Synthesis and transdermal permeation-enhancing activity of ketone, amide, and alkane analogs of Transkarbam 12

Article abstract of DOI:10.1016/j.bmc.2005.12.006

Transkarbam 12 (5-(dodecyloxycarbonyl)pentylammonium-5-(dodecyloxycarbonyl) pentylcarbamate, T12) is a highly active transdermal permeation enhancer. In this study, ketone, amide, and alkane analogs of T12 have been synthesized and evaluated for their permeation-enhancing activity using porcine skin and theophylline as a model drug. Replacement of ester by methylene and ketone, respectively, led to a significant decrease of activity. Amide analogs displayed lower activity in 60% propylene glycol and were comparable to T12 in isopropyl myristate. An intramolecular H-bond between ester and ammonium-carbamate group was suggested to be important for the permeation-enhancing activity of T12.

Full text of DOI:10.1016/j.bmc.2005.12.006

Bioorganic & Medicinal Chemistry 14 (2006) 2896–2903
Synthesis and transdermal permeation-enhancing activity
of ketone, amide, and alkane analogs of Transkarbam 12
*
´
´
´
´
´
Tomasˇ Holas, Katerˇina Vavrova, Jana Klimentova and Alexandr Hrabalek
Faculty of Pharmacy, Charles University, Heyrovske´ho 1203, 50005 Hradec Kra´love´, Czech Republic
Received 17 October 2005; revised 1 December 2005; accepted 2 December 2005
Available online 11 January 2006
Abstract—Transkarbam 12 (5-(dodecyloxycarbonyl)pentylammonium-5-(dodecyloxycarbonyl)pentylcarbamate, T12) is a highly
active transdermal permeation enhancer. In this study, ketone, amide, and alkane analogs of T12 have been synthesized and
evaluated for their permeation-enhancing activity using porcine skin and theophylline as a model drug. Replacement of ester by
methylene and ketone, respectively, led to a significant decrease of activity. Amide analogs displayed lower activity in 60% propylene
glycol and were comparable to T12 in isopropyl myristate. An intramolecular H-bond between ester and ammonium-carbamate
group was suggested to be important for the permeation-enhancing activity of T12.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
O
5a,b
9a-h
10
O
H3N
R
O
O
Transdermal drug delivery offers numerous advantages
over conventional routes of administration; however,
poor permeation of most drugs across the skin barrier
constitutes a serious limitation of this methodology.
One of the approaches used to enlarge the number of
transdermally applicable drugs uses permeation enhanc-
ers. These compounds promote drug permeation
through the skin by a reversible decrease of the barrier
resistance. The enhancers act by one or more ways of
these possibilities: interaction with the stratum corneum
lipids, interaction with protein structures and partition-
ing modification.1
O
O
O
N
HN
R
H
O
H2
C
T12
Figure 1. Transdermal permeation enhancer T12 (R = C12H23) and its
ketone (R = C11H21–C12H23), amide (R = C6H13–C18H37), and alkane
analogs (R = C12H23).
Diffusion experiments have demonstrated that the ammo-
nium-carbamate structure was responsible for the
enhancing activity; the parent amino ester was inactive.2
We have previously reported the permeation-enhancing
activity of 5-(dodecyloxycarbonyl)pentylammonium-5-
(dodecyloxycarbonyl)pentylcarbamate (Transkarbam
12, T12, Fig. 1). The substance enhanced skin transport
of a variety of drugs with a wide spectrum of physico-
chemical properties,2,3 displaying low toxicity and no
dermal irritability.4 Moreover, the susceptibility of the
compound to metabolic deactivation into nontoxic
substances was shown in vitro using porcine esterase.
To gain more information about the structure–activity
relationships and, consequently, the mode of action of
this promising enhancer, we aimed at investigating the
role of the ester bond in the T12 molecule. In this
preliminary study, ketone, amide, and alkane analogs
of T12 were prepared and their permeation-enhancing
activities evaluated.
2. Results
Keywords: Transdermal drug delivery; Percutaneous absorption;
Permeation enhancer; Carbamate; Hydrogen bonding; Structure–
activity relationships.
In this study, analogs of T12 with the ester group
replaced by amide, ketone, and methylene groups,
respectively, were synthesized. The procedure for the
preparation of the ketone analogs 5a,b is outlined in
*
Corresponding author. Tel.: +420 495 067497; fax: +420 495
514330; e-mail: katerina.vavrova@faf.cuni.cz
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.006

T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 2896–2903
2897
Scheme 1. 6-Bromohexanoyl chloride was prepared by
the opening of the caprolactone ring,5 substitution of
hydroxyl with HBr, and the reaction of the carboxyl
group with SOCl2. The ketone was formed by reaction
of the acyltributylphosphonium ion with the corre-
sponding Grignard reagent.6 The amino ketones were
prepared by Gabriel synthesis, and the phthalimide
hydrolyzed under acid catalysis.7 The resulting ammoni-
um chloride was alkalized, and the amine converted to
the pertinent carbamate by reaction with CO2.
tensity band at around 1650 cmꢀ1 was assigned to the
carbonyl stretching vibration of the carbamate group.10
The presence of the carbamate structure was further
confirmed by thermogravimetric analysis (TGA). An
example is given in Figure 2, showing a 6.9% decrease
of weight of the compound 9g at 135 °C, corresponding
to an equimolar release of CO2 from the carbamate
molecule.10
The enhancement ratio (ER) values for the permeation
of theophylline are summarized in Table 1. Theophylline
has been selected as a model drug of medium polarity
for comparison of the enhancing activities of the pre-
pared compounds. This model drug has been widely
studied previously using various enhancers,11–13 skin
species,14,15 used as a tool to study skin barrier proper-
ties,16,17 in mathematical models to predict skin perme-
ability,18 and for drug monitoring in premature
neonates.19 Typical permeation profiles for both tested
vehicles and enhancers with the same chain length are
shown in Figure 3. Propylene glycol/water 6:4 (Pg/W,
Fig. 3a) has been selected to represent a hydrophilic
vehicle and isopropyl myristate (IPM, Fig. 3b) a
lipophilic one.
The amide analogs were prepared via the mixed anhy-
dride method8 from 6-Boc-aminohexanoic acid. The
protective group was removed by HCl, and the resulting
amino amides reacted with CO2 to form the target
ammonium-carbamate salts 9a–h (Scheme 2).
The alkane analog 10 was prepared by introducing CO2
into the CHCl3 solution of octadecylamine.
