Synthesis, antimicrobial activity and cytotoxicity of triphenylphosphonium (TPP) conjugates of 1,2,3-triazolyl nucleoside analogues

Article abstract of DOI:10.1016/j.bioorg.2021.105328

Four new triphenylphosphonium (TPP) conjugates of 1,2,3-triazolyl nucleoside analogues were synthesized by coupling with 8-bromoctyl- or 10- bromdecyltriphenylphosphonium bromide and evaluated for the in vitro antibacterial activity against S. aureus, B.

Full text of DOI:10.1016/j.bioorg.2021.105328

Bioorganic Chemistry 116 (2021) 105328
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, antimicrobial activity and cytotoxicity of triphenylphosphonium
(TPP) conjugates of 1,2,3-triazolyl nucleoside analogues
Irina Yu. Strobykina, Alexandra D. Voloshina*, Olga V. Andreeva, Anastasiia S. Sapunova,
Anna P. Lyubina, Syumbelya K. Amerhanova, Mayya G. Belenok, Liliya F. Saifina,
Vyacheslav E. Semenov, Vladimir E. Kataev
Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center, Russian Academy of Sciences, Arbuzov str. 8, Kazan 420088, Russian Federation
A R T I C L E I N F O
A B S T R A C T
Keyword:
Four new triphenylphosphonium (TPP) conjugates of 1,2,3-triazolyl nucleoside analogues were synthesized by
coupling with 8-bromoctyl- or 10- bromdecyltriphenylphosphonium bromide and evaluated for the in vitro
antibacterial activity against S. aureus, B. cereus, E. faecalis, two MRSA strains isolated from patients and resistant
to fluoroquinolone antibiotic ciprofloxacin and β-lactam antibiotic amoxicillin, E. coli, antifungal activity against
T. mentagrophytes C. albicans and cytotoxicity against human cancer cell lines Mꢀ HeLa, MCF-7, A549, HuTu-80,
PC3, PANC-1 and normal cell line Wi-38. In these compounds a TPP cation was attached via an octyl or a decyl
linker to the N 3 atom of the heterocycle moiety (thymine, 6-methyluracil, quinazoline-2,4-dione) which was
bonded with 2′,3′,5′-tri- O - acetyl-greek beta-D-ribofuranose residue by the (1,2,3-triazol-4-il)methyl bridge. All
synthesized compounds showed high antibacterial activity against S. aureus within the range of MIC values
1.2–4.3 greek muM, and three of them appeared to be bactericidal with respect to tis bacterium at MBC values
4.1–4.3 greek muM. Two lead compounds showed both high antibacterial activity against the MRSA strains
resistant to Ciprofloxacin and Amoxicillin within the range of MIC values 1.0–4.3 greek muM and high cyto-
toxicity against human cancer cell lines HuTu-80 and MCF-7 within the range of IC50 values 6.4–10.2 greek
muM. This is one of the few examples when phosphonium salts exhibited both antibacterial activity and cyto-
toxicity against human cancer cell lines. According to the results obtained the bactericidal effect of the lead
compounds, unlike classical surfactants, was not caused by a violation of the integrity of the cytoplasmic
membrane of bacteria and their cytotoxic activity is most likely associated both with the induction of apoptosis
along the mitochondrial pathway and the arrest of the cell cycle in the G0/G1 phase.
Phosphonium salts
TPP-conjugates
Nucleoside analogues
Click chemistry
Antimicrobial activity
Cytotoxicity
1. Introduction
the length of the alkyl chain and with an increase in the number of
phosphonium groups [12,13,15,16]. As in the case of cytostatics,
Based on the facts that bulky lipophilic cations penetrate the mito-
chondrial membrane [1,2] and the mitochondria of cancer cells have a
more negative transmembrane potential compared to normal cells, a
triphenylphosphonium (TPP) cation is widely used for the targeted de-
livery of antioxidants and cytostatics to the mitochondria of cancer cells
[2–7]. The conjugation of such known drugs as doxorubicin, cisplatin,
chlorambucil, camptothecin and others with a TPP cation has provided
an increase in their cytotoxicity [5,8–10]. Besides, as a rule, phospho-
nium salts (especially TPP salts) with one, two or three alkyl chains
exhibit high antimicrobial activity, including against bacteria of the
ESKAPE group [11–15]. The activity increases both with an increase in
conjugation of known antibacterial agents with a TPP cation via an alkyl
linker leads to an increase in antibacterial activity [14]. Previously, the
mechanism of the antibacterial action of TPP salts was explained by
their destructive integration into the membranes of pathogens [17–19].
