Synthesis and in vitro evaluation of triphenylphosphonium derivatives of acetylsalicylic and salicylic acids: structure-dependent interactions with cancer cells, bacteria, and mitochondria

Article abstract of DOI:10.1007/s00044-020-02674-6

Salicylic acid (SA) remains one of the most fruitful natural compounds to generate drug molecules with versatile activities. In this study, effective synthesis of SA and acetylsalicylic acid (ASA) derivatives with a carrier triphenylphoshonium (TPP) group was proposed. A series of SA and ASA conjugates linked with the TPP group via alkyl chain linker (C₃-C₁₀) was synthesized. The conjugates showed enhanced TPP-mediated cytotoxicity towards MCF-7, Caco-2, PC-3 cells in proportion to the linker length. 7e, 8e (C₉), and 7f (C₁₀) were the most active against the cancer cells with IC₅₀ = 0.6–1.9 μM while were less toxic for HSF. Similarly, antibacterial (bactericidal) activity of the compounds against S. aureus increased with the linker elongation. The lowest MIC for SA and ASA derivatives were 4 and 1 μM, respectively. The TPP conjugates induced early linker length-dependent mitochondria depolarization and concurrent superoxide radical production in the cancer cells. The most lipophilic conjugates were found to specifically interact with ROS probe 2′,7′-dichlorofluorescin diacetate, forming mixed aggregates with the probe and inhibiting its fluorescence upon oxidation. These interactions were exploited to probe the compounds inside living cells. The results identify 7e and 7f as promising mitochondria-modulating and anticancer agents with increased cellular availability. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-020-02674-6

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:925–939
https://doi.org/10.1007/s00044-020-02674-6
ORIGINAL RESEARCH
Synthesis and in vitro evaluation of triphenylphosphonium
derivatives of acetylsalicylic and salicylic acids: structure-dependent
interactions with cancer cells, bacteria, and mitochondria
Olga V. Tsepaeva1 Taliya I. Salikhova2 Leysan R. Grigor’eva3 Denis V. Ponomaryov3 Trinh Dang2
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Rezeda A. Ishkaeva2 Timur I. Abdullin2 Andrey V. Nemtarev
Vladimir F. Mironov1,3
1,3
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Received: 9 September 2020 / Accepted: 21 November 2020 / Published online: 21 January 2021
© Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
Salicylic acid (SA) remains one of the most fruitful natural compounds to generate drug molecules with versatile activities.
In this study, effective synthesis of SA and acetylsalicylic acid (ASA) derivatives with a carrier triphenylphoshonium (TPP)
group was proposed. A series of SA and ASA conjugates linked with the TPP group via alkyl chain linker (C3-C10) was
synthesized. The conjugates showed enhanced TPP-mediated cytotoxicity towards MCF-7, Caco-2, PC-3 cells in proportion
to the linker length. 7e, 8e (C9), and 7f (C10) were the most active against the cancer cells with IC50 = 0.6–1.9 µM while were
less toxic for HSF. Similarly, antibacterial (bactericidal) activity of the compounds against S. aureus increased with the
linker elongation. The lowest MIC for SA and ASA derivatives were 4 and 1 µM, respectively. The TPP conjugates induced
early linker length-dependent mitochondria depolarization and concurrent superoxide radical production in the cancer cells.
The most lipophilic conjugates were found to specifically interact with ROS probe 2′,7′-dichlorofluorescin diacetate, forming
mixed aggregates with the probe and inhibiting its fluorescence upon oxidation. These interactions were exploited to probe
the compounds inside living cells. The results identify 7e and 7f as promising mitochondria-modulating and anticancer
agents with increased cellular availability.
Graphical Abstract
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Keywords Phosphonium salts Salicylic acid Acetylsalicylic acid Anticancer activity Anticancer activity Mitochondrial
potential
Introduction
These authors contributed equally: Olga V. Tsepaeva, Taliya I.
Salikhova
Polyhydroxybenzoates are widespread secondary metabo-
Supplementary information The online version of this article (https://
lites in plants with vital functions [1, 2]. Among them,
doi.org/10.1007/s00044-020-02674-6) contains supplementary
material, which is available to authorized users.
2
* Andrey V. Nemtarev
Institute of Fundamental Medicine and Biology, Kazan (Volga
a.nemtarev@mail.ru
Region) Federal University, 18 Kremlevskaya Str., 420008
Kazan, Russia
1
Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan
3
Alexander Butlerov Institute of Chemistry, Kazan (Volga Region)
Federal University, 18 Kremlevskaya Str., 420008 Kazan, Russia
Scientific Center of RAS, 8 Arbuzov Str., 420088 Kazan, Russia

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Medicinal Chemistry Research (2021) 30:925–939
Scheme 1 Synthesis of ω-bromoalkyl esters of acetylsalicylic (3a–f) and salicylic (4a–f) acids. Reagents and conditions: a K2CO3, CH3CN, Br
(CH2)nBr
ortho-hydroxybenzoic (salicylic) acid (SA) and its esters
(salicylates) are important medicinal compounds [2]. In
particular, acetylsalicylic acid (ASA, aspirin) introduced
over a century ago is still one of the most popular drugs
with versatile prescriptions, which cover cardiovascular,
inflammation and pain related diseases [3]. In recent years,
polyhydroxybenzoates such as SA and gallic acids and their
derivatives have attracted a great interest for preventing and
treating tumor diseases [4, 5].
powerful modifiers for small drug molecules (see reviews
and references within [24–27]). Acting as a delocalized
lipophilic cation, the TPP moiety effectively promotes cel-
lular and mitochondrial accumulation of the modified
compounds driven by the electrochemical gradient across
biomembranes. The conjugation with TPP cation has
proved to generate a range of bioactive molecules with
antibacterial [28, 29], antifungal [30, 31], cyto- and neu-
roprotective [32], anti-inflammatory [33, 34], and anticancer
[29, 35] properties.
Several preclinical and clinical trials of ASA supported
its antitumor potential [6, 7]. The mechanisms underlying
antitumor action of ASA interrelate with its anti-
inflammatory properties, which are generally result from
the inhibition of activity of cyclooxygenases [8, 9] along
with diminishing platelet activation and thrombosis
involved in the cancer progression and metastasis [10, 11].
Other reported mechanisms of ASA and its active meta-
bolite SA comprise the inhibition of IkB kinase/NF-kB
pathway in vitro and in vivo [12, 13], allosteric activation of
AMP-activated kinase [14], uncoupling of the respiratory
chain by transporting protons across the inner mitochondrial
membrane [15]. The latter activity inhibits oxidative phos-
phorylation, increases reactive oxygen species (ROS), thus
promoting apoptosis induction [16, 17]. In addition, ASA
was recently shown to induce the inhibition of acetyl-
transferase EP300, the suppressor of autophagy in cancer
cells [18].
In our group, the TPP derivatives of triterpenoids,
namely, betulin, betulinic, and betulonic acids were earlier
synthesized as highly-effective anticancer, antibacterial, and
mitochondria-modulating compounds [29, 35]. The TPP
group was shown to modulate cellular interactions and
protease resistance of YRFK motif-based peptides
depending on the peptide conformation and linker length;
TPP-C6-YrFK conjugate was highly resistant to enzymatic
degradation [32, 36].
Existing reports on the TPP derivatives of poly-
hydroxybenzoates deals with comparative in vitro evalua-
tion of decyl-TPP conjugated polyhydroxybenzoates, e.g.,
gallic, protocathechuic, salicylic, 2,3- and 2,5-dihydrox-
ybenzoic acids as mitochondria-targeted compounds against
breast cancer cells [27]. The alkyl gallate TPP derivatives
were shown to exhibit anticancer activity via increasing the
cytosolic ADP/ATP ratio, AMP level, and caspase-3
expression [37]. Furthermore, decyl-TPP gallate effec-
tively inhibited subcutaneous adenocarcinoma tumor in
mice when applied intraperitoneally in composition with
doxycycline due to combined interfering effect on mito-
chondria [37]. Some molecular mechanisms underlying the
inhibitory activity of decyl-TPP gallate on mitochondrial
Owing to their therapeutic uses, polyhydroxybenzoates
are considered important natural compounds to further
develop drug molecules with improved pharmacokinetics,
targeted properties, and new pharmacological activities [19–23].
To date, phosphonium salts and primarily triphenylpho-
sphonium (TPP) salt were identified as one of the most

