Cycloserine fatty acid derivatives as prodrugs: Synthesis, degradation and in Vitro skin permeability

Article abstract of DOI:10.1248/cpb.50.554

Various 4,5-dihydroisoxazol-3-yl fatty acid ester derivatives of cycloserine were synthesized to improve skin permeation of cycloserine. The ester derivatives were prepared by using the tert-butoxycarbonyl (t-Boc) protection strategy. The 4,5-dihydroisoxazol-3-yl esters were readily hydrolysed in an aqueous buffer solution, and the degradation profiles showed both specific acid and specific base catalysis. In 50% human serum the formation of cycloserine was observed, but enzymatic catalysis was limited. Delivery through hairless mouse skin was investigated, and the apparent permeability coefficient was measured based on the flux of cycloserine into the receptor phase. The skin permeation of cycloserine across the hairless mouse skin was increased up to 20-fold by the fatty acid esters. The 4,5-dihydroisoxazol-3-yl fatty acid esters of cycloserine can therefore be considered as new topical prodrugs with the potential use in treatment of various skin infections.

Full text of DOI:10.1248/cpb.50.554

554
Notes
Chem. Pharm. Bull. 50(4) 554—557 (2002)
Vol. 50, No. 4
Cycloserine Fatty Acid Derivatives as Prodrugs: Synthesis, Degradation
and in Vitro Skin Permeability
a
Thorsteinn THORSTEINSSON, Már MÁSSON, ,a Tomi JARVINEN,b Tapio NEVALAINEN,b and
Thorsteinn LOFTSSON
*
a
Faculty of Pharmacy, University of Iceland,a Hagi, Hofsvallagata 53, P.O. Box. 7210, IS-127 Reykjavik, Iceland and
Department of Pharmaceutical Chemistry, University of Kuopio,b P.O. Box 1627, FIN-70211 Kuopio, Finland.
Received December 21, 2001; accepted January 22, 2002
Various 4,5-dihydroisoxazol-3-yl fatty acid ester derivatives of cycloserine were synthesized to improve skin
permeation of cycloserine. The ester derivatives were prepared by using the tert-butoxycarbonyl (t-Boc) protec-
tion strategy. The 4,5-dihydroisoxazol-3-yl esters were readily hydrolysed in an aqueous buffer solution, and the
degradation profiles showed both specific acid and specific base catalysis. In 50% human serum the formation of
cycloserine was observed, but enzymatic catalysis was limited. Delivery through hairless mouse skin was investi-
gated, and the apparent permeability coefficient was measured based on the flux of cycloserine into the receptor
phase. The skin permeation of cycloserine across the hairless mouse skin was increased up to 20-fold by the fatty
acid esters. The 4,5-dihydroisoxazol-3-yl fatty acid esters of cycloserine can therefore be considered as new topi-
cal prodrugs with the potential use in treatment of various skin infections.
Key words prodrug; cycloserine; permeability; hydrolysis; hairless mouse skin
Cycloserine (Seromycin, 4(R)-amino-3-isoxazolidinone) is Results and Discussion
a well-known drug used for the treatment of tuberculosis and
All the compounds were synthesized according to the gen-
certain genitourinary infections, especially when caused by eral procedure described in Chart 1. The amino function of
Enterobacteria or E. coli.1) The drug was first isolated and cycloserine was protected with tert-butoxycarbonyl (t-Boc)
tested2) in 1955, but for the past few years it has also been (60% yields), and the 4,5-dihydroisoxazol-3-yl esters of
used in clinical trials against Alzheimer disease and to in- acetic acid, octanoic acid, lauric acid and oleic acid were
crease learning capabilities in animals.3) Systemic side ef- obtained by the 1-ethyl-3-(3-dimethylaminopropyl)-carbodi-
fects restrict the usage of cycloserine and topical application imide (EDAC) coupling strategy or reaction with the corre-
for local delivery has limited value because cycloserine is a sponding acetic anhydride (47—93% yields), followed by re-
highly water-soluble compound and poorly permeable moval of the protection group with trifluoroacetic acid (TFA)
through lipophilic membranes such as the skin. Only a few (69—81% yields). The prodrug structures were confirmed by
prodrugs of cycloserine have previously been synthesized for 1H- and 13C-NMR.
