Oleanolic acid oxime derivatives and their conjugates with aspirin modulate the NF-κB-mediated transcription in HepG2 hepatoma cells

Article abstract of DOI:10.1016/j.bioorg.2019.103326

The aim of this study was to evaluate the effect of new oleanolic acid oxime (OAO) derivatives and their conjugates with aspirin (ASP) on the expression and activation of NF-κB in human hepatoma HepG2 cells. OAO derivatives showed a stronger cytotoxic effect against HepG2 cells compared with their conjugates with aspirin. Moreover, conjugation of OAO with ASP led to enhanced downregulation of NF-κB expression and activation. Among the hybrids with ASP, compounds: 19, 3-(2-acetoxy)benzoyloxyiminoolean-12-en-28-oic acid morpholide and 13, 3-(2-acetoxy)benzoyloxyiminoolean-12-en-28-oic acid methyl ester, differing, respectively, in morpholide and methyl ester groups at the C-17 position of oleanolic acid (OA) molecule were the most efficient. COX-2 transcript and protein levels were also diminished after treatment with these compounds. The results of this study indicate that the new derivatives of OAO and particularly their conjugates with ASP, downregulate the expression of COX-2 in HepG2 cells by modulating the NF-κB signaling pathway and suggest their potential application in the prevention of liver inflammation and cancer.

Full text of DOI:10.1016/j.bioorg.2019.103326

BioorganicChemistry93(2019)103326
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Oleanolic acid oxime derivatives and their conjugates with aspirin modulate
the NF-κB-mediated transcription in HepG2 hepatoma cells
Violetta Krajka-Kuźniaka, Barbara Bednarczyk-Cwynarb, Jarosław Paluszczaka, Hanna Szaefera,
⁎
Maria Narożnaa, Lucjusz Zaprutkob, Wanda Baer-Dubowskaa,
a Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, Święcickiego 4, 60-781 Poznan, Poland
b Department of Organic Chemistry Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
The aim of this study was to evaluate the effect of new oleanolic acid oxime (OAO) derivatives and their con-
jugates with aspirin (ASP) on the expression and activation of NF-κB in human hepatoma HepG2 cells. OAO
derivatives showed a stronger cytotoxic effect against HepG2 cells compared with their conjugates with aspirin.
Moreover, conjugation of OAO with ASP led to enhanced downregulation of NF-κB expression and activation.
Among the hybrids with ASP, compounds: 19, 3-(2-acetoxy)benzoyloxyiminoolean-12-en-28-oic acid morpho-
lide and 13, 3-(2-acetoxy)benzoyloxyiminoolean-12-en-28-oic acid methyl ester, differing, respectively, in
morpholide and methyl ester groups at the C-17 position of oleanolic acid (OA) molecule were the most efficient.
COX-2 transcript and protein levels were also diminished after treatment with these compounds. The results of
this study indicate that the new derivatives of OAO and particularly their conjugates with ASP, downregulate the
expression of COX-2 in HepG2 cells by modulating the NF-κB signaling pathway and suggest their potential
application in the prevention of liver inflammation and cancer.
Oleanolic acid oximes derivatives
Aspirin oleanolate conjugates
NF-κB
HepG2 cells
1. Introduction
potential therapeutic activities of OA derivatives were associated with
their hydrophilicity when compared to the parent compound. In this
Naturally occurring triterpenoids have been shown to possess sev-
eral biological activities important for cancer chemoprevention and
therapy [1]. One of the best known and well-characterized re-
presentative of triterpenoids class is oleanolic acid (OA), 3-β-hydro-
xyolean-12-en-28-oic acid, produced by a variety of medicinal herbs
and other plants [1,2]. A number of studies have reported the anti-
carcinogenic activities of OA in different in vitro and in vivo models
[1]. An important factor contributing to these effects of OA is its anti-
inflammatory potential. Besides, OA possesses also well documented
hepatoprotective properties [3]. However, it has an absolute oral
bioavailability of only 0.7% because of its low permeability and aqu-
eous solubility [4]. The very low oral bioavailability of OA could be due
to poor absorption and extensive metabolic clearance [5].
regard, an earlier study showed that, in comparison to parent com-
pound, the OA oxime (OAO) derivative, methyl 3-octanoylox-
yiminoolean-12-en-28-oate, presented better anti-inflammatory activity
than that of the parent compound, which was demonstrated by its anti-
oedemic effects in rats with carrageenan-induced skin inflammation
[7]. Moreover, the pharmacological activity of OA derivatives can be
increased by conjugation with drugs such as aspirin (ASP, acet-
ylsalicylic acid). This type of conjugates may enhance the anti-in-
flammatory activity of OA derivatives, but also allows to avoid ASP-
induced gastrointestinal side effects [8]. The results of our previous
study demonstrated that in comparison to the parent compound, the
conjugate of 3-hydroxyiminoolean-12-en-28-oic acid morpholide with
ASP: 3-(2-acetoxy)benzoyloxyiminoolean-12-en-28-oic acid morpho-
lide (19) exerts strong anti-inflammatory activity in rats as result of
single or subchronic administration of OAO-ASP conjugate. The ad-
ministration of this compound lowered the blood concentration of in-
terleukin–6 (IL-6) and the level of IL-6 transcript level in blood lym-
phocytes, but the mechanism of its anti-inflammatory activity has not
been established yet [9]. Multiple signaling pathways might be
Over the past years, a large number of triterpenoids have been
chemically modified in order to improve their bioactivity and bioa-
vailability and to enhance their protective and/or therapeutic effects. In
many cases, the modified OA has been shown to possess better cyto-
protective and anti-inflammatory properties than that of the parent
compound [6]. The results of several studies pointed out that the
⁎ Corresponding author.