Medicinal Chemistry Research (2021) 30:925–939
927
dihalogenalkane (up to 4–6 eq.), mixed acetonitrile/DMFA
solvent, and 12 h reaction at room temperature.
Synthesis of phosphonium derivatives of SA and
ASA
Phosphonium derivatives of SA and ASA were obtained by
the reaction of the esters 3, 4 with triphenylphosphine
(PPh3) in acetonitrile upon heating for 12–18 h (Scheme 2).
The reaction progression was controlled by means of TLC
and 31Р NMR detection of PPh3 (δР 5 ppm) and the resultant
TPP salts of SA and ASA (δР 24 ppm), which were char-
acterized by almost quantitative yield.
Scheme 2 Synthesis of phosphonium derivatives of acetylsalicylic
(7a–f) and salicylic (8a–e) acids. Reagents and conditions: (a) PPh3,
CH3CN, reflux
bioenergetics were recently elucidated [26]. In addition, the
TPP derivatives of 2,4-dihydroxybenzoate and salicylhydrox-
amate [38], 4-hydroxybenzoate and 4-alkoxybenzaldehyde
[39], and gallate with C8-C12 alkyl linkers were [40] synthe-
sized and proved as antitrypanosomal agents.
Biology
Cytotoxic activity of TPP conjugates of SA and ASA
Table 1 shows IC50 values of the TPP conjugates 7a-f, 8a-c,
8e for the cancer cell lines, namely, PC-3 (human prostate
adenocarcinoma), MCF-7 (human breast adenocarcinoma),
Caco-2 (human colon adenocarcinoma) cells as well as
“normal” human skin fibroblasts (HSF). The SA and ASA
derivatives exhibited similar to each other cytotoxicity
profile, which increased by ca. ten times with the elongation
of alkyl linker of the TPP group from C3 to C9. The com-
pounds 7e and 8e were the most active against the cancer
cells with IC50 values of 0.6–1.9 µM and 0.7–1.7 µM,
respectively. Further increase of the linker length to C10 for
the ASA derivative 7f did not augment the cytotoxic effect.
The ω-bromoalkyl derivative of ASA 3d was 7.2–7.6 times
less active against the cancer cells than the corresponding
TPP conjugate 7d, confirming the enhancing effect of the
TPP cation. All TPP conjugates were significantly less toxic
for HSF than for the cancer cells. For instance, such a
The current data, which are mainly focused on gallic acid
derivatives, do not provide structure–bioactivity relation-
ships for the conjugated derivatives of salicylates and TPP
groups. To the best of our knowledge, the ASA-TPP con-
jugates have not been synthesized and evaluated to date in
spite of therapeutic importance of ASA and its availability
as a pharmaceutical substance. Herein, the TPP derivatives
of SA and ASA with aliphatic linker of variable length
(C3–C10) were synthesized and their interactions with living
cells and mitochondria were for the first time compared.
The conjugates with enhanced anticancer and antibacterial
properties were identified. In addition, we revealed linker-
dependent interactions of the conjugates with 2′,7′-
dichlorofluorescin diacetate (DCFDA), which allowed to
probe the compounds in solution and inside cells.
Chemistry
Table 1 IC50 values (µM) of acetylsalicylic and salicylic acids
derivatives (MTT assay, 72 h)
Results and discussion
Compound
PC-3
MCF-7
Caco-2
HSF
ω-Bromoalkyl esters of SA and ASA were synthesized
according to the procedure based on the reaction with 1,ω-
dihalogenalkanes in acetonitrile or DMFA with an addition
of potash [41]. At increased temperatures the yield of
desired monoesters 3, 4 was relatively low, whereas side
diesters 5, 6 amounted up to 80% of the total esterification
products (Scheme 1). The diesters 5, 6 became prevailed
products upon 2–4 h reaction at 90–100 °С in DMFA.
Based on intense experimentation, optimal conditions for
the effective synthesis of the monoesters of SA and ASA
were found. They require the use of excess of 1,ω-
7a
7b
7c
7d
7e
7f
7.9 1.2
6.1 1.7
2.0 0.4
3.2 0.3
0.6 0.2
1.1 0.3
24.4 1.2
6.6 0.6
2.9 0.6
3.0 0.7
0.7 0.2
12.4 3.8
4.9 0.7
5.4 1.1
2.6 0.6
1.9 0.1
0.7 0.1
18.8 1.3
11.0 1.8
5.9 0.9
5.0 0.2
1.7 0.4
10.7 1.5
4.6 0.9
3.5 0.7
1.6 0.3
0.7 0.1
0.9 0.1
11.9 3.6
10.9 2.9
8.1 1.0
5.4 0.5
1.2 0.2
20.4 0.9
27.8 4.8
11.9 2.3
15.8 1.5
4.9 0.5
3.8 0.4
3d
8a
8b
8c
8e
31.0 1.4
21.0 1.2
14.4 2.0
10.4 1.1
1.5 0.2

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Medicinal Chemistry Research (2021) 30:925–939
Fig. 1 Representative
distribution of fluorescence of A
TMRE and B, C MitoSOX
probes in PC-3 cells treated with
ASA-TPP conjugates 3d, 7b,
7d, 7f (A, B) and ASA-TPP
conjugates plus antimycin A
(C). Cells were exposed to
30 µM conjugates and 10 µM
antimycin A for 1 h in HBSS.
Ctrl shows untreated cells (A, B)
and cells treated with antimycin
A (C)
selectivity factor was up to 8.2 for the ASA derivative 7e
(Table 1), when compared to PC-3 cells.
(n < 10), presumably, due to increase in their lipophilic
properties. Further increase of the linker length does not
seem rational due to solubility and nonspecific cytotoxicity
issues. Interestingly, in contrast to the above conjugates, the
TPP derivatives of triterpenoids with shorter linkers pos-
sessed higher activity in vitro [29], suggesting the role of
optimal hydrophilic–lipophilic properties for each type of
conjugates. The requirements for an optimal aliphatic linker
to display anticancer and antiparasite activities were shown
The results show that the SA and ASA derivatives have
enhanced anticancer activity in vitro attributed to their
increased cellular/mitochondrial accumulation driven by the
TPP carrier group. Such an enhancing effect of the TPP
group was earlier reported for other conjugates of natural
compounds [24–27,42]. Cytotoxicity of the SA and ASA
derivatives was generally proportional to the linker length

Medicinal Chemistry Research (2021) 30:925–939
929
previously for the phosphonium salts of poly-
hydroxybenzoates [27, 43]. The phosphonium salts of gallic
acid were reported to have IC50 values in the range
0.4–1.6 µM for TA3/Ha, TA3-MTX-R, CCRF-CEM cancer
cells [44] and to exhibit lack of toxicity for normal tissues
in vivo [37]. Furthermore, their cytotoxic activity was
associated with decrease in the transmembrane potential of
mitochondria (ΔΨm) due to weak uncoupling effect
[27, 37].
Cytotoxicity of mono- and di-OH benzoate decyl ester
derivatives was earlier studied on breast cancer cell lines;
the structure of polyhydroxybenzoate scaffold did not
noticeably affect the activity of phosphonium salts [27].
Among the compounds, GATPP+C10 caused time-
dependent inhibition of mitochondrial bioenergetics, cell
cycle arrest in G1 phase and cell death in 24 and 48 h [26].
depolarizing effect by 2.7–4.5 times in proportion to the
linker length from C3 to C10 (Fig. 1A).
In addition, the level of mitochondrial ROS, i.e., super-
oxide radical, in the treated cells was measured with the aid
of MitoSOX probe. Antimycin A was used as a model
inhibitor of the mitochondrial complex III to promote ROS
generation [45]. The prooxidant effect of the compounds
was found to increase by 1.5–4.5 times in the order (mean
channel fluorescence, a.u.): control (219) <3d (327) <7f
(544) <7b (674) <7d (738) < antimycin A (981) (Fig. 1B).
The compositions of antimycin A with the TPP conjugates
did not enhance the ROS level in comparison with anti-
mycin A alone.
Altogether, the results show that the ASA-TPP con-
jugates profoundly decrease ΔΨm of PC-3 cells in com-
parison with the reference compound (3d) apparently due to
increased mitochondrial accumulation, uncoupling effect on
the inner membrane and concurrent overproduction of
superoxide radical due to electrons leakage across the
electron transport chain. The effect of TPP conjugates on
ΔΨm was generally proportional to the linker length (Fig. 1A).
However, there was a tendency in lowering the prooxidant
effect for 7f (C10) over 7d (C6) (Fig. 1B).
Effect of ASA-TPP conjugates on mitochondrial
potential and ROS level
To assess the interaction of TPP conjugates with mito-
chondria selected ASA derivatives 7b, 7d, 7f, and 3d and
PC-3 cells were used. To reveal early effect on ΔΨm, the
cells was exposed to the compounds for 1 h in HBSS at a
concentration of 30 µM, which did not reduce cell viability
(data not shown). According to cytofluorometry, the com-
pounds induced decrease in ΔΨm at a different extent in the
order (mean channel fluorescence, a.u.): control (172) >3d
(135)>7b (64)>7d (56)>7f (38). Thus, ω-bromoalkyl ester
3d moderately affected ΔΨm (by 1.3 times relative to the
untreated cells), whereas the TPP conjugates induced the
The inhibitory activity of different TPP derivatives on
mitochondria functioning were previously demonstrated
[46–50]. The TPP cation itself was shown to interact with
and diffuse across the inner mitochondrial membrane in
proportion to the electrochemical gradient allowing protons
to leak into the matrix and interfering with oxidative
phosphorylation [25]. The alkyl chains enhanced the
activity of TPP cation so that the decyl and dodecyl TPP
A
Fig. 2 Effect of selected TPP
conjugates on DCFDA
fluorescence induced by CoCl2/
H2O2. A Concentration-
7a
7f
150
125
100
75
50
25
0
7e
8e
125
100
75
50
25
0
dependent inhibition of DCFDA
oxidation in the presence of
compounds. B Emission spectra
of preoxidized DCFDA and its
compositions with TPP
conjugates (25 µM). 5 µM
DCFDA in PBS was used. For A
fluorescence signal (mean SD,
n = 3) was presented in %
relative to control oxidation
reaction in the absence of
conjugates (100%)
0.1
1
10
100
0.1
1
10
100
Concentration (µM)
Concentration (µM)
x103
50
B
Ctrl
7a
7e
7f
40
30
20
10
0
8e
520
540
560
580
600
Wavelength (nm)