The carbamate salts were not sufficiently soluble in any
solvent for recording the NMR spectra, except for
CDCl3 at elevated temperature, which, however, caused
decomposition of the carbamate. Even the method
employed previously for similar ammonium-carbamate
derivatives2,9,10 was not successful for detection of this
type of carbamate. The spectra recorded in CDCl3, how-
ever, confirmed the purity of the substances (as amino
compounds), and presence of the carbamate structure
has been confirmed by elemental analysis and FTIR
spectroscopy.
0,05
100
0,00
98
96
94
92
90
-0,05
-0,10
-0,15
-0,20
-0,25
-0,30
In the IR spectra of the prepared compounds, the band
of the NH stretching vibration of medium intensity
between 3300 and 3400 cmꢀ1, characteristic of the
carbamic acid salts, was observed. Weak, broad band
at around 2150 cmꢀ1 indicated the presence of ammoni-
release of CO2 = 6.9%
+
40
60
80
100 120 140 160
um ions (combination band of RNH3 torsion and
antisymmetrical RNH3 deformation). The medium-in-
+
Temperature (˚C)
Figure 2. The TGA curve of 9g representing an equimolar release of
CO2 from the ammonium-carbamate molecule.
O
5
ii
iii
i
Br COCl
Br
O
( )
5
( )
R
O
Table 1. Enhancement ratios of the T12 analogs
R–X–(CH2)5NHCOOꢀH3N+(CH2)5–X–R
1
2a,b
O
O
5
O
5
v
Cl H N
iv
3
( )
5a,b
R
( )
N
R
Compound
X
R
ER SD
4a,b
3a,b
O
Pg/W
IPM
Scheme 1. Synthesis of the ketone analogs. Reagents and conditions:
a: R = C11H23; b: R = C12H25; (i) 1. HBr/H2SO4, 2. SOCl2; (ii) Bu3P,
RMgBr; (iii) potassium phthalimide; (iv) 36% HCl, 175 °C, 10 bar;
(v) TEA/CO2.
5a
5b
9a
9b
9c
–CO–
C
11H23
C12H25
3.8 1.2*,** 2.6 0.2*,**
3.4 0.7*,** 1.1 0.1**
3.8 0.5*,** 4.9 0.8*
3.8 0.7*,** 6.8 0.4*
–CONH– C6H13
C7H15
Octan-2-yl
C8H17
C9H19
2.2 1.4**
5.2 0.7*
9d
9e
5.0 1.2*,** 7.0 0.4*
8.6 2.8*,** 8.6 1.3*
6.0 1.4*,** 6.7 1.5*
2.2 0.5*,** 5.1 0.7*
ii
i
BocHN COOH
( )5
H2N COOH
( )5
9f
C10H21
C12H25
C18H37
C12H25
C12H25
6
9g
9h
10
T12
0.9 0.4**
1.8 0.6**
22.8 1.1*
1.3 0.7**
1.2 0.4**
6.6 0.5*
O
( )5
N
O
iii
iv
9a-h
R
R
BocHN
Cl H3N
( )5
8a-h
N
–CH2–
–COO–
H
H
7
Scheme 2. Synthesis of the amide analogs. Reagents and conditions: a:
R = C6H13; b: R = C7H15; c: R = C8H17; d: R = C8H17; e: R = C9H19; f:
R = C10H21; g: R = C12H25; h: R = C18H37; (i) 1. Boc2, NaOH,
2. KHSO4; (ii) ClCOOC2H5, RNH2, TEA; (iii) HCl(gas); (iv) TEA/CO2.
n = 4–6 (skin fragments from at least two animals for one compound).
* Significantly different from control (theophylline suspension in the
given vehicle without an enhancer; p < 0.05).
** Significantly different from T12 (p < 0.05).

2898
T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 2896–2903
a
tested under argon showed that the ammonium-carba-
1000
800
600
400
200
0
mate structure is responsible for the enhancing effect
and the amino ester is completely inactive. The pH
dependence of its effect further supported this finding.2
control
10
5b
9g
T12
It was therefore hypothesized that it is only the ammo-
nium-carbamate polar head that bears the exceptional
enhancing properties of T12 and the rest of the molecule
(e.g., the chain length) only slightly modulates its activ-
ity. Further support was brought in by a series of
compounds with ÔreversedÕ ester group, that is, x-amin-
oalkan-1-ol derivatives, displaying equal permeation-en-
hancing activity.21
0
15
20
25
30
35
40
45
50
Time (h)
b
At first, we aimed to confirm this hypothesis by evaluat-
ing a simple alkylammonium-alkylcarbamate 10. Sur-
prisingly, the compound was not active. Therefore,
both carbamate salt and ester group in T12 participate
in its activity. The FTIR spectra of T12 showed a
doublet of the ester carbonyl vibration,22 suggesting dif-
ferent hydrogen bonding of the two ester groups in the
molecule. Thus, one ester group might be forming a
hydrogen bond with the ammonium-carbamate group,
which is somehow important for the activity of T12.
1200
1000
800
600
400
200
0
control
10
5b
9g
T12
0
15
20
25
30
35
40
45
50
We have further replaced the ester group in T12 by a ke-
tone, which is a stronger hydrogen bond acceptor. This,
however, resulted in activities an order of magnitude
lower. Monosubstituted amides are both hydrogen bond
donors and acceptors, and their activity was significant-
ly decreased in Pg/W and comparable to T12 in IPM.
None of these compounds displayed a doublet of car-
bonyl vibrations in FTIR spectra as T12. The carbonyl
groups of these compounds therefore do not form the
same hydrogen bonding as in T12, which may be the
reason for their decreased potency. The importance of
the hydrogen-bonding ability in skin permeation
enhancers has already been reported.23,24 Interestingly,
only T12 displayed markedly different activities in
Pg/W and IPM vehicles. This might be connected also
with different hydrogen bonding of T12 in these two
vehicles or a synergic action of T12 and Pg in the
stratum corneum.
Time (h)
Figure 3. The permeation profiles of theophylline through the porcine
skin using: (a) Pg/W and (b) IPM as the donor media and T12, and its
analogs with the same chain length as enhancers. Control samples
contained theophylline only in the pertinent vehicle.
Using Pg/W as the donor vehicle, the activity of the
compounds with the same hydrocarbon chain length de-
creased as follows: T12 (ester, ER = 22.8 1.1) > 5b
(ketone, ER = 3.4 0.7) > 9g (amide, ER = 2.2 0.5)
> 10 (methylene, ER = 1.8 0.6). When applied in a
hydrophobic IPM suspension, the activities of amides
were comparable with that of T12 (ERs 5.1 0.7 and
6.6 0.5 for 9g and T12, respectively), while the alkane
and ketones were almost inactive.
