Synthesis of Aromatic Retinoids and Curcuminoids and Evaluation of their Antiproliferative, Antiradical, and Anti-inflammatory Activities

Article abstract of DOI:10.1002/open.201600027

Natural retinoids and curcuminoids are known for their broad spectrum of biological properties, such as antioxidant, anti-inflammatory, antitumor, and so forth. In this work, a convenient synthesis of aromatic retinoids and curcuminoids from vinyl or allyl ketones, and the corresponding alcohols, using olefin metathesis as a key reaction, was elaborated. The best yields and diastereoselectivities were obtained from allylic or homoallylic alcohols by employing the two-step cross-metathesis/oxidation procedure. The synthesized analogues were tested for their antiproliferative activity on human cancer cell lines of various origin (leukemia CEM, adenocarcinoma MCF7, cervical carcinoma HeLa) as well as for their antioxidant and anti-inflammatory activity in vitro. All examined derivatives exhibited strong anti-inflammatory activity in vitro without affecting cell viability. They also showed strong cytotoxicity against leukemia cell line CEM, except for 18 and 35. The antioxidant activity of the tested compounds was rather weak.

Full text of DOI:10.1002/open.201600027

DOI: 10.1002/open.201600027
Synthesis of Aromatic Retinoids and Curcuminoids and
Evaluation of their Antiproliferative, Antiradical, and Anti-
inflammatory Activities
[a]
[b]
[b]
[a]
[a]
ˇ
Jacek W. Morzycki, Lucie Rµrovµ, Jiri Grfflz, Tomasz Sawczuk, Urszula Kiełczewska,
Leszek Siergiejczyk,[a] and Agnieszka Wojtkielewicz*[a]
Natural retinoids and curcuminoids are known for their broad
spectrum of biological properties, such as antioxidant, anti-in-
flammatory, antitumor, and so forth. In this work, a convenient
synthesis of aromatic retinoids and curcuminoids from vinyl or
allyl ketones, and the corresponding alcohols, using olefin
metathesis as a key reaction, was elaborated. The best yields
and diastereoselectivities were obtained from allylic or homoal-
lylic alcohols by employing the two-step cross-metathesis/oxi-
dation procedure. The synthesized analogues were tested for
their antiproliferative activity on human cancer cell lines of var-
ious origin (leukemia CEM, adenocarcinoma MCF7, cervical car-
cinoma HeLa) as well as for their antioxidant and anti-inflam-
matory activity in vitro. All examined derivatives exhibited
strong anti-inflammatory activity in vitro without affecting cell
viability. They also showed strong cytotoxicity against leukemia
cell line CEM, except for 18 and 35. The antioxidant activity of
the tested compounds was rather weak.
1. Introduction
Over the past few decades, olefin metathesis has emerged as
a powerful tool in organic synthesis. During this period, efforts
have been focused on the development of new metathesis
catalysts that show not only high activity, selectivity, and func-
tional-group tolerance, but also operational simplicity and
availability.[1–4] These investigations resulted in the discovery of
ruthenium complexes such as I, II, III, IV, V (Figure 1), which
have found successful application in the preparation of various
olefins, including natural and biologically active com-
pounds.[5–8] In this paper, we report the use of cross metathesis
for the synthesis of retinoids and curcuminoids. Both of these
compound families (Figure 2) exhibit beneficial biological activ-
immune response.[9–11] Curcuminoids, which are derivatives of
curcumin, a natural pigment isolated from the rhizome of Cur-
cuma longa, also show a broad range of biological activities,
including antioxidant, anti-inflammatory, antitumor, anti-HIV,
antibacterial, antiviral, and antifungal properties.[12] Additional-
ly, recent clinical trials have demonstrated that curcumin is
safe even in high doses.[13–17] Curcumin underwent clinical trials
for cancer[18] and Alzheimer’s disease.[15] However, its potential
use as a therapeutic agent is severely affected by its low water
solubility, rapid metabolism, and poor bioavailability.[19–21]
ities. Retinoids, which are natural and synthetic analogues of 2. Results and Discussion
retinoic acid, play an essential role in a variety of biological
2.1. Chemistry
processes, such as vision, reproduction, cell differentiation, and
Continuing our studies on the synthesis of aromatic reti-
noids[22] and other biologically active compounds,[23,24] we con-
ceived a strategy for the synthesis of retinoic acid analogues
with a carbonyl group instead of an ethenylene (ÀCH=CHÀ)
fragment in the polyene chains, as well as curcumin analogues
with an atypical arrangement of carbonyl groups. Both types
of compounds may be prepared from the same substrates,
that is, vinyl or allyl ketones, by employing cross metathesis
(CM) for the synthesis of retinoids and unsymmetrical curcumi-
noids, or self-metathesis (SM) in the case of symmetrical curcu-
minoids (Scheme 1).
[a] Prof. J. W. Morzycki, T. Sawczuk, U. Kiełczewska, Dr. L. Siergiejczyk,
Dr. A. Wojtkielewicz
Institute of Chemistry
University of Białystok
ul. K. Ciołkowskiego 1K, 15-245 Białystok (Poland)
E-mail: jeremy@uwb.edu.pl
[b] Dr. L. Rµrovµ, Dr. J. Grfflz
Department of Chemical Biology and Genetics
Centre of the Region Hanµ for Biotechnological and Agricultural Research
´
Palacky University
ˇ
˚
Slechtitelu 27, 783 71 Olomouc (Czech Republic)
Supporting Information for this article can be found under http://
dx.doi.org/10.1002/open.201600027.
To validate our strategy, we investigated the metathesis re-
actions of model vinyl and allyl ketones. The unsaturated ke-
tones 3, 4, 7, and 8 needed for synthesis were prepared from
commercially available benzaldehyde and cinnamaldehyde by
using the Grignard reaction with vinyl- or allylmagnesium bro-
mide followed by oxidation, as shown in Scheme 2.
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This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
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Figure 1. Examples of commercially available metathesis catalysts.
Figure 2. Natural and synthetic retinoids and curcuminoids.
on our previous studies related to the synthesis of etretinate
analogues,[22] we chose ethyl (2E,4E/Z)-3-methylhexa-2,4-dien-
oate (9) as a convenient partner for the cross-coupling reac-
tions. When the CM of ketone 3 was carried out under optimal
reaction conditions, as previously established for etretinate an-
alogues (3 equiv diene 9, 10 mol% catalyst III, toluene,
508C),[22] the desired product 10 was obtained in less than 5%
yield. The main product appeared to be ketone 11 formed
through an undesirable metathesis pathway (Scheme 3). This
reaction outcome could not be changed either by using differ-
ent amounts of the catalyst (5 or 20 mol%) or its type (com-
plex II). However, when the cross-coupling reaction was carried
out in dichloromethane instead of toluene, the desired oxoest-
er 10 was obtained in 18% yield. Although the product yield
was low, the reaction proved completely E-stereoselective. At-
tempts to optimize the reaction conditions did not result in
any further improvement.
