Simple amides of oleanolic acid as effective penetration enhancers

Article abstract of DOI:10.1371/journal.pone.0122857

Transdermal transport is now becoming one of the most convenient and safe pathways for drug delivery. In some cases it is necessary to use skin penetration enhancers in order to allow for the transdermal transport of drugs that are otherwise insufficiently skin-permeable. A series of oleanolic acid amides as potential transdermal penetration enhancers was formed by multistep synthesis and the synthesis of all newly prepared compounds is presented. The synthetized amides of oleanolic acid were tested for their in vitro penetration promoter activity. The above activity was evaluated by means of using the Fürst method. The relationships between the chemical structure of the studied compounds and penetration activity are presented.

Full text of DOI:10.1371/journal.pone.0122857

RESEARCH ARTICLE
Simple Amides of Oleanolic Acid as Effective
Penetration Enhancers
Barbara Bednarczyk-Cwynar1*, Danuta Partyka2¤, Lucjusz Zaprutko1
1 Department of Organic Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences,
Grunwaldzka Str. 6, 60–780 Poznan, Poland, 2 Department of Technology of Drug Forms, Faculty of
Pharmacy, Poznan University of Medical Sciences, Grunwaldzka Str. 6, 60–780 Poznan, Poland
¤ Current address: Department of Pharmaceutical Technology, Faculty of Pharmacy, Nicolaus Copernicus
University in Toruń, Jurasza Str. 2, 85–089 Bydgoszcz, Poland
* bcwynar@ump.edu.pl
Abstract
Transdermal transport is now becoming one of the most convenient and safe pathways for
drug delivery. In some cases it is necessary to use skin penetration enhancers in order to
allow for the transdermal transport of drugs that are otherwise insufficiently skin-permeable.
A series of oleanolic acid amides as potential transdermal penetration enhancers was
formed by multistep synthesis and the synthesis of all newly prepared compounds is pre-
sented. The synthetized amides of oleanolic acid were tested for their in vitro penetration
promoter activity. The above activity was evaluated by means of using the Fürst method.
The relationships between the chemical structure of the studied compounds and penetra-
tion activity are presented.
OPEN ACCESS
Citation: Bednarczyk-Cwynar B, Partyka D, Zaprutko
L (2015) Simple Amides of Oleanolic Acid as
Effective Penetration Enhancers. PLoS ONE 10(5):
e0122857. doi:10.1371/journal.pone.0122857
Academic Editor: A Ganesan, University of East
Anglia, UNITED KINGDOM
Introduction
Received: November 7, 2014
Accepted: February 15, 2015
Published: May 26, 2015
Administering active substances into the skin constitutes a fundamental pathway of their appli-
cation which has been known and used for many centuries in order to achieve local action. Be-
cause of its simplicity and convenience, this method can also be applied for a substance that
can act in the inner parts of the skin or—by being absorbed into the blood—it can cause the
systemic effect. However, this pathway is limited because only some substances can be trans-
ported through the skin. The substances can reach the deeper skin layers through the intercel-
lular pathway or through the transcellular pathway (by penetration) [1].
In order to improve the rate of transdermal penetration of drugs or cosmetics through the
horny layer, physical, chemical and biochemical methods are used [2–8] such as iontophoresis
[2], sonophoresis [3], microneedles [4], penetration enhancers [5,6] or liposomal vesicles
[7–9]. One of the above mentioned methods involves the addition of certain kinds of sub-
stances that can reversibly disrupt horny layer lipid organization, thus making it more perme-
able. These are known as promoters (accelerators) of transdermal penetration. Penetration
enhancers increase skin permeability via various mechanisms, including enhanced solubility,
increased partitioning into the stratum corneum, fluidization of its crystalline structure and
Copyright: © 2015 Bednarczyk-Cwynar et al. This is
an open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: The authors have no support or funding to
report.
Competing Interests: The authors have declared
that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
1 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
dissolution of stratum corneum lipids. Besides, deformable colloidal systems enhance migra-
tion across the stratum corneum under the influence of hydration or electric forces [6].
The most active enhancers include: hydrocarbons, lower alcohols (such as ethanol), fatty
acids (e.g. oleic acid, lauric acid), fatty alcohols (e.g. 1-octanol), fatty acid esters (e.g. isopropyl
mirystynate), amines and amides, sulfoxides and their analogues (e.g. DMSO), pyrrolidones (e.
g. N-methyl-2-pyrrolidone [NMP] and 2-pyrrolidone), tenzides, lipids, laurocapram (Azone)
and its derivatives, as well as terpenes and their derivatives [6,10–15].
1-Dodecylazacycloheptan-2-one (Azone) belongs to a large group of transdermal penetra-
tion accelerators and is one of the highly effective compound of such activity [6]. It possesses a
large polar head group and a lipid alkyl chain, both of which are thought to be necessary for its
activity. Its molecule, similarly as NMP, includes an amide nitrogen atom which is also present
in some derivatives of oleanolic acid that were synthetized in our laboratory. As is expected
from its chemical structure, Azone is a highly lipophilic compound with log P around 6.2. It
enhances skin transport of a wide variety of drugs with different lipophilicity [6,10–15].
During last years triterpenes became the subject of scientific research concerning this group
of species as active compounds incorporated into different types of vehicles enabling their
transdermal transport, but there are only a few works evaluating triterpenes and their deriva-
tives as a transdermal permeation enhancers.
Jaworska et al. [16] studied the formation of oil/water (O/W) nano-emulsions suitable for
cosmeceutical application. One of practically non-water soluble triterpenoic acid (name of
such compound was not given) was selected as an active compound and incorporated into the
stable formulation. The obtained results proved that the nanoemulsion based on caprylic/cap-
ric triglycerides with the oil/surfactant ratio O/S = 20:80 and the droplet size r = 25 nm was the
most stable one and additionally showed the highest solubilisation capacity for the
tested triterpene.
Chen et al. [17] prepared ursolic acid ethosomes and investigated the penetration character-
istics of such transdermal vehicles. The ursolic acid ethosomes were prepared by injection
method. The triterpene permeation tests in vitro through the rat’s skin were performed in TP-3
diffusion cel. The accumulated permeation amounts of ursolic acid were compared for 10% iso-
propanolic solution of ursolic acid, as well as for this triterpene liposomes and ethosomes. As
the scientists claimed, the encapsulation percentage of the ethosomes was satisfactory and the
stability of the ursolic acid ethosomes turned out to be very good. Ethosomes showed to be a
good method to enhance the diffusion rate of ursolic acid through the skin of rats.
In 2012 Pino and co-workers [18] tested transdermal permeation of avicins, a family of nat-
urally occurring glycosylated triterpene with a molecular weight more than 2,000 Da through
full-thickness porcine skin. As the results shown, avicines, which belong to a family of sapo-
nins, exhibit skin permeability comparable to those of small hydrophobic molecules, such as
estradiol. Pino and anothers [19] tested also avicines as transdermal permeation enhancers
with the appliance of local anesthetics such as hydrochlorides of lidocaine, prilocaine and bupi-
vacaine from aqueous vehicle, across full-thickness porcine skin. Avicins, the triterpenic sapo-
nins, in a concentration of 0.5–1%, caused from 2- to 5-fold in comparison to controls sample,
without enhancer.
In 1997 the influence of free oleanolic acid on liposomic membranes was described [20].
This triterpene was responsible for causing important changes within the above mentioned
membranes and caused their fluidity. Some new oleanolic acid derivatives with lactam and
thiolactam systems within the A- or C-ring were also prepared and tested as percutaneous
transport promoters in vitro. Their activity was comparable with that of N-dodecylcaprolactam
(Azone) [21].
