Synthesis and transdermal permeation-enhancing activity of carbonate and carbamate analogs of Transkarbam 12

Article abstract of DOI:10.1016/j.bmc.2006.08.014

Transkarbam 12 (5-(dodecyloxycarbonyl)pentylammonium-5-(dodecyloxycarbonyl)pentylcarbamate, T12) is a highly effective skin permeation enhancer. In this study, ester groups in the molecule of T12 were replaced by carbonate and carbamate ones, respectively. The in vitro permeation-enhancing activities were evaluated using porcine skin and compared with those of T12 and previously prepared series of amide, ketone, and alkyl analogs. According to the activities and behavior of the compounds in donor samples, ester group is essential for the activity of T12; its replacement not only decreases the enhancing potency, but is likely to change the mechanism of action.

Full text of DOI:10.1016/j.bmc.2006.08.014

Bioorganic & Medicinal Chemistry 14 (2006) 7671–7680
Synthesis and transdermal permeation-enhancing activity
of carbonate and carbamate analogs of Transkarbam 12
*
ˇ
Tom a´ sˇ Holas, Kate rˇ ina V a´ vrov a´ , Martin S ´ı ma, Jana Klimentov a´ and Alexandr Hrab a´ lek
Centre for New Antivirals and Antineoplastics, Faculty of Pharmacy, Charles University,
Heyrovsk e´ ho 1203, 50005 Hradec Kr a´ lov e´ , Czech Republic
Received 15 April 2006; revised 1 August 2006; accepted 11 August 2006
Available online 30 August 2006
Abstract—Transkarbam 12 (5-(dodecyloxycarbonyl)pentylammonium-5-(dodecyloxycarbonyl)pentylcarbamate, T12) is a highly
effective skin permeation enhancer. In this study, ester groups in the molecule of T12 were replaced by carbonate and carbamate
ones, respectively. The in vitro permeation-enhancing activities were evaluated using porcine skin and compared with those of
T12 and previously prepared series of amide, ketone, and alkyl analogs. According to the activities and behavior of the compounds
in donor samples, ester group is essential for the activity of T12; its replacement not only decreases the enhancing potency, but is
likely to change the mechanism of action.
ꢀ
2006 Elsevier Ltd. All rights reserved.
1
. Introduction
Replacement of ester with methylene and ketone
groups, respectively, resulted in a loss of enhancing
activity. Activities of the amide analogs were compa-
rable to that of T12 in a lipophilic vehicle; however,
in a hydrophilic one, where T12 reached its highest
Transdermal permeation enhancers are compounds,
which interact with skin constituents to promote drug
flux. To date, a vast array of chemicals has been evalu-
ated as enhancers, yet their inclusion into topical or
transdermal formulation is limited since the underlying
mechanisms of action of these agents are seldom clearly
defined. For the most recent reviews on enhancers, see
7
enhancement ratio, the amides were much less active.
In this study, we aimed at synthesizing two novel ser-
ies of T12 analogs, esters of carbonic or carbamic acid
with an additional heteroatom introduced into the
linking group (Fig. 1). The skin permeation-enhancing
activity of the analogs was evaluated under the same
1
2
for example, Williams and Barry, Purdon et al., and
3
V a´ vrov a´ et al.
7
5
-(Dodecyloxycarbonyl)pentylammonium-5-(dodecyl-
conditions as described in our previous study to al-
oxycarbonyl)pentylcarbamate (Transkarbam 12, T12,
Fig. 1) is a novel biodegradable permeation enhancer
with high activity toward a variety of drugs with a
low for comparison of the whole series. Furthermore,
solubility of the enhancers and their effect on solubil-
ity of the model drug, theophylline, in the donor sam-
ples were determined to gain more insights into the
mode of action of this structurally novel group of
enhancers.
4
,5
wide spectrum of physicochemical properties,
low
4
,6
toxicity, and no dermal irritability. The carbamate
polar head of T12 is essential for the permeation-
enhancing activity of the compound since the parent
4
amino ester has been completely inactive. In our ef-
fort to explain the mechanism of action of T12 and/
or to prepare more active analogs, we focused on
the role of ester linking group in T12 molecule.
Keywords: Transdermal drug delivery; Cutaneous absorption; Pene-
tration enhancer; Ammonium carbamate; Biodegradable.
Figure 1. Transdermal permeation enhancer T12 (n = 4; R = C12
and its carbamate (n = 4–6; R = C H17–C12H25), and carbonate
25
H )
*
Corresponding author. Tel.: +420 495 067497; fax: +420 495
14330; e-mail: katerina.vavrova@faf.cuni.cz
8
5
8
analogs (n = 5, 6; R = C H17, C10H21, C12H25).
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.08.014

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T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 7671–7680
2. Results
for routine characterization of the products. Neverthe-
less, when dissolved in chloroform or similar acidic
solvents, the products, that is, ammonium carbamate
derivatives, decompose into carbon dioxide and the
parent amino compounds, the NMR spectra of which
fully confirm the structure and purity of such com-
pounds. The presence of the ammonium carbamate
was then sufficiently proved by specific N–H vibration
in IR spectroscopy. For carbamic acid salts, moder-
ately intensive band of the NH stretching vibration
The carbonate analogs were prepared by reaction of
N-Boc protected amino alcohol with an appropriate
alkylchloroformate (Scheme 1). The protective group
was removed by acid hydrolysis and the amino carbon-
ates were subjected to a reaction with CO to yield the
2
target ammonium carbamate derivatives with carbonate
groups within the chains. In case of 4-aminobutanol
derivatives, that is, compounds having only 4-carbon
linking chain between nitrogen and the carbonate
group, no carbamate salts were obtained. Although pre-
cipitation of some material was observed at ꢀ20 ꢁC, and
we were able to isolate it at low temperature, it readily
decomposed at ambient temperature and the spectra
showed no presence of carbamic acid salt. Therefore,
these derivatives were not evaluated for their enhancing
activity.
ꢀ
1
between 3200 and 3400 cm was characteristic. Weak,
ꢀ
1
broad band around 2150 cm
ammonium ions (combination band of RNH torsion
indicated presence of
þ
3
and antisymmetrical RNH3 deformation). The medi-
þ
ꢀ
1
um-intensity band around 1650 cm was assigned to
8
amide I band of the carbamic acid salt. Moreover,
the existence of these substances in the form of an
ammonium–carbamate salt was confirmed by TGA
analysis, which showed that with increasing tempera-
ture, the substance releases exactly one equivalent of
carbon dioxide (Fig. 2a and b). Finally, CHN analysis
unambiguously confirmed the elemental composition
corresponding to the proposed structures.
For the preparation of the second series, the carbamate
analogs, isocyanates were prepared by the Curtius rear-
rangement. These intermediates reacted with hydrochlo-
rides of amino alcohols and the final products were
prepared by reaction with CO (Scheme 2).
2
Enhancement activity of the prepared compounds was
evaluated using theophylline as a model drug and three
vehicles of different polarity, water (W), propylene gly-
col/water 6:4 v/v (Pg/W), and isopropyl myristate
(IPM). For the enhancement ratios (ERs), see Table 1.
Due to the low solubility and instability of the final
lipophilic salts in acidic environment, characterization
by NMR spectra was problematic. The spectra were
recorded in CDCl3 saturated with triethylamine or
pyridine; however, only two members of the carbonate
series, 3c and 3f, gave satisfactory results. The other
When applied in aqueous suspensions, the carbonates
of the same chain length as T12 were approximately
equally active with ERs of 1.9 ± 0.3, 1.6 ± 0.3, and
2.5 ± 0.9 for T12, 3f, and 3c, respectively. Shorter-
chain carbonates 3a, b, d, and e displayed significantly
higher activity than T12 with ERs up to 5.1. Neither
of the carbamates showed significant enhancement of
theophylline permeation when applied in aqueous do-
nor vehicle.
spectra obtained in suspension were poor, showing
only
ꢀ
a
63.1 ppm (d1 = 8s, ct = 9200), and could not be used
weak signal of NHCOO
carbon at
1
The highest ER values were obtained with Pg/W as a do-
nor vehicle. In this vehicle, none of the novel enhancers
reached the activity of T12 (22.8 ± 1.1). Incorporation
of additional oxygen decreased the activity of the car-
bonates approximately twice (ERs from 7.1 to 12.4).
