Thiomorpholine Derivatives with Hypolipidemic and Antioxidant Activity

Article abstract of DOI:10.1002/ardp.201500147

A number of thiomorpholine derivatives that are structurally similar to some substituted morpholines possessing antioxidant and hypocholesterolemic activity were synthesized. The new compounds incorporate an antioxidant moiety as the thiomorpholine N-subs

Full text of DOI:10.1002/ardp.201500147

RCHH HARM
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Arch. Pharm. Chem. Life Sci. 2015, 348, 629–634
Archiv der Pharmazie
Full Paper
Thiomorpholine Derivatives with Hypolipidemic and
Antioxidant Activity
Kyriaki-Konstantina Tooulia, Panagiotis Theodosis-Nobelos, and Eleni A. Rekka
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki,
Thessaloniki, Greece
A number of thiomorpholine derivatives that are structurally similar to some substituted morpholines
possessing antioxidant and hypocholesterolemic activity were synthesized. The new compounds incorporate
an antioxidant moiety as the thiomorpholine N-substituent. The derivatives were found to inhibit the
ferrous/ascorbate-induced lipid peroxidation of microsomal membrane lipids, with IC50 values as low as
7.5 mM. In addition, these compounds demonstrate hypocholesterolemic and hypolipidemic action. The most
active compound (5) decreases the triglyceride, total cholesterol, and low-density lipoprotein levels in the
plasma of Triton WR-1339-induced hyperlipidemic rats, by 80, 78, and 76%, respectively, at 56 mmol/kg (i.p.).
They may also act as squalene synthase inhibitors. The above results indicate that the new molecules may be
useful as leads for the design of novel compounds as potentially antiatherogenic factors.
Keywords: Antioxidants / Cholesterol / Thiomorpholines / Triglycerides
Received: May 1, 2015; Revised: June 2, 2015; Accepted: June 23, 2015
DOI 10.1002/ardp.201500147
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
thus, they are still poorly controlled, mainly because the origin
of atherosclerosis is still not completely understood [3].
We have reported [4] that a number of properly substituted
Introduction
Atherosclerosis, a condition affecting arterial blood vessels, is
the main risk factor for cardiovascular disease, one of the most
common diseases in the modern western world. Hyperlipid-
emia, especially elevated levels of low-density lipoprotein
cholesterol (LDL), can lead to the formation of multiple
plaques within the artery. Furthermore, dyslipidemia is one of
the main alterations characterizing the metabolic syn-
drome [1]. Oxidation of LDL promotes inflammatory response,
which includes impaired vasomotor function, platelet, and
leukocyte adhesion to endothelial cells and increased free
radical production, leading to plaque vulnerability [2].
Dyslipidemias are not adequately diagnosed and treated,
morpholines acquired antioxidant activity and were potent
anti-dyslipidemic agents. Some of them possessed, in addition,
nitric oxide-donating activity. Selected structures strongly
inhibited squalene synthase activity and LDL oxidation. They
also inhibit atherosclerotic lesions in the cholesterol-fed rabbit.
In this investigation, we synthesized and studied a number of
substituted thiomorpholines, replacing the morpholine oxygen
by a sulfur atom. Further changes involve the introduction of an
antioxidantmoietyasthethiomorpholineN-substituent,inorder
to augment the antioxidant potential of these molecules, as well
as of a nicotinic acid residue, considering that nicotinic acid is a
potent treatment clinically available, since it possesses both anti-
dyslipidemic activity and lipid-independent actions on vascular
endothelial oxidative and inflammatory events [5].
Results and discussion
Correspondence: Dr. Eleni A. Rekka, Department of Pharmaceu-
tical Chemistry, School of Pharmacy, Aristotelian University of
Thessaloniki, Thessaloniki 54124, Greece
E-mail: rekka@pharm.auth.gr
Chemistry
The preparation of the compounds is demonstrated in
Fax: þ30-2310997852
Schemes 1–4.
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Scheme 1. Synthesis of compounds 1–3.