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
2 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
Fig 1. Syntesis of oleanolic acid amides 4a–4f and 5a–5f. Reagents and conditions: I: acetic anhydride,
pyridine, r.t.; II: thionyl chloride or oxalyl chloride, r.t.; III: benzene or dichloromethane, amine or glycine
ethylester hydrochloride and triethylamine, r.t.; IV: NaOH, ethanol, reflux.
doi:10.1371/journal.pone.0122857.g001
Materials and Methods
1. Chemistry
Oleanolic acid, isolated from a natural source (the mistletoe herb, Viscum album), was used as
the starting material for multistep synthesis of its derivatives. The transformations are pre-
sented in the Schemes 1–3. Amide derivatives of oleanolic acid were obtained on the basis of
methods known from literature data [22–24]. Oleanolic acid (1) was acetylated with a 10-fold
excess of acetic anhydride in dried pyridine. The product, with a reversibly blocked hydroxyl
group at C-3 position (3-O-acetyloleanolic acid, 2, R1 = CH3CO, R2 = OH), was subjected to
thionyl chloride action and the resulting 3-O-acetyloleanolic acid chloride in a crude form was
transformed into different amides. The reactions of the synthesis of cyclic amides of 3-O-acety-
loleanolic acid: 4a – 4c and aliphatic amides 4d, 4f were performed in dried benzene at room
temperature (Fig 1) and the amide 4e was obtained in dichloromethane at room temperature
with the use of glycine ethylester hydrochloride and triethylamine as an acceptor of the releas-
ing hydrochloride (Fig 1).
The obtained 3-O-acetylamides of oleanolic acid 4a – 4f were hydrolized with 5% sodium
hydroxide ethanolic solution with short heating (Fig 1). It was proven on the basis of spectral
data that in these alkaline conditions the ester bond at C-3 position as well as the ester group of
amide 4e underwent hydrolysis. Alkaline hydrolysis of compound 4e gave the product 5e,
which was insoluble in biological tests conditions.
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
3 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
Fig 2. Syntesis of 11-oxooleanolic acid amide 8d. Reagents and conditions: I: sodium dichromate, acetic
acid, heating; II: thionyl chloride, r.t.; III: dichloromethane, diethylamine hydrochloride, triethylamine, r.t.; IV:
NaOH, ethanol, reflux.
doi:10.1371/journal.pone.0122857.g002
The 11-oxoderivative of oleanolic acid 8d was obtained by oxidation of acetyloleanolic acid
(2) with sodium dichromate in acetic acid by applying method known from the literature [25],
followed by transformation of the obtained 11-oxoderivative into appriopriate acid chloride
(Fig 2) and then into respective amide [22–25].
The derivative of oleanolic acid with the C-3 carbonyl group and the morpholide function
(9a) was obtained by oxidation of an appropriate amide with free hydroxylic group (5a) with
Jones reagent (Fig 3) in acetone at room temperature on the basis of the method presented in
the literature [26,27].
The morpholides with the nitrile group formed from an opened A ring (11a) or with an ex-
panded seven-membered heterocyclic A ring (12a) were obtained as is presented in the Scheme
3. The 3-oxoderivative 9a was first subjected to reaction with hydroxylamine hydrochloride in
ethanol [27] and the obtained oxime 10a was transformed into nitrile 11a and lactam 12a
under the action of phosphoryl chloride in pyridine by the use of literature method [27].
2. In vitro screening of penetration enhancing activity
The aim of the performed test was to evaluate how well newly obtained nitrogen derivatives of
oleanolic acid could enhance penetration of progesterone from a hydrogel ointment that was
Fig 3. Syntesis of oleanolic acid morpholide derivatives 9a–12a. Reagents and conditions: I: Jones
reagent, acetone, r.t.; II: hydroxylamine hydrochloride, sodium acetate, ethanol, reflux; III: phosphoryl
chloride, pyridine, r.t.
doi:10.1371/journal.pone.0122857.g003
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
4 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
prepared on the basis of Carbopol 980. Progesterone was used as a model of a penetrating ac-
tive pharmaceutical substance whose pharmacological activity is known and which has a mod-
erate polarity (log P about 4.0). The above pharmaceutical form (hydrogel ointment) contained
1% of progesterone, and 0.5% or 1.0% (if the solubility allowed) of the triterpenic compound
(in both cases related to whole formulation) tested as a promoter.
The Fürst method [28] was applied in the in vitro tests of progesterone penetration into
membrane layers from the hydrogel ointment. This is a simple method allowing to perform
quick evaluation of penetration activity of tested compounds. This method consisted of quanti-
tatively determining of progesterone absorbed into the structure which was constructed of five
lipophilic membranes. The ointment was spread on the surface of the first membrane (4 cm2)
of the above mentioned system and left in a thermostat at 37°C for 1h. After this period of time
the content of the therapeutic substance was determined separately for each of the five lipophil-
ic membranes. The spectrophotometric method using the colour reaction of progesterone and
isonicotinic acid complex formation was applied in this order. The penetration tests were re-
peated 6 times.
The artificial lipophilic membranes were prepared in our laboratory and they were made
from dodecanol as a lipid phase and collodium as a colloxylinic matrix supporting the mem-
branes. The thickness of all of the obtained membranes was determined on the basis of the
Richter method [29] in which the spectrum of the interference wave was recorded for each
membrane in the infrared range.
Results and Discussion
In this work a series of oleanolic acid derivatives with the amide function was prepared as po-
tential transdermal penetration enhancers. These compounds’ ability to enhance the penetra-
tion of progesterone as a model substance through the system of five lipophilic membranes was
tested. All of the prepared oleanolic acid amide derivatives positively influenced the amount of
the model substance (progesterone) that was absorbed in five synthetic membranes. A marked-
ly increased amount of absorbed progesterone in all of the membranes, higher than for Azone,
was observed for amides: 4b, 5b (both with the N-methylpiperazine moiety and the acetoxy or
the hydroxy group at C-3 position), 4a, 5a (both with the morpholine ring), 4d (with the N,N-
diethylamide function) and 4f (having two nitrogen atoms within the amide function and the
acetoxy group at C-3 position). Because the amides with the morpholine ring (4a and 5a)
seemed to be particularly active compounds, further modifications of the C-3 hydroxyl func-
tion were performed and ketone (9a), oxime (10a), nitrile (11a) and lactam (12a) were ob-
tained. Among the above compounds, lactam 12a exhibited the best results. In most of the
described cases the increase of the promoter concentration in the ointment from 0.5% to 1.0%
caused an increase in the total amount of progesterone. In general, replacement of the C-3 ace-
tyl group with a free hydroxyl function caused an insignificant decrease in the total amount of
progesterone in the set of lipophilic membranes. Only the hydrolysis of the acetyl function of
compound 4c leading to compound 5c caused an increase in the total amount of absorbed pro-
gesterone in the five membranes. The introduction of an additional carbonyl group at C-11 po-
sition (compound 8d) did not cause a significant increase in the total amount of progesterone
in the set of membranes. Oxidation of the C-3 hydroxyl group of compound 5a, leading to ke-
tone 9a also had no important influence on the total amount of model substance absorbed into
the five membranes. The transformation of the 3-oxo function of derivative 9a to the 3-hydro-
xyimino group (within triterpene 10a) did not increase the amount of absorbed progesterone.
The rearrangement of oxime (10a) into lactam (12a) led to an improvement in promoting ac-
tivity of triterpene.
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
5 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
In order to compare the influence of triterpenes on the depth of progesterone penetration,
the amount of the absorbed model drug was determined for membranes 1–3 (meaning the “ex-
ternal layer” of the epidermis model), 4–5 (the „internal layer” of the epidermis model) and
1–5 (the „whole” epidermis model).