However, evidence has recently been received that TPP-conjugates do
not alter membrane permeability in some bacteria [20].
Since a TPP cation is capable of targeted penetration both into the
mitochondria of cancer cells and into the membranes of bacteria, it can
be assumed that TPP-conjugates should have dual activity - cytotoxic
against cancer cells and antibacterial. The literature has provided just a
few examples of such phenomenon. For example, the most active
* Corresponding author.
E-mail address: sobaka-1968@mail.ru (A.D. Voloshina).
https://doi.org/10.1016/j.bioorg.2021.105328
Received 10 June 2021; Received in revised form 28 August 2021; Accepted 31 August 2021
Available online 3 September 2021
0045-2068/© 2021 Published by Elsevier Inc.

I. Yu. Strobykina et al.
Bioorganic Chemistry 116 (2021) 105328
representatives of the series of 2-hydroxybenzylphosphonium salts
exhibited both antimicrobial activity at the micromolar level against
gram-positive bacteria S. aureus, B. cereus and fungi T.mentagrophytes
and C. ablicans and selective cytotoxicity against human cancer cell line
Mꢀ HeLa at the doxorubicin level, inducing mitochondrial apoptosis
[20].
cytotoxicity and antibacterial activity of TPP-conjugates of 1,2,3-tria-
zolyl nucleoside analogues with a wide range of alkyl chain lengths
and both with acylated and unprotected hydroxyl groups of the sugar
residue will soon follow this preliminary short communication.
2. Results and discussions
Recently, our group reported on the synthesis and cytotoxicity
evaluation of the first TPP-conjugates of uracil, thymine and 1,2,3-tria-
zolyl analogues of pyrimidine nucleosides in which the TPP cation was
attached to the N3 atom of the pyrimidine moiety by a butyl linker [21].
In this short communication, we report on the first TPP-conjugates of
1,2,3-triazolyl nucleoside analogues 1c, d; 2c; 4c possessing the TPP
cation attached to the N3 atom of the pyrimidine moiety by an octyl or
decyl linker which showed both high cytotoxicity against human cancer
cell lines HuTu-80, MCF-7 and high antibacterial activity against MRSA
strains resistant to ciprafloxacin and amoxicillin. The length of the
linkers was chosen in accordance with the data that conjugation with a
TPP cation by polymethylene linkers with n = 8, 10 or more provides
2.1. Chemistry
The target compounds 1c, d; 2c; 4c were synthesized in 2 stages. At
the first stage N1-propargyl derivatives of thymine 1a, 6-methyluracil
2a, quinazoline-2,4-dione 4a and azide 3 prepared by the methods
previously described [22] were involved in the Cu alkyne-azide cyclo-
addition (CuAAC) reaction which afforded 1,2,3-triazolyl analogues of
pyrimidine nucleosides 1b, 2b, 4b in very good yields (92%, 94%, and
94%, respectively). The spectral characteristics of 1b, 2b, 4b corre-
sponded to the literature ones [22] (Scheme 1). At the second stage
1,2,3-triazolyl nucleoside analogues 1b, 2b, 4b were reacted with 8-bro-
mooctyl- and 10-bromodecyltriphenylphosphonium bromides 5a and
higher antibacterial activity [15,19].
A full-scale study of the
Scheme 1. Synthesis of the target TPP-conjugates of 1,2,3-triazolyl nucleoside analogues 1c, 2c, 1d, 4c.
2

I. Yu. Strobykina et al.
Bioorganic Chemistry 116 (2021) 105328
5b obtained by heating 1,8-dibromoctane and 1,10-dibromodecane with
triphenylphosphine without solvent at an oil bath temperature of 90 ◦C
for 6 h similarly to the known procedure [23,24]. Target TPP-conjugates
of 1,2,3-triazolyl nucleoside analogues 1c, 2c, 1d, 4c were obtained in
36%, 15%, 26%, 31% yields, respectively (Scheme 1). In the last 10
years, so-called prodrug forms of nucleoside analogues with protected
polar groups were being synthesized to ensure better penetration into
cells through lipid-rich cell membranes [25]. That is why, in this study
we did not remove the acetyl protection of the hydroxyl groups of 1,2,3-
triazolyl nucleoside analogues 1c, 2c, 1d, 4c.
strain was resistant to fluoroquinolone antibiotic ciprofloxacin and
β-lactam antibiotic amoxicillin. The MRSA-2 strain was resistant only to
amoxicillin. The resulting data expressed as minimal inhibitory con-
centration (MIC), minimal bactericidal concentration (MBC), and min-
imal fungicidal concentration (MFC) are presented in Table 1.