3-Arylthiomorpholines were prepared by the hydrogenation
of the intermediate imine, obtained from the reaction of 2-
aminoethanethiol (1, 2) or D-penicillamine (3) with 2-bromo-
1-phenyl-ethanone or 2-bromo-1-(4-phenyl)phenyl-ethanone
in alkaline environment (Scheme 1). 2-Phenyl-thiomorpholine
(4), prepared as described in the literature, gave compounds 8
and 9, with reaction of the corresponding acid chlorides in the
presence of triethylamine (Scheme 2). Similarly, 3-aryl-
thiomorpholines or unsubstituted thiomorpholine reacted
with the related acyl or alkyl chlorides in the presence of
triethylamine, to give compounds 5–7 and 10–13 (Scheme 3).
Compounds 14–16 were prepared from 2-hydroxy-substituted
morpholine and the corresponding alcohol with reflux in
acidic environment (Scheme 4).
The highest hypolipidemic activity was offered by compound
5. Lower than 50% reduction of the lipidemic indices was
caused by 3, 12, and 16. The low activity of 3 and 16 could be
attributed to their low lipophilicity (Table 1). Compound 12,
however, is quite lipophilic, with a clogP value close to that of
the most active 5. We have reported that a biphenyl moiety at
position 2 of morpholines is needed for hypolipidemic
activity, since they are considered to inhibit squalene synthase
(SQS) activity [4]. This is further verified in this work,
compound 15 is a potent SQS inhibitor (IC50 0.05 mM), the
IC50 value of 14 is 19.0 mM, while 16 is lacking this activity.
The case may not be completely the same for the thiomorpho-
line derivatives, although 3-[(4-phenyl)phenyl]thiomorpho-
line (2) is found to inhibit SQS activity with IC50 0.40 mM, since
the monophenyl-substituted
8 and 9 are quite active
hypolipidemics. Still, both are very potent inhibitors of lipid
peroxidation (IC50 15 mM). The implication of antioxidant
activity in hypolipidemic potential is further supported by the
high hypolipidemic action of 13, comparable to that of 7, with
no phenyl substitution on the thiomorpholine ring. Com-
pound 13 is the most potent antioxidant in this series, and this
property may add to the hypolipidemic activity, since
oxidative stress plays an important role in LDL oxidation
and the pathogenesis of atherosclerosis [8–10].
Biological activity
The effect of the synthesized compounds on plasma lipids of
hyperlipidemic rats and on the in vitro peroxidation of rat
hepatic microsomal membrane lipids is shown in Table 1. Most
compounds reduced triglyceride, total cholesterol, and
LDL-cholesterol levels more than 50%. Furthermore, most
of them could effectively inhibit lipid peroxidation.
Atherosclerosis is a complex disorder of lipid metabolism
and a chronic inflammatory disease. Changes in lipoprotein
metabolism and hyperlipidemia play an important role in the
progression of atherosclerosis and are significant in predicting
the development of cardiovascular events as risk factors [6].
The Triton-1339-induced hyperlipidemia is characterized by
a dramatic increase of serum concentration of cholesterol
and especially triglycerides, the latter due to surfactant-
mediated inhibition of lipoprotein lipase activity. Triton-
induced hyperlipidemia occurs 24 h after the administration.
It has been shown that Triton produces significant increase
in the concentrations of atherogenic C-LDL, C-VLDL, and IDL in
mice and rats [7].
Compound 10, with hypolipidemic activity similar to that of
9, is a weaker antioxidant than 9, while 7, a very potent
hypolipidemic agent, has no antioxidant effect. Part of their
activity may be due to their N-substituent, a cinnamic acid
residue, since cinnamaldehyde [11], cinnamic acid [12], and
some cinnamic acid derivatives [13] have been reported to
possess antidyslipidemic activity.
In conclusion, properly substituted thiomorpholine
derivatives possess antidyslipidemic activity, which in most
cases seems to be connected with a biphenyl substituent.
