Antifungal activity of homoaconitate and homoisocitrate analogs

Article abstract of DOI:10.3390/molecules171214022

Thirteen structural analogs of two initial intermediates of the L-Aminoadipate pathway of L-lysine biosynthesis in fungi have been designed and synthesized, including fluoro- and epoxy-derivatives of homoaconitate and homoisocitrate. Some of the obtained compounds exhibited at milimolar range moderate enzyme inhibitory properties against homoaconitase and/or homoisocitrate dehydrogenase of Candida albicans. The structural basis for homoisocitrate dehydrogenase inhibition was revealed by molecular modeling of the enzyme-inhibitor complex. On the other hand, the trimethyl ester forms of some of the novel compounds exhibited antifungal effects. The highest antifungal activity was found for trimethyl trans-homoaconitate, which inhibited growth of some human pathogenic yeasts with minimal inhibitory concentration (MIC) values of 16-32 ?g/mL.

Full text of DOI:10.3390/molecules171214022

Molecules 2012, 17, 14022-14036; doi:10.3390/molecules171214022
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antifungal Activity of Homoaconitate and Homoisocitrate Analogs
Maria J. Milewska 1,*, Marta Prokop 1, Iwona Gabriel 2, Marek Wojciechowski 2 and
Sławomir Milewski 2
1
Department of Organic Chemistry, Gdańsk University of Technology, 11/12 Narutowicza Str.,
80-233 Gdańsk, Poland
2
Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology,
11/12 Narutowicza Str., 80-233 Gdańsk, Poland
* Author to whom correspondence should be addressed; E-Mail: mjm@chem.pg.gda.pl;
Tel.: +48-58-347-11-34; Fax: +48-58-347-11-44.
Received: 15 October 2012; in revised form: 14 November 2012 / Accepted: 15 November 2012 /
Published: 27 November 2012
Abstract: Thirteen structural analogs of two initial intermediates of the L--aminoadipate
pathway of L-lysine biosynthesis in fungi have been designed and synthesized, including
fluoro- and epoxy-derivatives of homoaconitate and homoisocitrate. Some of the obtained
compounds exhibited at milimolar range moderate enzyme inhibitory properties against
homoaconitase and/or homoisocitrate dehydrogenase of Candida albicans. The structural
basis for homoisocitrate dehydrogenase inhibition was revealed by molecular modeling of
the enzyme-inhibitor complex. On the other hand, the trimethyl ester forms of some of the
novel compounds exhibited antifungal effects. The highest antifungal activity was found
for trimethyl trans-homoaconitate, which inhibited growth of some human pathogenic
yeasts with minimal inhibitory concentration (MIC) values of 16–32 g/mL.
Keywords: homoisocitrate; homoaconitate; synthesis; inhibitors; molecular modeling;
antifungal activity
1. Introduction
Microbial resistance to antibiotics and synthetic drugs is an emerging challenge for clinicians
and the pharmaceutical industry. The multidrug-resistant fungi are the major cause of failure in
chemotherapy of disseminated mycoses [1,2]. Thus, the need for novel antifungals is especially urgent.

Molecules 2012, 17
14023
Among different strategies of searching for new potential drugs, identification of novel, unexploited
molecular targets and their inhibitors seems one of the most promising approaches. These days many
researchers are concentrated on fungal enzymes that catalyze essential biochemical reactions, specific
to pathogen cells, and absent from the host cells. Bioactive compounds that selectively block metabolic
pathways in human pathogenic fungi could be potential antifungals with low mammalian toxicity.
L-Lysine is an essential amino acid for humans, while bacteria, plants and fungi have developed
pathways of lysine biosynthesis. There are two versions of these anabolic routes: a diaminopimelic
acid pathway, which is characteristic for bacteria, plants and lower fungi and an -aminoadipate
pathway (AAP) for higher fungi, Ascomycetes and Basidomycetes, including most of the human
pathogenic yeasts and filamentous fungi. Enzymes that catalyze the reactions of the above mentioned
first pathway are considered promising molecular targets for antibacterial chemotherapy [3], while the
second pathway could be a source of new targets for antifungal chemotherapy [4]. Diminished virulence
in some systems of fungal cells lacking genes encoding enzymes of AAP was demonstrated [5,6], and
the antifungal activity of compounds targeting one of these enzymes was shown too [7].
In this paper we present results of our studies on the design, synthesis and evaluation of biological
properties of some novel structural analogs of homoaconitate and homoisocitrate, intermediates of the
AAP pathway.
2. Results and Discussion
Three initial reactions of the AAP (Scheme 1) are catalyzed by the enzymes that are unique to
fungi, so that their selective inhibitors may have antifungal potential. We have designed and
synthesized several compounds that are structural analogs of substrates/products of two of the
enzymes, homoaconitase (HA) and homoisocitrate dehydrogenase (HIcDH), i.e., cis-homoaconitate
and homoisocitrate. Whenever possible, the compounds that are di- or tricarboxylic acids were
obtained in two forms, potassium/sodium salts or methyl esters. It was expected that the ester forms of
potential enzyme inhibitors should better penetrate the fungal cell membrane than their salt
counterparts and after internalization could be cleaved by intracellular esterases.
Scheme 1. Details of the three initial reactions of AAP. HcS—homocitrate synthase;
HA—homoaconitase; HIcDH—homoisocitrate dehydrogenase.
AcCoA
O
OH
HA
CO2H
HcS
CO2H
HO2C
HO2C
HO2C
CO2H
CO2H
CO2H
H2O
HS-AcCoA
H2O
B
NAD+
OH
NH2
O
HIcDH
CO2H
CO2H
HO2C
HO2C
NH2
HO2C
CO2H
CO2
NADH

