Hybrid compounds strategy in the synthesis of oleanolic acid skeleton-NSAID derivatives

Article abstract of DOI:10.3390/molecules21040420

The current study focuses on the synthesis of several hybrid individuals combining a natural oleanolic acid skeleton and synthetic nonsteroidal anti-inflammatory drug moieties (NSAIDs). It studied structural modifications of the oleanolic acid structure by use of the direct reactivity of hydroxyl or hydroxyimino groups at position C-3 of the triterpenoid skeleton with the carboxylic function of anti-inflammatory drugs leading to new perspective compounds with high potential pharmacological activities. Novel ester- and iminoester-type derivatives of oleanolic unit with the different NSAIDs, such as ibuprofen, aspirin, naproxen, and ketoprofen, were obtained and characterized. Moreover, preliminary research of compounds obtaining structure stability under acidic conditions was examined and the PASS method of prediction of activity spectra for substances was used to estimate the potential biological activity of these compounds.

Full text of DOI:10.3390/molecules21040420

molecules
Article
Hybrid Compounds Strategy in the Synthesis of
Oleanolic Acid Skeleton-NSAID Derivatives
Anna Pawełczyk *, Dorota Olender, Katarzyna Sowa-Kasprzak and Lucjusz Zaprutko
Department of Organic Chemistry, Pharmaceutical Faculty, Poznan University of Medical Sciences,
Grunwaldzka 6, 60-780 Poznan´, Poland; dolender@ump.edu.pl (D.O.); kasik@ump.edu.pl (K.S.-K.);
zaprutko@ump.edu.pl (L.Z.)
* Correspondence: apaw@ump.edu.pl; Tel.: +48-618-546-672
Academic Editor: Derek J. McPhee
Received: 8 February 2016; Accepted: 23 March 2016; Published: 12 April 2016
Abstract: The current study focuses on the synthesis of several hybrid individuals combining a natural
oleanolic acid skeleton and synthetic nonsteroidal anti-inflammatory drug moieties (NSAIDs).
It studied structural modifications of the oleanolic acid structure by use of the direct reactivity
of hydroxyl or hydroxyimino groups at position C-3 of the triterpenoid skeleton with the carboxylic
function of anti-inflammatory drugs leading to new perspective compounds with high potential
pharmacological activities. Novel ester- and iminoester-type derivatives of oleanolic unit with
the different NSAIDs, such as ibuprofen, aspirin, naproxen, and ketoprofen, were obtained and
characterized. Moreover, preliminary research of compounds obtaining structure stability under
acidic conditions was examined and the PASS method of prediction of activity spectra for substances
was used to estimate the potential biological activity of these compounds.
Keywords: oleanolic acid; oleanolic oxime; NSAIDs; ibuprofen; aspirin; naproxen; ketoprofen;
hybrid compounds
1. Introduction
Many scientific reports on the future prospects for the design of potentially useful drugs have
been published. Among the many synthetic trends (dimers, heterodimers, heteromers, biooligomers,
dendrimers, dual-, bivalent- or multifunction agents, identical or non-identical twin drugs, mixed
or combo drugs, supramolecular particles, and various nanoindividuals) a clear leader is a hybrid
strategy [1]. A hybrid compound is a synthetic product combining two or more separate active
structures (or structure fragments) joined by at least one chemical bond. The unlimited possibilities
of combining natural or unnatural active compounds can be exploited to generate individuals with
unprecedented bioactivity and an additional potential synergic effect. Chemical association of two
structures into one new dual molecule depends on their functionality and cross-reactivity, and can
be implemented by way of various methods [2,3]; Generally, the most popular molecules integration
modes are: direct no-linker mode (fused hybrids) A; intermediate linker mode, B; and overlap mode
(merged hybrids), C (Figure 1). The use of hybrids for disease treatment could offer several advantages,
such as increased solubility, and several mechanisms of action in one molecule. This approach can be
used to optimize certain biological properties, like affinity and selectivity, but also to produce novel
biological activities distinct from those of the components.
Due to the great potential of natural products exhibiting pronounced biological activities, they
have been one of the major sources of components in hybrid molecules and, in practice, at least one
hybrid fragment should be natural. Terpenes are a widespread group of natural compounds with
considerable practical significance, which are found in most plants, as a result of their wide spectrum
of biological activities.
Molecules 2016, 21, 420; doi:10.3390/molecules21040420
www.mdpi.com/journal/molecules

Molecules 2016, 21, 420
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Figure 1. General integration modes of active dual structures. (A) Direct no-linker mode (fused
hybrids); (B) Intermediate linker mode; (C) Overlap mode (merged hybrids).
Inspired by the latest literature reports [
biologically-active compounds (NSAIDs), we decided to use the broadly advantageous structure
of naturally-occurring 3 -hydroxyolean-12-en-28-oic acid, commonly known as oleanolic acid ( ).
From a chemical point of view, it belongs to pentacyclic triterpene from the -amyrine group with
4–6] as a key modulator of the activity of other
β
1
β
five six-membered cycles in the molecule, which offers great powers both as a bioactive molecule
and as a carrier for other external activities. In nature, this compound exists either as a free acid or
as a component of triterpenoid saponins, in which it can be linked with one or more sugar chains.
This natural triterpene, widely distributed in plants, has shown several relevant biological activities
including the modulation of the immune inflammatory response [
possess several promising pharmacological activities, such as hepatoprotective and anti-inflammatory,
antiviral, antibacterial, fungicidal, antioxidant, or anticancer [ ]. Therefore, terpenes containing the
7]. Oleanolic acid and its derivatives
8
hydroxyl or hydroxyimino reactive groups can be used to prepare derivatives with the commercial
anti-inflammatory drugs with a carboxylic function, leading to new dual compounds. Recently,
its semi-synthetic acyloxyimino derivatives with analgesic and anti-inflammatory effects were also
reported [
9
,10]. In the last decade NSAID drugs have been extensively investigated for anticancer and
chemopreventive activity against various types of cancers [11
,12]. They appear to act via depression
of prostaglandin synthesis through inhibiting cyclooxygenase-2, which results in suppression of
proliferation, possibly throught enhancement of apoptosis [13]. However, NSAID drugs are known for
their side effects on the gastrointestinal system, particulary stomach ulceration, bleeding, perforation,
and action exerted by contact of drug with gastric mucosa [14]. Inflammation is not direct, but very
often the initial, trigger of several different diseases, it is supposed that fusing the anti-inflammatory
drugs and the oleanane active structure, resulting in combined compounds, showed an enhanced
biological effect with synergistic action and minimized toxicity or adverse reactions.
2. Results and Discussion
Our research examined the reactivity of oleanolic acid (
their corresponding oxime derivatives ( ) in the creation of new dual compounds. In order to
obtain the starting reactants, the carboxylic group (C-28) of oleanolic acid ( ) was converted into the
methyl ester group ( ) by use of dimethyl sulfate in an ethanolic solution of sodium hydroxide as
1), oleanolic acid methyl ester (2), and
5, 6
1
2
a methylation agent [15]. The secondary hydroxyl group at the C-3 position of the oleanolic acid
skeleton was reacted in the oxidation process with chromic anhydride in sulfuric acid (Jones reagent)
to give 3-oxooleanolic derivatives (3, 4) [16]. The carbonyl derivatives formed at the C-3 position
were reacted with hydroxylamine hydrochloride in the presence of sodium acetate and converted to
corresponding oximes (5, 6) [17] (Scheme 1).
The triterpene compounds mentioned above (
of oleanolic acid skeleton-NSAID hybrid derivatives. Racemic compounds, such as ibuprofen (
aspirin ( ), naproxen ( ), and ketoprofen (10) were applied as non-steroidal anti-inflammatory particles
(Figure 2).
1
,
2
,
5,
6) are useful starting reagents in the synthesis
7),
8
9