It can be seen that the TPP conjugates showed high selectivity for the
Gram-positive bacteria used, including the MRSA strains, showing very
low MIC values (1.0–4.3 μM). The lead compounds 1d and 4c demon-
strated high antibacterial activity against Gram-positive bacteria Sa, Bc,
MRSA-1, MRSA-2 (MIC = 1.0–2.0 μM) which exceeded the activity of
Procedures for the synthesis of 1c, 2c, 1d, 4c, their characterizations
including NMR spectra are presented in Supplementary data.
the reference compound – drug norfloxacin by 12 times in the case of Bc
and 250 times in the case of the resistant strain MRSA-1 (Table 1). TPP-
conjugate 1c also showed high antibacterial activity against Gram-
positive bacteria Sa, Ef, MRSA-2 (MIC = 2.1, 8.5, 4.3
μM, respec-
2.2. Biology
tively) which is comparable to the activity of the reference compound
norfloxacin (MIC = 7.5 μM). As for the resistant strain MRSA-1, 1c
2.2.1. Antimicrobial activity
inhibited its growth at the concentration 63 times lower than nor-
floxacin (Table 1). It should be noted that TPP-conjugates 1c and 1d
exhibited bactericidal action against Sa, MRSA-1, MRSA-2, and their
MIC and MBC values did not differ by >4 times. TPP-conjugate 2c was
Synthesized TPP-conjugates 1c, 2c, 1d, 4c were evaluated for their in
vitro antibacterial activity against Gram-positive bacteria Staphylococcus
aureus (Sa), Bacillus cereus (Bc), Enterococcus faecalis (Ef), MRSA-1 and
MRSA-2 strains isolated from patients of the Republic Clinical Hospital
(Kazan, Russian Federation), Gram-negative bacteria Escherichia coli
(Ec) and in vitro antifungal activity against Candida albicans (Ca) and
Trichophyton mentagrophytes var. gypseum (Tm). Both methicillin-
resistant S. aureus strains possessed high drug resistance. The MRSA-1
bactericidal only against Sa at MBC value of 4.3 μM (Table 1).
As it was reported previously, the antibacterial effect of quaternized
ammonium compounds and phosphonium salts is associated with
disruption of the bacterial cell membrane [17–19]. To examine whether
Table 1
In vitro antimicrobial activity of TPP-conjugates of 1,2,3-triazolyl nucleoside analogues 1c,2c,1d,4c.
Minimal inhibitory concentration (MIC, μM)
Compound
Structure
aSa
bBc
cEf
dMRSA-1
eMRSA-2
fEc
gTm
hCa
1c
2.1 ± 0.1
17.0 ± 1.3
8.5 ± 0.7
4.3 ± 0.3
4.3 ± 0.3
136 ± 12
na
na
2c
1d
4.3 ± 0.3
34 ± 2.3
272 ± 22
8.3 ± 0.6
17 ± 1.4
8.5 ± 0.7
272 ± 20
132 ± 10
na
na
na
na
1.0 0.08
2.0 0.1
1.0 0.08
1.0 0.06
4c
1.0 0.09
2.0 0.1
8.2 ± 0.7
1.0 0.06
1.0 0.07
na
na
na
Norfloxacin
Ketoconazol
7.5 ± 0.5
24.4 ± 2.1
7.5 ± 0.5
250 ± 21
7.5 ± 0.5
4.7 ± 0.02
na
na
nd
nd
nd
nd
nd
nd
7.3 ± 0.5
7.3 ± 0.5
Minimal bactericidal concentration (MBC,
μ
M)
Minimal fungicidal
concentration (MFC,
μ
M)
1c
4.3 0.3
34.0 ± 2.6
68.0 ± 5.7
4.3 0.3
4.3 0.3
272 ± 21
na
na
2c
1d
4.3 0.3
4.1 0.3
68.0 ± 5.5
na
17.0 ± 1.4
17.0 ± 1.3
na
na
na
na
na
na
33.0 ± 2.6
2.0 0.1
4.1 0.2
132 ± 11
4c
16.4 ± 1.3
131 ± 21
131 ± 19
1.0 0.08
1.0 0.07
131 ± 12
na
na
Norfloxacin
Ketoconazol
7.5 ± 0.6
24.4 ± 2.1
7.5 ± 0.6
nd
nd
7.5 ± 0.5
24.4 ± 2.1
nd
nd
nd
nd
nd
nd
nd
7.3 ± 0.5
7.3 ± 0.5
aSa - Staphylococcus aureus; bBc - Bacillus cereus; cEf - Enterococcus faecalis; dMRSA-1 – methicillin-resistant S. aureus resistant to fluoroquinolones and β-lactam anti-
biotics; eMRSA-2 - methicillin-resistant S. aureus resistant to β-lactam antibiotics; hEc - Escherichia coli; gTm - Trichophyton mentagrophytes; hCa - Candida albicans; na -
non active; nd - not determined. The experiments were repeated 3 times.
