Synthesis of benzylideneacetophenones and their inhibition of lipid peroxidation

Article abstract of DOI:10.1016/S0223-5234(00)00137-9

A series of benzylideneacetophenone derivatives have been synthesized and found to show potent inhibition of the lipid peroxidation (LPO) in rat liver microsomes. All 19 compounds prepared in this series are LPO inhibitors. The highest activity was found

Full text of DOI:10.1016/S0223-5234(00)00137-9

Eur. J. Med. Chem. 35 (2000) 449−457
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001379/SCO
449
Short communication
Synthesis of benzylideneacetophenones and their inhibition of lipid peroxidation
Indyah S. Artya, Henk Timmermanb, Mochammed Samhoedic, Sastrohamidjojod, Sugiyantoc,
Henk van der Gootb*
aFPMIPA IKIP Yogyakarta, Karangmalang, Yogyakarta, Indonesia
bLeiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Department of Pharmacochemistry, Vrije Universiteit,
1083 De Boelelaan, 1081 HV Amsterdam, The Netherlands
cFaculty of Pharmacy, Gadjah Mada University, Yogyakarta, Indonesia
dFMIPA, Gadjah Mada University, Yogyakarta, Indonesia
Received 25 May 1999; revised 15 November 1999; accepted 19 November 1999
Abstract – A series of benzylideneacetophenone derivatives have been synthesized and found to show potent inhibition of the lipid
peroxidation (LPO) in rat liver microsomes. All 19 compounds prepared in this series are LPO inhibitors. The highest activity was found in
para hydroxy derivatives with two meta tert-butyl substituents. © 2000 Éditions scientifiques et médicales Elsevier SAS
benzylideneacetophenones / chalcones / lipid peroxidation / antioxidant
1. Introduction
Currently no drugs have been introduced which exhibit
their anti-inflammatory activity solely based on the radi-
cal scavenging or antioxidant effects [1]. However, the
antioxidant effect may inhibit the oxidative process lead-
ing to inflammation because the antioxidant activity as an
elaborate defence system of the cell will blockade radical
formation in the membrane. As we know, in the case of
inflammation the radical production overwhelms the
defence system resulting in reactive oxygen species
which play a role in the inflammation process.
Figure 1. Structure of 1,3-diphenylpropen-1-ones.
A wide assortment of phenolic natural products of
various structural classes, such as flavonoids, display
many biological properties. Interestingly, many of these
compounds, particularly those with anti-inflammatory
activity, contain one or more phenolic hydroxyl groups.
Various derivatives of benzylidenacetophenone show anti-
inflammatory [2, 3], antibacterial [4], antiviral [5], and
gastric protectant activities [6].
inhibit lipid peroxidation (LPO). One of the modifica-
tions at the aromatic ring which was carried out by Lazer
et al. [8] resulted in the structure shown in figure 1 (R1 =
CH3, R2 = F).
This compound has an inhibitory activity on
5-lipoxygenase of 33% at a dose of 1 mM, whereas the
inhibitory activity on cyclo-oxygenase was 43% at the
same dose. Recently Rajakumar and Rao [9] reported the
inhibitory effect on lipid peroxidation using rat brain
homogenates of a related compound (figure 1, R1 = tert
butyl, R2 = H) with IC50 = 9.7 µM.
Sogawa et al. [7] found that benzylidenacetophenones
containing a 3,4-dihydroxycinnamoyl structure strongly
* Correspondence and reprints: vdgoot@chem.vu.nl

450
Figure 2. Synthesis of benzylideneacetophenones.
In order to evaluate the inhibition of LPO by this type
of compound we prepared a series of benzylidenac-
etophenones and established their inhibition of LPO.
ether was condensed with acetophenone, the yield was
increased to about 45%.
Interestingly, Herdan et al. [10] described the synthesis
of a small series of the title compounds under acidic
conditions in 75–80% yield.
2. Chemistry
In our study this improved method was used to
synthesize a series of benzylideneacetophenones (fig-
ure 2). In total, 19 compounds were synthesized from the
corresponding benzaldehydes and acetophenones (ta-
ble I). With TLC each isolated product gave a single spot.
Sogawa et al. [7] synthesized 3,4-dihydroxychalcone
under basic conditions using the Claisen-Schmidt con-
densation of acetophenone with 3,4-dihydroxy-
benzaldehyde. The yield was below 10%. However, when
3,4-dihydroxybenzaldehyde as the bis(tetrahydropyranyl)
Table I. Results of the synthesis of benzylideneacetophenones.
Number
R1
R2
R3
R4
R5
R6
Yield (%)
M.p. (°C)
Crystallized
from
1
2
3
4
5
6
7
8
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
t-Bu
t-Bu
CH3O
i-Pr
t-Bu
t-Bu
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
CH3O
H
H
H
CH3O
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
37
15
34
13
13
24
50
52
36
31
69
59
84
56
20
24
60
32
30
85–90
EtOH
EtOH/H2O
EtOH
EtOH
EtOH
CH3
CH3O
Cl
115–118
161–164
118–120
128–130
108–110
148–149
126–128
94–97
130–131
125–127a
163–165
124–127
147–152
147–148b
133–135
152–153c
102–104
123–124d
H
H
CH3O
CH3O
H
CH3
CH3O
OH
F
F
F
OH
OH
F
CH3
C2H5O
CH3O
OH
Cl
EtOH
EtOH/H2O
EtOH/H2O
EtOH/H2O
EtOH/H2O
EtOH/H2O
EtOH
EtOH/H2O
EtOH/H2O
EtOH
EtOH
EtOH
EtOH/H2O
EtOH
9
H
10
11
12
13
14
15
16
17
18
19
CH3O
t-Bu
t-Bu
H
i-Pr
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
H
b
c
d
a M.p. 117–118° [10]; m.p. 145–147° [10]; m.p. 149–150° [10]; m.p. 123° [10].