We suggest that part of the mechanism of action of
Scheme 2. Synthesis of compounds 4, 8, and
9.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 629–634
Hypolipidemic and Antioxidant Thiomorpholines
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Scheme 3. Synthesis of compounds 5–7 and 10–13.
biphenyl-morpholine or biphenyl-thiomorpholine deriva-
tives may be due to their squalene synthase inhibitory
activity [4]. One of the limiting stages of cholesterol
synthesis occurs in the endoplasmic reticulum and is
catalyzed by squalene synthase. Inhibition of squalene
synthase leads to a reduction of cholesterol synthesis
without affecting the synthesis of other nonsterol products
of the mevalonate metabolism. The biphenyl moiety may
mimic the lipophilic end of farnesyl pyrophosphate, as is
the case of 3-(biphenyl-4-yl)-3-hydroxyquinuclidine [14].
The hypolipidemic activity may be further enhanced by the
antioxidant properties of these compounds, expected to
inhibit LDL oxidation. Furthermore, compounds causing a
more than 50% reduction of total cholesterol have an
analogous effect on plasma triglyceride levels. This is
considered significant, because, currently, there is still a
need for a safe and effective treatment for the reduction of
both cholesterol and triglycerides, without the risk of
undesired effects, such as rhabdomyolysis. The synthesized
type of compounds may add to this direction.
Experimental
General
All commercially available reagents were purchased from
Merck or Sigma and used without further purification. The IR
spectra were recorded on a Perkin Elmer Spectrum BX FT-IR
spectrometer. The 1H NMR spectra were recorded using a
Bruker AC-400 MHz spectrometer. All chemical shifts are
reported in d (ppm) and signals are given as follows: s, singlet;
d, doublet; t, triplet; m, multiplet. Melting points (mp) were
determined with a MEL-TEMPII (Laboratory Devices, USA)
apparatus and are uncorrected. The microanalyses were
performed on a Perkin-Elmer 2400 CHN elemental analyzer
and were found within ꢀ0.4% of the theoretical values. Male
Fischer-344 rats (220–240 g body weight), about 3 months old,
were kept in a controlled temperature room (22 ꢀ 2°C), with
free access to laboratory chow and tap water, under a 12 h
light/dark cycle, and treated according to the European
Communities Council Directive of 24 November 1986
(86/609/EEC).
Scheme 4. Synthesis of compounds 14–16.
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Table 1. Effect of the synthesised compounds on lipid peroxidation, on lipidemic indices in hyperlipidemic rats and their
calculated lipophilicity (clogP).
Lipid
Percent reduction of lipidemic indicesa)
peroxidation
inhibition:
IC50 (mM)
Compound
TG
TC
LDL
clogP
2
3
5
6
7
8
9
10
11
12
13
14
15
16
450
30
200
100
–
62ꢃꢃ
–
35ꢃꢃ
14ꢃ
76ꢃꢃ
30ꢃ
3.83
3.04
8.71
4.24
6.09
6.39
4.06
2.50
4.84
9.05
5.41
4.26
1.87
ꢁ0.02
80ꢃꢃ
53ꢃꢃ
70ꢃꢃ
52ꢃꢃ
62ꢃꢃ
77ꢃꢃ
70ꢃꢃ
20ꢃ
78ꢃꢃ
65ꢃꢃ
70ꢃꢃ
80ꢃꢃ
50ꢃꢃ
85ꢃꢃ
66ꢃꢃ
20ꢃ
76ꢃꢃ
66ꢃꢃ
82ꢃꢃ
76ꢃꢃ
72ꢃꢃ
65ꢃꢃ
44ꢃ
15
15
350
>750
23
7.5
500
120
300
25ꢃ
62ꢃꢃ
35ꢃꢃ
85ꢃꢃ
45ꢃꢃ
82ꢃꢃ
36ꢃꢃ
63ꢃꢃ
20ꢃ
70ꢃꢃ
52ꢃꢃ
47ꢃꢃ
43ꢃꢃ
Asterisks indicate statistical significance (Student’s t-test) of the absorbance values as follows: ꢃꢃP < 0.005; ꢃP < 0.05. Each group is
composed of five to six rats. Results are from two to three independent experiments. TG, triglycerides; TC, total cholesterol; LDL,
low density lipoprotein cholesterol.
a)All determinations are performed at least in duplicate and SD is always within ꢀ10% of the absorbance values.
