The substantial improvement of amphotericin b selective toxicity upon modification of mycosamine with bulky substituents

Article abstract of DOI:10.2174/1573406415666181203114629

Background: It is assumed that the unfavorable selective toxicity of an antifungal drug Amphotericin B (AmB) can be improved upon chemical modification of the antibiotic molecule. Objective: The aim of this study was verification of the hypothesis that in

Full text of DOI:10.2174/1573406415666181203114629

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Medicinal Chemistry, 2020, 16, 128-139
RESEARCH ARTICLE
ISSN: 1573-4064
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The Substantial Improvement of Amphotericin B Selective Toxicity Upon
Modification of Mycosamine with Bulky Substituents
Edward Borowski1, Natalia Salewska1,2, Joanna Boros-Majewska1,3, Marcin Serocki3, Izabela
Chabowska1, Maria J. Milewska2, Dominik Ziętkowski1 and Sławomir Milewski3,*
1BLIRT S.A., Gdańsk, Poland, Trzy Lipy 3, 80-172 Gdańsk, Poland; 2Department of Organic Chemistry; 3Department of
Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdańsk University of Technology, 11/12
Narutowicza St., 80-233 Gdańsk, Poland
Abstract: Background: It is assumed that the unfavorable selective toxicity of an antifungal drug
Amphotericin B (AmB) can be improved upon chemical modification of the antibiotic molecule.
Objective: The aim of this study was verification of the hypothesis that introduction of bulky sub-
stituents at the amino sugar moiety of the antibiotic may result in diminishment of mammalian in
vitro toxicity of thus prepared AmB derivatives.
Methods: Twenty-eight derivatives of AmB were obtained upon chemical modification of the
amino group of mycosamine residue. This set comprised 10 N-succinimidyl-, 4 N-benzyl-, 5 N-
thioureidyl- and 9 N-aminoacyl derivatives. Parameters characterizing biological in vitro activity
A R T I C L E H I S T O R Y
of novel compounds were determined.
Received: August 27, 2018
Revised: November 13, 2018
Accepted: November 25, 2018
Results: All the novel compounds demonstrated lower than AmB antifungal in vitro activity but
most of them exhibited negligible cytotoxicity against human erythrocytes and three mammalian
cell lines. In consequence, the selective toxicity of majority of novel antifungals, reflected by the
selective toxicity index (STI = EH50/IC50) was improved in comparison with that of AmB, espe-
cially in the case of 5 compounds. The novel AmB derivatives with the highest STI, induced sub-
stantial potassium efflux from Candida albicans cells at concentrations slightly lower than IC50s
but did not trigger potassium release from human erythrocytes at concentrations lower than 100
μg/mL.
DOI:
10.2174/1573406415666181203114629
Conclusion: Some of the novel AmB derivatives can be considered promising antifungal drug
candidates.
Keywords: Antifungal agent, amphotericin B, chemical modification, selective toxicity, hemotoxicity, potassium efflux.
1. INTRODUCTION
mammalian toxicity, being a consequence of mechanism of
antibiotic action. According to the two hypotheses on this
mechanism, the “barrel-stave-pore” [3] and a “sterol sponge”
[4], binding of AmB to sterol present in the fungal or mam-
malian cell membrane is the necessary condition for its bio-
logical effect. In the “barrel-stave-pore” mechanism, Am-
B:sterol complexes assemble into transmembrane barrel-like
pores facilitating efflux of monovalent ions, especially K+.
This efflux, causing impairment of membrane barrier func-
tion, is considered a primary effect leading to cell death. A
slightly higher affinity of AmB to fungal ergosterol than to
mammalian cholesterol constitutes a molecular basis for the
selective toxicity of this antibiotic [5]. In consequence, the
therapeutic window of AmB is very narrow. This disadvan-
tage has been only in part overcome upon construction of
AmB complexes with lipids or liposomal formulations, such
as Abelcet®, Amphotec® or AmBisome®, at the price of a
substantial rise in treatment cost [6].
Polyene macrolide antibiotics constitute the group of the
most potent broad-spectrum antifungals. A member of this
group, heptaenic macrolide Amphotericin B (AmB), is the
drug of choice for the treatment of disseminated fungal in-
fections, especially in immunocompromised patients [1].
This antibiotic, consisting of a large polyene macrolide ring
and an amino sugar mycosamine, combines most of the fea-
tures expected for an “ideal” antifungal chemotherapeutic,
including the high antifungal efficacy at low drug concentra-
tion, broad antifungal spectrum covering the multidrug-
resistant fungal species, a fungicidal mode of action and a
very limited potential of inducing fungal specific resistance
[2]. The important disadvantage of AmB is its substantial
*Address correspondence to this author at the Department of Pharmaceuti-
cal Technology and Biochemistry, Faculty of Chemistry, Gdańsk University
of Technology, 80-233 Gdańsk, Poland; Tel/Fax: +4858 3472107/+4858
3471144; E-mail: slamilew@pg.edu.pl
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The Substantial Improvement of Amphotericin B Selective Toxicity
Medicinal Chemistry, 2020, Vol. 16, No. 1 129
Many examples of structural modifications of AmB
molecule aimed at the improvement of its selective toxicity
have been reported. Several AmB analogues modified in the
polyol region of the aglycone were obtained upon biosyn-
thetic engineering of the Nystatin-producing Streptomyces
noursei, resulting in a series of AmB-mimicking 28,29-
didehydronystatin derivatives, of which antibiotics S44HP
and BSG005 demonstrated advantages over AmB in the in
vivo tests [7, 8]. However, most modifications of AmB were
made by chemical reactions and concerned the carboxyl
group or the amino functionality of mycosamine. Some of
the AmB derivatives thus obtained demonstrated improved
selective toxicity, for example the AmB-benzoxaborole hy-
brids [9]. A substantial improvement was observed for N-D-
ornithyl-AMB [10], N-methyl-N-D-fructopyranosyl-ampho-
tericin B methyl ester [11] or N-piperidinepropionyl ampho-
tericin B methyl ester [12]. N, N-di- (3-aminopropyl) AmB
exhibited much higher than AmB antifungal activity and a
slightly lower mammalian toxicity [13]. Substantial reduc-
tion of mammalian toxicity in vitro with retention or mini-
mal decrease in antifungal potency has been achieved for 2’-
deoxyAmB [14] but the conversion of AmB into its 2’-deoxy
derivative is a multistep, costly procedure. The similar effect
was shown for the derivatives modified at the carboxyl func-
tionality, namely AmB urea derivatives [15] and conjugates
of AmB with “molecular umbrellas” [16].
