Synthesis, physico-chemical properties and penetration activity of alkyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrrolidin-1-yl)hexanoates as potential transdermal penetration enhancers

Article abstract of DOI:10.1016/j.bmc.2009.11.020

Skin penetration enhancers are used to allow formulation of transdermal delivery systems for drugs that are otherwise insufficiently skin-permeable. The series of seven esters of substituted 6-aminohexanoic acid as potential transdermal penetration enhanc

Full text of DOI:10.1016/j.bmc.2009.11.020

Bioorganic & Medicinal Chemistry 18 (2010) 73–79
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, physico-chemical properties and penetration activity of
alkyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrrolidin-1-yl)hexanoates as
potential transdermal penetration enhancers
Katerina Brychtova a, , Josef Jampilek a,b, Radka Opatrilova a, Ivan Raich c, Oldrich Farsa a, Jozef Csollei a
*
a Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
b Zentiva k.s., U kabelovny 130, 102 37 Prague, Czech Republic
c Department of Chemistry of Natural Compounds, Faculty of Food and Biochemical Technology, Institute of Chemical Technology, Technicka 5, 166 28 Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 April 2009
Revised 7 September 2009
Accepted 7 November 2009
Available online 12 November 2009
Skin penetration enhancers are used to allow formulation of transdermal delivery systems for drugs that
are otherwise insufficiently skin-permeable. The series of seven esters of substituted 6-aminohexanoic
acid as potential transdermal penetration enhancers was formed by multistep 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 ana-
lyzed using RP-HPLC method for the lipophilicity measurement and their lipophilicity (log k) was deter-
mined. Hydrophobicities (log P/C log P) of the studied compounds were also calculated using two
commercially available programs and 3D structures of the selected compounds were investigated by
means of ab initio/DFT calculations of geometry. All the synthesized esters were tested for their
in vitro transdermal penetration enhancer activity. The relationships between the lipophilicity and the
chemical structure (SLR) of the studied compounds as well as the relationships between their chemical
structure and transdermal penetration activity are mentioned.
Keywords:
Transdermal penetration enhancers
x
-Lactam derivatives
6-Aminohexanoic acid derivatives
Lipophilicity
Ab initio/DFT calculations
3D structures
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
tion enhancers and a number of potential sites and modes of action
were identified for them.3–5 In spite of the extensive research in
A transdermal route offers several advantages compared with
other routes for delivery of drugs with systemic activity. The advan-
tages include elimination of hepatic first-pass metabolism, easy use
and withdrawal in case of side effects, and continuous non-invasive
infusion of drugs having short biological half-times. However, the
outermost layer of skin, stratum corneum (SC), forms a strong barrier
for most of exogenous substances including drugs. The barrier func-
tion of the SC is attributed to its multilayered wall-like structure, in
which terminally differentiated keratin-rich epidermal cells (cor-
neocytes) are embedded in an intercellular lipid-rich matrix. Several
technological advances have been made in the recent decades to
overcome skin barrier properties. Examples include physical means
such as iontophoresis, sonophoresis, microneedles; chemical means
using penetration enhancers, and biochemical means such as lipo-
somal vesicles and enzyme inhibition.1,2
this field, chemical penetration enhancers have not reached their
full potential in transdermal or topical systems so far.
The penetration enhancers are generally classified as mem-
brane-acting types or co-solvent types. The membrane-acting
types achieve effects using small amounts by altering the condition
of the lipid membrane constituting the stratum corneum. The co-
solvent types, such as propylene glycol (PG), polyethylene glycol
and ethanol, achieve effects by improving the solubility of drugs
or enhancing permeation of drugs through the skin.6 It has been
suggested that the mechanisms by which these enhancers affect
skin permeability are (a) disruption of the ordered lipid bilayer
structures and eventual fluidization of the lipid environment of
the stratum corneum, (b) interaction with intracellular proteins of
the stratum corneum, and (c) improvement of partitioning and sol-
ubility of the drug in the stratum corneum.3,7,8
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 the topical dosage forms to blood circulation. Numerous com-
pounds (different chemical structures) were evaluated as penetra-
Besides the use of chemical penetration enhancers also skin
occlusion often, but not always, increases percutaneous absorption
of drugs. The occlusion of skin substantially changes many proper-
ties of the skin, including hydration, permeability of the skin bar-
rier to some but not all exogenous chemicals, epidermal lipid
composition, DNA synthesis, microbial flora, and other molecular
and cellular processes.9 The occlusion also increases penetration
of other chemicals and antigens, and hence may exacerbate irritant
* Corresponding author. Tel.: +420 5 41562924.