451
Figure 3. Absorbance at 535 nm versus incubation time in the
LPO test using various concentrations of compound 1.
Figure 4. Absorbance at 535 nm versus incubation time in the
LPO test using various concentrations of compound 19.
4. Results and discussion
Structures were confirmed with infrared spectroscopy,
Concerning the inhibition of LPO, it seems the para
hydroxyl group of the compounds prepared has the same
function as the hydroxyl group of α-tocopherol (figure 5).
The α-tocopherol molecule can be divided into two parts,
a chroman head and a phytyl chain [11].
nuclear magnetic resonance and mass spectrometry.
3. Pharmacology
It is generally accepted that the phytyl chain inter-
chelates with the fatty acid residues of phospholipids,
while the chroman head is responsible for the antioxidant
effect facing the cytosol, although the chroman ring is
still located in the hydrophobic zone of the lipid bilayer
[11]. The mechanism of the antioxidant activity of
benzylideneacetophenone analogues may involve scav-
enging of a radical (R·) by abstracting a hydrogen atom
from the aromatic hydroxyl group at the para position
(ArOH), thus forming a radical (ArO·).
Lipid peroxidation is mediated by radicals. The radi-
cals are formed in the inflammatory cascade as part of the
defence mechanism of the body. In this process polyun-
saturated fatty acids (PUFA’s) located in the membrane,
are peroxidized via a free radical mechanism. One of the
products formed is malondialdehyde (MDA). With
thiobarbituric acid it gives a pink colour; its concentration
can be measured spectrophotometrically at l = 535 nm
and is used as a parameter for lipid peroxidation.
The LPO inhibition test was used to determine the
effect of the benzylideneacetophenones prepared on the
Fe2+/Vit C induced lipid peroxidation in rat liver mi-
crosomes.
In this study, the LPO test was performed non-
enzymatically; the interaction between antioxidant with
enzymes present in the membranes is prevented by
heating the membranes used before incubation. The
percentage of inhibition was calculated when the control
had reached its maximum level. The profile of the control
curve showed that 90% of the maximum absorbance was
reached after 15 min of incubation (figures 3 and 4).
R· + ArOH → RH + ArO·
The radical (ArO·) is fairly stable due to delocalization
of the unpaired electron. Compounds with less orbital
overlap between a lone pair of the oxygen and the
aromatic π-system, were found to be less potent antioxi-
dants compared to tocopherols [12].
All the compounds prepared in this series are active as
inhibitors of lipid peroxidation; a remarkable common
structural feature is the hydroxyl group at the para
position in the one of the rings.
Figure 5. Structure of α-tocopherol.

452
From the profile of the absorbance versus time curve
(figures 3 and 4), a lag time was observed which is caused
by antioxidant consumption.
Based on the differences of the substituents R1, R2 and
R3, the synthetic compounds can be classified into four
groups as follows:
Group 3 contains compounds with substituents R1 = R3
= t-Bu, R2 = OH which is the case for compounds 11, 12,
15, 16, 17, 18 and 19. Their IC50 range is 0.18–2 µM after
15 min incubation, while after 30 min incubation their
IC50 range is 1.6–3.6 µM. Variation of R4 with F and CH3
did not affect the inhibitory activity after 30 min (11 ≈ 15).
Variation of R4 by OCH3, OC2H5, OH or Cl also did not
affect
the
inhibitory
activity
(compounds
Group 1 contains compounds with substituents R1 =
OCH3, R2 = OH and R3 = H which is the case for
compounds 1, 2, 3, 4, 5, 8, 9 and 13. Their IC50 range is
6–20 µM after 15 min incubation time, while after 30 min
incubation time their IC50 range is 7–22.5 µM. In general,
the range of IC50 values of compounds in this group after
15 min incubation did not differ from IC50 values after
30 min incubation.
16 ≈ 17 ≈ 18 ≈ 19). Remarkably, the introduction of an
additional OCH3 group in 18 affording 12 has no influ-
ence on the activity. Nevertheless substitutents at R4 like
OCH3, OC2H5, OH or Cl provide, in this small series, the
most active compounds (compounds 11 ≈ 15
12 ≈ 16 ≈ 17 ≈ 18 ≈ 19).
<
Group 4 may contain compounds with substituents R1
= R3 = i-Pr, R2 = OH, although there is only one example,
viz. compound 14. Its IC50 value is 2.43 µM after 15 min
incubation, while after 30 min incubation its IC50 value is
5.02 µM.
Group 2 contains compounds with substituents R1 = R3
= OCH3 and R2 = OH which is the case for compounds
6, 7 and 10. Their IC50 range is 0.2–4.24 µM after 15 min
incubation, while after 30 min incubation their IC50 range
is 1–5 µM. In general, the range of IC50 values of
compounds in this group after 15 min incubation did not
differ from IC50 values after 30 min incubation. Com-
pound number 6 (R4 = CH3, R5 = R6 = H) is the most
active compound in this group. After 30 min incubation
the IC50 value (1.28 µM) of this compound is six times its
IC50 value (0.21 µM) after 15 min incubation, while for
compounds 10 and 7 the ratios are 2 and 1.2, respectively.