In amide analogs, the effect of the chain length was fur-
ther investigated. A parabolic relationship was found
with maximum at nonyl derivatives in both vehicles.
Branching of the hydrocarbon chain decreased the activ-
ity markedly; the activity of the octyl derivative 9d and
its isomer octan-2-yl 9c applied in Pg/W was 5.0 1.2
and 2.2 1.4, respectively. Similar difference was
observed in IPM as a vehicle (Table 1).
The parabolic relationship between the chain length of
the amide analogs 9a–h and their enhancement activity
is in broad agreement with that found in other groups
of enhancers,21,25 suggesting a similar mode of action.
Also the negative effect of chain branching confirms
our previous findings.26
In conclusion, this preliminary study showed that not
only the carbamate polar head and a suitable chain
length were essential for the permeation-enhancing
activity of transkarbams, but also the character of the
linking group was of great importance. The reason for
the difference in activities of the present compounds
could be different steric ordering of the enhancer, hypo-
thetically via an intramolecular hydrogen bond, which
could bring different solubility in the donor medium,
and/or a specific action within the stratum corneum.
From the current comparison, ester bond seems to be
the best choice for its biodegradability and high activity;
3. Discussion
The novel group of skin permeation enhancers termed
transkarbams was originally prepared as 6-aminohexa-
noic acid esters,4 that is, open Azone20 analogs. The
compounds, however, were found to trap air carbon
dioxide to form two-chain ammonium-carbamate deriv-
atives. The direct comparison of the enhancing effect of
the carbamate T12 and the corresponding amino ester

T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 2896–2903
2899
however, this unusual behavior warrants further
investigation.
2849s, 1701s (m(CO)), 1471s, 1419m ðdsciðCOCH ÞÞ, 1384w,
2
1247m, 717w cmꢀ1
;
1H NMR (300 MHz, CDCl3): d
3.40 (t; 2H; J = 6.9 Hz; BrCH2), 2.41 (t; 2H;
J = 7.4 Hz; COCH2), 2.38 (t; 2H; J = 7.5 Hz; CH2CO),
1.87 (p; 2H; CH2CH2Br), 1.65–1.50 (m; 4H; CH2CH2-
COCH2CH2), 1.49–1.36 (m; 2H; CH2CH2CH2Br), 1.35–
1.14 (m; 16H; CH2), 0.88 (t; 3H; J = 6.6 Hz; CH3); 13C
NMR (75 MHz, CDCl3): d 211.2; 42.9; 42.4; 33.6;
32.5; 31.9; 29.6; 29.6; 29.5; 29.4; 29.3; 29.3; 27.7; 23.9;
22.8; 22.7; 14.1 ppm.
4. Experimental
4.1. Chemicals and instrumentation
All chemicals were purchased from Sigma–Aldrich (Sch-
nelldorf, Germany). Silica gel 60 (230–400 mesh) for col-
umn chromatography and TLC plates (silica gel 60 F254
,
aluminum back) were obtained from Merck (Darms-
tadt, Germany). IR spectra were recorded on a Nicolet
Impact 400 apparatus equipped with a DTGS detector
4.2.2.2. 1-Bromooctadecan-6-one (2b). White crystals;
mp = 28–33 °C, yield 43%; IR (CHCl3): mmax 2927s,
2855s, 1708s (m(CO)), 1464m, 1408w ðd
Þ, 1375w
1
with a resolution of 4 cmꢀ1. H and 13C NMR spectra
sciðCOCH2Þ
cmꢀ1 1H NMR (300 MHz, CDCl3): d 3.40 (t; 2H;
;
were measured on a Varian Mercury-Vx BB 300 instru-
J = 6.9 Hz; BrCH2), 2.41 (t; 2H; J = 7.0 Hz; COCH2),
2.38 (t; 2H; J = 7.3 Hz; CH2CO), 1.86 (p; 2H; J =
7.2 Hz; CH2CH2Br), 1.66–1.49 (m; 4H; CH2CH2-
COCH2CH2), 1.48–1.36 (m; 2H; CH2CH2CH2Br),
1.35–1.16 (m; 18H; CH2), 0.87 (t; 3H; J = 6.7 Hz;
CH3); 13C NMR (75 MHz, CDCl3): d 211.2; 42.9;
42.4; 33.6; 32.5; 31.9; 29.6; 29.6; 29.6; 29.5; 29.4; 29.3;
29.2; 27.7; 23.9; 22.8; 22.7; 14.1 ppm.
1
ment, operating at 300 MHz for H, 75 MHz for 13C.
Elemental analysis (C, H, and N) was performed on a
Fisons EA 1110 CHNS-O elemental analyzer. The melt-
ing point was measured with a Kofler apparatus and is
uncorrected. TGA was recorded using a Stanton Red-
croft TG 750 instrument.
4.2. Chemistry
The target compounds were synthesized according to
Scheme 1 (ketone analogs) and Scheme 2 (amide ana-
logs). The alkane analog was prepared by reaction of
octadecylamine with CO2.
4.2.3. General procedure for the preparation of N-(6-
oxoalkyl)phthalimides (3a,b)7. Solution of bromo ketone
2 in toluene was added to an anhydrous toluene suspen-
sion of potassium phthalimide (1.1 equiv) dropwise and
heated under reflux for 3 h. The reaction mixture was
kept at 4 °C overnight and precipitated, unreacted
potassium phthalimide filtered off. The mixture was
evaporated, dissolved in diethyl ether and extracted by
1 M NaOH solution and brine, and dried over Na2SO4.
The resulting orange oil was crystallized from methanol
to yield white crystals of 3a,b.
4.2.1. 6-Bromohexanoyl chloride (1). A mixture of capro-
lactone (236 g; 2.07 mol), 48% HBr (520 mL; 4.6 mol),
and 96% H2SO4 (235 mL) was heated at reflux for 3 h.
Five liters of water was added, the organic layer separat-
ed, and the aqueous layer extracted several times with
diethyl ether. The combined organic extracts were dis-
tilled at 150–155 °C/15 torr5 to yield a colorless liquid
6-bromohexanoic acid (305 g, 75%). To the resulting
acid (97 g, 0.5 mol) an excess of thionyl chloride
(71 mL; 1 mol) was added and stirred at 50 °C for 1 h.
The mixture was distilled at 113 °C/11 mbar to yield a
colorless liquid 1 (98 g, 93%).