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Medicinal Chemistry Research (2021) 30:925–939
derivatives were even more powerful uncouplers than
FCCP [51]. A mild uncoupling effect was established for
SA (and its prodrugs) [15], which can diffuse across lipid
membranes in the acidic form [42] and therefore act a as
protonophor. Considering potential enzymatic lability of the
ester bond in the TPP conjugates (Scheme 2), both the
initial conjugates and their metabolites may be potentially
responsible for mitochondria depolarizing and prooxidant
effects.
complexes with different DLS characteristics compared
with the constituents, indicating the formation of mixed
aggregates. Their mean hydrodynamic diameter (HD) con-
siderably decreased from ca. 830 to 220 nm with the linker
elongation in ASA derivatives from C3 (7a) to C10 (7f),
along with increase in their uniformity and positive zeta
potential (ζ) from ca. +6 to +40 mV (Table 2). This sug-
gests that longer alkyl linkers, i.e., C9/C10, favor the inter-
action of TPP conjugates with probe molecules resulting in
more compact and cationic aggregates. Even smaller and
more cationic aggregates were observed in the case of SA
derivative with C9 linker (8e) (Table 2).
Interaction of TPP conjugates with DCFDA probe
DCFDA dye, which is sensitive to intracellular ROS [52],
was studied as a molecular and cellular probe for the TPP
conjugates. We found that the fluorescence of DCFDA in
solution induced by prooxidant CoCl2/H2O2 system [53]
was effectively inhibited in the presence of conjugates with
longest linkers, i.e., 7e, 7f, 8e, in the concentration range
1–100 µM unlike smaller homologs such as 7a (Fig. 2A). 7e
and 8e above 10 µM concentration similarly to each other
induced ~50% decrease in the DCFDA signal, whereas 7f
showed the highest inhibitory effect with EC50 of 5.0
0.6 µМ (Fig. 2A). The compounds, however, did not show
radical scavenging activity in the DPPH assay at a con-
centration up to 5 mM (data not shown).
Altogether, the data show direct linker-dependent inter-
action of the TPP conjugates with DCFDA. Such a com-
plexation may effectively quench the probe fluorescence as
observed for the compounds with C9 and C10 linkers (Fig. 2).
Furthermore, in compact aggregates (Table 2) DCFDA
molecules could be additionally protected from the oxida-
tion. These results reveal structure-dependent inter-
molecular interactions of the TPP derivatives with DCFDA,
which should be considered in DCFDA based assays and
also can be used to probe new TPP compounds.
Probing TPP conjugates inside cells with DCFDA
The results show that the most lipophilic TPP conjugates
interfere with the oxidation and/or fluorescence of DCFDA.
Therefore, DCFDA probe was initially preoxidized and then
mixed with the compounds at an effective concentration of
25 µM. Under these conditions, 8e and 7f noticeably
quenched the fluorescence of DCFDA, whereas 7a and 7e
only modulated the signal intensity (Fig. 2B). This indicates
that the inhibition of DCFDA fluorescence (Fig. 2A) may
result from the direct probe-compound interaction.
Inhibition of DCFDA fluorescence by the TPP conjugates is
of practical interest to assess penetration of the compounds
into living cells. To verify feasibility of such an analysis,
adhered PC-3 cells were preloaded with DCFDA and sub-
jected to oxidative stress to activate the probe. Among
different oxidative agents we selected CoCl2, which induces
metal-mediated prooxidant reactions and ROS formation
[54] as well as phenazine methosulfate (PMS), which
enhances superoxide radical production via mediating O2
reduction by NAD(P)H [55]. Both agents provided repro-
ducible concentration-dependent increase in intracellular
ROS formation, however, PMS was more effective at
saturated concentrations (Fig. S92). The effect of TPP
conjugates (5 and 25 µM) added to the extracellular solution
on the above reactions was studied (Fig. 3).
Association of the TPP conjugates with DCFDA was
assessed by dynamic light scattering (DLS) technique
(Table 2). The compositions produced well-defined
Table 2 DLS data for the mixed aggregates formed by DCFDA
(50 µM) and TPP conjugates (250 µM) in aqueous solution
Compound
HD (nm)
PDI
ζ (mV)
The data demonstrate that the compounds 7e, 7f, and 8e,
but not 7a, inhibited DCFDA oxidation in the cells in
proportion to the concentration similarly to that observed in
the cell-free assay (Fig. 2A). The fluorescence signal
decreased to as low as 40% (CoCl2) and 10% (PMS) of the
control value (100%), indicating more effective prevention
of PMS-mediated oxidation of DCFDA by the conjugates.
The results suggest that sufficiently lipophilic derivatives of
both ASA (7e, 7f) and SA (8e) freely penetrate the plasma
membrane (presumably due to passive diffusion), reaching
effective (µM) intracellular levels to interact with DCFDA
molecules and inhibit their oxidation. The elongation of
alkyl linker from C9 (7e) to C10 (7f) significantly augmented
DCFDA*
7a
2333.7 288.6
144.7 4.8
672.6 31.1
188.5 24.3
200.3 8.4
833.3 144.1
291.7 2.5
220.2 1.6
201.8 0.2
0.84 0.11
0.42 0.01
0.34 0.06
0.27 0.04
0.31 0.03
0.36 0.04
0.16 0.01
0.12 0.01
0.08 0.02
–20.8 0.7
–17.2 1.8
–8.6 0.2
7e
7f
+10.4 6.9
+15.6 0.15
+6.2 0.2
+36.4 1.4
+40.2 1.6
+43.4 1.7
8e*
7a + DCFDA
7e + DCFDA
7f + DCFDA
8e + DCFDA
*All systems except those indicated by asterisk were of good DLS
quality

Medicinal Chemistry Research (2021) 30:925–939
931
A
B
120
100
80
60
40
20
0
120
5 µM
25 µM
5 µM
25 µM
***
**
**
100
80
60
40
20
0
*
***
*
7a
7e
7f
8e
7a
7e
7f
8e
Fig. 3 Detection of TPP conjugates inside PC-3 cells using DCFDA
probe. Extracellular concentration of conjugates was 5 and 25 µM;
cells were pre-stained with DCFDA (20 µM) and subjected to
oxidative stress by (A) CoCl2 and (B) PMS. Fluorescence signal
(mean SD, n = 3) was presented in % relative to control cells (100%)
Table 3 MIC values (µM) of acetylsalicylic and salicylic acids
derivatives (microdilution assay, 24 h)
the inhibitory effect, probably, due to enhanced interaction
of 7f with the DCFDA probe. Furthermore, the results show
that DCFDA–TPP conjugate interaction is not inhibited in
the cell interior compared with buffer solution, indicating a
specific character of such a binding.
Compound
E. coli
S. aureus
7a
125
125
125
125
125
62.5
125
250
125
125
125
62.5
1.5
31.25
15.6
15.6
7.8
7b
7c
Antibacterial activity of TPP conjugates
7d
7e
0.98 (2)*
0.98 (2)*
250
Considering interrelationships between anticancer and
antibacterial properties of TPP compounds [28, 29, 56, 57],
the bacteriostatic effect of synthesized conjugates was
additionally studied. Table 3 shows MIC values of the ASA
and SA derivatives against E. coli ATCC 25922, S. aureus
ATCC 29213. The compounds effectively prevented
growth of S. aureus at µM concentrations but not E. coli in
accordance with increased drug resistance of Gram-negative
over Gram-positive bacteria due to specifics of cell wall
structure [58, 59].
7f
3d
1
500
8a
15.6
7.8
8b
8c
7.8
8e
3.9
Ciprofloxacin
Amikacin
Ceftazidime
1.5
6.7
0.8
Clear increase in the bacteriostatic activity from ca. 31 to
1 µM (7a–7f) and 16 to 4 µM (8a–8e) was observed upon
increase in the linker length of compounds. Both unmodi-
fied ASA and ω-bromoalkyl ester 3d similarly were almost
inactive (MIC = 125–500 µM), indicating that the anti-
bacterial effect of the conjugates is determined by the TPP
group. The most lipophilic ASA derivatives (7e, 7f, MIC =
1 µM) were four-times more effective than the SA deriva-
tive 8e. The linker elongation from C9 (7e) to C10 (7f) did
not augment the effect. Therefore, the antibacterial activity
of the TPP conjugates reaches a maximum for the nonyl
linker. The corresponding MIC was as low as 1 µM, which
was comparable to that of existing antibiotics (Table 3).
Given that IC50 values of 7e and 7f for HSF was 4–5-
times higher than the corresponding MIC (Tables 1 and 3),
the compounds can be considered as potential anti-
staphylococcus agents. Minimal bactericidal concentration
(MBC) values of 7e and 7f were 2 µM (Table 3), i.e., two
times higher than the corresponding MIC values, suggesting
bactericidal mode of their action [60]. Similar antibacterial
activity was previously reported for different
0.9
14.3
*The values in parentheses correspond to MBC (µM)
organophosphorous derivatives of natural compounds, e.g.,
ammonium salts of dithiophosphoric acids with pyridoxine
and nicotine (MIC = 10 µM) [60], TPP-pyridoxine (MIC =
5 μg/mL) [28] and TPP-triterpenoid (MIC = 2 µM) con-
jugates [29], carboxylate phosphabetaines derivatives (zone
of inhibition = 8–28 mm) [61]. The alkyl derivatives of
dimethylsubstituted and trimethylsubstituted phosphonium
salts were earlier shown to possess the antibacterial activity
per se, which exceeded that of the corresponding ammo-
nium salts [62].
The general mechanism of their activity may involve cell
wall and membrane damage by amphiphilic organopho-
sphorous groups. Therefore, we additionally assessed the
interaction of 7e with the bacterial surface by measuring ζ
of the suspended bacteria (Fig. S93). The mean ζ of the
untreated bacteria was –16.6 0.4 (E. coli) and –19.0
0.4 mV (S. aureus). Following brief exposition (1 h,