3

I. Yu. Strobykina et al.
Bioorganic Chemistry 116 (2021) 105328
TPP-conjugates 1c, 2c, 1d, 4c can alter the integrity of the cell mem-
brane of S. aureus or not, its integrity has been verified by measuring the
absorption of hydrophobic crystal violet (CV) dye. The data obtained
(Supplementary data) allow to conclude that the mechanism of the
antibacterial effect of TPP-conjugates 1c, 2c, 1d, 4c differs from that of
classical surfactants [17–19], that is, it is not associated with a violation
of the integrity of the cytoplasmic membrane of bacteria.
some human cancer cell lines and moderate cytotoxicity against normal
Wi-38 cells of the lung embryo. The most significant results were ob-
tained for TPP-conjugates 1c, 4c and 1d. The first two compounds
showed selective cytotoxicity against HuTu-80 duodenal adenocarci-
noma cell line (IC50 = 8.3, 10.2
adenocarcinoma cell line (IC50 = 10.2, 7.3
compound 1d showed high selective cytotoxicity against HuTu-80 cell
line (IC50 = 6.4 M, SI > 16, Table 2, Table 3). Thus, TPP-conjugates 1c,
μ
M, respectively) and MCF-7 breast
μM, respectively). The lead
To assess the effect of TPP-conjugates 1c, 2c, 1d, 4c on the S. aureus
plasma membrane we used Molecular Probe LIVE/DEAD® BacLightTM
Bacterial Viability Kit method which is widely used for the qualitative
and quantitative analysis of viable and damaged cells after exposure to
antimicrobial agents [26]. The data obtained (Supplementary data)
confirmed the results of the absorption of crystal violet (CV) dye,
namely, TPP-conjugates 1c, 2c, 1d, 4c within the range of their MIC and
μ
1d, 4c showed cytotoxicity against HuTu-80 cell line which corre-
sponded to the cytotoxicity of the reference drug doxorubicin and
exceeded the cytotoxicity of the reference compound – drug fluorouracil
against the same cancer cell lines by 7–10 times. It should be emphasized
that the selectivity index of the lead compound 1d (SI > 16) is 23 times
higher than the selectivity index of fluorouracil (SI = 0.7) and 40 times
higher than the selectivity index of doxorubicin (SI = 0.4) (Table 3).
And, besides, TPP-conjugates 1c and 4c showed cytotoxicity against
MCF-7 cell line which corresponded to the cytotoxicity of the reference
drug doxorubicin and surpassed the cytotoxicity of the reference drug
tamoxifen against the same cancer cell line by 2–3 times (Table 2).
In addition to the already mentioned high selective cytotoxicity of
TPP-conjugates 1c, 1d, 4c, the analysis of Table 2 leads to two more
important conclusions. First, TPP-conjugate 2c with the 6-methyluracil
moiety was significantly less active against the used human cancer cell
lines than TPP-conjugates 1c, 1d with the thymine moiety and TPP-
conjugate 4c with the quinazoline-2,4-dione moiety. Second, the in-
crease in the length of the polymethylene chain linking the TPP cation
with the N3 atom of the heterocyclic moiety of TPP-conjugates of 1,2,3-
triazolyl nucleoside analogues is accompanied by a significant increase
in their cytotoxicity. This can be seen by comparing the cytotoxicity of
TPP-conjugates 1e, 1c, 1d against cancer cell lines Mꢀ HeLa, PC3,
PANC-1 presented in Table 2. In the series of compounds 1e, 1c, 1d with
the increase in the length of the polymethylene chain (n = 4, 8, 10,
MBC values (1.0–16.4 μM) have no significant effect on the permeability
of the cytoplasmic membrane of S. aureus. So, we can confidently sup-
pose that the mechanism of bacteriostatic and bactericidal action of TPP-
conjugates 1c, 2c, 1d, 4c on S. aureus is not associated with damage to
the bacterial cell wall and cytoplasmic membrane.
