Synthesis and antimicrobial properties of steroid-based imidazolium salts

Article abstract of DOI:10.1016/j.jsbmb.2019.02.006

Imidazolium salts reveal interesting biological properties, especially regarding antitumor and antimicrobial activities. Two series of imidazolium salts based on steroids were obtained in an efficient and convenient synthesis. They were biologically teste

Full text of DOI:10.1016/j.jsbmb.2019.02.006

Journal of Steroid Biochemistry and Molecular Biology 189 (2019) 65–72
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
Synthesis and antimicrobial properties of steroid-based imidazolium salts
a,⁎
a
b
c
Agnieszka Hryniewicka , Marta Malinowska , Tomasz Hauschild , Katarzyna Pieczul ,
a
Jacek W. Morzycki
a
b
c
Institute of Chemistry, University of Białystok, Ciołkowskiego Street 1K, 15-245, Białystok, Poland
Institute of Biology, University of Białystok, Ciołkowskiego Street 1J, 15-245, Białystok, Poland
Institute of Plant Protection, National Research Institute, Węgorka Street 20, 60-318, Poznań, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
Imidazolium salts reveal interesting biological properties, especially regarding antitumor and antimicrobial
activities. Two series of imidazolium salts based on steroids were obtained in an efficient and convenient
synthesis. They were biologically tested to evaluate their antibacterial and antifungal properties. The activities of
new salts, especially in relation to Gram-positive bacterial strains are comparable to the activities of known
antibiotics. The most promising activity was that against C. albicans, which exceeded the antifungal activity of
commonly used drugs. Some of the new salts exhibited improved antifungal activities against phytopathogenic
fungi: B. cinerea and C. beticola. Our research showed that new compounds could be potentially useful as an-
tifungal antibiotics or inhibiting agents against pathogenic fungi.
Imidazolium salt
Lithocholic acid
Antifungal activity
Antibacterial activity
1
. Introduction
Imidazolium salts are very important imidazole derivatives that
properties are connected with its large, rigid, and curved steroidal
skeleton, enantiomeric purity and unique amphiphilicity. The phar-
macological interest in lithocholic acid is directly related to the fact that
liver cells can specifically recognize such a natural ligand, which makes
LCA ideal building block for the synthesis of novel molecules that can
be recognized at the molecular level [24]. Derivatives of 1 with oxazole
fragment itself display some antifungal activity against Candida albicans
[25].
consist of discrete cation and anion pairs [1]. They are widely utilized
in organic synthesis, especially as ionic liquids [2] or precursors of N-
heterocyclic carbenes [3]. They have a tremendous potential in biolo-
gical applications, because of their antitumor [4–6] and antimicrobial
activities [7–9] or antioxidative properties [10,11]. They are also
widely utilized in bioengineering as drug/gene delivery systems [12] or
biosensors [13]. Imidazolium salts were also reported to exhibit fun-
gicidal activity [14]. These biological activities are related to their ionic
structure, the presence of azole core [15] and various substituents at-
tached to nitrogen atoms. The intrinsic biological activity of an azole
moiety is often expressed when it is introduced to some bioactive
compounds [16]. Moreover, it should be noted that combining two
bioactive molecules as a way to improve biological properties of
starting compounds is an emerging practice in medicinal chemistry. In
this context, it was expected that a hybrid compound formed by at-
taching an imidazole moiety to a biologically active steroid may en-
hance the biological properties of both fragments [17,18]. The basicity
and hydrophilicity of an azole moiety might alter the biological func-
tion of a steroid. Lithocholic acid (LCA, 1) was chosen because of its
wide range of biological activities such as α-2,3-sialyltransferase in-
hibition [19], vitamin D receptor modulation [20], antibacterial and
antifungal effect [21], and antitumor activity [22,23]. These interesting
Syntheses of some steroids with an imidazole ring attached to dif-
ferent positions of the skeleton were reported by substitution of halo-
geno- or epoxy-steroids with lithiated imidazole either under standard
conditions [26,27] or using microwave irradiation [28]. These com-
pounds exhibit cytotoxic activity against cancer cells [29,30], inhibit
17α-lyase [31], show potent skeletal muscle relaxant and neuromus-
cular blocking properties [32]. They can also be utilized as receptors for
fluoride ion recognition [33]. However, to the best of our knowledge,
there is no report about antimicrobial activity, including activity
against plant pathogens, of imidazolium salts, especially steroid deri-
vatives substituted in the side chain (22- or 24-imidazolo). We designed
and synthesized two series of imidazolium salts starting from litho-
cholic acid and a steroid compound similar to one of LCA metabolites
[34] with a 4-en-3-one group in ring A and a shorter side chain: 3-oxo-
23,24-dinorchol-4-en-22-al (2) (Fig. 1).
⁎
Corresponding author.
E-mail address: aga_h@uwb.edu.pl (A. Hryniewicka).
https://doi.org/10.1016/j.jsbmb.2019.02.006
Received 30 November 2018; Received in revised form 8 February 2019; Accepted 16 February 2019
Available online 20 February 2019
0960-0760/ © 2019 Elsevier Ltd. All rights reserved.

A. Hryniewicka, et al.
Journal of Steroid Biochemistry and Molecular Biology 189 (2019) 65–72
Fig. 1. The target imidazolium salts and starting steroids.
2.2.4. Synthesis of imidazolium salts – general procedure D
2
. Materials and methods
To the solution of N-imidazolyl steroid in dry CH
2
Cl (0.5–1 mL),
2
2.1. General remarks
alkyl bromide (excess) and NaI (2 eq) were added under argon. The
reaction was carried out at room temperature for 48 h and protected
from light. Then inorganic solid was filtered off and the filtrate was
Melting points were determined on an MP70 (Mettler Toledo) ap-
1
13
paratus and were uncorrected. H and C NMR spectra were recorded
on a Bruker Avance II spectrometer (400 and 100 MHz, respectively).
Spectra are referenced relative to the chemical shift of TMS. Mass
spectra were obtained with Micromass LCT TOF and Accurate-Mass Q-
TOF LC/MS 6530 spectrometers. IR spectra were recorded on a Nicolet
series II Magna-IR 550 FT-IR spectrometer. Column chromatography
evaporated. The remaining residue was dissolved in CH
2
Cl (2 mL), the
2
product was precipitated with Et
2
O (10 mL) and filtered. The crystal-
lization (CH Cl /Et O) was repeated two more times.