932
Medicinal Chemistry Research (2021) 30:925–939
250 μМ), 7e was found to decrease ζ of E. coli by almost
10 mV (to –5.9 0.3 mV, p < 0.001) and did not sig-
nificantly affect it for S. aureus (ζ = –18.6 0.6 mV). The
results may be explained by different mechanism of 7e
interaction with the surface of Gram-positive and Gram-
negative bacteria. In particular, the compounds are expected
to better retain at the cell wall of E. coli and more readily
penetrate across S. aureus surface.
Altogether, the data summarize linker length-dependent
antibacterial properties of the TPP conjugates of SA and
ASA (Table 3). Generally similar dependence was observed
for cytotoxic activity of the compounds against cancer cells
(Table 1). The nonyl and decyl linkers provide the maximal
activity of the compounds in vitro. Further linker elongation
is not advisable to design the corresponding TPP con-
jugates. While the effect of SA and ASA derivatives on
mammalian cells did not significantly differ, latter con-
jugates showed somewhat higher antibacterial activity
probably due to increased bacterial availability of the
acetylated SA derivatives.
Elemental analysis was accomplished with an automated
EuroVector EA3000 CHNS-O elemental analyzer (Euro-
Vector, Italy). The progress of reactions and the purity of
products were monitored by TLC on Sorbfil plates (IMID
Ltd., Russian Federation). The TLC plates were visualized
by treatment with phosphotungstic acid in ethanol, followed
by heating to 120 °C. The targeted compounds were iso-
lated using column chromatography on silica gel (60 A,
60–200 μm, Acros, Belgium). All solvents were dried
according to standard protocols.
General procedure for the synthesis of the
bromoalkane intermediates (3a–f, 4a, 4b, 4c, 4d, 4e)
and dimers (5a–f, 6a, 6b, 6c, 6d, 6e)
To a solution of benzoic acid 1, 2 (1.0 mmol) in DMF
(10 mL/g), MeCN (1 mL/g), α,ω-dibromoalkane (3.0 mmol),
and K2CO3 (1.0 mmol) were added. The reaction mixture
was stirred at 50 °C for the time indicated in each case. The
mixture was then poured into cold H2O (10× volume) and
extracted with CHCl3 (2 × 15 mL each). The combined
CHCl3 layers were washed with H2O (100 mL) and evapo-
rated. The crude residue was co-evaporated with H2O
(100 mL) to remove residual DMF. The product was purified
by column chromatography (petroleum ether (PE)/EtOAc;
150/10/50/50) to give compounds 3a–f, 4a, 4b, 4c, 4e and
not the 4-alkyloxy-substituted benzoic acid isomer.
Conclusion
TPP conjugates of SA and ASA can be regarded as pro-
mising anticancer compounds. In this work, the productive
synthesis of these conjugates was proposed. The structure of
compounds was optimized in respect of their cytotoxicity
for cancer and “normal” cells, mitochondria-disturbing and
antibacterial activities. Specific interactions of the TPP
conjugates with DCFDA, found in our study, are of interest
to assess the transportation of TPP compounds into mam-
malian and microbial cells and coaggregation of the com-
pounds under different conditions. The results encourage
further investigation of anticancer mechanisms of the
selected conjugates 7e and 7f.
3-Bromopropyl 2-(acetyloxy)benzoate, (3a)
Following the general procedure starting from 1,3-dibro-
mopropane (0.603 g, 3 mmol). Oil (159 mg, 53%). IR (KBr)
νmax: 2964, 2854, 1770, 1723, 1607, 1579, 1486, 1452,
1369, 1295, 1254, 1195, 1161, 1135, 1082, 1042, 1010,
1
960, 917, 876, 838, 815, 795, 753, 705, 672, 651 cm–1; H
NMR (CDCl3, 400 MHz): δ 2.24–2.31 (m, 2H), 2.34
(s, 3H), 3.51 (t, J = 6.5 Hz, 2H), 4.42 (t, J = 6.0 Hz, 2H),
7.1 (dd, J = 8.0, 0.9 Hz, 1H), 7.30 (ddd, J = 7.7, 7.7,
1.1 Hz, 1H), 7.56 (ddd, J = 8.0, 7.7, 1.7 Hz, 1H), 8.0 (dd,
J = 7.8, 1.6 Hz, 1H); anal. C, 47.74; H, 4.32; Br, 26.47%,
calcd for C12H13BrO4, C, 47.86; H, 4.35; Br, 26.53%.
Material and methods
All reagents and solvents were obtained from commercial
sources and used without further purification. Melting
points were determined on a Boetius compact heating table.
The IR spectra were recorded using a Bruker Tenzor
4-Bromobutyl 2-(acetyloxy)benzoate, (3b)
1
27 spectrometer. The H, 13C, and 31P NMR spectra were
Following the general procedure starting from 1,4-dibro-
mobutane (0.645 g, 3 mmol). Oil (179 mg, 57%). IR (KBr)
νmax: 2961, 2852, 1768, 1721, 1607, 1580, 1486, 1452,
1368, 1294, 1254, 1194, 1161, 1134, 1081, 1041, 1011,
959, 916, 877, 818, 794, 753, 704, 671, 650 cm–1; 1H NMR
(CDCl3, 400 MHz): δ 1.89–1.96 (m, 2H), 1.98–2.05(m,
2H), 2.36 (s, 3H), 3.47 (t, J = 6.5 Hz, 2H), 4.32 (t, J =
6.3 Hz, 2H), 7.12 (dd, J = 8.1, 1.0 Hz, 1H), 7.32 (ddd, J =
7.7, 7.5, 1.1 Hz, 1H), 7.57 (ddd, J = 7.5, 7.5, 1.7 Hz), 8.01
recorded using a Bruker Avance-400 NMR spectrometer.
Chemical shifts are referenced to the residual solvent peak
and reported in ppm (δ scale) and all coupling constant (J)
values are given in Hz. MALDI mass spectra were acquired
in the reflectron mode with Triton X-100 as a reference. A
mixture of Triton X-100 solution and analyte sample (1:1 v/v)
was used for internal calibration. 2,5-Dihydroxybenzoic
acid (5 mg/mL in methanol) was used as a matrix.

