PhSeZnCl in the synthesis of steroidal β-Hydroxy- phenylselenides having antibacterial activity

Article abstract of DOI:10.3390/ijms20092121

We report here the reaction of in situ prepared PhSeZnCl with steroid derivatives having an epoxide as an electrophilic functionalization. The corresponding ring-opening reaction resulted to be regio- and stereoselective affording to novel phenylselenium-substituted steroids. Assessment of their antibacterial properties against multidrug-resistant bacteria, such as Pseudomonas aeruginosa Xen 5 strain, indicates an interesting bactericidal activity and their ability to prevent bacterial biofilm formation.

Full text of DOI:10.3390/ijms20092121

International Journal of
Molecular Sciences
Communication
PhSeZnCl in the Synthesis of Steroidal
β-Hydroxy-Phenylselenides Having
Antibacterial Activity
Izabella Jastrzebska 1,*, Stefano Mellea 1, Valerio Salerno 1, Pawel Adam Grzes 1,
Leszek Siergiejczyk 1, Katarzyna Niemirowicz-Laskowska 2, Robert Bucki 2 , Bonifacio Monti 3
and Claudio Santi 3,
*
1
Institute of Chemistry, University of Białystok, ul. Ciołkowskiego 1K, 15-245 Białystok, Poland;
stefano.mellea@gmail.com (S.M.); vale_1192@hotmail.it (V.S.); felolix@gmail.com (P.A.G.);
nmrbial@uwb.edu.pl (L.S.)
Department of Microbiological and Nanobiomedical Engineering, Medical University of Białystok, ul.
Mickiewicza 2C, 15-222 Bialystok, Poland; katia146@wp.pl (K.N.-L.); buckirobert@gmail.com (R.B.)
Group of Catalysis and Organic Green Chemistry–Department of Pharmaceutical Sciences, University of
Perugia, Via del Liceo 1, 06132 Perugia, Italy; bonifaciomonti@gmail.com
Correspondence: i.jastrzebska@uwb.edu.pl (I.J.); claudio.santi@unipg.it (C.S.); Tel.: +48857388043 (I.J.);
+390755855106 (C.S.)
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 April 2019; Accepted: 24 April 2019; Published: 29 April 2019
Abstract: We report here the reaction of in situ prepared PhSeZnCl with steroid derivatives having
an epoxide as an electrophilic functionalization. The corresponding ring-opening reaction resulted to
be regio- and stereoselective affording to novel phenylselenium-substituted steroids. Assessment of
their antibacterial properties against multidrug-resistant bacteria, such as Pseudomonas aeruginosa
Xen 5 strain, indicates an interesting bactericidal activity and their ability to prevent bacterial
biofilm formation.
Keywords: steroids; selenium; antibacterial; antibiofilm
1. Introduction
The last few decades have seen a growing interest in the synthesis of organoselenium compounds
due to some promising biological activities, reported in the recent literature [1–3]. Selenium is present
in mammalian organisms in the form of selenoenzymes, embedded in the proteinogenic amino acid
selenocysteine. All the selenoproteins demonstrated redox properties and have a crucial role in the
redox modulation as well as in the control of the redox homeostasis of the living systems, playing also
a role in the cell’s protection against the oxidative stress [
compounds was recently described as an interesting property to be directed toward specific targets
developing antibacterial [ ], antiviral [ ], and antifungal agents [ ], hormetines [ ], and enzyme
inhibitors [ ]. Furthermore, direct and indirect interferences with the redox homeostasis and the redox
4]. The prooxidant activity of organoselenium
5
6
7
8
9
signaling can produce an anticancer effect affecting the differentiation, proliferation, senescence and
death pathways in the cells [10].
Even if many physiological and pathological mechanisms involving organoselenium derivatives
need to be clarified, the recent introduction of Ebselen® and Ebselen-like compounds in clinical trials
for the treatment of diabetes complications and non-small cell lung cancer, further demonstrate the
current interest in study these derivatives for medical purposes [11,12].
Till now, few examples of selenosteroids were reported in the literature and they were
tested and proved to be interesting antiproliferative agents following a prooxidant mechanism.
Int. J. Mol. Sci. 2019, 20, 2121; doi:10.3390/ijms20092121
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2019, 20, 2121
2 of 13
Some representative examples are reported in Figure 1 [13
–
15], selenium can be embedded as
selenourea, selenocyanate or alkyl/aryl selenide directly introduced in C-3 or C-6 through the SN2
reaction of a selenolates with a tosylate (VI) or an epoxide (V) affording, in this letter case to a
β-hydroxyselenide [16,17].
N
O
Se
N
OAc
OAc
Se
N
H
N
HO
H
I
II
O
O
H
H
SeCN
Br
OH
N
N
Se
O
HO
III
IV
O
RSe
HO
SeR
V
VI
Figure 1. Some representative selenosteroids with antiproliferative and prooxidant activity. Selenium
is directly bonded to the steroid (III, V and VI) selenium is imbedded in a different group (in blure)
linked to the steroid (I, II, IV).
During the last ten years, some of us deeply investigated the use of nucleophilic selenium reagents,
in the form of zinc selenolates, for the functionalization of electrophilic organic substrates [18]. Among
these reagents the PhSeZnCl, easily prepared through the oxidative insertion of elemental zinc into
Se-halogen bound of PhSeCl, it was the first bench stable organic selenolate [19], it is nowadays
commercially available, and someone recently named as Santi’s reagent [20].
These reagents demonstrate a broad range of applicability showing in several cases a strong rate
acceleration when the reaction is performed in “on water” conditions [21–23].
In addition, its use was recently described also in the ring opening reactions of aziridines [24
]
and epoxides for the optimization of a total synthesis [25] as well as in the functionalization of
preformed polymers [26], demonstrating the effective applicability of the method also starting from
polyfunctionalized substrates. The ring opening of epoxide with PhSeZnCl is generally highly
regioselective and the presence of the zinc as Lewis acid was demonstrated to be in some cases
important for the regioselective control of the reaction [19].
2. Results and Discussion
2.1. Chemistry
In the present work, we explored the use of PhSeZnCl for the functionalization of different steroid
derivatives having an epoxide as an electrophilic reactive center, in order to obtain new molecular
selenosteroids as prototypes for the investigation of the antibacterial activity. The optimization of
the reaction conditions was performed starting from the epoxide
isomer, respectively.
1 as a 4:1 mixture of the α and β