2.2.2. Cytotoxicity
TPP-conjugates of 1,2,3-triazolyl nucleoside analogues 1c, 2c, 1d, 4c
were also evaluated for in vitro cytotoxicity against six human cancer cell
lines: Mꢀ HeLa cervical epitheloid carcinoma, MCF-7 breast adenocar-
cinoma, A549 pulmonary adenocarcinoma, HuTu-80 duodenal adeno-
carcinoma, PC3 prostate adenocarcinoma, PANC-1 pancreatic
carcinoma as well as a diploid human cell strain WI-38 composed of
fibroblasts. The resulting data expressed as concentrations causing the
inhibition of the growth of 50% of cells in the experimental population
(IC50) and selectivity index (SI) which is the ratio IC50(normal cell)/
IC50(cancer cell) are presented in Table 2 and Table 3. As one can see in
Table 2, the tested TPP-conjugates demonstrated high activity against
Table 2
In vitro cytotoxicity of synthesized TPP-conjugates of 1,2,3-triazolyl nucleoside analogues against human cancer and human normal cell lines (IC50 values in μM with
standard errors).
Compound
Structure
Cancer cell lines
Normal
cell line
aMꢀ HeLa
81 ± 6.2h
bMCF-7
>100h
cA549
nd
dHuTu-80
nd
ePC3
>100h
fPANC-1
>100h
gWi-38
>250h
1e
1c
1d
50.2 ± 4.5
19.7 ± 1.6
10.2 0.8
57.9 ± 4.5
39.9 ± 2.7
8.3 0.6
6.4 0.4
>100
>100
55.5 ± 4.8
>100
48.7 ± 3.8
30.8 ± 2.3
60 ± 5.4
2c
4d
73.2 ± 6.3
>100h
55.4 ± 4.6
>100h
>100
35.7 ± 2.9
>100
>100
54.5 ± 4.6
>100h
nd
nd
>100h
>100h
4c
22.8 ± 1.9
7.3 0.5
>100
10.2 0.9
25.7 ± 1.7
27.5 ± 1.9
54.2 ± 4.3
Tamoxifen
28 ± 2.5
nd
25 ± 2.2
nd
nd
nd
nd
nd
45 ± 3.8
48 ± 4.1
1.3 ± 0.1
Fluorouracil
Doxorubicin
42.5 ± 3.6
65.2 ± 5.5
43 ± 3.6
39.3 ± 2.7
3.0 ± 0.2
3.0 ± 0.1
3.0 ± 0.2
3.0 ± 0.1
3.0 ± 0.1
7.0 ± 0.6
aMꢀ Hela is a human cervix epitheloid carcinoma; bMCF-7 is a human breast adenocarcinoma (pleural fluid); cA549 is a human lung carcinoma; dHuTu-80 is a human
duodenal adenocarcinoma; ePC3 is a human prostate adenocarcinoma; fPANC-1 is a human pancreatic carcinoma; gWi-38 is a diploid human embryo lung; hpreviously
published data [21]; ns no selectivity; nd not determined; the experiments were repeated 3 times.
4

I. Yu. Strobykina et al.
Bioorganic Chemistry 116 (2021) 105328
Table 3
Selectivity indices of TPP-conjugates 1c, 1d, 2c, 4c synthesized in this work and TPP-conjugates 1e, 4d previously published [21].