The carbamates showed activities even lower (ERs from
Scheme 1. Synthesis of the carbonate analogs. (a) n = 5, R = C
b) n = 5, R = C10 21; (c) n = 5, R = C12 25; (d) n = 6, R = C 17; (e)
n = 6, R = C10 21; (f) n = 6, R = C12 25. Reagents and conditions: (i)
8 17
H ;
(
H
H
8
H
H
H
1
—Boc anhydride, NaOH, THF, 2—alkylchloroformate, pyridine, N
2
1
.7 to 6.3).
atmosphere, 0 ꢁC; (ii) HCl/CHCl
TEA/CO
3
or CF COOH/CHCl
3
3
, 0 ꢁC; (iii)
2
.
With lipophilic donor vehicle, IPM, T12 enhanced per-
meation of theophylline 6.6 times. Only compound 3e
showed higher ER (9.7 ± 1.4) than T12; the other car-
bonates were less active (ERs from 2.2 to 4.0), as well
as the carbamates (ERs from 0.8 to 5.6).
The solubility of the model drug, theophylline, in the Pg/
W donor sample was 35 mg/mL. Addition of the
enhancers increased this value significantly, however,
no difference was observed between the individual com-
pounds (Table 2).
Scheme 2. Synthesis of the carbamate analogs. (a) n = 4, R = C
b) n = 4, R = C 19; (c) n = 4, R = C10 21; (d) n = 4, R = C11 23; (e)
n = 5, R = C10 21; (f) n = 5, R = C11 23; (g) n = 5, R = C12 25; (h)
n = 6, R = C10 21; (i) n = 6, R = C11 23; (j) n = 6, R = C12
Reagents and conditions: (i) 1—SOCl , 2—NaN
, 3—60 ꢁC; (ii)
n-hydroxyalkylammonium chloride; (iii) TEA/CO
8 17
H ;
(
9
H
H
H
H
H
H
The solubility of T12 and its analogs with the same
chain length including previously prepared ketone,
amide, and alkyl analogs in the Pg/W donor samples
H
H
25
H .
2
3
2
.

T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 7671–7680
7673
Figure 2. TGA analysis and derivation curves of compounds (a) 3b and (b) 5f, demonstrating an equimolar release of CO
2
from the ammonium–
carbamate structure.
Table 1. ER values of the prepared carbonates 3a–f and carbamates
a–j in three vehicles of different polarity (W, Pg/W, and IPM,
respectively)
the compounds, and their solubility in the donor sample,
with T12 excluded from the dataset.
5
Compound
ER ± SD
Pg/W
3. Discussion
W
IPM
*
*
*
,**
,**
*
*
*
*
3
3
3
a
b
c
4.1 ± 1.0
5.1 ± 1.1
2.5 ± 0.9
12.4 ± 2.6
11.8 ± 0.7
7.4 ± 0.2
2.2 ± 0.1
5.5 ± 1.6
2.9 ± 0.5
T12 was originally prepared as an open Azone analog,
6
dodecyl 6-aminohexanoate (DDEAC). Recently, we
*
*
have found that DDEAC traps carbon dioxide to form
two-chain ammonium–carbamate salt, T12. Direct com-
parison of these compounds revealed that only T12 is
responsible for the enhancing properties and DDEAC
*
*
*
,**
,**
*
*
*
*
3
3
3
d
e
f
4.6 ± 0.5
4.7 ± 0.5
1.6 ± 0.3
7.1 ± 0.4
7.9 ± 1.6
8.4 ± 3.3
3.4 ± 0.6
9.7 ± 1.4
4.0 ± 1.4
*,**
*
4
*
*
*
*
*
is inactive. The labile ammonium–carbamate salt with-
in the polar head was found to be essential and suggest-
5
5
5
5
a
b
c
1.6 ± 0.5
1.9 ± 0.6
2.1 ± 1.1
1.5 ± 0.2
3.2 ± 0.8
6.3 ± 1.8
5.6 ± 0.5
2.9 ± 0.4
4.8 ± 0.5
5.6 ± 0.3
3.6 ± 0.2
2.5 ± 0.2
9
*
*
ed to relate to the mode of action of T12. Thus, we
d
assumed that mainly the carbamate salt bears the
enhancing activity and the ester group only modulates
its solubility and/or partitioning into the stratum corne-
um. However, replacement of ester group by amide,
ketone, and methylene, respectively, resulted in com-
*
*
*
*
*
5
5
5
e
f
1.5 ± 0.5
1.0 ± 0.2
1.2 ± 0.3
5.3 ± 0.9
2.3 ± 0.5
1.7 ± 0.5
2.6 ± 0.4
2.2 ± 0.2
1.7 ± 0.3
g
*
*
*
*
5
5
5
h
i
1.2 ± 0.2
1.5 ± 0.6
1.3 ± 0.3
4.3 ± 0.6
3.7 ± 0.3
1.9 ± 0.3
3.1 ± 0.3
1.7 ± 0.3
0.8 ± 0.1
7
pounds with markedly decreased activity.
j
In this study, a series of carbonate and carbamate analogs
of T12 was evaluated. Both these groups have been widely
used as pH-sensitive biodegradable linkers for example, in
*
*
*
T12
1.9 ± 0.3
22.8 ± 1.1
6.6 ± 0.5
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).
10
11,12
surfactants
or carrier lipids for gene delivery.
Although the carbonates displayed significant perme-
ation-enhancing activities, none of the compounds reached
the potency of T12 in Pg/W donor vehicle. Replacement of
oxygen with nitrogen further decreased the activity, similar
to amide analogs. Generally, the 10-carbon-chain com-
pounds were the most active enhancers, which is in accor-
*
*
Significantly different from T12 (p < 0.05).
Table 2. Solubility of T12 and its analogs with the same chain length
and their effect on the drug solubility in Pg/W
Enhancer
Linking
group
ER
C
e
C
0
3,13
(mg/mL) (mg/mL)
dance with previous studies.
The length of the linking
chain between the ammonium–carbamate group and oxy-
gen in carbonate and carbamate group, respectively, had
only a little influence. However, certain length is probably
required as the carbonates with 4-carbon linking chain did
not form stabile ammonium–carbamates.
No enhancer
T12
—
1.0
— 35.0 ± 0.0
*
*
*
*
*
*
–COO–
–OCOO–
–OCONH–
22.8 ± 1.1 3.5 ± 0.1 42.8 ± 1.5
8.4 ± 3.3 6.4 ± 0.3 39.6 ± 0.9
1.9 ± 0.3 1.6 ± 0.0 37.8 ± 0.6
3
5
f
j
a
Ketone analog –CO–
Amide analog –CONH–
3.4 ± 0.7 1.0 ± 0.1 42.7 ± 3.9
a
2.2 ± 0.5 1.8 ± 0.0 40.6 ± 1.5
a
1.8 ± 0.6 0.5 ± 0.1 39.6 ± 1.6
Alkyl analog
–CH2–
Thus, ester group, which was originally selected for its
biodegradability, is essential for the activity of T12 as
well as the ammonium–carbamate polar head. Third
requirement is flexibility of the linking chain between
these functional groups. This is documented by de-
creased activity of tranexamic acid derivatives, which
differ from T12 only by presence of a cyclohexane ring
C
e
, solubility of the enhancer; C
7
Ketone, amide, and alkyl analogs were studied in our previous work .
0
, solubility of theophylline.
a
*
Significantly different from Control (without an enhancer; p < 0.05).
was determined by potentiometric titration and is shown
in Table 2. Linear correlation (p < 0.01; R = 0.96) was
found between the ER values, that is, the activity of
2
1
4
instead of the alkyl chain.

7674
T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 7671–7680
7
Drug permeation across the stratum corneum obeys
Fick’s first law (J = D · C · P/h) where steady-state
T12. Such hydrogen bond would bring different steric
ordering of the molecule, which could result in different
partitioning of T12 into the stratum corneum or, more
probably, a specific action within the barrier compared
to the analogs. The importance of hydrogen bonding
for the action of the skin permeation enhancers has al-
0
flux (J) is related to the diffusion coefficient (D), the drug
concentration in the vehicle (C ), the partition coeffi-
0
cient between the vehicle and the stratum corneum
(
P), and the stratum corneum thickness (h). Permeation
1
7,18
enhancement may be achieved by increasing D (enhanc-
er permeates into the stratum corneum and disorders the
barrier structures, for example, the intercellular lipid
ready been documented.