E-mail address: brychtovak@vfu.cz (K. Brychtova).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.020

74
K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 73–79
and allergic reactions.10 Also drug delivery from many transdermal
patches benefits from occlusion.
the mobile phase using end-capped non-polar C18 stationary RP
columns and calculating the capacity factor k. Log k, calculated
from the capacity factor k, is used as a lipophilicity index converted
to the log P scale.22
The multistep synthesis of seven alkyl-6-(2,5-dioxopyrrolidin-
1-yl)-2-(2-oxopyrrolidin-1-yl)hexanoates with C6–C12 linear alkyl
ester chains is described here. The lipophilicity (log k) of the com-
pounds was determined using RP-HPLC. Primary in vitro screening
of transdermal penetration activity of all synthesized esters was
evaluated using a Franz cell.23 The geometry of two selected com-
pounds was investigated by means of ab initio/DFT calculations,
and 3D structures are presented. The structure–activity relation-
ships of the studied compounds are discussed in this study.
Some of the important penetration enhancers are terpenes and
terpenoids, pyrrolidinones, fatty acids and esters, sulfoxides, alco-
hols and glycerides and miscellaneous enhancers including phos-
pholipids, cyclodextrin complexes, amino acid derivatives, lipid
synthesis inhibitors, clofibric acid, dodecyl-N,N-dimethylamino
acetate and enzymes.11 1-Dodecylazacycloheptan-2-one (AzoneÒ)
is one of the most studied penetration enhancers possessing a large
polar head group and a lipid alkyl chain which are thought to bee
necessary for its activity, see Figure 1. As it could be expected from
its chemical structure, AzoneÒ is a highly lipophilic material with
log P around 6.2. It enhances the skin transport of a wide variety
of drugs with different lipophilicity. Its possible mechanism of ac-
tion has been previously reported.3,12,13 The chemical structure of
AzoneÒ was considered to be a combination of pyrrolidone and
decylmethylsulfoxide, both of which are potential penetration
enhancers.14 To date, many Azone related compounds (including
modifications of heterocyclic moiety or/and lipophilic chain) have
been made and studied as penetration enhancers. 6-Aminohexa-
noic acid esters were also designed as acyclic Azone analogs15
and fragments of pyrrolidin-2,5-dione and pyrrolidin-2-one are
parts of described potent penetration enhancers as well.16
The traditional lipophilicity parameter log P is a well-known
physico-chemical descriptor largely used in QSAR analyses. In
some experimental studies of penetration enhancement the lipo-
philicity (non-polarity) of enhancers was measured and the corre-
sponding relationship between enhancer non-polarity and
penetration enhancement potency was investigated.17–21
The lipophilicity is one of the most important physical proper-
ties both for biologically active compounds and for excipients.
Classical methods for determination of the partition (log P) coeffi-
cient are time consuming and not always sufficiently reliable.
Therefore, reversed phase high performance liquid chromatogra-
phy (RP-HPLC) methods have become popular and widely used
for lipophilicity measurement. The general procedure is measure-
ment of directly accessible retention time under isocratic condi-
tions with various amounts of an organic modifier (methanol) in
2. Results and discussion
2.1. Chemistry
6-Aminohexanoic acid and succinic anhydride were used as the
starting materials for multistep synthesis, and by their reaction 6-
(2,5-dioxopyrrolidin-1-yl)hexanoic acid (1) was obtained. Under
the optimized Schwenk and Papa procedure24,25 acid 1 in the
one-pot synthesis gave ethyl-2-bromo-6-(2,5-dioxopyrrolidin-1-
yl)hexanoate (2).
The critical step of the synthesis was the C–N nucleophilic
coupling of pyrrolidin-2-one and ethyl-2-bromo-6-(2,5-dioxopyrr-
olidin-1-yl)hexanoate (2). This C–N bond-forming reaction of
a-bromocarboxylic compound 2 and a 5-membered x-lactam ring
was carried out under catalysis by powdered copper(I) oxide, and
ethyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrrolidin-1-yl)hexa-
noate (3) was obtained. Various conditions and yields of the C–N
coupling of non-aromatic N-heterocycle starting materials were re-
ported recently.26
The series of seven target alkyl-6-(2,5-dioxopyrrolidin-1-yl)-2-
(2-oxopyrrolidin-1-yl)hexanoates (4a–4g) was formed by conven-
tional base-catalyzed transesterification27 of the key intermediate
3 in excess of corresponding primary unbranched alcohol. The dis-
cussed compounds were synthesized according to Scheme 1.
2.2. Lipophilicity properties of the prepared compounds
N
Hydrophobicities (log P/C log P values) of the studied com-
pounds 3, 4a–4g were calculated using two commercially available
programmes (ChemDraw Ultra 10.0 and ACD/LogP DB) and mea-
sured by means of RP-HPLC determination of capacity factors k
Azone®
O
Figure 1. 1-Dodecylazacycloheptan-2-one or N-dodecylcaprolactam (AzoneÒ).