The presence of Azone as a transdermal promoter played a significant role in the increase of
the total amount of absorbed progesterone and the amount of progesterone that permeated
into the most external membranes (1–3), but at the same time it caused an important decrease
in the amount of progesterone in the internal membranes (4–5). Almost all of the obtained am-
ides of oleanolic acid caused an increase in the amount of the absorbed model substance in the
“external layer” of the epidermis model as well as in this model’s “internal layer”. Amide 4a in
a concentration of 1.0% caused the highest increase in the amount of absorbed progesterone in
membranes 1–3: the relative increase in the amount of the absorbed drug was over 40% in
comparison to about 32% for Azone. At the same time, two triterpenes in a concentration of
0.5% (4b and 5b) and four derivatives in a concentration of 1.0% (9a, 12a, 5c and 4d) caused a
more than 50% increase in the amount of the absorbed progesterone into more deeply located
membranes. Replacement of the C-3 hydroxyl group with the carbonyl one or with the hydro-
xyimino function led to an important improvement in promoting activity of triterpene within
the more internal membranes. A further increase in the amount of progesterone in the deeper
membranes was caused by the transformation of oxime 10a (about 40%) into lactam 12a (al-
most 66%).
Experimental
1. Chemistry
1.1. General. All solvents and reagents were pure for analysis grade and purchased from
Sigma-Aldrich. For reactions in anhydrous conditions the solvents were purified and dried by
using general methods described in literature [30]. The thin layer chromatography (TLC) anal-
ysis (reactions progress and the level of compounds purity) was conducted on HPTLC alumi-
num sheets covered with silicagel 60 F245 and benzene with ethyl acetate in a volume ratio of
4: 1 as the eluent. The chromatograms were visualized by being sprayed with 10% ethanolic so-
lution of sulfuric acid and heating the plates at about 110°C for a few minutes. The column
chromatography was performed using silicagel 60 (0.063–0.200 mm, 70–230 mesh).
Melting points were determined in open capillaries using the Büchi apparatus.
IR spectra were recorded using the Specord IR—75 spectrophotometer, for 0.5% mixtures
of the tested compounds and KBr. NMR spectra for hydrogen atoms (1H) and for carbon
atoms (13C) were recorded in deuterated chloroform or DMSO solutions on the Varian Gemini
300 VT apparatus, for frequencies 300 MHz and 75 MHz, respectively, with TMS as an internal
standard. The multiplicities of signals in 1H NMR spectra were marked as follows: s—singlet,
br/s—broad singlet, t—triplet, dd—doublet of doublets, m—multiplet. Mass spectra (MS) and
high resolution mass spectra (HR MS) were recorded using the AMD 402 spectrometer with
electroionization. The elucidation of the chemical structures was based on IR, 1H NMR, 13
NMR, MS and HR MS analysis.
C
1.2. General method for synthesis of amides 4a – 4d and 4f. Amine (0.01 mol) was
added dropwise to a vigorously stirred solution of crude 3β-acetoxyolean-12-en-28-oic acid
chloride 3 (obtained from 500 mg, 0.001 mol of 3β-acetoxyolean-12-en-28-oic acid 2) in dried
benzene (10 mL) and stirred up to the total consumption of the substrate (TLC control). The
resulting solid was filtered off, the filtrate was washed with water, dried and evaporated. The
obtained residue was crystallized from ethanol to afford the title amides 4a – 4d and 4f.
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
6 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
3β-Acetoxyolean-12-en-28-oic acid morpholide 4a: C36H57NO4; mol. mass: 567.85; yield:
549 mg (96.6%); m.p.: 238–239°C; IR (KBr, ν, cm-1): 1720 (C = O, CH3COO), 1630 (C = O,
amide); 1H NMR (CHCl3, δ, ppm): 5.27 (1H, t, J = 3.4 Hz, C12-H), 4.49 (1H, dd, J = 7.0 and 8.9
Hz, C3-Hα), 3.70–3.57 (8H, m, morpholine ring), 2.05 (3H, s, CH3COO), 1.14, 0.93, 0.92, 0.90,
0.86, 0.85 and 0.74 (21 H, 7 x s, 7 x CH3); 13C NMR (CHCl3, δ, ppm): 175.1 (Cq, C-28), 171.0
(Cq, CH3COO), 144.6 (Cq, C-13), 121.5 (CH, C-12), 80.9 (CH, C-3), 2 x 66.9, 46.3 and 46.0 (4 x
CH2, morpholine ring), 47.4 (Cq, C-17), 21.3 (CH3, CH3COO); DEPT: 8 x CH3, 14 x CH2, 5 x
CH (36 x C atoms); MS (m/z, %): 567.4 (68.8, M+•); HR MS: calcd. for C36H57NO4: 567.4287,
found: 567.4285.
3β-Acetoxyolean-12-en-28-oic acid N-methylpiperazide 4b: C37H60N2O3; mol. mass:
580.90; yield: 541 mg (93.2%); m.p.: 218–220°C; IR (KBr, ν, cm-1): 1710 (C = O, CH3COO),
1600 (C = O, amide); 1H NMR (CHCl3, δ, ppm): 5.26 (1H, t, J = 3.5 Hz, C12-H), 4.49 (1H, dd,
J = 7.0 and 8.8 Hz, C3-Hα), 3.70–3.59 and 2.41–2.31 (2 x 4H, 2 x m, piperazine ring), 2.29 (3H,
s, >N–CH3), 2.05 (3H, s, CH3COO), 1.13, 0.93, 0.92, 0.90, 0.86, 0.85 and 0.74 (21 H, 7 x s, 7 x
CH3); 13C NMR (CHCl3, δ, ppm): 174.9 (Cq, C-28), 170.9 (Cq, CH3COO), 144.8 (Cq, C-13),
121.3 (CH, C-12), 80.9 (CH, C-3), 55.2 x 2 and 38.0 x 2 (4 x CH2, piperazine ring), 47.6 (Cq, C-
17), 46.0 (CH3, >N–CH3), 21.3 (CH3, CH3COO); DEPT: 9 x CH3, 14 x CH2, 5 x CH (37 x C
atoms); MS (m/z, %): 580.7 (3.6, M+•); HR MS: calcd. for C37H60N2O3: 580.4604, found:
580.4600.
3β-Acetoxyolean-12-en-28-oic acid pyrrolidide 4c: C36H57NO3; mol. mass: 551.85; yield:
507 mg (91.9%); m.p.: 212–213°C; IR (ν, cm-1): 1720 (C = O, CH3COO), 1710 (C = O, amide);
1H NMR (CHCl3, δ, ppm): 5.26 (1H, t, J = 3.5 Hz, C12-H), 4.49 (1H, dd, J = 7.0 and 8.9 Hz, C3-
Hα), 3.55–3.44 and 1.73–1.54 (2 x 4H, 2 x m, pyrrolidine ring), 2.04 (3H, s, CH3COO), 1.14,
0.94, 0.92, 0.90, 0.86, 0.85 and 0.72 (21 H, 7 x s, 7 x CH3); 13C NMR (CHCl3, δ, ppm): 175.1
(Cq, C-28), 170.9 (Cq, CH3COO), 144.8 (Cq, C-13), 121.6 (CH, C-12), 80.9 (CH, C-3), 47.3 (Cq,
C-17), 46.1 x 2 and 27.5 x 2 (4 x CH2, pyrrolidine ring), 21.2 (CH3, CH3COO); DEPT: 8 x CH3,
14 x CH2, 5 x CH (36 x C atoms); MS (m/z, %): 551.6 (99.9, M+•); HR MS: calcd. for
C36H57NO3: 551.4338, found: 551.4340.
3β-Acetoxyolean-12-en-28-oic acid N,N-diethylamide 4d: C36H59NO3; mol. mass: 553.87;
yield: 512 mg (92.5%); m.p.: 224–226°C; IR (KBr, ν, cm-1): 1720 (C = O, CH3COO), 1705
(C = O, amide); 1H NMR (CHCl3, δ, ppm): 5.26 (1H, t, J = 3.6 Hz, C12-H), 4.49 (1H, dd, J = 7.0
and 8.9 Hz, C3-Hα), 3.49–3.26 and 1.99–1.81 (2 x 2H, 2 x m,—N< (CH2–CH3)2), 2.04 (3H, s,
CH3COO), 1.14 (6H, s,—N<(CH2–CH3)2), 1.12, 0.93, 0.92, 0.89, 0.86, 0.85 and 0.78 (21 H, 7 x
s, 7 x CH3); 13C NMR (CHCl3, δ, ppm): 174.9 (Cq, C-28), 170.9 (Cq, CH3COO), 145.0 (Cq, C-
13), 121.1 (CH, C-12), 80.9 (CH, C-3), 47.7 (Cq, C-17), 46.7 x 2 (CH2 x 2,—N<(CH2–CH3)2),
21.3 (CH3, CH3COO), 16.6 x 2 (CH3 x 2,—N<(CH2–CH3)2); DEPT: 10 x CH3, 12 x CH2, 5 x
CH (36 x C atoms); MS (m/z, %): 553.7 (34.7, M+•); HR MS: calcd. for C36H59NO3: 553.4492,
found: 553.4490.
