Synthesis of Non-Symmetrical Nitrogen-Containing Curcuminoids in the Pursuit of New Anticancer Candidates

Article abstract of DOI:10.1002/open.201800287

Curcumin is known to display pronounced anticancer effects and a variety of other biological activities. However, the low bioavailability and fast metabolism of this molecule present an issue of concern with respect to its medicinal applications. To addre

Full text of DOI:10.1002/open.201800287

DOI: 10.1002/open.201800287
Full Papers
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Synthesis of Non-Symmetrical Nitrogen-Containing
Curcuminoids in the Pursuit of New Anticancer Candidates
Atiruj Theppawong,[a] Tim Van de Walle,[a] Charlotte Grootaert,[b] Kristof Van Hecke,[c]
Nathalie Catry,[a] Tom Desmet,[d] John Van Camp,*[b] and Matthias D’hooghe*[a]
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Curcumin is known to display pronounced anticancer effects
and a variety of other biological activities. However, the low
bioavailability and fast metabolism of this molecule present an
issue of concern with respect to its medicinal applications. To
address this issue, structural modifications of the curcumin
scaffold can be envisioned as a strategy to improve both the
solubility and stability of this chemical entity, without compro-
mising its biological activities. Previous work in our group
targeted the synthesis of symmetrical azaheteroaromatic curcu-
minoids, which showed better solubility and cytotoxicity
profiles compared to curcumin. In continuation of that work, we
now focused on the synthesis of non-symmetrical nitrogen-
containing curcuminoids bearing both a phenolic and an
azaheteroaromatic moiety. In that way, we aimed to combine
good solubility, antioxidant potential and cytotoxic properties
into one molecule. Some derivatives were selected for further
chemical modification of their rather labile β-diketone scaffold
to the corresponding pyrazole moiety. In this way, thirteen new
non-symmetrical aza-aromatic curcuminoids and four pyrazole-
based analogues were successfully synthesized in a yield of 11–
69%. All newly synthesized analogues were evaluated for their
antioxidant properties, reactive oxygen species (ROS) produc-
tion, water solubility and anticancer activities. Several novel
derivatives displayed good cytotoxicity profiles compared to
curcumin, in combination with an improved water solubility
and stability, and were thus identified as potential hit scaffolds
for further optimization studies.
1. Introduction
E,[2i,7] which is known for its antioxidant effects and other
benefits in the human body. Considering these characteristics,
curcumin could be considered as a suitable candidate for
(anticancer) drug development. However, despite the afore-
mentioned benefits, the clinical application of curcumin is
severely limited because of its poor solubility and low stability/
fast metabolism under physiological, both neutral and basic,
conditions,[8] and curcumin has been classified as a PAINS (pan-
assay interference compounds) and an IMPS (invalid metabolic
panaceas) candidate.[9] In that respect, efforts to move away
from the classical approaches in curcumin research (usually
implying rather minor chemical modifications of the curcumin
scaffold) are recommended, hence our focus on the pursuit of
unprecedented types of nitrogen derivatives in the present
study. In addition, curcumin extensively undergoes in vitro and
in vivo Phase I and Phase II metabolism.[8a,10]
At present, multiple approaches are under investigation to
overcome these limitations. For example, chemical modification
of the curcumin structure has been studied and miscellaneous
analogues have been synthesized to improve the therapeutic
profile of the mother compound, curcumin. One advantage of
developing molecules around the curcumin scaffold is the
absence of toxicity, and it is believed that this unique property
of curcumin is due to its selective cytotoxic activity to cancer
cells only.[11] Surprisingly, large quantities of curcumin can be
consumed without inflicting toxicity, up to 12 g per day.[12]
Numerous analogues of curcumin have therefore been devel-
oped for therapeutic purposes to overcome the classical
limitations. Chemically, the structure of curcumin is composed
of two o-methoxy phenols, symmetrically attached at the two
terminal positions of an α,β-unsaturated β-diketone linker, that
Curcumin is an attractive compound with broad-spectrum
activities and is still used as a folk medicine to treat a variety of
illnesses such as liver disease (jaundice), indigestion, urinary
tract diseases, rheumatoid arthritis, and insect irritation.[1] From
a biological point of view, curcumin also exhibits a diversity of
interesting
properties
including
anticancer,[2]
anti-
inflammatory,[3] antioxidant,[4] antimicrobial[5] and antimalarial[6]
activities. Moreover, curcumin has been shown to display a
much stronger free-radical scavenger property than vitamin
[a] A. Theppawong, T. Van de Walle, N. Catry, Prof. Dr. M. D’hooghe
SynBioC Research Group, Department of Green Chemistry and Technology,
Faculty of Bioscience Engineering, Ghent University
Coupure Links 653, B-9000 Ghent, Belgium
E-mail: matthias.dhooghe@UGent.be
[b] Dr. C. Grootaert, Prof. Dr. J. Van Camp
Department of Food Technology, Safety and Health, Faculty of Bioscience
Engineering, Ghent University
Coupure Links 653, B-9000 Ghent, Belgium
E-mail: john.vancamp@UGent.be
[c] Prof. Dr. K. Van Hecke
XStruct, Department of Chemistry, Faculty of Science, Ghent University
Krijgslaan 281, S3, B-9000 Ghent, Belgium
[d] Prof. Dr. T. Desmet
Department of Biotechnology,
Faculty of Bioscience Engineering, Ghent University
Coupure Links 653, 9000 Ghent, Belgium
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/open.201800287
©2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution Non-Commercial NoDerivs License, which permits use and dis-
tribution in any medium, provided the original work is properly cited, the
use is non-commercial and no modifications or adaptations are made.
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stands in equilibrium with its enol tautomer.[13] In view of this,
several research groups have attempted to modify these
structural motifs of curcumin in order to slow down its
metabolism and achieve improvements in the potency and
efficacy of its anticancer activity.
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5
To address the previously mentioned shortcomings, our
former research focused on the synthesis of structurally
modified nitrogen-containing curcumin analogues, such as β-
enaminone and symmetrical azaheteroaromatic curcuminoids,
in order to improve their bioavailability.[2h,i] Interestingly, many
analogues exhibited good cytotoxicity profiles (low IC50 values)
as compared to curcumin. The most polar compounds also
showed much better water solubility than curcumin, albeit with
a lower cytotoxicity. Thus, a good balance between cytotoxicity,
water solubility and antioxidant properties must be pursued to
deliver the best candidates for pharmaceutical applications.
This work therefore focuses on the synthesis of non-sym-
metrical nitrogen-containing curcuminoids by introducing two
different aryl groups in the curcumin framework, with at least
one aryl group being azaheteroaromatic. Furthermore, some
derivatives underwent an extra modification by converting the
β-diketone moiety into its corresponding pyrazole framework.
This cyclization leads to a more rigid structure and elimination
of keto-enol tautomerism, making the resulting analogues less
prone to Phase I/II metabolism.[10a,14] In that respect, some
pyrazole curcuminoids have already been described in the
literature as promising anticancer candidates.[14–15] For instance,
one pyrazole analogue of curcumin was investigated for its
lipoxygenase inhibitory activity[16] and three other pyrazole
curcuminoids were evaluated with regard to endothelial cell
proliferation and cytotoxicity.[17] In continuation of our previous
work, these structural modifications should keep the pharmaco-
dynamic profile of the new compounds on a similar (or
improved) level as compared to curcumin. To validate the
concept of our research, biological evaluation, water solubility
and stability tests were conducted on all novel analogues and
the results were compared to the results of their corresponding
mother structures.
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Scheme 1. Synthesis of monoaryl curcumin analogues 2a–b.
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Table 1. Reaction conditions and yields for the synthesis of monoaryl
curcumin analogues 2a–b.
Compd
Ar
Eq. B2O3
Eq. Ar
Yield (%)
2a
2b
4-hydroxy-3-methoxyphenyl
indol-3-yl
0.8
1.0
0.9
0.8
32[a]
30[b]
[a] in EtOAc for 4 h. [b] in MeCN for 3 h.
ation with boron trioxide (B2O3) prevents the most acidic
methylene protons to be deprotonated, and therefore avoids
the formation of the Knoevenagel side product. The quantity of
base applied in the reaction is based on color change of the
reaction mixture from yellow to dark-brown. Careful monitoring
via either TLC or LC-MS analysis was necessary to stop the
reaction in time, although there was still starting material
present (30–44%), in order to reach the highest conversion
towards the desired monoaryl analogue with only minor
formation (3–12%) of the symmetrical diaryl side product. The
use of acetonitrile instead of ethyl acetate for the preparation
of analogue 2b resulted in a slightly higher yield. The crude
reaction products were purified using reversed phase column
chromatography to acquire monoaryl curcumin analogues 2a–
b in a yield of 32 and 30%, respectively.