The substitution of a methyl at R3 (6) by fluor to give
compound 10 or methoxy to give compound 7 decreased
the inhibitory activity (compounds 6 > 10 > 7).
5. Conclusions
LPO inhibitory activities of several compounds in
table II are in the same order of magnitude as the
well-known NDGA. The data mentioned above indicate
that the series prepared contained very potent com-
pounds. In more detail, the IC50 data showed that
introduction of isopropyl or tert-butyl groups at positions
R1 and R3 increases the inhibition of the LPO. This result
is in accordance with what has been reported earlier.
Particularly the introduction of tert-butyl groups ortho
towards the para hydroxyl group has been established as

453
Table II. Inhibition of lipid peroxidation by benzylideneacetophenones after 15 and 30 min incubation.
Number
R1
R2
R3
R4
R5
R6
LPO IC5015′* (µM ± LPO IC5030′**
SEM) n = 2
(µM ± SEM) n = 2
1
2
3
4
5
6
7
8
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
t-Bu
t-Bu
CH3O
i-Pr
t-Bu
t-Bu
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
CH3O
H
H
H
CH3O
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
7.10 ± 1.63
6.07 ± 7.25
17.06 ± 0.30
5.06 ± 0.60
10.55 ± 4.02
0.21 ± 0.02
4.24 ± 0.40
18.27 ± 1.06
19.08 ± 0.08
1.96 ± 0.04
1.97 ± 0.02
0.55 ± 0.05
14.28 ± 1.06
2.43 ± 0.50
1.84 ± 0.09
0.19 ± 0.01
1.27 ± 0.05
0.53 ± 0.05
0.22 ± 0.01
0.30 ± 0.01
17.93 ± 1.50
12.37 ± 0.94
18.51 ± 1.23
7.17 ± 2.50
14.84 ± 0.62
1.28 ± 0.51
5.06 ± 0.35
22.53 ± 2.07
20.44 ± 0.45
4.17 ± 0.04
3.61 ± 0.45
1.99 ± 0.02
19.76 ± 0.11
5.02 ± 0.56
3.59 ± 2.24
1.85 ± 0.03
1.95 ± 0.05
1.68 ± 0.05
1.82 ± 0.01
0.33 ± 0.00
CH3
CH3O
Cl
H
H
CH3O
CH3O
H
CH3
CH3O
OH
F
F
F
OH
OH
F
CH3
C2H5O
CH3O
OH
Cl
9
H
10
11
12
13
14
15
16
17
18
19
20***
CH3O
t-Bu
t-Bu
H
i-Pr
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
H
* Incubation time 15 min.
** Incubation time 30 min; reference compound NDGA (nordihydroguaiaretic acid).
favourable for LPO inhibition [13]. However, it is doubt-
ful whether the effect is caused by steric hindrance of the
phenolic OH group or by the lipophilic nature of the
tert-butyl group [13].
6. Experimental protocols
6.1. Chemistry
Concerning this aspect it is interesting to note that
replacement of the two tert-butyl groups (group 3) by two
methoxy groups (group 2) affords compounds with al-
most equal LPO inhibiting activity. Since the mono
methoxy derivatives (group 1) show much less LPO
inhibition, it may be concluded that the presence of two
substituents ortho to the phenolic OH group is favourable
for activity. At least in our series it seems that the
electronic nature of the substituents is of less importance.
6.1.1. General remarks
Melting points were obtained using a Mettler FP52
microscope and heating table and are uncorrected. TLC
(Merck UC alufolien) experiments were performed with
CH2Cl2 as mobile phase. IR spectra were obtained from
KBr pellets with a Mattson Galaxy 6030 FT-IR apparatus.
1H-NMR spectra were recorded on a Bruker 200 MHz
spectrometer with tetramethylsilane as an internal stan-
dard. Mass spectra were recorded using a Finnigan MAT
90 with EI ionisation (70 eV).
Variations of R4, R5 and R6 did not show strong effects
on LPO inhibitory activity.

454
+
6.1.2. General procedure
spectrum (EI, m/z): 284 (M+·, C17H16O4 , 100%), 283
+
+
Benzaldehyde derivatives (26.6 mmol) and acetophe-
none or acetophenone derivatives (30 mmol) were dis-
solved in 200 mL of ethanol saturated with HCl. HCl and
N2 gas were bubbled into the reaction mixture during the
reaction. Stirring was carried out for at least 6 h and each
hour the reaction mixture was analysed by TLC. Stirring
was stopped when the spot of the reaction product on the
TLC plates used was predominantly present. Then the
reaction mixture was poured into ice water. The mixture
was stirred until some crystals were produced. After
standing for 3 h the solid was filtered off and recrystal-
lized using a suitable solvent.
(M+·-H, C17H15O4 , 28%), 269 (M+·-CH3, C16H13O4+,
+
17%), 253 (M+·-OCH3, C16H13O3 , 16%), 135 (C8H7O2 ,
30%) mass units. Exact mass of molecular ion: m/z =
284.1048, calculated for C17H16O4: 284.1043.
