Hypocholesterolemic and hypolipidemic activity of some novel morpholine derivatives with antioxidant activity

Article abstract of DOI:10.1021/jm991039l

In this investigation, we study the synthesis and the evaluation of antioxidant and hypocholesterolemic activity of a number of 2-biphenylyl morpholine derivatives, which are structurally similar to some substituted morpholines possessing antioxidant activity, as well as to hypocholesterolemic 3-biaryl-quinuclidines. The novel derivatives are found to inhibit the ferrous/ascorbate induced lipid peroxidation of microsomal membrane lipids, the most potent derivative, 2-(4-biphenyl)-4-methyl- octahydro-1,4-benzoxazin-2-ol (compound 7), having an IC₅₀ value of 250 μM. In addition, these compounds demonstrate hypocholesterolemic and hypolipidemic action. The most active compound (7) decreases total cholesterol, low density lipoprotein, and triglycerides in plasma of Triton WR-1339 induced hyperlipidemic rats by 54%, 51%, and 49%, respectively, at 28 μmol/kg (ip). The above results indicate that the new molecules may be proven useful as leads for the design of novel compounds as potentially antiatherogenic factors.

Full text of DOI:10.1021/jm991039l

J . Med. Chem. 2000, 43, 609-612
609
Hyp och olester olem ic a n d Hyp olip id em ic Activity of Som e Novel Mor p h olin e
Der iva tives w ith An tioxid a n t Activity
Michael C. Chrysselis, Eleni A. Rekka, and Panos N. Kourounakis*
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki,
Thessaloniki 540 06, Greece
Received March 12, 1999
In this investigation, we study the synthesis and the evaluation of antioxidant and hypocho-
lesterolemic activity of a number of 2-biphenylyl morpholine derivatives, which are structurally
similar to some substituted morpholines possessing antioxidant activity, as well as to
hypocholesterolemic 3-biaryl-quinuclidines. The novel derivatives are found to inhibit the
ferrous/ascorbate induced lipid peroxidation of microsomal membrane lipids, the most potent
derivative, 2-(4-biphenyl)-4-methyl-octahydro-1,4-benzoxazin-2-ol (compound 7), having an IC50
value of 250 µM. In addition, these compounds demonstrate hypocholesterolemic and hypo-
lipidemic action. The most active compound (7) decreases total cholesterol, low density
lipoprotein, and triglycerides in plasma of Triton WR-1339 induced hyperlipidemic rats by 54%,
51%, and 49%, respectively, at 28 µmol/kg (ip). The above results indicate that the new molecules
may be proven useful as leads for the design of novel compounds as potentially antiatherogenic
factors.
Ch a r t 1
In tr od u ction
Hypercholesterolemia appears to be a serious risk
factor for atheromatosis. High levels of low-density
lipoprotein (LDL) are recognized as the initiating event
in atherogenesis. LDL can undergo extensive lipid
peroxidation, resulting in the generation of modified
LDL and the formation of atheromatic lesions. Conse-
quently, antioxidants such as R-tocopherol or caro-
tenoids have been found to reduce LDL oxidation.1
Furthermore, compounds that lower LDL and/or tri-
glyceride levels could be useful in the treatment of
atheromatosis.2,3 Considering the above, we found it
interesting to design molecules that would combine
antioxidant and hypocholesterolemic activity. We have
already studied the synthesis of several morpholine
derivatives and their action as potential inhibitors of
lipid peroxidation.4,5 Moreover, the hypocholesterolemic
potency of some substituted quinuclidines has been
reported.6 The latter compounds possess structural and
physicochemical similarities with the antioxidant mor-
pholines.
Resu lts a n d Discu ssion
Ch em istr y. The synthesis of the compounds is shown
in Scheme 1. The synthesis of the 2-hydroxy-2-(4-
biphenyl)morpholines 6-8 involves the production of an
intermediate hydroxyaminoketone, which undergoes
ring closure to the cyclic hemiketal (average yield 80%).
The derivatives 9-11 are formed from the hemiketals
in an acid catalyzed ketal formation process (overall
yield approximately 60%).
Structures are confirmed spectroscopically and by
elemental analysis. There is no evidence (TLC, IR, UV)
to suggest the presence of the keto alcohol tautomer of
compounds 6-8.
In this study, we report the synthesis of novel 2-bi-
phenylmorpholine derivatives and their antioxidant and
hypocholesterolemic activities. The examined param-
eters are the inhibition of lipid peroxidation in vitro and
their effect on plasma total cholesterol, LDL, and
triglyceride levels. To investigate the effect of lipophi-
licity on lipid peroxidation, we introduced in our study
the previously synthesized7,8 morpholines 1 and 2
(Chart 1). The objectives are to gain information about
the structural features which are important for the
specific actions and to draw further conclusions con-
cerning the potential use of antioxidants against athero-
matosis.
Lip id P er oxid a tion . The effect of different concen-
trations of the examined compounds on lipid peroxida-
tion is given in Figure 1A-D. The observed inhibition
of lipid peroxidation is time and concentration depend-
ent. After 30 min of incubation, compounds 1 and 2 (1
mM) cause 70% and 40% inhibition, respectively, while
compounds 6-11 (1 mM) can completely inhibit the
peroxidation reaction. At 0.5 mM, compounds 6 and 10
inhibit lipid peroxidation by 96% and 78%. The most
active antioxidant is compound 7, having an IC50 of 250
µM after 30 min of incubation, while at the above
concentration (250 µM) and under our experimental
conditions, probucol inhibits lipid peroxidation about
16%.
Hemiketals 6-8, although less lipophilic, are better
inhibitors of lipid peroxidation than the corresponding
* Author for correspondence. Tel: (3031) 997621. Fax: (3031)
997622. E-mail: xriselis@pharm.auth.gr.
10.1021/jm991039l CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/01/2000