CHCl3: MeOH: H2O (25:8:1, v/v); C CHCl3:MeOH:H2O
(30:8:1, v/v); D CHCl3:MeOH:H2O (10:6:1, v/v).
N-substituted maleimides were synthesized as described
previously [19]. Isothiocyanates were prepared following the
methodology described in previous works [20, 21]. N, N-
dialkyl amino acids were prepared upon reductive alkylation
of amino acid methyl esters with an appropriate aldehyde
(1:4 molar ratio), followed by ester hydrolysis [22].
The general methods of synthesis of N-succinimidyl-, N-
aminosuccinimidyl-, N-benzyl-, N-thioureidyl- and N-
aminoacyl derivatives of AmB were described previously
[23].
2.1.1. N-succinimidyl Derivatives of Amphotericin B
2.1.1.1. N-(N-benzylsuccinimidyl)-Amphotericin B ANS1
Yield 88%. TLC (C) Rf = 0.27; HPLC Rt = 15.480 min.,
purity 97.2%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
E1cm1% (MeOH, λ = 406 nm) = 1180; MS-ESI found m/z:
1109.3352 [M−H] −; calculated for C58H82N2O19 1110.1117.
2.1.1.2. N-(N-2,6-dimethylphenylsuccinimidyl)-Amphoteri-
cin B ANS2
Yield 92.2%. TLC (C) Rf = 0.30; HPLC Rt = 16.432
min., purity 95.3%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1147.4351 [M+Na]+; calculated for
C59H84N2O19 [M−H] − 1124.6663.
In our approach, we hypothesized that a substantial im-
provement of AmB selective toxicity could be achieved upon
introduction of bulky substituents at the amino group of my-
cosamine, with retention of a positive charge at this site.
Such an assumption was based on results of our previous
studies, indicating that AmB derivatives with bulky substitu-
ents at the amino group, should form complexes with ergos-
terol or cholesterol of different geometries which could re-
sult in differential abilities to assemble into lethal transmem-
brane channels in ergosterol- or cholesterol-containing
membranes [17]. On the other hand, the positive charge at
mycosamine or in its close vicinity is the well -documented
prerequisite for preservation of good antifungal in vitro ac-
tivity [18]. In this work, we report on synthesis and biologi-
cal properties of 28 AmB derivatives containing easily intro-
ducible bulky substituents at the amino group of mycosa-
mine, fulfilling the above mentioned structural requirements.
2.1.1.3. N-(N-2,4,6-trimethylphenylsuccinimidyl)-Amphote-
ricin B ANS3
Yield 89.9%. TLC (C) Rf = 0.32; HPLC Rt = 16.576
min., purity 98.2%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1137.4155 [M−H]−; calculated for
C60H86N2O19 1138.1753.
2.1.1.4. N-(N-2-tert-butylphenylsuccinimidyl)-Amphoteri-
cin B ANS4
Yield 91.9%. TLC (C) Rf = 0.22; HPLC Rt = 17.223
min., purity 99.1%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
−
MS-ESI found m/z: 1175.4531 [M−H] ; calculated for
C55H84N2O19 1076.6513.
2.1.1.5. N-(N-cyclohexylsuccinimidyl)-Amphotericin
ANS5
B
Yield 94.6%. TLC (B) Rf = 0.15; HPLC Rt = 17.567
min., purity 95.1%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1125.1167 [M+Na]+; calculated for
C57H86N2O19 1102.6112.
2. MATERIALS AND METHODS
2.1. Synthesis of AmB Derivatives
Purification of final reaction products was carried out by
preparative column liquid chromatography. Amphotericin B
was from Sigma-Aldrich Co. (St. Louis, MO). Fmoc amino
acids, amino acids and aromatic aldehydes were of commer-
cial origin. Commercial grade reagents and solvents were
used without further purification. The mobile phase was ace-
tonitrile/water (20-70% acetonitrile gradient) containing
0.5% HCOOH. HRMS-ESI spectra were recorded on
Aqilent Technologies 6540 UHD Accurate – Mass Q-TOF
LC/MS apparatus. UV-VIS spectra were recorded on
Lambda 45 Perkin Elmer spectrophotometer. HPLC analysis
was performed on Agilent 1200 Series apparatus, using the
Zorbax Eclipse XDB C18 column (4.6 × 150 mm). TLC sol-
vent systems: A CHCl3: MeOH: H2O (20:8:1, v/v); B
2.1.2. N-aminosuccinimidyl Derivatives of Amphotericin B
2.1.2.1. N-{N-[3-(N,N-dimethylamino)-2,2-dimethylpropyl]-
succinimidyl}-Amphotericin B ANS6
Yield 85.5%. TLC (A) Rf = 0.23; HPLC Rt = 10.682
min., purity 96.7%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1134.6259 [M+H]+; calculated for
C58H91N3O19 1133.6241.
2.1.2.2. N-{N-[2-(piperidin-1-yl)ethyl]succinimidyl}-Amph-
otericin B ANS7
Yield 82.6%. TLC (A) Rf = 0.23; HPLC Rt = 10.852
min., purity 92.8%. UV-vis: λmax (MeOH) 406; 382; 363 nm;

130 Medicinal Chemistry, 2020, Vol. 16, No. 1
Borowski et al.