Molecules 2016, 21, 420
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O
O
O
OH
c
b
R
R
HO
1
O
HON
5: R=OH
6: R=OCH3
3: R=OH
4: R=OCH3
a
O
OCH3
HO
2
Scheme 1. Oleanolic acid transformations. Reagents and condition: (a) (CH3)2SO4, NaOH, EtOH, reflux;
(b) Jones reagent, acetone, RT; and(c) NH2OH ˆ HCl, CH3COONa, EtOH, reflux.
O
O
OH
OH
O
O
O
O
O
OH
OH
O
7
10
9
8
Figure 2. NSAID molecules selected.
The main transformation planned to afford hybrid derivatives is a reaction of the secondary
hydroxyl group, or hydroxyimino group resulting from it, which is present in the terpene molecule at
the C-3 position with a free carboxylic moiety of the anti-inflammatory agents selected. These move in
two chemical directions (Scheme 2). Compounds 1 and 2 reacted by the esterification method lead to
the formation of ester-type hybrid derivatives (11
give an ester of oximes called as iminoester-type hybrid derivatives (19–26).
Oleanolic acid ( ) and its methyl ester were reacted respectively with appropriate NSAID
compounds ( 10) in the presence of dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino) pyridine
(DMAP) reagents. DCC is carboxylic acid activating and coupling agent, while strongly-nucleophilic
DMAP acts as an acyl transfer reagent in the Steglich esterification reaction [ ] which is a very useful
–18), while the corresponding terpene oximes (5,6)
1
2
7–
5,6
process in the conversion of sterically-demanding substrates. The water resulting from a DCC mediated
reaction reacts immediately with the carboimide applied and forms dicyclohexylurea (DHU), which is
insoluble in a reaction medium and precipitates as a white solid. It is, therefore, essential to carry out
the reaction in anhydrous conditions. High DMAP addition is also crucial for the efficient formation of
esters because in this case catalytic amounts of DMAP does not yield the product expected. The molar
ratio of terpene alcohol, NSAID, DCC, and DMAP reagents used in the reaction process is in the
sequence 1:4:5:3. In the case of the reaction of terpene oximes (
5, 6) with carboxylic anti-inflammatory
agents ( 10) towards oxime esters a similar procedure was applied, but the presence of DMAP is not
7–
crucial. The molar ratio of reagents used was 1:4:5 (terpene oxime, NSAID, and DCC). All reactions,
both towards ester and iminoester-type compounds, were carried out in an anhydrous chloroform
solution. The reagents were mixed together at 0 ˝C, and then stirred at room temperature for up to
two hours to achieve full conversion of the terpene substrate. Due to the fact that, during the reaction,
a urea derivative is precipitated as byproduct, the addition of hexane and cooling the reaction mixture
before filtration give the best final results, because the DHU is removed completely [5] (Scheme 2).
As a result of synthesis, sixteen structures hybridized with the formulas shown in Figure 3 were
produced. Further isolation and purification by column chromatography resulted in the desired pure
esters 11
–18 and iminoesters 19–26, with yields ranging from 62% to 89%. The structure of these hybrid
compounds 11–26 was confirmed by 1H- and 13C-NMR, DEPT, and MS data.

Molecules 2016, 21, 420
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O
O
R
R
HON
HO
NSAID
a
NSAID
b
5: R=OH
1: R=OH
6: R=OCH3
2: R=OCH3
O
O
O
R
O
R
NSAID
ON
NSAID
O
19,21,23,25: R=OH
20,22,24,26: R=OCH3
11,13,15,17: R=OH
12,14,16,18: R=OCH3
ESTER-TYPE HYBRIDS
IMINOESTER-TYPE HYBRIDS
Scheme 2. The main chemical directions of oleanolic acid-NSAID compounds synthesis. Reagents and
condition: (a) ester-type hybrids: DCC, DMAP, anhyd. CHCl3, RT; and (b) iminoester-type hybrid: DCC,
anhyd. CHCl3, RT.
O
O
O
O
O
R
R
R
R
O
ON
O
19: R=OH
20: R=OCH3
11: R=OH
12: R=OCH3
O
O
O
O
O
O
O
ON
21: R=OH
22: R=OCH3
13: R=OH
14: R=OCH3
O
O
O
O
O
O
O
R
R
R
O
O
ON
23: R=OH
24: R=OCH3
15: R=OH
16: R=OCH3
O
O
O
R
ON
O
O
17: R=OH
25: R=OH
18: R=OCH3
26: R=OCH3
Figure 3. Formulas of the final hybrid derivatives obtained.

Molecules 2016, 21, 420
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2.1. Preliminary Stability Studies
New potential therapeutic substances are susceptible to various transformations in human body
conditions. Compounds obtained as complex ester or iminoester individuals formed from triterpene
and anti-inflammatory units under suitable conditions may disintegrate into the compounds from
which they originated. Preliminary stability studies on the above compounds, in conditions outside
the human organism, were carried out using acidic hydrolysis conditions with hydrochloric acid in
an ethanolic solution (pH = 5). Compounds selected from each group, four esters (11
, 13, 15, 17) and
four iminoesters (19 21 23 25) were treated with an acidic ethanol solution and stirred. The test was
,
,
,
conducted at room temperature, and then in the case of stable compounds, at an elevated temperature
˝
(50 C). Stability control of the test compounds was performed with the TLC method using structural
reference substances after 1, 5, and 24 h. The tests clearly show that the resulting compounds are stable
under the conditions employed and do not lead to a substrate or do not show changes in its structure,
either at room temperature or at an elevated temperature. The results obtained are the initial data on
the stability of the esters and iminoesters studied which in the body, under the action of enzymes, does
not have to be stable. It is known that ester links are generally cleaved in the body by esterase leading
to the starting active components.
2.2. Prediction of Activity Spectra by the PASS Method
In this study, to estimate the biological activity of the substances obtained, the PASS method
(Prediction of Activity Spectra for Substance) was used. PASS Online [18] predicts over 4000 kinds of
biological activity, including pharmacological effects, mechanisms of action, toxic and adverse effects,
interaction with metabolic enzymes and transporters, or the influence on gene expression.
Biological activity, the Pa factor (probability “to be active”) and Pi factor (probability “to be
inactive”) spectrum for NSAID-triterpene derivatives using the structure-activity relationships were
calculated and predicted. All substances tested 11–26 showed a broad spectrum of potential activity.
Most of these compounds exhibit a very high probability of biological activity, with more than
70% occurring. Tables 1 and 2 summarize the results predicted biological activities and potential
mechanisms of action derivatives obtained.
Table 1. Predicted activity of ester-type derivatives 11–18 determined by the PASS method.
Pa Factor of Compounds 11–18 (Pa > 0.7, Pi ď 0.005)
Activity/Compounds Number
11
12
13
14
15
16
17
18
Insulin promoter
Hepatoprotectant
Lipid metabolism regulator
Chemopreventive
0.983
0.926
0.927
0.925
0.921
0.912
0.968
0.925
0.874
0.918
0.893
0.889
0.874
0.874
0.811
0.872
0.735
0.828
0.868
0.795
0.782
0.765
0.799
0.759
0.806
0.966
0.957
0.969
0.967
0.907
0.926
0.911
0.911
0.877
0.841
0.715
0.866
0.766
0.862
0.858
0.848
0.977
0.861
0.863
0.926
0.956
0.958
0.965
0.878
0.908
0.882
0.882
0.783
0.835
0.710
0.824
0.788
0.819
0.818
0.770
0.974
0.859
0.861
0.948
0.929
0.952
0.938
0.903
0.904
0.884
0.884
0.868
0.860
0.738
0.842
0.816
0.867
0.799
0.789
0.962
0.859
0.810
0.900
0.924
0.930
0.923
0.878
0.878
0.858
0.858
0.799
0.861
0.719
0.792
0.847
0.822
0.742
0.798
0.934
0.849
0.811
0.965
0.891
0.966
0.864
0.924
0.892
0.902
0.902
0.875
0.890
0.794
0.869
0.798
0.822
0.814
0.803
0.792
0.771
0.818
0.921
0.889
0.953
0.855
0.895
0.859
0.870
0.870
0.782
0.885
0.789
0.827
0.823
0.780
0.758
0.808
0.775
0.768
0.815
Oxidoreductase inhibitor
Membrane integrity antagonist
Transcription factor NF kappa B stimulant 0.905
Transcription factor stimulant
Mucomembranous protector
Anti-inflammatory
0.905
0.903
0.877
0.740
0.870
0.850
0.836
0.833
0.758
0.815
0.761
0.809
Non-steroidal-anti-inflammatory agent
Hypolipemic
Antisecretoric
Antinociceptive
Antiulcerative
Antineoplastic
Caspase 3 stimulant
Caspase 8 stimulant
Apoptosis agonist