4.2.3.1. N-(6-Oxoheptadecyl)phthalimide (3a). White
crystals; mp = 66–69 °C, yield 95%; IR (KBr): mmax
2918s, 2849s, 1772m (mas(NC@O)), 1698s (m(C@O)
,
s(NC@O)), 1614w, 1465m, 1435w, 1406m, 1374m,
m
1335w cmꢀ1
;
1H NMR (300 MHz, CDCl3): d 7.86–
7.79 (m; 2H; CH), 7.73–7.66 (m; 2H; CH), 3.66 (t; 2H;
J = 7.1 Hz; NCH2), 2.43–2.31 (m; 4H; CH2COCH2),
1.74–1.46 (m; 6H; CH2), 1.40–1.15 (m; 18H; CH2),
0.86 (t; 3H; J = 6.6 Hz; CH3); 13C NMR (75 MHz,
CDCl3): d 211.2; 168.4; 133.9; 132.1; 123.1; 42.8; 42.5;
37.8; 31.9; 29.7; 29.6; 29.6; 29.4; 29.3; 29.2; 28.3; 26.4;
23.9; 23.3; 22.6; 14.1 ppm.
4.2.2. General procedure for the preparation of 1-
bromoalkan-6-ones (2a,b)6. 6-Bromohexanoyl chloride
1
was dissolved in anhydrous THF, cooled to
ꢀ30 °C, and Bu3P (1.1 equiv) added under N2 atmo-
sphere. The resulting suspension was stirred for
30 min. An ethereal solution of the pertinent Grignard
reagent (1 equiv) was added slowly to the well-stirred
mixture. After stirring for 1 h at the same temperature,
the reaction was quenched by addition of 100 mL of
1 M HCl. The organic layer was separated and the
aqueous mixture was extracted three times with diethyl
ether. The combined organic layers were washed with
1% NaHCO3 and brine, and dried over Na2SO4. After
removal of the solvent, the residue was subjected to
column chromatography (petroleum ether/ethyl acetate
3:1) and subsequent crystallization afforded the
product.
4.2.3.2. N-(6-Oxooctadecyl)phthalimide (3b). White
crystals; mp = 70–72 °C, yield 91%; IR (KBr): mmax
2917s, 2849s, 1773m (mas(NC@O)), 1704s (m(C@O)), 1694s
(ms(NC@O)), 1615m, 1464m, 1435w, 1404m, 1375m,
1337w cmꢀ1 1H NMR (300 MHz, CDCl3): d 7.86–
;
7.78 (m; 2H; CH), 7.73–7.66 (m; 2H; CH), 3.66 (t; 2H;
J = 7.1 Hz; NCH2), 2.42–2.31 (m; 4H; CH2COCH2),
1.74–1.46 (m; 6H; CH2), 1.40–1.14 (m; 20H; CH2),
0.86 (t; 3H; J = 6.6 Hz; CH3); 13C NMR (75 MHz,
CDCl3): d 211.3; 168.4; 133.9; 132.1; 123.2; 42.9; 42.5;
37.8; 31.9; 29.6; 29.6; 29.6; 29.5; 29.4; 29.3; 29.2; 28.4;
26.5; 23.9; 23.3; 22.7; 14.1 ppm.
4.2.2.1. 1-Bromoheptadecan-6-one (2a). White crys-
tals; mp = 29–32 °C, yield 45%; IR (KBr): mmax 2918s,

2900
T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 2896–2903
4.2.4. General procedure for the preparation of 6-oxoalky-
lammonium-chlorides (4a,b)7. N-(6-Oxoalkyl)phthali-
mide 3 was hydrolyzed by 36% HCl in an autoclave
4.2.5.2. 6-Boc-aminohexanoic acid heptyl amide (7b).
White crystals; mp = 65–68 °C, yield 74%; IR (CHCl3):
mmax 3452m (m(NHBoc)), 1709s (m(NHC@OO)), 1661s
(m(NHC@O)), 1511s (d(1NH)), 1457w, 1393w (d(t-butyl)),
1367m (m(C–O)) cmꢀ1; H NMR (300 MHz, CDCl3): d
5.76 (s; 1H; NHamide), 4.62 (s; 1H; NH), 3.20 (q; 2H;
(Miniclave, Buchi AG Uster/Switzerland) at 175 °C/
¨
10 bar for 15 h. The product was mixed with toluene
and evaporated to remove the residual HCl. The result-
ing mixture was dissolved in CHCl3, filtered, and evapo-
rated. Crystallization from ethanol/acetone yielded a
white crystalline product.
J = 6.5 Hz;
CH2Namide),
3.13–3.00
(m;
2H;
CH2NHBoc), 2.14 (t; 2H; J = 7.5 Hz; CH2CONH),
1.69–1.55 (m; 2H; CH2), 1.53–1.11 (m; 23H; CH2),
0.84 (t; 3H; J = 6.6 Hz; CH3); 13C NMR (75 MHz,
CDCl3): d 172.9; 156.0; 79.0; 40.3; 39.5; 36.6; 31.7;
29.7; 29.6; 28.9; 28.4; 26.8; 26.3; 25.3; 22.5; 14.0 ppm.
4.2.4.1. 6-Oxoheptadecylammonium-chloride (4a).
White crystals; mp = 124–130 °C, yield 86%; IR (CHCl3):
þ
mmax 2927s, 2856s, 1708s (m(C@O)), 1615m ðd
ÞÞ,
asðNH3
þ
1521m ðdsðNH Þ, 1466m, 1408w ðdsciðCOCH ÞÞ, 1377m
4.2.5.3. 6-Boc-aminohexanoic acid octan-2-yl amide
(7c). White crystals; mp = 59–70 °C, yield 75%; IR
(CHCl3): mmax 3452m (m(NHBoc)) and 3438m and 3342w
(m(NHamide)), 1706s (m(NHC@OO)), 1655s (m(NHC@O)),
Þ
2
3
cmꢀ1
;
1H NMR (300 MHz, CDCl3): d 8.24 (s; 3H;
NH3+), 3.15–2.87 (m; 2H; CH2NH3+), 2.47–2.32 (m;
4H; CH2COCH2), 1.87–1.71 (m; 2H; CH2), 1.67–1.17
(m; 22H; CH2), 0.87 (t; 3H; J = 6.5 Hz; CH3); 13C NMR
(75 MHz, CDCl3): d 211.3; 42.9; 42.4; 39.8; 31.9; 29.6;
29.5; 29.4; 29.3; 29.3; 27.4; 26.0; 23.8; 22.9; 22.7; 14.1 ppm.
