The synthesis and biological activity of lipophilic derivatives of bicine conjugated with N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP)an inhibitor of glucosamine-6-phosphate synthase

Article abstract of DOI:10.3109/14756366.2011.582039

A series of bis-N,N-(2-hydroxyethyl)glycine (bicine) derivatives, conjugated with an inhibitor of glucosamine-6-phosphate synthase, have been synthesized and their lipophilic and antifungal properties have been tested. The obtained compounds demonstrated higher lipophilicity than free inhibitor (FMDP) and, in consequence, an increased potential to cross the cytoplasmic membrane. All the tested compounds show better antifungal activity than parent compound.

Full text of DOI:10.3109/14756366.2011.582039

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(2): 167–173
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.582039
RESEARCH ARTICLE
e synthesis and biological activity of lipophilic derivatives
of bicine conjugated with N3-(4-methoxyfumaroyl)-l-
2,3-diaminopropanoic acid (FMDP)—an inhibitor of
glucosamine-6-phosphate synthase
Dominik Koszel, Izabela Łącka, Katarzyna Kozłowska-Tylingo and Ryszard Andruszkiewicz
Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology, Gdańsk, Poland
Abstract
A series of bis-N,N-(2-hydroxyethyl)glycine (bicine) derivatives, conjugated with an inhibitor of glucosamine-6-
phosphate synthase, have been synthesized and their lipophilic and antifungal properties have been tested. The
obtained compounds demonstrated higher lipophilicity than free inhibitor (FMDP) and, in consequence, an increased
potential to cross the cytoplasmic membrane. All the tested compounds show better antifungal activity than parent
compound.
Keywords: Antifungal agents, FMDP derivatives, prodrug
Introduction
synthase that are analogues of glutamine. Representative
Infections caused mainly by human pathogenic fungi
are regarded as one of the most important problems to
be solved in modern chemotherapy. Only a very limited
number of antifungal chemotherapeutics are in clini-
cal use.1 One of the possible solutions to overcome this
problem is to consider an exploitation of new antifungal
targets, for example enzymes involved in the biosyn-
thesis pathway of the fungal cell wall.2 Glucosamine-6-
phosphate synthase (GlcN-6-P synthase, EC 2.6.1.16)3
catalyses the formation of D-glucosamine-6-phosphate
and therefore is one of the enzymes required for the bio-
synthesis of glucosamine-containing cell wall macromo
lecules:lipopolysaccharides and peptidoglycan in bac-
teria and mannoproteins and chitin in fungi.4 Inhibition
of this enzyme in fungal cells leads to morphological
changes and lysis,5,6 whereas in mammalian cells tempo-
rary depletion of enzyme activity is not lethal due to long
half-life of GlcN-6-P synthase and rapid expression of the
genes encoding it.7 us, GlcN-6-P synthase has been
proposed as a potential target for designing new antimi-
crobial agents.8 ere are many inhibitors of GlcN-6-P
for this group of compounds is N3-(4-methoxyfumaroyl)-
l-2,3-diaminopropanoic acid (FMDP), one of the most
potent and selective inhibitors of the enzyme.9,10 FMDP is
a non-peptide amino acid, therefore is poorly transported
into fungal cells by rather specific amino acid permeases
and exhibits only moderate antifungal activity. High
polarity of the inhibitor effectively hampered it transport
inside the cells. In order to overcome this problem, we
have modified FMDP molecule, aimed at the construc-
tion latent and lipophilic derivatives that might be able
to penetrate into the cells by free diffusion. Following
uptake, the modifying group could be removed intracel-
lularly. Modification of the carboxyl group involves the
formation of latent esters, such as acetoxymethyl ester.11
at approach is very common in penicillin group.12
On the other hand, formation of prodrugs by modifica-
tion of the amino group is relatively rare and difficult.13
In our approach, we have tried to apply bicine, that is,
bis-N,N-(2-hydroxyethyl)glycine as an acylating agent
of FMDP molecule. Bicine amide underwent, at physi-
ological pH, a facilitated cyclization (with t1/2 = 3 h) to a
Address for Correspondence: Ryszard Andruszkiewicz, Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of
Technology, 11/12 Narutowicza Street, 80-233 Gdańsk, Poland. Tel: 48 58 3471493. Fax: 48 58 3471144. E-mail: ryszarda@chem.pg.gda.pl
(Received 23 November 2010; revised 29 March 2011; accepted 13 April 2011)
167