Medicinal Chemistry Research (2021) 30:925–939
933
1
(dd, J = 7.8, 1.7 Hz, 1H); anal. C 49.38; H 4.73, Br 25.36%,
calcd for С13H15BrO4, C 49.54; H 4.80, Br 25.35%.
752, 704 cm–1; H NMR (CDCl3, 400 MHz): δ 1.3–1.4 (m,
12H), 1.7–1.8 (m, 2H), 1.8–1.9 (m, 2H), 2.3 (s, 3H), 3.4 (t,
J = 6.8 Hz, 2H), 4.2 (t, J = 6.7 Hz, 2H), 7.1 (dd, J = 8.0,
1.0 Hz, 1H), 7.3 (ddd, J = 7.6, 7.6, 1.0 Hz, 1H), 7.57 (ddd,
J = 8.0, 8.0, 1.7 Hz, 1H), 8.0 (dd, J = 7.8, 1.7 Hz, 1H); anal.
C 57.0; H 6.78, Br 20.0%, calcd for С19H27BrO4, C 57.15;
H 6.82, Br 20.01%.
5-Bromopentyl 2-(acetyloxy)benzoate, (3c)
Following the general procedure starting from 1,5-dibro-
mopentane (0.690 g, 3 mmol). Oil (154 mg, 47%). IR (KBr)
νmax: 2944, 2867, 1770, 1720, 1607, 1579, 1485, 1452,
1368, 1294, 1261, 1195, 1161, 1134, 1082, 1041, 1010,
Nonane-1,9-diyl bis(2-(acetyloxy)benzoate), (4e)
1
959, 916, 877, 856, 816, 795, 753, 705 cm–1; H NMR
(CDCl3, 400 MHz): δ 1.5–1.6 (m, 2H), 1.7–1.8 (m, 2H),
1.9-2.0 (m, 2H), 2.36 (s, 3H), 3.44 (t, J = 6.7 Hz, 2H), 4.3
(t, J = 6.6 Hz, 2H), 7.1 (dd, J = 8.0, 1.0 Hz, 1H), 7.3 (ddd,
J = 7.7, 7.7, 1.2 Hz, 1H), 7.57 (ddd, J = 8.0, 8.0, 1.7 Hz,
1H), 8.01 (ddd, J = 7.8, 1.6 Hz, 1H); anal. C 51.0; H 5.18,
Br 24.20%, calcd for С14H17BrO4, C 51.08; H 5.21, Br
24.27%.
Colorless solid (121 mg, 25%); mp 67 °C. IR (KBr) νmax:
2932, 2852, 1758, 1719, 1608, 1486, 1468, 1451, 1371,
1302, 1257, 1218, 1202, 1157, 1136, 1083, 1013, 956, 922,
867, 818, 799, 755, 708 cm–1; 1H NMR (CDCl3, 400 MHz):
δ 1.3-1.4 (m, 10H), 1.7–1.8 (m, 4H), 2.3 (s, 3H), 4.3 (t, J =
6.7 Hz, 4H), 7.1 (dd, J = 8, 0.9 Hz, 2H), 7.3 (ddd, J = 7.6,
7.4, 0.7 Hz, 2H), 7.58 (ddd, J = 8.0, 7.6, 1.6 Hz, 2H), 8.0
(dd, J = 7.8, 1.6 Hz, 2H); 13C NMR (CDCl3, 100 MHz): δ
21.0, 25.9, 28.6, 29.2, 29.4, 65.2, 123.8, 126.0, 131.7,
133.8, 150.5, 164.5, 169.7; anal. C 66.90; H 6.60%, calcd
for С27H32O8, C 66.93; H 6.66%.
6-Bromohexyl 2-(acetyloxy)benzoate, (3d)
Following the general procedure starting from 1,6-dibro-
mohexane (0.731 g, 3 mmol). Oil (143 mg, 42%). IR (KBr)
νmax: 2938, 2861, 1771, 1722, 1607, 1580, 1485, 1452,
1368, 1294, 1258, 1196, 1160, 1134, 1083, 1042, 1010,
3-Bromopropyl 2-hydroxybenzoate, (4a)
1
958, 916, 876, 816, 795, 753, 705 cm–1; H NMR (CDCl3,
Following the general procedure starting from 1,3-dibro-
mopropane (0.603 g, 3 mmol). Oil (168 mg, 65%). IR (KBr)
νmax: 3190, 2966, 1677, 1614, 1585, 1485, 1467, 1396,
1328, 1301, 1251, 1214, 1159, 1137, 1092, 1032, 997, 903,
864, 801, 759, 701, 668 cm–1; 1H NMR (CDCl3, 400 MHz):
δ 2.35 (m, 2H), 3.53 (t, J = 6.5 Hz, 2H), 4.52 (t, J = 6 Hz,
2H), 6.90 (t, J = 7.2 Hz, 1H), 7.0 (d, J = 8.3 Hz, 1H), 7.48
(td, J = 7.7, 1.6 Hz, 1H), 7.84 (dd, J = 7.9, 1.6 Hz, 1H),
10.7 (s, 1H); anal. C, 46.24; H, 4.32; Br, 30.47%, calcd for
C10H11BrO3, C, 46.36; H, 4.28; Br, 30.84%.
400 MHz): δ 1.4–1.5 (m, 4H), 1.6–1.7 (m, 2H), 1.8–1.9 (m,
2H), 2.3 (s, 3H), 3.37 (t, J = 6.7 Hz, 2H), 4.25 (t, J =
6.6 Hz, 2H), 7.0 (dd, J = 8.0, 1.0 Hz, 1H), 7.27 (ddd, J =
7.7, 7.7, 1.1 Hz, 1H), 7.51 (ddd, J = 8.0, 8.0, 1.7 Hz, 1H),
8.0 (dd, J = 7.8, 1.5 Hz, 1H); anal. C 52.30; H 5.51, Br
23.20%, calcd for С15H19BrO4, C 52.49; H 5.58, Br
23.28%.
9-Bromononyl 2-(acetyloxy)benzoate, (3e)
Following the general procedure starting from 1,9-dibro-
mononane (0.572 g, 2 mmol). Oil (146 mg, 38%). IR (KBr)
νmax: 2930, 2856, 1772, 1722, 1608, 1579, 1485, 1452,
1368, 1294, 1259, 1195, 1160, 1134, 1082, 1041, 1010,
958, 915, 816, 753, 705 cm–1; 1H NMR (CDCl3, 400 MHz):
δ 1.3–1.4 (m, 10H), 1.7–1.8 (m, 2H), 1.8–1.9 (m, 2H), 2.3
(s, 3H), 3.4 (t, J = 6.8 Hz, 2H), 4.2 (t, J = 6.7 Hz, 2H), 7.1
(dd, J = 8, 0.9 Hz, 1H), 7.3 (ddd, J = 7.6, 7.6, 1.0 Hz, 1H),
7.54 (ddd, J = 8.0, 8.0, 1.7 Hz, 1H), 8.0 (dd, J = 7.8,
1.6 Hz, 1H); anal. C 55.87; H 6.51, Br 20.73%, calcd for
С18H25BrO4, C 56.11; H 6.54, Br 20.74%.
4-Bromobutyl 2-hydroxybenzoate, (4b)
Following the general procedure starting from 1,4-dibro-
mobutane (0.645 g, 3 mmol). Oil (172 mg, 63%). IR (KBr)
νmax: 3187, 2962, 2924, 1675, 1614, 1586, 1468, 1469,
1395, 1326, 1302, 1250, 1215, 1158, 1138, 1092, 1033,
1
867, 801, 758, 701 cm–1; H NMR (CDCl3, 400 MHz): δ
1.9–2.0 (m, 4H), 3.5 (t, J = 6.5 Hz, 2H), 4.4 (t, J = 6 Hz,
2H), 6.90 (ddd, J = 8.0, 8.0, 1.0 Hz, 1H), 7.0 (dd, J = 8.4,
0.8 Hz, 1H), 7.5 (ddd, J = 7.8, 7.8, 1.7 Hz, 1H), 7.8 (dd,
J = 8.0, 1.7 Hz, 1H), 10.7 (s, 1H); anal. C, 48.24; H, 4.80;
Br, 28.74%, calcd for C11H13BrO3, C, 48.37; H, 4.80; Br,
29.26%.
10-Bromodecyl 2-(acetyloxy)benzoate, (3f)
Following the general procedure starting from 1,10-dibro-
5-Bromopentyl 2-hydroxybenzoate, (4c)
modecane (0.6 g, 2 mmol). Oil (140 mg, 35%). IR (KBr) νmax
:
2930, 2855, 1772, 1722, 1608, 1485, 1452, 1368, 1294,
1258, 1196, 1160, 1134, 1082, 1041, 1010, 958, 915, 816,
Following the general procedure starting from 1,5-dibro-
mopentane (0.690 g, 3 mmol). Oil (192 mg, 67%). IR (KBr)