Another possibility is that the hydrogen bond has an
influence on the stability of the ammonium–carbamate
polar head. Carbamic acid salts are, generally, unstable
in a mildly acidic environment. It has been hypothesized
that in the stratum corneum, which is acidic in nature,
T12 would be decomposed releasing carbon dioxide
and two molecules of DDEAC or, more precisely, its
matrix), C (enhancer increases solubility of a poorly
0
soluble drug in the vehicle), P (enhancer alters the sol-
vent nature of the skin barrier and thus increases parti-
tioning of a drug into the stratum corneum), or, less
likely, by decreasing h (providing a more permeable
shortcut).
9
ammonium salt. As DDEAC is inactive, the mode of
Increasing C , that is, maximizing the availability of
0
action could be related to the released carbon dioxide
or a conformational change connected to the decompo-
sition. Carbon dioxide present in the stratum corneum
could disrupt the lipid lamellae by changing the hydro-
gen bonding between their polar heads or create perme-
able pores. Studies aiming at investigating this
hypothesis are in progress.
drug for absorption, is a compromise between obtaining
a sufficient amount of poorly soluble drug in solution by
solubilization, and maintaining or inducing a high level
of thermodynamic activity to support the absorption
process. In case of T12 and its analogs, the donor sam-
ples were suspensions, thus the thermodynamic activity
of theophylline was at its maximum value in all samples.
Addition of the evaluated enhancers increased C , how-
0
In conclusion, this work described a novel series of T12
analogs. Although none of them reached the activity of
the parent compound, the results narrowed the number
of the possible mechanisms of action of this highly
active, nontoxic, nonirritant, and biodegradable skin
permeation enhancer.
ever, this increase was too small (approximately 20%) to
be responsible for the ERs observed. Moreover, there
was no difference between T12 and its analogs.
Partitioning of a drug into the stratum corneum is usu-
ally increased by polar solvents like ethanol and Pg. In
case of amphiphilic enhancers that enter the stratum
corneum in low concentrations, this mechanism is
4. Experimental
1
5
negligible.
4
.1. Chemicals and instrumentation
Therefore, we may assume that the main mode of action
of T12 and its analogs is increasing D, that is, a specific
action within the skin barrier. Since Pg is known to in-
crease P by changing the solvent properties of the stra-
tum corneum, the effects of T12 and Pg would be
synergic. This is consistent with the observed behavior;
the highest enhancement activity of T12 has been found
when applied in Pg/W donor vehicle.
All chemicals were purchased from Sigma–Aldrich
(Schnelldorf, Germany). Silica gel 60 (230–400 mesh)
for column chromatography and TLC plates (silica gel
60 F254, aluminum back) were obtained from Merck
(Darmstadt, Germany). IR spectra were recorded on a
Nicolet Impact 400 apparatus equipped with a DTGS
ꢀ
1
1
13
detector with a resolution of 4 cm
NMR spectra were measured on a Varian Mercury-Vx
.
H and
C
1
BB 300 instrument, operating at 300 MHz for H,
Disordering of the barrier structures requires certain
concentration of an enhancer in the site of action. For
the carbonate, carbamate, amide, ketone, and alkyl ana-
logs, a linear correlation was found between their solu-
bility in the donor sample and their activity,
suggesting an approximately equal extent of barrier-dis-
ordering ability. This could explain relatively high activ-
ity of the carbonate analogs compared to the other
derivatives. On the other hand, T12 is poorly soluble
in the vehicle; thus we hypothesize that ester group in
T12 is associated with a different mechanism of action.
1
3
75 MHz for C. Elemental analysis (C, H, N) was per-
formed on a Fisons EA 1110 CHNS-O elemental ana-
lyzer. The melting point was measured on a Kofler
apparatus and is uncorrected. TGA was recorded using
a Stanton Redcroft TG 750 instrument.
4.2. Chemistry
The target compounds were synthesized according to
Scheme 1 (carbonate analogs) and Scheme 2 (carbamate
analogs).
Previously, a doublet of ester carbonyl stretching has
1
6
been observed in FTIR spectra of T12, which could
be explained by different hydrogen bonding of the two
ester groups. No such splitting of carbonyl stretching
has been observed in the analogs. It has been suggested
that one ester group might be forming an intramolecular
hydrogen bond between ester and carbamate group of
4.2.1. General procedure for the preparation of carbonic
acid esters (1a–f). One hundred milliliters of Boc O
(0.24 mol) solution in THF was slowly dropped to an
aqueous solution (150 mL) of an aminoalcohol
(0.2 mol) and NaOH (0.2 mol) at 0 ꢁC. The mixture
was stirred overnight at room temperature. The reaction
2

T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 7671–7680
7675
mixture was evaporated in vacuum and extracted several
times with diethyl ether. The combined organic layers
were washed with diluted HCl and brine and dried over
Na SO . After removal of the solvent, the purity of
(75 MHz, CDCl ): d 155.9, 155.4, 79.1, 68.0, 67.7,
40.5, 31.8, 29.8, 29.5, 29.4, 29.2, 28.6, 28.3, 26.3, 25.6,
25.4, 22.6, 14.1 ppm.
3
2
4
product was checked by TLC. The yield of Boc protect-
9
ed aminoalcohol was quantitative.
4.2.1.5. 6-tert-Butoxycarbonylaminohexyl(decyl)car-
bonate (1e). Colorless liquid, yield 80%; H NMR
1
1
(
300 MHz, CDCl ): d 4.50 (s; 1H; NH), 4.09 (t; 4H;
3
An alkylchloroformate (4.4 mmol) was slowly added to
Boc-aminoalcohol (4 mmol) solution in freshly distilled
J = 6.8 Hz; CH OCOOCH ), 3.08 (t; 2H; J = 7.0 Hz;
2 2
CH NH), 1.57–1.70 (m; 4H; CH ), 1.17–1.52 (m; 29H;
2
2
1
3
dry pyridine (8 mL) at 0 ꢁC under N atmosphere. The
CH ; CH ), 0.86 (t; 3H; J = 6.5 Hz; CH ); C NMR
2 3 3
2
mixture was stirred overnight and then acidified by
1
(75 MHz, CDCl ): d 155.9, 155.4, 79.1, 68.0, 67.7,
3
0% HCl and extracted three times with diethyl ether.
40.5, 31.8, 29.9, 29.5, 29.4, 29.2, 29.2, 28.6, 28.5, 28.4,
26.3, 25.6, 25.4, 22.6, 14.1 ppm.
The combined organic layers were washed with brine
and the residue was subjected to column chromatogra-
phy (petroleum ether/ethyl acetate 8:2) to yield carbonic
4.2.1.6.
cyl)carbonate (1f). Colorless liquid, yield 52%;
6-tert-Butoxycarbonylaminohexyl(dode-
H
2
0
1
acid ester 1a–f.
NMR (300 MHz, CDCl ): d 4.54 (s; 1H; NH), 4.10 (t;
3
4.2.1.1. 5-tert-Butoxycarbonylaminopentyl(octyl)car-
bonate (1a). Colorless liquid, yield 78%; IR (CHCl3):
4H; J = 6.5 Hz; CH OCOOCH ), 3.08 (t; 2H;
J = 7.0 Hz; CH NH), 1.56–1.72 (m; 4H; CH ), 1.14–
2 2
2
2
tmax 3455m (t(N–H)), 1739s (t(OC@OO)), 1709s
1.52 (m; 33H; CH ; CH ), 0.86 (t; 3H; J = 6.6 Hz;
2
3
1
3
(
(
1
4
tas(HNC@OO)), 1508s (d(NH)), 1468m, 1457m, 1404m
tas(O–C(O)–O)), 1393m1 (d(tert-butyl)), 1367s (t(C–O)),
CH3); C NMR (75 MHz, CDCl ): d 155.9, 155.3,
3
79.1, 68.0, 67.7, 40.5, 31.7, 29.9, 29.8, 29.6, 29.5, 29.4,
29.3, 29.2, 28.6, 28.4, 28.4, 26.3, 25.6, 25.4, 22.6,
14.0 ppm.