2
2
2
2
.3. Synthesis of imidazolium salts
.3.1. 24-(N-imidazolyl)-5β-cholan-3α-ol (4)
was performed on silica gel 230–400 mesh. CH
2
Cl was dried by dis-
2
2
tillation over CaH , THF over Na/benzophenone. Steroidal compounds
2
General procedure
A was followed using imidazole (144 mg,
were synthesized according to literature procedures: 5β-cholane-3α,24-
diol [35], 24-p-toluenesulfonyloxy-5β-cholan-3α-ol and 24-iodo-5β-
cholan-3α-ol (3) [36,37], 22-hydroxy-23,24-dinorchol-4-en-3-one [38].
Other chemicals are commercially available and used as received.
2
2
.1 mmol), NaH (84 mg, 60% suspension in mineral oil, 2.1 mmol) and
4-iodo-5β-cholan-3α-ol (3, 200 mg, 0.42 mmol) to produce a white
solid in 100% yield (175 mg). Mp = 163–165 °C (from MeOH); IR
−
1 1
(
ATR) ν = 3104, 2927, 2852, 1506, 1445, 1363, 1067 cm
; H NMR
(
400 MHz, CDCl , 25 °C, TMS) δ 7.48 (s, 1H, Himidazole), 7.05 (s, 1H,
3
2
2
3
.2. General procedures A-D
H
imidazole), 6.91 (s, 1H, Himidazole), 3.83 (m, 2H, CH
2
3
N), 3.62 (m, 1H, H-
), 0.63 (s, 3H, CH
NMR (100 MHz, CDCl ) δ 137.0 (CHimidazole), 129.2
3
), 0.91 (s, 3H, CH
3
), 0.89 (d, J = 6.6 Hz, 3H, CH
3
)
1
3
.2.1. Synthesis of N-imidazolyl substituted steroid – general procedure A
Imidazole (5 eq) and NaH (5 eq) were stirred in dry THF (3 mL) for
0 min at room temperature under argon. To this mixture, the steroid
ppm;
C
3
(CHimidazole), 118.7 (CHimidazole), 71.6 (CH, C-3), 56.4 (CH), 55.9 (CH),
47.5 (CH
2
), 42.7 (C), 42.0 (CH), 40.3 (CH), 40.1 (CH
), 30.5 (CH
), 26.4 (CH
), 12.0 (CH
2
2
), 36.4 (CH
2
),
2
),
3
),
iodide (1 eq) in dry THF (5 mL) was added and the reaction was stirred
for 16 h at room temperature. Then water was added (20 mL) and the
35.8 (CH), 35.3 (CH), 34.5 (C), 32.7 (CH
2
), 29.6 (CH
28.2 (CH
2
2
), 27.6 (CH
2
3
), 27.1 (CH
2
2
), 24.1 (CH ), 23.3 (CH
2
mixture was extracted with CH
2
Cl
2
(3 x 15 mL). The combined extracts
20.8 (CH
), 18.5 (CH
3
) ppm; ESI-HRMS m/z: calcd for [M
+
+
were washed with brine (2 x 15 mL) and water (15 mL), dried over
+H]
C
27
H
45
N
2
O
413.3526, found 413.3513.
Na
2
SO , filtered and concentrated in vacuo. The resulting product was
4
sufficiently pure to use in the next step or was purified by flash chro-
matography.
2
.3.2. N-(5β-cholan-3α-ol-24-yl)-N’-methylimidazolyl iodide (5a)
General procedure B was followed using 4 (50 mg, 0.12 mmol),
methyl iodide (0.5 mL, 8 mmol) to produce a white salt in 83% yield
2
.2.2. Synthesis of imidazolium salts – general procedure B
(55 mg). Mp = 179–181 °C (from CH
2927, 2861, 1555, 1444, 1370, 1156, 1031 cm
CDCl , 25 °C, TMS) δ 10.12 (s, 1H, H-2imidazole), 7.44 (s, 1H, Himidazole),
7.34 (s, 1H, Himidazole), 4.29 (brs, 2H, CH N), 4.13 (s, 3H, NCH ), 3.62
(m, 1H, H-3), 0.93 (d, J = 6.6 Hz, 3H, CH ), 0.91 (s, 3H, CH ), 0.63 (s,
) δ 136.6 (CHimidazole), 123.7
2
Cl
2
/Et
2
O); IR (ATR) ν = 3342,
−1 1
To the solution of N-imidazolyl steroid in dry CH
2
Cl
2
(1 mL), alkyl
; H NMR (400 MHz,
iodide (excess) was added under argon. The reaction was carried out at
room temperature for 48 h, protected from light. Then the solvent and
excess of alkyl iodide were evaporated. The remaining residue was
3
2
3
3
3
3
1
3
dissolved in CH
2
Cl
2
(2 mL), the product was precipitated with Et
2
O
3H, CH
3
) ppm; C NMR (100 MHz, CDCl
(
10 mL) and filtered. The crystallization (CH
2
Cl
2
/Et
2
O) was repeated
(CHimidazole), 122.0 (CHimidazole), 71.6 (CH, C-3), 56.3 (CH), 55.7 (CH),
two more times.
50.6 (CH
2
2
2
), 42.6 (C), 41.9 (CH), 40.3 (CH), 40.0 (CH
2
2
), 37.1 (CH
3
),
2
),
2
),
3
3
2
6.3 (CH
), 35.7 (CH), 35.3 (CH), 34.4 (C), 32.2 (CH
), 30.4 (CH
0.3 (CH
3.3 (CH
), 28.3 (CH
2
2
), 27.1 (CH
2
), 27.0 (CH
), 12.0 (CH
427.3683, found 427.3695.
2
), 26.3 (CH ), 24.1 (CH
2
2
.2.3. Synthesis of imidazolium salts – general procedure C
N-Imidazolyl steroid was dissolved in alkyl iodide (excess) under
3
), 20.7 (CH
), 18.6 (CH
3
3
) ppm; ESI-HRMS m/z:
+
+
calcd for [M-I]
C
28
H
47
N O
2
argon. The reaction was carried out at room temperature for 48 h while
protected from light. Then the excess of alkyl iodide was evaporated.
The remaining residue was dissolved in CH
2
Cl
2
(2 mL), the product was
2.3.3. N-(5β-cholan-3α-ol-24-yl)-N’-ethylimidazolyl iodide (5b)
General procedure B was followed using 4 (50 mg, 0.12 mmol),
ethyl iodide (0.5 mL, 6.3 mmol) to produce a white salt in 64% yield
precipitated with Et
2
O (10 mL) and filtered. The crystallization from
CH Cl /Et O was repeated two more times.