The obtained monoaryl curcumin analogues 2a–b were
subsequently deployed for the synthesis of non-symmetrical
nitrogen-containing curcuminoids 3a–m by means of a second
condensation reaction with different azaheteroaromatic or
thiaheteroaromatic (for derivative 3m) aldehydes (Scheme 2,
Figure 1). Table 2 displays the optimized reaction conditions for
each analogue. As for the synthesis of monoaryl curcumin
analogues 2a–b, complexation with boron trioxide (B2O3) was
again conducted to prevent the formation of the Knoevenagel
side product. In comparison with the first condensation
reaction, the amount of aldehyde was increased to more than
one equivalent (1.1–1.5 equivalents), because full conversion of
2. Results and Discussion
The synthetic procedure for the preparation of non-symmetrical
curcuminoids is based on previously reported optimized
conditions for the synthesis of curcumin and symmetrical
curcumin analogues.[2i,18] In this work, however, two consecutive
condensation reactions with different aldehydes had to be
executed in order to obtain the desired non-symmetrical
curcuminoids. In a first step, monoaryl curcumin analogues 2a–
b were synthesized using vanillin and indole-3-carboxaldehyde,
respectively (Scheme 1). Vanillin was used to maintain the
antioxidant properties associated with the ortho-methoxy
phenol unit, while the rationale for using indole-3-carboxalde-
hyde is based on previous work, in which the symmetrical
indol-3-yl curcuminoid showed promising features for further
development. The optimized reaction conditions for both
monoaryl analogues can be found in Table 1. Boron complex-
Scheme 2. Synthesis of non-symmetrical nitrogen-containing curcuminoids
3a–m.
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Scheme 3. Synthesis of pyrazole-based non-symmetrical nitrogen-containing
curcuminoids 4a–d.
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Table 3. Reaction conditions and yields for the synthesis of pyrazole-based
non-symmetrical nitrogen-containing curcuminoids 4a–d.
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Compd Ar
Ar’
Eq.
Time Yield
H2NNH2 (min) (%)
H2O
4a
4b
4c
4d
4-hydroxy-
3-methoxyphenyl
4-hydroxy-
3-methoxyphenyl
4-hydroxy-
pyridin-3-yl
5
4
5
5
60
30
90
60
75
79
41
80
pyridin-4-yl
isoquinolin-5-yl
thiophen-2-yl
3-methoxyphenyl
indol-3-yl
Figure 1. Non-symmetrical nitrogen-containing curcuminoids 3.
fore, this cyclization of the rather labile β-diketone moiety to a
pyrazole scaffold renders more stable compounds for in vitro
and in vivo metabolism and therefore, it is expected that this
modification will lead to analogues with improved antiprolifer-
ative properties. Moreover, in previous literature studies, a
pyrazole-based curcuminoids demonstrated interesting biolog-
ical activity toward different cancer cells.[20] Scheme 3 shows the
synthesis of pyrazole-based analogues 4a–d through an acid-
catalyzed condensation reaction with hydrazine hydrate. Based
on a literature procedure, the reaction was first conducted in
ethanol as solvent with a catalytic amount of acetic acid.[21]
However, a switch to acetic acid as sole solvent resulted in
faster conversion and higher yields. Full conversion was
determined by means of either TLC or LC-MS analysis. Pyrazole-
based non-symmetrical nitrogen-containing curcuminoids 4a–d
were obtained after purification via reversed phase column
chromatography in a yield of 41–80% (Table 3, Figure 2).
Moreover, a thiophen-2-yl curcuminoid (5) was synthesized
(Figure 2, synthesis in supporting info) in order to compare its
the starting material was desirable in this case. Also, piperidine
seemed a better choice as base for the deprotonation of the
terminal carbon atom of monoaryl analogues 2a–b. A change
in color of the reaction mixture again indicated the amount of
base needed. It has been reported that piperidine can be used
in low catalytic amounts to effect condensation reactions due
to its high efficiency.[19] All reactions were followed up by either
TLC or LC-MS analysis and stopped when full conversion was
obtained. Only for analogues 3d, 3e and 3f, a maximum
conversion of approximately 80% could be reached. Purification
of the crude products with the use of reversed phase column
chromatography yielded non-symmetrical nitrogen-containing
curcuminoids 3a–m in 11–69% yield and high purity.
Four of the synthesized non-symmetrical curcuminoids (3a,
c, e, m) were further modified and converted into their
corresponding pyrazole-based derivatives. As mentioned be-
Table 2. Reaction conditions and yields for the synthesis of non-symmetrical nitrogen-containing curcuminoids 3a-m.
Compd
Ar
Ar’
Eq. B2O3
Eq. (nBuO)3B
Eq. Ar
Eq. piperidine
Time (min)[a]
Yield (%)
3a
3b
3c
3d
3e
3f
3g
3h
3i
4-hydroxy-3-methoxyphenyl
4-hydroxy-3-methoxyphenyl
4-hydroxy-3-methoxyphenyl
4-hydroxy-3-methoxyphenyl
4-hydroxy-3-methoxyphenyl
4-hydroxy-3-methoxyphenyl
4-hydroxy-3-methoxyphenyl
indol-3-yl
indol-3-yl
indol-3-yl
indol-3-yl
indol-3-yl
pyridin-3-yl
pyridin-2-yl
pyridin-4-yl
quinolin-4-yl
isoquinolin-5-yl
pyrrol-2-yl
1.1
1.1
1.1
1.1
1.1
1.0
1.1
1.0
1.0
1.0
1.0
1.0
1.0
1.2
2.2
1.2
1.2
1.2
2.2
2.2
1.2
1.2
1.2
1.2
1.2
2.0
1.1
1.1
1.1
1.1
1.1
1.2
1.2
1.1
1.1
1.1
1.1
1.1
1.5
0.016
0.05
0.016
0.020
0.048
0.1
0.2
0.1
0.1
0.1
20
10
30
60
180
90
10
10
15
20
30
60
15
65
60
12
11
41
37
49
49
16
19
43
62
69
indol-3-yl
pyridin-3-yl
pyridin-4-yl
pyridin-2-yl
quinolin-2-yl
pyrrol-2-yl
3j
3k
3l
0.15
0.2
0.15
3m
indol-3-yl
thiophen-2-yl
[a] Reaction time for step 3.
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Table 4. Determination of solubility experiments in phosphate buffer
(pH 6.8) and evaluation of chemical antioxidant capacity using FRAP assays,
n�3 triplicate independent experiments.
1
2
3
4
5
Compd
Solubility in 0.1 M
phosphate buffer
pH 6.8 (μM)
FRAP
(Trolox
equivalent
per μM)
FRAP
(curcumin
equivalent
per μM)
90 min
24 h
6
7
8
9
Cur
α-Tocopherol
Ascorbic acid
2.7�0.1
N/A
N/A
2.6�0.1
N/A
N/A
1.00
0.70
0.60
–
–
0.68
0.57
1.00
0.40
0.40
0.54
0.20
0.20
0.54
0.42
0.12
0.18
0.15
0.19
0.27
0.09
0.55
0.42
0.14
0.32
0.01
Trolox
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
4a
4b
4c
4d
5
N/A
N/A
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8.1�1.6
9.9�0.4
9.0�0.6
3.6�1.1
1.1 �0.2
20.9�1.7
7.6�0.9
5.3�0.1
7.8�0.1
10.7�0.4
8.1�0.1
12.2�0.3
1.27�0.0
8.8�0.4
16.8�1.6
6.4�0.2
13.7�0.5
10.7�0.2
14.5�1.1
11.6�0.6
12.8�1.1
4.4�1.3
1.4�0.2
18.3�0.4
13.2�0.3
5.4�0.1
7.7�0.6
7.9�0.2
9.6�0.8
12.0�0.7
1.25�0.0
7.7�0.7
12.8�1.4
7.0�0.2
11.7�2.3
12.8�1.4
0.42
0.41
0.56
0.20
0.21
0.56
0.44
0.13
0.19
0.16
0.20
0.29
0.10
0.57
0.43
0.15
0.33
0.01
Figure 2. Pyrazole-based curcuminoids 4 and thiophen-2-yl curcuminoid 5.
Figure 3. Molecular structure of 4b, determined by X-ray analysis, showing
thermal displacement ellipsoids at the 50% probability level.
bioactivity with compounds 3m and 4d, which contain one
thiophen-2-yl moiety. X-ray analysis was used to confirm the
structure of compound 4b (Figure 3). In the structure of 4b,
dimers are formed through hydrogen bonds between two
pyrazole rings (Figure S2, supporting info). These dimers are
further hydrogen bonded into chains via hydrogen bonds
between pyridine N-atoms and hydroxyl H-atoms (Figure S2,
supporting info), leading to a typical herringbone crystal
packing pattern (Figure S3, supporting info).
