Investigating the activity of 2-substituted alkyl-6-(2,5-dioxopyrrolidin-1- yl)hexanoates as skin penetration enhancers

Article abstract of DOI:10.1016/j.bmc.2010.10.025

Skin penetration enhancers are used in the formulation of transdermal delivery systems for drugs that are otherwise not sufficiently skin-permeable. We generated two series of esters by multi-step synthesis with substituted 6-aminohexanoic acid as potential transdermal penetration enhancers by multi-step synthesis. The synthesis of all newly prepared compounds is presented here. Structure confirmation of all generated compounds was accomplished by ¹H NMR, ¹³C NMR, IR and MS spectroscopy. All the prepared compounds were analyzed using RP-HPLC and their lipophilicity (log k) was determined. The hydrophobicity (log P/C log P) of the studied compounds was also calculated using two commercially available programs and 3D structures of the selected compounds were investigated by means of ab initio calculations of geometry and molecular dynamic simulations. All the synthesized esters were tested for their in vitro transdermal penetration-enhancing activity and showed higher enhancement ratios than oleic acid. The highest enhancement ratios were exhibited by compound 5f (C₍₂₎ substituted with piperidine-2-one, C₁₁ ester chain) and 5a (C₍₂₎ substituted with piperidine-2-one, C₆ ester chain). The series with a ω-lactam ring (piperidin-2-one; 5a-g), showed slightly higher activities than those with morpholine (6a-6g). All of the agents showed minimal anti-proliferative activity (IC₅₀ >6.25 μM), indicating they would have low cytotoxicity when administered as chemical penetration enhancers. The relationships between the lipophilicity and the chemical structure of the studied compounds, as well as the correlation between their chemical structure and transdermal penetration-enhancing activity, are discussed.

Full text of DOI:10.1016/j.bmc.2010.10.025

Bioorganic & Medicinal Chemistry 18 (2010) 8556–8565
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Investigating the activity of 2-substituted
alkyl-6-(2,5-dioxopyrrolidin-1-yl)hexanoates as skin penetration enhancers
Katerina Brychtova a, Radka Opatrilova a, Ivan Raich b, Danuta S. Kalinowski c, Lenka Dvorakova a,
a,d,
Lukas Placek d, Jozef Csollei a, Des R. Richardson c, , Josef Jampilek
⇑
⇑
a Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1/3, 612 42 Brno, Czech Republic
b Department of Chemistry of Natural Compounds, Faculty of Food and Biochemical Technology, Institute of Chemical Technology, Technicka 5, 166 28 Prague 6, Czech Republic
c Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia
d Zentiva k.s., U kabelovny 130, 102 37 Prague, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Skin penetration enhancers are used in the formulation of transdermal delivery systems for drugs that are
otherwise not sufficiently skin-permeable. We generated two series of esters by multi-step synthesis
with substituted 6-aminohexanoic acid as potential transdermal penetration enhancers by multi-step
synthesis. The synthesis of all newly prepared compounds is presented here. Structure confirmation of
all generated compounds was accomplished by 1H NMR, 13C NMR, IR and MS spectroscopy. All the pre-
pared compounds were analyzed using RP-HPLC and their lipophilicity (log k) was determined. The
hydrophobicity (log P/C log P) of the studied compounds was also calculated using two commercially
available programs and 3D structures of the selected compounds were investigated by means of ab initio
calculations of geometry and molecular dynamic simulations. All the synthesized esters were tested for
their in vitro transdermal penetration-enhancing activity and showed higher enhancement ratios than
oleic acid. The highest enhancement ratios were exhibited by compound 5f (C(2) substituted with
piperidine-2-one, C11 ester chain) and 5a (C(2) substituted with piperidine-2-one, C6 ester chain). The
Received 20 August 2010
Revised 8 October 2010
Accepted 11 October 2010
Available online 15 October 2010
Keywords:
x
-Lactam derivatives
6-Aminohexanoic acid derivatives
Lipophilicity
Ab initio calculations
Molecular dynamic simulations
3D structures
Transdermal penetration enhancers
Anti-proliferative activity
series with a
x
-lactam ring (piperidin-2-one; 5a–g), showed slightly higher activities than those with
morpholine (6a–6g). All of the agents showed minimal anti-proliferative activity (IC50 >6.25
lM), indicat-
ing they would have low cytotoxicity when administered as chemical penetration enhancers. The
relationships between the lipophilicity and the chemical structure of the studied compounds, as well
as the correlation between their chemical structure and transdermal penetration-enhancing activity,
are discussed.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
intercellular lipid-rich matrix. Several technological advances have
been made in the recent decades to overcome skin barrier
Developments in the field of pharmaceutical administration have
resulted in the discovery of highly sophisticated drug delivery sys-
tems that allow for the maintenance of a constant drug level in an
organism. Transdermal therapeutic systems are an excellent alter-
native to conventional pharmaceutical administration forms. How-
ever, the application of transdermal drug delivery faces the problem
of insufficient or no penetration of active pharmaceutical substances
through the skin. The outermost layer of skin, namely the stratum
corneum (SC), forms a barrier for most exogenous substances,
including drugs. The barrier function of the SC is attributed to its
multi-layered wall-like structure, in which terminally differentiated
keratin-rich epidermal cells (corneocytes) are embedded in an
properties. Examples include physical means such as iontophoresis,
sonophoresis, microneedles, chemical techniques using penetration
enhancers, and biochemical methods such as liposomal vesicles.1–3
Transdermal penetration enhancers (also called sorption pro-
moters or accelerants) are special pharmaceutical excipients that
interact with skin components to increase penetration of drugs
from topical application to entrance into the circulation. Numerous
compounds of different chemical varieties were evaluated as pen-
etration enhancers and a number of potential sites and modes of
action have been identified.3–6 In spite of the extensive research
in this field, chemical penetration enhancers have not reached
their full potential in transdermal or topical systems thus far.
Chemical penetration enhancers (CPEs) are compounds which
can partition into and interact with the SC constituents when
incorporated into a transdermal formulation, thereby reducing the
resistance of the skin to drug diffusion. Several possible mechanisms
of action of enhancers have been hypothesized, but the exact
⇑
Corresponding authors. Tel.: +61290366548; fax: +61290366549 (D.R.R.);
tel.: +420724139484; fax: +420272701331 (J.J.).
E-mail addresses: d.richardson@med.usyd.edu.au (D.R. Richardson), josef.jampi-
lek@zentiva.cz (J. Jampilek).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.025

K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 8556–8565
8557
Br
O
O
O
O
OH
OH
a
b
N
c
+
N
H2N
O
O
O
O
O
2
O
O
1
H
N
H
O
N
d
O
O
N
R = -C6H13 (5a)
-C7H15 (5b)
-C8H17 (5c)
-C9H19 (5d)
-C10H21 (5e)
O
N
O
N
O
O
O
e
OR
O
O
N
N
N
O
O
O
O
O
O
-C11 23
H
(5f)
3
4
5a-g
-C12H25 (5g)
e
O
R = -C6H13 (6a)
-C7H15 (6b)
-C8H17 (6c)
-C9H19 (6d)
-C10H21 (6e)
-C11H23 (6f)
-C12H25 (6g)
N
O
OR
N
O
O
6a-g
Scheme 1. Synthesis of target compounds 5a–g and 6a–g. Reagents: (a) acetone; (b) one pot synthesis: SOCl2, Br2, EtOH; (c) NaH, DMF, Cu2O; (d) toluene; (e) Na, R-OH.