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Medicinal Chemistry Research (2021) 30:925–939
νmax: 3185, 2941, 2865, 1673, 1614, 1586, 1486, 1467,
1400, 1326, 1302, 1251, 1215, 1158, 1138, 1091, 1033,
954, 866, 801, 758, 702 cm–1; 1H NMR (CDCl3, 400 MHz):
δ 1.6–1.7 (m, 2H), 1.8–1.9 (m, 2H), 1.9-2.0 (m, 2H), 3.4 (t,
J = 6.7 Hz, 2H), 4.3 (t, J = 6.5 Hz, 2H), 6.80 (ddd, J = 8.0,
8.0, 1.0 Hz, 1H), 7.0 (dd, J = 8.3, 0.8 Hz, 1H), 7.5 (ddd, J
= 7.8, 7.8, 1.7 Hz, 1H), 7.8 (dd, J = 8.0, 1.7 Hz, 1H), 10.8
(s, 1H); anal. C, 49.75; H, 5.25; Br, 27.64%, calcd for
С12H15BrO3, C, 50.19; H, 5.27; Br, 27.83%.
General procedure for synthesis of TPP-conjugates
of acetylsalicylic (7a–f) and salicylic acids (8a–e)
Triphenylphosphine (4-6 mmol) was added to the solu-
tion of bromide derivatives (3a-f, 4a-e) (1 mmol) in dry
acetonitrile under argon, and the mixture was stirred
under reflux for 6–8 h until the reaction was complete
according to TLC detection. Acetonitrile was removed
under reduced pressure, and the precipitate was washed
with hot petroleum ether (3 × 5 mL) and dissolved in
ethyl acetate. Petroleum ether (5 mL) was added to the
reaction mixture. The resulting precipitate was washed
with diethyl ether (3 mL) and dried in vacuo to give the
pure TPP conjugate.
6-Bromohexyl 2-hydroxybenzoate, (4d)
Following the general procedure starting from 1,6-
dibromohexane (0.731 g, 3 mmol). Oil (189 mg, 63%).
IR (KBr) νmax: 3185, 2941, 2865, 1673, 1614, 1586,
1486, 1467, 1400, 1326, 1302, 1251, 1215, 1158, 1138,
1091, 1033, 954, 866, 801 cm–1 H NMR (CDCl3,
400 MHz): δ 1.3–1.4 (m, 4H), 1.6–1.7 (m, 4H), 3.2 (m,
Hz, 2H), 4.2 (t, J = 6.5 Hz, 2H), 6.73 (dd, J = 12.5,
7.3 Hz, 1H), 6.8 (d, J = 8.4 Hz, 1H), 7.3 (t, J = 8.0 Hz,
1H), 7.7 (d, J = 7.9 Hz, 1H), 10.7 (s, 1H); anal. C, 51.77;
H, 5.65; Br, 26.48%, calcd for С13H17BrO3, C, 51.84; H,
5.69; Br, 26.53%.
(3-((2-(acetyloxy)benzoyl)oxy)propyl)
triphenylphosphonium bromide, (7a)
Oil; yield 92% 517 mg; IR (KBr) νmax: 2964, 2854, 1770,
1723, 1607, 1579, 1486, 1452, 1369, 1295, 1254, 1195,
1161, 1135, 1082, 1042, 1010, 960, 917, 876, 815, 753,
705, 672, 651 cm–1; 1H NMR (CDCl3, 400 MHz): δ 2.1 (m,
2H), 2.3 (s, 3H), 4.0 (m, 2H), 4.7 (t, J = 5.7 Hz, 2H), 7.0 (d,
J = 8.0 Hz, 1H), 7.3 (t, J = 7.4 Hz, 1H), 7.5 (t, J = 7.3 Hz,
1H), 7.6–7.8 (m, 15H), 7.9 (d, 7.7 = Hz, 1H); 13C NMR
(CDCl3, 100 MHz): 19.7 (d, J = 52.7 Hz), 21.3, 22.5, 64.07
(d, J = 17.7 Hz), 118.0 (d, J = 86.3 Hz), 123.1, 126.1, 130.5
(d, J = 12.5 Hz), 131.8, 133.8 (d, J = 10.0 Hz), 134.1, 135.1
(d, J = 3.0 Hz), 150.6, 164.2, 170.0; 31P NMR (CDCl3,
162 MHz): δ 24.8; anal. C, 63.09; H, 4.84; Br, 14.05; P,
5.43%, calcd for C30H28BrO4P, C, 63.95; H, 5.01; Br,
14.18; P, 5.50%.
9-Bromononyl 2-hydroxybenzoate, (4e)
Following the general procedure starting from 1,9-dibro-
mononane (0.572 g, 2 mmol). Oil (147 mg, 43%). IR (KBr)
νmax: 3181, 2930, 2856, 1674, 1614, 1586, 1486, 1467,
1396, 1326, 1302, 1251, 1214, 1187, 1158, 1138, 1091,
1
1033, 954, 758, 702 cm–1; H NMR (CDCl3, 400 MHz): δ
1.3–1.4 (m, 10H), 1.7–1.9 (m, 2H), 3.4 (t, J = 6.8 Hz, 2H),
4.3 (t, J = 6.6 Hz, 2H), 6.9 (ddd, J = 8.0, 8.0, 1.0 Hz, 1H),
7.0 (dd, J = 8.3, 0.8 Hz, 1H), 7.5 (ddd, J = 7.8, 7.8, 1.7 Hz,
1H), 7.9 (dd, J = 8.0, 1.7 Hz, 1H), 10.8 (s, 1H); anal. C,
55.57; H, 6.65; Br, 23.11%, calcd for С16H23BrO3, C,
55.99; H, 6.75; Br, 23.28%.
(4-((2-(acetyloxy)benzoyl)oxy)butyl)
triphenylphosphonium bromide, (7b)
Oil; yield 90% 519 mg; IR (KBr) νmax: 2866, 1764, 1717,
1606, 1485, 1438, 1368, 1295, 1255, 1196, 1161, 1113,
1085, 996, 752, 723, 690 cm–1; 1H NMR (CDCl3,
400 MHz): δ 1.7–1.8 (m, 2H), 2.2–2.25 (m, 2H), 2.3 (s,
3H), 3.9–4.0 (m, 2H), 4.35 (t, J = 6.0 Hz, 2H), 7.1 (dd, J =
8.0, 1.0 Hz, 1H), 7.27 (ddd, J = 7.7, 7.6, 1.0 Hz, 1H), 7.6
(ddd, J = 7.7, 7.7, 1.7 Hz, 1H), 7.6-7.9 (m, 16H); 13C NMR
(CDCl3, 100 MHz): 19.6 (d, J = 3.9 Hz), 21.0, 22.1 (d, J =
51 Hz), 28.9 (d, J = 16.9 Hz), 63.6, 118.0 (d, J = 88.8 Hz),
123.7, 125.9, 130.4 (d, J = 12.6 Hz), 131.4, 133.6 (d, J =
10.2 Hz), 133.8, 135.0 (d, J = 3.0 Hz), 150.6, 164.2, 169.6;
31P NMR (CDCl3, 162 MHz): δ 24.3; MALDIMS, m/z 497
[M–Br]+ (calcd m/z 497.19 [M–Br]+ for C31H30O4P); anal.
C, 63.87; H, 5.16; Br, 13.63; P, 5.28%, calcd for
C31H30BrO4P, C, 64.48; H, 5.24; Br, 13.84; P, 5.36%.
Nonane-1,9-diyl bis(2-hydroxylbenzoate), (6e)
Colorless solid (72 mg, 18%) mp 56 °C. IR (KBr) νmax
:
3143, 2964, 2925, 2856, 1678, 1614, 1587, 1488, 1468,
1399, 1325, 1302, 1252, 1215, 1194, 1157, 1134, 1092,
1033, 994, 950, 869, 815, 767, 757, 724, 695, 667 cm–1; 1H
NMR (CDCl3, 400 MHz): δ 1.3–1.4 (m, 10H), 1.7–1.8 (m,
4H), 4.35 (t, J = 6.6 Hz, 4H), 6.9 (t, J = 7.3 Hz, 2H), 7.0 (d,
J = 8.0 Hz, 2H), 7.5 (td, J = 8.6 1.6 Hz, 2H), 7.8 (dd, J =
8.0, 1.5 Hz, 2H); 10.8 (s, 2H); 13C NMR (CDCl3,
100 MHz): δ 25.9, 28.5, 29.1, 29.4, 65.4, 117.5, 119.0,
129.8, 135.5, 161.7, 170.2; anal. C 68.8; H 6.9%, calcd for
С23H28O6, C 68.98; H 7.05%.