Br
O
O
O
O
OH
OH
a
b
N
+
N
H2N
O
O
O
O
O
2
O
O
1
O
c
HN
R = -C6H13 (4a)
-C7H15 (4b)
-C8H17 (4c)
-C9H19 (4d)
-C10H21 (4e)
-C11H23 (4f)
-C12H25 (4g)
O
O
N
N
O
O
d
O
O
N
R
N
O
O
O
O
3
4a-g
Scheme 1. Synthesis of target
x-lactams 4a–4g. Reagents and conditions: (a) acetone, 25 °C, 24 h; (b) one-pot synthesis: SOCl2, Br2, EtOH; (c) NaH, DMF, Cu2O; (d) Na, R–OH.

K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 73–79
75
with subsequent calculation of log k. The results are shown in
Table 1.
Of course, many of them would be prohibited due to close contacts
of some atoms. Anyway, the number of possible conformations
precludes the use of systematic mapping of conformational space.
Calculations with an explicit solvent or even a solvent mixture are
generally not feasible at ab initio level and the dielectric contin-
uum solvation model29 must be used. It is not so sensitive to the
dielectric constant and thus water as a dielectric medium is a good
approximation for geometry optimizations. The initial scan of
geometries was performed using the Conformational Search utility
in HyperChem and corresponds to vacuo. 39,682 and 30,214 initial
conformations were explored for compounds 4a and 4g, respec-
tively. Structures within 3 kcal/mol were then reoptimized in
water at HF/4-31G ab initio level in GAUSSIAN 03W.30,31 For both
compounds this resulted in more than 100 possible conformations.
Global optima for S-enantiomers of compounds 4a and 4g are illus-
trated in Figures 2 and 3, respectively. Used S-configuration does
not influence the predicted properties. However, their populations
would be no more than 1%. According to Figures 2 and 3 it can be
assumed, that all the presented compounds show folded confor-
mation, that is, all the compounds represent coned (coiled)
molecules.
As expected, ethyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrr-
olidin-1-yl)hexanoate (3) showed the lowest lipophilicity, whereas
dodecyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrrolidin-1-yl)hex-
anoate (4g) possessed the highest lipophilicity. It can be assumed,
that the calculated log P/C log P data and the determined log k val-
ues correspond to the expected lipophilicity increasing within the
series of the evaluated compounds (ethyl n hexyl < hep-
tyl < nonyl < decyl < undecyl < dodecyl derivatives). This depen-
dence is approximately linear. Log k data specify lipophilicity
within this series of the discussed compounds.
2.3. Geometries—3D structures
The chemical structure as well as exact 3D geometries of the
studied penetration enhancers under study influence their phys-
ico-chemical properties and thus moderate their penetration activ-
ity. Furthermore, they are also used for the derivation of a series of
structural descriptors like molecular dimensions, volume and sur-
face, dipole moment etc., which are often used for in silico screen-
ing and design of new penetration enhancers.28 For this reason,
structures of compounds 4a and 4g as the shortest and the longest
potential enhancers within the series were investigated. Unfortu-
nately, the determination of 3D structures of both compounds
was a complicated task due to their large conformational flexibility
and complex media. If only three orientations were taken into ac-
count for each torsion in the molecule, the number of initial con-
formations would be 1,594,323 and 1,162,261,467, respectively.
2.4. In vitro screening of penetration enhancing activity
The enhancement activity of the prepared compounds was eval-
uated using theophylline as a model penetrant and propylene gly-
col/water 1:1 v/v as a donor vehicle. Theophylline was used as a
model drug of medium polarity (log P À0.06; log D8 À0.05)32,33
as it has been extensively studied in transdermal penetration
experiments.34–36 Most of the studies involved the use of propyl-
ene glycol (PG) or its mixture with water or ethanol as a donor
vehicle. Previous works has 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.37–39 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.40,41 The porcine
skin has shown to be histologically and biochemically similar to
the human skin. The same as full-thickness pig ear skin it was used
in numerous percutaneous absorption studies.42 Nevertheless for
testing of hydrophobic penetrants dermatomated skin is highly
recommended.43 The skin permeation experiments were performed
Table 1
Comparison of calculated lipophilicities (log P/C log P) with determined log k values
Compd
Log k
Log P/C log P ChemOffice
Log P ACD/LogP DB
3
4a
4b
4c
4d
4e
4f
4g
À1.0629
À0.3005
À0.1314
0.0349
0.2013
0.3640
0.5285
0.6906
À0.39/0.1972
1.35/2.3132
1.77/2.8422
2.19/3.3371
2.60/3.9002
3.02/4.4292
3.44/4.9582
3.86/5.4872
6.29/7.7860
7.07/6.5400
–0.16 0.56
1.88 0.56
2.39 0.56
2.90 0.56
3.41 0.56
3.92 0.56
4.43 0.56
4.94 0.56
7.42 0.20
7.82 0.35
a
Oleic acid
—
—
AzoneÒ
a
Log k value was not determined by the described method.
Figure 2. 3D structure of S-enantiomer of hexyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-
oxopyrrolidin-1-yl)hexanoate (4a).
Figure 3. 3D structure of S-enantiomer of dodecyl-6-(2,5-dioxopyrrolidin-1-yl)-2-
(2-oxopyrrolidin-1-yl)hexanoate (4g).