265sbr (t(C–N,C–O)); H NMR (300 MHz, CDCl ): d
3
.54 (s; 1H; NH), 4.10 (t; 4H; J = 6.5 Hz; CH OCO-
2
OCH ), 3.10 (t; 2H; J = 6.9 Hz; CH NH), 1.59–1.73
2
2
(
m; 4H; CH ), 1.17–1.55 (m; 23H; CH ; CH ), 0.86 (t;
2
4.2.2. General procedure for the preparation of 5-
alkoxycarbonyloxypentylammonium salts (2a–f). Method
A: Ten equivalents of CF COOH was added dropwise
3
2
3
1
3
3
1
2
H; J = 6.5 Hz; CH3); C NMR (75 MHz, CDCl ): d
3
2
1
55.9, 155.2, 79.1, 68.1, 67.6, 40.4, 31.7, 29.7, 29.1,
8.6, 28.3, 28.3, 25.6, 23.0, 22.6, 14.1 ppm.
in a solution of compound 1a–c in dry CHCl at 0 ꢁC.
3
The reaction was monitored by TLC (petroleum ether/
ethyl acetate 8:2). The reaction mixture was evaporated
in vacuum and the residual CF COOH was removed by
4
.2.1.2. 5-tert-Butoxycarbonylaminopentyl(decyl)car-
bonate (1b). Colorless liquid, yield 72%; IR (CHCl ): t
3
max
3
3
455m (t(N–H)), 1740s (t(OC@OO)), 1710s (tas(HNC@OO)),
a stream of N to yield yellowish liquid 2a–c (quant.).
2
1
1
508s (d(NH)), 1468m, 1457m, 1404m (tas(O–C(O)–O)),
393m (d(tert-butyl)), 1367s (t(C–O)), 1266sbr (t(C–N,C–O));
Method B: Dry HCl was slowly introduced into a
solution of compound 1d–f (4 mmol) in 10 mL of
dry CHCl3 at ꢀ10 ꢁC for 30 min. The reaction was
monitored by TLC (petroleum ether/ethyl acetate
8:2). The reaction mixture was evaporated in vacuum
1
H NMR (300 MHz, CDCl ): d 4.55 (s; 1H; NH), 4.09
3
(
J = 6.9 Hz; CH NH), 1.59–1.72 (m; 4H; CH ), 1.15–
t; 4H; J = 6.7 Hz; CH OCOOCH ), 3.10 (t; 2H;
2
2
2
2
1
.53 (m; 27H; CH ; CH ), 0.86 (t; 3H; J = 6.6 Hz; CH );
2 3 3
C NMR (75 MHz, CDCl ): d 155.9, 155.2, 79.1, 68.1,
1
3
and crystallized from CHCl /diethyl ether. The yield
3
3
6
2
7.6, 40.4, 31.8, 29.7, 29.5, 29.4, 29.2, 29.1, 28.6, 28.3,
8.3, 25.6, 23.0, 22.6, 14.0 ppm.
of white crystalline compound 2d–f was approximately
75%.
4
cyl)carbonate (1c). Colorless liquid, yield 62%; IR
.2.1.3.
5-tert-Butoxycarbonylaminopentyl(dode-
4.2.2.1. 5-Octyloxycarbonyloxypentylammonium-tri-
fluoroacetate (2a). Colorless liquid, IR (CDCl ): t
3
max
ꢀ
1741s (t(OC@OO)), 1676s (tas
(CF COO
3
(
(
(
1
4
CHCl ): t
3455m (t(N–H)), 1740s (t(OC@OO)), 1710s
)), 1529m dsNH3 ,
þ
3
max
tas(HNC@OO)), 1507s (d(NH)), 1467m, 1457m, 1403m
tas(O–C(O)–O)), 1393m1 (d(tert-butyl)), 1367s (t(C–O)),
267sbr (t(C–N,C–O)); H NMR (300 MHz, CDCl ): d
1469m, 1459m, 1436m, 1407m (tas(O–C(O)–O)), 1380w,
1
1269s (t
3H; NH3 ), 4.06–4.17 (m; 4H; CH OCOOCH ), 2.94–
); H NMR (300 MHz, CDCl ): d7.50 (s;
3
(C–O)
þ
3
2
2
þ
.57 (s; 1H; NH), 4.08 (t; 4H; J = 6.5 Hz; CH OCO-
3.08 (m; 2H; CH
1.17–1.53 (m; 12H; CH ), 0.87 (t; 3H; J = 6.8 Hz;
CH3); C NMR (75 MHz, CDCl ): d 162.0, 161.6,
2
NH3 ), 1.59–1.78 (m; 6H; CH2),
2
OCH ), 3.09 (t; 2H; J = 6.9 Hz; CH NH), 1.56–1.73
2
2
2
1
3
(
m; 4H; CH ), 1.14–1.56 (m; 31H; CH ; CH ), 0.86 (t;
2
2
3
3
1
3
3
1
2
1
H; J = 6.5 Hz; CH3); C NMR (75 MHz, CDCl ): d
161.1, 160.5, 155.5, 121.0, 117.3, 113.5, 109.4, 68.5,
67.3, 40.1, 31.7, 29.1, 29.1, 28.5, 27.8, 26.8, 25.6, 22.6,
22.4, 14.1 ppm.
3
55.9, 155.3, 79.1, 68.1, 67.6, 40.4, 31.7, 29.7, 29.6,
9.5, 29.4, 29.3, 29.1, 28.6, 28.3, 28.3, 25.6, 23.0, 22.6,
4.1 ppm.
4
.2.2.2. 5-Decyloxycarbonyloxypentylammonium-tri-
H
1
4.2.1.4. 6-tert-Butoxycarbonylaminohexyl(octyl)car-
bonate (1d). Colorless liquid, yield 75%; H NMR
fluoroacetate (2b). Colorless liquid,
(300 MHz, CDCl ): d 7.61 (s; 3H; ), 4.11 (t; NH3 4H;
NMR
þ
1
3
(
J = 6.6 Hz; CH OCOOCH ), 3.08 (t; 2H; J = 6.9 Hz;
300 MHz, CDCl ): d 4.50 (s; 1H; NH), 4.09 (t; 4H;
J = 6.5 Hz; CH OCOOCH ), 2.92–3.06 (m; 2H;
2
3
2
þ
CH
16H; CH ), 0.87 (t; 3H; J = 6.8 Hz; CH ); C NMR
2
NH3 ), 1.59–1.77 (m; 6H; CH ), 1.18–1.51 (m;
2
2
2
1
3
CH NH), 1.59–1.72 (m; 4H; CH ), 1.13–1.50 (m; 25H;
2
2
2
3
1
CH ; CH ), 0.86 (t; 3H; J = 6.6 Hz; CH ); C NMR
3
(75 MHz, CDCl ): d 162.2, 161.7, 161.2, 160.7, 155.4,
3
2
3
3

7
676
T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 7671–7680
Table 3. Boiling points of the prepared acyl chlorides and alkyl
isocyanates
1
2
1
21.3, 117.5, 113.7, 109.8, 68.5, 67.3, 40.0, 31.8, 29.5,
9.5, 29.3, 29.2, 28.6, 27.9, 26.8, 25.6, 22.6, 22.4,
4.1 ppm.
R=
Bp (RCOCl)
Bp (RNCO)
Octyl
Nonyl
Decyl
118 ꢁC/45 mbar
133 ꢁC/35 mbar
145 ꢁC/36 mbar
163 ꢁC/40 mbar
121 ꢁC/5 mbar
114 ꢁC/50 mbar
128 ꢁC/40 mbar
138 ꢁC/35 mbar
150 ꢁC/36 mbar
114 ꢁC/5 mbar
4.2.2.3. 5-Dodecycarbonyloxypentylammonium-triflu-
oroacetate (2c). Colorless liquid, H NMR(300 MHz,
1
þ
CDCl ): d 7.36 (s; 3H; NH3 ), 4.08–4.18 (m; 4H;
Undecyl
Dodecyl
3
þ
CH OCOOCH ), 2.98–3.10 (m; 2H; CH
2
NH3 ),
.59–1.78 (m; 6H; CH ), 1.17–1.51 (m; 20H; CH ),
2
2
1
0
2
2
1
.87 (t; 3H; J = 6.7 Hz; CH3); C NMR (75 MHz,
3
CDCl ): d 162.2, 161.7, 161.2, 160.7, 155.6, 121.3,
4.2.4. General procedure for the preparation of
alkylcarbamoyloxyalkylammonium-chlorides 5a–j. n-
Hydroxyalkylammonium chloride in acetonitrile was
added to alkyl isocyanate (1.05 equiv) and heated under
reflux for 12 h. Precipitated solid was filtered off and
recrystallized from ethanol/acetonitrile to yield white
3
1
2
1
17.5, 113.7, 109.8, 68.6, 67.4, 40.3, 31.9, 29.6, 29.5,
9.5, 29.3, 29.2, 28.5, 27.8, 26.8, 25.6, 22.7, 22.3,
4.1 ppm.