Table 1
possible mechanism of action has been reported.3,4,7,8 The chemical
Comparison of calculated lipophilicities (log P/C log P) with determined log k values
structure of AzoneÒ is a combination of pyrrolidone and decylmeth-
ylsulfoxide, both of which are potential penetration enhancers.9 To
date, many AzoneÒ-related compounds (including modifications of
heterocyclic moiety and/or lipophilic chain) have been made and
studied as penetration enhancers.3 The 6-aminohexanoic acid esters
were also designed as acyclic AzoneÒ analogs3 and pyrrolidin-2,5-
dione, piperidin-2-one and morpholine are also fragments of
described potent penetration enhancers. The novelty of the new
compounds described herein is that they represent a combination
of drug fragmentswhichshowed high efficiencyas transdermalpen-
etration enhancers.3,10,11
The multi-step synthesis of a series of seven alkyl-6-(2,5-dioxo-
pyrrolidin-1-yl)-2-(2-oxopiperidin-1-yl)hexanoates and seven
alkyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(morpholin-4-yl)hexanoates
with C6–C12 linear alkyl ester chains as novel CPEs is described here-
in. The traditional lipophilicity parameter, log P, is a well-known
physico-chemical descriptor largely used for quantitative struc-
ture–activity relationship analysis. In some experimental studies
of penetration enhancement, the lipophilicity (non-polarity) of
enhancers was measured and the corresponding relationship be-
tween enhancer lipophilicity and penetration enhancement potency
was investigated.12–16 Therefore, we have examined both the exper-
imental lipophilicity log k data17 and calculated lipophilicity log P/
C log P of all compounds. The geometry of four selected compounds
was investigated by means of ab initio calculations and molecular
dynamic simulations and their 3D structures are presented. This is
significant, as physico-chemical properties such as planarity can
influence transdermal penetration. Primary in vitro screening of
transdermal penetration-enhancing activity of all synthesized
esters was evaluated using a Franz cell.18 Compounds possessing
carbonyl moieties are potential iron chelating agents with possible
anti-proliferative activity.19 Therefore, all the discussed compounds
were evaluated for their anti-proliferative activity against the
human SK-N-MC neuroepithelioma cell line. The structure–activity
relationships of the studied compounds are discussed.
Compd
Log k
Log P/C log P (ChemOffice)
Log P (ACD/Log P DB)
3
4
5a
5b
5c
5d
5e
5f
5g
6a
6b
6c
6d
6e
6f
6g
ꢀ0.9565
ꢀ0.7951
ꢀ0.2356
ꢀ0.0685
0.0973
0.2638
0.4285
0.5894
0.7413
ꢀ0.0736
0.0934
0.2613
0.4177
0.5834
0.7588
0.9221
0.03/0.7562
ꢀ0.20/0.55
1.77/2.8722
2.19/3.4012
2.60/3.9302
3.02/4.4592
3.44/4.9882
3.86/5.5172
4.27/6.0462
1.54/2.606
1.95/3.195
2.37/3.724
2.79/4.253
3.21/4.782
3.62/5.311
4.02/5.84
0.37 0.56
ꢀ0.33 0.48
2.41 0.56
2.92 0.56
3.43 0.56
3.94 0.56
4.45 0.56
4.96 0.56
5.47 0.56
1.71 0.48
2.22 0.45
2.73 0.48
3.23 0.48
3.74 0.48
4.25 0.48
4.76 0.48
7.42 0.20
a
Oleic acid
—
6.29/7.7860
a
Log k value was not determined.
mechanisms have not been elucidated. It is almost certain that they
have multiple effects. Generally, it can be stated that CPEs can
(i) interact with the intercellular lipid matrix (especially ceramides),
(ii) interact with protein structures (influencing the conformation of
keratin in the corneocytes or proteins in desmosomes), or
(iii) promote partitioning (influencing the SC nature leading to an
increase in the penetrant concentrationgradient and thus increasing
the flux, i.e., increasing the concentration of the drug in the
skin).3
Dodecylazacycloheptan-2-one (AzoneÒ) is one of the most stud-
ied penetration enhancers and possesses a large polar head group
and a lipid alkyl chain which are thought to be necessary for its activ-
ity.3,4 As it could be expected from its chemical structure, AzoneÒ is a
highly lipophilic chemical with a log P of 6.2.3,4 It enhances the skin
transport of a wide variety of drugs with different lipophilicities. Its

8558
K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 8556–8565
Figure 1. 3D structure of the S-enantiomer of hexyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopiperidin-1-yl)hexanoate (5a) and hexyl-6-(2,5-dioxopyrrolidin-1-yl)-
2-(morpholin-4-yl)hexanoate (6a).
Figure 2. 3D structure of the S-enantiomer of dodecyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopiperidin-1-yl)hexanoate (5g) and dodecyl-6-(2,5-dioxopyrrolidin-1-yl)-
2-(morpholin-4-yl)hexanoate (6g).
Table 2
tained (Scheme 1). Using an optimised Schwenk and Papa
procedure,20,21
1 gave ethyl-2-bromo-6-(2,5-dioxopyrrolidin-1-
Exocyclic torsion angles (ExTors) of compounds 5a and 5g from molecular dynamics
(MD) and ab initio (AI) calculations
yl)hexanoate (2) through a one-pot synthesis, which we have
reported previously.22
ExTor [°]
5a (MD)
5a (AI)
5g (MD)
5g (AI)
The key intermediate, ethyl-6-(2,5-dioxopyrrolidin-1-yl)-
2-(2-oxopiperidin-1-yl)hexanoate (3), was prepared from the
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
180
182
311
177
195
293
294
105
264
183
64
66
176
67
180
—
—
180
172; 133; 7
175; 63
181; 77; 289
181; 67
178; 64; 296
271; 72
177
241
61
174
181
57
254
290
182
67
289
60
188
290
187
171
69
179
182
178
56; 179
175; 69
182; 292
182; 292
183; 296; 63
109
a-bromocarboxylic compound, 2, and piperidin-2-one using the
heterogeneous catalyst, powdered copper(I) oxide. The preparation
of a morpholine adduct, 4, was carried out under conventional
conditions.23
The series of desired alkyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-
oxopiperidin-1-yl)hexanoates (5a–g) and alkyl-6-(2,5-dioxopyrr-
olidin-1-yl)-2-(morpholin-4-yl)hexanoates (6a–g) were formed
by conventional base-catalyzed transesterification24 of the key
intermediates, 3 or 4, in excess of the corresponding primary un-
branched alcohol, according to Scheme 1.
244
64
180; 73; 288
299; 64; 180
180; 69
181; 67; 290
180; 292
300; 60; 180
—
178; 72; 286
301; 179
181; 290
180; 295; 65
180; 290; 67
180; 64;295
179; 297; 3
118; 58;298
182; 357
182; 357
180; 64; 295
59; 298; 180
—
—
—
—
—
—
—
—
2.2. Lipophilicity of the prepared compounds
The hydrophobicity (log P/C log P values) of the studied com-
pounds 3, 4, 5a–5g, and 6a–6g were calculated using two commer-
cially available programs (ChemDraw Ultra 10.0 and ACD/Log P
DB). Their hydrophobicities were measured experimentally by RP-
HPLC determination of their capacity factors (k) with subsequent
calculation of log k. This procedure measures the retention times
of the compounds under isocratic conditions with various amounts
of an organic modifier (methanol) in the mobile phase. Using an
end-capped non-polar C18 stationary RP column, the capacity factor
k can be calculated. log k is used as a lipophilicity index converted to
the log P scale.17 The results are shown in Table 1.