3β-Acetoxyolean-12-en-28-oic acid N’,N’-dimethylaminoethylamide 4f: C36H60N2O3;
mol. mass: 568.88; yield: 535 mg (94.1%); m.p.: 163–165°C; IR (KBr, ν, cm-1): 3400 (N–H),
1720 (C = O, CH3COO), 1630 (C = O, amide); 1H NMR (DMSO, δ, ppm): 7.71 (1H, t, J = 5.2
Hz, N–H), 5.23 (1H, s, C12-H), 4.49 (1H, dd, J = 4.6 and 11.1 Hz, C3-Hα), 2.72 (6H, s,—NH–
CH2–CH2–N<(CH3)2), 2.02 (3H, s, CH3COO), 1.62–1.44 (4H, m,—NH–CH2–CH2–N<
(CH3)2), 1.11, 0.89, 0.88, 0.87, 0.81, 0.80, 0.67 (21 H, 7 x s, 7 x CH3); 13C NMR (DMSO,
δ, ppm): 177.1 (Cq, C-28), 170.9 (Cq, CH3COO), 143.9 (Cq, C-13), 121.5 (CH, C-12), 79.9 (CH,
C-3), 56.0 x 2 (2 x CH2,—NH–CH2–CH2–N<(CH3)2), 45.4 (Cq, C-17), 42.5 x 2 (2 x CH3,—
NH–CH2–CH2–N<(CH3)2), 21.0 (CH3, CH3COO); DEPT: 10 x CH3, 12 x CH2, 5 x CH (36 x
C atoms); MS (m/z, %): 568.4 (6.01, M+•); HR MS: calcd. for C36H60N2O3: 568.4603, found:
568.4606.
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
7 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
1.3. Synthesis of amide 4e. A solution of glycine ethylester hydrochloride (0.004 mol) and
triethylamine (0.008 mol) in dichloromethane (10 mL) was added to a vigorously stirred solu-
tion of crude 3β-acetoxyolean-12-en-28-oic acid chloride 3 (obtained from 500 mg, 0.001 mol
of 3β-acetoxyolean-12-en-28-oic acid 2) in dried dichloromethane (10 mL) and left at room
temperature overnight (TLC control). The resulting mixture was washed with water, dried and
subjected to column chromatography with silicagel (benzene and ethyl acetate, volume ratio of
4:1). After evaporation the obtained residue was crystallized from ethanol to afford the title
amide 4e.
3β-Acetoxyolean-12-en-28-oic acid ethoxycarbonylmethylamide 4e: C36H57NO5; mol.
mass: 583.85; yield: 424 mg (72.7%); m.p.: 169–170°C (ethanol); IR (KBr, ν, cm-1): 3390 (N–
H), 1750 (C = O, COOC2H5), 1720 (C = O, CH3COO), 1630 (C = O, amide); 1H NMR
(CHCl3, δ, ppm): 6.57 (1H, dd, J = 3.7 and 5.1 Hz, N–H), 5.46 (1H, t, J = 3.3 Hz, C12-H), 4.49
(1H, dd, J = 7.9 and 7.9 Hz, C3-Hα), 4.22 (2H, dd, J = 7.1 and 14.3 Hz,—NH–CH2–COO–CH2–
CH3), 3.74 (2H, dd, J = 5.2 and 15.5 Hz,—NH–CH2–COO–CH2–CH3), 2.05 (3H, s, CH3COO),
1.29 (3H, s,—NH–CH2–COO–CH2–CH3), 1.24, 1.16, 0.92, 0.92, 0.86, 0.85, 0.70 (21 H, 7 x s, 7
x CH3); 13C NMR (CHCl3, δ, ppm): 178.2 (Cq, C-28), 171.0 and 170.0 (2 x Cq, CH3COO and—
NH–CH2–COO–CH2–CH3), 144.2 (Cq, C-13), 123.1 (CH, C-12), 80.8 (CH, C-3), 61.4 (CH2,—
NH–CH2–COO–CH2–CH3), 46.5 (CH2,—NH–CH2–COO–CH2–CH3), 46.2 (Cq, C-17), 21.2
(CH3, CH3COO), 14.1 (CH3,—NH–CH2–COO–CH2–CH3); DEPT: 9 x CH3, 12 x CH2, 5 x
CH (36 x C atoms); MS (m/z, %): 583.5 (11.7, M+•); HR MS: calcd. for C36H57NO5: 583.4236,
found: 583.4230.
1.4. General metod for synthesis of amides 5a – 5f. A saturated solution of 3β-acetoxyo-
lean-12-en-28-oic acid amide 4a – 4f (0.001 mol) in 5% ethanolic solution of NaOH was heated
under reflux until the total consumption of the substrate (TLC control, about 30 min). The re-
sulting mixture was cooled and poured into a 5-fold volume of water slightly acidified with
HCl. The obtained precipitate was filtered off, washed with water and dried. The received resi-
due was crystallized from ethanol or precipitated with water from ethanolic solution to afford
the title amides 5a – 5f.
3β-Hydroxyolean-12-en-28-oic acid morpholide 5a: C34H55NO3; mol. mass: 525.82; yield:
502 mg (95.5%); m.p.: 230–231°C (ethanol with water); IR (KBr, ν, cm-1): 3445 (O–H), 1625
(C = O); 1H NMR (CHCl3, δ, ppm): 5.27 (1H, t, J = 3.4 Hz, C12-H), 3.70–3.58 (8H, m, morpho-
line ring), 3.22 (1H, dd, J = 3.8 and 7.7 Hz, C3-Hα), 1.14, 0.99, 0.93, 0.90, 0.89, 0.78, 0.73 (21 H,
7 x s, 7 x CH3); 13C NMR (CHCl3, δ, ppm): 175.0 (Cq, C-28), 144.5 (Cq, C-13), 121.6 (CH, C-
12), 79.0 (CH, C-3), 2 x 67.0, 46.5 and 46.2 (4 x CH2, morpholine ring), 47.9 (Cq, C-17); DEPT:
7 x CH3, 14 x CH2, 5 x CH (34 x C atoms); MS (m/z, %): 525.4 (63.7, M+•); HR MS: calcd. for
C34H55NO3: 525.4182, found: 525.4189.
3β-Hydroxyolean-12-en-28-oic acid N-methylpiperazide 5b: C35H58N2O2; mol. mass:
538.86; yield: 512 mg (95.1%); m.p.: 244–245°C; IR (KBr, ν, cm-1): 3450 (O–H), 1600 (C = O);
1H NMR (CHCl3, δ, ppm): 5.26 (1H, t, J = 3.5 Hz, C12-H), 3.69–3.60 and 2.40–2.30 (2 x 4H, 2 x
m, piperazine ring), 3.21 (1H, dd, J = 3.8 and 7.7 Hz, C3-Hα), 2.29 (3H, s, >N–CH3), 1.13, 0.93,
0.92, 0.90, 0.86, 0.85, 0.74 (21 H, 7 x s, 7 x CH3); 13C NMR (CHCl3, δ, ppm): 174.8 (Cq, C-28),
144.9 (Cq, C-13), 121.3 (CH, C-12), 79.0 (CH, C-3), 55.2 x 2 and 38.0 x 2 (4 x CH2, piperazine
ring), 47.5 (Cq, C-17), 46.0 (CH3, >N–CH3); DEPT: 8 x CH3, 14 x CH2, 5 x CH (35 x C
atoms); MS (m/z, %): 538.5 (3.9, M+•); HR MS: calcd. for C35H58N2O2: 538.4498, found:
538.4501.