4
.2.2.4. 6-Octyloxycarbonyloxyhexylammonium-chlo-
2
3
ride (2d). Mp = 88–90 ꢁC, IR (CHCl ): t 1739s
crystalline compound 4a–j.
3
max
(
1
t
), 1614m (dasNH3 ), 1522m (dsNH3 ), 1468m,
þ þ
OC@OO)
1
(
404m (tas(O–C(O)–O)), 1380w, 1268s (t(C–O)); H NMR
300 MHz, CDCl ): d8.23 (s; 3H; NH3 ), 4.09 (t; 4H;
4.2.4.1. 4-Octylcarbamoyloxybutylammonium-chloride
þ
(
J = 6.5 Hz; CH OCOOCH ), 2.90–3.06 (m; 2H;
CH NH3 ), 1.58–1.83 (m; 6H; CH ), 1.18–1.49 (m;
2
(4a). Mp = 135–138 ꢁC, yield 90%; IR (nujol): t
3
max
3334m (t
), 2034, 1685s (t
), 1611w (dasNH3 ),
þ
(C@O)
1
2
2
(N–H)
þ
1537m (d(N–H)), 1275m, 1252m, 1145m; H NMR
2
1
4H; CH ), 0.86 (t; 3H; J = 6.8 Hz; CH ); C NMR
3
þ
1
(300 MHz, DMSO): d 8.13 (s; 3H; NH3 ), 7.18 (t; 1H;
2
3
(
2
75 MHz, CDCl ): d 155.3, 68.1, 67.5, 39.8, 31.7, 29.1,
9.1, 28.6, 28.3, 27.4, 26.0, 25.6, 25.1, 22.6, 14.0 ppm.
J = 5.2 Hz; NH), 3.97 (t; 2H; J = 6.0 Hz; CH O), 2.88–
2.97 (m; 2H; CH NH), 2.79 (t; 2H; J = 6.8 Hz;
3
2
2
þ
CH NH ), 1.84 (p; 2H; J = 6.4 Hz; CH ), 1.09–1.43
2
3
(m; 14H; CH ), 0.84 (t; 3H; J = 6.2 Hz; CH );
2
1
3
4.2.2.5. 6-Decyloxycarbonyloxyhexylammonium-chlo-
C
2
3
ride (2e). Mp = 96–97 ꢁC, IR (CHCl ): t 1740s
NMR(75 MHz, DMSO): d 156.3, 61.2, 40.4, 36.4,
31.5, 29.6, 28.9, 28.9, 28.7, 27.1, 26.5, 22.3, 14.2 ppm.
3
max
(
t
), 1615m (das
þ
), 1522m (ds ), 1467m,
þ
NH3
(
OC@OO)
NH3
1
1
404m (tas(O–C(O)–O)), 1380w, 1267s (t(C–O)); H NMR
þ
(
J = 7.0 Hz; CH OCOOCH ), 2.91–3.05 (m; 2H;
300 MHz, CDCl ): d 8.24 (s; 3H; NH3 ), 4.08 (t; 4H;
4.2.4.2.
chloride (4b). Mp = 120–123 ꢁC, yield 86%; IR (nujol):
t 3333m (t ), 2034, 1686s (t ), 1611w
max
4-Nonylcarbamoyloxybutylammonium-
3
2
2
þ
CH
1
2
NH3 ), 1.57–1.84 (m; 6H; CH ), 1.17–1.50 (m;
2
(N–H)
(C@O)
1
8H; CH ), 0.85 (t; 3H; J = 6.6 Hz; CH ); C NMR
3
þ
(NH3 ), 1542m (d(N–H)), 1298m, 1269m, 1246m,
1145m; H NMR (300 MHz, DMSO): d 8.12 (s; 3H;
NH3 ), 7.16 (t; 1H; J = 5.5 Hz; NH), 3.97 (t; 2H;
2
3
1
(
75 MHz, CDCl ): d 155.3, 68.0, 67.4, 39.7, 31.8, 29.4,
3
þ
2
1
9.4, 29.2, 29.2, 28.6, 28.3, 27.4, 26.0, 25.6, 25.0, 22.6,
4.0 ppm.
J = 6.0 Hz; CH O), 2.92 (q; 2H; J = 6.2 Hz; CH NH),
2
2
þ
2
.79 (t; 2H; J = 7.4 Hz; CH
2
NH3 ), 1.84 (p; 2H;
4
.2.2.6. 6-Dodecylcarbonyloxyhexylammonium-chlo-
J = 6.4 Hz; CH ), 1.05–1.43 (m; 16H; CH ), 0.83 (t;
2 2
1
3
ride (2f). Mp = 100–102 ꢁC, IR (CHCl ): t 1740s
3H; J = 6.1 Hz; CH3); C NMR (75 MHz, DMSO): d
156.3, 61.2, 40.4, 36.4, 31.5, 29.6, 29.2, 29.1, 29.0, 28.9,
27.0, 26.5, 22.3, 14.2 ppm.
3
max
(
1
t
), 1615m (dasNH3 ), 1522m (dsNH3 ), 1467m,
þ þ
OC@OO)
1
(
404m (tas(O–C(O)–O)), 1380w, 1266s (t(C–O)); H NMR
þ
(300 MHz, CDCl ): d 8.24 (s; 3H; NH3 ), 4.09 (t; 4H;
J = 6.5 Hz; CH OCOOCH ), 2.90–3.05 (m; 2H;
3
4.2.4.3.
chloride (4c). Mp = 128–131 ꢁC, yield 78%; IR (nujol):
3334m (t ), 2021, 1684s (t ), 1598w
4-Decylcarbamoyloxybutylammonium-
2
2
þ
CH NH3 ), 1.57–1.83 (m; 6H; CH ), 1.17–1.47 (m;
2
2
1
3
2H; CH ), 0.85 (t; 3H; J = 7.0 Hz; CH ); C NMR
2
t
max
2
3
(N–H)
(C@O)
(
2
2
75 MHz, CDCl ): d 155.3, 68.1, 67.4, 39.8, 31.8, 29.6,
9.6, 29.5, 29.4, 29.3, 29.2, 28.6, 28.3, 27.4, 26.0, 25.6,
5.1, 22.6, 14.1 ppm.
(d
), 1531m (d(N–H)), 1286m, 1262m, 1240m,
as(NH3+)
1
3
1143m; H NMR (300 MHz, DMSO): d 7.81 (s; 3H;
NH3 ), 7.18 (t; 1H; J = 5.5 Hz; NH), 3.96 (t; 2H;
J = 5.8 Hz; CH O), 2.92 (q; 2H; J = 6.2 Hz; CH NH),
2.79 (t; 2H; J = 7.2 Hz; CH NH3 ), 1.84 (p; 2H;
J = 6.5 Hz; CH ), 1.11–1.41 (m; 18H; CH ), 0.83 (t; 3H;
2 2
J = 5.8 Hz; CH3); C NMR (75 MHz, DMSO): d 156.3,
61.2, 40.4, 36.4, 31.5, 29.6, 29.2, 29.2, 29.0, 28.9, 27.0,
26.5, 22.3, 14.2 ppm.
þ
2
2
þ
4.2.3. General procedure for the preparation of alkyl
isocyanates. An acyl chloride (prepared from the corre-
sponding carboxylic acid by reaction with SOCl and
2
purified by vacuum distillation, Table 3) in acetone
was added dropwise to a saturated aqueous solution
of NaN3 (1.4 equiv) at temperature not exceeding
2
1
3
5
ꢁC. The organic upper phase was removed by a syr-
4.2.4.4.