—
2. Result and discussion
2.1. Chemistry
6-Aminohexanoic acid and succinic anhydride were used as the
starting materials for the multi-step synthesis and by their
reaction, 6-(2,5-dioxopyrrolidin-1-yl)hexanoic acid (1) was ob-

K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 8556–8565
8559
Figure 3. Exocyclic torsion angles (ExTors) from molecular dynamic calculations.
The data obtained with all the compounds show that the exper-
imentally-determined lipophilicities (log k) of compounds 3–6g
are lower than those indicated by the calculated log P/C log P. Con-
trary to our experimental results, the calculated data showed that
the morpholine series, 6a–6g, possess lower lipophilicity than the
piperidin-2-one series, 5a–5g. This is probably caused by intra-
molecular interactions between the lactam carbonyl moiety and
other sterically close carbonyl groups.
As expected, 3 and 4 showed the lowest lipophilicity, whereas
dodecyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopiperidin-1-yl)

8560
K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 8556–8565
hexanoate (5g) and dodecyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(mor-
pholin-4-yl)hexanoate (6g) were the most lipophilic within each
series. The calculated log P/C log P data and the determined log k
values correspond to the expected trend in lipophilicity, increasing
within each series of evaluated compounds (ethyl n hexyl < hep-
tyl < nonyl < decyl < undecyl < dodecyl derivatives). This depen-
dence is approximately linear. The log k data corresponds to the
lipophilicity within the series of compounds discussed.
molecules were deleted so as to preserve the composition and
macroscopic density. The whole molecular system was optimized,
heated to room temperature and equilibrated. A series of MD runs
for compounds 5a and 5g were started from several best conforma-
tions after the conformational search.
Mean values of all exocyclic torsions from MD simulations are
summarized in Table 2 (in decreasing order of frequency) and their
graphical representations for compound 5g are given in Figure 3.
For comparison, global minima from ab initio calculations are also
given in Table 2. Designation of exocyclic torsions are illustrated in
Figure 4.
2.3. Geometries—3D structures
The chemical structure as well as exact 3D geometries of the
studied penetration enhancers under study influence their
physico-chemical properties and, thus, moderate their penetra-
tion-enhancing activity. Furthermore, they are also used for the
derivation of a series of structural descriptors like molecular
dimensions, volume and surface, dipole moment etc., which are of-
ten used for in silico screening and design of new penetration
enhancers.3,25 One of several possible mechanisms of action of
AzoneÒ-like enhancers is that they intercalate between ceramides
in the SC.3,7 By doing this, they disrupt ceramide-ceramide
H-bonds and bind firmly to the ceramide molecule, forming a
‘‘channel” in the SC.3,7 As intercalation potency is dependent on a
planar conformation, understanding the 3D structures of the stud-
ied compounds is of importance. Therefore, this section describes
the ab initio calculations of geometry and molecular dynamic
(MD) simulations. To correlate penetration-enhancing activity
and other physico-chemical properties with structure, the exact
3D geometries of the (S)-enantiomers of compounds 5a and 5g,
as well as 6a and 6g were studied by computational methods.
The reason why these compounds were chosen is that they repre-
sent the shortest and the longest potential enhancers within each
series under investigation. Due to the size of the molecules, which
was discussed previously,11 as well as for a better representation of
the solvent media, a combination of molecular mechanics and ab
initio quantum chemical methods were used to examine the geom-
etries of our compounds.
MD simulations starting from different conformers led usually to
the same torsions with more or less different frequencies and
consecutive MD runs produced only slightly different frequencies.
This means that longer MD runs should be used for more realistic
frequencies, but otherwise the qualitative picture is satisfactory.
The MD torsion values agree well with ab initio values in Table 2.
However, conformational flexibility is best revealed from the graphs
in Figure 3 which show independent changes of almost all torsions.
It is clearly evident that the conformer lifetimes are of the order of
only few tens of picoseconds (Fig. 3) and for derivatives with longer
alkyl chains it would be even less.
It can be concluded in this respect that MD simulations with ex-
plicit solvent molecules provide appropriate sampling of available
conformations of the studied compounds which is far beyond the
possibilities of ab initio calculations at present. A detailed analysis
of all torsions and their changes as well as of hydrogen bonds
which were observed during simulations would be interesting,
but beyond the scope of this paper.
Considering the above discussed data, the compounds exam-
ined in this study show a non-planar conformation, the conse-
quence of which can be lower penetration activity. Indeed, a
planar configuration is advantageous due to preferable interactions
with skin components, especially ceramides.3,7
2.4. In vitro screening of transdermal penetration-enhancing
activity
The initial scan of geometries for all compounds was performed
in vacuo using the conformational search utility in the program,
HyperChem. For structures 5a, 5g, 6a and 6g, 28671, 49564,
39286 and 19091 initial conformations were explored, respec-
tively. For subsequent ab initio calculations, this number was
reduced and only conformations with relative energies within
2 kcal/mole were considered. These conformations were then
re-optimized in water at HF/4-31G level in GAUSSIAN 09 W.26,27
Global minima for all compounds are illustrated in Figures 1 and 2.
While ab initio calculations are appropriate for prediction of ex-
act geometries of a limited number of conformers and their spec-
tral (NMR, IR) or chiroptical properties (optical rotation, VCD) in
a gas phase, they are not suitable for the scanning of a large num-
ber of conformers, as it is a time-consuming process. As noted pre-
viously,11 the solvent media are usually quite poorly represented
by ab initio calculations. For the above calculations at the ab initio
level, the actual solvent used for experiments (propylene glycol/
water 1:1 v/v) was simplified so that propylene glycol molecules
were omitted and water molecules were replaced by a dielectric
continuum using the CPCM (conductor-like polarizable continuum
model) solvation model.
The penetration-enhancing activity of the prepared compounds
was evaluated using theophylline as a model penetrant and pro-
pylene glycol/water 1:1 v/v as a donor vehicle. Theophylline has
been used as a model drug of medium polarity (log P ꢀ0.06; log D8
ꢀ0.05),28,29 as it has been extensively studied in transdermal pen-
etration experiments.30,31 Most of the studies involved the use of
propylene glycol (PG) or its mixture with water or ethanol as a do-
nor vehicle. Previous studies have indicated that PG by itself (or a
PG/water co-solvent system) does not interfere with membranes,
but rather exhibits a synergistic effect in combination with other
penetration enhancers.32–34 Porcine ear skin was selected for initial
evaluation of enhancement activity of prepared compounds as this
tissue is a suitable in vitro model for human skin.35,36 Porcine skin
has been shown to be histologically and biochemically similar to
human skin and full-thickness pig ear skin has been used in
numerous percutaneous absorption studies.37 Nevertheless, for
testing of hydrophobic penetrants, dermatomed skin has been rec-
ommended.38 The skin permeation experiments were performed
using static Franz diffusion cells.18
As the first step for a better representation of the solvent at the
ab initio level, as well as a better scan of possible conformations
and their dynamics, we performed a series of molecular MD runs
at the molecular mechanics level with explicit solvent molecules
under periodic boundary conditions. For this purpose, a periodic
box was created and filled with a mixture of (R)-propylene
glycol/(S)-propylene glycol/water 0.5:0.5:1 v/v. After pre-equili-
bration, a solute molecule was soaked and selected solvent
18 17 16
H3C20 19
O
N
O
8
2
15
6
5
4
3
10 11 12 13 14
1 O 9
N 7
O
O
Figure 4. Designation of exocyclic torsion angles (ExTors).

K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 8556–8565
8561
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Compounds
Figure 5. Enhancement ratios (ERs) of the prepared compounds 5a–g, 6a–g and oleic acid (OA). Control experiments used theophylline in the donor vehicle without any
enhancer. Results are expressed as mean SD (n = 3 experiments).