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K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 73–79
using static Franz diffusion cells.23 Generally, the use of static cells
would yield the same results as the use of flow-through systems
when it is not necessary to maintain skin viability. The effect of
the perfusion rate in the receptor medium has shown to influence
results particularly if a permeant is very lipophilic in nature.44–46
The effect of alkyl esters 4a–4g on the penetration of theophyl-
line through the porcine skin is presented in Figure 4. All prepared
compounds 4a–4g enhanced the penetration of theophylline
through the porcine skin and showed better enhancement ratios
(ERs) than oleic acid, which was used for comparison and belongs
to the most studied enhancers of the group of fatty acids.47 Control
experiments were run with only theophylline in donor vehicle and
without any enhancer. The highest enhancement ratios (ERs) were
obtained with compounds 4d (nonyl) with ER 2.45 and 4g (dode-
cyl) with ER 2.38. The ERs are presented in Table 2.
4a and 4b, which were detected in the receptor phase in 24 h.
The action mechanism seems to be in the interaction of enhancers
with intercellular lipids of the stratum corneum.
4. Experimental
4.1. Chemistry
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. 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 vapor. The melting points were determined on a Boetius
PHMK apparatus (Nagema, Germany) and are uncorrected. Infrared
(IR) spectra were recorded on a Smart MIRacle™ ATR ZnSe for
Nicolet™ 6700 FT-IR spectrometer (Thermo Scientific, USA). The
spectra were obtained by accumulation of 256 scans with 2 cmÀ1
resolution in the region of 4000–600 cmÀ1. All 1H and 13C NMR
spectra were recorded on a Bruker Avance-500 FT-NMR spectrom-
eter (500 MHz for 1H and 125 MHz for 13C, Bruker Comp., Kar-
lsruhe, Germany). Chemicals shifts are reported in ppm (d) to
internal Si(CH3)4, when diffused easily exchangeable signals are
omitted. Mass spectra were measured using a LTQ Orbitrap Hybrid
Mass spectrometer (Thermo Electron Corporation, USA) with direct
injection into APCI source (400 °C) in the positive mode.
The penetration of the enhancers through the skin was further
investigated. Only compounds 4a and 4b were detected in the
receptor compartment after 24 h since the start of the experiment.
All other alkyl esters 4c–4g were not detected, and it was assumed
that due to their higher lipophilicity they did not get through the
skin barrier or remained incorporated in the skin structures.
3. Conclusion
In this work a series of seven alkyl-6-(2,5-dioxopyrrolidin-1-
yl)-2-(2-oxopyrrolidin-1-yl)hexanoates with C6–C12 linear alkyl
ester chain as potential transdermal penetration enhancers was
prepared. The ability of these compounds to enhance the penetra-
tion of theophylline through porcine skin was tested. All prepared
compounds showed higher ERs than oleic acid. The highest
enhancement ratios (ERs) in this study were exhibited by com-
pound 4d (C9 ester chain) and 4g (C12 ester chain). The prepared
compounds did not get through the skin, except for compounds
4.1.1. 6-(2,5-Dioxopyrrolidin-1-yl)hexanoic acid (1)
The solution of succinic anhydride (45.0 g, 450.0 mmol) in ace-
tone (230 mL) was added dropwise to the suspension of 6-amino-
hexanoic acid (34.4 g, 262.0 mmol) in acetone (140 mL). The
reaction mixture was stirred at room temperature for 24 h, after
which it was filtered and a pure white powder product was washed
with acetone. Yield: 82%. Mp 100–102 °C. IR (cmÀ1): 3315, 2929,
1688, 1560, 1414, 1250, 1183. 1H NMR (500 MHz, DMSO-d6), d:
12.05 (s, 1H, OH), 3.02 (t, 2H, J = 7.0 Hz, NCH2), 2.90 (s, 4H,
OCCH2CH2CO), 2.41 (t, 2H, J = 6.0 Hz, OOCCH2), 2.32–2.15 (m, 4H,
CH2), 1.52–1.24 (m, 2H, CH2). 13C NMR (125 MHz, DMSO-d6), d:
174.25, 173.66, 38.27, 33.52, 28.72, 28.62, 25.83, 24.11. HR-MS:
for C10H16O4N [M+H]+ calcd 214.2378 m/z, found 214.2377 m/z.