Molecules 2012, 17
14024
The following compounds were synthesized: cis-homoaconitate analogs: trans-homoaconitates 1a
and 2, methyl ester and potassium salts of trans-1,2-epoxybutane-1,2,4-tricarboxylate (compounds 3
and 4); homoisocitrate analogs: (2S,3R)-2-hydroxy-3-propylsuccinate (5a); (2S,3R)-2-hydroxy-3-
butylsuccinate (5b); (2S,3R)-2-hydroxy-3-allylsuccinate (5c); (2R,3S)-2-fluoro-3-propylsuccinate (6a);
(2R,3S)-2-fluoro-3-butylsuccinate (6b); (2R,3S)-2-fluoro-3-allylsuccinates 6c and 7c; (2R,3S)-2-fluoro-2-
deoxyhomoisocitrates 8–10.
2.1. Synthesis of Homoaconitate and Homoisocitrate Analogs
Two stereoisomers of homoaconitate were synthesized from (R)-homocitric lactone following the
strategy shown in Scheme 2. The lactone was first converted into the respective dichloride. This
compound was then brominated at the -methylene position. Heating in methanol promoted the
conversion of the acid chlorides into methyl esters, ring opening and HBr elimination. In this way, a
mixture of cis and trans isomers of homoaconitate (molar ratio 3:1) was obtained with the overall yield
of 37%. The stereoisomers could be effectively separated by flash chromatography. Both were easily,
almost quantitatively converted into respective tripotassium salts upon treatment with potassium
trimethylsilanolate.
Scheme 2. Synthetic route to compounds 1 and 2.
CO2H
SOCl2/toluene
COCl
COCl
1. Br2 (1eq)
CO2Me
CO2Me
O

2. MeOH, 
O
O
O
CO2H
O
O
Br
MeO2C
MeO2C
H
H
CO2Me
CO2
H
TMSOK/Et2O
24h, rt
+
CO2Me
MeO2C
CO2Me
O2C
CO2
1a
1b
2
Treatment of trans-homoaconitate with H2WO4/H2O2, as shown in Scheme 3, afforded
corresponding trans-epoxide in high yield.
Scheme 3. Synthesis of epoxy derivatives 3 and 4.
H
CO2
O
CO2Me
O
1. H2WO4, 30% H2O2
2.HCl
3. CH2N2/Et2O
H
CO2
H
TMSOK/Et2O
24h, rt
MeO2C
O2C
O2C
CO2
CO2Me
2
CO2
4
3
Rather unexpectedly, the same methodology applied to tripotassium cis-homoaconitate as a
substrate, resulted in formation of trans-epoxide as the only product in 33% yield. The stereochemistry

Molecules 2012, 17
14025
1
of this compound was unequivocally confirmed by comparison of its H- and 13C-NMR spectra to
those of reference samples of cis- and trans-1,2-epoxy-propane-1,2,3-carboxylate.
Exchange of the hydroxyl substituent for fluorine for the purpose of preparation of deoxyfluoro
analogs of homoisocitrate was performed by use of N,N-(diethylamino)sulfur trifluoride (DAST) as
fluorinating agent. Surprisingly, the reaction in which homoisocitrate was a substrate, afforded a
mixture of homoaconitate stereoisomers as the only products. Therefore, an alternative multistep
strategy shown in Scheme 4 was applied.
Scheme 4. Strategy of synthesis of deoxyfluoro homoisocitrate analogs 5–10.
R
R
MeO2C
DAST, Ar
CH2Cl2, -78°C
1. LDA, THF, Ar, -78°C
RBr
MeO2C
MeO2C
CO2Me
CO2Me
CO2Me
OH
F
OH
5a - c
6a R = CH2H2CH3
6b R = CH2CH2CH2CH3
6c R = CH2CH=CH2
.
1. BH3 Me2S
2. PDC/DMF
TMSOK/Et2O
24h, rt
CO2
CO2Me
CO2H
TMSOK/Et2O
24h, rt
CH2N2/Et2O
R
O2C
O2C
MeO2C
MeO2C
CO2
CO2
CO2Me
CO2Me
F
F
10
F
9
F
8
7c
Dimethyl (S)-malate was first alkylated with an appropriate alkyl/allyl bromide. This reaction was
highly stereoselective, as expected for a Seebach-type alkylation [8] and resulted in formation of single
diastereoisomers of compounds 5a–c. The stereochemistry of these compounds was assigned as
(2S,3R), based on the known stereospecificity of the Seebach alkylation of (S)-malate [8,9]. With
dimethyl (R)-malate as a substrate, (2R,3S) stereoisomers were formed as the only products (data not
shown). Treatment of compounds 5a–c with DAST afforded single diastereoisomers of the succinate
derivatives 6a–c. This reaction, as a nucleophilic substitution of the SN2 type [10], lead to the inversion
of configuration at the hydroxyl-substituted C2 atom. Obviously, there was no inversion of the actual
absolute configuration at C3 in this reaction but its stereochemical notation changed, due to the
introduction of fluorine at C2. Thus the (2R,3S) configuration could be assigned to compounds 6a–c.
Compound 6c was converted into dimethyl (2R,3S)-2-fluoro-2-deoxyhomoisocitrate in a two-step
reaction. The trimethyl ester 9 was obtained upon treatment of dimethyl (2R,3S)-2-fluoro-2-
deoxyhomoisocitrate 8 with an ethereal solution of diazomethane and could be converted into the
respective tripotassium salt 10. All the compounds 1–10 were fully characterized using their
spectroscopic data.
2.2. Biological Activity of Novel Compounds
Compounds 1–10 were tested for their in vitro antifungal activity against six yeast strains. MICs of
the studied compounds determined by the microplate serial dilution method are shown in Table 1.

Molecules 2012, 17
14026
None of the salts (compounds 2, 4, 7c, 10) showed any antifungal activity. The highest activity was
found for trimethyl trans-homoaconitate (1a) that inhibited the growth of all tested yeast strains, with
MIC values in the 16–32 g/mL range. The corresponding epoxide 3 inhibited growth of four out of
the six yeast strains, but its MIC values were much higher (128–512 g/mL). Among the deoxyfluoro
analogs of homoisocitrate, some antifungal activity against selected strains was noted for dimethyl
(2R,3S)-2-fluoro-3-allylsuccinate (6c) and trimethyl (2R,3S)-2-fluoro-2-deoxyhomoisocitrate (9)
(MICs 64-512 g/mL). In all cases the MIC values were higher than those determined for the known
antifungal fluconazole, however trans-homoaconitate 1a was only slightly less effective.
Table 1. Antifungal in vitro activity of compounds 1–10.
MIC (g/mL)
C. albicans C. glabrata C. tropicalis
Comp.
S. cerevisiae
32
C. lusitaniae
16
C. krusei
32
1a
2
16
>512
256
16
32
>512
128
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
4
>512
>512
512
>512
>512
>512
>512
>512
>512
>512
>512
512
3
128
4
>512
>512
>512
>512
>512
256
>512
>512
>512
>512
>512
>512
128
>512
>512
>512
>512
512
5a
5b
5c
6a
6b
6c
7c
9
>512
>512
>512
>512
512
128
256
256
256
>512
512
>512
128
>512
>512
64
>512
256
256
>512
>512
4
10
Flu *
>512
4
>512
>512
2
>512
8
8
* Flu = Fluconazole.
It was expected that the novel compounds in the free acid or salt form could inhibit activity of
homoaconitase or homoisocitrate dehydrogenase. To check this possibility, the effect of compounds
1–10 on activity of recombinant homoaconitase and homoisocitrate dehydrogenase from Candida
albicans was quantified. According to the expectations, none of the ester derivatives: 1a, 3, 5a–c, 6a–c
and 9 had any effect on enzyme activity at concentrations up to 10 mM. Effects noted for salts 2, 4, 7c
and 10 are summarized in Table 2.
Table 2. Enzyme inhibitory potential of compounds 2, 4, 7c and 10. Values are the means
of three independent determinations ± SD.
IC50 (mM)
Compound
HA
HIcDH
> 10 mM
> 10 mM
6.78  0.56
3.22  0.55
2
4
1.58  0.11
5.76  0.34
>10 mM
>10 mM
7c
10