1510s (d(NH)), 1456m, 1393m (d(t-butyl)), 1367s (m(C–O)
)
cmꢀ1 1H NMR (300 MHz, CDCl3): d 5.46 (s; 1H;
;
NHamide), 4.58 (s; 1H; NH), 3.95 (p; 1H; J = 7.0 Hz;
CH), 3.16–2.99 (m; 2H; CH2NBoc), 2.15 (t; 2H;
J = 7.7 Hz; CH2CONH), 1.63 (p; 2H; J = 7.5 Hz;
CH2), 1.54–1.14 (m; 23H; CH2), 1.10 (d; 3H;
J = 6.6 Hz; CH3), 0.86 (t; 3H; J = 6.3 Hz; CH3); 13C
NMR (75 MHz, CDCl3): d 172.2; 156.0; 79.1; 45.2;
40.3; 36.9; 36.6; 31.7; 29.7; 29.1; 28.4; 28.3; 26.0; 25.4;
22.5; 21.0; 14.0 ppm.
4.2.4.2. 6-Oxooctadecylammonium-chloride (4b).
White crystals; mp = 120–130 °C, yield 78%; IR (CHCl3):
þ
mmax 2927s, 2856s, 1708s (m(C@O)), 1615m ðd
ÞÞ,
asðNH3
þ
1522m ðdsðNH Þ, 1466m, 1408w ðdsciðCOCH ÞÞ, 1377m
Þ
2
3
cmꢀ1
;
1H NMR (300 MHz, CDCl3): d 8.07 (s; 3H;
NH3+), 3.13–2.97 (m; 2H; CH2NH3+), 2.48–2.33 (m;
4H; CH2COCH2), 1.90–1.75 (m; 4H; CH2), 1.67–1.46
(m; 4H; CH2), 1.46–1.33 (m; 2H; CH2), 1.32–1.14 (m;
16H; CH2), 0.87 (t; 3H; J = 6.5 Hz; CH3); 13C NMR
(75 MHz, CDCl3): d 211.6; 43.0; 42.2; 39.9; 31.9; 29.7;
29.6; 29.5; 29.4; 29.3; 29.3; 27.2; 26.0; 23.9; 22.9; 22.7;
14.1 ppm.
4.2.5.4. 6-Boc-aminohexanoic acid octyl amide (7d).
White crystals; mp = 74–75 °C, yield 88%; IR (CHCl3):
mmax 3452s (m(NHBoc)
) and 3351w (m(N–H)), 1706s
(m(NHC@OO)), 1661s (m(NHC@O)), 1511s (d(NH)), 1457w,
1393m (d(t-butyl)), 1367s (m(C–O)
)
cmꢀ1 1H NMR
;
(300 MHz, CDCl3): d 5.68 (s; 1H; NHamide), 4.58 (s;
1H; NH), 3.210 (q; 2H; J = 6.5 Hz; CH2Namide), 3.13–
3.01 (m; 2H; CH2NHBoc), 2.15 (t; 2H; J = 7.6 Hz;
CH2CONH), 1.69–1.56 (m; 2H; CH2), 1.53–1.13 (m;
25H; CH2), 0.85 (t; 3H; J = 6.6 Hz; CH3); 13C NMR
(75 MHz, CDCl3): d 172.9; 156.0; 79.0; 40.3; 39.5; 36.6;
31.7; 29.7; 29.6; 29.3; 28.9; 28.4; 26.8; 26.3; 25.3; 22.5;
14.0 ppm.
4.2.5. General procedure for the preparation of 6-Boc-
aminohexanoic acid alkyl amides (7)8. 6-Boc-amino-
hexanoic acid (1.15 g; 5 mmol, prepared as described
previously27,28) in 20 mL of dry 1,2-dimethoxyethane
was mixed with dry TEA (5 mmol) at ꢀ15 °C. After
15 min, ethylchloroformate (5 mmol), and, after addi-
tional 10 min, 20% solution of the corresponding amine
(5 mmol) in dry 1,2-dimethoxyethane were slowly add-
ed. The mixture was stirred for 2 h at laboratory temper-
ature and then triethylammonium-chloride was filtered
off. The solution was evaporated, dissolved in ethyl ace-
tate, and washed with 10% HCl, 5% NaHCO3 and twice
with water. The organic layer was evaporated and the
product crystallized from a mixture of diethyl ether/
petroleum ether.
4.2.5.5. 6-Boc-aminohexanoic acid pentyl amide (7e).
White crystals; mp = 73–76 °C, yield 74%; IR (CHCl3):
mmax 3452m (m(NHBoc)), 3351w (m(NHamide)), 1708s
(m(NHC@OO)), 1660s (m(NHC@O)), 1511s (d(NH)), 1457m,
1393m (d(t-butyl)), 1367s (m(C–O)
) ;
cmꢀ1 1H NMR
(300 MHz, CDCl3): d 5.63 (s; 1H; NHamide), 4.55 (s;
1H; NH), 3.27–3.18 (m; 2H; CH2Namide), 3.09 (t; 2H;
J = 6.0 Hz; CH2NH), 2.17 (t; 2H; J = 7.5 Hz;
CH2CONH), 1.70–1.58 (m; 2H; CH2), 1.54–1.17 (m;
27H; CH2), 0.87 (t; 3H; J = 6.8 Hz; CH3); 13C NMR
(75 MHz, CDCl3): d 172.9; 156.0; 79.1; 40.3; 39.6;
36.5; 31.8; 29.8; 29.6; 29.5; 29.3; 29.2; 28.4; 26.9; 26.3;
25.3; 22.6; 14.1 ppm.
4.2.5.1. 6-Boc-aminohexanoic acid hexyl amide (7a).
White crystals; mp = 43–50 °C, yield 83%; IR (CHCl3):
mmax 3452m (m(NHBoc)) and 3350w (m(N–H)), 1708s
(m(NHC@OO)), 1661s (m(NHC@O)), 1511s (d(NH)), 1456m,
1393m (d(t-butyl)), 1367s (m(C–O)
) ;
cmꢀ1 1H NMR
(300 MHz, CDCl3): d 5.74 (s; 1H; NHamide), 4.62 (s;
1H; NH), 3.22 (q; 2H; J = 6.4 Hz; CH2Namide), 3.14–
3.02 (m; 2H; CH2NHBoc), 2.16 (t; 2H; J = 7.8 Hz;
CH2CONH), 1.63 (p; 2H; J = 7.6 Hz; CH2), 1.53–1.14
(m; 21H; CH2), 0.86 (t; 3H; J = 6.5 Hz; CH3); 13C
NMR (75 MHz, CDCl3): d 173.0; 156.0; 79.0; 40.3;
39.5; 36.5; 31.4; 29.7; 29.5; 28.4; 26.5; 26.3; 25.3; 22.5;
14.0 ppm.
4.2.5.6. 6-Boc-aminohexanoic acid decyl amide (7f).