2
2
2
66

A. Hryniewicka, et al.
Journal of Steroid Biochemistry and Molecular Biology 189 (2019) 65–72
(
44 mg). Mp = 176–178 °C (from CH
2
Cl
2
/Et
2
O); IR (ATR) ν = 3380,
42.4 (C), 39.2 (CH
2
), 38.5 (C), 36.8 (CH), 35.7 (CH
2
2
), 35.6 (CH), 33.9
), 21.0 (CH ), 20.9
) ppm, ESI-HRMS m/z: calcd
−
1 1
2
920, 2858, 1561, 1444, 1378, 1166, 1035 cm
;
H NMR (400 MHz,
, 25 °C, TMS) δ 10.14 (s, 1H, H-2imidazole), 7.56 (s, 1H, Himidazole),
N), 4.30 (brs, 2H,
), 0.90 (brs, 6H, 2CH ),
NMR (100 MHz, CDCl 136.1
(CH
(CH
2
2
), 32.8 (CH
2
), 31.9 (CH
2
), 27.5 (CH
2
), 24.0 (CH
2
CDCl
3
), 20.7 (CH
3
), 17.3 (CH
3
), 12.7 (CH
3
+
+
7
.44 (s, 1H, Himidazole), 4.45 (d, J = 6.6 Hz, 2H, CH
2
for [M+H]
C
22
H
34IO 441.1659, found 441.1649.
CH
2
N), 3.62 (brs, 1H, H-3), 1.63 (brs, 3H, CH
3
3
1
3
0
.62 (s, 3H, CH
3
)
ppm;
C
3
)
δ
2.3.7. 22-(N-Imidazolyl)-23,24-dinorchol-4-en-3-one (7)
(
CHimidazole), 122.0 (2CHimidazole), 71.6 (CH, C-3), 56.4 (CH), 55.7 (CH),
General procedure A was followed using imidazole (155 mg,
5
3
3
2
0.5 (CH
2
2
), 45.4 (CH
2
), 42.6 (C), 42.0 (CH), 40.3 (CH), 40.0 (CH
), 30.5 (CH
), 24.1 (CH
), 12.0 (CH
2
2
2
),
),
),
2.3 mmol), NaH (92 mg, 60% suspension in mineral oil, 2.3 mmol) and
22-iodo-23,24-dinorchol-4-en-3-one (6, 200 mg, 0.45 mmol) to pro-
duce, after column chromatography (FC, dichloromethane – methanol,
v/v 12:1), a white solid in 53% yield (91 mg). Mp = 176–177 °C (from
MeOH); IR (ATR) ν = 2935, 2864, 1668, 1614, 1506, 1445, 1378, 1228
6.3 (CH
), 35.7 (CH), 35.3 (CH), 34.5 (C), 32.2 (CH
2
0.4 (CH
2
3
), 28.3 (CH
2
2
), 27.1 (CH
2
), 27.0 (CH
2
), 26.3 (CH
2
3.3 (CH
), 20.7 (CH
), 18.6 (CH
3
), 15.7 (CH
3
3
) ppm; ESI-
+
+
HRMS m/z: calcd for [M-I]
C
29
H
49
N
2
O
441.3839, found 441.3851.
−1 1
cm ; H NMR (400 MHz, CDCl , 25 °C, TMS) δ 7.41 (s, 1H, Himidazole),
3
2
.3.4. N-(5β-cholan-3α-ol-24-yl)-N’-pentylimidazolyl iodide (5c)
7.03 (s, 1H, Himidazole), 6.86 (s, 1H, Himidazole), 5.71 (s, 1H, CH = C),
General procedure D was followed using 4 (50 mg, 0.12 mmol),
4.00 (dd, J = 13.8, 3.6 Hz, 1H, CH
2
N), 3.52 (dd, J = 13.8, 9.3 Hz, 1H,
), 0.84 (d, J = 6.6 Hz, 3H, CH ), 0.75 (s, 3H,
) ppm; C NMR (100 MHz, CDCl ) δ 199.4 (C, C = O), 171.1 (C,
pentyl bromide (0.5 mL, 4 mmol) and NaI (36 mg, 0.24 mmol) to pro-
duce a pale yellow salt in 48% yield (35 mg). Mp = 182–184 °C (from
CH
CH
2
3
N), 1.17 (s, 3H, CH
3
3
1
3
3
CH
2
Cl
2
/Et
2
O) (decomposition); IR (ATR) ν = 3324, 2925, 2859, 1561,
C=CH), 137.7 (CHimidazole), 129.2 (CHimidazole), 123.8 (CH, C=CH),
−
1 1
1
448, 1374, 1161, 1037 cm ; H NMR (400 MHz, CDCl
δ 10.37 (s, 1H, H-2imidazole), 7.43 (s, 1H, Himidazole), 7.40 (s, 1H,
imidazole), 4.35 (m, 4H, CH N), 3.61 (brs, 1H, H-3), 0.89 (brs, 6H,
3
, 25 °C, TMS)
119.3 (CHimidazole), 55.5 (CH), 53.6 (CH
(C), 39.3 (CH ), 38.5 (CH), 38.4 (C), 35.6 (CH
32.8 (CH ), 31.9 (CH ), 28.2 (CH ), 24.2 (CH
16.9 (CH ), 11.9 (CH
2
), 53.5 (CH), 52.7 (CH), 42.6
2
2
), 35.5 (CH), 33.9 (CH
2
3
),
H
2
2
2
2
2
), 20.9 (CH ), 17.3 (CH
2
),
1
3
+
2
CH
3
), 0.61 (s, 3H, CH
3
) ppm; C NMR (100 MHz, CDCl
3
) δ 137.2
3
3
) ppm; ESI-HRMS m/z: calcd for [M+H]
+
(
CHimidazole), 121.9 (CHimidazole), 121.7 (CHimidazole), 71.6 (CH, C-3),
C
25
H
37
N O
2
381.2900, found 381.2915.