The effect of alkyl esters, 5a–g and 6a–g, on the penetration of
theophylline through porcine skin is presented in Figure 5. All pre-
pared compounds, 5a–g and 6a–g, enhanced the penetration of
theophylline through the porcine skin and showed better enhance-
ment ratios (ERs) than oleic acid (OA), which was used for compar-
ison and belongs to the most studied enhancers in the fatty acid
group of compounds.39 Control experiments were run with only
theophylline in the donor vehicle in the absence of any enhancer.
The highest enhancement ratios (ERs) were obtained with
compounds 5f (undecyl, piperidin-2-one) and 5a (hexyl, piperi-
din-2-one), with ERs of 2.36 and 2.33, respectively. The ERs are
presented in Table 3.
According to the above presented data (Table 3, Fig. 5), it can be
concluded that all the discussed compounds showed only moderate
penetration activity. This moderate activity may be predicted from
the 3D structures of the selected compounds (Figs. 1 and 2) in the
medium propylene glycol/water 1:1 v/v, where non-planar
(coned/coiled) conformations of compounds are illustrated. Due to
the non-planar conformations of all the discussed compounds, their
intercalation potency is decreased, contrary to planar (open) confor-
mations, which can cause their moderate penetration activity.3,7
The penetration of the enhancers through the skin was further
investigated. Only compounds 5a, 5b, 6a and 6b were detected in
the receptor compartment after 24 h. All other alkyl esters 5c–5g
and 6c–g were not detected and it was assumed that they did
not get through the skin barrier or remained incorporated in skin
structures due to their high lipophilicity.
2.5. In vitro anti-proliferative activity
Carbonyl-containing moieties are potential iron chelating
agents, with many demonstrating anti-proliferative activity due
to their ability to bind cellular iron which is essential for prolifer-
ation.40 Therefore, we examined the anti-proliferative effects of our
novel CPEs against the human SK-N-MC neuroepithelioma cell line
to examine if there may be any undesired cytotoxic side-effects
associated with CPE treatment (Table 3). The SK-N-MC cell line
was chosen as the effect of iron chelators on their proliferation
has been extensively examined.41,42 The anti-proliferative activity
of these novel compounds was assessed in comparison to the
well-known clinically used iron chelator, desferrioxamine (DFO),
and the highly cytotoxic chelator, di-2-pyridylketone 4,4-di-
methyl-3-thiosemicarbazone (Dp44mT).40
All alkyl esters, 5a–g and 6a–g, demonstrated poor anti-prolif-
erative effects against the SK-N-MC cell line, with IC50 values great-
er than 6.25
studies,41,42 the iron chelator, DFO, demonstrated poor anti-cancer
activity, with an IC50 value of 17.07 M, while the cytotoxic chela-
tor, Dp44mT (IC50: 0.01 M), showed potent anti-proliferative ef-
lM (Table 3). As expected from our previous
l
l
fects (Table 3). These results suggest that the poor anti-
proliferative activity of our alkyl ester, 5a–g and 6a–g, will lead
to limited cytotoxicity when used in vivo as CPEs.
3. Conclusions
In the current study, a series of seven alkyl-6-(2,5-dioxopyrroli-
din-1-yl)-2-(2-oxopiperidin-1-yl)hexanoates and seven alkyl-6-
(2,5-dioxopyrrolidin-1-yl)-2-(morpholin-4-yl)hexanoates with
C6–C12 linear alkyl ester chains were prepared as potential transder-
mal penetration enhancers. The ability of these compounds to en-
hance the penetration of theophylline through porcine skin was
examined. All prepared compounds showed higher enhancement
ratios (ERs) than oleic acid. The highest ERs in this study were exhib-
ited by compound 5f (C(2) substituted with piperidine-2-one, C11 es-
ter chain) and 5a (C(2) substituted with piperidine-2-one, C6 ester
Table 3
Enhancement ratios (ERs) of the prepared compounds, 5a–5g and 6a–6g, and oleic
acid (OA) and in vitro anti-proliferative activity of the studied compounds using
SK-N-MC neuroepithelioma cells
Compd
ER
SK-N-MC IC50 (lM)
5a
5b
5c
5d
5e
5f
5g
6a
6b
6c
6d
6e
6f
6g
OA
Dp44mT
DFO
2.33 0.18
2.17 0.15
1.88 0.21
1.81 0.23
1.63 0.15
2.36 0.25
1.75 0.16
2.11 0.13
1.26 0.22
1.80 0.29
1.49 0.21
1.71 0.17
1.61 0.25
1.92 0.20
1.13 0.18
—
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
—
chain). We can conclude that the series with a
x-lactam ring (piper-
idin-2-one), 5a–g, showed slightly higher activities than those with
morpholine 6a–6g. Most of the prepared compounds did not pene-
trate through the skin, except for compounds 5a, 5b, 6a and 6b
which were detected in the receptor phase in 24 h. All the com-
pounds were additionally evaluated for their anti-proliferative
activity against the human SK-N-MC neuroepithelioma cell line.
None of the studied compounds showed any significant anti-prolif-
erative activity, suggesting that these compounds would have low
cytotoxic side-effects when administered as CPEs. Thus, our novel
alkyl esters, 5a and 5f, show promise as CPEs with minimal
cytotoxicity.
0.01 0.01
17.07 3.77
—
ERs data are expressed as mean SD (n = 3 experiments) and anti-proliferative
activity of the compounds are expressed as mean SD (n = 3 experiments).

8562
K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 8556–8565
(80%). 1H NMR (500 MHz, CDCl3), d: 4.18 (q, J = 7.1 Hz, 2H,
4. Experimental
4.1. Chemistry
COOCH2), 3.74–3.63 (m, 4H, OCH2 morph.), 3.50 (t, J = 7.4 Hz, 2H,
NCH2), 3.10 (t, J = 7.4 Hz, 1H, CH), 2.71 (s, 4H, OCCH2CH2CO),
2.66–2.48 (m, 4H, NCH2 morph.), 1.80–1.52 (m, 4H, CH2), 1.45–
1.27 (m, 2H, CH2), 1.29 (t, J = 7.1 Hz, 3H, CH3). 13C NMR
(125 MHz, CDCl3), d: 177.07, 171.72, 67.54, 67.38, 60.19, 49.95,
38.57, 28.29, 28.13, 27.38, 23.32, 14.48. IR (cmꢀ1): 2948, 2855,
1697, 1400, 1150, 1114. HR-MS: for C16H27O5N2 [M+H]+ calcd
327.1914 m/z, found 327.1915 m/z.
All reagents were purchased from Sigma-Aldrich (Schnelldorf,
Germany) and Merck (Darmstadt, Germany). Kieselgel 60, 0.040–
0.063 mm (Merck, Darmstadt, Germany) was used for column
chromatography. Thin layer chromatography (TLC) experiments
were performed on alumina-backed Silica Gel 40 F254 plates
(Merck, Darmstadt, Germany). The plates were illuminated under
UV (254 nm) and evaluated in iodine vapour. The melting points
were determined on a Mikro-Heiztisch System PolyTherm A appa-
ratus (Wagner & Munz, Munich and Hund, Wetzlar, Germany) and
are uncorrected. Infrared (IR) spectra were recorded on a Smart
MIRacle™ ATR ZnSe for Nicolet™ 6700 FT-IR Spectrometer (Ther-
mo Scientific, USA). The spectra were obtained by accumulation
of 256 scans with 2 cmꢀ1 resolution in the 4000–600 cmꢀ1 region.