Scheme 1. Retrosynthesis of etretinate and curcumin analogues.
In the first experiments, the metathesis reactions of phenyl
vinyl ketone (3) were studied. Having in mind the synthesis of
oxoretinoids and curcumin analogues, two series of reactions
of 3, that is, its CM and SM reactions were investigated. Based
Then, to test the usefulness of phenyl vinyl ketone for the
synthesis of symmetrical curcuminoids, the SM of 3 in the pres-
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Scheme 2. Preparation of substrates for metathesis reactions. Reagents and conditions: a) 1: 85%, 2: 90%, b) 3: 53%, [O]=PDC, 4: 77%, [O]=DMP, c) 5:
91%, 6: 98%, d) 7: 60%, [O]=MnO2, 8: 67%, [O]=DMP.
Scheme 3. CM reaction between phenyl vinyl ketone (3) and ethyl (2E,4E/Z)-3-methylhexa-2,4-dienoate (9). Reagents and conditions: a) 10 mol% of catalyst III,
toluene, 508C, 10: <5%, 11: 30%, b) 10 mol% of catalyst III, CH2Cl2, reflux, 10: 18%, 11: <2%.
As these initial results of the vinyl and allyl ketone metathe-
sis reactions appeared unsatisfactory, in view of the planned
retinoid and curcuminoid synthesis, we decided to modify our
strategy. In the next reactions, we used the corresponding al-
cohols 1 and 2 as substrates, which were expected to be more
reactive, instead of the unsaturated ketones. Allyl alcohol 1
was subjected to CM with diene 9 under various reaction con-
ditions. The results of these experiments are summarized in
Table 1.
Scheme 4. SM reactions of unsaturated ketones 3 and 4. Reagents and con-
ditions: a) 15 mol% of catalyst III, CH2Cl2, reflux, 16 h, 12: 45%, E/Z=17:1,
13: 49%, E/Z=23:1.
When the reaction was carried out with three equivalents of
diene 9 in the presence of a second-generation catalyst (II or
III) in toluene at room temperature, product 14 was formed in
low yields (ca. 10%), but with complete E selectivity (Table 1,
entries 1 and 2). Additionally, homocoupled alcohol and un-
reacted substrates were observed in the reaction mixture. By
using dichloromethane as a solvent and increasing the temper-
ature to 408C, better yields of the desired product 14 were ob-
tained (Table 1, entries 4–8). Interestingly, in this solvent, slow
SM of ethyl 3-methylhexa-2,4-dienoate (9) was observed,
which never occurred in toluene. The best product yield was
obtained when alcohol 1 was reacted with five equivalents of
diene 9 in the presence of catalyst III in refluxing dichlorome-
thane (Table 1, entry 6). When alcohol 1 was used in excess,
a significant decrease in the yield of product 14 was observed
(Table 1, entry 7). This result may suggest that SM of alcohol 1
is faster than its reaction with 9, and the corresponding dimer
is resistant to secondary metathesis reactions. By changing the
solvent from dichloromethane to toluene or 1,2-dichloroethane
and increasing the reaction temperature to 658C, the yield de-
creased from 71 to 22 or 36%, respectively (Table 1, entries 3
and 9). It seems that lower yields of 14 at elevated tempera-
ture can be attributed to faster catalyst and product decompo-
sition. In all cases, catalyst III proved to be more efficient in
promoting this transformation than complex II. The best reac-
ence of III in CH2Cl2 or toluene (Scheme 4) was studied. Under
optimal conditions (15 mol% III, CH2Cl2, reflux), alkene 12 was
obtained in a moderate yield (45%). In the case of symmetrical
1,2-disubstituted olefins, analysis of the E/Z product configura-
tion by using the NMR method is a challenging task. The cou-
pling constant 3J(H,H) is not directly accessible from the
1H NMR spectrum, owing to the equivalence of the alkene pro-
tons. Determining a double-bond configuration requires a new
method to be employed, which uses the low natural abun-
dance of the NMR-active 13C isotope to break the symmetry of
the carbon-bound vicinal protons.[25] The 2D NMR technique
uses a combination of INEPT and selective TOCSY pulse se-
quences to retrieve the coupling constant value of chemically
equivalent alkene protons. Analysis of the correlation spectra
for ketone 12 allowed us to identify the alkene proton signals
and then to determine, from the peak areas, that the alkene
was formed as a mixture of E and Z isomers in a ratio of 17:1.
The SM of allyl ketone 4 was also investigated (Scheme 4). De-
spite expecting that this substrate should be more reactive
than the electron-deficient vinyl ketone 3, product 13, in the
presence of catalyst III, was only produced in a 49% yield as
a mixture of E and Z isomers in a ratio of 23:1, as determined
by using the same method.
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Table 1. CM reaction between 1-phenylprop-2-en-1-ol (1) and ethyl 3-methylhexa-2,4-dienoate (9) under various reaction conditions.
Entry
Equiv of 9
Catalyst[a]
Solvent
T [8C]
Product yield[b,c] [%]
1
2
3
4
5
6
7
8
9
10
3
3
3
3
3
5
0.2
5
5
III
II
III
III
II
III
III
II
III
II
toluene
toluene
toluene
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
rt
rt
12
10
22
29
25
71
26
43
36
22
65
40
40
40
40
40
65
65
[d]
C2H4Cl2
C2H4Cl2
5
[a] In all cases, 10 mol% of the catalyst was used and reaction was carried out for 16 h. [b] Product was obtained as all-E isomer. [c] The remaining material
comprised mainly unreacted substrates and homocoupled alcohol. [d] C2H4Cl2-1,2-dichlotoroethane.
Scheme 5. CM reaction between homoallyl alcohol 2 and ethyl (2E,4E/Z)-3-methylhexa-2,4-dienoate (9). Reagents and conditions: a) 10 mol% of catalyst III,
5 equiv of 9, CH2Cl2, reflux, 16 h, 15: 80%, 100% E.
tion conditions were also optimal for the CM of homoallylic al-
cohol 2 with ethyl 3-methylhexa-2,4-dienoate (Scheme 5), af-
fording product 15 in 80% yield. In both cases, the desired
products (14 or 15) were formed with complete E stereo-
selectivity.
to be an easy goal, as these substrates could be considered as
type II and type I olefins, respectively, according to the Grubbs
model of olefin reactivity.[26] Indeed, the optimized yields of SM
products 16 and 17, as reported in Table 2, were satisfactory.