4-Undecylcarbamoyloxybutylammonium-
inge, slowly dropped to dry toluene at 60 ꢁC, and stir-
red at the same temperature for 1 h. Toluene was
evaporated in vacuum and the residue was purified
by distillation under reduced pressure (Table 3) yield-
ing 65–75% of alkyl isocyanate (40% for
chloride (4d). Mp = 131–134 ꢁC, yield 80%; IR (CHCl ):
3
þ
t
3453w (t
), 1712s (t
), 1604w (NH3 ),
max
(N–H)
(C@O)
1
1537m (d(N–H)), 1417m, 1363m; H NMR (300 MHz,
DMSO): d 8.23 (s; 3H; NH3 ), 7.17 (t; 1H; J = 4.9 Hz;
þ
NH), 3.96 (t; 2H; J = 5.8 Hz; CH O), 2.91 (q; 2H;
þ
2
2
2
undecylisocyanate).
J = 5.8 Hz; CH NH), 1.85–2.72 (m; 2H; CH
2
2
NH3 ),

T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 7671–7680
7677
þ
1
1
0
.85 (p; 2H; J = 6.3 Hz; CH ), 1.07–1.42 (m; 20H; CH2),
2
(NH3 ), 1519s (d(N–H)), 1467m, 1378w; H NMR
1
.83 (t; 3H; J = 5.8 Hz; CH3); C NMR (75 MHz,
3
þ
(300 MHz, CDCl ): d 8.23 (s; 3H; NH ), 4.96 (t; 1H;
3
3
J = 5.6 Hz; NH), 4.02 (t; 2H; J = 6.3 Hz; CH O), 3.13
DMSO): d 156.3, 61.2, 40.4, 36.4, 31.6, 29.6, 29.3,
9.2, 29.0, 29.0, 27.0, 26.5, 22.4, 14.2 ppm.
2
2
(q; 2H; J = 6.6 Hz; CH NH), 3.00 (t; 2H; J = 7.4 Hz;
2
þ
þ
CH
2
NH3 ), 1.78 (p; 2H; J = 7.3 Hz; CH
1.68 (m; 2H; CH ), 1.16–1.54 (m; 22H; CH ), 0.87 (t;
2
NH3 ), 1.55–
4
.2.4.5. 5-Decylcarbamoyloxypentylammonium-chlor-
2
2
1
3
ide (4e). Mp = 127–133 ꢁC, yield 79%; IR (KBr): t
3
1
3H; J = 6.6 Hz; CH3); C NMR (75 MHz, CDCl ): d
156.8, 64.4, 41.0, 39.8, 31.9, 30.0, 29.6, 29.6, 29.3, 28.6,
27.4, 26.8, 26.0, 25.3, 22.7, 14.1 ppm.
max
3
327m (t(
), 2957w, 2922s, 2852s, 1686s (t
),
N–H)
þ
(C@O)
623w (NH3 ), 1541m (d(N–H)), 1468w, 1284m, 1263m
1
(
t(N-CO-O)), 1145m; H NMR (300 MHz, CDCl ): d
3
þ
8
.21 (s; 3H; NH3 ), 5.16 (t; 1H; J = 5.5 Hz; NH), 4.04
t; 2H; J = 6.3 Hz; CH O), 3.12 (q; 2H; J = 6.6 Hz;
4.2.4.10.
6-Dodecylcarbamoyloxyhexylammonium-
(
CH NH), 3.01 (t; 2H;J = 7.6 Hz; CH NH ), 1.83 (p;
chloride (4j). Mp = 120–123 ꢁC, yield 80%; IR (CHCl ):
2
3
þ
t
3452m (t
), 2928s, 2856s, 1706s (t
), 1616w
(C@O)
2
2
3
NH3 ), 1.56–1.70 (m; 2H; CH2),
max
(N–H)
þ
þ
1
2
1
3
1
2
H; J = 7.6 Hz; CH
2
(NH3 ), 1519s (d(N–H)), 1466m, 1378w; H NMR
þ
.47 (p; 4H; CH ), 1.16–1.36 (m; 14H; CH ), 0.87 (t;
2
(300 MHz, CDCl ): d 8.25 (s; 3H; NH ), 4.90–5.02
2
3
3
(m; 1H; NH), 3.97–4.13 (m; 2H; CH O), 3.06–3.19 (m;
1
3
H; J = 6.6 Hz; CH3); C NMR (75 MHz, CDCl ): d
3
2
þ
56.8, 64.2, 41.0, 39.8, 31.9, 30.0, 29.5, 29.3, 28.2, 27.1,
6.8, 22.9, 22.6, 14.1 ppm.
2H; CH NH), 3.00 (t; 2H; J = 7.4 Hz; CH
2
2
NH3 ),
NH3 ), 1.54–1.68 (m; 2H;
þ
1.70–1.84 (m; 2H; CH
CH2), 1.16–1.54 (m; 24H; CH2), 0.87 (t; 3H;
2
1
3
4
.2.4.6.
5-Undecylcarbamoyloxypentylammonium-
J = 6.6 Hz; CH3);
C NMR (75 MHz, CDCl ): d
3
chloride (4f). Mp = 141–145 ꢁC, yield 77%; IR (KBr):
156.8, 64.4, 41.0, 39.8, 31.9, 30.0, 29.6, 29.6, 29.3, 28.6,
27.4, 26.8, 26.0, 25.3, 22.7, 14.1 ppm.
t
3328m (t
), 2957w, 2922s, 2852s, 1687s
þ
max
(N–H)
(
1
t(C@O)), 1623w (NH3 ), 1541s (1d(N–H)), 1468m, 1276m,
258m (t(N–CO–O)), 1145m;
H
NMR (300 MHz,
4.2.5. General procedure for the preparation of the
carbamic acid salts 3a–f and 5a–j. Ammonium salt 2a–f
and 5a–j, respectively, was dissolved in water, alkalized
by 1.5 equiv of triethylamine, and extracted three times
with diethyl ether. The ethereal extracts were dried over
Na SO and then dry CO slowly bubbled through the
þ
CDCl ): d 8.20 (s; 3H; NH3 ), 5.17 (t; 1H; J = 5.5 Hz;
3
NH), 4.03 (t; 2H; J = 5.9 Hz; CH O), 3.11 (q; 2H;
J = 6.6 Hz; CH NH), 3.01 (t; 2H; J = 7.4 Hz;
2
2
þ
þ
2
NH3 ), 1.56–
CH
1
1
2
NH3 ), 1.82 (p; 2H; J = 7.7 Hz; CH
.70 (m; 2H; CH ), 1.47 (p; 4H; CH ), 1.20–1.33 (m;
2
2
2
4
2
1
6H; CH ), 0.86 (t; 3H; J = 6.6 Hz; CH ); C NMR
3
solution for approximately 20 min. The precipitated
ammonium–carbamate was filtered off through dense fil-
ter paper and dried in vacuum over P O .
2
3
(
75 MHz, CDCl ): d 156.8, 64.2, 41.0, 39.8, 31.9, 30.0,
3
2
9.6, 29.6, 29.3, 28.2, 27.0, 26.8, 23.0, 22.6, 14.1 ppm.
4
10
4
.2.4.7. 5-Dodecylcarbamoyloxypentylammonium-
4.2.5.1. 5-Octyloxycarbonyloxypentylammonium-5-
octyloxycarbonyloxypentylcarbamate (3a). Mp = 45–
chloride (4g). Mp = 128–130 ꢁC, yield 76%; IR (KBr):
max
t
(
1
3331m (t
), 2957w, 2921s, 2851s, 1687s
þ
47 ꢁC, yield 50%; IR (nujol): t
3381m (t(N–H)),
), 1652w, 1591m, 1533m
(OC@OO)
(N–H)
max
t(C@O)), 1621w (NH3 ), 1540m (d
270m, 1252m (t(N-CO-O)), 1145m; H NMR (300 MHz,
CDCl ): d 8.21 (s; 3H; NH3 ), 5.16 (t; 1H; J = 5.5 Hz;
NH), 4.04 (t; 2H; J = 6.2 Hz; CH O), 3.11 (q; 2H;
J = 6.6 Hz; CH NH), 3.01 (t; 2H; J = 7.6 Hz;
), 1468m, 1378w,
2178wbr, 1742s (t
(N–H)
1
(dsNH3
þ
), 1271s (t(C–O,C–N)); CHN analysis for
þ
C H N O (found/calculated): 61.66/61.89, 10.62/
10.39, 5.10/4.98.