3β-Hydroxyolean-12-en-28-oic acid pyrrolidide 5c: C34H55NO2; mol. mass: 509.82; yield:
484 mg (94.9%); m.p.: 227–229°C; IR (KBr, ν, cm-1): 3480 (O–H), 1695 (C = O); 1H NMR
(CHCl3, δ, ppm): 5.27 (1H, t, J = 3.5 Hz, C12-H), 3.55–3.44 and 1.73–1.54 (2 x 4H, 2 x m, pyrro-
lidine ring), 3.20 (1H, dd, J = 3.8 and 7.8 Hz, C3-Hα), 1.16, 0.94, 0.90, 0.89, 0.86, 0.85, 0.70 (21
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
8 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
H, 7 x s, 7 x CH3); 13C NMR (CHCl3, δ, ppm): 175.3 (Cq, C-28), 144.4 (Cq, C-13), 121.8 (CH,
C-12), 78.9 (CH, C-3), 47.3 (Cq, C-17), 46.0 x 2 and 27.4 x 2 (4 x CH2, pyrrolidine ring),
DEPT: 7 x CH3, 14 x CH2, 5 x CH (34 x C atoms); MS (m/z, %): 509.6 (95.9, M+•); HR MS:
calcd. for C34H55NO2: 509.4232, found: 509.4237.
3β-Hydroxyolean-12-en-28-oic acid N,N-diethylamide 5d: C34H57NO2; mol. mass:
511.83; yield: 481 mg (93.9%); m.p.: 198–199°C; IR (KBr, ν, cm-1): 3480 (O–H), 1700 (C = O);
1H NMR (CHCl3, δ, ppm): 5.26 (1H, t, J = 3.5 Hz, C12-H), 3.50–3.26 and 1.99–1.82 (2 x 2H, 2 x
m,—N<(CH2–CH3)2), 3.20 (1H, dd, J = 3.8 and 7.8 Hz, C3-Hα), 1.14 (6H, s,—N<(CH2–
CH3)2), 1.12, 0.92, 0.91, 0.90, 0.86, 0.84, 0.78 (21 H, 7 x s, 7 x CH3); 13C NMR (CHCl3, δ, ppm):
175.0 (Cq, C-28), 144.9 (Cq, C-13), 121.3 (CH, C-12), 78.9 (CH, C-3), 47.7 (Cq, C-17), 46.6 x 2
(CH2 x 2,—N<(CH2–CH3)2), 16.7 x 2 (CH3 x 2,—N<(CH2–CH3)2); DEPT: 9 x CH3, 12 x
CH2, 5 x CH (34 x C atoms); MS (m/z, %): 511.7 (35.2, M+•); HR MS: calcd. for C34H57NO2:
511.4389, found: 511.4392.
3β-Hydroxyolean-12-en-28-oic acid hydroxycarbonylmethylamide 5e: C32H51NO4; mol.
mass: 513.76; yield: 487 mg (94.9%); m.p.: 238–241°C (precipit. with water from ethanolic sol.);
IR (KBr, ν, cm-1): 3490 (O–H), 3400 (N–H), 1710 (C = O, COOH), 1635 (C = O, amide); 1H
NMR (DMSO, δ, ppm): 12.37 (1H, br/s, COOH), 7.58 (1H, t, J = 5.4 Hz, N–H), 5.19 (1H, s,
C12-H), 3.74 (1H, dd, J = 5.9 and 17.3 Hz) and 3.54 (1H, dd, J = 5.2 and 17.2 Hz,—NH–CH2–
COOH), 2.99 (1H, dd, J = 6.3 and 9.0 Hz, C3-Hα), 1.08, 0.89, 0.88, 0.88, 0.84, 0.67, 0.64 (21 H, 7
x s, 7 x CH3); 13C NMR (DMSO, δ, ppm): 176.7 (Cq, C-28), 171.4 (Cq,—NH–CH2–COOH),
143.9 (Cq, C-13), 121.5 (CH, C-12), 76.8 (CH, C-3), 46.0 (CH2,—NH–CH2–COOH), 45.2 (Cq,
C-17); DEPT: 7 x CH3, 11 x CH2, 5 x CH (32 x C atoms); MS (m/z, %): 513.5 (20.7, M+•); HR
MS: calcd. for C32H51NO4: 513.3818, found: 513.3812.
3β-Hydroxyolean-12-en-28-oic acid N’,N’–dimethylaminoethylamide 5f: C34H58N2O2;
mol mass: 526.85; yield: 486 mg (92.2%); m.p.: 122–124°C; IR (KBr, ν, cm-1): 3485 (O–H),
3400 (N–H), 1630 (C = O); 1H NMR (DMSO, δ, ppm): 7.75 (1H, t, J = 5.3 Hz, N–H), 5.24 (1H,
s, C12-H), 3.23 (1H, d, J = 4.6 Hz, C3-Hα), 2.74 (6H, s,—NH–CH2–CH2–N<(CH3)2), 1.67–1.46
(4H, m,—NH–CH2–CH2–N<(CH3)2), 1.09, 0.89, 0.88, 0.87, 0.85, 0.68, 0.67 (21 H, 7 x s, 7 x
CH3); 13C NMR (DMSO, δ, ppm): 177.4 (Cq, C-28), 143.9 (Cq, C-13), 121.7 (CH, C-12), 78.9
(CH, C-3), 56.1 x 2 (2 x CH2,—NH–CH2–CH2–N<(CH3)2), 45.4 (Cq, C-17), 42.7 x 2 (2 x CH3,
—NH–CH2–CH2–N<(CH3)2); DEPT: 9 x CH3, 12 x CH2, 5 x CH (34 x C atoms); MS (m/z,
%): 526.4 (19.5, M+•); HR MS: calcd. for C34H58N2O2: 526.4498, found: 526.4491.
1.5. Synthesis of amide 8d. Synthesis was performed from 3β-acetoxy-11-oxoolean-
12-en-28-oic acid chloride (7) as described in 1.2. The crude product was subjected to column
chromatography with silicagel (benzene and ethyl acetate, volume ratio of 9:1). After evapora-
tion the obtained residue was crystallized to afford the title amide 8d.
3β-Acetoxy-11-oxoolean-12-en-28-oic acid N,N-diethylamide 8d: C36H57NO4; mol mass:
567.85; yield: 378 mg (66.6%); m.p.: 270–272 oC (decomp.); IR (KBr, ν, cm-1): 1720 (C = O,
CH3COO); 1710 (C = O, amide), 1650 (C = O at C-11); 1H NMR (CHCl3, δ, ppm): 5.66 (1H, s,
C12-H), 4.50 (1H, dd, J = 7.0 and 8.8 Hz, C3-Hα), 3.50–3.26 and 2.01–1.81 (2 x 2H, 2 x m,—N<
(CH2–CH3)2), 2.05 (3H, s, CH3COO), 1.14 (6H, s,—N<(CH2–CH3)2), 1.11, 0.92, 0.92, 0.90,
0.86, 0.82, 0.75 (21 H, 7 x s, 7 x CH3); 13C NMR (CHCl3, δ, ppm): 199.8 (Cq, C-11); 175.0 (Cq,
C-28), 170.7 (Cq, CH3COO), 168.3 (Cq, C-13); 127.9 (CH, C-12), 81.0 (CH, C-3), 47.6 (Cq, C-
17), 46.6 x 2 (CH2 x 2,—N<(CH2–CH3)2), 21.2 (CH3, CH3COO), 16.5 x 2 (CH3 x 2,—N<
(CH2–CH3)2); DEPT: 10 x CH3, 11 x CH2, 5 x CH (36 x C atoms); MS (m/z, %): 567.6 (32.1,
M+•); HR MS: calcd. for C36H57NO4: 567.4287, found: 567.4292.