3.00
2.50
2.00
1.50
1.00
0.50
0.00
4.1.2. Ethyl-2-bromo-6-(2,5-dioxopyrrolidin-1-yl)hexanoate (2)
To organic acid 1 (45.8 g, 214.8 mmol), held at 30 °C, thionyl
chloride (29.4 g, 247.0 mmol, 17.9 mL) was added slowly dropwise
and the mixture was stirred at 60–80 °C until the gas evolution
essentially stopped. At 80 °C Br2 (36.1 g, 225.5 mmol, 11.6 mL)
was added dropwise at approximately the rate, at which Br2 was
consumed. Stirring continued for several hours until the evolution
of HBr nearly stopped. Absolute ethanol (27 mL) was added slowly
to the crude acid chloride at 20–30 °C. After stirring overnight, the
mixture was evaporated until dry in a vacuum and the residue was
dissolved in diethyl ether (50 mL). The solution was washed with
dilute NaHSO3 and water, the organic layer was dried over anhy-
drous MgSO4, filtered and the organic solvent was removed under
rotary evaporation. The crude product (yield: 94%) was purified by
vacuum distillation using a Vigreux column (bp 160–165 °C/
0.35 mbar) to yield 81% of a colourless oil. RF 0.44 (propan-2-ol
100%). IR (cmÀ1): 2939, 1730, 1692, 1436, 1399, 1143, 818. 1H
NMR (500 MHz, CDCl3), d: 4.16 (q, 2H, J = 7.0 Hz, OCH2), 4.11 (t,
1H, J = 7.3 Hz, BrCH), 3.44 (t, 2H, J = 7.2 Hz, NCH2), 2.64 (s, 4H,
OCCH2CH2CO), 1.99 (q, 2H, J = 7.3 Hz, CHCH2), 1.54 (qi, 2H,
J = 7.0 Hz, CH2), 1.43–1.15(m, 2H, CH2), 1.23 (t, 3H, J = 7.1 Hz,
CH3). 13C NMR (125 MHz, CDCl3), d: 176.94, 169.43, 61.84, 45.63,
38.23, 34.17, 28.08, 26.77, 24.43, 13.82. HR-MS: for C12H19O4NBr
[M+H]+ calcd 320.0492 m/z, found 320.0491 m/z.
control OA 4a
4b
4c
4d
4e
4f
4g
Compounds
Figure 4. Enhancement ratios (ERs) of the prepared compounds 4a–4g and oleic
acid (OA), control is with theophylline only without any enhancer. Error bars
correspond to the standard deviation of three experiments.
Table 2
Enhancement ratios (ERs) of the prepared compounds 4a–4g and oleic acid (OA), data
are expressed as means SD (n = 3)
Compd
ER SD
4a
4b
4c
4d
4e
4f
2.15 0.19
1.86 0.35
1.60 0.35
2.45 0.24
1.56 0.34
1.86 0.27
2.38 0.15
1.13 0.18
4g
OA

K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 73–79
77
4.1.3. Ethyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrrolidin-1-
4.1.4.3. Octyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrrolidin-
yl)hexanoate (3)
1-yl)hexanoate (4c).
Light yellow oil. Yield 62%. RF 0.25 (ethyl
Pyrrolidin-2-one (4.0 g, 46.9 mmol) was added slowly to the
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 evolution of hydrogen gas stopped. Compound 3 (10.0 g,
31.2 mmol) and Cu2O (1.1 g, 7.8 mmol) were then added, and the
mixture was refluxed 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
anhydrous MgSO4, filtered and the organic solvent was removed
under rotary evaporation. The crude product was purified by flash
chromatography on silica gel (ethyl acetate/petroleum ether 3:1)
provided a light yellow oil. Yield 66%. RF 0.45 (propan-2-ol 100%).
IR (cmÀ1): 2927, 1767, 1687, 1401, 1284, 1187, 1153, 1027. 1H
NMR (500 MHz, CDCl3), d: 4.66 (dd, 1H, J1 = 5.0 Hz, J2 = 10.6 Hz,
CH), 4.16 (q, 2H, J = 7.1 Hz, OCH2), 3.50 (t, 2H, J = 7.2 Hz, NCH2),
3.54–3.29 (m, 2H, CH2pyrr.), 2.70 (s, 4H, OCCH2CH2CO), 2.42 (t,
2H, J = 8.0 Hz, CH2pyrr.), 2.17–1.95 (m, 2H, CH2pyrr. and 1H from
CH2–CH), 1.78–1.56 (m, 2H, CH2 and 1H from CH2–CH), 1.34–
1.28 (m, 2H, CH2), 1.26 (t, 3H, J = 7.1 Hz, CH3). 13C NMR
(125 MHz, CDCl3), d: 177.13, 175.78, 170.76, 61.12, 53.51, 43.53,
38.26, 30.73, 28.08, 27.03, 23.35, 18.21, 14.07. HR-MS: for
C16H25O5N2 [M+H]+ calcd 325.1758 m/z, found 325.1757 m/z.