After we successfully obtained the desired products, we
further investigated the physicochemical properties of these
non-symmetrical curcuminoids. In that respect, water solubility
tests were performed on all newly synthesized compounds
following a similar procedure as in a previous research.[2h,i] An
excess amount of solid was dissolved in 0.1 M phosphate buffer
(pH 6.8) using the shake flask method.[22] The solubility values
were obtained via linear regression of each standard curve,
which was experimentally triplicated. In general, all azaheter-
N/A:No analysis (commercially available compounds).
designed. One series kept the ortho-methoxy phenol unit of
curcumin, combined with an azaheteroaromatic part (3a–g),
whereas in the other series (3h–m), the indol-3-yl moiety was
used as fixed aromatic part and joined together with different
azaheteroaromatics. The reason behind the applied structure
modifications was to retain antioxidant/antiproliferative proper-
ties. All newly synthesized derivatives were assessed for their
antioxidant effects using the ferric reducing antioxidant power
(FRAP) assay.[2i,24] The FRAP assay is generally used to evaluate
chemical antioxidant activity, which is expressed as either
Trolox equivalents or curcumin equivalents. The known positive
controls are α-tocopherol and ascorbic acid. Compounds 3a–g
showed around 50% reduced activity when compared to either
curcumin or Trolox (3c, f showed activity comparable to
ascorbic acid), whereas 3h–m showed only 10–20% antioxidant
properties compared to either curcumin or Trolox. Compounds
4a–d illustrated 15–57% antioxidant properties compared to
curcumin (4a showed effects comparable to ascorbic acid),
which could be explained by the fact that the free NH
functionality on either the indol-3yl moiety or on another
azaheteroaromatic ring can be equipotent to the OH moiety on
the curcumin scaffold in that respect. Thiophenyl curcuminoid
(5) showed no interesting antioxidant properties (0.01 Trolox
equivalent, 1% compared to curcumin). In other words,
preservation of the 4-hydroxy-3-methoxyphenyl moiety (3a–g,
4a–c) may provide interesting properties when combined with
different azaheteroaromatics.
oaromatic non-symmetrical analogues
3 (except 3e) and
pyrazole-based non-symmetrical compounds (4) showed better
solubility than curcumin, their mother compound (Table 4).
Moreover, the thiophenyl curcuminoid (5) also showed better
solubility than 3d–e, h, m, 4c and curcumin (Table 4). From
these results, we can conclude that through the synthesis of
non-symmetrical analogues bearing at least one azaheteroar-
omatic moiety, we successfully achieved one of our main
research aims, which was to produce new curcumin analogues
with a better aqueous solubility. This higher aqueous solubility
solves the problem of in vitro precipitation in biological
medium.
Curcumin is also known to display interesting antioxidant
properties.[23] To investigate the effect of our structural mod-
ifications on the antioxidant properties, two different series of
non-symmetrical azaheteroaromatic curcumin analogues were
To evaluate anticancer properties, a combination of assays
was performed in order to reveal essential information concern-
ing these newly synthesized analogues (3–5). Compounds 3–5
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Table 5. Cell growth inhibition (expressed as IC50 values) of compounds 3–5 measured after 72 hours treatment as tested by mitochondrial activity (MTT)
and protein content (SRB) assays.[a]
1
2
3
Compd
HepG2
MTT (μM)
HT-29
MTT (μM)
Caco-2
MTT (μM)
EA.hy926
MTT (μM)
CHO-K1
MTT (μM)
SRB (μM)
SRB (μM)
SRB (μM)
SRB (μM)
SRB (μM)
4
3a
3b
3c
3d
3e
3f
3g
3h
3i
14.1�0.3
24.6�4.8
3.2�0.8
44.8�5.5
4.7�0.2
31.7�0.6
19.5�0.6
5.0�0.3
48.5�1.7
38.9�0.8
56.2�1.6
16.5�0.4
4.7�0.2
11.2�0.1
56.7�0.5
6.7�0.4
34.9�0.8
>75
16.8�0.9
20.6�1.4
9.8�0.6
55.4�0.3
7.3�0.3
7.1�0.2
13.5�1.1
10.2�0.4
>75
34.0�2.8
54.1 �1.0
9.8�0.3
10.8�1.6
>75
54.6�0.3
28.3�4.7
49.5�2.1
66.0�2.6
22.1�2.6
17.1�1.0
1.3�0.3
17.9�0.1
34.3�2.7
4.9�0.3
47.0�1.0
1.3�0.5
33.9�0.4
12.9�0.9
6.4�0.8
42.0�1.0
33.8�3.5
42.0�2.0
10.5�0.1
6.9�0.0
>75
49.0�0.7
37.7�1.3
4.2�0.4
>75
43.4�4.6
49.7�4.4
4.8�0.1
20.9�0.9
34.8�1.2
48.8�1.8
47.2�2.2
10.7�1.6
36.1�2.3
40.5�4.3
34.6�0.8
64.2�2.76
46.0�3.0
43.8�0.9
10.0�0.1
4.8�0.3
14.6�2.0
22.3�0.9
7.1�0.4
25.0�1.8
25.8�1.1
12.0�0.7
40.8�1.6
17.7�3.0
15.9�2.0
37.9�1.8
10.3�0.1
29.8�2.3
40.2�1.9
47.5�0.2
10.1�0.5
5.7�0.2
8.5�0.4
1.1�0.1
6.1�0.1
42.3�1.2
5.2�0.2
45.6�0.9
19.0�0.4
6.2�0.3
34.9�1.3
1.4�0.2
38.3�1.7
3.3�0.1
4.4�0.4
18.5�1.3
35.7�0.1
38.8�0.4
6.0�0.9
43.7�1.2
46.8�0.7
17.2�1.1
4.1�0.1
3.8�0.0
3.1�0.0
3.7�0.2
60.2�10.4
16.2�0.6
33.8�1.4
45.1�2.6
2.9�0.1
>75
3.7�0.2
53.2�1.3
4.2�0.3
8.6�0.5
60.7�0.7
51.8�0.3
>75
21.4�1.3
25.2�1.5
13.3�2.6
22.3�1.4
7.6�0.3
36.1�1.3
30.9�1.1
17.7�1.4
19.3�0.7
32.6�0.4
49.0�1.3
23.3�3.3
24.3�4.4
66.3�6.3
34.7�2.8
43.3�0.4
9.64�1.2
11.1�0.4
13.6�0.3
41.8�0.7
7.9�0.5
5
6
7
8
46.3�3.7
6.9�0.3
17.1�0.4
5.8�0.3
24.1 �1.6
26.4�2.4
9.2�0.6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
6.7�0.4
47.1�1.6
35.5�0.4
44.7�2.1
6.5�0.2
42.9�0.4
22.3�1.2
41.6�1.7
11.0�0.5
5.9�0.1
3j
3k
3l
3m
4a
4b
4c
4d
5
Cur
Bis
Dox
0.5�0.3
24.0�1.6
47.4�0.2
>75
53.7�6.1
39.3�1.2
58.7�2.0
19.6�4.0
42.0�2.6
47.6�1.5
17.3�0.7
13.0�0.5
18.7�1.2
46.5�0.5
60.3�3.5
21.1�0.3
46.0�0.9
39.1�0.1
24.5�3.3
9.2�0.5
13.2�0.7
32.2�0.9
8.4�0.0
9.7�0.5
9.5�0.4
59.9�1.5
58.4�4.2
>75
0.9�0.0
16.2�1.0
54.4�4.1
38.8�0.3
31.0�3.5
2.4�0.3
59.1�3.3
52.3�3.8
65.6�5.2
2.7�0.1
44.0�2.4
44.9�0.6
31.0�2.5
2.5�0.1
25.3�0.9
17.8�0.3
1.8�0.1
3.4�0.4
[a] Data are presented as mean � standard deviation. Combination data of MTT and SRB (n�3).
were evaluated for in vitro cytotoxicity against four cancer cell
lines, HT-29, Caco-2, EAhy.926 and HepG2, and non-carcinoma
cells (CHO-K1) by mitochondrial activity (MTT) and protein
content (SRB) assays, as well as the measurement of intracellular
reactive oxygen species (ROS) production. For the MTT and SRB
assays, doxorubicin (Dox) was used as positive control and
curcumin was taken as standard reference. In addition, N-acetyl-
L-cysteine (NAC) and doxorubicin[25] were used as controls for
respectively decreased and increased intracellular ROS produc-
tion. The results are described as IC50 values in Table 5. From
these results, it could be observed that most non-symmetrical
analogues demonstrated higher mitochondrial activity com-
pared to curcumin. Moreover, the SRB results pointed out a
similar trend as the MTT results, and confirmed that azaheter-
oaromatic non-symmetrical analogues (3a–m) and pyrazole
non-symmetrical analogues (4a–d) exerted stronger cell growth
inhibition effects than curcumin. In contrast, compound 5
showed comparable activity to its mother compound, curcumin.
Interestingly, compounds 3c, e, h, l and 4c even displayed
comparable activity to the commercial doxorubicin drug.
Previous work demonstrated that the introduction of azaheter-
oaromatic moieties leads to higher antiproliferative
properties.[2i] Therefore, with the intention to combine antiox-
idant and antiproliferative properties, compounds 3–4 were
designed in this work, and these compounds indeed showed an
improved antiproliferative capacity, in addition to a better water
solubility.
and will sequentially cause damage to lipids, proteins and DNA,
which could further increase the risk of cancer development.[26]
Moreover, higher ROS production may trigger cell apoptosis
pathways by stimulating intrinsic mitochondrial pathways.[27]
The experiment was conducted using 2’,7’-dichlorodihydrofluor-
escein diacetate (DCFH-DA). Two concentrations (10 and 1 μM)
were examined, parallel with previous work.[2g–i] Moreover, ROS
data were normalized on SRB measurements of the same well,
in order to correct for differences in cell density. One of the
controls in these experiments was NAC, a ROS inhibitor,[28]
which induced a significant decrease in ROS generation in four
different cancer cell lines, namely HepG2, HT-29, Caco-2 and
EA.hy926, which was also observed in previous research.[2i]
Moreover, doxorubicin induced a significant increase in ROS
generation in all five cell lines. Both decreased and increased
intracellular ROS levels were observed, and this was compound
and cell line dependent (Table 6). In the HepG2 cell line,
compounds 3a and 4c showed a significantly decreased ROS
signal, whereas compounds 3c, f, h, i, k–m, 4b and 4d
exhibited a significantly increased ROS production. In the HT-
29 cell line, compounds 3a, d–e, g–i and 4a showed a
significantly decreased ROS signal, whereas compounds 3f, k–
m, 4d and 5 showed a higher ROS signal. In the Caco-2 cell line,
compounds 3e and 4a showed significantly decreased ROS
production, whereas compounds 3a, f–g, k–m, 4d and 5
showed significantly increased ROS production, which have
similar trends concerning intestinal carcinoma, represented by
the HT-29 cell line. In the EA.hy926 cell line, only 4b showed
decreased intracellular ROS, whereas compounds 3a–b, d–e, f–
g, i–m and 4d showed significantly increased ROS production.