Int. J. Mol. Sci. 2019, 20, 2121
3 of 13
The reaction of 5,6-epoxycholestane
1
with solid PhSeZnCl was firstly investigated using the
conditions reported for the opening of epoxide both in water suspension and THF solution at room
temperature [19] but these procedures did not afford the desired product. Differently, when the
reagent was prepared in situ in THF and the epoxide
1 was refluxed for 3 h with the reagent the
β
-hydroxyselenide was obtained in 60% yield (75% on converted material), as depicted in Scheme 1.
2
The nucleophilic attack to the epoxide ring occurs exclusively on the less sterically hindered carbon (C6)
due to the presence of axial C-19 methyl group, indicating that an SN2 mechanism is involved in the
process and affording the corresponding trans-hydroxy selenides
was observed and the unreacted 5 ,6 -epoxide was quantitatively recovered after chromatographic
purification. The reason of the non-reactivity of 5 ,6 -epoxide with nucleophilic reagents was recently
2. Only the trans hydroxyl selenide 2
β
β
β
β
explained and deal with the unfavorable formation of a constrained structure that should arise from
the trans-diaxial opening at C6 with the hydroxyl at C5 in the syn position with regard to the C19 [27].
A separate experiment with epoxycholestane
After 3 h, TLC monitoring showed the consumption of a part of the starting material
1H NMR spectral analysis, it was established that only the
-epoxide reacted while the
-hydroxy-5 ,6 -epoxycholestane (20%) was
1
and (PhSe)2 in the presence of NaBH4 was performed.
. On the basis of
-isomer of
1
α
1
β
1
remained intact. Moreover, after 3 h of the reaction, 3
β
α α
still present in the reaction mixture.
Compound 2 was fully characterized by NMR, IR and MS spectroscopies and the collected data
correspond to those previously reported by Rodrigues et al. [16].
In the optimized protocol, the zinc (1.0 equivalent) activated by treatment with HCl 10%, was
added to the solution of PhSeCl (1.0 equivalent) in anhydrous THF and heated at the reflux temperature
till the discoloration of the solution that is assumed to be indicative for the formation in situ of
PhSeZnCl. At this point, the substrate 1 (1.0 equiv) dissolved in THF was added. The reaction was
refluxed for an additional 3 h and monitored by thin-layer chromatography (TLC).
Zn, THF
reflux
PhSeZnCl
PhSeCl
Se
HO
OH
2 (75%)
THF, reflux
3h
O
HO
4:1 mixture
O
1 (20%)
1 (5α,6α)
HO
O
1 (5β,6β)
HO
sterically
unfavored
sterically
unfavored
favored
O
HO
O
HO
unstability
of the product
Scheme 1. Reaction of 3β-hydroxy-5α,6α-epoxycholestane (1) with in situ formed PhSeZnCl.
Using the above-described conditions, the scope of epoxy substrates 1, 3, 5, 6 and
8
was
explored. The results are collected in Table 1. Interestingly the mild conditions of the procedure
resulted compatible with the sensitive spiroacetal system [28] in and the sterically hindered
3

Int. J. Mol. Sci. 2019, 20, 2121
4 of 13
epoxide
situ generated organoselenium reagent gave 1
,3 -dihydroxy-1 -phenylselenylcholestane (9
5
[
29] was totally unreactive. The reactions of 1,2-epoxysteroids
,3 -dihydroxy-2
), respectively. For the accurate determination of the
6
and
8
with the in
β
β
β-phenylselenylcholestane (7) and
2
β
α
α
stereochemistry of the centers formed in the C-1 and C-2 positions, two-dimensional NMR experiments
were performed (COrrelation SpectrocpY (COSY), heteronuclear single quantum correlation (HSQC),
and heteronuclear multiple bond correlation (HMBC)) in 400 MHz instrument. For compound 7, the
coupling system inferred from the correlation spectra indicates that phenylselenyl group is bonded
to C-2, since 2-H is coupled to both adjacent 1-H and 3-H. In addition, the 3-H signal has the width
at half-height of 24 Hz, which clearly indicates that it is axial, and therefore, 2-H must be equatorial
because its width at half-height is small (10 Hz). The downfield shift of C-1 (78.4) shows that the
configuration of the OH group is
inferred from COSY, HSQC, and HMBC spectra of compound
group is bonded to C-1. Proton 3-H clearly has configuration (equatorial) since its width at half-height
is 8 Hz. In turn, 2-H has configuration (equatorial) since long range coupling to 4-Heq is observed in
β
(axial), and therefore, 1-H is equatorial. The correlation pattern
9
allows us to state that phenylselenyl
β
α
COSY spectrum filling the marks of the H-C-C-C-H W-shape structure (Figure 2) [30].
Figure 2. Relevant data from COrrelation SpectrocpY (COSY and heteronuclear multiple bond
correlation (HMBC) experiments for the structure of the compound
HMBC correlations–dashed line, – means no relation).
9 (COSY correlations – plain line,
Table 1. Scope of the reaction
Entry
Substrate
Time (h)
Product
Yield
1
3
75%
2
HO
HO
Se
1 (5α,6α)
HO
O
2
3
–
–
1 (5β,6β)
HO
O