acetate/petroleum ether 5:1). IR (cmÀ1): 3380, 1688, 1592, 1512,
1432, 1320, 1160, 1096, 984, 824, 632. 1H NMR (500 MHz, CDCl3),
d: 4.71 (dd, J1 = 10.74 Hz, J2 = 4.92 Hz, 1H, CH), 4.06 (t, J = 6.67 Hz,
2H, COOCH2), 3.47 (t, J = 7.23 Hz, 2H, NCH2), 3.39 (dq,
J1 = 15.34 Hz, J2 = 7.7 Hz, 2H, CH2pyrr.), 2.69 (s, 4H, COCH2), 2.41
(t, J = 7.90 Hz, 2H, CH2pyrr.), 2.11–1.86 (m, 4H, CH2pyrr., CHCH2),
1.76–1.48 (m, 4H, CH2), 1.33–1.20 (m, 12H, CH2), 0.86 (t,
J = 6.40 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.13,
175.68, 170.87, 65.33, 54.45, 53.59, 43.57, 38.33, 31.70, 30.77,
29.08, 28.50, 28.19, 28.13, 27.10, 25.82, 23.42, 22.56, 18.27,
13.99. HR-MS: for C22H37O5N2 [M+H]+ calcd 409.2697 m/z, found
409.2696 m/z.
4.1.4.4. Nonyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrrolidin-
1-yl)hexanoate (4d).
Light yellow oil. Yield 55%. RF 0.25
(ethyl acetate/petroleum ether 5:1). IR (cmÀ1): 3380, 3020, 1816,
1736, 1672, 1592, 1528, 1432, 1352, 1288, 1192, 1160, 1032,
856, 824, 728, 632. 1H NMR (500 MHz, CDCl3), d: 4.71 (dd,
J1 = 10.78 Hz, J2 = 4.92 Hz, 1H, CH), 4.07 (t, J = 6.72 Hz, 2H,
COOCH2), 3.48 (t, J = 7.24 Hz, 2H, NCH2), 3.39 (dq, J1 = 15.34 Hz,
J2 = 7.7 Hz, 2H, CH2pyrr.), 2.69 (s, 4H, COCH2), 2.41 (t, J = 8.11 Hz,
2H, CH2pyrr.), 2.12–1.86 (m, 4H, CH2pyrr., CHCH2), 1.79–1.52 (m,
4H, CH2), 1.33–1.21 (m, 14H, CH2), 0.86 (t, J = 6.40 Hz, 3H, CH3).
13C NMR (125 MHz, CDCl3), d: 177.21, 175.75, 170.89, 65.36,
53.54, 43.54, 38.33, 31.79, 30.79, 29.41, 29.17, 28.48, 28.14,
27.11, 25.82, 23.42, 22.61, 18.26, 14.06. HR-MS: for C23H39O5N2
[M+H]+ calcd 423.2853 m/z, found 423.2854 m/z.
4.1.4. General procedure for preparation alkyl-6-(2,5-
dioxopyrrolidin-1-yl)-2-(2-oxopyrrolidin-1-yl)hexanoates (4a–g)
The mixture of ethyl ester 3 (7.7 mmol), appropriate primary
alcohol (38.5 mmol) and metallic sodium (3.85 mmol) was stirred
at 90 °C in the oil bath until sodium was dissolved completely, then
the mixture was heated at 130 °C for 5–7 h and during the reac-
tion ethanol was distilled off as formed. The excess of longer-chain
alkyl alcohol was distilled off under reduced pressure and the
rest was extracted with acetic acid (0.5 M) and diethylether,
organic layer was dried over anhydrous MgSO4, filtered and evap-
orated. The crude product was purified by column chromatography
on silica gel using ethyl acetate/petroleum ether (5:1) as the
eluent.
4.1.4.5. Decyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrrolidin-
1-yl)hexanoate (4e).
Light yellow oil. Yield 58%. RF 0.23 (ethyl
acetate/petroleum ether 5:1). IR (cmÀ1): 3380, 2980, 1752, 1688,
1592, 1432, 1272, 1160, 1080, 840, 728. 1H NMR (500 MHz, CDCl3),
d: 4.69 (dd, J1 = 10.71 Hz, J2 = 4.94 Hz, 1H, CH), 4.05 (t, J = 6.59 Hz,
2H, COOCH2), 3.45 (t, J = 7.22 Hz, 2H, NCH2), 3.37 (dq,
J1 = 15.39 Hz, J2 = 9.27 Hz, 2H, CH2pyrr.), 2.67 (s, 4H, COCH2), 2.38
(t, J = 7.98 Hz, 2H, CH2pyrr.), 2.09–1.84 (m, 4H, CH2pyrr., CHCH2),
1.77–1.50 (m, 4H, CH2), 1.31–1.15 (m, 16H, CH2), 0.84 (t,
J = 6.40 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.09,
175.65, 170.85, 65.31, 53.58, 43.56, 38.31, 31.80, 30.75, 29.43,
29.20, 29.11, 28.49, 28.12, 27.08, 25.80, 23.40, 22.58, 18.26,
13.99. HR-MS: for C24H41O5N2 [M+H]+ calcd 437.3010 m/z, found
437.3010 m/z.
4.1.4.1. Hexyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrrolidin-
1-yl)hexanoate (4a).