1.6. Synthesis of amide 9a. Jones’ reagent was added dropwise with small excess to a vig-
orously stirred solution of 3β-hydroxyolean-12-en-28-oic acid morpholide (5a, 0.001 mol) in
acetone at room temperature. The resulting mixture was stirred up to the total consumption of
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
9 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
the substrate (TLC control, about 30 min), then isopropyl alcohol was added dropwise and the
obtained suspension was filtered off; the filtrate was poured into a 5-fold volume of water
slightly acidified with HCl. The formed precipitate was filtered off, washed with water and
dried. The obtained residue was crystallized from ethanol to afford the title amide 9a.
3-Oxoolean-12-en-28-oic acid morpholide 9a: C34H53NO3; mol mass: 523.80; yield: 495
mg (94.5%); m.p.: 215–218°C; IR (KBr, ν, cm-1): 1690 (C = O, C-3), 1625 (C = O, amide); 1H
NMR (CHCl3, δ, ppm): 5.29 (1H, t, J = 3.4 Hz, C12-H), 3.71–3.57 (8H, m, morpholine ring),
1.15, 1.09, 1.04, 1.03, 0.93, 0.90, 0.79 (21 H, 7 x s, 7 x CH3); 13C NMR (CHCl3, δ, ppm): 217.0
(Cq, C-3), 175.0 (Cq, C-28), 144.6 (Cq, C-13), 121.4 (CH, C-12), 66.9 x 2, 46.4 and 46.2 (4 x
CH2, morpholine ring), 47.5 (Cq, C-17); DEPT: 7 x CH3, 14 x CH2, 4 x CH (34 x C atoms); MS
(m/z, %): 523.5 (100.0, M+•); HR MS: calcd. for C34H53NO3: 523.4025, found: 523.4022.
1.7. Synthesis of amide 10a. Hydroxylamine hydrochloride (350 mg, 0.005 mol) and sodi-
um acetate (660 mg, 0.008 mol) were added to a saturated, hot solution of 3-oxoolean-12-en-
28-oic acid morpholide (9a, 0.001 mol) in ethanol and the resulting mixture was refluxed up to
total consumption of the substrate (TLC control, about 30 min), cooled and poured into a
5-fold volume of water slightly acidified with HCl. The formed precipitate was filtered off,
washed with water and dried. The obtained residue was crystallized from ethanol to afford the
title amide 10a.
3-Hydroxyiminoolean-12-en-28-oic acid morpholide 10a: C34H54N2O3; mol mass:
538.81; yield: 467 mg (86.7%); m.p.: 210–211°C; IR (KBr, ν, cm-1): 3445 (O–H), 1625 (C = O);
1H NMR (CHCl3, δ, ppm): 9.03 (1H, s, N-OH); 5.28 (1H, t, J = 3.1 Hz, C12-H), 3.71–3.57 (8H,
m, morpholine ring), 1.16, 1.12, 1.05, 1.03, 0.93, 0.90, 0.77 (21 H, 7 x s, 7 x CH3); 13C NMR
(CHCl3, δ, ppm): 175.2 (Cq, C-28), 167.0 (Cq, C-3), 144.7 (Cq, C-13), 121.5 (CH, C-12), 66.9 x
2, 46.3 and 46.0 (4 x CH2, morpholine ring), 47.4 (Cq, C-17); DEPT: 7 x CH3, 14 x CH2, 4 x
CH (34 x C atoms); MS (m/z, %): 538.6 (100.0, M+•); HR MS: calcd. for C34H54N2O3:
538.4134, found: 538.4132.
1.8. Synthesis of amides 11a and 12a. Phosphoryl chloride (POCl3, 0.28 mL, 460 mg,
0.003 mol) was added dropwise with stirring to a saturated solution of 3-hydroxyiminoolean-
12-en-28-oic acid morpholide (10a, 539 mg, 0.001 mol) in anhydrous pyridine. The resulting
mixture was left in room temperature overnight, poured into a 5-fold volume of water slightly
acidified with HCl. The precipitate was filtered off, washed with water and dried. The obtained
residue was subjected to column chromatography with silicagel (benzene and acetone, volume
ratio of 1:1, next acetone). After evaporation the obtained residues were crystallized to afford
the title amides: 11a and 12a.
3-Nitrile-3,4-sekoolean-4(23),12-dien-28-oic acid morpholide 11a (afforded from ben-
zene and acetone fraction): C34H52N2O2; mol mass: 520.80; yield: 107 mg (20.5%); m.p.: 195–
197°C (n-hexane); IR (KBr, ν, cm-1): 3060 (C = C), 2240 (CꢀN), 1630 (C = O); 1H NMR
(CHCl3, δ, ppm): 5.28 (1H, t, J = 3.1 Hz, C12-H), 4.90 (1H, s, C23-H), 4.66 (1H, s, C23-H), 3.72–
3.52 (4 x 2H, m, morpholine ring), 1.74 (3H, s, C24-H), 1.15, 0.97, 0.92, 0.90, 0.80 (15 H, 5 x s, 5
x CH3); 13C NMR (CHCl3, δ, ppm): 175.1 (Cq, C-28), 146.8 (Cq, C-4), 144.9 (Cq, C-13), 120.8
(CH, C-12), 120.2 (Cq, C-3), 114.0 (CH2, C-23); 66.9 x 2, 46.54 and 47.0 (4 x CH2, morpholine
ring), 47.4 (Cq, C-17); DEPT: 6 x CH3, 15 x CH2, 4 x CH (34 x C atoms); MS (m/z, %): 520.7
(19.1, M+•); HR MS: calcd. for C34H52N2O2: 520.4028, found: 520.4021.
3-Oxo-3a-aza-A-homoolean-12-en-28-oic acid morpholide 12a (afforded from acetone
fraction): C34H54N2O3; mol mass: 538.81; yield: 329 mg (61.0%); m.p.: 277–280°C (ethanol);
IR (KBr, ν, cm-1): 3450 (N-H), 1670 (C = O, lactam), 1630 (C = O, amide); 1H NMR (CHCl3,
δ, ppm): 5.69 (1H, s, N–H, lactam), 5.28 (1H, t, J = 3.4 Hz, C12-H), 3.70–3.54 (4 x 2H, m, mor-
pholine ring), 1.27, 1.24, 1.14, 1.10, 0.93, 0.90, 0.78 (21 H, 7 x s, 7 x CH3); 13C NMR (CHCl3,
δ, ppm): 176.9 (Cq, C-3, lactam); 174.9 (Cq, C-28), 144.2 (Cq,C-13), 121.4 (CH, C-12), 66.9 x 2,
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
10 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
46 4 and 47.0 (4 x CH2, morpholine ring), 47.4 (Cq, C-17); DEPT: 7 x CH3, 14 x CH2, 4 x CH
(34 x C atoms); MS (m/z, %): 538.9 (23.3, M+•); HR MS: calcd. for C34H54N2O3: 538.4132,
found: 538.4134.
2. In vitro tests of penetration enhancing activity
2.1. Preparation of isonicotinic acid hydrazide solution. Isonicotinic acid hydrazide (0.1
g) was dissolved in methanol (15 mL) in volumetric flask (of 25 mL). Then concentrated hy-
drochloric acid (0.12 mL) was added and the obtained solution was filled up with methanol to
25.0 mL.
2.2. Preparation of ointments with progesterone. Progesterone (1.0 g) was dissolved in
ethanol (25.0 mL) and water (38.0 mL). Then Carbopol 980 (1.0 g) was added and the resulting
mixture was stirred up for 120 minutes at room temperature in order to hydrate the acrylic
acid. The excess of this acid was neutralized with a 10.0% solution of trometamol and stirred
for a while. The model ointment, without progesterone, was prepared according to the above
method without the usage of progesterone.
2.3. Preparation of ointments with penetration enhancer. 329 or 161 mg of the tested
enhancer was dissolved in 10 mL of ethanol. Then 1.5 g (1.9 mL) of such solution was mixed
with 4.5 g of ointment with progesterone described in 2.2.