6.1.2.4. 1-(4-Chlorophenyl)-3-(4-hydroxy-
3-methoxyphenyl)-2-propen-1-one 4
IR (cm–1): 1 651 (C=O), 3 508 (OH, phenolic), 2 950
(=C–H, aliphatic); 3 050 (=C–H, aromatic), 1 579 and
1 512 (-CH=CH-, aromatic). NMR CDCl3, δ (ppm): 3.95
(s. 3H, -OCH3), 5.95 (s, 1H, -OH), 7.35 (d, 1H, J = 13 Hz,
=CHa), 7.75 (d, 1H, J = 13 Hz, =CHb) 7.33 (s, 1H, Ar
2′-H), 7.15 (m, 2H, Ar 5′,6′-H), 7.45 (d, 2H, Ar3″,5″-H),
7.95 (d, 2H, Ar 2″,6″-H). Mass spectrum (EI, m/z): 290
(M+· + 2, C16H13O3Cl+, 28.3%), 288 (M+·, C16H13O3Cl+,
100%), 287 (M+.-H, C16H12O3Cl+, 41.5%), 271 (M+-OH,
C16H12O2Cl+, 15%), 257 (M+·-OCH3, C15H10O2Cl+,
6.1.2.1. 3-(4-Hydroxy-3-methoxyphenyl)-
1-phenyl-2-propen-1-one 1
IR (cm–1): 1 655 (C=O), 3 323 (OH, phenolic), 3 100
(=C–H, alkene; =C–H, aromatic). NMR CDCl3, δ (ppm):
3.95 (s, 3H, -OCH3), 5.90 (s, 1H, -OH), 7.35 (d, 1Ha, J =
13 Hz, =CH), 7.75 (d, 1Hb, J = 13 Hz, =CH), 7.1 (s, 1H,
+
15%), 253 (M+.-Cl, C16H13O3 , 51%), 139 (C7H4ClO+,
26%), 111 (C6H4Cl+, 34%) mass units. Exact mass of
Ar 2′-H), 6.95 (d, H, Ar 5′-H), 7.2 (d, H, Ar 6′-H), 7.50
(m, 3H, Ar 3″,4″,5″-H), 8.00 (d, 2H, Ar 2″,6″-H). Mass
molecular ion: m/z
=
288.0548, calculated for
C16H13O3Cl: 288.0553.
+
spectrum (EI, m/z): 254 (M+·, C16H14O3 , 100%), 253
+
+
(C16H13O3 , 39%), 177 (C10H9O3 , 23%), 105 (C7H5O+,
6.1.2.5. 3-(4-Hydroxy-3-methoxyphenyl)-
1-(2-hydroxyphenyl)-2-propen-1-one 5
+
+
34.%), 77 (C6H5 , 66%), 51 (C4H3 , 27%) mass units.
Exact mass of molecular ion: m/z = 254.0941, calculated
for C16H14O3: 254.0943.
IR (cm–1): 1 637 (C=O), 3 414 (OH, phenolic), 1 578
and 1 472 (-CH=CH-, aromatic). NMR CDCl3, δ (ppm):
3.95 (s. 3H, -OCH3), 5.95 (s, 1H, Ar 4-OH), 7.35 (d, 1H,
J = 13 Hz, =CHa), 7.75 (d, 1H, J = 13 Hz, =CHb), 7.33
(s, 1H, Ar 2′-H), 7.15 (m, 2H, Ar 4″,5″-H), 7.45 (d, 2H,
Ar-3″,6″H), 7.95 (d, 2H, Ar 5′,6′-H), 12.9 (s, 1H, Ar
2″-OH). Mass spectrum (EI, m/z): 271 (M+· + 1,
6.1.2.2. 3-(4-Hydroxy-3-methoxyphenyl)-
1-(4-methylphenyl)-2-propen-1-one 2
IR (cm–1): 1 655.0 (-C=O) 3 402.6 (-OH, aromatic),
1 464 and 1 431 (CH3), 1 583 and 1 514 (-CH=CH-,
aromatic). NMR CDCl3, δ (ppm): 2.43 (s, 3H, -CH3),
3.95 (s, 3H, -OCH3), 6.00 (s, 1H, -OH), 7.35 (d,1Ha, J =
13 Hz, =CH) 7.75 (d, 1Hb, J = 13 Hz, =CH), 6.95 (d, 2H,
Ar 3″,5″-H), 7.92 (d, 2H, Ar 2″,6″-H), 7.28 (s, 1H, Ar
2′-H), 7.15 (m, 2H, Ar 5′,6′-H). Mass spectrum (EI, m/z):
+
+
C16H14O4 , 13.59%), 270 (M+·, C16H14O4 , 100%), 269
+
(M+·-H, C16H13O4 , 51.5%), 253 (M+·-OH, C16H13O+,
+
15%), 239 (M+·-OCH3, C15H11O3 , 5%) mass units.
Exact mass of molecular ion: m/z = 270.0890, calculated
for C16H14O4: 270.0892.
+
+
269 (M+· + 1, C17H16O3 , 16.7%), 268 (M+·, C17H16O3 ,
+
+
100%), 267 (C17H15O3 , 38%), 177 (C10H9O3 , 14.7%),
6.1.2.6. 3-(4-Hydroxy-3,5-dimethoxyphenyl)-
+
119 (C8H7O+, 32%), 91 (C7H7 , 40.9%) mass units.
1-(4-methylphenyl)-2-propen-1-one 6
Exact mass of molecular ion: m/z = 268.1100, calculated
for C17H16O3: 268.1100.