All 1H and 13C NMR spectra were recorded on a Bruker Avance-500
FT-NMR spectrometer (500 MHz for 1H and 125 MHz for 13C, Bru-
ker Comp., Karlsruhe, Germany). Chemicals shifts are reported in
ppm (d) using internal Si(CH3)4 as the reference, with diffuse, easily
exchangeable signals being omitted. Mass spectra were measured
using a LTQ Orbitrap Hybrid Mass Spectrometer (Thermo Electron
Corporation, USA) with direct injection into an APCI source
(400 °C) in the positive mode.
4.1.5. General procedure for preparation alkyl-6-(2,5-
dioxopyrrolidin-1-yl)-2-(2-oxopiperidin-1-yl)hexanoates (5a–g)
and alkyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(morpholin-4-yl)
hexanoates (6a–g)
The mixture of ethyl ester 3 or 4 (7.7 mmol), appropriate pri-
mary alcohol (38.5 mmol) and metallic sodium (3.85 mmol) was
stirred at 90 °C in an oil bath until the sodium was completely dis-
solved. The mixture was then heated at 130 °C for 5–7 h and etha-
nol was distilled off during the reaction as it formed. The excess of
longer-chain alkyl alcohol was distilled off under reduced pressure
and the rest was extracted with AcOH (0.5 M) and Et2O. The organ-
ic layer was dried over MgSO4, filtered and evaporated. The crude
product was purified by column chromatography on silica gel
using ethyl acetate/petroleum ether (5:1) as the eluent.
4.1.5.1. Hexyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopiperidin-1-
yl)hexanoate (5a). Colorless oil, yield 39%. 1H NMR (500 MHz,
CDCl3), d: 5.16 (dd, J1 = 10.4 Hz, J2 = 5.3 Hz, 1H, CH), 4.18–4.03 (m,
2H, COOCH2), 3.50 (t, J = 7.3 Hz, 2H, NCH2), 3.27–3.17 (m, 2H, NCH2
pip.), 2.71 (s, 4H, OCCH2CH2CO), 2.48–2.41 (m, 2H, CH2pip.),
2.05–1.53 (m, 10H, CH2), 1.36–1.26 (m, 8H, CH2), 0.89 (t, J = 6.5 Hz,
3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.16, 171.26, 170.44,
65.22, 55.79, 44.19, 38.45, 32.24, 31.33, 28.49, 28.14, 27.49, 27.25,
25.54, 23.49, 23.14, 22.48, 21.01, 13.92. IR (cmꢀ1): 2932, 2868,
1731, 1697, 1640, 1401, 1166. HR-MS: for C21H35O5N2 [M+H]+ calcd
395.2540 m/z, found 395.2541 m/z.
4.1.1. 6-(2,5-Dioxopyrrolidin-1-yl)hexanoic acid (1)
This was synthesised as described previously by Brychtova
et al.11,22
4.1.2. Ethyl-2-bromo-6-(2,5-dioxopyrrolidin-1-yl)hexanoate (2)
This was synthesised as described by Brychtova et al.11,22
4.1.3. Ethyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopiperidin-1-yl)
hexanoate (3)
Piperidin-2-one (46.9 mmol) was added slowly to a suspension
of NaH (51.5 mmol, 60% dispersion in mineral oil) in dry DMF
(100 mL). The mixture was stirred for a few minutes until the evolu-
tion of hydrogen gas ceased. Compound 2 (10.0 g, 31.2 mmol) and
Cu2O (1.1 g, 7.8 mmol) were then added and the mixture was re-
fluxed under argon for 9 h. The cooled mixture was poured onto
ice, filtered and extracted with chloroform. The combined organic
extracts were washed with water, dried over MgSO4, filtered and
the organic solvent was removed under vacuum. The crude product
was purified by flash chromatography on silica gel (ethyl acetate/
petroleumether/TEA10:1:0.1). Thisprovided a light yellowoil. Yield
7.3 g (70%). 1H NMR (500 MHz, CDCl3), d: 5.14 (dd, J1 = 5.0 Hz,
J2 = 10.3 Hz, 1H, CH), 4.22–4.06 (m, 2H, COOCH2), 3.50 (t,
J = 7.1 Hz, 2H, NCH2), 3.29–3.17 (m, 2H, NCH2pip.), 2.70 (s, 4H,
OCCH2CH2CO), 2.48–2.42 (m, 2H, CH2pip.), 2.01–1.53 (m, 8H, CH2),
1.37–1.23 (m, 2H, CH2), 1.26 (t, J = 7.2 Hz, 3H, CH3). 13C NMR
(125 MHz, CDCl3), d: 177.13, 171.16, 170.47, 61.00, 55.72, 44.13,
38.42, 32.21, 28.13, 27.48, 27.22, 23.46, 23.12, 20.98, 14.15. IR
(cmꢀ1): 2941, 2868, 1731, 1698, 1637, 1401, 1178. HR-MS: for
4.1.5.2. Heptyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopiperidin-1-
yl)hexanoate (5b). Colorless oil, yield 45%. 1H NMR (500 MHz,
CDCl3), d: 5.16 (dd, J1 = 10.4 Hz, J2 = 5.2 Hz, 1H, CH), 4.15–4.02 (m,
2H, COOCH2), 3.50 (t, J = 7.3 Hz, 2H, NCH2), 3.28–3.17 (m, 2H,
NCH2pip.), 2.71 (s, 4H, OCCH2CH2CO), 2.48–2.41 (m, 2H, CH2pip.),
2.05–1.52 (m, 10H, CH2), 1.41–1.21 (m, 10H, CH2), 0.88 (t,
J = 6.5 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.16, 171.25,
170.44, 65.22, 55.78, 44.18, 38.45, 32.23, 31.67, 28.81, 28.53,
28.14, 27.49, 27.26, 25.83, 23.49, 23.14, 22.52, 21.01, 13.99. IR
(cmꢀ1): 2931, 2859, 1731, 1697, 1640, 1401, 1166. HR-MS: for
C
22H37O5N2 [M+H]+ calcd 409.2697 m/z, found 409.2697 m/z.
4.1.5.3. Octyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopiperidin-1-
yl)hexanoate (5c). Colorless oil, yield 53%. 1H NMR (500 MHz,
CDCl3), d: 5.15 (dd, J1 = 10.4 Hz, J2 = 5.3 Hz, 1H, CH), 4.15–4.02 (m,
2H, COOCH2), 3.50 (t, J = 7.3 Hz, 2H, NCH2), 3.27–3.17 (m, 2H,
NCH2pip.), 2.70 (s, 4H, OCCH2CH2CO), 2.48–2.41 (m, 2H, CH2pip.),
2.03–1.53 (m, 10H, CH2), 1.37–1.21 (m, 12H, CH2), 0.88 (t,
J = 6.4 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.09, 171.21,
170.38, 65.18, 55.79, 44.17, 38.42, 32.21, 31.70, 29.09, 28.52,
28.12, 27.48, 27.23, 25.85, 23.46, 23.13, 22.55, 20.99, 13.98. IR
(cmꢀ1): 2928, 2857, 1732, 1697, 1641, 1401, 1166. HR-MS: for
C
17H27O5N2 [M+H]+ calcd 339.1914 m/z, found 339.1914 m/z.
4.1.4. Ethyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(morpholin-4-
yl)hexanoate (4)
Morpholine (5.4 g, 62.4 mmol) was dissolved in toluene (50 mL)
and compound 2 (10.0 g, 31.2 mmol) was added. The mixture was
refluxed under argon for 5 h. The solvent was evaporated and the
residue was suspended in Et2O. The solid was filtered, washed with
Et2O and the filtrate was concentrated under reduced pressure. The
crude product was purified by flash chromatography on silica gel
(CH2Cl2/MeOH 95:5). This provided a yellow oil. Yield: 8.1 g
C
23H39O5N2 [M+H]+ calcd 423.2853 m/z, found 423.2854 m/z.