5
6.4 (CH), 55.8 (CH), 50.4 (CH
2
), 50.0 (CH
2
), 42.6 (C), 42.0 (CH), 40.3
(
(
(
(
CH), 40.1 (CH
2
), 36.3 (CH
2
2
2
), 35.7 (CH), 35.29 (CH), 35.27 (CH
), 30.0 (CH ), 28.3 (CH ), 28.2 (CH
), 24.1 (CH ), 23.3 (CH ), 22.0 (CH
), 12.0 (CH ) ppm; ESI-HRMS m/z: calcd
2
2
2
), 34.5
), 27.1
), 20.7
2.3.8. N-(3-Oxo-23,24-dinorchol-4-en-22-yl)-N’-methylimidazolyl iodide
(8a)
C), 32.2 (CH
2
), 30.4 (CH
2
2
3
CH
CH
2
2
), 27.0 (CH
2
), 26.3 (CH
2
General procedure C was followed using 7 (50 mg, 0.13 mmol),
methyl iodide (1 mL, 16 mmol) to produce a white salt in quantitative
), 18.5 (CH
3
), 13.8 (CH
3
3
+
+
for [M-I]
C
32
H
55
N
2
O
483.4309, found 483.4321.
yield (68 mg). Mp = 223–225 °C (from CH
2
Cl
2
/Et O); IR (ATR)
2
−1 1
ν = 3418, 2933, 1666, 1610, 1575, 1446, 1383, 1170 cm
(400 MHz, CDCl , 25 °C, TMS) δ 10.07 (s, 1H, H-2imidazole), 7.57 (s, 1H,
imidazole), 7.37 (s, 1H, Himidazole), 5.69 (s, 1H, CH = C), 4.34 (dd, J =
13.6, 3.6 Hz, 1H, CH N), 4.13 (s, 3H, NCH ), 4.00 (dd, J = 13.6, 9.7 Hz,
1H, CH N), 1.16 (s, 3H, CH ), 0.96 (d, J = 6.5 Hz, 3H, CH ), 0.76 (s,
3H, CH ) ppm; C NMR (100 MHz, CDCl ) δ 199.4 (C, C = O), 171.2
(C, C=CH), 137.2 (CHimidazole), 123.7 (CH, C=CH), 123.6 (CHimidazole),
122.5 (CHimidazole), 55.5 (CH ), 55.4 (CH), 53.4 (CH), 53.2 (CH), 42.8
(C), 39.2 (CH ), 38.4 (C), 37.5 (CH), 37.0 (CH ), 35.6 (CH ), 35.4 (CH),
), 32.7 (CH ), 31.8 (CH ), 28.1 (CH ), 24.1 (CH ), 20.8 (CH ),
), 16.7 (CH ), 12.1 (CH ) ppm; ESI-HRMS m/z: calcd for [M-
; H NMR
2
.3.5. N-(5β-cholan-3α-ol-24-yl)-N’-hexylimidazolyl iodide (5d)
3
General procedure D was followed using 4 (50 mg, 0.12 mmol),
H
hexyl bromide (0.5 mL, 3.6 mmol) and NaI (36 mg, 0.24 mmol) to
produce a pale yellow salt in 43% yield (32 mg). Mp = 129–130 °C
2
3
2
3
3
3
1
3
(
from CH
2
Cl
2
/Et
2
O) (decomposition); IR (ATR) ν = 3320, 2924, 2860,
3
−
1 1
1
562, 1441, 1368, 1152 cm ; H NMR (400 MHz, CDCl , 25 °C, TMS)
3
δ 10.44 (s, 1H, H-2imidazole), 7.40 (s, 1H, Himidazole), 7.38 (s, 1H,
2
H
imidazole), 4.37 (m, 4H, CH
2
N), 3.62 (m, 1H, H-3), 0.92 (d, J = 7.0 Hz,
2
3
2
) ppm; 1 C NMR (100 MHz,
3
33.9 (CH
3
H, CH
3
), 0.90 (s, 3H, CH
3
), 0.62 (s, 3H, CH
3
2
2
2
2
2
2
CDCl
3
) δ 137.3 (CHimidazole), 121.8 (CHimidazole), 121.7 (CHimidazole),
17.3 (CH
3
3
3
+
7
4
3
2
2
1.6 (CH, C-3), 56.4 (CH), 55.8 (CH), 50.5 (CH
2
), 50.1 (CH
2
), 42.7 (C),
I] 395.3057, found 395.3069.
2.0 (CH), 40.3 (CH), 40.1 (CH
4.5 (C), 32.2 (CH ), 31.0 (CH ), 30.5 (CH
7.1 (CH ), 27.0 (CH
2.3 (CH ), 20.7 (CH
2
), 36.4 (CH
2
), 35.8 (CH), 35.3 (CH),
2
2
2
), 30.2 (2CH
2
), 28.3 (CH
), 23.3 (CH
), 12.0 (CH
2
3
),
),
2.3.9. N-(3-Oxo-23,24-dinorchol-4-en-22-yl)-N’-ethylimidazolyl
iodide
2
2
2
2
), 26.4 (CH
2
), 25.8 (CH
2
), 24.1 (CH
2
(8b)
), 18.6 (CH
3
), 13.9 (CH
3
3
) ppm; ESI-
General procedure C was followed using 7 (50 mg, 0.13 mmol),
+
+
HRMS m/z: calcd for [M-I]
C
33
H
57
N
2
O
497.8315, found 497.8328.
ethyl iodide (1 mL, 12.5 mmol) to produce a pale yellow salt in 90%
yield (63 mg). Mp = 151–152 °C (from CH
2
Cl
2
/Et O); IR (ATR)
−1 1
2
2
.3.6. 22-Iodo-23,24-dinorchol-4-en-3-one (6)
ν = 3440, 2935, 1661, 1561, 1445, 1352, 1164 cm
(400 MHz, CDCl , 25 °C, TMS) δ 10.31 (s, 1H, H-2imidazole), 7.49 (s, 1H,
imidazole), 7.31 (s, 1H, Himidazole), 5.72 (s, 1H, CH = C), 4.47 (q, J
= 7.3 Hz, 2H, CH N), 4.39 (dd, J = 13.6, 3.4 Hz, 1H, CH N), 4.01 (dd,
J = 13.5, 9.9 Hz, 1H, CH N), 1.63 (t, J = 7.3 Hz, 3 H), 1.18 (s, 3H,
CH ), 0.94 (d, J = 6.4 Hz, 3H, CH
(100 MHz, CDCl ) δ 199.2 (C, C = O), 171.1 (C, C=CH), 136.2
;
H NMR
To the solution of triphenylphosphine (1.09 g, 4.17 mmol, 1.5 eq) in
3
dry CH
2
Cl
2
cooled to 0 °C, iodine (1.06 g, 4.17 mmol, 1.5 eq) and imi-
H
dazole (0.28 g, 4.17 mmol, 1.5 eq) were added. After stirring for 30 min
at room temperature while protected from light, a solution of 22-hy-
droxy-23,24-dinorchol-4-en-3-one (0.92 g, 2.78 mmol, 1 eq) in dry
2
2
2
1
3
3
3
), 0.79 (s, 3H, CH ) ppm; C NMR
3
CH
2
Cl
2
was added. The reaction was carried out at room temperature
3
for 30 min. Then an aqueous solution of Na
and the mixture was extracted with CH Cl (3 × 15 mL). The combined
extracts were washed with aqueous solution of NaHCO (2 x 15 mL) and
water (15 mL), dried over Na SO , filtered and concentrated in vacuo.