610 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Chrysselis et al.
Sch em e 1a
sclerosis.11 Atheromatosis is connected with increased
levels of cholesterol, especially LDL, as well as trigly-
cerides. Since LDL constitutes the main cholesterol
carrier of the plasma, it influences cholesterol plasma
concentrations and is closely related to the aetiology of
atherosclerosis. Oxygen derived radicals attack LDL,
and this oxidatively modified LDL leads to the formation
of foam cells and atheromatic plaques.
The synthesized derivatives can considerably inhibit
lipid peroxidation. They also possess a significant hy-
pocholesterolemic and hypolipidemic effect. Probucol, a
lipophilic antioxidant agent used in the treatment of
atheromatosis,12 demonstrates a minor effect on cho-
lesterol, triglycerides, and LDL, in our experimental
protocols. Therefore, it could be concluded that the
design of these molecules, combining hypolipidemic and
antioxidant activities, could be proven useful in the
development of new, potentially antiatherogenic agents.
In addition, further work is underway to assess the
inhibitory activity after per os administration of the
synthesized compounds.
a
Reagents and conditions: (i) 4-bromoacetylbiphenyl, acetone,
room temperature, 15 h; (ii) gaseous HCl in Et2O, EtOH, or
n-PrOH, reflux, 12-15 h.
ketals 9-11. However, it is known that the lipophilic
character of antioxidant molecules is an important
factor for protection against lipid peroxidation.9
Ketals 1, 9, and 2 have increasing clogP values10 (3.98,
4.47, 5.06, respectively) and different substituents on
the phenyl ring (Br, phenyl, H). It is found that the most
lipophilic (2) is the weakest antioxidant. Furthermore,
hemiketals 7 and 8, with about the same clogP as ketal
9, are significantly more potent inhibitors of lipid
peroxidation than 9. Therefore, it could be assumed that
other structural characteristics of the studied com-
pounds are more important than lipophilicity. We
propose as a possible explanation of their antioxidant
activity the formation of an intermediate radical gener-
ated during oxygen radical attack.
P la sm a Levels of Ch olester ol, LDL, a n d Tr igly-
cer id es. The percent decrease of plasma total choles-
terol, LDL, and triglyceride levels after the administra-
tion of the studied compounds and probucol to hyper-
lipidemic rats is demonstrated in Table 1. Compounds
6-11 significantly reduce cholesterol and triglyceride
concentrations in plasma.
Compounds are administered by the same route to
rats at a dose of 56 µmol/kg, twice in 24 h. All animals
appeared normal macroscopically and by autopsy.
The most active of the examined compounds is 7,
which decreases cholesterol and triglyceride concentra-
tions by ca. 50% at the lowest administered dose (28
µmol/kg, 10 mg/kg). Compounds 6, 8-11 demonstrate
no significant effect at the dose of 28 µmol/kg. Although
the hemiketals 6-8 exert roughly the same effect on
total cholesterol and triglyceride levels as the corre-
sponding ketals 9-11, derivatives 6-8 have a signifi-
cantly higher effect on LDL levels than their ketal