168 D. Koszel et al.
4-(2-hydroxyethyl)morpholin-2-one with the releasing of
ammonia molecule. Formation of the six-membered ring
is the driving force in this reaction. e t1/2 for hydrolysis
of glycinamide at physiological pH was determined to
be ~7 years.14 erefore, the amide bond between bicine
and FMDP should be cleaved as a result of intramolecu-
lar alcoholysis, thus leading to the formation of FMDP
and 4-(2-hydroxyethyl)morpholin-2-one. To increase
stability and lipophilicity of prodrug, esterification of
hydroxyl groups in bicine was carried out, according to
the published procedure.15 e fully modified FMDP
prodrug should penetrate into the fungal cells by free
diffusion, and inside, the ester bonds should be cleaved
by intracellular enzymes followed by cyclization of the
bicine residue to a 4-(2-hydroxyethyl)morpholin-2-one
and generation of free FMDP (Scheme 3). As a conse-
quence of that reaction, inhibition of fungal cells should
be observed. Moreover, we have also evaluated lipophi-
licity of the novel prodrugs by measuring their affinity to
biological membranes.
N NaHCO3 (3 × 10 mL), water (10 mL) and dried (MgSO4).
e solvent was evaporated in vacuo. Tert-butyl ester of
bis-N,N-(2-acyloxyethyl)glycine (2a–d) was further puri-
fied by silica gel column chromatography. Compounds
2a–d were treated with TFA for 4 h at room temperature.
Excess TFA was evaporated in vacuo, the residue was trit-
urated with diethyl ether and the precipitate was filtered
off and dried in vacuo over KOH pellets.
Bis-N,N-(2-acetoxyethyl)glycine tifluoroacetate (3a) Tert-
butyl ester of bis-N,N-(2-acetoxyethyl)glycine (2a) was
purified by column chromatography on silica gel (ethyl
acetate:petroleum ether, 1:7) to give 2a (0.25g, 91%).
Compound 3awas obtained as waxy solid (0.29g, 97%). 1H
NMR (200MHz, CDCl3): δ 2.05 (s, 6H, 2x(CH3C(O))); 3.01
(t, 4H, J=5.8 Hz, 2x(CH2N)); 3.41 (s, 2H, NH2C(O)); 4.15
(t, 4H, J=5.8 Hz, 2x(OCH2)). MS(FAB) m/z: 248 (MH+).
Bis-N,N-(2-propionyloxyethyl)glycine
tifluoroacetate
(3b) Tert-butyl ester of bis-N,N-(2-propionyl)glycine
(2b) was purified by column chromatography on silica
gel (ethyl acetate:petroleum ether, 1:8) to give 2b (0.23 g,
79%). Compound 3b was obtained as waxy solid (0.265 g,
95%).1H NMR (500 MHz, CDCl3): δ 1.13 (t, 6H, J= 7.5 Hz,
2x(CH3)); 2.33 (q, 6 H, J= 7.5 Hz, 2x(CH2)); 3.00 (m, 4H,
2x(CH2N)); 3.42 (s, 2H, NH2C(O)); 4.17 (m, 4H, 2x(OCH2)).
MS(FAB) m/z: 276 (MH+).
Experimental
Chemistry
All reagents were purchased from Aldrich Chemical
Co. in layer chromatography (TLC) was performed
using Merck aluminum backed plates (Kieselgel 60 F254
)
and visualized by ultraviolet (UV) light. Separations by
column chromatography were achieved using silica
gel (0.063–0.200 mm). MS spectrum was recorded on
a Quadrupolic Mass Spectrophotometer Trio-3 (FAB
Bis-N,N-(2-butyryloxyethyl)glycine
tifluoroacetate
(3c) Tert-butyl ester of bis-N,N-(2-butyryloxyethyl)
glycine (2c) was purified by column chromatography
on silica gel (ethyl acetate:petroleum ether, 1:10) to give
2c (0.23 g, 73%). Compound 3c was obtained as waxy
solid (0.269 g, 97%). 1H NMR (500 MHz, CDCl3): δ 0.94 (t,
6H, J= 7.4 Hz, 2x(CH3)); 1.66 (q, 4H, J= 7.4 Hz, 2x(CH2));
2.28 (t, 4 H, J= 7.4 Hz, 2x(CH2C(O))); 2.98 (t, 4H, J= 6 Hz,
2x(CH2N)); 3.39 (s, 2H, NH2C(O)); 4.13 (t, 4H, J= 6 Hz,
2x(C(O)CH2)). MS (FAB) m/z: 304 (MH+).