Synthesis
General method for the synthesis of 3-aryl-
arom.). Anal. calcd. for C16H18ClNS: C 65.85, H 6.22, N 4.80;
Found: C 65.80, H 6.25, N 4.60%.
thiomorpholines
2-Amino-ethanethiol hydrochloride (0.02 mol) was added to a
solution of potassium hydroxide (0.04 mol) in 50 mL of
methanol cooled to 0–5°C under nitrogen. To this mixture,
a solution of 2-bromo-1-aryl-ethanone (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 hydro-
chloric acid. After stirring for an additional 1 h at 0°C, sodium
borohydride (0.04 mol) was slowly added and the mixture was
stirred for 30 min. The hydrolysis was carried 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 saturated solution of sodium bicarbon-
ate and extracted with chloroform. The extracts were dried
(K2CO3) and the solvent was removed at reduced pressure to
give the final products.
2,2-Dimethyl-5-[(4-phenyl)phenyl]thiomorpholine-3-
carboxylic acid (3)
Obtained as the hydrochloride from acetone-diethyl ether,
starting from 2-bromo-1-(4-phenyl)phenyl-ethanone and
D-penicillamine ((2S)-2-amino-3-methyl-3-sulfanyl-butanoic
acid) hydrochloride. White solid, yield 88%, mp 234–236°C.
IR (Nujol): 3389, 2450, 1744, 1606 cmꢁ1. 1H NMR (DMSO-d6) d:
1.5 (s, 6H, CH3), 2.7 (d, 1H, C-6ax), 3.55 (t, 1H, C-5), 3.4–3.9 (bs,
1H, NH), 4.15 (s, 1H, C-3), 4.4 (d, 1H, C-6eq), 7.2–7.4 (m, 3H,
arom), 7.5–7.6 (m, 4H, arom), 7.75–7.85 (d, 2H, arom). Anal.
calcd. for C19H22ClNO2S ꢂ 0.545H2O: C 61.06, H 6.23, N 3.75;
Found: C 61.05, H 6.04, N 3.69%.
2-Phenyl-thiomorpholine (4)
Prepared as described in the literature [16], from 2-amino-
ethanethiol hydrochloride and methyl a-bromophenyl ace-
tate, followed by reduction of the intermediate lactam with
sodium borohydride. Yield 90%, mp 59–60°C.
3-Phenyl-thiomorpholine (1)
Prepared from 2-bromo-1-phenyl-ethanone. White solid, yield
52%, mp 62–64°C. IR (Nujol): 2705, 2641, 2598, 2449 cmꢁ1 [15].
General method for the preparation of amide derivatives
of thiomorpholines
3-[(4-Phenyl)phenyl]thiomorpholine (2)
Obtained as the hydrochloride from acetone-diethyl ether,
To a solution or suspension of the appropriate thiomorpho-
line (4 mmol) in dry diethylether (20 mL) triethylamine
(4.4 mmol) was added. Then, the corresponding acyl chloride
(4 mmol), in dry ether (40 mL) was added, the mixture was
stirred at room temperature (3–12 h), filtered and the filtrate
starting from 2-bromo-1-(4-phenyl)phenyl-ethanone. White
solid, yield 78%, mp 191–193°C. IR (Nujol): 2452, 1577 cmꢁ1
.
1H NMR (CDCl3) d: 2.1–2.7 (d, 2H, C-6), 2.9–3.5 (m, 4H, C-2, C-5),
4.0–4.3 (t, 1H, C-3), 7.2–7.3 (m, 5H, arom.), 7.4–7.6 (m, 4H,
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was concentrated, washed with water, dried (K2CO3), the
solvent was distilled off and the final product was received
with flash chromatography of recrystallization.
4-Cinnamoyl-thiomorpholine (10)
Prepared from thiomorpholine and cinnamoyl chloride,
recrystallized from dichloromethane and diethyl ether.