Int. J. Mol. Sci. 2019, 20, 2121
5 of 13
Table 1. Cont.
Entry
Substrate
Time (h)
Product
Yield
O
O
O
O
3
6
8
62
4
AcO
HO
Se
3 (5α,6α)
AcO
O
O
4
–
–
5
AcO
OAc
OH
O
5
6
2
2
54
53
Se
6
7
HO
HO
Se
O
HO
HO
8
9
HO
Based on these spectroscopic evidence, stereogenic centers at C-1 and C-2 have the absolute
configuration depicted in structure and , and a plausible mechanism for their formation starting
from 1,2-epoxysteroids and with PhSeZnCl is proposed in Scheme 2. In both cases, the nucleophilic
7
9
6
8
attack proceeds in order to minimize the steric hindrance of the approaching selenium reagent with
the substituents, following a pseudo-axial attack according to the Fürst-Plattner rule [31]. Furthermore,
the interaction of zinc with the hydroxyl group at C-3 could increase the selenium nucleophilicity and
cooperate on driving the observed regioselectivity. Steric factors and the lack of a suitable pseudo-axial
approach resulted to be particularly detrimental in the reactivity of PhSeZnCl with the epoxy-steroids
and could explain all the observed failures.
sterically
unfavored
+δ
Cl
Se
Se
Zn
-δ
HO
O
O
H
OH
7
6
OH
O
H
Se
-δ
Se
8
O
OH
9
sterically
unfavored
H
Zn
Cl
+δ
Scheme 2. The proposed mechanism for ring opening of
6 and 8 according to a pseudo-axial ring
opening approach.
2.2. Biological Activity
A further purpose of this study was to investigate the biological properties of the prepared
selenosteroids. The search for new compounds that exhibit antimicrobial properties is a big challenge

Int. J. Mol. Sci. 2019, 20, 2121
6 of 13
and an emerging need in modern medicine, especially in the context of the growing number of
infections caused by multidrug-resistant bacteria. Furthermore, the antibacterial activity against the
biofilm formation of Pseudomonas aeruginosa, which usually causes opportunistic infections, particularly
in immunocompromised patients, can be considered a promising characteristic for the development of
a new class of antibiotics [32].
The changes of Pseudomonas aeruginosa, Xen 5 luminescence provide an easy way to assess bacteria
cell viability and metabolic function. [33] Accordingly, Figure 3A–E shows that all tested agents
affect the viability of employed bacteria strain. However, activity of compound
compared to other tested agents. More than 95% of decrease in P. aeruginosa Xen 5 chemiluminescence,
was observed after 10 min of incubation when highest dose of was tested. This inhibitory effect is
7 was stronger when
7
comparable to activity of standard antibiotic agents used in concentrations corresponding to a 100-fold
increase of MIC value (Figure 3E). The obtained results suggest that the position of phenylselenyl
group might affect and modulate the antimicrobial activity. Additionally, the activity of synthesized
compounds, was compared to that of colistin, currently used in the treatment of P. aeruginosa infections.
Interestingly, it was observed that the use of colistin at a dose corresponding to 1xMIC value does not
affect the metabolic function of P. aeruginosa infections caused by MDR strains. In turn, application of
5-fold MIC concentration disturbs the cell function at 60%.
Figure 3. Phenylselenium-substituted steroids decrease the metabolic activity of MDR Pseudomonas
aeruginosa strain. Panels
Xen 5 in comparison to colistin (
A
–
D
show activity of tested agents against planktonic form of P. aeruginosa
). Statistical significance for the samples treated by tested compounds
E
compared to control was marked by (*), p ≤ 0.05. Results from 3 measurements ±SD.
In a different set of experiments, luminometric measurements was applied to determine the
ability of the tested phenylselenium-substituted steroids on preventing bacterial biofilm formation.
Figure 4A–C illustrates that the tested agents are able to inhibit biofilm formation and effectively kill
bacteria embedded into the biofilm matrix in time and dose-dependent manners. However, after 24 h,
in the case of compound
formation. In the case of mature biofilm, formed after 48 and 72 h treatment with the tested agents
(> 20 g/mL), decreases biofilm viability by ~50%, for compound and respectively, and ~ 90% for
compound . In the case of colistin, their use in 1 and 5- fold MIC was insufficient to restrict biofilm
7, high doses (>50 µg/mL) were required to obtain ~50% inhibition of biofilm
µ
2
4
7
metabolic activity. Therefore, the observed proprieties of the tested agents might help in developing of
effective strategies against Pseudomonas aeruginosa biofilm formation, which is directly associated with