1
technique). H NMR spectra were recorded on a Varian
Unity Plus spectrometer operating at 500 MHz using
deuterated solvent (CDCl3). Chemical shifts are given
in part per million (ppm) relative to internal standard
tetramethylsilane.
Tert-butyl ester of bis-N,N-(2-hydroxyethyl)glycine (1)
A solution of tert-butyl bromoacetate (5.40 g, 27.70 mmol)
and diethanolamine (11.60 g, 110.80 mmol) in anhydrous
methylene chloride (DCM, 250 mL) was stirred at room
temperature for 18 h. e reaction mixture was washed
with water (4 × 50 mL), and the organic layer dried over
anhydrous sodium sulphate, followed by filtration and
removal of the solvent in vacuo to yield 1 (4.05 g, 67%). 1H
NMR (500 MHz, CDCl3): δ 1.45 (s, 9H, C(CH3)3), 2.89 (t,
4H, J= 4.4 Hz, 2x(CH2N)). MS (FAB) m/z: 220 (MH+).
Bis-N,N-(2-benzoyloxyethyl)glycine
tifluoroacetate
(3d) Tert-butyl ester of bis-N,N-(2-benzoyloxyethyl)
glycine (2d) was purified by column chromatography on
silica gel (ethyl acetate:petroleum ether, 1:12) to give 2d
(0.225 g, 58%). Compound 3d was obtained as waxy solid
1
(0.23 g, 92%). H NMR (500 MHz, CDCl3): δ 3.25 (t, 4H,
J= 5.8 Hz, 2x(CH2N)); 3.58 (s, 2H, NH2C(O)); 4.46 (t, 4H,
J= 5.8 Hz, 2x(C(O)CH2)); 7.36–7.58 (m, 6H,); 7.99–8.05
(m, 4H). MS (FAB) m/z: 372 (MH+).
General method for the preparation of bis-N,N-(2-
acyloxyethyl)glycine trifluoroacetate (3a–d)
General method for the preparation of FMDP acyloxyalkyl
esters (6a–f)
A
solution of compound 1 (0.20 g, 0.91 mmol),
4,4′-(dimethylamino)pyridine (DMAP, 0.11 g, 0.91
mmol) and appropriate carboxylic acid (0.91 mmol)
in anhydrous DCM (15 mL) was cooled to 0°C.
Dicyclohexylcarbodiimide (DCC, 0.206 g, 1.00 mmol)
was added and the reaction mixture was stirred at 0°C for
30 min and then at room temperature for 12 h. e reac-
tion mixture was filtered, the filtrate was washed with 0.5
To a solution of BocFMDP (4) (2.00 g, 6.33 mmol), 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU, 0.96 g, 6.33 mmol)
and sodium iodide (0.35 g, 1.90 mmol) in anhydrous
acetonitrile (25 mL), the corresponding 1-chloroalkyl
ester (5a–f) was added (7.60 mmol) and the mixture
was refluxed with stirring for 5 h. e solvent was evapo-
rated, the residue was diluted with ethyl acetate, washed
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and biological activity of lipophilic derivatives of bicine conjugated with FMDP 169
with 0.5 N NaHCO3 (2 × 20 mL), water (20 mL) and dried
N-hydroxysuccinimide (0.114g, 1 mmol) in tetrahydro-
furan (THF) (10mL) was cooled to 0°C and DCC (0.216g,
1.05 mmol) was added. After 12h, the dicyclohexylurea
was filtered off and the filtrate was added drop wise to
solution of corresponding acyloxyalkyl ester of FMDP (1
mmol) and triethylamine (0.20g, 2 mmol) in THF (7mL).
e mixture was stirred at 0°C for 30min, and then at
room temperature for 14h. e solvent was evaporated,
the residue was dissolved in ethyl acetate (20mL), washed
with 0.5 N NaHCO3 (2×15mL), water (10mL) and dried
(MgSO4). e solvent was evaporated in vacuo and the
residue was purified by silica gel column chromatography
(ethyl acetate:petroleum ether, 3:1). e product was dis-
solved in ethyl acetate and equimolar amount of 2.00M
HCl in diethyl ether was added. e precipitate was fil-
tered off and dried in vacuo over KOH pellets.