MS-ESI found m/z: 1132.5898 [M+H]+; calculated for
C58H89N3O19 1131.6090.
MS-ESI found m/z: 1060.5225 [M+H]+; calculated for
C53H77N3O17 S 1059.5184.
2.1.2.3. N-[N-(2-morpholin-1-yl-ethyl)succinimidyl]-Amph-
otericin B ANS8
2.1.4.3. N-{3-[3-(N,N-dimethylamino)propyl]-thioureidyl}-
Amphotericin B ATU3
Yield 78.5%.TLC (A) Rf = 0.17; HPLC Rt = 10.627 min.,
purity 94.6%. UV-vis: λmax (MeOH) 406; 382; 363 nm; MS-
ESI found m/z: 1134.5891 [M+H]+; calculated for
C57H87N3O20 1133.5833.
Yield 87.5%. TLC (A) Rf = 0.38; HPLC Rt = 11.952
min., purity 95.0%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1068.4765 [M+H]+; calculated for
C53H85N3O17 S 1067.6157.
2.1.2.4. N-{N-[2-(4-methylpiperazin-1-yl)ethyl]succinimi-
dyl}-Amphotericin B ANS9
2.1.4.4. N-{3-[2-(N,N-diethylamino)ethyl]-thioureidyl}-Am-
photericin B ATU4
Yield 80.4%. TLC (A) Rf = 0.22; HPLC Rt = 10.077
min., purity 96.3%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1147.6147 [M+H]+; calculated for
C58H90N4O19 1146.6199.
Yield 89.7%. TLC (A) Rf = 0.32; HPLC Rt = 12.021
min., purity 97.8%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1096.5578 [M+H]+; calculated for
C55H89N3O17 S 1095.6009.
2.1.2.5. N-{N-[(2-pyridin-4-yl)-methyl]succinimidyl}-Amph-
otericin B ANS10
2.1.4.5. N-{3-[(2-piperidin-1-yl)ethyl]-thioureidyl}-Ampho-
tericin B ATU5
Yield 89.7%. TLC (A) Rf = 0.25; HPLC Rt = 10.599
min., purity 92.2%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1113.5337 [M+H]+; calculated for
C57H87N3O19 1112.5337.
Yield 92.8%. TLC (A) Rf = 0.40; HPLC Rt = 12.082
min., purity 92.2%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1094.5634 [M+H]+; calculated for
C55H87N3O17 S 1093.6112.
2.1.3. N-benzyl Derivatives of Amphotericin B
2.1.3.1. N-benzyl-Amphotericin B AB1
2.1.5. N-aminoacyl and N-(N′-alkylamino)acyl Derivatives
of Amphotericin B
2.1.5.1. N-D-phenylglycyl-Amphotericin B AAA1
Yield 86.9%. TLC (D) Rf = 0.81; HPLC Rt = 14.771
min., purity 96.5%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1012.3333 [M-H]+; calculated for
C54H79NO17 1013.6501.
Yield 87.0%TLC (A) Rf = 0.35; HPLC Rt = 13.323 min.,
purity 97.2%. UV-vis: λmax (MeOH) 406; 382; 363 nm; MS-
ESI found m/z: 1081.3461 [M+Na]+; calculated for
C55H80N2O18 1056.5530.
2.1.3.2. N-[(pyridin-2-yl)methyl]Amphotericin B AB2
2.1.5.2. N-[β-(pyridine-1-yl)-D-alanyl]Amphotericin
AAA2
B
Yield 91.6%. TLC (D) Rf = 0.42; HPLC Rt = 13.750
min., purity 94.5%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1013.3258 [M-H]-; calculated for
C53H78N2O17 1014.5226.
Yield 85.3%. TLC (A) Rf = 0.70; HPLC Rt = 8.193 min.,
purity 92.6%. UV-vis: λmax (MeOH) 406; 382; 363 nm; MS-
ESI found m/z: 1072.9115 [M-H]-; calculated for
C55H83N3O18 1073.6561.
2.1.3.3. N-(4-tert-butylbenzyl)Amphotericin B AB3
Yield 90.7%. TLC (D) Rf = 0.87; HPLC Rt = 18.322
min., purity 99.7%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1068.5211 [M-H]-; calculated for
C58H87NO17 1069.6511.
2.1.5.3. N-(Oγ-tert-butyl-D-glutamyl)Amphotericin B AAA3
Yield 90.3%. TLC (A) Rf = 0.26; HPLC Rt = 14.861
min., purity 90.9%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1107.3614 [M-H]-; calculated for
C56H88N2O20 1108.6122.
2.1.3.4. N-(4-N,N-diethylaminobenzyl)Amphotericin B AB4
Yield 89.0%. TLC (D) Rf = 0.75; HPLC Rt = 12.175
min., purity 93.4%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1083.3544 [M-H]-; calculated for
C58H88N2O17 1084.6177.
2.1.5.4. N-(O-tert-butyl-D-seryl)Amphotericin B AAA4
Yield 93.3%. TLC (A) Rf = 0.35; HPLC Rt = 13.720
min., purity 93.2%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1065.3437 [M-H]-; calculated for
C54H86N2O19 1066.6444.
2.1.4. N-Thioureidyl Derivatives of Amphotericin B
2.1.4.1. N-(3-phenyl)-Thioureidyl-Amphotericin B ATU1
2.1.5.5. N-(Oβ-tert-butyl-D-aspartyl)Amphotericin B AAA5
Yield 85.5%. TLC (A) Rf = 0.85; HPLC Rt = 15.385
min., purity 87.2%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1057.5432 [M−H]−; calculated for
C54H78N2O17 S 1058.5088.
Yield 88.4%. TLC (A) Rf = 0.35; HPLC Rt = 14.714
min., purity 92.5%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1093.4937 [M-H]-; calculated for
C55H86N2O20 1094.6504.