Molecules 2012, 17
14027
Trans-homoaconitate 2 and the epoxy derivative 4 inhibited homoaconitase, while the 2-fluoro-2-
deoxy analogs of homoisocitrate, i.e., compounds 7c and 10, affected the activity of homoisocitrate
dehydrogenase. The IC50 values were in the milimolar range and as such were much higher than those
of the most active inhibitors of homoisocitrate dehydrogenase known so far that inhibited the
S. cerevisiae enzyme at micromolar concentrations [9,10]. Nevertheless, one can clearly see a
correlation between the enzyme inhibitory properties of compounds 2, 4, 7c and 10 and the antifungal
activity of their respective ester derivatives 1a, 3, 6 and 9. It is very likely therefore that the ester
compounds penetrate the fungal cell membrane and are metabolized intracellularly to release the active
enzyme inhibitors. Such a correlation has been demonstrated for the first time for inhibitors of the
AAP. The previous reports concerned either enzymatic inhibitors without any data on their
antifungal properties [11,12], or described antifungal action of methyl esters of carboxyalkyl- and
carboxyaryl-substituted D-malic acid derivatives without identification of any enzymatic target [7].
2.3. Molecular Modeling of Enzyme: Inhibitor Interactions
Compounds 7c and 10 are structural analogs of homoisocitrate. Despite the fact that the real 3D
arrangement of respective substituents of the C3 atom of these compounds is opposite to that in the
HIcDH dehydrogenase physiological substrate, i.e., in (2R,3S)-homoisocitrate (despite the identity of
their formal stereochemical notations), they both inhibit enzyme activity. In order to elucidate a
molecular basis for this inhibition, we have studied a pattern of interactions of 7c docked into the
modeled C. albicans HIcDH active site. This active centre is in fact the only site for inhibitor binding,
since in HIcDH there are no other specific ligand binding sites, including those for possible allosteric
inhibitors [13]. Results of these studies are shown in Figure 1.
Figure 1. Compound 7c docked at the active site of modeled CaHIcDH. The most probable
two orientations of the ligand are shown. Mg(II) ion of the active site is displayed as a
violet sphere and a fluorine atom of 7c is presented as a green ball.

Molecules 2012, 17
14028
The C1 carboxylate of the inhibitor is bound to the active site magnesium ion, while the C7
carboxylate is bound by the receptor’s Arg112 and Arg140 guanidine moieties. The divalent cations
Arg112 and Arg140 were previously identified as those involved in binding of the respective
carboxylate functions of the homoisocitrate substrate [13,14]. On the other hand, in the most probable
orientation, the allyl substituent at C3 of 7c points toward the small, partially hydrophobic pocket
formed by the Val214, Ile246 residues and the nonpolar part of the Lys211 side chain. This pocket is
supposed to be the binding site of the NADH substrate [13]. The hydrophobic character of the niche
accommodating the C3 substituent explains the slightly higher inhibitory potential of 7c (the allyl
derivative) in comparison to 10, bearing the carboxylate function. It should be noted therefore, that the
third carboxylate function of (2R,3S)-homoisocitrate docked at the active site of C. albicans HIcDH is
bound by the -amino group of Lys149 [14], so that one may assume that binding of 7c should be
weaker than that of the substrate, due to the substitution of relatively strong electrostatic interactions
between carboxylate of the substrate and Lys149 by the much weaker hydrophobic effect.
Nevertheless, binding of 7c and 10 at the active site at the orientation partially overlapping that of the
homoisocitrate substrate, seems the most plausible reason of its weak CaHIcDH inhibitory properties.
3. Experimental
3.1. Materials and Reagents
Solvents and reagents were purchased from Sigma-Aldrich, St. Louis, MO. USA. Melting points
are uncorrected. NMR spectra were recorded on the Varian Gemini 200 and 500 MHz spectrometers,
using TMS as an internal standard in 1H and 13C experiments and CFCl3 in 19F experiments. Thin layer
chromatography (TLC) was performed on silica gel plates (Merck, Darmstadt, Germany) using the
following solvent systems as eluents: (A) hexane/ethyl acetate (9:5); (B) benzene/ethyl acetate/ethanol
(85:20:5). Elementary analysis (EA) was run on a Carlo Erba, type EA 1108 analyzer. Optical
rotations were measured on a Rudolph Autpol II polarimeter at 589 nm.
3.2. Chemical Synthesis
Trisodium (R)-homocitrate, dimethyl (S)-methylenehomocitrate and (R)-homocitric lactone were
synthesized as described previously [15]. (2R,3S)-Homoisocitric acid was synthesized according to the
procedure of Ma and Palmer [9].
Trimethyl homoaconitate (1a and 1b). Thionyl chloride (9.6 mL; 132 mmol) was added dropwise to a
solution of (R)-homocitric lactone (13.5 g; 66 mmol) in toluene (400 mL) and the resulting mixture
was heated under reflux for 48 h. The solvent was then removed by evaporation under reduced
pressure to give the corresponding dichloride as an oily residue (13 g; 54 mmol; 82%). Anhydrous
bromine (3 mL; 54 mmol) was then added dropwise and the mixture was heated under reflux for 1 h.
An additional portion of bromine (1 mL) was then added and heating was continued overnight.
Anhydrous methanol (500 mL) was added to the cooled reaction mixture and the solution was heated
under reflux for about 4 h. The solvent was then distilled off, the residue was dissolved in ethyl acetate
and sequentially washed with aqueous 2% Na2CO3 solution, water, 3% Na2CO3 solution and water.