Medicinal Chemistry Research (2021) 30:925–939
935
(5-((2-(acetyloxy)benzoyl)oxy)pentyl)
triphenylphosphonium bromide, (7c)
121.2, 123.5, 128.0 (d, J = 12.5 Hz), 129.2, 131.1 (d, J =
10.0 Hz), 131.2, 132.4 (d, J = 2.7 Hz), 148.1, 162.0, 167.1;
31P NMR (CDCl3, 162 MHz): δ 24.3; MALDIMS, m/z 567
[M–Br]+ (calcd m/z 567.69 [M–Br]+ for C36H40O4P); anal.
C, 66.58; H, 6.07; Br, 12.19; P, 4.62%, calcd for
C36H40BrO4P, C, 66.77; H, 6.23; Br, 12.34; P, 4.78%.
Oil; yield 93% 550 mg; IR (KBr) νmax: 2907, 1766, 1718,
1607, 1588, 1485, 1451, 1439, 1368, 1295, 1257, 1197,
1162, 1114, 1083, 1041, 1011, 997, 918, 751, 724,
1
691 cm–1; H NMR (CDCl3, 400 MHz): δ 1.6–1.8 (m, 6H),
2.3 (s, 3H), 3.8–3.9 (m, 2H), 4.2 (t, J = 6.0 Hz, 2H), 7.0
(dd, J = 8.0, 0.9 Hz, 1H), 7.29 (ddd, J = 7.6, 7.6, 1.0 Hz,
1H), 7.54 (ddd, J = 7.5, 7.9, 1.7 Hz, 1H), 7.6-7.9 (m, 15H),
7.95 (dd, J = 8.0, 0.9 Hz, 1H); 13C NMR (CDCl3,
100 MHz): 21.1, 22.2 (d, J = 3.8 Hz), 22.8 (d, J = 50.3 Hz),
26.8 (d, J = 16.2 Hz), 28.1, 64.5, 118.2 (d, J = 86.0 Hz),
123.5, 126.7, 130.5 (d, J = 12.5 Hz), 131.7, 133.6 (d, J =
10.0 Hz), 133.8, 135.0 (d, J = 3.0 Hz), 150.6, 164.3, 169.6;
31P NMR (CDCl3, 162 MHz): δ 24.5; MALDIMS, m/z 511
[M–Br]+ (calcd m/z 511.58 [M–Br]+ for C32H32O4P); anal.
C, 64.79; H, 5.31; Br, 13.41; P, 5.18%, calcd for
C32H32BrO4P, C, 64.98; H, 5.45; Br, 13.51; P, 5.24%.
(10-((2-(acetyloxy)benzoyl)oxy)decyll)
triphenylphosphonium bromide, (7f)
Oil; yield 85% 561 mg IR (KBr) νmax: 3392, 2928, 2856,
1768(С=О), 1719, 1606, 1587, 1485, 1438, 1368, 1294,
1258, 1196, 1161, 1114, 1082, 1041, 1010, 997, 918, 794,
752, 724, 692 cm–1; 1H NMR (CDCl3, 400 MHz): δ 1.2–1.3
(m, 10H), 1.6–1.7 (m, 6H), 2.3 (s, 3H), 3.73 (m, 2H), 4.2
(t, J = 6.7 Hz, 2H), 7.0 (dd, J = 8.0, 0.9 Hz, 1H), 7.3 (ddd,
J = 7.8, 7.7, 0.7 Hz 1H), 7.5 (ddd, J = 8.0, 8.0, 1.7 Hz, 1H),
7.6–7.8 (m, 15H), 7.9 (dd, J = 7.9, 1.6 Hz, 1H); 13C NMR
(CDCl3, 100 MHz): 21.0, 22.5 (d, J = 52.5 Hz), 22.7, 25.8,
28.6, 29.1, 29.3, 30.3 (d, J = 15.3 Hz), 65.2, 118.4 (d, J =
85.7 Hz), 123.5, 123.7, 126.0, 130.5 (d, J = 12.4 Hz),
131.7, 133.7 (d, J = 9.4 Hz), 135.0, 135.0, 150.6, 164.5,
169.6; 31P NMR (CDCl3, 162 MHz): δ 24.9; anal. C, 68.93;
H, 6.27; Br, 11.56; P, 4.59%, calcd for C37H42BrO4P, C,
67.17; H, 6.40; Br, 12.08; P, 4.68%.
(6-((2-(acetyloxy)benzoyl)oxy)hexyl)
triphenylphosphonium bromide, (7d)
Oil; yield 91% 550 mg IR (KBr) νmax: 2936, 2863, 1767,
1718, 1607, 1588, 1485, 1451, 1439, 1368, 1295, 1260,
1197, 1161, 1114, 1082, 1042, 997, 922, 751, 725,
692 cm–1; 1H NMR (CDCl3, 400 MHz): δ 1.4 (m, 2H),
1.6–1.7 (m, 6H), 2.3 (s, 3H), 3.7–3.8 (m, 2H), 4.2 (t, J =
6.4 Hz, 2H), 7.0 (d, J = 8.0 Hz, 1H), 7.3 (m, 1H), 7.5 (t,
J = 8.4 Hz, 1H), 7.6–7.8 (m, 15H), 7.9 (d, J = 7.8 Hz, 1H);
13C NMR (CDCl3, 100 MHz): 21.07, 22.6, 22.75 (d, J =
50.2 Hz), 25.5, 28.3, 29.8 (d, J = 15.7 Hz), 64.9, 118.3 (d,
J = 85.8 Hz), 123.4, 126.0, 130.5 (d, J = 12.1 Hz), 131.7,
133.6 (d, J = 10.0 Hz), 133.7, 135.0 (d, J = 3.0 Hz), 150.6,
164.4, 169.6; 31P NMR (CDCl3, 162 MHz): δ 24.8; MAL-
DIMS, m/z 525 [M–Br]+ (calcd m/z 525.6 [M–Br]+ for
C33H34O4P); anal. C, 65.28; H, 5.42; Br, 13.05; P, 5.18%,
calcd for C33H34BrO4P, C, 65.46; H, 5.66; Br, 13.20;
P, 5.00%.
(3-((2-hydroxybenzoyl)oxy)propyl)
triphenylphosphonium bromide, (8a)
Oil; yield 91% 474 mg; IR (KBr) νmax: 3190, 2966, 1677,
1614, 1585, 1485, 1467, 1396, 1328, 1301, 1251, 1214,
1159, 1137, 1092, 1032, 997, 903, 864, 801, 759, 701,
668 cm–1; 1H NMR (CDCl3, 400 MHz): δ 2.1 (m, 2H), 4.1
(m, 2H), 4.7 (t, J = 6.2 Hz, 2H), 6.8 (td, J = 8.0, 1.0 Hz,
1H), 6.9 (dd, J = 8.3, 0.6 Hz 1H), 7.4 (ddd, J = 7.8, 7.7,
1.7 Hz, 1H), 7.6–7.8 (m, 16H), 10.5 (s, 1H); 13C NMR
(CDCl3, 100 MHz): 19.8 (d, J = 53.0 Hz), 22.2 (d, J =
3.1 Hz), 64.3 (d, J = 17.8 Hz), 112.0, 117.5, 118.0 (d,
J = 86.3 Hz), 119.3, 129.9, 130.6 (d, J = 12.6 Hz), 133.6
(d, J = 10.6 Hz), 135.2 (d, J = 3.3 Hz), 135.9, 161.6,
169.7; 31P NMR (CDCl3, 162 MHz): δ 26.0; MALDIMS,
m/z 441 [M–Br]+ (calcd m/z 441.16 [M–Br]+ for
C28H26O3P); anal. C, 64.37; H, 4.89; Br, 15.09; P, 5.82%,
calcd for C28H26BrO3P, C, 64.50; H, 5.03; Br, 15.33;
P, 5.94%.
(9-((2-(acetyloxy)benzoyl)oxy)nonyl)
triphenylphosphonium bromide, (7e)
Oil; yield 87% 562 mg IR (KBr) νmax: 2928, 2857, 1767,
1719, 1670, 1608, 1587, 1485, 1438, 1368, 1296, 1251,
1197, 1159, 1114, 1084, 997, 922, 918, 750, 723, 691 cm–1;
1H NMR (CDCl3, 400 MHz): δ 1.2–1.3 (m, 8H), 1.6–1.7
(m, 6H), 2.3 (s, 3H), 3.75 (m, 2H), 4.2 (t, J = 6.7 Hz, 2H),
7.0 (dd, J = 8.0, 0.9 Hz, 1H), 7.3 (ddd, J = 7.8, 7.7, 0.7 Hz
1H), 7.5 (ddd, J = 8.0, 8.0, 1.7 Hz, 1H), 7.6-7.8 (m, 15H),
8.0 (dd, J = 7.9, 1.6 Hz, 1H); 13C NMR (CDCl3, 100 MHz):
18.5, 20.1, 20.3 (d, J = 48 Hz), 23.3, 26.0, 26.5, 26.5, 27.8
(d, J = 15.5 Hz), 28.4, 62.7, 115.9 (d, J = 85.8 Hz), 121.0,
(4-((2-hydroxybenzoyl)oxy)butyl)
triphenylphosphonium bromide, (8b)
Oil; yield 92% 492 mg; IR (KBr) νmax: 3353, 1664, 1610,
1484, 1439, 1396, 1328, 1298, 1248, 1222, 1159, 1113,
1
771, 751, 723, 691 cm–1; H NMR (CDCl3, 400 MHz): δ
1.7–1.8 (m, 2H), 2.1-2.3 (m, 2H), 3.9–4.0 (m, 2H), 4.4 (t,