4.1.5.4. Nonyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopiperidin-1-
yl)hexanoate (5d). Colorless oil, yield 45%. 1H NMR (500 MHz,
CDCl3), d: 5.16 (dd, J1 = 10.4 Hz, J2 = 5.3 Hz, 1H, CH), 4.15–4.02
(m, 2H, COOCH2), 3.50 (t, J = 7.3 Hz, 2H, NCH2), 3.27–3.17 (m, 2H,

K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 8556–8565
8563
NCH2pip.), 2.70 (s, 4H, OCCH2CH2CO), 2.48–2.41 (m, 2H, CH2pip.),
2.03–1.53 (m, 10H, CH2), 1.37–1.23 (m, 14H, CH2), 0.88 (t,
J = 6.4 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.12,
171.25, 170.43, 65.22, 55.81, 44.20, 38.46, 32.25, 31.82, 29.44,
29.18, 28.55, 28.15, 27.51, 27.26, 25.88, 23.50, 23.16, 22.62,
21.03, 14.03. IR (cmꢀ1): 2926, 2855, 1732, 1698, 1641, 1401,
1166. HR-MS: for C24H41O5N2 [M+H]+ calcd 437.3010 m/z, found
437.3011 m/z.
(t, J = 6.5 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.06,
171.84, 67.58, 67.39, 64.42, 49.94, 38.56, 31.66, 28.81, 28.74,
28.31, 28.13, 27.39, 25.92, 23.34, 22.52, 13.99. IR (cmꢀ1): 2930,
2855, 1722, 1698, 1400, 1152, 1116. HR-MS: for C21H36O5N2
[M+H]+ calcd 397.2697 m/z, found 397.2696 m/z.
4.1.5.10. Octyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(morpholin-4-yl)
hexanoate (6c). Light yellow crystalline compound, mp. 40.4-
41.6 °C, yield 58%. 1H NMR (500 MHz, CDCl3), d: 4.11 (t,
J = 6.7 Hz, 2H, COOCH2), 3.71–3.62 (m, 4H, OCH2 morph.), 3.50 (t,
J = 7.4 Hz, 2H, NCH2), 3.10 (t, J = 7.4 Hz, 1H, CH), 2.70 (s, 4H,
OCCH2CH2CO), 2.64–2.55 (m, 4H, NCH2 morph.), 1.76–1.52 (m,
6H, CH2), 1.46–1.23 (m, 12H, CH2), 0.88 (t, J = 6.5 Hz, 3H, CH3).
13C NMR (125 MHz, CDCl3), d: 177.10, 171.87, 67.59, 67.41, 64.45,
49.95, 38.58, 31.76, 29.14, 28.75, 28.33, 28.15, 27.41, 25.98,
23.36, 22.61, 14.05. IR (cmꢀ1): 2930, 2854, 1718, 1692, 1402,
1149, 1117. HR-MS: for C22H38O5N2 [M+H]+ calcd 411.2853 m/z,
found 411.2855 m/z.
4.1.5.5. Decyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopiperidin-1-
yl)hexanoate (5e). Colorless oil, yield 47%. 1H NMR (500 MHz,
CDCl3), d: 5.16 (dd, J1 = 10.5 Hz, J2 = 5.4 Hz, 1H, CH), 4.14–4.02
(m, 2H, COOCH2), 3.50 (t, J = 7.4 Hz, 2H, NCH2), 3.26–3.17 (m, 2H,
NCH2pip.), 2.70 (s, 4H, OCCH2CH2CO), 2.48–2.40 (m, 2H, CH2pip.),
2.02–1.52 (m, 10H, CH2), 1.41–1.21 (m, 16H, CH2), 0.88 (t,
J = 6.5 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.14,
171.27, 170.45, 65.24, 55.81, 44.20, 38.47, 32.26, 31.86, 29.50,
29.26, 29.19, 28.56, 28.16, 27.51, 27.27, 25.90, 23.51, 23.17,
22.65, 21.04, 14.06. IR (cmꢀ1): 2925, 2854, 1732, 1698, 1642,
1401, 1166. HR-MS: for C25H43O5N2 [M+H]+ calcd 451.3166 m/z,
found 451.3167 m/z.
4.1.5.11. Nonyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(morpholin-4-yl)
hexanoate (6d). Light yellow crystalline compound, mp 42.3-
43.1 °C, yield 51%. 1H NMR (500 MHz, CDCl3), d: 4.10 (t,
J = 6.6 Hz, 2H, COOCH2), 3.71–3.65 (m, 4H, OCH2 morph.), 3.50 (t,
J = 7.4 Hz, 2H, NCH2), 3.10 (t, J = 7.4 Hz, 1H, CH), 2.70 (s, 4H,
OCCH2CH2CO), 2.64–2.55 (m, 4H, NCH2 morph.), 1.75–1.52 (m,
6H, CH2), 1.42–1.20 (m, 14H, CH2), 0.88 (t, J = 6.4 Hz, 3H, CH3).
13C NMR (125 MHz, CDCl3), d: 177.10, 171.86, 67.58, 67.40, 64.45,
49.94, 38.58, 31.82, 29.44, 29.19, 28.75, 28.32, 28.14, 27.40,
25.98, 23.35, 22.63, 14.06. IR (cmꢀ1): 2916, 2850, 1728, 1699,
1400, 1158, 1117. HR-MS: for C23H40O5N2 [M+H]+ calcd 425.3010
m/z, found 425.3011 m/z.
4.1.5.6.
Undecyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopiperi-
din-1-yl)hexanoate (5f). Colorless oil, yield 37%. 1H NMR
(500 MHz, CDCl3), d: 5.16 (dd, J1 = 10.5 Hz, J2 = 5.3 Hz, 1H, CH),
4.15–4.02 (m, 2H, COOCH2), 3.50 (t, J = 7.3 Hz, 2H, NCH2), 3.25–
3.16 (m, 2H, NCH2pip.), 2.71 (s, 4H, OCCH2CH2CO), 2.48–2.41 (m,
2H, CH2pip.), 2.05–1.50 (m, 10H, CH2), 1.40–1.18 (m, 18H, CH2),
0.88 (t, J = 6.4 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.13,
171.25, 170.42, 65.22, 55.79, 44.19, 38.45, 32.24, 31.87, 29.55,
29.48, 29.28, 29.18, 28.55, 28.15, 27.49, 27.26, 25.88, 23.49,
23.15, 22.64, 21.02, 14.05. IR (cmꢀ1): 2924, 2854, 1732, 1698,
1642, 1401, 1166. HR-MS: for C25H43O5N2 [M+H]+ calcd 465.3323
m/z, found 465.3323 m/z.
4.1.5.12. Decyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(morpholin-4-yl)
hexanoate (6e). Light yellow crystalline compound, mp 45.5-
46.1 °C, yield 53%. 1H NMR (500 MHz, CDCl3), d: 4.10 (t,
J = 6.7 Hz, 2H, COOCH2), 3.71–3.65 (m, 4H, OCH2 morph.), 3.50 (t,
J = 7.4 Hz, 2H, NCH2), 3.10 (t, J = 7.4 Hz, 1H, CH), 2.70 (s, 4H,
OCCH2CH2CO), 2.64–2.55 (m, 4H, NCH2 morph.), 1.76–1.52 (m,
6H, CH2), 1.42–1.20 (m, 16H, CH2), 0.88 (t, J = 6.4 Hz, 3H, CH3).