various purposes, such as improving biological effects,4) pre-
Cycloserine (1) is relatively stable in the pH range investi-
venting dimerization in water,5) enhancing delivery through gated. No degradation at 60 °C was observed over 24 h (Table
biological membranes6) and for possible use as anticancer 1). All the protected compounds 3—6 showed similar pH/
drugs.7) These prodrugs were based on chemical modification chemical stability dependence with specific base catalysis
of the nitrogen atoms. In the present study we have synthe- above pH 5 and the maximum stability between pH 3 and 5
sized fatty acid derivatives on the 3-oxo group yielding a (Fig. 1). The pH rate profiles for compounds 7—10 were
more lipophilic prodrugs. Previously, we have synthesized similar. The ester derivatives were formed on an enol form of
fatty acid derivatives of metronidazole, and we were able to the relatively acid 3-isoxazolidone (pKaϭ4.4).1) The hydroly-
achieve a 40-fold increase in the delivery of the drug through sis of the prodrugs was therefore rapid (Fig. 2). In the case of
hairless mouse skin.8) In this case, the fatty acids were used cycloserine laurate the neutral hydrolysis was about 10 times
as transport moieties to achieve increased absorption of the
drug. An additional benefit of the use of fatty acids as trans-
port moieties is that fatty acids can act as penetration en-
hancers, and the prodrug can therefore facilitate its own ab-
sorption. Furthermore, fatty acids can also have some biolog-
ical activity of their own, especially antimicrobial activity.9)
Using a fatty acid as a transport moiety can therefore be ex-
pected to potentiate the activity of an antimicrobial drug.
In the present study we report investigations of 4,5-dihy-
droisoxazol-3-yl fatty acid esters of cycloserine, as possible
prodrugs to enhance dermal delivery of cycloserine. System-
atic side effects of the oral drug treatment of certain skin in-
fections could therefore be avoided by topical application
with cycloserine prodrugs. For example Mycobacterium
avium skin infection in the dermis, which is currently
treated10) by surgery, followed by antibiotic treatment with
combination of cycloserine, isoniazid and clarithromycin.
Chart 1. Total Synthesis of the Prodrugs
© 2002 Pharmaceutical Society of Japan
To whom correspondence should be addressed. e-mail: mmasson@hi.is

April 2002
555
Table 1. Rate of Degradation (MeanϮS.E.; nϭ3) of Compounds 2—10 in Table 2. The Apparent Permeability Coefficients (MeanϮS.E.; nϭ3) and
Aqueous Buffers (pH 2.0, pH 7.0) at 60 °C Calculated log P Values for Compounds 1—10
kobs. (hϪ1
pH 7.0
)
kobs. (hϪ1
pH 2.0
)
Estimateda)
Permeability
Compounds
Compounds
MW (g/mol)
log P
(cm/h)ϫ103
1
2
3
4
5
6
7
8
9
No degr.
Not determ.
12.2Ϯ1.1
No degr.
0.82Ϯ0.14
2.69Ϯ0.01
2.08Ϯ0.08
0.42Ϯ0.05
Not determ.
0.82Ϯ0.14
0.93Ϯ0.09
1.43Ϯ0.21
1.25Ϯ0.12
1
2
3
4
5
6
7
8
9
102
202
244
328
400
466
144
228
284
366
Ϫ1.7
Ϫ0.3
0.5
3.3
4.9
7.1
Ϫ0.8
1.9
3.6
5.7
0.5Ϯ0.4
Not determ.
1.3Ϯ0.6
5.5Ϯ2.7
9.0Ϯ1.7
11.9Ϯ4.4
1.7Ϯ0.5
3.5Ϯ1.1
4.5Ϯ1.1
6.2Ϯ1.6
15.2Ϯ1.2
1.44Ϯ0.28
1.03Ϯ0.32
1.06Ϯ0.26
1.33Ϯ0.39
4.26Ϯ1.30
3.99Ϯ1.62
10
10
a) log P calculated with CS Chemdraw Ultra from CambridgeSoft.
the electron-deficient 4,5-dihydroisoxazoline ring system are
poor substrates for hydrolytic enzymes. In contrast, there was
more than a 100% increase in the degradation rate of metron-
idazolyl fatty acid esters in the presence of human serum.8)
Compound 1 had activity against Staph. aureus with mini-
mum inhibition concentration (MIC)ϭ32 mg/ml and mini-
mum lethal concentration (MLC)ϭ64 mg/ml, and there was
also activity against E. coli (MICϭ16 mg/ml). Anti-bacterial
activity was not present in the derivatives (MICϾ500 mg/ml).