Molecules 2016, 21, 420
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Table 2. Predicted activity of iminoester-type derivatives 19–26 determined by the PASS method.
Pa Factor of Compounds 19–26 (Pa > 0.5, Pi ď 0.005)
Activity/Compounds Number
19
20
21
22
23
24
25
26
Insulin promoter
Hepatoprotectant
Chemopreventive
0.942
0.702
-
0.836
0.815
0.853
0.706
-
0.734
0.718
0.835
0.835
0.846
0.743
0.727
0.831
0.867
0.884
0.884
0.725
-
-
0.760
0.717
-
-
0.842
-
-
0.841
0.719
0.877
0.877
-
-
-
0.741
0.717
0.721
0.813
0.843
0.843
0.711
-
0.701
-
-
Oxidoreductase inhibitor
Membrane integrity antagonist
Transcription factor NF kappa B stimulant 0.881
0.776
0.789
0.851
0.851
0.751
0.742
0.806
0.716
-
0.744
-
0.829
0.829
-
0.815
0.815
0.748
-
0.757
-
-
Transcription factor stimulant
Anti-inflammatory
Antinociceptive
0.881
0.776
0.700
-
-
-
0.762
0.802
0.790
-
-
-
-
-
-
-
-
-
-
-
-
Caspase 3 stimulant
Caspase 8 stimulant
Apoptosis agonist
0.830
0.752
0.790
0.815
0.749
0.786
“-”—potential activity in the Pa factor range of 50%–70%.
If the value of Pa factor is greater than 70%, there is a high probability that the substance will be
active in the conditions of in vitro experiment, whereas if the Pa factor is within the 50%–70% range,
there is a slightly lower probability confirming the activity of the substance in the biological studies.
Therefore, in Tables 1 and 2 only the results for the highest value of the Pa factor above 70% were
presented. Ester-type connections, compared to the corresponding iminoester-type compounds, are
potentially more active individuals with a much higher Pa factor above 70%. Related iminoesters also
demonstrate potential activity, although many of them are in the Pa factor range of 50%–70%.
The resulting derivatives, particularly ester-type compounds, have a high potential to
become effective pharmaceutical individuals: e.g., as new anti-inflammatory agents that may
show a remarkable increase in activity and decrease toxicity, compared with a single synthetic
anti-inflammatory drug or terpene. Analyzing the data obtained using the PASS method, it is believed
that the formation of both ester and iminoester moieties is certainly a good and useful strategy in
the design of potential new therapeutic compounds that will minimize disease-associated morbidity
and mortality.
3. Materials and Methods
3.1. Equipment and General Procedures
The melting points of all compounds used in this study were determined on a Boetius apparatus
and were uncorrected. The 1H- and 13C-NMR spectra were recorded using a Varian Gemini 300 VT
spectrometer, 300 and 75 MHz respectively (Agilent Technologies, Santa Clara, CA, USA). Chemical
shifts (δ) were expressed in parts per million (ppm), relative to tetramethylsilane (TMS) as an internal
standard, using CDCl3 as solvent. Coupling constants (J) are expressed in Hertz (Hz). Splitting patterns
are designated as follows: s, singlet; d, doublet; dd, double doublet; t, triplet; and m, multiplet.
MS spectra were recorded on a 402 AMD INTECTRA apparatus (AMD Intectra GmbH, Harpstedt,
Germany) by the electron impact technique (EI), operating at 75 eV. The infrared (IR) spectra were
recorded in KBr tablets using a Specord 75–IR spectrophotometer (Carl Zeiss, Jena, Germany) and
were expressed in the cm´1 scale. The progress of reactions and purity of products were checked
using the TLC method on silica gel plates (60 F254 from Merck, Darmstadt, Germany) in hexane/ethyl
acetate (4:1, v/v) as a developing system. The TLC spots on the plates were observed in UV light
(
λ = 254 nm) and were visualized by spraying the chromatograms with 10% H2SO4 in ethanol and
˝
heating the plates at 110 C for approximately 3 min. Silica gel 60 (63–200
used for column chromatography.
µm particle size, Merck) was
Oleanolic acid (Bio-Tech, Beijing, China, plant material: Olea europea), DCC, DMAP, racemic
NSAID compounds (ibuprofen, aspirin naproxen, ketoprofen), and solvents (chloroform, ethyl acetate,

Molecules 2016, 21, 420
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hexane) were commercial products (Sigma-Aldrich, St. Louis, MO, USA). The compounds used for the
synthesis were of a purity higher than 98%. Chloroform was additionally dried over anhydrous CaCl2
and distilled. Other solvents were used without further purification.
3.2. Preparation of Oleanolic Acid- and Their Me-Ester-NSAID Hybrid Derivatives
3.2.1. Starting Oleanolic Acid Derivatives 2–6
First, oleanolic acid (
and methyl oleanolate ( ) was obtained with a high yield [19]. Next, oleanolic acid and its methyl ester
were oxidized with Jones reagent in acetone at room temperature [16]. The 3-oxoderivatives obtained
) were subjected to a reaction with hydroxylamine hydrochloride in ethanol, in accordance with
the procedure known for many different oxotriterpenoids. Heating oleanolic ketones ( ) with
hydroxylamine hydrochloride produces the corresponding oleanolate oximes ( ) [17]. The spectral
1) was methylated with dimethyl sulfate in an ethanolic solution of NaOH
2
(3, 4
3, 4
5, 6
data of all the starting compounds (2–6) were confirmed the data from the literature [16,17,19,20].
3.2.2. Synthesis of Ester-Type Hybrid Compounds 11–18
Oleanolic acid (1) or oleanolic acid methyl ester (2) (1 mmol), DCC (1.03 g, 5 mmol), DMAP (0.37 g,
3 mmol) and anti-inflammatory drugs: ibuprofen, aspirin, naproxen, or ketoprofen (4 mmol) were
mixed at 0 ˝C and then stirred at room temperature in dry CHCl3 (12 mL) for 2 h. Hexane (8 mL)
was added to the reaction mixture and cooled. Next, the resulting precipitate of dicyclohexylurea
was filtered. The organic phase was washed with 5% aqueous hydrochloric acid, 5% aqueous sodium
bicarbonate and water and dried over anhydrous sodium sulfate. After removal of the drying agent
and solvent evaporation, the crude new esters (11
–18) were purified with column chromatography on
silica gel using a hexane–ethyl acetate (4:1, v/v) mixture as an eluent.
Oleanoyl ibuprofenate (11) [ ]: after purification by silica gel column chromatography yield: 0.56 g (87%)
of 11 (Rf = 0.48, hexane EtOAc 4:1) as colorless solid, mp: 228–230 C; IR
(COOH), 1710 (C=O, ester ibu-olean), 1695 (COOH), 1170 (C–O, ester ibu-olean); 1H-NMR (CDCl3):
Oleanoyl moiety: = 11.01 (1H, s, COOH), 5.26 (1H, t, J = 3.4, CH-12), 4.43 (1H, m, CH-3), 2.80 (1H, dd,
J = 14.1, 3.4, CH-18), 1.11–0.54 (21H, s, 7 CH3); Ibuprofen moiety: = 7.20 (2H, d, J = 8.4, CH-arom),
7.07 (2H, d, J = 8.0, CH-arom), 3.69 (1H, m, CH), 2.44 (2H, d, J = 4.0, CH2), 2.25 (1H, m, CH), 1.49 (3H,
d, J = 7.0, CH3), 0.87 (3H, s, CH3), 0.86 (3H, s, CH3); 13C-NMR (CDCl3): Oleanoyl moiety:
= 183.50
4
˝
/
ν
max (KBr, cm´1) = 3300–3420
δ
ˆ
δ
δ
(C-28), 143.58 (C-13), 122.53 (C-12), 80.85 (C-3), 55.30 (C-5), 47.60 (C-9), 46.72 (C-17), 45.85 (C-19), 41.72
(C-14), 41.30 (C-18), 39.50 (C-8), 38.10 (C-1), 37.90 (C-4), 36.91 (C-10), 33.88 (C-21), 33.15 (C-29), 32.70
(C-7), 32.52 (C-22), 30.70 (C-20), 29.81 (C-15), 27.96 (C-23), 27.80 (C-2), 25.98 (C-27), 23.66 (C-30), 23.55
(C-16), 23.45 (C-11), 18.20 (C-6), 17.88 (C-26), 16.85 (C-24), 15.30 (C-25); Ibuprofen moiety:
(C=O ester ibu-olean), 140.39, 137.74, 137.48, 129.25, 127.33, 127.10 (6 C-arom), 44.93 (CH2), 41.59
CH3, 11 CH2, 11 CH; MS
δ = 174.35
ˆ
(CHCOO), 27.55 (CH(CH3)), 22.30, 22.20, 19.60 (3
(EI) m/z = 645.0 [M]+ (C43H64O4).
ˆ
CH3); DEPT: 10
ˆ
ˆ
ˆ
Oleanoyl ibuprofenate methyl ester (12) [
yield: 0.53 g (80%) of 12 (Rf = 0.85 (hexane
(KBr, cm´1) = 1720 (C=O, ester ibu-olean), 1700 (COOCH3), 1170 (C–O, ester ibu-olean); H-NMR
4
]: after purification by silica gel column chromatography
˝
/
EtOAc 4:1) as colorless solid, mp:139–141 C; IR
ν
max
1
(CDCl3): Oleanoyl moiety:
(1H, dd, J = 13.6, 3.3, CH-18), 1.11–0.54 (21H, s, 7
δ
= 5.27 (1H, t, J = 3.4, CH-12), 4.42 (1H, m, CH-3), 3.67 (3H, s, OCH3), 2.85
CH3); Ibuprofen moiety: = 7.20 (2H, d, J = 8.4,
ˆ
δ
CH-arom), 7.07 (2H, d, J = 8.0, CH-arom), 3.67 (1H, m, CH), 2.43 (2H, d, J = 4.0, CH2), 2.26 (1H, m, CH),
1.50 (3H, d, J = 7.0, CH3), 0.87 (3H, s, CH3), 0.86 (3H, s, CH3); 13C-NMR (CDCl3): Oleanoyl moiety:
δ = 178.39 (C-28), 143.70 (C-13), 122.39 (C-12), 80.90 (C-3), 55.63 (C-5), 51.59 (COOCH3), 47.77 (C-9),
46.70 (C-17), 45.87 (C-19), 41.70 (C-14), 41.20 (C-18), 39.51 (C-8), 38.20 (C-1), 37.24 (C-4), 36.70 (C-10),
33.75 (C-21), 33.01 (C-29), 32.75 (C-7), 31.40 (C-22), 30.23 (C-20), 28.45 (C-15), 27.90 (C-23), 27.45 (C-2),
25.80 (C-27), 23.78 (C-30), 23.55 (C-16), 23.48 (C-11), 18.29 (C-6), 17.40 (C-26), 16.90 (C-24), 15.30 (C-25);