Yellow solid, yield 95%, mp. 144–148°C. IR (Nujol): 1642,
1595 cmꢁ1. 1H NMR (CDCl3) d: 2.60–2.75 (m, 4H, C-2, C-6), 3.86–
3-[(4-Phenyl)phenyl]-4-(3,5-di-t-butyl-4-hydroxy-benzoyl)-
thiomorpholine (5)
–
–
4.05 (m, 4H, C-3, C-5), 6.80–6.90 (d, 1H, –CH CH–phenyl), 7.25–
–
Prepared from 2 and 3,5-di-t-butyl-4-hydroxy-benzoyl chlo-
ride and isolated with flash chromatography, using petroleum
ether/ethyl acetate 2:1. White solid, yield 60%, mp 131–134°C.
7.74 (m, 6H, 5Harom, –CH CH–phenyl). Anal. calcd. for
–
C
13H15NOS: C 66.92, H 6.48, N 6.00; Found: C 67.16, H 6.83,
N 5.69%.
IR (Nujol): 3615, 1680, 1598 cmꢁ1 1H NMR (CDCl3) d: 1.5 (s.
.
18H, CH3), 3.4–3.55 (m, 4H, C-2, C-6), 3.6–3.7 (m, 1H, C-5ax),
3.8–3.9 (m, 1H, C-5eq), 4.5–4.7 (m, 1H, C-3), 5.7 (s, 1H, OH), 7.2–
7.4 (m, 3H, arom), 7.5–7,6 (m, 4H, arom), 7.8–7.9 (d, 2H, arom),
8.15 (s, 2H, arom). Anal. calcd. for C31H37NO2S: C 76.35, H 7.65,
N 2.87; Found: C 76.31, H 7.70, N 2.85%.
2-[(4-Phenyl)phenyl]-2-(2-methylthio)ethoxy-4-
methylmorpholine (14)
Prepared by heating (12 h) 2-[(4-phenyl)phenyl-4-methylmor-
pholin-2-ol [17] (0.01 mol) and 2-(methylthio)ethanol
(0.06 mol) in dry, acidified acetone (60 mL) and obtained as
hydrochloride. White solid, yield 85%, mp 157–158°C. 1H NMR
(DMSO-d6) d: 1.7 (m, 1H), 2.1 (s, 3H, S-CH3), 2.7 (d, 1H), 2.8–3.0
(m, 5H), 3.3 (m, 2H), 3.6 (m, 3H), 4.1 (d, 1H), 4.6 (t, 1H), 7.2–7.65
(m, 9Harom). Anal. calcd. for C20H26ClNO2S: C 63.22, H 6.90, N
3.69; Found: 62.73, H 7.03 N 3.56.
3-[(4-Phenyl)phenyl]-4-nicotinoylthiomorpholine (6)
Prepared from 2 and nicotinoyl chloride and isolated with
flash chromatography, using petroleum ether/ethyl acetate
1:1. White solid, yield 70%, mp 139–142°C. IR (Nujol): 1680,
1598 cmꢁ1. 1H NMR (CDCl3) d: 2.4–2.6 (m, 2H, C-6), 2.8–3.0 (m,
2H, C-2), 3.1–3.2 (m, 1H, C-5ax), 3.4–3.5 (m, 1H, C-5eq), 3.9–4.1
(t, 1H, C-3), 7.3–7.8 (m, 13H, arom). Anal. calcd. for
The following compounds: 4-cinnamoyl-thiomorpholine
(10) [18], (3,5-di-t-butyl-4-hydroxy-benzoyl)thiomorpholine
(11),
2,6-di-t-butyl-4-(3-phenyl-thiomorpholin-4-ylmethyl)-
C
22H20N2OS: C 73.30, H 5.59, N 7.77; Found: C 73.40, H 5.69,
phenol (12), 2,6-di-t-butyl-4-thiomorpholin-4-ylmethyl-phe-
nol (13) [15], 2-(4-biphenyl)-2-(3-nitrooxypropoxy)-4-methyl-
morpholine (15), and 2-phenyl-2-(3-nitrooxypropoxy)-4-
methylmorpholine (16) [19] were prepared as reported
earlier.
N 7.40%.