E-mail address: baerw@ump.edu.pl (W. Baer-Dubowska).
https://doi.org/10.1016/j.bioorg.2019.103326
Received 19 July 2019; Received in revised form 26 September 2019; Accepted 26 September 2019
Availableonline27September2019
0045-2068/©2019PublishedbyElsevierInc.

V. Krajka-Kuźniak, et al.
BioorganicChemistry93(2019)103326
involved in the injury-inflammation-regeneration response. Classical
NF-κB signaling is one of the most important pathways that is activated
particularly during inflammation-related carcinogenesis. IL-6 is one of
the many genes which is regulated by this transcription factor. NF-κB
commonly refers to a p50-p65 heterodimer, which represents the major
Rel/NF-κB complex in most of the cells. In the latent state, NF-κB is
sequestered in the cytosol by its inhibitor, IκB protein. Stimulation with
a variety of signals, including cytokines, pathogens and injuries leads to
the activation of IκB kinase (IKK) which phosphorylates IκB. This results
in the proteasomal degradation of IKK and the release of NF-κB for
nuclear translocation and activation of transcription of genes such as
COX-2, which encodes cyclooxygenase-2 [10]. Hepatocellular carci-
noma (HCC), the major form of primary liver cancer, represents the
quintessential example of inflammation-driven cancers. Inflammation is
relevant as a risk factor and also as the consequence of carcinogenesis
induction in HCC [11].
carboxyl moiety of ASP provided a signal at δ of about 162 ppm. All
signals were present in 1H and 13C NMR spectra of conjugates 13, 15,
17 and 19, which confirm the presence of oleanolate skeleton and its
characteristic functional groups.
2.2. Cell viability
The impact of OA, OAO derivatives and their conjugates with ASP
on the viability of human immortalized hepatocytes (THLE-2) and he-
patoma cells (HepG2) was evaluated using the MTT assay. In the con-
centration range 0.2–150 µM, all tested compounds reduced the viabi-
lity of both cells types in a dose-dependent manner (Fig. 1). As shown in
Fig. 1A, most of the synthesized derivatives demonstrated moderate to
favorable anti-proliferative activities in HepG2 cells, whereas three
compounds (12, 16 and 18) exhibited stronger cytotoxic activity than
that of the parent compound (1). The cytotoxicity of OAO derivatives
conjugates with ASP (13, 15, 17 and 19) and ASP itself in HepG2 cells
was significantly lower than that of OA (1) or OAO (12, 14, 16 and 18).
ASP and its OAO derivatives conjugates (13, 15, 17 and 19) were not
toxic in the tested concentration range, although the cytotoxicity of
compound 19 was slightly higher than that of other conjugates. Its
dose-response curves are presented in Fig. 1B. Fig. 1C and D show the
dose–response curves for THLE-2 cells. Basically, the trend was similar
in both HepG2 and THLE-2 cells. However, the comparison of IC50
values indicates that OA and its oximes are more cytotoxic toward
HepG2 cells in comparison to THLE-2 cells. There were no differences
in the cytotoxic effects of ASP and its conjugates with OAO on both cell
types (Table 1). These data indicate that OAO derivatives themselves,
particularly compound 19 (morpholide derivative), may be more effi-
cient as therapeutic agents, while their conjugates with aspirin may be
important for chemoprevention purposes.
Overexpression of COX-2, related to increased cell growth and in-
vasiveness, is observed in human HCC. Therefore, the suppression of
COX-2 activity can be an important approach to prevent hepatic in-
flammation. Moreover, designing the inhibitors of NF-κB inhibitors, is
an interesting approach to develop new cancer chemopreventive agents
or therapeutics [12].
Therefore, the aim of this study was the further elucidation of the
mechanism of the anti-inflammatory activity of OAO-derivatives and
their conjugates with ASP, through the evaluation of their effect on the
expression and activation of NF-κB in human hepatoma HepG2 cells. In
order to select the most potent modulator of this pathway, a series of
new OAO-ASP hybrids were designed and synthesized.
2. Results and discussion
2.1. Synthesis and NMR spectroscopic characteristics of the OAO and their
conjugates with aspirin
2.3. Effect of OA, OAO and their ASP conjugates on the expression of NF-
κB p50 and p65 genes
OAO derivatives and their conjugates were synthesized according to
the previously described procedures [7]. Schemes 1 and 2 present an
overview of the synthesis. The chemical structures of the synthesized
compounds were elucidated based on the analysis of infrared (IR)
spectroscopy, 1H and 13C nuclear magnetic resonance (NMR) spectro-
scopy, mass spectroscopy (MS) and high-resolution mass spectroscopy
(HRMS). The 1H NMR spectra of conjugates 13, 15, 17 and 19 show
that the protons of aromatic ring of ASP moiety formed two doublet of
doublets (observed at about δ 7.97 and 7.14 ppm) and two triplets of
doublets (observed at about δ 7.57 and 7.32 ppm). The presence of a
strong signal, located at about δ 2.33 ppm, of intensity 3H, confirmed
the presence of acetylsalicylic moiety within the molecules of com-
pounds 13, 15, 17 and 19. The chemical structures of the synthesized
compounds were elucidated based on 1H and 13C NMR spectra. The 1H
NMR spectra of conjugates 13, 15, 17 and 19 show that the protons of
aromatic ring of acetylsalicylic moiety formed two doublet of doublets
(observed at about δ 7.97 and 7.14 ppm) and two triplets of doublets
(observed at about δ 7.57 and 7.32 ppm). The presence of a strong
signal, located at about δ 2.33 ppm, of intensity 3H, confirmed the
presence of acetyl group in acetylsalicylic moiety within the molecules
Fig. 2 presents the results of quantitative PCR analysis of the tran-
script levels of p50 and p65 NF-κB subunits, the major components of
active Rel/NF-κB complex in the cell nucleus. Panel A shows the effects
of treatment with OA and OAO, while Panel B illustrates the changes in
NF-κB subunits expression as the result of treatment of HepG2 cells with
OAO conjugates. Incubation with all OAO derivatives decreased the NF-
κB p50 and NF-κB p65 transcript levels in comparison to the control
group. Derivatives 12, 16 and 18 at a concentration of 20 µM sig-
nificantly decreased the expression of NF-κB p50 by ~23%, 20% and
30%, respectively. Similarly the expression of NF-κB p65 subunit was
diminished by compounds 12, 14, 16 and 18 by ~27%, 21%, 32% and
45%, respectively. Treatment with OAO conjugates 13 and 19 also
decreased the expression of NF-κB p50 and NF-κB p65 by ~20–49% at a
concentration of 10 and 20 µM, while conjugates 15 diminished the
expression of these NF-κB subunits by ~20–27% only at a concentration
of 20 µM. Thus, the morpholide derivative of OAO and its conjugate
with ASP diminished the expression of NF-κB subunits to the most ex-
tent. Aspirin, in comparable concentrations, showed weaker effect on
the transcription of NF-κB, suggesting potentially higher anti-in-
flammatory activity of its hybrid compounds.