Molecules 2012, 17
14029
The organic layer was then dried over anhydrous MgSO4 and the solvent was subsequently removed
by evaporation in vacuo. The crude oily product (5.27 g, 37%, mixture of cis and trans isomers) was
resolved by silica gel (70–230 mesh) column chromatography. The column was developed with
hexane/AcOEt (5:1). The following oily products were isolated: trimethyl trans-homoaconitate
(1a, 1.12 g, 8%) and trimethyl cis-homoaconitate (1b, 3.54 g, 24.5%).
Trimethyl trans-homoaconitate [trimethyl (1E)-but-1-ene-1,2,4-tricarboxylate, 1a]. Rf = 0.54 (solvent
1
system A). H-NMR (CDCl3) δ [ppm]: 2.54 (t, J = 7.9 Hz, 2H, CH2CH2CO); 3.09 (t, J = 7.7 Hz, 2H,
CH=CCH2); 3.67 (s, 3H, OCH3); 3.78 (s, 3H, OCH3); 3.82 (s, 3H, OCH3); 6.82 (s, 1H, =CH-);
13C-NMR (CDCl3) δ[ppm]: 23.58; 33.28; 51.92; 52.14; 52.91; 127.94; 146.12; 166.01; 167.10; 173.08.
Elemental anal. (%), calcd. for C10H14O6: C, 52.17; H, 6.09; found: C, 52.14; H, 5.93.
Trimethyl cis-homoaconitate [trimethyl (1Z)-but-1-ene-1,2,4-tricarboxylate 1b]. Rf = 0.47 (solvent
1
system A). H-NMR (CDCl3) δ [ppm]: 2.55 (t, J = 7.2 Hz, 2H, CH2CH2CO); 2.69 (t, J = 7.5 Hz, 2H,
CH=CCH2); 3.69 (s, 3H, OCH3); 3.73 (s, 3H, OCH3); 3.83 (s, 3H, OCH3); 5.90 (s, 1H, =CH-);
13C-NMR (CDCl3) δ [ppm]: 29.16; 31.49; 51.86; 51.91; 52.44; 120.86; 147.67; 165.21; 168.51;
172.08. Elemental anal. (%), calcd. for C10H14O6: C, 52.17; H, 6.09; found: C, 52.07; H, 5.97.
Tripotassium trans-homoaconitate [tripotassium (1E)-but-1-ene-1,2,4-tricarboxylate 2]. Trimethyl
trans-homoaconitate (1a, 276 mg, 1.2 mmol) was dissolved in anhydrous diethyl ether (20 mL).
Potassium trimethylsilanolate (510 mg, 4 mmol) was added to the solution and the mixture was stirred
for 24 h. The solvent was removed by evaporation under reduced pressure, the residue was washed
with anhydrous diethyl ether and finally dried under reduced pressure. The product was obtained as a
1
slightly brownish powder (322 mg, 89%). H-NMR (D2O) δ [ppm]: 2.54 (t, J = 7.9 Hz, 2H, CH2,);
13
2.74 (t, J = 8.1 Hz, 2H, CH2,); 6.51 (s, CH=C, 1H). C-NMR (D2O) δ [ppm]: 23.54; 33.32; 51.82;
52.18; 52.99; 127.90; 146.02. Elemental anal. (%), calcd. for C7H5O6K3: C, 27.63; H, 2.30; found: C,
27.69; H, 2.27.
Trimethyl (1E)1,2-epoxy-butane-1,2,4-tricarboxylate (3). Tripotassium trans-homoaconitate (2,
302 mg; 1 mmol) was dissolved in water (5 mL) and tungstic acid (35 mg; 0.14 mmol) and 30% H2O2
(300 μL) were added. The mixture was stirred for 2 h at 85 °C. Then 10 M HCl (0.35 mL) was added
and the reaction mixture was subjected to continuous extraction with diethyl ether for several hours.
The organic solvent was removed from the extract by evaporation, to give 172 mg of a slightly
yellowish oil. The ethereal solution of diazomethane was then added dropwise at 0 °C until pH of the
solution reached 7.0, then the mixture was left overnight at room temperature. After solvent
evaporation, 216 mg of the crude oily product was obtained, that was further purified by column silica
gel chromatography eluting with hexane/AcOEt (5:1), followed by AcOEt. Finally, after solvent
evaporation, a slightly yellow oily product was obtained (202 mg; 82%); Rf = 0.57 (solvent system B).
1H-NMR (CDCl3) δ [ppm]: 2.39–2.33 (m, 1H, CH2CH2CO); 2.63–2.53 (m, 2H, CH2CH2CO);
2.75–2.67 (m, 1H, CH2CH2CO); 3.67 (s, 3H, OCH3); 3.69 (s, 3H, OCH3); 3.70 (s, 3H, OCH3); 3.74
(s, 1H, CH); 13C-NMR (CDCl3) δ [ppm]: 31.57; 45.64; 52.19; 52.92; 52.97; 53.21; 60.05; 168.16;
170.82; 173.23. Elemental anal. (%), calcd. for C10H14O7: C, 48.78; H, 5.69; found: C, 48.67; H, 5.63.