The obtained solid was purified by flash chromatography
hexane–AcOEt, v/v 9:1) to give a white solid in 81% yield (996 mg).
Mp = 142–144 °C (from MeOH); IR (ATR) ν = 2924, 2850, 1666, 1611,
2
S
2
O
3
was added (20 mL)
(CHimidazole), 123.5 (CH, C=CH), 122.6 (CHimidazole), 122.0
2
2
(CHimidazole), 55.2 (CH
2
), 55.2 (CH), 53.3 (CH), 53.1 (CH), 45.2 (CH
), 37.3 (CH), 35.4 (CH ), 35.2 (CH), 33.7
), 27.8 (CH ), 24.0 (CH ), 20.7 (CH ), 17.1
), 12.0 (CH ) ppm; ESI-HRMS m/z: calcd
2
),
3
42.6 (C), 39.0 (C), 38.3 (CH
2
2
3
2
2
4
(CH
(CH
2
3
), 32.5 (CH
2
), 31.6 (CH
2
2
2
), 16.4 (CH
3
), 15.0 (CH
3
+
+
(
for [M-I]
C
27
H
41
N
2
O
409.3213, found 409.3225.
−
1
1
1
447 cm
;
H NMR (400 MHz, CDCl
3
, 25 °C, TMS) δ 5.72 (s, 1H,
I), 3.16 (dd, J = 9.4,
), 0.75
2.3.10. N-(3-Oxo-23,24-dinorchol-4-en-22-yl)-N’-pentylimidazolyl iodide
(8c)
CH = C), 3.32 (dd, J = 9.5, 1.4 Hz, 1H, CH
2
4
.5 Hz, 1H, CH I), 1.18 (s, 3H, CH ), 1.02 (d, 3H, J = 5.3 Hz, CH
2
3
3
General procedure D was followed using 7 (50 mg, 0.13 mmol),
pentyl bromide (1 mL, 8 mmol) and NaI (39 mg, 0.26 mmol) to produce
1
3
(
s, 3H, CH
3
) ppm; C NMR (100 MHz, CDCl ) δ 199.5 (C, C = O),
3
1
71.3 (C, C=CH), 123.8 (CH, C=CH), 55.5 (CH), 55.3 (CH), 53.6 (CH),
a yellow salt in 78% yield (59 mg). Mp = 143–145 °C (from CH
2
Cl /
2
67

A. Hryniewicka, et al.
Journal of Steroid Biochemistry and Molecular Biology 189 (2019) 65–72
Et
561, 1448, 1378, 1163 cm ; H NMR (400 MHz, CDCl
δ 10.44 (s, 1H, H-2imidazole), 7.50 (s, 1H, Himidazole), 7.37 (s, 1H,
imidazole), 5.68 (s, 1H, CH = C), 4.35 (m, 3H, CH N), 4.04 (m, 1H,
CH N), 1.15 (s, 3H, CH ), 0.88 (m, 7 H), 0.75 (s, 3H, CH
) δ 199.4 (C, C = O), 171.1 (C, C=CH), 137.5
CHimidazole), 123.7 (CH, C=CH), 122.4 (CHimidazole), 122.0
CHimidazole), 55.4 (CH), 55.3 (CH ), 53.4 (CH), 53.3 (CH), 50.0 (CH ),
2
O) (decomposition); IR (ATR) ν = 3433, 2933, 2853, 1663, 1613,
with the final solution composition being 95% MHB and 5% DMSO by
volume. The salts were then serially two-fold diluted to obtain con-
centrations ranging from 512 to 0.125 μg/mL in wells containing MHB.
Then each diluted sample (50 μL) was mixed with 50 μL of inoculums of
the tested microorganisms to achieve an initial inoculum of approxi-
−
1 1
1
3
, 25 °C, TMS)
H
2
1
3
2
3
3
) ppm;
C
6
NMR (100 MHz, CDCl
3
mately 10 CFU/mL and incubated at 35 °C for 24 h. The experiments
(
(
were performed in duplicates. The MIC value was determined as the
lowest concentration of the salt that inhibits visible growth after in-
cubation. Four reference strains were tested: Staphylococcus aureus
ATCC 25923, Bacillus cereus ATCC 11778, Escherichia coli DSM 10233
and Candida albicans ATCC 10231.
2
2
4
2.8 (C), 39.2 (CH
2
), 38.4 (C), 37.5 (CH), 35.5 (CH
2
), 35.4 (CH), 33.8
(
(
(
CH
2
2
3
), 32.7 (CH
2
2
), 31.8 (CH
2
2
), 29.9 (CH
2
3
), 28.1 (CH
2
), 27.9 (CH
2
3
), 24.1
), 12.1
CH
CH
), 21.9 (CH
), 20.8 (CH
), 17.2 (CH
), 16.5 (CH
3
), 13.8 (CH
+
+
) ppm; ESI-HRMS m/z: calcd for [M-I]
C
30
H
47
N
2
O
451.3683,
Activities of imidazolium salts on growth inhibition level of plant
pathogenic fungi were tested on four widespread species – Alternaria
alternata, Botrytis cinerea, Cercospora beticola and Fusarium culmorum.
The strains were provided by Mycology Department of Plant Protection
Institute – National Research Institute (Poznań, Poland). All the isolates
were incubated on a PDA medium (Potato Dextrose Agar; Becton,
found 451.3682.