Thus, the derivatives could be considered true prodrugs,
lacking biological activity of the parent compound.
When the prodrugs where applied to hairless mouse skin
in a propylene glycol donor phase the parent drug appeared
in the receptor phase. The cycloserine prodrugs were rather
unstable and therefore three types of mechanism could be
considered: degradation of the prodrug in the donor phase
and subsequent permeation of the parent drug through the
skin; permeation of the prodrug through the skin and degra-
dation in the receptor phase; or degradation of the prodrug
within skin tissue. The first mechanism could not explain the
presence of cycloserine in the receptor phase as no degrada-
tion of prodrug in the donor phase was observed after 48 h.
The second mechanism is unlikely as no trace of prodrug
(less than 0, 1 mg/ml) could be measured in the receptor
phase. Therefore it was assumed that the main mechanism
was degradation of prodrug within the skin. The apparent
permeation coefficient was calculated on the basis of flux of
cycloserine into the receptor phase. The presence or absence
of the t-Boc protection group did not have a significant effect
on permeation through the skin. Thus, the compounds with
the protection group could also be considered as prodrugs for
dermal delivery. The permeability coefficient increased 2 to
20-fold, relative to compound 1 (Table 2). The enhanced per-
meability correlated with increasing lipophilicity (calculated
log P values), whereas the differences in degradation rates
(Table 1) where much smaller and did not correlate with the
permeability differences. When the prodrugs degraded
within the skin the fatty acid component, which has absorp-
tion enhancing properties, was released. The absorption en-
hancing effects of the free fatty acids could also have con-
tributed to the fact that the permeation was higher with fatty
acid derived prodrugs (compounds 4—6, 8—10) than with
acetic acid derived prodrugs (compounds 3, 7). However a
larger study will be required to quantify the contribution of
this absorption enhancement.
Fig. 1. The pH-Degradation Rate Profile of Compounds 3 (᭺), 4 (ᮀ), 5
(᭹) and 6 (᭿) at 60 °C
Fig. 2. Degradation of Prodrug 10 (᭹) and Formation of Cycloserine (᭺)
in Buffer Solution (pH 8.0) at 60 °C
faster than the hydrolysis of the prodrug metronidazolyl lau-
rate.8)
The enzymatic hydrolysis of the prodrugs by bovine serum
esterase was investigated. In the presence of bovine serum
esterase there was no increase in the hydrolysis rate relative
to chemical hydrolysis in buffer solutions. However, 50%
human serum did have some catalytic effect, with a 14% in-
crease (kobs.ϭ0.133 hϪ1) in the formation of cycloserine from
compound 10 relative to the reference solution (kobs.ϭ0.116
hϪ1). Thus, it could be concluded that the fatty acid esters of

556
Vol. 50, No. 4
(7) 4-Amino-4,5-dihydroisoxazol-3-yl Acetate (Cycloserine Acetate):
Clear oil (74% yield). 1H-NMR (DMSO-d6) d: 6.74 (bs, 2H), 3.93 (bs, 2H),
3.75 (m, 1H), 2.39 (s, 3H). 13C-NMR (DMSO-d6) d: 171.8, 153.5, 64.4,
51.6, 22.8.
Conclusion
The present study describes the synthesis of 4,5-dihy-
droisoxazol-3-yl fatty acid esters of cycloserine. These ester
derivatives are prodrugs that can improve cycloserine skin
(8) 4-Amino-4,5-dihydroisoxazol-3-yl Octate (Cycloserine Octanoate):
1
permeation up to 20-fold as compared to the unmodified par- Yellowish oil (81% yield). H-NMR (DMSO-d6) d: 6.75 (bs, 1H), 3.92 (bs,
2H), 3.76 (m, 1H), 2.00 (m, 2H), 1.48 (m, 2H), 1.24 (s, 8H), 0.86 (t, 3H,
ent drug. These prodrugs can therefore be considered for
local delivery of cycloserine, especially for the treatment of
skin infections. Thus the systematic side effects of cycloser-
ine could be avoided.