Light yellow oil. Yield 62%. RF 0.27
(ethyl acetate/petroleum ether 5:1). IR (cmÀ1): 3300, 2980, 1848,
1736, 1688, 1592, 1448, 1336, 1272, 1192, 1096, 920, 712, 632.
1H NMR (500 MHz, CDCl3), d: 4.70 (dd, J1 = 10.61 Hz, J2 = 4.79 Hz,
1H, CH), 4.06 (t, J = 6.61 Hz, 2H, COOCH2), 3.52–3.24 (m, 2H,
CH2pyrr.), 3.46 (t, J = 6.98 Hz, 2H, NCH2), 2.68 (s, 4H, COCH2),
2.40 (t, J = 7.95 Hz, 2H, CH2pyrr.), 2.15–1.85 (m, 4H, CH2pyrr.,
CHCH2), 1.77–1.51 (m, 4H, CH2), 1.35–1.18 (m, 8H, CH2), 0.86 (t,
J = 6.15 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.23,
175.76, 170.82, 65.33, 53.55, 43.56, 38.28, 31.34, 30.75, 28.50,
28.39, 28.10, 27.05, 25.43, 22.42, 20.63, 18.21, 13.89. HR-MS: for
C20H33O5N2 [M+H]+ calcd 381.2384 m/z, found 381.2385 m/z.
4.1.4.6. Undecyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrroli-
din-1-yl)hexanoate (4f).
Light yellow oil. Yield 54%. RF 0.24
(ethyl acetate/petroleum ether 7:1). IR (cmÀ1): 3379, 3020, 1736,
1672, 1592, 1528, 1432, 1352, 1288, 1192, 1160, 1032, 824, 632.
1H NMR (500 MHz, CDCl3), d: 4.70 (dd, J1 = 10.70 Hz, J2 = 4.96 Hz,
1H, CH), 4.06 (t, J = 6.57 Hz, 2H, COOCH2), 3.53–3.24 (m, 2H,
CH2pyrr.), 3.47 (t, J = 7.22 Hz, 2H, NCH2), 2.68 (s, 4H, COCH2),
2.40 (t, J = 7.90 Hz, 2H, CH2pyrr.), 2.11–1.78 (m, 4H, CH2pyrr.,
CHCH2), 1.74–1.52(m, 4H, CH2),1.33–1.17 (m, 18H), 0.86 (t,
J = 6.45 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.05,
175.60, 170.89, 65.33, 53.67, 43.62, 38.36, 31.86, 30.78, 29.53,
29.45, 29.25, 29.15, 28.56, 28.25, 28.15, 27.13, 25.85, 23.45,
22.61, 18.31, 13.99. HR-MS: for C25H43O5N2 [M+H]+ calcd
451.3166 m/z, found 451.3168 m/z.
4.1.4.2. Heptyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrrolidin-
1-yl)hexanoate (4b).
Light yellow oil. Yield 59%. RF 0.28 (ethyl
acetate/petroleum ether 5:1). IR (cmÀ1): 3380, 3020, 1768, 1736,
1672, 1640, 1432, 1352, 1192, 1160, 1032, 808, 632. 1H NMR
(500 MHz, CDCl3), d: 4.67 (dd, J1 = 10.70 Hz, J2 = 4.97 Hz, 1H, CH),
4.03 (t, J = 6.59 Hz, 2H, COOCH2), 3.44 (t, J = 7.22 Hz, 2H, NCH2),
3.36 (dq, J1 = 15.40 Hz, J2 = 9.27 Hz, 2H, CH2pyrr.), 2.65 (s, 4H,
COCH2), 2.37 (t, J = 7.84 Hz, 2H, CH2pyrr.), 2.08–1.82 (m, 4H,
CH2pyrr., CHCH2), 1.75–1.45 (m, 4H, CH2), 1.30–1.19 (m, 10H, CH2),
0.83 (t, J = 6.60 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d: 177.13,
175.66, 170.78, 65.25, 53.46, 43.47, 38.22, 31.53, 30.69, 28.69,
28.38, 28.05, 27.00, 25.68, 23.31, 22.41, 18.16, 13.90. HR-MS: for
C21H35O5N2 [M+H]+ calcd 395.2540 m/z, found 395.2542 m/z.
4.1.4.7. Dodecyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrroli-
din-1-yl)hexanoate (4g).