of compounds 13, 15, 17 and 19. This fact was further confirmed by 13
C
NMR spectra. Aromatic ring carbons formed two quaternary signals (δ
about 150 and 123 ppm) and four tertiary signals (δ about 134, 131,
126 and 124 ppm). The presence of acetoxy group within the acet-
ylsalicylic moiety was proved on the basis of two signals: one that was
detected at δ 170 ppm (Cq) and the other that was detected at δ
21.0 ppm. In 13C NMR spectra of oximes 12, 14, 16 and 18, a signal
derived from C-3 atom was detected at about 167 ppm [13]. Replace-
ment of hydrogen atom within hydroxyimino group of acetylsalicylic
moiety resulted in the shifting of value of chemical shift for C-3 atom
toward higher values and for compounds 13, 15, 17 and 19 signal for C-
3 atom was present at δ about 176 ppm. The carbon atom in the
2.4. Effect of OA, OAO derivatives and their ASP conjugates on NF-κB
activation
Fig. 3 presents the results of the assessment of the binding of p50
and p65 NF-κB subunits to their immobilized consensus site. All OAO
derivatives at the whole range of tested concentrations diminished the
content of NF-κB p50 and NF-κB p65 subunits in the DNA-binding
complex extracted from the nuclei. However, significant changes
(by ~ 20–28% and 25–40% for the binding of p50 and p65, respec-
tively) were observed only for higher concentrations of these
2

V. Krajka-Kuźniak, et al.
BioorganicChemistry93(2019)103326
Scheme 1. Synthesis of intermediates 2–11 for the synthesis of oleanolic acid oximes (OAO) and conjugates 12–19. Reagents and conditions: (a) dimethyl sulfate,
NaOH, ethanol, reflux; (b) benzyl chloride, DMF, K2CO3, heating; (c) Jones reagent, acetone, r.t.; (d) acetic anhydride, reflux; (e) thionyl chloride, r.t; (f) morpholine,
benzene, r.t.; (g) NaOH, ethanol, reflux.
compounds. Again, compound 18 was shown to be the most effective
inhibitor of NF-κB activation. ASP, in the highest concentration and its
conjugates with OAO derivatives significantly reduced the binding of
p65 to its consensus sequence. Conjugate of ASP with morpholide de-
rivative of OAO (19) was the most effective and reduced the binding
level of p65 by ~ 49%, followed by methyl derivative (13). Binding of
p50 subunit to its consensus sequence was less affected, but compounds
13 and 19 diminished it by ~ 30% and 25% respectively at the highest
concentration. The NF-κB p50 subunit serves only as a helper in NF-κB
DNA binding, whereas the p65 subunit is responsible for initiating
transcription [14]. The disproportionate increase in the activated p65
and subsequent transactivation of effector molecules is integral to the
pathogenesis of many diseases including cancer. Hence, the NF-κB p65
signaling pathway is considered a pivotal point for the drug discovery
Scheme 2. Synthesis of OAO (12, 14, 16 and 18) and their conjugates with aspirin (ASP) (13, 15, 17 and 19). Reagents and conditions: (a) NH2OH × HCl, ethanol,
reflux; (b) aspirin, dioxane, DCC, r.t.
3

V. Krajka-Kuźniak, et al.
BioorganicChemistry93(2019)103326
Fig. 1. The effect of the OA, OAO derivatives (12, 14, 16, and 18; Panels A and C) and OAO conjugates with ASP (13, 15, 17 and 19; Panels B and D) on the viability
of HepG2 and THLE-2 cells. Data are expressed as means
SEM from three separate measurements.
2.5. Effect of OA, OAO derivatives and their ASP conjugates on the IKK
protein
Table 1
Comparison of OAO derivatives cytotoxicity in non-tumorigenic (THLE-2) and
hepatoma cells (HepG2).
NF-κB activation involves IκB kinase (IKK) activation which leads to
IκB phosphorylation and subsequent degradation of ubiquitin-depen-
dent IκB by 26S proteasome complex. Therefore, phosphorylation of IκB
by IKK complex (α and β) is a critical regulatory step in the activation
of NF-κB [16]. Moreover, both IKK α and β can phosphorylate the RelA/
p65 subunit to promote transactivation potential [17]. Consequently, in
this study, the most active OAO derivatives, 12 and 18, and their
conjugates with ASP (13 and 19) significantly (by 20–38%) reduced the
IKKα/β protein level (Fig. 5). Thus, decreased activation of NF-κB
might result principally from a lower amount of IKK available for IκB
phosphorylation and subsequent inhibition of NF-κB transactivation.