White crystals; mp = 59–61 °C, yield 47%; IR (CHCl3):
mmax 3452m (m(NHBoc)), 3350w (m(NHamide)), 1708s
(m(NHC@OO)), 1661s (m(NHC@O)), 1511s (d(NH)), 1457m,
1393m (d(t-butyl)), 1367s (m(C–O)
(300 MHz, CDCl3): d 5.63 (s; 1H; NHamide), 4.58 (s;
1H; NH), 3.26–3.17 (m; 2H; CH2Namide), 3.14–3.03
) ;
cmꢀ1 1H NMR

T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 2896–2903
2901
(m; 2H; CH2NH), 2.16 (t; 2H; J = 7.6 Hz; CH2CONH),
1.70–1.57 (m; 2H; CH2), 1.54–1.19 (m; 29H; CH2), 0.86
(t; 3H; J = 7.0 Hz; CH3); 13C NMR (75 MHz, CDCl3): d
172.9; 156.0; 79.1; 40.3; 39.6; 36.5; 31.9; 29.7; 29.6; 29.5;
29.3; 28.4; 26.9; 26.3; 25.3; 22.6; 14.1 ppm.
J = 8.4 Hz; NH), 3.69 (h; 1H; J = 6.6 Hz; CH), 2.70 (t;
2H; J = 7.1 Hz; CH2NH3+), 2.01 (t; 2H; J = 7.2 Hz;
CH2), 1.60–1.39 (m; 4H; CH2), 1.38–1.09 (m; 12H;
CH2), 0.84 (t; 3H; J = 6.6 Hz; CH3); 13C NMR
(75 MHz, DMSO): d 171.2; 44.1; 38.8; 36.3; 35.4; 31.5;
28.8; 26.9; 25.9; 25.7; 25.1; 22.3; 21.1; 14.2 ppm.
4.2.5.7. 6-Boc-aminohexanoic acid dodecyl amide (7g).
White crystals; mp = 68–70 °C, yield 48%; IR (CHCl3):
mmax 3451m (m(NHBoc)), 3350w (m(NHamide)), 1708s
(m(NHC@OO)), 1661s (m(NHC@O)), 1511s (d(NH)), 1457m,
4.2.6.4. 5-Octylcarbamoylpentylammonium-chloride
(8d). White crystals; mp = 161–165 °C; IR (Nujol): mmax
3280m (m(N–H)), 1632s (m(NHC@O)), 1547m (d(NH)), 1524m
1
cmꢀ1
;
1H NMR
ðd
ÞÞ, 1152w cmꢀ1; H NMR (300 MHz, CDCl3): d
þ
1393m (d(t-butyl)), 1368s (m(C–O)
)
ðNH3
(300 MHz, CDCl3): d 5.64 (s; 1H; NHamide), 4.56 (s;
1H; NH), 3.26–3.17 (m; 2H; CH2Namide), 3.14–3.03
(m; 2H; CH2NH), 2.16 (t; 2H; J = 7.5 Hz; CH2CONH),
1.70–1.58 (m; 2H; CH2), 1.54–1.17 (m; 33H; CH2), 0.87
(t; 3H; J = 6.8 Hz; CH3); 13CNMR (75 MHz, CDCl3): d
172.9; 156.0; 79.1; 40.3; 39.6; 36.5; 31.9; 29.8; 29.6; 29.6;
29.5; 29.3; 29.3; 28.4; 26.9; 26.3; 25.3; 22.7; 14.1 ppm.
8.06 (s; 3H; NH3+), 7.84 (t; 1H; J = 5.2 Hz; NH), 2.98
(q; 2H; J = 6.5 Hz; CH2NH), 2.71 (t; 2H; J = 7.5 Hz;
CH2NH3+), 2.03 (t; 2H; J = 7.2 Hz; CH2), 1.62–1.12
(m; 18H; CH2), 0.85 (t; 3H; J = 6.6 Hz; CH3); 13C
NMR (75 MHz, DMSO): d 171.9; 38.8; 38.6; 35.3;
31.5; 29.4; 29.2; 28.6; 27.0; 26.6; 25.8; 25.0; 22.3;
14.2 ppm.
4.2.5.8. 6-Boc-aminohexanoic acid octadecyl amide
(7h). White crystals; mp = 69–74 °C, yield 37%; IR
(KBr): mmax 3359br m, 3323br m, 1687s (m(NHC@OO)),
1637s (m(NHC@O)), 1538s (d(NH)), 1469m, 1390m
4.2.6.5. 5-Nonylcarbamoylpentylammonium-chloride
(8e). White crystals; mp = 165–167 °C; IR (Nujol): mmax
3292s (m(N–H)), 1632s (m(NHC@O)), 1561m (d(NH)), 1523m
1
ÞÞ, 1152m cmꢀ1; H NMR (300 MHz, DMSO):
þ
ðd
ðNH3
(d(t-butyl)), 1366m (m(C–O)) cmꢀ1
.
d 8.05 (s; 3H; NH3+), 7.83 (t; 1H; J = 5.5 Hz; NH),
3.03–2.94 (m; 2H; CH2NH), 2.72 (t; 2H; J = 7.4 Hz;
CH2NH3+), 2.04 (t; 2H; J = 7.4 Hz; CH2), 1.61–1.41
(m; 4H; CH2), 1.39–1.14 (m; 16H; CH2), 0.85 (t; 3H;
J = 6.9 Hz; CH3); 13C NMR (75 MHz, DMSO): d
171.9; 38.8; 38.6; 35.3; 31.5; 29.4; 29.2; 29.0, 28.9; 26.9;
26.6; 25.7; 25.0; 22.3; 14.2 ppm.
4.2.6. General procedure for the preparation of 5-
alkylcarbamoylpentylammonium-chlorides (8). The Boc-
derivatives 7a–h were dissolved in dry CHCl3 and
deprotected by introducing dry HCl into the mixture
for 1 h. The dissolved HCl was removed by flushing
the mixture with dry N2. Crystallization from ethanol/
diethyl ether gave white crystalline products. The yields
were quantitative.
4.2.6.6. 5-Decylcarbamoylpentylammonium-chloride
(8f). White crystals; mp = 162–165 °C; IR (Nujol): mmax
3297s (m(N–H)), 1633s (m(NHC@O)), 1560m (d(NH)),
þ
4.2.6.1. 5-Hexylcarbamoylpentylammonium-chloride
(8a). White crystals; mp = 157–159 °C; IR (Nujol): mmax
3276s (m(N–H)), 1640s (m(NHC@O)), 1560m (d(NH)), 1523m
1523m ðd
ÞÞ, 1152m cmꢀ1
;
1H NMR (300 MHz,
ðNH3
DMSO):
d
8.02 (s; 3H; NH3+), 7.83 (t; 1H;
J = 5.5 Hz; NH), 2.99 (q; 2H; J = 6.5 Hz; CH2NH),
2.72 (t; 2H; J = 7.4 Hz; CH2NH3+), 2.04 (t; 2H;
J = 7.4 Hz; CH2), 1.60–1.41 (m; 4H; CH2), 1.39–1.14
(m; 18H; CH2), 0.85 (t; 3H; J = 6.6 Hz; CH3); 13C
NMR (75 MHz, DMSO): d 171.9; 38.5; 38.3; 35.1;
31.3; 29.1; 29.0; 28.9; 28.7; 28.7; 26.7; 26.4; 25.5;
24.8; 22.1; 13.9 ppm.