IR (cm–1): 1 655 (C=O), 3 400 (OH, phenolic), 3 050
(=C–H, aromatic), 2 950 (C–H, aliphatic), 1 581 and
1 460 (-CH=CH-, aromatic). NMR CDCl3, δ (ppm): 2.5
(s, 3H, CH3), 4.00 (s. 6H, -OCH3), 5.83 (s, 1H, -OH), 7.4
(d, 1H, J = 13 Hz, =CHa), 7.75 (d, 1H, J = 13 Hz, =CHb),
6.9 (s, 2H, Ar 2′,6′-H), 7.3 (d, 2H, Ar 3″,5″-H), 7.95 (d,
2H, Ar 2″,6″-H). Mass spectrum (EI, m/z): 298 (M+·,
6.1.2.3. 3-(4-Hydroxy-3-methoxyphenyl)-
1-(4-methoxyphenyl)-2-propen-1-one 3
IR (cm–1): 3 418 until 3 352 (-OH, hydroxyl aromatic),
3 100 and 3 025 (-C–H, aromatic), 2 960 and 2 850
(-C–H, aliphatic), 1 651 (-C=O), 1 591 and 1 514
(CH=CH, aromatic). NMR CDCl3, δ (ppm): 3.83 (s, 3H,
-OCH3), 3.93 (s, 3H, -OCH3), 6.00 (s, 1H, -OH), 7.20 (d,
1H, J = 13 Hz, =CHa) 7.35 (d, 1H, J = 13 Hz, =CHb), 6.95
(d, 2H, Ar 3″,5″-H), 8.00 (d, 2H, Ar 2″,6″-H), 7.1 (s, 1H,
Ar 2′-H), 6.90 (d, 1H, Ar 5′-H), 7.2 (d,1H, Ar 6′-H). Mass
+
C18H18O4, 100%), 297 (M+.-H, C18H17O4 , 20%), 267
+
+
(M+·-OCH3, C17H15O3 , 41%), 154 (C10H11O3 , 13.7%),
+
119 (C8H7O+, 18%), 91 (C7H7 , 34%) mass units. Exact
mass of molecular ion: m/z = 298.1202, calculated for
C18H18O4: 298.1205.

455
6.1.2.7. 3-(4-Hydroxy-3,5-dimethoxyphenyl)-
1-(4-methoxy-phenyl)-2-propen-1-one 7
6.1.2.10. 1-(4-Fluorophenyl)-3-(4-hydroxy-
3,5-dimethoxyphenyl)-2-propen-1-one 10
IR (cm–1): 1 651 (C=O), 3 439 (OH, phenolic),
3 053(=C–H, aromatic), 2 946 (C–H, alkyl), 1 599 and
1 577 (-C=C-, aromatic). NMR CDCl3, δ (ppm): 3.95 (s.
6H, -OCH3), 5.85 (s, 1H, -OH), 7.22 (d, 1H, J = 13 Hz,
=CHa), 7.30 (d, 1H, J = 13 Hz, =CHb), 6.88 (s, 2H, Ar
2′,6′-H), 7.15 (d, 2H, Ar 3″,5″-H), 8.05 (d, 2H, Ar
2″,6″-H). Mass spectrum (EI, m/z): 303 (M+· + 1,
C17H15O4F+, 15.1%), 302 (M+·, C17H15O4F+, 100%), 271
(M+·-OCH3, C16H12O3F+, 50.5%), 123 (C7H4OF+,
37.1%), 95 (C6H4F+, 45.5%) mass units. Exact mass of
molecular ion: m/z = 302.095, calculated for C17H15O4F:
302.094.
IR (cm–1): 3 660–3 089 (-OH, hydroxyl aromatic),
3 053 (=C–H, aromatic), 2 928 and 2 964 (-C–H, ali-
phatic), 1 643 (-C=O), 1 597 and 1 425 (CH=CH, aro-
matic). NMR CDCl3, δ (ppm): 3.88 (s, 3H, -OCH3), 3.97
(s, 6H, 2 -OCH3), 5.8 (s, 1H, -OH), 7.35 (d,1H, J = 13 Hz,
=CHa), 7.7 (d, 1H, J = 13 Hz, =CHb), 6.95 (d, 2H, Ar
3″,5″-H), 8.00 (d, 2H, Ar 2″6″-H), 6.85 (s, 2H, Ar
2′,6′-H). Mass spectrum (EI, m/z): 315 (M+· + 1,
+
+
C18H18O5 , 16.8%), 314 (M+·, C18H18O5 , 100%), 299
+
(M+·-CH3, C17H15O5 , 8.9%), 283 (M+·-OCH3,
+
+
C17H15O4 , 35%), 135 (C8H7O2 , 35.2%), 107 (C7H7O+,
+
6.7%), 92 (C6H4O+, 16%), 64 (C5H4 , 8%) mass units.
Exact mass of molecular ion: m/z = 314.1158, calculated
for C18H18O5: 314.1154.