Jϭ3.75 Hz). 13C-NMR (DMSO-d6) d: 171.7, 153.5, 64.5, 51.7, 35.1, 31.3,
28.9, 28.6, 23.5, 22.3, 13.8.
(9) 4-Amino-4,5-dihydroisoxazol-3-yl Laurate (Cycloserine Laurate):
White crystals (69% yield); mp 38—41 °C. 1H-NMR (DMSO-d6) d: 4.07
(bs, 1H), 3.37 (bs, 4H), 1.50 (m, 2H), 1.20 (bs, 16H), 0.85 (t, 3H,
Jϭ7.5 Hz). 13C-NMR (DMSO-d6) d: 171.6, 153.4, 64.6, 51.7, 31-22, 13.9.
(10) 4-Amino-4,5-dihydroisoxazol-3-yl Oleate (Cycloserine Oleate):
White crystals (76% yield); mp 36—39 °C. 1H-NMR (DMSO-d6) d: 5.31
(bs, 2H), 4.17 (bs, 2H), 3.76 (s, 1H), 3.43 (bs, 2H), 1.98, 1.97 (2s, 4H), 1.50
(m, 2H), 1.24 (bs, 20H), 0.85 (t, 3H, Jϭ7.5 Hz). 13C-NMR (DMSO-d6) d:
167.3, 164.7, 129.5, 72.8, 53.0, 40-22, 13.8.
Experimental
General Methods 1H- and 13C-NMR spectra were measured on either a
400 MHz Bruker AM 400 spectrometer or a 250 MHz Bruker AC 250 P
NMR spectrometer with tetramethylsilane (Me4Si) as an internal reference
and CDCl3 or D2O as a solvent. Both 1H- and 13C-NMR spectral data are re-
ported in parts per million (d) relative to Me4Si.
1
(1) 4-Amino-3-isoxazolidone (Cycloserine): H-NMR (D2O) d: 4.54 (dd,
HPLC Measurements The HPLC system consisted of a Merck Hitachi
L-7100 gradient solvent delivery system with a L-7400 UV detector. For all
sample separations a Waters reverse-phase C8 column (150ϫ4.6 mm, 5 mm)
was used. The detection was done at a 230-nm wavelength, and the flow rate
was 1.0 ml/min. The mobile phases consisted of MeOH and 0.015% octan-
sulfonic acid in H2O. The volume fractions and retention times for each
compound were as follows: (1) 10 (MeOH) : 90 (0.015% octansulfonic acid)
(250 mm column), 3.0 min; (2) (15 : 85), 3.2 min; (3) (60 : 40), 3.2 min; (4)
(60 : 40), 3.0 min; (5) (65 : 35), 2.8 min; (6) (70 : 30), 3.0 min; (7) (34 : 66),
3.1 min; (8) (40 : 60), 2.9 min; (9) (60 : 40), 3.8 min; (10) (65 : 35), 3.3 min.
Degradation Rate Studies The degradation rates of the compounds
were determined in Theorell–Stenhagen buffer systems11) (acetate, phos-
phate, borate, NaOH; pH values from 2 to 10) at 60 °C. The rate constants
(kobs.) were obtained by linear regression of the logarithm of HPLC peak in-
tensity. The enzymatic degradation was studied with porcine liver esterase
(Sigma numbers E-2884) in a 20 mM phosphate buffer (pH 7.4) at 37 °C. A
blank medium with no enzyme was used as a control. Degradation in 50%
human serum was studied. A blank medium with no human serum was used
as a control.
Antibacterial Test The antibacterial tests were performed at the Ice-
landic National Hospital, Department of Microbiology. The bacteria in a
growth media were exposed to diluted solutions of the compounds; the MIC
and MLC were measured according to the dilution series and expressed in
mg/ml of compound needed. The reaction was incubated for 24 h at 37 °C.