Molecules 2012, 17
14030
Tripotassium (1E)1,2-epoxy-butane-1,2,4-tricarboxylate (4). Trimethyl (1E)1,2-epoxybutane-1,2,4-
tricarboxylate (3, 148 mg, 0.6 mmol) was dissolved in anhydrous diethyl ether (10 mL). Potassium
trimethylsilanolate (255 mg, 2 mmol) was added to the solution and the mixture was stirred for 24 h.
The solvent was removed by evaporation under reduced pressure, the residue was washed with
anhydrous diethyl ether and finally dried under reduced pressure. The product was obtained as a
yellow amorphous powder (156 mg, 82%). 1H-NMR (D2O) δ [ppm]: 2.29–2.35 (m, 1H, CH2CH2CO);
2.57–262 (m, 2H, CH2CH2CO); 2.66–2.71 (m, 1H, CH2CH2CO); 3.78 (s, 1H, CH); 13C-NMR (CDCl3)
δ [ppm]: 31.32; 45.68; 51.88; 53.15; 53.24; 53.48; 61.60. Elemental anal. (%), calcd. for C7H5O7K3: C,
27.27; H, 1.62; found: C, 27.27; H, 1.65.
General procedure for the synthesis of compounds 5a–c. n-BuLi (20.8 mL, 52.2 mmol, 2.5 M in
hexane) was added dropwise to a stirred solution of diisopropylamine (62.6 mmol) in THF (38 mL) at
−78 C under argon. After 1 h, a solution of dimethyl (S)-malate (3.38 g, 20.9 mmol) in THF (6 mL)
was added, while the temp. was kept at −78 C. The mixture was then warmed to −25 C and stirred
for 1 h. After that time, the mixture was again chilled to −78 C and propyl, butyl or allyl bromide
(53 mmol) was added dropwise. The resulting solution was stirred for 2 h at −25 C and then left
overnight at 4 C. The mixture was chilled to −78 C, acetic acid (6.4 mL in 10 mL of Et2O) was
added and the resulting mixture was combined with Et2O (250 mL) and water (30 mL). The organic
layer was washed with the saturated solution of sodium carbonate and with saline. Aqueous layers
were combined and extracted three times with Et2O. Combined ethereal layers were dried over
anhydrous MgSO4 and the solvent was evaporated. The crude products were purified by flash silica-gel
chromatography with hexane/AcOEt (5:1) as an eluent to afford the final products.
Dimethyl (2S,3R)-2-hydroxy-3-propylsuccinate (5a). Oil, final yield 58%. []2D0 +19.7 (c1.33; CHCl3).
1H-NMR (CDCl3) δ [ppm]: 0.95 (t, 3H, CH2CH2CH3), 1.41 (m, 2H, CH2CH2CH3), 1.66 (m, 1H,
CH2CH2CH3), 1.83 (m, 1H, CH2CH2CH3), 3.69 (s, 3H, OCH3); 3.80 (s, 3H, OCH3); 4.29 (d,
J = 3.9 Hz, 1H, CHOH); 13C-NMR (CDCl3) δ [ppm]: 14.08; 20.79; 30.36; 48.54; 52.18; 52.93; 71.22;
173.74; 174.18. Elemental anal. (%), calcd. for C9H16O5: C, 52.94; H, 7.84; found: C, 52.87; H, 7.90.
Dimethyl (2S,3R)-2-hydroxy-3-butylsuccinate (5b). Oil, final yield 65%. []2D0 +12.8 (c1.15; CHCl3).
1H-NMR (CDCl3) δ [ppm]: 0.94 (t, 3H, CH2CH2CH2CH3), 1.39 (m, 2H, CH2CH2CH2CH3), 1.68 (m,
2H, CH2CH2CH2CH3), 1.89 (m, 1H, CH2CH2CH2CH3), 3.70 (s, 3H, OCH3); 3.77 (s, 3H, OCH3); 4.29
13
(d, J = 4.2 Hz, 1H, CHOH); C-NMR (CDCl3) δ [ppm]: 12.01; 18.45; 21.01; 30.63; 48.91; 51.65;
52.98; 70.88; 173.11; 174.55. Elemental anal. (%), calcd. for C10H18O5: C, 55.05; H, 8.26; found: C,
54.98; H, 8.30.
Dimethyl (2S,3R)-2-hydroxy-3-allylsuccinate (5c). Oil, final yield 62%. []2D0 +16.1 (c1.24; CHCl3).
1H-NMR (CDCl3) δ [ppm]: 2.45 (m, 1H, CH2CH=CH2); 2.63 (m, 1H, CH2CH=CH2); 2.99 (m, 1H,
CH(OH)CHCO2CH3); 3.69 (s, 3H, OCH3); 3.81 (s, 3H, OCH3); 4.31 (d, J = 3 Hz, 1H,
CH(OH)CHCO2CH3); 5.19–5.11 (dd, J = 17 Hz and 10 Hz, 2H, CH2CH=CH2); 5.82 (m, 1H,
CH2CH=CH2); 13C-NMR (CDCl3) δ [ppm]: 32.36; 48.38; 52.27; 52.99; 70.36; 118.30; 134.91; 172.78;
174.21. Elemental anal. (%), calcd. for C9H14O5: C, 53.47; H, 6.93; found: C, 53.37; H, 7.01.