Compound
Structure
Cancer cell lines
aM-HeLa
>3g
bMCF-7
nsg
cA549
nd
dHuTu-80
nd
ePC3
nsg
fPANC-1
nsg
1e
1c
1d
1.1
5.4
0.9
6.7
ns
ns
>5
>2
>3
>16
>3
>1.7
2c
4d
0.7
nsg
1.0
nsg
ns
1.5
nd
ns
ns
nd
nsg
nsg
4c
2.4
7.4
ns
5.3
2.1
2.0
Tamoxifen
1.6
nd
1.8
nd
nd
nd
nd
nd
Fluorouracil
1.1
0.7
1.1
1.2
Doxorubicin
0.4
0.4
0.4
0.4
0.4
0.2
aM-Hela is a human cervix epitheloid carcinoma; bMCF-7 is a human breast adenocarcinoma (pleural fluid); cA549 is a adenocarcinomic human alveolar basal epi-
thelial cell line; dHuTu-80 is a duodenal adenocarcinoma; ePC3 is a human prostate adenocarcinoma; fPANC-1 is a human pancreatic cancer cell line isolated from a
pancreatic carcinoma of ductal cell origin; gpreviously published data [21]; ns no selectivity; nd not determined.
respectively), the IC50 values decrease (Table 2). So, if TPP-conjugate 1e
in which the TPP cation is attached to the N3 atom of the thymine
moiety by the butyl chain was inactive against PC3 and PANC-1 cancer
specific ligands bind to death receptors on the cell membrane surface,
belonging to the superfamily of tumor necrosis factor receptor (TNFR),
with their respective protein TNF family ligands [27]. In the case of
mitochondrial apoptosis, cell death occurs as a result of irreparable
damage to DNA. For this reason, the cell starts an internal apoptotic
cascade. The internal pathway of apoptosis induction is accompanied by
the destruction of the mitochondrial membrane, which leads to a
decrease in its potential, which is a key indicator of the state of cells
[28]. The results obtained with the help of flow cytometry using JC-10
fluorescent dye from the Mitochondria Membrane Potential Kit showed
that the lead compound 1d induced apoptosis along the mitochondrial
pathway (Supplementary data).
cell lines and showed moderate cytotoxicity (IC50 = 81 μM) against
Mꢀ HeLa, then TPP-conjugate 1d with decyl chain between the TPP
cation and the N3 atom of the thymine moiety demonstrated moderate
to high cytotoxicity against these cancer cell lines with IC50 values of
30.8, 60, and 19.7
μM, respectively (Table 2). Similarly, if TPP-
conjugate 4d in which the TPP cation is attached to the N3 atom of
the quinazoline-2,4-dione moiety via the butyl chain was inactive
against Mꢀ HeLa, MCF-7, PC3, and PANC-1 cancer cell lines, then TPP-
conjugate 4c possessing the decyl chain between the TPP cation and the
quinazoline-2,4-dione moiety showed moderate cytotoxicity against
The mechanism of action of cytotoxic agents, as a rule, is associated
with a violation of the passage of cells of the cell cycle, leading to syn-
chronization and slowing down of proliferation of rapidly multiplying
cells. Cell cycle analysis by quantifying the DNA content of a cell is a
reliable method to assess at which phase the cell cycle has been stopped.
We have carried out such investigation using fluorescent dye propidium
iodide, which binds in proportion to the amount of DNA present in the
cell. The results of the analysis of the cell cycle of a cell line HuTu-80
after its treatment with the lead compound 1d for 24 h (Supplemen-
tary data) showed that the mechanism of its action is most likely asso-
ciated not only with the induction of apoptosis along the mitochondrial
pathway but also with the arrest of the cell cycle in the G0/G1 phase.
these cancer cell lines with IC50 values of 22.8, 7.3, 25.7, and 27.5 μM,
respectively (Table 2). So, the lengthening of the polymethylene linker
between the TPP cation and the pyrimidine moiety in the studied TPP-
conjugates considerably increases not only antimicrobial activity but
also cytotoxicity against some human cancer cell lines.
Currently, apoptosis is one of the key mechanisms used in the
development of new anticancer agents. In this regard, it was of consid-
erable interest to study the ability of the lead compound 1d to induce
apoptosis in HuTu-80 cells. According to the data of flow cytometry, it
can be seen that as a result of 24 h incubation of HuTu-80 cells in the
presence of 1d, a dose-dependent induction of the apoptosis process is
observed (Fig. 1, Fig. 2). Moreover, apoptotic effects were manifested
only at the stage of early apoptosis.