In the CHO-K1 cell line, only 5 showed decreased intracellular
ROS, whereas compounds 3b–c, d, f, h, j–m, 4b and d showed
significantly increased ROS. The ROS reduction effects were
In the next set of experiments, intracellular ROS determi-
nation was performed in different cell lines in order to
investigate their possible role in cytotoxicity-related pathways.
An imbalance between ROS and antioxidants could lead to cell
damage, which is implicated in a range of pathologies. Over-
production of reactive oxygen species is considered as toxic
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related to the compounds that contained the 4-hydroxy-3-
methoxyphenyl moiety (3a–g), which demonstrated approx-
imately 50% antioxidant properties in comparison with curcu-
min based on the FRAP assay (Table 4). Moreover, non-sym-
metrical curcuminoids with an indol-3yl aromatic moiety (3h– Experimental Section
m) induced higher intracellular ROS production, especially
properties and remarkable bioactivities, and thus represent
interesting hit compounds for further in depth investigations.
1
2
3
4
5
6
7
8
9
compounds 3k–m showing promising results in this work,
despite their 13–30% antioxidant properties based on the FRAP
assay. Results from our previous work also announced higher
ROS productions in pyrol-2yl and indol-3yl curcuminoid
analogues,[2i] with similar trends as azaheteroaromatic non-
symmetrical derivatives 3 h–m. Therefore, our data for 3h–m
showed pro-oxidative effects of non-symmetrical derivatives. As
we compared the results with the positive controls (Dox), the
increased ROS production was observed in the non-symmetrical
indol-3-yl curcuminoids (3h–m) and 3f–g, which may undergo
similar intrinsic mitochondrial pathways leading to cell apopto-
sis. However, some compounds showed different pro-oxidative
and antioxidative effects depending on the cell characteristics.
For instance, HepG2 are liver cancer cells which display robust-
ness and a detoxification mechanism,[29] whereas intestinal cells
serve two main functions: absorbing useful substances into the
body and restricting the entry of harmful substances.[30]
Chemistry
1H NMR spectra were recorded at 400 MHz (Bruker Avance III
Nanobay) with CDCl3, DMSO-d6 or MeOD-d4 as solvent. 13C NMR
spectra were recorded at 100.6 MHz (Bruker Avance III Nanobay)
with CDCl3, DMSO-d6 or MeOD-d4 as solvent. Low resolution mass
spectra were recorded via injection on an Agilent 1100 Series LC/
MSD type SL mass spectrometer with electrospray ionization (ESI
70 eV) and using a mass-selective detector (quadrupole). When
crude reaction mixtures were analyzed, the mass spectrometer was
preceded by a HPLC reversed phase column (Ascentis® Express C18,
HPLC column 3 cm×4.6 mm, 2.7 μm) with a diode array UV/VIS
detector. IR spectra were measured with a Fourier Transform
Infrared spectrophotometer (The IRaffinity-1S). Melting points of
crystalline compounds were measured with a Kofler Bench, type
WME Heizbank of Wagner & Munz. The purity of all tested
compounds was assessed by 1H NMR analysis and/or HPLC analysis,
confirming a purity of�95%.
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Representative Procedure for the Synthesis of Monoaryl
Curcumin Analogues 2a–b
The synthesis of (1E,4Z)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)
hexa-1,4-diene-3-one 2a will be described as a representative
example for the synthesis of monoaryl curcumin analogues 2a–b.
The reaction conditions for monoaryl analogue 2b can be found in
Table 1.
3. Conclusions
In summary, we successfully produced thirteen new non-
symmetrical nitrogen-containing curcuminoids, four pyrazole-
based curcuminoids and one thiophen-2-yl curcuminoid. To
obtain the desired derivatives, the synthesis started from
monoaryl curcuminoids 2a–b (4-hydroxy-3-methoxyphenyl for
3a–g and indol-3-yl for 3h–m). The preparation was performed
by complexation of either 2a or 2b, followed by a condensation
reaction with other aldehydes in order to furnish the expected
compounds in moderate to good yields. Furthermore, the β-
diketo functionality in some derivatives was transformed into
the corresponding pyrazole moiety, resulting in compounds
4a–d. All synthesized compounds (3–5) demonstrated im-
proved water solubility when compared to curcumin, their
mother compound. In this study, several new analogues
showed a good bio-accessibility as expressed by improved
water solubility and cytotoxicity (MTT and SRB, low IC50 values).
Compounds 3–4 exhibited notably good antiproliferative
activity towards different cancer cell lines (HepG2, HT-29, Caco-
2 and EA.hy926) compared to curcumin. On the basis of our
analysis, the azaheteroaromatic non-symmetrical curcuminoids
showed higher solubility (2–7 fold) than curcumin. Compounds
3a, c, e, g–h, l–m, 4a, and 4c–d demonstrated good
antiproliferative activities towards four different cell lines
(HepG2, EA.hy926, HT-29 and Caco-2). Furthermore, activity
against non-carcinogenic cells (CHO-K1) mostly showed higher
IC50 values as compared to carcinogenic cancer cell lines, which
implied that newly synthesized compounds may be less harmful
towards healthy cells. Therefore, these non-symmetrical ana-
logues offer a desirable balance between good physicochemical
Acetylacetone (1; 40 mmol, 4.11 mL) was added to a solution of 0.8
equivalents boron trioxide (32 mmol, 2.23 g) in ethyl acetate
(30 mL). The mixture was stirred at reflux for 1 h. Then, 0.9
equivalents vanillin (36 mmol, 5.48 g) and 1.2 equivalents tributyl
borate (48 mmol, 12.95 mL) were added and the reaction mixture
was stirred for an additional 20 min at reflux conditions. Afterwards,
0.5 equivalents of n-butylamine (20 mmol, 1.98 mL) were added
dropwise. The reaction mixture was further stirred for 4 h at reflux
conditions until maximum conversion was reached according to
°
either TLC or LC-MS analysis. After cooling down to 50 C,
decomplexation of the boron complex was performed by adding
15 mL 1 N HCl to the reaction mixture and letting it stir for 30 min.
The reaction was washed with a saturated solution of sodium
bicarbonate (3×30 mL), after which the aqueous phase was
extracted with ethyl acetate (3×30 mL). The combined organic
fractions were washed with H2O and brine and then dried with
magnesium sulphate, filtrated and evaporated under reduced
pressure. Purification by reversed phase column chromatography
(acetonitrile/water, gradient conditions from 15 to 100%) yielded
(1E,4Z)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)hexa-1,4-diene-3-
one 2a (2.70 g, 32%).
For both monoaryl curcumin analogues 2a–b, the obtained spectra
corresponded with those described in the literature.[31]
Representative Procedure for the Synthesis of
Non-Symmetrical Nitrogen-Containing Curcuminoids 3a–m.
The synthesis of (1E,4Z,6E)-5-hydroxy-1-(4-hydroxy-3-methoxyphen-
yl)-7-(pyridin-3-yl)hepta-1,4,6-triene-3-one 3a will be described as a
representative example for the synthesis of non-symmetrical nitro-
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gen-containing curcuminoids 3a–m. The reaction conditions for
analogues 3b–m can be found in Table 2.