This method was developed by NCCLS (National Committee for Clinical
Laboratory Standards).12) The bacterial strains, Enterococcus faecalis
(ATCC 29212), Staph. aureus (ATCC 25923), E. coli (ATCC 25922) and
Pseudomonas aeruginosa (ATCC 27853) were chosen because they are the
most common cause of infection at hospitals. ATCC is American Type Cul-
ture Collection of standard bacteria strains.
1H, Jϭ8, 8 Hz, CHAH), 4.32 (dd, 1H, Jϭ8, 8 Hz, CH), 4.22 (dd, 1H, Jϭ8,
8 Hz, CHHB). 13C-NMR (D2O) d: 173.8 (CϭO), 73.5 (CH), 57.5 (CH2).
(2) N-tert-Butoxycabonyl-4-amino-3-isoxazolidone (t-Boc-Cycloserine):
2.00 g (19 mmol) of compound 1 dissolved in 50 ml of 50% aq. tetrahydrofu-
ran (THF), 2.10 g (20 mmol) of triethylamine added to the solution. 4.10 g
(19 mmol) of di-tert-butyldicarbonate dissolved in 15 ml of THF was added
dropwise to the solution over a period of 1 h and stirred for 5 h at room tem-
perature. After removal of the solvent in vacuum, the compound was puri-
fied by silica gel column chromatography. Elution with ethyl acetate/acetone
mixture (4 : 1) gave 2.30 g of white crystals (58% yield). mp 134—136 °C.
1H-NMR (CDCl3) d: 5.37 (bs, 1H, NH), 4.76 (t, 1H, Jϭ8 Hz, CHAH), 4.65
(bs, 1H, CH), 4.10 (t, 1H, Jϭ8 Hz, CHHB), 1.49 (s, 9H, CH3). 13C-NMR
(CDCl3) d: 171.4 (CϭO), 156.4 (CϭO), 79.8 (C), 75.5 (CH), 53.5 (CH2),
26.3 ((CH3)3).
(3) N-tert-Butoxycarbonyl-4-amino-4,5-dihydroisoxazol-3-yl Acetate (t-
Boc-Cycloserine-Acetate): 2.01 g (10 mmol) of compound 2, 1.30 g (13
mmol) of acetic acid anhydride and 100 mg of 4-(dimethylamino)pyridine
(DMAP) was dissolved in 30 ml of CH2Cl2 and stirred at room temperature
overnight. The solution was evaporated, and the crude was purified by silica
gel chromatography. Elution with ethyl acetate afforded 1.94 g of white crys-
tals (79% yield); mp 86—89 °C. 1H-NMR (CDCl3) d: 5.34 (bs, 1H), 4.69
(bs, 2H), 4.12 (dd, 1H, Jϭ15, 12 Hz), 2.39 (s, 3H), 1.39 (s, 9H). 13C-NMR
(CDCl3) d: 166.7, 163.4, 155.0, 81.1, 71.6, 53.5, 28.1, 22.9.
General Procedure for Compounds 4—6 Compound 2 (between 5
and 10 mmol) and the carboxylic acid were dissolved in 25 ml of CH2Cl2 in
the presence of 10% excess of EDAC and 50 mg of DMAP and the solution
was refluxed overnight. The solution was then washed 3ϫ with 25 ml 5%
NaHCO3, and the solvent was evaporated in vacuo and the crude product
was purified by silica gel chromatography. Finally the compound was dried
under reduced pressure (0.02 mbar) to obtain solid material.
Permeation Studies Female hairless mice, (3CH/Tif h/h) obtained from
Bommice (Denmark) were sacrificed by cervical dislocation and their full-
thickness skins were removed. The outer surface of the skin was rinsed with
35% (v/v) methanol in water and subsequently with distilled water to re-
move any contamination. The skin was placed in Franz diffusion cells of
type FDC 400 15 FF (Vangard International Inc., U.S.A.). The receiver com-
partment had a volume of 12.3 ml. The surface area of the skin in the diffu-
sion cell was 1.77 cm2. The receptor phase consisted of phosphate buffer
saline pH 7.4 (Ph.Eur., 2nd Ed., VII.1.3.) containing 0.3% (w/v) Brij-58 to
ensure sufficient drug solubility in the receptor phase. The receptor phase
was sonicated under vacuum prior to usage to remove dissolved air. The skin
diffusion cells were stirred with a magnetic bar and kept at 37 °C by circulat-
ing water through an external jacket. The donor phase consisted of a
1 mg/ml solution of the prodrug in propylene glycol, which also prevent
degradations during the experiment. The solution in the donor chamber was
analyzed to determine degradation after 48 h. 2 ml of the donor phase were
applied to the skin surface and the donor chamber covered with parafilm.