analogues 9-11. This could be considered as an indica-
tion that the hydroxy derivatives may act selectively on
LDL. It has been reported for a number of 3-biphenyl-
3-hydroxyquinuclidines, compounds with structures
similar to those of the present study, that the 3-hydroxyl
is a critical structural element for their hypocholester-
olemic action.6
Exp er im en ta l Section
Ma ter ia ls. All chemicals are of the highest commercially
available purity. 2-Thiobarbituric acid and diagnostic kits for
total cholesterol, LDL, and triglyceride determination are
purchased from Sigma Chemical Co. (St. Louis, MO). All other
reagents are purchased from Aldrich-Chemie (Steinheim,
Germany). Commercial 2-piperidinemethanol (Aldrich) corre-
sponds to the racemate.
Syn th esis. Melting points (mp) are obtained on a MEL-
TEMPII (Laboratory Devices USA) apparatus and are uncor-
rected. Infrared (IR) spectra are recorded on a Perkin-Elmer
597 infrared spectrophotometer. Proton nuclear magnetic
resonance (1H NMR) spectra are obtained with a Bruker AW-
80 MHz and a Bruker 400 MHz spectrometer. Chemical shifts
are reported in parts per million (δ) relative to tetramethyl-
silane (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.
Gen er a l P r oced u r e for th e P r ep a r a tion of 2-Hyd r oxy-
m or p h olin es 6, 7, a n d 8 a n d Th eir Alcoxy Der iva tives 9,
10, a n d 11. A solution of 4-bromoacetylbiphenyl (10 mmol)
and 2-methylaminoethanol 3, or 2-methylamino-cyclohexanol
4, or 2-piperidinemethanol 5 (22 mmol) in acetone (150 mL)
was maintained at room temperature for 15 h.7 Then, acetone
was evaporated in vacuo, ether was added to the residue, the
mixture was washed with saturated sodium chloride solution
and dried (K2CO3), and the product was isolated as a salt.
Conversion into alcoxy derivatives 9, 10, and 11 was carried
out by refluxing in acidified ethanol or n-propanol during 12-
15 h.
2-(4-Bip h en yl)-4-m et h yl-m or p h olin -2-ol, H yd r och lo-
r id e, 6. It is isolated as a white solid and recrystallized from
acetone and ether. Yield 67%, mp 138-140 °C. IR 2450, 1600
cm-1 1H NMR (DMSO-d6) δ 2.8 (3H, s), 3.05-3.65 (5H, m),
.
4.0 (1H, m), 4.1 (1H, m), 7.3-7.6 (3H, m), 7.65-7.9 (6H, m).
Analysis (C17H20ClNO2) C, H, N.
2-(4-Bip h en yl)-4-m et h yl-oct a h yd r o-1,4-b en zoxa zin -2-
ol, Hyd r och lor id e, 7. The product is isolated as a white solid
and recrystallized from acetone and ether. Yield 75%, mp 161-
163 °C. IR 3150, 2450, 1600 cm-1. 1H NMR (DMSO-d6) δ 1.2-
1.6 (5H, m), 1.7-1.9 (3H, m), 2.23 (1H, d, J ) 10.6 Hz), 2.8
(3H, s), 3.12 (1H, t, J ) 18.0 Hz), 3.30 (1H, d, J ) 12.5 Hz),
3.60, (1H, d, J ) 12.5 Hz), 4.05-4.3 (1H, m), 7.3-7.6 (3H, m),
7.65-7.9 (6H, m). Analysis (C21H26ClNO2) C, H, N.
3-(4-Bip h en yl)-oct a h yd r o-1,4-p yr id o[2,1-c]oxa zin -3-
ol, Hyd r och lor id e, 8. The product is isolated as a white solid,
recrystallized from acetone and ether. Yield 78%, mp 175-
177 °C. IR 3300, 2550, 1600 cm-1. 1H NMR (DMSO-d6) δ 1.35-
1.8 (6H, m), 2.8-3.0 (2H, m), 3.10 (1H, t, J ) 11.2 Hz), 3.2-
Lipid peroxidation is particularly recognized to be a
major causative factor in the pathogenesis of athero-