2.1.4.2. N-[3-(pyridin-3-yl)-thioureidyl]-Amphotericin
ATU2
B
2.1.5.6. N-(2-fluoro-D-phenylalanyl)Amphotericin B AAA6
Yield 87.6%. TLC (A) Rf = 0.41; HPLC Rt = 14.143
min., purity 90.2%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
Yield 95.8%. TLC (A) Rf = 0.83; HPLC Rt = 12.186
min., purity 92.8%. UV-vis: λmax (MeOH) 406; 382; 363 nm;

The Substantial Improvement of Amphotericin B Selective Toxicity
Medicinal Chemistry, 2020, Vol. 16, No. 1 131
MS-ESI found m/z: 1087.2517 [M-H]-; calculated for
C54H81FN2O18 1088.5561.
values were read, which were the interpolated concentrations
of a tested compound, at which, the A531 value was exactly
50% of the A531 value for the control sample. MIC was de-
fined as the lowest drug concentration that gave at least 80%
decrease in turbidity, relative to that of the drug-free growth
control.
2.1.5.7. N-(N-methyl-α-methylalanyl)Amphotericin B AA-
A7
Yield 91.1%. TLC (A) Rf = 0.20; HPLC Rt = 9.276 min.,
purity 90.7%. UV-vis: λmax (MeOH) 406; 382; 363 nm; MS-
ESI found m/z: 1021.3418 [M]+; calculated for C52H81N2O18
1021.6155.
2.1.5.8. N-(N-methyl-Oγ-tert-butyl-L-glutamyl)Amphoter-
icin B AAA8
2.4. Determination of Mammalian in vitro Cytotoxicity
Hemolysis assay and cytotoxicity determination were
performed exactly as described previously [26].
2.5. Determination of Potassium Efflux
Yield 89.9%. TLC (A) Rf = 0.24; HPLC Rt = 9.895 min.,
purity 97.2%. UV-vis: λmax (MeOH) 406; 382; 363 nm; MS-
ESI found m/z: 1121.4531 [M-H]-; calculated for
C57H90N2O20 1022.6831.
A. Yeast cells. C. albicans ATCC 10231 cells were
grown overnight in YPD liquid medium at 30oC with vigor-
ous shaking. The cells were harvested by centrifugation for
10 minutes at 1700 × g, washed three times with sterile TBS
(Tris-HCl, pH 7.4 containing 0.9% NaCl) and dissolved in
TBS to the final concentration of 2.0 mg of dry weight per
mL. Kinetics of potassium leakage was followed with a
potassium-selective combined electrode (PerfectION™,
Mettler Toledo) linked with CPI-505 pH-ionmeter
(Elmetron) in 25 mL cell suspensions. After 10 minutes of
signal stabilization, 0.25 mL solutions of compounds tested
in DMSO (100 × concentrated) were added and the
measurement was continued for 30 minutes. The maximal
potassium efflux (100%) was determined for cell suspension
pre-boiled for 15 min (positive control).
2.1.5.9. N-[(3-piperidin-1-yl)propionyl]amphotericin B (A-
AA9)
Yield 87.9%. TLC (A) Rf = 0.27; HPLC Rt = 11.681
min., purity 94.4%. UV-vis: λmax (MeOH) 406; 382; 363 nm;
MS-ESI found m/z: 1061.4053 [M-H]-; calculated for
C55H86N2O18 1062.6734.
2.2. Microbial Strains and Culture Conditions
The reference strains used in this study were: Candida al-
bicans ATCC 10231, Candida albicans SC 5314, Candida
glabrata DSM 11226, Candida krusei DSM 6128, Candida
parapsilosis DSM 5784, Candida stellatoidea CBS 1905,
Candida dubliniensis CBS 7987, Candida lusitaniae DSM
70102, Candida guiliermondi DSM 11947, Candida tropi-
calis DSM 11953, Cryptococcus gatti 8395, Cryptococcus
neoformans 8398, Trichoderma viride LOCK E159, Asper-
gillus fumigatus 10507, Aspergillus niger LOCK E201,
Fusarium solani, 13932, Mucor circinelloides 9898 and
Rhisopus oryzae 8542. C. albicans B3, B4, Gu4 and Gu5
clinical isolates were kindly provided by Joachim
Morschhäuser, Würzburg, Germany. Gu4 and B3 are flu-
conazole-sensitive isolates obtained from early infection
episodes, while Gu5 and B4 are the corresponding flucona-
zole-resistant isolates obtained from later episodes in the
same patients treated with fluconazole [24]. Strains were
grown at 30°C in YPD medium (2% glucose, 1% Yeast Ex-
tract and 1% Bacto Peptone) and stored on YPD agar plates
containing 2% agar.
B. Erythrocytes. To the freshly prepared human erythro-
cytes (RBC) suspensions (50 mL, 2 × 107 cells/ mL, hemocy-
tometer count) in saline, 100 × concentrated solutions of
compounds tested in 0.5 mL of DMSO were added. Suspen-
sions were incubated at 37°C for 30 min. Samples of 5 mL
were collected at 5 min intervals and immediately filtered
through the Whatman GF/A filters and filtrates were col-
lected. Potassium concentration in filtrates was determined
with the BWB-1 flame photometer. Erythrocyte suspension
containing 0.1% Triton X-100 and 1% DMSO served as a
positive control (100% efflux) and erythrocyte suspension
containing 1% DMSO as a negative control (0% efflux).
The percent of K+ ions released from C. albicans of cells
or erythrocytes after 30 min in comparison with positive
controls were plotted against log of compound concentration
and EK50 values, i.e. compound concentration inducing 50%
potassium release after 30 min, were determined from the
curves obtained.
2.3. Determination of Antifungal in vitro Activity
The in vitro growth inhibitory activity of antifungals was
quantified by determination of MIC values by the serial two-
fold dilution method, using the 96-well microtiter plates.
AmB and its derivatives were dissolved in DMSO and 5 μL
aliquots of serial two-fold dilutions were added to individual
wells so that the final DMSO concentration was 0.5% (v/v).