2
.3.11. N-(3-Oxo-23,24-dinorchol-4-en-22-yl)-N’-hexylimidazolyl iodide
8d)
General procedure D was followed using 7 (50 mg, 0.13 mmol),
hexyl bromide (1 mL, 7 mmol) and NaI (39 mg, 0.26 mmol) to produce
a yellow salt in 42% yield (32 mg). Mp = 148–149 °C (from CH Cl
O) (decomposition); IR (ATR) ν = 3730, 3393, 2933, 2854, 1665,
(
2
Dickinson and Co.) for 1 week at 21 °C. Mycelial plugs (0.5 cm ) taken
2
2
/
from the edge of the colonies and placed on a PDA medium containing
imidazolium salts at a concentration of: 0.1, 1, 5, 10, 25, 50 and
100 μg/mL and on PDA without amendment. The salts were dissolved
in methanol at 5 mg/mL before being added to the medium. The sam-
ples were incubated for 5–8 days at 21 °C. The assays were repeated
three times and the mean percentage of growth inhibition rate and
EC50 value (half maximal effective concentration) of the tested isolates
was calculated.
Et
2
−
1
1
1
559, 1444, 1161 cm
;
H NMR (400 MHz, CDCl
3
, 25 °C, TMS) δ
1
0.58 (s, 1H, H-2imidazole), 7.37 (s, 1H, Himidazole), 7.27 (s, 1H,
H
imidazole), 5.72 (s, 1H, CH = C), 4.39 (m, 3H, CH
2
N), 4.05 (m, 1H,
CH
CH
2
3
N), 1.17 (s, 3H, CH
3
), 0.88 (d, J = 6.6 Hz, 3H, CH ), 0.78 (s, 3H,
3
1
3
) ppm; C NMR (100 MHz, CDCl ) δ 199.5 (C, C = O), 171.1 (C,
3
C=CH), 137.8 (CHimidazole), 123.8 (CH, C=CH), 122.2 (CHimidazole),
1
21.7 (CHimidazole), 55.5 (CH ), 55.4 (CH), 53.5 (CH), 53.3 (CH), 50.2
2
(
CH
2
), 42.8 (C), 39.3 (CH
2
), 38.5 (C), 37.6 (CH), 35.6 (CH
2
), 35.5 (CH),
3. Results and discussion
3
2
1
3.9 (CH
2
2
), 32.7 (CH
), 24.2 (CH
2
2
), 31.8 (CH
), 22.3 (CH
2
2
), 31.0 (CH
), 20.9 (CH
2
2
), 30.2 (CH
), 17.3 (CH
2
3
), 28.0 (CH
2
3
),
),
5.8 (CH
), 16.5 (CH
3.1. Synthesis of imidazolium salts
+
3.9 (CH
3
), 12.1 (CH ) ppm; ESI-HRMS m/z: calcd for [M-I]
3
+
C
31
H
49
N
2
O
465.3839, found 465.3837.
Two series of imidazolium iodides based on lithocholic acid (1,
Scheme 1) and 3-oxo-23,24-dinorchol-4-en-22-al (2, Scheme 2) were
2
.3.12. Bis-N,N’-(3-oxo-23,24-dinorchol-4-en-22-yl)imidazolyl
iodide
prepared. LCA was subjected to reduction with LiAlH [35] followed by
4
(
8e)
p-tosylation of the 24-hydroxy group. Selective tosylation was achieved
Imidazole (15 mg, 0.22 mmol, 1 eq) and NaH (9 mg, 60% suspen-
sion in mineral oil, 0.22 mmol, 1 eq) were stirred in dry THF (2 mL) for
0 min at room temperature under argon. To this mixture, steroid 6
using Et N as a base at 0–4 °C in THF [36,37]. The crude product
3
needed chromatographic purification due to contamination by
3,24-ditosylate (20%) then was isolated in 60% yield. The 24-iodide (3,
Scheme 1) was synthesized by substitution of pure 24-tosylate with NaI
[36]. The reaction of 3 with an excess of anion generated in situ from
imidazole with NaH afforded N-steroid substituted imidazole (4) in
quantitative yield. The product was subjected to reaction with alkyl
iodide or bromide of different chain lengths. In the case of alkyl
bromides, an activator (NaI) was added. As a result of these reactions,
four imidazolium salts 5a-d were obtained. The most efficient reaction
was for short-chain alkyl halides (e.g. for preparation of 5a) and yields
slightly decreased with increasing chain lengths of the substituent.
3
(
200 mg, 0.45 mmol, 2 eq) in dry THF (3 mL) was added and the re-
action was refluxed for 16 h. Then the inorganic solid was filtered off
and the filtrate was evaporated. The residue was dissolved in DCM
(
2 mL), then the product was precipitated with Et O (10 mL) and fil-
2
tered. The crystallization from DCM/Et
2
O was repeated two more times
to produce a pale yellow salt in 34% yield (64 mg). Mp = 287–289 °C
(
from CH
2
Cl
447, 1379, 1233 cm
0.45 (s, 1H, H-2imidazole), 7.26 (s, 2H, Himidazole), 5.73 (s, 2H, CH = C),
.43 (dd, J = 13.6, 3.6 Hz, 2H, CH N), 4.04 (dd, J = 13.6, 9.8 Hz, 2H,
N), 1.18 (s, 6H, 2CH ), 0.94 (d, J = 6.4 Hz, 6H, 2CH ), 0.80 (s, 6H,
) ppm; C NMR (100 MHz, CDCl ) δ 199.4 (C, C = O), 171.1 (C,
C=CH), 137.4 (CHimidazole), 123.7 (CH, C=CH), 122.5 (CHimidazole),
2
/Et
2
O) (decomposition); IR (ATR) ν = 2937, 2851, 1681,
−
1
1
1
1
4
;
H NMR (400 MHz, CDCl , 25 °C, TMS) δ
3
2
3-Iodo-23,24-dinorchol-4-en-22-al (6) was obtained upon NaBH
4
CH
2
CH
3
3
reduction of steroid 2 followed by the Appel iodination (Scheme 2).
1
3
2
3
3
Selective reduction of the 22-carbonyl group 2 was achieved using one
equivalent of NaBH (r.t. 30 min) [38]. Subsequent transformations of
4
5
5.5 (CH
2
), 55.4 (CH), 53.5 (CH), 53.4 (CH), 42.8 (C), 39.3 (CH
2
2
3
), 38.4
iodide 6 were analogous with those for LCA derivatives. Although the
synthesis of N-steroid substituted imidazole 7 was less efficient, the
formation of salts 8a-d proceeded in higher yields than those of LCA
derivatives. Additionally, symmetrically substituted steroid salt 8e was
obtained directly from iodide 6.