However, both products were formed as inseparable mixtures
of diastereoisomers (RS, RR/SS, both E or Z), although the ste-
reochemical issue will not be discussed here. In the case of re-
action of homoallylic alcohol 2, the best yields (over 90%) of
As in the case of unsaturated ketones, allylic (1) and homoal-
lylic (2) alcohols were also subjected to SM. Metathetic homo-
coupling of secondary allylic and homoallylic alcohols seemed
Table 2. SM reactions of 1-phenylprop-2-en-1-ol (1) and 1-phenylbut-3-en-1-ol (2).
Entry
Substrate
Catalyst (amount)
Reaction conditions[a]
SM product (yield)
1
2
3
4
5
6
7
8
2
2
2
2
2
2
1
1
1
1
1
1
1
1
III (3 mol%)
III (5 mol%)
II (3 mol%)
II (5 mol%)
I (5 mol%)
VI[27] (5 mol%)
III (10 or 5 mol%)
III (10 mol%)
III (10 mol%)
III (10 mol%)
III (5 mol%)
II (5 mol%)
CH2Cl2, 408C
CH2Cl2, 408C
CH2Cl2, 408C
CH2Cl2, 408C
CH2Cl2, 408C
CH2Cl2, 408C
CH2Cl2, 408C
CH2Cl2, rt
toluene, 508C
toluene, rt
toluene, rt
CH2Cl2, 408C
toluene, rt
CH2Cl2, 408C
17 (31%)
17 (94%)
17 (52%)
17 (90%)
17 (77%)
17 (66%)
16 (0%)+18 (40%)
16 (42%)+18 (40%)
16 (0%)+18 (31%)
16 (66%)+18 (23%)
16 (66%)+18 (<10%)
16 (78%)
9
10
11
12
13
14
II (5 mol%)
VI[27] (5 mol%)
16 (55%)+18 (<5%)
16 (69%)
[a] In all cases, reaction was carried out for 16 h.
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product 17 were achieved with 5 mol% of complexes III or II
(Table 2, entries 2 and 4). Lowering the catalyst loading from 5
to 3 mol% decreased the product yield from 94 to 31% for
complex III and from 90 to 52% for complex II (Table 2, en-
tries 1 and 3). Other catalysts, such as I and VI (Figure 3), also
proved effective (Table 2, entries 5 and 6).
ing the promoter for compound II or VI (Figure 3) and lower-
ing the catalyst loading from 10 to 5 mol% allowed us to
hamper the isomerization process completely. The desired
product 16 was produced in good yields (78% in the presence
of II, 69% in the presence of VI) without a trace of the unde-
sired isomerized product 18 (Table 2, entries 12 and 14).
When planning the synthesis of unsymmetrical curcumi-
noids, homoallylic alcohol 2 was coupled with allylic alcohol 1
in the presence of a second-generation catalyst (II or III). Al-
though there are examples of selective CM reactions between
type I and type II olefins,[26] in the case of the reaction of two
model substrates, a mixture of products was formed as a result
of CM and SM of both substrates, even if the allylic alcohol
was used in excess. When we used the less reactive vinyl
ketone 3 instead of allylic alcohol 1 as a cross partner for 2,
the reaction proved to be much more selective, and the de-
sired product 19, under optimized reaction conditions, was ob-
tained in 77% yield with high diastereoselectivity (E>98%,
Scheme 6). The result of this reaction suggested that phenyl
vinyl ketone 3 may be efficiently coupled with more reactive
olefins of type 1 or 2, according to the Grubbs classification.
However, in the case of a less reactive partner (as diene 9), the
cross-coupled product is formed only in low yields (Table 1). In
subsequent experiments, we investigated the reactions of ke-
tones 7 and 8 as well as of alcohols 5 and 6 derived from cin-
namaldehyde (Scheme 2). The presence of an additional inter-
nal double bond in these substrates makes their CM reactions
more challenging. However, it could be expected that the ter-
minal monosubstituted double bond is more reactive. A series
of reactions was carried out for olefin 5 with ethyl sorbate 9
(Table 3). The main product of reactions in toluene (entries 1–
4) was 1-phenylpent-1-en-3-one (21), produced by ruthenium
complex-catalyzed isomerization of substrate 5. Even the addi-
tion of chloroborocatechol or 1,4-benzoquinone[28–30] to pre-
vent isomerization was unsuccessful. Only traces of the expect-
ed product 20 were formed. The same happened when 1,2-di-
chloroethane was used as a solvent at 658C (entries 9 and 10).
However, reactions carried out in CH2Cl2 (entries 5–7) or in
ethyl ether (entry 8) at reflux produced mostly product 20,
albeit in low yields. Interestingly, the influence of the catalyst
type, its concentration, and the reaction temperature on the
reaction course proved to be less important. The above CM ap-
peared to be regioselective—the product of the internal
double-bond reaction in substrate 5 was formed in negligible
amounts.
Figure 3. New modified ruthenium metathesis catalyst with polyether clamp
embracing N,N’-2,4-dimethylphenyl substituents in the NHC ligand.[27]
Homometathesis of allylic alcohol 1 turned out to be more
challenging. When the coupling reaction was performed with
catalyst III (10 mol%) in DCM at reflux, instead of the desired
diol 16, compound 18 (40%, Figure 4) was isolated (Table 2,
entry 7), which was formed through SM followed by isomeriza-
Figure 4. Isomerization product formed in the SM reaction of 1-phenylprop-
2-en-1-ol (1).
tion, in addition to a small amount of the isomerized substrate,
propiophenone. Different reaction conditions were attempted
to avoid the formation of product 18 and to improve the yield
of the SM product (Table 2, entries 8–14). At first, the influen-
ces of temperature, catalyst amount, and solvent were exam-
ined for reactions promoted by catalyst III (Table 2, entries 7–
11). It was found that the yield of by-product 18 could be de-
creased by lowering both the reaction temperature (Table 2,
entries 8 and 10) and catalyst loading (Table 2, entry 11). From
entries 8 and 10/11 in Table 2 it can be seen that the choice of
solvent is also important. When the reaction was carried out in
the presence of catalyst III in toluene, the conversion was
higher and less of the isomerization product was formed. How-
ever, this solvent effect was not obvious for reactions catalyzed
by complex II (Table 2, entries 12 and 13). Fortunately, chang-
The problem of the competitive isomerization process was
also encountered during the study of the SM of 5. Even the re-
actions of compound 5 in the presence of catalyst II carried
out in refluxing CH2Cl2 led to the formation of ketone 21 as
Scheme 6. CM reaction between phenyl vinyl ketone (3) and 1-phenylbut-3-en-1-ol (2). Reagents and conditions: a) 10 mol% of catalyst III, 4 equiv of 3,
CH2Cl2, reflux, 16 h, 19: 77%, E>98%.