Light yellow oil. Yield 56%. RF 0.24
(ethyl acetate/petroleum ether 7:1). IR (cmÀ1): 3380, 2940, 1752,
1672, 1624, 1592, 1432, 1272, 1192, 1160, 1016, 952, 904, 824,
744, 648. 1H NMR (500 MHz, CDCl3), d: 4.71 (dd, J1 = 10.77 Hz,
J2 = 4.84 Hz, 1H, CH), 4.06 (t, J = 6.72 Hz, 2H, COOCH2), 3.53–3.24

78
K. Brychtova et al. / Bioorg. Med. Chem. 18 (2010) 73–79
(m, 2H, CH2pyrr.), 3.47 (t, J = 7.25 Hz, 2H, NCH2), 2.69 (s, 4H,
COCH2), 2.40 (t, J = 7.97 Hz, 2H, CH2pyrr.), 2.11–1.86 (m, 4H,
CH2pyrr., CHCH2), 1.78–1.50 (m, 4H, CH2), 1.40–1.15 (m, 20H),
0.86 (t, J = 6.35 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3), d:
177.08, 175.64, 170.87, 65.34, 53.65, 43.62, 38.35, 35.35, 31.87,
30.78, 29.58, 29.53, 29.46, 29.28, 29.15, 28.55, 28.23, 28.15,
27.12, 25.85, 23.44, 22.62, 18.31, 14.01. HR-MS: for C26H45O5N2
[M+H]+ calcd 465.3323 m/z, found 465.3323 m/z.
stirred using magnetic stirring bar. The skin was kept in the contact
with receptor phase for 0.5 h prior to experiment. The donor sam-
ples were prepared by dissolving of tested enhancer (20 mg) in PG
(0.5 mL) and the solution of theophylline (5 mg) in water (0.5 mL)
was added. This mixture was shaken vigorously and then sonicated
for 10 minutes at 40 °C, than this stable system was applicated on
the skin surface, and the donor compartment of the cell was cov-
ered by ParafilmÒ. The control samples were prepared likewise
without enhancers. Samples (0.5 mL) of the receptor phase were
withdrawn at six predetermined 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. Minimal three determinations for each
compound were performed using skin fragments from minimal
two animals for each compound. The samples were stored at
À18 °C until HPLC analysis.
4.2. Lipophilicity HPLC determination (capacity factor k/
calculated log k)
The HPLC separation system Agilent 1200 series instrument was
used, equipped with a diode array detection (DAD) system, a quar-
ternary model pump, and an automatic injector (Agilent Technolo-
gies, Germany). Data acquisition was performed using the
ChemStation chromatography software. The chromatographic col-
umn Zorbax Eclipse XDB (Agilent Technologies, Germany), C18
4.5. Sample analysis
5
l
m, 4.6 Â 150 mm, was used. The 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, injection 10 L, col-
Analysis of samples for theophylline content was performed
using the HPLC system Agilent 1200 series instrument, equipped
with a diode array detection (DAD) system, quarternary model
pump, and automatic injector (Agilent Technologies, Germany).
Data acquisition was performed using the ChemStation chroma-
tography software. The chromatographic column Waters Symme-
l
umn temperature 25 °C. The detection wavelength of 204 nm and
the bandwidth of 8 nm were chosen. The KI methanolic solution
was used for dead time (tD) determination. Retention times (tR)
were measured in minutes.
The capacity factors k were calculated using the ChemStation
chromatography software according to the formula k = (tR À 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.22 The log k values of the individual compounds
are shown in Table 1.
try, C8
5
l
m, 4.6 mm  250 mm, was used. The mixture of
Acetonitrile-HPLC grade (50.0%) and H2O-HPLC grade (50.0%) was
used as a mobile phase. The total flow of the column was 0.5 mL/
min, injection 10 lL, column temperature 25 °C. The detection
wavelength of 280 nm, the bandwidth of 8 nm, was chosen. The
retention time (tR) of theophylline was 5.07 0.05 min.
The cumulative amounts of theophylline penetrated across the
skin into receptor compartment (
moval were plotted against time (hours). An approximately linear
l
g/cm2) corrected for sample re-
4.3. Lipophilicity and geometry calculations
dependence was found (R2 P 0.98) and steady state fluxes (
lg/
cm2/h) were calculated from the linear region of the plots.
Enhancement ratios (ERs) were calculated as the ratio of the flux
of theophylline with and without the enhancer.
Log P values, that is, the logarithm of the partition coefficient for
n-octanol/water, were predicted using the CS ChemOffice Ultra ver.
10.0 (CambridgeSoft, Cambridge, MA, USA) and the ACD/LogP DB
software (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 by means of the CS ChemOffice Ultra
ver. 10.0 (CambridgeSoft, Cambridge, MA, USA) software. The re-
sults are shown in Table 1.
The results are summarized in Table 2.
Determination of the penetrated enhancer was performed using
HPLC method described in Section 4.2, injection 10 lL, and the
samples of the receptor phase withdrawn at the mentioned inter-
vals were analyzed.
Acknowledgments
The initial scan of geometries was performed using the Confor-
mational Search utility in the HyperChem (HyperChem for Win-
dows, ver. 8.0.3, Hypercube, Inc., Gainesville, 2007). Structures
within 3 kcal/mol were then reoptimized in water at HF/4-31G
ab initio level in GAUSSIAN 03W.30 Water was simulated using the
CPCM polarizable conductor calculation solvation model.31 The re-
sults are illustrated in Figures 2 and 3.
This study was supported by IGA VFU 128/2008/FaF and by the
Ministry of Education of the Czech Republic MSM 6215712403 and
MSM 6046137305.
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