Molecules 2012, 17
14031
General procedure for the synthesis of compounds 6a–c. DAST (0.76 mL, 5.9 mmol) was dissolved in
anhydrous methylene chloride (30 mL) and the resulting solution was chilled to −78 °C under argon.
An analogously chilled solution of one of the compounds 5a–c (4.4 mmol) in methylene chloride
(10 mL) was added dropwise using a cannulated syringe and the resulting mixture was stirred for 1 h at
−78°C and for 2 h at room temperature. Chloroform (40 mL) was added and the resulting solution was
washed with 5% NaHCO3, water and saline. The solvent was evaporated and the crude products were
purified by flash silica-gel chromatography with hexane/AcOEt (9:1) to afford the final products.
Dimethyl (2R,3S)-2-fluoro-3-propylsuccinate (6a). Oil, final yield 92%. []2D0 +6.2 (c1.5; CHCl3).
1H-NMR (CDCl3) δ [ppm]: 0.93 (t, 3H, CH2CH2CH3), 1.32 (m, 1H, CH2CH2CH3), 1.43 (m, 1H,
CH2CH2CH3), 1.62 (m, 1H, CH2CH2CH3), 1.82 (m, 1H, CH2CH2CH3), 2.98 (m, 1H, FCHCHCO2CH3),
3.74 (s, 3H, OCH3); 3.82 (s, 3H, OCH3); 5.16 (dd, 2JHF = 47.4 Hz, 3JHH = 5.9 Hz, 1H, FCH); 13C-NMR
(CDCl3) δ [ppm]: 14.07; 20.67; 29.30; 47.91 (2JFC = 23.8 Hz); 52.43; 52.85; 88.92 (JFC = 197.3 Hz);
169.04; 172.21. 19F-NMR (CDCl3) δ[ppm]: −197.51 (dd, 3JHF = 23 Hz, 2JHF = 47 Hz). Elemental anal.
(%), calcd. for C9H15O4F: C, 52.43; H, 7.28; found: C, 52.34; H, 7.35.
Dimethyl (2R,3S)-2-fluoro-3-butylsuccinate (6b). Oil, final yield 88%. []2D0 +4.9 (c1.3; CHCl3).
1H-NMR (CDCl3) δ [ppm]: 0.93 (t, 3H, CH2CH2CH2CH3), 1.24 (m, 1H, CH2CH2CH2CH3), 1.32 (m,
1H, CH2CH2CH2CH3), 1.41 (m, 1H, CH2CH2CH2CH3), 1.53 (m, 1H, CH2CH2CH2CH3), 1.62 (m, 1H,
CH2CH2CH2CH3); 1.82 (m, 1H, CH2CH2CH2CH3); 2.99 (m, 1H, FCHCHCO2CH3), 3.74 (s, 3H,
2
3
13
OCH3); 3.82 (s, 3H, OCH3); 5.17 (dd, 1H, JHF = 42 Hz, JHH = 5.9 Hz, FCH); C-NMR (CDCl3)
δ [ppm]: 12.01; 18.45; 21.01; 30.63; 48.91; 51.65; 52.98; 70.88; 173.11; 174.5514.07; 20.67; 29.30;
47.91 (2JFC = 23.8 Hz); 52.43; 52.85; 88.92 (JFC = 197.3 Hz); 169.04; 172.21. 19F-NMR (CDCl3)
δ [ppm]: −197.46 (dd, 3JHF = 19.2 Hz and 2JHF = 46.8 Hz). Elemental anal. (%), calcd. for C10H17O4F:
C, 54.55; H, 7.73; found: C, 54.48; H, 7.73.
Dimethyl (2R,3S)-2-fluoro-3-allylsuccinate (6c). Oil, final yield 89%. []2D0 +2.5 (c1.7; CHCl3).
1H-NMR (CDCl3) δ [ppm]: 2.46 (m, 1H, CH2CH=CH2); 2.60 (m, 1H, CH2CH=CH2); 3.10 (m, 1H,
CH(OH)CHCO2CH3), 3.73 (s, 3H, OCH3); 3.83 (s, 3H, OCH3); 5.11 (m, 1H, FCHCHCO2CH3); 5.18
13
(m, 1H, CH2CH=CH2); 5.24 (m, 1H, CH2CH=CH2); 5.76 (m, 1H, CH2CH=CH2); C-NMR (CDCl3)
δ [ppm]: 38.21; 47.74 (2JFC = 21.5 Hz); 52.13; 52.62; 88.20 (JFC = 190.2 Hz); 119.57; 133.08; 165.68;
19
3
2
169.06; F NMR (CDCl3) δ[ppm]: -198.16 (dd, JHF = 21 Hz; JHF = 47 Hz). Elemental anal. (%),
calcd. for C9H13O4F: C, 52.94; H, 6.37; found: C, 53.02; H, 6.40.
Dipotassium (2R,3S)-2-fluoro-3-allylsuccinate (7c). Dimethyl (2R,3S)-2-fluoro-3-allylsuccinate
(6c, 41 mg; 0.2 mmol) was dissolved in anhydrous diethyl ether (20 mL). Potassium trimethylsilanolate
(56 mg, 0.44 mmol) was added to the solution and the mixture was stirred for 24 h. The solvent was
removed by evaporation under reduced pressure, the residue was washed with anhydrous diethyl ether
and finally dried under reduced pressure. The product was obtained as a slightly yellow amorphous
1
powder (35 mg; 70%). H-NMR (CDCl3) δ [ppm]: 2.44 (m, 1H, CH2CH=CH2); 2.59 (m, 1H,
CH2CH=CH2); 3.14 (m, 1H, CH(OH)CHCO2CH3), 5.11 (m, 1H, FCHCHCO2CH3); 5.20 (m, 1H,
13
CH2CH=CH2); 5.25 (m, 1H, CH2CH=CH2); 5.75 (m, 1H, CH2CH=CH2); C-NMR (CDCl3) δ[ppm]:
19
38.44; 47.07 (2JFC = 23 Hz); 52.35; 53.62; 88.20 (JFC = 192 Hz); 118.22; 135.69; F-NMR (CDCl3)

Molecules 2012, 17
14032
3
2
δ [ppm]: −197.55 (dd, JHF = 18 Hz; JHF = 44 Hz). Elemental anal. (%), calcd. for C7H7O4FK2: C,
33.33; H, 2.78; found: C, 33.39; H, 2.79.
Dimethyl (2R,3S)-2-fluoro-2-deoxyhomoisocitrate (8). BH3  SMe2 (0.51 mL, 5.05 mmol) was added
dropwise to a solution of dimethyl (2R,3S)-2-fluoro-3-allyl succinate (6c, 1.03 g, 5.05 mmol) in
methylene dichloride (8 mL) at 0 C. The reaction mixture was left at room temperature with stirring
for 2 h. Then pyridinium dichromate (PDC, 14.6 g, 38 mmol) in anhydrous DMF (30 mL) was slowly
added and stirring was continued for 48 h. After that time, the mixture was transferred to the flask
containing water (300 mL) and the whole solution was stirred for another 16 h. The resulting mixture
was extracted with diethyl ether (3  250 mL) and the aqueous layer was further extracted with ethyl
acetate (2  170 mL). The organic layers were combined and dried over anhydrous MgSO4. The drying
agent was removed and the organic solvents were evaporated under reduced pressure to give 1.2 g of
the crude product that was further purified by flash chromatography on silica gel with stepwise elution
1
with hexane/AcOEt (5:1), AcOEt and MeOH to give 0.87 g (73%) of the purified product. H-NMR
(CDCl3) δ [ppm]: 2.02 (m, 1H, CH2CH2COOH); 2.21 (m, 1H, CH2CH2COOH); 2.48 (m, 2H,
CH2CH2COOH ); 3.03 (m, 1H, FCHCOOMe); 3.76 (s, 3H, OCH3); 3.83 (s, 3H, OCH3); 5.32–5.22 (dd,
2
13
3JHH = 4.4 Hz, JHF = 47.4 Hz, CHF); C-NMR (CDCl3) δ [ppm]: 23.59; 31.80; 47.77; 51.98; 52.34;
53.09; 172.66; 173.52. 19F-NMR (CDCl3) δ [ppm]: −196.23 (dd, JHF = 21 Hz; JHF = 47 Hz).
3
2
Elemental anal. (%), calcd. for C9H13O6F: C, 45.76; H, 5.50; found: C, 45.71; H, 5.47.
Trimethyl
(2R,3S)-2-fluoro-2-deoxyhomoisocitrate
[trimethyl
(1R,2S)-1-fluorobutane-1,2,4-
tricarboxylate, 9]. Dimethyl (2R,3S)-2-fluoro-2-deoxyhomoisocitrate (8, 203 mg, 0.9 mmol) was
treated with diazomethane solution in diethyl ether under argon. The mixture was left overnight at
room temperature. The solvent was then removed by evaporation under reduced pressure. The residue
was washed twice with diethyl ether. The crude oily product was purified by flash chromatography on
silica gel with hexane/AcOEt (2:1) as an eluent. The final product was obtained as a colorless oil
(228 mg, 95%). []2D0 +12.5 (c2.0; CHCl3). 1H-NMR (CDCl3) δ [ppm]: 2.04 (m, 1H, CH2CH2COOMe);
2.21 (m, 1H, CH2CH2COOMe); 2.48 (m, 2H, CH2CH2COOMe); 3.03 (m, 1H, FCHCOOMe); 3.71
13
(s, 3H, OCH3); 3.72 (s, 3H, OCH3); 3.83 (s, 3H, OCH3); 4.63 (d, J = 3.4 Hz, 1H, FCH); C-NMR
19
(CDCl3) δ [ppm]: 23.59; 31.80; 47.77; 51.98; 52.34; 53.09; 71.34; 172.66; 173.52; 173.84. F-NMR
3
2
(CDCl3) δ [ppm]: −193.44 (dd, JHF = 22 Hz; JHF = 49 Hz). Elemental anal. (%), calcd. for
C10H15O6F: C, 50.00; H, 6.25; found: C, 50.07; H, 6.22.
Tripotassium (2R,3S)-2-fluoro-2-deoxyhomoisocitrate [tripotassium (1R,2S)-1-fluorobutane-1,2,4-
tricarboxylate, 10]. Conversion of the trimethyl ester 9 into the respective tripotassium salt was
achieved upon treatment with potassium trimethylsilanolate under condition described above for
preparation of compound 4. The product was obtained as a yellowish amorphous powder (309 mg,
1
93%). H-NMR (D2O) δ [ppm]: 2.02 (m, 1H, CH2CH2COOK); 2.25 (m, 1H, CH2CH2COOK); 2.52
(m, 2H, CH2CH2COOK); 3.01 (m, 1H, FCHCOOK); 4.68 (d, J = 3.4 Hz, 1H, FCH); 13C-NMR
(CDCl3) δ [ppm]: 23.48; 31.85; 47.72; 51.95; 52.38; 53.00; 71.31. 19F-NMR (CDCl3) δ[ppm]: −192.58
(dd, 3JHF = 20 Hz; 2JHF = 51 Hz). Elemental anal. (%), calcd. for C7H6O6FK3: C, 48.58; H, 4.86; found:
C, 48.51; H, 4.92.