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Table 3. CM of 1-phenylpenta-1,4-dien-3-ol (5) with ethyl 3-methylhexa-2,4-dienoate (9) under various conditions.
Entry
Catalyst (amount)
Reaction conditions[a]
Product yield [%]
20[b]
21
1
2
3
4
5
6
7
8
9
10
II (10 mol%)
II (5 mol%)
3 equiv of 9, toluene, rt
3 equiv of 9, toluene, rt
chloroborocatechol, 3 equiv of 9, toluene, rt
3 equiv of 9, toluene, rt
3 equiv of 9, CH2Cl2, 408C
5 equiv of 9, CH2Cl2, 408C
5 equiv of 9, CH2Cl2, 408C
Cu2I2 (15 mol%), 3 equiv of 9, Et2O, 358C, 5 h
5 equiv of 9, C2H4Cl2, 658C
5 equiv of 9, C2H4Cl2, 658C
<2
<2
<2
0
12
15
12
13
<2
3
70
64
70
65
7
6
7
4
43
40
II (10 mol%)
III (5 mol%)
II (10 mol%)
II (10 mol%)
III (10 mol%)
II (10 mol%)
II (10 mol%)
III (10 mol%)
[a] In all cases, reaction was carried out for 16 h. [b] E-isomer of product 20 was formed (>97%).
Table 4. CM of 1-phenylpenta-1,4-dien-3-one (7) and ethyl 3-methylhexa-2,4-dienoate (9) under various reaction conditions.
Entry
Catalyst (amount)
Reaction conditions[a]
Product yield[b] [%]
22
23
1
2
3
4
5
6
7
8
III (10 mol%)
III (10 mol%)
III (10 mol%)
III (10 mol%)
IV (10 mol%)
IV (10 mol%)
IV (10 mol%)
II (10 mol%)
II (10 mol%)
II (10 mol%)
VI[27] (10 mol%)
2 equiv of 9, toluene, 508C
2 equiv of 9, CH2Cl2, 408C
1 equiv of 9, CH2Cl2, 408C
2 equiv of 9, CH2Cl2, rt
2 equiv of 9, CH2Cl2, 408C
2 equiv of 9, CH2Cl2, rt
2 equiv of 9, toluene, 508C
2 equiv of 9, CH2Cl2, rt
2 equiv of 9, CH2Cl2, 408C
Cu2I2 (15 mol%), 2 equiv of 9, Et2O, 358C, 5 h
2 equiv of 9, CH2Cl2, 408C
15
35
25
15
40
30
12
15
30
18
30
29
22
10
16
26
13
16
29
66
51
<1
9
10
11
[a] In all cases, reaction was carried out for 16 h. [b] In all cases, E-isomers of products were formed >96%.
the main product, in addition to the unreacted substrate. In
subsequent experiments, to overcome the problem of isomeri-
zation, the corresponding unsaturated ketone 7 was subjected
to CM and SM reactions. The results of reaction between vinyl
ketone 7 and ethyl 3-methylhexa-2,4-dienoate 9 in the pres-
ence of various catalysts are summarized in Table 4. In all reac-
tions, the desired product 22 was accompanied by ester 23, re-
sulting from the CM reaction on the internal double bond and
products of SM of the starting ketone on both double bonds,
although to a much lesser extent.
such as III or IV, unselective scission also occurred; however,
these complexes favored the formation of the desired ester 22
over the short-chain ester 23 (Table 4, entries 2–6). A complete-
ly regioselective reaction was observed when complex VI
(entry 11) was used as a catalyst (Figure 3). The retinoid ana-
logue 22 was obtained in 30% yield with high diastereoselec-
tivity. The catalyst’s bulkiness likely prevented a reaction on
the internal, more hindered double bond. The considerable re-
activity of the internal double bond in 7 can be explained as
follows. As the steric factors favor a reaction on the less hin-
dered terminal double bond, the electronic effects seemed to
be responsible for the increased reactivity of this ketone’s in-
ternal double bond. The conjugation of benzene p electrons
with the a,b-unsaturated ketone system caused higher elec-
tron density on this double bond, as compared with the elec-
tron-deficient terminal double bond. This assumption seems to
be confirmed by the high reactivity of cinnamaldehyde in CM
The highest conversion was obtained in reactions promoted
by catalyst II in refluxing CH2Cl2 or under Lipshutz conditions
(II, Cu2I2, refluxing ether)[31] (Table 4, entries 9 and 10). However,
this complex promoted the metathesis reaction on both
double bonds present in the substrate. To our surprise, the
product on the internal double bond 23 dominated in the re-
action mixture. With the use of other metathesis catalysts,
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reactions[22] as well as by the resistance of the analogous
double bond in the corresponding alcohol (no conjugation) to
the metathesis reactions, as described above (Table 2).
the two-step procedure consisting of CM followed by oxida-
tion (Scheme 8). In both cases (substrates 1 and 26), CM reac-
tions gave the desired products (14 and 27) in high yields and
with high E stereoselectivity. Oxidation of CM products by PDC
yielded retinoids 10 and 28 in satisfactory yields.
The initial studies proved that a similar strategy should also
be optimal for the preparation of symmetrical curcuminoids.
Allylic and homoallylic alcohols 2, 26, and 31 were subjected
to SM reactions followed by oxidation to afford the desired an-
alogues of curcumin (Scheme 9). The unsymmetrical curcumi-
noid 34 was obtained through the CM reaction between
phenyl vinyl ketone (3) and homoallylic alcohol 31 in a good
yield and with high E selectivity (Scheme 10).
Then, we attempted metathesis reactions of allylic ketone 8.
Although its CM with ethyl sorbate 9 was carried out under
various conditions, the reactions proved to be very sluggish
and a complex mixture of products was obtained as a result of
competitive CM, SM, and isomerization processes. When the
corresponding alcohol 6 was subjected to a coupling reaction
with the same diene 9, the desired product 24 was obtained,
accompanied by SM products and unreacted substrates. The
best product yield was achieved by employing the Lipshutz[31]
procedure; ester 24 was then obtained in 38% yield and with
high diastereoselectivity (Scheme 7). SM of the same substrate
proceeded in the highest product yield (total yield of the mix-
ture of diastereoisomers: 82%) in the presence of catalyst I
(Scheme 7).