Conditions of the assay were the same as outlined in the
CLSI recommendations [25], except for the end-point read-
out that was done by spectrophotometric determination of
cell density at 531 nm. Turbidity in individual wells was
measured with a microplate reader (Victor3; Perkin Elmer).
On the basis of obtained results, the diagrams of the
relationship between A531 values and concentration of exam-
ined compound were drawn. From these graphs, the IC50
3. RESULTS
3.1. Chemistry
The synthetic strategies of AmB modification at its
amino group applied in this work comprised: a/ the forma-
tion of N-succinimidyl analogs upon Michael-type addition
of N-substituted maleimide derivatives; b/ the formation of
N-benzyl analogs upon reductive amination with benzalde-
hyde derivatives; c/ the formation of N-thioureidyl analogs
upon reaction with isothiocyanates; d/ the formation of N-
aminoacyl analogs upon DCC-driven acylation with respec-
tive amino acid N-hydroxy-succinimides. All these reactions
were performed under mild, preservative conditions, with no
need for prior protection of any functionalities of the AmB

132 Medicinal Chemistry, 2020, Vol. 16, No. 1
Borowski et al.
OH
OH
O
O
OH
O
O
OH
OH
OH
OH
OH
OH
HO
H
O
H
O
AmB, R = H
HO
NHR
N-succinimidyl
N-aminoacyl
N-thioureidyl
R =
NH2
NH2
Symbol
Symbol
R =
R =
Symbol
AAA1
O
O
O
O
N
ATU1
ANS1
ANS2
S
O
O
N
O
O
ATU2
ATU3
ATU4
N
N
AAA2
AAA3
AAA4
S
O
O
O
O
O
nBu
O
ANS3
ANS4
N
S
S
N
NH2
NH2
O
O
tBu
O
O
N
NEt2
But
O
O
O
O
F
O
ATU5
tBu
ANS5
N
AAA5
N
NH2
NH2
O
S
O
O
O
N-benzyl
ANS6
ANS7
N
AAA6
AAA7
Symbol
AB1
R =
O
O
O
O
H
N
N
N
N
O
O
O
N
tBu
AB2
AB3
O
ANS8
N
AAA8
NH
O
O
O
O
ANS9
N
N
AAA9
tBu
N
N
O
O
ANS10
AB4
N
NEt2
O
N
Fig. (1). Structures of AmB and its derivatives.
molecule. The yields were generally good, in 78-98% range.
The final products (28 compounds, Fig. (1) were purified by
HPLC and their identity was unequivocally confirmed by
HRMS and UV-vis spectroscopy. Almost all AmB deriva-
tives specified in Fig. (1) are novel, except for the previously
described N-benzyl-AMB (AB1) [27].

The Substantial Improvement of Amphotericin B Selective Toxicity
Medicinal Chemistry, 2020, Vol. 16, No. 1 133
Table 1. Parameters of anticandidal in vitro activity and hemolytic activity and selective toxicity indexes of AmB and its novel de-
rivatives. The IC50 and EH50 data are the means of 3 independent determinations SD.
Anticandidal in vitro Activity
Hemolysis EH50
-
STI (EH50 / IC50)
[μg/mL]
MIC [μg/mL]
IC50 [μg/mL]
AmB
ANS1
ANS2
ANS3
ANS4
ANS5
ANS6
ANS7
ANS8
ANS9
ANS10
AB1
0.25
4.0
2.0
4.0
4.0
2.0
4.0
4.0
4.0
1.0
8.0
1.0
0.5
4.0
1.0
8.0
2.0
1.0
2.0
1.0
2.0
2.0
4.0
2.0
4.0
2.0
0.5
4.0
0.5
0.19 0.02
3.11 0.05
1.55 0.07
2.87 0.08
3.22 0.07
1.48 0.05
3.11 0.02
3.05 0.03
2.95 0.07
0.87 0.01
5.26 0.09
0.76 0.01
0.32 0.02
3.28 0.89
0.66 0.03
5.87 0.56
1.48 0.07
0.75 0.03
1.57 0.04
0.65 0.02
1.76 0.12
1.14 0.09
3.28 0.08
1.07 0.11
2.86 0.14
1.56 0.07
0.34 0.02
3.02 0.18
0.39 0.02
2.06 0.06
>100
10.8
>32.2
>64.5
>34.8
>32.0
>67.6
>32.2
>32.8
13.0
>100
>100
>100
>100
>100
>100
38.36 0.87
>100
>114.9
>19.0
4.6
>100
3.50 0.12
3.93 0.08
>100
AB2
12.3
AB3
>30.5
>151.5
>17.4
>67.6
55.8
AB4
>100
ATU1
ATU2
ATU3
ATU4
ATU5
AAA1
AAA2
AAA3
AAA4
AAA5
AAA6
AAA7
AAA8
AAA9
>100
>100
41.85 1.32
>100
>63.7
>153.9
>56.8
>87.7
>30.5
>93.5
>35.0
>64.1
36.9
>100
>100
>100
>100
>100
>100
>100
12.55 0.78
>100
>33.1
36.0
14.03 0.66
3.2. Biological Evaluation
EH50, i.e. concentration of compound tested causing lysis of
50% of erythrocytes. The MIC, IC50 and EH50 values deter-
mined for all compounds are shown in Table 1.
AmB and all its derivatives were tested for antifungal in
vitro activity against the model C. albicans ATCC 10 231
strain and hemotoxicity against human RBC. The former
was characterized by the MIC and IC50 values, determined in
RPMI-1640 medium under conditions recommended by
CLSI [25]. The quantitative parameter of hemotoxicity was
The fungistatic in vitro activity of all derivatives was
lower than that of AmB and their MIC values were 2 – 32
times higher than that of the mother antibiotic but for 16 out
of 28 derivatives, this increment was in 2 – 8-fold range

134 Medicinal Chemistry, 2020, Vol. 16, No. 1
Borowski et al.
Table 2. Spectrum of antifungal in vitro activity of AmB and its selected 5 derivatives.