(
(
(
C), 37.5 (CH), 35.6 (CH
2
), 35.4 (CH), 33.9 (CH
2
), 32.7 (CH ), 31.8
CH
CH
2
3
), 28.0 (CH
2
), 24.2 (CH
2
), 20.9 (CH
2
), 17.3 (CH
3
), 16.5 (CH
), 12.2
+
+
) ppm, ESI-MS m/z: 693 (M-I ). ESI-HRMS m/z: calcd for [M-I]
+
C
47
H
69
N
2
O
2
693.5354, found 693.5345.
It should be emphasized that the synthesis of new salts proved very
efficient and quite convenient, especially the last step, where chroma-
tographic purification was not needed. Salts with sufficient purity were
obtained after triple crystallization. They were air-stable solids and may
be handled under normal laboratory conditions.
2.4. Anti-microbial studies
Antibacterial and antifungal activities of imidazolium salts were
evaluated by broth microdilution assay in 96 well plates. The two-fold
serial microdilution assay, described by the Clinical and Laboratory
Standards Institute, was performed for the measurements of the
minimal inhibitory concentrations (MICs) expressed in μg/mL. The
imidazolium salt was first dissolved in DMSO and incorporated into
Mueller-Hinton broth (MHB) to obtain a concentration of 1024 μg/mL
3.2. The activity of imidazolium salts against human pathogens
Several reports have recently shown that imidazolium salts possess
promising biological activities [1]. Encouraged by these results, we
68

A. Hryniewicka, et al.
Journal of Steroid Biochemistry and Molecular Biology 189 (2019) 65–72
Scheme 1. Synthesis of imidazolium salts based on lithocholic acid.
evaluated the antimicrobial activities of steroidal imidazolium salts
against bacterial species causing nosocomial and healthcare-associated
infections (S. aureus, E. coli). A severe causative agent for food poi-
soning (S. aureus, B. cereus) and C. albicans, an opportunistic fungal
pathogen that is most frequently isolated from immunocompromised
patients, were also used to examine effects of imidazolium salts.
The results of MIC experiments showed that all the tested imida-
zolium salts have a great effective activity against bacteria with MIC
values from 0.5 to 64 μg/mL and 2- to 32-fold more potent activity
against fungi with MIC values from 0.25 to 2 μg/mL (Table 1). Com-
paring both series of imidazolium salts, those based on lithocholic acid
The antimicrobial activity of new salts presented as a relationship
between the number of carbon atoms in the alkyl substituent and the
log MIC values are shown in Fig. 2 (LCA derivatives) and Fig. 3 (steroid
2 derivatives). In general, the antibacterial efficacy of all tested imi-
dazolium salts increased with increasing chain length of the substituent.
However, the chain length of the substituent had no significant effect on
the antifungal activity of both series of imidazolium salts. It should be
noted that in both cases antifungal activity is even better than anti-
bacterial one. The obtained MIC values for C. albicans of all tested salts
were significantly lower than that for commercial Fluconazole. In ad-
dition, in the case of LCA derivatives, the antifungal effect was better
than that observed for Amphotericin B, which is the most effective but
highly toxic antifungal drug. Therefore, new salts seem to be very
promising compounds.
(
salts 5a–d) demonstrated better activity than those based on 3-oxo-
3,24-dinorchol-4-en-22-al (salts 8a–d) against S. aureus, B. cereus and
C. albicans, respectively. However, compounds 8a–d were found to be
2
2
- to 4-fold more effective against E. coli than compounds 5a–d. The
symmetrically substituted disteroidal imidazolium salt (8e) exhibited
excellent activity against S. aureus, B. cereus and C. albicans, but mod-
erate to weak activity against E. coli.
3.3. Evaluation of activity against phytopathogenic fungi
Encouraged by the bioassays above, especially promising results
Scheme 2. Synthesis of imidazolium salts based on 3-oxo-23,24-dinorchol-4-en-22-al.
69

A. Hryniewicka, et al.
Journal of Steroid Biochemistry and Molecular Biology 189 (2019) 65–72
Table 1
MIC values of imidazolium salts.
MIC
Imidazolium salt
Staphylococcus aureus
μg/mL]
Bacillus cereus
Escherichia coli
Candida albicans
[
[μM]
[μg/mL]
[μM]
[μg/mL]
[μM]
[μg/mL]
[μM]
5
5
5
5
8
8
8
8
8
a
b
c
4
7.2
3.5
1.6
0.8
61
32
58
28
6.5
3
16
16
16
16
16
8
29
0.25
0.5
0.5
0.5
2
0.5
0.9
0.8
0.8
3.8
1.9
3.5
1.7
2.4
–
2
16
28
1
4
26
d
a
b
c
0.5
2
26
32
64
122
60
7
31
16
30
32
15
1
4
7
4
4
7
2
d
e
2
3.4
1.2
0.7-2.9
–
2
3.4
1.2
0.7-1.4
–
4
6.7
1
1
1
64
0.8-2
–
78
2
†
Ampicillin
0.25-1
0.25-0.5
5.7-22.9
–
‡
Fluconazole
–
–
–
–
–
–
2
6.5
1.1
‡
Amphotericin B
–
–
–
1
†
‡
Clinical and Laboratory Standards Institute (CLSI), approved guideline M45A2E and supplement M100-S23.
http://www.eucast.org/clinical_breakpoints.
against C. albicans, antifungal activity was additionally assessed by in
vivo experiments on a set of phytopathogenic fungi causing widespread
diseases on crops (Botrytis cinerea, Fusarium culmorum, Alternaria alter-
nata, Cercospora beticola). The activity data for compounds 5a–d and
8
a–d at a concentration of 0.1 mg/mL against different fungi are re-
ported in Table 2. The best activity of all new compounds was observed
against B. cinerea, which causes gray mold. It is a very common disease
that affects about 200 species of different botanical families and has
shown resistance to some currently used fungicides [39].
Due to the fact that the salts activity at a concentration of 0.1 mg/
mL (0.16-0.19 mM) was very high, we also evaluated the activity at
lower concentrations: 0.1, 1, 5, 10, 25 and 50 μg/mL (representative
test for salt 5b is presented in Fig. 4). Although new compounds showed
no significant activity against F. culmorum and A. alternata at a con-
centration of less than 0.1 mg/mL, their activity against B. cinerea was
surprisingly high. Additionally, EC50 vales were determined and the
results of the selected salts are summarized in Table 3. The most active
compounds were 5a and 5b, which showed 45.1% and 86.7% of growth
inhibition for B. cinerea, respectively, at a concentration as low as 5 μg/
mL. Compounds 5c and 8b showed high activity (> 70%) at a con-
centration of 10 μg/mL. The trend in results obtained for B. cinerea is
Fig. 2. Antimicrobial activity of salts 5a–d.