Molecules 2016, 21, 420
8 of 14
Ibuprofen moiety:
δ
= 174.36 (C=O ester ibu-olean), 140.37, 137.73, 137.48, 129.10 127.25 (6
ˆ
C-arom),
127.08, 44.92 (CH2), 41.60 (CHCOO), 27.56 (CH(CH3)), 22.32, 22.30, 19.29 (3
11 ˆ CH2, 11 ˆ CH; MS (EI) m/z = 658.6 [M]+ (C44H66O4).
ˆ
CH3); DEPT: 11
ˆ
CH3,
Oleanoyl aspirinate (13): after purification by silica gel column chromatography yield: 0.38 g (62%) of 13
˝
(Rf = 0.45, hexane
/
EtOAc 4:1) as colorless solid, mp: 182–184 C; IR
ν
(KBr, cm´1) = 3350 (COOH),
max
1
1710 (C=O, ester asp-olean), 1700 (COOH), 1650 (C=O asp), 1175 (C–O, ester asp-olean); H-NMR
(CDCl3): Oleanoyl moiety:
2.86 (1H, dd, J = 14.1, 3.4, CH-18), 1.12–0.56 (21H, s, 7
7.09 (4H, m, CH-arom), 2.33 (3H, s, CH3); 13C-NMR (CDCl3): Oleanoyl fragment:
δ
= 11.01 (1H, COOH), 5.28 (1H, t, J = 3.4, CH-12), 4.49 (1H, m, CH-3),
CH3); Aspirin moiety: = 7.50, 7.43, 7.22,
= 180.83 (C-28),
ˆ
δ
δ
143.76 (C-13), 122.30 (C-12), 82.17 (C-3), 52.44 (C-5), 47.67 (C-9), 46.79 (C-17), 45.33 (C-19), 41.97 (C-14),
41.23 (C-18), 39.00 (C-8), 38.11 (C-1), 37.79 (C-4), 36.98 (C-10), 33.81 (C-21), 33.10 (C-29), 32.56 (C-22),
32.12 (C-7), 30.75 (C-20), 27.98 (C-15), 27.69 (C-23), 27.54 (C-2), 25.79 (C-27), 23.60 (C-30), 23.51 (C-16),
23.34 (C-11), 18.20 (C-6), 16.82 (C-26), 16.73 (C-24), 15.36 (C-25); Aspirin moiety:
δ
= 170.96 (C=O ester
aspir-olean), 169.58 (COOCH3), 150.63, 137.33, 130.38, 125.80, 121.92, 116.13 (6
DEPT: 8 ˆ CH3, 10 ˆ CH2, 9 ˆ CH; MS (EI) m/z = 618. 9 [M]+ (C39H54O6).
ˆ
Carom), 21.12 (CH3);
Oleanoyl aspirinate methyl ester (14): after purification by silica gel column chromatography yield: 0.45 g
˝
(72%) of 14 (Rf = 0.65 (hexane/EtOAc 4:1) as colorless solid, mp: 223–226 C; IR ν
max (KBr, cm´1) = 1725
(C=O, ester asp-olean), 1710 (COOCH3), 1660 (C=O asp), 1175 (C–O, ester asp-olean); 1H-NMR (CDCl3):
Oleanoyl moiety: 5.28 (1H, t, J = 3.4, CH-12), 4.48 (1H, m, CH-3), 3.62 (3H, s, COOCH3), 2.85 (1H, dd,
J = 13.6, 4.04, CH-18), 1.12–0.58 (21H, s, 7
ˆ
CH3); Aspirin moiety:
δ
= 7.52, 7.31, 7.21, 7.00 (4H, m,
= 178.31 (C-28), 143.78 (C-13),
CH-arom), 2.06 (3H, s, CH3); 13C-NMR (CDCl3): Oleanoyl moiety:
δ
122.25 (C-12), 80.91 (C-3), 55.27 (C-5), 51.52 (COOCH3), 47.52 (C-9), 46.71 (C-17), 45.81 (C-19), 41.60
(C-14), 41.26 (C-18), 39.25 (C-8), 38.07 (C-1), 37.66 (C-4), 36.90 (C-10), 33.82 (C-21), 33.08 (C-29), 32.56
(C-7), 31.36 (C-22), 30.68 (C-20), 28.00 (C-15), 27.65 (C-23), 27.52 (C-2), 25.88 (C-27), 23.62 (C-30), 23.50
(C-16), 23.38 (C-11), 18.19 (C-6), 16.81 (C-26), 16.67 (C-24), 15.34 (C-25); Aspirin moiety:
(C=O ester asp-olean), 170.04 (COOCH3), 150.04, 137.92, 131.38, 127.80, 121.99, 117.03 (6
21.15 (CH3); DEPT: 9 ˆ CH3, 10 ˆ CH2, 9 ˆ CH; MS (EI) m/z = 632.9 [M]+ (C40H56O6).
δ
= 170.31
C-arom),
ˆ
Oleanoyl naproxenate (15) [
of 15 (Rf = 0.44, hexane EtOAc 4:1) as colorless solid, mp: 257–258 C; IR
(COOH), 1725 (C=O, ester napx-olean), 1690 (COOH), 1175 (C–O, ester napx-olean); 1H-NMR (CDCl3):
Oleanoyl moiety: = 10.95 (1H, COOH), 5.25 (1H, t, J = 3.4, CH-12), 4.47 (1H, m, CH-3), 2.80 (1H, dd,
J = 13.6, 4.0, CH-18), 1.11–0.57 (21H, s, 7 CH3); Naproxen moiety: = 7.70 (1H, d, J = 8.8, CH-arom),
7.68 (1H, s, CH-arom), 7.64 (1H, d, J = 8.8, CH-arom), 7.42 (1H, d, J = 8.4, CH-arom), 7.40 (1H, d, J = 8.4
CH-arom), 7.14 (1H, d, J = 11, CH-arom), 3.91 (3H, s, OCH3), 3,84 (1H, m, CH), 1.57 (3H, d, J = 7.0,
CH3); 13C-NMR (CDCl3): Oleanoyl moiety:
= 183.24 (C-28), 143.53 (C-13), 122.51 (C-12), 81.06 (C-3),
4]: after purification by silica gel column chromatography yield: 0.58 g (89%)
˝
/
ν
max (KBr, cm´1) = 3300–3420
δ
ˆ
δ
,
δ
55.26 (C-5), 47.46 (C-9), 46.49 (C-17), 45.79 (C-19), 41.56 (C-14), 40.95 (C-18), 39.21 (C-8), 37.98 (C-1),
37.84 (C-4), 36.89 (C-10), 33.76 (C-21), 33.02 (C-29), 32.48 (C-7), 32.39 (C-22), 30.63 (C-20), 29.72 (C-15),
27.68 (C-23), 27.61 (C-2), 25.85 (C-27), 23.53 (C-30), 23.49 (C-16), 23.35 (C-11), 18.17 (C-6), 17.99 (C-26),
16.52 (C-24), 15.27 (C-25); Naproxen moiety:
δ
= 174.25 (C=O ester napx-olean), 157.51, 136.02, 133.60,
C-arom), 55.3 (OCH3), 45.9 (CH), 17.04 (CH3);
129.23, 128.90, 127.46, 126.38, 125.99, 118.85, 105.56 (10
DEPT: 9 ˆ CH3, 10 ˆ CH2, 12 ˆ CH; MS (EI) m/z = 668.5 [M]+ (C44H60O5).
ˆ
Oleanoyl naproxenate methyl ester (16) [
4]: after purification by silica gel column chromatography yield:
˝
´1
0.53 g (78%) of 16 (Rf = 0.60, hexane
/
EtOAc 4:1) as colorless solid, mp: 158–160 C; IR
ν
max (KBr, cm
)
1
= 1725 (C=O, ester napx-olean), 1700 (COOCH3), 1185 (C–O, ester napx-olean); H-NMR (CDCl3):
Oleanoyl moiety:
(1H, dd, J = 13.6, 4.0, CH-18), 1.10–0.56 (21H, s, 7
CH-arom), 7.68 (1H, s, CH-arom), 7.67 (1H, d, J = 8.8, CH-arom), 7.42 (1H, d, J = 8.4, CH-arom), 7.40
(1H, d, J = 8.4, CH-arom), 7.14 (1H, d, J = 11, CH-arom), 3.91 (3H, s, OCH3), 3.83 (1H, m, CH), 1.58 (3H,
δ
= 5.26 (1H, t, J = 3.6, CH-12), 4.46 (1H, m, CH-3), 3.61 (3H, s, COOCH3), 2.84
CH3); Naproxen moiety: = 7.70 (1H, d, J = 8.8,
ˆ
δ