MIC [μg/mL]
-
AmB
ANS9
AB4
ATU5
AAA2
AAA4
C. albicans
SC 5314
0.25
1
0.5
2
1
1
C. albicans Gu4
C. albicans Gu5
C. albicans B3
C. albicans B4
C. glabrata
0.125
0.5
0.5
1
1
1
0.25
2
1
4
1
1
0.125
0.25
1
1
2
0.25
4
0.5
4
1
1
1
1
2
2
4
4
4
C. lusitaniae
C. krusei
0.125
0.5
0.5
2
1
1
2
2
2
2
4
4
C. parapsilosis
C. tropicalis
C. stellatoidea
C. dubliniensis
C. guiliermondi
C. gatti
0.5
1
2
2
2
2
0.25
0.25
0.25
0.25
0.25
0.25
0.5
1
2
1
2
4
2
1
1
2
2
4
0.5
2
1
1
1
4
1
1
4
1
1
2
2
4
C. neoformans
T. viride
0.5
2
1
2
2
2
4
2
4
4
A. niger
0.25
1
1
1
2
1
1
A. fumigatus
F. solani
8
4
8
8
16
>16
4
0.25
0.25
1
4
2
16
2
>16
2
M. circinelloides
R. oryzae
2
2
4
1
4
4
4
only. A much more significant difference between AmB and
the derivatives was found for their hemotoxicity. Whereas
AmB caused 50% lysis of erythrocytes at 2.06 μg/mL, only
for 6 out of 28 derivatives their EH50 values were lower than
100 μg/mL. For the remaining 22 compounds, the EH50 val-
ues could not be actually determined, since the extent of
hemolysis at 100 μg/mL was below 50% (9 – 22%) and poor
solubility of these compounds at concentrations exceeding
100 μg/mL precluded determination of EH50 values. One
may thus conclude that these derivatives were poorly hemo-
lytic.
AA4). In all these cases, a relatively low MIC (1 or 2
μg/mL) was combined with poor hemolytic properties (EH50
> 100 μg/mL).
The antifungal spectrum of 5 derivatives exhibiting the
best selective toxicity, i.e. the highest STI values, was com-
pared to that of AmB against 15 yeasts (9 reference strains of
the Candida genus, 4 clinical strains of C. albicans, 2 Cryp-
tococci) and 6 filamentous fungi. Results of this comparison
are shown in Table 2.
Activity of 5 novel derivatives against human pathogenic
yeasts was good (MICs in the 0.25 – 4 μg/mL range), al-
though worse than that of AmB. The multidrug-resistant C.
albicans strains Gu5 and B4, overproducing the drug-efflux
pumps Cdr1p or Mdrp1, were equally or only slightly less
sensitive than their respective counterparts Gu4 and B3, but
the same regularity was found for AmB. The novel deriva-
tives were less active than AmB against filamentous fungi
and especially high MIC values (8 - >16 μg/mL) were found
for ATU5, AAA2 and AAA4 against Aspergillus fumigatus
and Fusarium solani.
For all but one derivative, the EH50/IC50 ratio (STI, selec-
tive toxicity index), which is a rational quantitative measure
of selective toxicity under in vitro conditions, was higher
than that of AmB. Outstanding STIs (close to 100 or higher)
were found for 5 derivatives, namely N- {N-[2-(4-methy-
lpiperazin-1-yl)ethyl]succinimidyl}-amphotericin B (ANS9),
N-(4-N,N-diethylam-inobenzyl)amphotericin
B (A-B4),N-
{3-[(2-piperidin-1-yl)ethyl]-thioureidyl}-amphoteri-cin B (A
TU5), N-[β-(pyridine-1-yl)-D-alanyl]amphoter-icin B (A-
AA2) and N-(O-tert-butyl-D-seryl)amp-hotericin B (A-

The Substantial Improvement of Amphotericin B Selective Toxicity
Medicinal Chemistry, 2020, Vol. 16, No. 1 135
Table 3. Cytotoxicity of AmB and some of its derivatives toward three mammalian cell lines in tissue culture. Data are the means
of 3 independent determinations SD.
IC50 [μg/mL]
Compounds
HepG2
LLC-PK1
CCRF-CEM
AmB
ANS1
ANS2
ANS3
ANS4
ANS5
ANS6
ANS7
ANS9
ANS10
AB3
5.4 1.05
>100
19.7 8.05
>100
4.30 0.86
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
73.3 1.20
>100
>100
50.1 1.30
>100
>100
>100
70.7 2.30
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
AB4
>100
>100
>100
ATU1
ATU2
ATU4
ATU5
AAA1
AAA2
AAA3
AAA4
AAA5
AAA6
AAA8
30.0 5.06
>100
84.15 3.66
>100
0.78 0.093
>100
>100
>100
80.9 2.30
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Cytotoxicity of 22 out of 28 novel derivatives demon-
strating low hemolytic activity (EH50>100 μg/mL, Table 1)
was assessed against Hep G2, LLC-PK1 and HEK-293T
cells. Results shown in Table 3 clearly indicate that most of
the derivatives tested were not cytotoxic (IC50 > 100
μg/mL). A substantial cytotoxic effect against all cell lines
was noted only for AmB and ATU1. ANS5 was cytotoxic
against two cell lines, ANS7 exhibited cytotoxicity against
LLC-PK1 cells and ATU4 against CCRF-CEM cells.
selective toxicity (ANS9, AB4, ATU5, AAA2 and AAA4)
and AmB + 7 derivatives demonstrating the lowest STI
(ANS8, ANS10, AB1, AB2, ATU1, AAA7 and AAA9).
Cells suspended in TBS (C. albicans) or saline (RBC) were
exposed to compounds tested and changes in the extracellu-
lar potassium level were monitored with potassium-selective
electrode (RBC) or by flame photometry (C. albicans).