1
þ
ðd
ÞÞ, 1146m cmꢀ1; H NMR (300 MHz, CDCl3):
ðNH3
d 8.06 (s; 3H; NH3+), 7.84 (t; 1H; J = 4.9 Hz; NH),
2.98 (q; 2H; J = 5.9 Hz; CH2NH), 2.70 (t; 2H;
J = 7.2 Hz; CH2NH3+), 2.03 (t; 2H; J = 6.8 Hz; CH2),
1.61–1.13 (m; 14H; CH2), 0.84 (t; 3H; J = 6.5 Hz;
CH3); 13C NMR (75 MHz, DMSO): d 171.9; 38.8;
38.6; 35.3; 31.2; 29.3; 27.0; 26.3; 25.8; 25.0; 22.3;
14.2 ppm.
4.2.6.7. 5-Dodecylcarbamoylpentylammonium-chloride
(8g). White crystals; mp = 160–164 °C; IR (Nujol): mmax
3295s (m(N–H)), 1632s (m(NHC@O)), 1561m (d(NH)), 1523m
4.2.6.2. 5-Heptylcarbamoylpentylammonium-chloride
(8b). White crystals; mp = 159–164 °C; IR (Nujol): mmax
3296s (m(N–H)), 1633s (m(NHC@O)), 1562m (d(NH)), 1524w
1
þ
ðd
ÞÞ, 1152m cmꢀ1; H NMR (300 MHz, DMSO):
ðNH3
d 8.07 (s; 3H; NH3+), 7.84 (t; 1H; J = 5.5 Hz; NH),
2.99 (q; 2H; J = 6.5 Hz; CH2NH), 2.72 (t; 2H;
J = 7.7 Hz; CH2NH3+), 2.04 (t; 2H; J = 7.1 Hz; CH2),
1.61–1.41 (m; 4H; CH2), 1.39–1.15 (m; 22H; CH2),
0.84 (t; 3H; J = 6.9 Hz; CH3); 13C NMR (75 MHz,
DMSO): d 171.9; 38.8; 38.6; 35.3; 31.5; 29.4; 29.2;
29.0; 29.0; 27.0, 26.7; 25.8; 25.0; 22.3; 14.2 ppm.
1
þ
ðd
ÞÞ, 1152m cmꢀ1; H NMR (300 MHz, CDCl3):
ðNH3
d 8.08 (s; 3H; NH3+), 7.85 (t; 1H; J = 5.0 Hz; NH),
2.98 (q; 2H; J = 6.4 Hz; CH2NH), 2.70 (t; 2H;
J = 7.8 Hz; CH2NH3+), 2.03 (t; 2H; J = 7.1 Hz; CH2),
1.62–1.11 (m; 16H; CH2), 0.84 (t; 3H; J = 6.5 Hz;
CH3); 13C NMR (75 MHz, DMSO): d 171.9; 38.8;
38.6; 35.3; 31.5; 29.4; 28.6; 27.0; 26.6; 25.8; 25.0; 22.3;
14.2 ppm.
4.2.6.8. 5-Octadecylcarbamoylpentylammonium-chlo-
ride (8h). White crystals; mp = 144–149 °C; IR (Nujol):
mmax 3294s (m(N–H)), 1632s (m(NHC@O)), 1559m (d(NH)),
þ
4.2.6.3.
5-(Octan-2-ylcarbamoyl)pentylammonium-
chloride (8c). White crystals; mp = 129–131 °C; IR (Nu-
1523w ðd
ÞÞ, 1152m cmꢀ1
.
ðNH3
jol): mmax 3270m (m(N–H)), 1632s (m(NHC@O)), 1548m
þ
(d(NH)), 1523m ðd
ÞÞ, 1153w cmꢀ1
;
1H NMR
4.2.7. General procedure for the preparation of carba-
mates (5, 9, and 10). The appropriate ammonium-chlo-
ðNH3
(300 MHz, CDCl3): d 8.05 (s; 3H; NH3+), 7.63 (d; 1H;

2902
T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 2896–2903
ride was dissolved in water, alkalized by 1.5 equiv of
TEA, and extracted three times with diethyl ether. The
ethereal extracts were dried over Na2SO4 and then dry
CO2 was slowly bubbled through the solution for
approximately 20 min. The precipitated carbamate was
filtered off through dense filter paper and dried in vacuo
over P4O10.
C33H68N4O4 (found/calculated): 67.88/67.76; 11.73/
11.72; 9.67/9.58.
4.2.7.9. 5-Dodecylcarbamoylpentylammonium-5-dode-
cylcarbamoylpentylcarbamate (9g). White crystals;
mp = 95–100 °C, yield 61%; IR (Nujol): mmax 3369m
ꢀ
ðm
ðm
ÞÞ, 3253s (m(CON–H)), 2128w, 1654m
ðN–HCOO
ðNHC@OO
ÞÞ, 1626s (m(NHC@O)), 1553s,br (d(NH)) cmꢀ1
;
ꢀ
4.2.7.1.
6-Oxoheptadecylammonium-6-oxoheptade-
CHN analysis for C37H76N4O4 (found/calculated):
69.48/69.33; 11.64/11.95; 8.93/8.74.
cylcarbamate (5a). White crystals; mp = 82–86 °C, yield
59%; IR (Nujol): mmax 3307m (m(N–H)), 2196wbr, 1705s
ꢀ
þ
(m(C@O)), 1696w, 1640m ðm
ÞÞ, 1610m ðd
ÞÞ
4.2.7.10. 5-Octadecylcarbamoylpentylammonium-5-
octadecylcarbamoylpentylcarbamate (9h). The starting
ðNHC@OO
ðNH3
cmꢀ1; CHN analysis for C35H70N2O4 (found/calculat-
ed): 71.68/72.11; 12.00/12.10; 4.86/4.81.
ammonium chloride was insoluble in water; therefore, it
was dissolved in chloroform, mixed with TEA and water,
and extracted with chloroform. White crystals; mp = 93–
ꢀ
4.2.7.2. 6-Oxooctadecylammonium-6-oxooctadecylc-
arbamate (5b). White crystals; mp = 78–85 °C, yield
49%; IR (Nujol): mmax 3305m (m(N–H)), 2183wbr, 1705s
105 °C, yield 49%; IR (Nujol): mmax 3369m ðm
(m(NHC@O)), 1553s,br (d(NH)) cmꢀ1; CHN analysis for
C49H100N4O4 (found/calculated): 73.18/72.72; 11.94/
12.45; 6.93/6.92.