13C NMR (125 MHz, CDCl3), d: 177.06, 171.82, 67.54, 67.36, 64.41,
49.91, 38.53, 31.83, 29.46, 29.23, 29.15, 28.71, 28.28, 28.11,
27.37, 25.95, 23.31, 22.61, 14.04. IR (cmꢀ1): 2924, 2854, 1699,
1400, 1155, 1117. HR-MS: for C24H42O5N2 [M+H]+ calcd 439.3166
m/z, found 439.3166 m/z.
4.1.5.7. Dodecyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopiperidin-
1-yl)hexanoate (5g). Colorless oil, yield 42%. 1H NMR (500 MHz,
CDCl3), d: 5.16 (dd, J1 = 10.5 Hz, J2 = 5.3 Hz, 1H, CH), 4.15–4.02
(m, 2H, COOCH2), 3.50 (t, J = 7.3 Hz, 2H, NCH2), 3.25–3.17 (m, 2H,
NCH2pip.), 2.71 (s, 4H, OCCH2CH2CO), 2.48–2.41 (m, 2H, CH2pip.),
2.05–1.50 (m, 10H, CH2), 1.41–1.12 (m, 20H, CH2), 0.88 (t,
J = 6.5 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.12,
171.24, 170.41, 65.22, 55.79, 44.19, 38.45, 32.23, 31.87, 29.58,
29.53, 29.48, 29.29, 29.17, 28.54, 28.14, 27.49, 27.25, 25.88,
23.48, 23.15, 22.64, 21.02, 14.05. IR (cmꢀ1): 2923, 2853, 1732,
1698, 1642, 1401, 1166. HR-MS: for C27H47O5N2 [M+H]+ calcd
479.3479 m/z, found 479.3480 m/z.
4.1.5.13. Undecyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(morpholin-4-
yl)hexanoate (6f). Light yellow crystalline compound, mp 48.9-
49.7 °C, yield 52%. 1H NMR (500 MHz, CDCl3), d: 4.10 (t, J = 6.7 Hz,
2H, COOCH2), 3.71–3.65 (m, 4H, OCH2 morph.), 3.50 (t, J = 7.4 Hz,
2H, NCH2), 3.10 (t, J = 7.4 Hz, 1H, CH), 2.70 (s, 4H, OCCH2CH2CO),
2.64–2.55 (m, 4H, NCH2 morph.), 1.76–1.52 (m, 6H, CH2), 1.42–
1.17 (m, 18H, CH2), 0.88 (t, J = 6.4 Hz, 3H, CH3). 13C NMR
(125 MHz, CDCl3), d: 177.08, 171.84, 67.57, 67.39, 64.43, 49.93,
38.56, 31.87, 29.55, 29.48, 29.29, 29.17, 28.74, 28.31, 28.13, 27.39,
25.97, 23.34, 22.64, 14.06. IR (cmꢀ1): 2924, 2853, 1699, 1401,
1156, 1117. HR-MS: for C25H44O5N2 [M+H]+ calcd 453.3323 m/z,
found 453.3323 m/z.
4.1.5.8. Hexyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(morpholin-4-yl)
hexanoate (6a). Light yellow oil, yield 53%. 1H NMR (500 MHz,
CDCl3), d: 4.03 (t, J = 6.6 Hz, 2H, COOCH2), 3.63–3.57 (m, 4H,
OCH2 morph.), 3.43 (t, J = 7.4 Hz, 2H, NCH2), 3.03 (t, J = 7.4 Hz,
1H, CH), 2.63 (s, 4H, OCCH2CH2CO), 2.57–2.47 (m, 4H, NCH2
morph.), 1.69–1.45 (m, 6H, CH2), 1.38–1.15 (m, 8H, CH2), 0.82 (t,
J = 6.5 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 176.98,
171.70, 67.43, 67.25, 64.27, 49.80, 38.40, 31.20, 28.56, 28.17,
28.01, 27.25, 25.49, 23.20, 22.35, 13.81. IR (cmꢀ1): 2932, 2856,
1722, 1697, 1401, 1149, 1116. HR-MS: for C20H34O5N2 [M+H]+
calcd 383.2540 m/z, found 383.2541 m/z.
4.1.5.14. Dodecyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(morpholin-4-
yl)hexanoate (6g). Light yellow crystalline compound, mp 52.6-
53.4 °C, yield 55%. 1H NMR (500 MHz, CDCl3), d: 4.10 (t,
J = 6.7 Hz, 2H, COOCH2), 3.71–3.65 (m, 4H, OCH2 morph.), 3.50 (t,
J = 7.4 Hz, 2H, NCH2), 3.10 (t, J = 7.4 Hz, 1H, CH), 2.70 (s, 4H,
OCCH2CH2CO), 2.64–2,55 (m, 4H, NCH2 morph.), 1.76–1.52 (m,
6H, CH2), 1.42–1.17 (m, 20H, CH2), 0.88 (t, J = 6.4 Hz, 3H, CH3).
13C NMR (125 MHz, CDCl3), d: 177.09, 171.86, 67.58, 67.39, 64.45,
4.1.5.9. Heptyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(morpholin-4-yl)
hexanoate (6b). Light yellow oil, yield 55%. 1H NMR (500 MHz,
CDCl3), d: 4.10 (t, J = 6.6 Hz, 2H, COOCH2), 3.71–3.65 (m, 4H,
OCH2 morph.), 3.50 (t, J = 7.4 Hz, 2H, NCH2), 3.10 (t, J = 7.4 Hz,
1H, CH), 2.70 (s, 4H, OCCH2CH2CO), 2.64–2.55 (m, 4H, NCH2
morph.), 1.76–1.52 (m, 6H, CH2), 1.42–1.20 (m, 10H, CH2), 0.89

8564
K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 8556–8565
49.94, 38.57, 31.89, 29.60, 29.55, 29.49, 29.31, 29.19, 28.75, 28.31,
28.14, 27.40, 25.98, 23.35, 22.65, 14.07. IR (cmꢀ1): 2919, 2852,
1717, 1694, 1401, 1155, 1117. HR-MS: for C26H46O5N2 [M+H]+
calcd 467.3479 m/z, found 467.3480 m/z.
(PermeGear Inc., USA), with a donor surface area of 0.635 cm2 and
receptor volume of 5.2 mL. The skin was mounted between the do-
nor and receptor compartments of the diffusion cell with the epider-
mal side up. The receptor compartment was filled with phosphate
buffered saline (pH 7.4) and was maintained at 37 0.5 °C through
the use of a circulating water bath. The receptor compartment was
continuously stirred using a magnetic stirring bar. The skin was kept
in contact with the receptor phase for 0.5 h prior to the experiment.
The donor samples were prepared by dissolving the tested enhancer
(20 mg) in PG (0.5 mL) and a solution of theophylline (5 mg) in water
(0.5 mL) was added. This mixture was shaken vigorously and then
sonicated for 10 min at 40 °C. This stable system was applied to
the skin surface and the donor compartment of the cell was covered
by ParafilmÒ. The control samples were prepared in the same man-
ner without enhancers. Samples (0.5 mL) of the receptor phase were
withdrawn at six pre-determined time intervals over 24 h (1, 2, 4, 8,
12 and 24 h) and the cell was refilled with an equivalent amount of
fresh buffer solution. A minimum of three determinations were per-
formed using skin fragments from a minimum of two animals for
each compound. The samples were stored at ꢀ18 °C until analyzed
by HPLC.