Int. J. Mol. Sci. 2019, 20, 2121
7 of 13
hospital infections via the colonization of medical devices, as well as a major cause of the recurrence
and chronic infections, such as pneumonia in cystic fibrosis patients. Due to amphipathic nature
of tested phenylselenium-substituted steroids and their similarity to ceragenins, which possess a
broad spectrum of antimicrobial activity, further studies are needed to determine a full antimicrobial
spectrum of the tested agents and to establish their mode of action [34].
Figure 4. Anti-biofilm activity of phenylselenium-substituted steroids against P. aeruginosa strain.
Ability of phenylselenium-substituted steroids to prevent biofilm-formation of P. aeruginosa after 24, 48
and 72 h. Results from 3 measurements ±SD.
The analysis of 77Se-NMR chemical shift of
consideration. In compounds and the presence of hydroxyl groups in a suitable position to establish
a non-bonding interaction with the selenium atom produce an evident upfield of the chemical shift
60 ppm for 116 ppm for respect and ). This correspond to a higher electron density on the
2,4,7 and 9 (reported in Figure 5) afforded interesting
7
9
(
−
9
;
−
7
2
4
selenium atom of 7 respect to the other derivatives and, consequently, a lower electrophilicity to which
correspond a reduced prooxidant activity and, reasonably, a lower general toxicity.
2
δ = 364 ppm
HO
HO
Se
Se
HO
HO
O
O
δ = 304 ppm
9
4
δ = 363 ppm
AcO
OH
HO
Se
Se
HO
δ = 248 ppm
7
upfield effect on 77SeNMR chemical shift = increase electron density of Selenium atom
electrophilicity and prooxidant activity
Figure 5. 77SeNMR Chemical shift as parameter to predict biological aspects.
Considering that Rodrigues et al. reported that derivatives similar to
important prooxidant activity we explored the redox behavior of as well as its GPx-like activity
using the 77Se-NMR and NMR-DTT coupled test [16 17]. Initially, we investigated by 77Se-NMR
2 are characterized by an
2
,
spectroscopy, the actual intermediates involved in GPx-like cycle. With the addition of hydrogen
peroxide (5 eq), we observed the formation of a couple of diastereomeric selenoxides (δ 77Se-NMR in
CD3OD: 901 and 866 ppm), that can be readily reduced to selenide by the addition of a stoichiometric
amount (5 eq) of reduced dithiotreitol (DTTred). The experiment was performed directly into the NMR
tube (Scheme 3). The kinetics of the peroxide reduction catalyzed by
2 was measured studying via

Int. J. Mol. Sci. 2019, 20, 2121
8 of 13
1H-NMR the oxidation of DTT. This process resulted particularly slow producing, after 19 h, only
22% of oxidized DTT. This value is particularly low respect those reported in literature for other GPx
mimetics and we believe that it could be due to the steric hindrance around the selenium atom of
selenoxide, that prevented the fast attack of the thiol and consequently the reduction of the selenoxide.
H2O2
H2O
O
Se
HO
HO
OH
Se
HO
O
Se
OH
OH
HO
HO
HO
HO
S
S
SH
SH
DTTred
DTTox
Scheme 3. Proposed redox cycle of 3β,5α-dihydroxy-6β-phenylselenylcholestane (2).
3. Materials and Methods
3.1. Chemistry
3.1.1. General Methods
Reagent-grade chemicals were purchased and used as received. Methylene chloride was freshly
distilled. Flash column chromatography and flash chromatography were performed with silica gel,
pore size 40A (70–230 mesh), unless otherwise stated. All reactions were monitored by TLC on silica
1
gel plates 60 F254. H (400 MHz) and 13C NMR (100 MHz) spectra for all compounds were recorded
at ambient temperature and were referenced to TMS (0.0 ppm) and CDCl3 (77.0 ppm), respectively,
unless otherwise noted. NMR resonance multiplicities were reported using the following abbreviations:
b = broad, s = singlet, d = doublet, t = triplet, q = quartet, and m = multiplet; coupling constants J were
reported in Hz. IR spectra were obtained in a CHCl3 solution with an FT-IR spectrometer, and data are
reported in cm−1. Melting points were determined by a Kofler bench (Boetius type) apparatus and are
uncorrected. (Spectra of the synthetized compounds are collected in the Supplementary Materials)
3.1.2. General Procedure
To a solution of the PhSeCl (1.0 equiv.) in anhydrous THF (1 mL/mmol) under argon atmosphere
and at reflux, the activated zinc (1.0 equiv) was added. The formation of PhSeZnCl was indicated
by the formation of colourless solution. Then, a solution of the starting material (1.0 equiv) in THF
under argon atmosphere was added. The reaction was stirred at reflux for 3–8 h. Then THF was
removed under vacuum. The yellow oil obtained was dissolved in CH2Cl2 and washed 3 times with
H2O. The organic layers were dried with Na2SO4, filtered and the solvent removed under vacuum.
The products were purified by flash chromatography.