2.2. Biological Tests
The synthesized analogues of etretinate and curcumin were
screened against various tumor cells. The T-lymphoblastic leu-
kemia CEM cell line proved to be the most sensitive to the reti-
noids and curcuminoids (Table 5) in the micromolar range. The
most effective antiproliferative activity on all of the tested cell
lines was displayed by compound 22 (IC50 0.9–2.8). However,
retinoids and curcuminoids also appeared toxic toward normal
With these results in hand, we examined the application of
the method to retinoid and curcuminoid syntheses. As the CM
reaction of model 1-phenylprop-2-en-1-ol (1) with ethyl 3-
methylhexa-2,4-dienoate (9) was much more efficient than CM
of the corresponding vinyl ketone 3, the synthesis of retinoids
10 and 28 from allylic alcohols was designed by employing
Scheme 7. CM and SM reactions of unsaturated alcohol 6. Reagents and conditions: a) 10 mol% of catalyst II, 15 mol% of Cu2I2, Et2O, reflux, 3 h, 24: 38%, E/
Z=15:1, b) 5 mol% of catalyst I, CH2Cl2, reflux, 16 h, 25: 82%.
Scheme 8. Synthesis of oxoretinoids 10 and 28. Reagents and conditions: a) 10 mol% of catalyst III, CH2Cl2, reflux, 16 h, 14: 71%, 27: 73%, b) PDC, CH2Cl2, rt,
6 h, 10: 70%, 28: 67%.
Scheme 9. Synthesis of symmetrical curcuminoids 13, 30, and 33. Reagents and conditions: a) 10 mol% of catalyst III, CH2Cl2, reflux, 16 h, 29: 58%, 32: 78%,
17: 94%, b) 30: 78%, E>98%, [O]=MnO2, 33: 63%, E>99%, [O]=DMP 13: 68%, E> 94%, [O]=DMP.
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Scheme 10. Synthesis of unsymmetrical curcuminoid 34. Reagents and conditions: a) 10 mol% of catalyst III, CH2Cl2, 4, eqiuv of 3, reflux, 16 h, 34: 94%,
E>97%.
Table 5. IC50 [mM] values obtained from the Calcein AM assays with tested cancer and normal cell lines.[a]
Entry
Compound
CEM
MCF7
HeLa
BJ
Positive control
staurosporine
0.023Æ0.064
0.023Æ0.002
0.175Æ0.007
0.002Æ0.000
Retinoids
1
2
3
4
5
10
28
22
35
36
6.9Æ0.1
3.5Æ0.0
0.9Æ0.1
>50
24.1Æ5.9
9.0Æ3.5
2.8Æ0.1
>50
>50
28.5Æ4.9
8.0Æ0.0
1.0Æ0.0
>50
18.9Æ7.5
2.5Æ0.2
>50
17.3Æ0.3
27.2Æ0.7
34.2Æ6.2
14.6Æ3.5
Curcuminoids
6
7
8
9
10
11
11
18
12
30
13
33
19
34
35.0Æ0.1
3.6Æ0.1
5.6Æ0.3
6.8Æ0.7
7.5Æ0.2
1.8Æ0.4
6.5Æ0.7
>50
>50
>50
>50
>50
>50
32.4Æ15.1
41.3Æ3.0
34.1Æ7.9
>50
6.4Æ0.1
24.2Æ0.1
8.6Æ0.1
18.0Æ8.9
>50
>50
>50
12.7Æ4.0
40.9Æ4.9
6.3Æ0.2
22.4Æ0.6
[a] Cells were treated for 72 h with increasing serial compound concentrations. Mean values ÆSD were obtained from three independent experiments per-
formed in triplicates.
thoxy groups in the curcuminoid benzene ring decreased the
cytotoxicity.
The oxygen radical absorbance capacity (ORAC) is the ability
of compounds to scavenge free peroxy radicals in vitro.[32] The
synthesized compounds were found to be moderate antioxi-
dants whose activities reached ꢀ7.9% of trolox activity on an
equimolar basis (Table 6). In detail, 19 was the most active radi-
cal scavenger, whereas the activities of 35, 18, and 28 were
below the detection limit.
Figure 5. Phenyl and trimethoxyphenyl analogues of ethyl retinoate 35 and
36.[22]
Table 6. Oxygen radical absorbance capacity given as a ratio between
compound and trolox on an equimolar basis.[a]
Entry
Compound
ORAC (compound/trolox)
human fibroblasts (BJ) at a similar concentration. The cytotoxic
activity results obtained for oxoretinoids (10, 22, 28) and the
analogues of ethyl retinoate (35 and 36)[22] (Figure 5) proved
that the presence of a carbonyl group in the polyene chain sig-
nificantly improves antitumor properties (Table 5, entries 1–5).
Among the oxoretinoids, higher antiproliferative activity was
observed for the compound possessing the methoxy group in
the benzene ring (28) and a longer polyenic chain (22). The
polyene chain length seems to be essential. In the curcuminoid
series, analogues with the double bond in the chain, especially
conjugated to the carbonyl group, exhibited a stronger cyto-
toxic effect (Table 5, compare entries 6 vs. 7 vs. 11, and 7 vs. 8,
and 9 vs. 10). In contrast to the retinoids, the presence of me-
Curcuminoids
1
2
3
4
5
6
7
18
12
30
13
33
19
34
nd[b]
0.065Æ0.004
0.02Æ0.003
0.052Æ0.005
0.063Æ0.001
0.079Æ0.002
nd
Retinoids
8
9
10
28
22
10
nd<
0.041Æ0.001
0.026Æ0.001
[a] Data are expressed as mean ÆSD (n=4). [b] Not detected.
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Figure 6. E-selectin (ELAM) expression (4 h) in TNFa-induced HUVECs pretreated with retinoids and curcuminoids (30 min) as compared to cytotoxicity deter-
mined by Calcein AM.
The in vitro anti-inflammatory properties of retinoids and
curcuminoids were studied by using enzyme-linked activity
assays (ELISAs) in pretreated human umbilical vein endothelial
Cells (HUVECs), in which these compounds could inhibit NF–
kB. E-selectin (ELAM) expression was induced by TNFa, which
is indicative of NF–kB activation. The observed reduction of
ELAM expression upon treatment of the HUVECs with 10 or
30 mm of retinoids and curcuminoids was significant for all of
the tested compounds in all concentrations in a dose-depen-
dent manner. The most active derivatives were 22 and 19, as
compared to 10 mm curcumin as a positive control (Figure 6).