(MgSO4). e solvent was evaporated in vacuo and the
residue was purified by silica gel column chromatogra-
phy (ethyl acetate:petroleum ether, 1:2). e product was
treated with TFA for 2 h at room temperature. Excess TFA
was evaporated in vacuo, the residue was triturated with
diethyl ether and the precipitate was filtered off, dried in
vacuo over KOH pellets.
Acetoxymethyl ester of N3-(4-methoxyfumaroyl)-(L)-2,3-
diaminopropanoic acid trifluoroacetate (6a) Yield 1.42 g
(56%). 1H NMR (500 MHz, CDCl3): δ 2.15 (s, 3H, (O)
CCH3); 3.82 (s, 5H, OCH3, NCH2); 4.45 (m, 1H, CH);
5.81 (dd, 2H, J= 5 Hz, OCH2O); 6.90 (ABq, 2H, J= 15 Hz,
CH=CH). MS(FAB) m/z: 389 (MH+).
(Isobutyryloxy)methyl ester of N3-(4-methoxyfumaroyl)-(L)-
2,3-diaminopropanoic acid trifluoroacetate (6b) Yield
1.58 g (58%). H NMR (500 MHz, CDCl3): δ 1.18 (d, 6H,
J= 6.5 Hz, 2x(CH3)); 2.64 (m, 1H, CH); 3.80 (s, 5H, OCH3,
NCH2); 5.75 (dd, 2H, J= 5 Hz, OCH2O); 6.86 (ABq, 2H,
J = 15 Hz, CH=CH). MS (FAB) m/z: 417 (MH+).
Acetoxymethyl ester of N2-(bis-N,N-(2-acetoxyethyl)glycinyl)-
N3-(4-methoxyfumaroyl)-(L)-2,3-diaminopropanoic
acid hydrochloride (8a) Yield 0.23 g (41%). 1H NMR
(500 MHz, CDCl3): δ 2.09 (s, 6H, 2x(CH3C(O)); 2.12 (s, 3H,
CH3C(O)); 2.88 (m, 4H, 2x(CH2N)); 3.31 (d, 2H, J= 5.4 Hz,
NCH2C(O)); 3.78 (s, 5H, OCH3, HNCH2); 4.10–4.35 (m,
4H, 2x((CH2OC(O)); 4.70 (m, 1H, NHCH); 5.75 (dd, 2H,
J= 5.6 Hz, OCH2O); 6.90 (ABq, 2H, J= 15.4 Hz, CH=CH).
MS (FAB) m/z: 518 (MH+).
1
(2-Phenylacetoxy)methyl ester of N3-(4-methoxyfumaroyl)-
(L)-2,3-diaminopropanoic acid trifluoroacetate (6c) Yield
1
1.91 g (63%). H NMR (500 MHz, CDCl3): δ 3.71 (s, 2H,
CH2); 3.80 (s, 5H, OCH3, NCH2); 4.42 (m, 1H, CH); 5.75 (dd,
2H, J= 5 Hz, OCH2O); 6.86 (ABq, 2H, J= 15 Hz, CH=CH);
7.32 (m, 5H, Ar). MS (FAB) m/z: 465 (MH+).
Acetoxymethyl ester of N2-(bis-N,N-(2-propionyloxyethyl)
glycinyl)-N3-(4-methoxyfumaroyl)-(L)-2,3-diaminopro-
panoic acid hydrochloride (8b) Yield 0.28 g (48%). 1H NMR
(500 MHz, CDCl3): δ 1.13 (t, 6H, J= 7.5 Hz, 2x(CH3); 2.12
(s, 3H, CH3C(O)); 2.36 (q, 4H, J= 7.5 Hz, 2x(CH2C(O)));
2.88 (m, 4H, 2x(CH2N)); 3.29 (s, 2H, NCH2C(O)); 3.78 (s,
5H, OCH3, HNCH2); 4.15–4.35 (m, 4H, 2x((CH2OC(O));
4.70 (m, 1H, NHCH); 5.75 (dd, 2H, J= 5.6 Hz, OCH2O);
6.90 (ABq, 2H, J= 15.4 Hz, CH=CH). MS (FAB) m/z: 546
(MH+).