6.1.2.11. 3-(3,5-Ditertbutyl-4-hydroxyphenyl)-
1-(4-fluorophenyl)-2-propen-1-one 11
IR (cm–1): 1 655 (C=O), 3 537–3 214 (OH, phenolic),
1 601 and 1 568 (-C=C-, aromatic). NMR CDCl3, δ
(ppm): 1.50 (s. 18H, tert-C4H9), 5.65 (s, 1H, -OH), 7.30
(d, 1H, J = 13 Hz, =CHa), 7.75 (d, 1H, J = 13 Hz, =CHb)
7.47 (s, 2H, Ar 2′,6′-H), 7.25 (d, 2H, Ar 3″,5″-H), 8.05 (d,
2H, Ar 2″,6″-H). Mass spectrum (EI, m/z): 355 (M+· + 1,
C23H27O2F+, 18.2%), 354 (M+·, C23H27O2F+, 79.3%),
339 (M+·-CH3, C22H24O2F+, 100%), 297 (M+·-C4H9,
C19H18O2F+, 9%), 123 (C7H4OF+, 93.3%), 95 (C6H4F+,
6.1.2.8. 1,3-Bis(4-hydroxy-3-
methoxyphenyl)-2-propen-1-one 8
IR (cm–1): 1 645 (-C=O) 3 589–3 151 (-OH, aromatic),
3 294 (=C–H, aromatic), 1 464 and 1 431 (CH3), 1 593
and 1 427 (-CH=CH-, aromatic). NMR CDCl3, δ (ppm):
3.98 (s, 6H, 2-OCH3), 5.95 (s, 1H, -OH), 6.1 (s, 1H,
-OH), 7.35 (d, 1H, J = 13 Hz, =CHa), 7.75 (d,1H, J = 13
Hz, =CHb), 6.98 (s, 1H, Ar 2′-H), 7.1 (d, 1H, Ar 6′-H), 7.6
(s, 1H, Ar 2″-H), 7.62 (d, 2H, Ar 6″-H), 7.15 (d, 1H, Ar
5′-H), 7.22 (d, 1H, Ar 5″-H). Mass spectrum (EI, m/z):
+
45.6%), 57 (C4H9 , 27.5%) mass units. Exact mass of
molecular ion: m/z = 354.1988 calculated for C23H27O2F:
354.1995.
+
+
301 (M+· + 1, C17H16O5 , 15.5%), 300 (M+·, C17H16O5 ,
+
100%), 299 (M+·-H, C17H15O3 , 18%), 285 (M+.-CH3,
6.1.2.12. 3-(3,5-Ditertbutyl-4-hydroxyphenyl)-
+
+
C16H13O5 , 14.7%), 283 (M+- OH, C17H15O4 , 6.8%),
1-(4-hydroxy-3-methoxy-phenyl)-2-propen-1-one 12
IR (cm–1): 1 655 (C=O), 3 603–3 329 (OH, phenolic);
2 951 (C–H, aliphatic), 1 587 and 1 572 (-C=C-, aro-
matic). NMR CDCl3, δ (ppm): 1.45 (s. 18H, tert-C4H9),
3.95 (s, 3H, OCH3), 5.55 (s, 1H, -OH), 6,13 (s, 1H, -OH),
7.35 (d, 1H, J = 13 Hz, =CHa), 7.80 (d, 1H, J = 13 Hz,
=CHb), 7.45 (s, 2H, Ar 2′,6′-H), 7.60 (m, 2H, Ar 2″,6″-H),
7.00 (d, 1H, Ar 5″-H). Mass spectrum (EI, m/z): 382 (M+,
+
+
269 (M+·-OCH3, C16H13O4 , 20.5%), 177 (C10H9O3 ,
+
32%), 151 (C8H7O3 , 23.6%). Exact mass of molecular
ion: m/z = 300.1001, calculated for C17H16O5: 300.0998.
6.1.2.9. 1-(4-Fluorophenyl)-3-(4-hydroxy-
3-methoxyphenyl)-2-propen-1-one 9
+
+
C24H30O4 , 100%), 367 (M+-CH3, C23H27O4 , 56.6%),
IR (cm–1): 1 637 (C=O), 3 439 (OH, phenolic), 1 597
and 1 444 (-C=C-, aromatic). NMR CDCl3, δ (ppm): 3.98
(s. 3H, -OCH3), 5.95 (s, 1H, -OH), 7.25 (d, 1H, J = 13 Hz,
=CHa), 7.75 (d, 1H, J = 13 Hz, =CHb), 7.35 (s, 1H, Ar
2′-H), 7.18 (d, 1H, Ar 6′-H), 6.95 (d, 1H, Ar 5′-H), 7.15
(m, 2H, Ar 3″,5″-H), 8.00 (m, 2H, Ar 2″,6″-H). Mass
spectrum (EI, m/z): 273 (M+· + 1, C16H13O3F+, 15%), 272
(M+·, C16H13O3F+, 100%), 271 (M+·-H, C16H12O3F+,
45.4%), 255 (M+·-OH, C16H12O2F+, 13.6%), 241
(C15H10O2F+, 15.7%), 257 (M+·-CH3, C15H9O3F+, 6%),
123 (C7H4FO+, 44%), 95 (C6H4F+, 54.7%) mass units.
Exact mass of molecular ion: m/z = 272.0846, calculated
for C16H13O3F: 272.0849.
+
+
325 (M+- C4H9, C20H21O4 , 12%), 151 (C8H7O3 ,
41.4%), 123 (C7H7O2, 6.5%) mass units. Exact mass of
molecular ion: m/z = 382.2147, calculated for C21H30O4:
382.2144.