The exemplary kinetics of potassium efflux from yeast
and erythrocytes induced by AmB, AB1 and AB4 are shown
in Fig (2AB). AmB and AB1 induced rapid efflux from both
cell types, although the concentration-dependent difference
between yeast cells and RBC was observed. On the contrary,
for AB4 the rapid efflux at low compound concentration was
noted from yeast cells only.
According to the “barrel-stave-pore” mechanism of ac-
tion of AmB and its previously described derivatives, the
selective toxicity of these compounds was to a large extent
correlated with their differential potential of induction of
potassium efflux from fungal and mammalian cells [5]. In
this study, we measured the time- and concentration-
dependent K+ release from C. albicans and RBC induced by
AmB and 12 selected novel derivatives. The set of com-
pounds tested comprised 5 derivatives exhibiting the highest
The EK50 values, i.e. concentrations causing loss of 50%
of intracellular potassium, estimated from dose response
curves (not shown), are presented in Table 4. All compounds

136 Medicinal Chemistry, 2020, Vol. 16, No. 1
Borowski et al.
Fig. (2). Kinetics of potassium efflux from C. albicans (A) and RBC (B) induced by AmB, AB1 and AB4. Compounds concentrations were
1 μg/mL in A and 10 μg/mL in B, respectively. (A higher resolution / colour version of this figure is available in the electronic copy of the
article).
Table 4. Induction of potassium efflux from C. albicans cells and RBC by AmB and its selected derivatives. The data presented are
the means of 3 independent determination of EK50 values SD.
EK50 [μg/mL]
Compounds
C. albicans
ATCC 10 231
Erythrocytes
AmB
ANS8
ANS9
ANS10
AB1
0.09 0.01
1.24 0.22
15.34 1.87
>100
2.64 0.15
0.69 0.06
4.89 0.35
0.58 0.06
0.22 0.06
0.54 0.04
5.51 0.48
0.56 0.07
0.99 0.08
1.01 0.09
0.28 0.02
0.34 0.03
89.54 2.84
2.65 0.13
3.14 0.18
>100
AB2
AB4
ATU1
ATU5
AAA2
AAA4
AAA7
AAA9
95.76 4.11
>100
>100
>100
8.56 0.76
10.49 0.9
tested induced substantial potassium efflux from C. albicans
cells at low concentrations (EK50 in the 0.09 – 5.51 μg/mL
range). In all cases, the EK50 values were lower than the IC50
values presented in Table 1. A substantial difference was
found for potassium efflux from RBC. For the 5 AmB de-
rivatives characterized by high STI values, EK50 could not be
actually determined, because at the highest concentration
applied, potassium efflux was lower than 50% (13 - 28%
range). For 8 other compounds tested, the EK50 values were
lower than the respective EH50 data presented in Table 1.
this toxicity and increase solubility. Numerous AmB deriva-
tives have been obtained by chemical modifications of C16
carboxyl (methyl ester and amides) or the amino group of
mycosamine (N-acyl- and N-alkyl analogs) [5, 28]. It has
been demonstrated that modification of the C16 carboxyl
group of AmB or its analogue, 28,29-didehydronystatin,
leading to charge suppression, reduces toxicity to mammal-
ian cells, with little effect on antifungal activity [2, 29-31]
but substantial improvement of selective toxicity has been
achieved only recently for urea derivatives and conjugates
with molecular umbrellas [15, 16]. None of modifications of
the amino functionality of mycosamine, suppressing the
positive charge at this site, resulted in improvement of selec-
tive toxicity of AmB [5, 28] but this effect has been achieved
due to the bisalkylation with aminopropyl groups [13]. The
early works of Wright et al. and results of previous studies in
our group suggested that introduction of bulky substituents at
4. DISCUSSION
Several approaches have been used previously to address
the problem of high mammalian toxicity and poor solubility
of Amphotericin B. A number of semisynthetic analogs have
been generated over the past 20 years in an attempt to reduce

The Substantial Improvement of Amphotericin B Selective Toxicity
Medicinal Chemistry, 2020, Vol. 16, No. 1 137
the amino group with concomitant charge retention, may
lead to the improvement of selective toxicity of AmB and its
methyl ester [10-12]. This assumption was based on the
hypothesis that AmB derivatives with bulky substituents at
the amino group should form AmB derivative:sterol com-
plexes with geometry different from that of AmB:sterol
complexes. This could result in the differential ability to ag-
gregate into lethal transmembrane channels in ergosterol and
cholesterol-containing membranes [17, 32]. On the other
hand, the importance of the positive charge at the amino
group for AmB:ergosterol interaction was previously shown
[33].
lesterol. In consequence, this difference would constitute a
molecular basis for slightly diminished antifungal activity
and substantially reduced hemotoxicity of novel AmB de-
rivatives in comparison with the mother antibiotic. The simi-
lar explanation could be also valid for the previously ob-
served improved selective toxicity of N-methyl-N-fructosyl-
AmB methyl ester, developed in our group several years ago
[11].
These features of novel AmB derivatives are in contrast
with the biological properties of compounds obtained by
Carreira and coworkers by bisalkylation of mycosamine [13,
18, 38]. Several AmB derivatives of this type demonstrated
improved selective toxicity due to the higher than that of
AmB antifungal in vitro activity (MIC values even 10-fold
lower) and slightly lower hemotoxicity. It seems therefore,
that the molecular basis of high STI of bisalkylated AmB
derivatives might be different from that suggested above for
the sterically hindered novel compounds.