Samples (100 ml) of receptor phase were removed from the cells at various
time intervals of up to 48 h and replaced with a fresh buffer solution. The
samples were kept frozen until analysed by HPLC. The apparent permeabil-
ity coefficient was calculated from the flux of cycloserine into the receptor
phase divided by the concentration of the prodrug in the donor phase.
(4) N-tert-Butoxycarbonyl-4-amino-4,5-dihydroisoxazol-3-yl Octanoate
(t-Boc-Cycloserine-Octanoate): (93% yield); mp 66—68 °C. 1H-NMR
(CDCl3) d: 5.48 (bs, 1H), 4.67 (bs, 2H), 4.13 (dd, 1H, Jϭ10, 8 Hz), 2.68 (t,
2H, Jϭ7.5 Hz), 1.60 (m, 2H), 1.38 (s, 8H), 1.22 (s, 9H), 0.82 (t, 3H,
Jϭ3.75 Hz). 13C-NMR (CDCl3) d: 166.8, 166.5, 155.0, 80.8, 71.4, 53.5,
28.0, 35.1, 31.4, 28.8, 28.7, 23.7, 22.3, 13.8.
(5) N-tert-Butoxycarbonyl-4-amino-4,5-dihydroisoxazol-3-yl Laurate (t-
1
Boc-Cycloserine-Laurate): White crystals (82% yield); mp 26—30 °C. H-
NMR (CDCl3) d: 5.13 (bs, 1H), 4.82 (t, 1H, Jϭ8 Hz), 4.74 (bs, 1H), 4.15
(dd, 1H, Jϭ11, 8 Hz), 2.78 (t, 2H, JϭHz), 1.68 (m, 2H), 1.46 (s, 9H), 1.26
(s, 16H), 0.88 (t, 3H, Jϭ8 Hz). 13C-NMR (CDCl3) d: 166.8, 166.4, 155.0,
80.9, 71.6, 53.6, 28.1, 35.3, 31.8, 29.6, 29.4, 29.1, 29.0, 28.8, 27.0, 23.8,
22.5, 14.0.
(6) N-tert-Butoxycarbonyl-4-amino-4,5-dihydroisoxazol-3-yl Oleate (t-
Boc-Cycloserine-Oleate): White crystals (47% yield); mp 36—39 °C. 1H-
NMR (CDCl3) d: 5.33 (bs, 1H), 4.77 (bs, 2H), 4.16 (dd, 1H, Jϭ12, 12 Hz),
2.76 (t, 2H, Jϭ7.5 Hz), 2.01, 1.99 (2s, 4H), 1.66 (m, 2H), 1.45 (s, 9H), 1.26
(bs, 20H), 0.87 (t, 3H, Jϭ7.5 Hz). 13C-NMR (CDCl3) d: 166.8, 166.4, 155.0,
129.9, 129.6, 81.0, 71.8, 53.6, 28.1, 35.3, 31.8, 29.6, 29.4, 29.2, 29.1, 29.0,
28.9, 28.8, 27.1, 27.0, 23.8 , 22.5, 14.0.
General Procedure for Compounds 7—10 One gram of the parent
compound with the t-Boc protection group was dissolved in 5 ml of CH2Cl2.
Then 3 ml of TFA was added to the solution and stirred at room temperature
for 1 h. The solution was evaporated, and the crude product was purified by
silica gel chromatography.
Acknowledgements The authors thank Dr. Karl G. Kristinsson and
Martha A. Hjálmarsdóttir at the Icelandic National Hospital, Department of
Microbiology for their antimicrobial analysis. This study was supported in

April 2002
557
part by a research grant from the Icelandic Research Council, University of
Iceland Research Fund and Academy of Finland.
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