ÞÞ,
ðN–HCOO
ðNHC@OO
ꢀ
3253s (m(CON–H)), 2128w, 1654m ðm
ÞÞ, 1626s
ꢀ
þ
(m(C@O)), 1641m ðm
ÞÞ, 1609m ðd
ÞÞ cmꢀ1
;
ðNHC@OO
ðNH3
CHN analysis for C37H74N2O4 (found/calculated):
72.82/72.73; 12.22/12.21; 4.57/4.58.
4.2.7.3. 5-Hexylcarbamoylpentylammonium-5-hexylc-
arbamoylpentylcarbamate (9a). White crystals; mp = 93–
97 °C, yield 70%; IR (Nujol): mmax 3294s (m(N–H)), 1632s
þ
4.2.8. Octadecylammonium-octadecylcarbamate (10).
Octadecylamine was dissolved in dry CHCl3 and treated
with CO2 likewise. White crystals; mp = 85–88 °C, yield
71%; IR (Nujol): mmax 3331s (m(N–H)), 2152w, 1647m
ꢀ
(m(NHC@O)), 1559m (d(NH)), 1523w ðd
ÞÞ, 1152m
ðNH3
cmꢀ1; CHN analysis for C25H52N4O4 (found/calculat-
ed): 63.37/63.52; 11.45/11.09; 11.89/11.85.
ðm
ÞÞ, 1567s (d(NH)), 1314s, 1157m, 816w cmꢀ1
;
ðNHC@OO
CHN analysis for C37H78N2O2 (found/calculated):
75.91/76.22; 13.45/13.48; 4.69/4.80.
4.2.7.4. 5-Heptylcarbamoylpentyammonium-5-heptylc-
arbamoylpentylcarbamate (9b). White crystals; mp = 93–
97 °C, yield 64%; IR (Nujol): mmax 3294s (m(N–H)), 1632s
þ
4.3. Skin preparation
(m(NHC@O)), 1559m (d(NH)), 1523w ðd
ÞÞ, 1152m
Porcine ears were purchased from a local slaughter-
house. The full-thickness dorsal skin was collected and
hairs were removed using a clipper. The skin was then
immersed in 0.05% sodium azide solution in saline for
5 minutes for preservation. The skin fragments were
stored vacuum-sealed at ꢀ18 °C for maximum of 2
months. The skin samples were thawed immediately
before use.
ðNH3
cmꢀ1; CHN analysis for C27H56N4O4 (found/calculat-
ed): 65.33/64.76; 11.63/11.27; 11.41/11.19.
4.2.7.5. 5-(Octan-2-ylcarbamoyl)pentylammonium-5-
(octan-2-ylcarbamoyl)pentylcarbamate (9c). White crys-
tals; mp = 75–80 °C, yield quantitative; IR (Nujol): mmax
3294s (m(N–H)), 1632s (m(NHC@O)), 1559m (d(NH)), 1523w
þ
ðd
ÞÞ, 1152m cmꢀ1; CHN analysis for C29H60N4O4
ðNH3
(found/calculated): 65.66/65.87; 11.56/11.44; 10.24/10.59.
4.4. Permeation experiments
4.2.7.6. 5-Octylcarbamoylpentylammonium-5-octylc-
arbamoylpentylcarbamate (9d). White crystals; mp = 96–
The permeation-enhancing activities of the prepared
compounds were evaluated in vitro using the Franz dif-
fusion cells and theophylline as a model permeant. The
donor samples were prepared by dispersing theophylline
(5%) and the tested enhancer (1%) in Pg/W 3:2 v/v or
IPM. At this concentration, both theophylline and the
enhancers were suspended, with partial dissolution.
The control samples were prepared likewise in the
respective vehicle without addition of the enhancers.
The suspensions were stirred at 50 °C for 5 min, and
allowed to equilibrate at 37 °C for 24 h, and re-dispersed
before application on the skin. The skin was cut into
fragments and mounted into the cells dermal side down
to leave a diffusion area of 1 cm2. The acceptor compart-
ment of the cells was filled with ca 17 mL of phosphate-
buffered saline at pH 7.4 with 0.03% sodium azide as a
preservative and allowed to equilibrate at 32 °C for
30 min. The donor sample of 200 lL volume was ap-
plied on the skin surface and occluded with a cover
glass. The acceptor phase was kept at 32 °C with stirring
ꢀ
98 °C, yield 60%; IR (Nujol): mmax 3356sðm
ÞÞ,
ðN–HCOO
3232s (m(CON–H)), 2121w, 1626s (m(NHC@O)), 1560s,br
(d(NH)) cmꢀ1; CHN analysis for C29H60N4O4 (found/cal-
culated): 65.49/65.87; 11.62/11.44; 10.42/10.59.
4.2.7.7. 5-Nonylcarbamoylpentylammonium-5-nonyl-
carbamoylpentylcarbamate
(9e).
White
crystals;
mp = 94–100 °C, yield 63%; IR (Nujol): mmax 3372m
ꢀ
ꢀ
ðm
ÞÞ, 3255m (m(CON–H)), 1656m ðm
ÞÞ,
ðN–HCOO
ðNHC@OO
1628s (m(NHC@O)), 1556s,br (d(NH)) cmꢀ1; CHN analysis
for C31H64N4O4 (found/calculated): 67.04/66.86; 11.94/
11.58; 10.17/10.06.
4.2.7.8. 5-Decylcarbamoylpentylammonium-5-decylc-
arbamoylpentylcarbamate (9f). White crystals; mp = 98–
ꢀ
102 °C, yield 57%; IR (Nujol): mmax 3369m ðm
ÞÞ,
ðN–HCOO
ðNHC@OO
ꢀ
3255m (m(CON–H)), 2144w, 1656w ðm
ÞÞ, 1628s
(m(NHC@O)), 1556s,br (d(NH)) cmꢀ1; CHN analysis for

T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 2896–2903
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Acknowledgments
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This work was supported by the Grant Agency of the
Czech Republic (GACR 203/04/P042) and the Ministry
of Education of the Czech Republic (MSM0021620822).
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