3-[(4-Phenyl)phenyl]-4-cinnamoylthiomorpholine (7)
Prepared from 2 and cinnamoyl chloride ((E)-3-phenylprop-2-
enoic acid chloride) and purified with recrystallization from
dichloromethane and diethyl ether. White solid, yield 65%,
Biological activity
mp 137–140°C. IR (KBr): 1643, 1597 cmꢁ1
.
1H NMR (CDCl3) d:
In vivo evaluation of hypolipidemic activity
An aqueous solution of Triton WR 1339 was administered i.p.
to rats (200 mg/kg), [19] and 1 h later the examined
compounds (56 mmol/kg) dissolved in saline or saline only
were given i.p. In all cases, 24 h after the administration of
Triton, blood was taken from the aorta and used for the
determination of plasma total cholesterol, LDL, and triglycer-
ide concentrations.
3.45–3.55 (m, 4H, C-2, C-6), 3.7–3.8 (m, 1H, C-5ax), 3.8–3.9 (m,
1H, C-5eq), 4.8–4.95 (m. 1H, C-3), 7.2–7.65 (m, 16H, 14arom,
–CH CH–). Anal. calcd. for C25H23NOS: C 77.89, H 6.01, N 3.63;
–
–
Found: C 77.92, H 6.06, N 3.50%.
2-Phenyl-4-(3,5-di-t-butyl-4-hydroxy-benzoyl)-
thiomorpholine (8)
Prepared from 3 and 3,5-di-t-butyl-4-hydroxy-benzoyl chlo-
ride and recrystallized from acetone and diethyl ether. White
solid, yield 80%, mp 147–149°C. IR (Nujol): 3615, 1651,
1598 cmꢁ1. 1H NMR (CDCl3) d: 1.4 (s, 18H, CH3), 3.4–3.8 (m, 7H,
C-2, C-3, C-5, C-6), 5.3 (s, 1H, OH), 7.2–7.5 (m, 7H, arom). Anal.
calcd. for C25H33NO2S: C 72.95, H 8.08, N 3.40; Found: C 72.92,
H 8.50, N 3.13%.
In vitro lipid peroxidation
The incubation mixture contained heat-inactivated hepatic
microsomal fraction from untreated rats (corresponding to
2.5 mg of hepatic protein per milliliter or 4 mM fatty acid
residues) [20], ascorbic acid (0.2 mM) in Tris-HCl/KCl buffer
(50 mM, 150 mM, pH 7.4), and the studied compounds in
dimethyl sulfoxide (DMSO) at various concentrations. The
peroxidation reaction was started with FeSO4 solution (10 mM),
and aliquots were taken from the incubation mixture (37°C) at
various time intervals for 45min. Lipid peroxidation was
assessed by spectrophotometric (535 against 600 nm) determi-
nation of the 2-thiobarbituric acid reactive material consisting
mainlyofmalondialdehyde,anendproductofpolyunsaturated
lipidperoxidation[19]. Allcompoundsandsolventsweretested
and not found to interfere with the assay.
2-Phenyl-4-cinnamoylthiomorpholine (9)
Prepared from 3 and cinnamoyl chloride, recrystallized from
dichloromethane and diethyl ether. White solid, yield 70%,
mp 145–148°C. IR (KBr): 1643, 1597 cmꢁ1 1H NMR (CDCl3) d:
.
2.5–2.65 (m, 2H, C-6), 2.9–3.0 (m. 2H, C-5), 3.3-3.4 (m, H, C-3ax),
3.5–3.7 (m. H, C-3eq), 4.0–4.15 (t, 1H, C-2), 7.3–7.5 (m, 12H,
–
10arom, –CH CH–). Anal. calcd. for C19H19NOS: C 73.75, H
–
6.19, N 4.35; Found: C 73.85, H 6.39, N 4.25%.
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K.-K. Tooulia et al.
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In vitro squalene synthase activity evaluation
Squalene synthase (SQS) activity was evaluated by determin-
ing the amount of [3H]farnesyl pyrophosphate converted to
squalene as previously described [21]. IC50 values of the tested
compounds 2, 14, 15, 16 represent the mean concentration of
compounds that inhibits the activity of the enzyme by 50%.
All standard errors are within 10% of the respective reported
values.
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