Compound
IC50 (µM)
SEM*
THLE-2
HepG2
OA
12
14
16
18
ASP
13
15
17
19
80
46
3.71
1.61
41
3.31
1.7
34
> 150
148
34
1.36
38
0.93
1.58
2.55
105
2.13
0.67
3.53
25
> 150
146
> 150
> 150
> 150
> 150
> 150
> 150
141
> 150
* The IC50 values are expressed as mean
SEM obtained from three in-
2.6. Effect of the OA, OAO derivatives and their ASP conjugates on the
COX-2 transcript and protein level
dependent experiments and calculated based on the dose-response curves as-
sessed by the MTT assay.
COX-2 gene is under the transcriptional control of NF-κB, thus in the
next step the mRNA and protein level of this enzyme was determined.
In agreement with the data concerning the effect of new OAO deriva-
tives and their conjugates with aspirin on the activation of NF-κB, COX-
2 mRNA transcript and protein levels were the most reduced as result of
treatment with OAO derivative 18 and its conjugate with ASP, 19. The
effect of the conjugate was more pronounced on mRNA level, but still
conjugates 19 and 13 were more effective than ASP alone (Fig. 6).
These results confirm that OAO derivatives, through inhibition of NF-κB
and development [15]. Thus, taking the level of cytotoxicity into con-
sideration, the morpholide derivative of OAO might be considered as
one of the candidates for new therapeutics, while its conjugate with
ASP as a chemopreventive agent. A similar pattern, as in the case of NF-
κB p65 DNA binding capability, was observed in the reduction of the
p65 nuclear protein level (Fig. 4). This further confirms the effect of
these compounds on the activation of NF-κB.
4

V. Krajka-Kuźniak, et al.
BioorganicChemistry93(2019)103326
Fig. 2. The effect of the OA, OAO derivatives (12, 14, 16 and 18, panel A) and OAO conjugates with ASP (13, 15, 17 and 19, panel B) on the expression of NF-κB in
HepG2 cells. Data (means SEM) from three separate experiments. The values were calculated as mRNA level in comparison with control cells (expression equals
1). The asterisk (*) above the bar denotes statistically significant difference from the control group, p < 0,05.
activation, diminish the expression of one of the key elements of in-
flammatory response. Moreover, conjugation of ASP particularly with
OAO morpholide derivatives (19) strengthens this effect.
further supports the conclusion on the potential application of these
compounds in the prevention of liver inflammation and cancer.
3. Conclusion
4. Experimental section
The results of this study provided the elucidation of the molecular
basis of previously observed in vivo anti-inflammatory activity of
oleanolic acid oxime morpholide conjugated with ASP. Inhibition of
NF-κB expression and activation, as a result of the reduced amount of
IKKα/β, can be responsible for this effect. The specificity of OAO
structure substitution seems to be crucial for the NF-κB modulation and
subsequent anti-inflammatory effect. In this regard, comparison with
parent compound OA (1) as well as its oxime (16) clearly indicates that
the substitution within carboxyl group at position C-17 increases their
ability to modulate the NF-κB signaling pathway. Substitution with
morpholine (18) followed by the substitution with a methyl group (12)
showed to be the most effective, whereas substitution with benzyl
group did not differ in its activity from OA (1) or OAO (16) in NF-κB
modulation. Moreover, conjugation of OAO substituted with morpho-
line or methyl group at C-28 with ASP inhibited NF-κB activation and
subsequently COX-2 expression to a greater extent not only in com-
parison with OAO, but also ASP.
4.1. Synthesis of triterpenic derivatives 2–19
General information. For reactions in anhydrous conditions, the sol-
vents were purified and dried by using common procedures. Thin layer
chromatographic (TLC) analysis (reactions progress and the level of
compounds purity) was conducted on HPTLC aluminum sheets covered
with silicagel 60 F245. Benzene ethyl acetate (1:1, 2:1, 4:1, or 9:1, v:v)
was used as an eluent. The chromatograms were visualized by spraying
with 10% ethanolic solution of sulfuric acid and heating the plates at
about 110 °C for a few minutes. Column chromatography was per-
formed using silicagel 60 (0.063–0.200 mm, 70–230 mesh) and me-
thylene chloride as eluent. Melting points were measured with a Büchi
apparatus in an open capillary and are uncorrected. IR spectra were
recorded for KBr disks (0.5% mixture of sample in KBr) using a Specord
IR-75 spectrophotometer. NMR spectra for hydrogen atoms (1H) and for
carbon atoms (13C) were recorded in deuterated chloroform or deut-
erated pyridine solutions with tetramethylsilane (TMS) as an internal
standard with the application of Varian Gemini 300 VT spectrometer.
The multiplicities of signals in 1H NMR spectra were marked as follows:
s–singlet, br/s–broad singlet, d–doublet, t–triplet, td–triplet of doublets,
dd–doublet of doublets and m–multiplet. The most representative sig-
nals are given in the spectral data (1H and 13C NMR) listed below. MS
were recorded using an AMD 402 spectrometer with electroionization.
The elemental analyses (C, H and N) were performed using a 2400 CHN
analyzer (PerkinElmer, MA, USA).
Conjugation with ASP significantly decreased the cytotoxicity of
OAO derivatives in human hepatoma cells. Therefore, the combination
of reduced cytotoxicity with diminished NF-κB expression and activity
in comparison not only with the OAO, but also ASP might be particu-
larly important for the application of these hybrids in liver cancer
chemoprevention and/or in adjuvant therapy. Since the overexpression
of COX-2, related to increased cell growth and invasiveness, is observed
in human HCC, its suppression by OAO derivatives conjugates with ASP
5

V. Krajka-Kuźniak, et al.
BioorganicChemistry93(2019)103326
Fig. 3. The effect of the OA, OAO derivatives (12, 14, 16 and 18, Panel A) and OAO conjugates with ASP (13, 15, 17 and 19, Panel B) on NF-κB binding to DNA in
HepG2 cells. Data (means SEM) from three separate experiments. The values were calculated as the level of protein in comparison with control cells (expression
equals 100%). The asterisk (*) above the bar denotes statistically significant differences from the control group, p < 0,05.