Molecules 2012, 17
14033
3.3. Determination of Antifungal Activity
Antifungal in vitro activity was determined by the modified M27-A3 method specified by the CLSI [16]
in RPMI-1640 medium buffered to pH 7.0. The test micro-organisms were: Candida albicans ATCC
10231, Candida glabrata DSM 11226, Candida tropicalis KKP 334 Candida krusei DSM 6128,
Candida lusitaniae DSM 70102 and Saccharomyces cerevisiae ATCC 9763. Wells containing serially
diluted examined compounds and compound-free controls were inoculated with 12 h cultures of tested
strains to the final concentration of 104 colony-forming units (cfu)/mL. Plates were incubated for 24 h
at 37 °C and growth was then quantified by measuring an optical density at 531 nm, using a microplate
reader (Victor3V; Perkin Elmer, Centre of Excellence ChemBioFarm). The MIC was defined as the
lowest drug concentration at which at least 80% decrease in turbidity, in comparison to the drug-free
control, was observed.
3.4. Determination of Enzyme Inhibition by Novel Compounds
Recombinant C. albicans homoaconitase (HA) and homoisocitrate dehydrogenase (HIcDH)
containing oligoHis N-terminal tags were constructed and purified to near homogeneity as described
earlier [14,17,18]. Both recombinant proteins exhibited enzymatic activities identical to their wild type
counterparts. Recombinant HA demonstrated KM = 1.5 mM for (R)-homocitrate and recombinant
HIcDH showed KM = 74 M for (2R, 3S)-homoisocitrate and KM = 1.1 mM for NAD+.
Reaction catalyzed by homoisocitrate dehydrogenase (HIcDH) was monitored by measuring the
NADH absorption at 340 nm. The standard assay mixture contained 10 mM KCl, 5.0 mM MgCl2,
5 mM NAD+ and 0.1 mM (2R,3S)-homoisocitrate in 50 mM Hepes-NaOH (pH 7.8) buffer. The
reaction mixture with all required components except for the enzyme was preincubated for 3 min and
the reaction was started by an addition of the recombinant His6-HIcDH (50 g). The formation of
NADH was measured for 30 s at 20 °C to determine initial reaction velocity. The initial rates were
determined using a linear portion of the reaction progress curve.
Activity of homoaconitase (HA) was measured by a coupled reaction with HIcDH by monitoring
the reduction of NAD+ to NADH at 340 nm [19]. Reaction mixtures containing 50 mM Tris-HCl
(pH 8.5), 50 mM KCl, 5 mM MgCl2, 20 mM dithiothreitol, 1 mM NAD+ and 30 g/mL of
His6-HIcDH in a total volume of 0.9 mL were degassed under argon for 10 min, after which 0.05 mL
of His6-HAC solution was added to a final concentration of 10 g/mL. The reaction was initiated with
addition of 0.05 mL of 50 mM (R)-homocitrate. The formation of NADH was measured for 30 s at
20 °C to determine initial reaction velocity. The initial rates were determined using a linear portion of
the reaction progress curve.
The effects of novel compounds on the HA and HIcDH activity was determined by measuring the
enzyme activity in standard assay mixtures containing various concentration of an inhibitor (0–10 mM).
Inhibitory potential was expressed as IC50 values, i.e., concentrations causing 50% inhibition of
enzyme activity in relation to control. All determinations were performed in triplicate.