6.1.2.13. 3-(4-Hydroxy-3-methoxyphenyl)-
1-(4-hydroxyphenyl)-2-propen-1-one 13
IR (cm–1): 1 641 (C=O), 3 600 and 3 321 (OH, phe-
nolic), 1 591 and 1 512 (-C=C-, aromatic). NMR DMSO,
δ (ppm): 3.89 (s. 3H, -OCH3), 10.00 (s, 2H, 2 x-OH), 7.6
(d, 1H, J = 13 Hz, =CHa), 7.75 (d, 1H, J = 13 Hz, =CHb),
6.80 (d, 1H, Ar 6′-H), 7.23 (d, 1H, Ar 5′-H), 7.50 (s, 1H,
Ar 2′-H), 6.90 (d, 2H, Ar 3″,5″-H, 8.05 (d, 2H, Ar

456
2″,6″-H). Mass spectrum (EI, m/z): 271 (M+ + 1,
6.1.2.17. 3-(3,5-Ditertbutyl-4-hydroxyphenyl)-
1-(4-methoxyphenyl)-2-propen-1-one 17
+
+
C16H14O4 , 14.56%), 270 (M+, C16H14O4 , 100%), 269
+
(M+-H, C16H13O4 , 34.5%), 253 (M+-OH, C16H13O+,
IR (cm–1): 1 656 (C=O), 3 593 (broad, OH, phenolic),
3 028 (CH, aromatic), 2 960 and 2 912 (-CH, aliphatic),
1 601 and 1 570 (-C=C-, aromatic). NMR CDCl3, δ
(ppm): 1.50 (s. 18H, tert-C4H9), 3.90 (s, 3H, -OCH3),
5.55 (s, 1H, -OH), 7.38 (d, 1H, J = 13 Hz, =CHa), 7.78 (d,
1H, J = 13 Hz, =CHb), 7.48 (s, 2H, Ar 2′,6′-H), 7.00 (d,
2H, Ar 3″,5″-H), 8.05 (d, 2H, Ar 2″,6″-H). Mass spectrum
+
10.6%), 239 (M+-OCH3, C15H11O3 , 9.8%), 121
+
(C7H5O2 , 45.9%), 93 (C6H5O+, 15.1%) mass units.
Exact mass of molecular ion: m/z = 270.0895, calculated
for C16H14O4: 270.0892.
6.1.2.14. 1-(4-Fluorophenyl)-3-(4-hydroxy-
3,5-diisopropyl phenyl)-2-propen-1-one 14
+
(EI, m/z), 367 (M+· + 1, C24H30O3 , 19.5%), 366 (M+·,
IR (cm–1): 1 653 (C=O), 3 522–3 246 (OH, phenolic);
1 597 and 1 560 (-C=C-, aromatic). NMR CDCl3, δ
(ppm): 1.3 (d, 12H, 4 x-CH3), 3.15 (septet, 2H, 2 x-CH),
5.28 (s, 1H, -OH), 7.35 (d, 1H, J = 13 Hz, =CHa), 7.78 (d,
1H, J = 13 Hz, =CHb), 7.33 (s, 2H, Ar 2′,6′-H), 7.15 (d,
2H, Ar 3″,5″-H), 8.0 (d, 2H, Ar 2″,6″-H). Mass spectrum
(EI, m/z): 327 (M+· + 1, C21H23O2F+, 10.61%), 326 (M+·,
C21H23O2F+, 50.4%), 283 (M+·-C3H7, C18H16O2F+,
100%), 123 (C7H4OF+, 61.3%), 95 (C6H4F+, 29%) mass
units. Exact mass of molecular ion: m/z = 326.1681,
calculated for C21H23O2F: 326.1682.
+
+
C24H30O3 , 100%), 351 (M+·-CH3, C23H27O3 , 61%),
+
335 (M+·-OCH3, C23H27O2 , 3.55%), 309 (M+·-C4H9,
+
+
C20H21O3 , 7.9%), 135 (C8H7O2 , 34.9%), 107 (C7H7O+,
+
2.4%), 77 (C6H5 , 0.2%) mass units. Exact mass of
molecular ion: m/z = 366.2199, calculated for C24H30O3:
366.2195.
6.1.2.18. 3-(3,5′Ditertbutyl-4-hydroxyphenyl)-
1-(4-hydroxyphenyl)-2-propen-1-one 18
IR (cm–1): 1 649 (C=O), 3 491 (broad, OH, phenolic),
3 003 (CH, aromatic), 2 955 and 2 912 (-CH, aliphatic),
1 591 and 1 566 (-C=C-, aromatic). NMR CDCl3, δ
(ppm): 1.50 (s. 18H, tert-C4H9), 5.58 (s, 1H, -OH), 7.25
(s, 1H, -OH), 7.35 (d, 1H, J = 13 Hz, =CHa), 7.80 (d, 1H,
J = 13 Hz, =CHb), 7.50 (s, 2H, Ar 2′,6′-H), 6.95 (d, 2H,
Ar 3″,5″-H), 8.00 (d, 2H, Ar 2″,6″-H). Mass spectrum
6.1.2.15. 3-(3,5-Ditertbutyl-4-hydroxyphenyl)-
1-(4-methylphenyl)-2-propen-1-one 15
IR (cm–1): 1 653 (C=O), 3 350 (broad, OH, phenolic),
2 958 (-CH, aliphatic), 1 585 and 1 566 (-C=C-, aro-
matic). NMR CDCl3, δ (ppm): 1.50 (s. 18H, tert-C4H9),
2.45 (s, 3H, -CH3), 5.55 (s, 1H, -OH), 7.45 (d, 1H, J = 13
Hz, =CHa), 7.80 (d, 1H, J = 13 Hz, =CHb), 7.50 (s, 2H,
Ar 2′, 6′-H), 7.30 (d, 2H, Ar 3″,5″-H), 7.95 (d, 2H, Ar
2″,6″-H). Mass spectrum (EI, m/z): 351 (M+· + 1,
+
(EI, m/z): 353 (M+· + 1, C23H28O3 , 23.5%), 352 (M+·,
+
+
C23H28O3 , 100%), 337 (M+·-CH3, C22H25O3 , 77%),
+
+
295 (M+·-C4H9, C19H19O3 , 9.46%), 121 (C7H5O2 ,
+
+
C24H30O2 , 27.3%), 350 (M+·, C24H30O2 , 100%), 335
+
38.5%), 93 (C6H5O+, 4.14%), 57 (C4H9 , 11.57%) mass
+
(M+·-CH3, C23H27O2 , 84.4%), 293 (M+·-C4H9,
units. Exact mass of molecular ion: m/z = 352.2039,
calculated for C23H28O3: 352.2038.