4.2. Lipophilicity HPLC determination (capacity factor
k/calculated log k)
An Agilent 1200 series HPLC system was used equipped with a
diode array detection (DAD) system, a quaternary model pump and
an automatic injector (Agilent Technologies, Germany). Data acqui-
sition was performed using ChemStation chromatography soft-
ware. The C18
5
l
m, 4.6 ꢁ 150 mm Zorbax Eclipse XDB
chromatographic column (Agilent Technologies, Germany) was
used for these studies. A mixture of MeOH (HPLC grade, 85.0%)
and H2O (HPLC grade, 15.0%) was used as a mobile phase. The total
flow of the column was 0.4 mL/min. An injection volume of 10 lL
and column temperature of 25 °C were used. A detection wave-
length of 204 nm and the bandwidth of 8 nm were chosen. A KI
methanolic solution was used for dead time (tD) determination.
Retention times (tR) were measured in minutes.
The capacity factors k were calculated using ChemStation chro-
matography software according to the formula k = (tR ꢀ tD)/tD,
where tR is the retention time of the solute, whereas tD denotes
the dead time obtained via an unretained analyte. Log k, calculated
from the capacity factor k, is used as a lipophilicity index converted
to the log P scale.17 The log k values of the individual compounds
are shown in Table 1.
4.5. Sample analysis
Analysis of samples for theophylline content from the transder-
mal penetration-enhancing activity studies above was performed
using an Agilent 1200 series HPLC system. This was equipped with
a diode array detection (DAD) system, quaternary model pump and
automatic injector (Agilent Technologies, Germany). Data acquisi-
tion was performed using ChemStation chromatography software.
4.3. Lipophilicity and geometry calculations
A Waters Symmetry, C8 5
l
m, 4.6 ꢁ 250 mm chromatographic col-
Log P values (i.e., the logarithm of the partition coefficient for
n-octanol/water) were predicted using CS ChemOffice Ultra ver.
10.0 (CambridgeSoft, Cambridge, MA, USA) and ACD/Log P DB soft-
ware (ACD/Labs, ver. 11.01, Advanced Chemistry Development,
Inc., Canada, Toronto, 2007). C log P values (the logarithm of n-oct-
anol/water partition coefficient based on established chemical
interactions) were generated using CS ChemOffice Ultra ver. 10.0
(CambridgeSoft, Cambridge, MA, USA) software. The results are
shown in Table 1.
The initial scan of geometries was performed using the confor-
mational search utility in HyperChem (HyperChem for Windows,
ver. 8.0.3, Hypercube, Inc., Gainesville, 2007). Structures within
2 kcal/mole were then re-optimized in water at HF/4-31G ab initio
level in GAUSSIAN 09 W.26 Water was simulated using the CPCM
polarizable conductor calculation solvation model.27 The results
are illustrated in Figures 1 and 2.
umn was used. A mixture of acetonitrile (HPLC grade, 50.0%) and
H2O (HPLC grade, 50.0%) was used as the mobile phase. The total
flow of the column was 0.5 mL/min, with an injection volume of
10 lL and a column temperature of 25 °C. A detection wavelength
of 280 nm with a bandwidth of 8 nm was chosen. The retention
time (tR) of theophylline was 5.07 0.05 min.
The cumulative amounts of theophylline that penetrated across
the skin into the receptor compartment (
corrected for sample removal, were plotted against time (h). A linear
dependence was found (R2 P 0.98) and steady state fluxes ( g/cm2/
h) were calculated using the linear region of the plots. Enhancement
ratios (ERs) were calculated as the ratio of the flux of theophylline
with and without the enhancer. The results are summarized in
Table 3.
Determination of the penetrated enhancer was performed using
the HPLC method described in Section 4.2. An injection volume of
10 lL was utilized and the samples of the receptor phase with-
l
g/cm2), which were
l
MD simulations were performed in HyperChem using the MM+
0
force field. A periodic box with dimensions a = b = c = 20 AÅ was filled
with solvent molecules using a published method43 and adjusted
using our own code. The resulting system consisted of 15 molecules
of (R)-propylene glycol, 15 molecules of the (S)-enantiomer, 120
molecules of water and a solute molecule. The resulting macroscopic
density was 1.02 g/cm3. MD data were accumulated from a series of
500 ps runs with a 1 fs time step at 298 K within a thermostatically
controlled water bath. Data were collected every 10 steps.
drawn at the mentioned intervals were then analysed.
4.6. Cell culture and in vitro anti-proliferative activity
The human SK-N-MC neuroepithelioma cell type was obtained
from the American Type Culture Collection (Manassas, VA, USA)
and was cultured in minimum essential medium (MEM; Gibco,
Melbourne Australia) containing 10% (v/v) FBS, 1.0 mM sodium
pyruvate (Gibco), 1% (v/v) non-essential amino acids (Gibco),
4.4. In vitro screening of transdermal penetration-enhancing
activity
2 mM
L-glutamine (Gibco), 100 U/mL penicillin (Gibco), streptomy-
cin (Gibco) and 0.28
l
g/mL fungizone (Squibb Pharmaceuticals,
Montreal, Canada). Cells were cultured under standard conditions
at 37 °C, in a humidified atmosphere at 5% CO2.
The effect of the compounds on cellular proliferation were
determined by the MTT [1-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyl tetrazolium] assay using standard techniques.41,42 Previous
studies have demonstrated that MTT reduction is proportional to
Skin samples were obtained from porcine ear. Full thickness dor-
sal skin was cut in fragments and stored at ꢀ20 °C until utilized. Skin
samples were then slowly thawed (at 4 °C overnight and then at
ambient temperature) before each experiment. The penetration-
enhancing activity of newly synthesized compounds 5a–5g and
6a–6g were evaluated in vitro using a vertical Franz diffusion cell

K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 8556–8565
8565
viable cell counts using SK-N-MC cells.44 The SK-N-MC cells were
15. Kim, N.; El-Kattan, A. F.; Asbill, C. S.; Kennette, R. J.; Sowell, J. W.; Latour, R.;
Michniak, B. B. J. Controlled Release 2001, 73, 183.
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der Waterbeemd, H., Eds., 1st ed.; Methods and Principles in Medicinal
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seeded in 96-well microtiter plates at 1.5 ꢁ 104 cells/well in med-
ium containing human diferric transferrin (Tf) at 1.25 lM
([Fe] = 2.5
l
M) and compounds at
a
range of concentrations
(0–25 M). Control samples contained medium with Tf (1.25
l
l
M)
without the compounds. The chelators, DFO and Dp44mT, were
also included as internal controls, as their effects are well charac-
terized in this cell line.41,42 The cells were incubated at 37 °C in a
humidified atmosphere containing 5% CO2 and 95% air for 72 h.
After 72 h, 10
added to each well and incubated for 2 h at 37 °C. After solubiliza-
tion of the cells with 100 L of 10% SDS-50% isobutanol in 0.01 M
lL of MTT solution (stock solution: 5 mg/mL) was
l
HCl, the plates were read at 570 nm using a scanning multi-well
spectrophotometer. The inhibitory concentration (IC50) was de-
fined as the compound concentration necessary to reduce the
absorbance to 50% of the untreated control.
24. Otera, J. Chem. Rev. 1993, 93, 1449. and references cited therein.
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Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.;
Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.;
Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven,
T.; Montgomery, Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.;
Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.;
Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.;
Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.;
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.;
Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.;
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Acknowledgments
This study was supported by IGA VFU Brno 20/2010/FaF and by
the Ministry of Education of the Czech Republic MSM 6215712403,
MSM 6046137305. D.R.R. was supported by a NHMRC Senior
Principal Research Fellowship and Project grants and an ARC Dis-
covery Grant. D.S.K. was supported by a Cancer Institute New
South Wales Early Career Development Fellowship and Innovation
Grant.
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