Hypocholesterolemic Activity of Morpholine Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 611
F igu r e 1. Time course of lipid peroxidation, as affected by the test compounds (panel A, 7; B, 8; C, 9; D, 11): control, open
squares; 0.1 mM, filled squares; 0.2 mM, open circles; 0.4 mM, filled circles; 0.5 mM, open triangles; 1 mM, filled triangles.
Ta ble 1. Effect of the Examined Compounds and Probucol on
Plasma Total Cholesterol (TC), Triglyceride (TG), and Low
Density Lipoprotein (LDL) Levels and clog P Values
(5H, m), 4.05-4.3 (1H, m), 7.3-7.6 (3H, m), 7.65-7.9 (6H, m).
Analysis (C24H32ClNO2‚H2O) C, H, N.
3-(4-Bip h en yl)-3-p r op oxy-octa h yd r o-1,4-p yr id o[2,1-c]-
oxa zin e, Hyd r och lor id e, 11. The product is isolated as a
white solid and recrystallized from n-propanol and ether. Yield
percent decrease compared to controlsa
dose
clog
compd (µmol/kg, ip)
TC
TG
LDL
P10
1
71%, mp 170 °C (dec). IR 2500, 1600 cm-1. H NMR (DMSO-
6
7
8
9
10
11
56
28
56
56
56
56
56
32**
42**
32*
3.28
4.72
4.20
4.47
6.43
5.91
10.75
d6) δ 0.88 (3H, t, J ) 14.8 Hz), 1.35-1.85 (7H, m), 1.95-2.15
(1H, m), 2.8-3.0 (2H, m), 3.11 (1H, t, J ) 11.4 Hz), 3.2-3.4
(3H, m), 3.7-3.9 (2H, m), 4.03 (1H, d, J ) 10.4 Hz), 7.41 (1H,
t, J ) 7.32 Hz), 7.50 (2H, t, J ) 15.2 Hz), 7.58, (2H, d, J )
8.2), 7.71 (2H, d, J ) 7.6 Hz), 7.78, (2H, d, J ) 8.2 Hz). Analysis
(C23H30ClNO2‚1/10H2O) C, H, N.
54***
22**
30**
50**
36**
18**
49**
20**
39**
41**
34**
11NS
51***
51**
22**
28**
17NS
18NS
probucol
a
In Vitr o Lip id P er oxid a tion . A heat-inactivated hepatic
microsomal fraction from untreated male Fischer-344 rats
(180-220 g) is prepared.13 All compounds and the solvents are
tested and found not to interfere with the assay. The incuba-
tion mixture contains the microsomal fraction corresponding
to 2.5 mg of hepatic protein/mL (final concentration), or 4 mM
fatty acid residues,14 ascorbic acid (0.2 mM) in Tris-HCl/KCl
buffer (50 mM, 150 mM, pH 7.4), and the studied compounds
dissolved in dimethyl sulfoxide (DMSO) at various concentra-
tions (0.1-1.0 mM). The peroxidation reaction is initiated by
the addition of a freshly prepared FeSO4 solution (10 µM), and
the mixture is incubated at 37 °C for 45 min. Aliquots are
taken from the incubation mixture at various time intervals,
and lipid peroxidation is assessed by spectrophotometric (535
against 600 nm) determination of the 2-thiobarbituric acid
reactive material.13,15
Each group is composed of 4-6 rats. Results are from two to
three independent experiments. All determinations are performed
at least in duplicate, and SD is always within (10% of the
absorbance values. Asterisks indicate statistical significance
(Student’s t-test) as follows: ***P < 0.005, **P < 0.05, *P < 0.1,
NSnot significant (P > 0.1).
3.4 (3H, m), 3.7-3.9 (1H, m), 4.0 (1H, d, J ) 10.4 Hz), 7.41
(1H, t, J ) 7.32 Hz), 7.50 (2H, t, J ) 15.2 Hz), 7.58, (2H, d, J
) 8.2 Hz), 7.71 (2H, d, J ) 7.6 Hz), 7.78, (2H, d, J ) 8.2 Hz).
Analysis (C20H24ClNO2‚H2O) C, H, N.
2-(4-Bip h en yl)-2-eth oxy-4-m eth ylm or p h olin e, Hyd r o-
ch lor id e, 9. It is isolated as a white solid and recrystallized
from acetone and ether. Yield 68%, mp 150-151 °C. IR 2450,
1
1600 cm-1. H NMR (DMSO-d6) δ 0.9 (3H, t, J ) 8.9 Hz), 2.8
(3H, s), 3.0-3.4 (6H, m), 4.0 (1H, m), 4.1 (1H, m), 7.3-7.6 (3H,
m), 7.65-7.9 (6H, m). Analysis (C19H24ClNO2) C, H, N.
In Vivo Eva lu a tion of Hyp och olester olem ic a n d Hy-
p olip id em ic Activity. An aqueous solution of Triton WR
1339 is administered ip (200 mg/kg) to male Fischer-344 rats
(230-280 g),16 and 1 h later the examined compounds (28 or
56 µmol/kg) dissolved in saline or saline only is given ip. After
24 h, blood is taken from the aorta and used for the determi-
2-(4-Bip h en yl)-2-p r op oxy-4-m eth yl-octa h yd r o-1,4-ben -
zoxa zin e, Hyd r och lor id e, 10. The product is isolated as a
white solid, recrystallized from acetone and ether. Yield 70%,
1
mp 115-116 °C. IR 2350, 1600 cm-1. H NMR (CDCl3) δ 0.9
(3H, t, J ) 14.5 Hz), 1.2-1.8 (10H, m), 2.8 (3H, s), 2.9-3.4

612 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
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Ack n ow led gm en t. This work is a part of the Ph.D.
thesis of MCC, supervised by EAR. MCC acknowledges
the Greek State Scholarship Foundation for a grant. We
also acknowledge ELPEN Pharm. Co. (Athens) for
support.
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