2.4. Preparation of lipophilic memebranes. Dodecanol (1.0 g) and a 4% solution of collo-
dium in diethyl ether (the same volume as 1.0 g of dodecanol) were mixed. Then 1.5 mL of the
above mixture of collodium was inserted into the cylinders of 50 mm diameter and left for 24
hours at room temperature. Next, 5 mL of bidistilled water was added into each cylinder and
after 30 minutes the membranes were taken out and dried.
2.5. Penetration of progestrone tests. A set of 5 artificial, stabilized lipophilic membranes
were arranged consecutively on a plexiglass plate and covered with a plate-template. The tested
ointment (0.01 g) was applied on the surface of the first membrane as defined by the stencil
cavity. The set of membranes was inserted in the Fürst apparatus [28] and put into a thermo-
state in 37°C for 60 minutes. Next the membranes were separated and each membrane was sep-
arately dissolved in methanol (3.0 mL). The isoniazide solution (0.5 mL) was added to each of
the obtained methanolic solutions and the mixtures were left in darkness for 30 minutes. The
absorption of UV light with a wavelength λmax of 395 nm (the absorption maximum of the re-
sulted complex) was measured against the reference sample, which was preparared as above
but without progesterone.
The percentage of progesterone in each membrane was estimated on the basis of the equata-
tion [28]:
A ꢁ v
C ¼
ꢁ 100%
1%
1cm
a
ꢁ M ꢁ m
C—percentage of progesterone absorbed within the membrane
A—absorance of the tested sample
a11%cm —specific absorbance of progesterone (410.9)
v—volume of the tested sample (3.5 mL)
M—concentration of progesterone in an ointment [%]
m—mass of ointment applied on the membrane [g]
All of the obtained compounds containing the amide function at C-17 position were exam-
ined in vitro in order to estimate their ability to penetration activity of a model therapeutic sub-
stance into a system of synthetic lipophilic membranes simulating the corneous skin layer. An
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
11 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
Table 1. The average percentage of progesterone absorbed from oinment into membranes 1–3, 4–5 and 1–5.
The percentage of progesterone absorbed in membranes (%)
nr of promoter and its concentr.
Σ 1–3
Σ 4–5
Σ 1–5
relative increase of absorbed
progesterone
Σ 1–3
Σ 4–5
Σ 1–5
without promoter
Azone (0.5%)
4a (0.5%)
4a (1.0%)
5a (0.5%)
5a (1.0%)
9a (1.0%)
10a (1.0%)
12a (1.0%)
4b (0.5%)
4b (1.0%)
5b (0.5%)
5b (1.0%)
4c (1.0%)
5c (1.0%)
4d (0.5%)
4d (1.0%)
8d (1.0%)
4e (0.5%)
4e (1.0%)
4f (0.5%)
56.76 0.70
74.83 1.54
59.90 0.73
79.54 1.68
56.97 1.31
71.89 1.56
65.04 0.99
59.75 1.13
68.85 1.57
67.43 1.38
70.37 1.54
66.12 1.59
66.67 1.59
65.70 1.27
58.09 1.65
58.49 1.42
68.27 0.82
62.67 1.38
56.47 1.22
67.46 0.57
66.28 1.29
70.01 1.84
13.02 0.50
10.73 1.61
14.62 1.37
18.12 1.36
12.59 1.88
14.42 1.61
20.32 1.55
18.30 1.80
21.57 1.80
21.16 1.28
13.51 1.52
20.73 1.31
16.56 1.62
10.58 1.42
21.44 0.75
18.29 0.61
19.56 0.82
15.89 1.22
19.39 0.62
13.50 0.95
11.10 1.66
18.59 1.37
69.78 0.96
85.56 1.30
74.52 1.82
97.66 1.54
69.56 1.75
86.31 2.13
85.36 1.82
78.05 1.47
90.42 1.95
88.59 1.85
83.88 0.39
86.85 2.27
83.23 2.05
76.28 1.52
79.53 2.29
76.78 1.28
87.83 0.91
78.56 1.59
75.86 0.99
80.96 1.00
77.38 1.42
88.60 1.59
-
-
-
+ 31.83
+ 5.53
+ 40.13
+ 0.37
+ 26.66
+ 14.59
+ 5.27
+ 21.30
+ 18.80
+ 23.98
+ 16.49
+ 17.46
+ 15.75
+ 2.34
+ 3.05
+ 20.28
+ 10.41
– 0.51
- 17.59
+ 12.29
+ 39.17
- 3.30
+ 22.61
+ 6.79
+ 39.95
- 0.31
+ 10.75
+ 56.07
+ 40.55
+ 65.67
+ 61.75
+ 3.76
+ 23.69
+ 22.33
+ 11.85
+ 29.58
+ 26.96
+ 20.21
+ 24.46
+ 19.27
+ 9.31
+ 59.22
+ 27.19
- 18.74
+ 64.67
+ 40.48
+ 50.23
+ 22.04
+ 48.92
+ 3.69
+ 13.97
+ 10.03
+ 25.87
+ 12.58
+ 8.71
+ 18.85
+ 16.77
+ 23.34
+ 16.02
+ 10.89
+ 26.97
- 14.75
+ 42.78
4f (1.0%)
doi:10.1371/journal.pone.0122857.t001
analogous test was performed for the standard substance, Azone, for a comparison. The results
are presented in Table 1.
Conclusions
It was proven, on the basis of the conducted analyses of the dependence of the obtained com-
pounds’ structure and their biological activity as transdermal delivery promoters, that replacing
of the carboxylic group at C-17 position with the amide function led to a group of compounds
of high activity. Almost all of the obtained compounds in both concentrations increased the
total amount of the model substance in a set of artificial membranes simulating epidermis, par-
ticularly in the external part of this model (stratum corneum). In most cases the increased con-
centration of the triterpenic promoter caused better penetration of progesterone into these
memebrane leyers. In general, alkalyne hydrolysis of the acetoxy function within the acetoxya-
mides led to compounds with lower activity as transdermal penetration promoters in compari-
son to the appropriate amides of acetyloleanolic acid. Introducing an additional 11-oxo
function into the molecule of triterpenic amide did not have an influence on the total amount
of progesterone that was absorbed into the set of five membranes.
Our results proved that the most advantageous amide group positively influencing the total
amount of progesterone absorbed in the whole system of lypophilic membranes were the N-
methylpiperazine, the morpholine and the N,N-diethylamine moieties. A markedly increased
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
12 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
amount of absorbed progesterone in all of the membranes, higher than in the case of applying
of Azone, was observed for amides: 4a, 5a (both with the morpholine ring), 4b, 5b (both with
the N-methylpiperazine ring and the acetoxy or the hydroxy group at C-3 position), 4d (with
the N,N-diethyl function and the acetoxy function at C-3 position) and 4f (having two nitrogen
atoms within the amide function and the acetoxy group at C-3 position). Particularly active
penetration promoters belonging to the group of oleanolic or acetylooleanolic acid amides
turned out to be morpholides. Further activation of promoting activity of these compounds
was performed by transforming of the C-3 hydroxy function, and the most active compound
was seven-membered A-lactam.
Among the tested triterpenes, morpholide 4a in a 1.0% concentration caused an over 40%
increase in the progesterone amount in the external part of the human skin model (membranes
1–3). Two of the tested enhancers in a concentration of 0.5% (4b and 5b) and four derivatives
in a concentration of 1.0% (9a, 12a, 5c and 4d) caused an over 50% increase in the amount of
the absorbed progesterone in the deeper parts of the stratum corneum model (membranes 4
and 5). Replacement of the C-3 hydroxyl group with the carbonyl or the hydroxyimino func-
tion as well as transformation of the last group into a lactam system resulted in better penetra-
tion of progesterone into the deeper membranes, with the best results for the compound with
lactam system 12a (65.67% for membranes 4 and 5).
Author Contributions
Conceived and designed the experiments: BBC DP LZ. Performed the experiments: BBC DP
LZ. Analyzed the data: BBC DP LZ. Contributed reagents/materials/analysis tools: BBC DP LZ.