Int. J. Mol. Sci. 2019, 20, 2121
9 of 13
β,5α-Dihydroxy-6β-Phenylselenylcholestane (2)
The reaction with 3β-hydroxy-5
ζ
,6
ζ
-epoxycholestane [35] (1, 4
α
:1β, 100 mg, 0.2 mmol) was
carried out (reaction time 3 h). Silica gel column chromatography gave pure compound 2 as a white
solid (69 mg; 60%) eluted with ethyl acetate/hexane 1:4, and 3
recovered.
: m.p. 159–162 ◦C (CH2Cl2/hexane). IR
β
-hydroxy-5
β
,6
β
-epoxycholestane was
1
2
ν
(cm−1): 3554, 3407, 1064. H NMR
δ
7.56
max
(m, 2H), 7.27 (m, 3H), 4.08 (m, 1H), 3.10 (m, 1H), 2.46 (dd, J = 11.2, 13.3 Hz, 1H), 1.18 (s, 3H), 0.92 (d,
J = 6.5 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H), 0.74 (s, 3H). 13C NMR
134.0 (CH 2),
132.6 (C), 129.1 (CH 2), 129.1 (CH), 78.1 (C), 68.1 (CH), 56.3 (CH), 55.5 (CH), 54.2 (CH), 46.1 (CH),
δ
×
×
55.7 (CH), 45.4 (CH), 44.2 (CH2), 42.8 (C), 39.9 (CH2), 39.5 (CH2), 39.3 (C), 36.2 (CH2), 35.8 (CH), 34.5
(CH2), 32.6 (CH2), 31.3 (CH2), 30.8 (CH2), 28.2 (CH2), 28.0 (CH), 24.2 (CH2), 23.8 (CH2), 22.8 (CH3), 22.5
(CH3), 21.3 (CH2), 18.6 (CH3), 17.6 (CH3), 12.2 (CH3). 77Se NMR
560.3133, found: 560.3120.
δ 365.8. HRMS calcd. for C33H52O2Se:
(25R)-5α-Hydroxy-6β-Phenylselenylspirostan-3β-ol Acetate (4)
The reaction with (25R)-5α,6α-epoxyspirostan-3β-ol acetate [36] (3, 85 mg, 0.2 mmol) was carried
out (reaction time 6 h). Silica gel column chromatography gave pure compound 4 as a white solid (70 mg;
62%) eluted with ethyl acetate/hexane 5:95. Colorless crystals: m.p. 270–273 ◦C (CH2Cl2/hexane). IR,
1
ν
max (cm−1) 3427, 3005, 1710, 1064. H NMR: 7.54 (m, 2H), 7.27 (m, 3H), 5.15 (m, 1H), 4.39 (m, 1H), 3.47
(dd, J = 2.6, 9.4 Hz, 1H), 3.37 (m, J = 10.9 Hz, 1H), 3.06 (m, 1H), 2.47 (dd, J = 11.3, 13.4 Hz), 2.05 (s,
3H), 1.20 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.85 (s, 3H). 0.79 (d, J = 6.3 Hz, 3H); 171.8 (C),
13C NMR:
134.7 (CH 2), 131.9 (C), 129.1 (CH 2), 127.1 (CH), 109.2 (C), 80.7 (CH), 71.2 (CH), 66.8 (CH2), 62.2
δ
δ
×
×
(CH), 55.2 (CH), 54.0 (CH), 45.7 (CH), 40.8 (C), 40.4 (CH2), 39.9 (CH2), 39.5 (C), 36.6 (CH2), 33.9 (CH2),
32.3 (CH2), 31.7 (CH2), 31.4 (CH2), 30.8 (CH), 30.3 (CH), 28.8 (CH2), 26.6 (CH2), 21.4 (CH3), 21.0 (CH2),
17.5 (CH3), 17.1 (CH3), 16.6 (CH3), 14.5 (CH3). 77Se NMR
631.2902, found: 631.2877.
δ
363.1. HRMS calcd. for [C35H50O5SeH]+:
1α,3β-Dihydroxy-2β-Phenylselenylcholestane (7)
The reaction with 3
for 2 h. Silica gel column chromatography gave pure compound
15:85 as oil (60 mg; 54%). IR
β
-hydroxy-1
α
,2
α
-epoxycholestane [37] (
6
) (80 mg; 0.2 mmol) was carried out
eluted with ethyl acetate/hexane
7.64 (m, 2H), 7.26 (m, 3H), 4.39
7
1
ν
(cm−1): 3429, 3055, 1458. H NMR
δ
max
(s, 1H), 4.09 (m, 1H), 3.78 (t, J = 2.5 Hz, 1H), 2.34 (d, J = 11.2 Hz, 1H), 0.96 (s, 3H), 0.91 (d, J = 6.5 Hz,
3H), 0.87 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H), 0.67 (s, 3H). 13C NMR
132.8 (C), 132.7 (CH 2),
129.2 (CH 2), 127.3 (CH), 78.4 (CH), 67.4 (CH), 59.5 (CH), 56.3 (CH 2), 47.7 (CH), 42.6 (C), 40.1 (C),
δ
×
×
×
39.8 (CH2), 39.5 (CH2), 39.0 (CH), 36.1 (CH2), 35.9 (CH2), 35.7 (CH), 34.9 (CH), 31.5 (CH2), 28.2 (CH2),
28.0 (CH2), 27.9 (CH), 24.2 (CH2), 23.8 (CH2), 22.8 (CH3), 22.5 (CH3), 20.9 (CH2), 18.7 (CH3), 13.5 (CH3),
12.1 l(CH3). 77Se NMR δ 248.4 ppm. HRMS calcd. for C33H52O2Se: 560.3133, found: 560.3121.
2β,3α-Dihydroxy-1α-Phenylselenylcholestane (9)
The reaction with 3
α-hydroxy-1β,2β-epoxycholestane [38] (8) was carried out for 2 h. Silica gel
column chromatography gave pure compound
9
eluted with ethyl acetate/hexane 1:4. as white solid
1
(60 mg, 53%); m.p. 174–175 ◦C (CH2Cl2/hexane); IR
ν
max (cm−1): 3565, 3351, 1437. H NMR
δ
, ppm
7.54 (m, 2H), 7.26 (m, 3H), 4.39 (m, 1H), 4.09 (s, 1H), 3.50 (d, J = 2.6 Hz, 1H), 2.47 (bs, 1H), 2.11 (bs, 1H),
1.19 (s, 3H), 0.91 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H), 0.68 (s, 3H). 13
NMR 133.8 (CH 2), 130.2 (C), 129.2 (CH 2), 127.4 (CH), 73.9 (CH), 67.9 (CH), 57.4 (CH), 56.2 (CH),
C
δ
×
×
56.1 (C), 52.4 (CH), 42.6 (C), 41.3 (CH), 39.6 (CH2), 39.5 (CH2), 36.1 (CH2), 35.7 (CH), 35.1 (CH), 32.9
(CH2), 31.5 (CH2), 28.7 (CH2), 28.2 (CH2), 27.9 (CH), 24.2 (CH2), 23.8 (CH2), 22.8 (CH3), 22.5 (CH3),
20.7 (CH2), 18.6 (CH3), 15.9 (CH3), 12.1 (CH3). 77Se NMR
560.3133, found: 560.3139.
δ 304.5 ppm. HRMS calcd. for C33H52O2Se:

Int. J. Mol. Sci. 2019, 20, 2121
10 of 13
3.2. Biological Activity
3.2.1. Antibacterial Testing
To assess the biological activities of synthesized compounds, we have chosen to determine their
antibacterial activity against Gram-negative, multidrug resistant (MDR) Pseudomonas aeruginosa Xen 5
strain. Briefly, a bacterial culture was grown to mid-log phase at 37 ◦C, re-suspended in LB, and brought
to 108 CFU/mL. Then 100
range 1–100
µ
L of bacteria suspensions were added to tested agents at a concentration
µ
g/mL. Colistin was use as a control, since it represents “last treatment option” in some
infections caused by MDR Pseudomonas aeruginosa. Upon bacteria addition the chemiluminescence
intensity were registered using Labsystems Varioscan Flash (Thermo Scientific Waltham, MA, USA)
over a period of 1h.
3.2.2. Anti-Biofilm Activity
The biomass of biofilm, formed by Pseudomonas aeruginosa Xen 5 in the presence of tested molecules,
was evaluated using a luminescence technique as described previously [39]. Chemiluminescence
intensity of Pseudomonas aeruginosa Xen 5 biomass was measured after 24, 48, and 72 h of growth.
In this setting, time-dependent biofilm mass and viability was determined
3.2.3. GPx-Like Activity by NMR
In a 5mm NMR tube containing 0.15 mmol of DTTred and 0.015 of catalyst (
2) dissolved in 0.55 mL
of CD3OD, 0.15 mmol of H2O2 (30%) was added and 1H-NMR experiments at 200 MHz were recorded
at regular interval for a period of 24 h. Ratios between DTTred/DTTox were determined by comparison
of integrals at 2.88 ppm and 2.64 ppm, respectively.
4. Conclusions
In conclusion we demonstrated that in situ formed PhSeZnCl can be used for the functionalization
of steroids with selenium moieties. The obtained novel compounds were tested for their potential
antibacterial properties against Pseudomonas aeruginosa Xen 5 strain, evidencing a promising bactericidal
activity associated to a biofilm formation prevention.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/9/2121/
s1.
Author Contributions: Conceptualization, I.J. and C.S.; methodology, I.J., C.S. and R.B..; investigation, S.M, V.S.,
P.A.G., L.S., K.N-L.; B.M.; resources, I.J.; data curation I.J; R.B. writing—experimental design of antibacterial
activity, original draft preparation, I.J. and C.S.; writing—review and editing, I.J. and C.S and K.N.-L.; supervision,
I.J. and C.S.; funding acquisition, I.J.
Funding: This research was funded by National Science Center, Poland, grant number UMO-2015/17/B/ST5/02892.
Acknowledgments: The authors would like to thank Erasmus + Program (SM and VS). The project was performed
under the umbrella of “SeS Redox & Catalyst Network”. Part of the study was conducted with the use of
equipment purchased by Medical University of Białystok as part of the RPOWP 2007-2013 funding, Priority I,
Axis 1.1, contract No. UDA-RPPD.01.01.00-20-001/15-00 dated 26.06.2015. CS also thanks the inter-universities
consortium C.I.N.M.P.I.S.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Abbreviations
COSY COrrelation SpectrocpY
DTT
GPx
DiThioTreitol
Glutathione Peroxidase
HMBC Heteronuclear Multiple Bond Correlation
HSQC Heteronuclear Single Quantum Correlation