+
+
C20H21O2 , 15.4%), 119 (C8H7O+, 36%), 91 (C7H7 ,
17.99%) mass units. Exact mass of molecular ion: m/z =
350.2244, calculated for C24H30O2: 350.2246.
6.1.2.19. 3-(3,5-Ditertbutyl-4-hydroxyphenyl)-
1-(4-chlorophenyl)-2-propen-1-one 19
6.1.2.16. 3-(3,5-Ditertbutyl-4-hydroxyphenyl)-
1-(4-ethoxyphenyl)-2 propen-1-one 16
IR (cm–1): 1 655 (C=O), 3 551 (broad, OH, phenolic),
2 955 (-CH, aliphatic), 1 591 and 1 574 (-C=C-, aro-
matic). NMR CDCl3, δ (ppm): 1.50 (s. 18H, tert-C4H9),
5.62 (s, 1H, -OH), 7.30 (d, 1H, J = 13 Hz, =CHa), 7.78 (d,
1H, J = 13 Hz, =CHb), 7.50 (s, 2H, Ar 2′, 6′-H), 7.48 (d,
2H, Ar 3″,5″-H), 7.98 (d, 2H, Ar 2″,6″-H). Mass spectrum
(EI, m/z): 372 (M+· + 2, C23H27O2Cl+, 29.12%), 371 (M+·
+ 1, C23H27O2Cl+, 21.93%), 370 (M+·, C23H27O2Cl+,
85.37%), 355 (M+.-CH3, C22H24O2Cl+, 94.87%), 313
(M+·-C4H9, C19H18O2Cl+, 10.7%), 139 (C7H4ClO+,
IR (cm–1): 1 651 (C=O), 3 470 (broad, OH, phenolic),
2 957 and 2 914 (-CH, aliphatic), 1 587 and 1 566 (-C=C-,
aromatic). NMR CDCl3, δ (ppm): 1.50 (s. 18H, tert-
C4H9), 1.45 (t, 3H, -CH3), 4.15 (q, 2H, -CH2), 5.55 (s,
1H, -OH), 7.45 (d, 1H, J = 13 Hz, =CHa), 7.8 (d, 1H, J
= 13 Hz, =CHb), 7.48 (s, 2H, Ar 2′,6′-H), 6.98 (d, 2H, Ar
3″,5″-H), 8.02 (d, 2H, Ar 2″,6″-H). Mass spectrum (EI,
+
m/z), 381 (M+· + 1, C25H32O3 , 26.1%), 380 (M+·,
+
+
C25H32O3 , 100%), 365 (M+·-CH3, C24H29O3 , 46.9%),
+
+
351 (M+·-C2H5, C23H27O3 , 7.11%), 149 (C9H9O2 ,
16.5%), 121 (C8H9O+, 15.5%) mass units. Exact mass of
molecular ion: m/z = 380.2354, calculated for C25H32O3:
380.2351.
+
43.21%), 111 (C6H4Cl+, 13.9%), 57 (C4H9 , 27.9%) mass
units. Exact mass of molecular ion: m/z = 370.1699,
calculated for C23H27O2Cl: 370.1700.

457
6.2. Pharmacology
absorbance of the supernatant was measured at 535 nm,
and the presence of protein was checked at 600 nm [14].
IC50 values were calculated by interpolation between two
concentrations, one giving less and the other more than
50% inhibition. Experiments were done in duplicate.
6.2.1. Determination of the inhibition
of lipid peroxidation
Microsomes obtained from male Wistar rats, 250–350 g,
(1 mL cup comprises of 17 mg protein/mL in phosphate
buffer) were thawed and assimilated in approximately 30
volumes containing Tris-HCl buffer (50 mM, pH 7.4) and
150 mM KCl. The phosphate buffer was removed by
centrifugation at 115 000 g for 45 min and then the pellet
was heated in a water bath at 100 °C for 2 s. In order to
dilute the microsomes to 0.9 mg protein/mL, the pellet
was diluted in 18 mL of Tris-HCl buffer (50 mM, pH 7.4,
and 150 mM KCl) at 4 °C. The ascorbic acid solution and
ferrous sulfate solution had been prepared just before the
incubation was started. Test compounds were dissolved in
DMSO. The final concentrations of test compound used
were 1, 3 and 10 µM in 3 000 µL of the incubation
mixture or 10, 15 and 30 µM in 3 000 µL of the
incubation mixture. After pre-incubation of the mixture,
consisting of 1 805 µL Tris buffer, 1 000 µL microsome,
75 µL test compound and 100 µL (100 µM) ascorbic acid,
in plastic incubation tubes for approximately 10 min at
37 °C, the reaction was started by adding Fe2+ giving
10 µM final concentration (t = 0). Samples of 0.3 mL
were taken out from the incubation mixture at t = 0, 5, 10,
15, 30, 45, and 60 min and the reactions were stopped
directly by adding 0.3 mL of the incubation mixture into
2 mL of ice cold mixtures of TBA in TCA (4.16 mg/mL)
– BHT in EtOH (1.5 mg/mL) (ratio 10:1) in glass
incubation tubes. Then the reaction mixtures were first
heated for 15 min at 80 °C and then chilled on ice. The
denatured proteins were separated from the mixture by
centrifugation at 3 000 rpm for 15 min (4 °C). The
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