Molecules 2016, 21, 420
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d, J = 7.0, CH3); 13C-NMR (CDCl3): Oleanoyl moiety:
δ
= 178.30 (C-28), 143.77 (C-13), 122.23 (C-12),
81.04 (C-3), 55.27 (C-5), 51.51 (COOCH3), 47.46 (C-9), 46.69 (C-17), 45.96 (C-19), 41.59 (C-14), 41.25
(C-18), 39.22 (C-8), 38.00 (C-1), 37.84 (C-4), 36.86 (C-10), 33.82 (C-21), 33.08 (C-29), 32.51 (C-7), 32.35
(C-22), 30.67 (C-20), 29.69 (C-15), 27.67 (C-23), 27.63 (C-2), 25.87 (C-27), 23.62 (C-30), 23.50 (C-16), 23.39
(C-11), 18.15 (C-6), 16.78 (C-26), 16.54 (C-24), 15.27 (C-25); Naproxen moiety:
napx-olean), 157.49, 136.02, 133.58, 129.24, 128.88, 126.94, 126.39, 125.98, 118.84, 105.53 (10
δ
= 174.25 (C=O ester
C-arom),
CH; MS (EI) m/z = 682.7
ˆ
55.20 (OCH3), 45.80 (CH), 18.04 (CH3); DEPT: 10
[M]+ (C45H62O5).
ˆ
CH3, 10
ˆ
CH2, 12
ˆ
Oleanoyl ketoprofenate (17): after purification by silica gel column chromatography yield: 0.27 g
(79%) of 17 (Rf = 0.32, hexane EtOAc 4:1) as colorless resin (with a tendency to crystallize); IR
(KBr, cm´1) = 3300–3420 (COOH), 1725 (C=O, ester ketopr-olean), 1690 (COOH), 1175 (C–O, ester
ketopr-olean); 1H-NMR (CDCl3): Oleanoyl moiety:
= 10.95 (1H, COOH), 5.27 (1H, t, J = 3.4, CH-12),
4.47 (1H, m, CH-3), 2.80 (1H, dd, J = 13.6, 3.6, CH-18), 1.21–0.61 (21H, s 7 CH3); Ketoprofen moiety:
= 7.80 (1H, d, J = 8.0, CH-arom), 7.73 (1H, s, CH-arom), 7.64 (1H, d, J = 7.2, CH-arom), 7.58 (1H, d,
J = 6.8, CH-arom), 7.57 (1H, d, J = 6.0, CH-arom), 7.53 (1H, d, J = 6.4, CH-arom), 7.49 (1H, d, J = 7.6
CH-arom), 7.47 (1H, d, J = 8.8, CH-arom), 7.43 (1H, d, J = 7.6, CH-arom), 3.78 (2H, m, CH), 1.59
(3H, d, J = 7.2, CH3); 13C-NMR (CDCl3): Oleanoyl moiety:
= 178.28 (C-28), 143.79 (C-13), 122.22
/
ν
max
δ
ˆ
δ
,
δ
(C-12), 81.34 (C-3), 55.22 (C-5), 47.50 (C-9), 46.70 (C-17), 45.95 (C-19), 41.61 (C-18), 41.28 (C-14), 39.26
(C-8), 37.85 (C-1), 37.74 (C-4), 36.62 (C-10), 33.84 (C-21), 33.08 (C-29), 32.54 (C-7), 32.36 (C-22), 30.66
(C-20), 28.43 (C-15), 27.72 (C-23), 27.65 (C-2), 25.88 (C-27), 23.47 (C-30), 23.38 (C-16), 23.05 (C-11), 18.09
(C-6), 18.03 (C-26), 16.62 (C-24), 16.53 (C-25); Ketoprofen moiety:
δ
= 196.57 (C=O), 173.57 (C=O, ester
C-arom),
ketopr-olean), 141.22, 140.95, 137.88, 137.57, 130.00, 129.26, 128.83, 128.44, 128.39, 128.28 (12
ˆ
45.83 (CH), 15.27 (CH3); DEPT: 8 ˆ CH3, 10 ˆ CH2, 15 ˆ CH; MS (EI) m/z = 692.9 [M]+ (C46H60O5).
Oleanoyl ketoprofenate methyl ester (18): after purification by silica gel column chromatography yield:
0.31 g (88%) of 18 (Rf = 0.50, hexane
max (KBr, cm´1) = 1725 (C=O, ester ketopr-olean), 1700 (COOCH3), 1185 (C–O, ester ketopr-olean);
1H-NMR (CDCl3): Oleanoyl moiety:
= 5.27 (1H, t, J = 3.6, CH-12), 4.46 (1H, m, CH-3), 3.62 (3H, s,
COOCH3), 2.81 (1H, dd, J = 13.6, 3.6, CH-18), 1.21–0.61 (21H, s 7 CH3); Ketoprofen moiety: = 7.80
/EtOAc 4:1) as colorless resin (with a tendency to crystallize); IR
ν
δ
ˆ
δ
(1H, d, J = 8.0, CH-arom), 7.73 (1H, s, CH-arom), 7.64 (1H, d, J = 7.2, CH-arom), 7.58 (1H, d, J = 6.8,
CH-arom), 7.57 (1H, d, J = 6.0, CH-arom), 7.53 (1H, d, J = 6.4, CH-arom), 7.49 (1H, d, J = 7.6, CH-arom),
7.47 (1H, d, J = 8.8, CH-arom), 7.43 (1H, d, J = 7.6, CH-arom), 3.78 (2H, m, CH), 1.59 (3H, d, J = 7.2,
CH3); 13C-NMR (CDCl3): Oleanoyl moiety:
δ = 178.28 (C-28), 143.79 (C-13), 122.22 (C-12), 81.34 (C-3),
55.22 (C-5), 51.49 (COOCH3), 47.50 (C-9), 46.70 (C-17), 45.95 (C-19), 41.61 (C-18), 41.28 (C-14), 39.26
(C-8), 37.85 (C-1), 37.74 (C-4), 36.62 (C-10), 33.84 (C-21), 33.08 (C-29), 32.54 (C-7), 32.36 (C-22), 30.66
(C-20), 28.43 (C-15), 27.72 (C-23), 27.65 (C-2), 25.88 (C-27), 23.47 (C-30), 23.38 (C-16), 23.05 (C-11), 18.09
(C-6), 18.03 (C-26), 16.62 (C-24), 16.53 (C-25); Ketoprofen moiety:
δ
= 196.57 (C=O), 173.57 (C=O, ester
C-arom),
ketopr-olean), 141.22, 140.95, 137.88, 137.57, 130.00, 129.26, 128.83, 128.44, 128.39, 128.28 (12
ˆ
45.83 (CH), 15.27 (CH3); DEPT: 9 ˆ CH3, 10 ˆ CH2, 15 ˆ CH; MS (EI) m/z = 706.9 [M]+ (C47H62O5).
3.2.3. Synthesis of Iminoester-Type Hybrid Compounds 19–26
Oleanolic acid oxime (5) or its methyl ester (6) (1 mmol), DCC (1.03 g, 5 mmol) and
anti-inflammatory drugs: ibuprofen, aspirin, naproxen, or ketoprofen (4 mmol), were mixed at 0 ˝C
and then stirred at room temperature in dry CHCl3 (12 mL) for 2 h. Hexane (8 mL) was added to
the reaction mixture and cooled. Next, the resulting precipitate of dicyclohexylurea was filtered. The
organic phase was washed with 5% aqueous hydrochloric acid, 5% aqueous sodium bicarbonate
and water and dried over anhydrous sodium sulfate. After removal of the drying agent and solvent
evaporation, the crude new iminoesters 19
–26 were purified with column chromatography on silica
gel using hexane–ethyl acetate (4:1, v/v) mixture as a eluent.