Int. J. Mol. Sci. 2019, 20, 2121
11 of 13
IR
InfraRed
MIC
MS
Minimum Inhibitory Concentration
Mass
NMR
SD
THF
TLC
Nuclear Magnetic Resonance
Standard Deviation
TetraHydroFuran
Thin Layer Chromatography
References
1.
2.
3.
Sk, U.H.; Sharma, A.K.; Ghosh, S.; Bhattacharya, S. Synthesis and biological evaluation of novel spiro
6-methoxytetralin-1,30-pyrrolidine based organoselenocyanates against cadmium-induced oxidative and
hepatic damage in mice. Eur. J. Med. Chem. 2010, 45, 3265–3273. [CrossRef] [PubMed]
de Souza, D.; Mariano, D.O.C.; Nedel, F.; Schultze, E.; Campos, V.F.; Seixas, F.; da Silva, R.S.; Munchen, T.S.;
Ilha, V.; Dornelles, L.; et al. New Organochalcogen Multitarget Drug: Synthesis and Antioxidant and
Antitumoral Activities of Chalcogenozidovudine Derivatives. J. Med. Chem. 2015, 58, 3329–3339. [CrossRef]
Barcellos, A.M.; Abenante, L.; Sarro, M.T.; Leo, I.D.; Lenardao, E.J.; Perin, G.; Santi, C. New Prospective for
Redox Modulation Mediated by Organo selenium and Organotellurium Compounds. Curr. Org. Chem. 2017
,
21, 2044–2061. [CrossRef]
4.
5.
Brigelius-Floh
1830, 3289–3303. [CrossRef]
Sancineto, L.; Piccioni, M.; De Marco, S.; Pagiotti, R.; Nascimento, V.; Braga, A.L.; Santi, C.; Pietrella, D.
Diphenyl diselenide derivatives inhibit microbial biofilm formation involved in wound infection.
BMC Microbiol. 2016, 16, 220. [CrossRef] [PubMed]
é, R.; Maiorino, M. Glutathione peroxidases. Biochim. Biophys. Acta Gen. Subj. 2013
,
6.
7.
8.
9.
Rossato, L.; Loreto, E.S.; Venturini, T.P.; de Azevedo, M.I.; Al-Hatmi, A.M.S.; Santurio, J.M.; Alves, S.H.
In vitro combination between antifungals and diphenyl diselenide against Cryptococcus species. Mycoses
2019. [CrossRef]
Sancineto, L.; Mariotti, A.; Bagnoli, L.; Marini, F.; Desantis, J.; Iraci, N.; Santi, C.; Pannecouque, C.; Tabarrini, O.
Design and Synthesis of DiselenoBisBenzamides (DISeBAs) as Nucleocapsid Protein 7 (NCp7) Inhibitors
with anti-HIV Activity. J. Med. Chem. 2015, 58, 9601–9614. [CrossRef]
Bartolini, D.; Commodi, J.; Piroddi, M.; Incipini, L.; Sancineto, L.; Santi, C.; Galli, F. Glutathione S-transferase
pi expression regulates the Nrf2-dependent response to hormetic diselenides. Free Radic. Biol. Med. 2015
88, 466–480. [CrossRef] [PubMed]
,
Bartolini, D.; Piroddi, M.; Tidei, C.; Giovagnoli, S.; Pietrella, D.; Manevich, Y.; Tew, K.D.; Giustarini, D.;
Rossi, R.; Townsend, D.M.; et al. Reaction kinetics and targeting to cellular glutathione S-transferase of
the glutathione peroxidase mimetic PhSeZnCl and its d,l-polylactide microparticle formulation. Free Radic.
Biol. Med. 2015, 78, 56–65. [CrossRef]
10. Bartolini, D.; Sancineto, L.; Fabro de Bem, A.; Tew, K.D.; Santi, C.; Radi, R.; Toquato, P.; Galli, F.
Selenocompounds in Cancer Therapy: An Overview. In Advances in Cancer Research; Elsevier: San
Diego, CA, USA, 2017; pp. 259–302. ISBN 978-0-12-812016-3.
11. Tan, S.M.; Sharma, A.; Stefanovic, N.; de Haan, J.B. Late-intervention study with ebselen in an experimental
model of type 1 diabetic nephropathy. Free Radic. Res. 2015, 49, 219–227.
12. He, J.; Li, D.; Xiong, K.; Ge, Y.; Jin, H.; Zhang, G.; Hong, M.; Tian, Y.; Yin, J.; Zeng, H. Inhibition of thioredoxin
reductase by a novel series of bis-1,2-benzisoselenazol-3(2H)-ones: Organoselenium compounds for cancer
therapy. Bioorg. Med. Chem. 2012, 20, 3816–3827.
13. Romero-Hernandez, L.L.; Merino-Montiel, P.; Montiel-Smith, S.; Meza-Reyes, S.; Vega-B
áez, J.L.; Abasolo, I.;
Schwartz, S., Jr.; Lopez, O.; Fernandez-Bolanos, J.G. Diosgenin-based thio (seleno) ureas and triazolyl
glycoconjugates as hybrid drugs. Antioxidant and antiproliferative profile. Eur. J. Med. Chem. 2015
99, 67–81. [CrossRef]
,

