nujol): 3581, 1765, 1620, 1602 cmÀ1
.
1H
NMR (CDCl3): d 1.37 (s, 18H, CH3) d 1.82–2.26 (m,
4H, CH2CH2) d 3.56–3.67 (m, 2H, NCH2) d 3.70 (s,
The incubation mixture contained heat-inactivated rat
hepatic microsomal fraction (corresponding to 2.5 mg
of hepatic protein per millilitre or 4 mM fatty acid
residues), ascorbic acid (0.2 mM) in Tris–HCl/KCl
buffer (50 mM/150 mM, pH 7.4) and the studied com-
pounds in dimethylsulfoxide at concentrations of
5 lM–1.0 mM. The peroxidation was started with the
3H, COOCH3)
d
4.55–4.59 (t, J = 2.0 Hz, 1H,
NCHCOOCH3) d 5.40 (s, 1H, OH) d 7.39 (s, 2H arom).
Anal. Calcd for C21H31NO4: C, 69.78; H, 8.64; N, 3.87.
Found: C, 69.31; H, 8.44; N, 3.62.
4.3. Carrageenan paw edema
addition of
a
freshly prepared FeSO4 solution
(10 lM), and aliquots were taken from the incubation
mixture (37 °C) at various time intervals for 45 min.
Lipid peroxidation was assessed by spectrophotomet-
ric (535 against 600 nm) determination of the 2-thio-
An aqueous solution of carrageenan was prepared
(1% w/v) and 0.1 mL of this was injected into the
right hind paw of male rats, the left paw serving

5624
G. N. Ziakas et al. / Bioorg. Med. Chem. 14 (2006) 5616–5624
17. Song, Y.; Connor, D. T.; Doubleday, R.; Sorenson, R. J.;
Sercel, A. D.; Unangst, P. C.; Roth, B. D.; Gilbertsen, R.
B.; Chan, K.; Schrier, D. J.; Guglietta, A.; Bornemeier, D.
A.; Dyer, R. D. J. Med. Chem. 1999, 42, 1151.
18. Song, Y.; Connor, D. T.; Sercel, A. D.; Sorenson, R. J.;
Doubleday, R.; Unangst, P. C.; Roth, B. D.; Beylin, V. G.;
Gilbertsen, R. B.; Chan, K.; Schrier, D. J.; Guglietta, A.;
Bornemeier, D. A.; Dyer, R. D. J. Med. Chem. 1999, 42,
1161.
19. Inagaki, M.; Tsuri, T.; Jyoyama, H.; Ono, T.; Yamada,
K.; Kobayashi, M.; Hori, Y.; Arimura, A.; Yasui, K.;
Ohno, K.; Kakudo, S.; Koizumi, K.; Suzuki, R.; Kato,
M.; Kawai, S.; Matsumoto, S. J. Med. Chem. 2000, 43,
2040.
barbituric acid reactive material. All compounds and
solvents were tested and found not to interfere with
the assay.33
4.8. In vitro interaction with the stable radical DPPH
Compounds, dissolved in absolute ethanol, at concentra-
tions of 25–200 lM, were added to an equal volume of an
ethanolic solution of DPPH (final concentration 200 lM)
at room temperature (22 2 °C). Absorbance (517 nm)
was recorded at different time intervals for 4 h.34
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