4.1.1. Oleanolic acid (OA) esterification
was performed according to the protocol given in the literature.
Methyl oleanolate (2). Synthesis of methyl oleanolate (2) from
1.0 mmol of OA (1) was performed as presented in the literature.
Physical and spectral data agree with those available in the literature
data [18].
Physical and spectral data agree with those available in the literature
[18].
Oleanolic acid morpholide (10). Synthesis of oleanolic acid
morpholide (10) from 1.0 mmol of acetyloleanolic acid morpholide (9)
was performed according to the protocol given in the literature.
Physical and spectral data agree with those available in the literature
[18].
Benzyl oleanolate (3). K2CO3 (210 mg, 1.5 mmol) was added to a
stirred and heated (120 °C) solution of OA (1) and, (456 mg, 1.0 mmol)
in dried N,N-dimethylformamide (DMF) (8.0 mL). The resulted sus-
pension was stirred and heated at 120 °C for 30 min and to this, benzyl
chloride (150 mg, 1.2 mmol) was added dropwise. After further stirring
and heating for 30 min, the solution was poured into a container with a
5-time excess volume of water. The formed precipitate was filtered off,
washed with water and dried. C37H54O3; mol. mass: 546.4073; yield:
520 mg (95.1%); m.p.: 190 °C (EtOH); spectral data agree with those
available in the literature [19].
4.1.3. Jones oxidation of compounds 2, 3, 1, and 10
Methyl oleanonate (4). Synthesis of methyl oleanonate (4) from
1.0 mmol of methyl oleanolate (2) was performed according to the
protocol given in the literature [13,18]. Physical and spectral data
agree with those available in the literature [13].
Benzyl oleanonate (5). Synthesis of benzyl oleanonate (5) from
benzyl oleanolate (3) was performed according to the protocol given in
the literature [13]. Physical and spectral data agree with those avail-
able in the literature [21].
4.1.2. Multistep synthesis of OA morpholide (10)
Acetyloleanolic acid (7). Synthesis of acetyloleanolic acid (7) from
1.0 mmol of oleanolic acid (1) was performed according to the protocol
given in the literature [18]. Physical and spectral data agree with those
available in the literature [20].
Oleanonic acid (6). Synthesis of oleanonic acid (6) from 1.0 mmol
of oleanolic acid (1) was performed according to the protocol given in
the literature [13,18]. Physical and spectral data agree with those
available in the literature [21].
Acetyloleanolic acid chloride (8). Synthesis of acetyloleanolic
acid chloride (8) from 1.0 mmol of acetyloleanolic acid (7) was per-
formed according to the protocol given in the literature [18].
Acetyloleanolic acid morpholide (9). Synthesis of acetyloleanolic
acid morpholide (9) from 1.0 mmol of acetyloleanolic acid chloride (8)
Oleanonic acid morpholide (11). Synthesis of oleanonic acid
morpholide (11) from 1.0 mmol of oleanolic acid morpholide (10) was
performed according to the protocol given in the literature. Physical
and spectral data agree with those available in the literature [18].
6

V. Krajka-Kuźniak, et al.
BioorganicChemistry93(2019)103326
A
NF-κB p50 protein
NF-κB p65 protein
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
*
*
*
*
NF-κB p50
NF-κB p65
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
Lamin
B
NF-κB p50 protein
NF-κB p65 protein
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
*
*
*
NF-κB p50
NF-κB p65
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
Lamin
Fig. 4. The effect of the OA, OAO derivatives (12, 14, 16 and 18, Panel A) and OAO conjugates with ASP (13, 15, 17 and 19, Panel B) on NF-κB protein level in
HepG2 cells. Data (means
SEM) of Western blot analysis of the nuclear content of p50 and p65 from three separate experiments and representative immunoblots
are shown. The sequence of the bands corresponds to the sequence of bars in the graph. Lamin content was used for the normalization of results. The values were
calculated as the level of protein in comparison with control cells (expression equals 100%). The asterisk (*) above the bar denotes statistically significant differences
from the control group, p < 0,05.
4.1.4. Synthesis of oximes 12, 14, 16 and 18
dd, J = 4.0 and 13.7 Hz, C18-Hβ); 1.15; 1.10; 1.05; 1.00; 0.92; 0.89;
0.64 (7 × 3H, 7 × s, 7 × CH3); 13C NMR (100 MHz, CDCl3): 177.4 (Cq,
–COO-CH2-Ar); 168.4 (Cq, C-3); 143.7 (Cq, C-13); 136.4 (Cq, –COO-CH2-
Ar), 128.4 × 2, 127.9 × 2 and 127.8 (5 × CH, –COO-CH2-Ar); 122.3
(CH, C-12); 65.9 (CH2, –COO-CH2-Ar); 47.6 (Cq, C-17). MS/EI (m/z):
559.41 (45.1%, M•+). Elemental analysis calculated: C 79.38%, H
9.54% and N 2.50%, found: C 79.40%, H 9.55% and N 2.50%.
Oxime of oleanonic acid (16). Synthesis of oxime of oleanonic acid
(16) from 1.0 mmol of oleanonic acid (6) was performed according to
the protocol given in the literature [13,18]. Physical and spectral data
agree with those available in the literature [22].
Oxime of methyl oleanonate (12). Synthesis of oxime of methyl
oleanonate (12) from 1.0 mmol of methyl oleanonate (4) was per-
formed according to the protocol given in the literature [13,18]. Phy-
sical and spectral data agree with those available in the literature [13].