Molecules 2016, 21, 420
10 of 14
Oleanoyl oxime ibuprofenate (19): after purification by silica gel column chromatography yield:
0.46 g (74%) of 19 (Rf = 0.45; hexane
EtOAc 4:1) as colorless solid, mp: 163–165 ˝C; IR
(KBr, cm´1) = 3300–3420 (COOH), 1715 (C=O, iminoester ibu-olean), 1695 (COOH), 1170 (C–O,
iminoester ibu-olean); 1H-NMR (CDCl3): Oleanoyl moiety:
= 11.17 (1H, COOH), 5.53 (1H, t, J = 3.4
CH-12), 2.84 (1H, dd, J = 13.4, 3.4, CH-18), 1.35–0.72 (21H, s, 7 CH3); Ibuprofen moiety: δ = 7.18
/
ν
max
δ
,
ˆ
(2H, d, J = 8.4, CH-arom), 7.08 (2H, d, J = 8.0, CH-arom), 3.85 (1H, m, CH), 2.43 (2H, d, J = 7.0, CH2),
2.25 (1H, m, CH), 1.55 (3H, d, J = 7.0, CH3), 0.89 (3H, s, CH3), 0.88 (3H, s CH3); 13C-NMR (CDCl3):
δ = Oleanoyl moiety:
δ = 178.30 (C-28), 172.60 (C-3), 143.53 (C-13), 126.60 (C-12), 55.30 (C-5), 49.50
(C-9), 47.61 (C-17), 45.89 (C-19), 41.74 (C-14), 41.30 (C-18), 39.58 (C-8), 38.10 (C-1), 37.92 (C-4), 36.92
(C-10), 33.15 (C-29), 32.54 (C-22), 33.86 (C-21), 32.72 (C-7), 30.73 (C-20), 29.82 (C-15), 27.95 (C-23), 27.80
(C-2), 25.95 (C-27), 23.65 (C-30), 23.58 (C-16), 23.43 (C-11), 18.20 (C-6), 17.80 (C-26), 16.86 (C-24), 15.30
(C-25); Ibuprofen moiety:
126.69 (6
δ = 174.76 (C=O iminoester ibu-olean), 140.52, 138.40, 137.48, 128.98, 126.88,
ˆ
C-arom), 44.59 (CH2), 40.18 (CHCOO), 27.56 (CH(CH3)), 22.32 , 18.58, 15.09 (3
ˆ
CH3);
DEPT: 10 ˆ CH3, 11 ˆ CH2, 10 ˆ CH; MS (EI) m/z = 657.6 [M]+ (C43H63NO4).
Oleanoyl oxime ibuprofenate methyl ester (20): after purification by silica gel column chromatography
yield: 0.51 g (77%) of 20 (Rf = 0.67, hexane EtOAc 4:1) as colorless resin; IR
max (KBr, cm´1) = 1710
(C=O, iminoester ibu-olean), 1170 (C–O, iminoester ibu-olean); 1700 (COOCH3); 1H-NMR (CDCl3):
Oleanoyl moiety: = 5.28 (1H, t, J = 3.4, CH-12), 3.62 (3H, s, OCH3), 2.84 (1H, dd, J = 13.7, 3.4,
CH-18), 1.49–0.72 (21H, s, 7 CH3); Ibuprofen moiety: = 7.23 (2H, d, J = 8.0, CH-arom), 7.08 (2H, d,
J = 8.0, CH-arom), 3.85 (1H, m, CH), 2.45 (2H, d, J = 7.0, CH2), 2.25 (1H, m, CH), 1.55 (3H, d, J = 7.0
CH3), 0.88 (3H, s, CH3), 0.87 (3H, s, CH3); 13C-NMR (CDCl3): Oleanoyl moiety:
= 178.30 (C-28),
/
ν
δ
ˆ
δ
,
δ
172.44 (C-3), 143.90 (C-13), 122.30 (C-12), 55.30 (C-5), 51.59 (COOCH3), 49.51 (C-9), 47.65 (C-17), 45.90
(C-19), 41.72 (C-14), 40.19 (C-18), 39.58 (C-8), 38.19 (C-1), 37.90 (C-4), 36.69 (C-10), 33.10 (C-29), 32.51
(C-22), 33.81 (C-21), 32.78 (C-7), 30.27 (C-20), 29.68 (C-15), 27.99 (C-23), 27.58 (C-2), 25.69 (C-27), 23.86
(C-30), 23.59 (C-16), 23.45 (C-11), 18.29 (C-6), 17.38 (C-26), 16.88 (C-24), 15.39 (C-25); Ibuprofen moiety:
δ = 175.78 (C=O iminoester ibu-olean), 140.56, 137.62, 137.48, 129.22, 127.35, 127.10, 44.50 (CH2), 40.19
(CHCOO), 27.52 (CH(CH3)), 22.30, 18.59, 15.20 (3
(EI) m/z = 671.6 [M]+ (C44H65NO4).
ˆ
CH3); DEPT: 11
ˆ
CH3, 11
ˆ
CH2, 10
ˆ
CH; MS
Oleanoyl oxime aspirinate (21): after purification by silica gel column chromatography yield: 0.39 g (62%)
of 21 (Rf = 0.39, hexane
EtOAc 4:1) as colorless solid, mp: 104–107 ˝C; IR (KBr, cm´1) = 3350
(COOH), 1750 (C=O iminoester asp-olean), 1710 (COOH), 1695 (C=O asp.), 1175 (C–O, iminoester
asp-olean); 1H-NMR (CDCl3): Oleanoyl moiety:
= 10.80 (1H, COOH), 5.32 (1H, t, J = 3.4, CH-12), 3.31
(1H, m, CH-18), 1.33–0.83 (21H, s, 7 CH3); Aspirin moiety: = 7.50, 7.43, 7.22, 7.10 (4H, m, CH-arom),
2.35 (3H, s, CH3). 13C-NMR (CDCl3): Oleanoyl moiety:
= 178.26 (C-28), 176.40 (C-3), 143.96 (C-13),
/
ν
max
δ
ˆ
δ
δ
122.13 (C-12), 55.39 (C-5), 47.63 (C-9), 46.76 (C-17), 45.83 (C-19), 41.75 (C-14), 41.39 (C-18), 40.95 (C-8),
38.19 (C-1), 37.99 (C-4), 36.98 (C-10), 33.11 (C-29), 32.55 (C-22), 33.82 (C-21), 32.71 (C-7), 30.72 (C-20),
29.89 (C-15), 27.92 (C-23), 27.80 (C-2), 25.90 (C-27), 23.69 (C-30), 23.58 (C-16), 23.45 (C-11), 18.29 (C-6),
17.89 (C-26), 16.81 (C-24), 15.32 (C-25); Aspirin moiety: δ = 171.12 (C=O iminoester asp-olean), 165.54
(COOCH3), 154.04, 131.38, 129.58, 127.79, 125.92, 121.90 (6
10 ˆ CH2, 8 ˆ CH; MS (EI) m/z = 631. 9 [M]+ (C39H53NO6).
ˆ
C-arom), 21.30 (CH3); DEPT: 8
ˆ
CH3,
Oleanoyl oxime aspirinate methyl ester (22): after purification by silica gel columnchromatography yield:
˝
0.42 g (67%) of 22 (Rf = 0.47, hexane
cm´1) = 1750 (C=O asp), 1710 (COOCH3), 1650 (C=O asp), 1175 (C-O iminoester asp-olean); 1H-NMR
(CDCl3): Oleanoyl moiety: = 5.28 (1H, t, J = 3.4, CH-12), 3.63 (3H, s, COOCH3), 2.99 (1H, dd, J = 13.7,
3.4, CH-18), 1.20–0.77 (21H, s, 7 CH3); Aspirin moiety: = 7.97, 7.57, 7.32, 7.14 (4H, m, CH-arom),
2.35 (3H, s, CH3); 13C-NMR (CDCl3): Oleanoyl moiety:
= 178.26 (C-28), 176.40 (C-3), 143.96 (C-13),
/
EtOAc 4:1) as colorless solid, mp: 179–180 C; IR
ν
(KBr,
max
δ
ˆ
δ
δ
122.13 (C-12), 55.78 (C-5), 51.59 (COOCH3), 47.14 (C-9), 46.72 (C-17), 45.79 (C-19), 41.73 (C-14), 41.61
(C-18), 41.32 (C-8), 39.29 (C-1), 38.74 (C-4), 36.99 (C-10), 33.82 (C-21), 33.09 (C-29), 32.32 (C-22), 31.67
(C-7), 30.68 (C-20), 30.36 (C-15), 27.64 (C-23), 27.12 (C-2), 26.07 (CH3), 25.85 (C-27), 24.32 (C-30), 23.61