(1E,4Z,6E)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(quinolin-4-
yl)hepta-1,4,6-triene-3-one (3d): Dark-orange solid (11%); reversed
phase column chromatography, tR =3.12 min (gradient conditions,
15% acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
1
2
3
4
5
6
7
8
9
(1E,4Z)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)hexa-1,4-diene-3-
one 2a (1 mmol, 234.25 mg) was added to a solution of 1.1
equivalents boron trioxide (1.1 mmol, 76.58 mg) in acetonitrile
(10 mL) at reflux. After stirring for 1 h at reflux, 1.1 equivalents
pyridin-3-carboxaldehyde (1.1 mmol, 0.10 mL) and 1.2 equivalents
tributyl borate (1.2 mmol, 0.32 mL) were added. The reaction
mixture was stirred for 20 min, after which 0.016 equivalents
piperidine (0.016 mmol, 0.0016 mL), dissolved in acetonitrile (1:10),
were added. After 20 min of stirring at reflux conditions, full
conversion was determined by either TLC or LC-MS analysis, and
1
1 mLminÀ 1); H NMR (400 MHz, CDCl3): δ=3.96 (3H, s), 5.92 (1H, s),
6.55 (1H, d, J=15.8 Hz), 6.81 (1H, d, J=15.6 Hz), 6.95 (1H, d, J=
8.2 Hz), 7.08 (1H, d, J=1.7 Hz), 7.16 (1H, dd, J=8.2, 1.7 Hz); 7.58 (1H,
d, J=4.5 Hz), 7.62–7.66 (1H, m), 7.67 (1H, d, J=15.8 Hz), 7.75–7.80
(1H, m), 8.16 (1H, d, J=8.4 Hz), 8.22 (1H, d, J=8.4 Hz), 8.37 (1H, d,
J=15.6 Hz), 8.95 ppm (1H, d, J=4.5 Hz); 13C NMR (100 MHz, CDCl3):
δ=56.0, 102.4, 109.7, 114.9, 117.8, 121.8, 123.4, 123.5, 126.2, 127.2,
127.4, 129.8, 129.9, 130.2, 133.8, 140.8, 142.2, 146.9, 148.3, 148.8,
150.1, 179.0, 186.6 ppm; IR (ATR): ν 2922 (br), 1626, 1580, 1508,
10
11
12
13
14
15
16
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19
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°
the reaction mixture was cooled down to 50 C. 3 N HCl (0.5 mL)
1425, 1273, 1209, 1125, 1030, 962, 758 cmÀ 1; MS (70 eV): m/z (%)=
°
was added and the mixture was stirred during 30 min at 50 C. The
+
°
374 [M+1] (100); Mp=192 C.
reaction mixture was washed with a saturated solution of sodium
bicarbonate (3×10 mL), after which the aqueous phase was
extracted with ethyl acetate (3×10 mL). The combined organic
fractions were washed with H2O and brine. Then, the solution was
dried over MgSO4, filtrated and evaporated under reduced pressure.
After reversed phase column chromatography (acetonitrile/water,
gradient conditions from 15 to 100%), (1E,4Z,6E)-5-hydroxy-1-(4-
hydroxy-3-methoxyphenyl)-7-(pyridin-3-yl)hepta-1,4,6-triene-3-one
3a (210.18 mg, 65%) could be obtained.
(1E,4Z,6E)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(isoquinolin-
5-yl)hepta-1,4,6-triene-3-one (3e): Light-brown solid (41%); re-
versed phase column chromatography, tR =3.53 min (gradient
conditions, 15% acetonitrile in water to 100% acetonitrile, 5 min,
flow rate: 1 mLminÀ 1); 1H NMR (400 MHz, CDCl3): δ=3.96 (3H, s),
5.89 (1H, s), 6.53 (1H, d, J=15.7 Hz), 6.73 (1H, d, J=15.6 Hz), 6.95
(1H, d, J=8.3 Hz), 7.07 (1H, d, J=1.7 Hz), 7.15 (1H, dd, J=8.2,
1.7 Hz), 7.63–7.67 (2H, m), 7.65 (1H, d, J=15.7 Hz), 8.01–8.04 (3H,
m), 8.37 (1H, d, J=15.6 Hz), 8.63 (1H, d, J=6.1 Hz), 9.29 ppm (1H, s);
13C NMR (100 MHz, CDCl3): δ=56.0, 102.0, 109.7, 114.9, 116.4, 121.8,
123.2, 126.9, 127.4, 127.5, 128.5, 128.9, 129.6, 131.7, 134.2, 134.7,
141.6, 143.9, 146.9, 148.2, 153.3, 180.7, 185.4 ppm; IR (ATR): ν 2924
(1E,4Z,6E)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(pyridin-3-
yl)hepta-1,4,6-triene-3-one (3a): orange solid (65%); reversed phase
column chromatography, tR =3.31 min (gradient conditions, 15%
acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
(br), 1626, 1582, 1510, 1283, 1136 cmÀ 1; MS (70 eV): m/z (%)=374
1
1 mLminÀ 1); H NMR (400 MHz, CDCl3): δ=3.96 (3H, s), 5.85 (1H, s),
+
[M+1]+ (100), 396 [M+Na] (10); Mp=204 C.
°
6.51 (1H, d, J=15.7 Hz), 6.67 (1H, d, J=15.9 Hz), 6.94 (1H, d, J=
8,1 Hz), 7.06 (1H, d, J=1.5 Hz); 7.14 (1H, dd, J=8.1, 1.5 Hz), 7.34 (1H,
dd, J=7.9, 4.8 Hz), 7.62 (1H, d, J=15.9 Hz), 7.63 (1H, d, J=15.7 Hz),
7.85 (1H, dt, J=7.9, 1.7 Hz), 8.59 (1H, dd, J=4.8, 1.7 Hz), 8.78 ppm
(1H, d, J=1.7 Hz); 13C NMR (100 MHz, CDCl3): δ=56.0, 101.8, 109.7,
114.9, 121.7, 123.2, 123.8, 125.9, 127.4, 130.9, 134.2, 136.1, 141.7,
146.9, 148.2, 149.5, 150.5, 180.4, 185.4 ppm; IR (ATR): ν 2924 (br),
1624, 1508, 1429, 1273, 1213, 1179, 1125 cmÀ 1; MS (70 eV): m/z (%)
(1E,4Z,6E)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(1H-pyrrol-
2-yl)hepta-1,4,6-trien-3-one (3f): dark-red solid (37%); reversed
phase column chromatography, tR =3.43 min (gradient conditions,
15% acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
1 mLminÀ 1); H NMR (400 MHz, [D4]MeOD): δ=3.90 (s, 3H), 5.84 (s,
1
1H), 6.23 (dd, J=3.3, 2.4 Hz, 1H), 6.36 (d, J=15.7 Hz, 1H), 6.56–6.60
(m, 2H), 6.82 (d, J=8.2 Hz, 1H), 6.98 (dd, J=2.4, 1.2 Hz, 1H), 7.09
(dd, J=8.2, 1.7 Hz, 1H), 7.19 (d, J=1.7 Hz, 1H), 7.52 ppm (dd, J=
15.7, 4.5 Hz, 2H); 13C NMR (100 MHz, [D4]MeOD): δ=55.0, 100.4,
110.2, 110.3, 114.8, 115.2, 116.78, 116.81, 120.7, 120.8, 122.5, 123.2,
127.3, 129.3, 130.9, 140.0, 148.0, 148.8, 181.26, 181.32, 185.0,
185.1 ppm; IR (ATR): ν 3296 (br), 1620, 1566, 1508, 1425, 1261,
1157 cmÀ 1; MS (70 eV): m/z (%)=312 [M+1]+(100), 334 [M+Na]+
=324 [M+1]+ (46), 102 (40), 86 (100); Mp=195 C.
°
(1E,4Z,6E)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(pyridin-2-
yl)hepta-1,4,6-triene-3-one (3b): Orange solid (60%); reversed phase
column chromatography, tR =3.42 min (gradient conditions, 15%
acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
1 mLminÀ 1); 1H NMR (400 MHz, CDCl3): δ=3.88 (3H, s), 5.83 (1H, s),
6.47 (1H, d, J=15.7 Hz), 6.90 (1H, d, J=8.0 Hz), 7.03 (1H, s), 7.08 (1H,
d, J=8.0 Hz), 7.12 (1H, d, J=15.5 Hz), 7.23 (1H, dd, J=7.5, 4.7 Hz),
7.38 (1H, d, J=7.5 Hz), 7.58 (1H, d, J=15.7 Hz), 7.59 (1H, d, J=
15.5 Hz), 7.69 (1H, td, J=7.5, 1.3 Hz), 8.62 ppm (1H, dd, J=4.7,
1.3 Hz); 13 C NMR (100 MHz, CDCl3): δ=56.0, 102.6, 109.8, 115.2,
122.0, 123.4, 124.0, 124.8, 127.4, 127.9, 137.0, 138.0, 141.9, 147.2,
148.5, 150.1, 153.5, 179.7, 186.6 ppm; IR (ATR): ν 3065 (br), 1628,
1584, 1510, 1468, 1429, 1132, 970 cmÀ 1; MS (70 eV): m/z (%)=324
°
(80); Mp=162 C.
(1E,4Z,6E)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(1H-indol-3-
yl)hepta-1,4,6-trien-3-one (3g): dark red solid (49%); reversed phase
column chromatography, tR =3.51 min (gradient conditions, 15%
acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
1
1 mLminÀ 1); H NMR (400 MHz, CDCl3): δ=3.95 (s, 3H), 5.82 (s, 1H),
5.87 (brs, 1H), 6.49 (d, J=15.8 Hz, 1H), 6.69 (d, J=15.8 Hz, 1H), 6.94
(d, J=8.2 Hz, 1H), 7.05 (d, J=1.6 Hz, 1H), 7.12 (dd, J=8.2, 1.6 Hz,
1H), 7.27–7.32 (m, 2H), 7.42–7.45 (m, 2H), 7.54 (d, J=2.4 Hz, 1H),
7.58 (d, J=15.8 Hz, 1H), 7.94 (d, J=15.8 Hz, 1H), 7.94–7.98 (m, 1H),
8.54 ppm (brs, 1H); 13 C NMR (100 MHz, CDCl3): δ=56.0, 101.1,
109.5, 111.9, 114.5, 114.8, 120.2, 120.6, 121.6, 121.8, 122.7, 123.5,
125.4, 127.9, 129.1, 134.6, 137.2, 139.7, 146.8, 147.6, 181.6,
[M+1]+ (100); Mp=162 C.