Oxime of benzyl oleanonate (14). Synthesis of oxime of benzyl
oleanonate (14) from 1.0 mmol of benzyl oleanonate (5) was performed
according to the protocol in the literature [13,18]. C37H53NO3; mol.
mass: 559.4025; yield: 510.7 mg (91.3%); m.p.: 154–156 °C
(EtOH + H2O). IR (KBr, cm−1): 3240 (OH, NOH), 2905 (CH, -COO-
CH2Ar), 1705 (C]O, COOCH2Ar); 1H NMR (400 MHz, CDCl3): 9.34
(1H, br/s, =NOH); 7.36 – 7.30 (5H, m, –COO-CH2-Ar); 5.29 (1H, t,
J = 3.5 Hz, C12-H); 5.08 (2H, d, J = 12.6 Hz, –COO-CH2-Ar); 2.91 (1H,
Oxime of oleanonic acid morpholide (18). Synthesis of oxime of
oleanonic acid morpholide (18) from oleanonic acid morpholide (11)
7

V. Krajka-Kuźniak, et al.
BioorganicChemistry93(2019)103326
B
A
IKK protein
*
IKK protein
*
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
*
IKK
IKK
1
2
3
3
4
4
5
6
7
8
9
10 11 12 13 14 15 16
1
2
3
4
5
6
7
7
8
9
10 11 12 13 14 15 16
10 11 12 13 14 15 16
Actin
Actin
1
1
2
3
4
5
6
8
9
2
5
6
7
8
9
10 11 12 13 14 15 16
Fig. 5. The effect of the OA, OAO derivatives (12, 14, 16 and 18, Panel A) and OAO conjugates with ASP (13, 15, 17 and 19, Panel B) on IκB kinase (IKK) protein
level in HepG2 cells. Data (means
SEM) of Western blot analysis of the cytosolic content of IKK from three separate experiments and representative immunoblots
are shown. The sequence of the bands corresponds to the sequence of bars in the graph. Actin content was used for the normalization of results. The values were
calculated as the level of protein in comparison with control cells (expression equals 100%). The asterisk (*) above the bar denotes statistically significant differences
from the control group, p < 0,05.
was performed according to the protocol given in the literature [18].
Physical and spectral data agree with those available in the literature
[13,18].
51.5 (CH3, –COOCH3); 46.6 (Cq, C-17); 21.0 (CH3, CH3OCO-Ar-
COON]); Ar = aromatic ring. MS/EI (m/z): 645.45 (13.2%, M•+).
Elemental analysis calculated: C 74.39%, H 8.58% and N 2.17%, found:
C 74.41%, H 8.57% and N 2.16%.
4.1.5. Synthesis of conjugates of oximes 12, 14, 16 and 18 with aspirin
(synthesis of compounds 13, 15, 17 and 19)
Conjugate of oxime of benzyl oleanonate and aspirin (=3-(2-
acetoxy)benzoyloxyiminoolean-12-en-28-oic acid benzyl ester, 15).
General method: To a saturated solution of oxime (12, 14, 16, or
18, 1.0 mmol) in dioxane, aspirin (1.3 mmol) and dicyclohex-
ylcarbodiimide (DCC, 1.5 mmol) were added and stirred. The stirring
was continued at room temperature until the total consumption of
oxime (TLC control). The resulting precipitate was filtered, washed
with dioxane and rejected. The filtrate was poured into a container with
5-times excess water and the precipitated solid was collected and ex-
tracted with methylene chloride (5 × 5 mL). The organic solution was
washed with a 5% solution of HCl (5 × 5 mL) and water (3 × 5 mL) and
then dried and subjected to column chromatography on silica gel. After
evaporating the solvent the crude product was dissolved in ethanol and
precipitated with water.
C46H59NO6; mol. mass: 721.434 yield: 673 mg (93.3%), m.p.: 90–95 °C.
IR (KBr, cm−1): 2900 (CH, -COOCH2Ar), 1775 (C]O, CH3OCO-Ar-
COON]C), 1755 (C]O, CH3OCO-Ar-COON = C), 1725 (N]C,
CH3OCO-Ar-COON = C), 1705 (C]O, COOCH2Ar); 1H NMR (400 MHz,
CDCl3): 7.97 (1H, dd, J = 1.6 and 7.8 Hz, CH3OCO-Ar-COON]), 7.56
(1H, td, J = 1.2 and 7.8 Hz, CH3OCO-Ar-COON = ), and 7.13 (1H, dd,
J = 1.0 and 8.1 Hz, CH3OCO-Ar-COON]); 7.36 – 7.30 (1H + 5H, m,
–CH3OCO-Ar-COON = and –COO-CH2-Ar); 5.30 (1H, t, J = 3.5 Hz, C12
-
H); 5.09 (2H, d, J = 12.5 Hz, –COO-CH2-Ar); 2.91 (1H, dd, J = 4.0 and
13.8 Hz, C18-Hβ), 2.32 (3H, s, CH3OCO-Ar-COON]), 1.20, 1.17, 1.14,
1.01, 0.80, 0.77 and 0.68 (7 × 3H, 7 × s, 7 CH3 groups); Ar = aromatic
ring. 13C NMR (100 MHz, CDCl3): 177.1 (Cq, –COOCH3); 176.3 (Cq, C-
3); 169.6 (Cq, CH3OCO-Ar-COON]); 161.8 (Cq, CH3OCO-Ar-COON]);
150.5 (Cq, CH3OCO-Ar-COON]), 133.6, 131.2, 125.8, 124.0 (4 × CH,
CH3OCO-Ar-COON]) and 122.8 (Cq, CH3OCO-Ar-COON]); 143.6 (Cq,
C-13); 136.1 (Cq, COO-CH2-Ar), 128.2 × 2, 127.8 × 2 and 127.7
(5 × CH, –COO-CH2-Ar); 122.3 (CH, C-12); 65.7 (CH2, –COO-CH2-Ar);
47.5 (Cq, C-17); 21.0 (CH3, CH3OCO-Ar-COON]); Ar = aromatic ring.