[(2-Ethylbutanoyl)oxy]methyl
ester
of
N3-(4-
methoxyfumaroyl)-(L)-2,3-diaminopropanoic acid trif-
luoroacetate (6d) Yield 2.17 g (75%).1H NMR (500 MHz,
CDCl3): δ 0.86 (t, 6H, J= 7.4 Hz, 2x(CH3)); 1.62 (m, 4H,
2x(CH2)); 2.35 (m, 1H, CH); 3.79 (s, 5H, OCH3, NCH2);
4.42 (m, 1H, CH); 5.78 (dd, 2H, J= 5.6 Hz, OCH2O); 6.88
(ABq, 2H, J= 15 Hz, CH=CH). MS (FAB) m/z: 445 (MH+).
1-Acetoxypropyl ester of N3-(4-methoxyfumaroyl)-(L)-2,3-
diaminopropanoic acid trifluoroacetate (6e) Yield 1.44 g
(53%).1H NMR (500 MHz, CDCl3): δ 0.99 (m, 3H, CH3);
1.84 (m, 2H, CH2); 2.11 (s, 3H, CH3C(O)); 3.80 (s, 5H,
OCH3, NCH2); 4.45 (m, 1H, CH); 5.65 (m, 1H, OCHO);
6.86 (m, 2H, CH=CH). MS (FAB) m/z: 417 (MH+).
Acetoxymethyl ester of N2-(bis-N,N-(2-butyryloethyl)glycinyl)-
N3-(4-methoxyfumaroyl)-(L)-2,3-diaminopropanoic
acid
hydrochloride (8c) Yield 0.28 g (46%). 1H NMR (500 MHz,
CDCl3): δ 0.94 (t, 6H, J= 7.3 Hz, 2x(CH3)); 1.64 (m, 4H,
2x(CH2)); 2.12 (s, 3H, CH3C(O)); 2.32 (t, 4 H, J= 7.5 Hz,
2x(CH2C(O))); 2.89 (t, 4H, J= 5.6 Hz 2x(CH2N)); 3.31 (s,
2H, NCH2C(O)); 3.78 (s, 5H, OCH3, HNCH2); 4.14–4.30 (m,
4H, 2x((CH2OC(O)); 4.72 (m, 1H, NHCH); 5.76 (dd, 2H,
J= 5.6 Hz, OCH2O); 6.88 (ABq, 2H, J= 15.4 Hz, CH=CH).
MS (FAB) m/z: 574 (MH+).
1-[(Ethoxycarbonyl)oxy]ethylesterofN3-(4-methoxyfumaroyl)-
(L)-2,3-diaminopropanoic acid trifluoroacetate (6f) Yield
1
1.44g (51%). H NMR (500MHz, CDCl3): δ 1.35 (m, 3H,
CH3); 1.58 (m, 2H, CH2); 3.80 (s, 5H, OCH3, NCH2); 4.28
(m, 2H, CH2); 4.45 (m, 1H, CH); 5.78 (m, 1H, OCHO); 6.86
(m, 2H, CH=CH). MS (FAB) m/z: 433 (MH+).
Acetoxymethyl ester of N2-(bis-N,N-(2-benzoyloxyethyl)
glycinyl)-N3-(4-methoxyfumaroyl)-(L)-2,3-diaminopro-
panoic acid hydrochloride (8d) Yield 0.28 g (41%). 1H
NMR (500 MHz, CDCl3): δ 2.09 (s, 3H, CH3C(O)); 3.18 (t,
J= 5.4 Hz, 4H, 2x(CH2N)); 3.53 (s, 2H, NCH2C(O)); 3.72 (s,
5H, OCH3, HNCH2); 4.53 (m, 4H, 2x((CH2OC(O)); 4.58 (m,
1H, NHCH); 5.83 (dd, 2H, J= 5.6 Hz, OCH2O); 6.82 (ABq,
2H, J= 15.4 Hz, CH=CH); 7.35–7.90 (m, 10H). MS (FAB)
m/z: 642 (MH+).
General method for the preparation of acyloxyalkyl
ester of N2-(bis-N,N-(2-acyloxyethyl)glycinyl)-N3-(4-
methoxyfumaroyl)-(L)-2,3-diaminopropanoic acid
hydrochloride (8a–i)
A solution of bis-N,N-(2-acyloxyethyl)glycine trifluoroac-
etate (3a–d) (1 mmol), triethylamine (0.20g, 2 mmol) and
© 2012 Informa UK, Ltd.