In parallel, the cytotoxicity of all compounds was investigated.
These derivatives had a very slight influence on cell viability.
The obtained results provide evidence that the NF–kB pathway
was targeted by the retinoids and curcuminoids.
tivity of the internal double bond that led to unselective meta-
thesis of such substrates. The most likely reason for this was
the high electron density of the double bond, owing to conju-
gation with both the benzene ring and the carbonyl group.
Taking into account the obtained results, a convenient synthet-
ic route to various retinoids and curcuminoids was elaborated.
The method consists of a two-step procedure starting from al-
lylic and homoallylic alcohols with the CM reaction as the first
step, followed by mild oxidation of alcohols to ketones. The
desired oxoretinoids and curcuminoids were obtained in good
yields and with high diastereoselectivity. The synthesized com-
pounds were tested for antiproliferative, antioxidant, and anti-
inflammatory activity in vitro. All examined derivatives exhibit-
ed strong cytotoxicity against leukemia cell line CEM, except
for 18 and 35. However, the tests demonstrated that they are
also toxic towards the normal human fibroblasts (BJ). The anti-
oxidant activity was weak for all of the tested compounds. In
contrast, they exhibited strong anti-inflammatory activity in vi-
tro without affecting cell viability.
3. Conclusions
We studied the metathesis reactions of vinyl and allyl ketones
as well as that of the corresponding alcohols under various
conditions to find the best route for the synthesis of oxoreti-
noids and curcuminoids. Our studies proved that the CM of
unsaturated alcohols is much more efficient than that of unsa-
turated ketones. The exception was 1-phenylpenta-1,4-dien-3-
ol (5), which showed a high tendency for isomerization under
metathesis conditions. Although second-generation catalysts
are known to be able to promote metathesis reactions of elec-
tron-deficient substrates such as a,b-unsaturated carbonyl
compounds, in the case of the unsaturated ketone reactions
we examined, the desired products were obtained with only
low-to-moderate yields, albeit with high diastereoselectivity.
For vinyl and allyl ketones with an additional double bond
conjugated to the ketone moiety, we observed increased reac-
Experimental Section
Chemistry
The melting points presented here were determined by using
Toledo Mettler-MP70 apparatus. NMR spectra were recorded with
Bruker Avance II 400 or Avance DPX 200 spectrometers operating
at 400 and 200 MHz, respectively, using CDCl3 solutions with TMS
1
as the internal standard (only selected signals in the H NMR spec-
tra are reported). Infrared spectra (in chloroform solution) were re-
corded by using a Nicolet series II Magna-IR 550 FTIR spectrometer.
Mass spectra were obtained at 70 eV with an AMD-604 spectrome-
ter. The reaction products were isolated by column chromatogra-
phy, performed using 70–230 mesh silica gel (J. T. Baker) or by
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semi-preparative HPLC. The purity of final compounds was found
to be ꢁ95% as determined by HPLC analysis. HPLC analysis was
performed with a LabAlliance apparatus comprised of pumps (III
Pump series), a UV/Vis detector (525 Dual-wavelength), and an in-
jection valve (Rheodyne Model 7725i). Analytical HPLC was carried
out with a Supelco-Si column (5 mm), 0.46”25 cm, and semi-prep-
arative HPLC with a SMT-Si column (5 mm), 1.0”25 cm.
the crude product was purified directly by using silica gel column
chromatography.
Dimer 32 [1H NMR (400 MHz, CDCl3): d=2.52 (m, 4H), 3.78 (s, 6H),
3.81 (s, 6H), 4.92 (m, 2H), 5.57 (m, 2H), 6.78 (m, 4H), 6.95 ppm (m,
2H); 13C NMR (100 MHz, CDCl3): d=40.90 (CH2), 55.7 (2CH3), 40.92
(CH2), 55.76 (CH3), 55.83 (CH3), 69.2 (CH), 69.3 (CH), 111.5 (2”CH),
112.5 (2”CH), 112.8 (2”CH), 129.9 (CH), 130.0 (CH), 150.4 (2”C),
153.7 (2<M” >C), 153.8 ppm (2”C); IR (CHCl3): n˜ =3601, 3546,
3010, 1591, 1498, 1277,1047, 811 cmÀ1; MS ESI [MNa+] m/z: 411.0]
was eluted with hexane/ethyl acetate (6:4) in 78% yield (22 mg). In
the next step, the solution of Dess–Martin periodinane (3 equiv,
66 mg) in dichloromethane (6 mL) was added to the solution of al-
cohol 32 (20 mg), cooled to 08C, in dichloromethane (3 mL). The
reaction mixture was stirred at 08C. After completion of the reac-
tion (TLC control, 0.5 h), the reaction mixture was passed through
a small pad of Celite and silica gel, and eluted with a mixture of
hexane/ethyl acetate (8:2). The filtrate was evaporated under re-
duced pressure to give the desired product 33 in 99% yield
(19 mg, isomer E>94%), which needed no further purification.
Example Procedure for Retinoid Synthesis
The solution of 1-phenylprop-2-en-1-ol (1, 20 mg; obtained from
benzaldehyde through a Grignard reaction with vinylmagnesium
bromide in a routine manner) in dry dichloromethane (0.5 mL) was
added drop-wise to a solution of ethyl (2E,4E/Z)-3-methylhexa-2,4-
dienoate (9, 5 equiv, 115 mg, 0.11 mL) and a Hoveyda–Grubbs
second-generation catalyst (III, 10 mol%, 9 mg) in dry dichlorome-
thane (0.5 mL). The reaction mixture was stirred at 408C under an
argon atmosphere for 16 h. Then, the mixture was concentrated in
vacuo and purified directly by using silica gel column chromatog-
raphy.
(3E)-1,6-Di(2,5-dimethoxyphenyl)hex-3-ena-1,4-dione (33): 1H NMR
(400 MHz, CDCl3): 3.72 (s, 6H), 3.73 (m, 4H), 3.79 (s, 6H), 5.76 (m,
2H), 6.82 (d, J=9.0, 2H), 6.95 (dd, J=9.0, 3.2, 2H), 7.18 ppm (d, J=
3.2, 2H); 13C NMR (100mHz, CDCl3): d=47.5 (CH2), 53.4 (CH2), 55.8
(2”CH3), 56.0 (2”CH3), 113.1 (2”CH), 114.1 (2”CH), 120.1 (2”CH),
126.7 (2”CH), 128.2 (2”C), 153.1 (2”C), 153.5 (2”C), 200.1 ppm
(2”C); IR (CHCl3): n˜ =1672, 1609 1497 cmÀ1; MS ESI: 407.0 (MNa+),
791.0 (2MNa+); HRMS ESI calculated for C22H24O6Na: 407.1471,
found: 407.1473.