Int. J. Mol. Sci. 2019, 20, 2121
12 of 13
14. Fuentes-Aguilar, A.; Romero-Hern
á
ndez, L.L.; Arenas-Gonz
ález, A.; Merino-Montiel, P.; Montiel-Smith, S.;
Meza-Reyes, S.; Fernández-Bolaños, J.G. New selenosteroids as antiproliferative agents. Org. Biomol. Chem.
2017, 15, 5041–5054. [CrossRef]
15. Cui, J.G.; Qi, B.B.; Gan, C.F. Synthesis and in vitro antiproliferative evaluation of some B-norcholesteryl
benzimidazole and benzothiazole derivatives. Mar. Drugs 2015, 13, 2488–2504. [CrossRef]
16. Affeldt, R.F.; Santos, F.P.; da Silva, R.S.; Rodrigues, O.E.D.; Wessjohann, L.A.; Lüdtke, D.S. Stereoselective
glycoconjugation of steroids with selenocarbohydrates. RSC Advances 2016, 6, 93905–93914. [CrossRef]
17. Rodrigues, O.E.D.; de Souza, D.; Soares, L.C.; Dornelles, L.; Burrow, R.A.; Appelt, H.R.; Alves, C.F.; Alves, D.;
Braga, A.L. Stereoselective synthesis of selenosteroids. Tetrahedron Letters 2010, 51, 2237–2240. [CrossRef]
18. Santi, C.; Capoccia, L.; Monti, B. Zinc-Selenium reagents in organic synthesis. Phys. Sci. Rev. 2018, 3.
Available online: https://www.degruyter.com/view/j/psr.2018.3.issue-12/psr-2017-0129/psr-2017-0129.xml
(accessed on 11 August 2018). [CrossRef]
19. Santi, C.; Santoro, S.; Battistelli, B.; Testaferri, L.; Tiecco, M. Preparation of the First Bench-Stable Phenyl
Selenolate: An Interesting “On Water” Nucleophilic Reagent. Eur. J. Org. Chem. 2008, 2008, 5387–5390.
[CrossRef]
20. Perin, G.; Alves, D.; Jacob, R.G.; Barcellos, A.M.; Soares, L.K.; Lenardão, E.J. Synthesis of Organochalcogen
Compounds using Non-Conventional Reaction Media. ChemistrySelect 2016, 1, 205–258. [CrossRef]
21. Santi, C.; Battistelli, B.; Testaferri, L.; Tiecco, M. On water preparation of phenylselenoesters. Green Chemistry
2012, 14, 1277. [CrossRef]
22. Battistelli, B.; Lorenzo, T.; Tiecco, M.; Santi, C. “On-Water” Michael-Type Addition Reactions Promoted by
PhSeZnCl. Eur. J. Org. Chem. 2011, 2011, 1848–1851. [CrossRef]
23. Santoro, S.; Battistelli, B.; Testaferri, L.; Tiecco, M.; Santi, C. Vinylic Substitutions Promoted by PhSeZnCl:
Synthetic and Theoretical Investigations. Eur. J. Org. Chem. 2009, 2009, 4921–4925. [CrossRef]
24. Salman, S.; Schwab, R.; Alberto, E.; Vargas, J.; Dornelles, L.; Rodrigues, O.; Braga, A. Efficient Ring Opening
of Protected and Unprotected Aziridines Promoted by Stable Zinc Selenolate in Ionic Liquid. Synlett 2011
,
2011, 69–72. [CrossRef]
25. Nagasawa, T.; Shimada, N.; Torihata, M.; Kuwahara, S. Enantioselective total synthesis of idesolide via
NaHCO3-promoted dimerization. Tetrahedron 2010, 66, 4965–4969. [CrossRef]
26. Jiang, H.; Pan, X.; Li, N.; Zhang, Z.; Zhu, J.; Zhu, X. Selenide-containing high refractive index polymer
material with adjustable refractive index and Abbe’s number. React. Funct. Polym. 2017, 111, 1–6. [CrossRef]
27. Paillasse, M.R.; Saffon, N.; Gornitzka, H.; Silvente-Poirot, S.; Poirot, M.; de Medina, P. Surprising unreactivity
of cholesterol-5,6-epoxides towards nucleophiles. J. Lipid Res. 2012, 53, 718–725. [CrossRef]
28. Jastrzebska, I. Chemistry of Steroidal Sapogenins-New Advances in a Classical Field. Curr. Org. Chem. 2012
16, 353–372. [CrossRef]
,
29. Petrow, V.A. 212. Steroids and related compounds. Part III. The constitution of Westphalen’s diol. J. Chem.
Soc. (Resumed) 1939, 998. [CrossRef]
30. Kasal, A.; Budesinsky, M.; Griffiths, W.J. Spectroscopic Methods of Steroid Analysis. In Steroid Analysis,
2nd ed.; Makin, H.L.J., Gower, D.B., Eds.; Springer: London, UK, 2010; pp. 62–106.
31. Fürst, A.; Plattner, P.A. 2α, 3α-und 2β, 3β-Oxido-chlolestane; Konfiguration der 2-Oxy-cholestane. Helvetica
Chimica Acta 1949, 32, 275–283. [CrossRef] [PubMed]
32. Veesenmeyer, J.L.; Hauser, A.R.; Lisboa, T.; Rello, J. Pseudomonas aeruginosa virulence and therapy: Evolving
translational strategies. Crit. Care Med. 2009, 37, 1777–1786. [CrossRef] [PubMed]
33. Kadurugamuwa, J.L.; Sin, L.; Albert, E.; Yu, J.; Francis, K.; DeBoer, M.; Rubin, M.; Bellinger-Kawahara, C.;
Parr, T.R.; Contag, P.R. Direct Continuous Method for Monitoring Biofilm Infection in a Mouse Model.
Infect. Immun. 2003, 71, 882–890.
34. Epand, R.M.; Epand, R.F.; Savage, P.B. Ceragenins (Cationic Steroid Compounds), a novel class of antimicrobial
agents. Drug News Perspect. 2008, 21, 307. [CrossRef]
35. Mori, K.; Nakayama, T.; Sakuma, M. Synthesis of some analogues of blattellastanoside A, the steroidal
aggregation pheromone of the German cockroach. Bioorganic Med. Chem. 1996, 4, 401–408. [CrossRef]
36. Shawakfeh, K.Q.; Al-Said, N.H. Synthesis of new symmetrical bis-steroidal pyrazine analogues from
diosgenin. Steroids 2011, 76, 232–237. [CrossRef]
37. Weissenberg, M.; Lavie, D.; Glotter, E. Studies on epoxides—VI. Tetrahedron 1973, 29, 353–358. [CrossRef]

Int. J. Mol. Sci. 2019, 20, 2121
13 of 13
38. Weissenberg, M.; Glotter, E. Steric course of reduction with sodium borohydride of steroidal αβ-epoxy-ketones.
J. Chem. Soc. Perkin Trans. 1978, 1, 568–571. [CrossRef]
39. Niemirowicz, K.; Piktel, E.; Wilczewska, A.; Markiewicz, K.; Durnas´, B.; Wa˛tek, M.; Puszkarz, I.;
Wróblewska, M.; Niklin´ska, W.; Savage, P.; et al. Core-shell magnetic nanoparticles display synergistic
antibacterial effects against Pseudomonas aeruginosa and Staphylococcus aureus when combined with
cathelicidin LL-37 or selected ceragenins. Int. J. Nanomed. 2016, 11, 5443–5455. [CrossRef]
©
2019 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
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).