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(C-16), 23.45 (C-11), 19.88 (C-6), 18.96 (C-26), 16.80 (C-24), 15.11 (C-25); Aspirin moiety:
δ
= 171.12 (C=O
iminoester asp-olean), 165.54 (COOCH3), 154.04, 131.39, 129.60, 127.80, 125.97, 121.99 (6
21.08 (CH3); DEPT: 9 ˆ CH3, 10 ˆ CH2, 8 ˆ CH; MS (EI) m/z = 645. 9 [M]+ (C40H55NO6).
ˆ
Carom),
Oleanoyl oxime naproxenate (23): after purification by silica gel column chromatography yield: 0.55 g
˝
(81%) of 23 (Rf = 0.36, hexane
= 3300–3420 (COOH), 1725 (C=O, iminoester napx-olean), 1690 (COOH), 1175 (C–O, iminoester
napx-olean); 1H-NMR (CDCl3): Oleanoyl moiety:
= 9.78 (1H, COOH), 5.59 (1H, t, J = 3.4, CH-12),
2.50 (1H, dd, J = 13.6, 4.0, CH-18), 1.32–0.57 (21H, s, 7 CH3); Naproxen moiety: = 7.70 (1H, d, J = 8.8,
/
EtOAc 4:1) as colorless solid, mp: 160–162 C; IR
ν
(KBr, cm´1
)
max
δ
ˆ
δ
CH-arom), 7.68 (1H, s, CH-arom), 7.64 (1H, d, J = 8.8, CH-arom), 7.38 (2H, dd, J = 8.4, 8.4 CH-arom),
7.15 (1H, d, J = 11, CH-arom), 3.91 (3H, s, OCH3), 3.85 (1H, m, CH), 1.52 (3H, d, J = 7.0, CH3); 13C-NMR
(CDCl3): Oleanoyl moiety:
δ = 178.25 (C-28), 175.60 (C-3), 143.5 (C-13), 125.65 (C-12), 55.68 (C-5), 49.92
(C-9), 46.51 (C-17), 45.83 (C-19), 41.55 (C-14), 41.05 (C-18), 39.26 (C-8), 38.02 (C-1), 37.87 (C-4), 36.94
(C-10), 33.80 (C-21), 33.14 (C-29), 32.66 (C-7), 32.38 (C-22), 30.44 (C-20), 29.10 (C-15), 26.28 (C-23), 26.12
(C-2), 25.38 (C-27), 24.60 (C-30), 23.89 (C-16), 23.39 (C-11), 18.03 (C-6), 17.10 (C-26), 16.56 (C-24), 15.32
(C-25); Naproxen moiety:
δ = 174.18 (C=O iminoester napx-olean), 154.06, 136.69, 133.51, 129.12, 128.92,
127.46, 125.99, 125.65, 119.07, 105.52 (10
ˆ
C-arom), 55.28 (OCH3); 45.52 (CH), 18.12 (CH3); DEPT:
9 ˆ CH3, 10 ˆ CH2, 11 ˆ CH; MS (EI) m/z = 681.9 [M]+ (C44H59NO5).
Oleanoyl oxime naproxenate methyl ester (24): after purification by silica gel column chromatography
yield: 0.52 g (75%) of 24 (Rf = 0.51, hexane
(KBr, cm´1) = 1710 (C=O, iminoester ibu-olean), 1695 (COOCH3), 1170 (C–O, iminoester ibu-olean);
1H-NMR (CDCl3): Oleanoyl moiety:
= 5.27 (1H, t, J = 3.6, CH-12), 3.62 (3H, s, OCH3), 2.86 (1H, dd,
J = 13.6, 4.2, CH-18), 1.23–0.73 (21H, s, 7 CH3); Naproxen moiety: = 7.70 (1H, d, J = 8.8, CH-arom),
/ ν
EtOAc 4:1) as colorless solid, mp: 110–112 ˝C; IR
max
δ
ˆ
δ
7.69 (1H, s, CH-arom), 7.42 (1H, d, J = 8.4, CH-arom), 7.39 (1H, d, J = 8.4, CH-arom), 7.14 (1H, d, J = 11,
CH-arom), 3.91 (3H, s, OCH3), 4.04 (1H, m, CH), 1.59 (3H, d, J = 7.0, CH3); 13C-NMR (CDCl3): Oleanoyl
moiety:
δ = 178.26 (C-28), 172.51 (C-3), 143.93 (C-13), 122.23 (C-12), 55.77 (C-5), 51.59 (COOCH3), 49.91
(C-17), 46.69 (C-9), 45.79 (C-19), 41.45 (C-14), 41.30 (C-18), 39.24 (C-8), 38.60 (C-1), 37.90 (C-4), 36.9
(C-10), 33.80 (C-29), 33.08 (C-22), 32.90 (C-21), 32.42 (C-7), 32.31 (C-23), 31.16 (C-20), 30.68 (C-15), 27.60
(C-2), 26.93 (C-27), 25.31 (C-30), 23.60 (C-16), 22.99 (C-11), 18.87 (C-6), 18.76 (C-26), 16.76 (C-24), 15.01
(C-25); Naproxen moiety:
δ = 175.73 (C=O iminoester napx-olean), 157.59, 136.74, 133.64, 129.16, 128.96,
127.49, 126.38, 125.99, 118.85, 105.58 (10
ˆ
C-arom), 55.30 (OCH3), 45.59 (CH), 18.75 (CH3); DEPT:
10 ˆ CH3, 10 ˆ CH2, 11 ˆ CH; MS (EI) m/z = 695.9 [M]+ (C45H61NO5).
Oleanoyl oxime ketoprofenate (25): after purification by silica gel column chromatography yield: 0.26 g
(73%) of 25 (Rf = 0.18; hexane
/
EtOAc 4:1) as colorless resin; IR
ν
(KBr, cm´1) = 3300–3420 (COOH),
max
1715 (C=O, iminoester ketopr-olean), 1695 (COOH), 1170 (C–O, iminoester ketopr-olean); 1H-NMR
(CDCl3): Oleanoyl moiety:
CH-18), 1.25–0.74 (21H, s, 7
(1H, s, CH-arom.), 7.64 (1H, d, J = 7.2, CH-arom.), 7.58 (1H, d, J = 6.8, CH-arom.), 7.57 (1H, d, J = 6.0
δ
= 11.05 (1H, COOH), 5.28 (1H, s, CH-12), 2.84 (1H, dd, J = 13.4, 3.4,
CH3); Ketoprofen moiety: = 7.80 (1H, d, J = 8.0, CH-arom.), 7.75
ˆ
δ
,
CH-arom.), 7.53 (1H, d, J = 6.4, CH-arom.), 7.49 (1H, d, J = 7.6, CH-arom), 7.47 (1H, d, J = 8.8, CH-arom.),
7.43 (1H, d, J = 7.6, CH-arom.), 3.75 (2H, m, CH), 1.60 (3H, d, J = 7.2, CH3); 13C-NMR (CDCl3): Oleanoyl
moiety:
δ = 178.25 (C-28), 171.94 (C-3), 143.90 (C-13), 122.00 (C-12), 55.77 (C-5), 47.07 (C-9) 46.72 (C-17),
44.46 (C-19), 41.70 (C-18), 41.32 (C-14), 39.27 (C-8), 38.66 (C-1), 38.61 (C-4), 36.93 (C-10), 33.83 (C-21),
33.08 (C-29), 32.61 (C-7), 32.32 (C-22), 30.68 (C-20), 27.62 (C-23), 26.96 (C-15), 25.83 (C-2), 24.67 (C-27),
23.62 (C-30), 23.42 (C-16), 23.02 (C-11), 19.44 (C-6), 18.41 (C-26), 16.78 (C-24), 15.15 (C-25); Ketoprofen
moiety:
δ = 196.46 (C=O), 175.93 (C=O, ester ketopr-olean), 140.66, 140.59, 137.89, 137.48, 132.49, 131.59,
130.04, 129.35, 129.21, 128.99, 128.52, 128.31 (12
ˆ
C-arom), 45.52 (CH), 15.05 (CH3); DEPT: 8
ˆ
CH3,
10 ˆ CH2, 14 ˆ CH; MS (EI) m/z = 705.9 [M]+ (C46H59NO5).
Oleanoyl oxime ketoprofenate methyl ester (26): after purification by silica gel column chromatography
yield: 0.30 g (84%) of 26 (Rf = 0.38, hexane EtOAc 4:1) as colorless resin; IR
/
ν
max (KBr, cm´1) = 1710