MS/EI (m/z): 721.39 (22.1%, M•+). Elemental analysis calculated: C
76.53%, H 8.24% and N 1.94%, found: C 76.52%, H 8.25% and N
1.93%.
Conjugate of oxime of methyl oleanonate and aspirin (=3-(2-
acetoxy)benzoyloxyiminoolean-12-en-28-oic acid methyl ester, 13).
C
40H55NO6; mol. mass: 645.403; yield: 606 mg (93.9%), m.p.:
95–100 °C. IR (KBr, cm−1): 1775 (C]O, CH3OCO-Ar-COON = C), 1745
(C]O, CH3OCO-Ar-COON = C), 1730 (N]C, CH3OCO-Ar-COON]C),
1715 (C]O, –COOCH3); 1H NMR (400 MHz, CDCl3): 7.97 (1H, dd,
J = 1.6 and 7.8 Hz, CH3OCO-Ar-COON]), 7.57 (1H, td, J = 1.2 and
7.8 Hz, CH3OCO-Ar-COON]), 7.32 (1H, td, J = 1.2 and 7.6 Hz,
CH3OCO-Ar-COON]) and 7.19 (1H, dd, J = 1.0 and 8.1 Hz, CH3OCO-
Ar-COON]), 5.30 (1H, t, J = 3.5 Hz, C12-H); 3.63 (3H, s, –COOCH3);
2.89 (1H, dd, J = 4.0 and 13.8 Hz, C18-Hβ), 2.34 (3H, s, CH3OCO-Ar-
COON]), 1.30, 1.20, 1.15, 1.05, 0.85, 0.81 and 0.76 (7 × 3H, 7 × s, 7
CH3 groups); Ar = aromatic ring. 13C NMR (100 MHz, CDCl3): 178.2
(Cq, –COOCH3); 176.3 (Cq, C-3); 170.1 (Cq, CH3OCO-Ar-COON]);
162.0 (Cq, CH3OCO-Ar-COON]); 150.0 (Cq, CH3OCO-Ar-COON]),
133.7, 131.2, 125.9, and 124.0 (4 × CH, CH3OCO-Ar-COON]) and
122.7 (Cq, CH3OCO-Ar-COON]); 143.8 (Cq, C-13); 121.9 (CH, C-12);
Conjugate of oxime of oleanonic acid and aspirin (=3-(2-
acetoxy)benzoyloxyiminoolean-12-en-28-oic acid, 17).
C39H53NO6;
mol. mass: 631.387; yield: 612 mg (96.9%); m.p.: 125–128 °C, white
powder. IR (KBr, cm−1): 3350 (OH, –COOH), 1770 (C]O, CH3OCO-Ar-
COON]C), 1750 (C]O, CH3OCO-Ar-COON = C), 1725 (N]C,
CH3OCO-Ar-COON = C), 1700 (C]O, COOH); 1H NMR (400 MHz,
CDCl3): 7.97 (1H, dd, J = 1.6 and 7.8 Hz, CH3OCO-Ar-COON = ), 7.57
(1H, td, J = 1.2 and 7.8 Hz, CH3OCO-Ar-COON]), 7.33 (1H, td,
8

V. Krajka-Kuźniak, et al.
BioorganicChemistry93(2019)103326
A
COX-2 mRNA
COX-2 mRNA
1.6
1.4
1.2
1
1.6
1.4
1.2
1
*
*
*
*
0.8
0.6
0.4
0.2
0
*
*
0.8
0.6
0.4
0.2
0
*
*
*
B
COX-2 protein
COX-2 protein
*
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
*
COX-2
COX-2
1
2
3
4
5
6
6
7
7
8
9
10 11 12 13 14 15 16
1
2
3
4
5
6
7
7
8
9
10 11 12 13 14 15 16
Actin
Actin
1
2
3
4
5
8
9
10 11 12 13 14 15 16
1
2
3
4
5
6
8
9
10 11 12 13 14 15 16
Fig. 6. The effect of the OA, OAO derivatives (12, 14, 16 and 18) and OAO conjugates with ASP (13, 15, 17 and 19) on COX-2 mRNA (Panel A) and protein (Panel B)
level in HepG2 cells. Panel A - Data (means
(expression equals 1). Panel B - Data (means
SEM) from three separate experiments. The values were calculated as mRNA level in comparison to control cells
SEM) of Western blot analysis of the cytosolic content of COX-2 from three separate experiments and representative
immunoblots are shown. The sequence of the bands corresponds to the sequence of bars in the graph. Actin content was used for the normalization of results. The
values were calculated as the level of protein in comparison with control cells (expression equals 100%). The asterisk (*) above the bar denotes statistically significant
differences from the control group, p < 0.05.
J = 1.2 and 7.5 Hz, CH3OCO-Ar-COON = ) and 7.14 (1H, dd, J = 1.1
and 8.1 Hz, CH3OCO-Ar-COON]), 5.30 (1H, t, J = 3.4 Hz, C12-H); 2.84
(1H, dd, J = 3.5 and 13.7 Hz, C18-Hβ), 2.33 (3H, s, CH3OCO-Ar-
COON]), 1.14, 1.12, 1.10, 1.09, 0.90, 0.86 and 0.77 (7 × 3H, 7 × s, 7
CH3 groups); Ar = aromatic ring. 13C NMR (100 MHz, CDCl3): 183.5
(Cq, –COOH); 176.2 (Cq, C-3); 170.0 (Cq, CH3OCO-Ar-COON]); 162.0
(Cq, CH3OCO-Ar-COON = ); 150.0 (Cq, CH3OCO-Ar-COON]), 133.7,
131.3, 125.9, 124.0 (4 × CH, CH3OCO-Ar-COON]) and 122.8 (Cq,
CH3OCO-Ar-COON]); 146.8 (Cq, C-13); 122.1 (CH, C-12); 46.5 (Cq, C-
17); 21.0 (CH3, CH3OCO-Ar-COON]); Ar = aromatic ring. MS/EI (m/
z): 631.31 (15.9%, M•+). Elemental analysis calculated: C 74.14%, H
8.45% and N 2.22%, found: C 74.15%, H 8.44% and N 2.21%.