3. Conclusion
There are two mechanisms for the induction of apoptosis: the
external apoptotic pathway through death receptors and the internal
apoptotic pathway (mitochondria-dependent apoptosis). The external
pathway triggers apoptosis in response to extrinsic stimuli, during which
Four new triphenylphosphonium (TPP) conjugates of 1,2,3-triazolyl
nucleoside analogues were synthesized and evaluated for the in vitro
antibacterial activity against S. aureus, B. cereus, E. faecalis, two MRSA
5

I. Yu. Strobykina et al.
Bioorganic Chemistry 116 (2021) 105328
Fig. 1. Apoptosis induction in HuTu-80 cells incubated with 5 µM and 10 µM of compound 1d for 24 h. Quantification of dot plots expressed as percentage of total
cells (mean ± SD (n = 3); (*) P < 0.01 compared to control).
TPP-conjugates of 1,2,3-triazolyl nucleoside analogues with a wide
range of alkyl chain lengths and both with acylated and unprotected
hydroxyl groups of the sugar residue will soon follow this preliminary
short communication.
Funding
The biological part of the work and the synthesis of the TPP-
conjugates of 1,2,3-triazolyl nucleoside analogues were financial sup-
ported by the Russian Science Foundation (grant no. 19–13-00003).
Pyrimidine derivatives bearing ω-alkyne substituents at the N-1 atom as
well as 2,3,5-tri-O-acetyl-β-D-ribofuranosyl azide were synthesized
under financial support from the government assignment for FRC Kazan
Scientific Center of RAS.
Fig. 2. Representative histograms for the number of cells (% of the total) at the
early and late stages of apoptosis for the control and experimental groups after
treatment with 1d. The values are presented as the mean ± SD (n = 3); (*) P <
0.01 compared to control.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
strains isolated from patients and resistant to fluoroquinolone antibiotic
ciprofloxacin and β-lactam antibiotic amoxicillin, E. coli, antifungal ac-
tivity against T. mentagrophytes, C. albicans and cytotoxicity against
human cancer cell lines Mꢀ HeLa, MCF-7, A549, HuTu-80, PC3, PANC-1
and normal cell line Wi-38. All synthesized compounds showed high
antibacterial activity against S. aureus within the range of MIC values
Acknowledgements
The authors are grateful to the Assigned Spectral-Analytical Center of
FRC Kazan Scientific Center of RAS for technical assistance in research.
1.2–4.3
μ
M, and three of them appeared to be bactericidal with respect
to this bacterium at MBC values of 4.1–4.3
μM. Moreover, TPP-
conjugates 1c and 1d in which the TPP cation was attached to the N3
atom of the thymine moiety via an octyl (or a decyl) linker respevtively
and TPP-conjugate 4c in which the TPP cation was attached to the N3
atom of quinazoline-2,4-moiety via an octyl linker showed both high
antibacterial activity against the MRSA strains resistant to ciprofloxacin
Appendix A. Supplementary material
This Appendix includes the data about the examination of the ability
of TPP-conjugates 1c, 2c, 1d, 4c to alter the integrity of the cell mem-
brane of S. aureus; the data about the qualitative and quantitative
analysis of viable and damaged cells S. aureus after exposure to TPP-
conjugates 1c, d; 2c, 4c; the data about the estimation of the possibil-
ity of the lead compound 1d to induce apoptosis in a cell line HuTu-80
along the mitochondrial pathway; the data about the analysis of the cell
cycle of a cell line HuTu-80 after its treatment with the lead compound
1d; general procedures for the synthesis of TPP-conjugates 1c, 2c, 1d,
4c and theit characterizations including 1H, 13C and 31P spectra; detailes
of all biological experiments. Supplementary data to this article can be
found online at https://doi.org/10.1016/j.bioorg.2021.105328.
and amoxicillin within the range of MIC values 1.0–4.3
cytotoxicity against human cancer cell lines HuTu-80 and MCF-7 within
the range of IC50 values 6.4–10.2 M. This is one of the few examples
μM and high
μ
when phosphonium salts exhibited both antibacterial activity and
cytotoxicity against human cancer cell lines. According to the results of
crystal violet dye assay and LIVE/DEAD BacLightTM Bacterial Viability
Method the synthesized TPP-conjugates within the range of their MIC
and MBC values, unlike classical surfuctants, do not effect the integrity
of the cytoplasmic membrane of S. aureus. Therefore, one can suppose
that the mechanism of antibacterial action of these TPP-conjugates is
significantly different from that of classical antibacterial agents i.e. is
not associated with damage to the bacterial cell wall and cytoplasmic
membrane. It was found that cytotoxic activity of the lead compound 1d
is due both to induction of apoptosis proceeding along the mitochondrial
pathway and the arrest of the cell cycle in the G0/G1 phase.
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