Fig. 3. Antimicrobial activity of salts 8a–d.
Table 2
Percent inhibition of mycelial growth induced by imidazolium salts (con-
†
centration 0.1 mg/mL) .
Imidazolium salt Botrytis cinerea Cercospora
Alternaria
alternata
Fusarium
culmorum
beticola
5
5
5
5
8
8
8
8
a
b
c
100 ±
0
66.2 ± 3.4
69.0 ± 2.0
65.3 ± 4.6
58.0 ± 2.3
54.1 ± 5.1
46.1 ± 7.0
61.9 ± 2.3
28.6 ± 1.1
50.5 ± 7.2
66.0 ± 3.9
44.2 ± 3.7
49.6 ± 2.8
27.5 ± 6.8
2.2 ± 0.8
29.4 ± 1.2
6.0 ± 3.5
13.7 ± 3.4
52.4 ± 2.7
49.0 ± 9.0
44.8 ± 1.0
94.1 ± 5.8
100 ±
94.1 ±
100 ±
100 ±
100 ±
100 ±
0
d
a
b
c
0
0
0
0
0
83.3 ±
0
75.6 ± 4.2
67.4 ±
68.2 ± 2.1
0
d
†
Values are the mean ± standard deviation of three replicates.
Fig. 4. Evaluation of the activity of 5b against B. cinerea at different con-
centrations.
70

A. Hryniewicka, et al.
Journal of Steroid Biochemistry and Molecular Biology 189 (2019) 65–72
Table 3
†
EC50 values and percent inhibition of mycelial growth B. cinerea and C. beticola induced by selected salts .
Imidazolium salt
concentration
Botrytis cinerea
Cercospora beticola
[
μg/mL]
[μM]
% of inhibitiona
EC50
% of inhibitiona
EC50
5
5
5
8
8
a
b
c
5
1
2
5
1
5
1
2
5
1
5
1
2
5
1
5
1
2
5
1
5
1
2
5
1
9
45.1 ± 2.5
87.4 ± 2.4
92.5 ± 0.7
5.5 μg/mL (10 μM)
51.6 ± 5.2
53.8 ± 1.9
60.6 ± 6.2
65.0 ± 1.9
66.2 ± 3.4
47.6 ± 4.1
53.5 ± 3.6
55.9 ± 4.1
64.3 ± 3.6
69.0 ± 2.0
42.7 ± 2.3
48.0 ± 4.0
57.3 ± 2.3
57.4 ± 4.6
65.3 ± 4.6
49.9 ± 5.6
59.7 ± 6.3
65.8 ± 5.6
67.0 ± 3.6
75.6 ± 4.2
36.5 ± 4.2
53.6 ± 2.1
60.9 ± 4.2
63.4 ± 3.7
68.2 ± 2.1
4.6 μg/mL (8 μM)
0
18
45
90
180
9
5
0
100 ±
100 ±
0
0
00
86.7 ± 2.4
93.7 ± 0.7
94.5 ± 0.7
89.8 ± 8.8
94.1 ± 5.9
17.6 ± 2.0
77.6 ± 4.7
3.0 μg/mL (5 μM)
7.2 μg/mL (12 μM)
6.8 μg/mL (13 μM)
18.5 μg/mL (31 μM)
6.0 μg/mL (11 μM)
12.0 μg/mL (20 μM)
5.0 μg/mL (9 μM)
8.0 μg/mL (13 μM)
0
18
44
88
176
8
5
0
00
0
16
41
82
164
9
5
100 ±
100 ±
100 ±
0
0
0
0
00
b
d
27.4 ± 4.3
73.3 ± 1.9
0
18
47
93
186
8
5
100 ±
100 ±
100 ±
0
0
0
0
00
0.6 ± 0.6
5.8 ± 1.2
72.5 ± 6.5
94.5 ± 0.7
0
17
42
84
169
5
0
00
100 ±
0
†
Values are the mean ± standard deviation of three replicates.
similar to that of C. albicans, where LCA-based salts were better than
steroid 2 based ones. In the case of compounds 8a–d, salts with an even
number of carbon atoms in the alkyl substituent (8b and 8d) showed
better activity than those with an odd amount (8a and 8c). In Table 3,
the results of salts against C. beticola were at a similar level and did not
exceed 70%, with one exception; 8b at a concentration of 100 μg/mL
showed 75.6% of growth inhibition.
fungicides against phytopathogenic fungi. In contrast, salts with longer
alkyl chains were highly active against Gram-positive bacteria. In
summary, new compounds showed a broad spectrum of activity and
had promising antimicrobial activities, especially antifungal effect
against either human or plant pathogens, at a level comparable to
commercial drugs and fungicides.
In summary, new salts showed similar or even greater activity than
the agricultural fungicides used against B. cinerea. For example, com-
mercial drazoxolon inhibited mycelium growth in 91% at 42 μM [40],
whereas salt 5b showed similar inhibition at a concentration more than
Acknowledgement
The authors gratefully acknowledge financial support from the
National Science Centre, Poland, Grant No. UMO-2015/17/B/ST5/
02892.
4
times lower (9 μM). Another known fungicide, procymidone, com-
pletely inhibited the growth at 88 μM [41] just like salts 5c and 8b. The
EC50 values of imidazole-based fungicide, imazalil, against B. cinerea
Appendix A. Supplementary data
(
isolates collected from table and wine grapes) ranged from 0.4 to
3
4 μM [42] and were similar to those of salts 5a-c, 8b and 8d. It should
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jsbmb.2019.02.006.
be noted that the ethyl substituent is optimal for achieving the highest
antifungal activity (5b and 8b). Although tetraconazole, showing total
inhibition at 27 μM [41], proved to be more effective against C. beticola
than new salts, it should be noted that 5a–c, 8b and 8d displayed EC50
in the range of 8 to 20 μM. To our knowledge, this is the first report on
the possibility of using steroid-based imidazolium iodides as fungicides.
It should be noted that new iodides are very stable, easy to handle and
soluble in organic solvents (e.g. in alcohols). In addition, imidazolium
salts generally show low toxicity to humans. New salts may serve as
potential fungicides or at least as structural templates in the search for
new and highly efficient fungicides.
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