°
(1E,4Z,6E)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(pyridin-4-
yl)hepta-1,4,6-triene-3-one (3c): Dark-orange solid (12%); reversed
phase column chromatography, tR =3.32 min (gradient conditions,
15% acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
185.3 ppm ; IR (ATR): ν 3336 (br), 1566, 1508, 1454, 1423, 1242,
1
1 mLminÀ 1); H NMR (400 MHz, CDCl3): δ=3.96 (3H, s), 5.87 (1H, s),
+
1124 cmÀ 1; MS (70 eV): m/z (%)=362 [M+1] (100); Mp=174 C.
°
6.52 (1H, d, J=15.8 Hz), 6.75 (1H, d, J=16.0 Hz), 6.95 (1H, d, J=
8.2 Hz), 7.07 (1H, d, J=1.8 Hz), 7.15 (1H, dd, J=8.2, 1.8 Hz), 7.38 (2H,
dd, J=4.6, 1.4 Hz), 7.54 (1H, d, J=16.0 Hz), 7.65 (1H, d, J=15.8 Hz),
8.65 ppm (2H, dd, J=4.6, 1.4 Hz); 13C NMR (100 MHz, CDCl3): δ=
56.0, 102.3, 109.7, 114.9, 121.7, 121.8, 123.3, 127.4, 128.1, 136.6,
142.1, 142.4, 146.8, 148.3, 150.6, 179.1, 186.5 ppm; IR (ATR): ν 2928
(br), 1624, 1593, 1512, 1423, 1281, 1251, 1126 cmÀ 1; MS (70 eV): m/z
(1E,4Z,6E)-5-Hydroxy-1-(1H-indol-3-yl)-7-(pyridin-3-yl)hepta-1,4,6-tri-
en-3-one (3h): orange solid (49%); reversed phase column
chromatography, tR =3.45 min (gradient conditions, 15%
acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
1 mLminÀ 1); H NMR (400 MHz, [D6]DMSO): δ=6.20 (s, 1H), 6.82 (d,
1
J=15.8 Hz, 1H), 7.02 (d, J=16.0 Hz, 1H), 7.20–7.27 (m, 2H), 7.46 (dd,
J=7.9, 4.4 Hz, 1H), 7.50 (dd, J=6.8, 1.7 Hz, 1H), 7.60 (d, J=16.0 Hz,
(%)=324 [M+1]+ (100); Mp=210 C.
°
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1H), 7.99 (d, J=15.8 Hz, 1H), 8.02 (d, J=1.4 Hz, 1H), 8.03 (s, 1H), 8.15
(d, J=7.9 Hz, 1H), 8.57 (d, J=4.4 Hz, 1H), 8.88 (s, 1H), 11.93 ppm
(brs, 1H); 13C NMR (100 MHz, [D6]DMSO): δ=102.1, 113.0, 113.2,
118.5, 120.7, 121.6, 123.3, 124.4, 125.4, 126.6, 131.2, 133.5, 134.7,
135.5, 137.1, 138.1, 150.1, 150.8, 178.7, 187.7 ppm; IR (ATR): ν 3221
(br), 3039 (br), 1608, 1550, 1415, 1242, 1114 cmÀ 1; MS (70 eV): m/z
(%)=317 [M+1]+ (100), 339 [M+Na]+(30), 355 [M+K]+(10) ; Mp=
(1E,4Z,6E)-5-Hydroxy-1-(1H-indol-3-yl)-7-(thiophen-2-yl)hepta-1,4,6-
trien-3-one (3m): bright red solid (69%); reversed phase column
chromatography, tR =3.84 min (gradient conditions, 15%
acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
1
2
3
4
5
6
7
8
9
1
1 mLminÀ 1); H NMR (400 MHz, [D6]DMSO): δ=6.18 (s, 1H), 6.55 (d,
J=15.6 Hz, 1H), 6.75 (d, J=15.8 Hz, 1H), 7.16 (dd, J=5.0, 3.6 Hz,
1H), 7.20–7.27 (m, 2H), 7.49–7.51 (m, 2H), 7.71 (d, J=5.0 Hz, 1H),
7.75 (d, J=15.6 Hz, 1H), 7.95 (d, J=15.8 Hz, 1H), 7.99–8.01 (m, 2H),
11.89 ppm (s, 1H); 13C NMR (100 MHz, [D6]DMSO): δ=101.5, 113.0,
113.2, 118.5, 120.6, 121.6, 123.2, 123.4, 125.4, 129.1, 129.8, 131.8,
132.1, 133.2, 136.5, 138.0, 140.6, 179.7, 186.6 ppm; IR (ATR): ν 3221
(br), 1606, 1502, 1359, 1288, 1242, 1115 cmÀ 1; MS (70 eV): m/z (%)=
°
198 C.
(1E,4Z,6E)-5-Hydroxy-1-(1H-indol-3-yl)-7-(pyridin-4-yl)hepta-1,4,6-tri-
en-3-one (3i): orange solid (16%); reversed phase column chroma-
tography, tR =3.46 min (gradient conditions, 15% acetonitrile in
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
1
water to 100% acetonitrile, 5 min, flow rate: 1 mLminÀ 1); H NMR
+
+
°
322 [M+1] (100), 343 [M+Na] (35); Mp=188 C.
(400 MHz, [D6]DMSO): δ=6.26 (s, 1H), 6.83 (d, J=15.8 Hz, 1H), 7.18
(d, J=16.1 Hz, 1H), 7.21–7.28 (m, 2H), 7.49–7.51 (m, 1H), 7.54 (d, J=
16.1 Hz, 1H), 7.75–7.78 (m, 2H), 8.00 (d, J=15.8 Hz, 1H), 8.01–8.06
(m, 2H), 8.67–8.69 (m, 2H), 11.97 ppm (brs, 1H); 13C NMR (100 MHz,
[D6]DMSO): δ=102.8, 113.0, 113.2, 118.6, 120.7, 121.7, 122.3, 123.3,
125.4, 129.0, 133.8, 135.8, 137.6, 138.1, 142.6, 150.8, 177.3,
Representative Procedure for the Synthesis of Pyrazole-Based
Non-Symmetrical Nitrogen-Containing Curcuminoids 4a–d.
The synthesis of 2-methoxy-4-{(E)-2-[5-((E)-2-(pyridin-3-yl)vinyl)-1H-
pyrazol-3-yl]vinyl}phenol 4a will be described as a representative
example for the synthesis of pyrazole-based non-symmetrical
nitrogen-containing curcuminoids 4a–d. The reaction conditions
for the synthesis of analogues 4b–d can be found in Table 3.
188.7 ppm; IR (ATR): ν 3037 (br), 1587, 1545, 1413, 1269, 1116 cmÀ 1
;
MS (70 eV): m/z (%)=317 [M+1]+(100), 339 [M+Na]+(10); Mp=
°
178 C.
(1E,4Z,6E)-5-Hydroxy-1-(1H-indol-3-yl)-7-(pyridin-2-yl)hepta-1,4,6-tri-
en-3-one (3j): orange solid (19%); reversed phase column chroma-
tography, tR=3.56 min (gradient conditions, 15% acetonitrile in
(1E,4Z,6E)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(pyridin-3-
yl)hepta-1,4,6-triene-3-one 3a (1 mmol, 323.25 mg) was dissolved
in 7.5 mL acetic acid, after which 5 equivalents hydrazine hydrate
(5 mmol, 0.24 mL) were added. The reaction mixture was stirred for
1 h at reflux conditions. After cooling down, the reaction mixture
was diluted in 10 mL ethyl acetate and washed with a saturated
solution of sodium bicarbonate (3×5 mL). The organic phase was
washed with water, dried over MgSO4, filtrated and evaporated
under reduced pressure. Purification by means of reversed phase
column chromatography (acetonitrile/water, gradient conditions
from 15 to 100%), afforded 2-methoxy-4-{(E)-2-[5-((E)-2-(pyridin-3-
yl)vinyl)-1H-pyrazol-3-yl]vinyl}phenol 4a (239.37 mg, 75%).
1
water to 100% acetonitrile, 5 min, flow rate: 1 mLminÀ 1); H NMR
(400 MHz, [D6]DMSO): δ=6.31 (s, 1H), 6.82 (d, J=15.7 Hz, 1H), 7.21
(d, J=15.5 Hz, 1H), 7.20–7.27 (m, 2H), 7.38 (ddd, J=7.6, 4.8, 0.9 Hz,
1H), 7.49–7.51 (m, 1H), 7.58 (d, J=15.5 Hz, 1H), 7.70 (dd, J=7.7,
0.9 Hz, 1H), 7.86 (ddd, J=7.7, 7.6, 1.4 Hz, 1H), 8.01 (d, J=15.7 Hz, 1
H), 8.01–8.03 (m, 2H), 8.66 (dd, J=4.8, 1.4 Hz, 1H), 11.93 ppm (s,
1H); 13C NMR (100 MHz, [D6]DMSO): δ=102.7, 113.0, 113.2, 118.8,
120.7, 121.6, 123.3, 124.6, 125.0, 125.5, 127.8, 133.6, 137.4, 137.6,
137.7, 138.0, 150.5, 153.4, 177.5, 188.7 ppm; δ=IR (ATR): ν 3159
(br), 1612, 1575, 1483, 1442, 1288, 1174 cmÀ 1; MS (70 eV): m/z (%)=
+
+
°
317 [M+1] (100), 339 [M+Na] (20); Mp=204 C.