3
29 58
2
8
2
2
þ
þ
CH NH3 ), 1.70–1.89 (p; 2H; CH NH3 ), 1.56–1.70 (m;
2
4.2.5.2. 5-Decyloxycarbonyloxypentylammonium-5-
decyloxycarbonyloxypentylcarbamate (3b). Mp = 48–
2
2
0
H; CH ), 1.47 (p; 4H; CH ), 1.18–1.35 (m; 18H; CH ),
2
2
2
1
.86 (t; 3H; J = 6.6 Hz; CH3); C NMR (75 MHz,
3
53 ꢁC, yield 62%; IR (nujol): t
3381w (t(N–H)),
), 1652w, 1591m, 1534m
(OC@OO)
max
CDCl ): d 156.8, 64.2, 41.0, 39.8, 31.9, 30.0, 29.6, 29.6,
2180wbr, 1742s (t
3
2
9.5, 29.3, 28.2, 27.1, 26.8, 23.0, 22.8, 14.1 ppm.
(dsNH3
þ
), 1269s (t(C–O,C–N)), 958m; CHN analysis for
C H N O (found/calculated): 63.58/64.04, 10.76/
10.75, 4.58/4.53.
3
3
66
2
8
4
.2.4.8. 6-Decylcarbamoyloxyhexylammonium-chlor-
ide (4h). Mp = 135–138 ꢁC, yield 80%; IR (CHCl ): t
3
max
3
453m (t(
), 2956m, 2929s, 2857s, 1706s (t
),
H
4.2.5.3. 5-Dodecyloxycarbonyloxypentylammonium-5-
dodecyloxycarbonyloxypentylcarbamate (3c). Mp = 56–
N–H)
þ
(C@ O1 )
1
616w (NH3 ), 1519s (d(N–H)), 1467m, 1378w;
þ
NMR (300 MHz, CDCl ): d 8.23 (s; 3H; NH3 ), 4.96
59 ꢁC, yield 43%; IR (nujol): t
3383m (t(N–H)),
3
max
(
CH O), 3.13 (q; 2H; J = 6.5 Hz; CH NH), 3.00 (t; 2H;
t; 1H; J = 5.5 Hz; NH), 4.02 (t; 2H; J = 6.3 Hz;
2177wbr, 1742s (t(
), 1653w, 1592m, 1533m
OC@OO)
1
(dsNH3 ), 1272s (t(C–O, C–N)), 1258w, 1168w, 953m; H
þ
2
2
þ
þ
J = 7.4 Hz; CH NH3 ), 1.70–1.83 (m; 2H; CH NH ),
2
NMR (300 MHz, CDCl +triethylamine (TEA)): d 10.57
2
3
.55–1.67 (m; 2H; CH ), 1.16–1.54 (m; 20H; CH ),
3
þ
1
0
(s; 3H; NH3 ), 4.60 (s; 1H; NH), 4.04 (t; 8H; J = 6.7 Hz;
þ
2
2
1
.87 (t; 3H; J = 6.6 Hz; CH3); C NMR (75 MHz,
3
CH COCH ), 2.99–3.11 (m; 2H; CH
2
2
NH3 ), 2.58–2.71
(t; 2H; J = 7.0 Hz; CH NH), 1.10–1.67 (m; 52H; CH ),
2 2
2
CDCl ): d 156.8, 64.4, 41.0, 39.8, 31.9, 30.0, 29.5, 29.3,
3
1
0.81 (t; 6H; J = 6.6 Hz; CH3); C NMR (75 MHz,
3
2
8.6, 27.3, 26.8, 26.0, 25.3, 22.6, 14.1 ppm.
CDCl +TEA): d 162.0, 155.3, 67.9, 67.7, 41.1, 31.8,
3
4
.2.4.9. 6-Undecylcarbamoyloxyhexylammonium-chlor-
30.1, 29.5, 29.4, 29.4, 29.2, 29.1, 28.5, 28.3, 25.6, 23.0,
22.5, 14.0 ppm; CHN analysis for C H N O (found/
calculated): 66.24/65.84, 11.22/11.05, 4.15/4.15.
ide (4i). Mp = 134–137 ꢁC, yield 91%; IR (CHCl ): t
452m (t(
3
max
37 74
2
8
3
), 2928s, 2856s, 1707s (t
), 1613w
(C@O)
N–H)

7678
T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 7671–7680
4
.2.5.4.
6-Octyloxycarbonyloxyhexylammonium-6-
octyloxycarbonyloxyhexylcarbamate (3d). Mp = 40–
4.2.5.11. 5-Decylcarbamoyloxypentylammonium-5-
decylcarbamoyloxypentylcarbamate (5e). Mp = 77–
4
2
1
7 ꢁC, yield 49%; IR (nujol): t
3326m (t(N–H)),
), 1683w, 1630w, 1556m,
79 ꢁC, yield 89%; IR (nujol): t
3362s (t(N–H)), 3350s
max
max
106wbr, 1748s (t
(t
1530s, 1493m, 1258s, 1144m; CHN analysis for
), 1687s (t
), 1641m (t
ꢀ ), 1574w,
(NHC@OO )
(
OC@OO)
(N–H)
as(C@O)
260s (t(C–O,C–N)), 950m; CHN analysis for
C H N O (found/calculated): 62.65/63.02, 10.68/
1
C H N O (found/calculated): 64.37/64.25, 11.35/
33 68
11.11, 9.25/9.08.
3
1
62
2
8
4
6
0.58, 4.89/4.74.
4
.2.5.5.
6-Decyloxycarbonyloxyhexylammonium-6-
decyloxycarbonyloxyhexylcarbamate (3e). Mp = 52–
4.2.5.12. 5-Undecylcarbamoyloxypentylammonium-5-
undecylcarbamoyloxypentylcarbamate (5f). Mp = 77–
5
2
1
5 ꢁC, yield 55%; IR (nujol): t
3239m (t(N–H)),
), 1650w, 1631w, 1579m,
80 ꢁC, yield 85%; IR (nujol): t
3355ws (t(N–H)),
), 1617m, 1537s,
max
max
192wbr, 1748s (t
1686s (t
), 1650w (t
ꢀ
(
OC@OO)
as(C@O)
(NHC@OO )
268s (t(C–O,C–N)); CHN analysis for C H N O
1496m, 1257m, 1143m; CHN analysis for C H N O
35 72 4 6
35
70
2
8
(
found/calculated): 64.42/64.98, 10.96/10.91, 4.35/4.33.
(found/calculated): 65.14/65.18, 11.43/11.25, 8.73/8.69.
4.2.5.6. 6-Dodecyloxycarbonyloxyhexylammonium-6-
dodecyloxycarbonyloxyhexylcarbamate (3f). Mp = 54–
4.2.5.13. 5-Dodecylcarbamoyloxypentylammonium-5-
dodecylcarbamoyloxypentylcarbamate (5g). Mp = 76–
5
2
1
7 ꢁC, yield 54%; IR (nujol): tmax 3239m (t(N–H)),
80 ꢁC, yield 90%; IR (nujol): t
3364s (t(N–H)), 3353s
max
192wbr, 1748s (t ), 1649w, 1630w, 1577m,
(t
), 1687s (t
), 1652m (t
ꢀ ), 1617m,
(NHC@OO )
1576w, 1532s, 1493m, 1249m, 1143m; CHN analysis
(
OC@OO)
(N–H)
as(C@O)
1
277s (t(C–O,C–N)); H NMR (300 MHz, CDCl +pyri-
3
þ
dine (Py)): d6.91 (s; 3H; NH3 ), 4.64 (s; 1H; NH),
for C H N O (found/calculated): 66.20/66.03, 11.77/
6
37
76
4
4
J = 6.7 Hz; CH
.05 (t; 8H; J = 6.5 Hz; CH COCH ), 3.00 (t; 2H;
þ
11.38, 8.50/8.32.