Conjugate of oxime of oleanonic acid morpholide and aspirin
(19). Physical and spectral data agree with those available in the lit-
erature [9].
4.2. Biological assays
4.2.1. Cell culture and viability assay
HepG2 (ATCC HB 8065) and THLE-2 (ATCC CRL-2706) cells were
provided by American Type Culture Collection (ATCC, USA). HepG2
cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM,
Sigma-Aldrich, USA) containing 10% fetal bovine serum (EURx,
Poland) and 1% antibiotics solution (Sigma-Aldrich, USA) while THLE-
2 were cultured in BEGM medium supplemented with Bullet Kit (Lonza,
Germany) and additionally 10% fetal bovine serum, 5 ng/mL epidermal
growth factor (EGF), 70 ng/mL phosphoethanolamine at 37 °C, in a
humidified atmosphere with 5% CO2. To assess the effect of OA, OAO
derivatives and ASP with its conjugates on measured parameters,
1 × 106 cells were seeded per 100 mm culture dish and after 24 h of
initial incubation the cells were treated with 2 µM, 10 µM and 20 µM of
OA, its oximes (12, 14, 16 and 18), ASP and its conjugates (13, 15, 17
9

V. Krajka-Kuźniak, et al.
Table 2
BioorganicChemistry93(2019)103326
Primers used in RT-PCR.
Gene
Forward primer
Reverse primer
NF-ĸB p50
NF-ĸB p65
COX-2
5′ATCATCCACCTTCATTCTCAA
5′CGCCTGTCCTTTCTCATC
5′CCTGTGCCTGATGATTGC
5′TCAGATAGCATACAAGAGACC
5′GGCACCACTCCACTGTATC
5′AATCCTCCACCACATCTTCC
5′ACCTCAATGTCCTCTTTCTG
5′CAGCCCGTTGGTGAAAGC
5′TGGAATGTTACGAGCAGTG
5′GGGATTATATTCGGCGTTTCG
PBGD
TBP
and 19) and 0.1% dimethyl sulfoxide (DMSO) as the control solution.
Incubation lasted for 24 h and the cells were harvested.
visualized with BioRad AP Conjugate Substrate Kit NBT/BCIP. The
amount of immunoreactive product in each lane was determined using
Quantity One software (BioRad Laboratories, USA). Values were cal-
culated as relative absorbance units (RQ) per mg protein. All the ex-
periments were repeated three times.
The effect of OA and its tested derivatives on cell viability was as-
sessed by MTT assay, following the standard protocol. Briefly, THLE-2
and HepG2 cells were seeded (104 per well) in 96-well plates. After 24 h
of pre-incubation in the complete medium, compounds were added in
various concentrations and cells were incubated for the next 24 h. Later,
cells were washed twice with phosphate buffered saline (PBS) and
further incubated for 4 h with medium containing 0.5 mg/mL 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT).
Then, the formazan crystals were dissolved in acidic isopropanol and
the absorbance was measured at 570 nm and 690 nm. All experiments
were repeated three times. In the following experiments, we used non-
toxic concentrations of compounds (with viability level of above 70%):
2, 10, and 20 μM of oleanolic acid (OA), its derivatives (12, 14, 16 and
18), aspirin (ASP) and conjugates of oximes with ASP (13, 15, 17 and
19).
4.2.6. NF-κB binding assay
NF-κB p50 and NF-κB p65 activation was assessed by enzymatic
immunoassay (Transcription Factor ELISA Assay Kit Active Motif,
Belgium) according to manufacturer’s instructions. Activated NF-κB
was measured as the amount of p65 and p50 subunits contained in
DNA-binding complex. The oligonucleotides containing (5′-GGGACTT
TCC-3′) a consensus site for NF-κB were immobilized in/on microplates
as bait. Nuclear fractions were incubated with oligonucleotides for 1 h,
then wells were washed and DNA-bound subunits were detected by the
specific primary antibody and secondary antibody conjugated with the
horseradish peroxidase (HRP). The results were expressed as the nor-
malized level of absorbance (OD450 nm per mg of protein).
4.2.2. Nuclear, cytosolic and total protein lysates preparation
The subcellular extracts were prepared using the Nuclear/Cytosol
Fractionation Kit (BioVision Research, CA, USA). The concentration of
proteins was assessed by the Lowry method and then samples were
stored at –80 °C for further analysis.
4.2.7. Statistical analysis
Statistical analysis was performed using the GraphPad Instat. The
statistical significance between the experimental groups and their re-
spective controls was assessed by the Student-Newman-Keuls-test.
Acknowledgements
4.2.3. Total RNA isolation and cDNA synthesis
Extraction of total RNA was performed using GeneMatrix Universal
DNA/RNA/Protein Purification Kit (EURx, Poland) and samples were
subsequently subjected to reverse transcription by RevertAid First
Strand cDNA Synthesis Kit (ThermoFisher Scientific, USA) according to
manufacturer’s instructions. All experiments were repeated three times.
This study was supported by the by Polish National Science Centre,
grant no 2016/21/B/NZ7/01758.
Declaration of Competing Interest
The authors declare no conflicts of interest
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4.2.4. Quantitative real-time PCR
For real-time PCR analyses, the Maxima SYBR Green Kit (Fermentas,
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11