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1
(C=O, iminoester ketopr-olean), 1170 (C–O, iminoester ketopr-olean); 1700 (COOCH3); H-NMR
(CDCl3): Oleanoyl moiety:
CH-18), 1.27–0.74 (21H, s, 7
(1H, s, CH-arom.), 7.64 (1H, d, J = 7.2, CH-arom.), 7.58 (1H, d, J = 6.8, CH-arom.), 7.57 (1H, d, J = 6.0
δ
= 5.28 (1H, s, CH-12), 3.62 (3H, s, OCH3), 2.84 (1H, dd, J = 13.4, 3.3,
CH3); Ketoprofen moiety: = 7.80 (1H, d, J = 8.0, CH-arom.), 7.75
ˆ
δ
,
CH-arom.), 7.53 (1H, d, J = 6.4, CH-arom.), 7.49 (1H, d, J = 7.6, CH-arom), 7.47 (1H, d, J = 8.8, CH-arom.),
7.43 (1H, d, J = 7.6, CH-arom.), 3.75 (2H, m, CH), 1.60 (3H, d, J = 7.2, CH3); 13C-NMR (CDCl3): Oleanoyl
moiety:
δ = 178.25 (C-28), 171.84 (C-3), 143.92 (C-13), 122.02 (C-12), 55.77 (C-5), 51.52 (COOCH3), 47.07
(C-9) 46.72 (C-17), 44.46 (C-19), 41.70 (C-18), 41.32 (C-14), 39.27 (C-8), 38.66 (C-1), 38.61 (C-4), 36.93
(C-10), 33.83 (C-21), 33.08 (C-29), 32.61 (C-7), 32.32 (C-22), 30.68 (C-20), 27.62 (C-23), 26.96 (C-15), 25.83
(C-2), 24.67 (C-27), 23.62 (C-30), 23.42 (C-16), 23.02 (C-11), 19.44 (C-6), 18.41 (C-26), 16.78 (C-24), 15.05
(C-25); Ketoprofen moiety:
δ = 196.46 (C=O), 175.93 (C=O, ester ketopr-olean), 140.66, 140.59, 137.89,
137.48, 132.49, 131.59, 130.04, 129.35, 129.21, 128.99, 128.52, 128.31 (12
ˆ
C-arom), 45.82 (CH), 15.02
(CH3); DEPT: 9 ˆ CH3, 10 ˆ CH2, 14 ˆ CH; MS (EI) m/z = 719.9 [M]+ (C47H61NO5).
3.3. Evaluation of Hydrolytic Stability
The mixture of hybrid compound (esters: 11
and ethanol solution of HCl (pH = 5, 10 mL) was stirred at room temperature. After 1, 5, and 24 h, the
mixture was sampled and analyzed using the TLC method with the use of hexane EtOAc (4:1, v/v) as
, 13, 15, 17 and iminoesters: 19, 21, 23, 25) (0.1 mmol)
/
a eluent. The mixture was stirred at room temperature and next sampled and analyzed using the TLC
method after 1, 5, and 24 h, respectively. Since all of the compounds were stable in the test at room
temperature, another test was conducted at an elevated temperature (50 ˝C), and then sampled and
analyzed using the TLC method after 1, 5, and 24 h respectively.
4. Conclusions
Starting from naturally-occurring oleanolic acid, its oxime, and their methyl esters, as well as
from four synthetic anti-inflammatory drugs (ibuprofen, aspirin, naproxen, ketoprofen), 16 new
derivatives were synthesized, combining the oleanolic acid active skeleton and anti-inflammatory
structure into new single-hybrid chemical individuals. Oleanolic acid and its methyl ester were
reacted with NSAIDs containing a non-blocked COOH group in the presence of DCC and DMAP in
towards ester-type hybrids, while oleanolic oxime and its methyl ester were reacted with NSAIDs
in the presence of DCC only in towards to iminoester-type hybrids. The structure of compounds
obtained was confirmed by the spectral method. The presence of some functional groups in the
terpene skeleton and NSAID structures could improve their potential biological activity. The resulting
derivatives were determined and analyzed by the PASS method and they have great potential to
become effective pharmaceutical individuals as anti-inflammatory, chemopreventive, hypolipemic,
antisecretoric, antinociceptive, antiulcerative, or antineoplastic agents, various stimulants, inhibitor,
antagonists, promoters, or regulators. Ester and iminoester moieties in these molecules are also a useful
strategy for designing new therapeutic compounds and it should be noted that ester-type derivatives
are more active agents using the PASS method. Ester connections exhibit very high probability of the
occurrence of biological activity greater than 70%. A preliminary stability test showed that the resulting
structures (11–26) are stable and not susceptible to degradation under acidic hydrolysis conditions.
Furthermore, the importance and potential application of synthetic oleanolic acid derivatives are
highlighted and the prospects for research on oleanolic acid are given. The hybridization method of
natural and synthetic compounds is one of the most promising and fundamentally novel approaches
for the design of new lead structures and the discovery of new and potent drugs in the field of
medicinal chemistry.
Acknowledgments: The study was financially supported by the NCN (National Science Centre) on the basis of
decision DEC-2013/09/B/NZ7/01279.

Molecules 2016, 21, 420
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Author Contributions: A.P., D.O., K.S.-K. were responsible for synthesis, purification and determination of the
structures and properties of the compounds obtained, A.P. and D.O. wrote manuscript, L.Z. designed the study
and contributed with valuable discussion and revision of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).