170 D. Koszel et al.
(Isobutyryloxy)methyl ester of N2-(bis-N,N-(2-acetoxyethyl)
glycinyl)-N3-(4-methoxyfumaroyl)-(L)-2,3-diaminopro-
panoic acid hydrochloride (8e) Yield 0.23 g (40%). 1H
NMR (500 MHz, CDCl3): δ 1.16 (d, 6H, J= 6.5 Hz, 2x(CH3));
2.09 (s, 6H, 2x(CH3C(O))), 2.69 (m, 1H, CH); 2.98 (m, 4H,
2x(CH2N)); 3.75 (s, 5H, OCH3, HNCH2) 4.10–4.40 (m, 4H,
2x((CH2OC(O)); 4.69 (m, 1H, NHCH); 5.78 (dd, 2H, J= 5.4
Hz, OCH2O); 6.89 (ABq, 2H, J= 15.4 Hz, CH=CH). MS
(FAB) m/z: 546 (MH+).
width 300 Å. e chromatographic system consisted
of a UV/vis detector Model G1315B, Vacuum degas-
ser Model G1322A, Quaternary pump Model G1311A
(Agilent Technologies 1200 Series, Palo Alto, CA) .
0.1 M Sörensen buffer (K2HPO4/KH2PO4, pH 7.2)/
acetonitrile was used in proportions 50:50, 60:40, 65:35,
70:30, 75:25, 80:20 (v/v) as mobile phase. e injection
volume was 10 μL; the flow rate was 1 mL/min; the sam-
ples were detected at 254 nm. e dead volume of the col-
umn was determined by the retention time of citric acid
(aqueous solution, 50 μg/mL) and was used to calculate
capacity factors k′IAM. e dependence of logk′IAM versus
concentration of acetonitrile was linear. e logk′IAM,0 was
obtained by extrapolation of logarithmic capacity factor
to concentration of acetonitrile equal to 0. A standard,
commercially available statistical package for regression
analysis was applied on a personal computer.
(2-Phenylacetoxy)methyl
ester
of
N2-(bis-N,N-(2-
acetoxyethyl)glycinyl)-N3-(4-methoxyfumaroyl)-(L)-2,3-
diaminopropanoic acid hydrochloride (8f) Yield 0.23 g
(37%). 1H NMR (500 MHz, CDCl3): δ 2.09 (s, 6H,
2x(CH3C(O))); 3.05 (m, 4H, 2x(CH2N)), 3.71 (s, 2H,
CH2); 3.78 (s, 5H, OCH3, HNCH2); 4.10–4.40 (m, 4H,
2x((CH2OC(O)); 4.65 (m, 1H, NHCH), 5.75 (dd, 2H,
J = 5.4 Hz, OCH2O); 6.88 (ABq, 2H, J = 15.4 Hz, CH=CH).
MS (FAB) m/z: 594 (MH+).
Antifungal susceptibility tests
Minimal inhibitory concentrations (MICs) of the examined
compounds were determined by the serial 2-fold dilution
microtitreplatemethod,intheminimalliquidyeastnitrogen
base (YNB) medium without amino acids and ammonium
sulphate containing 2% glucose and l-proline (4mg/mL).
Wellscontainingseriallydilutedtestcompoundsandcontrol
were inoculated with 104 cells/mL of an overnight culture of
fungal cells and the microtitre plates were incubated for 24h
at 30°C. Fungal growth was measured using the microplate
reader (Labsystem Multiscan Biochromatic) Please provide
city and state for Labsystem Multiscan Biochromatic at
λ=595nm. e MIC was defined as the inhibitor concentra-
tion preventing at least 80% of fungal growth, as compared
with the inhibitor-free control.
[(2-Ethylbutanoyl)oxy]methyl ester of N2-(bis-N,N-(2-
acetoxyethyl)glycinyl)-N3-(4-methoxyfumaroyl)-(L)-2,3-
diaminopropanoic acid hydrochloride (8g) Yield 0.26 g
(43%). 1H NMR (500 MHz, CDCl3): δ 0.87 (t, 6H, J= 7.4 Hz,
2x(CH3)); 1.57 (m, 6H, 2x(CH2)); 2.09 (s, 6H, 2x(CH3C(O));
2.27 (m, 1H, CHC(O)); 2.89 (m, 4H, 2x(CH2N)); 3.30 (m,
2H, NCH2C(O)); 3.78 (s, 5H, OCH3, HNCH2); 4.10–4.40 (m,
4H, 2x((CH2OC(O)); 4.70 (m, 1H, NHCH); 5.80 (dd, 2H,
J= 5.5 Hz, OCH2O); 6.90 (ABq, 2H, J= 15.6 Hz, CH=CH).