2
2
2
NH3 ), 2.71 (t; 2H; J = 7.0 Hz;
CH NH), 1.14–1.67 (m; 56H; CH ), 0.82 (t; 6H;
4.2.5.14. 6-Decylcarbamoyloxyhexylammonium-6-dec-
ylcarbamoyloxyhexylcarbamate (5h). Mp = 86–87 ꢁC,
yield 83%; IR (nujol): tmax 3331s (t(N–H)), 3246w
(t(N–H)), 2210wbr, 1684s (tas(C@O)), 1655w (t
1629w, 1578m, 1537s, 1529s, 1149m; CHN analysis for
2
2
1
3
J = 6.5 Hz; CH3); C NMR (75 MHz, CDCl , Py): d
3
1
2
2
62.2, 155.2, 67.9, 67.5, 41.3, 40.0, 31.8, 30.2, 29.5,
9.5, 29.4, 29.4, 29.2, 29.1, 28.5, 28.4, 26.5, 26.2,
5.6, 25.4, 25.2, 22.5, 14.0 ppm; CHN analysis for
ꢀ ),
(NHC@OO )
C H N O (found/calculated): 66.13/66.63, 11.48/
8
C H N O (found/calculated): 65.76/65.18, 11.32/
6
3
9
78
2
35 72
4
1
1.18, 3.88/3.98.
11.25, 8.58/8.69.
4
.2.5.7. 4-Octylcarbamoyloxybutylammonium-4-octyl-
4.2.5.15. 6-Undecylcarbamoyloxyhexylammonium-6-
undecylcarbamoyloxyhexylcarbamate (5i). Mp = 82 ꢁC,
carbamoyloxybutylcarbamate (5a). Mp = 83–86 ꢁC,
yield 72%; IR (nujol): tmax 3366m (t(N–H)), 3337s
t(N–H)), 3272wbr, 2183wbr, 1682s (t(C@O)), 1622w,
yield 84%; IR (nujol): t
3329s (t
), 3245m
), 1629w,
max
(N–H)
(
(t(N–H)), 1684s (tas(C@O)), 1655m (t
ꢀ
(NHC@OO )
1
1
584m, 1563m, 1538m, 1523m (d(NH)), 1276m,
1578m, 1538s, 1255s, 1149m; CHN analysis for
C H N O (found/calculated): 65.47/66.03, 11.45/
11.38, 8.28/8.32.
249m, 1150m; CHN analysis for C H N O
2
7
56
4
6
37 76
4
6
(
found/calculated): 60.80/60.87, 10.68/10.59, 11.01/
0.52.
1
4
.2.5.16. 6-Dodecylcarbamoyloxyhexylammonium-6-
dodecylcarbamoyloxyhexylcarbamate (5j). Mp = 74–
76 ꢁC, yield 87%; IR (nujol): t 3332s (t(N–H)), 3246s
4
.2.5.8. 4-Nonylcarbamoyloxybutylammonium-4-non-
ylcarbamoyloxybutylcarbamate (5b). Mp = 80–83 ꢁC,
yield 80%; IR (nujol): tmax 3365m (t(N–H)), 3336s (t
max
(t
), 1684s (t
), 1655w (t
ꢀ ), 1615w,
(NHC@OO )
(N–
(N–H)
as(C@O)
H)), 3270wbr, 2180wbr, 1683s (t(C@O)), 1657w, 1625w,
1577m, 1538s, 1251s, 1149m; CHN analysis for
C H N O (found/calculated): 66.35/66.81; 11.59/
11.50; 7.86/7.99.
1
585m, 1563m, 1539m, 1522m (d(NH)),1270m, 1244m,
39
80
4
6
1
ed): 61.67/62.11, 11.09/10.78, 10.40/9.99.
149m; CHN analysis for C H N O (found/calculat-
2
9
60
4
6
4
.3. Skin preparation
4.2.5.9. 4-Decylcarbamoyloxybutylammonium-4-dec-
ylcarbamoyloxybutylcarbamate (5c). Mp = 83–86 ꢁC,
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 min 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.
yield 78%; IR (nujol): tmax 3369m (t(N–H)), 3341s (t
(N–
H)), 3274wbr, 2184wbr, 1683s (t(C@O)), 1656w, 1625w,
1
584m, 1563m, 1538m, 1522m (d(NH)), 1257m, 1238m,
1
ed): 63.03/63.23, 11.28/10.95, 9.95/9.51.
149m; CHN analysis for C H N O (found/calculat-
3
1
64
4
6
4.2.5.10. 4-Undecylcarbamoyloxybutylammonium-4-
undecylcarbamoyloxybutylcarbamate (5d). Mp = 73–
4.4. Donor samples
7
3
1
1
6 ꢁC, yield 65%; IR (nujol): t
3367m (t(N–H)),
max
338s (t(N–H)), 3274wbr, 2182wbr, 1684s (t(C@O)),
654, 1623w, 1582m, 1538m, 1521m (d(NH)), 1254m,
233w, 1148m; CHN analysis for C H N O (found/
Donor samples were prepared by suspending theophyl-
line (5%) and/or the tested enhancer (1%) in water
(W), propylene glycol/water 3:2 (Pg/W) or isopropyl
myristate (IPM). The suspensions were stirred for
3
3
68
4
6
calculated): 63.79/64.25, 11.22/11.11, 9.47/9.08.

T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 7671–7680
7679
5
min at 50 ꢁC and then were left at 37 ꢁC for 24 h to
plot. The ER value was calculated as the ratio of the flux
of theophylline with an enhancer and the flux of the per-
meant alone.
equilibrate. Before application to the skin, the samples
were carefully resuspended.
Supernatants from the Pg/W donor samples, prepared
as described above, were filtered through 0.22 lm filter.
The filtrate was appropriately diluted with the mobile
phase and analyzed for theophylline solubility by
HPLC. Each sample was analyzed four times.
The data are presented as means ± SD (n = 4–6) ob-
tained using the skin fragments from at least two ani-
mals. The statistical significance of the differences was
analyzed using Student’s t-test. A value of p < 0.05
was considered significant.
Solubility of the enhancers in the donor sample was
determined by a potentiometric titration. The pertinent
enhancer (1%) was dispersed in Pg/W (3:2) and treated
like the donor samples for the permeation experiment.
After equilibration, the supernatants from the samples
were filtered through 0.22 lm filter. Six microliters of
each filtrate was diluted with 10 mL of water, acidified
to pH 3 by 0.1 M HCl, and titrated with 0.1 M NaOH
to pH 10. Enhancer concentration was calculated from
the consumption of NaOH between two inflections.
For each enhancer, three samples were separately pre-
pared and analyzed.
Acknowledgments
This work was supported by the Centre for New
Antivirals and Antineoplastics (1M0508), the Grant
Agency of the Czech Republic (203/04/P042), and
the Ministry of Education of the Czech Republic
(
MSM0021620822).
References and notes
4
.5. Permeation experiments
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. Purdon, C. H.; Azzi, C. G.; Zhang, J.; Smith, E. W.;
The skin permeability was evaluated in vitro using the
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meant. Skin samples ca 2 · 2 cm (obtained from the
same porcine ear for one experiment) were mounted
into the Franz diffusion cells to leave an area of
1
Maibach, H. I. Crit. Rev. Ther. Drug Carrier Syst. 2004,
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3
4
2
cm . The acceptor compartments of the cells were
filled with ca 17 mL of phosphate-buffered saline, pH
.4, with 0.03% sodium azide as a preservative and al-
7
5. Hrab a´ lek, A.; Dole zˇ al, P.; Farsa, O.; Krebs, A.; Kroutil,
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volume of the acceptor phase in each cell was mea-
sured and included into calculation. Then 200 lL of
a donor sample was applied and occluded with a cov-
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at 32 ꢁC throughout the experiment. Samples of the
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vals during 48 h.
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38.
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.6. HPLC determination
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1
1
1
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was determined by HPLC using the LCP high-pressure
pump (ECOM, Prague, Czech Republic), autosampler
(
4
ECOM, Prague, Czech Republic), LiChroCART 250-
column (LiChrospher 100, RP 18. 5 lm, Merck,
Darmstadt, Germany), an SP 8440 UV detector (Spectra
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0
.1 M NaH PO 6:4 v/v was used as the mobile phase
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ment, was plotted against time. The steady-state flux
351.
ˇ
1
2
(
lg/cm /h) was calculated from the linear region of the

7680
T. Holas et al. / Bioorg. Med. Chem. 14 (2006) 7671–7680
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2
2