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Medicinal Chemistry Research (2021) 30:925–939
J = 6.0 Hz, 2H), 6.8 (ddd, J = 7.5, 7.6, 1.0 Hz, 1H), 6.9 (dd,
J = 8.3, 0.6 Hz 1H), 7.4 (ddd, J = 7.3, 7.2, 1.7 Hz, 1H), 7.6
(dd, J = 8.0, 1.6 Hz, 1H), 7.6–7.9 (m, 15H), 10.6 (s, 1H);
13C NMR (CDCl3, 100 MHz): 19.1 (d, J = 3.8 Hz), 22.1 (d,
J = 50.8 Hz), 28.9 (d, J = 16.9 Hz), 63.9, 112.2, 117.5,
118.5 (d, J = 86.0 Hz), 119.1, 129.7, 130.5 (d, J = 12.5 Hz),
133.6 (d, J = 10.0 Hz), 135.0 (d, J = 3.0 Hz), 135.7, 161.5,
169.9; 31P NMR (CDCl3, 162 MHz): δ 24.6; MALDIMS,
m/z 455 [M–Br]+ (calcd m/z 455.52 [M–Br]+ for
C29H28O3P); anal. C, 64.91; H, 5.13; Br, 14.83; P, 5.69%,
calcd for C29H28BrO3P, C, 65.06; H, 5.27; Br, 14.92;
P, 5.78%.
(9-((2-hydroxybenzoyl)oxy)nonyl)
triphenylphosphonium bromide, (8e)
Oil; yield 87% 526 mg; IR (KBr) νmax: 3386, 2927, 2856,
1670, 1613, 1587, 1485, 1466, 1438, 1397, 1327, 1300,
1249, 1216, 1159, 1114, 1091, 1031, 996, 751, 723,
691 cm–1; 1H NMR (CDCl3, 400 MHz): δ 1.2–1.3 (m, 10H),
1.6-1.7 (m, 4H), 3.7 (m, 2H), 4.3 (t, J = 6.6 Hz, 2H), 6.8
(dd, J = 7.8, 7.3, Hz, 1H), 6.9 (dd, J = 8.3, 0.6 Hz, 1H), 7.4
(dd, J = 7.2, 7.2, Hz, 1H), 7.6–7.8 (m, 16H), 10.7 (s, 1H);
13C NMR (CDCl3, 100 MHz): 22.6, 23.0 (d, J = 50.0 Hz),
25.8, 28.4, 29.0, 30.3 (d, J = 15.5 Hz) 65.4, 112.6, 117.4,
118.3 (d, J = 85.8 Hz), 119.1, 129.9, 130.5 (d, J =
12.5 Hz),133.6 (d, J = 10.0 Hz), 135.0 (d, J = 3.0 Hz),
135.5, 161.65 170.2; 31P NMR (CDCl3, 162 MHz): δ 25.3;
MALDIMS, m/z 525 [M–Br]+ (calcd m/z 525.65 [M–Br]+
for C34H38O3P); anal. C, 67.32; H, 6.21; Br, 13.08; P,
4.99%, calcd for C34H38BrO3P, C, 67.44; H, 6.33; Br,
13.20; P, 5.11%.
(5-((2-hydroxybenzoyl)oxy)pentyl)
triphenylphosphonium bromide, (8c)
Oil; yield 90% 494 mg; IR (KBr) νmax: 3185, 2941, 2865,
1673, 1614, 1586, 1486, 1400, 1326, 1302, 1251, 1215,
1158, 1138, 1090, 1033, 954, 866, 801, 758, 731, 702,
667 cm–1; 1H NMR (CDCl3, 400 MHz): δ 1.7 (m, 2H), 1.8
(m, 4H), 3.9–4.0 (m, 2H), 4.3 (t, J = 6.0 Hz, 2H), 6.8
(ddd, J = 8.0, 8.0, 1.0 Hz, 1H), 6.9 (dd, J = 8.3, 0.6 Hz,
1H), 7.4 (ddd, J = 7.8, 7.8, 1.7 Hz, 1H), 7.6 (dd, J = 8.0,
1.6 Hz, 1H), 7.4–7.9 (m, 15H), 10.7 (s, 1H); 13C NMR
(CDCl3, 100 MHz): 22.3, 22.9 (d, J = 50.0 Hz), 26.8 (d,
J = 16.1 Hz), 28.1, 64.8, 112.5, 117.4, 118.8 (d, J =
86.0 Hz), 119.2, 129.9, 130.5 (d, J = 12.5 Hz), 133.8 (d,
J = 10.0 Hz), 135.0 (d, J = 3.0 Hz), 135.6, 161.6, 170.1;
31P NMR (CDCl3, 162 MHz): δ 24.6; MALDIMS, m/z
469.0 [M–Br]+ (calcd m/z 469.54 [M–Br]+ for
C30H30O3P); anal. C, 65.39; H, 5.42; Br, 14.45; P, 5.58%,
calcd for C30H30BrO3P, C, 65.58; H, 5.50; Br, 14.54;
P, 5.64%.
Biological assay
2′,7′-dichlorofluorescin diacetate (DCFDA), 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2′-
diphenyl-1-picrylhydrazyl (DPPH), tetramethylrhodamine
ethyl ester perchlorate (TMRE), antimycin A, phenazine
methosulfate (PMS) were purchased from Sigma-Aldrich.
MitoSOX Red Mitochondrial Superoxide Indicator was
purchased from ThermoFisher Scientific. Milli-Q grade
water (Milli-Q Advantage A10, Merck Millipore) was used
to prepare buffers and solutions. Materials for cell culturing
were obtained from PanEco company (Russia).
MCF-7, PC-3, and Caco-2 cancer cell lines (ATCC) were
used. The cells were cultured aseptically in DMEM con-
taining 10% fetal bovine serum (FBS), 2 mM L-glutamine,
100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C
in humidified air atmosphere with 5% CO2. Human skin
fibroblasts (HSF) were isolated as described earlier [35].
HSF were cultured under the same conditions but in α-
MEM with 10% FBS.
(6-((2-hydroxybenzoyl)oxy)hexyl)
triphenylphosphonium bromide, (8d)
Colorless solid (462 mg, 82%); mp 132–133 °C; IR (KBr)
νmax: 3437, 2936, 2864, 2798, 1672, 1612, 1587, 1484,
1466, 1439, 1394, 1324, 1298, 1252, 1215, 1197, 1159,
1134, 1112, 1089, 993, 950, 889, 868, 748, 722, 690 cm–1;
1H NMR (CDCl3, 400 MHz): δ 1.4–1.5 (m, 2H), 1.6–1.7
(m, 2H), 1.7–1.8 (m, 4H), 3.8 (m, 2H), 4.3 (t, J = 6.4 Hz,
2H), 6.9 (t, J = 7.6 Hz, 1H), 7.0 (d, J = 8.3 Hz, 1H), 7.4 (t,
J = 7.4 Hz, 1H), 7.7–7.8 (m, 16H), 10.8 (s, 1H); 13C NMR
(CDCl3, 100 MHz): 22.17, 23.1 (d, J = 50.0 Hz), 25.2, 27.8,
29.6 (d, J = 16.0 Hz), 64.9, 112.0, 116.9, 117.7 (d, J =
85.8 Hz), 118.9, 129.6, 130.3 (d, J = 12.5 Hz), 133.3 (d,
J = 9.2 Hz), 134.8, 135.4, 161.0, 169.8; 31P NMR (CDCl3,
162 MHz): δ 24.1; anal. C, 65.92; H, 5.64; Br, 14.03; P,
5.41%, calcd for C31H32BrO3P, C, 66.08; H, 5.72; Br,
14.18; P, 5.50%.
Cytotoxicity study
Cytotoxic activity of the compounds was evaluated by means
of the MTT proliferation assay as detailed previously [35]
using an Infinite 200 PRO microplate analyzer (TECAN).
Stock solutions of the compounds were prepared in DMSO
and re-dissolved in HBSS; the cytotoxicity of residual
amounts of DMSO was verified. The cells were cultured in
the presence of compounds in the concentration range from
48.9 nM to 100 µM for 72 h. Сell viability was presented
relative to control cells grown without compounds (100%
viability). Half-maximal inhibitory concentrations (IC50)

Medicinal Chemistry Research (2021) 30:925–939
937
were calculated from concentration–viability relationships
using OriginPro software.
turbidity/precipitate and, in addition, by the change in color
of phenol red indicator (18 µg/mL). Minimal inhibitory
concentration (MIC) values were determined as minimal
concentrations of a compound prevented bacteria growth.
To determine minimal bactericidal concentration (MBC), an
aliquot of the treated bacterial culture was transferred onto
Mueller–Hinton agar. MBC was the minimal concentration
at which bacterial colonies were not detected.
DPPH and DCFDA assays
The measurements were performed in a 96-well plate on an
Infinite 200 PRO microplate analyzer. In the DPPH-assay
serially diluted compounds were mixed with 0.25 mM
DPPH in PBS supplemented with Triton X100 (8 mg/mL)
followed by the colorimetric detection of the unreacted
DPPH at λ = 515 nm [53]. The Fenton system used to
oxidize DCFDA (5 µM) fluorescent probe comprised
0.23 mM CoCl2 and 22 mM H2O2 in PBS. The fluorescence
intensity of DCFDA upon oxidation was detected at λex =
488 nm and λem = 535 nm [53]. The effect of compounds on
the DCFDA was evaluated in the concentration range
0.1–100 µM signal.
For cell based DCFDA assay pre-grown PC-3 cells were
stained with 20 µM DCFDA for 30 min, washed with PBS
and subjected to the oxidative stress by addition of CoCl2
(0.002–4 mM) or PMS (0.003–0.1 mM) in PBS followed by
the detection of bottom fluorescence at λex/λem = 488/
535 nm. The compounds were added to the extracellular
solution at a concentration of 25 µM.
Dynamic light scattering (DLS) analysis
The HD, particle dispersion index (PDI), and ζ of molecular
aggregates of DCFDA (50 µM), the compounds (250 µM)
and their compositions in milliQ water were measured with
the aid of DLS technique on a Zetasizer NanoZS analyzer
(Malvern Instruments). To determine ζ of bacteria, the night
culture was diluted with PBS at 109 CFU/mL and mixed
with a compound (1 mM) or PBS (control) followed by
incubation for 1 h at 37 °C upon shaking. The treated bac-
teria were collected by centrifugation, resuspended in
50 mM HEPES buffer (pH = 7) and subjected to the
analysis.
Statistical analysis
Analysis of mitochondrial potential and ROS
Quantitative data were presented as mean SD (n ≥ 3). The
normality of the data distribution and the significance of
differences were assessed using GraphPad Prism 5.0 soft-
ware (Student’s t test, p < 0.05).
The analysis was performed on a BD FACSCalibur flow
cytometer (BD Biosciences). Briefly, PC-3 cells were col-
lected with the aid of trypsin-EDTA dissociation solution,
washed with and suspended in HBSS at a density of 106
cells/mL. Then the cells were mixed with the compounds
(30 µM) and incubated for 1 h at 37 °C in the CO2-incu-
bator. The treated cells were stained with 0.1 µM TMRE for
20 min at 37 °С to analyze transmembrane potential of
mitochondria. To evaluate mitochondrial ROS 5 µM Mito-
SOX was added to the cell suspension and incubated for
20 min at 37 °С in the CO2-incubator. The prestained cells
were diluted to a density of 3 × 105 cells/mL, then mixed
with the synthesized compounds (30 µM) and/or antymicin
A (10 µM) and incubated at 37 °С for 1 h. Cell viability was
additionally verified by bright-filed microscopy and the
MTT assay in parallel samples.
Acknowledgements This work was supported by the Ministry of
Education and Science of the Russian Federation (subsidies allocated
to FRC Kazan Scientific Center of RAS and Kazan Federal University
for the state assignments in the sphere of scientific activities (0671-
2020-0063)), the Russian Foundation for Basic Research (grant no. 20-
33-70194), and also performed according to the Russian Government
Program of Competitive Growth of Kazan Federal University. RI
acknowledges RFBR project No 19-34-90139 (analysis of oxidative
stress in cell monolayers). The authors gratefully acknowledge the
CSF-SAC FRC KSC RAS and Interdisciplinary Centre for Shared Use
of Kazan Federal University.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Study of antibacterial activity
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Escherichia coli and Staphylococcus aureus ATCC 29213
were used. Bacteriostatic activity was studied by the
microdilution method in Mueller–Hinton broth [60]. Sterile
serially diluted compounds (250–0.5 µM) were mixed with
the bacterial inoculum in 96-well plates and incubated for
24 h at 37 °C under moderate shaking. Bacterial growth was
analyzed visually in three parallel wells by appearance of
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