CM product 14 [1H NMR (400 MHz, CDCl3): d=1.30 (t, J=7.1 Hz,
3H), 2.06 (bs, 1H), 2.28 (s, 3H), 4.18 (q, J=7.1 Hz, 2H), 5.35 (d, J=
4.0, 1H), 5.82 (s, 1H), 6.26 (dd, J=15.6, 5.9 Hz, 1H), 6.41 (d, J=
15.6 Hz, 1H), 7.33 (m, 1H), 7.39 ppm (m, 4H); 13C NMR (100 MHz,
CDCl3): d=13.9 (CH3), 14.3 (CH3), 59.8 (CH2), 74.7 (CH), 120.2 (CH),
126.3 (2”CH), 128.1 (CH), 128.7 (2”CH), 133.5 (CH), 137.0 (CH),
142.3 (C), 151.2 (C), 166.9 ppm (C); IR (CHCl3): n˜ =3599, 1705, 1614,
1239, 1161 cmÀ1; MS EI: [M+] m/z (%): 246 (8), 228 (6), 171 (20), 158
(24), 105 (100), 77.0 (48)] was eluted with hexane/ethyl acetate
(85:15) in 71% yield (26 mg).In the next step, the solution of alco-
hol 14 (20 mg) in dry dichloromethane (5 mL) was added to PDC
(3 equiv, 90 mg) under argon at room temperature. The reaction
mixture was stirred for 6 h until all starting material was consumed.
The mixture was filtered through a small pad of Celite and silica
gel with hexane/ethyl acetate (95:5), and the filtrate was evaporat-
ed under reduced pressure to give the desired product 10 in 70%
(14 mg), which needed no further purification.
Compounds 12, 13, and 30 were also prepared by using this
method; the details together with the analytical data of products
are provided in the Supporting Information.
Example Procedure for Unsymmetrical Curcuminoid
Synthesis
Ethyl
(2E,4E)-3-methyl-6-oxo-6-phenylhexa-2,4-dienoate
(10):
1H NMR (400 MHz, CDCl3): d=1.33 (t, J=7.2 Hz, 3H), 2.42 (s, 3H),
4.23 (q, J=7.2 Hz, 2H), 6.15 (s, 1H), 7.28 (d, J=15.5 Hz, 1H), 7.41
(d, J=15.5 Hz, 1H), 7.52 (m, 2H), 7.61 (m, 1H), 7.98 ppm (m, 2H);
13C NMR (100 MHz, CDCl3): d=13.8 (CH3), 14.2 (CH3), 60.3 (CH2),
126.5 (CH), 126.6 (CH), 128.5 (2”CH), 128.7 (2”CH), 133.1 (CH),
137.8 (C), 146.9 (CH), 149.2 (C), 166.2 (C), 190.2 ppm (C); IR (CHCl3):
n˜ =1710, 1664, 1610, 1234, 1165 cmÀ1; MS EI: [M+] m/z (%): 244.2
(16), 171.2 (72), 139.1 (31), 105 (100), 77.0 (56); HRMS EI calculated
for C15H16O3: 244.1099, found: 244.1106.
The solution of unsaturated alcohol 31 (12 mg) in dry dichlorome-
thane (0.5 mL) was added drop-wise to the solution of phenyl vinyl
ketone 3 (4 equiv, 30 mg) and the Hoveyda–Grubbs second-gener-
ation catalyst (III, 10 mol%, 4 mg) in dry dichloromethane (0.5 mL).
The reaction mixture was stirred at 408C under argon atmosphere
for 16 h. Then, the mixture was concentrated in vacuo and purified
directly by using silica gel column chromatography. Product 34
was eluted with hexane/ethyl acetate (7:3) in 94% yield (16 mg,
E>97%).
(2E)-5-(2,5-Dimethoxyphenyl)-5-hydroxy-1-phenylpent-2-en-1-one
(34): H NMR (400 MHz, CDCl3): d=2.71 (m, 1H), 2.79 (m, 2H), 3.78
Compound 28 was also prepared by using this method. Com-
pound 22 was obtained in a CM reaction of 1-phenylpenta-1,4-
dien-3-one (7) and ethyl 3-methylhexa-2,4-dienoate (9) in the pres-
ence of catalyst VI (10 mol%); details of the procedure together
with analytical data are provided in the Supporting Information.
1
(s, 3H), 3.82 (s, 3H), 5.10 (m, 1H), 6.81 (m, 2H), 6.91 (m, 1H), 6.97
(d, J=2.8, 1H), 7.07 (dt, J=15.4, 7.2; 1H), 7.46 (m, 2H), 7.55 (m,
1H), 7.89 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=40.8 (CH2),
55.7 (CH3), 55.8 (CH3), 69.5 (CH), 11.5 (CH), 112.91 (CH), 112.93 (CH),
128.3 (CH), 128 (2”CH), 128.6 (2”CH), 132.4 (C), 132.6 (CH), 137.8
(C), 145.8 (CH), 150.4 (C), 153.8 (C), 190.9 ppm (C); IR (CHCl3): n˜ =
3600, 3009, 1670, 1622, 1228,1047 cmÀ1; MS EI [M+] (%): 311.9 (8),
293.9 (9), 166.9 (100), 145.9 (70), 105 (47); HRMS EI calculated for
C19H20O4: 312.1362, found: 312.1358.
Example Procedure for Symmetrical Curcuminoid Synthesis
The solution of unsaturated alcohol (31, 30 mg) in dry dichlorome-
thane (1 mL) was added to the Hoveyda–Grubbs second-genera-
tion catalyst (III, 5 mol%, 5 mg) in dry dichloromethane (0.5 mL).
The reaction mixture was stirred at 408C under an argon atmos-
phere for 16 h. Then, the mixture was concentrated in vacuo and
Compound 19 was also prepared by this method; the details to-
gether with the analytical data of product are provided in the Sup-
porting Information.
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485 nm, Em. 510 nm) was read every 2 min for 60 min. The net
area under the curve was used to calculate the ORAC, which was
expressed as a ratio between the tested compound and trolox on
an equimolar basis.