Molecules 2012, 17
14034
3.5. Molecular Modeling and Docking Calculations
Model of the receptor (CaHIcDH) was built using the template of Schisosaccharomyces pombe
HIcDH [13], as described elsewhere [12]. Docking calculations of all ligands were performed with the
AutoDock software version 4.2 [20]. Initial geometries of the ligands were built by means of the
Accelrys Discovery Studio. Final AutoDock topologies of ligands and receptors in the pdbqt format,
including the appropriate for the program atom types and partial charges, were calculated and assigned
with AutoDock accompanied scripts. Docking grid was centered in the middle of the binding site at the
Arg122 guanidine moiety of HIcDH. Semi flexible docking methodology was used (with fixed
receptor and flexible ligands) and Lamarckian genetic algorithm (LGA) was chosen as the search
engine. The initial size of the population was set to 150 random solutions and the number of
generations was limited to 27,000. To ensure the convergence of the search procedure number of
objective function evaluations was raised to 25,000,000. Docking procedure was repeated 50 times and
the resulting 50 ligand poses were clustered with the tolerance of 1.8 Å. The most abundant and the
lowest energy clusters were selected for analysis.
4. Conclusions
Two analogs of cis-homoaconitate, namely the tripotassium salts of trans-homoaconitate and
(1E)1,2-epoxybutane-1,2,4-tricarboxylate were found to inhibit activity of C. albicans HA, while
tripotassium (2R,3S)-2-fluoro-3-allylsuccinate and tripotassium (2R,3S)-2-fluoro-2-deoxyhomo-
isocitrate inhibited HIcDH from the same source. The trimethyl ester derivatives of these compounds
inhibited growth of some human pathogenic yeast, including C. albicans. We have been thus able to
demonstrate for the first time that inhibitors of particular enzymes catalyzing initial steps of
fungi-specific AAP pathway may exhibit antifungal activity after conversion into more lipophilic,
membrane-permeable forms. The compounds described in this work may serve as leads for further
studies on the design of compounds targeting AAP as potential antifungals. In light of our results, it
seems very likely that the ester forms of strong inhibitors of the enzymes of AAP may exhibit good
antifungal potential, so that a further search for such compounds as potential antifungal pro-drugs is
undoubtedly worth pursuing.
Acknowledgments
Financial support of these studies by the Foundation for Polish Science is gratefully acknowledged.
Calculations were carried out at the Academic Computer Centre in Gdańsk (TASK).
Conflict of Interests
The authors declare no conflict of interests.
References
1. Pfaller, M.A.; Diekema, D.J. Epidemiology of invasive candidiasis: A persistent public health
problem. Clin. Microbiol. Rev. 2007, 20, 133–163.

Molecules 2012, 17
14035
2. Monk, B.C.; Goffeau, A. Outwitting multidrug resistance to antifungals. Science 2008, 321, 367–369.
3. Hutton, C.A.; Southwood, T.J.; Turner, J.J. Inhibitors of lysine biosynthesis as antibacterial
agents. Mini Rev. Med. Chem. 2003, 3, 115–127.
4. Xu, H.; Andi, B.; Qian, J.; West, A.H.; Cook, P.F. The alpha-aminoadipate pathway for lysine
biosynthesis in fungi. Cell Biochem. Biophys. 2006, 46, 43–64.
5. Schöbel, F.; Jacobsen, I.D.; Brock, M. Evaluation of lysine biosynthesis as an antifungal drug
target: Biochemical characterization of Aspergillus fumigatus homocitrate synthase and virulence
studies. Eukaryotic Cell 2010, 9, 878–893.
6. Liebmann, B.; Mühleisen, T.W.; Müller, M.; Hecht, M.; Weidner, G.; Braun, A.; Brock, M.;
Brakhage, A.A. Deletion of the Aspergillus fumigatus lysine biosynthesis gene lysF encoding
homoaconitase leads to attenuated virulence in a low-dose mouse infection model in invasive
aspergillosis. Arch. Microbiol. 2004, 181, 378–383.
7. Palmer, D.R.J.; Balogh, H.; Ma, G.; Zhou, X.; Marko, M.; Kaminskyj, S.G.W. Synthesis and
antifungal properties of compounds which target the aminoadipate pathway. Pharmazie 2004,
59, 93–98.
8. Seebach, D.; Aebi, J.; Wasmuth, D. Diastereoselective -alkylation of -hydroxycarboxylic esters
through alkoxide enolates: diethyl (2S,3R)-(+)-3-allyl-2-hydroxysuccinate from diethyl
(S)-(−)-malate. Org. Synth. 1985, 63, 109–120.
9. Ma, G.; Palmer, D.R.J. Improved asymmetric syntheses of (R)-(−)-homocitrate and (2R,3S)-(−)-
homoisocitrate, intermediates in the alpha-aminoadipate pathway of fungi. Tetrahedron Lett.
2000, 41, 9209–9212.
10. Kirk, K.L. Fluorination in medicinal chemistry: Methods, strategies and recent developments.
Org. Proc. Res. Dev. 2008, 12, 305–321.
11. Yamamoto, T.; Miyazaki, K.; Eguchi, T. Substrate specificity analysis and inhibitor design of
homoisocitrate dehydrogenase. Bioorg. Med. Chem. 2007, 15, 1346–1355.
12. Yamamoto, T.; Eguchi, T. Thiahomoisocitrate: a highly potent inhibitor of homoisocitrate
dehydrogenase involved in the alpha-aminoadipate pathway. Bioorg. Med. Chem. 2008, 16,
3372–3376.
13. Bulfer, S.L.; Hendershot, J.M.; Trievel, R.C. Crystal structure of homoisocitrate dehydrogenase
from Schizosaccharomyces pombe. Proteins 2012, 80, 661–666.
14. Gabriel, I.; Vetter, N.D.; Palmer, D.R.J.; Milewska, M.J.; Wojciechowski, M.; Milewski, S.
Homoisocitrate dehydrogenase from Candida albicans: Properties, inhibition and targeting by an
antifungal pro-drug. FEMS Yeast Res. 2012, submitted for publication.
15. Prokop, M.; Milewska, M.J. An improved synthesis of trisodium (R)-homocitrate from citric acid.
Polish J. Chem. 2009, 83, 1317–1322.
16. Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution
Antifungal Susceptibility Testing of Yeasts, Approved Standard, 3rd ed.; CLSI document M27-A3;
CLSI: Wayne, PA, USA, 2008.
17. Kur, K. Homocitrate synthase and homoaconitase of Candida albicans as potential molecular
targets for antifungal chemotherapy. Ph.D. Thesis, Gdansk University of Technology, Gdańsk,
Poland, 2009.

Molecules 2012, 17
14036
18. Gabriel, I.; Szweda, P.; Kur, K.; Prokop, P.; Milewska, M.J.; Milewski, S. Enzymes of the lysine
biosynthetic pathway as targets for antifungals? FEBS J. 2010, 277, 94–95.
19. Drevland, R.M.; Jla, Y.; Palmer, D.R.J.; Graham, D.E. Methanogen homoaconitase catalyzes both
hydrolyase reactions in coenzyme B biosynthesis. J. Biol. Chem. 2008, 283, 28888–28896.
20. Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J.
AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility.
Comput. Chem. 2009, 30, 2785–2791.
Sample Availability: Samples of the compounds 1–10 are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).