MS (FAB) m/z: 574 (MH+).
1-Acetoxypropyl ester of N2-(bis-N,N-(2-acetoxyethyl)
glycinyl)-N3-(4-methoxyfumaroyl)-(L)-2,3-diaminopro-
panoic acid hydrochloride (8h) Yield 0.24 g (41%). 1H
NMR (200 MHz, CDCl3): δ 0.97 (m, 3H, CH3); 1.82 (m, 2H,
CH2); 2.11 (s, 3H, CH3C(O)); 2.13 (s, 6H, 2x(CH3C(O));
2.92 (m, 4H, 2x(CH2N)); 3.40 (m, 2H, NCH2C(O)); 3.79 (s,
5H, OCH3, HNCH2); 4.10–4.35 (m, 4H, 2x((CH2OC(O));
4.69 (m, 1H, NHCH); 6.70 (m, 2H, OCHO); 6.80 (m, 2H,
CH=CH). MS (FAB) m/z: 546 (MH+).
Results and discussion
Chemistry
e syntheses of compounds 3a–d (Scheme 1) was
started with preparation of tert-butyl ester of bis-N,N-(2-
hydroxyethyl)glycine (1). Compound 1 was prepared by
a displacement reaction using tert-butyl bromoacetate.
Condensation of compound 1 with corresponding car-
boxylic acids was carried out in the presence of DCC/
DMAP17 to give appropriate tert-butyl ester of bis-N,N-
(2-acyloxyethyl)glycine (2a–d). Deprotection of the car-
boxyl group was performed in anhydrous trifluoroacetic
acid. e bicine derivatives 3a–d were prepared as trif-
luoroacetate salts. In the case of the FMDP acyloxyalkyl
esters synthesis (Scheme 2), BocFMDP (4) was treated
with an appropriate chloroalkyl esters18 in the presence
of DBU and NaI.19 e reaction proceeds smoothly in
a polar solvent such as acetonitrile. e products were
purified by silica gel column chromatography with ethyl
acetate:petroleum ether (1:2) as a mobile phase. After
deprotection of the amino group using anhydrous TFA in
CH2Cl2, compounds 6a–f were obtained as hygroscopic
trifluoroacetate salts. e synthesis of compounds 8a–i
was performed using standard synthetic procedures
1-[(Ethoxycarbonyl)oxy]ethyl ester of N2-(bis-N,N-(2-
acetoxyethyl)glycinyl)-N3-(4-methoxyfumaroyl)-(L)-2,3-
diaminopropanoic acid hydrochloride (8i) Yield 0.23 g
1
(38%). H NMR (500 MHz, CDCl3): δ 1.32 (m, 3H, CH3);
1.58 (m, 2H, CH2); 2.09 (s, 6H, 2x(CH3C(O)); 2.89 (m, 4H,
2x(CH2N)); 3.28 (m, 2H, NCH2C(O)); 3.75 (s, 5H, OCH3,
HNCH2); 4.10–4.35 (m, 4H, 2x((CH2OC(O)); 4.22 (m, 2H,
CH2); 4.71 (m, 1H, NHCH); 6.75 (m, 1H, OCHO); 6.90 (m,
2H, CH=CH). MS (FAB) m/z: 562 (MH+).
Determination of the affinity to the artificial
biological membrane
Interactions between the tested compounds and
immobilized artificial membrane were investigated
using HPLC column IAM.PC.DD2 (Regis Technologies,
Inc., Morton Grove, IL).16 e column dimensions
were 3 cm × 4.6 mm, particle diameter 10 μm and pore
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and biological activity of lipophilic derivatives of bicine conjugated with FMDP 171
(Scheme 3). e compounds 3a–d were activated with
Membrane affinity
N-hydroxysuccinimide (HOSu) and DCC20 to give the
active esters (7a–d) and condensed with appropriate
acyloxyalkyl esters of FMDP (5a–f). e products were
purified by silica gel column chromatography with ethyl
acetate:petroleum ether (3:1) as a mobile phase. Using
anhydrous hydrochloride in diethyl ether, compounds
8a–i were obtained as amorphous hydrochloride salts.