Structures of most of the novel AmB derivatives de-
scribed in this work were designed following the assump-
tions formulated above. All derivatives were synthesized
using the unmodified AmB as a substrate and substituents at
the amino group were introduced by simple reactions per-
formed under mild conditions, namely the Michel-type addi-
tion of N-substituted maleimides (ANS1-10), the reductive
amination of benzaldehydes (AB1-4), the addition to isothio-
cyanates (ATU1-5) and the DCC-driven acylation (AAA1-
9). Two out of four reaction types have been already previ-
ously used for the preparation of AmB derivatives different
from those reported in this work (except for AB1) [13, 18,
26, 34], N-aminoacyl derivatives of AmB methyl ester but
not of AmB are known [35] and addition to isocyanates to
form thioureidyl derivatives has been now used for the first
time for derivatization of the AmB amino group. In each of
the four sets of derivatives, there are 1 or 2 compounds in
which the substituents introduced at the amino functionality
are expected to provide the least steric hindrance (ANS1,
AB1, AB2, ATU1, ATU2 AA1 and AA2) and the remaining
ones, in which the local steric hindrance is provided due to
the bulky substituents, including t-butyl and cyclohexyl
functionalities or the heterocyclic rings.
The biological properties of N-succinimidyl and N-
benzyl derivatives of AmB described in this work could be
compared to those of the similar compounds containing less
bulky substituents, described earlier. N-(N-ethylsuc-cinimid-
yl) AmB and N-(N-dimethylaminopropyl-succini-midyl)-
amphotericin B demonstrated slightly lower antifungal activ-
ity and were slightly less hemolytic than AmB [34]. The
antifungal activity of 11 N-benzyl derivatives of AmB de-
scribed by Belakhov and Shenin was the same or lower than
that of AmB but their hemolytic properties are not known
[27].
Low in vitro cytotoxicity of most of the novel AmB de-
rivatives against hepatocytes and kidney cell lines seems
especially advantageous for them as the drug candidates in
light of the well-known nephrotoxicity of AmB and its lipid
preparations [39]. Moreover, these compounds should be
much cheaper than known liposomal AmB preparations
demonstrating lower nephrotoxicity than AmB. Another ad-
vantage is their better solubility in aqueous solutions than
that of the mother antibiotic. However, further study, espe-
cially under in vivo conditions, is needed to warrant the use
of these compounds for clinical applications. Such studies
are now in progress.
The expected improvement of the selective toxicity index
in comparison with that of AmB has been achieved for all
novel AmB derivatives containing bulky substituents and for
5 of them, their STI was at least 10-fold higher than that of
AmB. Notably, the increase of STI was mainly due to the
substantial reduction of hemotoxicity, whereas the antifungal
in vitro activity of all novel derivatives was slightly lower
than that of AmB. Accordingly, potassium efflux from C.
albicans cells was triggered by the novel derivatives allow
concentrations, while that from RBC at much higher ones. A
possible molecular basis of STI increase may be a conse-
quence of the steric hindrance generated in the vicinity of
mycosamine amino group. Presence of any bulky substitu-
ent at this group could affect the interaction of the neighbor-
ing 2’-OH group with 3β-OH of cholesterol and ergosterol.
This interaction was postulated to be important for the for-
mation of AmB:sterol complexes [36, 37] but results of
another later study showed that it is absolutely crucial for the
formation of cholesterol:AmB complex, since 2’-
deoksyAmB did not bind cholesterol at all, while its binding
to ergosterol was only slightly disturbed [14]. Assuming that
formation of the antibiotic:sterol complex is the prerequisite
for both antifungal and hemolytic activity of AmB and its
derivatives, it seems possible that presence of bulky sub-
stituents at the amino group of mycosamine decreases
slightly AmB affinity to ergosterol and markedly that to cho-
CONCLUSION
Introduction of bulky substituents at the amino group of
mycosamine residue in Amphotericin B generates deriv-
atives exhibiting substantially improved selective toxicity,
resulting mainly from the strongly reduced hemolytic poten-
tial. This effect is most likely due to the induced steric hin-
drance which may affect the creation of the hydrogen bond
between 2’-OH of mycosamine and 3β-OH of cholesterol
present in the mammalian cell membranes, thus preventing
the formation of the AmB derivativ-e:cholesterol complex
and assembly of the potassium-perme-able transmembrane
channels. Facility and simplicity of synthesis and the advan-
tageous biological properties of some of the novel AmB de-
rivatives make them promising drug candidates for antifun-
gal chemotherapy.

138 Medicinal Chemistry, 2020, Vol. 16, No. 1
Borowski et al.
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ETHICS APPROVAL AND CONSENT TO PARTICI-
PATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
[http://dx.doi.org/10.1016/j.chembiol.2008.08.009]
19022180]
[PMID:
No Animals/Humans were used for studies that are the
basis of this research.
Tevyashova, A.N.; Korolev, A.M.; Trenin, A.S.; Dezhenkova,
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Not applicable.
[10]
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AVAILABILITY OF DATA AND MATERIALS
[http://dx.doi.org/10.7164/antibiotics.35.911] [PMID: 6757233]
Grzybowska, J.; Sowiński, P.; Gumieniak, J.; Zieniawa, T.;
The data that support the findings of this study are avail-
able from the corresponding author, [SM], upon reasonable
request.
Borowski, E. N-methyl-N-D-fructopyranosylamphotericin
B
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FUNDING
[12]
Financial support of these studies by the POIG.01.04.00-
22-064/09-00 grant from the National Centre for Research
and Development to BLIRT S.A. and by the UMO2014/
13/B/NZ7/02305 grant from the National Science Centre to
MJM is gratefully acknowledged.
[13]
[14]
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
[http://dx.doi.org/10.1021/ja403255s] [PMID: 23718627]
Davis, S.A.; Vincent, B.M.; Endo, M.M.; Whitesell, L.; Marchillo,
K.; Andes, D.R.; Lindquist, S.; Burke, M.D. Nontoxic antimicrobi-
als that evade drug resistance. Nat. Chem. Biol., 2015, 11(7), 481-
487.
ACKNOWLEDGEMENTS
[15]
[16]
[17]
Declared none.
[http://dx.doi.org/10.1038/nchembio.1821] [PMID: 26030729]
Janout, V.; Schell, W.A.; Thévenin, D.; Yu, Y.; Perfect, J.R.; Re-
gen, S.L. Taming amphotericin B. Bioconjug. Chem., 2015, 26(10),
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