Wrote the paper: BBC.
References
1. Barry BW. Novel mechanisms and devices to enable successful transdermal drug delivery. Eur J
Pharm Sci. 2001; 14:101–111. PMID: 11500256
2. Yan G, Li SK, Higuchi WI. Evaluation of constant current alternating current iontophoresis for transder-
mal drug delivery. J Control Release. 2005; 110:141–150. PMID: 16289410
3. Ogura M, Paliwal S, Mitragotri S. Low-frequency sonophoresis: Current status and future prospects.
Adv Drug Delivery Rev. 2008; 60:1218–1223. doi: 10.1016/j.addr.2008.03.006 PMID: 18450318
4. Demir YK, Akan Z, Kerimoglu O. Characterization of Polymeric Microneedle Arrays for Transdermal
Drug Delivery. PLoS ONE. 2013; 8(10):e77289. doi: 10.1371/journal.pone.0077289 PMID: 24194879
5. Ibrahim SA, Li SK. Chemical enhancer solubility in human stratum corneum lipids and enhancer mech-
anism of action on stratum corneum lipid domain. Int J Pharm. 2010; 383:89–98 doi: 10.1016/j.ijpharm.
2009.09.014 PMID: 19747970
6. Williams AC, Barry BW. Penetration enhancers. Adv Drug Deliv Rev. 2004; 56:603–618. PMID:
15019749
7. Honeywell-Nguyen PL, Bouwstra JA. Vesicles as a tool for transdermal and dermal delivery. Drug Dis-
cov Today Technologies. 2005; 2:67–74. doi: 10.1016/j.ddtec.2005.05.003 PMID: 24981757
8. Ch Sinico, Manconi M, Peppi M, Lai F, Valenti D, Fadda AM. Liposomes as carriers for dermal delivery
of tretinoin: in vitro evaluation of drug permeation and vesicle-skin interaction. J Control Release. 2005;
103:123–136. PMID: 15710506
9. Marepally S, Boakye CHA, Shah PP, Etukala JR, Vemuri A, Singh M. Design, Synthesis of Novel Lipids
as Chemical Permeation Enhancers and Development of Nanoparticle System for Transdermal Drug
Delivery. PLoS ONE. 2013; 8(12):e82581. doi: 10.1371/journal.pone.0082581 PMID: 24349315
10. Karande P, Mitragotri S. Enhancement of transdermal drug delivery via synergistic action of chemicals.
Biochim Biophys Acta. 2009; 1788:2362–2373. doi: 10.1016/j.bbamem.2009.08.015 PMID: 19733150
11. Hadgraft J, Peck J, Williams DG, Pugh WJ, Allan G. Mechanisms of action of skin penetration enhanc-
ers retarders: Azone and analogues. Int J Pharm. 1996; 141:17–25.
12. Asbill ChS, Michniak BB. Percutaneous penetration enhancers: local versus transdermal activity.
Pharm Sci Technol Today. 2000; 3:36–41. PMID: 10637599
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
13 / 14

Derivatives of Oleanolic Acid and Their Biological Activity
13. Harrison JE, Groundwater PW, Brain KR, Hadgraft J. Azone induced fluidity in human stratum cor-
neum. A fourier transform infrared spectroscopy investigation using the perdeuterated analogue. J
Control Release. 1996; 41:283–290.
14. Godwin DA, Michniak BB, Player MR, Sowell JW Sr. Transdermal and dermal enhancing activity of pyr-
rolidones in hairless mouse skin. Int J Pharm. 1997; 155:241–250.
15. Kim N, El-Kattan AF, Asbill CS, Kennette RJ, Sowell JW Sr, Latour R et al. Evaluation of derivatives of
3-(2-oxo-1-pyrrolidine) hexahydro-1H-azepine-2-one as dermal penetration enhancers: side chain
length variation and molecular modeling. J Control Release, 2001; 73:183–196. PMID: 11516496
16. Jaworska M, Sikora E, Zielina M, Ogonowski J. Studies on the formation of O/W nano-emulsions, by
low-energy emulsification method, suitable for cosmeceutical applications. Acta Biologica Polonica.
2013; 60:779–782. PMID: 24432331
17. Chen Y, Wiu Q, Zhang Zh, Zhou L, Liu X, Du M et al. Preparation and transdermal diffusion of ursolic
acid ethosomes. China J Chin Materia Med. 2011; 36:4–7.
18. Pino ChJ, Gutterman JU, Vonwil D, Mitragotri S, Shastri VP. Glycosylation facilitates transdermal trans-
port of macromolecules. PNAS. 2012; 109:21283–21288. doi: 10.1073/pnas.1200942109 PMID:
23236155
19. Pino ChJ, Scherer MA, Shastri VP. Investigation of the transdermal transport of charged local anesthet-
ics in the presence of triterpene saponin glycosides. Drug Deliv and Transl Res. 2014; 4:131–138. doi:
10.1007/s13346-013-0186-3 PMID: 25786727
20. Han SK, Ko YI, Park SJ, Jin IJ, Kim YM. Oleanolic acid and ursolic acid stabilize liposomal membranes.
Lipids. 1997; 32:769–773. PMID: 9252966
21. Zaprutko L, Partyka D, Bednarczyk-Cwynar B. Triterpenoids. Part 21: Oleanolic acid azaderivatives as
percutaneous transport promoters. Bioorg Med Chem Lett. 2004; 14:4723–4726. PMID: 15324895
22. Wrzeciono U, Małecki I, Budzianowski J, Kieryłowicz H, Beimcik E, Zaprutko L et al. Nitrogenous triter-
pene derivatives. 9. 3-Acetyloleanol acid-28-amides and their antiulcer effect. Pharmazie. 1984;
39:683–686. PMID: 6522444
23. Yasue M, Sakakibara J, Kaiya T. Synthesis of nitrogen-containing triterpenes. III. derivatives of betuli-
nic acid and oleanolic acid. Yakugaku Zasshi. 1974; 94:1468–1474. PMID: 4478177
24. Wrzeciono U, Małecki I, Budzianowski J, Kieryłowicz H, Zaprutko L, Beimcik E et al. Nitrogenous triter-
pene derivatives. Part 10: Hemisuccunates of some derivatives of oleanolic acid and their antiulcer ef-
fects. Pharmazie. 1985; 40:542–544. PMID: 4080798
25. Zaprutko L. Triterpenoids. Part 9. Structure elucidation of a new sodium dichromate oxidation product
of methyl oleanolate and some of its derivatives. Polish J Chem. 1995; 69:1003–1012.
26. Pungitore CR, Garcia M, Gianello JC, Sosa ME, Tonn CE. Insecticidal and antifeedant effects of Junel-
lia aspera (Verbenaceae) triterpenes and derivatives on Sitophilus oryzae (Coleoptera: Curculionidae).
J Stored Prod Res. 2005; 41:433–443.
27. Bednarczyk-Cwynar B, Zaprutko L, Froelich A. Beckmann Rearrangement of Oxime Obtained from
Oleanolic Acid. Structure Elucidation of the Initial Oxime. J Mol Struct. 2013; 1053:115–121.
28. Fürst W, Neubert R, Wildner R. Drug permeation through synthetic lipid membranes. Part 10: The ef-
fects of diffusion layers on the permeation process. Pharmazie 1980; 35:106–109. PMID: 7384178
29. Richter H, Morbitzer B, Finkt K, Winter K, Fürst W. Zur Bestimmung der Schichtdicke von künstlichen
Membranen. Pharmazie. 1978; 33:523–525. PMID: 724782
30. Vogel AI, Tatchell AR, Furnis BS, Hannaford AJ, Smith PWG. Vogel’s Textbook of Practical Organic
Chemistry, Longman Scientific & Technical copublished with John Wiley & Sons, Inc., 1989, New
York, pp. 395–409.
PLOS ONE | DOI:10.1371/journal.pone.0122857 May 26, 2015
14 / 14