e affinity of a drug for biological membrane is an
important factor facilitating its diffusion into fungal
cell. For compounds 8a–i, their interaction with a
biological membrane model was investigated using
an HPLC chromatographic column immobilized
artificial membrane (IAM) PC.DD 2 with stationary
phase mimicking the cell membrane. e retention
Scheme 1. Syntheses of bis-N,N-(2-acyloxyethyl)glycine trifluoroacetate (3a–d). (i) diethanolamine, CH2Cl2; (ii) RCOOH, DMAP, DCC and
(iii) TFA.
Scheme 2. Syntheses of FMDP acyloxyalkyl esters. (i) CH3CN, DBU, NaI. * denotes salt formation.
Scheme 3. Syntheses of acyloxyalkyl ester of N2-(bis-N,N-(2-acyloxyethyl)glycinyl)-N3-(4-methoxyfumaroyl)-(l)-2,3-diaminopropanoic
acid hydrochloride (8a–i), (i) HOSu/DCC and (ii) HCl/Et2O and hypothetical degradation pathway. * denotes salt formation.
© 2012 Informa UK, Ltd.

172 D. Koszel et al.
Table 1. Antifungal activity of compounds 8a–i and their affinity to biological membrane.
MIC (μg/mL)
Compound
R
CH3
R1
CH3
R2
C. albicans
500
C. glabrata
C. tropicalis
1000
500
S. cerevisiae logk′IAM,0
8a
H
500
1000
500
1000
1000
1000
500
0.91
1.01
1.36
2.11
1.34
1.76
1.72
1.55
1.26
−0.85
8b
8c
CH3CH2
CH3(CH2)2
C6H5
CH3
CH3
H
1000
1000
500
CH3
H
H
250
8d
8e
CH3
125
500
CH(CH3)2
CH2C6H5
CH(CH2CH3)2
CH3
H
125
125
62.5
31.3
125
8f
CH3
H
31.3
125
62.5
8g
CH3
H
31.3
250
125
500
8h
8i
CH3
CH3CH2
CH3
—
62.5
125
125
250
CH3
OCH2CH3
—
125
250
15.6
125
FMDP
—
>2000
>2000
>2000
>2000
time determined for the examined compounds was
expressed as logk′IAM and extrapolated to logk′IAM,0. e
highest logk′IAM,0 value was determined for compound
8d (containing an aromatic and methyl substituents),
whereas the lowest one was determined for compound
8a (with only methyl substituents). ese values com-
prise not only the lipophilic properties but also other
interactions with membrane such as hydrogen bond
formation or electrostatic interactions. erefore, the
logk′IAM,0 values reflect rather membrane affinity than
lipophilic properties.
Acknowledgments
is work was supported by the Ministry of Education
and Science (Grant No. NN 405 1829 35). e HPLC
instrument (which was used in this study) is a part
of equipments of e Research Laboratory Center of
Excellence “ChemBioFarm.” Its purchase was partially
funded by the European Union Structural Funds under
the Sectoral Operational Programme—Improvement of
the Competitiveness of Enterprises.
Declaration of interest
Antifungal activity
e authors report no conflicts of interest.
MICs were determined in YNB medium with proline
as carbon source. Table 1 present activity against few
species. Most of the synthesized compounds exhibited
moderate antifungal activity in the range from 15.6 to
1000 μg/mL and their activity is not correlated with
apparent lipophilicity. e lack of correlation between
the logk′IAM,0 and MIC values may be attributed to the
slow releasing of FMDP molecule from bicine conjugate.
Compounds 8a–d displayed almost the same antifungal
activity thus showing that the type of substituent in “R”
position is not important for the activity. On the other
hand, the change of latent ester of FMDP in compounds
8e–i (position “R1” and “R2”) improves antifungal activ-
ity of these compounds. is may be explained by differ-
ent rates of intracellular hydrolysis of these compounds
resulting in higher level of FMDP inside the cells. All the
obtained compounds have better lipophilic properties
than free inhibitor—FMDP. e logk′IAM,0 values between
0.91 and 2.11 show that compounds 8a–i may be able to
cross the cell membrane by free diffusion.
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