2-Methoxy-4-{(E)-2-[5-((E)-2-(pyridin-3-yl)vinyl)-1H-pyrazol-3-yl]vinyl}
phenol (4a): beige solid (75%); reversed phase column chromatog-
raphy, tR =3.04 min (gradient conditions, 15% acetonitrile in water
to 100% acetonitrile, 5 min, flow rate: 1 mLminÀ 1); 1H NMR
(400 MHz, [D6]DMSO): δ=3.84 (3H, s), 6.72 (1H, s), 6.78 (1H, d, J=
8.2 Hz), 6.94 (1H, d, J=16.0 Hz), 6.94 (1H, dd, J=8.2, 1.7 Hz), 7.08
(1H, d, J=16.0 Hz), 7.15 (1H, d, J=1.7 Hz), 7.17 (1H, d, J=16.1 Hz),
7.26 (1H, d, J=16.1 Hz), 7.40 (1H, dd, J=7.9, 4.4 Hz), 8.03 (1H, dt,
J=7.9, 1.6 Hz), 8.46 (1H, d, J=4.4 Hz), 8.73 (1H, d, J=1.6 Hz), 9.16
(1H, brs), 13.00 ppm (1H, brs); 13C NMR (100 MHz, [D6]DMSO): δ=
56.0, 100.6, 110.0, 116.1, 120.1, 124.3, 125.9, 128.6, 130.4, 133.0,
147.4, 148.4, 148.6, 148.9 ppm; IR (ATR): ν 3360 (br), 3244 (br), 1641,
1510, 1277, 957 cmÀ 1; MS (70 eV): m/z (%)=320 [M+1]+ (100);
(1E,4Z,6E)-5-Hydroxy-1-(1H-indol-3-yl)-7-(quinolin-2-yl)hepta-1,4,6-
trien-3-one (3k): orange solid (43%); reversed phase column
chromatography, tR =3.84 min (gradient conditions, 15%
acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
1 mLminÀ 1); H NMR (400 MHz, [D6]DMSO): δ=6.40 (s, 1H), 6.84 (d,
1
J=15.8 Hz, 1H), 7.22–7.28 (m, 2H), 7.38 (d, J=15.8 Hz, 1H), 7.50 (d,
J=7.2 Hz, 1H), 7.64 (dd, J=7.3, 7.3 Hz, 1H), 7.72 (d, J=15.8 Hz, 1H),
7.81 (dd, J=7.4, 7.4 Hz, 1H), 7.95 (d, J=8.5 Hz, 1H), 7.99–8.05 (m,
4H), 8.01 (d, J=15.8 Hz, 1H), 8.44 (d, J=8.5 Hz, 1H), 11.95 ppm (s,
1H); 13C NMR (100 MHz, [D6]DMSO): δ=103.0, 113.0, 113.3, 118.9,
120.7, 121.6, 121.7, 123.3, 125.5, 127.6, 128.1, 128.4, 129.6, 129.7,
130.7, 133.7, 137.4, 137.7, 138.0, 138.1, 148.1, 154.1, 176.9,
189.1 ppm; IR (ATR): ν 3184 (br), 1598, 1564, 1502, 1226, 1116 cmÀ 1
;
°
Mp=208 C.
MS (70 eV): m/z (%)=367 [M+1]+(100), 389 [M+Na]+(5); Mp=
2-Methoxy-4-{(E)-2-[5-((E)-2-(pyridin-4-yl)vinyl)-1H-pyrazol-3-yl]vinyl}
phenol (4b): pale green-yellow solid (79%); reversed phase column
chromatography, tR =3.05 min (gradient conditions, 15%
acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
°
204 C.
(1E,4Z,6E)-5-Hydroxy-1-(1H-indol-3-yl)-7-(1H-pyrrol-2-yl)hepta-1,4,6-
trien-3-one (3l): dark-red solid (62%); reversed phase column
chromatography, tR =3.56 min (gradient conditions, 15%
acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
1 mLminÀ 1); 1H NMR (400 MHz, [D4]MeOD): δ=5.87 (s, 1H), 6.23 (dd,
J=3.4, 2.8 Hz, 1H), 6.37 (d, J=15.7 Hz, 1H), 6.55 (dd, J=3.4, 0.8 Hz,
1H), 6.70 (d, J=15.8 Hz, 1H), 6.97–6.98 (m, 1H), 7.19–7.26 (m, 2H),
7.44–7.46 (m, 1H), 7.50 (d, J=15.7 Hz, 1H), 7.68 (s, 1H), 7.92 (d, J=
15.8 Hz, 1H), 7.95–7.96 ppm (m, 1H); 13C NMR (100 MHz, [D4]MeOD):
δ=110.1, 111.8, 113.4, 114.2, 116.7, 118.2, 119.7, 120.7, 122.5, 122.8,
125.3, 129.4, 130.0, 130.5, 134.9, 137.9, 182.5, 184.3 ppm; δ=IR
(ATR): ν 3367 (br), 3244 (br), 1602, 1566, 1496, 1369, 1242 cmÀ 1; MS
1
1 mLminÀ 1); H NMR (400 MHz, [D6]DMSO): δ=3.82 (s, 3H), 6.75 (s,
1H), 6.76 (d, J=7.7 Hz, 1H), 6.91 (d, J=17.1 Hz, 1H), 6.92 (d, J=
7.7 Hz, 1H), 7.08 (d, J=17.1 Hz, 1H), 7.12 (d, J=16.7 Hz, 1H), 7.12 (s,
1H), 7.41 (d, J=16.7 Hz, 1H), 7.53 (d, J=5.8 Hz, 2H), 8.54 ppm (d, J=
5.8 Hz, 2H); 13C NMR (100 MHz, [D6]DMSO): δ=56.0, 101.0, 110.0,
114.3, 116.2, 120.8, 121.1, 124.6, 126.8, 127.8, 130.8, 144.6, 148.6,
150.5 ppm; IR (ATR): ν 3176 (br), 3080 (br), 1587, 1510, 1267,
1118 cmÀ 1; MS (70 eV): m/z (%)=320 [M+1]+ (100); Mp=180 C.
°
2-Methoxy-4-{(E)-2-[5-((E)-2-(isoquinolin-5-yl)vinyl)-1H-pyrazol-3-yl]
vinyl}phenol (4c): Light-brown solid (41%); reversed phase column
chromatography, tR =3.21 min (gradient conditions, 15%
+
+
°
(70 eV): m/z (%)=305 [M+1] (100) 327 [M+Na] (40); Mp=172 C.
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acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
1 mLminÀ 1); 1H NMR (400 MHz, [D6]DMSO): δ=3.85 (3H, s), 6.79 (1H,
d, J=8.1 Hz), 6.94 (1H, brs), 6.96 (1H, dd, J=8.1, 1.3 Hz), 6.97 (1H, d,
J=16.5 Hz), 7.09 (1H, d, J=16.5 Hz), 7.17 (1H, d, J=1.3 Hz), 7.29
(1H, d, J=16.3 Hz), 7.72 (1H, dd, J=7.9, 7.5 Hz), 7.96 (1H, d, J=
16.3 Hz), 8.08 (1H, d, J=7.9 Hz), 8.17 (1H, d, J=7.5 Hz), 8.25 (1H, d,
J=6.0 Hz), 8.60 (1H, d, J=6.0 Hz), 9.35 ppm (1H, s); 13C NMR
(100 MHz, [D6]DMSO): δ=56.0, 110.0, 116.1, 116.9, 120.6, 124.2,
127.0, 127.9, 129.1, 130.4, 133.5, 143.6, 147.3, 148.4, 153.5 ppm; IR
(ATR): ν 3179 (br), 2932 (br), 1584, 1514, 1277 cmÀ 1; MS (70 eV): m/z
(PBS-, no calcium and no magnesium) were obtained from Life
Technologies and fetal bovine serum (FBS) from Greiner Bio-one. All
cell lines, Caco-2 (colorectal adenocarcinoma), HepG2 (hepatocel-
lular carcinoma), CHO (Chinese hamster ovary), HT-29 (colorectal
adenocarcinoma) and EA.hy926 (endothelial) were obtained from
ATCC. These cell lines were cultivated and maintained in a growth
medium containing DMEM+glutamax, 1% NEAA, 1% P/S and 10%
fetal bovine serum (FBS). During the MTT experiment, serum-free
medium was used to avoid interferences. Each compound or
positive control was dissolved in DMSO in order to prepare the
25 mM stock solution for both cytotoxicity and antioxidant tests.
The stock solutions were aliquoted and stored in a refrigerator until
treatment of the cells.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
(%)=370 [M+1]+ (100), 282 (20); Mp=214 C.