Biological Tests
Cell Culture
Stock solutions (10 mmolLÀ1) of retinoids and curcuminoids were
prepared by dissolving an appropriate quantity of each substance
in dimethyl sulfoxide (DMSO). Dulbecco’s modified Eagle’s medium
(DMEM, RPMI 1640 medium), fetal bovine serum (FBS), l-glutamine,
penicillin, and streptomycin were purchased from Sigma (MO,
USA). Calcein AM was obtained from Molecular Probes (Life Tech-
nologies, CA, USA).
Anti-inflammatory Activity In Vitro
CD62E (E-selectin, ELAM) Induction Assays
Each well of the 96-well plate was coated with collagen G for
15 min at 378C. The outer wells (A1-A12, H1-H12, 1-H1, and A12-
H12) contained only 200 mL per well medium and served as an
evaporation barrier. A total of 1”104 of HUVECs was seeded in
each of the other wells in 200 mL medium and grown for 48 h to
optimal confluence. Increasing concentrations of compounds were
then added to the HUVEC-containing wells in triplicate, and the
cells were incubated for 30 min, after which 10 ngmLÀ1 TNFa was
added per well to stimulate NF–kB, and thus ELAM. After further
4 h incubation, levels of ELAM in each of the HUVEC-containing
wells were determined by using ELISAs, as described below.
The screening cell lines (T-lymphoblastic leukemia CEM cell line,
breast carcinoma cell line MCF7, cervical carcinoma cell line HeLa
and BJ human fibroblasts) were obtained from the American Type
Culture Collection (Manassas, VA, USA). The CEM cell line was cul-
tured in RPMI 1640 medium and the others in DMEM medium
(Sigma, MO, USA); both media were supplemented with 10% fetal
bovine serum, 2 mmolLÀ1 l-glutamine, 10000 U penicillin, and
10 mgmLÀ1 streptomycin. The cell lines were maintained under
standard cell culture conditions at 378C and 5% CO2 in a humid
environment. Cells were sub-cultured twice or three times a week
by using the standard trypsinization procedure.
Cell-Surface ELISA ELAM
HUVECs were cultured in ECGM medium (endothelial cell growth
medium, Provitro, Berlin, Germany), supplemented with 10% fetal
bovine serum (Sigma Aldrich, Munich, Germany). Cells were main-
tained under standard cell culture conditions at 378C and 5% CO2
in a humid environment. The cells were sub-cultured twice or
three times a week by using the standard trypsinization procedure.
The HUVECs were a kind gift from Prof. Jitka Ulrichovµ (Medical
Faculty, Palacky University, Olomouc).
Cells were washed once with phosphate-buffered saline (PBS) and
fixed with 0.1% glutaraldehyde (Sigma–Aldrich, Munich, Germany)
for 15 min at room temperature. Then, the cells were washed three
times with 200 mL PBS/0.05% Tween 20 per well, blocked with
200 mL 5% BSA/PBS per well for 1 h, and washed again three times
with 200 mL PBS/0.05% Tween 20 per well. Then, the anti-ELAM an-
tibody (clone BBA-1, R&D Systems, Minneapolis, MN, USA), diluted
1:5000 in 0.1% BSA/PBS (100 mL per well) was added for 1 h at
room temperature and washed five times with 200 mL PBS/0.05%
Tween 20 per well. Subsequently, goat anti-mouse HRP antibody
(Sigma–Aldrich, Munich, Germany), diluted 1:10000 in 0.1% BSA/
PBS (100 mL per well), was applied and the cells were incubated for
1 h in the dark at room temperature and, after decanting, washed
five times with 200 mL, PBS/0.05% Tween 20 per well. HRP activity
of the cells in each of the wells was estimated by using fast-OPD
(o-phenylenediaminedihydrochloride) (Sigma–Aldrich, Munich, Ger-
many) assay, as described,[33] and absorbance was measured at
OD492nm in a vertical spectrophotometer.
Calcein AM Assay
Suspensions of the tested cell lines (ca. 1.0”105 cellsmLÀ1) were
placed in 96-well microtiter plates; after 24 h of stabilization (time
zero), the tested compounds were added (in three 20 mL aliquots)
in serially diluted concentrations in DMSO. Control cultures were
treated with DMSO alone, and the final concentration of DMSO in
the incubation mixtures never exceeded 0.6%. The tested com-
pounds were typically evaluated at six three-fold dilutions, and the
highest final concentration was generally 50 mm. After 72 h incuba-
tion, 100 mL of Calcein AM solution (Molecular Probes, Invitrogen,
CA, USA) was added, and incubation was continued for a further
1 h. Fluorescence of viable cells was then quantified by using a Flu-
oroskan Ascent instrument (Labsystems, Finland). The percentage
of surviving cells in each well was calculated by dividing the inten-
sity of the fluorescence signals from the exposed wells by the in-
tensity of signals from the control wells and multiplying it by 100.
These ratios were then used to construct dose-response curves,
from which IC50 values, that is, the concentrations of respective
compounds that were lethal to 50% of the tumor cells, were calcu-
lated. The results obtained for selected compounds are shown in
Table 5.
Cytotoxicity Testing
For the ELAM expression assay, the toxicity of the tested com-
pounds was assessed in the HUVECs by Calcein AM (Molecular
Probes, Invitrogen, Karlsruhe, Germany) cytotoxicity assays in 96-
well microtiter plates.[34] 20 mL portions of each of the compound
concentrations were added in triplicate to the cells, which were
then incubated at 378C in an atmosphere containing 5% CO2 for
4 h, after which Calcein AM solution was added for 1 h according
to the manufacturer’s instructions. The fluorescence of viable cells
was quantified by using a Fluoroskan Ascent reader (Lab-systems,
Finland) and cytotoxic concentrations were calculated on the basis
of triplicate experiments.
Determination of Oxygen Radical Absorbance Capacity
The ORAC was determined according to Ou et al.[32] Briefly, 100 mL
of 500 nm fluorescein and 25 mL of diluted solutions of the tested
compounds were pipetted into each working well of a (96-well)
microplate, pre-incubated at 378C. Then, 25 mL of 250 mm AAPH
was added and the microplate was shaken for 5 s in a fluorimeter,
Infinite 200 (Tecan, Mannedorf, Switzerland). Fluorescence (Ex.
Acknowledgements
Financial support from the Polish National Science Centre (UMO-
2011/02A/ST5/00459) is gratefully acknowledged. This work was
also financed by a Czech Ministry of Education grant from the
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Received: March 18, 2016
Published online on June 15, 2016
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