Synthesis and transdermal penetration-enhancing activity of carbonic and carbamic acid esters - Comparison with transkarbam 12

Article abstract of DOI:10.1016/j.bmcl.2005.12.086

Transkarbam 12 (T12) is a novel transdermal penetration enhancer with high activity. Its polar head group is formed by carbamic acid salt that is unstable in acidic environment and releases CO₂. To find out whether this property influences its high activity, two series of compounds with CO ₂ stronger bound in the polar head have been prepared-carbonic and carbamic acid esters. The carbamate salt in the polar head was found to be essential for the enhancing activity and its decomposition in an acidic environment suggested relating to the mode of action of T12.

Full text of DOI:10.1016/j.bmcl.2005.12.086

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1981–1984
Synthesis and transdermal penetration-enhancing activity
of carbonic and carbamic acid esters—Comparison
with transkarbam 12
*
´
´
ˇ
´
´
´ˇ
ˇ
Jana Klimentova, Alexandr Hrabalek, Katerina Vavrova, Tomas Holas and Ales Kroutil
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
Heyrovske´ho 1203, 500 05 Hradec Kra´love´, Czech Republic
Received 3 November 2005; revised 20 December 2005; accepted 20 December 2005
Available online 30 January 2006
Abstract—Transkarbam 12 (T12) is a novel transdermal penetration enhancer with high activity. Its polar head group is formed by
carbamic acid salt that is unstable in acidic environment and releases CO2. To find out whether this property influences its high
activity, two series of compounds with CO2 stronger bound in the polar head have been prepared—carbonic and carbamic acid
esters. The carbamate salt in the polar head was found to be essential for the enhancing activity and its decomposition in an acidic
environment suggested relating to the mode of action of T12.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Over the past few years major advances have been made
in the field of transdermal drug delivery (TDD). Increas-
ing numbers of drugs are being added to the list of
therapeutic agents that can be delivered to the systemic
circulation via skin.1–3 The advantages of TDD are
well documented and include the following: avoidance
of first-pass metabolism by the liver, reduction of side
effects, extended duration of activity, reduction of
fluctuations of drug concentration in the blood and
convenient termination of drug administration.4
x-Amino acid derivatives form an important group
of transdermal penetration enhancers,10,11 for a review
´
´
of the structure–activity relationships, see Vavrova
et al.12 It has been found that a group of carbamic acid
salts derived from x-amino acid esters (so-called
transkarbams) have very high transdermal penetration-
enhancing activity. The most active substance amongst
this group was a carbamic acid salt formed by reaction
of 6-aminohexanoic acid dodecylester (DDEAC) with
CO2, termed transkarbam 1213 (T12, Fig. 1). Mode of
its enhancing action is unclear and the aim of this study
was to contribute to its elucidation.
The major limitation to TDD is the skin itself. The bar-
rier function of the skin is firmly attributed to the stra-
tum corneum (SC), the outermost layer of the skin
that is formed from several layers of dead cells embed-
ded in a lipid matrix.5 The use of chemical penetration
enhancers seems to be an effective way to reduce the bar-
rier properties of SC. Current research focuses particu-
larly on non-irritant, non-toxic6, and biodegradable7
enhancers. The activity of some of these compounds
arises from their interactions with the lipids of the SC,
especially with ceramides;8,9 however, the detailed mech-
anisms are not properly elucidated yet.
We assume that the mechanism of action of T12 is more
specific than simply increasing the drug partitioning into
the SC or drug solubility in the vehicle. Being a carbam-
ic acid salt, T12 is very unstable in a mildly acidic envi-
ronment. Thus, we hypothesize that in the SC, where
fatty acids comprise approximately 10% of the SC lipids
and the average pH is 5.5, its polar head would be
O
H
O
N
O
O
+
O
O
Keywords: Percutaneous absorption; Penetration enhancers; Carbon-
ate; Carbamate; Carbon dioxide.
H3N
*
Corresponding author. Tel.: +420 495 067 402; fax: +420 495 514
330; e-mail: alexandr.hrabalek@faf.cuni.cz
Figure 1. Transkarbam 12.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.086

´
J. Klimentova et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1981–1984
1982
decomposed releasing a molecule of CO2.7 The CO2 re-
lease in the SC could cause the disruption of the highly
ordered lipid lamellae: (a) by changing the hydrogen
bonding within the polar heads of the lipids and (b) by
conformational changes in the molecule of T12. Such
disordering of the lipid lamellae would consequently
result in an easier drug permeation through the skin
barrier. This theory is further supported by the fact
that the free amine DDEAC was inactive. For the
R1
O
O
O
R1
O
R2 NH2
R2
a
+
HO
N
H
n
n
n
80-93 %
1-4
C12H25
O
b, c
32-51 %
O
HO
O
n
5, 6
No.
1
n
R1
H
H
R2
No.
5
6
n
4
5
4
decyl
dodecyl
nonyl
conformational changes and phase behaviour of T12,
14
2
3
4
4
3
2
´
see Zbytovska et al.
H
CH3 dodecyl
To confirm that the lability of the CO2-containing polar
head of T12 is the reason for its exceptional activity, two
series of compounds with CO2 covalently bound in the
polar head, namely carbonic acid esters 7–20 (Fig. 2)
and carbamic acid esters 21–26 (Fig. 3), were prepared
from their precursors 1–6 and DDEAC. Their transder-
mal penetration-enhancing activities were evaluated
in vitro and compared with that of T12. The structures
were designed to resemble that of T12; the most similar
were compounds 19, 20, 25 and 26.
Scheme 1. (a) MeOÀNa+, 120 ꢁC, 1–2 h, (b) 1 M NaOH, reflux, 2 h, (c)
TBAI, dodecylbromide, 120 ꢁC, 2 h.
ysis of the appropriate lactones in the presence of cata-
lytic amount of sodium methoxide.15,16 The hydroxy
esters (5, 6) were prepared by the reaction of sodium salt
of the corresponding x-hydroxy acid with dodecyl bro-
mide in the presence of tetrabutylammonium iodide
(TBAI) as a phase transfer catalyst.17,18 The sodium
salts of x-hydroxy acids were prepared by the hydrolysis
of their lactones. The commercially unavailable x-hept-
anolide was obtained by the oxidation of cyclohepta-
none by H2O2.19
The target esters of carbonic and carbamic acid were pre-
pared by the reactions of hydroxy (1–6) or amino (DDE-
AC) compounds with the corresponding chloroformates.
Scheme 1 shows the preparation of the precursors 1–6.
The hydroxy amides (1–4) were prepared by the aminol-
Scheme 2 shows the synthesis of the products 7–26. The
carbonic acid esters 7–17 were prepared by adding the
commercially available alkyl chloroformate to the pyri-
dine solution of the hydroxy compounds 1–5.20 The
preparation of the carbamic acid esters 21–24 from
DDEAC was similar. DDEAC was prepared by decom-
position of T12 in CHCl3 prior to the reaction.21
O
R1
O
C12H25
X
O
X
4
O
X
R2
n
O
O
n
C12H25
O
O
R3
O
18-20
7-17
Symmetrical carbonates 18 and 19 were prepared by
adding twofold molar excess of the hydroxy compound
2 or 5 to the solution of trichloromethyl chloroformate
(diphosgene) in anhydrous tetrahydrofurane (THF).
Unsymmetrical carbonate 20 was prepared in two steps.
First, the commercially unavailable chloroformate was
prepared by adding 6 to the THF solution of an equimo-
lar amount of diphosgene22 and the resulting chlorofor-
No.
7
8
X
n
4
4
4
4
3
2
R1
H
H
H
H
H
CH3
R2
decyl
decyl
decyl
decyl
nonyl
dodecyl
dodecyl
dodecyl
dodecyl
R3
ethyl
octyl
decyl
dodecyl
ethyl
ethyl
NH
NH
NH
NH
NH
NH
9
10
11
12
13
14
15
NH
NH
NH
2
2
2
CH3
CH3
CH3
octyl
decyl
dodecyl
16
17
18
19
20
O
O
NH
O
4
4
3
3
4
H
H
dodecyl
dodecyl
ethyl
dodecyl
R: ethyl
O
a
octyl
decyl
dodecyl
7-17
R
1-5
+
Cl
O
67-80 %
O
c, d
b
Figure 2. Carbonic acid esters.
20
18, 19
6
2,5
20 %
63-66 %
O
e
O
21-24
+
R
DDEAC
O
Cl
O
C12H25
O
78-92 %
HN
O
4
HN
O C12H25
4
c, f
O
O
C12H25
n
O
O R
25, 26
5, 6
O
46-47 %
25, 26
No.
21-24
No.
R
n
Scheme 2. (a) Pyridine, À20 ꢁC for 1 h, room temperature (rt)
overnight, (b) diphosgene, THF, pyridine, À20 ꢁC for 2 h, rt overnight,
21 ethyl
22 octyl
23 decyl
24 dodecyl
25
26
3
4
(c) diphosgene, THF, activated charcoal, rt, 1.5 h adding
6 to
diphosgene, 1 h stirring, (d) 5, pyridine, À20 ꢁC for 1 h, rt overnight,
(e) CHCl3, pyridine, room temperature, 3 h, (f) DDEAC, CHCl3,
pyridine, rt overnight.
Figure 3. Carbamic acid esters.

´
J. Klimentova et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1981–1984
1983
mate reacted with the pyridine solution of 5 immediately
without any purification following the same procedure
as in the carbonates 7–17.
the contrary, when PG/W was used as the vehicle, the
ER value was an order of magnitude higher (22.8).
The large difference between its activities from these
two vehicles was surprising; no such difference was ob-
served in the previous study using the human cadaver
skin (ERs were 39.3 and 35.0, when applied in water
and PG/W, respectively).10 The compounds 7, 16, 17
and 25 were more active than T12 from aqueous suspen-
sions (p < 0.05).
The preparation of carbamates 25 and 26 was similar to
the previous one. In the first step, the unavailable chlo-
roformate was prepared from hydroxy esters 5 or 6 and
added to freshly prepared DDEAC in chloroform with
pyridine.
All these reactions were carried out under nitrogen
atmosphere. Yields, melting points, FT-IR, H NMR
However, the activities of the prepared compounds were
significantly lower than those of T12 in both PG/W and
IPM on porcine skin. PG and IPM are used widely in
dermatological formulations as solvents or co-solvents
for a wide range of drugs and they can influence the
activity of the enhancer24 as shown for T12. However,
no such effect was observed in the prepared compounds.
Their lack of activity in IPM and approximately an or-
der of magnitude lower ERs in PG/W supports our
hypothesis about the importance of the labile carbamate
group for the activity of transkarbams.
1
and 13C NMR spectra of the products and intermediates
are available as supporting information.
The transdermal penetration-enhancing activity was
evaluated in vitro using Franz diffusion cell and the-
ophylline as a model permeant. Porcine ear skin of full
thickness was employed. Donor samples (200 lL/cm2)
were prepared as 5% w/w suspensions of theophylline
with 1% w/w of the suspended enhancer in three donor
vehicles of different polarity: (a) water, (b) mixture of
propylene glycol/water 6:4 (PG/W) and (c) isopropyl-
myristate (IPM). Control samples were prepared like-
wise without the addition of enhancer. Theophylline
in the acceptor phase was determined by HPLC. The
detailed preparation of the skin, donor samples and
the theophylline determination have been described
elsewhere.23
Figure 4 represents cumulative amounts of theophylline
permeated through the porcine skin plotted against
time. In this case, PG/W was used as a donor medium
and T12 and two compounds with the most similar
structure (19 and 25) were used as enhancers. The per-
meation profiles clearly showed the difference between
an ammonium-carbamate salt (T12), carbonate (19)
and carbamate (25).
Cumulative amounts of theophylline (lg/cm2) were plot-
ted against time and fluxes were calculated (lg/cm2/h).
The transdermal penetration-enhancing activity was
then expressed as the enhancement ratio (ER), which
is the ratio of flux of theophylline from the donor
sample with the addition of the enhancer and without
the enhancer (control).
The ERs of the prepared compounds ranged from 0.77
to 3.37 and 0.47 to 3.70 in water and PG/W, respective-
ly. Using IPM as a donor medium, no significant
enhancing activity was detected. Because of the low
activities, there were no relevant structure–activity rela-
tionships within the group of the prepared enhancers.
The maximum activities were observed in the com-
pounds, where both chains contained ester or amide
group in contrast to those with one simple alkyl chain,
either carbonates (18, 19) or carbamates (25). On con-
trary, the lowest enhancing effect was achieved in the
compound 24 (carbamate with one simple dodecyl
chain), which could be considered to be a permeation
retardant with theophylline flux significantly lower than
Table 1 shows the ER values of selected compounds in
three vehicles of different polarity compared with T12.
Using porcine skin, T12 was only slightly active
(ER = 1.9) when applied in an aqueous suspension. On
Table 1. Enhancement ratios of selected compounds and T12
Compound
ER SD
PG/W
Water
IPM
1200
1000
800
600
400
200
0
control
T12
19
7
10
12
15
16
17
18
19
20
21
24
25
26
T12
3.37 0.66*,
1.13 0.14
1.48 0.50
1.70 0.48*
2.40 1.15
2.49 1.13*,
0.91 0.24
1.76 0.63
0.80 0.03
1.83 0.37*
0.77 0.16
2.27 0.36*,
1.22 0.72
1.90 0.27*
1.36 0.31
2.66 0.74*
1.45 0.89
1.11 0.19
2.13 0.03*
1.13 0.36
3.29 1.24*
3.70 1.14*
1.24 0.81
1.52 0.50
0.47 0.10
2.63 0.06*
1.02 0.46
22.80 1.08*
1.75 0.91
1.48 0.22*
1.77 0.54
1.15 0.17
1.07 0.37
1.29 0.57
1.23 0.36
1.39 0.38
1.49 0.68
1.01 0.12
0.57 0.20
1.29 0.56
1.07 0.47
6.56 0.52*
25
15
20
25
30
time [h]
35
40
45
50
Figure 4. Example permeation profiles of theophylline through
porcine skin using PG/W as a donor medium and T12 or its analogues
(19 and 25) as enhancers.
* p < 0.05 versus control.
p < 0.05 versus T12.

´
J. Klimentova et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1981–1984
1984
´
ˇ
13. Hrabalek, A.; Dolezal, P.; Farsa, O.; Krebs,A.; Roman,
that of control (ER < 1 in all three vehicles). However,
the mechanism of its retarding activity is not known
and the effect should be evaluated for a wider spectrum
of permeants.
ˇ
M.; Sklubalova, Z. U.S. Patent 6,187,938, 2001.
´
14. Zbytovska, J.; Raudenkolb, S.; Wartewig, S.; Hubner, W.;
´
¨
´
ˇ
Rettig, W.; Pissis, P.; Hrabalek, A.; Dolezal, P.; Neubert,
R. H. H. Chem. Phys. Lipids 2004, 129, 97.
15. Mizushima, H.; Takahashi, M.; Yamamuro, A.; Matsuo,
T.; Yahagi, K. U.S. Patent 5,599,483, 1997.
In conclusion, this study showed that the permeation-en-
hancing activity of T12 is connected with the ammoni-
um-carbamate polar head; the most similar carbonates
19 and 20 and carbamates 25 and 26 did not reach its
activity. The results suggest that the exceptional activity
of T12 is connected with the lability of its polar head and
very probably arises from its ability to release CO2 in SC.
Further studies are to be done to describe the detailed
interaction of T12 with SC lipids.
16. General procedure for the preparation of the hydroxy-
amides 1–4: To a mixture of lactone (17.5 mmol) and
n-alkyl amine (19.3 mmol), 1 M methanolic solution of
MeONa (0.1–0.2 mL) was added. The mixture was heated
to 120 ꢁC for 1–2 h. After cooling to room temperature,
the products were purified by precipitating the methanolic
or ethanolic solution with 10% HCl.
17. Barry, J.; Bram, G.; Decodts, G.; Loupy, A.; Pigeon, P.;
Sansoulet, J. Tetrahedron Lett. 1982, 23, 5407.
18. General procedure for the preparation of the hydroxy-
esters
5
and 6: sodium salt of x-hydroxyacid
Acknowledgments
(6.5 mmol) and TBAI (0.65 mmol) were suspended in
dodecylbromide (8.3 mmol) and heated to reflux for
2 h. After cooling to room temperature, the mixture
was diluted with diethyl ether (10 mL) and filtered. The
filtrate was dried with Na2SO4, filtered and evaporated.
The resulting yellowish oil was chromatographed on
SiO2 (n-hexane/EtOAc 3:2).
The work was financially supported by the Ministry of
Education of the Czech Republic (Project Nos. 715/
G6/2005 and MSM0021620822).
19. Heyne, H. W.; Jones, H. J. Am. Chem. Soc. 1951, 73, 1361.
20. General procedure for the preparation of the compounds
7–17: a solution of hydroxy compound 1–5 (1 mmol) in
anhydrous pyridine (5 mL) was cooled to À20 ꢁC and
alkyl chloroformate (1.25 mmol) was added dropwise. The
mixture was stirred at À20 ꢁC for 1 h and warmed to room
temperature overnight. Then it was diluted with CHCl3
(10 mL), washed with water (3· 5 mL) and the organic
layer was dried over Na2SO4 and filtered. The pyridine
was removed by azeotropic distillation with toluene in
vacuo. The residue was dried in vacuo over H2SO4 for 1
day. The crude material was chromatographed on SiO2
(mixtures of n-hexane/EtOAc) to give the desired
products.
21. General procedure for the preparation of the compounds
21–24: to a solution of T12 (0.6 mmol) in dry CHCl3
(5 mL) and pyridine (1 mL) alkyl chloroformate
(1.5 mmol) was added drop wise. The mixture was stirred
at room temperature for 3 h. Then it was diluted with
CHCl3 (10 mL), washed with water (3· 5 mL) and the
organic layer was dried over Na2SO4 and filtered. The
pyridine was removed by azeotropic distillation with
toluene in vacuo. The residue was dried in vacuo over
H2SO4 for 1 day. The crude material was chromato-
graphed on SiO2 (mixtures of n-hexane/EtOAc) to give the
desired products.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2005.12.086.
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