°
3-{(E)-2-[5-((E)-2-(thiophen-2-yl)vinyl)-1H-pyrazol-3-yl]vinyl}-1H-
indole (4d): pale brown solid (80%); reversed phase column
chromatography, tR =3.53 min (gradient conditions, 15%
acetonitrile in water to 100% acetonitrile, 5 min, flow rate:
Cytotoxicity Activity: MTT Experiments
1
1 mLminÀ 1); H NMR (400 MHz, [D6]DMSO): δ=6.70 (s, 1H), 6.84 (d,
Throughout the experiment, standard procedures were used to
J=16.2 Hz, 1H), 6.96 (d, J=16.5 Hz, 1H), 7.06–7.08 (m, 1H), 7.12–
7.21 (m, 3H), 7.32 (d, J=16.2 Hz, 1H), 7.35 (d, J=16.5 Hz, 1H), 7.43–
7.47 (m, 2H), 7.65 (s, 1H), 7.89 (d, J=7.6, 1H), 11.38 (s, 1H),
12.75 ppm (brs, 1H); 13C NMR (100 MHz, [D6]DMSO): δ=99.2, 111.8,
112.5, 113.6, 119.9, 120.3, 122.3, 122.6, 124.2, 125.5, 125.6, 126.55,
126.60 126.9, 128.5, 137.5, 142.5 ppm; IR (ATR): ν 3178 (br), 1710,
1689, 1550, 1462, 1244 cmÀ 1; MS (70 eV): m/z (%)=318 [M+1]+
°
maintain all cell lines at 37 C with 95% humidity and 10% CO2. The
MTT assay was performed to determine the number of viable cells
in this assay. Briefly, 20000 cells suspended in the DMEM medium
(200 μL) were first inoculated into each well of 96-well microplates
and incubated for 24 hours. Then, the medium in each well was
removed and an equal volume of serum free medium (200 μL/well)
containing either test compounds or positive control (doxorubicin
hydrochloride, Dox) at various concentrations was added for
72 hours. Each compound was performed in triplicate. Afterwards,
the cell viability was determined by removing 100 μL of medium
and adding 20 μL of MTT solution (5 mg/mL in PBS) followed by
2 hours of incubation. Finally, the DMEM medium with MTT solution
was then removed and replaced with DMSO to dissolve the
formazan crystal. The 96-well microplate was then measured at
570 nm using a Spectramax (Molecular Devices) microplate spec-
trophotometer. For the data analysis, the percentage of surviving
cells after a 72 hours exposure to various concentrations of each
test compound was calculated to obtain the IC50 value of each
compound.
°
(100); Mp=202 C.
23 X-Ray Crystal Structural Determinations
24
25
For the structure of 4b, X-ray intensity data were collected at 100 K,
on a Rigaku Oxford Diffraction Supernova Dual Source (Cu at zero)
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
diffractometer equipped with an Atlas CCD detector using ω scans
and CuKα (λ=1.54184 Å) radiation. The images were interpreted
and integrated with the program CrysAlisPro.[32] Using Olex2,[33] the
structure was solved by direct methods using the ShelXS structure
solution program and refined by full-matrix least-squares on F2
using the ShelXL program package.[34] Non-hydrogen atoms were
anisotropically refined and the hydrogen atoms in the riding mode
and isotropic temperature factors fixed at 1.2 times U(eq) of the
parent atoms (1.5 times for methyl and hydroxyl groups). The
pyrazole and hydroxyl hydrogen atoms were located from a
difference Fourier electron density map.
Protein Content (SRB) Analysis
The SRB assay is based on the measurement of cellular proteins.
Sulforhodamine B binds electrostatically with basic amino acid
residues if the cells are fixed with trichloroacetic acid (TCA) and can
be solubilized by weak bases. Because of this quantitative staining
capacity of SRB, the assay is used to screen for cytotoxicity and cell
density. The cells were seeded at a concentration of 20000 cells per
well, incubated for 24 hours and treated with or without (control)
compounds. Three days after this treatment the cells were fixed by
addition of 50 μL of 50% TCA in Milli-Q water for 1 hour in the cold
Crystal data for compound 4b. C19H17N3O2, M=319.36, monoclinic,
space group P21/n (No. 14), a=16.6763(3) Å, b=5.40376(11) Å, c=
3
°
17.7393(4) Å, β=96.9999(19) , V=1586.66(6) Å , Z=4, T=100 K,
1
calc =1.337 gcmÀ 3
,
μ(CuÀ Kα)=0.718 mmÀ 1
, F(000)=672, 26796
reflections measured, 3278 unique (Rint =0.0491) which were used
in all calculations. The final R1 was 0.0387 (I >2σ (I)) and wR2 was
0.1103 (all data).
°
room, 4 C. The plate was washed at least 3 times with tap water
and dried, after which the cells were stained with an SRB solution
CCDC 1877188 contains the supplementary crystallographic data
for this paper and can be obtained free of charge via www.ccdc.ca-
m.ac.uk/conts/retrieving.html (or from the Cambridge Crystallo-
graphic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax:
+44-1223-336033; or deposit@ccdc.cam.ac.uk).
°
(0.4% sulforhodamine B in 1% glacial acetic acid) at 4 C. After
30 minutes, the plate was rinsed for 5 times with 1% glacial acetic
acid and dried. Sequentially, Tris buffer in a concentration of 10 mM
was used to re-dissolve the stain. Finally, the absorbance was
measured using the microplate spectrophotometer at a wavelength
of 490 nm. Each condition was performed in triplicate.
Biology
General
Reactive Oxygen Species (ROS) Assay
MEM non-essential amino acid solution (NEAA), 2,2-diphenyl-1-
picrylhydrazyl (DPPH), penicillin/streptomycin (P/S), Trolox and 2,7-
dichlorofluorescein diacetate were purchased from Sigma-Aldrich,
°
The experiments were performed in an incubator at 37 C with 10%
CO2. Seeding of a concentration of 20000 cells per well in a DMEM
medium was performed in a black 96-well plate with transparent
bottom. After 24 hours, the confluent cells were equally treated
with or without the compounds at 10 and 1 μM in serum-free
medium (200 μL). Then, after removal of the medium, the cells
whereas
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) and trypan blue were obtained from Amresco.
Trypsin/EDTA solution and Dulbecco’s phosphate-buffered saline
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Full Papers
were washed with PBS buffer followed by the addition of 2’,7’-
dichlorofluorescein diacetate (DCFH-DA, 20 μM) for a 30-minute
incubation period. Thereafter, the DCFH-DA was removed and the
cells were washed with PBS buffer. Afterwards, the DMEM without
phenol red medium was added in equal volume for one hour of
incubation. Finally, the black 96-well plates were measured for
support. KVH thanks the Hercules Foundation (project AUGE/11/
029 “3D-SPACE: 3D Structural Platform Aiming for Chemical
Excellence”) and the Special Research Fund (BOF) – UGent (project
01N03217) for funding. The authors also would like to thank ir.
Sari Deketelaere from Ghent University for her assistance in this
manuscript preparation.
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fluorescence with
a Gemini XPS Microplate Reader with an
excitation of 485 nm and an emission of 535 nm. Afterwards, an
SRB assay was applied to the cells as described above, in order to
normalize the results, for cell density.
Conflict of Interest
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The authors declare no conflict of interest.
Chemical Antioxidant Capacity: Ferric Reducing Ability of
Plasma (FRAP) Assay
An acetate buffer of 300 mM was prepared by adding 3.1 g of
sodium acetate trihydrate to 16 mL of acetic acid, which was
diluted to 1000 mL with Milli-Q water. The TPTZ (2,4,6-tripyridyl-s-
triazine) solution of 10 mM was prepared by adding 0.156 g of TPTZ
to 50 mL of ethanol. Lastly, a 20 mM solution of iron(III) chloride
hexahydrate was prepared by mixing 0.5404 g of FeCl3.6H2O with
2 mL 37% HCl and 98 mL Milli-Q water. The TPTZ and iron solutions
were freshly prepared on the day of the assay. These 3 mixtures
were added in a 10:1:1 ratio to obtain the FRAP reagent (acetate
buffer:TPTZ: FeCl3.6H2O). Finally, 100 μL of the samples were mixed
with 900 μL of the FRAP reagent and after 4 minutes the
absorbance was measured at 593 nm in a Spectramax microplate
spectrophotometer. Trolox was used as a standard and the FRAP
value was calculated as Trolox equivalent (μM) via a linear
regression of the Trolox standard curve or curcumin standard curve.
Keywords: curcumin · cytotoxicity · water solubility · anticancer
activity · antioxidant activity
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Water Solubility Measurement
The shake flask method is commonly used to measure aqueous
solubility.[22b,35] The test compounds were first measured for
maximum absorbance using the Liquid Chromatography (LC,
Agilent 1100 Series LC/MSD type SL mass spectrometer with
electrospray ionization (ESI 70 eV)) technique using Ascentis®
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bath at 37 C and the second one at 30 minutes under a sonicator
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bath at 37 C followed by 23 hours of incubation at 300 rpm and a
further 30 minutes’ sonication. Both time points of each sample
were centrifuged at 14,000 rpm for 5 minutes. Then, all compounds
°
were filtered (0.2 micron) at 37 C. Subsequently, the solutions were
diluted in methanol to avoid the precipitation of the compounds at
room temperature as using in previous procedures.[2h,i] Each
compound was individually measured at its maximum absorbance.
Finally, the solubility values were calculated from the linear
equations of each standard curve. The experiment was independ-
ently triplicated.
Acknowledgements
The authors are indebted to Ghent University and to the Lotus+
Erasmus Mundus Programme of the European Union for financial
ChemistryOpen 2019, 8, 236–247
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© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA

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Manuscript received: December 13, 2018
Revised manuscript received: February 5, 2019
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© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA